Interleukin-7 produced by intestinal epithelial cells in response to Citrobacter rodentium infection plays a major role in innate immunity against this pathogen.

Interleukin-7 Produced by Intestinal Epithelial Cells in Response to Citrobacter rodentium Infection Plays a Major Role in Innate Immunity against This Pathogen Jun-O Jina

Shanghai Public Health Clinical Center, Shanghai Medical College, Fudan University, Shanghai, Chinaa; Department of Immunology and Infectious Diseases, The Forsyth Institute, Cambridge, Massachusetts, USAb

Interleukin-7 (IL-7) engages multiple mechanisms to overcome chronic viral infections, but the role of IL-7 in bacterial infections, especially enteric bacterial infections, remains unclear. Here we characterized the previously unexplored role of IL-7 in the innate immune response to the attaching and effacing bacterium Citrobacter rodentium. C. rodentium infection induced IL-7 production from intestinal epithelial cells (IECs). IL-7 production from IECs in response to C. rodentium was dependent on gamma interferon (IFN-␥)-producing NK1.1ⴙ cells and IL-12. Treatment with anti-IL-7R␣ antibody during C. rodentium infection resulted in a higher bacterial burden, enhanced intestinal damage, and greater weight loss and mortality than observed with the control IgG treatment. IEC-produced IL-7 was only essential for protective immunity against C. rodentium during the first 6 days after infection. An impaired bacterial clearance upon IL-7R␣ blockade was associated with a significant decrease in macrophage accumulation and activation in the colon. Moreover, C. rodentium-induced expansion and activation of intestinal CD4ⴙ lymphoid tissue inducer (LTi) cells was completely abrogated by IL-7R␣ blockade. Collectively, these data demonstrate that IL-7 is produced by IECs in response to C. rodentium infection and plays a critical role in the protective immunity against this intestinal attaching and effacing bacterium.

C

itrobacter rodentium is a mouse extracellular enteric pathogen that mimics human-enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E coli (EHEC). These bacterial pathogens attach intimately to intestinal epithelium and cause subcellular attaching and effacing lesions, which lead to severe diarrhea, vomiting, and fever, with high rates of fatality (1). C. rodentium infection of mice causes epithelial hyperplasia in the colon and cecum, goblet cell loss, and mucosal infiltration with macrophages, lymphocytes, and neutrophils (2). Therefore, this is an ideal model to dissect how immune cells interact with gut epithelial pathogens. The innate immune cells, including macrophages, dendritic cells (DCs), natural killer (NK) cells, neutrophils, and innate lymphoid cells (ILCs) have been shown to play an essential role in the clearance of C. rodentium infection (3–6). Moreover, the adaptive immune cells, mostly T and B cells, are also required for the clearance of this pathogen (7, 8). Furthermore, the cytokines interleukin-6 (IL-6), IL-12, IL-17, IL-22, IL-23, and gamma interferon (IFN-␥) are upregulated in the colon tissues of C. rodentium-infected mice and are necessary for an effective immune defense against this pathogen (3–5, 7, 9). IL-7 is a stroma-derived cytokine that can be secreted by fetal liver cells, stromal cells in the bone marrow and thymus, and intestinal epithelial cells (IECs) (10). IL-7 acts on various cells through its receptor, a heterodimer consisting of an alpha-chain (IL-7R␣) and the common cytokine receptor gamma chain. The IL-7 receptor is expressed on lymphoid T and B precursors, innate lymphoid cells, antigen-presenting cells (APCs), and mature T cells (11). At physiological levels, IL-7 is integral to T and B cell development in primary lymphoid organs and plays an essential role in supporting normal T cell development and homeostasis (12, 13). Moreover, IL-7 supports CD4⫹ lymphoid tissue inducer (LTi) cell survival and function (14). Furthermore, IL-7 induces proliferation of naive and memory T cells (15) and enhances ef-

August 2015 Volume 83 Number 8

fector T cell responses, preferentially T helper 1 (Th1) and Th17 responses (16–18). These functional effects of IL-7 on T cells make IL-7 a critical enhancer of protective immunity, as well as of autoimmunity and inflammation (16, 17, 19, 20). In the case of bacterial infections, gastric tissue biopsy specimens from patients infected with Helicobacter pylori (21) or mice infected with Mycobacterium tuberculosis have increased expression of IL-7 (22). In addition, administration of exogenous IL-7 enhanced the survival of M. tuberculosis-infected mice (23). However, the role of IL-7 in intestinal bacterial infection and the related inflammation has not been well characterized. Moreover, the function of IL-7 in immune responses against bacterial pathogens is much less understood than its role in antiviral immunity. In the present study, we investigated the in vivo role of IL-7 in host responses against C. rodentium infection. We found that C. rodentium infection induces expression of IL-7 in intestinal epithelial cells. We hypothesized that IL-7 may play a crucial role in the innate immune activation required for the clearance of C. roden-

Received 9 March 2015 Returned for modification 18 April 2015 Accepted 24 May 2015 Accepted manuscript posted online 1 June 2015 Citation Zhang W, Du J-Y, Yu Q, Jin J. 2015. Interleukin-7 produced by intestinal epithelial cells in response to Citrobacter rodentium infection plays a major role in innate immunity against this pathogen. Infect Immun 83:3213–3223. doi:10.1128/IAI.00320-15. Editor: S. M. Payne Address correspondence to Jun-O Jin, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.00320-15

Infection and Immunity

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Wei Zhang,a Jiang-Yuan Du,a Qing Yu,b

Zhang et al.

