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PGE2 production at sites of tissue injury promotes an anti-inflammatory neutrophil phenotype and determines the outcome of inflammation resolution in vivo

Neutrophil removal from inflammatory sites is regulated by lipid mediator signals between macrophages and neutrophils.

INTRODUCTION
Inflammation is a critical process that maintains normal tissue homeostasis following injury or infection, by removing potential pathogens and beginning the process of wound repair and healing. Neutrophils are the first immune cells recruited to a site of injury or infection, where they carry out a number of functions (1). They destroy foreign pathogens, control the spread of infection, and thereby minimize tissue damage. Neutrophils engulf bacteria and cell debris at the site of inflammation through phagocytosis and release antimicrobial molecules by degranulation. Along with the release of reactive oxygen species, this activity aids in the destruction of pathogens (2) and permits neutrophil penetration into otherwise inaccessible tissues (3). Having completed their role, neutrophils must then be removed from the inflammatory site for successful inflammation resolution to occur. If this tightly controlled physiological process is disrupted, then failure of inflammation resolution can lead to inflammatory diseases such as chronic obstructive pulmonary disease.
There are several mechanisms by which neutrophils can be removed from sites of inflammation. Caspase-dependent neutrophil apoptosis and subsequent efferocytosis by macrophages have been shown to contribute to the successful resolution of inflammation in mouse (4), human (5), and zebrafish (6). Natural killer cells can also induce granulocyte apoptosis as a proresolving mechanism (7).
Neutrophil apoptosis is tightly coupled to uptake by macrophages to prevent tissue damage (8). Macrophages phagocytose pathogens and apoptotic cells and contribute to inflammation resolution (9), tissue remodeling, and regeneration (9,10); however, their influence on inflammation resolution is incompletely defined.
More recently, it has been demonstrated that neutrophils can also undergo reverse migration away from sites of inflammation, which, in some circumstances, might contribute to the dissipation of the inflammatory burden and hence to inflammation resolution (11,12). In the zebrafish, reverse migration is anti-inflammatory and is suppressed by proinflammatory stimuli such as hypoxia (13). Variations on this process have also been seen in mouse models (14,15) and in human neutrophils (16,17). Understanding the molecular mechanisms that govern neutrophil removal, either via efferocytosis or via reverse migration, is essential to developing new therapeutics for inflammation resolution.
Macrophages are well known to be key mediators in determining the outcome of the inflammatory response (18). Macrophages and neutrophils secrete a range of pro-and anti-inflammatory cytokines, dependent on the local inflammatory environment. Studies in mammalian systems (19,20) have shown that upon uptake of apoptotic cells, macrophages regulate pro-and anti-inflammatory cytokine production (21). When fed with apoptotic neutrophils, lipopolysaccharide-stimulated human monocyte-derived macrophages down-regulate key proinflammatory cytokines and up-regulate antiinflammatory mediators transforming growth factor-1 (TGF1), prostaglandin E2 (PGE 2 ), and platelet activating factor (PAF). Prostaglandins, such as PGE 2 , are not stored in cells, but rather produced following arachidonic acid metabolism and therefore can be produced by almost all cells in the body (22). PGE 2 is the most abundant prostaglandin in humans (23) generated via the action of cyclooxygenase enzymes. While PGE 2 is known to have proinflammatory properties under defined conditions (24), it has also been shown to have anti-inflammatory functions (25). In zebrafish cancer models, inhibition of PGE 2 production led to a change in macrophage behavior and enhanced proinflammatory activity (26), consistent with PGE 2 directing macrophages toward an anti-inflammatory phenotype (27). In human peripheral blood neutrophils, PGE 2 switches lipid mediator biosynthesis from predominantly proinflammatory leukotriene B 4 [LTB 4 ; 5-lipoxygenase (5-LO)-initiated pathway] to lipoxin A 4 (LXA 4 ), which reduces neutrophil infiltration into exudates (28). These studies support a role for PGE 2 in influencing immune cell phenotype and led us to ask whether macrophages might be altering local lipid mediator signaling as a mechanism for influencing inflammation resolution in vivo.
Using a zebrafish model of in vivo inflammation resolution, we examined the potential role of the lipid mediator PGE 2 in driving inflammation resolution. We demonstrate that macrophage uptake of apoptotic cells is necessary for successful inflammation resolution; PGE 2 is produced and acts via EP4 receptors to drive inflammation resolution by reverse migration.

Macrophage clearance of apoptotic cells is necessary for successful resolution of neutrophilic inflammation in vivo
Inflammation requires the presence and activity of both neutrophils and macrophages. To test whether neutrophil dynamics were influenced by the presence of macrophages, we ablated macrophages using tissue-specific bacterial nitroreductase expression and by treatment with metronidazole as previously described (29,30). Injury was performed on 3 days post fertilization (dpf) macrophage-ablated larvae, and neutrophil and macrophage numbers were assessed at the time points indicated (Fig. 1A). In control larvae, neutrophil numbers peaked between 4 and 6 hours post injury (hpi), with numbers returning to basal levels by 24 hpi, demonstrating spontaneous resolution of inflammation. Macrophages were recruited to the site of injury at a lower rate, with numbers plateauing at 12 hpi and remaining high until at least 24 hpi. In metronidazole-treated larvae, macrophages were not seen at the wound throughout the inflammatory time course, confirming successful ablation. Neutrophil numbers at the wound site were significantly increased at 24 hpi in macrophagedepleted larvae compared to control larvae, suggesting failed inflammation resolution (Fig. 1, A and B). In contrast to wound-site neutrophils, whole-body neutrophil counts were unaltered (Fig. 1C). These data demonstrate that macrophages are necessary at the site of tissue injury for normal inflammation resolution to occur.
In many models of inflammation, including the zebrafish, apoptosis of neutrophils contributes to inflammation resolution (6,31). It might therefore be expected that macrophage depletion would leave apoptotic neutrophils at the wound site. To test whether the persistence of apoptotic corpses explained the increase in neutrophil numbers, we ablated macrophages by treating transgenic zebrafish with metronidazole at 7 dpf, injuring the tailfin as before at 8 dpf and then fixing at 24 hpi in 4% paraformaldehyde for TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) staining. Older larvae were used to increase cell numbers involved in the inflammatory response and to increase our ability to detect apoptotic events. We costained for endogenous peroxidase activity of neutrophils using a modification of the tyramide signal amplification (TSA) system (6). Double-stained cells corresponding to apoptotic neutrophils were then counted in macrophage-depleted larvae. Although the number of neutrophils was higher in the metronidazole-treated fish, the percentage of these neutrophils that were apoptotic was not different in the metronidazole-treated group (Fig. 1D), suggesting that persisting apoptotic neutrophil corpses did not account for the increase in neutrophil numbers in the absence of macrophages. During analysis of these images, we did notice a striking and unexpected increase in TUNEL-positive, TSA-negative cells in macrophagedepleted larvae (Fig. 1E), corresponding to apoptotic cells from nonneutrophil lineages. Representative images of stained cells are shown in Fig. 1F. This finding demonstrates that macrophages have an important role in removing apoptotic cells arising from tissue injury and that neutrophil persistence is not due to uncleared apoptotic neutrophils. This suggested to us that the majority of neutrophils might be removed from the wound by an alternative mechanism, such as reverse migration (12).

Inflammation resolution is not exclusively dictated by direct macrophage-neutrophil interaction
Neutrophil-macrophage interaction has been observed during the inflammatory response (32) and, under some circumstances, this has been shown to alter neutrophil migratory behavior in ways that could explain the observed failure of inflammation resolution in the absence of macrophages. We therefore sought to analyze neutrophil migration before and after interaction with macrophages to see whether that interaction caused neutrophils to migrate away from the wound, thereby dissipating the inflammatory burden. We tracked individual transgenically labeled macrophages and neutrophils, monitoring their interaction with other inflammatory cells. We tracked neutrophils and macrophages during an inflammatory response and plotted their x,y coordinates ( Fig. 2A). Interaction between red (macrophage) and green (neutrophil) tracks was identified, and for each interaction, we identified whether contact between a macrophage and a neutrophil induced a significant change in behavior of the neutrophil. Direct cell contact was defined as when membranes of both cell types could be seen to touch. Interaction with a macrophage made no significant difference in the neutrophil meandering index (Fig. 2B), suggesting that interaction did not lead to a change in migratory behavior. Moreover, analysis of vector maps of neutrophils before and after interaction with a macrophage showed no clear difference between the two vector maps. The vector maps show the overall direction a neutrophil takes during the analyzed period (Fig. 2C). When analyzed, the percentage of reverse-migrated neutrophils that did not interact with a macrophage was 71%, compared to 65% of neutrophils reverse-migrating following a macrophage interaction (Fig. 2D). There was no significant difference in reverse migration behavior following macrophage interaction (P = 0.78, Fisher's exact test). Direct contact between macrophages and neutrophils is therefore not sufficient to completely explain the observed changes in neutrophil direction, suggesting that an additional soluble factor is involved.

