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Role of Calcium Signaling in GA101-Induced Cell Death in Malignant Human B Cells

GA101/obinutuzumab is a novel type II anti-CD20 monoclonal antibody (mAb), which is more effective than rituximab (RTX) in preclinical and clinical studies when used in combination with chemotherapy. Ca2+ signaling was shown to play a role in RTX-induced cell death. This report concerns the effect of GA101 on Ca2+ signaling and its involvement in the direct cell death induced by GA101. We reveal that GA101 triggered an intracellular Ca2+ increase by mobilizing intracellular Ca2+ stores and activating Orai1-dependent Ca2+ influx in non-Hodgkin lymphoma cell lines and primary B-Cell Chronic Lymphocytic Leukemia (B-CLL) cells. According to the cell type, Ca2+ was mobilized from two distinct intracellular compartments. In Raji, BL2, and B-CLL cells, GA101 induced a Ca2+ release from lysosomes, leading to the subsequent lysosomal membrane permeabilization and cell death. Inhibition of this calcium signaling reduced GA101-induced cell death in these cells. In SU-DHL-4 cells, GA101 mobilized Ca2+ from the endoplasmic reticulum (ER). Inhibition of ER replenishment, by blocking Orai1-dependent Ca2+ influx, led to an ER stress and unfolded protein response (UPR) which sensitized these cells to GA101-induced cell death. These results revealed the central role of Ca2+ signaling in GA101’s action mechanism, which may contribute to designing new rational drug combinations improving its clinical efficacy.

Introduction
The anti-CD20 monoclonal antibody (mAb), rituximab (RTX), combined with chemotherapy, became the standard regimen for treating non-Hodgkin lymphomas (NHL), such as diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL) [1][2][3]. However, a significant number of patients with DLBCL and most patients with FL or CLL are resistant or relapse [4]. Given that CD20 is highly and specifically expressed on the surface of B cells, it constitutes an ideal therapeutic target in NHL. Therefore, the development of a new anti-CD20 mAb was the subject of intensive research over the past few years.
Currently, anti-CD20 mAbs are generally divided into two distinct classes. Type I mAbs, such as rituximab, induce antibody-dependent cell-mediated cytotoxicity (ADCC), complement dependent

Calcium Responses Induced by GA101 in NHL Cell Lines
The addition of GA101 triggered a cytosolic Ca 2+ increase in all cell lines recorded in HBSS (Hank's Balanced Salt Solution) containing 2 mM Ca 2+ , consisting of multi-peaks and/or a sustained plateau phase, but of lower amplitude and frequency in BL2 and Raji cell lines (Figure 1, Supplemental Figure S1A). To determine the origin of these Ca 2+ responses, cells were recorded in Ca 2+ -free medium. Ca 2+ responses were maintained, but the areas under the curves were statistically smaller in all cell types ( Figure 1; Figure S1A), suggesting that these responses resulted from both intracellular Ca 2+ store mobilization and extracellular Ca 2+ influx. To identify the membrane ion channels involved in the GA101-induced calcium influx, SU-DHL-4 and BL2 cells stably expressing a short hairpin RNA (shRNA) Orai1 were generated ( Figure S2). Once again, Ca 2+ responses induced by GA101 were significantly attenuated in Orai1 knockdown cells compared to cells expressing a non-targeting shRNA (sh NT) ( Figure 1). Moreover, cells pretreated with BTP2, a commonly-used SOCE inhibitor, exhibited significantly lower GA101-induced Ca 2+ responses ( Figure S1B). These results revealed that GA101 was able to induce an Orai1-dependent Ca 2+ influx in NHL-B cells. The next step was to determine the origin of the intracellular Ca 2+ store mobilization induced by GA101. To this end, cells were pretreated with either thapsigargin (TG, a sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor, emptying the ER Ca 2+ pool) or Ned-19 (an inhibitor of two pore channels expressed on lysosome) and recorded in Ca 2+ -free medium. Pretreatment of SU-DHL-4 cells with TG, but not with Ned-19, dramatically decreased the Ca 2+ response in terms of percentage and amplitude ( Figure 1A). In contrast, in BL2 and Raji cells, Ca 2+ mobilization was unaffected by TG, but abolished by Ned-19 ( Figure 1B; Figure S1A). These results revealed that GA101 mobilized Ca 2+ from ER in SU-DHL-4 cells or lysosomes in BL2 and Raji cells, and subsequently activated Ca 2+ influx by Orai1-dependent Ca 2+ channels in all cell lines.
Cancers 2019, 11, x 3 of 18 channels involved in the GA101-induced calcium influx, SU-DHL-4 and BL2 cells stably expressing a short hairpin RNA (shRNA) Orai1 were generated ( Figure S2). Once again, Ca 2+ responses induced by GA101 were significantly attenuated in Orai1 knockdown cells compared to cells expressing a non-targeting shRNA (sh NT) ( Figure 1). Moreover, cells pretreated with BTP2, a commonly-used SOCE inhibitor, exhibited significantly lower GA101-induced Ca 2+ responses ( Figure S1B). These results revealed that GA101 was able to induce an Orai1-dependent Ca 2+ influx in NHL-B cells. The next step was to determine the origin of the intracellular Ca 2+ store mobilization induced by GA101.
To this end, cells were pretreated with either thapsigargin (TG, a sarco/endoplasmic reticulum Ca 2+ ATPase (SERCA) inhibitor, emptying the ER Ca 2+ pool) or Ned-19 (an inhibitor of two pore channels expressed on lysosome) and recorded in Ca 2+ -free medium. Pretreatment of SU-DHL-4 cells with TG, but not with Ned-19, dramatically decreased the Ca 2+ response in terms of percentage and amplitude ( Figure 1A). In contrast, in BL2 and Raji cells, Ca 2+ mobilization was unaffected by TG, but abolished by Ned-19 ( Figure 1B; Figure S1A). These results revealed that GA101 mobilized Ca 2+ from ER in SU-DHL-4 cells or lysosomes in BL2 and Raji cells, and subsequently activated Ca 2+ influx by Orai1dependent Ca 2+ channels in all cell lines.

Role of Calcium Influx in GA101-Induced Cell Death
Given that type II anti-CD20 mAbs cause a strong homotypic adhesion leading to cell aggregation, it was suggested by Golay et al. [25] that the analysis of the cell death induced by these Abs using flow cytometry should be interpreted with caution. Other studies clearly showed that cell death could be detected after GA101 treatment by various techniques including flow cytometry [5,26]. In a preliminary approach, we analyzed and compared cell death induced by GA101 by microscopy and flow cytometry after propidium iodide (PI) labeling, two conventional techniques. As shown in Figure S3A, GA101 triggered cell death in all cell lines tested, and the increase in dead cells detected by both methods was of the same order. Thus, regardless of the cell death detection technique used, we observed that BL2 cells were the most sensitive to GA101-induced cell death, while SU-DHL-4 cells were the least. Flow cytometry allowed a rapid analysis of thousands of cells; in the further experiments, cell death was measured using this technique.
Orai1-dependent Ca 2+ influx was reported to exert a negative feedback on RTX-induced apoptosis [27]. Therefore, we examined whether the same type of mechanism was activated by GA101. In BL2 and Raji cells, Orai1 knockdown or BTP2 pretreatment had no effect on GA101induced cell death (Figure 2A; Figure S3B). In contrast, BTP2 and, to a lesser extent, the downregulation of Orai1 improved the efficacy of GA101 for inducing cell death in SU-DHL-4 cells, Ca 2+ responses to GA101 (10 µg/mL) were measured using Fluo2-Leak Resistant-Acetoxy Methyl ester (Fluo2-LR-AM) Ca 2+ dye and recorded by videomicroscopy (Zeiss LSM 510) using a 25× objective. Black arrows indicate GA101 addition. Each trace represents the response of one cell and data are representative of at least three independent experiments. Data were processed using OriginPro 7.5 (Origin Lab) or GraphPad prism. Cells were recorded in extracellular Hank's Balanced Salt Solution (HBSS) containing 2 mM Ca 2+ (2Ca) or in Ca 2+ -free HBSS (0Ca). Cells were preincubated with 100 nM thapsigargin (TG) for 45 min and recorded in Ca 2+ -free HBSS (0Ca + TG) or with 10 µM Ned-19 for 1 h and recorded in Ca 2+ -free HBSS (0Ca + Ned19). Calcium responses to GA101 in cells expressing Non Targeting shRNA (sh NT) or sh Orai1 were recorded in HBSS containing 2 mM Ca 2+ . Histograms represent areas under curves (AUC) calculated, under various recording conditions, between the application time of GA101 and t = 2000 s; * p < 0.05.

