The FASEB Journal article fj.15-271700. Published online April 15, 2015.

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Simulated microgravity disrupts intestinal homeostasis and increases colitis susceptibility Pingping Li,*,1 Junxiu Shi,*,1 Peng Zhang,†,1 Ke Wang,* Jinglong Li,‡ Hongju Liu,† Yu Zhou,* Xi Xu,* Jie Hao,* Xiuyuan Sun,* Xuewen Pang,* Yan Li,* Hounan Wu,§ Xiaoping Chen,†,2 and Qing Ge*,2 *Key Laboratory of Medical Immunology, Ministry of Health, Department of Immunology, School of Basic Medical Sciences, and §Peking University Medical and Health Analytical Center, Peking University Health Sciences Center, Beijing, P. R. China; †State Key Laboratory of Space Medicine Fundamentals and Application, Chinese Astronaut Research and Training Center, Beijing, P. R. China; and ‡College of Life Sciences and Key Laboratory of Modern Teaching Technology, Shaanxi Normal University, Xian, P.R. China The immune systems can be altered by spaceflight in many aspects, but microgravity-related mucosal immune changes and its clinical significance have not been well studied. The purpose of this study was to investigate whether simulated microgravity influences the intestinal homeostasis and increases the susceptibility to colon inflammation. The hindlimb unloading (HU) mouse model was used to simulate the microgravity condition. Three percent dextran sulfate sodium (DSS) was given to mice to induce colitis. Compared to ground control (Ctrl) mice, the HU ones revealed an impaired intestinal homeostasis and increased susceptibility to DSS-induced colitis. This includes an early-onset, 4-fold expansion of segmented filamentous bacteria (SFB), more than 2-fold decrease in regulatory T cell (Treg) numbers and IL-10 production, ∼2-fold increase in colonic IL-1b expression, 2-fold increase in circulating neutrophils, and colonic neutrophil infiltration. The application of antibiotics ameliorated the Treg and IL-10 reductions but did not significantly dampen neutrophilia and elevated expression of colonic IL-1b. These results indicate that the intestinal microflora and innate immune system both respond to simulated microgravity and together, contribute to the proinflammatory shift in the gut microenvironment. The data also emphasize the necessity for evaluating the susceptibility to inflammatory bowel diseases (IBDs) in distant space travels.—Li, P., Shi, J., Zhang, P., Wang, K., Li, J., Liu, H., Zhou, Y., Xu, X., Hao, J., Sun, X., Pang, X., Li, Y., Wu, H., Chen, X., Ge, Q. Simulated microgravity disrupts intestinal homeostasis and increases colitis susceptibility. FASEB J. 29, 000–000 (2015). www.fasebj.org ABSTRACT

SPACEFLIGHT, IN PARTICULAR MICROGRAVITY, CHANGES the innate and adpative immune system in many aspects (1–3). For example, astronauts returning from spaceflights have neutrophilia and eosinopenia (4–6). The phagocytic capabilities of circulating neutrophils obtained from these postflight astronauts were decreased, whereas the adhesion to endothelial cells was increased (5, 6). Monocytes from the blood also showed altered pro- and antiinflammatory cytokine production, increased TLR4 but decreased CD14 expressions, reduced ability to engulf pathogenic Escherichia coli, and an inability to generate an oxidative burst (4, 7–9). Altered T cell distribution and cytokine production, decreased T cell activation, proliferation, cytotoxic functions, and decreased NK functions were also observed (4, 10, 11). This systemic immune dysfunction under microgravity may render hosts more susceptible to pathogenic infections (4, 12–14). Spaceflight also affects properties of microorganisms. Increased growth rate, lengthened exponential growth phase, increased virulence, and increased resistance to several environmental stressors have been shown in bacteria cultured under microgravity (15–19). Thus, it is expected that mucosal microbiota, coexisting with animals and playing an important role in normal development and health, are also influenced by microgravity (20, 21). However, the initial analysis of a limited number of mice aboard the International Space Station had not confirmed a significant change in the diversity of gut microflora (22). However, it remains to be determined how alterations in the immune system and microbiota during spaceflight affect the complex interactions between mutualistic

Key Words: IL-10 • neutrophil • microbiota 1

These authors contributed equally to this work. Correspondence: Q.G., Dept. of Immunology, School of Basic Medical Sciences, Peking University Health Sciences Center, 38 Xue Yuan Rd., Beijing, 100191, P. R. China. E-mail: [email protected]; X.C., Chinese Astronaut Research and Training Center, Beijing, 100094, P. R. China. E-mail: [email protected] doi: 10.1096/fj.15-271700 This article includes supplemental data. Please visit http:// www.fasebj.org to obtain this information. 2

Abbreviations: APC, allophycocyanin; CD62L, cluster of differentiation 62 ligand; CMF, Ca2+/Mg2+ free; Ct, comparative threshold; Ctrl, control; Ctrl-D, control with 3% dextran sulfate sodium in water; DSS, dextran sulfate sodium; FCS, fetal calf serum; Foxp3, forkhead box p3; H&E, hematoxylin and eosin; HU, hindlimb unloading; HU-D, hindlimb (continued on next page)

