Interleukin-23 drives innate and T cell–mediated intestinal inflammation

IL-23 is essential for innate intestinal inflammation

We initially examined whether H. hepaticus–induced innate immune typhlocolitis was associated with excessive production of IL-12 or IL-23 using real-time quantitative PCR (Q-PCR) to assay the expression of these cytokines in the normal and inflamed intestine. As illustrated in Fig. 1 A, significantly increased expression of IL-23p19 and IL-12p40 mRNA, but not of IL-12p35, was observed in the cecum and colon of H. hepaticus–infected (Hh ⁺) 129SvEvRAG^−/− mice. The increased expression of IL-23p19 was restricted to the intestine, as it was not observed in either the mesenteric LN (MLN) or spleen (Fig. 1 A).

As increased IL-17 expression has been associated with several IL-23–driven autoimmune T cell–mediated pathologies (16), we analyzed whether increased IL-17 expression was also a feature of T cell–independent innate intestinal pathology. Somewhat unexpectedly, we observed a dramatic increase in expression of IL-17 in the inflamed cecum of Hh ⁺ 129SvEvRAG^−/− mice (Fig. 1 B), which was again restricted to the intestine, as it was not increased in spleen (Fig. 1 B). To confirm that these differences in mRNA expression correlated with increased cytokine secretion, we cultured colon explants from Hh ⁺ or control uninfected 129SvEvRAG^−/− mice overnight and quantified cytokine release into the medium. As shown in Fig. 1 C, significantly higher amounts of both IL-23 and IL-17 were produced by colon explants from Hh ⁺ 129SvEvRAG^−/− mice, but there was no increase in IL-12 secretion. To identify the innate sources of IL-17 in the intestine, we used cell sorting to isolate different populations of lamina propria leukocytes (LPLs) from Hh ⁺ 129SvEvRAG^−/− mice. Three major subpopulations could be readily identified within the LPLs: Gr1⁺CD11b⁺ (predominantly granulocytes), Gr1⁻CD11b⁺ (monocytic cells), and Gr1⁻CD11b⁻ cells (Fig. 1 D). Q-PCR analysis also revealed that all leukocyte subpopulations isolated from the intestine expressed high levels of IL-17 mRNA (Fig. 1 E). In contrast, consistent with the results obtained using whole spleen samples (Fig. 1 B), leukocyte subpopulations isolated from the spleen of Hh ⁺ 129SvEvRAG^−/− mice expressed only very low levels of IL-17, around 100–1,000-fold lower than their intestinal counterparts (Fig. 1 E). Collectively, these results indicated that increased local production of IL-23 and IL-17 correlated with innate intestinal pathology.

To formally assess the requirement for IL-23 in innate intestinal pathology, we used an anti–IL-23p19 monoclonal antibody to neutralize IL-23 in vivo. Although Hh ⁺ 129SvEvRAG^−/− mice exhibited extensive inflammation in both the cecum and colon, treatment with anti-p19 throughout the course of the experiment resulted in highly attenuated intestinal pathology (Fig. 2 A). Anti-p19–treated mice had markedly reduced levels of inflammatory infiltrates and epithelial hyperplasia in the cecum and colon (Fig. 2 B). These results demonstrate that IL-23 plays an essential role in H. hepaticus–induced innate immune typhlocolitis.

IL-23 is essential for H. hepaticus–triggered systemic innate inflammatory responses

As Hh ⁺ 129SvEvRAG^−/− mice also exhibit marked systemic inflammatory responses (30), we next examined the effect of anti–IL-23p19 treatment on these disease parameters. As shown in Fig. 3 (A and B), anti–IL-23p19 treatment significantly reduced H. hepaticus–induced splenomegaly in terms of both spleen weights and total spleen cell numbers. FACS analysis of spleen cells revealed that anti–IL-23p19 treatment significantly reduced the accumulation of granulocytes and monocytes that was found in Hh ⁺ 129SvEvRAG^−/− mice (Fig. 3, C and D). Moreover, although scattered small inflammatory foci were present in the livers of Hh ⁺ 129SvEvRAG^−/− mice, these were not observed in mice that received anti–IL-23p19 (Fig. 2 B). These results demonstrate that the systemic innate immune activation triggered by H. hepaticus infection is also dependent on IL-23.