The eicosanoid PGE 2 is necessary for timely inflammation resolution
Previous studies in vitro have shown that, following uptake of apoptotic cells, macrophages can alter both their profile of cytokine production and their phenotype, from pro-to anti-inflammatory (21). It has been reported that levels of TGF1 and PGE 2 increase following 3 of 13 phagocytosis of apoptotic cells, whereas levels of cytokines such as interleukin-10 and tumor necrosis factor- fail to increase further. When PGE 2 production was blocked by indomethacin, TGF1 levels remained low, suggesting that PGE 2 might be an upstream regulator of macrophage phenotype following uptake of apoptotic cells (21). We therefore sought to establish whether PGE 2 could be a potential mediator in enhancing inflammation resolution following tail injury in vivo.
Microsomal PGE synthase, Ptges, is necessary for the conversion of prostaglandin H 2 into PGE 2 (33). PGE 2 levels can be significantly reduced when Ptges expression is inhibited through the use of a morpholino antisense oligo (34). We used a previously published morpholino to knock down ptges to test the effect of reduced PGE 2 levels on neutrophil behavior during inflammation. Reverse transcription polymerase chain reaction (PCR) demonstrated a significant reduction in correctly spliced ptges transcript at 4 dpf (fig. S1). Neutrophil counts throughout the inflammatory response revealed a significant increase in neutrophil recruitment to the wound site. At the later time points, during the resolution phase, significantly more neutrophils were retained at the site of injury (Fig. 3A). Since that interacted with a macrophage reverse-migrated, whereas 71% neutrophils without a macrophage interaction also reverse-migrated away from the wound site. Chi-squared Fisher's exact test indicated no significant difference in reverse migration with or without macrophage contact. P = 0.78. PGE 2 has been shown to be critical in hematopoiesis (35), we looked at total neutrophil number in ptges morphants. Knockdown of ptges significantly reduced the number of neutrophils in the whole zebrafish ( Fig. 3B), demonstrating that the increase in neutrophil numbers seen during inflammation is not due to an overall increase in neutrophils within the zebrafish. These data demonstrate that Ptges activity plays an important role in the resolution of inflammation.
Genetic knockdown of ptges by morpholino has indicated that PGE 2 production is essential for successful inflammation resolution. Because of the well-documented nonspecific effects of some morpholinos (36), we wanted to confirm our findings using a second approach. To address this, we have used CRISPR/Cas9 technology to support our findings (37). We designed a guide RNA (gRNA) that targets the ATG of ptges. Successful mutation by Cas9 protein deletes an MwoI restriction site, allowing the efficiency of the guide to be assessed and the larvae to be genotyped ( fig. S4) with an uncut mutant band present at 293 base pairs (bp). Using our tailfin injury model, we have injected Tg(mpx:EGFP)i114 embryos at the one-cell stage with transactivating crRNA (tracrRNA) and Cas9 protein alone or in combination with a control gRNA, targeting tyrosinase (tyr) or a gRNA targeting ptges. Tyrosinase is the gene required for converting tyrosine into the pigment melanin, enabling visual identification of successful Cas9 knockdown (38). Genetic knockdown of ptges using Cas9 protein also leads to persisting neutrophils at the wound site compared to tyr-injected larvae (Fig. 3C), phenocopying the ptges morphant. Representative images were taken of each group to show this (Fig. 3D). These data strongly support the essential role of PGE 2 for successful neutrophil removal from wound sites. PGE 2 is sufficient to drive inflammation resolution PGE 2 has both anti- (39) and proinflammatory effects dependent on the timing of its production and concentration (24,40). To test whether PGE 2 is sufficient to drive inflammation resolution, we immersed 3 dpf transgenic zebrafish larvae in a range of PGE 2 concentrations at 8 hours after tailfin transection, after peak neutrophil recruitment had occurred. Wound neutrophil numbers were then assessed at 12 hpi to look for accelerated inflammation resolution. Twelve hours post injury is a time point where untreated larvae still have relatively high neutrophil numbers, and therefore, an enhanced reduction in neutrophil numbers would be detectable. Neutrophil numbers were significantly decreased in a dose-dependent manner when treated with PGE 2 , with 1 M chosen as the optimal concentration (Fig. 3E). Exogenous PGE 2 was able to rapidly enhance neutrophil removal from the site of injury to return neutrophil numbers to basal levels, thereby promoting successful inflammation resolution. As PGE 2 reduced neutrophil numbers at the site of injury, we tested whether PGE 2 accelerated neutrophil apoptosis at the wound site. Three days post fertilization larvae were injured, treated with PGE 2 , and fixed at 12 hpi for dual TUNEL (apoptosis) and TSA (neutrophil) staining. There was no difference in apoptotic cell numbers in larvae treated with PGE 2 compared to control ( fig. S2A). This suggests that neutrophils are removed through a mechanism other than apoptosis.
Since exogenous PGE 2 could enhance inflammation resolution in our model, we investigated whether PGE 2 could rescue the persisting neutrophil phenotype observed with macrophage ablation. Zebrafish larvae with nitroreductase-expressing macrophages and green fluorescent protein (GFP)-expressing neutrophils were treated with metronidazole at 2 dpf, injured at 3 dpf, and treated with PGE 2 at 8 hpi. Cell counts were performed at 12 and 24 hpi. PGE 2 was able to significantly reduce neutrophil numbers at the wound in metronidazole-treated larvae at 12 hpi compared to metronidazoleonly larvae, with means of 4.6 ± 0.4 and 9.6 ± 0.8, respectively ( fig. S2B). At 24 hpi, neutrophil numbers remained significantly reduced (Fig. 3F). These data demonstrate that PGE 2 can drive inflammation resolution in the absence of macrophages, thus correcting the resulting inflammatory phenotype.
Neutrophil migration pattern, but not speed, is modulated by PGE 2 During inflammation resolution, neutrophils can be removed through multiple mechanisms, including neutrophil apoptosis (41) and reverse migration (13,42). Macrophage depletion did not result in a detectable difference in apoptotic neutrophil numbers compared to control, due to either residual macrophage activity or the infrequency of neutrophil apoptosis in the zebrafish model. Nonetheless, PGE 2 can still reduce neutrophil numbers in these larvae, implying that neutrophils involved in the inflammatory response are being removed from the site of injury through a mechanism other than efferocytosis. We therefore investigated whether PGE 2 could enhance inflammation resolution by acceleration of neutrophil reverse migration in tail-transected zebrafish larvae. Using well-established photoconversion protocols (43,44) in transgenic zebrafish expressing the photoconvertable fluorophore kaede in neutrophils, we labeled neutrophils at the wound site at 8 hours after tailfin transection and observed the effects of addition of PGE 2 . Neutrophils at the wound site were tracked during the resolution phase of inflammation for 2 hours, from 10 to 12 hpi. Red, photoconverted neutrophils could be seen migrating away from the site of injury more readily in PGE 2 -treated larvae (Fig. 4, A to C). PGE 2 significantly increased the number of neutrophils moving away from the wound area (Fig. 4D). This equates to there being a 50% difference in the number of reverse-migrated neutrophils in the PGE 2 -treated fish at 140 min after conversion, with a significant P value of 0.0015. These data show that PGE 2 drives inflammation resolution by enhanced reverse migration away from the injury site.
We then asked whether this enhanced migration from the wound could be due to an increased migration speed of the neutrophils or to them leaving the site of injury sooner. Neutrophil migration speed away from the site of injury was measured in the same reverse migration assays described above (Fig. 4E). There was no significant difference in migration speed, implying that PGE 2 may enhance release of neutrophils from their patrolling behavior at the wound site. The timing of PGE 2 release is therefore critical to allow neutrophils to perform their role and then drive them away without compromising the inflammatory response.
Signaling through EP4 receptors contributes to inflammation resolution PGE 2 can signal through four prostanoid receptors: EP1, EP2, EP3, and EP4 (45). The expression and distribution of these receptors vary between different tissues and cell types. EP4 is considered a stimulatory receptor and is expressed on human macrophages, with studies showing that blockage of EP4 signaling inhibits cytokine release by macrophages (46). All four EP receptors are present in zebrafish, with multiple paralogs of each receptor (47). EP4b is the most abundant in adult zebrafish and is closest to human EP4 phylogenetically (48). To test whether PGE 2 could be signaling in vivo through EP receptors, we used a previously published antagonist, AH23848, to inhibit EP4 receptor signaling (49). As we were assessing endogenous PGE 2 signaling during a natural inflammatory response, where neutrophil numbers reduce from around 6 hpi, we performed 24 hpi counts for inhibitor assays to be able to detect neutrophil persistence over normal basal levels. Neutrophil counts at 24 hpi showed a significant increase in number when EP4 signaling was blocked, suggesting that endogenous PGE 2 may act via this receptor. Addition of PGE 2 at standard doses did not lead to significant abrogation of this effect (Fig. 5F). These data suggest that PGE 2 signals predominantly through the EP4 receptor, contributing significantly to inflammation resolution. However, it remained unclear how EP4 signaling might lead to altered neutrophil behavior.