Role of Calcium Influx in GA101-Induced Cell Death
Given that type II anti-CD20 mAbs cause a strong homotypic adhesion leading to cell aggregation, it was suggested by Golay et al. [25] that the analysis of the cell death induced by these Abs using flow cytometry should be interpreted with caution. Other studies clearly showed that cell death could be detected after GA101 treatment by various techniques including flow cytometry [5,26]. In a preliminary approach, we analyzed and compared cell death induced by GA101 by microscopy and flow cytometry after propidium iodide (PI) labeling, two conventional techniques. As shown in Figure S3A, GA101 triggered cell death in all cell lines tested, and the increase in dead cells detected by both methods was of the same order. Thus, regardless of the cell death detection technique used, we observed that BL2 cells were the most sensitive to GA101-induced cell death, while SU-DHL-4 cells were the least. Flow cytometry allowed a rapid analysis of thousands of cells; in the further experiments, cell death was measured using this technique.
Orai1-dependent Ca 2+ influx was reported to exert a negative feedback on RTX-induced apoptosis [27]. Therefore, we examined whether the same type of mechanism was activated by GA101. In BL2 and Raji cells, Orai1 knockdown or BTP2 pretreatment had no effect on GA101-induced cell death (Figure 2A; Figure S3B). In contrast, BTP2 and, to a lesser extent, the downregulation of Orai1 improved the efficacy of GA101 for inducing cell death in SU-DHL-4 cells, ( Figure 2B); however, only Orai1 knockdown increased their sensitivity for GA101 (half maximal efficacy concentration (EC 50 ) Control = 0.037 ± 0.005 vs. BTP2 = 0.036 ± 0.002 µg/mL, p > 0.05; EC 50 Sh NT = 0.040 ± 0.002 vs. Sh Orai1 = 0.018 ± 0.002 µg/mL, p < 0.05) which is likely attributable to the higher specificity of Sh Orai1 than BTP2 to inhibit Ca 2+ influx. The effects of Orai1 inhibition on GA101-induced cell death in SU-DHL-4 were not due to CD95 engagement since, unlike RTX [27], GA101 was unable to induce CD95 capping formation, a hallmark of CD95 pathway activation ( Figure S4).  Figure 2B); however, only Orai1 knockdown increased their sensitivity for GA101 (half maximal efficacy concentration (EC50) Control = 0.037 ± 0.005 vs. BTP2 = 0.036 ± 0.002 μg/mL, p > 0.05; EC50 Sh NT = 0.040 ± 0.002 vs. Sh Orai1 = 0.018 ± 0.002 μg/mL, p < 0.05) which is likely attributable to the higher specificity of Sh Orai1 than BTP2 to inhibit Ca 2+ influx. The effects of Orai1 inhibition on GA101-induced cell death in SU-DHL-4 were not due to CD95 engagement since, unlike RTX [27], GA101 was unable to induce CD95 capping formation, a hallmark of CD95 pathway activation ( Figure S4). Disruption of ER Ca 2+ homeostasis by SERCA inhibition (TG) or Ca 2+ influx inhibition leads to the accumulation of unfolded proteins and causes ER stress likely to promote cell death [28]. To envisage the involvement of Orai1 inhibition-dependent ER stress in the potentiation of the cell death induced by GA101, we investigated the impact of GA101 on the activation of UPR in cells expressing sh NT or sh Orai1 (SU-DHL-4 and BL2) or after treatment with BTP2 (Raji). To this end, we studied eIF2 phosphorylation and the expression of BIM, one of the targets transcriptionally regulated by CHOP. Our results revealed an increase in eIF2 phosphorylation in under-expressing Orai1 SU-DHL-4 cells treated with GA101. In contrast, no effect of Orai1 under-expression or inhibition was observed in BL2 or Raji cells, respectively ( Figure 3; Figure S5A). In agreement with these data, we found that BIM expression increased in SU-DHL-4, while, in BL2 or Raji cells treated with GA101, it decreased with time ( Figure 3; Figure S5A). Moreover, we showed that tunicamycin, a main ER stress inducer, sensitized the SU-DHL-4 cell line to GA101-induced cell death but not BL2 or Raji cell lines ( Figure S5B). These results reveal a distinctive role of Ca 2+ entry in GA101-induced cell death, according to the cell line studied. In SU-DHL-4, Ca 2+ entry repressed cell death by preventing ER stress activation, while, in BL2 and Raji cell lines, Ca 2+ influx was not, apparently, involved in GA101induced cell death. Disruption of ER Ca 2+ homeostasis by SERCA inhibition (TG) or Ca 2+ influx inhibition leads to the accumulation of unfolded proteins and causes ER stress likely to promote cell death [28]. To envisage the involvement of Orai1 inhibition-dependent ER stress in the potentiation of the cell death induced by GA101, we investigated the impact of GA101 on the activation of UPR in cells expressing sh NT or sh Orai1 (SU-DHL-4 and BL2) or after treatment with BTP2 (Raji). To this end, we studied eIF2α phosphorylation and the expression of BIM, one of the targets transcriptionally regulated by CHOP. Our results revealed an increase in eIF2α phosphorylation in under-expressing Orai1 SU-DHL-4 cells treated with GA101. In contrast, no effect of Orai1 under-expression or inhibition was observed in BL2 or Raji cells, respectively ( Figure 3; Figure S5A). In agreement with these data, we found that BIM expression increased in SU-DHL-4, while, in BL2 or Raji cells treated with GA101, it decreased with time ( Figure 3; Figure S5A). Moreover, we showed that tunicamycin, a main ER stress inducer, sensitized the SU-DHL-4 cell line to GA101-induced cell death but not BL2 or Raji cell lines ( Figure S5B). These results reveal a distinctive role of Ca 2+ entry in GA101-induced cell death, according to the cell line studied. In SU-DHL-4, Ca 2+ entry repressed cell death by preventing ER stress activation, while, in BL2 and Raji cell lines, Ca 2+ influx was not, apparently, involved in GA101-induced cell death. (A) Cells expressing sh NT or sh Orai1 were treated with GA101 (1 μg/mL) for varying lengths of time. After lysis, phosphorylated eukaryotic translation initiation factor alpha (P-eIF2), eIF2, and BIM expression levels were assessed by immunoblot analysis. GAPDH was used as a loading control. (B) Quantification of Western blots is given as means ± SE of three to five independent experiments; * p < 0.05.

Role of Lysosomes in GA101-Induced Cell Death
Previous works reported the key role of lysosomal membrane permeabilization (LMP) in type II anti-CD20 mAb-induced cell death [9,29,30], and our data suggest that GA101 mobilized lysosomal Ca 2+ in the BL2 and Raji but not the SU-DHL-4 cell line. To confirm the effect of GA101 on lysosomes, LMP was measured in all cell lines by the decrease in lysotracker fluorescence using videomicroscopy. GA101 provoked a drop in lysotracker fluorescence in BL2 and Raji cells, while no effect was observed in SU-DHL-4 cells or when HBSS was applied to the cell preparation instead of GA101 ( Figure 4(Aa,Ab,Ba,Bb); Figure S6A,B). These observations were confirmed after quantification of the fluorescent signal, by calculating the slope of the fluorescence leakage before and after GA101 application. This analysis clearly revealed that GA101 induced a significant increase in the slope ratio in BL2 and Raji but not SU-DHL-4 cells, confirming the activation of LMP in BL2 and Raji but not SU-DHL-4 cells (Figure 4(Ac,Bc); Figure S6C). Moreover, the pre-incubation of BL2 or Raji cells with Ned-19 significantly reversed GA101-induced LMP (Figure 4(Ab,Ac,Bb,Bc); Figure  S6B,C). These data suggested that Ca 2+ efflux from lysosomes by TPC was upstream of LMP and participated in this process. To determine whether the lysosomes released their contents in response to GA101, we performed immunofluorescence staining of cathepsin B, a releasable component of lysosomes. As expected, these experiments confirmed an increase in cathepsin B release in the cytosol of BL2 cells, similar to that observed with the lysosomotropic agent, siramesine, used as a positive control (Figure 4Ad). Note that no cathepsin B release was observed in SU-DHL-4 cells (Figure 4Bd). Pretreatment of BL2 cells with Ned-19 inhibited the GA101-induced cytosolic release of cathepsin B (Figure 4Ad). Finally, we confirmed that LMP was involved in cell death induced by GA101 in BL2 and Raji but not SU-DHL-4 cells, since pretreatment with Ned-19 and E64D, a cathepsin B inhibitor, reduced GA101-induced apoptosis (Figure 4(Ae,Be); Figure S6D). (A) Cells expressing sh NT or sh Orai1 were treated with GA101 (1 µg/mL) for varying lengths of time. After lysis, phosphorylated eukaryotic translation initiation factor alpha (P-eIF2α), eIF2α, and BIM expression levels were assessed by immunoblot analysis. GAPDH was used as a loading control. (B) Quantification of Western blots is given as means ± SE of three to five independent experiments; * p < 0.05.

Role of Lysosomes in GA101-Induced Cell Death
Previous works reported the key role of lysosomal membrane permeabilization (LMP) in type II anti-CD20 mAb-induced cell death [9,29,30], and our data suggest that GA101 mobilized lysosomal Ca 2+ in the BL2 and Raji but not the SU-DHL-4 cell line. To confirm the effect of GA101 on lysosomes, LMP was measured in all cell lines by the decrease in lysotracker fluorescence using videomicroscopy. GA101 provoked a drop in lysotracker fluorescence in BL2 and Raji cells, while no effect was observed in SU-DHL-4 cells or when HBSS was applied to the cell preparation instead of GA101 ( Figure 4(Aa,Ab,Ba,Bb); Figure S6A,B). These observations were confirmed after quantification of the fluorescent signal, by calculating the slope of the fluorescence leakage before and after GA101 application. This analysis clearly revealed that GA101 induced a significant increase in the slope ratio in BL2 and Raji but not SU-DHL-4 cells, confirming the activation of LMP in BL2 and Raji but not SU-DHL-4 cells (Figure 4(Ac,Bc); Figure S6C). Moreover, the pre-incubation of BL2 or Raji cells with Ned-19 significantly reversed GA101-induced LMP (Figure 4(Ab,Ac,Bb,Bc); Figure  S6B,C). These data suggested that Ca 2+ efflux from lysosomes by TPC was upstream of LMP and participated in this process. To determine whether the lysosomes released their contents in response to GA101, we performed immunofluorescence staining of cathepsin B, a releasable component of lysosomes. As expected, these experiments confirmed an increase in cathepsin B release in the cytosol of BL2 cells, similar to that observed with the lysosomotropic agent, siramesine, used as a positive control (Figure 4Ad). Note that no cathepsin B release was observed in SU-DHL-4 cells (Figure 4Bd). Pretreatment of BL2 cells with Ned-19 inhibited the GA101-induced cytosolic release of cathepsin B (Figure 4Ad). Finally, we confirmed that LMP was involved in cell death induced by GA101 in BL2 and Raji but not SU-DHL-4 cells, since pretreatment with Ned-19 and E64D, a cathepsin B inhibitor, reduced GA101-induced apoptosis (Figure 4(Ae,Be); Figure S6D).
In agreement with our calcium-signaling investigations, we revealed that, in BL2 and Raji cells, GA101 activated the release of lysosomal Ca 2+ leading to the LMP induction and cathepsin B release responsible for cell death. In contrast, in the SU-DHL-4 cell line, GA101 activated a signaling pathway, involving Ca 2+ influx and ER stress, without the lysosomal pathway playing a major role.
In agreement with our calcium-signaling investigations, we revealed that, in BL2 and Raji cells, GA101 activated the release of lysosomal Ca 2+ leading to the LMP induction and cathepsin B release responsible for cell death. In contrast, in the SU-DHL-4 cell line, GA101 activated a signaling pathway, involving Ca 2+ influx and ER stress, without the lysosomal pathway playing a major role.

Effect of GA101 on Primary B-CLL
Given that the most encouraging results in clinical trials with GA101 were obtained in patients with B-CLL and that B-CLL is closely related to lymphocytic lymphoma, which is classified as indolent non-Hodgkin's lymphoma, we tested the effect of GA101 on the death of B-CLL cells harvested from patient blood samples (Table 1). GA101 induced significant cell death in 15 out of 23 B-CLL samples tested ( Table 1). The induction of cell death by GA101 is apparently dependent on CD20 expression, since CD20 expression was higher in responsive than non-responsive B-CLL ( Figure 5A). We further investigated the effects of GA101 on [Ca 2+ ]i of 10 CLLs responsive to GA101induced cell death. GA101 triggered an intracellular Ca 2+ increase in all CLLs tested. These Ca 2+ responses were due to both intracellular Ca 2+ mobilization and extracellular Ca 2+ entry ( Figure 5B,C). We then determined the origin of GA101-induced Ca 2+ mobilization. To this end, cells from six B-CLL samples were pretreated with Ned-19 or TG and then recorded in Ca 2+ -free medium. In three CLL samples, Ned-19 completely blocked GA101-induced Ca 2+ mobilization, while it was unchanged after TG pre-treatment. In contrast, in the three other B-CLL samples tested, Ca 2+ response was inhibited after TG pretreatment but not after Ned-19 treatment ( Figure 5B,C). These data suggested that the Ca 2+ response to GA101 of some CLL samples was like that of BL2 and Raji cell lines, while others behaved like SU-DHL-4 cells. To decipher the molecular mechanisms responsible for the activation of lysosomal Ca 2+ release or not in cells, we investigated the expression of CD20, CD38, TPC1, and TPC2 proteins. However, no difference was observed between GA101-induced lysosomal Ca 2+ release cells or not ( Figure 5D; Figure S7).

