Interleukin-7 Produced by Intestinal Epithelial Cells in Response toCitrobacter rodentium Infection Plays a Major Role in InnateImmunity against This Pathogen

Wei Zhang,a Jiang-Yuan Du,a Qing Yu,b Jun-O Jina

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

Interleukin-7 (IL-7) engages multiple mechanisms to overcome chronic viral infections, but the role of IL-7 in bacterial infec-tions, especially enteric bacterial infections, remains unclear. Here we characterized the previously unexplored role of IL-7 in theinnate immune response to the attaching and effacing bacterium Citrobacter rodentium. C. rodentium infection induced IL-7production from intestinal epithelial cells (IECs). IL-7 production from IECs in response to C. rodentium was dependent ongamma interferon (IFN-�)-producing NK1.1� cells and IL-12. Treatment with anti-IL-7R� antibody during C. rodentium infec-tion resulted in a higher bacterial burden, enhanced intestinal damage, and greater weight loss and mortality than observed withthe control IgG treatment. IEC-produced IL-7 was only essential for protective immunity against C. rodentium during the first 6days after infection. An impaired bacterial clearance upon IL-7R� blockade was associated with a significant decrease in macro-phage accumulation and activation in the colon. Moreover, C. rodentium-induced expansion and activation of intestinal CD4�

lymphoid tissue inducer (LTi) cells was completely abrogated by IL-7R� blockade. Collectively, these data demonstrate that IL-7is produced by IECs in response to C. rodentium infection and plays a critical role in the protective immunity against this intesti-nal attaching and effacing bacterium.

Citrobacter rodentium is a mouse extracellular enteric pathogenthat mimics human-enteropathogenic Escherichia coli (EPEC)

and enterohemorrhagic E coli (EHEC). These bacterial pathogensattach intimately to intestinal epithelium and cause subcellularattaching and effacing lesions, which lead to severe diarrhea, vom-iting, and fever, with high rates of fatality (1). C. rodentium infec-tion of mice causes epithelial hyperplasia in the colon and cecum,goblet cell loss, and mucosal infiltration with macrophages, lym-phocytes, and neutrophils (2). Therefore, this is an ideal model todissect how immune cells interact with gut epithelial pathogens.The innate immune cells, including macrophages, dendritic cells(DCs), natural killer (NK) cells, neutrophils, and innate lymphoidcells (ILCs) have been shown to play an essential role in the clear-ance of C. rodentium infection (3–6). Moreover, the adaptive im-mune cells, mostly T and B cells, are also required for the clearanceof 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 miceand are necessary for an effective immune defense against thispathogen (3–5, 7, 9).

IL-7 is a stroma-derived cytokine that can be secreted by fetalliver cells, stromal cells in the bone marrow and thymus, and in-testinal epithelial cells (IECs) (10). IL-7 acts on various cellsthrough its receptor, a heterodimer consisting of an alpha-chain(IL-7R�) and the common cytokine receptor gamma chain. TheIL-7 receptor is expressed on lymphoid T and B precursors, innatelymphoid cells, antigen-presenting cells (APCs), and mature Tcells (11). At physiological levels, IL-7 is integral to T and B celldevelopment in primary lymphoid organs and plays an essentialrole 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 inducesproliferation of naive and memory T cells (15) and enhances ef-

fector T cell responses, preferentially T helper 1 (Th1) and Th17responses (16–18). These functional effects of IL-7 on T cells makeIL-7 a critical enhancer of protective immunity, as well as of au-toimmunity and inflammation (16, 17, 19, 20).