tium in vivo, and we undertook the current study to test this hypothesis. MATERIALS AND METHODS

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Animals. C57BL/6 mice were purchased from Shanghai Public Health Clinical Center, and C57BL/6 Rag1⫺/⫺ mice were purchased from Jackson Laboratory and kept under pathogen-free conditions. All experiments were carried out under the guidelines of the Institutional Animal Care and Use Committee at the Shanghai Public Health Clinical Center. The protocol was approved by the committee on the Ethics of Animal Experiments of the Shanghai Public Health Clinical Center (animal protocol SYXK-2010-0098). Bacterial infections. C. rodentium DBS100 wild type was cultured in brain heart infusion broth (Sigma-Aldrich, St. Louis, MO) for 16 h at 37°C in air with shaking (180 rpm). Bacteria were pelleted by centrifugation, washed with phosphate-buffered saline (PBS), and centrifuged again before a final resuspension in PBS to an optical density at 600 nm of 1.0. The number of viable bacteria was determined after serial dilution and plating onto agar. Mice were orally treated via gavage with 2 ⫻ 109 CFU of C. rodentium in 0.1 ml PBS. Analysis of CFU from overnight cultures of mechanically homogenized whole colons was determined via serial dilutions on MacConkey’s agar. In vivo administration of antibody. Female C57BL/6 mice were injected intraperitoneally (i.p.) with 100 ␮g of control IgG or anti-IL-7R␣ antibody (Ab) every 2 days, starting at the time of infection with C. rodentium. Some mice, prior to infection, were injected i.p. with 200 ␮g antiNK1.1 Ab for depletion of NK cells. Mice were then sacrificed and organs were harvested for analysis. Histology and immunofluorescence staining. Colon samples were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a 5-␮m thickness. Sections were then stained with hematoxylin and eosin (H&E) and examined for tissue damage. Colon sections were evaluated to obtain pathology scores for evidence of inflammatory damage, such as goblet cell loss, crypt elongation, mucosal thickening, and epithelial injury, including hyperplasia and enterocyte shedding into the gut lumen. Scores were determined on a scale of 0 to 3 (0, none; 1, mild; 2, moderate; 3, severe). Some paraffin sections were subjected to deparaffinization, rehydration, and antigen retrieval. These sections were then incubated with biotin-conjugated anti-cytokeratin antibody (AE1/AE3; Abcam) and rabbit anti-mouse IL-7 antibody (M-19), followed by Alexa Fluor 488streptavidin and Alexa Fluor 647-conjugated secondary Abs. The stained samples were examined with a laser scanning confocal microscope (Leica Microsystems). Antibodies and cytokines. Cells were stained and analyzed on a FACSAria II system (Becton Dickinson), with dead cells excluded based on forward light scatter. The following fluorescence-conjugated Abs were used: CD3 (17A2), CD11b (M1/70), CD11c (N418), CD86 (GL-1), CD90 (OX-1), CD103 (2E7), CD127 (A7R34), CCR6 (29-2L17), CXCR3 (G025H7), F4/80 (BM8), major histocompatibility complex (MHC) class II (M5/114.15.2), Ly-6G (1A8), NK1.1 (PK136), IL-17 (TC11-18H10.1), and IL-22 (poly5164) were from BioLegend; purified blockade or depilation anti-IFN-␥ (XMG1.2), IFNAR1 (MAR1-5A3), and NK1.1 (PK136) were from BioLegend; purified monoclonal rat anti-mouse IL-7R␣ (A7R34) and its isotype control, rat IgG2a (2A3), were from BioXcell. Preparation of colon single-cell suspensions. Colons were isolated and washed twice in ice-cold PBS. The tissues were cut open longitudinally, and mucus and gross debris were quickly removed by covering the specimen with dry paper towels. The samples were cut in to 0.5- to 1-cm pieces. IECs were separated from intestinal pieces by incubating in 0.15% dithiothreitol–Hanks’ balanced salt solution buffer with shaking for 30 min at 37°C. IECs were collected by filtering through a mesh screen. After epithelial removal, lamina propria cells were collected by mincing the remaining tissue into, followed by digestion with 2% fetal bovine serum (FBS) containing collagenase with shaking for 30 min at 37°C. The cells were filtered through a 100-␮m nylon mesh and washed, and the resulting