Anti–IL-23p19 treatment decreases proinflammatory cytokine production in the intestine

As the main producers of IL-23 are activated DCs and macrophages(16), we reasoned that high levels of IL-23 production might trigger a cascade of inflammatory cytokines that drives chronic intestinal inflammation. We therefore measured the levels of several proinflammatory cytokines in intestinal tissue homogenates. Although colon homogenates prepared from Hh ⁺ 129SvEvRAG^−/− mice contained markedly elevated levels of IL-6, monocyte chemoattractant protein–1 (MCP-1), IFN-γ, TNF-α, mouse chemokine CXCL1 (KCs), and IL-1β, samples isolated from Hh ⁺ 129SvEvRAG^−/− mice that had received anti–IL-23p19 expressed similar low levels of proinflammatory cytokines to uninfected controls (Fig. 4 A). Treatment of Hh ⁺ 129SvEvRAG^−/− mice with anti–IL-23p19 also led to a similar reduction in IL-17 levels in the colon (Fig. 4 B), indicating that innate IL-17 production is also controlled by IL-23 in vivo. To confirm that anti–IL-23p19 was preventing intestinal pathology by inhibiting host innate immune activation and not through antibacterial effects, we used Q-PCR to assess H. hepaticus infection levels. As shown in Fig. 4 C, treatment with anti–IL-23p19 had no effect on the level of H. hepaticus colonization in 129SvEvRAG^−/− mice. These results indicate that neutralization of IL-23 prevents intestinal inflammation by inhibiting innate immune responses and suggest that high production of IL-23 in the intestine triggers a proinflammatory cytokine cascade that mediates local and systemic pathology.

IL-23 is essential for T cell–mediated intestinal inflammation but not systemic inflammatory responses

To determine whether IL-23 was a general mediator of intestinal inflammation, we next evaluated the roles of IL-12 and IL-23 in a well-established T cell–dependent model of IBD (35, 36). Thus, naive CD4⁺CD45RB^high T cells isolated from C57BL/6 mice were adoptively transferred into age-matched cohorts of syngeneic RAG^−/− recipient mice, p40^−/−RAG^−/− mice (lacking both IL-12 and IL-23), p35^−/−RAG^−/− mice (lacking only IL-12), or p19^−/−RAG^−/− mice (lacking only IL-23), and development of intestinal inflammation was monitored. As illustrated in Fig. 5, the severe colitis induced by naive CD4⁺CD45RB^high T cell transfer into RAG^−/− recipients was highly attenuated in p40^−/−RAG^−/− recipients, confirming that IL-12p40 was essential for disease. Strikingly, although p35^−/−RAG^−/− recipients developed colitis of similar severity to that found in control RAG^−/− recipients, intestinal inflammation was highly attenuated in p19^−/−RAG^−/− recipients (Fig. 5). These results clearly indicate that IL-23, but not IL-12, is required for the development of T cell–mediated colitis.

In addition to colitis, naive CD4⁺ T cell reconstitution of RAG^−/− recipients also leads to systemic immune pathology, including splenomegaly and hepatic inflammation (35, 36). We therefore analyzed these parameters to examine the role of IL-23 in T cell–mediated systemic immune pathology. T cell–mediated colitis was accompanied by marked splenomegaly in RAG^−/− recipients and, even though this was significantly decreased in p40^−/−RAG^−/− recipients, there was no significant decrease in splenomegaly in either p35^−/−RAG^−/− or p19^−/−RAG^−/− recipients (Fig. 6, A and B). FACS analysis confirmed that the adoptively transferred CD4⁺ T cells had reconstituted the spleens of all groups of RAG^−/− mice and, even though this was reduced in p40^−/−RAG^−/− recipients, there was no significant reduction in CD4⁺ T cells in p35^−/−RAG^−/− or p19^−/−RAG^−/− recipients (Fig. 6 C). A similar pattern was observed with respect to liver pathology, with numerous prominent inflammatory foci present in RAG^−/−, p35^−/−RAG^−/−, and p19^−/−RAG^−/− recipients but virtually absent in p40^−/−RAG^−/− recipients (Fig. 6 D). Collectively, these results show that IL-23 is not required for CD4⁺ T cell–mediated systemic inflammatory responses and further indicate that either IL-12 or IL-23 alone is able to facilitate CD4⁺ T cell–dependent inflammatory responses in the liver and spleen.

