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. Author manuscript; available in PMC: 2018 Nov 1.
Published in final edited form as: Inflamm Bowel Dis. 2017 Nov;23(11):1983–1995. doi: 10.1097/MIB.0000000000001274

Interleukin 27 is a potential rescue therapy for acute severe colitis via interleukin-10 dependent, T cell independent attenuation of colonic mucosal innate immune responses

Mairi H McLean 1,2, Caroline Andrews 1, Miranda L Hanson 1, Walter A Baseler 1, Miriam R Anver 3, Emilee Senkevitch 1, Aleksandra K Staniszewska 1,2, Christopher Smith 1,2, Luke C Davies 1, Julie Hixon 1, Wenqeng Li 1, Wei Shen 1, Lothar Steidler 4, Scott K Durum 1
PMCID: PMC5796760  NIHMSID: NIHMS903449  PMID: 29019857

Abstract

Background

IBD patients with acute severe colitis face systemic anti-TNF biologic rescue therapy or colectomy, if treatment with intravenous steroids fail. Interleukin (IL)-27 is a cytokine with an immunosuppressive role in adaptive immune responses. However, the IL-27 receptor complex is also expressed on innate immune cells, and there is evidence that IL-27 can impact the function of innate cell subsets, although this particular functionality in vivo is not understood. Our aim was to define the efficacy of IL-27 in acute severe colitis and characterize novel IL-27 driven mechanisms of immunosuppression in the colonic mucosa.

Methods

We assessed oral delivery of Lactococcus lactis expressing an IL-27 hyperkine on the innate immune response in vivo in a genetically intact, non-infective acute murine colitis model induced by intra-rectal instillation of 2,4,6-Trinitrobenzenesulfonic acid in SJL/J mice.

Results

IL-27 attenuates acute severe colitis through reduction of colonic mucosal neutrophil infiltrate associated with a decreased CXC chemokine gradient. This suppression was T cell-independent and IL-10-dependent, initially featuring enhanced mucosal IL-10. IL-27 was associated with a reduction in colonic pro-inflammatory cytokines and induced a multifocal strong positive nuclear expression of phosphorylated STAT-1 in mucosal epithelial cells.

Conclusion

We have defined novel mechanisms of IL-27 immunosuppression towards colonic innate immune responses in vivo. Mucosal delivery of IL-27 has translational potential as a novel therapeutic for IBD and is a future mucosal directed rescue therapy in acute severe inflammatory bowel disease.

Keywords: interleukin-27, cytokine, IBD, colitis

Introduction

Inflammatory bowel disease is a progressive relapsing remitting inflammation of the gastrointestinal tract 1 and is increasing in incidence globally 2. Biologic therapy has revolutionized patient management 3, 4. Despite this and the use of other immunosuppressant drugs, there remains a significant cohort of patients with refractory or relapsing disease, or those that cannot tolerate available treatments. The systemic nature of current immunosuppressant therapies is associated with an appreciable risk of significant adverse events and carries a financial burden. Novel, safer, and more affordable treatment strategies are required for maintenance therapies.

Acute severe colitis requires hospital admission for intravenous steroid therapy and close monitoring, due to potential life threatening complications such as perforation and toxic dilatation 5–7. Up to 15% of patients present with acute severe disease. Clinical and laboratory severity markers predict the need for emergent colectomy and 85% of patients with >8 stools/day or 3–8 stools/day and raised CRP >45mg/L on day 3 need urgent consideration of escalated medical therapy with rescue infliximab anti-TNF biologic therapy or intravenous ciclosporin, or surgery. If medical therapy fails by day 7 or life threatening complications occur, emergency colectomy with ileostomy is required as a lifesaving treatment. This clinical need for life saving and life changing surgery unfolds rapidly and can be associated with post-operative psychosocial morbidity, especially in adolescent and young adult patients 8–10. There is a need for novel rescue therapies for the treatment of acute severe colitis.