In the case of bacterial infections, gastric tissue biopsy speci-mens from patients infected with Helicobacter pylori (21) or miceinfected with Mycobacterium tuberculosis have increased expres-sion of IL-7 (22). In addition, administration of exogenous IL-7enhanced the survival of M. tuberculosis-infected mice (23). How-ever, the role of IL-7 in intestinal bacterial infection and the re-lated inflammation has not been well characterized. Moreover, thefunction of IL-7 in immune responses against bacterial pathogensis much less understood than its role in antiviral immunity. In thepresent study, we investigated the in vivo role of IL-7 in host re-sponses against C. rodentium infection. We found that C. roden-tium infection induces expression of IL-7 in intestinal epithelialcells. We hypothesized that IL-7 may play a crucial role in theinnate immune activation required for the clearance of C. roden-

Received 9 March 2015 Returned for modification 18 April 2015Accepted 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 intestinalepithelial cells in response to Citrobacter rodentium infection plays a major role ininnate 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, Junojin1@gmail.com.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00320-15

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tium in vivo, and we undertook the current study to test this hy-pothesis.

MATERIALS AND METHODSAnimals. C57BL/6 mice were purchased from Shanghai Public HealthClinical Center, and C57BL/6 Rag1�/� mice were purchased from Jack-son Laboratory and kept under pathogen-free conditions. All experimentswere carried out under the guidelines of the Institutional Animal Care andUse Committee at the Shanghai Public Health Clinical Center. The pro-tocol was approved by the committee on the Ethics of Animal Experi-ments of the Shanghai Public Health Clinical Center (animal protocolSYXK-2010-0098).

Bacterial infections. C. rodentium DBS100 wild type was cultured inbrain heart infusion broth (Sigma-Aldrich, St. Louis, MO) for 16 h at 37°Cin air with shaking (180 rpm). Bacteria were pelleted by centrifugation,washed with phosphate-buffered saline (PBS), and centrifuged again be-fore a final resuspension in PBS to an optical density at 600 nm of 1.0. Thenumber of viable bacteria was determined after serial dilution and platingonto 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 ofmechanically homogenized whole colons was determined via serial dilu-tions on MacConkey’s agar.

In vivo administration of antibody. Female C57BL/6 mice were in-jected 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. roden-tium. Some mice, prior to infection, were injected i.p. with 200 �g anti-NK1.1 Ab for depletion of NK cells. Mice were then sacrificed and organswere harvested for analysis.

Histology and immunofluorescence staining. Colon samples werefixed in 4% paraformaldehyde, embedded in paraffin, and sectioned to a5-�m thickness. Sections were then stained with hematoxylin and eosin(H&E) and examined for tissue damage. Colon sections were evaluated toobtain pathology scores for evidence of inflammatory damage, such asgoblet cell loss, crypt elongation, mucosal thickening, and epithelial in-jury, 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 incubatedwith biotin-conjugated anti-cytokeratin antibody (AE1/AE3; Abcam) andrabbit anti-mouse IL-7 antibody (M-19), followed by Alexa Fluor 488-streptavidin and Alexa Fluor 647-conjugated secondary Abs. The stainedsamples were examined with a laser scanning confocal microscope (LeicaMicrosystems).

Antibodies and cytokines. Cells were stained and analyzed on aFACSAria II system (Becton Dickinson), with dead cells excluded basedon forward light scatter. The following fluorescence-conjugated Abs wereused: 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) classII (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 depila-tion 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 isolatedand washed twice in ice-cold PBS. The tissues were cut open longitudi-nally, and mucus and gross debris were quickly removed by covering thespecimen with dry paper towels. The samples were cut in to 0.5- to 1-cmpieces. IECs were separated from intestinal pieces by incubating in 0.15%dithiothreitol–Hanks’ balanced salt solution buffer with shaking for 30min at 37°C. IECs were collected by filtering through a mesh screen. Afterepithelial removal, lamina propria cells were collected by mincing theremaining tissue into, followed by digestion with 2% fetal bovine serum(FBS) containing collagenase with shaking for 30 min at 37°C. The cellswere filtered through a 100-�m nylon mesh and washed, and the resulting

pellet was resuspended in RPMI 1640 medium and layered over His-topaque-1.077 (Sigma-Aldrich). After centrifugation at 1,700 � g for 10min, the low-density fraction (�1.077 g/cm3) was collected. The singlecells were resuspended in culture medium.

