s2fieldsofstudy: Biology Medicine

text:
Human bone marrow mesenchymal stem cell-derived extracellular vesicles attenuate neuroinflammation evoked by focal brain injury in rats

Background Ischemic stroke is the major cause of long-term severe disability and death in aged population. Cell death in the infarcted region of the brain induces immune reaction leading to further progression of tissue damage. Immunomodulatory function of mesenchymal stem cells (MSCs) has been shown in multiple preclinical studies; however, it has not been successfully translated to a routine clinical practice due to logistical, economical, regulatory, and intellectual property obstacles. It has been recently demonstrated that therapeutic effect of intravenously administered MSCs can be recapitulated by extracellular vesicles (EVs) derived from them. However, in contrast to MSCs, EVs were not capable to decrease stroke-induced neuroinflammation. Therefore, the aim of the study was to investigate if intra-arterial delivery of MSC-derived EVs will have stronger impact on focal brain injury-induced neuroinflammation, which mimics ischemic stroke, and how it compares to MSCs. Methods The studies were performed in adult male Wistar rats with focal brain injury induced by injection of 1 μl of 50 nmol ouabain into the right hemisphere. Two days after brain insult, 5 × 105 human bone marrow MSCs (hBM-MSCs) labeled with Molday ION or 1.3 × 109 EVs stained with PKH26 were intra-arterially injected into the right hemisphere under real-time MRI guidance. At days 1, 3, and 7 post-transplantation, the rats were decapitated, the brains were removed, and the presence of donor cells or EVs was analyzed. The cellular immune response in host brain was evaluated immunohistochemically, and humoral factors were measured by multiplex immunoassay. Results hBM-MSCs and EVs transplanted intra-arterially were observed in the rat ipsilateral hemisphere, near the ischemic region. Immunohistochemical analysis of brain tissue showed that injection of hBM-MSCs or EVs leads to the decrease of cell activation by ischemic injury, i.e., astrocytes, microglia, and infiltrating leucocytes, including T cytotoxic cells. Furthermore, we observed significant decrease of pro-inflammatory cytokines and chemokines after hBM-MSC or EV infusion comparing with non-treated rats with focal brain injury. Conclusions Intra-arterially injected EVs attenuated neuroinflammation evoked by focal brain injury, which mimics ischemic stroke, and this effect was comparable to intra-arterial hBM-MSC transplantation. Thus, intra-arterial injection of EVs might be an attractive therapeutic approach, which obviates MSC-related obstacles.

Background
Stroke is the third cause of death in people after 60 years old in the developed countries and the main cause of disability. Every year, 15 millions of people worldwide suffer from stroke, 5 million of whom die and 5 million stay permanently disabled [1]. According to available data, therapy for people after stroke uses from 3% to 7% of total healthcare expenditures in the developed countries [2]. Ischemic stroke represents 87% of all stroke cases and typically originates from obstruction of oxygen and nutrient delivery to the brain due to intravascular clot formation [1]. Cell death in the ischemic region results in production of DAMPs (damage-associated molecular patterns) and induction of the local immune response in the brain. In the first few minutes after ischemic stroke, astrocytes and microglial cells are activated, which lead to production of pro-inflammatory cytokines, blood-brain barrier (BBB) injury, and progression of tissue damage [3]. Local inflammatory response activates endothelial cells, which facilitate infiltration of leucocytes to the nervous tissue and to further increase the damaged region in the brain [4]. On the other hand, the switch of astrocytes and microglia/macrophages' phenotype from pro-inflammatory to anti-inflammatory may lead to production of anti-inflammatory cytokines and activation of T regulatory lymphocytes which have neuroprotective effect on injured tissue. Thus, there is a need to find a therapy which will modulate the immune reaction and limit the damage in the region injured by ischemia.
Many studies have shown that mesenchymal stem cells have immunomodulatory properties and can be potentially used in central nervous system therapies including ischemic stroke [5][6][7][8]. The past decade has seen an explosion of research directed toward better understanding of the mechanisms of MSC function during rescue of injured organs and tissues. On the contrary to the initial assumption, MSCs may not primary exert their functions in a cellular but rather in a paracrine manner. Recent studies suggest that the signals responsible for these therapeutic effects are at least partially linked to the production of extracellular vesicles (EVs).
The discovery of extracellular vesicles, naturally released by almost all cells, through which protein-, lipid-, and nucleic acid-based regulatory factors are transferred is a great promise for ischemic stroke treatment and could be an alternative for using stem cells. Despite the positive effects of stem cells in the models of experimental stroke, some researchers have demonstrated the risk of their application, including graft rejection after allogeneic transplantation [9], the possibility of small blood vessel obstruction [10], or tumor transformation [11]. Extracellular vesicles derived from stem cells do not have limits such as graft rejection, tumor formation, or small blood vessels' blockage. Moreover, EVs have the ability to cross blood-brain barrier [12] and show greater stability during storage, and the procedure of their transplantation is easier than cells. Until now, the secretome of different stem cells has been shown to be effective in promoting neuroprotection and recovery in cell and animal models of central nervous system (CNS) disorders.
Previously, the characteristics of EVs derived from human MSCs were examined by us in vitro [13]. It has been recently demonstrated that therapeutic effect of intravenously administered MSCs can be recapitulated by extracellular vesicles derived from them. However, in contrast to MSCs, EVs were not capable to decrease stroke-induced neuroinflammation. Therefore, the aim of the study was to investigate if intra-arterial delivery of MSC-derived EVs will have more impact on focal brain injury-induced neuroinflammation, which mimics ischemic stroke, and how it compares to MSCs.
Using the focal brain injury model, EVs or MSCs were transplanted intra-arterially into adult Wistar rats and the grafts have been analyzed in the host brain. Moreover, the cellular immune response in the brain of transplant recipients was evaluated by immunohistochemical staining and the cytokine and chemokine levels were measured by multiplex immunoassay.

Cell culture
Commercially available human bone marrow mesenchymal stem cells (hBM-MSCs) (Lonza Inc., Walkersville, MD, USA) were plated in 75 cm 2 polystyrene tissue culture flasks (Thermo Scientific) at a density of 5 × 10 3 cells/cm 2 with 10 ml of Mesenchymal Stem Cell Growth Medium (MSCGM™, Bullet Kit, Lonza). Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO 2 . Cells were subsequently maintained in MSCGM™ medium and passaged at 80% confluence in a ratio 1:3 in trypsin/EDTA solution (Gibco, Life Technologies) with the culture medium changed thrice a week.

EV isolation from hBM-MSCs
The isolation of EVs was performed from conditioning media of non-labeled hBM-MSCs. 5 × 10 6 of hBM-MSCs (passages 4-6) were cultured in 75 cm 2 polystyrene tissue flasks to reach 50-60% confluence, then the culture medium was changed, and the cells were incubated for additional 48-72 h to the confluence of 70-80%. Cell culture supernatants were collected and centrifuged at 200g for 10 min, then at 500g for 10 min at 4°C, aliquoted and frozen at − 70°C for further use. In order to isolate EVs, hBM-MSC culture supernatants were thawed, spun down at 2000g for 20 min to remove cellular debris, and then centrifuged at 100,000g for 75 min at 4°C using a Thermo Scientific Type 865 Fixed Angle Rotor. The pellets were washed with deionized phosphate-buffered saline (DPBS) and subjected to an additional centrifugation at 100,000g for 75 min at 4°C using a Thermo Scientific Type 865 Fixed Angle Rotor. Then, the supernatant was discarded and the pellet was re-suspended in 1 ml of DPBS and stored at − 70°C until needed.

Labeling of hBM-MSCs
The labeling of cells with Molday ION consisted of superparamagnetic iron oxide nanoparticles (SPIO) and rhodamine B purchased from BioPAL (Worcester, USA) was performed as previously described by us. Briefly, 100 μl of Molday ION was added to the 5 × 10 5 hBM-MSCs cultured in 10 ml Mesenchymal Stem Cell Growth Medium and incubated over 16 h at 37°C in a humidified atmosphere containing 5% CO 2 . After that, medium with label was removed, cells were washed with phosphate-buffered saline (PBS), fresh medium was added, and cells were cultured 48 or 72 h.

Labeling of EVs using PKH26
EVs isolated from non-labeled hBM-MSCs were tagged with PKH26 (Red Fluorescent Cell Linker Kits MINI26; Sigma-Aldrich Co., St Louis, MO, USA) at room temperature (RT) for 5 min in the dark and blocked with fetal bovine serum (FBS), according to manufacturer's instructions. The unincorporated labels were removed by EV centrifugation at 100,000×g for 75 min at 4°C using a Thermo Scientific T-865 Fixed Angle Rotor Thermo Scientific Sorvall WX Ultracentrifuge Series. EVs were washed with DPBS and subjected to additional centrifugations. Then, the pellet was re-suspended in 1 ml DPBS for further use.

NanoSight particle tracking analysis of EVs isolated from hBM-MSCs
The size and concentration of EVs were analyzed using NanoSight NS300 system (Malvern, UK), configured with sCMOS camera and blue 488 nm laser. For Nano-Sight analysis, extracellular vesicles were diluted in 1 ml DPBS and collected and analyzed by NanoSight tracking analysis (NTA) software version 3.2. Each of EV samples from the different isolations was recorded three times for 60 s at constant temperature 23°C creating three replicable histograms which were averaged.