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pathogens and their hosts, in particular, the mucosal immune system. With the use of a well-accepted, groundbased spaceflight analog—HU of mice—Zhou et al. (23) recently reported that simulated microgravity led to a transient elevation of circulating LPS and an activation of the innate immune system. Currently, the impact of these changes on the intestine is not known, but the inflammation in the gastrointestinal tract was indeed reported in Apollo crew members (6), and reduced length of villi and depth of crypts were found in rats flown on the COSMOS 2044 mission (24). IBD is a chronic inflammatory disorder of the gastrointestinal tract that jeopardizes a patient’s quality of life and daily performance. IBD patients also have an increased risk of developing other chronic inflammatory disorders, such as psoriasis and primary sclerosing cholangitis (25, 26). Altered host-microbiome interaction with genetic susceptibility plays a large part in IBD pathogenesis (27, 28). This includes enhanced proinflammatory cytokine production by a variety of innate and adaptive immune cells, reduced number/function of Tregs, massive infiltration of neutrophils in the mucosa and epithelial crypts, and shifts in the composition of commensal flora (28–30). Although astronauts must meet high standards of physical health, the dysregulated, systemic immune surveillance, changes in pathogens, and reported case of intestinal inflammation in Apollo crew members all suggest the need to evaluate the risks of IBD in astronauts/animals during spaceflights or simulated microgravity. Thus, a DSS-induced colitis model was used in combination with an HU model to investigate whether simulated microgravity increases the susceptibility to colonic inflammation. The pathology, cellular distribution, cytokine expressions, and microbiota changes in HU mice, with or without DSS treatment, were evaluated. HU of different duration was also examined to look for possible causes of these changes.

Biomedicals, Santa Ana, CA, USA), dissolved in drinking water until the body weight of mice reduced to 70% of initial weight, which lasted 6–7 days.

Study design All mice, according to weight, were divided randomly into 4 groups: Ctrl, HU, Ctrl with 3% DSS in water (Ctrl-D), and HU with 3% DSS in water (HU-D). The mice in HU and HU-D groups were hindlimb unloaded from day 0 until the end of the experiment. On day 7, the mice in Ctrl-D and HU-D groups were given 3% DSS to induce colitis, whereas the mice in the Ctrl and HU groups got plain water. The mice were weighed daily and monitored for the appearance of diarrhea and blood in stool. On day 14, fresh stool from Ctrl and HU mice was collected. The animals were then killed, and arterial blood was withdrawn for cytokine assay and flow cytometry analysis. Under an aseptic technique, a laparotomy was performed through a midline incision, and peritoneal cells and mesenteric lymph nodes (MLNs) were collected. The entire colorectum from the colocaecal junction to the anal verge was also excised and cut for histologic section, RNA isolation, homogenization, and culture. Animals Female C57BL/6 mice, at 8–10 weeks of age, were purchased from Peking University Health Science Center and Vital River Lab Animal Technology Company (Beijing, China). The mice were kept in a specific pathogen-free animal facility at Peking University Health Science Center. The study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the U.S. National Institutes of Health. The experimental procedures on use and care of animals had been approved by the Ethics Committee of Peking University Health Science Center. Histology and immunohistochemistry

MATERIALS AND METHODS HU Individual mice were suspended by the tail at 15 degrees headdown tilt with no load bearing on the hindlimbs (31). Access to food and water was ensured by use of water bottles, gel packs, and food distributed around the floor of the cage. Animals demonstrated no adverse effects or pronounced weight loss. Groups of 3–6 mice/treatment/experiment were used as a result of hindlimb suspension cage limitations, and each experiment was repeated at least 3 times. Induction of colitis Acute colitis was induced by feeding the animals ad libitum with 3% (wt/vol) DSS (molecular weight 36–50 kDa; MP (continued from previous page) unloading with 3% dextran sulfate sodium in water; IBD, inflammatory bowel disease; IL-1ra, IL-1 receptor antagonist; LP, lamina propria; MLN, mesenteric lymph node; MPO, myeloperoxidase; PE, phycoerythrin; PerCP, peridinin chlorophyll protein complex; RPMI, Roswell Park Memorial Institute medium; SFB, segmented filamentous bacteria; TCR, T cell receptor; Treg, regulatory T cell

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Specimens (1 cm long), from the most distal part of the colon or “swiss roll” of the entire colon, were fixed in 4% neutral-buffered formalin, dehydrated, and embedded in paraffin. Serial sections were prepared and stained with hematoxylin and eosin (H&E). Histopathologic scoring was performed in a blinded fashion, as a combined score of inflammatory cell infiltration (score 0–3) and tissue damage (score 0–3). For the evaluation of inflammatory cell infiltration, rare inflammatory cells in the lamina propria (LP) were counted as 0; increased numbers of inflammatory cells, including neutrophils in the LP, as 1; confluence of inflammatory cells and extending into submucosal area as 2; and a score of 3 was given for transmural extension of inflammatory cell infiltration. For the evaluation of epithelial damage, absence of mucosal damage was counted as 0, discrete focal lymphoepithelial lesions were counted as 1, mucosal erosion/ulceration as 2, and a score of 3 was given for extensive mucosal damage and extension through deeper structures of the bowel wall. The localization of neutrophils in the colon of Ctrl and HU mice was analyzed by staining of serial colon sections with myeloperoxidase (MPO)-specific antibody (Dako, Glostrup, Denmark).