Effect of GA101 on Primary B-CLL
Given that the most encouraging results in clinical trials with GA101 were obtained in patients with B-CLL and that B-CLL is closely related to lymphocytic lymphoma, which is classified as indolent non-Hodgkin's lymphoma, we tested the effect of GA101 on the death of B-CLL cells harvested from patient blood samples (Table 1). GA101 induced significant cell death in 15 out of 23 B-CLL samples tested ( Table 1). The induction of cell death by GA101 is apparently dependent on CD20 expression, since CD20 expression was higher in responsive than non-responsive B-CLL ( Figure 5A). We further investigated the effects of GA101 on [Ca 2+ ] i of 10 CLLs responsive to GA101-induced cell death. GA101 triggered an intracellular Ca 2+ increase in all CLLs tested. These Ca 2+ responses were due to both intracellular Ca 2+ mobilization and extracellular Ca 2+ entry ( Figure 5B,C). We then determined the origin of GA101-induced Ca 2+ mobilization. To this end, cells from six B-CLL samples were pretreated with Ned-19 or TG and then recorded in Ca 2+ -free medium. In three CLL samples, Ned-19 completely blocked GA101-induced Ca 2+ mobilization, while it was unchanged after TG pre-treatment. In contrast, in the three other B-CLL samples tested, Ca 2+ response was inhibited after TG pretreatment but not after Ned-19 treatment ( Figure 5B,C). These data suggested that the Ca 2+ response to GA101 of some CLL samples was like that of BL2 and Raji cell lines, while others behaved like SU-DHL-4 cells. To decipher the molecular mechanisms responsible for the activation of lysosomal Ca 2+ release or not in cells, we investigated the expression of CD20, CD38, TPC1, and TPC2 proteins. However, no difference was observed between GA101-induced lysosomal Ca 2+ release cells or not ( Figure 5D; Figure S7). The next step was to investigate the involvement of Ca 2+ signaling in GA101-induced cell death in B-CLL samples. Due to the toxicity of pre-treatments on some B-CLL samples, the effects of BTP2 and Ned-19 on GA101-induced cell death were analyzed in nine B-CLL samples. In one out of nine B-CLL samples, no treatment had any effect on GA101-induced cell death (CLL 19). In four CLL samples (CLL5, CLL17, CLL23, and CLL24), Ned-19 statistically inhibited cell death ( Figure 5E). Finally, we observed that BTP2 had no effect (CLL 19 and CLL 23) or inhibited GA101-induced cell death (CLL17, CLL20, CLL14, CLL13, CLL5 and CLL22) ( Figure 5E,F). We hypothesized that this latter result may be due to B-CLL resistance to ER stress. Thus, we tested the effects of tunicamycin on basal and GA101-induced cell death in five B-CLL samples. Our results revealed that tunicamycin did not induce cell death in B-CLL or sensitize cells to GA101 ( Figure 5G), suggesting that CLL may be resistant to ER stress.  were incubated with GA101 1 µg/mL for 24 h, and cell death was assessed by measuring the loss of mitochondrial membrane potential (∆Ψm), using TMRM as a fluorescent dye, and analyzed in CD19pos cells by flow cytometry. Cells were considered responders when GA101 induced a statistically significant increase in cell death (Mann-Whitney U; p < 0.05 considered as significant). Representative Ca 2+ responses to GA101 (10 µg/mL) in CLL involving (B) TG-sensitive (CLL20) or (C) Ned-19-sensitive Ca 2+ pools (CLL23) recorded in HBSS containing 2 mM Ca 2+ (2Ca), in Ca 2+ -free extracellular medium (0Ca), after pretreatment with TG (100 nM, 45 min) and recorded in Ca 2+ -free extracellular medium (0Ca + TG), or after pretreatment with Ned-19 (10 µM, 1 h) and recorded in Ca 2+ -free extracellular medium (0Ca + Ned19). Histograms represent areas under curves calculated under various recording conditions, between the application time of GA101 and t = 2000 s; * p < 0.05. Ca 2+ responses were recorded as described in Figure 1 In GA101-responsive B-CLL samples, cells were incubated with GA101 (1 µg/mL) in the presence or absence of tunicamycin 10 ng/mL for 24 h. Cell death was assessed as described above; * p < 0.05.

Discussion
The role of Ca 2+ in cell death was extensively investigated [31]; however, its pro-or anti-apoptotic effects seem highly dependent on the stimulus and cell type, as well as the Ca 2+ toolkit that enables cells to specifically regulate Ca 2+ -dependent cell death. Here, we investigated the effect of GA101 on Ca 2+ signaling and its involvement in the direct cell death evoked by GA101. We identified some key findings: (1) GA101 triggered an intracellular Ca 2+ increase by mobilizing intracellular Ca 2+ stores and activating Orai1-dependent Ca 2+ influx in the three studied NHL cell lines; (2) lysosomal Ca 2+ release and subsequent LMP were responsible for the cell death induced by GA101 in BL2 and Raji but not in SU-DHL-4 cells; (3) in SU-DHL-4 cells, cell death was repressed by Orai1-dependent Ca 2+ influx and, consequently, enhanced by Orai1 inhibitors through the activation of UPR in response to ER stress; (4) in B-CLL, GA101 triggered an intracellular Ca 2+ increase involving mobilization of either lysosomal or ER Ca 2+ pools and a Ca 2+ influx, which are differentially implicated in GA101-induced cell death. Taken together, these results revealed the central role of GA101-induced Ca 2+ signaling in its action mechanism.
GA101 was found to activate two distinct Ca 2+ responses: one similar to that described with RTX [27], involving ER Ca 2+ store mobilization and capacitive Ca 2+ influx in SU-DHL-4 cells, and another involving lysosomal Ca 2+ release and extracellular Ca 2+ influx, in BL2 and Raji cell lines. Lysosomes express Ca 2+ channels, including TPC1 and TPC2, which are activated by NAADP (Nicotinic acid adenine dinucleotide phosphate) [32][33][34]. In BL2 and Raji cells, Ned-19, a NAADP receptor antagonist, was found to suppress Ca 2+ mobilization triggered by GA101, suggesting that GA101 induces NAADP formation and TPC-dependent Ca 2+ release from lysosome. Moreover, this lysosomal Ca 2+ release is probably involved in LMP, since LMP was significantly inhibited by pre-treatment with Ned-19. Interestingly, these two Ca 2+ signaling pathways were also observed in B-CLL with a ratio of 50/50. In order to identify which molecular mechanism determined the type of Ca 2+ responses activated by GA101 in cells, we evaluated the expression of various proteins in NHLB cell lines and B-CLL. Unlike for the induction of cell death by GA101 which is related to CD20 expression level, no difference in CD20 expression was observed between lysosomal responsive cells or not. Another explanation could be that CD38 expression, which is responsible for the transformation of NAD+ (Nicotinamide adenine dinucleotide) to the active second messengers NAADP and cADPr (cyclic ADP ribose) [35], may be differentially expressed in B-NHL and B-CLL cells, constituting a switch between the two GA101-activated signaling pathways; however, this hypothesis was not confirmed by our results obtained in cell lines and tested B-CLL. Similarly, TPC1 and TPC2 expression was unchanged between cells. However, these data should be confirmed in a larger cohort of B-CLL. Finally, we cannot exclude that the different Ca 2+ responses induced by GA101 are due to the origin of the NHL (DLBCL or Burkitt lymphoma) or B-CLL. Further experiments will be necessary to identify the molecular process responsible for activating or not LMP pathway in these cells.
We and others [36,37] showed that RTX-induced Ca 2+ response in NHLB was due to IP3dependent intracellular Ca 2+ mobilization and Orai1-dependent Ca 2+ influx, while we demonstrated that GA101 was able to trigger at least two different molecular pathways for Ca 2+ signaling according to the cell type, which could contribute to its better clinical efficacy than RTX. Indeed, type I CD20 antibody RTX and type II CD20 antibody GA101 differ fundamentally in their interaction with CD20, which may account for activation of various transduction pathways, including Ca 2+ signal kinetics and/or more or less antibody efficiency [38].
In human primary B-CLL samples, we observed heterogeneity in cell death responses induced by GA101, which may be due to individual variability in CD20 expression or to BCR (B-cell receptor) functional signaling and/or to IGHV (immunoglobulin heavy-chain variable region) mutational status. Indeed, CLL IGHV mutational status reveals bifurcation of responses toward proliferation or anergy [39], and one can hypothesize that anergic mutated IGHV CLL cells could be more sensitive to GA101-induced cell death than proliferative unmutated IGHV CLL because their increased susceptibility to apoptosis. Further experiments should be necessary to elucidate this point.
It was previously shown that GA101 triggered LMP and the subsequent cathepsin release responsible for protein cleavage (including caspases) and cell death [9,40]. We confirmed this mechanism and suggest that LMP and cell death are, at least partly, initiated by lysosomal Ca 2+ release, since Ned-19 inhibited LMP and GA101-induced cell death in BL2, Raji cells, and in half of B-CLL samples. In contrast, in the SU-DHL-4 cell line, GA101 was unable to trigger LMP. Based on the slope of lysotracker fluorescence in resting conditions (before GA101 application) being higher in SU-DHL-4 than those observed in BL2 and Raji cells, it could be possible that LMP could not be observed in these cells. However, this absence of LMP is correlated with the lack of lysosomal Ca 2+ mobilization, with the absence of cathepsin release in response to GA101 and with no effect of Ned-19 or E64D on cell death induced by GA101 suggesting that lysosomal cell death is not activated by GA101 in these cells. In SU-DHL-4 cells, Orai1-dependent Ca 2+ influx was clearly shown to repress sensitivity to GA101-induced cell death, since pharmacological or genetic inhibition of Orai1 activity improved the effect of GA101. This repressive effect of the Orai1-dependent Ca 2+ influx on GA101-induced cell death is similar to that observed with RTX; however, in contrast to RTX, this effect was not attributable to Ca 2+ inhibition of CD95 engagement [27,41], but rather the impediment of ER stress. Indeed, Orai1 inhibition increased the phosphorylation level of eIF2α, as well as the expression of BIM, two ER-stress markers [28]. In the SU-DHL-4 cell line, GA101 triggered ER Ca 2+ store release, but activated capacitive Ca 2+ influx, thus refilling the Ca 2+ stores and preserving Ca 2+ homeostasis and the efficiency of ER. When this Ca 2+ influx was inhibited, the reticular Ca 2+ concentration remained low, inducing accumulation of unfolded proteins, causing ER stress and BIM expression to increase, which promoted cell death [28,42] In BL2, Raji, and B-CLL cells, although GA101 triggered a Ca 2+ influx, its inhibition did not sensitize cells to GA101-induced cell death. Two hypotheses may explain this result. The first is that BL2, Raji, and B-CLL cells are resistant to ER stress, as suggested by experiments where ER-stress inducers were unable to induce cell death or potentiate GA101-induced cell death. This resistance to ER stress may be due to Bcl-2 overexpression, as already described in B-CLL [43,44], which, by interacting with IP3R [45], may repress ER Ca 2+ release and, in turn, protect cells from ER stress. The second possibility is that Orai1-dependent Ca 2+ entry is not the main source of Ca 2+ influx in these cells, but other Ca 2+ channels, such as TRPC (Transient Receptor Potential channels Canonical) [46] or TRPV (Transient Receptor Potential channels Vanilloid) [47] channels, may be significant contributors to Ca 2+ entry.

Short Hairpin RNA Lentivirus Transduction
The short hairpin RNA (shRNA) lentivirus transduction approach was used to knockdown orai1 gene expression. pLKO1 lentiviral vectors expressing DNA sequences encoding for Orai1 shRNA (TRCN0000165044 and TRCN0000161221) were purchased from Sigma-Aldrich. Non-targeting shRNA (sh NT) was used as a lentivirus cell transduction control. Lentiviruses were produced as previously described [27]. The titer of each lentiviral batch was determined on various cell lines. Transduced cells were selected by puromycin treatment.

