Lyn deficiency leads to increased microbiota-dependent intestinal inflammation and susceptibility to enteric pathogens.

The Journal of Immunology

Lyn Deficiency Leads to Increased Microbiota-Dependent Intestinal Inflammation and Susceptibility to Enteric Pathogens Morgan E. Roberts,*,1 Jennifer L. Bishop,*,1 Xueling Fan,* Jennifer L. Beer,* Winnie W. S. Kum,*,† Danielle L. Krebs,* Morris Huang,* Navkiran Gill,*,† John J. Priatel,‡ B. Brett Finlay,*,† and Kenneth W. Harder* The Lyn tyrosine kinase governs the development and function of various immune cells, and its dysregulation has been linked to malignancy and autoimmunity. Using models of chemically induced colitis and enteric infection, we show that Lyn plays a critical role in regulating the intestinal microbiota and inflammatory responses as well as protection from enteric pathogens. Lyn2/2 mice were highly susceptible to dextran sulfate sodium (DSS) colitis, characterized by significant wasting, rectal bleeding, colonic pathology, and enhanced barrier permeability. Increased DSS susceptibility in Lyn2/2 mice required the presence of T but not B cells and correlated with dysbiosis and increased IFN-g+ and/or IL-17+ colonic T cells. This dysbiosis was characterized by an expansion of segmented filamentous bacteria, associated with altered intestinal production of IL-22 and IgA, and was transmissible to wild-type mice, resulting in increased susceptibility to DSS. Lyn deficiency also resulted in an inability to control infection by the enteric pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Lyn2/2 mice exhibited profound cecal inflammation, bacterial dissemination, and morbidity following S. Typhimurium challenge and greater colonic inflammation throughout the course of C. rodentium infection. These results identify Lyn as a key regulator of the mucosal immune system, governing pathophysiology in multiple models of intestinal disease. The Journal of Immunology, 2014, 193: 5249–5263.

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he human intestinal tract houses up to 100 trillion microorganisms that exist as complex communities collectively termed the commensal microbiota (1). These microbes provide many benefits to their host, including aiding in digestion, protection from colonization by pathogens, as well as the appropriate development of the immune system (2). However, the close proximity of potentially pathogenic microorganisms with *Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada; †Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada; and ‡Child and Family Research Institute, Vancouver, British Columbia V5Z 4H4, Canada 1

M.E.R. and J.L. Bishop contributed equally to this work.

Received for publication October 21, 2013. Accepted for publication September 4, 2014. This work was funded by grants from the Canadian Institutes of Health Research (to K.W.H. and B.B.F.) and the Canadian Breast Cancer Foundation (to K.W.H. and D.L.K.). K.W.H. holds a Tier II Canada Research Chair. Studentship and fellowship support was provided by the Canadian Institutes of Health Research Transplantation Training Program (to M.E.R.), the Canadian Association of Gastroenterology, the Crohn’s and Colitis Foundation Canada, and the Canadian Institutes of Health Research (to J.L. Bishop), the Canadian Institutes of Health Research (to M.E.R. and N.G.), and the Michael Smith Foundation for Health Research (to N.G.). B.B.F. is a Canadian Institutes of Health Research Distinguished Investigator, a Howard Hughes Medical Institute International Research Scholar, and a University of British Columbia Peter Wall Distinguished Professor. Address correspondence and reprint requests to Dr. Kenneth W. Harder, University of British Columbia, Life Sciences Centre, 2350 Health Sciences Mall, Vancouver, BC V6T 1Z3, Canada. E-mail address: [email protected] The online version of this article contains supplemental material. Abbreviations used in this article: ABS, adult bovine serum; BM, bone marrow; BoyJ mice, B6.SJL-Ptprca Pepcb/BoyJ mice; DC, dendritic cell; DSS, dextran sulfate sodium; IBD, inflammatory bowel disease; ILC, innate lymphoid cell; MTPBS, mouse tonicity PBS; PRR, pattern recognition receptor; Rorc, retinoic acid–related orphan receptor C; SFB, segmented filamentous bacteria; SFK, Src family kinase; S. Typhimurium, Salmonella enterica serovar Typhimurium; wt, wild-type. Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302832

the internal environment puts the host at risk for serious infection. The gut, similar to other mucosal sites, is therefore reinforced by an elaborate immune system charged with the unique task of containing and tolerating the microbiota while maintaining an ability to recognize and defend against enteric pathogens. This immune system is continuously exposed to microbial products from the luminal flora, which triggers innate responses via pattern recognition receptors (PRRs) (3, 4). These interactions play a major role in regulating the intestinal inflammatory environment, which in turn affects the composition of the microbiota (5). This can have profound implications for the outcome of intestinal inflammation. For example, mice deficient in NLRP6, ASC, IL18, or Caspase-1 possess distinct microbiotas associated with exacerbated colitis induced by the chemical irritant dextran sodium sulfate (DSS) (6). The colitogenic effects of dysbiosis are transferable, as cohousing or cross-fostering of mutant mice with wild-type (wt) mice results in enhanced susceptibility of wt mice to DSS (6). This phenomenon extends to susceptibility to infection by enteric pathogens such as Citrobacter rodentium, in that transfer of microbiota from resistant strains of mice into susceptible hosts can both increase resistance to infection as well as decrease inflammation-induced damage (7, 8). The mechanisms that affect the dynamics between the host immune system and commensal microbiota are still largely unclear, as is the impact of perturbations of these interactions on the outcome of inflammation and susceptibility to enteric infection. An important regulator of immune homeostasis and PRRinduced responses is the Lyn tyrosine kinase. Lyn is a member of the Src family of nonreceptor tyrosine kinases expressed throughout the hematopoietic system with the exception of T cells. The kinase is activated upon ligand binding to a wide variety of cell surface receptors that are essential for initiating or limiting immune responses (9, 10). Studies of Lyn gain-of-function (Lynup/up) and

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Lyn PROTECTS FROM COLITIS AND ENTERIC INFECTION

Lyn-deficient (Lyn2/2) mice have shown that Lyn is an important regulator of B cell responses (11–14), proliferation and degranulation of mast cells (15, 16), integrin signaling in neutrophils (17), and M2 macrophage polarization (18), and that Lyn is a critical regulator of dendritic cell (DC) function and NK cell activation following TLR stimulation (19, 20). Furthermore, we have recently shown that Lyn activation promotes IL-22 production and protects mice from DSS colitis, phenotypes associated with enhanced TLR-dependent DC activation of group 3 innate lymphocytes (ILCs) (21). In this study we show that Lyn deficiency results in increased susceptibility to intestinal damage–induced inflammation and enteric infection. Lyn2/2 mice exhibited enhanced susceptibility to DSS-induced colitis, increased colonic pathology following C. rodentium infection, and were highly susceptible to bacterial colonization and inflammation in the gut during gastroenteritis and typhoid models of salmonellosis. Susceptibility to DSS in Lyn2/2 mice was associated with exaggerated production of IL-17 and IFN-g by colonic T cells and was dependent on the presence of the adaptive immune system excluding CD20+ B cells. The development of dysbiosis in Lyn2/2 mice, which included the expansion of segmented filamentous bacteria (SFB), was associated with altered IL-22 and IgA production and susceptibility to DSS colitis that was transferable to wt mice via cohousing. Taken together, these results demonstrate that Lyn plays an important role in controlling intestinal inflammatory responses by regulating both the nature of the immune response and the composition of the microbiota.

Bacterial infections

Materials and Methods Animals Experiments were performed with age- (8–14 wk) and sex-matched C57BL/6 and C57BL/6 background Lyn2/2 mice (.10 generations). Mice were bred in-house and housed in specific pathogen-free microisolator cages and fed autoclaved food and reverse osmosis water. Experiments were conducted in accordance with the guidelines set by the University of British Columbia Animal Care Committee and the Canadian Council of Animal Care. Lyn2/2 mice have been previously described (12, 22) and were crossed with Rag12/2 (C57BL/6 background) mice to generate Rag2/2Lyn2/2 mice.