Up-regulation of lipoxygenase by PGE 2 is necessary for inflammation resolution
The metabolism of the membrane lipid arachidonic acid is a complex pathway, producing not only prostaglandins but also other eicosanoids such as proresolving lipoxins and proinflammatory leukotrienes. Lipoxygenases (LOXs) are essential for the production of these metabolites, with select isozymes being key for particular pathways. Previous studies have shown that arachidonate 12-lipoxygenase (12-LO) or 15-lipoxygenase (15-LO) activity, in combination with arachidonate 5-lipoxygenase (5-LO), can produce lipoxins, and 5-LO activity alone forms leukotrienes (50). Increasing concentrations of PGE 2 inhibit 5-LO translocation from the cytoplasm to the nucleus, abrogating leukotriene synthesis (51) and skewing the balance between pro-and Plot showing the number of neutrophils moving away from the wound over 10 to 12 hpi, preincubated with or without PGE 2 from 8 to 9 hpi. PGE 2 -treated neutrophils migrate away from the site of injury between 10 and 12 hpi more readily. Line of best fit shown is calculated by linear regression. P value shown is for the difference between the two slopes. (E) The speed of neutrophils moving away from the site of injury is not significantly different in the presence of exogenous PGE 2 , indicating that neutrophils migrate away sooner rather than at a greater speed in PGE 2 -treated larvae. (F) Neutrophil counts at 24 hpi show a significant increase in neutrophil number when EP4 signaling is blocked using the antagonist AH23848. **P < 0.01, ****P < 0.0001. Addition of PGE 2 does not lead to significant abrogation of this effect, implying PGE 2 signals through the EP4 receptor to promote neutrophil removal. All data are presented as means ± SEM, from n = 18 larvae for (D) and (E) and n = 53 for (F) from three experimental repeats. Images were taken using ×10 magnification on a TE2000U inverted microscope (Nikon).
anti-inflammatory mediators toward increased lipoxin synthesis-a process termed "lipid mediator class switching" (28). Zebrafish have the three key LOX genes for lipid mediator synthesis: alox12, alox5, and alox15b genes (52)(53)(54). To assess the contribution of lipoxin production during inflammation resolution in the zebrafish, we immersed injured transgenic larvae in PD146176, a specific 15-LO inhibitor (55). Again, as we hypothesized that there would be a delay in inflammation resolution, we performed 24 hpi counts for inhibitor assays to be able to detect neutrophil persistence over normal basal levels. We found that inhibiting 15-LO activity during inflammation led to a significant increase in neutrophil numbers at 24 hpi compared to control larvae (Fig. 5A), indicating persisting inflammation at the wound site. This effect was dose-dependent (Fig. 5B). Addition of exogenous PGE 2 following 15-LO inhibition was unable to abrogate this effect, with neutrophil numbers remaining high at the wound site 24 hpi (Fig. 5C), supporting a mechanism of PGE 2 acting upstream of 15-LO in this pathway.
The oxygenation of arachidonic acid by LOXs can occur at varying positions along the fatty acid carbon chain (50) and can determine the functionality of the protein according to which carbon is oxygenated and which amino acid residue is present at specific sites. In zebrafish, LOX genes are annotated in Ensembl as alox12 and alox15b; we predict that zebrafish Alox12 should function predominantly in the role of a 15-LO due to the presence of phenylalanine (F) at position 353 and a bulky valine (V) at position 418 [in com-parison to the rabbit leukocyte 12-LO protein sequence (56) and associated with predominantly 15-LO activity in mammalian LOXs (57); fig. S3]. We therefore assessed alteration of expression of alox12 (the zebrafish putative 15-LO) by quantitative PCR (qPCR) during injury-induced inflammation. There was a significant linear increase in 12-LO expression up to 6 hpi (Fig. 5D), demonstrating that inflammation caused by injury induces a change in LOX activity. This suggests a plausible mechanism for the induction of reverse migration through increased lipoxin production during inflammation resolution.

Lipoxin A 4 drives inflammation resolution via reverse migration
LXA 4 is a proresolving product of arachidonic acid metabolism that inhibits neutrophil chemotaxis, transmigration, superoxide generation, and the production of proinflammatory cytokines (58). Because alox12 expression was increased during injury-induced inflammation, we next determined whether its key product LXA 4 plays a role in inflammation in the zebrafish model. First, we added exogenous LXA 4 to the injured larvae and assessed neutrophil number at 6 hpi. Our data demonstrate a protective effect of LXA 4 acting to inhibit neutrophil chemotaxis, significantly reducing neutrophil recruitment to the wound site (Fig. 6A). This provides evidence that exogenous LXA4 is able to promote a response in our in vivo model. If PGE 2 production following injury increases alox12 activity during inflammation resolution, and thereby increases endogenous LXA 4 production, we would predict that LXA 4 could allow removal of neutrophils already present at the wound site due to loss of sensitivity to local wound signals. To assess this, we performed reverse migration experiments in the presence of exogenous LXA 4 . LXA 4 caused neutrophils to move away from the wound earlier than in untreated larvae (Fig. 6B), without altering neutrophil migration speed (Fig. 6C) or the path that the neutrophils take (Fig. 6D). Our data are best explained by proposing that production of endogenous LXA 4 by Alox12 following injury-induced PGE 2 production is the mechanism that promotes inflammation resolution in vivo.

DISCUSSION
Restoration of tissue homeostasis following infection or injury requires successful resolution of inflammation. Inflammation resolution should not occur before the threat is ablated, nor should inflammation persist longer than necessary. The process is therefore tightly regulated. Neutrophils and macrophages as a population are well positioned to assess the threat to the organism and determine the duration of the inflammatory response. Once the resolution is decided, neutrophils can either die at inflammatory sites by apoptosis and be cleared by macrophages (5) or can be removed from inflammatory sites by altered migratory patterns (reverse migration) (42). If this tightly controlled inflammatory process is disrupted, failure of inflammation resolution can lead to the tissue damage seen in chronic inflammatory diseases (59). The molecular mechanisms con-trolling the decisions on duration of inflammation are complex and yet to be fully determined. Existing evidence indicates that lipid mediator secretion is likely to be important in signaling inflammation resolution (60), but it is unknown which mediators regulate neutrophil removal from sites of inflammation in vivo. We have shown, for the first time, that the lipid mediator signaling molecule PGE 2 is essential for determining the outcome of neutrophil inflammation in response to tissue injury. Moreover, our data suggest that alterations in LOX activity lead to production of LXA 4 . This lipid mediator class switching appears to be the critical mechanistic step by which macrophage PGE 2 release after uptake of apoptotic cells promotes inflammation resolution in vivo.
Previous in vitro studies have suggested that phagocytosis of apoptotic neutrophils by macrophages might contribute toward successful inflammation resolution (21,61). Ingestion of apoptotic cells by macrophages promotes an anti-inflammatory environment, suppressing pro inflammatory cytokine production and promoting LXA 4 production (21). While mice lacking macrophages and neutrophils show healing without excessive inflammation or scar formation, in macrophage-only depleted mice, neutrophils populated the wound area, with impaired wound morphology and delayed healing (62). We have shown that in the absence of macrophages, apoptotic bodies accumulate at the wound site, resulting in neutrophil persistence and pro longed inflammation. This finding de monstrates the importance of understanding macrophage-neutrophil interactions during inflammation resolution to prevent further damage to healing tissues.
Recent work has shown that reverse migration is a contributor to neutrophil removal from inflammatory sites (13,15,42,63). It has been shown that macrophage recruitment and redox-SFK (Src family kinase) signaling play key roles in neutrophil-mediated inflammation resolution (32). These studies also suggest a direct contact mechanism between neutrophils and macrophages, which may direct neutrophil reverse migration; however, in the absence of macrophage contact, neutrophils are still able to reverse-migrate, suggesting that other, unknown factors may be involved. Tracking analysis of macro phage and neutrophil contact in our model of inflammation demonstrates that direct contact is not an essential requirement for reverse migration. Furthermore, our studies support the importance of a soluble factor, the lipid mediator PGE 2 , for successful inflammation resolution. Our data suggest that this lipid mediator is a key signaling molecule between macrophages and neutrophils during inflammation, leading to production of LXA 4 , enhancing neutrophil reverse migration and, hence, speeding inflammation resolution. PGE 2 is known to be involved in many cellular processes, including cell proliferation, apoptosis, angiogenesis, showing the number of neutrophils reverse-migrating away from the wound over a time course of 6 to 15 hpi, preinjected at 4 hpi with or without LXA 4 . LXA 4 -treated neutrophils migrate away from the site of injury sooner than control neutrophils. Line of best fit shown is calculated by linear regression. P value shown is for the difference between the two slopes. n = 21 larvae. (C) The speed of neutrophils moving away from the site of injury was measured and is not significantly different in the presence of exogenous LXA 4 . (D) The path the neutrophils take (meandering index) when moving away from the wound was also not significantly different when supplemented with LXA 4 . All data are presented as means ± SEM, from three experimental repeats.
inflammation, and immune surveillance (64). The production of PGE 2 from prostaglandin synthases is essential for the development of organisms past the gastrulation stage (34) and is crucial for hematopoietic stem cell homeostasis (35). However, to our knowledge, PGE 2 has not been shown to be necessary for resolution of neutrophilic inflammation. We have shown by genetic and pharmacological approaches that the production of PGE 2 is essential for inflammation to resolve and that PGE 2 is sufficient to drive neutrophil removal from wound sites. PGE 2 can promote neutrophil removal from wound sites in the absence of macrophages, implying an important connection between macrophages and the lipid mediator PGE 2 . This could prove to be a useful target for therapeutic intervention in chronic inflammatory disease. PGE 2 acts via EP receptors present on a range of cell types, including neutrophils, macrophages, and epithelial and endothelial cells. Neutrophils predominantly express EP2 and EP4 (65), both of which, upon activation, cause an increase in adenosine 3′,5′-monophosphate (cAMP) levels within the cell. An increase in intracellular cAMP can "reprogram" the cell to switch from 5-LO products to the production of 15-LO products (28). Our studies show PGE 2 signaling through EP4 receptors to be important in the resolution of neutrophilic inflammation and that this activity is mediated via 15-LO products, most likely the anti-inflammatory mediator LXA 4 .
Human polymorphonuclear leukocytes in vitro markedly increase expression of 15-LO mRNA in the presence of PGE 2 (28). Our data from whole animals show a significantly increased expression of the annotated zebrafish 12-LO mRNA following tailfin injury relative to time. Human blood neutrophils exposed to PGE 2 inhibit LTB 4 biosynthesis and demonstrate reduced neutrophil migration (66). Our data confirm the regulation of neutrophil migration by PGE 2 and LXA 4 in an in vivo model of inflammation resolution. Neutrophils more readily migrate away from the wound site when exposed to exogenous PGE 2 or LXA 4 . The addition of PGE 2 after the maximum recruitment of neutrophils to the wound is able to markedly enhance the reverse migration of neutrophils. The mechanism of this step is still unknown, but PGE 2 alteration of LO activity would fit with its known role in inhibiting neutrophil recruitment (67). In our studies, exogenous LXA 4 causes neutrophils to leave the wound sooner. LXA 4 has been shown to inhibit chemokine signaling in neutrophils in vitro (68), suggesting a mechanism by which LXA 4 facilitates neutrophil reverse migration from sites of inflammation. Mathematical modeling data from in vivo neutrophil migration following zebrafish tailfin wounding suggest that neutrophil reverse migration is a phenomenon of stochastic redistribution of neutrophils in the tissues (69). This suggests a model whereby neutrophils are retained at wounds by a molecular "retention signal" and that decay of this signal leads to reverse migration (12). Chemokine receptor desensitization would be a strong candidate for explaining how these retention signals might be initiated and subsequently decayed. LXA 4 interference with these signals might be a potential mechanism by which PGE 2 ultimately alters neutrophil migration patterns, leading to inflammation resolution.
In this context, an in vivo reporter would be useful to further elucidate the presence and activity of PGE 2 . Understanding the molecular mechanisms involved in neutrophil removal from wound sites, specifically during reverse migration, would greatly improve our understanding of inflammatory disease and provide potential strategies and targets for much needed therapeutic intervention.