IEC analysis. Isolated IECs (1 � 106) were incubated with 10 �g/mlbrefeldin A (Biolegend) for 4 h. Cells were then intracellularly stained withAlexa Fluor 488-conjugated anti-cytokeratin (AE1/AE3; eBioscience) andrabbit anti-mouse IL-7 (M-19) Abs, followed by Alexa Fluor 647-conju-gated anti-rabbit secondary Abs. Dead cells were excluded based on for-ward light scatter. At least 100,000 events were collected from each sampleby gating on live cells, and data were analyzed using FlowJo software(TreeStar Inc.).

Macrophage and cDC analysis. Single-cell suspensions from colons andmesenteric lymph nodes (mLNs) were incubated for 30 min with the follow-ing 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-conjugatedCD45 (30-F11). Among CD45� lineage� cells, the CD103� CD11c� cellsand CD103� CD11c� cells were defined as migratory cDCs and residentcDCs, respectively (24, 25). The CD11c� F4/80� cells were defined as mac-rophages. Analysis was carried out on a FACSAria II system (Becton Dickin-son).

CD4� LTi cell analysis. Single-cell suspensions from colons andmLNs were stimulated directly ex vivo by incubation for 4 h with 50 ng/mlphorbol myristate acetate, 1 �M ionomycin, 10 �g/ml brefeldin A (allobtained from BioLegend), and 10 ng/ml recombinant IL-23 (Peprotech).Cells were stained with surface antibodies to the following markers: FITC-conjugated CD3 (17A2), CD5 (53-7.3), and CD11c (N418); Pacific blue-conjugated 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 byusing 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 Light-Cycler 480 real-time PCR system (Roche). Primer sequences were thefollowing: -actin forward, 5=-TGGATGACGATATCGCTGCG-3=, andreverse, 5=-AGGGTCAGGATACCTCTCTT-3=; IL-7 forward, 5=-GGAACTGATAGTAATTGCCCG-3=, and reverse, 5=-TTCAACTTGCGAGCAGCACG-3=; IFN-� forward, 5=-ACCTCAGGAACAAGAGAGCC-3=, andreverse, 5=-CTGCGGGAATCCAAAGTCCT-3=; IFN- forward, 5=-TAAGCAGCTCCAGCTCCAAG-3=, and reverse, 5=-CCCTGTAGGTGAGGTTGATC-3=; IFN-� forward, 5=-GGATGCATTCATGAGTATTGC-3=, andreverse, 5=-CTTTTCCGCTTCCTGAGG-3=; CCL2 forward, 5=-TCCCAATGAGTAGGCTGGAGAGC-3=, and reverse, 5=-TCCCCCAAGCATTGACAGT-3=; F4/80 forward, 5=-GAGGCTTCCTGTCCAGCAAT-3=, and re-verse, 5=-GGACCACAAGGTGAGTCACT-3=; MIF forward, 5=-TTTCTGTCGGAGCTCACCCA-3=, and reverse, 5=-CGCTAAAGTCATGAGCTGGT-3=; IL-6 forward, 5=-ACGATGATGCACTTGCAGA-3=, and reverse,5=-GAGCATTGGAAATTGGGGTA-3=; IL-12p40 forward, 5=-CACATCTGCTGCTCCACAAG-3=, and reverse, 5=-CCGTCCGGAGTAATTTGGTG-3=; IL-23p19 forward, 5=-CTCTCG GAATCTCTGCATGC-3=, andreverse, 5=-ACCATCTTCACACTGGATACG-3=; IL-22 forward, 5=-AAGCTGCATGCTCACAGTGC-3=, and reverse, 5=-GGAGGTGGTACCTTTCCTGA-3=.

ELISA. The mouse IL-7 concentration in whole colonic homogenateswas determined using enzyme-linked immunosorbent assay (ELISA) kitsaccording to the manufacturer’s protocols (R&D Systems). The assayrange of the IL-7 ELISA kit was 31.2 to 2,000 pg/ml.