DSS colitis Unless otherwise indicated, mice were treated with 2.5% (w/v) of DSS (36,000–50,000 Da [MP Biomedicals] or 40,000–50,000 Da [Affymetrix], with both inducing similar disease) in drinking water for the indicated time. Body weight and rectal bleeding were monitored daily. Rectal bleeding was scored as follows: 0, no blood, visible or occult; 1, occult blood detected; 2, mild rectal bleeding; 3, moderate rectal bleeding; 4, severe rectal bleeding. Intestinal pathology was scored as follows: 1, mild mucosal; 2, mild multifocal; 3, moderate multifocal; 4, severe multifocal. Detailed scoring criteria are available upon request. For cohousing experiments, female mice were cohoused for 4 wk prior to DSS treatment.

Animal rederivation, chimerism, and cell depletions For rederivation (performed by the Centre for Disease Modeling Transgenic Core Facility, University of British Columbia), Lyn2/2 embryos were harvested from pregnant females 2.5 d postcoitum and were surgically implanted in specific pathogen-free pseudo-pregnant CD1 foster mothers. Pups were born naturally and were kept with foster mothers until time of weaning. For bone marrow (BM) chimeric mice, B6.SJL-Ptprca Pepcb/BoyJ (BoyJ) recipient mice were subjected to lethal irradiation (650 rads, two doses, 4 h apart) and 1 d later were injected i.v. with 5 3 106 C57BL/6 or Lyn2/2 BM cells. Mice were allowed to reconstitute for at least 14 wk before DSS treatment. For B cell depletion experiments, mice were treated with 100 mg i.v. of anti-CD20 mAbs (clone 5D2, isotype IgG2a, Genentech) or an isotype control (anti-human OKT3, IgG2a, made in-house) on days 24 and 22 prior to DSS treatment. B cell depletion was confirmed by flow cytometry at the end of the experiment (day 7).

Bacterial preparation. Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SL1344 and C. rodentium strain DBS100 were grown overnight in 3 and 5 ml Luria broth, respectively, at 37˚C with shaking (200 rpm). Bacteria were then washed and resuspended in HEPES buffer for infection by oral gavage. Salmonella-induced gastroenteritis. Twenty-four hours prior to infection, mice were treated orally with 20 mg streptomycin sulfate (Sigma-Aldrich) diluted in HEPES buffer followed by oral infection with 3 3 106 S. Typhimurium SL1344. Typhoid model. Mice were infected orally with 1 3 106 S. Typhimurium strain SL1344. C. rodentium. Mice were infected orally with 1 3 108 C. rodentium DBS100.

Histology and pathological scoring Cross-sections of distal colon were fixed in 10% neutral buffered formalin, paraffin embedded, and stained with H&E. An Olympus BX61 microscope was used to capture bright-field microscopy images, using 34 and 310 apochromatic objective lenses, and an Olympus color CCD camera. All images were acquired and processed using the cellSens Dimension (Olympus) and Picasa (Google) software, respectively. Pathological scores were determined for S. Typhimurium– and C. rodentium–induced pathology as previously described (23, 24).

Western blot analysis Colon sections (0.75 cm) were harvested and homogenized in RIPA buffer plus 1 mM sodium orthovanadate, 10 mM sodium fluoride, and a complete protease inhibitor mixture (Roche, Mississauga, ON, Canada) using a gentleMACS tissue dissociator. Western blotting was performed as previously described using Abs against Lyn (made in-house), phospho-Lyn (Y507), phospho–Src family (Y418), and b-actin (Cell Signaling Technology) (21).

Intestinal permeability assay (FITC-dextran) Four hours prior to sacrifice, DSS-treated mice were orally gavaged with 200 ml 400 ng/ml FITC-dextran (Sigma-Aldrich; FD4) in mouse tonicity PBS (MTPBS). Mice were euthanized and blood was collected by cardiac puncture and immediately added to 50 ml 3% acid citrate dextrose (20 mM citric acid, 100 nM sodium citrate, 5 mM dextrose). Serum was collected and fluorescence was quantified at excitation 485 nm, emission 530 nm for 0.1 s (Wallac Victor; PerkinElmer Life Sciences).

Cytokine analysis from colon explant cultures Freshly harvested colons were opened longitudinally and washed thoroughly of fecal matter in RPMI 1640 (Lonza). Sections of distal colon (0.5 cm) were cultured for 24 h at 37˚C and 5% CO2 in RPMI 1640 supplemented with penicillin (100 U/ml, Life Technologies), streptomycin (100 mg/ml, Sigma-Aldrich), and gentamicin (100 mg/ml, Sigma-Aldrich). Supernatants were removed and cleared of debris by centrifugation. Cytokine levels were assessed by ELISA (eBioscience) or Luminex protein assay (Life Technologies) according to the manufacturers’ instructions.

Cytokine and transcription factor quantitative PCR Distal colon tissue (0.5 cm) from freshly harvested colons was stored in RNA later at 280˚C. Tissue was homogenized in buffer RLT (Qiagen) using a gentleMACS tissue dissociator (Miltenyi Biotec). RNA was isolated using an RNeasy kit (Qiagen) according to the manufacturer’s instructions, and genomic DNA was removed by treatment with DNAse I. Alternatively, RNA was isolated using TRIzol reagent (Sigma-Aldrich) followed by oligo(dT) purification of mRNA as previously described (25). Synthesis of cDNA was performed using an iScript cDNA synthesis kit. Quantitative real-time PCR was done using iQ SYBR Green Supermix or SsoFast EvaGreen Supermix (Bio-Rad) and the Bio-Rad CFX96 realtime system. Primer sequences were previously described (21).

Bacterial quantitative PCR Fecal pellets were isolated from mice, homogenized using a mixer mill (Retsch), and total DNA was isolated from fecal samples using the QIAamp DNA stool kit (Qiagen) according to the manufacturer’s instructions. Quantitative PCR was performed on a 7500 fast real-time system (Applied Biosystems) using QuantiTect SYBR Green Master Mix (Qiagen). Alternatively, fecal DNA was isolated by two sequential phenol-chloroform extractions, and quantitative PCR was done using SsoFast EvaGreen

The Journal of Immunology Supermix and the Bio-Rad CFX96 real-time system. In all figures, groupspecific bacterial abundance was determined relative to eubacteria and is expressed as a relative abundance. Primer sequences are available upon request.

IgA quantification IgA concentrations were measured in serum and feces by ELISA (Affymetrix/eBioscience) according to the manufacturer’s instructions. Fecal protein suspensions were obtained by resuspending fecal pellets, previously frozen at 280˚C, in 10 ml/mg feces of MTPBS plus 0.1 mg/ml soybean trypsin inhibitor (type II-S, Sigma-Aldrich) plus 13 protease inhibitor mixture (Roche). Samples were then spun at 16,000 relative centrifugal force for 15 min to remove debris.

Isolation of colonic lamina propria leukocytes Freshly isolated colons were opened longitudinally, washed thoroughly in MTPBS plus 5% heat-inactivated adult bovine serum (ABS), and cut into 0.5 cm pieces. The epithelium was removed by three consecutive washes with 37˚C MTPBS plus 5% ABS and 2 mM EDTA with rocking for 30 min. Tissue was washed in RPMI 1640 plus 5% ABS, then minced and digested in RPMI 1640 plus 5% ABS and 580 U/ml collagenase type VIII for 30 min at 37˚C with shaking (200 rpm). Following digestion, supernatant was strained through a 70-mm cell strainer and remaining tissue was collected and redigested under the same conditions. Material was strained again and cells from both digests were combined. Cells were washed in RPMI 1640 plus 5% ABS, and colonic lamina propria leukocytes were prepared for flow cytometric analysis.