Apoptosis Assays
B-cells were untreated or pretreated with various pharmacological agents for 1 h and then incubated with or without GA101 for 24 h. Apoptosis was detected by the loss of mitochondrial membrane potential using TMRM as a fluorescent dye. TMRM is a fluorescent lipophilic cation which accumulates in hyperpolarized mitochondria of living cells, but not in depolarized mitochondria of apoptotic cells [48]. Thus, after treatment, cells were incubated with 200 nM TMRM for 20 min, and the loss of cell fluorescence, corresponding to apoptotic cells, was measured by flow cytometry. Apoptosis was also assessed by measuring the percentage of cells with active caspase 3, detected by the FAM-FLICA in vitro caspase detection kit, used according to the manufacturer's instructions. Apoptotic cells were analyzed by flow cytometry using a FacsCalibur cytometer and Cell Quest software (BD biosciences, Le Pont de Claix, France).

Intracellular Calcium Measurement
For single-cell [Ca 2+ ] i measurement cells were loaded with 7.5 µM Fluo2-LR-AM (λex: 488 nm, λem 515 nm) calcium dye, which exhibits limited compartmentalization in intracellular stores and is leakage resistant, in the presence of 0.02% pluronic F127 in HBSS at room temperature for 25 min. The cells were rinsed with HBSS and incubated without Ca 2+ probe for 15 min to complete de-esterification of the dye. For B-CLL, cells were previously labeled with anti-human CD19 APC (Allophycocyanin) at room temperature for 20 min, to ensure that Ca 2+ recordings originated from B cells. In some experiments, cells were placed in a Ca 2+ -free HBSS medium, to which 100 µM EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N ,N -tetraacetic acid) was added to chelate any residual Ca 2+ ions. This medium was added to the cells just before recording to avoid leaks from the intracellular calcium stores.
Fluorescence was recorded using an inverted fluorescence confocal microscope (Zeiss LSM 510, Göttingen, Germany) with a 25× oil immersion objective. Images were recorded at constant 10-s intervals. Regions of interest corresponding to cells recorded were drawn to analyze the fluorescence signal. To overcome putative differences of Ca 2+ probe concentration between cells, we normalized the fluorescence signal (F) of each cell by its starting basal fluorescence (F0). Data were processed using OriginPro 7.5 (Origin Lab, Paris, France) and Prism 6.0 (Graphpad, San Diego, CA, USA) software.

Assessement of Lysosomal Membrane Permeabilization
Lysosomal membrane permeabilization (LMP) and lysosomal cathepsin B release were assessed by lysotracker staining and immunofluorescence, respectively, and analyzed by fluorescence microscopy. For LMP detection, cells were stained with lysotracker red (100 nM) in HBSS at 37 • C for 45 min. Cells were then rinsed twice in HBSS. Fluorescence micrograph images were obtained using an inverted fluorescence microscope with an ApoPLAN 63× objective. Images were recorded at constant 1-min intervals. Regions of interest corresponding to cells recorded were drawn to analyze the fluorescence signal, and its slope was calculated using GraphPad Prism software.
To visualize cathepsin B release in the cytosol, cells were untreated or treated with GA101 or siramesine (a lysosomotropic agent), and immunofluorescence was performed. Cells were fixed in Phosphate Buffer Saline (PBS) containing 4% w/v paraformaldehyde at 4 • C for 10 min and then permeabilized in PBS supplemented with 5% Bovine Serum Albumin (BSA)/0.1% saponin for 5 min. Cells were incubated with anti-cathepsin B in PBS/1% w/v BSA at 4 • C overnight. Cathepsin B was revealed using secondary Alexa594-coupled donkey anti-goat Ab. Nuclei were stained using Hoechst 33258. Images were acquired using a Zeiss LSM 510 meta confocal microscope with an ApoPLAN 63× objective.

Western Blotting
Cell lysates were prepared using a lysis buffer containing protease and phosphatase inhibitors (Cell Signaling Technology). The lysates were separated by SDS-PAGE and transferred to Polyvinylidene fluoride (PVDF) membrane using an iBlot Gel Transfer System (Thermofisher Scientific). The membranes were incubated with 5% nonfat milk w/v in PBS buffer for 1 h and then reacted with the primary Ab in PBS/Tween buffer with 5% nonfat milk w/v by shaking at 4 • C overnight. The appropriate second Abs conjugated to HRP was used to detect the protein of interest via Enhanced Chemiluminescence (ECL) (Millipore).

Statistical Analysis
All data were expressed as means ± SE. The significance of differences was calculated using the parametric t-test or one-way ANOVA test or the non-parametric Mann-Whitney U or Wilcoxon tests, as appropriate (* p < 0.05).

Conclusions
In conclusion, this study provides new mechanistic insights into GA101-induced cell death, including the important, complex role of Ca 2+ signaling. The source of Ca 2+ increase, as well as its role in GA101-induced cell death, differed according to the cell type studied. Further investigations are required to identify the biomarkers of Ca 2+ involvement in GA101-induced cell death, in order to design new, rational drug combinations to improve the clinical efficacy of GA101.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/11/3/291/s1: Figure S1: Effect of GA101 on intracellular Ca 2+ concentration in the Raji cell line, Figure S2: Validation of SU-DHL-4 and BL2 cell lines infected with non-targeting or Orai1-targeting shRNA lentiviruses, Figure S3: Cell death induced by GA101 in various cell lines measured by microscopy or flow cytometry, Figure S4: Unlike rituximab, GA101 is unable to induce CD95 capping, Figure S5: Involvement of ER stress in GA101-induced cell death, Figure S6: Effect of GA101 on LMP in the Raji cell line, Figure


extfieldsofstudy: Medicine Biology

text:
A Review of Macrophage MicroRNAs’ Role in Human Asthma

There is an imbalance in asthma between classically activated macrophages (M1 cells) and alternatively activated macrophages (M2 cells) in favor of the latter. MicroRNAs (miRNAs) play a critical role in regulating macrophage proliferation and differentiation and control the balance of M1 and M2 macrophage polarization, thereby controlling immune responses. Here we review the current published data concerning miRNAs with known correlation to a specific human macrophage phenotype and polarization, and their association with adult asthma. MiRNA-targeted therapy is still in the initial stages, but clinical trials are under recruitment or currently running for some miRNAs in other diseases. Regulating miRNA expression via their upregulation or downregulation could show potential as a novel therapy for improving treatment efficacy in asthma.

Introduction
Although it was recently reported that there is a spectrum of human macrophage activation states, two main polarized states have been described for macrophages-pro-inflammatory classically activated M1 and anti-inflammatory alternatively activated M2 macrophages [1][2][3]. Interferon-γ (alone or in combination with microbial stimuli and/or inflammatory cytokines) induces M1 macrophages, and cytokines such as IL-4 and IL-13 induce the alternatively activated M2 macrophages. Macrophage functions depend on polarization status: M1 macrophages are characterized by a pro-inflammatory phenotype and functions, whereas M2 macrophages display anti-inflammatory functions [1]. It has been reported that resident macrophages nest in various tissues including the lungs. They express a number of M2 markers, including Ym1, IL-10, IL-4, CD206, Fizz1, and Arg1, suggesting that tissue-resident macrophages are already polarized towards an anti-inflammatory M2 phenotype under normal physiological conditions [4].
Allergic asthma is a chronic inflammatory disease characterized by an imbalance between exaggerated type 2 and deficient type 1 immune responses, including a predominant M2 macrophage phenotype [5]. At the moment, based on inflammatory cell count (eosinophilic, neutrophilic, and paucigranulocytic) in tissue and blood, two main subtypes of type 2 inflammation have been defined in asthma: T helper type 2 cell high (T2-high) and T helper type 2 cell low (T2-low). T2-high phenotypes have been classified into three groups: early-onset allergic asthma, late-onset eosinophilic asthma, and aspirin-exacerbated respiratory disease. The non-T2-high (T2-low) endotype is characterized by neutrophilic or paucigranulocytic (i.e., normal sputum levels of both eosinophils and neutrophils) inflammation and a lack of response to corticosteroid therapy [6,7]. In a recent report from the U-BIOPRED about an adult asthmatic study, four asthma phenotypes were identified: phenotype 1, moderate-to-severe asthma; and three severe asthma phenotypes, namely, phenotype 2 (T2) with severe late-onset eosinophilic asthma; phenotype 3 (T3) with severe asthma, oral corticosteroid therapy-dependent; and phenotype 4 (T4) with severe asthma, female predominance, obesity. The severe asthma clusters (T2 to T4) had higher sputum eosinophilia than cluster T1, with no difference in sputum neutrophil counts, exhaled nitric oxide and serum IgE levels, and received oral corticosteroids frequently [8].
The microRNA (miRNA) family is a class of small (20-30 nucleotides in length) non-protein-coding RNAs. MiRNA sequences are transcribed by RNA polymerase II into primary miRNAs (pri-miRNAs) that generate precursor miRNAs (pre-miRNAs) which are exported into the cytoplasm where they inhibit target gene expression by mRNA degradation and/or mRNA inhibition of translation to target protein. MiRNAs are abundant in the circulation and some have been proposed as useful tools for diagnosis, prognosis, and monitoring of treatment outcomes in cancer and other diseases. Deregulation of certain miRNAs is thought to play a role in asthma pathogenesis. Because miRNA functions demonstrated in animal studies may not reflect their role in human asthma, this review focuses mainly on miRNAs with relevance for human adult asthma [9][10][11].

MiRNAs Relevant for Asthma
Current reports about miRNA levels in asthma are contradictory, with some reporting upregulation and others reporting downregulation for the same miRNAs. It appears, however, that miRNA dysregulation could be associated with asthma pathogenesis and responsiveness to treatment in asthma. MiRNAs are present in lung cells (airway biopsies, epithelial brushings), sera/plasma, and other extracellular fluids (sputum, bronchoalveolar lavage (BAL), exosomes [19], exhaled breath condensate). Their levels could be measured and correlated with clinical, biological, and functional parameters in asthma.