Western blot analysis
hBM-MSC or EV pellets were re-suspended in RIPA lysis buffer. Protein concentrations were determined using a Bio-Rad DC™protein assay kit (Bio-Rad) in the supernatant as well as in the pellet solution. Samples of the pellet were ran on 10-15% SDS-PAGE gels and transferred onto nitrocellulose membranes (Amersham Bioscience). After blocking, membranes were probed with calnexin anti-body (Millipore) and then incubated with horseradish peroxidaseconjugated secondary IgG antibodies (Sigma-Aldrich). Immunoblot signals were visualized using ECL chemiluminescence kit (GE Healthcare Life Sciences). The β-actin antibody was used as an internal control.

Rat model of focal brain injury
Adult male Wistar rats, weighing 250 g and housed in cages with a 12-h light-dark cycle and free access to food and water, were used in all experiments. Rats were anesthetized with a mix of Bioketan (Vetoquinol; 53.6 mg/kg) and Domitor (Orion Pharma; 0.4 mg/kg) by i.p. injection and placed in a stereotaxic apparatus (Stoelting). The model of brain infarct was performed as previously described by us [14]. Briefly, a burr hole was drilled in the skull and the needle (length 15 mm, gage 33), connected to a 10-μl syringe (Hamilton, Switzerland), was lowered into the right striatum (coordinates A 0.5, L 3.8, D 4.7 mm). Then, 1 μl of 5 nmol ouabain (Sigma, Poland) was injected into the brain at a speed of 1 μl/min using a microinfusion pump (Stoelting, USA). The needle was then withdrawn, and the skin was closed with a suture. After the procedure, all animals were treated with an antibiotic (Baytril; Bayer; 0.4 mg/ml) and an analgesic (Rycarfa; Krka; 5 mg/ml).

Intra-arterial transplantation of hBM-MSCs or EVs
Intra-arterial infusion of hBM-MSCs or EVs was performed 48 h after the induction of focal brain injury as previously described by us. Briefly, under general anesthesia (2% isoflurane), the common carotid artery (CCA), the external carotid artery (ECA), and the internal carotid artery (ICA) were exposed, the occipital artery branching off the ECA was coagulated, and the pterygopalatine artery branching off the ICA was ligated, as well as the proximal segments of the ECA and CCA. Then, the vascular clip (FT 180T, Aesculap, Center Valley, PA, USA) was applied to the ICA proximal to the pterygopalatine artery to prevent backflow, the incision into the CCA was performed distal to the ligature, and the catheter was inserted into the CCA, followed by the suture tightening on the artery over the catheter to prevent bleeding. Then, the clip was removed, the animal was placed inside the gantry of the MR scanner, and 5 × 10 5 hBM-MSCs labeled with Molday ION or 1.3 × 10 9 EVs stained with PKH26 were transplanted into the right internal carotid artery of Wistar rats in 1 ml of PBS at a safe speed of 0.2 ml/min.

Brain tissue collection and preparation
One, three, or seven days after the intra-arterial transplantation of hBM-MSCs or EVs, rats were deeply anesthetized by intraperitoneal administration of Bioketan (53.6 mg/kg) and Domitor (0.4 mg/kg) and decapitated. The brains were isolated, immediately frozen on dry ice, and stored at − 70°C. For immunohistochemical analysis, the brains were incubated at − 20°C, cut on cryostat into coronal tissue sections 25 μm thick, placed on microscope slides, and frozen at − 70°C. For cytokine expression detection, the brains were incubated at 4°C, homogenized in RIPA lysis buffer containing 20 mM Tris-HCl (pH = 7.5), 150 mM NaCl, 1 mM PMSF, 0.05 Tween-20, and proteinase and phosphatase inhibitor cocktail (Sigma). The lysates were clarified by centrifugation at 11,500 rpm for 10 min at 4°C, and the supernatants were collected and re-suspended in RIPA lysis buffer.

Immunohistochemistry
The following antibodies (source and final dilution) were used for brain tissue staining: mouse monoclonal antihuman CD44 (Santa Cruz, 1:100), mouse monoclonal antihuman STEM121 (Cellartis, 1:100), mouse monoclonal antirat ED1 (Serotec, 1:100), rabbit monoclonal anti-rat glial fibrillary acidic protein (GFAP) (Dako, 1:200), mouse monoclonal anti-rat CD45RA (Serotec, 1:100), and mouse monoclonal anti-rat CD8 (Serotec, 1:300). Coronal cryostat sections of the brain (25 μm thick) were cut in serial order to create 10 series sections. Double fluorescent immunohistochemistry was performed. After blocking for unspecific reactivity, adjacent series of sections were stained for a specific cell-lineage marker. Tissue sections were rinsed in PBS and then incubated in 10% goat serum in PBS containing 0.25% Triton X-100 and 0.1% BSA for 60 min in RT. Next, the sections were washed with PBS and incubated with primary antibodies overnight at 4°C. The following day, tissue sections underwent the washing procedure, and the primary antibodies were revealed by applying appropriate secondary antibodies (Alexa Fluor, 1:500) for 60 min at room temperature and in the dark. Nuclei were subsequently labeled with the fluorescent dye 5 μM Hoechst 33258 (Life Technologies). Labeling was verified using a confocal laser scanning microscope (LSM 780, Carl Zeiss, Germany) and Cell Observer SD (Carl Zeiss, Germany) using × 20 or × 40 objectives. A helium-neon laser (543 nm) was utilized in the excitation of Alexa Fluor 546, while an argon laser (488 nm) was applied in the excitation of FITC. ZEN software was used for quantitative analysis of immunoreactivity in all sections. Six animals per group were analyzed. Images from two sections per animal were taken, and the number of positivelabeled cells was assessed.

Statistical analysis
The results were shown as mean ± SD. The statistical analysis was performed using the one-way ANOVA and the Tukey test. In all calculations, the program Graph-Pad Prism 3.0 was used. The significance level less than 0.05 was considered as statistically significant.

Analysis of EVs isolated from hBM-MSCs
NanoSight particle tracking analysis revealed that EVs derived from hBM-MSCs represented heterogeneous population consisted of exosomes/smaller microvesicles with the average peak at 111.7 ± 11.5 nm and larger microvesicles with the average peak at 398.3 ± 49.3 nm (Fig. 1a). The average concentration of EVs isolated from 5 × 10 6 cells was 1.3 × 10 9 ± 1.8 × 10 7 /ml which corresponded to 110.2 ± 24.0 particles/frame and 134.9 ± 37.6 centers/frame. The expression of specific proteins was assessed in EVs and hBM-MSCs by Western blot confirming the presence of cell-associated calnexin, a calcium-binding, ER-associated protein in hBM-MSCs but the absence of calnexin in EVs (Fig. 1b).

MRI of hBM-MSCs after intra-arterial transplantation
The MRI analysis enables visualization of transplanted cells labeled with Molday ION as dark spots (Fig. 2bd, green arrows). The region of injured brain is seen as brighter area (Fig. 2e-h, red arrows). The results of our studies showed that hBM-MSCs stained with Molday ION injected into the right internal carotid artery were routed into focal injury of the rat right hemisphere (Fig. 2b, f). After 24 h, the signal generated from transplanted cells significantly decreased (Fig. 2c, g). Seven days after hBM-MSC intra-arterial infusion, only single dark spots were observed in some rats (Fig. 2d, h).

Immunohistochemistry of hBM-MSCs transplanted into the right hemisphere
To characterize hBM-MSCs present in the rat brain after their intra-arterial transplantation, immunohistochemical staining for human stem cell markers CD44 and STEM121 was performed. The analysis revealed that hBM-MSCs stained with Molday ION seen in the right hemisphere co-expressed CD44 marker 24 h (Fig. 3a), 3 days (Fig. 3b), and 7 days (Fig. 3c) after transplantation. Furthermore, hBM-MSCs labeled with Molday ION observed in the rat brain were positive for STEM121 24 h (Fig. 3d), 3 days (Fig. 3e), and 7 days (Fig. 3f) after their intra-arterial injection. The analysis of EV presence in the right hemisphere after intra-arterial injection To detect EVs labeled with PKH26 in the rat brain after transplantation into the right internal carotid artery, the analysis in confocal microscope was performed. The study revealed that EVs stained with PKH26 were observed in the rat right hemisphere 24 h after intraarterial transplantation (Fig. 4a-c).