T cell–dependent intestinal proinflammatory cytokine production is attenuated in the absence of IL-23

As the T cell transfer colitis model has been associated with increased local Th1 responses (8), we also examined the levels of several proinflammatory cytokines in intestinal tissue homogenates from the various groups of RAG^−/− recipients. Although colon homogenates isolated from control RAG^−/− recipients contained elevated levels of TNF-α, IFN-γ, IL-6, MCP-1, IL-1β, and KC, these levels were markedly decreased in p40^−/−RAG^−/− recipients (Fig. 7). As expected, p35^−/−RAG^−/− recipients with colitis expressed similarly elevated levels of proinflammatory cytokines as control RAG^−/− recipients, whereas these were again attenuated in p19^−/−RAG^−/− recipients (Fig. 7). These results demonstrate that IL-23, but not IL-12, is required for the efficient expression of proinflammatory cytokine cascades in the intestine.

As IL-23 has been strongly associated with proinflammatory IL-17–secreting CD4⁺ T cells (Th17), we also examined the levels of IL-17 in the colon homogenates and assayed for Th17 cells using intracellular FACS analysis. The highest levels of IL-17 were detected in colon homogenates isolated from p35^−/−RAG^−/− recipients (Fig. 8 A). In contrast, only low levels of IL-17 were present in colon homogenates from p40^−/−RAG^−/− or p19^−/−RAG^−/− recipients (Fig. 8 A). CD4⁺ T cells isolated from the MLN and LPLs of RAG^−/− or p35^−/−RAG^−/− recipients contained a high proportion of IFN-γ⁺ cells and a small population of IL-17⁺ cells (Fig. 8 B and not depicted). Interestingly, CD4⁺ T cells isolated from p40^−/−RAG^−/− or p19^−/−RAG^−/− recipients showed a marked decrease in the frequency of IFN-γ⁺ cells but still contained a small population of IL-17⁺ cells (Fig. 8 B). These results show that T cell–mediated colitis correlates with increased frequencies of IFN-γ–secreting Th1 cells in intestinal lymphoid tissue and increased levels of IL-17 in the colon, suggesting that both Th1 and Th17 cells may contribute to pathogenesis. They further show that neither IL-12 nor IL-23 is absolutely required for the differentiation of Th17 cells from naive CD4⁺ T cells in vivo.

Mice.

129SvEvRAG2^−/− mice and wild-type B6, control B6RAG1^−/−, and IL-12/23–deficient strains on a B6RAG1^−/− background (IL-12p40^−/−RAG^−/−, IL-12p35^−/−RAG^−/−, and IL-23p19^−/−RAG^−/− mice) were bred and maintained under specific pathogen-free conditions in accredited animal facilities at the University of Oxford. Experiments were conducted in accordance with the UK Scientific Procedures Act of 1986. Mice were routinely screened for the presence of Helicobacter spp. and were >6 wk old when first used.

Bacteria.

H. hepaticus NCI-Frederick isolate 1A (strain 51449; American Type Culture Collection) was grown on blood agar plates containing trimethoprim, vancomycin, and polymixin B (all obtained from Oxoid) under microaerophilic conditions as previously described (30, 55). For H. hepaticus infections, bacterial viability was confirmed using fluorescent microscopy with a bacterial live/dead kit (BacLight; Invitrogen), and 129SvEvRAG2^−/− mice were fed three times on alternate days with ∼5 × 10⁷–2 × 10⁸ CFU H. hepaticus.

Antibody treatment.

Anti–IL-23p19 monoclonal antibody (PAB1106) was produced and characterized as previously described (56). Antibody treatment was commenced on the day of the first inoculation with H. hepaticus, and mice received weekly i.p. injections of 1 mg anti–IL-23p19 or isotype control for the duration of the experiment.

Induction of colitis with naive CD4⁺CD45RB^high T cells.