pellet was resuspended in RPMI 1640 medium and layered over Histopaque-1.077 (Sigma-Aldrich). After centrifugation at 1,700 ⫻ g for 10 min, the low-density fraction (⬍1.077 g/cm3) was collected. The single cells were resuspended in culture medium. IEC analysis. Isolated IECs (1 ⫻ 106) were incubated with 10 ␮g/ml brefeldin A (Biolegend) for 4 h. Cells were then intracellularly stained with Alexa Fluor 488-conjugated anti-cytokeratin (AE1/AE3; eBioscience) and rabbit anti-mouse IL-7 (M-19) Abs, followed by Alexa Fluor 647-conjugated anti-rabbit secondary Abs. Dead cells were excluded based on forward light scatter. At least 100,000 events were collected from each sample by gating on live cells, and data were analyzed using FlowJo software (TreeStar Inc.). Macrophage and cDC analysis. Single-cell suspensions from colons and mesenteric lymph nodes (mLNs) were incubated for 30 min with the following fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies (MAbs) as lineage⫹ cells: anti-CD3 (17A2), anti-Thy1.1 (OX-7), anti-CD49b (DX5), and anti-TER-119 (TER-119), or phycoerythrin-Cy7-conjugated CD45 (30-F11). Among CD45⫹ lineage⫺ cells, the CD103⫹ CD11c⫹ cells and CD103⫺ CD11c⫹ cells were defined as migratory cDCs and resident cDCs, respectively (24, 25). The CD11c⫹ F4/80⫹ cells were defined as macrophages. Analysis was carried out on a FACSAria II system (Becton Dickinson). CD4ⴙ LTi cell analysis. Single-cell suspensions from colons and mLNs were stimulated directly ex vivo by incubation for 4 h with 50 ng/ml phorbol myristate acetate, 1 ␮M ionomycin, 10 ␮g/ml brefeldin A (all obtained from BioLegend), and 10 ng/ml recombinant IL-23 (Peprotech). Cells were stained with surface antibodies to the following markers: FITCconjugated CD3 (17A2), CD5 (53-7.3), and CD11c (N418); Pacific blueconjugated CD4 (GK1.5); allophycocyanin-conjugated CD90 (OX-1). The CD3⫺ CD5⫺ CD11c⫺ CD4⫹ CD90⫹ cells were defined as LTi cells. Analysis was carried out on a FACSAria II system (Becton Dickinson). Real-time PCR. Total RNA was reverse transcribed into cDNA by using oligo(dT) and Moloney murine leukemia virus reverse transcriptase (RT; Promega). The cDNA was subjected to real-time PCR amplification (Qiagen) for 40 cycles, with annealing and extension at 60°C, on a LightCycler 480 real-time PCR system (Roche). Primer sequences were the following: ␤-actin forward, 5=-TGGATGACGATATCGCTGCG-3=, and reverse, 5=-AGGGTCAGGATACCTCTCTT-3=; IL-7 forward, 5=-GGAAC TGATAGTAATTGCCCG-3=, and reverse, 5=-TTCAACTTGCGAGCAG CACG-3=; IFN-␣ forward, 5=-ACCTCAGGAACAAGAGAGCC-3=, and reverse, 5=-CTGCGGGAATCCAAAGTCCT-3=; IFN-␤ forward, 5=-TAA GCAGCTCCAGCTCCAAG-3=, and reverse, 5=-CCCTGTAGGTGAGGT TGATC-3=; IFN-␥ forward, 5=-GGATGCATTCATGAGTATTGC-3=, and reverse, 5=-CTTTTCCGCTTCCTGAGG-3=; CCL2 forward, 5=-TCCCAA TGAGTAGGCTGGAGAGC-3=, and reverse, 5=-TCCCCCAAGCATTGA CAGT-3=; F4/80 forward, 5=-GAGGCTTCCTGTCCAGCAAT-3=, and reverse, 5=-GGACCACAAGGTGAGTCACT-3=; MIF forward, 5=-TTTCTG TCGGAGCTCACCCA-3=, and reverse, 5=-CGCTAAAGTCATGAGCTG GT-3=; IL-6 forward, 5=-ACGATGATGCACTTGCAGA-3=, and reverse, 5=-GAGCATTGGAAATTGGGGTA-3=; IL-12p40 forward, 5=-CACATC TGCTGCTCCACAAG-3=, and reverse, 5=-CCGTCCGGAGTAATTTGG TG-3=; IL-23p19 forward, 5=-CTCTCG GAATCTCTGCATGC-3=, and reverse, 5=-ACCATCTTCACACTGGATACG-3=; IL-22 forward, 5=-AAG CTGCATGCTCACAGTGC-3=, and reverse, 5=-GGAGGTGGTACCTTT CCTGA-3=. ELISA. The mouse IL-7 concentration in whole colonic homogenates was determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocols (R&D Systems). The assay range of the IL-7 ELISA kit was 31.2 to 2,000 pg/ml. Statistical analysis. Results are expressed as the means ⫾ standard errors of the means (SEM). The statistical significance of differences between experimental groups was calculated using analysis of variance with a Bonferroni posttest or an unpaired Student’s t test. Additional statistical analysis for more than two variables was done with a multivariate analysis

IL-7 Promotes the Clearance of C. rodentium

of variance (MANOVA) test. All P values of ⬍0.05 were considered significant.

RESULTS

IECs express IL-7 in response to C. rodentium infection. To investigate the function of IL-7, we first examined the expression of IL-7 in colons of mice infected with C. rodentium. C57BL/6 mice were infected with C. rodentium for 1, 3, or 5 days and their mRNA or protein levels of IL-7 in the colon were measured. We found that infection with C. rodentium led to a marked increase in IL-7 mRNA levels in the colon (Fig. 1A). Consistent with mRNA levels, protein levels of IL-7 in the colon homogenates from animals infected with C. rodentium increased with time (Fig. 1B). To determine what type of cells produced IL-7, we harvested colons and analyzed intracellular IL-7 production by using multicolor flow cytometry analysis on day 3 postinfection with C. rodentium. IL-7


was produced in cells that express cytokeratin, a housekeeping gene for IECs (Fig. 1C). Moreover, as determined by immunofluorescence staining, IL-7-positive cells were mainly located among IECs in the C. rodentium-infected mice (Fig. 1D). These data indicate that IECs produce IL-7 in response to C. rodentium infection. IIFN-␥-producing NK1.1ⴙ cells contribute to IL-7 production in an IL-12-dependent fashion in response to C. rodentium infection. It has been reported that type 1 interferons and IFN-␥ are involved in IL-7 production in hepatocytes and IECs (18, 26). We therefore next investigated whether C. rodentium-induced IL-7 expression in the colon was dependent on IFNs. We pretreated C57BL/6 mice with a neutralizing anti-IFNAR1 or antiIFN-␥ Ab before inoculation with C. rodentium. C. rodentiuminduced upregulation of IL-7 mRNA levels in the colon was markedly reduced by anti-IFN-␥ treatment, whereas anti-