MiRNAs Polarizing Macrophages Towards a Pro-Inflammatory M1 Phenotype
The miRNAs that polarize macrophages towards a pro-inflammatory M1 phenotype (Table 1, Figure 1) are important in balancing the anti-inflammatory/regulatory M2 macrophages which promote Th2 immune responses. In addition, pro-inflammatory M1 macrophages promote T helper/cytotoxic type 1 antiviral responses, and virus infections are the most common cause of asthma exacerbations. An excessive macrophage pro-inflammatory response could, however, play a role in severe asthma and, particularly, in neutrophilic asthma.
Let-7f appears to polarize macrophages towards an M1 phenotype.
In the THP1 cell line, pri-let-7f levels were increased in response to LPS while the corresponding mature miRNA let-7f levels were reduced. The overexpression of let-7f in THP-1 differentiated macrophages led to a reduction in SOCS4 protein levels [32].
When individual members of the let-7 family were measured, the expression of let-7f was reported to be higher in cultured epithelial brushings from asthmatic donors [20].
IL-17A is associated with severe asthma and requires IL-23R signaling, which is negatively regulated by let-7f miRNA in CD4+ lymphocytes [33]. Let-7f was negatively regulated by estrogen receptor signaling in the MCF-7 breast cancer cell line [34]. Women have an increased prevalence of severe asthma compared with men. In patients with severe asthma, IL-17A production was increased to a greater degree in TH17 cells from women compared with those from men [35]. Let-7f expression was lower and IL-23R expression was higher in TH17-differentiated cells from women compared with men with severe asthma [35].
MiRNA-9 levels were synergistically increased following IFNγ/LPS co-exposure in isolated macrophages and in vivo, in an IFN-γ/LPS-induced mouse model of steroid-resistant airway hyperresponsiveness (AHR) [21]. MiRNA-9 production decreased protein phosphatase 2A (PP2A) activity and also inhibited steroid-induced glucocorticoid receptor (GR) nuclear translocation. AntagomiRNA-mediated inhibition of miRNA-9 increased both PP2A activity and GR nuclear translocation in macrophages, and restored steroid sensitivity in isolated macrophages and in several mouse models of steroid-resistant AHR [21].
The pro-inflammatory miRNA-9 was found to be increased in sputum samples from patients with neutrophilic asthma, which is often resistant to corticosteroid therapy, compared with eosinophilic asthma [21]. Animal studies suggest that targeting miRNA-9 could be an effective treatment for steroid-resistant asthma.
MiRNA-19 has a direct role in upregulating NF-κB signaling and pro-inflammatory cytokine production. miRNA-19a-3p is capable of inducing an M1 phenotype by targeting Fos-related antigen-1 (Fra-1) transcription factor which plays a key role in the polarization of the M1 to the M2 phenotype and, consequently, decreasing the expression of the Fra-1 downstream genes VEGF, STAT3, and pSTAT3 [39].
The available studies concerning the levels of individual miRNAs of the miRNA-17-92 cluster in different biological specimens in asthma report decreased levels of miRNA-17, miRNA-18, miRNA 19b, and miRNA-20. On the contrary, one study reported increased miRNA-19a levels. miRNA-17-5p was reported downregulated in brushing bronchial epithelial cells cultured in vitro from severe asthmatics compared to cells from healthy donors [26]. Downregulation of miRNA-18a was observed in nasal mucosa biopsies in patients with asthma in comparison with control subjects [23], and in bronchial epithelial cells, obtained by brushing and cultured in vitro, from patients with asthma with different degrees of severity compared to cells from healthy donors [22]. Upregulation of miRNA-19a was found in epithelial cells isolated from biopsies of subjects with severe asthma (only 6) compared with cells from subjects with mild asthma (9) and healthy controls (9), and miRNA-19a was shown to enhance proliferation of bronchial epithelial cells from patients with severe asthma by targeting the TGF-β receptor 2 gene [24]. The levels of miRNA-19b-3p were found to be decreased in bronchial cells obtained by bronchoscopic brushing from patients with steroid-naïve asthma [25] and in brushing bronchial epithelial cells from severe asthmatics cultured in vitro compared to cells from healthy donors [26]. miRNA-20a-5p levels were found to be downregulated in brushing bronchial epithelial cells cultured in vitro from severe asthmatics compared to cells from healthy donors [26]. In another study, miRNA-20 was expressed at the same level in primary bronchial epithelial cells of patients with severe or difficult-to-treat asthma, treated with inhaled corticosteroids and subjects without asthma [40].
All available studies report decreased levels of miRNA-26 in asthma. miRNA-26a has been found to be decreased in bronchial epithelial cells obtained by brushing from patients with steroid-naïve asthma [25]. Levanen and colleagues demonstrated for the first time the presence of miRNAs in exosomes from BAL fluid of both healthy and asthmatic subjects with mild intermittent stable disease [28]. They showed that exosomal miRNA profiles at baseline are different, and in asthma the expression profiles of the identified miRNAs were highly correlated with forced expiratory volume in 1 s (FEV1). Levels of miRNA-26a were downregulated in BAL fluid exosomes in patients with mild asthma [28]. Decreased expression of serum miRNA-26a was observed in a very small group of mild persistent or moderate persistent asthmatic patients (n = 10, 8/10 allergic, smokers 4/10), compared to control subjects (n = 10, 3 being allergic, smokers 5/10) [44]. In another study of the same group, the plasma levels of miRNA-26b, but not miRNA-26a, were decreased in patients with asthma [27].
The expression levels of miRNA-27a and miRNA-27b-3p in freshly isolated bronchial epithelial brushings were lower in patients with steroid-naïve asthma and steroid-using asthma, compared to healthy control subjects [25]. miRNA-27a and miRNA-27b-3p expression was downregulated in bronchial epithelial cells obtained by brushing and cultured in vitro from patients with asthma with different degrees of severity, compared to cells obtained from healthy donors [22].
Circulating plasma miRNA-125b levels were most predictive of asthmatic status, being increased in patients with asthma who had high eosinophil counts [27].

MiRNA-155
MiRNA-155 was increased in M1 and was downregulated in IL-10-polarized human M2c macrophages [2,15,47,48]. miRNA-155 promotes pro-inflammatory classical M1 activation by blocking anti-inflammatory signals and transcription factors, but it can also prevent excessive TLR signaling. Early during TLR activation, miRNA-155 expression is upregulated and inhibits the expression of the negative regulators, such as IL-10, allowing TLR signal transduction and type I IFN-mediated antiviral response. Later on, the increase in anti-inflammatory miRNA-21 induces IL-10 production, and increased IL-10 reduces miRNA-155 expression, limiting the TLR signaling pathways [49]. miRNA-155 also enhances M1-polarization by the repression of negative regulators of pro-inflammatory responses including SOCS1, SH2 domain-containing inositol 5-phosphatase 1 (SHIP1), and BCL6 in various diseases. The serine-threonine kinases Akt contribute to macrophage polarization, and miRNA-155 was found to be essential in serine-threonine kinase Akt-dependent M1/M2 polarization of macrophages (with Akt1 involved in M2-and Akt2 in M1-polarization) by targeting CCAAT/enhancer binding protein-β (C/EBPβ), a key regulator of M2 polarization [50]. SOCS2, a marker of M2, is an essential controller of macrophage activation and function, and also regulates SOCS1 and SOCS3 expression levels through proteasomal degradation [51].
Data regarding the levels of miRNA-155 in asthma are divergent. In primary non-asthmatic and asthmatic human airway smooth muscle cells (hASMCs) isolated from non-transplantable donor lungs or resected lung tissue by enzymatic digestion, miRNA-155 expression was higher in IL-1β/TNF-α/IFN-γ-treated asthmatic cells as compared to normal cells [53].
Levels of miRNA-155 were lower in asthmatic bronchial epithelial cells obtained by brushing and cultured in vitro, than in cells from healthy donors [22]. miRNA-155 expression was also found downregulated in the nasal mucosa [23], in exhaled breath condensates [30], and in cell-free induced sputum in patients with asthma in comparison to control subjects [29]. Low levels of miRNA-155 were reported also in plasma of patients with asthma [27]. In a recent report, patients with severe asthma had higher plasma levels of miRNA-155 when compared with mild-to-moderate asthmatics and non-asthmatic control subjects, suggesting that miRNA-155 may contribute to the severity of inflammation [54].
Recent animal data showed increased expression of miRNA-155 in an ovalbumin (OVA)-induced mouse model of asthma, and lentiviral vector-delivered small interfering (si)RNA targeting miRNA-155 resulted in reduced AHR, airway inflammation, and Th2 cytokine production [55]. These data suggest that miRNA-155 could be involved in asthma severity and that targeting miRNA-155 could be a novel approach for the treatment of allergic asthma [55].
In conclusion, pro-inflammatory miRNA-9, miRNA-18/19, miRNA-26, miRNA-27, miRNA-125b, and miRNA-155 have been shown in vitro to polarize macrophages towards a pro-inflammatory M1 phenotype and their levels were generally found to be decreased in various tissues in asthma. Theoretically, M1-polarized macrophages balance M2 macrophages and play a positive role in both normal individuals and in patients with asthma. However, these pro-inflammatory miRNAs in excess may play a detrimental role in severe asthma.
MiRNA-9 was increased in patients with neutrophilic asthma and the targeting of miRNA-9 has been suggested for the treatment of steroid-resistant asthma.
MiRNA-125b and miRNA-155 were increased in severe asthma, which would suggest that in this situation targeting these miRNAs could be an additional tool in its treatment.

MiRNAs Polarizing Macrophages towards an Anti-Inflammatory/Suppressor M2 Phenotype
Certain miRNAs may prove to play an important role in the pathogenesis of allergic inflammation in asthma by polarizing macrophages towards the M2 phenotype (Table 2, Figure 2). MiRNA-125b and miRNA-155 were increased in severe asthma, which would suggest that in this situation targeting these miRNAs could be an additional tool in its treatment.

MiRNAs Polarizing Macrophages towards an anti-Inflammatory/Suppressor M2 Phenotype
Certain miRNAs may prove to play an important role in the pathogenesis of allergic inflammation in asthma by polarizing macrophages towards the M2 phenotype (Table 2, Figure 2).   The miRNA let-7 family members are generally implicated in regulating the TLR/NF-κB signal pathway and are known as tumor suppressor miRNAs, participating in cell differentiation, proliferation, and apoptosis [61]. The levels of let-7a/b/c/e were found upregulated in tumor-associated macrophages (TAMs), which displayed characteristics of anti-inflammatory M2 macrophages [62].
Let-7 decreased the expression of TLR4 in various cell types [63,64]. Levels of let-7a were significantly decreased in exosomes isolated from BAL fluid in patients with mild intermittent asthma [28] and in exhaled breath condensates from patients with asthma [30]. Serum levels of miRNA-7a were reported to be decreased in patients with asthma [44].
In contrast to reports of mouse studies, TLR7/TLR8 activation triggered monocytes to differentiate into a M2 macrophage surface phenotype with a mixed M1/M2 cytokine secretion profile [66].
Treatment with antagomiRNA against let-7b in a house dust mite-induced model of AHR did not suppress features of the disease [67].
Plasma let-7b expression was found increased in patients with asthma who had high eosinophil counts [27].
Levels of let-7c were found decreased in bronchial cells obtained by bronchoscopic brushing from patients with steroid-naïve asthma [25]. let-7c levels were reported increased in plasma in asthmatic patients with high eosinophil counts [27].
Serum miRNA let-7d levels were reported decreased in patients with asthma [44]. let-7e targeted STAT3 inhibitor SOCS1 in human suppressor antigen-presenting cells (obtained by monocyte stimulation with the TLR7/8-and NLR-activator R848 (Resiquimod)) [68]. In primary nasal epithelial cells from patients with allergic rhinitis, one pathway of the anti-inflammatory effect of miRNA-let-7e was via activation of the JAK1/STAT3 pathway, which caused a decrease in SOCS4 expression [69].
In the nasal mucosa of patients with asthma, let-7e expression was lower than in control subjects [23], but plasma let-7e levels were reported increased in asthmatic patients with high eosinophil counts [27].
By targeting transcripts of proteins that regulate cell division and apoptosis, such as PTEN and programmed cell death 4 (PDCD4) (negative regulators of cell death and cell cycle, respectively) miRNA-21 is considered an oncomiRNA. miRNA-21 inhibited NF-kB and the production of IL-6 and increased IL-10 indirectly, via downregulation of the pro-inflammatory molecule tumor suppressor PDCD4, a negative regulator of AP-1 and IL-10, in a macrophage cell line and bone marrow-derived macrophages [71,72].
Upregulation of miRNA-21 was observed in diphenylcyclopropenone-challenged skin biopsies in human allergic contact dermatitis [73].
The data available concerning miRNA-21 levels in human adult asthma are conflicting, with different degrees of modulation being observed in different cell types and tissues. Levels of miRNA-21 were found decreased in exosomes from BAL and exhaled breath condensates from patients with asthma [28,30].
In contrast, miRNA-21 expression was increased in cultured epithelial cell specimens collected with a tracheal swab in patients with asthma regardless of treatment with inhaled corticosteroids (ICS) compared to the normal subjects, and was significantly higher in the ICS non-treated than the ICS-treated patients with asthma [56]. When these epithelial cells were cultured in vitro with IL-13, miRNA-21 expression increased with increasing IL-13 concentration.
Upregulation of plasma miRNA-21 was also reported in eosinophilic asthma, and it was suggested that miRNA-21 represents a profile of type 2 airway inflammation [27].
The miRNA-21 seems to reflect the type 2 environment characteristic to asthma, and targeting miRNA-21 could be beneficial in asthma [56].