Maintenance and breeding of zebrafish
To visualize both macrophages and neutrophils within the same zebrafish larva, triple transgenic fish Tg(cfms:Gal4)i186;Tg(UAS: nfsB-mCherry)i149;Tg(mpx:EGFP)i114, as previously described (29), were incrossed. Tg(mpx:gal4)sh267;Tg(UASkaede)i222 zebrafish were used for photoconversion and neutrophil tracking experiments, subsequently termed mpx:kaede. Tg(mpx:EGFP)i114 zebrafish were used to study neutrophils during inflammation. Zebrafish strains were maintained according to standard protocols. Adult fish were maintained on a 14:10-hour light/dark cycle at 28°C in UK Home Officeapproved facilities in The Bateson Centre aquaria at the University of Sheffield, under AWERB (Animal Welfare and Ethical Review Bodies) and UK Home Office-approved protocols.

Inflammation assay
Inflammation was induced in zebrafish embryos by tail transection as described previously (70). Embryos were anesthetized at 3 or 8 dpf by immersion in tricaine (0.168 mg/ml; Sigma-Aldrich), and tail transection was performed using a microscalpel (World Precision Instruments). Neutrophils and macrophages were counted at the site of transection at various time points including 2, 4, 6, 8, and 24 hpi using a fluorescence dissecting stereomicroscope (Leica).

In vivo cell-specific ablation
Metronidazole (Sigma-Aldrich) was dissolved to a concentration of 2.5 mM in E3 with 0.2% DMSO. Solutions of metronidazole were made fresh on the day of use with vigorous agitation until fully dissolved. Tg(cfms:Gal4)i186;Tg(UAS:nfsB-mCherry)i149;Tg(mpx:EGFP)i114 were immersed in metronidazole or E3 and 0.2% DMSO at 2 or 7 dpf overnight at 28°C for 15 hours. Larvae were wrapped in foil during the experiment. At 3 or 8 dpf, larvae were checked for macrophage depletion on a fluorescence dissecting stereomicroscope (Leica).

Neutrophil and apoptotic cell labeling
Rates of neutrophil apoptosis were assessed by dual staining with Rhodamine-TUNEL and FITC-TSA. Transgenic zebrafish larvae were injured at 8 dpf and fixed at 24 hpi in 4% paraformaldehyde. TUNEL (ApopTag Red; Millipore Corp.) staining labeled apoptotic cells with red fluorescence, and TSA (TSAplus kit; Fluorescence Systems, PerkinElmer Life and Analytical Sciences) staining labeled neutrophils with fluorescein green fluorescence. Neutrophils at the wound were imaged on an Eclipse TE2000-U inverted compound fluorescence microscope (Nikon), and neutrophil-specific apoptosis was assessed by the percentage of TSA-positive neutrophils labeled with TUNEL. Red TUNEL-positive-only cells were assessed for total apoptosis rates.

Cell tracking
To assess neutrophil migration behavior before and after interaction with a macrophage, transgenic larvae Tg(mpx:EGFP)i114 were crossed to Tg(mpeg1:mCherry-CAAX)sh378. Three days post fertilization larvae were injured, and images were taken every 3 min between 0 and 12 hpi using an Eclipse TE2000-U inverted compound fluorescence microscope (Nikon). Direct contact was defined as when cell membranes could be seen to touch. Comparisons of migration behavior were made over a 30-min period before and after interaction.

Compound treatment of zebrafish larvae
Following tail transection at 3 dpf, zebrafish larvae were screened at 4 hpi for neutrophil recruitment. Selected larvae were immersed in 1 M PGE 2 (Sigma-Aldrich) at 8 hpi with counts performed at 12 hpi to look for accelerated inflammation resolution and 24 hpi for correction of phenotype assays. Concentrations of PGE 2 ranged from 0.01 to 10 M for dose-response assays. The EP4 antagonist AH23848 (Cayman Chemicals) was used at a final concentration of 1 M. Fish were incubated at 2 dpf, and tail transection was performed at 3 dpf. PD146176, a 15-LO inhibitor (Sigma-Aldrich), was injected at 6 hpi into the Duct of Cuvier to a final concentration of 27 nM in 2 dpf larvae. LXA 4 (Cayman Chemicals) was injected into the Duct of Cuvier to a final concentration of 27 nM at 2 dpf, followed by tail transection and 6 hpi counts.

Reverse migration assay
Tail transection of Tg(mpx:gal4)sh267;Tg(UAS:kaede)i222 larvae was performed at 3 dpf. Embryos were raised to 8 hpi, incubated in 1 M PGE 2 for 1 hour, and mounted in 0.8% low-melting-point agarose (Sigma-Aldrich). An UltraVIEW PhotoKinesis device on an UltraVIEW VoX spinning disk confocal microscope (PerkinElmer Life and Analytical Sciences) was used to photoconvert Kaede-labeled cells at the wound site using 120 pulses of the 405-nm laser at 40% laser power. Embryos were transferred to an Eclipse TE2000-U inverted compound fluorescence microscope (Nikon), where a 1394 ORCA-ERA camera (Hamamatsu Photonics Inc) was used to capture a timelapse series with 2.5-min intervals for 2 hours. Tracking analysis of red-fluorescing neutrophils was performed in Volocity 6 (Improvision; PerkinElmer Life and Analytical Sciences), using the intensity of fluorescence to identify individually labeled neutrophils over time. LXA 4 was injected at 4 hpi at a final concentration of 27 nM, neutrophils at the wound site was photoconverted at 5 hpi, and larvae were time-lapsed between 6 and 15 hpi. Speed data were calculated using tracking time lapses, where larvae were incubated in 10 M LXA 4 . We refer to the track a neutrophil takes as the meandering index of the neutrophil, where a direct movement in a straight line from A to B is given the value of 1. Tracks that are not a straight line fall between zero and 1.

Morpholino studies
All morpholinos were obtained from GeneTools LLC. ptges splice blocking morpholino is described by Feng et al. (26). Standard control morpholino (CoMo; 0.5 nl of 0.4mM) or ptges morpholino was injected into embryos at the one-cell stage previously described (26). Injected larvae were incubated in 5 M PGE 2 at the onset of epiboly to compensate for the PGE 2 required for normal development up to 42 hours post fertilization (hpf ). PGE 2 was then withdrawn from the larvae at 48 hpf. At 3 dpf, larvae were injured, and neutrophil counts were performed at 4, 8, and 24 hpi. Knockdown was assessed in the same fish at 48 hpf and 4 dpf using reverse transcription PCR, and primer sequences were PTGESF1 5′-CTCGGGAGCGACATACAGTT-3′ and PTGESR1 5′-AGCAGATATGCAACGCTGTG-3′. ptges splice blocking morpholino leads to deletion of exon 2 and a loss of 83 bp.

Quantitative PCR
Three days post fertilization larvae were injured, and total RNA was extracted using TRIzol at various time points after injury. Complementary DNA synthesis was performed using SuperScript II (Thermo Fisher Scientific), and qPCR experiments using a DyNAmo Flash SYBR green qPCR kit (Thermo Fisher Scientific) were carried out. CT was calculated using elongation factor 1 alpha as a reference gene. Relative expression levels were plotted after determining Ct by normalizing to a single sample with a high CT value. Primer sequences for alox12 gene were alox12F ccacggaatatcaccagatgga and alox12R attcagagtcgttgtgggtatc.

gRNA design and CRISPR/Cas9 injection
The online web tool CHOPCHOP was used to design a specific gRNA spanning the ATG of ptges. Purified gRNA, Cas9 protein, and tracrRNA were purchased from Sigma-Aldrich. The ptges guide sequence is gauagaggcucaagaugcucguuuuagagcuaugcuguuuug, targeting sequence gatagaggctcaagatgctcggg of the gene. Each embryo was injected with 1 nl of 6.6 nM gRNA, tracrRNA, and Cas9 protein at the one-cell stage. Genomic DNA was extracted from injected larvae, and PCR amplification was performed, generating a 345-bp product spanning the ATG of the ptges gene (ENSDARG00000020136). Primers used were ptges guide F1 gccaagtataatgaggaatggg and ptges guide R1 aatgtttggattaaacgcgact. Genotype was determined by digestion with MwoI. Wild-type PCR products produce 184-, 109-, and 52-bp bands. Mutant forms produce 293-and 52-bp bands.