We previously demonstrated that oral delivery of Lactococcus lactis (L. lactis) expressing an IL-27 hyperkine (LL-IL-27), composed of both EBI3 and p28 subunits, a linker molecule and secretary peptide, is immunosuppressive in murine chronic enterocolitis induced by CD4+CD45Rbhi T cell transfer 11. In this T cell driven colitis model, representing adaptive immune responses, LL-IL-27 led to significant histological improvement and survival advantage, through induction of IL-10 derived from intra-epithelial mucosal T cells. We also described an immunosuppressive action on acute DSS colitis 11, although the mechanism was not explored. We then hypothesized that IL-27 may exert an immunosuppressive role in innate immune responses.

IL-27, a heterodimeric cytokine composed of EBI3 and p28 subunits, is secreted from antigen presenting cells and signals via a heterodimeric receptor complex composed of the widely expressed Gp130, and the specific IL-27Rα 12–14. IL-27 was initially considered pro-inflammatory through promotion of Th1 responses 15. There is now appreciation that IL-27 has a wide functional repertoire, including profound anti-inflammatory effects through promotion of IL-10 secreting Tr1 regulatory T cells and inhibition of Th2 and Th17 responses 12–14. Knowledge of IL-27 functionality has largely come from T cell biology, and its role in the adaptive immune response is well described 12–14. However, the IL-27 receptor complex is also expressed on other immune cell types, including granulocytes 16 and macrophages 17, indicating that the functional capabilities of IL27 may be even wider and in particular IL27 may play a role in mediating innate immunity. Indeed, IL-27 can impact the function of innate cell subsets, including macrophages 18, neutrophils 16, 19, and dendritic cells 20–22. It has been shown that IL-27 influences innate responses to bacterial challenge in the context of systemic sepsis in genetic manipulation models such as IL-27Rα knock-out murine strains. 23,24. To date, there is no report on the immunosuppressive mechanism of IL27 in acute inflammation of the colon driven by innate cells in a genetically intact, non-infective environment.

Here, by demonstrating an immunosuppressive effect of IL-27 on the innate, rather than adaptive, immune response in vivo in a genetically intact, non-infective acute murine colitis model induced by intra-rectal instillation of 2,4,6-Trinitrobenzenesulfonic acid (TNBS), we offer novel mechanistic insights into the biology of IL-27 in vivo. Our data highlights the immunosuppressive role of IL-27 in innate immune responses offering translational potential as a novel rescue therapy in acute severe colitis.

Materials and Methods

Study approval

Animal experiments were conducted under approved protocols by the NCI Animal Care and Use Committee, in keeping with federal regulations governing care and use of animals in biomedical research. Frederick National Laboratory is accredited by AAALAC International and follows the Public Health Service Policy for the Care and Use of Laboratory Animals. Animal care was provided in accordance with the procedures outlined in the “Guide for Care and Use of Laboratory Animals” (National Research Council; 2011; National Academies Press; Washington, D.C.).

Animals

Experiments were performed on 6–10 week old male SJL/J, B6 Rag−/− (01BJ2 - B6.129S7-Rag1), and B6 IL-10−/− (01IL6 - IL-10 GFP/C) mice, maintained within the NCI-Frederick animal facility. Mice were fed normal chow, had free access to water and exposure to a 12 hourly light/dark cycle.

Acute colitis induction

100 µl TNBS (Sigma, ~1M in H2O, stored at −20°C, 92823) in 45% ethanol vehicle was administered intra-rectally to isoflurane anaesthetized mice with a 3cm flexible catheter. The administered dose was optimized within our facility and dependent on the sensitivity of the mouse strain; 2mg for SJL/J, 4mg for B6/Rag−/− and 6mg for B6/IL-10−/−. The mice were held vertically for > 30secs post instillation to ensure retention. Mice were given supportive care (wet food/ice chips/heat pad) throughout the protocol. Prior to colitis induction, mice were fasted to solid food overnight and given access to 5% sucrose water.