Flow cytometry Cells were resuspended in FACS buffer (MTPBS plus 5% ABS, 0.05% NaN3 and 2.5 mM EDTA) and incubated with 2.4G2 mAb (Fc block) and then stained with fluorochrome-conjugated Abs. Dead cells were excluded by propidium iodide uptake or Fixable Viability Dye (eBioscience), and red cells were excluded by size or by gating on CD45+ cells. For intracellular cytokine staining, 5 3 106 cells (splenocytes) or the colonic lamina propria cells from half a colon per well in a 24-well plate were stimulated for 4 h in RPMI 1640 plus 10% heat-inactivated FBS, penicillin G (100 U/ml), streptomycin (100 mg/ml), GlutaMAX (2 mM), 2-ME (10 mm), 13 nonessential amino acids, 20 mM HEPES, and 1 mM sodium pyruvate plus brefeldin A (10 mg/ml) with or without PMA (50 ng/ml) and ionomycin (750 ng/ml) at 37˚C with 5% CO2. After washing in FACS buffer, cells were stained for surface markers as described above, fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin in FACS buffer, and stained with anti-cytokine Abs. Cells were analyzed using an LSR II flow cytometer and FACSDiva software (BD Biosciences). Flow cytometry data were analyzed using FlowJo analysis software (Tree Star). Abs were purchased from eBioscience, BD Biosciences, and the Biomedical Research Centre (Vancouver, BC, Canada).

Statistical analysis Survival data from in vivo experiments were analyzed by a log-rank test performed on curves generated by GraphPad Prism 4.0 (Software MacKiev). For all other analysis, two-tailed, unpaired Student t tests with a 95% confidence interval performed on graphs generated in GraphPad Prism were used. Error bars represent the SEM. A p value ,0.05 was considered statistically significant.

Results Lyn deficiency results in increased susceptibility to experimental colitis We recently identified a protective role for Lyn in intestinal inflammation, showing that hypersensitivity to PRR ligands in Lyn gain-of-function mice led to increased protective responses during DSS treatment, a phenotype that was independent of the adaptive immune system (21). In the present study, we used both enteric infection and intestinal damage models of disease to better understand the role of Lyn in regulating intestinal inflammation and the mucosal immune system. Lyn2/2 and wt mice were challenged with increasing concentrations of DSS for 7 d followed by recovery, and morbidity (mice were euthanized after $20% body weight loss) was assessed throughout the experiment. At intermediate (2.5%) and high (5%) doses of DSS, survival was sig-

5251 nificantly reduced in Lyn2/2 mice. A dose of 5% DSS caused 100% morbidity of both wt and Lyn2/2 mice, with wt mice surviving a maximum of 8 d and Lyn2/2 mice a maximum of 6 d. Decreasing the dose to 2.5% extended survival of wt mice to a maximum of 11 d, whereas Lyn2/2 mice survived only until day 9. At a dose of 1.5% DSS, 100% of Lyn2/2 and wt mice survived the entire treatment period (Fig. 1A). Colon length was measured for each mouse at the experimental endpoint ($20% weight loss or 14 d). Lyn2/2 colons were shorter than those of wt mice after 1.5% DSS treatment and significantly shorter following 2.5 and 5% DSS treatment, and there was no significant difference in colon length of untreated mice (Fig. 1B). Rectal bleeding scores in Lyn2/2 mice also indicated increased susceptibility to disease and increased with DSS dose (Fig. 1C). Colonic changes at early time points during DSS challenge, as well as altered cytokine production, correlate with increased susceptibility to disease in Lyn2/2 mice To investigate changes in the colon throughout DSS treatment, we first sought to investigate whether Src family kinases (SFKs), including Lyn, were activated during DSS treatment. Western blot analysis revealed an increase in active SFKs in the colons of DSStreated mice compared with untreated controls, based on an increase in the phosphorylation of the activating tyrosine residue (Y418 for Src and Y396 for Lyn). An increase in Lyn activity was supported by a modest decrease in the phosphorylation of the inhibitory tyrosine (Y507) (Fig. 1D). We then challenged wt and Lyn2/2 mice with 2.5% DSS for 2, 4, or 7 d and monitored disease progression. Lyn2/2 mice lost significantly more body weight than did wt controls on days 4 and 7 of treatment (Fig. 2A). Additionally, as early as day 2 after DSS treatment, Lyn2/2 mice had significantly shorter colons than did wt mice, with colon length in both strains decreasing progressively during treatment (Fig. 2B). Shorter colons in Lyn2/2 mice correlated with modestly elevated pathology scores at days 2 and 4, and significantly higher scores at day 7 of DSS treatment, compared with wt mice (Fig. 2C). Assessment of epithelial integrity during DSS treatment by measuring FITC-dextran diffusion into the bloodstream showed that permeability of the intestinal epithelium was significantly greater in Lyn2/2 mice at days 2 and 4 after DSS treatment (Fig. 2D). This increase in barrier permeability was associated with increased cellular proliferation in Lyn2/2 colons. After 2 d of DSS treatment, Ki67+ cells were observed significantly farther up the colonic crypts compared with those of wt controls (Fig. 2E). Furthermore, there were significantly increased numbers of Ki67+ cells per crypt in Lyn2/2 mice on day 7 after DSS treatment (Fig. 2F, 2H). Consistent with increased numbers of proliferating epithelial cells, crypt hyperplasia was a feature of Lyn2/2 colons after DSS treatment, with the greatest differences in crypt length observed at day 7 (Fig. 2G). Representative histological sections of colons taken at each time point are shown in Fig. 2H and indicate increased pathology, including immune cell infiltration, epithelial injury, crypt hyperplasia, and edema, in Lyn2/2 mice by day 7 of DSS. Taken together, these data demonstrate an increase in kinetics and severity of intestinal damage in Lyn2/2 mice following DSS exposure. Cytokine production in explant cultures was assessed, as the cytokine environment plays a major role in the development and outcome of intestinal inflammation. Although Lyn2/2 mice had more severe inflammation and intestinal damage, we found no significant differences in the production of the proinflammatory cytokines IL-1b, TNF-a, or IL-6 in DSS-treated wt and Lyn2/2 mice (Fig. 2I). IL-17, IL-22, and IFN-g also play important roles

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FIGURE 1. Lyn deficiency increases susceptibility to DSS-induced colitis. Increasing concentrations of DSS (1.5, 2.5, and 5%) were administered to wt (Lyn+/+) and Lyn2/2 mice for 7 d followed by a recovery period. (A) Animals were monitored daily for percentage body weight loss as an indicator of morbidity (experimental endpoint was equivalent to $20% loss of starting weight). (B) Once 20% weight loss was reached, or on day 14, mice were sacrificed and colon length was measured. (C) Rectal bleeding was scored daily throughout the treatment. Representative data of two independent experiments are shown (n = 4–6). Error bars represent SEM. *p , 0.05, **p , 0.01. (D) Colonic protein lysates from naive (day 0) and DSS-treated (days 3 and 6) mice were analyzed by Western blotting for total Lyn and phospho-Lyn (inhibitory, pY507) and phospho-Src family (activating, pY418). b-actin was used as a loading control.