Isolation of colonic LP cells Isolation of colonic LP and epithelial cells was performed by following a method established previously with slight modifications

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(32). After washing off fecal contents and mucus, mouse colons were cut into 1.5 cm-long pieces and incubated in Ca2+/Mg2+ free (CMF)-HBSS and 1 mM DTT, twice with 5 mM EDTA, in an orbital shaker at 250 rpm for 20 minutes at 37°C, and the colon pieces were washed thoroughly with CMF-PBS and then digested twice in 0.5 mg/ml collagenase IV (Roche, Basel Schweiz, Switzerland) and 0.2 mg/ml DNase-containing medium with 5% fetal bovine serum in Roswell Park Memorial Institute medium (RPMI) 1640 at 200 rpm for 20 min at 37°C. The digested cell suspension was then washed with CMF-PBS and passed through a 100 mm cell strainer, followed by a 40 mm cell strainer. The supernatants were centrifugated, and the cell pellet was considered as LP cells. RNA isolation and real-time PCR Total RNA was extracted from colon tissues by use of the RNeasy Mini kit (Qiagen, Hilden, Germany). The purity of RNA was verified spectrophotometrically at 260/280 nm. The RNA samples (2 mg) were reversed transcribed into cDNAs by use of the FastQuant RT kit (Tiangen, Beijing, China), according to the manufacturer’s instructions. Quantitative real-time PCR was performed by use of FastStart Universal SYBR Green Master (Roche) on an iCycler real-time PCR system (Bio-Rad, Hemel Hempstead, United Kingdom), with each sample in triplicate. The sequences of primers are listed in Supplemental Table 1. PCRs were performed for 45 cycles with an initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 10 seconds. The quantification was based on DD comparative threshold (Ct) calculations and were normalized to glyceraldehyde 3-phosphate dehydrogenase as loading Ctrls. The DDCt value of each HU mouse was calculated relative to the mean DCt value of Ctrl mice at the same time-point. Colon homogenization Specimens (2 cm long) from the distal part of colon were cut into strips of ;1 mm2 and placed in 2 ml radioimmunoprecipitation assay buffer (Beyotime, Beijing, China) containing 13 protease inhibitor cocktail (Roche) on ice. The samples were gently homogenized for 6 times, 10 seconds for each time, by use of a FastPrep-24 instrument (MP Biomedicals). The homogenate was then centrifuged at 2000 g for 10 minutes at 4°C, and the supernatant was collected and stored at 280°C. Colon tissue culture A segment of colon was cut open longitudinally and washed in PBS containing penicillin and streptomycin. The colon segment was then cut into strips of ;1 mm2 and placed in 24-well, flatbottom plates containing 1 ml of RPMI, supplemented with penicillin and streptomycin. The strips were incubated at 37°C for 24 hours, and the culture supernatants were harvested and assayed for cytokines.

Antibiotic treatment The antibiotic cocktail was given according to the protocol described by Qiu et al. (34). In brief, the mice were supplied with autoclaved water, supplemented with or without antibiotics (1 g/L ampicillin, 1 g/L gentamicin, 1 g/L metronidazole, 1 g/L neomycin, and 0.5 g/L vancomycin) from 3 days before HU to 14 days post-HU initiation. Cell culture Single-cell suspensions from MLNs were prepared by passing cells through a 100 mm strainer. The cells were plated at a density of 1 3 106/ml and were activated by use of 2 mg/ml plate-bound anti-CD3 and 1 mg/ml soluble anti-CD28 antibodies (BD PharMingen, San Diego, CA, USA) in RPMI, supplemented with 10% fetal calf serum (FCS). In the experiments with MLN cells from 3-, 7-, and 14-day HU mice, the cells were stimulated with 0.5 mg/ml anti-CD3 and 0.25 mg/ml anti-CD28. The peritoneal cells were cultured in the presence or absence of 1 mg/ml LPS (SigmaAldrich, St. Louis, MO, USA) in RPMI, supplemented with 10% FCS at a density of 1 3 106/ml. The LP cells were cultured unstimulated or in the presence of 1 mg/ml LPS or activated by use of 0.5 mg/ml plate-bound anti-CD3 and 0.25 mg/ml soluble anti-CD28 antibodies in RPMI, supplemented with 10% FCS at a density of 4 3 105/ml. The cells were cultured at 37°C for 24 hours before supernatant collection. ELISA Supernatants from peritoneal cell cultures, colon homogenization, and colon cultures were collected and analyzed by ELISA for IL-1b, IL-6, and IL-10. The supernatants from lymphocyte cultures were analyzed for IL-17, IFN-g, and IL-10, according to the manufacturer’s instructions. All ELISA kits were purchased from eBioscience (San Diego, CA, USA). Flow cytometry Peripheral blood mononuclear cells and peritoneal cells were stained with Gr1-FITC, B220-phycoerythrin (PE), T cell receptor (TCR)-b-allophycocyanin (APC), Ly6C-peridinin chlorophyll protein complex (PerCP)-Cy5.5, and CD11b-PE-Cy7 at 4°C for 30 minutes and then analyzed by flow cytometry (Gallios; Beckman Coulter, Brea, CA, USA). Cells from MLNs were stained with cluster of differentiation 62 ligand (CD62L)-PE, CD44-APC, CD4-PerCP-Cy5.5, and CD8-PE-Cy7 and then analyzed by flow cytometry. For Treg detection, lymphocytes were incubated with CD4-FITC and CD25-APC at 4°C for 30 minutes, fixed in paraformaldehyde, permeabilized in Perm/Fix solution, and finally stained intracellularly with forkhead box p3 (Foxp3)-PE. The antibodies and fixation/permeabilization agents were purchased from QuantoBio (Beijing, China), BioLegend (San Diego, CA, USA), BD PharMingen, or eBioscience. Statistics