Naive CD4⁺CD45RB^high T cells were isolated from spleens of C57BL/6 mice using FACS sorting as previously described (57). In brief, single cell suspensions were depleted of CD8⁺, MHC class II⁺, Mac-1⁺, and B220⁺ cells by negative selection using a panel of rat monoclonal antibodies, followed by sheep anti–rat–coated Dynabeads (Dynal). After staining with Cy-Chrome–conjugated anti-CD4, PE-conjugated anti-CD25, and FITC–anti-CD45RB (all obtained from BD Biosciences), naive CD4⁺CD25⁻CD45RB^high T cells were purified (∼99%) by cell sorting with a cell sorter (MoFlo; DakoCytomation). Sex-matched control or IL-12/23–deficient B6RAG^−/− mice received 4 × 10⁵ CD4⁺CD45RB^high T cells by i.p. injection, and development of intestinal inflammation was monitored as described in the next paragraph.

Assessment of intestinal inflammation.

Mice were killed when symptoms of clinical disease (weight loss or diarrhea) became apparent in control groups, usually 6–8 wk after initiation of experiments. Samples of liver, cecum, and proximal, mid-, and distal colon were immediately fixed in buffered 10% formalin. 4–5 μm of paraffin-embedded sections was stained with hematoxylin and eosin, and inflammation was assessed as previously described (30, 57). Each sample was graded semiquantitatively from 0 to 4, and typical features of each grade are as follows: 0 = normal; 1 = mild epithelial hyperplasia; 2 = pronounced hyperplasia with substantial inflammatory infiltrates; 3 = severe hyperplasia and infiltration with marked decrease in goblet cells; and 4 = severe hyperplasia, severe transmural inflammation, ulceration, crypt abscesses, and severe depletion of goblet cells. Ceca and colons were assessed separately, and three separate sections from each sample were examined. The total colonic score was obtained by adding the individual scores from the sections of proximal, mid-, and distal colon.

Flow cytometry.

Aliquots of 1–5 × 10⁵ cells were stained in FACS buffer (HBSS, 0.1% BSA, 5 mM EDTA; both obtained from Sigma-Aldrich) using the following panel of monoclonal antibody to mouse cell surface molecules (all obtained from BD Biosciences): biotinylated anti–NK cells (DX5), biotinylated anti-Gr1 (Ly6G), allophycocyanin-conjugated anti-CD11b, PE–anti-CD11c, PerCP-conjugated anti-CD4, and PE–anti–mouse TCRβ. Biotinylated antibodies were detected using PE-, allophycocyanin-, or FITC-conjugated streptavidin (all obtained from BD Biosciences). Samples were first incubated for 10 min with Fc block (eBioscience) to block any nonspecific binding, and subsequent staining steps were performed for 20 min on ice, followed by washing with FACS buffer. Samples were fixed in FACS buffer containing 2% paraformaldehyde (Biolab) acquired using a FACSCalibur (Becton Dickinson) and analyzed with software (FlowJo; Tree Star, Inc.). For intracellular cytokine analysis, cells were incubated in complete RPMI 1640 (10% heat-inactivated FCS, 2 mM l-glutamine, 100 U/ml penicillin and streptomycin [obtained from Invitrogen], and 0.05 mM 2-mercaptoethanol [obtained from Sigma-Aldrich]) containing 0.1 μM PMA and 1 μM ionomycin (both obtained from Sigma-Aldrich), plus 10 μg/ml Brefeldin A (Sigma-Aldrich), for 4 h at 37°C in a humidified incubator with 5% CO₂. Cell surface staining was performed as described previously in this paper, and, after fixing, the samples were incubated in permeabilization buffer (PBS, 0.1% BSA, 0.5% saponin; Sigma-Aldrich) containing allophycocyanin–anti–mouse IFN-γ, PE–anti–mouse IL-17 or appropriate isotype controls, allophycocyanin–rat IgG1, and PE–rat IgG2b (all obtained from BD Biosciences) for 30 min at room temperature, washed in permeabilization buffer, and analyzed as described in this paper.

Isolation of leukocyte subpopulations.