Characterization of immune cells in the rat brain
The number of immune cells was counted in native control rats, in experimental animals with focal brain injury,    Human bone marrow mesenchymal stem cells (hBM-MSCs) or their extracellular vesicles (EVs) transplanted intra-arterially alleviate GFAP+ cells in the rat brain evoked by focal brain injury. a Representative images of GFAP staining in the rat brain of control rats, focal brain injured rats left intact, and those receiving hBM-MSC or EV transplantation. Scale bars 20 μm. b Quantitative analysis of the host cells positive for GFAP observed in the rat brains of focal brain injured rats 24 h, 3 days, and 7 days after hBM-MSC or EV transplantation versus untreated or control rats. All data reflect mean ± SD from 6 animals. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 and those which additionally received hBM-MSC or EV transplantation. Immunohistochemical analysis of the rat brain revealed that injection of ouabain into the rat striatum caused the increase of immune cells in the right hemisphere comparing to intact animals, such as microglial/macrophages ED1 + (Fig. 5b), astrocytes GFAP + (Fig. 6b), leucocytes CD45RA + (Fig. 7b), and T CD8 + lymphocytes (Fig. 8b).
Intra-arterial infusion of hBM-MSCs or EVs into the rat brain 48 h after induction of brain injury reduced the number of ED1 + microglia/macrophages in the rat brain. The effect was much stronger after extracellular vesicle than hBM-MSCs infusion (Fig. 5b). Transplantation of hBM-MSCs or EVs produced the reduction of astrocyte activation; however, statistically significant difference was noticed only 24 h after hBM-MSC infusion (Fig. 6b).
This effect was accompanied by the decrease of leukocyte number (CD45RA + ) after hBM-MSC or EV injection. Significant loss was observed 24 h and 7 days after EV transplantation (Fig. 7b). Among this population, the number of T lymphocytes expressing CD8 marker was found lower in rats receiving hBM-MSC or EV graft compared with the ischemic brain injured animals left without transplantation, although not in all time points of observation the differences were statistically significant (Fig. 8b).
The expression of most of pro-inflammatory cytokines was reduced after hBM-MSC or EV injection. Infusion of hBM-MSCs or EVs led to the decrease of IL-1α; however, statistically significant difference was noticed 24 h after hBM-MSC (p < 0.05) and EV (p < 0.01) transplantation (Fig. 9a). The reduction of IL-1β was observed 24 h after hBM-MSC or EV infusion (p < 0.001); the statistically significant decrease was seen after 3 and 7 days of observation in rats receiving hBM-MSC or EV transplantation (Fig. 9b). Also, the level of IL-6 was significantly lower after injection of hBM-MSCs or EVs in each time point (p < 0.0001) (Fig. 9c). Concomitantly, the lower expression of TGF-β2 was detected after hBM-MSC or EV infusion; statistically significant difference was observed 3 and 7 days after cell or vesicle transplantation (p < 0.001) (Fig. 9d).
Transplantation of mesenchymal stem cells or extracellular vesicles resulted in the decrease of chemokines expression. The infusion of hBM-MSCs or EVs led to the reduction of CXCL1, highly statistically significant in each time point (p < 0.0001) (Fig. 10a). The difference in MCP-1 level was also statistically significant after hBM-MSC or EV infusion, which was observed stronger due to the longer time of observation after transplantation (Fig. 10b). We also noticed the strong reduction of the MIP-1α level after hBM-MSC or EV infusion in each time point (Fig. 10c). Moreover, the expression of MIP-3α Fig. 7 Human bone marrow mesenchymal stem cells (hBM-MSCs) or their extracellular vesicles (EVs) transplanted intra-arterially alleviate CD45RA+ cells in the rat brain evoked by focal brain injury. a Representative images of CD45RA staining in the rat brain of control rats, focal brain injured rats left intact, and those receiving hBM-MSC or EV transplantation. Scale bars 20 μm. b Quantitative analysis of the host cells positive for CD45RA observed in the rat brains of focal brain injured rats 24 h, 3 days, and 7 days after hBM-MSC or EV transplantation versus untreated or control rats. All data reflect mean ± SD from 6 animals. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 decreased after stem cell or EV transplantation and its level was similar to this detected in control rats (Fig. 10d).