Microbiota analysis Fresh fecal samples were collected at death and frozen at 220°C. Bacterial DNA was extracted by use of a Qiagen stool DNA kit (Qiagen), following the manufacturer’s instructions. DNA was amplified by real-time PCR, as described above, and primers of analyzed bacterial species are described in Supplemental Table 2 (33). The results were presented as percentage expression of each species relative to total bacteria (Eubacteria).

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The data are presented as mean values of 3–6 mice in each group 6 SD. The statistical analysis of the results was performed by use of GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Statistical significance between 2 groups was evaluated by 2tailed unpaired Student’s t test. The difference of mouse survival between Ctrl-D and HU-D groups was calculated by log-rank test. Throughout the text, figures, and figure legends, statistical significance is indicated.

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RESULTS Mice under simulated microgravity had increased susceptibility to DSS-induced colitis To investigate whether a microgravity condition could increase the susceptibility to DSS-induced inflammatory responses in colon, the mice with (HU) or without (Ctrl) 14 day HU were each divided into 2 groups; 1 received plain water (Ctrl and HU), and the other received 3% DSS in water (Ctrl-D and HU-D) for 1 week, starting from 1 week after the initiation of HU. The 14 day HU alone did not result in changes in body weight and colon length compared with the ground Ctrl (Fig. 1A, B). All of the mice that received 1 week of DSS treatment revealed decreased body weight and shortened colon length (Fig. 1A, B). However, the HU-D group showed more weight loss than the Ctrl-D group, starting from 72 hours post-DSS treatment (Fig. 1A). Fifty percent of the HU-D mice died within 6–9 days of DSS treatment, whereas only 7% of the Ctrl-D mice died (Fig. 1C; P = 0.0223). Rectal bleeding was found in all of the HU-D mice compared with only 58% of Ctrl-D mice. These results suggest that the mice under a simulated microgravity condition develop more severe DSS-induced colitis. The colons of all the mice were analyzed further by H&E staining. Significant hyperplasia, inflammatory cell infiltration, and loss of crypts were observed in all of the colon sections obtained from DSS-treated mice (Fig. 1D, E). However, compared with Ctrl-D mice, the pathology of colon samples from HU-D mice revealed more severe tissue damage, as measured by histopathologic scores (Fig. 1E), confirming that mice under microgravity are more susceptible to DSS-induced colitis. Notably, the colon

section of HU mice without DSS treatment also had tissue damage, as immune cell infiltration could be found (Fig. 1D, E), suggesting that microgravity alone may lead to the transmigration of immune cells to the colon. Increased circulating neutrophils and enhanced colonic neutrophil infiltration in HU mice To determine whether increased susceptibility to DSSinduced intestinal inflammation is a result of an altered immune cell distribution under a microgravity condition (1, 9, 35, 36), the changes of immune cells in the peripheral blood were first investigated. Consistent with previous findings during spaceflight or under simulated microgravity (1, 9, 35, 36), the ratio of B cells (B220+) decreased, whereas that of myeloid cells (CD11b+) increased in the peripheral blood of 14-day HU mice compared with Ctrl ones (Fig. 2A). The difference in circulating T cell percentages between these 2 groups did not reach significance. Within myeloid cells, more neutrophils (Gr1+) were found in HU mice than Ctrl ones (Fig. 2A). Likewise, more neutrophils were shown in the blood of HU-D than Ctrl-D mice that were treated with DSS (Fig. 2A). To investigate whether HU-derived neutrophilia occurs before DSS induction at Day 7, the mice that received HU for 3, 7, and 14 days were examined. As shown in Fig. 2B, the increase in circulating neutrophils was found only in 14-day HU mice but not 3 or 7 day ones. Interestingly, the phenotype of these CD11b+ myeloid cells, in particular, Gr1+ neutrophils, revealed a decrease in CD62L and an increase in CD11b fluorescence intensity on 3 days post-HU initiation (Fig. 2C, D). A similar CD62L downregulation was also reported in human peripheral monocytes following short-duration spaceflight (9). As CD62L

Figure 1. HU renders the mice more susceptible to DSS-induced colitis. C57BL/6 mice were divided into 4 groups: 2 HU groups that received 14 days of HU (HU and HU-D) and 2 ground Ctrl groups (Ctrl and Ctrl-D). Each group had 4–5 mice. The HU-D and Ctrl-D groups received 3% (wt/vol) DSS in drinking water, 1 week after the initiation of HU. The body weight of these mice was monitored daily after DSS administration and was shown as a percentage of their own weight on the second day of DSS treatment and 8 days post-HU initiation (A). The statistical significance between HU and Ctrl or HU-D and Ctrl-D at any timepoint was calculated by 2-tailed unpaired Student’s t test with a 95% confidence interval. Significant difference was found between HU-D and Ctrl-D groups. The colon length of each mouse was measured and photographed (B). Each symbol represents 1 mouse. The horizontal bars represent the mean value. The survival of HU-D and Ctrl-D mice was compared, and logrank test was used to calculate the statistical significance (C). H&E staining was performed on the colon section (D), and the histopathologic score (E) was given blindly by a pathologist. The representative sections were shown. Four independent experiments were performed, and similar results were obtained. *P , 0.05; **P , 0.01; ***P , 0.005.