Statistical analysis. Results are expressed as the means standarderrors of the means (SEM). The statistical significance of differences be-tween experimental groups was calculated using analysis of variance witha Bonferroni posttest or an unpaired Student’s t test. Additional statisticalanalysis for more than two variables was done with a multivariate analysis

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of variance (MANOVA) test. All P values of �0.05 were considered sig-nificant.

RESULTSIECs express IL-7 in response to C. rodentium infection. To in-vestigate the function of IL-7, we first examined the expression ofIL-7 in colons of mice infected with C. rodentium. C57BL/6 micewere infected with C. rodentium for 1, 3, or 5 days and their mRNAor protein levels of IL-7 in the colon were measured. We foundthat infection with C. rodentium led to a marked increase in IL-7mRNA levels in the colon (Fig. 1A). Consistent with mRNA levels,protein levels of IL-7 in the colon homogenates from animalsinfected with C. rodentium increased with time (Fig. 1B). To de-termine what type of cells produced IL-7, we harvested colons andanalyzed intracellular IL-7 production by using multicolor flowcytometry analysis on day 3 postinfection with C. rodentium. IL-7

was produced in cells that express cytokeratin, a housekeepinggene for IECs (Fig. 1C). Moreover, as determined by immunoflu-orescence staining, IL-7-positive cells were mainly located amongIECs in the C. rodentium-infected mice (Fig. 1D). These data in-dicate that IECs produce IL-7 in response to C. rodentium infec-tion.

IIFN-�-producing NK1.1� cells contribute to IL-7 produc-tion in an IL-12-dependent fashion in response to C. rodentiuminfection. 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-inducedIL-7 expression in the colon was dependent on IFNs. We pre-treated C57BL/6 mice with a neutralizing anti-IFNAR1 or anti-IFN-� Ab before inoculation with C. rodentium. C. rodentium-induced upregulation of IL-7 mRNA levels in the colon wasmarkedly reduced by anti-IFN-� treatment, whereas anti-

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-7mRNA 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 theaverages 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-7expression in colon tissue from C57BL/6 mice at day 3 postinfection with C. rodentium. Data are representative of analyses of 3 independent experiments (totaln � 6). (D) Immunofluorescence staining of IL-7 in colon sections from C57BL/6 mice at day 3 postinfection with C. rodentium. Data are representative ofanalyses of 3 independent experiment (total n � 6).

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IFNAR1 Ab did not affect IL-7 expression (Fig. 2A). We nextexamined whether IL-7 could reciprocally affect the expression oftype 1 IFNs and IFN-�. We pretreated mice with a neutralizinganti-IL-7R� Ab before C. rodentium infection and assessed themRNA levels of IFNs 24 h later. We found that gene expression ofIFN-� was markedly upregulated in the colon 24 h after C. roden-tium infection, whereas levels of IFN-� and IFN- were not al-tered (Fig. 2B). Moreover, the induction of IFN-� was not reducedby IL-7R� blockade (Fig. 2B). Since NK1.1� cells produce IFN-�

at early time points after C. rodentium infection (5), we examinedwhether NK1.1� cells are required for IL-7 production in re-sponse to C. rodentium infection. As expected, C. rodentium-in-duced 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 wasalso reduced in the colons of NK1.1� cell-depleted mice (Fig. 2C).We next examined whether IL-12, a promoter of IFN-� produc-tion 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 priorC. 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 independentexperiment (total n � 6). (B) Mice were pretreated with anti-IL-7R� before C. rodentium infection. After 24 h, colons were analyzed for expression of theindicated 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 Ab24 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 ofanalyses 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 micewere 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� cellsin 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.

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pretreated mice with a neutralizing anti-IL-12p40 Ab before C.rodentium infection. C. rodentium-induced expression of IL-7 andIFN-� was almost completely abrogated in the colons of anti-IL-12p40 Ab-treated mice compared to IgG-treated control mice(Fig. 2D). Moreover, C. rodentium infection-induced IFN-� pro-duction in intestinal NK1.1� cells was also inhibited by anti-IL-12p40 Ab treatment (Fig. 2E). Therefore, these data indicate thatC. rodentium-induced production of IL-7 is at least partially de-pendent on IL-12- and IFN-�-producing NK1.1� cells.