Discussion
Stroke is accompanied with inflammatory and immune reactions which have been shown to be activated at each stage of disease from the early destructive events to the latest events of impaired brain tissue repair and vascular regeneration. Neuroinflammation has been suggested as an attractive treatment target in stroke, since it offers a broader therapeutic window in comparison to currently established thrombolytic approaches. Mesenchymal stem cells have recently emerged as promising candidates for cell-based therapy in neurological disorders. It might be that the extracellular vesicles isolated from MSCs which cross blood-brain barrier and easily migrate into the brain seem to be a promising alternative. In the current study, we highlighted the immunomodulation effect of human bone marrow-derived mesenchymal stem cells or extracellular vesicles released from these cells after their transplantation into focal brain injured rats. One of the critical questions in cell transplantation is about the route of graft delivery in terms of its efficacy and safety. The intravascular route of stem cell infusion has met the increasing interest because of less invasive procedure comparing to stereotactic injection. Unfortunately, the evaluation of cell distribution after intravenous infusion in neurologic disorders has shown that most of the cells are initially entrapped within the lungs and do not travel to the brain [15]. Recent studies have shown that an intra-arterial approach would be a more efficient way of cell delivery to CNS [16][17][18][19]. In neurological disorders, the transplantation procedure into internal carotid artery seems to be an effective way of cell injection into the brain. In the current study, we performed hBM-MSC or EV infusion into the right internal carotid artery ipsilateral to the focal rat brain insult.
The results of our experiments have proved that hBM-MSCs stained with Molday ION injected intra-arterially were observed in the injured hemisphere of graft Fig. 8 Human bone marrow mesenchymal stem cells (hBM-MSCs) or their extracellular vesicles (EVs) transplanted intra-arterially alleviate CD8+ cells in the rat brain evoked by focal brain injury. a Representative images of CD8 staining in the rat brain of control rats, focal brain injured rats left intact, and those receiving hBM-MSC or EV transplantation. Scale bars 20 μm. b Quantitative analysis of the host cells positive for CD8 observed in the rat brains of focal brain injured rats 24 h, 3 days, and 7 days after hBM-MSC or EV transplantation versus untreated or control rats. All data reflect mean ± SD from 6 animals. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 recipients. The presence of intra-arterially injected cells in the host brain was confirmed by other authors. Namestnikova et al. showed that MSCs labeled with SPIO transplanted intra-arterially were detected in the basal ganglia and cerebral cortex of rat brain immediately after cell transplantation [8]. Walczak et al. observed MSCs stained with SPIO in the injured hemisphere from 2 to 24 h after intra-arterial infusion [20]. This phenomenon was also described by Janowski's group where glial-restricted progenitors (GRPs) labeled with Molday ION infused intra-arterially were detected by MRI in the rat brain just after transplantation [17]. To prove the donor origin of cells observed in MRI, additional methods, i.e., bioluminescence or immunohistochemically, are used. Namestnikova et al. performed the co-localization studies of iron nanoparticles conjugated Fig. 9 Human bone marrow mesenchymal stem cells (hBM-MSCs) or their extracellular vesicles (EVs) transplanted intra-arterially reduced proinflammatory cytokines observed in the rat brain after focal brain injury. Quantitation of IL-1α (a), IL-1β (b), IL-6 (c), and TGF-β2 (d) protein levels detected in the rat brains of focal brain injured rats 24 h, 3 days, and 7 days after hBM-MSC or EV transplantation versus untreated or control rats. All data reflect mean ± SD from 6 animals. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 Fig. 10 Human bone marrow mesenchymal stem cells (hBM-MSCs) or their extracellular vesicles (EVs) transplanted intra-arterially reduced the elevated level of chemokines noticed in the rat brain after focal brain injury. Quantitation of CXCL1 (a), MCP-1 (b), MIP-1α (c), and MIP-3α (d) protein levels detected in the rat brains of focal brain injured rats 24 h, 3 days, and 7 days after hBM-MSC or EV transplantation versus untreated or control rats. All data reflect mean ± SD from 6 animals. *p < 0.1; **p < 0.01; ***p < 0.001; ****p < 0.0001 with fluorescence magnetic polymers MC03F and membrane marker PKH26 [21]. Walczak et al. employed the marker of proliferating cells BrdU to show MSCs infused intra-arterially in the donor tissues [20]. In our studies, to identify transplanted hBM-MSCs labeled with Molday ION in the rat brain, the co-expression of mesenchymal stem cell marker CD44 or human stem cell marker STEM121 was applied. The positively stained hBM-MSCs visible in the brain of graft recipients were localized in the vessels in the area of brain lesion during 7 days of our observation. This was also described previously by other researchers who found that hBM-MSCs infused intra-arterially were present in the brain inside blood vessels and do not migrate into parenchyma [18,22]. There are not many studies describing EV localization in the brain after their systemic transplantation. Xin et al. observed the transfer of exosomes enriched with green fluorescence protein (GFP) from MSCs into neurons and astrocytes in the rat brain [23]. Similarly, Chen et al. visualized exosomes stained with Dil injected intra-venously in the rat brain after ischemia [12]. Recently, Lapchak et al. detected extracellular vesicles stained with DiD near the injured area of the rabbit brain after their systemic infusion [24]. Our findings are in agreement with above studies. We were able to identify EVs labeled with PKH26 in the right hemisphere of focal brain injured rats 24 h after intra-arterial transplantation.
The abilities to stimulate neurogenesis and neuroprotective properties of EVs derived from MSCs in the models of ischemic stroke have been revealed in some studies. Xin e al. showed that exosomes from MSCs promote functional recovery, induce neurite remodeling, and activate neurogenesis and angiogenesis in rats after stroke [25]. Similarly, Otero-Ortega et al. demonstrated that EVs derived from MSCs stimulate axonal growth, white matter restoration, and signal transduction and reduce motor deficits in the rats with subcortical stroke [26]. Moreover, the administration of EVs from MSCs reduces the lesion area and improves functions impaired after ischemic stroke in sheep fetuses [27]. Additionally, EVs derived from MSCs have neuroprotective effect and module peripheral immune response in a model of ischemic stroke [28]. However, the detailed role of EVs from MSCs on local immune response in the brain after ischemic stroke has not been investigated so far. After the brain injury activation of astrocytes, microglia cells and infiltration of leucocytes into the damaged tissue were detected [3,29]. In our studies, focal brain insult induced by ouabain injection into rat striatum was accompanied with the local activation of astrocytes and microglia/macrophages and the influx of leucocytes, including T cytotoxic cells into ipsilateral hemisphere. We have noticed that transplantation of hBM-MSCs caused the decrease of the number of activated astrocytes 24 h after cell injection. It is known that MSC infusion may not only inhibit astrocytes activation but mostly promote their polarization into anti-inflammatory phenotype which leads to glutamate capture and production of neurotrophic factors [30]. In our experiments, we also observed the reduction of microglia/macrophages after intra-arterial transplantation of hBM-MSCs into rats with focal brain injury. Previously, the inhibition of microglial cells or promotion of their polarization due to MSC presence has been detected by other researchers during in vitro studies [31,32]. Our in vivo experiments revealed that intra-arterial EV transplantation into focal brain injury rats diminishes the number of microglia/ macrophages evoked by the insult. The effect was much stronger after extracellular vesicle infusion than after transplantation of their counterparts. Similar to our findings, Ruppert et al. demonstrated that extracellular vesicles derived from MSCs reduced the number of microglia pro-inflammatory cells in spinal cord injury [33]. Activation of astrocytes and microglia caused by brain injury is often accompanied with the influx of leucocytes from the periphery. In our studies, hBM-MSC transplantation led to the inhibition of leukocyte infiltration into injured rat hemisphere. Our findings are in agreement with another study demonstrating MSC infusion in stroke mice, resulting in reduced brain leukocyte infiltration [8]. Moreover, we noticed that intra-arterial injection of EVs into focal brain injured rats caused the lower number of leucocytes visible in damaged hemisphere of graft recipients. This was in contrast to the previous study of Doeppner et al. in which EVs derived from MSCs transplanted intraventricular in focal brain ischemic mice did not diminish brain leukocyte infiltration [28]. Among different types of leucocytes, T lymphocytes play an important role in immunological defense. We noticed the decrease of T CD8 + cytotoxic cells after hBM-MSC or EV injection. This was in accordance with the other studies where the reduction of T cytotoxic cells after MSC infusion was found [34,35]. The inhibitory effect of EVs in vivo has not been studied up to now. However, the induction of apoptosis in activated T cells by EVs derived from mesenchymal stem cells was demonstrated in vitro [36,37].
Brain injury induces the release of cytokines accompanied with cellular immune response. Many studies showed that interleukin-1 produced few hours after the brain ischemic damage stimulated the production of cytokines, chemokines, and cell adhesion molecules which led to blood-barrier damage [38,39]. Previous studies revealed that transplantation of mesenchymal stem cells caused the decrease of IL-1 production by microglia due to their inhibition and T helper cells polarization into anti-inflammatory phenotype [32,40]. In our studies, we observed the significant decrease in the level of IL-1α and IL-1β evoked by focal brain injury after MSC or EV infusion. Ebrahim et al. showed that EVs isolated from MSCs reduced the level of interleukin-1 in a model of intrauterine adhesions [41]; however, the impact of extracellular vesicles on IL-1 production in brain-injured EV recipients has not been studied so far. Another cytokine involved in pro-inflammatory response after ischemic stroke is interleukin-6. Our studies revealed that transplantation of mesenchymal stem cells or their extracellular vesicles caused statistically significant decrease of IL-6 level in focally injured rat brain probably due to the inhibitory effect of hBM-MSCs and EVs on local immunologically effector cells, i.e., microglia cells, macrophages, and neurons. Similarly, Karlupia et al. referred that MSCs injected intra-arterially led to the decrease of IL-6 level in rat model of ischemic stroke [6]. Recent studies revealed that despite of IL-1 and IL-6, different isoforms of transforming growth factors-β (TGFβ) may be efficient as pro-inflammatory factors in immune response after ischemic stroke. Xin et al. showed the increased production of TGF-β1 in the nervous tissue injured by ischemic stroke and reduced release of this cytokine after MSCs transplantation [42]. Our studies revealed significant increase of TGF-β2 production in rat brain after striatal focal injury and its significant decrease 3 and 7 days after hBM-MSC or EV infusion.
Brain injury is accompanied by high expression of chemokines that can be critical for the outcome in neurological disorders due to its widely exerted effects on cellular activity, proliferation, and survival. Silva et al. showed that ischemic stroke causes the release of chemokine C-X-C motif ligand-1 (CXCL-1) which leads to the loss of neural cells in injured tissue [43]. In our studies, we observed the elevation of CXCL-1 in the rat brain 48 h after focal injury. Intra-arterial transplantation of hBM-MSCs or EVs strongly inhibited the production of CXCL-1 in our experimental model which was probably related to the reduction of microglial/macrophages. Similar effect was observed by Donizetti-Oliveira et al.; the authors noticed that adipose tissue-derived mesenchymal stem cells (AD-MSC) injection resulted in the decrease of CXCL-1 in a model of renal disease [44]. Another chemokine reported to be released after ischemic stroke is monocyte chemattractant protein-1 (MCP-1) which induces monocyte infiltration, activates proinflammatory cytokines, and leads to the progression of tissue damage [45,46]. In our studies, we observed the increase of MCP-1 in the focally injured rat brain and the statistically significant reduction of MCP-1 level after hBM-MSC or EV transplantation. This was correleted with the decrease of microglia/macrophages and astrocyte activation. Our observation was concomitant with the results of Yoo et al. who showed that injection of mesenchymal stem cells diminished MCP-1 production in a model of ischemic stroke [8]. Brain injury results also in increase of macrophage chemoattract proteins: MIP-1α and MIP-3α. It is known that macrophage chemoattract proteins produced after ischemic stroke stimulated IL-1β release and activated nitric oxide synthase which caused the further progression of tissue damage. Inhibition of MIP-3α caused reduction of the lesion in the rat brains injured by ischemic stroke [47]. Previous studies showed that MIP-1α lead to the activation of astrocytes and microglia as well as monocyte infiltration into injured tissue [48]. In our experimental model, focal striatal injury induced high level of MIP-1α and MIP-3α in the rat brain and hBM-MSC or EV injection led to reduction of MIP-1α and MIP-3α level in each time point of observation. Similar effect was referred by Sun et al.'s studies where EV transplantation in a murine model of spinal cord injury caused the decrease of MIP-1α expression [49].
As shown here, focal brain injury induced by ouabain injection into rat striatum is accompanied with the local immune reaction visible in the damaged tissue after the insult. The activation of the innate effectors and the influx of acquired immune cells from the peripheral blood are observed in the ipsilateral hemisphere of brain disorders. Intra-arterial transplantation of human bone marrow-derived mesenchymal stem cells or their extracellular vesicles infused to the internal carotid artery enables their migration into the brain. The donor cells are localized in the damaged area; however, they do not migrate into the brain parenchyma being captured inside the microvessels during 7 days of observation. The infusion of hBM-MSCs or their EVs alleviates the local immune response in rat brain evoked by the insult. The decrease of astrocyte and microglia/macrophages' activation and the number of T lymphocytes while declining the level of pro-inflammatory cytokines and chemokines was detected. Interestingly, the suppressive effect of EVs was comparable to their counterparts, and in some situations, the reduction of immunological response in the host brain is even more efficient than observed after hBM-MSC graft.

Conclusions
Our studies revealed that human bone marrow mesenchymal stem cells or extracellular vesicles derived from them transplanted intra-arterially modulate immune response in the brain caused by focal brain injury. In our experimental model, EVs present similar immunomodulatory properties as their cells of origin so they can be potentially used in stroke treatment instead of MSCs.


s2fieldsofstudy:
Biology
Agricultural And Food Sciences

text:
Characterization of the Intestinal Microbiota and Metabolites of Broiler Breeders with Different Egg Laying Rate