MiRNA-34
The miRNA-34 family is comprised of miRNA-34a, -34b, and -34c, of which miRNA-34a was the first identified tumor suppressor gene [74] and was shown to promote M2 polarization in vitro [2]. The expression of miRNA-34a-5p was found upregulated during in vitro maturation of monocytes to macrophages (unpolarized macrophages, M1, M2a, and a little more in M2c) compared with monocytes [15].
No data are available on miRNA-34a in asthma, but miRNA-34b-5p and miRNA-34c-5p levels in freshly isolated bronchial epithelial brushings were lower in patients with steroid-naïve and steroid-using asthma, compared with healthy control subjects [25]. IL-13 stimulation in vitro decreased levels of miRNA-34b-5p and miRNA-34c-5p in human bronchial epithelial cells (obtained from airways of three lungs donated for lung transplantation and cultured at air-liquid interface (ALI) for 21 days) [25]. Serum levels of miRNA-34 were decreased in patients with asthma, concurrently with the upregulation of autophagy-related proteins and increased levels of pro-inflammatory factors and fibrosis-related proteins [57].
In primary non-asthmatic and asthmatic hASMCs isolated from non-transplantable donor lungs or resected lung tissue by enzymatic digestion, no differences in basal miRNA-146a or miRNA-146b expression were observed between asthmatic and non-asthmatic cells at 0 or 20 hours [86]. Differences in expression between non-asthmatic and asthmatic cells were evident when the cells were treated with the IL-1β/TNF-α/IFN-γ multicytokine cocktail/cytomix that mimics the inflammatory milieu [86]. Induction with cytomix was greater than induction with individual cytokines, and asthmatic ASMCs exhibited higher levels of miRNA-146a expression following IL-1β/TNF-α/IFN-γ cytomix treatment than non-asthmatic cells [86]. In another study, however, IL-1β induced lower miRNA-146a expression in ASMCs derived from patients with asthma compared with control subjects [58].
Williams and colleagues found no differences in miRNA-146a or miRNA-146b expression in airway biopsies from patients with mild asthma compared with control subjects [87]. However, in a recent study, decreased miRNA-146a expression was reported in vivo in bronchial biopsies from patients with asthma compared with healthy donors [58].
MiRNA-146a levels were reported to be increased in adults with asthma [27], and associated with indices related to worse asthma outcomes, including elevated blood eosinophil counts, higher asthma control questionnaire scores, and need for higher doses of ICS [88]. Overexpression of miRNA-146a in A549 human lung carcinoma cells combined with dexamethasone had greater anti-inflammatory effects than either alone, suggesting that miRNA-146a could be used to boost the anti-inflammatory effects of glucocorticosteroids in steroid-insensitive asthmatics [88].
Negative correlation was demonstrated between miRNA-146 expression and that of genes involved in immunity and inflammation (e.g., IRF7 and TLR4). MiRNA-146b modulates the TLR4 signaling pathway by direct targeting of multiple elements, including the LPS receptor TLR4 and the key adaptor/signaling proteins such as myeloid differentiation primary-response protein 88 (MyD88), IRAK-1, and TRAF6 [14,83]. The enforced expression of miRNA-146b in human monocytes led to a significant reduction in the TLR (LPS)-dependent production of several pro-inflammatory cytokines and chemokines [14]. In normal non-transformed cells, NF-kB activation increased STAT3 and consequently miRNA-146b which, as an endogenous negative feedback regulator, attenuated the activation of NF-kB and decreased STAT3 activation [90,91].
There are no studies reporting levels of miRNA-146b-5p in adults with asthma.

MiRNA-223-3p
As monocytes differentiate into macrophages, miRNA-223 expression decreases [15,92]. In vitro differentiation of human monocytes into macrophages with GM-CSF (or M-CSF) was accompanied by a decrease in the expression of miRNA-223, which led to a significant increase in the serine-threonine kinase IKKα protein expression (not of IKKβ or IKKγ).
In addition of macrophage regulation, miRNA-223 controls also eosinophils and neutrophils [93]. The upregulation of miRNA-223 was observed in diphenylcyclopropenone-challenged skin biopsies in human allergic contact dermatitis [73]. The most abundant miRNA in freshly isolated human neutrophils was miRNA-223 [94]. Neutrophil transfer of miRNA-223 to lung epithelial cells was reported [95], and miRNA-223 was expressed in cultured human bronchial epithelial cells, where it also downregulates NF-κB signaling [96]. The administration of miRNA-223 mimics could thus restrict the migration of inflammatory cells to the site of infection and thereby diminish inflammation.
The expression of miRNA-223-3p was significantly upregulated in the sputum of patients with severe asthma compared with that in healthy control subjects, and miRNA-223-3p expression was associated with sputum neutrophilia and also with airway obstruction (FEV1/forced vital capacity) [59]. Plasma miRNA-223 levels have been reported to be increased in patients with asthma [27].
Plasma miRNA-511-3p levels were significantly lower in patients with allergic asthma compared with those with allergic rhinitis, and with subjects without asthma or allergy [60].
In conclusion, the anti-inflammatory miRNAs have been shown in vitro to polarize macrophages towards an anti-inflammatory M2 phenotype. M2 macrophages, together with other type 2 cells, have an important role in allergic asthma. However, the published data concerning the levels of anti-inflammatory miRNAs in asthma are divergent. This could be attributed to few studies, heterogeneous asthma groups, different asthma phenotype, medication received, etc. It is only in recent years that asthma phenotypes have been reported, and criteria to identify them have been clarified. We also do not know yet how different medication alters miRNA levels in different biological samples.
In one study, the levels of let-7b, let-7c, let-7e, miRNA-21, miRNA-146a, and miRNA-223 were found to be increased in plasma in patients with eosinophilic asthma. In another study of patients with severe asthma, miRNA-223 was reported increased in sputum (where macrophages are the predominant type of cells) and correlated with neutrophil counts.

Discussions: microRNAs as Diagnostic Markers and Therapeutic Targets
There is an imbalance in allergic asthma between pro-inflammatory type 1 cells (T cells, B cells, dendritic cells, macrophages, etc.) and anti-inflammatory type 2 cells, in the favor of type 2 cells. Through their involvement in the differentiation, activation, and polarization of monocytes/macrophages, miRNAs could play an important role in the pathogenesis of asthma. Alteration of miRNA levels in macrophages should thus be able to influence the switch towards M1 and M2 phenotypes and between subtypes.
As presented above, according to the current available studies, some miRNAs (i.e., miRNA-9, miRNA-27, miRNA-155, and miRNA-125b) can promote M1 polarization, while other anti-inflammatory miRNAs (miRNA-21, miRNA-223, miRNA-34, let-7c, miRNA-146a, and miRNA-511) can promote anti-inflammatory responses and M2 polarization in macrophages, in both circulatory monocytes and tissue-resident macrophages. The data available on miRNA levels in human adult asthma are conflicting, with different modulation demonstrated in different cell types and tissues. Their levels have been found to be either increased or decreased in various tissues in asthma.
The hypothesis that M1-promoting miRNAs would be decreased and M2-promoting miRNAs would be increased in asthma is not supported by the current data. It is known, however, that the M1 and M2 phenotypes are not inflexible, but rather reversible phenotypes. Plasticity is a fundamental characteristic of macrophages, and M1 and M2 are two extremes in a continuum of the polarization state [17].
Further study of miRNAs in polarized macrophages could help to identify specific miRNAs as markers for M1 or M2 macrophages, and miRNAs could be also proved to be valuable biomarkers for asthma diagnosis and assessment of asthma severity, and to help to discriminate between different asthma phenotypes, eosinophilic or neutrophilic. They may also be a useful adjuvant in asthma therapy and the monitoring of treatment.
Specifically, certain miRNAs which have been found to correlate with disease severity could be proved to be useful as diagnostic biomarkers for asthma [99]. miRNAs could also be used to identify eosinophilic and neutrophilic asthma. Circulating miRNAs levels and, in particular, the plasma level of miRNA-125b, miRNA-21, and let-7b/c/e appear to be predictive of asthmatic status, being increased in patients with asthma who have high eosinophil counts [27]. The pro-inflammatory miRNA-9 was increased in sputum samples from patients with neutrophilic asthma, which is often more resistant to corticosteroid therapy than eosinophilic asthma, and animal studies suggest that targeting miRNA-9 could be effective for the treatment of steroid-resistant asthma [21].
Because of the M2/M1 imbalance in asthma, repolarizing M2 to M1 macrophages with miRNAs could be a possible approach for asthma treatment. Nevertheless, how to achieve macrophage-targeted miRNA delivery constitutes a challenge.
The expression of miRNAs can be controlled by miRNA inhibitors (antagomiRNA) or miRNA agonists (miRNA mimics). The knockdown of miRNAs is an effective method to use for characterizing the functions of miRNAs in vivo. AntagomiRNAs are chemically modified oligonucleotides complementary to individual miRNAs used to transiently inhibit miRNA function. The antisense oligonucleotide binds to the corresponding miRNA and blocks the interaction with its target rather than inducing its degradation.
miRNA-21 appears to reflect the type 2 environment characteristic of asthma, and targeting miRNA-21 could be beneficial in asthma therapy [56]. In a mouse model of acute bronchial asthma, miRNA-21 antagomiRNAs suppressed the development of allergic airway inflammation, inhibiting Th2 activation [100].
The pro-inflammatory miR-125b and miR-155 were observed to be increased in severe asthma, which would suggest that, in this situation, targeting these miRNAs could be an added tool in the treatment of severe asthma [55].
Earlier studies have identified miRNA-155 as a crucial positive regulator of Th1 immune response in autoimmune diseases, but also as a suppressor of Th2 immunity in allergic disorders.
More recent studies provided new evidence that miRNA-155 plays an indispensable role in allergic asthma. When miRNA-155-5p was targeted by using a specific antagomiRNA in an OVA-induced allergic airways disease model [101], the antagomiRNA failed to alter the disease phenotype, although it effectively targeted myeloid cell populations. The author explained the failure of this miRNA-155-5p antagomiRNA to be due to limited ability to target lung lymphocytes. In another OVA-induced mouse model of acute asthma animal study, where miRNA-155 was upregulated, the inhibition of miRNA-155 using a lentiviral vector-delivered small interfering (si)RNA targeting miRNA-155 alleviated airway inflammation, AHR, and Th2 cytokine release [55]. The success of the lentiviral vector-mediated gene delivery system is explained by the high efficiency of gene transduction into a wide variety of dividing and non-dividing cells, and by long-term infection [55].
A first-in-human phase 1 clinical trial of cobomarsen, an oligonucleotide inhibitor of miRNA-155 (antimir-155, MRG-106, miRagen Therapeutics) in patients with certain lymphomas and leukemias is currently underway to measure the safety, tolerability, and pharmacokinetics of this miRNA-155 inhibitor [103].
In another study, it was suggested that miRNA-146a could be used to boost the anti-inflammatory effects of glucocorticosteroids in steroid-insensitive asthmatics [88].
Recently, it has been shown that anti-inflammatory miRNA-125b can reprogram M2 into a pro-inflammatory (M1) phenotype [104]. Following intraperitoneal administration, the CD44 targeting hyaluronic acid-poly(ethylenimine) (HA-PEI)-based nanoparticles encapsulating miRNA-125b nanoparticles were shown to accumulate in the macrophage-ablated lung tissues of both naïve and KRAS/p53 double mutant genetically engineered (KP-GEM) non-small cell lung cancer (NSCLC) mouse model. In addition, a more than 6-fold increase in the M1 to M2 macrophage ratio was observed as well as a 300-fold increase in the iNOS (M1 marker)/Arg-1 (M2 marker) ratio in TAMs, compared to the untreated control group. Successful pro-tumor (M2) phenotype repolarization towards the M1 phenotype has significant implications in anticancer immunotherapy and, by association, for asthma.