Blockade of IL-7R� impairs clearance of C. rodentium. Wenext examined whether IL-7 is required for protection from C.rodentium infection. We injected i.p. 100 �g anti-IL-7R� or itsisotype control IgG into C. rodentium-infected C57B/6 mice every2 days. C. rodentium infection has been shown to be associatedwith goblet cell loss, colonic crypt hyperplasia, and mucosal in-flammation. We measured distal colonic weight to indirectly as-sess epithelial hyperplasia. Distal colons from anti-IL-7R�-treatedmice were significantly heavier (P � 0.01) on day 10 postinfectionthan those from control IgG-treated mice, suggesting increasedhyperplasia 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). More-over, anti-IL-7R�-treated mice began to lose weight and die byday 14 postinfection, whereas control IgG-injected mice did notlose weight and all survived throughout the observation period of28 days (Fig. 3C). Histological analysis and scoring revealed thatblockade of IL-7R� enhanced the structural disruption and in-flammation of the colon epithelium during C. rodentium infec-tion, consistent with the higher bacterial counts and colon weights(Fig. 3D and E). Furthermore, anti-IL-7R�-treated mice hadsmaller and significantly lower numbers of goblet cells at days 5and 10 postinfection than control mice (Fig. 3F). Together, thesedata demonstrated that IL-7 plays a crucial role in the host muco-sal defense against the intestinal pathogen C. rodentium.

IL-7 contributes to early immune activation for clearance ofC. rodentium. To further define the temporal requirements forIL-7 in host defense against C. rodentium, anti-IL-7R� Ab wasadministered to mice at different time points following infectionwith C. rodentium. The C57BL/6 mice that received anti-IL-7R�starting on day 0 or day 2 postinfection succumbed to infection byday 18 (Fig. 4A and B). However, blockade of IL-7R� starting onday 4 postinfection resulted in an intermediate degree of mortality

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 infectedwith 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 platesspotted 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 independentexperiments (total n � 10). (D) Representative histology of H&E-stained colons at day 10 postinfection. Magnification, �20. (E) Pathology scores were measuredas 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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(Fig. 1C). Furthermore, mice receiving anti-IL-7R� Ab beginningon day 6 postinfection did not exhibit mortality after C. rodentiuminfection (Fig. 1D), suggesting that IL-7 is only essential for pro-tective immunity against C. rodentium during the first 6 days ofinfection. Thus, these data demonstrate that during C. rodentiuminfection, innate immune cells are the main targets of IL-7 forearly resistance to these pathogens.

Tissue recruitment and activation of macrophages in re-sponse to C. rodentium require IL-7 signaling. To examinewhether IL-7 is required for the optimal activation of innate im-mune cells against C. rodentium infection, we analyzed the migra-tion and activation of intestinal macrophages and DCs during thisbacterial infection. Flow cytometry analysis showed that the fre-quency and number of intestinal F4/80� macrophages were sig-nificantly increased at day 5, and these increases were substantiallyreduced by anti-IL-7R� treatment (Fig. 5A and B). To understandthe mechanisms by which IL-7 increases the number of macro-phages in the colon, we measured mRNA levels of chemokine(C-C motif) ligand 2 (CCL2), an important chemokine for mac-rophage tissue migration (27). Infection with C. rodentium sub-stantially increased CCL2 mRNA levels in the colon at day 5postinfection, and this increase was abrogated by IL-7R� blockade(Fig. 5C, left panel). Correlating with the changes in CCL2 expres-sion and numbers of macrophages in the colon, C. rodentium-induced upregulation of F4/80 and macrophage migration inhib-itory factor (MIF) was also significantly inhibited by IL-7R�blockade (Fig. 5C, right panel). Furthermore, colon expression ofIL-6, IL-12p40, and IL-23p19, which are mainly produced bymacrophages and DCs, were upregulated by C. rodentium infec-

tion, and this upregulation was reduced by IL-7R� blockade(Fig. 5D).