Background The aim of the present study was to determine differences in the intestinal microbiota and metabolites of broiler breeders with different egg laying rate; and its possible relationships between intestinal microbiota and intestinal metabolites were also explored. Methods A total of 200 AA+ parent broiler breeders (41-week-old) were separated into two groups according to their different egg laying rate: average reproductive group (AR: 77.26%±0.88%) and high reproductive group (HR: 86.67%±0.75%), with 10 replicates and 10 birds each 42-days study. Results Feed conversion ratio (FCR), ovary cell apoptosis rate (ApoCR) and abdominal fat pad weight percentage were lower (p < 0.05), while the hatchability of qualied egg rate (HQR) was higher (p < 0.05) in HR group than that in AR group. At phylum level, Firmicutes and Proteobacteria were the dominant bacteria in small intestines, while Bacteroidetes was dominant bacteria in cecum. At genus level, Lactobacillus was dominant in small intestines while Bacteroides were dominant in cecum. In HR Phascolarctobacterium were lower (p < 0.05) in ileum. A positive correlation was observed between Actinobacteria in the small intestines and ApoCR (r = 0.34 & p < 0.05). A positive correlation was observed between FCR and Spirochaetes in cecum (r = 0.54 & p < 0.05). The metabolomics analysis on ileal digesta content revealed a higher concentration of Octadecanoic acid, 9,12-Octadecadienoic acid, 11-trans-Octadecenoic acid, d-Ribose and Inositol in HR broiler breeders. Moreover, inositol was enriched in "Phosphatidylinositol signaling system", "Inositol phosphate metabolism", "Ascorbate and aldarate metabolism", "Galactose metabolism", "ABC transporters", and Octadecanoic acid was enriched in "Fatty acid biosynthesis", of unsaturated fatty acids". results suggest that glycerol, inositol modulate energy partitioning in fat metabolism to inuence reproductive performances. Microbiota, such as Actinobacteria (phylum), Spirochaetes (phylum), and Phascolarctobacterium (genus) may have negative relationship with broiler breeders’ reproductive performances. The present study obtained a total of 5,328,583 high quality 16S rRNA gene sequences from duodenum (1,253,578), jejunum (1,320,806), ileum (1,347,555), cecum (1,406,644), with an average of 83,259 sequences per sample and a range of 67,290 to 95,053. A total of 36,679 OTUs were identied at 97% sequence identity. The Chao1 and ACE indexes were lower in duodenum and ileum of HR (p < 0.05) (Fig 2 a,b) and Shannon index was lower in ileum of HR (p < 0.05) (Fig 2 c). The Venn and Flower diagrams were used to describe common and unique OTUs between goups. In the duodenum, jejunum, ileum and cecum of the AR and HR birds, we observed, respectively, 713, 595 756 and 1046 common OTUs (Fig 3). inositol play a signicant role in fat metabolism though by modulating energy partitioning which can subsequently inuence reproductive performances. Also, higher diversity and richness of microbial communities in the small intestines could induce nutrient competition with the host, negatively impacting reproduction. What’s more, the higher relative abundance of Phascolarctobacterium (genus), Actinobacteria (phylum) and Spirochaetes (phylum) in the AR birds seems to be correlated to their lower reproduction performances.

Sample Collection and Measurements
Egg production, broiler breeder mortality, quali ed hatching egg production and feed consumption were recorded daily. Hatching performance was recorded at day 42. At end of the study (day 42), birds were sacri ced by cervical dislocation and the intestines were removed for the measurement of duodenum (Duo), jejunum (Jej), ileum (Ile), cecum (Cec) weight and length. The content of the duodenum, jejunum, ileum and cecum was immediately transferred into 1.5ml sterile centrifuge tubes for microbiota and metabolomics analysis. The ovaries were collected and placed into 4% paraformaldehyde (pH = 7.2) for cell apoptosis analysis.

Follicle Apoptosis by TUNEL Method
Sections were histochemically stained using the TUNEL technique by an in situ apoptosis detection kit (Roche, Switzerland; [24]. The follicle section of ovary was aseptically removed and placed immediately into a solution of 3-ethoxy-3-aminopropyl silane (Zhongshan Golden Bridge Biotechnology Co., Ltd. Beijing, China). Set into oven to 60 °C for 60 min. Dewaxing and hydration. After an incubation with proteinase K (Boehringer Mannheim, Germany), rinse thrice with PBS. With 50 μL TUNEL in the reaction mixture specimens, with slide in the dark wet box in response to 37 °C for 1 h. Rinse thrice with PBS. Add 50 μL (converter-POD) in liquid samples, or sealing membrane reaction 37 °C in the dark wet box with glass for 30 min. Rinse thrice with PBS. 50-100 μL DAB chromogenic agent, reaction of 25 °C, 10 min. Rinse thrice with PBS Hematoxylin staining, rinse water after a few seconds; alcohol dehydration, xylene transparent, neutral balata. Using BA200Digital (Mike Audi Industrial Group Co., Ltd.) to image acquisition. Apoptotic color is brown yellow, and negative expression is blue with white background.

DNA Extraction and Microbiota Analysis
Total microbial genomic DNA was extracted from the chyme of duodenum, jejunum, ileum and cecum, using the TIANamp Bacteria DNA isolation kit (DP302-02, Tiangen, Beijing, China) according to the manufacturer's instructions. 16S rRNA genes of distinct regions (16S V4) were ampli ed used the speci c primers 515F (5'-GTTTCGGT GCCAGCMGCCGCGGTAA-3') and 806R (5'-GCCAA TGGACTACHVGGGTWTCTAAT-3') with the barcode. All PCR reactions were carried out with Phusion® High-Fidelity PCR Master Mix (New England Biolabs). PCR products, a bright main strip between 400-450 bp, were mixed in equal density ratios for puri cation with Qiagen Gel Extraction Kit (Qiagen, Germany). Sequencing libraries were generated via the TruSeq®DNA PCR-Free Sample Preparation Kit (Illumina, USA) following manufacturer's recommendations, index codes were added. Finally, the library was sequenced on an IlluminaHiSeq2500 platform and 250 bp paired-end reads were generated. Splicing sequences were using FLASH (V1.2.7) to assemble [25], and then quality ltration was using QIIME (V1.7.0) [26] to nish. Chimera tags were removed using UCHIME algorithm [27]. Sequence analysis were performed via Uparse software (Uparse v7.0.1001) [28]. The original GC/MS data were converted into les in netCDF format [32] and subsequently preprocessed, including the identi cation, ltration, and alignment of peaks by XCMS software (www.bioconductor.org). Finally, a data matrix of information, including mass-to-charge ratios (m/z), retention times, and peak intensities, was obtained. Identi cation of metabolites was performed using the AMIDS system (Automated Mass Spectral Deconvolution and Identi cation System) by searching the National Institute of Standards and Technology library (http://srdata.nist.gov/gateway/) [33].
The resultant GC-MS data matrix were imported into the MetaX software package (http://metax.genomics.cn) for multivariate analysis, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). PCA was utilized to visualize global clustering and display differences in metabolic pro les between samples. To achieve the maximum separation between samples, PLS-DA was used to identify differential metabolites that explained the separation between groups, missing values were imputed using a k-Nearest Neighbor (KNN) method [34]. The model quality was evaluated using the R2X, R2Y, and Q2 parameters. The R2X and R2Y parameters, which represent the fractions of explained X-variation and Y-variation, respectively, can be used to evaluate the quality of the model. The Q2 parameter represents the predictive ability of the model. In general, the model is acceptable when the values of R 2 X, R 2 Y, and Q 2 are greater than 0.5 [35]. In addition to cross-validation, the PLS-DA models were veri ed via 200 repeated permutation tests.
As described above, PLS-DA was employed to identify differential metabolites between groups. Furthermore, variable importance in the projection (VIP) values in the PLS-DA model were used to rank the metabolites based on their importance indiscriminating between groups. Metabolites with the highest VIP values are the most powerful group discriminators. Traditionally, VIP values > 1 are signi cant. In this study, metabolites with VIP values (VIP > 1.0) in the PLS-DA model, signi cance (p < 0.05) in Student's t-test (R package) and fold change < 0.83 or > 1.2 were selected as differential metabolites. Then, receiver operating characteristic (ROC) curves analysis was performed (R package pROC), and the areas under the curves (AUCs) were calculated to determine the diagnostic value of these differential metabolites [36]. The discriminatory power of the differential metabolites was ranked and visualized using heat maps [36].
Metabolic pathways were obtained for these identi ed differential metabolites from the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/kegg).

Statistical Analysis
Data was analyzed by independent-sample T-test in the SPSS version 25.0 statistical software package (IBM® SPSS® Statistics, New York, USA), differences among treatments were considered signi cant at p < 0.05. Alpha indexes were analyzed by Wilcox rank sum test, differences among treatments were considered signi cant at p < 0.05. Adonis test was based on Bray-Curtis distance, differences among treatments were considered signi cant at p < 0.05.

Reproductive Performance
Average daily feed intake (ADFI) in both HR and AR birds; similarly, no differences in egg weight, quali ed and unquali ed egg rates were observed between the two groups. Feed conversion ratio (FCR), however, was lower in the HR birds (p < 0.05). As expected, the hatchability of quali ed egg rate (HQR) was higher in the HR group (p < 0.05) ( Table 1).

Digestive Organs
Compared to the AR groups, birds in the HR group presented a lower abdominal fat content (p < 0.05), however no differences were observed for chicken weight, relative weight of crop, proventriculus, gizzard, intestines (duodenum, jejunum, ileum, cecum) ( Table 2) and length of intestines (Supplementary Table  1, Additional File 2).

Ovary cell apoptosis
Ovary cell apoptosis rate (ApoCR) in the ovaries of the HR birds was lower than to that observed in the AR group (p < 0.05) (Fig 1).

Summary of High-Throughput Sequencing and Alpha Diversity
The present study obtained a total of 5,328,583 high quality 16S rRNA gene sequences from duodenum (1,253,578), jejunum (1,320,806), ileum (1,347,555), cecum (1,406,644), with an average of 83,259 sequences per sample and a range of 67,290 to 95,053. A total of 36,679 OTUs were identi ed at 97% sequence identity. The Chao1 and ACE indexes were lower in duodenum and ileum of HR (p < 0.05) (Fig 2 a,b) and Shannon index was lower in ileum of HR (p < 0.05) (Fig 2 c). The Venn and Flower diagrams were used to describe common and unique OTUs between goups. In the duodenum, jejunum, ileum and cecum of the AR and HR birds, we observed, respectively, 713, 595 756 and 1046 common OTUs (Fig 3).