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FIG 1 C. rodentium induces IL-7 production from IECs. C57BL/6 mice were infected with 2 ⫻ 109 CFU of C. rodentium. (A) Real-time PCR analysis of IL-7 mRNA levels in colons from uninfected or C. rodentium-infected mice. (B) Mean IL-7 levels in whole-colon homogenates, measured in an ELISA. Data are the averages of analyses of 3 independent experiments (total n ⫽ 6). Data shown are means ⫾ SEM. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. (C) Intracellular IL-7 expression in colon tissue from C57BL/6 mice at day 3 postinfection with C. rodentium. Data are representative of analyses of 3 independent experiments (total n ⫽ 6). (D) Immunofluorescence staining of IL-7 in colon sections from C57BL/6 mice at day 3 postinfection with C. rodentium. Data are representative of analyses of 3 independent experiment (total n ⫽ 6).

Zhang et al.

IFNAR1 Ab did not affect IL-7 expression (Fig. 2A). We next examined whether IL-7 could reciprocally affect the expression of type 1 IFNs and IFN-␥. We pretreated mice with a neutralizing anti-IL-7R␣ Ab before C. rodentium infection and assessed the mRNA levels of IFNs 24 h later. We found that gene expression of IFN-␥ was markedly upregulated in the colon 24 h after C. rodentium infection, whereas levels of IFN-␣ and IFN-␤ were not altered (Fig. 2B). Moreover, the induction of IFN-␥ was not reduced by IL-7R␣ blockade (Fig. 2B). Since NK1.1⫹ cells produce IFN-␥

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at early time points after C. rodentium infection (5), we examined whether NK1.1⫹ cells are required for IL-7 production in response to C. rodentium infection. As expected, C. rodentium-induced expression of IFN-␥ was significantly decreased in NK1.1⫹ cell-depleted mice compared to IgG-treated control mice (Fig. 2C). Importantly, C. rodentium-induced expression of IL-7 was also reduced in the colons of NK1.1⫹ cell-depleted mice (Fig. 2C). We next examined whether IL-12, a promoter of IFN-␥ production in NK and T cells, was required for IL-7 production. We




FIG 2 IL-7 production from IECs is dependent of IFN-␥, IL-12, and NK1.1⫹ cells. (A) C57BL/6 mice were treated with anti-IFNAR1 or anti-IFN-␥ Ab 2 h prior C. rodentium infection. After 24 h, relative IL-7 mRNA levels in the colon were measured by real-time RT-PCR. Data are the averages of analyses of 3 independent experiment (total n ⫽ 6). (B) Mice were pretreated with anti-IL-7R␣ before C. rodentium infection. After 24 h, colons were analyzed for expression of the indicated genes. Data from 3 independent experiments (total n ⫽ 6) are shown. (C) NK1.1⫹ cells were depleted in C57BL/6 mice by injection of anti-NK1.1 Ab 24 h prior C. rodentium infection. After 24 h, relative IL-7 and IFN-␥ mRNA levels in colon tissue were measured by real-time RT-PCR. Data are the averages of analyses of 3 independent experiments (total n ⫽ 6). (D) IL-7 and IFN-␥ mRNA levels were measured after treatment with anti-IL-12p40 Abs. (E) C57BL/6 mice were treated with anti-IL-12p40 Ab 2 h prior to C. rodentium infection. After 24 h, intracellular IFN-␥ production levels were measured in NK1.1⫹ CD3⫺ cells in the colon. Data are representative of analyses of 3 independent experiments (total n ⫽ 6). Data shown are means ⫾ SEM. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.


pretreated mice with a neutralizing anti-IL-12p40 Ab before C. rodentium infection. C. rodentium-induced expression of IL-7 and IFN-␥ was almost completely abrogated in the colons of anti-IL12p40 Ab-treated mice compared to IgG-treated control mice (Fig. 2D). Moreover, C. rodentium infection-induced IFN-␥ production in intestinal NK1.1⫹ cells was also inhibited by anti-IL12p40 Ab treatment (Fig. 2E). Therefore, these data indicate that C. rodentium-induced production of IL-7 is at least partially dependent on IL-12- and IFN-␥-producing NK1.1⫹ cells. Blockade of IL-7R␣ impairs clearance of C. rodentium. We next examined whether IL-7 is required for protection from C. rodentium infection. We injected i.p. 100 ␮g anti-IL-7R␣ or its isotype control IgG into C. rodentium-infected C57B/6 mice every 2 days. C. rodentium infection has been shown to be associated with goblet cell loss, colonic crypt hyperplasia, and mucosal inflammation. We measured distal colonic weight to indirectly assess epithelial hyperplasia. Distal colons from anti-IL-7R␣-treated mice were significantly heavier (P ⫽ 0.01) on day 10 postinfection than those from control IgG-treated mice, suggesting increased hyperplasia after anti-IL-7R␣ treatment (Fig. 3A). Anti-IL-7R␣treated mice also had significantly higher (P ⬍ 0.001) numbers of