in the pathogenesis of inflammatory bowel disease (IBD) and experimental models of colitis (26). Levels of IL-17A/F were variable but trended toward a slight increase in Lyn2/2 mice, which was consistent with a significant increase in the expression of retinoic acid–related orphan receptor C (Rorc; encoding RORgt), a transcription factor required for the generation of Th17 cells (27) and IL-17–producing ILCs (28). Increased IFN-g was also observed in Lyn2/2 mice during DSS treatment; however, IL22 was not consistently different between genotypes (Fig. 2J, 2K). The adaptive immune system is required for increased susceptibility of Lyn2/2 mice to DSS The innate immune system plays a critical role in dictating the nature and magnitude of intestinal immune responses and susceptibility to DSS colitis. We therefore analyzed the innate immune compartments in the colonic lamina propria of wt and Lyn2/2 mice before and after DSS challenge. No consistent differences were found in the frequency of colonic macrophages (CD11b+F4/80+) or granulocytes/neutrophils (CD11b+Gr1hi) in untreated or DSStreated Lyn2/2 and wt mice. However, we observed a small but consistent increase in frequency in DCs (CD11c+MHC class II+) in untreated Lyn2/2 mice, and this difference was significant after DSS treatment (Supplemental Fig. 1A). Further examination of the DC compartment revealed significantly decreased frequency of the immunoregulatory CD11b2CD103+ DCs in the colons of DSS-treated Lyn2/2 mice (Supplemental Fig. 1B). No major differences were observed in MHC class II, CD80, or CD86 expression in DCs from naive mice; however, after DSS treatment, Lyn2/2 DCs expressed modestly elevated levels of CD80 and CD86 (Supplemental Fig. 1B). Overall, the observed changes in colonic DC populations in Lyn2/2 mice correlated with increased intestinal inflammation in these mice. To gain further insight into the mechanism of susceptibility to DSS in Lyn2/2 mice, we questioned whether the adaptive immune

system was required for disease severity. Lyn2/2, Rag2/2, and Rag2/2Lyn2/2 mice were treated with DSS for 7 d. Lyn2/2 mice exhibited the greatest susceptibility to disease, but Rag2/2Lyn2/2 mice were modestly resistant to DSS colitis compared with their Rag2/2 counterparts (Fig. 3A). No major differences were observed in rectal bleeding between the groups; however, the Rag2/2 Lyn2/2 mice exhibited the least amount of rectal bleeding, particularly between days 2 and 4 of DSS treatment (Fig. 3B). Additionally, Lyn2/2 mice had significantly shorter colons following DSS treatment than did both immunodeficient groups, which did not differ from each other (Fig. 3C). Consistent with increased inflammation, immunocompetent Lyn2/2 mice exhibited significantly elevated IL-17 and IFN-g production and expression of Rorc compared with Rag2/2Lyn2/2 mice. Lyn2/2 mice also expressed significantly increased levels of Rorc mRNA and trended toward more IL-17 production compared with Rag2/2 mice. Interestingly, in the absence of an adaptive immune system (Rag2/2), Lyn-deficient (Rag2/2Lyn2/2) mice no longer exhibited increased IFN-g or type 17 response markers (IL17 and Rorc) compared with their Lyn wt (Rag2/2) counterparts (Fig. 3D, 3E). Lyn2/2 mice are know to have an altered B cell compartment involving both defects in B cell development as well as hyperactivity of B cells that results in autoimmune disease in aged mice (11–14). Consistent with published reports on systemic B cell populations, naive Lyn2/2 mice had significantly fewer B cells in the colonic lamina propria than did wt mice. Following DSS, this difference was diminished but still trended toward decreased B cells (Fig. 3F). We therefore questioned whether altered B cell responses, perhaps through differences in IL-10 or other immunoregulatory cytokines, were contributing to increased DSS colitis in Lyn2/2 mice. However, B cell depletion had no impact on the disease severity of wt or Lyn2/2 mice, suggesting that these cells are not directly contributing to disease etiology in our mice


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FIGURE 2. Lyn deficiency leads to exacerbated DSS colitis, barrier permeability, crypt hyperplasia, and distinct cytokine and transcription factor expression profiles. Wt (Lyn+/+) and Lyn2/2 mice were treated with 2.5% DSS and animals were sacrificed on days 2, 4, and 7 after the start of treatment. (A) Body weight was monitored daily. (B) Colon length was measured at the experimental endpoint. (C) Pathology scoring and (G) crypt length were assessed from (H) cross-sections of distal colon stained with H&E or Ki67 (green) and DAPI (blue). (D) At the experimental endpoint, mice were administered 400 ng/g FITC-dextran by oral gavage and sacrificed 4 h later. Blood was collected for analysis of FITC serum levels as an indicator of intestinal permeability. Quantitation of Ki67+ cells are expressed either as (E) percentage length of crypt containing Ki67+ cells or as (F) total numbers of Ki67+ cells per crypt. (I and J) At the experimental endpoint, colons were harvested and cytokines were measured in colon explant supernatants. (K) After 7 d of DSS, RNA was extracted from distal colon tissue and Rorc mRNA expression was assessed by quantitative PCR. Target gene expression (Norm. Exp.) was normalized to Gapdh. Representative data from more than three independent experiments are shown for (A) (n = 3). Pooled data from two to three independent experiments are shown in (B)–(D) (n = 6–11; untreated, n = 3). For (E)–(G), representative data of one to two independent experiments are shown for DSStreated mice (n = 3–6). Representative data from three independent experiments are shown for (I) and (K) (n = 4–6). Pooled data from three independent experiments are shown for cytokine production in (J) (n = 6–9). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

(Fig. 3H–J). Taken together, these data suggest that Lyn deficiency drives pathogenic adaptive immune responses, potentially involving exaggerated IL-17– and IFN-g–producing T cells. Increased susceptibility to DSS in Lyn2/2 mice is dependent on the microbiota The ability of the immune system to shape the composition of the microbiota has been highlighted in a number of recent studies.

For example, dysbiosis is a feature of Tbx212/2 Rag22/2 (29), Pycard2/2 (encodes ASC), and Nlrp62/2 mice (6). Importantly, the dysbiosis that develops in these mutant mice is sufficient to drive spontaneous colitis (29) or increased susceptibility to experimental colitis (6). We therefore sought to determine whether Lyn is a direct negative regulator of intestinal inflammation or, alternatively, whether Lyn deficiency might lead to the emergence of a distinct microbiota predisposing Lyn2/2 mice to colitic dis-

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FIGURE 3. The adaptive immune system is required for susceptibility to DSS colitis in Lyn2/2 mice. (A–E) Lyn2/2, Rag2/2, and Rag2/2Lyn2/2 mice were treated with 2.5% DSS and (A) body weight and (B) rectal bleeding were monitored during 7 d. At the experimental endpoint (day 7), mice were sacrificed and (C) colon length was measured. (D) Cytokine production was measured in colon explant cultures. (E) RNA was extracted from distal colon and mRNA expression was assessed by quantitative PCR. Target gene expression was normalized to Rps29. Representative data from three independent experiments for Rag2/2 and Rag2/2Lyn2/2 and an independent experiment including Lyn2/2 are shown (n = 6–7). (F) Total B cell numbers (B220+ or CD19+) were assessed by flow cytometry in naive and DSS treated wt (Lyn+/+) and Lyn2/2 mice. Graph represents data pooled from three to four experiments (n = 6–8). (G–J) Wt (Lyn+/+) and Lyn2/2 mice were treated with anti-CD20 depleting mAb (aCD20) or isotype control Abs (Iso) 4 and 2 d prior to treatment with 2.5% DSS for 7 d. (G) B cell (B220+CD32) depletion in spleen and colonic lamina propria were assessed by flow cytometry. Representative plots are shown and numbers represent mean frequency per group 6 SD. (H and I) Body weight and rectal bleeding were monitored daily throughout the DSS treatment. After 7 d of DSS treatment, mice were sacrificed and (J) colon length was measured. Data from an independent experiment are shown (n = 6–7). (A and B) **p , 0.01, ***p , 0.001, differences between Rag2/2 and Rag2/2Lyn2/2. #p , 0.05, ##p , 0.01, ###p , 0.001, differences between Lyn2/2 and Rag2/2Lyn2/2. (D)–(F) *p , 0.05, **p , 0.01. (H–J) *p , 0.05, **p , 0.01, ***p , 0.001, differences between Lyn+/+ (Iso) and Lyn2/2 (Iso). #p , 0.05, ##p , 0.01, ###p , 0.001, differences between Lyn+/+ (aCD20) and Lyn2/2 (aCD20).