Statistical analysis
Data were analyzed (Prism 7.0; GraphPad Software) using unpaired, two-tailed t tests for comparisons between two groups and one-way ANOVA (with appropriate posttest adjustment) for other data. A  2 Fisher's exact test was used to assess statistical significance in Fig. 2. Linear regression analysis was performed on qPCR data in Fig. 6.


extfieldsofstudy: Chemistry Medicine

text:
Structure-function analysis and therapeutic efficacy of antibodies to fungal melanin for melanoma radioimmunotherapy

Metastatic melanoma remains difficult to treat despite recent approvals of several new drugs. Recently we reported encouraging results of Phase I clinical trial of radiolabeled with 188Re murine monoclonal IgM 6D2 to melanin in patients with Stage III/IV melanoma. Subsequently we generated a novel murine IgG 8C3 to melanin. IgGs are more amenable to humanization and cGMP (current Good Manufacturing Practice) manufacturing than IgMs. We performed comparative structural analysis of melanin-binding IgM 6D2 and IgG 8C3. The therapeutic efficacy of 213Bi- and 188Re-labeled 8C3 and its comparison with anti-CTLA4 immunotherapy was performed in B16-F10 murine melanoma model. The primary structures of these antibodies revealed significant homology, with the CDRs containing a high percentage of positively charged amino acids. The 8C3 model has a negatively charged binding surface and significant number of aromatic residues in its H3 domain, suggesting that hydrophobic interactions contribute to the antibody-melanin interaction. Radiolabeled IgG 8C3 showed significant therapeutic efficacy in murine melanoma, safety towards healthy melanin-containing tissues and favorable comparison with the anti-CTLA4 antibody. We have demonstrated that antibody binding to melanin relies on both charge and hydrophobic interactions while the in vivo data supports further development of 8C3 IgG as radioimmunotherapy reagent for metastatic melanoma.

In our search for new treatments for metastatic melanoma that would not rely on specific genotypes, biochemical pathways or the variability of an individual's immune response, we turned to radioimmunotherapy (RIT) targeting melanin. RIT has emerged as a useful anti-tumor therapy via the targeting of radionuclides with antibodies against tumor-associated antigens 11 . RIT has historically proven successful in the treatment of primary, recurrent, and refractory non-Hodgkin's lymphomas using anti-CD20 mAbs coupled to two β-emitters, Yttrium-90 and Iodine-131 12 . In the last decade the use of α-emitting radionuclides is gaining momentum both in clinical trials and in the preclinical studies which is driven by the advantages of α-emitters over β-emitters, including very specific targeting of the diseased cells due to the α-particles' short 50-80 µm tissue range, and increased killing efficiency due to high linear energy transfer 13,14 .
Melanoma owes its name to the presence of the melanin pigment. Pigmentation is a very important factor in melanoma biology. The presence or absence of pigment affects melanoma patients survival, radiotherapy, chemotherapy and immune therapy, as was found in experimental models and in melanoma patient population [15][16][17][18][19][20][21] . Pigmentation is also related to hypoxic conditions and HIF-1α expression 22 , and plays a role in the regulation of physiological and pathological conditions 19 .
Historically, melanin was not considered a target for RIT because it is an intracellular pigment contained in organelles called melanosomes, and thus assumed not to be accessible to Ab. However, melanomas are rapidly growing tumors with high rates of cellular turnover and cell necrosis releases melanin into the extracellular space, where it is accessible to radiolabeled mAbs to melanin. Such antibodies deliver cytotoxic radiation to nearby malignant cells via the so called "cross-fire" effect. Furthermore, this strategy is attractive because melanin in normal tissues with very little cellular turnover is not accessible to mAbs by virtue of its "safe" intracellular location. Several IgM isotype mAbs to fungal melanin, such as 6D2 mAb, have been generated in our laboratories in the process of studying Cryptococcus neoformans melanogenesis in vivo and were shown to bind to melanins of different origins 23 . Experimental results have established the feasibility of targeting melanin released from dead melanoma cells in tumors with melanin-binding 6D2 mAb labeled with the beta-emitting radionuclide 188-Rhenium ( 188 Re) 24,25 . Moreover, a phase Ia/Ib clinical trial of 188 Re-6D2 mAb in patients with Stage III/IV melanoma demonstrated safety of RIT, and the trial data was indicative of its efficacy, as there was a prolongation in patients' median survival 26 . However, the IgMs are difficult to produce under the cGMP (current Good Manufacturing Practice) conditions, to purify and to radiolabel. For this reason, the continuous development of RIT targeting melanin towards the clinical product calls for IgG isotype. IgGs are generally more amenable to pre-clinical development including chimerization or humanization and large scale manufacturing for clinical trials compared to mAbs with IgM isotype. More recently, our laboratories generated an IgG type mAb (8C3) to melanin from another pathogenic fungus, Paracoccidioides brasiliensis 27 . Here we report the results of the comparative structural investigation of the novel 8C3 IgG and the previous generation 6D2 IgM and 8C3 evaluation as a potential RIT agent in aggressive murine melanoma.

Results
IgM 6D2 and IgG 8C3 to melanin manifest similar V regions. The sequences of the IgM 6D2 and IgG 8C3 mAbs were deposited at NCBI GenBank (accession numbers KX346262 and KX346264, respectively). The complementarity determining regions (CDRs) of both mAbs contain high percentage of positively charged amino acids (Fig. 1). This finding suggests that these V regions were selected in part by negative charges on the fungal melanin molecules 28,29 and that electrostatic attractions contribute significantly to the binding of the mAbs to melanin. Overall, blast analysis showed that the two mAbs have 53% similarity and 47% identity for the VL and 55% and 40% for the VH. Both mAbs have a remarkable abundance of aromatic residues.
We further compared the structures of 6D2 IgM and 8C3 IgG via molecular modeling which revealed additional contributions to mAbs affinity to melanin. Molecular models of the 6D2 and 8C3 V regions were generated based on the most similar known H and L chain antibody structures (Fig. 2). Alignment of these models revealed pronounced structural differences in the CDR L1 and CDR H3 loops ( Fig. 2A). Visualization of the 6D2 binding surface indicated a slightly positively charged paratope with a negatively charged pocket (Fig. 2B). The 6D2 paratope also contains several aromatic residues, with most located in CDRs L1, L3, H1, and H3 (Fig. 2C). The 6D2 CDR H3 contains aromatic residues Tyr107, Tyr108, Tyr109, Trp111, and Phe112. The 8C3 model has a negatively charged binding surface, mostly originating from residues in and near the 8C3 VL CDRs: Asp1, Glu27, Asp30, Glu109 (Fig. 2D). There are, however, some notable regions of shared positive charge between 8C3 and 6D2 including CDR H2 and several framework regions. The 8C3 model also shows a highly aromatic paratope from residues in CDRs L1, L3, H1, and H3 (Fig. 2E). The CDR H3 in particular contains aromatic residues Tyr109, Tyr110, Tyr113, Phe115, and Phe116.
Altering the immunoglobulin constant region while leaving the variable region unchanged can cause changes in binding affinity and specificity. A recent review of this phenomenon found that antibodies described in the literature whose binding specificities changed after alterations to the C region clustered around similar V region gene families 30 . While the number of unique murine antibodies in that analysis was relatively small (n = 13), we repeated the phylogenetic clustering here including the 6D2 V region sequences (Fig. 3). The 6D2 sequences are labeled and the numbering of the other sequences corresponds to the numbering in the previous review. The grouping of the 6D2 sequences is consistent with the previous grouping and with the notion that certain V region gene families may differ in being permissive of specificity changes following alteration of the C region. However, firm conclusions cannot be drawn at this time due to the small size of the sample and the limited number of non-permissive sequences.  (Fig. 4A). The mAb started to clear from the blood-rich organs at 24 hrs and, in general its biodistribution pattern was typical for a murine IgG. Importantly, the uptake of 111 In-8C3 into the melanin-containing healthy tissues such black eyes and melanized skin on the tails of C57BL6 mice was negligible which demonstrates that this mAb cannot penetrate the membranes of the intact cells and, therefore, cannot damage healthy melanized tissues. To compare the uptake of the melanin-binding antibody into the skin/hair follicles and any ectopic melanin deposits we have compared the uptake of 111 In-8C3 to that of the irrelevant control IgG (Fig. 4B). The uptake of the irrelevant antibody into the melanized tail was equal to that that melanin-binding 8C3 (Fig. 4B). We have also performed the microSPECT/CT imaging of the B16F10 tumor-bearing mice 72 hr post injection of 111 In-8C3 -no uptake into the hair follicles, eyes, brain or anywhere in the body except for the tumor and residual activity in blood pool was seen on the microSPECT/CT data (Fig. 4C).

IgG 8C3 bound to tumor melanin in vivo
RIT with radiolabeled 8C3 was effective in reducing melanoma lesions in the lungs and compared favorably to immunotherapy. Treatment of B16-F10 tumor-bearing C57BL6 mice with 213 Bi-6D2 or 213 Bi-8C3 mAb demonstrated significant (p < 0.01) reduction in metastases-like nodules in the lungs of mice compared to untreated mice or those administered unlabeled 6D2 or 8C3 (Fig. 5A). The IgG Mab 213 Bi-8C3, however, was significantly more effective than the IgM Mab 213 Bi-6D2 in eliminating melanoma lesions in the lungs (p = 0.03) (Fig. 5A). This might be due to better penetration and accumulation in the tumor mass by the IgG antibody. The positive therapeutic results were not only quantitatively significant, but visually impressive. Figure 5B shows the microphotographs of the lungs from a mouse given unlabeled 8C3 mAb (left panel) and a mouse treated with 213 Bi-8C3 mAb. Importantly, the radiolabeling of 8C3 with 213 Bi was accomplished with the high radiolabeling yield of ≥ 95% and required no post radiolabeling purification. In contrast, 213 Bi-6D2 required extensive purification from unreacted 213 Bi, further emphasizing advantages of developing the IgG isotype over that of the IgM for RIT. The comparison of the beta-versus alpha-emitting radiolabel for RIT demonstrated practically the equivalence of 213 Bi-8C3 and 188 Re-8C3 efficacy, both achieving significance compared to untreated or unlabeled 8C3 controls (p < 0.01) in the metastatic B16-F10 melanoma model (Fig. 5C). 188 Re-8C3 appeared to be more effective than 213 Bi-8C3, however, this trend did not reach statistical significance (p = 0.06). Finally, we compared 213 Bi-8C3 RIT with anti-CTLA4 immunotherapy as well as investigated the effect of their combination on the metastasis-like B16-F10 lesions in mouse lungs. The time line of the treatment is shown in Fig. 6A. These results demonstrate that anti-CTLA-4 mAb 9D9 alone could not contain the growth of tumors in this aggressive melanoma model. Both the RIT and RIT plus immunotherapy groups showed significantly fewer lung metastases with n = 28 (±16.9) and n = 28.6 (±3.2), respectively, in comparison with the untreated control group (p = 0.045). However, there was no statistical difference between the efficacy of RIT alone and the combination of RIT plus immunotherapy groups (p = 0.956), suggesting that the effect on the tumor in tumor in the combination treatment group may have resulted solely from RIT.