C. rodentium in the colons than did control mice (Fig. 3B). Moreover, anti-IL-7R␣-treated mice began to lose weight and die by day 14 postinfection, whereas control IgG-injected mice did not lose weight and all survived throughout the observation period of 28 days (Fig. 3C). Histological analysis and scoring revealed that blockade of IL-7R␣ enhanced the structural disruption and inflammation of the colon epithelium during C. rodentium infection, consistent with the higher bacterial counts and colon weights (Fig. 3D and E). Furthermore, anti-IL-7R␣-treated mice had smaller and significantly lower numbers of goblet cells at days 5 and 10 postinfection than control mice (Fig. 3F). Together, these data demonstrated that IL-7 plays a crucial role in the host mucosal defense against the intestinal pathogen C. rodentium. IL-7 contributes to early immune activation for clearance of C. rodentium. To further define the temporal requirements for IL-7 in host defense against C. rodentium, anti-IL-7R␣ Ab was administered to mice at different time points following infection with C. rodentium. The C57BL/6 mice that received anti-IL-7R␣ starting on day 0 or day 2 postinfection succumbed to infection by day 18 (Fig. 4A and B). However, blockade of IL-7R␣ starting on day 4 postinfection resulted in an intermediate degree of mortality


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FIG 3 IL-7 is required for the clearance of C. rodentium from the colon. C57BL/6 mice were treated with anti-IL-7R␣ or control IgG every 2 days and infected with 2 ⫻ 109 CFU of C. rodentium. (A) The colon weights and lengths were determined and are expressed as colon weight/length ratios. (B) CFU from plates spotted with homogenates from colons of the indicated groups was determined. Data are the averages of analyses of 3 independent experiments (total n ⫽ 6). (C) Average body weight changes (left) and survival (right) in control IgG- or anti-IL-7R␣-injected mice. Data are the averages of analyses of 3 independent experiments (total n ⫽ 10). (D) Representative histology of H&E-stained colons at day 10 postinfection. Magnification, ⫻20. (E) Pathology scores were measured as described in Materials and Methods. (F) Means of the total number of goblet cell per square millimeter at day 10 postinfection, based on 6 mice/time point. Data are representative of or the average of analyses of 3 independent experiments (total n ⫽ 6). Data shown are means ⫾ SEM. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.

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Blocked IL-7R␣ Ab was administered to C57BL/6 mice at days 0 (A), 2 (B), 4 (C), and 6 (D) following C. rodentium infection. Antibody treatment was initiated on the indicated day and continued every 2 days. All data are representative of 2 independent experiments with 5 mice per group or time point.

(Fig. 1C). Furthermore, mice receiving anti-IL-7R␣ Ab beginning on day 6 postinfection did not exhibit mortality after C. rodentium infection (Fig. 1D), suggesting that IL-7 is only essential for protective immunity against C. rodentium during the first 6 days of infection. Thus, these data demonstrate that during C. rodentium infection, innate immune cells are the main targets of IL-7 for early resistance to these pathogens. Tissue recruitment and activation of macrophages in response to C. rodentium require IL-7 signaling. To examine whether IL-7 is required for the optimal activation of innate immune cells against C. rodentium infection, we analyzed the migration and activation of intestinal macrophages and DCs during this bacterial infection. Flow cytometry analysis showed that the frequency and number of intestinal F4/80⫹ macrophages were significantly increased at day 5, and these increases were substantially reduced by anti-IL-7R␣ treatment (Fig. 5A and B). To understand the mechanisms by which IL-7 increases the number of macrophages in the colon, we measured mRNA levels of chemokine (C-C motif) ligand 2 (CCL2), an important chemokine for macrophage tissue migration (27). Infection with C. rodentium substantially increased CCL2 mRNA levels in the colon at day 5 postinfection, and this increase was abrogated by IL-7R␣ blockade (Fig. 5C, left panel). Correlating with the changes in CCL2 expression and numbers of macrophages in the colon, C. rodentiuminduced upregulation of F4/80 and macrophage migration inhibitory factor (MIF) was also significantly inhibited by IL-7R␣ blockade (Fig. 5C, right panel). Furthermore, colon expression of IL-6, IL-12p40, and IL-23p19, which are mainly produced by macrophages and DCs, were upregulated by C. rodentium infec-

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FIG 4 IL-7 is essential for immunity to C. rodentium during early infection.