In addition to promoting the tissue recruitment of macro-phages, we speculate that IL-7 also directly affects intestinal mac-rophage activation. To address this possibility, we examinedwhether these macrophages expressed IL-7R�, and we found thatMHC class II� CD103� F4/80� macrophages expressed high lev-els of surface IL-7R� (Fig. 5E). Moreover, C. rodentium-inducedupregulation of CD86 in intestinal F4/80� macrophages was in-hibited by anti-IL-7R� treatment (Fig. 5F). In addition, C. roden-tium-induced upregulation of CD86 and MHC class II expressionon CD11b� F4/80� cells in mLNs was also markedly decreased byblockade of IL-7R� (Fig. 5G). Collectively, these results suggestthat IL-7 acts directly on intestinal macrophages to enhance theiractivation and function.

In contrast to macrophages, the C. rodentium infection-in-duced increase in intestinal CD103� migratory cDCs was not af-fected by anti-IL-7R� treatment (Fig. 5A). Moreover, C. roden-tium-induced upregulation of CD86 in intestinal cDCs was notaltered by IL-7R� treatment (Fig. 5F). In addition, intestinal mi-gratory cDCs did not express surface IL-7R� (Fig. 5E). Thus, thesedata indicate that IL-7 is required for recruitment and full activa-tion of macrophages, but not cDCs, in the colon in response to C.rodentium.

IL-7 is required for the expansion and function of CD4� LTicells during C. rodentium infection. To more thoroughly definethe function of IL-7 in the activation of innate immune cellsagainst C. rodentium infection, we also examined whether IL-7promotes CD4� LTi cell activation, since CD4� LTi cells contrib-ute to protective immunity against C. rodentium infection (28).We found that blockade of IL-7R� inhibited IL-23 expression, adominant inducer of IL-22 expression in CD4� LTi cells. In com-parison to uninfected mice, C. rodentium-infected mice had sub-stantial increases in the percentages and numbers of CD4� LTi cellpopulations (CD3� CD5� CD11c� CD90� CD4�) in the colonand mLNs, and these increases were almost completely abolishedby IL-7R� blockade (Fig. 6A, B, and C). CD4� LTi cells in thecolon and mLNs of infected mice exhibited an increase in thefrequency of Ki-67� cells, which was also inhibited by blockade ofIL-7R�, suggesting that C. rodentium infection-induced prolifer-ation of CD4� LTi cells is dependent on IL-7 (Fig. 6D). In addi-tion, C. rodentium-induced IL-22 production in CD4� LTi cells inthe colon and mLNs was decreased by IL-7R� blockade (Fig. 6E).Furthermore, the C. rodentium-induced increase in IL-22 mRNAlevels in the colon was also abrogated by IL-7R� blockade (Fig.6F). Taken together, these data indicate that IL-7 is required forthe 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 administeredeither isotype control IgG or anti-IL-7R� Ab. C. rodentium infec-tion in Rag1�/� mice caused an increase in IL-7 protein levels incolonic homogenates (Fig. 6G) and an increase in the frequency ofthe CD4� LTi population (Fig. 6H). The IL-7R� blockade com-pletely abolished C. rodentium-induced expansion of CD4� LTicells (Fig. 6H). Moreover, administration of anti-IL-7R� Ab toinfected Rag1�/� mice led to a reduction in IL-22 and IL-23 geneexpression in the colon (Fig. 6I). Finally, compared to Rag1�/�

mice treated with control IgG, those treated with anti-IL-7R� suc-cumbed to infection at earlier time points (Fig. 6J). These data

FIG 4 IL-7 is essential for immunity to C. rodentium during early infection.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 ini-tiated on the indicated day and continued every 2 days. All data are represen-tative of 2 independent experiments with 5 mice per group or time point.

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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 wereinfected 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 thecolon 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, andIL-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� residentcDCs, 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 MHCclass 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 averageof analyses of 3 independent experiment (total n � 6). Data shown are the means SEM. *, P � 0.05; **, P � 0.01.