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Figure 2. Neutrophilia and colonic neutrophil infiltration in HU mice. A) Phenotypic analysis of T, B, and myeloid cells in the peripheral blood by flow cytometry. Representative data of Ctrl and HU mice were shown on the left. The mean percentages of circulating T cells (TCR-b+), B cells (B220+), and myeloid cells (CD11b+) are shown in the middle. The average percentage of neutrophils (Gr1+) in myeloid cells is shown on the right. The error bars represent the SD. B) Increased circulating myeloid cells and neutrophils in 14-day HU mice. C57BL/6 mice received HU for 3, 7, and 14 days. Three mice were in each group. The average percentage of myeloid cells and neutrophils in the blood of HU and Ctrl mice at each time-point is compared. C) Early decrease in surface CD62L expression in HU neutrophils. MFI, Mean fluorescence intensity. D) Early up-regulation of CD11b in myeloid cells and neutrophils in HU mice. E) More neutrophils in colonic LP of HU mice. Colonic LP cells were harvested from Ctrl and HU mice at different time-points and stained for CD11b and Gr1. The phenotype of LP cells was shown on the left, and the numbers of Gr1+ LP cells were shown on the right. F) More neutrophil infiltration in the colon tissues of HU mice. The colon section from Ctrl and 14-day HU mice were stained for MPO (red) and DAPI dilactate (blue). The representative sections were shown (3200 magnification). Statistical significance between any 2 groups at a given time-point was calculated by Student’s t test. The experiment was repeated for 2–3 times, and similar results were seen. *P , 0.05; **P , 0.01; ***P , 0.005.

and CD11b are both important molecules regulating leukocyte adhesion to endothelium, these data suggest that under a microgravity condition, some neutrophils may quickly lose secondary lymphoid organ-homing capability and migrate to other tissues, such as the intestinal mucosa. To examine directly whether HU mice have more neutrophils migrating to the gut, myeloid cells from colonic LP were analyzed. As shown in Fig. 2E, significantly more Gr1+ neutrophils were found in 3- and 14-day HU mice than in Ctrl ones. Notably, more than half of these Gr1+ LP cells lost CD11b expression in 14 but not 3 day HU mice, implicating the possibility of a 2-wave neutrophil infiltration in the colon. The MPO staining of colon tissue sections INTESTINAL CHANGES UNDER MICROGRAVITY

further confirmed the colonic neutrophil infiltration in HU mice (Fig. 2F). These results suggest that simulated microgravity not only increases neutrophils in the blood but also promotes the early infiltration of these cells into colonic mucosa. Thus, it may predispose HU mice to neutrophil-mediated epithelial injury and inflammation. Reduced Tregs in the draining lymph nodes and LP of HU mice As T cells, in particular, Tregs play a critical role in the maintenance of gut immune homeostasis and tolerance, 5

the changes of T cells in gut draining lymph nodes (MLNs) and LP were examined. Compared with Ctrl mice, the 14-day HU mice showed significantly reduced numbers of total CD8+ and CD4+ T cells in MLNs (Fig. 3A, B). CD44lo (na¨ıve) and CD44hi (activated/memory) T cells were decreased in HU mice (Fig. 3B). Within the CD4+ T cell population, the percentage of Tregs (CD4+CD25+ Foxp3+) was not different between HU and Ctrl groups, whereas the numbers of Tregs in 14-day HU mice were significantly lower than those in Ctrl ones (Fig. 3A, B). In those mice that received DSS treatment, dramatically reduced T cells, including Tregs, were only found in the MLN of HU-D mice but not in that of Ctrl-D ones (Fig. 3B). Notably, when mice with different HU durations were analyzed, the decrease in MLN Treg numbers in HU mice was not only seen in 14-day but also in 7-day post-HU initiation (Fig. 3C). The change of Tregs was examined further in LP, and significantly less Tregs were again revealed in 7- and 14-day HU mice (Fig. 3D). This indicates that T cell-mediated intestinal homeostasis is altered under microgravity, likely resulting in defective T cell responses to DSS-induced colonic inflammation.

Down-regulated IL-10 and up-regulated IL-1b expressions in the colon of HU mice The balance of pro- and anti-inflammatory cytokines plays a fundamental role in maintaining intestinal homeostasis (29). Thus, we investigated the changes of colonic cytokine milieu under microgravity condition. Compared with ground Ctrls, the 14-day HU mice revealed no significant difference in the protein levels of proinflammatory cytokines IL-1b and IL-6 in colon extracts (Fig. 4A) and colon tissue cultures (Fig. 4B). The productions of IL-1b and IL-6 were induced dramatically, whereas that of IL-10 was suppressed in the colon samples of DSS-treated animals (HU-D and Ctrl-D; Fig. 4A, B). Notably, the level of IL-1b in HU-D mice was significantly higher than that in Ctrl-D ones (Fig. 4A, B), confirming the increased severity of colitis in HU-D mice. We also used quantitative RT-PCR to examine various pro- and anti-inflammatory cytokines at mRNA level. Different from the results at the protein level, a significant upregulation of IL-1b mRNA was seen in the colon of 14-day HU mice without DSS treatment (Fig. 4C and Supplemental Fig. 1A). The mice with 3 or 7 days of HU did not