ease. A basic examination of the microbiota revealed no significant difference in relative abundance of Clostridium coccoides or Lactobacillus/Lactococcus between wt and Lyn2/2 mice; however, Lyn2/2 mice had a significantly elevated population of SFB (Fig. 4A). Our mouse colonies had previously tested positive for

colitogenic Helicobacter species, including H. hepaticus, but no difference in abundance was observed between wt and Lyn2/2 mice (Fig. 4A), and Helicobacter bilis was undetectable in our colony (data not shown). To determine whether differences in microbiota contributed to DSS susceptibility in Lyn2/2 mice, wt


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FIGURE 4. DSS susceptibility in Lyn2/2 mice is associated with a distinct microbiota. (A) Bacterial DNA was isolated from fecal pellets from naive wt (Lyn +/+ ) and Lyn 2/2 mice and the relative abundance of SFB, C. coccoides (Clost.), Lactobacillus/Lactococcus (Lacto.), and H. hepaticus (H. hep.) DNA was quantified by quantitative PCR. Target bacterial DNA was normalized to total eubacterial DNA. Representative data from three independent experiments are shown (n = 8). The y-axis on the right represents the scale for abundance of H. hepaticus. (B–E) Wt and Lyn2/2 mice were cohoused for 4 wk prior to treatment with 2.5% DSS. (B) Body weight and (C) rectal bleeding were monitored daily during DSS treatment. (D) At the experimental endpoint (day 7), mice were sacrificed and colon length was measured. (E) Fresh feces were collected after 0, 2, and 4 wk of cohousing and relative abundance of SFB DNA was assessed by quantitative PCR using total eubacteria as a reference. (B–E) Representative data from two independent experiments are shown (n = 45). (F–H) Wt and Lyn2/2 mice were rederived by surgical implantation of embryos into CD1 female recipient mice. Rederived mice were treated with 3.5% DSS for 7 d and (F) body weight and (G) rectal bleeding were monitored daily. (H) At the experimental endpoint (day 7), mice were sacrificed and colon length was measured. (F–H) Representative data from three independent experiments are shown (n = 4). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

and Lyn2/2 mice were cohoused for 4 wk prior to DSS treatment. Strikingly, changes in microbiota had a dramatic effect on DSS susceptibility. Cohousing of wt mice with Lyn2/2 mice increased DSS-induced disease in wt mice and provided limited protection in Lyn2/2 mice, as both genotypes displayed an intermediate DSS susceptibility phenotype based on weight loss and colon length (Fig. 4B, 4D). Following cohousing, the differences in weight loss between wt and Lyn2/2 mice were greatly reduced and both genotypes were indistinguishable based on colon length and rectal bleeding assessments (Fig. 4C, 4D). These changes in disease susceptibility correlated with changes in SFB abundance. Prior to cohousing, Lyn2/2 mice had a significantly increased (∼20-fold) abundance of SFB compared with wt mice, which had low to undetectable levels of SFB DNA. Cohousing resulted in an increase in SFB abundance in wt mice and a concurrent decrease in Lyn2/2 mice, with similar levels of SFB DNA detected in fecal pellets of both genotypes following 4 wk of cohousing (Fig. 4E). To further determine the role of the microbiota in Lyn-mediated protection from DSS, Lyn2/2 mice were rederived into a higher barrier specific pathogen-free facility by surgical implantation of embryos into CD1 mice (which provided the initial source of microbiota). Following rederivation, no SFB DNA could be detected in feces of either wt or Lyn2/2 mice (data not shown). Consistent with the cohousing data, rederived Lyn2/2 mice did not exhibit increased susceptibility to DSS. In fact, Lyn2/2 mice exhibited slightly less weight loss than did wt mice, and no major

differences were observed in rectal bleeding or colon length following DSS treatment (Fig. 4F–H). The contribution of the microbiota to DSS susceptibility was further confirmed in radiation chimeric mice in which congenic (CD45.22CD45.1+) BoyJ mice were reconstituted with either wt or Lyn2/2 BM (CD45.2+ CD45.12). The use of BoyJ hosts allowed for the normalization of the microbiota between recipients of wt or Lyn2/2 BM. Chimerism was confirmed in the spleen and colonic lamina propria by flow cytometry and at least 95% of total CD45+ cells were of donor origin (CD45.2+CD45.12) (data not shown). No differences were observed in the microbial communities assayed between the groups of chimeric mice, which were all negative for SFB and H. hepaticus (Fig. 5A). Chimeric wt and Lyn2/2 mice were treated with DSS for 7 d and were either euthanized following DSS treatment or were left to recover for an additional 7 d. Consistent with results of the rederived Lyn2/2 mice, Lyn2/2 chimeric mice did not show increased susceptibility to DSS compared with wt mice, based on weight loss, rectal bleeding, colon length, and survival (Fig. 5B–E). Interestingly, Lyn2/2 chimeric mice trended toward a slight advantage during the recovery period in terms of weight loss and rectal bleeding (Fig. 5B, 5C). This supports the role for an altered microbiota in driving increased susceptibility to DSS in Lyn2/2 mice, although a potential contribution of Lyn expressing radio-resistant cells cannot be excluded. Taken together, the results from these three sets of experiments suggest that Lyn deficiency alters an intimate cross-talk between the micro-

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Lyn PROTECTS FROM COLITIS AND ENTERIC INFECTION Finally, we sought to identify a potential mechanism through which Lyn might regulate the microbiota. Using Lyn gain-offunction mice, we recently showed that Lyn regulates systemic and intestinal production of IL-22 in response to microbial products such as LPS (21). Because IL-22 can regulate the intestinal microbiota, including SFB (30–33), we questioned whether Lyn deficiency would have a negative impact on IL-22 production. In response to IL-23, Lyn2/2 splenocytes produced significantly less IL-22 than did wt splenocytes (Fig. 6A). In response to systemic administration of LPS, we observed decreased IL-22 production by Lyn2/2 ileal explant cultures, but no differences were observed in blood or colon explant cultures (Fig. 6B). Diminished IL-22 production was not due to a lack of intestinal or splenic ILCs, which are major producers of IL-22 (Fig. 6C–E). IgA is another immune effector molecule known to impact the microbiota, including SFB, and a previous study identified altered IgA production in serum of Lyn2/2 mice (13). Consistent with this study, we observed increased IgA concentrations in the blood and feces of Lyn2/2 mice (Fig. 6F). Lyn2/2 mice from both original (SFB+) and rederived (SFB2) colonies had significantly increased IgA in their blood. Fecal IgA concentrations were only significantly increased in the rederived (SFB2) Lyn2/2 mice compared with their wt counterparts; however, IgA concentrations were significantly higher in both original (SFB+) colonies compared with their rederived controls. Taken together, these data suggest the possibility that Lyn may regulate the microbiota via diverse mechanisms, including IL-22 and IgA production. Increased T cell production of IFN-g and IL-17 in Lyn2/2 mice and the impact of the microbiota on colonic T cell accumulation

FIGURE 5. Lyn-deficient BM radiation chimeric mice do not exhibit increased susceptibility to DSS. Wt (Lyn+/+) or Lyn2/2 BM (CD45.2+) was transplanted into lethally irradiated BoyJ mice (CD45.1+). Four months later mice were treated with DSS for 7 d followed by sterile drinking water for an additional 7 d. (A) Prior to DSS treatment, bacterial DNA was isolated from fecal pellets from naive wt (Lyn+/+) and Lyn2/2 mice and the relative abundance of SFB, C. coccoides (Clost.), Lactobacillus/Lactococcus (Lacto.), and H. hepaticus (H. hep.) DNA was quantified by quantitative PCR. Target bacterial DNA was normalized to total eubacterial DNA. Representative data are shown (n = 4–5). (B) Body weight and (C) rectal bleeding were monitored for 14 d. (D) At the experimental endpoint ($20% weight loss) mice were sacrificed. (E) Some mice were sacrificed on day 7 and colon length was measured. (B–E) Representative data from an independent 14 d and two independent 7 d experiments are shown (n = 5–6). Error bars represent SEM.

biota and immune system, which regulates intestinal microbial composition and the nature of the inflammatory response to intestinal damage.