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demonstrated that IL-7 is required for the expansion and functionof CD4� LTi cells and the protective innate immunity against C.rodentium.

Blockade of IL-7 does not affect the number of T cells andTreg cells during C. rodentium infection. Previous studiesshowed that anti-IL-7R� (clone A7R34) inhibits binding of IL-7Rto cell surface IL-7R� and does not deplete IL-7R-expressing cells(29, 30). In this study, we also confirmed that treatment withanti-IL-7R� during C. rodentium infection did not significantlyreduce the overall T cell numbers in either spleens or mLNs (Fig.7A). Moreover, blockade of IL-7R� did not change the expressionlevels of Foxp3, the key transcription factor controlling regulatoryT cell development and function, or the number of Foxp3� CD4�

T cells in response to C. rodentium infection (Fig. 7B). These datasuggest that the effects of IL-7R� blockade on macrophages andCD4� LTi cells in response to C. rodentium infection are notcaused by altered numbers of T cell or Treg cells.

DISCUSSION

The gastrointestinal tract and the intestinal mucus have efficientmechanisms that protect the epithelium from pathogenic bacteriaby promoting bacterial clearance and separating bacteria from the

mucosal immune cells, thereby inhibiting inflammation and in-fection (31). In this study, we found that infection with the entericrodent pathogen C. rodentium promoted IL-7 production fromIECs, in agreement with a published study suggesting that H. py-lori infection causes increased intestinal IL-7 in humans (32).Therefore, intestinal epithelium-derived IL-7 may be one of theimportant molecules that inhibit inflammation and infectioncaused by various bacteria.

We demonstrated that one important mechanism by whichIL-7 increases the number of macrophages in the colon is by pro-moting the expression of CCL2, a critical chemoattractant of mac-rophages. In addition to promoting macrophage recruitment tothe colon, IL-7 may also directly enhance the activation and func-tion of macrophages, which is supported by the observations thatintestinal F4/80� macrophages express high levels of IL-7R� andthat the IL-7R� signaling pathway is required for optimal activa-tion of macrophages both in the colon and in mLNs. Thus, IL-7enhances both tissue migration and activation of macrophagesduring C. rodentium infection. In contrast to macrophages,CD103� migratory cDCs in the colon did not express IL-7R�. Inresponse to C. rodentium, the frequency of CD103� migratorycDCs in the colon and mLNs were markedly increased, suggestingthe migration of C. rodentium-activated cDCs to these tissues (4,33). However, the activation and migration of CD103� cDCs wereindependent of the IL-7R� signaling pathway, consistent with thelack of IL-7R� expression in colon CD103� cDCs. Hence, theIL-7R� signaling pathway does not contribute to the activationand migration of intestinal cDCs.

CD4� LTi cells have the capacity to promote lymphoid tissueorganogenesis 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� blockadereduced the CD4� LTi cell population in colon and mLN cells ofC. rodentium-infected mice, which was accompanied by decreasedproliferation of these cells in the absence of the IL-7R� signalingpathway. Moreover, previous studies have demonstrated thatCD4� LTi cells express IL-22 (28, 35), an important cytokine ineliciting an antimicrobial immune response and maintaining mu-cosal barrier integrity within the intestine (36, 37), in an IL-23-dependent manner (28, 37). We showed that IL-7R� blockade inC. rodentium-infected mice led to a significant decrease in IL-23expression in the colon, which may in turn inhibit the productionof IL-22 by CD4� LTi cells. Previous studies demonstrated thatmacrophages and DCs are the main producers of IL-23 (3, 28).Our data suggested that IL-7R� signaling is required for the opti-mal recruitment and activation of macrophages, but not cDCs, in

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 wereinfected 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 infectedmice. Populations were gated on live lineage� CD4� CD90� cells. Lineage markers included CD3, CD5, and CD11c. (C) Absolute CD4� LTi cell numbers incolon (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 colonand 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 representativeof 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 administeredan 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, measuredby ELISA. (H) Frequencies of CD4� CD90� cells in lineage� gated splenocytes from antibody-treated Rag1�/� mice. (I) IL-22 and IL-23p19 mRNA levels incolons 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 foreach 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.