Figure 3. Decreased Treg numbers in the MLN and colonic LP of HU mice. A) Phenotypic analysis of singlecell suspensions collected from the MLNs. CD44lo and CD44hi cells in CD4+ or CD8+ cells represent na¨ıve and activated/memory T cells, respectively. CD4+CD25+Foxp3+ cells represent Tregs. B) Comparison of the numbers of total T and T cell subsets among Ctrl, HU, Ctrl-D, and HU-D mice at 14 days postHU initiation. Statistical significance between any 2 groups was calculated by Student’s t test. C) Comparison of MLN Treg numbers in Ctrl and 3, 7, and 14 day HU mice. D) Comparison of colonic LP Treg numbers in Ctrl and 3-, 7-, and 14-day HU mice. Data shown are means 6 SD and derived from 3–4 mice in each group. The experiments have been performed for 2–3 times, and similar results were obtained. *P , 0.05; **P , 0.01.

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Figure 4. Increased proinflammatory cytokine IL-1b and decreased anti-inflammatory cytokine IL-10 in the colon tissues of HU mice. A) Comparison of IL-1b and IL-6 production in colon-tissue extracts. ELISA was performed to measure the cytokine levels in colon extracts and was normalized to total protein amount in each sample. B) Comparison of IL-1b, IL-6, and IL-10 levels in the colon-tissue culture. C) Changes in pro- and anti-inflammatory cytokine expression in the colons of 3-, 7-, and 14-day HU mice. Total RNA was extracted from colon tissues, and quantitative RT-PCR was performed. D) Comparison of IL-1b and IL-10 expression in colonic LP cells, with or without LPS (1 mg/ml) stimulation. E) Comparison of IL-17A and IL-10 transcription in activated LP T cells (0.5 mg/ml anti-CD3 and 0.25 mg/ml anti-CD28). Data shown represent mean values 6 SD and are derived from 3 mice/group, each analyzed in duplicate. The experiment was repeated 3 times, and similar results were obtained. The statistical significance between any 2 groups at a given time-point was calculated by Student’s t test. *P , 0.05; **P , 0.01; ***P , 0.005.

show IL-1b up-regulation. The administration of DSS further enhanced IL-1b expression, resulting in significantly more IL-1b in the colon of HU-D than Ctrl-D mice (Supplemental Fig. 1A). Except for a mild increase in colonic CCL2 mRNA in 14 day HU mice, the mRNA levels of other proinflammatory cytokines, including IL-17A, IL-18, IL-22, IL-23, IL-6, and CXCL1, were not significantly different between HU and Ctrl mice (Fig. 4C, Supplemental Fig. 1A, B, and data not shown). The levels of anti-inflammatory cytokines were also measured, and a significant downregulation of colonic IL-10 mRNAs was found in 7- and 14day HU mice (Fig. 4C). The expression of IL-1 receptor antagonist (IL-1ra), which limits the signaling of proinflammatory IL-1, was also reduced significantly in 14-day HU mice. No overt difference in TGF-b was revealed INTESTINAL CHANGES UNDER MICROGRAVITY

between HU and Ctrl (Fig. 4C). Together, the upregulation of IL-1b and down-regulation of IL-10 and IL1ra suggest a proinflammatory shift in the colon of HU mice, likely promoting the exacerbation of DSS-induced colitis in HU mice. The cytokine expression was examined further in LP cells. As shown in Fig. 4D, significantly increased IL-1b mRNA was found in unstimulated or LPS-stimulated LP cells at 14 days post-HU initiation. A mild up-regulation of this cytokine was also found in LP cells collected from 7-day HU mice (Fig. 4E). The expressions of IL-23 and IL-22 were similar between HU and Ctrl groups (data not shown), whereas that of IL-10 was reduced in LP cells from 14-day HU mice (Fig. 4D). Consistently, a significant IL-10 protein reduction was also found in LPS-stimulated peritoneal cells 7