Increased susceptibility of Lyn2/2 mice to DSS colitis correlated with the presence of SFB as well as a moderate increase in colonic IFN-g and IL-17 production that was dependent on the adaptive immune system. We therefore questioned whether these cytokines were T cell derived and whether the presence of SFB was driving the exaggerated cytokine production in Lyn2/2 mice. Wt and Lyn2/2 mice from the original (SFB+) and rederived (SFB2) colonies were left untreated or were treated with DSS for 7 d, and IFN-g and IL-17 production by splenic and colonic lamina propria T cells was assessed by flow cytometry. Untreated Lyn2/2 mice from the original but not the rederived colony had a 3- to 4-fold increase in colonic CD4+ and CD8+ T cells compared with their wt counterparts (Fig. 7B); however, the same mice trended toward a decrease in splenic T cell populations (Supplemental Fig. 2). Furthermore, naive Lyn2/2 mice from both the original (SFB+) and rederived (SFB2) colonies had increased frequencies and numbers of colonic IFN-g+ and IL-17+ CD4+ T cells compared with their wt counterparts. However, only Lyn2/2 mice from the original (SFB+) but not the rederived (SFB2) colony also had increased frequencies and numbers of IFN-g+CD8+ T cells and IFN-g+IL-17+CD4+ T cells compared with wt mice. Correlating with susceptibility to DSS, SFB-containing Lyn2/2 mice, and not the rederived (SFB2) mice, maintained their increased numbers of IFN-g– and IL-17–producing colonic T cells following DSS (Fig. 7). In the spleen, only the rederived (SFB2) Lyn2/2 mice had an increase in IL-17+CD4+ T cell numbers and this difference was observed in both naive and DSS-treated mice. Furthermore, naive Lyn2/2 mice from both colonies had increased numbers of IFN-g+ T cells; however, following DSS treatment this difference was lost in the original colonies (Supplemental Fig. 2). Of note, the increases in the number of cytokine-producing colonic T cells in DSS compared with naive mice is reflective of changes in the total


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FIGURE 6. Lyn2/2 mice have altered IL-22 and IgA responses. (A) Total splenocytes from naive Lyn+/+ and Lyn2/2 mice were left untreated or were stimulated with IL-23, and IL-22 production was quantified by ELISA. Pooled data from four independent experiments are shown (untreated, n = 4; +IL-23, n = 8). (B) Lyn+/+ and Lyn2/2 mice were treated with LPS and IL-22 in ileum, colon and blood was assessed. Data from three representative experiments are shown for blood and colon and an independent experiment for ileum (n = 6–8). (C) Splenic, (D) colonic, and (E) mesenteric lymph node ILCs (lineage2 CD90+) from naive Lyn+/+ and Lyn2/2 mice were analyzed by flow cytometry. Graphs and plots represent data pooled from three independent experiments (n = 9). Numbers on plots represent mean frequency 6 SEM. (F) Blood and fecal IgA concentrations in naive Lyn+/+ and Lyn2/2 mice from the original (Orig.) and rederived (Red.) colonies were assessed by ELISA (n = 6–8). *p , 0.05, **p , 0.01, ***p , 0.001.

numbers of T cells more than an increase proportion of cytokineproducing T cells. This suggests that the polarization of the inflammatory T cell response occurs at steady-state and that the magnitude of inflammatory T cell responses as opposed to altered polarization are being regulated during DSS-induced inflammation. Therefore, steady-state regulation of the intestinal T cell populations, influenced by the commensal microbiota, may predispose Lyn2/2 mice to susceptibility to inflammatory diseases that are driven by production of IL-17 and/or IFN-g. Interestingly, total T cells as well as IFN-g– and IL-17–producing T cell numbers were increased in the original (SFB+) compared with the rederived (SFB2) Lyn2/2 mice, both at the steady-state and following DSS treatment. We therefore sought to confirm whether this was a direct result of changes in the microbiota. Lyn2/2 mice from the rederived (SFB2) colony were housed in cages supplemented twice weekly with dirty bedding from either rederived (SFB2) or original (SFB+) Lyn2/2 mice for 4 wk prior to DSS treatment. All the mice housed in original Lyn2/2 bedding, but none of the mice with the rederived bedding, became SFB+ (data not shown) and are referred to as either SFB+ or SFB2, respectively. Following DSS treatment, the production of IFN-g and IL-17 by T cells in the colonic lamina propria was assessed by flow cytometry. The introduction of microbes from the original Lyn2/2 colony to the rederived Lyn2/2 mice led to a significant increase in total and IFN-g+ CD4+ and CD8+ T cells as well as IL17+ and IL-17+IFN-g+CD4+ T cells (Supplemental Fig. 3D). This was also associated with a trend toward increased susceptibility to DSS based on rectal bleeding and colon length, although differ-

ences in weight loss were not observed (Supplemental Fig. 3A–C). Taken together, these data suggest that loss of Lyn is sufficient to induce increased IL-17 and IFN-g production by T cells irrespective of microbiota status; however, in the presence of SFB, both single-positive (IL-17+IFN-g2, IL-172IFN-g+) and doublepositive (IL-17+IFN-g+) cytokine-producing T cells were increased in the colons of Lyn2/2 mice, correlating with DSS susceptibility. Increased susceptibility to enteric pathogens and SFB expansion in Lyn2/2 mice Finally, we sought to investigate whether the dysbiosis in Lyn2/2 mice resulted from an acute inability to control potentially pathogenic bacteria. Because the presence of increased SFB in Lyn2/2 mice was associated with their susceptibility to DSS and sustained production of IFN-g and IL-17 by colonic T cells, we questioned whether Lyn2/2 mice were impaired in their ability to control SFB. Wt and Lyn2/2 mice from the rederived colonies (SFB2) were housed in cages supplemented twice weekly with dirty bedding from either their own colony (SFB2) or bedding from original Lyn2/2 mice (SFB+). The presence of fecal SFB was assessed weekly for 3 wk. Indeed, throughout the course of the experiment, Lyn2/2 mice trended toward increased fecal SFB abundance compared with wt mice, suggesting an impaired ability to control intestinal SFB populations (Supplemental Fig. 4). An inability to control colonization by intestinal microbes can leave the host susceptible to infection by enteric pathogens. We therefore questioned whether Lyn-deficient mice would exhibit a general susceptibility to enteric pathogens including C. rodentium