Discussion
Targeted radionuclide therapy of melanoma is experiencing a resurgence 31 and can be offered to patients who are not responding to current standard of care immunotherapies or used in concert with the immunotherapy. This   Unique VH (A) and VL (B) amino acid sequences for 13 of these murine antibodies were described previously 30 . Leaf labels in the dendrograms are colored according to whether changes in the constant region for that antibody were permissive (green) or non-permissive (red) of specificity changes. Numbering of the sequences corresponds to their numbering in the previous review 30 . prompted us to investigate the structure-function relationship and the therapeutic potential of an IgG mAb to fungal melanin generated in our laboratories.
Although immunization of mice with fungal melanin particles elicits an Ab response that includes IgM and IgG, the mechanism by which the immune system recognizes insoluble highly charged melanin particles is unknown 32 . Only a few mAbs to melanin to have been reported, of which mAb 6D2 is the best characterized. This mAb helped to prove that C. neoformans melanizes in vivo 23 , demonstrated that Abs to melanin have antifungal properties 33 , and subsequently was utilized in melanoma RIT [24][25][26] . The high number of aromatic amino acids in the CDR regions suggests that these residues could potentially interact with melanin through aromatic interactions since melanin pigment is composed of cross-linked subunits with high aromatic content 34,35 . Given melanin's negative charge, it is likely that aromatic interactions predominate for 8C3 binding. The structural differences between the two models also suggest different melanin epitopes for the two mAbs. Compared to 6D2, the highly aromatic CDR H3 loop of 8C3 is flipped toward the center of the binding groove, blocking an interior pocket present in the 6D2 structure. Additionally, the 8C3 CDR L1 loop is pulled away from the binding groove but contains the addition of aromatic residues Phe38 along the paratope edge and Tyr34, which protrudes above and back toward the paratope. In contrast, the 6D2 CDR L1 lines the wall of the binding groove with aromatic residues His31, Tyr38, and His109.
The IgG isotype of 8C3 facilitated quantitative radiolabeling with 213 Bi and 188 Re, which is in striking contrast with the IgM 6D2 used in previous pre-clinical and clinical work, which required extensive post-radiolabeling purification. The ability of radiolabeled 8C3 to bind melanin in vivo clearly resulted from its structure and its many common features with the 6D2 IgM to melanin. Both 213 Bi and 188 Re radionuclides transformed the 8C3 mAb into a therapeutic agent with an impressive efficacy in the very aggressive B16-F10 melanoma model. Importantly, 8C3 was not taken up by the healthy melanin containing tissues such as melanized skin or hair  213 Bi-8C3 mAbs demonstrated significant (p < 0.01) reduction in metastases-like nodules compared to untreated mice or those administered unlabeled 6D2 or 8C3. 213 Bi-8C3 was significantly more effective than 213 Bi-6D2 (p = 0.03). Both 213 Bi-8C3 and 188 Re-8C3 achieved significance compared to untreated or unlabeled 8C3 controls (p < 0.01) in treatment of B16-F10 melanoma. 188 Re-8C3 appeared to be more effective than 213 Bi-8C3, however, this trend did not reach statistical significance (p = 0.06).
SCIENTIfIC REPORTS | (2018) 8:5466 | DOI:10.1038/s41598-018-23889-z follicles due to its inability to cross the cellular membrane and access intracellular melanin. This important feature will ensure its safety towards healthy tissues when used for radioimmunotherapy. Performing this safety evaluation was important, because melanoma cells often secretes the pigment into the environment, where it can be phagocyted by macrophages, or even some melanoma cells may undergo fusion with macrophages. Macrophages may spread the tumor-produced melanin around the organism which created additional danger for normal tissues 16,[36][37][38] . In our future evaluation of the safety of RIT with the humanized version of 8C3 mAb to melanin we are planning to utilize a model of cyclophosphamide-induced alopecia in C57BL6 mice described in 38 which can damage hair follicles and make melanin accessible to the 8C3 mAb, thus emulating a situation of a melanoma patient treated with chemotherapy before being treated with RIT. Effectiveness of 213 Bi-8C3 mAb is particularly encouraging as the interest in, and the use of targeted systemic oncologic therapies with high relative biological effectiveness (RBE) alpha-particle emitters is currently burgeoning worldwide [11][12][13][14] .
As part of the evaluation of 8C3 mAb as a potential RIT agent, we performed its side by side comparison with one of the standards of care drugs for metastatic melanoma treatment -anti-CTLA4 mAb. For this we utilized anti murine-CTLA4 mAb 9D9 which has been tested previously in the B16F10 murine melanoma model 39,40 . In addition, we investigated if preceding the immunotherapy with RIT would increase the efficacy of the former, potentially via an additive or synergistic effect. In this regard, combination treatments have been performed in the treatment of multiple myeloma using alpha-RIT followed by adoptive T-cell transfer (ACT), and results have demonstrated a significant decrease in tumor size compared to control mice or mice that received RIT alone, and those that received ACT alone 41 . The results of our experiment demonstrated that while RIT was quite effective in reducing the melanoma nodules in the lungs of the mice, no reduction was observed in anti-CTLA4 mAb group. The latter result was somewhat expected, since prior studies using the same B16F10 model had demonstrated that the anti-CTLA4 mAbs alone were not very effective 39,40 . Similarly, in clinical practice, though anti-CTLA-4 mAb ipilimumab has been approved as a single agent treatment for patients with metastatic melanoma, currently it is primarily being used in combination with other agents including anti-PD-1 antibodies, anti-cancer vaccines, chemotherapy and radiation 6,7,42,43 . Interestingly, in these studies we were unable to demonstrate that preceding anti-CTLA-4 mAb administration with RIT provided a boost to immune system evidenced by the lack of reduction in the number of lesions in the combination therapy group. This was, most likely, due to the remarkable efficacy of RIT alone and the complete lack of anti-CTLA4 mAb efficacy. In this regard, anti-CTLA4 therapy often needs cooperation from the host microbiome which might not be present in the mice from every vendor 44 . Taken together, these results of the side by side comparison of RIT with anti-CTLA4 immunotherapy demonstrate the advantages of RIT and emphasize its independence of the immune status of the host or state of its microbiome. We hope that future experiments on combining RIT with anti-PD1 and/or anti-PDL-1 mAbs might produce synergistic results. In conclusion, two mAbs to fungal melanin -IgM 6D2 and IgG 8C3 -generated a decade apart, showed remarkable similarity in their variable region and CDR as well as an abundance of aromatic residues that may influence their capacity to bind melanin. The encouraging preliminary results on using 8C3 as a targeting molecule for RIT of experimental metastatic melanoma provide the impetus for the development of melanin-targeted IgG isotype mAbs for clinical applications in treatment of metastatic melanoma. Future attempts to generate chimeric Abs or humanize the murine melanin-binding Abs need to consider that these V regions are permissive to C-mediated changes, suggesting that additional engineering would be required to insure that the murine-derived Vs bind melanin.

Materials and Methods
Data availability statement. The antibodies sequences are available at NCBI GenBank (accession numbers KX346262 and KX346264). The authors will make materials, data and associated protocols promptly available to readers upon publication in Scientific Reports without undue qualifications in materials transfer agreements. mAbs, cell lines and radionuclides. Melanin-binding 6D2 IgM was produced by Goodwin Biotechnologies (Plantation, FL, USA) as described in Dadachova et al. 24 and 8C3 IgG was produced in our laboratories as described in Urán et al. 27 . Anti-murine CTLA4 mAb 9D9 for the immunotherapy treatment arm was purchased from BioXCell (West Lebanon, NH, USA) and IgG control mAb -from Creative Diagnostics (NYC, NY, USA). The pigmented murine melanoma cell line B16-F10 was obtained from American Type Culture Collection (Manassas, VA, USA). 213-Bismuth ( 213 Bi), the alpha particle emitting radionuclide, was eluted from the 225 Ac/ 213 Bi radionuclide generator system, produced by the Directorate for Nuclear Safety and Security, Karlsruhe, Germany. 188Rhenium ( 188 Re) beta emitter was eluted from the 188 W/ 188 Re radionuclide generator (ITG, Garching, Germany). 111Indium ( 111 In) in form of 111 In chloride was purchased from MDS Nordion (Vancouver, BC, Canada). The CHXA" bifunctional chelating agent for labeling the mAbs with 111 In and 213 Bi was obtained from Macrocyclics (Plano, TX, USA).

Sequencing of 6D2 IgM and 8C3 IgG1. 6D2 sequencing was performed by Fusion Antibodies Ltd
(Belfast, Northern Ireland). 6D2 mRNA was extracted from the cell pellets using Fusion Antibodies in-house RNA extraction protocol. 8C3 was sequenced at the Albert Einstein College of Medicine from cDNA reverse transcribed from the RNA using an oligo(dT) primer. PCR reactions were designed to amplify the variable regions of the cell line. The constant and variable region PCR products were cloned into the sequencing vector pCR2.1 (Invitrogen) for 6D2 or into pGEM T easy vector (Promega) for 8C3, and then transformed into TOP10 Escherichia coli (Thermo Fisher Scientific) for the selection of positive transformants. Selected colonies were picked and analyzed through sequencing by both vbase2 (http://www.vbase2.org/vbase2.php) and NCBI blast (http://www.ncbi.nlm.nih.gov/igblast/). Sequences of the mAbs were compared by Clustal Omega (http://www. ebi.ac.uk/Tools/psa/emboss_needle/).