FIG 7 Anti-IL-7R� treatment does not affect the numbers of total T cells orTregs in the spleen and mLNs during C. rodentium infection. Anti-IL-7R� Ab-or 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 andmLNs (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 perexperiment, total of 3 independent experiments).

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response to C. rodentium. Although further investigation is re-quired, macrophages may be the main producers of IL-23 in re-sponse to C. rodentium. Furthermore, our data showed that block-ade of IL-7R� in C. rodentium-infected Rag1�/� mice resulted inreduced CD4� LTi cells, reduced expression of IL-22 and IL-23,and a more rapid onset of host mortality. Collectively, these dataidentify a previously unrecognized role of IL-7 in the expansionand function of CD4� LTi cells in the innate immune responsesagainst enteric bacterial infection.

The IL-17-mediated immune response has been shown to berequired for protection against C. rodentium infection (8). Previ-ous studies identified IL-7 as an important promoter of IL-17production 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 block-ade of IL-7R� significantly downregulated expression of IL-23, apotent promoter of Th17 differentiation and stabilization. Con-sistent with this, we found that colon-infiltrating Th17 cells weredecreased by blockade with IL-7R� at day 10 of infection (data notshown). These results indicated that the Th17 response during C.rodentium infection is also suppressed by IL-7R� blockade, possi-bly as a result of impaired IL-23 production. Future studies willfurther define the cellular and molecular mechanisms by whichthe IL-7R� blockade causes an impaired Th17 response during C.rodentium infection.

We found that suppression of innate immune responses byIL-7R� blockade during C. rodentium infection led to increasedbacterial burden and colon inflammation. The increased inflam-mation 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 thecolon was observed in response to C. rodentium infection in anti-IL-7R�-treated mice (data not shown). Although neutrophils arethe first cells to be recruited to the site of bacterial infection andhave a potent antibacterial function through the production ofoxidants and proteinases, excessive recruitment and accumula-tion of activated neutrophils in the intestine under pathologicalconditions is associated with mucosal injury (40). Hence, in theabsence of IL-7R� signaling, the defective immune responseagainst C. rodentium leads to an increased and persistent bacterialburden, which may be the cause of increased neutrophils in thecolon, resulting in subsequent colon inflammation and damage.Further investigation will address this possibility and elucidate thespecific mechanisms by which IL-7 affects neutrophil activitiesand colon inflammation during C. rodentium infection.

In this study, we injected C57BL/6 mice with anti-IL-7R� Absduring C. rodentium infection. Administration of an anti-IL-7R�Ab has the advantage that IL-7 in mice can be only depleted duringinfection. In comparison, IL-7R� knockout mice have dramati-cally reduced mature T cells and innate lymphoid cells, due todefective development and homeostasis of these cells, making itdifficult 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 re-sponse to C. rodentium infection than normal control mice, whichwill be investigated in a future study. On the other hand, overex-pression of IL-7 promotes the development and homeostasis of Tcells, innate lymphoid cells, and macrophages and promotes tissueinflammation (43–45). Hence, in future research, we will also ex-

amine whether administration of exogenous IL-7 during C. roden-tium infection can facilitate the clearance of this pathogen.

In summary, the present study demonstrates the crucial pro-tective functions of IL-7 in C. rodentium infection. This knowl-edge will enable further investigation of the role of IL-7 in otherbacterial infections and a comprehensive understanding of itsfunction in various aspects of bacterial infections, in order to de-velop novel therapeutic strategies.

ACKNOWLEDGMENTS

We thank the Shanghai Public Health Clinical Center animal facility formaintaining animals and the histology lab for helping with immunofluo-rescence assays.

This study was supported by the Research Fund for InternationalYoung Scientists from the National Natural Science Foundation of China(81450110090).

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