Taxonomic Composition of GIT microbiota
A total top 10 phyla and top 10 genera were based on the identi ed OTUs. At phylum level, Firmicutes, Proteobacteria in duodenum, Firmicutes, Cyanobacteria in jejunum and ileum, Firmicutes, Bacteroidetes in cecum were the predominant phyla (Fig 4, a) ( The principal coordinate analysis (PCoA) based on weighted unifrac distance was applied to examine differences in taxonomic community composition and structure in the duodenum, jejunum, ileum and cecum. The PCoA analysis indicates that the bacterial community composition of the duodenum, jejunum, ileum and cecum of the HR birds can be separated from that of the AR group (Fig 5), however this separation did not reach signi cant difference (Table 4).
Spearman correlation analysis was employed to explore correlations between microbiota richness and performance parameters (EPR, FCR, HQR and ApoCR) measured in this study. At phylum level, Actinobacteria in the small intestines were positively correlated with ApoCR. Euryarchaeota in the small intestines were negatively correlated with EPR and HQR and positively correlated with FCR. At genus level, Phyllobacterium, Romboutsia, Megamonas, Coprococcus_1 in the small intestines were negatively correlated with EPR; Phyllobacterium in the small intestines were positively correlated with FCR (Table 5-1). At phylum level, Spirochaetes in the cecum was positive correlated with FCR while Planctomycetes in the cecum was negatively correlated with HQR. At genus level, Sphaerochaeta in the cecum was negatively correlated with EPR (Table 5-2).
A total of 71 metabolites were identi ed in both ileal and caecal samples. Ileal metabolites presented a higher level of expression than the caecal ones (Fig 6,  a). In the ileum Octadecanoic acid, 9,12-Octadecadienoic acid, 11-trans-Octadecenoic acid, d-Ribose and inositol were expressed at higher level in the HR birds, according to their VIP values in the PLS-DA model (VIP > 1) and p-value from Student's t-test (p < 0.05): (Fig 6, b; Table 6). In the cecum, we observed that inositol was lower in the HR birds (Table 6). A ROC analysis was carried out to determine the ability of the identi ed ileal metabolites to discriminate between the AR and HR groups. The heatmap (Fig 6, c) reports the ileal metabolites ranked in order of their AUC values, and indicated that the ve metabolites were effective in discriminating between the HR and AR birds. The KEGG pathway database was utilized to analyze the metabolic pathways relating to the ve metabolites identi ed in the ileum. We observed that inositol was enriched in the "Phosphatidylinositol signaling system", "Inositol phosphate metabolism", "Ascorbate and aldarate metabolism", "Galactose metabolism", "ABC transporters", while octadecanoic acid was enriched in the "Fatty acid biosynthesis", "Biosynthesis of unsaturated fatty acids" pathways (Fig 6, d).

Reproductive performances
The main function of the GIT is to digest feed and absorb nutrients. In the small intestine (duodenum, jejunum and ileum), the host absorbs mostly the nutrients of dietary origin [37], while in the cecum, the host absorbs nutrients that product by microbial fermentation [38,39]. It is well accepted that productive and reproductive performances are in uenced by absorption and utilization of nutrients in poultry [40,41]. In the present study, even though both AR and HR birds were fed the same diet, we observed that abdominal fat was lower in the HR birds, but both body and organ weight was not different from that of AR birds (Table 2). Moreover, FCR was lower in HR birds (Table 1), which suggests that energy and nutrients were used for production purposes rather than being diverted to abdominal fat depots as observed in the HR birds. It has been reported that changes in ovarian function are accompanied by increased abdominal adiposity [42][43][44][45][46] and that body fat is negatively correlated with fertility [47]. These observations support our ndings that ApoCR was higher in the AR birds (Fig 1).

Metabolites
In the present study, with the KEGG enrichment pathway analysis, we found that in "Fatty acid biosynthesis" and "Biosynthesis of unsaturated fatty acids" pathways, hexadecanoic acid (palmitic acid) was lower while octadecanoic acid (stearic acid) was higher in the HR birds ( Supplementary Fig 1, Fig 2, Additional File 2). In agreement with our observations are the nding of Xie et al (2012) where broiler hens had increased abdominal fat pad weight and reduced egg production. it has been observed that high concentrations of palmitic acid can be toxic to granulosa cells [48]. Palmitic acid is a saturated fatty acid that can induce cellular apoptosis and exert cytotoxic effect as it is a precursor of the complex lipid ceramide, which has been reported to induce apoptosis in granulosa cells of hens [49]. Palmitic acid can also induce cellular apoptosis through a ceramide-independent pathway [50]. Finally, palmitic acid and stearic acid could induce apoptosis in human ovarian granulosa cells [51]. In our study we observed higher stearic acid expression in the HR birds, however in light of the lower ApoCR and levels of palmitic acid observed in this group, it is likely that the higher stearic acid expression was not su cient to exert any negative effect on ovarian function in the HR broiler breeders.
We observed that in the "Inositol phosphate metabolism" and "Galactose metabolism" pathways, glycerol was lower while inositol (D-myo-inositol) was higher in the HR birds ( Supplementary Fig 3, Fig 4, Additional File 2). Glycerol can be converted into glycerol-3-phosphate and glyceric acid via glycolysis to supply energy [52] which probably was diverted to the abdominal fat depots in the AR birds. In the "Inositol phosphate metabolism" pathway, phosphatidyl-1D-myoinositol, an intermediate in inositol phosphate metabolism, was also indirectly linked to the "Glycerophospholipid metabolism", which plays an important role in fat metabolism [53]. Although in present study, we observed that inositol was lower in the cecum of the HR birds (Table 6), the overall expression of inositol in the GIT of HR birds was higher than that observed in the AR birds.
Overall, our ndings suggest that intestinal metabolites play a signi cant role in fat metabolism though by modulating energy partitioning which can subsequently in uence reproductive performances.

Microbiota
The nutritional requirements of the gastrointestinal microbiota are species-dependent as bacteria need different dietary substrates for their growth and metabolic activity [39]. Therefore it is obvious that in the gastrointestinal there is a resources competition between the microbiota and the host. In present study, we observed that the duodenum and jejunum microbiota of AR birds has more richness than that of the HR ones (Fig 2 a, b), and the ileum microbiota was more diverse in the AR birds than in the HR one (Fig 2 c). The above mentioned higher diversity and richness of bacteria in the AR birds could have resulted in an increased nutrients competition with the host and the microbiota in the small intestines; the higher FCR observed in the AR birds could support this hypothesis. In present study, Firmicutes and Proteobacteria were the dominant phyla in the small intestines while Bacteroidetes was the dominant bacteria in the cecum. Lactobacillus was thedominant genus in the small intestines while Bacteroides was the dominant genus in the cecum. Similar observations have been made in broiler chickens and laying hens [54,55]. Diet is a well known factor that can in uence the microbiota composition and metabolic activity [56,57]. For example, an altered abundance of Bacteriodetes and Firmicutes has been observed in animals fed a high fat diet [58][59][60], however as the same diet was to the fed AR and HR birds this could probably explain the lack of differences in bacterial communities between the two groups. Indeed, while the PCoA analysis suggested that there might be distinct bacteria communities along the different segments of the gastrointestinal tract of AR and HR birds (Fig 5), these were not signi cant (Table 4).
It is worth noting that we observed a higher relative abundance of Phascolarctobacterium in the ileum of AR birds (Fig 4, c). A higher abundance of Phascolarctobacterium has been correlated with elevated oxidative stress in sows during late gestation and at parturition [61]. Moreover the higher abundance of Phascolarctobacterium has been linked to intestinal in ammation and pathology [62]. Finally, Phascolarctobacterium seems to be correlated with increased body weight and fat mass, and altered metabolic pro le [63]. Therefore, in present study, the higher abundance of Phascolarctobacterium observed in the AR birdsmight be linked to the lower production and reproductive performances observed in the AR birds.
It has been reported that the microbiota can also metabolize steroids and produce hormones [64,65]. For example, Actinobacteria can regulate the sterols: sterol uptake, eliminate the aliphatic side chain at C17, oxidize the steroid nucleus [66] and synthetize testosterone [67,68]. In our study, Actinobacteria abundance in the small intestines was positively correlated with ApoCR (Table 5-1), suggesting that the increased apoptosis cell rate in ovaries could be ascribed to a modi cation in steroids metabolism induced by the increased abundance of Actinobacteria.
In our study we observed that Spirochaetes (phlum) abundance in the cecum was positively correlated with FCR (Table 5-2). Spirochaetes are responsible for infectious typhlitis in hens[69] which is characterized by decreased egg production and increased the feed consumption [70]. Broiler breeders infected by Spirochaetes had poorer feed conversion and the impact of the disease was also extended to their offspring, with chicks being weak, slow growing and with impaired gastrointestinal functionality [71]. In this study, the lower productive and reproductive performances observed in the AR birds could be partially ascribed to the higher relative abundance of Spirochaetes in the cecum of the AR birds (

Conclusions
Overall, our ndings suggest that intestinal metabolites: glycerol, inositol play a signi cant role in fat metabolism though by modulating energy partitioning which can subsequently in uence reproductive performances. Also, higher diversity and richness of microbial communities in the small intestines could induce nutrient competition with the host, negatively impacting reproduction. What's more, the higher relative abundance of Phascolarctobacterium (genus), Actinobacteria (phylum) and Spirochaetes (phylum) in the AR birds seems to be correlated to their lower reproduction performances.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.             Principal coordinate analysis (PCoA) of microbial comunities in the duodenum (a), jejunum (b), ileum (c), cecum (d), of AR and HR broiler breeders. Figure 6 a: Expression and distribution density of metabolites in ileum and cecum of AR and HR broiler breeders; the Y axis represented the peak-area (log 10), and the X axis represented each groups. b: volcano diagram showing different metabolites in the ileum of AR and HR birds; the Y axis represents the p-value (-log 10), and the X axis represents the fold change (log 2) of metabolites. The size of the spots represent the metabolite VIP value; black spots represent no-different metabolites; red spots represent up-different metabolites. c: heatmap clusering of different metabolites in the ileum of AR and HR broiler breeders. d: KEGG enrichment pathway analysis from different metabolites in ileum of AR and HR broiler breeders; the Y axis represents the enriched pathwat terms, and the X axis represents the ratio of individual metaboltes to the whole identi ed metabolites. The size of the spots represents the number of different metaboltes enriched in each pathway, while the color shade of the spot represented the p-value (-log10) of each pathway.