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FIGURE 7. Accumulation of IL-17– and/or IFN-g–producing colonic T cells correlates with DSS susceptibility and the presence of a distinct microbiota in Lyn2/2 mice. Wt (Lyn+/+) and Lyn2/2 mice from the original (Orig.) or rederived colonies (Red.) were left untreated or were treated with 2.5% DSS for 7 d. Colonic lamina propria cells were isolated and stimulated ex vivo with PMA, ionomycin, and brefeldin A for 4 h and production of IL-17A and IFN-g in CD4+ and CD8+ T cells (CD3+) was assessed by intracellular flow cytometry. Cells were first gated on live CD45+ populations. (A) Representative plots are shown and numbers represent mean frequency. (B) Graphs represent total cell numbers per colon. Representative data from two independent experiments are shown (n = 3–4/experiment). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

and S. Typhimurium. C. rodentium is a natural murine pathogen that attaches to colonic epithelium, effacing the barrier and causing inflammation. Infection persists for ∼1 mo in the colon, where the bacterial load increases to the height of infection at weeks 1–2 postinfection and then decreases until bacteria are eliminated by week 4 (34). Bacteria can move to systemic sites but do not typically cause overt disease leading to morbidity. Monitoring of progression of C. rodentium infection in Lyn2/2 and wt mice revealed a significant increase in colonic pathology at 6, 12, 21, and 28 d postinfection in Lyn2/2 mice (Fig. 8A). This coincided with a significantly higher bacterial load in the colons of Lyn2/2 mice on day 21 postinfection (Fig. 8B), but no significant differences in systemic bacterial number were found in spleen or liver at any time point (Fig. 8C, 8D). Histological examination revealed increased colonic pathology, including hyperplasia, immune cell infiltration, edema, and epithelial damage at each time point during infection in Lyn2/2 mice (Fig. 8A, 8E). S. Typhimurium is a human-adapted pathogen that causes gastroenteritis in mice pretreated with a high dose of oral streptomycin, which perturbs the endogenous microbiota allowing efficient bacterial colonization of the colon. Susceptibility to disease

can be monitored by bacterial enumeration and assessment of cecal pathology, as well as by assessing morbidity and systemic bacterial dissemination (35). Lyn2/2 mice were highly susceptible to S. Typhimurium, with all mice becoming moribund significantly earlier after infection (day 6) compared with wt mice (day 13) (Fig. 8F). On day 4 postinfection, Lyn2/2 mice had significantly higher bacterial loads in the cecum and spleen (Fig. 8G), correlating with significantly greater cecal pathology (Fig. 8H). The ceca of Lyn2/2 mice showed complete loss of crypt architecture, severe edema, and more extensive luminal inflammatory infiltrates compared with wt mice (Fig. 8I). Lyn2/2 mice were also highly susceptible to systemic infection with S. Typhimurium. In this “typhoid-like” model of disease, mice are not pretreated with antibiotic, and therefore rather than colonizing the gut, bacteria rapidly translocate through the intestinal epithelium, disseminating and replicating systemically, leading to organ failure and morbidity (35). Similar to our results in streptomycin-treated mice, all Lyn2/2 animals became moribund significantly earlier than did wt mice (day 5 versus day 7) following S. Typhimurium infection (Fig. 8J), and this was associated with significantly higher bacterial loads in the colon and a trend toward increased loads in the liver and spleen (Fig. 8K).


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FIGURE 8. Lyn deficiency increases susceptibility to enteric infection–induced inflammation. Wt (Lyn+/+) and Lyn2/2 mice were infected with C. rodentium and sacrificed at the indicated days after infection. (A) Histopathology was scored from H&E-stained sections of distal colon. The remainder of the (B) colon, as well as the (C) spleen and (D) liver, was homogenized and bacterial CFU per organ were determined. (E) Representative H&E sections from each time point are shown. Pooled data from two independent experiments are shown for (A)–(D) (infected, n = 6; naive, n = 3). Representative sections from two independent experiments are shown for (E) (n = 6). (F–I) Wt (Lyn+/+) and Lyn2/2 mice were treated with streptomycin 24 h prior to infection with S. Typhimurium. (F) Mice were monitored daily for signs of disease and sacrificed when moribund. (G) Four days postinfection, mice were sacrificed and the cecum and spleen were homogenized for enumeration of bacterial CFU. (H and I) Ceca were stained with H&E and slides were scored to quantify levels of inflammation. Representative data from two independent experiments are shown for (F) and (I) (n = 6–8). (J and K) Mice were infected with S. Typhimurium and were (J) monitored daily for signs of disease and sacrificed when moribund. (K) Four days postinfection, mice were sacrificed and the colon, spleen, and liver were homogenized for enumeration of bacterial CFU. (J) Pooled and (K) representative data from two independent experiments are shown (n = 6 and n = 3, respectively). Error bars represent SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

Discussion The Lyn tyrosine kinase is a critical regulator of signal transduction pathways regulating the development and function of a number of

different leukocyte populations involved in intestinal immune and inflammatory responses. Until recently, the only evidence implicating a role for Lyn in intestinal disease had been clinical studies

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showing colitis as a side effect in cancer patients treated with the broad-spectrum tyrosine kinase inhibitor dasatinib (36–38). Although Dasatinib may affect a number of distinct tyrosine kinase families, including SFKs, of which Lyn is a member, these results nevertheless suggest a protective role for tyrosine kinase signaling pathways in the gut. However, we recently demonstrated that Lyn gain-of-function mutant mice were protected from DSS colitis, which was independent of the adaptive immune system and involved hypersensitivity to TLR ligands (21). Understanding the biological role of Lyn is complicated by the opposing roles of Lyn in both activating and inhibitory cell signaling pathways and by potential redundancy with other members of the SFK family. Our studies of Lyn gain-of-function mice allowed us to dissect the consequence of increased Lyn activity as well as the functions of Lyn that may be redundant with other SFKs. In this study, we sought to understand how loss of Lyn leads to susceptibility to colitis, focusing on identifying nonredundant roles for Lyn using Lyn2/2 mice. Our work clearly shows a protective role of Lyn in limiting intestinal inflammation induced by intestinal epithelial damage as well as during enteric infection. In the present study, we investigated the requirement for Lyn in regulating inflammatory responses to infection by two enteric pathogens and investigated the mechanism of susceptibility to DSS in Lyn2/2 mice. Importantly, the data presented in this study highlight how Lyn deficiency affects both innate and adaptive immune responses, resulting in perturbations in the composition of the microbiota and the outcome of intestinal inflammation. Intestinal homeostasis is mediated by a number of immune and environmental factors, including the production of cytokines by innate and adaptive immune cells. Importantly, the microbiota has recently emerged as a critical factor in intestinal homeostasis and inflammation in both humans and mice (26, 39–42). Sequencing of 16S rRNA genes has identified differences in the microbiota of healthy controls compared with IBD patients, with a loss of diversity being a hallmark of disease (43–45). Inflammation in itself results in changes in bacterial composition (48), which makes the interpretation of dysbiosis in IBD difficult to decipher. However, the effective use of antibiotics (47–50) and probiotics (51) to control IBD along with the increased incidence of IBD observed in children previously treated with multiple courses of antibiotics (52) suggest an important role for dysbiosis in IBD pathogenesis. Lyn plays a critical role in immune homeostasis and PRRinduced responses, which are both involved in the maintenance of the microbiota. We therefore questioned whether the increased inflammation in Lyn2/2 mice resulted indirectly from changes in the microbiota or directly through exaggerated inflammatory responses following barrier disruption. Cohousing experiments demonstrated that Lyn2/2 mice foster a proinflammatory microbiota that was transferable to wt mice, resulting in their increased susceptibility to DSS colitis. Interestingly, the Lyn2/2 phenotype was also partially rescued by cohousing with wt mice. Furthermore, rederivation of Lyn2/2 mice completely rescued these mice from DSS susceptibility. Taken together, this suggests that Lyn2/2 mice may harbor a microbiota not only enriched for colitogenic microorganisms but may also lack tolerance-inducing species. Our limited analysis of the microbiota identified an increased representation of SFB DNA in Lyn2/2 feces. Interestingly, SFB abundance correlated with susceptibility to DSS of both wt and Lyn2/2 mice following cohousing, and we are currently investigating the specific contribution of SFB to the exaggerated intestinal inflammation observed in Lyn2/2 mice. Changes in intestinal microbiota can have profound effects on the tone of T cell responses both locally at mucosal sites and systemically. For example, colonization by a defined group of