harvested from HU mice, and a more profound suppression of IL-10 in peritoneal cells was observed in HU mice treated with DSS (HU-D) compared with the Ctrls (Ctrl-D; Supplemental Fig.1C). Thus, simulated microgravity affects the functions of LP cells, likely myeloid cells, and results in the imbalance of pro- and anti-inflammatory cytokine expression. Activated T cells (anti-CD3 and anti-CD28 stimulation for 24 hours) from MLN and LP were also examined for their cytokine production. As shown in Supplemental Fig. 2A, B, MLN T cells from 14-day HU and Ctrl mice secreted similar levels of IFN-g and IL-17A, whereas those from DSStreated HU mice (HU-D) produced slightly less IFN-g and more IL-17A than Ctrls (Ctrl-D). A trend of IL-17A upregulation was seen in activated LP T cells from 14-day HU mice (Fig. 4E). Consistent with Treg reduction in MLN and LP of HU mice, a significant down-regulation of IL-10 was revealed in activated MLN and LP T cells in HU mice (Fig. 4E and Supplemental Fig. 2B). Thus, the reduction of Treg numbers and T cell-derived IL-10 production under simulated microgravity may disrupt T cell-mediated intestinal homeostasis and lead to the expression of proinflammatory cytokines by other immune cells in colonic mucosa. Altered intestinal microbiota in HU mice It has been well accepted that commensal bacteria actively modulate the immune system. Altered patterns of gutassociated microbial communities are closely associated with various types of colitis (37, 38). Thus, we investigated whether intestinal microbiota is altered under simulated microgravity and whether the dysbiosis subsequently affects colonic immunity. Several of the most common bacterial groups comprising the microbiota were analyzed by real-time PCR (33). Compared with Ctrl mice, a shift in gut-associated microflora composition, with a significant elevation in the SFB group, could be seen in the feces of HU mice as early as 3 days post-HU initiation (Fig. 5A, B), suggesting that intestinal microflora may respond to microgravity rapidly, days before the occurrence of most of the immune changes. To determine whether the alteration in commensal microbiota contributes to the phenotype found in HU mice, antibiotics were applied to the animals before and during the 14-day HU. As shown in Fig. 5, HU mice, with and without antibiotics, revealed similar neutrophilia in the blood (Fig. 5C). Compared with the groups without antibiotics, the ones with antibiotics showed much smaller difference in MLN Treg numbers between HU and Ctrl mice (Fig. 5D). The colonic IL-10 reduction in HU mice almost disappeared in antibiotic-treated ones (Fig. 5E). The trend of colonic IL-1b up-regulation in HU mice was not changed by antibiotics, although the difference between HU and Ctrl mice with antibiotics was smaller than that without antibiotics (Fig. 5E). These results suggest that HU-induced intestinal microbiota changes may be responsible for the declined Treg numbers and IL-10 production in the HU colon, but the increased neutrophil infiltration and IL-1b may be caused more directly by HU or/and indirectly by impaired intestinal homeostasis. 8

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DISCUSSION Intestinal homeostasis is maintained by complex and dynamic cross-regulation of commensal microbiota and a mucosal immune system, within which commensal bacteria contribute to immune system development, Tregs and Th17 differentiation, pro- and anti-inflammatory cytokine balance, and intestinal barrier functions, whereas the effective mucosal immune system contains these mutualistic pathogens and responds rapidly to alterations in microbiota (39). A disruption of intestinal homeostasis, including dysbiosis and dysfunction in the mucosal immune system, is closely associated with IBD and some extraintestinal autoimmune diseases. Whether the intestinal homeostasis of human and animals could be affected by space travel is not well examined. The present study applied a simulated weightlessness mouse model of HU to investigate the changes in the mucosal immune system and to test whether these alterations could increase the susceptibility to DSS-induced acute colitis. The results revealed a disruption of intestinal homeostasis in HU mice, characterized by an expansion of SFB, decreased intestinal production of anti-inflammatory cytokine IL-10, increased expression of proinflammatory cytokine IL-1b, declined Treg numbers in intestinal lymphoid tissues, and augmented colonic neutrophil infiltration. These changes predispose the animals to a low level of intestinal inflammation, which is exacerbated in DSS-induced colitis. Spaceflight has been shown to increase the growth rate and virulence of multiple types of bacteria, but whether it affects commensal microbiota, a crucial arm of intestinal homeostasis, has not been thoroughly investigated (22). We present here that simulated microgravity could change the diversity of commensal bacteria within just 3 days, resulting in a dominant expansion of SFB. As SFBcontaining colonic cytokines, including IL-23, IL-22, and IL-10 (40–42), showed no significant changes in mice at 3 days post-HU initiation, it is possible that SFB colonization is induced directly by HU and is promoted further by a relatively late-occurring IL-10 reduction at 7 days postHU. SFB has been shown to facilitate Th17 differentiation (39). In the current study, the outgrowth of SFB in HU mice was not accompanied by the elevation of IL-17 in T cells from MLN and colonic LP, supporting others’ findings that SFB dysregulation may have more impact on small intestinal Th17, as well as that SFB alone may not be sufficient in mediating Th17 development (42–44). However, the expansion of SFB at the expense of other commensal bacteria substantially altered the composition of intestinal microflora and likely commensal components and metabolites, which may affect Treg development and immune cell-derived IL-10 production (39, 45). Thus, our finding of the relatively later occurrence of Treg/IL-10 reduction (7 days post-HU) than of microbiota changes (3 days post-HU) and the dampened Treg/IL-10 reduction upon antibiotic treatment indicates that dysbiosis contributes significantly to the decrease in anti-inflammatory IL10 and Tregs (45). However, a direct microgravity-induced Treg reduction cannot be excluded completely, as a mild decrease in MLN Tregs can still be seen in antibiotic-treated HU mice. Gut microbiota is also one of the main factors triggering the activation of the host’s innate and adaptive immune