Molecular modeling of 6D2 IgM and 8C3 IgG.
Nucleotide sequences for the H and L chains of 6D2 and 8C3 were analyzed using version 3.4.0 of the IMGT/V-QUEST tool for murine immunoglobulin sequences 45 . This tool was used to identify the closest germline V region gene for each sequence and infer the V region family. The IMGT unique numbering scheme reported by the IMGT/V-QUEST tool was used when referring to the residue numbers in the molecular models. The translated amino acid sequence of each V region was used to generate structural models of 6D2 and 8C3 using the Prediction of Immunoglobulin Structure (PIGS) server 46 . PIGS models were generated based on the best H and L chain templates from known structures. CDR loops were modeled based on loops with similar canonical structure, while CDR H3 was grafted. Molecular graphics, structural alignment of models, and calculation of surface electrostatic potential were performed with UCSF Chimera, which is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311) 47 . Highlighted aromatic residues consisted of Phe, Tyr, Trp, and His.
We also compared the V region elements of the melanin Abs to sequences of Abs that had shown changes in specificity when the C region was changed (permissive) and those that had not (non-permissive). A dendrogram showing the similarity of 6D2 antibody V region sequences with other permissive and non-permissive antibody sequences was created as previously described 30 . Briefly, immunoglobulins identified in the literature with differing C regions and identical V regions were grouped according to whether or not specificity changes were observed after altering the constant region. Germline genes for each antibody were identified using the IgBLAST sequence analysis tool 48 . Dendrograms were constructed through hierarchical average-linkage clustering with pairwise sequence similarity calculated as the Levenshtein or edit distance. The 6D2 V region sequences were included in this analysis while the other 13 murine sequences are identical to those described in the previous review 30 . Biodistribution and microSPECT/CT of 111 In-8C3 mAb in subcutaneous B16-F10 melanoma. The 8C3 and control IgG mAbs were conjugated to the bifunctional chelator CHXA" and radiolabeled with 111 In as described in McFarren et al. 51 . The resulting specific activity was 2 µCi/µg mAb. The mice were injected intravenously (IV) via tail vein with 33 µCi 111 In-8C3 or 111 In-IgG in 100 µL PBS. At the predetermined time points of 4 and 24 hrs the groups of 5 mice were humanely sacrificed, their tumors and major organs removed, blotted from blood, weighed, counted for radioactivity in a gamma counter, and percentage of injected dose per gram (ID/g) organ/tissue was calculated. For microSPECT/CT (micro single photon emission computer tomography/computer tomography) imaging some of the tumor-bearing mice were injected IV with 200 µCi 111 In-8C3 in 100 µL PBS and imaged 72 hrs post administration. The imaging was performed on microSPECT/ CT (VECTor4CT, Utrecht, NL).
Evaluation of the radiolabeled 8C3 IgG as an RIT agent in murine melanoma model and its comparison with immunotherapy. In the first series of therapy experiments the efficacy of 8C3 mAb radiolabeled with 213 Bi as described in McFarren et al. 51 was compared with that of 6D2 mAb. The mAbs were radiolabeled with 100 µCi 213 Bi and administered intraperitoneally to C57BL6 mice on Day 4 post B16-F10 intravenous tumor cells injection. Mice treated with unlabeled ("cold") 8C3 and 6D2 mAbs as well as untreated mice served as control groups. The mice were sacrificed on Day 14 post tumor cell injection and the metastases-like melanoma lesions in their lungs were enumerated with the 20 x −40 x Binocular Stereo Dissecting Microscope with USB Camera (AmScope). In the second series of experiments the efficacy of 213 Bi-and 188 Re-labeled 8C3 was compared. For this purpose 8C3 was radiolabeled with 200 and 400 µCi 213 Bi and 188 Re as described in McFarren et al. 51 and Dadachova et al. 25 , respectively, and mice were treated on Day 4 post tumor cells injection as described above. Finally, in the third series of experiments we compared the efficacy of RIT with 213 Bi-8C3 to that of immunotherapy with anti-CTLA4 mAb 9D9. The four groups included untreated mice, RIT alone, immunotherapy with anti-CTLA4 9D9 mAb alone, and the combination of RIT and immunotherapy. On day 4 after cell injection, the RIT alone and combination treatment groups received 150 μCi/100 μL 213 Bi-8C3 as intraperitoneal (IP) injection. The same volume of PBS was given IP to the untreated control group. On days 5, 9 and 11 after cell injection, the immunotherapy alone and combination treatment groups received IP injection of 100 μg/100 μL of 9D9 mAb following the treatment protocol described in Curran et al. 39 .
Statistical analysis. Power was estimated using PASS version 11 (NCSS, Inc., Kaysville, UT, http://www. ncss.com) using simulations of different tumor volumes based on our pilot data and conservative assumptions regarding the groups treated with the radiolabeled antibodies. All simulations showed power of at least 83% with only five animals per group because of the large differences between treated and untreated animals. Thus, 5 mice per group were utilized in all studies. The differences between the groups was analyzed using Kruskal-Wallis test.


s2fieldsofstudy: Biology Medicine

text:
Relationship between IGF2BP2 and IGFBP3 polymorphisms and susceptibility to non-small-cell lung cancer: a case–control study in Eastern Chinese Han population

Background IGF2BP2 and IGFBP3 polymorphisms may be associated with cancer risk. Methods With an aim to determine the association of variations in IGF2BP2 and IGFBP3 genes with risk of non-small-cell lung cancer (NSCLC), IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A polymorphisms were selected and genotyped in 521 NSCLC patients and 1,030 controls. Results We found that there was no difference in IGF2BP2 and IGFBP3 genotype distribution among the NSCLC patients and controls. The stratified analyses suggested that IGF2BP2 rs1470579 A>C polymorphism decreased the risk of NSCLC in some subgroups (female subgroup: CC vs AA: adjusted P=0.032 and CC vs AC/AA: adjusted P=0.028; <60 years subgroup: CC vs AA: adjusted P=0.012 and CC vs AC/AA: adjusted P=0.013; and never drinking subgroup: CC vs AA: adjusted P=0.046 and CC vs AC/AA: adjusted P=0.031). The stratified analyses also found that IGF2BP2 rs4402960 G>T polymorphism decreased the risk of NSCLC in some subgroups (female subgroup: TT vs GG: adjusted P=0.031 and TT vs GT/GG: adjusted P=0.026; <60 subgroup: TT vs GG: adjusted P=0.037 and TT vs GT/GG: adjusted P=0.038; and never drinking subgroup: TT vs GT/GG: adjusted P=0.046). Haplotype analysis indicated Ars1470579Crs2270628Grs3110697Grs4402960Ars6953668 haplotype decreased susceptibility of NSCLC (P=0.007). Conclusion Our study suggests that IGF2BP2 rs1470579 A>C, rs4402960 G>T single-nucleotide polymorphisms are candidates for decreased susceptibility to NSCLC among female, <60 years, and never drinking subgroups. In the future, more case–control studies with functional analysis are needed to confirm these preliminary findings.

Introduction
Lung cancer (LC) is the most common malignancy worldwide. It was reported that 1.8 million new LC patients were diagnosed in 2012, which accounted for about 13% of total cancer cases. 1 Because of aging, air pollution, smoking, and exposure to occupational and/or environmental carcinogens, LC constitutes a burden all over the world. Some risk factors mentioned above might contribute to the development of LC; however, other susceptibility factors could also increase the incidence of LC. Nowadays, genetic variants were supported to influence the risk of LC, especially non-small-cell lung cancer (NSCLC), which was a common subtype of LC.
In humans, insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is a protein which is encoded by IGF2BP2 gene. 2

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Chen et al factor 2 (IGF2) translation by binding to the 5′ UTR of IGF2 mRNA. 3 Gu et al reported that IGF2BP2 was overexpression in both ovarian cancer and ovarian low malignant potential tumor samples compared to either normal ovary or ovarian adenomas samples. 4 A previous study also found that IGF2BP2/IGF-1/ IGF-1 receptor signaling pathways might involve in cancermediated endothelial recruitment, which was an important feature of metastatic cancer in the tumor microenvironment. 5 In addition, Liu et al found that an lncRNA (IGF2BP2-AS1) was associated with better overall survival in lung squamous cell carcinoma. 6 In view of these previous studies, we thought that IGF2BP2 might influence the development of LC.
IGF family involves IGF ligands, IGF receptors, and IGFbinding proteins (IGFBPs). IGF-1 is a potent mitogen and regulates mitogenesis and antiapoptosis. 7 IGFBP3, a major binding protein of IGF-1, interacts with IGF-1, regulates its biological activity, and may play important roles in antiproliferation and proapoptosis. 8 Papadimitrakopoulou et al reported that IGFBP3 downregulation is an early event during head and neck carcinogenesis. 9 Adenoviral IGFBP3 and farnesyltransferase inhibitor might decrease Akt expression and promote NSCLC cell apoptosis in vitro and in vivo. 10 Results of a previous study highlighted that IGFBP3 could mediate LC progression. In addition, overexpression of IGFBP3 might induce apoptosis of NSCLC cells and promote cisplatin response in vitro. 11 Several case-control studies focused on the association of IGF2BP2 and IGFBP3 polymorphisms with risk to cancer. Results of previous studies indicated that IGF2BP2 rs4402960 G>T single-nucleotide polymorphism (SNP) was associated with the development of breast cancer. 12 Terry et al found that IGFBP3 rs2270628 variants increased IGF1 levels in plasma and was associated with the risk of ovarian cancer. 13 In addition, IGFBP3 rs3110697 variants were significantly associated with IGFBP-3 levels in a multiethnic populations, 14 and IGFBP3 rs3110697 AA genotypes increased the risk of death among Chinese postmenopausal women with breast cancer. 15 However, the relationship between IGF2BP2 and IGFBP3 polymorphisms and NSCLC risk was unclear. With an aim to determine the potential association of genetic variations in IGF2BP2 and IGFBP3 genes with risk of NSCLC in Eastern Chinese Han populations, IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A SNPs were selected and genotyped in 521 NSCLC patients and 1,030 cancer-free controls.