Spleen cells and LPLs were isolated from 6–12 pooled Hh ⁺129SvEvRAG2^−/− mice as previously described (30). Cells were stained with fluorescent antibodies, and leukocyte subpopulations were purified using FACS sorting as outlined previously in this paper. Cells were sorted into distinct subpopulations on the basis of size (forward scatter [FSC]) and granularity (side scatter [SSC]) in combination with surface marker expression (58). Characteristics of subpopulations were as follows: granulocytes, FSC^HiSSC^HiGr1^HiCD11b^HiCD11c⁻; monocytes, FSC^HiSSC^LoGr1^IntCD11b^HiCD11c⁻; DCs, FSC^HiSSC^LoGr1⁻CD11c^Hi; and NK cells, FSC^LoSSC^LoDX-5^Hi.

Quantitation of cytokine gene expression using real-time PCR.

RNA was purified from frozen tissue samples or sorted cells using RNAeasy kits (QIAGEN). Homogenization was performed using a Polytron Homogenizer (Kinematica AG). RNA purity and quantification was determined using a Nanodrop spectrophotometer (Nanodrop Technologies). cDNA synthesis was performed using a reverse transcriptase kit (Superscript III) with Oligo dT (both obtained from Invitrogen). Q-PCR reactions were performed using the following primers, together with FAM/TAMRA- or VIC/TAMRA-labeled probes: HPRT primers, 5′-GACCGGTCCCGTCATGC-3′ and 5′-TCATAACCTGGTTCATCATCGC-3′, and probe, 5′-ACCCGCAGTCCCAGCGTCGTC-3′; IL-23p19 primers, 5′-AGCGGGACATATGAATCTACTAAGAGA-3′ and 5′-GTCCTAGTAGGGAGGTGTGAAGTTG-3′, and probe, 5′-CCAGTTCTGCTTGCAAAGGATCCGC-3′; IL-12p35 primers, 5′-TACTAGAGAGACTTCTTCCACAACAAGAG-3′ and 5′-TCTGGTACATCTTCAAGTCCTCATAGA-3′, and probe, 5′-AGACGTCTTTGATGATGACCCTGTGCCT-3′; IL-12p40 primers, 5′-GACCATCACTGTCAAAGAGTTTCTAGAT-3′and 5′-AGGAAAGTCTTGTTTTTGAAATTTTTTAA-3′, and probe, 5′-CCACTCACATCTGCTGCTCCACAAGAAG-3′; and IL-17A primers, 5′-GCTCCAGAAGGCCCTCAG-3′ and 5′-CTTTCCCTCCGCATTGACA-3′, and probe, 5′-ACCTCAACCGTTCCACGTCACCCTG-3′.

cDNA samples were assayed in triplicate using a detection system (Chromo4; GRI), and cytokine gene expression levels for each individual sample were normalized relative to HPRT using ΔCt calculations (59).

Quantitation of cytokine protein levels in intestinal tissues.

In initial experiments, small pieces of colon (∼5 mm of mid-colon) were isolated and rinsed in HBSS/BSA and weighed. Colon explants were cultured overnight in 24-well tissue culture plates (Costar) in 500 μl complete RPMI 1640 at 37°C in an atmosphere containing 5% CO₂. After centrifugation at 10,000 g to pellet debris, culture supernatants were transferred to fresh tubes and stored at −20°C. Cytokine concentrations were measured using specific sandwich ELISAs (IL-23, R&D Systems; IL-17, Bio-Rad Laboratories) and were normalized to the weight of the colon explant.

For more comprehensive analysis, frozen intestinal tissue samples were homogenized in PBS containing a cocktail of protease inhibitors (Protease Inhibitor Cocktail Tablets; Roche) using a Polytron Homogenizer. After centrifugation at 10,000 g to pellet debris, concentrations of a panel of proinflammatory cytokines in supernatants were measured either using the cytometric bead assay (BD Biosciences) or the Luminex 100 assay (Bio-Rad Laboratories). In all cases, cytokine levels were normalized to the total protein level in each sample, as measured using the Bradford assay (Bio- Rad Laboratories).

Quantitation of H. hepaticus using real-time PCR.

DNA was purified from cecal contents taken from H. hepaticus–infected mice using the DNA Stool kit (QIAGEN). H. hepaticus DNA was determined using a Q-PCR method based on the cdtB gene and performed with a Chromo4 detection system, as previously described (30, 60).

Statistics.

The nonparametric Mann-Whitney test was used for comparing pathology scores and Q-PCR data, and an unpaired t test was used to examine spleen weights and cell counts. Differences were considered statistically significant when P < 0.05.