Conclusions
Macrophage polarization is a highly plastic physiological process that responds to a variety of factors, by changing macrophage phenotype and function. Human data concerning miRNAs as modeling elements of the macrophage phenotype are frequently contradicted, however, by the in vivo data obtained from animal studies. Although the initial studies show promising results, further studies are required to elucidate the role of lung macrophages as a cellular source of miRNAs in the lung in asthma and to determine whether they may serve as biomarkers and potential therapeutic targets.
Author Contributions: All authors were involved in the design of the review, and in writing or revising the manuscript. All authors approved the submitted version. All authors agreed to be personally accountable for their own contributions and for ensuring that questions related to the accuracy or integrity of any part of the work, even those in which the author was not personally involved, are appropriately investigated, resolved, and documented in the literature.

Conflicts of Interest:
The authors declare no conflict of interest.


text:
Chi3l3 induces oligodendrogenesis in an experimental model of autoimmune neuroinflammation

In demyelinating diseases including multiple sclerosis (MS), neural stem cells (NSCs) can replace damaged oligodendrocytes if the local microenvironment supports the required differentiation process. Although chitinase-like proteins (CLPs) form part of this microenvironment, their function in this differentiation process is unknown. Here, we demonstrate that murine Chitinase 3-like-3 (Chi3l3/Ym1), human Chi3L1 and Chit1 induce oligodendrogenesis. In mice, Chi3l3 is highly expressed in the subventricular zone, a stem cell niche of the adult brain, and in inflammatory brain lesions during experimental autoimmune encephalomyelitis (EAE). We find that silencing Chi3l3 increases severity of EAE. We present evidence that in NSCs Chi3l3 activates the epidermal growth factor receptor (EGFR), thereby inducing Pyk2-and Erk1/2- dependent expression of a pro-oligodendrogenic transcription factor signature. Our results implicate CLP-EGFR-Pyk2-MEK-ERK as a key intrinsic pathway controlling oligodendrogenesis.

A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted Give P values as exact values whenever suitable.

For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings
For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated

Clearly defined error bars
State explicitly what error bars represent (e.g. SD, SE, CI) Our web collection on statistics for biologists may be useful.

Software and code
Policy information about availability of computer code Data collection n/a Data analysis n/a For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors/reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Research guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A list of figures that have associated raw data -A description of any restrictions on data availability The data that support the findings of this study are available from the corresponding author upon reasonable request. All studies must disclose on these points even when the disclosure is negative.

Sample size
No sample size calculation was performed. Sample size was chosen based on previous experiments and significance levels determined as described in the individual Figure legends.
Data exclusions No data were excluded.

Replication
In vitro experiments were repeated at least two times with at least n=3 independent technical replicates or three times with at least n=2 independent technical replicates. In vivo experiments included at least n=3 mice. Individual repeats and sample sizes as well as significance levels are indicated in the Figure

Blinding
The investigators were blinded as to experimental groups during data collection and analysis. Specifically, for in vivo experiments, EAE scoring and subsequent tissue analysis was performed by an independent researcher, who was blinded to experimental treatment groups.
Reporting for specific materials, systems and methods  Authentication -293T cells were authenticated by Life Technologies.
-H9 hESC derived NSCs (H9, N7800-100) were authenticated by the vendor and additional human genome sequencing and microarray analysis was performed (data not shown) -BV2 cells were positively tested for microglial markers, however no further authentication was performed.

Mycoplasma contamination
Testing for mycoplasma contamination was not performed.
Commonly misidentified lines (See ICLAC register) n/a

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research

Laboratory animals
Female SJL/J/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). All animals were housed in pathogenfree facilities. All experiments were performed with the approval of the local animal welfare committees (Harvard Medical Area Standing Committee on Animals, and LAGeSo, Berlin) and in accordance with national and international guidelines to minimize discomfort to animals (NIH and 86/609/EEC).

Wild animals are not included
Field-collected samples was not performed.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
Mice were perfused under anesthesia with cold PBS (without Mg+2 and Ca+2), the brain and spleen were extracted and kept in RPMI medium on ice. Each brain hemisphere was processed separately. In brief, the brain tissue was mechanically homogenized with a syringe plug through a 70μm cell strainer (Corning) and washed with RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (FCS) and antibiotics. The myelin rich cell suspension was resuspended in 37% Percoll (Sigma-Aldrich), the myelin-free non-neuronal cells were collected from the pellet after centrifugation at 2800g, and washed two times in medium. Single cell suspension of spleen was obtained by homogenizing the tissue though a 100μm cell strainer; erythrocytes were lysed for 10 minutes with 0.15 M ammonium chloride and washed two times. Flow cytometry staining was performed at 4°C in 1ml Micronic tubes in PBS without calcium and magnesium (Gibco) containing 0.5% BSA (Serva). First, the cells were incubated for 15 minutes with anti-mouse CD16/CD32 (clone 2.4G2, BD Biosciences) to


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Psychology

text:
Differential effects of D-cycloserine and amantadine on motor behavior and D2/3 receptor binding in the nigrostriatal and mesolimbic system of the adult rat

D-cycloserine (DCS) and amantadine (AMA) act as partial NMDA receptor (R) agonist and antagonist, respectively. In the present study, we compared the effects of DCS and AMA on dopamine D2/3R binding in the brain of adult rats in relation to motor behavior. D2/3R binding was determined with small animal SPECT in baseline and after challenge with DCS (20 mg/kg) or AMA (40 mg/kg) with [123I]IBZM as radioligand. Immediately post-challenge, motor/exploratory behavior was assessed for 30 min in an open field. The regional binding potentials (ratios of the specifically bound compartments to the cerebellar reference region) were computed in baseline and post-challenge. DCS increased D2/3R binding in nucleus accumbens, substantia nigra/ventral tegmental area, thalamus, frontal, motor and parietal cortex as well as anterodorsal and posterior hippocampus, whereas AMA decreased D2/3R binding in nucleus accumbens, caudateputamen and thalamus. After DCS, ambulation and head-shoulder motility were decreased, while sitting was increased compared to vehicle and AMA. Moreover, DCS increased rearing relative to AMA. The regional elevations of D2/3R binding after DCS reflect a reduction of available dopamine throughout the mesolimbocortical system. In contrast, the reductions of D2/3R binding after AMA indicate increased dopamine in nucleus accumbens, caudateputamen and thalamus. Findings imply that, after DCS, nigrostriatal and mesolimbic dopamine levels are directly related to motor/exploratory activity, whereas an inverse relationship may be inferred for AMA.

In rats, systemic AMA (100 mg/kg i.p.) augmented striatal acetylcholine (ACh) and nigral and striatal GABA, starting immediately post-injection 21 . Increases of striatal DA and serotonin (5-HT) concentrations were detectable, but not significant 22 . Also Maj et al. 19 observed no effect on striatal DA after application of lower AMA doses (10 to 80 mg/kg i.p.). Other scientific groups, however, reported a significant augmentation of striatal DA after systemic AMA (40 mg/kg s.c. 27 ; 46 or 92 mg/kg i.p. 28 ; 100 mg/kg i.p. 29 ), also starting immediately post-injection 29 . Likewise, intrastriatal infusion of AMA (0.1 mM or 1 mM) elevated the striatal release of both DA and GLU 29 .
After systemic DCS, so far, no in vivo imaging studies of D 2 R-like binding have been performed on either humans or rats. After chronic treatment with AMA (200 mg/day for at least 10 days), two in vivo imaging studies of striatal D 2 R-like binding have been conducted on Parkinsonian patients, using [ 11 C]raclopride as radioligand 30,31 . Both studies reported a significant elevation of striatal D 2 R-like binding, implying that, at least in Parkinsonian patients, AMA did not increase synaptic DA to an extent sufficient to effect a detecable competition with the exogenous radioligand. Contrarily, in our previous study on healthy rats 32 , AMA challenge with 40 mg/ kg i.p. reduced D 2 R-like binding in nucleus accumbens (NAC), caudateputamen (CP) and thalamus (THAL) relative to baseline, while 10 mg/kg diminished D 2 R-like binding in the anterodorsal HIPP (aHIPP).
In the present investigation, we assessed the effects of systemic DCS (20 mg/kg i.p.) on motor/exploratory behaviors and on D 2 R-like binding in regions of the rat nigrostriatal and mesolimbic systems, which are related to motor as well as cognitive and emotional functioning ( [33][34][35] . Effects of DCS on D 2 R-like binding were compared to our previous findings on the NMDAR antagonist AMA 32 .

Materials and Methods
Animals. Imaging studies of D 2 R-like binding sites were conducted on 38 adult male Wistar rats (ZETT, Heinrich-Heine University, Düsseldorf, Germany), weighing 397 ± 49 g (mean ± standard deviation [SD]; age: 3-4 months). The animals underwent morphological MRI, SPECT measurements in baseline and after injection of DCS or AMA, and behavioral testing after injection of DCS (20 mg/kg: n = 16) or AMA (40 mg/kg: n = 22). MRI and SPECT measurements in baseline and after pharmacological challenges were performed in randomized order and were separated by at least 3 days. Due to seizures or cardial arrest after the administation of the anaesthetic, 5 rats merely underwent behavioral measurements without subsequent D 2 R-like imaging. Behavioral data obtained after DCS and AMA were compared to the behavioral data obtained after vehicle (0.9% saline) in 16 further male rats of the same strain, age (3-4 months) and weight (418 ± 63 g). Behavioral data obtained after 40 mg/kg AMA and saline have been previously published 32,36 .
Rats were kept as previously described (e.g. 32 ). The study was performed in accordance with the German Law on the Protection of Animals and the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978). The protocol was approved by the regional authority (Landesamt für Natur, Umwelt und Verbraucherschutz, Nordrhein-Westfalen, Recklinghausen, Germany).
SPECT studies. D 2 R-like imaging in baseline and after challenge with DCS or AMA was conducted as previously described 32,36,37,40 . Also the employed small animal SPECT ("TierSPECT"; field of view: 90 mm; sensitivity: 22 for 123 I; spatial resolution: 3.4 mm for 123 I) employed in the present study was described in detail elsewhere 41 .
Behavioral studies. Immediately after administration of DCS, AMA or saline, motor and exploratory behaviors were assessed in an open field (Phenotyper ® , Noldus Information Technology, Wageningen, The Netherlands; dimensions: 45 × 45 × 56 cm) with EthoVision XT (Noldus Information Technology, Wageningen, The Netherlands) as previously described 32,36,37,40 . Durations (s) and frequencies (n) of ambulation, sitting (as a measure of "passive immobility" 45 ), rearing, head-shoulder motility and grooming were rated in blocks of 5 min for a total of 30 min. Subsequent to behavioral tests, rats were anaesthetized as described above and injected [ 123 I] IBZM.
Evaluation of SPECT imaging studies. D 2/3 R imaging data were evaluated with PMOD (version 3.5, PMOD Technologies Ltd., Zürich, Switzerland) as previously described 32,37 . Briefly, for each rat, SPECT and MR images were coregistered. Then, the MR image of each rat was coregistered with the Paxinos standard rat brain MRI 46 provided by PMOD. The necessary mathematical transformations were used to re-import the SPECT image previously coregistered with the MRI. On the individual overlays of each rat brain SPECT with the Paxinos standard rat brain MRI volumes of interest (VOIs) were defined, as previously described 32,37 . Thereby, the maximum VOI diameters were either in the range of or beyond the spatial resolution of the employed small animal SPECT. Regional BPs were estimated according to the simplified reference tissue model 47 by computing ratios of radioactivity counts obtained in the specifically-bound compartments (NAC, CP, THAL, SN/VTA, FC, MC, PC, aHIPP and pHIPP) to radioactivity counts in the cerebellar reference VOI.