tion, and this upregulation was reduced by IL-7R␣ blockade (Fig. 5D). In addition to promoting the tissue recruitment of macrophages, we speculate that IL-7 also directly affects intestinal macrophage activation. To address this possibility, we examined whether these macrophages expressed IL-7R␣, and we found that MHC class II⫹ CD103⫺ F4/80⫹ macrophages expressed high levels of surface IL-7R␣ (Fig. 5E). Moreover, C. rodentium-induced upregulation of CD86 in intestinal F4/80⫹ macrophages was inhibited by anti-IL-7R␣ treatment (Fig. 5F). In addition, C. rodentium-induced upregulation of CD86 and MHC class II expression on CD11b⫹ F4/80⫹ cells in mLNs was also markedly decreased by blockade of IL-7R␣ (Fig. 5G). Collectively, these results suggest that IL-7 acts directly on intestinal macrophages to enhance their activation and function. In contrast to macrophages, the C. rodentium infection-induced increase in intestinal CD103⫹ migratory cDCs was not affected by anti-IL-7R␣ treatment (Fig. 5A). Moreover, C. rodentium-induced upregulation of CD86 in intestinal cDCs was not altered by IL-7R␣ treatment (Fig. 5F). In addition, intestinal migratory cDCs did not express surface IL-7R␣ (Fig. 5E). Thus, these data indicate that IL-7 is required for recruitment and full activation of macrophages, but not cDCs, in the colon in response to C. rodentium. IL-7 is required for the expansion and function of CD4ⴙ LTi cells during C. rodentium infection. To more thoroughly define the function of IL-7 in the activation of innate immune cells against C. rodentium infection, we also examined whether IL-7 promotes CD4⫹ LTi cell activation, since CD4⫹ LTi cells contribute to protective immunity against C. rodentium infection (28). We found that blockade of IL-7R␣ inhibited IL-23 expression, a dominant inducer of IL-22 expression in CD4⫹ LTi cells. In comparison to uninfected mice, C. rodentium-infected mice had substantial increases in the percentages and numbers of CD4⫹ LTi cell populations (CD3⫺ CD5⫺ CD11c⫺ CD90⫹ CD4⫹) in the colon and mLNs, and these increases were almost completely abolished by IL-7R␣ blockade (Fig. 6A, B, and C). CD4⫹ LTi cells in the colon and mLNs of infected mice exhibited an increase in the frequency of Ki-67⫹ cells, which was also inhibited by blockade of IL-7R␣, suggesting that C. rodentium infection-induced proliferation of CD4⫹ LTi cells is dependent on IL-7 (Fig. 6D). In addition, C. rodentium-induced IL-22 production in CD4⫹ LTi cells in the colon and mLNs was decreased by IL-7R␣ blockade (Fig. 6E). Furthermore, the C. rodentium-induced increase in IL-22 mRNA levels in the colon was also abrogated by IL-7R␣ blockade (Fig. 6F). Taken together, these data indicate that IL-7 is required for the expansion and optimal function of CD4⫹ LTi cells during C. rodentium infection. To further evaluate the effect of IL-7 on CD4⫹ LTi cells, Rag1⫺/⫺ mice were infected with C. rodentium and administered either isotype control IgG or anti-IL-7R␣ Ab. C. rodentium infection in Rag1⫺/⫺ mice caused an increase in IL-7 protein levels in colonic homogenates (Fig. 6G) and an increase in the frequency of the CD4⫹ LTi population (Fig. 6H). The IL-7R␣ blockade completely abolished C. rodentium-induced expansion of CD4⫹ LTi cells (Fig. 6H). Moreover, administration of anti-IL-7R␣ Ab to infected Rag1⫺/⫺ mice led to a reduction in IL-22 and IL-23 gene expression in the colon (Fig. 6I). Finally, compared to Rag1⫺/⫺ mice treated with control IgG, those treated with anti-IL-7R␣ succumbed to infection at earlier time points (Fig. 6J). These data

Downloaded from http://iai.asm.org/ on November 17, 2015 by KUNGL. TEKNISKA HOGSKOLAN FIG 5 Blockade of IL-7R␣ inhibits recruitment and activation of macrophages in response to C. rodentium. Anti-IL-7R␣ Ab- or IgG-treated C57BL/6 mice were infected with C. rodentium and euthanized at day 5. (A) Percentage of CD103⫹ F4/80⫺ cDCs, CD103⫺ F4/80⫺ cDCs, and CD103⫺ F4/80⫹ macrophages in the colon in control; (B) absolute cell numbers of these cells in the colon. (C) Relative CCL2, F4/80, and MIF mRNA levels in colon. (D) Relative IL-6, IL-12p40, and IL-23p19 mRNA levels in colon. (E) Flow cytometric analysis of IL-7R␣ expression on the gated CD11c⫹ CD103⫺ cDCs, CD11c⫹ CD103⫺ cDCs, or CD11c⫹ F4/80⫹ macrophages in CD11c⫹ MHC class II⫹ cells in colons from naive mice. (F) Expression levels of CD86 in CD103⫹ migratory cDCs, CD103⫺ resident cDCs, or CD103⫺ F4/80⫹ macrophages in the colons of control IgG- or anti-IL-7R␣-treated mice euthanized at day 5. (G) Expression levels of CD86 and MHC class II in CD11b⫹ F4/80⫹ macrophages in the mLNs in control IgG- or anti-IL-7R␣-treated mice euthanized at day 5. All data are representative of or the average of analyses of 3 independent experiment (total n ⫽ 6). Data shown are the means ⫾ SEM. *, P ⬍ 0.05; **, P ⬍ 0.01.



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Tregs in the spleen and mLNs during C. rodentium infection. Anti-IL-7R␣ Abor IgG-treated C57BL/6 mice were infected orally with 2 ⫻ 109 CFU of C. rodentium. (A) Absolute numbers of CD4 and CD8 T cells in spleens and mLNs (n ⫽ 6). (B) Foxp3 mRNA levels (left) and absolute numbers of Foxp3⫹ Tregs in mLNs (right). Data are averages of analyses of 6 mice (2 mice per experiment, total of 3 independent experiments).

demonstrated that IL-7 is required for the expansion and function of CD4⫹ LTi cells and the protective innate immunity against C. rodentium. Blockade of IL-7 does not affect the number of T cells and Treg cells during C. rodentium infection. Previous studies showed that anti-IL-7R␣ (clone A7R34) inhibits binding of IL-7R to cell surface IL-7R␣ and does not deplete IL-7R-expressing cells (29, 30). In this study, we also confirmed that treatment with anti-IL-7R␣ during C. rodentium infection did not significantly reduce the overall T cell numbers in either spleens or mLNs (Fig. 7A). Moreover, blockade of IL-7R␣ did not change the expression levels of Foxp3, the key transcription factor controlling regulatory T cell development and function, or the number of Foxp3⫹ CD4⫹ T cells in response to C. rodentium infection (Fig. 7B). These data suggest that the effects of IL-7R␣ blockade on macrophages and CD4⫹ LTi cells in response to C. rodentium infection are not caused by altered numbers of T cell or Treg cells. DISCUSSION