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Figure 5. Early expansion of SFB and the resultant Treg and IL-10 reduction in HU mice. A) Changes in overall commensal baceteria in the feces of 3-, 7-, and 14-day HU mice. Bact, Bacterioides sp.; C. perf, Clostridium perfringens; Erec, Eubacterium rectale/ Clostridium coccoides; Helicobacter, Helicobacter pylori; Lac, Lactobacillus sp.; MIB, mouse intestinal Bacterioids. B) Specific comparison of the levels of SFB (left), Clostridium leptum (middle), and C. coccoides (right) in the feces of Ctrl and HU mice at 3, 7, and 14 days post-HU initiation. C) The increase in circulating neutrophils in 14-day HU mice was not altered by the administration of antibiotics (Abx). D) Antibiotics treatment ameliorated the reduction of MLN Tregs in HU mice. E) The application of antibiotics in HU mice ameliorated colonic IL-10 reduction but did not alter IL-1b up-regulation significantly. *P , 0.05; **P , 0.01; ***P , 0.005.

responses in the gut and beyond (46). Thus, it likely contributes to the proinflammatory shift in the HU intestine, which is characterized by colonic IL-1b induction, IL-10, and IL-1ra suppression and neutrophil infiltration at 14 days post-HU initiation. Among them, IL-10 is one of the most important anti-inflammatory cytokines and has a broad effect on immunomodulation, tolerance, and gut defense. Impaired IL-10 signaling in macrophages and defects in IL-10 expression are closely associated with early and aggressive IBD (47, 48). In an HU mouse model, we found that myeloid cell-derived and T cell-derived IL-10 productions were decreased significantly. As a similar downregulation of monocyte-derived IL-10 was seen in Space Shuttle crewmembers during and after a short-duration spaceflight (9, 10), this indicates that IL-10 reduction in mucosal and circulating immune cells is a common feature during spaceflight and under simulated microgravity, which likely results in the disturbance of intestinal homeostasis and enhanced susceptibility to colonic inflammation. Our antibiotic results further suggest that microgravity-induced intestinal microbiota changes may be responsible for this IL-10 reduction. The proinflammatory cytokine IL-1 plays pivotal roles at multiple levels in regulating inflammatory responses to INTESTINAL CHANGES UNDER MICROGRAVITY

infections and sterile insults (49), resulting in direct tissue destruction, induction of secondary inflammatory cytokines and chemokines, infiltration, and transmigration of neutrophils across the mucosa. An increase in IL-1b was found readily in human and animals under microgravity, although the clinical significance remains unclear (10). For instance, IL-1 up-regulation was observed in mice that had been exposed to the space environment for 91 days (50) and in humans during head-down bed rest (a microgravity analog) studies (36, 51, 52). Our data that use an HU mouse model specifically revealed an elevated IL-1b mRNA level in colonic LP cells in HU mice. The initial sensing of the threat for IL-1b transcription and release in the HU model or even in spaceflight is not known. The administration of antibiotics in HU mice did not significantly ameliorate colonic IL-1b up-regulation, suggesting that dysbiosis may not be the only factor in HU-induced IL-1b transcription. Whether microgravity itself or microgravity-induced changes in body fluid and immune cell distribution contribute to the induction of IL-1b awaits further investigation. Neutrophilia has been found in astronauts returning from spaceflights (4–6), but the reason for increased circulating neutrophils, the migration of these cells, and the 9

clinical significance of neutrophilia was not clear. Our simulated microgravity model revealed increased migration of neutrophils into the colon tissue. This colonic neutrophil infiltration may work together with elevated IL-1b to perpetuate the inflammatory environment and subsequently increase the susceptibility to DSS-induced colitis (53). Intestinal dysbiosis is one of the main factors activating the host’s immune responses in the gut and recruiting inflammatory cells, including neutrophils. However, the administration of antibiotics did not ameliorate neutrophilia and colonic neutrophil infiltration. Together with the finding of early changes of CD62L and CD11b expression on circulating neutrophils and an early increase of neutrophils in colonic LP, it is possible that microgravity and its related body-fluid redistribution contribute significantly to the early infiltration of neutrophils in the colon. Taken together, our results reveal that HU of the mouse, an analog of spaceflight, results in altered intestinal microflora composition, imbalance of pro- and anti-inflammatory factors, increased colonic neutrophil infiltration, and increased susceptibility to colon inflammation. As microbiota and the mucosal immune system are highly correlated, and their response individually to microgravity is unknown, it is currently difficult for us to distinguish strictly which factor is influenced directly by microgravity and which is affected indirectly. In addition, it is not yet known whether these factors in humans can be affected during a short- or long-duration spaceflight. However, the current findings emphasize the necessity for a more comprehensive understanding of microgravity’s impacts on the intestinal homeostasis and its association with intestinal and generalized inflammation. It also urges the in-depth evaluation of the clinical significance of these changes in the gut during long-duration spaceflight and the development of countermeasures to improve the mucosal immune functions in astronauts. In addition, these findings may shed light on a better understanding of the complex regulation of intestinal homeostasis and the progression of intestinal inflammation in general. The authors thank Professors Xiaohuan Guo (Qinghua University), Zhihua Liu (Institute of Biophysics, Chinese Academy of Sciences), Yu Zhang, Wenling Han, and Lu Wang (Peking University Health Science Center) for helpful discussions. This work was supported by grants from the Natural Basic Research Program of China (2011CB711000 and 2010CB945300; to Q.G.), National Natural Science Foundation of China (31270935 and 81471525, to Q.G.; 31171144 and 81272177, to X.C.), Beijing Natural Science Foundation (5152010; to Q.G.), and State Key Laboratory Grant of Space Medicine Fundamentals and Application (SMFA10A01; to X.C.). The authors declare no conflicts of interest.

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