Statistical analysis. D 2/3 R imaging studies.
Distributions of both regional BPs and behavioral data were tested for normality with the non-parametric Kolmogorov-Smirnov test (α ≤ 0.05). Regional BPs were neither uniformly distributed in baseline, nor after DCS or AMA (0.002 ≤ p ≤ 0.200).
Medians and interquartile ranges (25-/75-and 5-/95-percentiles) of regional BPs were computed for both compounds. Moreover, percentual differences of BPs after DCS or AMA relative to baseline were calculated. Regional BPs were compared between baseline and challenge (20 mg/kg DCS or 40 mg/kg mg/kg AMA) with the non-parametric Wilcoxon signed rank test for paired samples (two-tailed, α ≤ 0.05).
Behavioral studies. Behavioral variables (duration and frequencies of ambulation, sitting, rearing, head-and-shoulder motility and grooming) were evaluated with two-way analyses of variance (ANOVAs) with the factors "time" (denoting the individual 5-min time bins) and "treatment" (denoting challenge with either 20 mg/kg DCS, 40 mg/kg AMA or saline). In the majority of comparisons over time bins and treatments, the Shapiro-Wilk normality test failed (p < 0.050). Post hoc pairwise comparisons between treatment groups were performed for each variable in the individual time bins with the Holm-Sidak test (overall α ≤ 0.05). Furthermore, Spearman rank correlation coefficients (r; α ≤ 0.05) were calculated for regional radioligand binding and behavioral parameters in the individual time frames (min 1-5, 6-10, 11-15, 16-20, 21-25 and 26-30).
Statistical analysis was performed with IBM SPSS Statistics 23 (IBM SPSS Software Germany, Ehningen, Germany). and SigmaStat (version 3.5, Systat Software Inc., Erkrath, Germany). Figures 1 and 2 show images of the Paxinos standard rat brain MRI atlas 46 at different positions from Bregma together with the standard VOI templates provided by PMOD (left columns). The next columns show characteristic images of regional [ 123 I]IBZM accumulations on coronal slices in baseline (middle) and after challenge (right) with 20 mg/kg DCS ( Fig. 1) and 40 mg/kg AMA (Fig. 2), respectively, at the positions from Bregma depicted in the left columns 48 . Baseline and post-challenge scans after both treatments stem from the same rat.
In precedent studies on rats, systemic treatment with the DA precursor L-DOPA 36 , the DA reuptake inhibitor methylphenidate 49 , and the GABA A R agonist muscimol 37,40 diminished [ 123 ]IBZM binding to the rat D 2/3 R. The augmentation of DA concentrations in the synaptic cleft is common to all of these compounds. Since [ 123 ]IBZM competes with endogenous DA molecules for D 2/3 R binding sites, the observed decreases of D 2/3 R binding may be conceived to reflect increased levels of synaptic DA 50 . Therefore, it may be surmised that, also in the present study, the AMA-induced regional reductions of D 2/3 R binding were due to elevated DA concentrations in these areas, whereas the observed regional increases of D 2/3 R binding after pre-treatment with DCS indicate reductions of available DA. www.nature.com/scientificreports www.nature.com/scientificreports/ This is the first study, which assessed the effects of DCS on subcortical and neocortical DA in rats with a non-invasive in vivo imaging approach. Until now, the effect of DCS challenge on DA has only been studied in the rat CP, where either no effect 23 or a significant elevation of DA efflux 22 was observed. The latter is in contrast with our findings, which did not show an alteration of D 2/3 R binding in the CP after 20 mg/kg DCS. Likely reasons for this inconsistency are the differences in methods: firstly, we performed in vivo SPECT, while Bennett and Gronier 22 assessed striatal homogenates with high pressure liquid chromatography; secondly, we administered DCS systemically, while Bennett and Gronier 22 incubated striatal slices; and, thirdly, we used adult rats with a mean weight of 397 ± 49 g, while Bennett and Gronier 22 employed adolesent animals, weighing between 250 and 350 g.
The present result of significantly decreased ambulation after challenge with DCS compared to saline contradicts previous studies, which reported either no effect or merely a slight depression of spontaneous locomotor activity after systemic treatment with 0.3 to 65 mg/kg DCS [15][16][17][18] . Also the finding of decreased ambulation after challenge with AMA does not agree with previous fndings, showing an elevation of locomotor activity after systemic treatment with 40 to 100 mg/kg AMA [19][20][21] . Only the present finding of unaffected grooming duration confirms previous results obtained after 0.3 and 3 mg/kg DCS 17 .
Pharmacological effects on motor function in rodents are strongly dependent on age: the DA precursor L-DOPA, for instance, increased motor activity in neonatal (5 to 8 days old 51 ) and immature rats (18 to 20 days old 52 ) www.nature.com/scientificreports www.nature.com/scientificreports/ after doses of 12.5 to 50 mg/kg and 150 mg/kg, respectively, whereas motor activity was diminished in adult animals (25 to 30 days of age 52 ). In the present study, rats were considerably older (approximately 4 months old and weighing 397 ± 49 g) compared to the other investigations on DCS and AMA, in which adolescent (250-300 g 15 ; 250 ± 50 g 16 ; 200-250 g 17 ; 200 g 18 ; 110-115 g 19 ; 100-120 g 20 ; average weight of 250 g 21 ) animals were used. As a consequence, the present discrepancies may be accounted for by the difference of ages between samples in conjunction with NMDAR agonistic and antagonistic action on synaptic DA levels. The effects of DCS and AMA on both regional D 2/3 R binding and motor/exploratory parameters must be assessed in future investigations in rats of different ages in order to shed further light on this matter.
It is not surprising that the NMDAR agonist DCS and the NMDAR antagonist AMA exert opposite actions on D 2/3 R binding. Striking, however, are the differences in regional contributions: while AMA affects merely NAC, CP and THAL, DCS acts on the site of origin of DA fibers (SN/VTA) as well as on target regions of DAergic projections throughout the mesolimbic and nigrostriatal system (NAC, THAL, neocortex, HIPP) with the exception of the CP. www.nature.com/scientificreports www.nature.com/scientificreports/ In rodents, DCS increased GABA efflux in the mouse whole brain 18,24 . Moreover, decreases of GLU levels were observed in the rat amygdala 25 as well as in the mouse whole brain 18 , whereas no effect was detected in the rat FC 26 . This implies that overall alterations of GABAergic and GLUergic input to the nigrostriatal and mesolimbic target regions of ascending and descending fibers incurred a net decline of DA, reflected by the observed increases of D 2 R binding in NAC, SN/VTA, THAL, neocortex and HIPP.
As far as can be inferred from precedent investigations, the major difference between DCS and AMA action on cortical and subcortical DA levels is that GLU appears to be either unaffected or decreased by the former 18,25,26 but increased by the latter 29 . As previously outlined in more detail 32 , it may be assumed that AMA (contrarily to DCS) increases GLUergic input to the target regions of corticostriatal and corticomesolimbic projections, thus augmenting DA efflux in CP, NAC and THAL. It may be hypothesized that these differences in GLUergic and DAergic activation are related to the observed behavioral differences, namely decreased rearing duration and frequency (min 1-10, each) and increased sitting frequency (min [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] after AMA compared to DCS. Moreover, also in the second half of the testing time, after AMA, exploration was primarily performed by the sitting animal merely moving its head and shoulders (min [11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30]. In the direct pathway (CP -pars reticulata of the SN/internal globus pallidus) DA disinhibits GABAergic neurons, incurring an activiation of the mesencephalic, diencephalic and brainstem motor centers, whereas, in the indirect pathway (CP -external globus pallidus/subthalamic nucleus -pars recticulata of the SN/internal globus pallidus), GABAergic neurons are inhibited by DA, resulting in a suppression of motor activity 53 . Moreover, the NAC with its afferents to the limbic system and its efferents to the GP acts as a limbic-motor interface, which is pivotal for the translation of emotional and motivational states into action 54 . Correlation analysis revealed that, after DCS, high sitting frequency (at 15 min post-challenge) predicted high D 2/3 R binding (and low DA) in CP, FC and MC (at 75 min post-challenge). Moreover, a low frequency of head-shoulder motility (immediately post-challenge) predicted high D 2/3 R binding (and low DA) in NAC, CP, THAL, SN/VTA, FC and pHIPP (at 75 min post-challenge). Contrarily, after AMA, a high sitting frequency (at 6 min post-challenge) predicted low D 2/3 R binding in NAC and CP (at 75 min post-challenge), whereas low durations and/or frequencies of ambulation and head-shoulder motility (immediately post-challenge) predicted low D 2/3 R binding in NAC, CP, THAL and SN/VTA (at 75 min post-challenge). This infers that after DCS and AMA, the altered levels of DA in the individual regions of the nigrostriatal and mesolimbic pathway within the first 15 min post-injection differentially affected motor neurons in conjunction with emotional/motivational states. Thereby, after DCS, lower regional DA concentrations produced the effect that rats were less able (and/or less "motivated") to ambulate or explore their environment by head-shoulder movements, but more able (and/or more "motivated") to exhibit rearing behavior. In contrast, after AMA, higher regional DA levels induced a general behavioral depression characterized by decreased rearing and increased sitting, while, consistently, exploration mainly consisted of head-and-shoulder movements. The question still remains to be solved, however, in as much the regional BPs (and DA levels) at the time of in vivo imaging correspond to the DA levels at the time of data acquisition in the open field. Future investigations are needed, in which behavior are assessed for a longer time than 30 min post-challenge. Moreover, regional D 2/3 R binding should be determined in different sets of animals at various times after [ 123 I]IBZM application.
In the present study, findings may have been biased by the employment of the NMDAR antagonist ketamine as anaesthetic. Since ketamine has previously been shown to enhance DA release in rats (e.g. [55][56][57], it can not be excluded that DA release elicited by ketamine actually reduced the amounts of visible regional D 2/3 receptor binding after both DCS and AMA. Effects on neostriatal and/or ventrostriatal DA, however, are exerted by practically all known anaesthetics, including pentobarbital, propofol, halothane, chloral hydrate and isoflurane (for review see 58 ). Therefore, we decided to maintain the usage of ketamine, which was employed in all our previous investigations. Since also this possible pitfall concerns the outcome of SPECT measurements both in baseline and post-challenge, the obtained BPs remain comparable between conditions.

Conclusion
Taken together, in adult rats, DCS increased D 2/3 R binding in NAC, SN/VTA, THAL, FC, MC, PC, aHIPP and pHIPP, whereas AMA decreased D 2/3 R binding in NAC, CP and THAL. The elevations of D 2/3 R binding after DCS reflect a reduction of available DA throughout the nigrostriatal and mesolimbic system, while the reductions of D 2/3 R binding after AMA indicate an increased availability of DA in NAC, CP and THAL. Findings imply a direct relationship between nigrostriatal and mesolimbic DA levels and motor/exploratory activity after DCS, but an inverse relationship after AMA.