The gastrointestinal tract and the intestinal mucus have efficient mechanisms that protect the epithelium from pathogenic bacteria by promoting bacterial clearance and separating bacteria from the

FIG 6 IL-7 is required for the expansion and activation of CD4⫹ LTi cells during C. rodentium infection. Anti-IL-7R␣ Ab- or IgG-treated C57BL/6 mice were

infected with C. rodentium on day 0 and sacrificed on day 5. (A and B) Frequencies of CD4⫹ LTi cells in the colon (A) and mLNs (B) of uninfected and infected mice. Populations were gated on live lineage⫺ CD4⫹ CD90⫹ cells. Lineage markers included CD3, CD5, and CD11c. (C) Absolute CD4⫹ LTi cell numbers in colon (left) and mLNs (right). (D) Frequency of Ki-67⫹ CD4⫹ LTi cells in the colon and mLNs. (E) Intracellular IL-22 production in CD4⫹ LTi cells in the colon and mLNs. (F) IL-22 mRNA levels in the colon were measured by real-time RT-PCR, and data are presented relative to those for ␤-actin. Data are representative of or the averages of analyses of 6 independent samples (2 mice per experiment, total of 3 independent experiments). (G to J) C57BL/6 Rag1⫺/⫺ were administered an isotype control MAb or an anti-IL-7R␣ Abs every 2 days and infected with C. rodentium on day 0. (G) Mean IL-7 levels in whole-colon homogenates, measured by ELISA. (H) Frequencies of CD4⫹ CD90⫹ cells in lineage⫺ gated splenocytes from antibody-treated Rag1⫺/⫺ mice. (I) IL-22 and IL-23p19 mRNA levels in colons was measured by real-time RT-PCR, and data are presented relative to those for ␤-actin. Data are the averages of analyses of 6 independent samples for each group (3 samples per experiment, total of 2 independent experiments). (J) Survival rates in control IgG- or anti-IL-7R␣-injected C57BL/6 Rag1⫺/⫺ mice (n ⫽ 5). Data shown are means ⫾ SEM. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001.



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FIG 7 Anti-IL-7R␣ treatment does not affect the numbers of total T cells or

mucosal immune cells, thereby inhibiting inflammation and infection (31). In this study, we found that infection with the enteric rodent pathogen C. rodentium promoted IL-7 production from IECs, in agreement with a published study suggesting that H. pylori infection causes increased intestinal IL-7 in humans (32). Therefore, intestinal epithelium-derived IL-7 may be one of the important molecules that inhibit inflammation and infection caused by various bacteria. We demonstrated that one important mechanism by which IL-7 increases the number of macrophages in the colon is by promoting the expression of CCL2, a critical chemoattractant of macrophages. In addition to promoting macrophage recruitment to the colon, IL-7 may also directly enhance the activation and function of macrophages, which is supported by the observations that intestinal F4/80⫹ macrophages express high levels of IL-7R␣ and that the IL-7R␣ signaling pathway is required for optimal activation of macrophages both in the colon and in mLNs. Thus, IL-7 enhances both tissue migration and activation of macrophages during C. rodentium infection. In contrast to macrophages, CD103⫹ migratory cDCs in the colon did not express IL-7R␣. In response to C. rodentium, the frequency of CD103⫹ migratory cDCs in the colon and mLNs were markedly increased, suggesting the migration of C. rodentium-activated cDCs to these tissues (4, 33). However, the activation and migration of CD103⫹ cDCs were independent of the IL-7R␣ signaling pathway, consistent with the lack of IL-7R␣ expression in colon CD103⫹ cDCs. Hence, the IL-7R␣ signaling pathway does not contribute to the activation and migration of intestinal cDCs. CD4⫹ LTi cells have the capacity to promote lymphoid tissue organogenesis and maintenance of lymphoid tissues (34). CD4⫹ LTi cells are defined, based on a panel of surface markers, as Lin⫺ c⫺ kit⫹ CD4⫹ CD44⫹ CD127(IL-7R␣)⫹ CD25⫹ CD90⫹ CCR6⫹, and IL-7 has been shown to regulate their survival and function (14). Consistent with this, we also found that IL-7R␣ blockade reduced the CD4⫹ LTi cell population in colon and mLN cells of C. rodentium-infected mice, which was accompanied by decreased proliferation of these cells in the absence of the IL-7R␣ signaling pathway. Moreover, previous studies have demonstrated that CD4⫹ LTi cells express IL-22 (28, 35), an important cytokine in eliciting an antimicrobial immune response and maintaining mucosal barrier integrity within the intestine (36, 37), in an IL-23dependent manner (28, 37). We showed that IL-7R␣ blockade in C. rodentium-infected mice led to a significant decrease in IL-23 expression in the colon, which may in turn inhibit the production of IL-22 by CD4⫹ LTi cells. Previous studies demonstrated that macrophages and DCs are the main producers of IL-23 (3, 28). Our data suggested that IL-7R␣ signaling is required for the optimal recruitment and activation of macrophages, but not cDCs, in

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amine whether administration of exogenous IL-7 during C. rodentium infection can facilitate the clearance of this pathogen. In summary, the present study demonstrates the crucial protective functions of IL-7 in C. rodentium infection. This knowledge will enable further investigation of the role of IL-7 in other bacterial infections and a comprehensive understanding of its function in various aspects of bacterial infections, in order to develop novel therapeutic strategies. ACKNOWLEDGMENTS We thank the Shanghai Public Health Clinical Center animal facility for maintaining animals and the histology lab for helping with immunofluorescence assays. This study was supported by the Research Fund for International Young Scientists from the National Natural Science Foundation of China (81450110090).