Supplementary Files
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text:
Upregulation of contactin-1 expression promotes prostate cancer progression

Contactin-1 (CNTN-1) has been reported to serve an oncogenic role in several cancer types. However, detailed mechanisms describing the influence of CNTN-1 in prostate cancer progression have not yet been elucidated. The present study aimed to determine the clinical significance of CNTN-1 expression in prostate cancer progression, and also to investigate the regulatory role of CNTN-1 in the proliferation, migration and invasive ability of prostate cancer cells. The results of the present study indicated that expression levels of CNTN-1 were significantly higher in prostate cancer tissues compared with adjacent normal tissues. Moreover, a high expression level of CNTN-1 was positively correlated with tumor size, stage and metastasis, as well as a poorer prognosis in patients with prostate cancer. Furthermore, CNTN-1-knockdown in prostate cancer cells (using short hairpin RNA) resulted in the significant inhibition of cancer cell proliferation, colony formation, migration and invasiveness. Silencing of CNTN-1 expression also suppressed epithelial-mesenchymal transition in prostate cancer cells via the upregulation of E-cadherin, and the downregulation of N-cadherin and vimentin expression. Inhibition of CNTN-1 expression also reduced the activity of the PI3K/AKT signaling pathway in prostate cancer cells. Thus, it was demonstrated that CNTN-1 expression is upregulated, and plays an oncogenic role, in prostate cancer cells. The results of the current study suggest that CNTN-1 may represent a promising therapeutic target, potentially improving the treatment of patients with prostate cancer.

Introduction
Prostate cancer is a highly prevalent malignancy and represents the second leading cause of cancer-related mortality in elderly men (1,2). Moreover, it is estimated that there would be 161,360 new prostate cancer diagnoses and 26,730 prostate cancer-associated mortalities in 2015 (1,2). The molecular heterogeneity of prostate cancer can make its early diagnosis and treatment problematic; thus, the identification of accurate molecular biomarkers and potential therapeutic targets (at various disease stages) may result in improved patient outcome (3,4). It is therefore important to investigate the molecular mechanisms underpinning the development and progression of prostate cancer, as this may catalyze the identification of novel therapeutic targets (5,6).
Contactin-1 (CNTN-1), a member of the immunoglobulin superfamily is a glycosylphosphatidylinositol (GPI)-anchored neuronal membrane protein that facilitates cell adhesion (7,8). It may also influence the formation of axon connections in the developing nervous system (7). Mikami et al (7) also reported that CNTN-1 was a functional receptor for neuroregulatory chondroitin sulfate-E. Additionally, Lamprianou et al (8) discovered a complex (formed from CNTN-1 and protein tyrosine phosphatase receptor type Z1) that mediated the development of oligodendrocyte precursor cells. CNTN-1 is upregulated in several common types of human cancer, and promotes the progression of lung (9) and gastric cancer (10), and esophageal (11) and oral squamous cell carcinoma (12). For example, the upregulation of CNTN-1 expression is correlated with more advanced clinical stages and lymph node metastasis in patients with esophageal squamous cell carcinoma (11). Moreover, CNTN-1 expression is upregulated in oral squamous cell carcinoma, and is associated with lymph node metastasis, as well as a poor prognosis (12). Su et al (9) discovered that the knockdown of CNTN-1 expression inhibited the invasion and metastasis of lung adenocarcinoma, suggesting that it may represent a promising therapeutic target for the treatment of patients with the disease.
Furthermore, Yan et al (13) reported that the knockdown of CNTN1 inhibited stem-like, cell-mediated tumor initiation in prostate cancer. It was also reported that the overexpression of CNTN1 promoted cellular invasion in vitro, as well as enhancing xenograft tumor formation and lung metastasis in vivo (13). Taken together, these findings suggest that CNTN-1 may promote prostate cancer progression. The present study aimed to investigate the clinical significance of CNTN-1 expression in prostate cancer progression, and to determine the mechanism of CNTN-1 regulation of the malignant phenotypes of prostate cancer cells.

Materials and methods
Tissue collection. A total of 56 prostate cancer tissues and matched adjacent paracancerous tissues were obtained from patients with primary prostate cancer at the First Affiliated Hospital of Jishou University (Jishou, China) between April 2011 and September 2013 and stored at -80˚C until use. The patients were aged between 58 and 79 years (mean age, 66.5 years). The clinicopathological features of all patients are presented in Table I. Follow-up occurred for 60 months after surgery by phone calls. Written informed consent was obtained from all patients prior to surgery, and the experimental procedures were approved by the Ethics Committee of the First Affiliated Hospital of Jishou University.
Reverse transcription-quantitative (RT-q)PCR. Total RNA was extracted from the tissues and cell lines using TRIzol ® reagent (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The RevertAid First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc.) was used to reverse transcribe the RNA into cDNA according to the manufacturer's protocol, for which the primer sequences are as follows: CNTN-1 forward, 5'-TGT TCA GCA AAT TCA TCC CA-3' and reverse, 5'-TCT ACC CAC TCA GGG AAT GC-3'; and GAPDH forward, 5'-ACG GAT TTG GTC GTA TTG GGC G-3'; and reverse, 5'-CTC CTG GAA GAT GGT GAT GG-3'. ABI Power SYBR ® Green PCR Master mix (Thermo Fisher Scientific, Inc.) was subsequently used to perform qPCR according to the manufacturer's protocol. The reaction conditions were 95˚C for 3 min, followed by 35 cycles of 95˚C for 15 sec, 58˚C for 15 sec, and 72˚C for 15 sec. The relative expression levels were quantified using the 2 -∆∆Cq method (14) and normalized to those of GADPH.
Cell proliferation assay. Transfected cells (5x10 4 cells per well) were seeded into 96-well plates, and cultured at 37˚C for 0, 24, 48 or 72 h. Subsequently, 10 µl CCK-8 solution (Dojindo Molecular Technologies, Inc.) was added to each well, and the cells were incubated at 37˚C for 4 h. The absorbance at 450 nm was measured using a microplate reader (Thermo Labsystems).
Colony formation assays. Transfected cells (1x10 3 cells/well) were seeded into 12-well plates and cultured for 7 days. Crystal violet (0.1%; Thermo Fisher Scientific) was then used to stain the cells before images were captured using a light microscope (magnification, x200). The number of colonies was determined using ImageJ (v. 1.46; National Institutes of health).
Wound-healing assay. Transfected cells (5x10 5 cells/well) were seeded into 6-well plates, and cultured at 37˚C until ~100% confluence was achieved. A sterile 200-µl pipette tip was used to scratch a wound line in each well. The transfected cells were washed twice with Dulbecco's phosphate-buffered saline (Gibco; Thermo Fisher Scientific) and resuspended in serum-free DMEM, before being incubated at 37˚C for 24 h. Cells were then imaged at 0 and 24 h using a light microscope (magnification, x40). The wound closure between the 0 to 24 h time points was measured using ImageJ (v. 1.8; NIH) and relative wound closure was determined.
Cell invasion assay. Transfected cells (5x10 4 cells/well) were resuspended in serum-free DMEM and seeded into the upper chamber of 8-µm Transwell inserts (BD Biosciences), which had been pre-coated with Matrigel (BD Biosciences) at 37˚C for 30 min. The lower chamber was plated with DMEM supplemented with 10% FBS, and the cells were incubated at 37˚C for 24 h. The cells were then fixed using 4% paraformaldehyde at room temperature for 30 min, and then stained using crystal violet at room temperature for 5 min, before being imaged under a light microscope (magnification, x100). The number of invading cells was counted in five random non-overlapping fields.
Western blotting. Total protein was extracted from the transfected cells using RIPA buffer (Thermo Fisher Scientific Inc.). The total protein was quantified using a BCA method with a Pierce BCA Protein assay kit (Thermo Fisher Scientific Inc.). The proteins (50 µg/lane) were separated by 10% SDS-PAGE gel, and transferred onto PVDF membranes (Thermo Fisher Scientific Inc.). The membranes were blocked using 5% non-fat milk at room temperature for 3 h, and then incubated with rabbit anti-human antibodies against: CNTN- and GAPDH (1:500; cat. no. ab8245) at room temperature for 3 h, followed by further incubation with horse radish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. ab6721) at room temperature for 1 h. All antibodies were purchased from Abcam. The protein bands were visualized using the Pierce™ ECL western blotting substrate (Thermo Fisher Scientific, Inc.) and ImageJ software (v. 1.46; National Institutes of health) was used to conduct densitometric analysis.
Statistical analysis. All experiments were repeated ≥3 times. The data are presented as the mean ± SD and were analyzed using SPSS (v.20.0; IBM Corp.). Differences between 2 groups were analyzed using the paired or unpaired Student's t-test, and differences between multiple groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. The log-rank test was used to compare patient survival times between high-and low-CNTN1 expression groups, and the χ 2 test was used to analyze the results presented in Table I. P<0.05 was considered to indicate a statistically significant difference.