Materials and methods ethics statement
This case-control study conformed to the Helsinki declaration and was approved by the Institutional Review Board of Fujian Medical University. A written consent was obtained from each participant.

subjects
In our study, a total of 521 sporadic NSCLC cases and 1,030 age-and gender-matched controls were enrolled. All participants were recruited from the Department of Thoracic Surgery in Affiliated Union Hospital of Fujian Medical University and Affiliated People's Hospital of Jiangsu University. All NSCLC patients (mean age at 59.76±10.71 years) were diagnosed by pathology. The corresponding information was retrieved from medical files ( Table 1). The cancer-free controls were well-matched to NSCLC patients by age (mean age at 60.34±9.11 years) and sex (P=0.453). Individuals without any history of personal malignancy or autoimmune disorder were included as controls. Both NSCLC cases and controls were hereditarily unrelated and were from Eastern Chinese Han population. Each participant was informed about the study protocols and a written consent was obtained. A body mass index (BMI) ≥24 kg/m 2 was considered as the criterion of Chinese individuals with obesity and overweight. 16,17 The definitions of "ever smokers" and "ever drinkers" were described in our previous study. 18

selection of snPs
To assess the relationship between IGF2BP2 and IGFBP3 SNPs and NSCLC risk, we selected polymorphisms in IGF2BP2 and IGFBP3 gene according to the publications, which were associated with the development of cancer. 12,13,[19][20][21] Dna extraction and genotyping Using a universal Promega DNA kit (Promega Corporation, Fitchburg, WI, USA), genomic DNA was extracted from whole blood sample which was stored with EDTA-anticoagulation tube. IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, 22 rs3110697 G>A and rs6953668 G>A genotypes were determined by a custom-by-design 48-Plex PCR (SNPscan TM kit; Genesky Biotechnologies Inc., Shanghai, China). 23 We used ABI 3730XL sequencer to obtain genotypes. The data were read out by

Baseline characteristics
A total of 521 NSCLC cases were included in this study.  Table 1. The primary information for IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A SNPs was shown in Table 2. The successful ratio was >99.00% for each SNP. Minor allele frequency (MAF) of IGF2BP2 and IGFBP3 SNPs was similar to the data in Chinese database (Table 2). In controls, the genotype frequencies for IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A polymorphisms were in HWE ( Table 2).
association of IGF2BP2 rs1470579 a>C, rs4402960 g>T and IGFBP3 rs2270628 C>T, rs3110697 g>a, and rs6953668 g>a polymorphisms with nsClC Table 3 showed the frequencies of IGF2BP2 and IGFBP3 genotypes in different NSCLC subgroups and control group.
Results of the single locus analyses were summarized in Table 4. We found that there was no difference in IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A genotype distribution among overall NSCLC patients and controls. In addition, similar findings were also identified among different NSCLC subtype and controls.

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Chen et al association of igF2BP2 rs1470579 a>C, rs4402960 g>T and igFBP3 rs2270628 C>T, rs3110697 g>a, and rs6953668 g>a polymorphisms with nsClC in a stratification analysis As shown in Table 5, the stratified analyses suggested that IGF2BP2 rs1470579 A>C polymorphism decreased the risk   However, we found no significant difference in IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A genotype distribution among NSCLC cases and controls (Tables 7-9, respectively).

snP haplotypes
We harnessed an online SHEsis software 31 to establish haplotypes of IGF2BP2 and IGFBP3 gene (Table 10). Finally, 19 haplotypes of IGF2BP2 and IGFBP3 genes were constructed. When A rs1470579 C rs2270628 G rs3110697 G rs4402960 G rs6953668 haplotype was used as reference, A rs1470579 C rs2270628 G rs3110697 G rs4402960 A rs6953668 haplotype decreased susceptibility to NSCLC (P=0.007, Table 10).

Discussion
In this study, we explored the potential relationship of IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A SNPs with susceptibility to NSCLC. We found that IGF2BP2 rs1470579 A>C, rs4402960 G>T and IGFBP3 rs2270628 C>T, rs3110697 G>A, and rs6953668 G>A polymorphisms might not confer risk to overall NSCLC. However, in stratified analyses, we found significant associations between IGF2BP2 rs1470579 A>C, rs4402960 G>T polymorphisms and decreased risk of NSCLC in female, <60 years, and never drinking subgroups. We also found that A rs1470579 C rs2270628 G rs3110697 G rs4402960 A rs6953668 haplotype decreased susceptibility to NSCLC. To our knowledge, the present study was the first investigation to identify the correlation between IGF2BP2 rs1470579 A>C, rs4402960 G>T polymorphisms and the decreased risk of NSCLC in Asians. Dai et al reported that IGF2BP2 is a tumor promoter which promotes malignancy proliferation through its client mRNAs IGF2 and high mobility group A1. 32 In many human malignancies, the gene encoding IGF2BP2 was found to be amplified and overexpressed. Recently, Barghash et al found that elevated expression of IGF2BP2 was associated with a shorter survival and metastasis in esophageal adenocarcinoma. 33 Several case-control studies reported that IGF2BP2 rs4402960 G>T polymorphism was associated with the risk of T2DM and might affect the therapeutic efficacy of antidiabetic in Chinese population. 34,35 We found that the IGF2BP2 rs4402960 TT genotype was associated with the decreased susceptibility of NSCLC among female,    years, and never drinking patients. Previous report showed that IGF2BP2 rs4402960 G>T polymorphism was associated with the increased risk of breast cancer in a Chinese population. 12 The other case-control study did not find any association between IGF2BP2 rs4402960 G>T polymorphism and colorectal cancer. 36 We identified that IGF2BP2 rs4402960 T allele might probably be a protective factor for NSCLC, which was not consistent with the findings of previous studies. It is believed that there are some LC-related driver genes possessing low frequency variant, which modify the states of chromatin or DNA. 37 In addition, intronic region could bind to some proteins and even directly alter special gene transcription. [37][38][39] rs4402960 G>T polymorphism is located in the intron region of IGF2BP2 gene, which may influence

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Chen et al the post-transcription process. IGF2BP2 rs4402960 G>T polymorphism may accordingly alter the risk of NSCLC through post-transcription process mechanisms, and our study suggested that IGF2BP2 rs4402960 TT genotype and T allele play an important role in lung carcinogenesis. In the future, the function of the IGF2BP2 rs4402960 G>T    40 and Huang et al found that this SNP was a risk factor for T2DM in a Chinese population. 35 In addition, a quantitative assessment demonstrated that this common polymorphism was associated with the development of T2DM. Therefore, whether the A-to-C variant in the intron region of IGF2BP2 gene does influence the expression of IGF2BP2 gene needs to be further studied. Using SHEsis software, 31 we constructed 19 haplotypes to assess the potential inherited patterns of IGF2BP2 and IGFBP3 genes. Compared with A rs1470579 C rs2270628 G rs3110697 G rs-4402960 G rs6953668 haplotype, we found that A rs1470579 C rs2270628 G rs-3110697 G rs4402960 A rs6953668 haplotype significantly decreased the risk of NSCLC (P=0.007, Table 10). To the best of our knowledge, we first identified the relationship of this haplotypes with susceptibility to NSCLC. However, this rare haplotype only influenced a very minor fraction (<1%) of the studied populations. In the future, more studies with a larger sample size and an adequate methodological quality should be performed to confirm or refute these primary findings.
Some limitations in the current study should be acknowledged. First, we selected only some functional polymorphisms in IGF2BP2 and IGFBP3 genes. In the future, a fine-mapping study should be conducted to further study the potential relationship of GF2BP2 and IGFBP3 polymorphisms with risk of NSCLC. Second, in this case-control study, the sample size of NSCLC patients was relatively limited, which might lead to lack of sufficient power to identify true correlation, especially in the subgroup analysis. In the future, more NSCLC cases and controls should be enrolled, and a replicated study should be carried out. Third, this case-control study was hospital-based. The cancer-free controls recruited from local hospitals might not completely represent a general Eastern Chinese Han population. Fourth, the genotype frequencies of IGF2BP2 rs1470579 A>C and rs4402960 G>T polymorphisms were not in HWE, which might lead to bias. Fifth, a functional experimentation was not performed. Finally, because of the lack of the information on survival of NSCLC, we did not further analyze the role of GF2BP2 and IGFBP3 variants on NSCLC prognosis.

Conclusion
Our study suggests that IGF2BP2 rs1470579 A>C, rs4402960 G>T polymorphisms are candidates for decreased susceptibility to NSCLC in Eastern Chinese Han population among female, <60 years, and never drinking subgroups. Compared with A rs1470579 C rs2270628 G rs3110697 G rs4402960 G rs6953668 Cancer Management and Research 2018:10 submit your manuscript | www.dovepress.com

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Chen et al haplotype, A rs1470579 C rs2270628 G rs3110697 G rs4402960 A rs6953668 haplotype significantly decreased risk of NSCLC. In the future, more case-control studies with comprehensive resequencing or SNP functional analysis are needed to confirm these preliminary findings.