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response to C. rodentium. Although further investigation is required, macrophages may be the main producers of IL-23 in response to C. rodentium. Furthermore, our data showed that blockade of IL-7R␣ in C. rodentium-infected Rag1⫺/⫺ mice resulted in reduced CD4⫹ LTi cells, reduced expression of IL-22 and IL-23, and a more rapid onset of host mortality. Collectively, these data identify a previously unrecognized role of IL-7 in the expansion and function of CD4⫹ LTi cells in the innate immune responses against enteric bacterial infection. The IL-17-mediated immune response has been shown to be required for protection against C. rodentium infection (8). Previous studies identified IL-7 as an important promoter of IL-17 production in T helper (Th) and ␥␦ T cells (38, 39). Therefore, blockade of IL-7R␣ may also affect the IL-17 response during C. rodentium infection. Indeed, we showed in this study that blockade of IL-7R␣ significantly downregulated expression of IL-23, a potent promoter of Th17 differentiation and stabilization. Consistent with this, we found that colon-infiltrating Th17 cells were decreased by blockade with IL-7R␣ at day 10 of infection (data not shown). These results indicated that the Th17 response during C. rodentium infection is also suppressed by IL-7R␣ blockade, possibly as a result of impaired IL-23 production. Future studies will further define the cellular and molecular mechanisms by which the IL-7R␣ blockade causes an impaired Th17 response during C. rodentium infection. We found that suppression of innate immune responses by IL-7R␣ blockade during C. rodentium infection led to increased bacterial burden and colon inflammation. The increased inflammation of the colon in mice with impaired immunity against C. rodentium may be caused by the persistence of this pathogen. Concurrent with this idea, increased neutrophil infiltration in the colon was observed in response to C. rodentium infection in antiIL-7R␣-treated mice (data not shown). Although neutrophils are the first cells to be recruited to the site of bacterial infection and have a potent antibacterial function through the production of oxidants and proteinases, excessive recruitment and accumulation of activated neutrophils in the intestine under pathological conditions is associated with mucosal injury (40). Hence, in the absence of IL-7R␣ signaling, the defective immune response against C. rodentium leads to an increased and persistent bacterial burden, which may be the cause of increased neutrophils in the colon, resulting in subsequent colon inflammation and damage. Further investigation will address this possibility and elucidate the specific mechanisms by which IL-7 affects neutrophil activities and colon inflammation during C. rodentium infection. In this study, we injected C57BL/6 mice with anti-IL-7R␣ Abs during C. rodentium infection. Administration of an anti-IL-7R␣ Ab has the advantage that IL-7 in mice can be only depleted during infection. In comparison, IL-7R␣ knockout mice have dramatically reduced mature T cells and innate lymphoid cells, due to defective development and homeostasis of these cells, making it difficult to evaluate the effect of IL-7 specifically during infection (41, 42). However, it is still important to test whether IL-7R␣ knockout mice have a higher susceptibility and mortality in response to C. rodentium infection than normal control mice, which will be investigated in a future study. On the other hand, overexpression of IL-7 promotes the development and homeostasis of T cells, innate lymphoid cells, and macrophages and promotes tissue inflammation (43–45). Hence, in future research, we will also ex-


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S100A4 contributes to colitis development by increasing the adherence of Citrobacter rodentium in intestinal epithelial cells.

DOCK2 confers immunity and intestinal colonization resistance to Citrobacter rodentium infection.

E pathogenesis initiation to an early window of opportunity.

Galectin-3 Plays an Important Role in Innate Immunity to Gastric Infection by Helicobacter pylori.

MyD88 signaling in dendritic cells and the intestinal epithelium controls immunity against intestinal infection with C. rodentium.

Citrobacter rodentium: infection, inflammation and the microbiota.

The role of Galectin-1 and Galectin-3 in the mucosal immune response to Citrobacter rodentium infection.

Mitochondrial UPR-regulated innate immunity provides resistance to pathogen infection.

Protein Malnutrition Modifies Innate Immunity and Gene Expression by Intestinal Epithelial Cells and Human Rotavirus Infection in Neonatal Gnotobiotic Pigs.

Dysbiosis caused by vitamin D receptor deficiency confers colonization resistance to Citrobacter rodentium through modulation of innate lymphoid cells.

The Serine Protease Autotransporter Pic Modulates Citrobacter rodentium Pathogenesis and Its Innate Recognition by the Host.

Intestinal macrophages arising from CCR2(+) monocytes control pathogen infection by activating innate lymphoid cells.

Innate immunity against HIV-1 infection.

The Role of Sphingolipids on Innate Immunity to Intestinal Salmonella Infection.

In this issue: autoimmunity and innate immunity.

Respiratory epithelial cells orchestrate pulmonary innate immunity.

Retinoic Acid and Its Role in Modulating Intestinal Innate Immunity.

Cutting Edge: Expression of IRF8 in Gastric Epithelial Cells Confers Protective Innate Immunity against Helicobacter pylori Infection.

A novel mechanism linking memory stem cells with innate immunity in protection against HIV-1 infection.

Regulatory T cells promote a protective Th17-associated immune response to intestinal bacterial infection with C. rodentium.

IL-6-dependent manner.

mTOR is critical for intestinal T-cell homeostasis and resistance to Citrobacter rodentium.

Attenuation of intestinal inflammation in interleukin-10-deficient mice infected with Citrobacter rodentium.

HRS plays an important role for TLR7 signaling to orchestrate inflammation and innate immunity upon EV71 infection.