Results
Upregulation of CNTN-1 is associated with prostate cancer progression. In the present study, the expression levels of CNTN-1 in prostate cancer tissues were compared with those of matched adjacent paracancerous tissues. The mRNA and protein expression levels of CNTN-1 were significantly higher in prostate cancer tissues than in the corresponding adjacent tissues (Fig. 1A and B; P<0.01). Patients were then divided into high-and low-CNTN-1 expression groups, and the clinical significance of CNTN-1 expression in the progression of prostate cancer was investigated. The high-expression group was significantly associated with a larger tumor size, more advanced tumor state and risk of metastasis (Table I). Moreover, it was determined that the high-CNTN-1 expression level group exhibited shorter overall survival times compared with the low-expression group, suggesting that the upregulation of CNTN-1 may predict poor prognosis in patients with prostate cancer (Fig. 1C).
Knockdown of CNTN-1 inhibits prostate cancer cell proliferation. The influence of CNTN-1 expression on prostate cancer progression was also examined in vitro. To investigate whether CNTN-1 expression was upregulated in prostate cancer cells, cells were transfected with shRNA to knockdown CNTN-1 expression. Following transfection, the mRNA and protein levels of CNTN-1 were significantly reduced when compared with those of the control group. However, transfection with NC shRNA did not affect CNTN-1 expression in prostate cancer cells ( Fig. 2A and B; P<0.01). The function of CNTN-1 in prostate cancer cell proliferation was then investigated. As shown in Fig. 2C-E, CNTN-1-knockdown significantly inhibited the proliferation and colony formation of prostate cancer cells (P<0.01), suggesting that CNTN-1 promotes prostate cancer cell proliferation.

CNTN-1-knockdown suppresses the migration and invasion abilities of prostate cancer cells.
To further characterize the function of CNTN-1 in prostate cancer metastasis, wound-healing and Transwell assays were performed to examine the effects of CNTN-1-knockdown on the migration and invasiveness of prostate cancer cells. As shown in Fig. 3A and B, prostate cancer cell migration was significantly inhibited in the CNTN-1-knockdown group, compared with the control group (P<0.01). Knockdown of CNTN-1 significantly suppressed the invasive capacity of PC3 and LNCaP cells ( Fig. 3C and D; P<0.01). Therefore, CNTN-1 is suggested to promote the regulation of migration and invasion in prostate cancer cells.

Inhibition of CNTN-1 represses epithelial-mesenchymal transition (EMT) and PI3K/AKT signaling in prostate cancer cells.
The effect of CNTN-1-knockdown on EMT (a mechanism facilitating cancer cell migration and invasion) in prostate cancer cells was also investigated. As exhibited in  Fig. 4A and B, knockdown of CNTN-1 significantly increased the expression of E-cadherin, but decreased the protein levels of N-cadherin and Vimentin in prostate cancer cells (P<0.01), indicating that EMT was inhibited. Therefore, it was suggested that CNTN-1-knockdown may suppress prostate cancer metastasis via the inhibition of EMT. The molecular mechanism of CNTN-1 in prostate cancer progression was then investigated. PI3K/AKT signaling has been reported to influence prostate tumor growth and metastasis. Therefore, the function of CNTN-1 in the regulation of the PI3K/AKT signaling pathway was assessed. As indicated in Fig. 4C and D, the expression levels of phosphorylated PI3K and AKT were significantly reduced in the shCNTN-1 group compared with those the shNC group, indicating that CNTN-1-knockdown reduced PI3K/AKT signaling in prostate cancer cells (P<0.01). Therefore, the results of the current study suggest that the PI3K/AKT signaling pathway may be involved in the function of CNTN-1, by regulating the malignant phenotypes of prostate cancer cells.

Discussion
The function and clinical significance of CNTN-1 expression in prostate cancer has not yet been fully elucidated. In the present study, it was observed that the expression level of CNTN-1 was significantly higher in prostate cancer tissues compared with adjacent paracancerous tissues. Moreover, high expression of CNTN-1 was positively correlated with cancer progression, as well as poor prognosis in patients with prostate cancer. CNTN-1-knockdown resulted in significant inhibitory effects on prostate cancer cell proliferation, colony formation, migration and invasiveness. Moreover, the CNTN-1-knockdown inhibited EMT and modulated PI3K/AKT signaling in prostate cancer cells.
CNTN-1 has been discovered to promote prostate cancer cell invasion in vitro, as well as tumor growth and lung metastasis in vivo (13). In the present study, the expression pattern and function of CNTN-1 in prostate cancer was investigated, and the data suggested that the expression levels of CNTN-1 were significantly higher in prostate cancer tissues compared with those in adjacent paracancerous tissues. Moreover, upregulation of CNTN-1 expression was significantly associated with a larger tumor size, a more advanced clinical stage and metastasis in patients with prostate cancer. Consistent with the results of the present study, Yan et al (13) reported that the expression level of CNTN-1 was significantly higher in prostate cancer cells from primary tumors, lymph nodes and bone metastases, compared with paracancerous prostate gland tissues. The present study indicated that patients with high-CNTN-1 expression exhibited shorter overall survival times when compared with the low-expression group, suggesting that CNTN-1 expression may be used as a predictive biomarker of prostate cancer. Yan et al also showed that following radical prostatectomy, CNTN1 expression was associated with a shorter recurrence-free survival time in patients with prostate cancer (13). In the current study, transfection with shRNA was used to knockdown the expression of CNTN-1 in prostate cancer cell lines, which resulted in significantly reduced proliferation, colony formation, migration and invasiveness.
This further supports the hypothesis that CNTN-1 promotes the progression of prostate cancer, and suggests that CNTN-1 may represent a promising therapeutic target for the treatment Figure 2. CNTN-1-knockdown inhibits prostate cancer cell proliferation. PC3 and LNCaP prostate cancer cell were transfected with negative control shRNA or CNTN-1 shRNA. Following transfection, (A) reverse transcription-quantitative PCR and (B) western blotting were used to examine the mRNA and protein expression of CNTN-1. A CCK-8 assay was performed to study the proliferation of (C) PC3 and (D) LNCaP cells. The colony formation rate of (E) PC3 and (F) LNCaP was also examined. ** P<0.01 vs. shNC. CNTN-1, contactin-1; shRNA, short hairpin RNA; shNC, short hairpin negative control; OD, optimal density. of the disease. Yan et al (13) used the DU145 cell line to study the function of CNTN-1 in vitro. In the present study, two different cell lines (PC3 and LNCaP) were used, providing further validation of these results (13). Moreover, in the study conducted by Yan et al (13), only CNTN-1 cell invasion was investigated. In the present study wound-healing assays were performed to further elucidate the function of CNTN-1 in prostate cancer cell migration.
EMT is tightly regulated by several internal and external stimuli that orchestrate the transition from an epithelial-like to a mesenchymal phenotype (15)(16)(17). EMT facilitates tumor cell invasiveness and metastatic capacity, and is thus a principal mediator of cancer progression and metastasis (18)(19)(20).
Yan et al (13) only detected the expression of E-cadherin, and thus did not reveal the function of CNTN-1 in EMT in prostate cancer. In the present study, elucidation of the role of CNTN-1 in EMT was a key objective. Thus, the effect of CNTN-1-knockdown significantly increased the expression of E-cadherin, while inhibiting the expression of N-cadherin and vimentin in prostate cancer cells, indicating that EMT was suppressed. The current findings indicated that the CNTN-1-mediated promotion of prostate cancer cell migration and invasion might be the result of EMT regulation.
The PI3K/AKT signaling pathway promotes tumor cell proliferation, migration and invasion, and EMT in multiple human cancers (21)(22)(23)(24). In the present study, it was  cancer (26). Therefore, the interaction between CNTN-1 and the PI3K/AKT signaling pathway may represent a commonality shared by multiple cancer types. Animal experiments may help to further clarify the exact function, and validate the regulatory mechanisms of CNTN-1 in prostate cancer, in vivo.
In summary, the present study demonstrated that CNTN-1 was significantly upregulated in prostate cancer compared with adjacent paracancerous tissues, and that the knockdown of CNTN-1 inhibited proliferation, migration, invasiveness and EMT in prostate cancer cells. Taken together, the results suggest that CNTN-1 may represent a potential therapeutic target for the treatment of prostate cancer.