Clostridium species promotes intestinal regulatory T cell development and function (53), whereas the introduction of microbiota containing closely related SFB induces the differentiation of CD4+ T cells that express IL-17 and IFN-g (54, 55). Furthermore, the presence of SFB is sufficient to drive increased pathology associated with increased Th17 responses in murine models of multiple sclerosis and arthritis (54, 56). Alternatively, SFB have been associated with protection from diabetes in female mice (57). Interestingly, we found increased IL-17 and IFN-g production by certain T cell populations in both rederived (SFB2) and original (SFB+) Lyn2/2 mice, indicating a predisposition toward these T cell responses independent of the microbiome, possibly through increased BAFF or IL-6 production in Lyn2/2 mice (14, 58). However, disease severity was associated with an increase of these T cell populations as a result of altered microbiota characterized by the presences of SFB. Although early reports indicated a type 2 cytokine skewing in Lyn2/2 mice (59), the data presented in the present study are consistent with more recent reports that also showed an increase in IFN-g production by CD4+ and CD8+ T cells in Lyn2/2 mice (58). IL-17 and IFN-g, as well as the cells that produce them, play important roles in the etiology of IBD. For example, IL-17– and IFN-g–producing Th17 cells are located in the lamina propria and IL-17 levels are elevated in the colons and blood of patients with both Crohn’s disease and ulcerative colitis (60–62). IFN-g is also elevated in Crohn’s disease patients (63). Additionally, polymorphisms resulting in hyperactive IL-17 production are associated with IBD (64). However, whether IL-17 promotes inflammation in murine models of colitis remains controversial. In DSS colitis the use of IL-17–neutralizing Abs increases susceptibility to DSS, implicating a protective role for this cytokine in disease (65). However, mounting evidence suggests that IL-17 indeed promotes inflammation in the colon. For example, IL17A–deficient mice have reduced epithelial damage and inflammation of the colon during DSS treatment (66) and show a reduction in chronic inflammation and tumor development in a model of colitis-associated cancer (67). Accordingly, when we investigated the role of adaptive immune responses in susceptibility to DSS in Lyn2/2 mice, we found that unlike immunosufficient background mice, Rag2/2Lyn2/2 mice were modestly protected from colitis. Furthermore, no differences were observed in IL-17 production or Rorc expression in Rag2/2Lyn2/2 compared with Rag2/2 mice, and IFN-g was even decreased in Rag2/2 Lyn2/2 mice, correlating with decreased disease severity. Importantly, although Lyn is an important regulator of B cell development and function, B cells did not account for the adaptive immune component that was necessary to drive enhanced sensitivity to DSS colitis in Lyn2/2 mice. A number of recent studies have described the complex web of interactions between the microbiota and host immune system. However, much of the work has focused on how commensals (including SFB) impact the immune system (68–70), and less is understood about what immune mechanisms influence SFB colonization and maintenance. Nonetheless, an aryl hydrocarbon receptor/IL-22/antimicriobial peptide axis has emerged as a pathway that regulates the microbiota and SFB (33, 71). We previously showed that increased Lyn activity promoted IL-22 production. In the present study, we found that Lyn deficiency resulted in a decreased capacity to produce IL-22 in the ileum of Lyn2/2 mice. Given that the ileum is the primary site of SFB colonization, this suggests a possible mechanism to explain the dysbiosis and SFB expansion in Lyn2/2 mice. Another possible mechanism through which Lyn may impact the microbiota is by affecting IgA production (72). Interestingly, this may be directly due to B cell–

The Journal of Immunology intrinsic loss of Lyn, or may indirectly involve altered T cell populations, as both Th17 and regulatory T cells have been implicated in intestinal T cell–dependent IgA production (73–75). In addition to alterations in DSS-induced disease, changes in the microbiota in Lyn2/2 mice likely also dictate the outcome of infection with S. Typhimurium and C. rodentium. Access to the gut epithelium during enteric infection is limited by the microbiota, a phenomenon called colonization resistance. This is best exemplified in the S. Typhimurium gastroenteritis model of infection, where pretreatment with streptomycin transiently reduces gut commensal number and diversity (35), thereby opening a niche for S. Typhimurium to colonize the cecum and colon. Other studies have shown that low intestinal microbial complexity reduces colonization resistance during Salmonella infection (76). Interestingly, we found that even in the typhoid model of Salmonella infection, bacterial burden in the colons of Lyn2/2 mice after 4 d was significantly higher than those in wt mice, suggesting that Lyn deficiency may alter the microbiota in a way that reduces colonization resistance. Previous studies suggested that the presence of SFB correlated with protection from C. rodentium infection (55). However, our data are consistent with a more recent study that showed that decreased IL-22 production, which is known to increase susceptibility to C. rodentium infection, results in the expansion of SFB (33). Overall, these data suggest that Lyn deficiency does indeed alter the composition of commensal communities in the gut that can impact enteric infections. Although the exploration of the mechanisms behind susceptibility to these pathogens is outside the scope of this work, there are a myriad of factors that can determine the outcome of infection and may involve alterations in immune responses in Lyn2/2 mice. For example, both T and B cell responses play roles in the clearance of S. Typhimurium (77) and C. rodentium infections (34). Although the early (2 d postinfection) pathology observed in Lyn2/2 mice infected with S. Typhimurium suggests that Lyn deficiency in innate cells contributes to colonization susceptibility and inflammation, the increased inflammation observed throughout the duration of C. rodentium infection in Lyn2/2 mice could be mediated by both innate and adaptive mechanisms. Interestingly, whereas IL-17A/F–deficient mice are more susceptible to C. rodentium infection (78), other studies have shown that colonic inflammation is associated with IL-17 and IFN-g production early after infection (79). This response may be exaggerated in Lyn2/2 mice, leading to increased inflammation at day 6 postinfection. In summary, we have identified Lyn as a critical regulator of the microbiota as well as intestinal inflammation in murine models of IBD and enteric infection. We speculate that increased abundance of SFB in Lyn2/2 mice, together with a genetic predisposition to increased numbers of IFN-g– and IL-17–producing T cells, contributes to exaggerated IFN-g and IL-17 production by colonic T cells and enhanced intestinal inflammation in Lyn2/2 mice. Thus, as is the case in human IBD, complex interactions between host genotype and intestinal microbial composition are key to disease etiology. As drugs targeting Lyn are entering the clinic and are being actively pursued to treat neoplastic disease, this work suggests that these agents be thoroughly investigated for untoward dysbiotic and gastrointestinal effects, as well as drug-induced enteric pathogen susceptibility.

Acknowledgments We thank Drs. Bruce Vallance and Amit Bhavsar for technical advice and thoughtful discussions, and Manreet Chehal and Alex Sio for excellent technical support.

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Disclosures The authors have no financial conflicts of interest.

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Carbonic anhydrase 2 deficiency leads to increased pyelonephritis susceptibility.

C-Raf deficiency leads to hearing loss and increased noise susceptibility.

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B cell-specific loss of Lyn kinase leads to autoimmunity.

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Multicopy Single-Stranded DNA Directs Intestinal Colonization of Enteric Pathogens.

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European genetic diversity and susceptibility to pathogens.

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Apoplastic Nucleoside Accumulation in Arabidopsis Leads to Reduced Photosynthetic Performance and Increased Susceptibility Against Botrytis cinerea.

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