Bacterial handling and administration

Lactococcus lactis (L. lactis) expressing IL-27 (LL-IL-27) and L. lactis empty vector control (LL-C) were prepared as described previously 11. Briefly, L. lactis strain MG1363 was used for bacterial preparations. A bacterial research bank was prepared and IL-27 secretion confirmed by IL-27p28 ELISA (R&D systems, M2728) before storage in 50% glycerol at −80°C. For oral administration, stock bacteria were cultured 1:1000 in Difco M17 broth (BD Biosciences, 218561) supplemented with 0.5% glucose and 5mg/ml erythromycin (Sigma, E5389) (GM17E) at 30°C for 16 hours. The bacteria were harvested by centrifugation and resuspended in buffered M9 salt media. Each mouse was administered 100 µl of this bacterial suspension by oral gavage at 24 hour intervals within the treatment protocol, commencing on the day of colitis induction following recovery from anesthesia.

Clinical assessment of colitis activity

Disease activity index was assessed daily as previously reported 11. This is a composite score (maximum 12) of three parameters: weight loss from baseline (≤1% 0, 1<≤5% 1, 5<≤10% 2, 10<≤20% 3, >20% 4), consistency of stool (normal pellets 0, soft-semi formed 2, diarrhea 4) and presence of fecal blood (none 0, occult blood positive 2, overt blood 4). Macroscopic colitis score was assessed by colon weight (mg) and length (cm) immediately after harvest. Histological colitis score (maximum 14) was reported by a veterinary pathologist (MRA) based on multiple parameters: severity of inflammatory cell infiltrate (none 0, mild 1, moderate 2, severe 3), goblet cell depletion (none 0, mild 1, moderate 2, severe 3), crypt hyperplasia (none 0, mild 1, moderate 2, severe 3), degree of ulceration of epithelium, (none 0, erosion 1, mild 2, moderate 3, severe 4 ulceration), presence (1) or absence (0) of granulomas.

Measurement of systemic inflammatory response

Blood was collected on day 2 post TNBS instillation in a serum separator tube, centrifuged and the supernatant stored at −80°C for analysis. Serum C-reactive protein (CRP) was measured using ELISA kit (Life Diagnostics Inc, 2210-1) as per instructions.

Gene expression

RNA was extracted from cells or snap frozen distal colon tissue with the RNeasy mini kit (Qiagen, 74104) as per instructions. Quality and yield were assessed by Nanodrop spectrophotometry. cDNA was synthesized using the QuantiTect reverse transcription kit (Qiagen, 205310) with inclusive genomic DNA wipeout buffer. Further details of gene expression assays are available in the supplementary materials.

Total protein extraction and protein expression assays

Total protein was extracted by mechanical homogenization from distal colon tissue in 1X RIPA buffer (Cell signaling 10X, 9806) containing 1:1000 protease inhibitors at 4°C. Following 20 min incubation on ice, tissue homogenates were centrifuged at 14,000rpm for 10 minutes and the supernatant stored at −80°C for analysis. Total protein concentration was established using the Pierce BCA protein assay kit (23225) as compared to albumin standard. Further details of protein expression ELISA assays are available in the supplementary materials.

Colonic lamina propria cell isolation

Single cell suspension from freshly harvested distal colon tissue was extracted with a lamina propria dissociation kit (Miltenyi Biotec, 130-097-410) on the gentleMACS dissociator as per instructions, incorporating chemical and physical dissociation.

Flow cytometry & cell sorting

Nonspecific staining of cells was blocked by 20 minute incubation with anti-mouse CD16/CD32 Fc block (1:100 dilution) (BD Pharminogen, 553142). Macrophages and neutrophils were identified by cell surface expression of F4/80 and Ly6G using anti-mouse APC-F4/80 (eBiosciences, 17–4801-82, clone BM8) and anti-mouse Pacific Blue-Ly6G (Biolegend, 127611) on 4% paraformaldehyde fixed cells using the BD FACSCanto II flow cytometer. Single stain positive and unstained negative cell controls were included. F4/80+ cells were collected using a FACSAria II (BD Biosciences) cell sorter. Data was analyzed using FlowJo software (Tree Star, Inc, Ashland, OR).

Assessment of mucosal inflammatory cell phenotype, proliferation index and phosphorylated STAT-1 response

Immunohistochemistry was performed on 10% NBF fixed, paraffin embedded tissue sections of distal colon. Further details of immunohistochemistry protocols are available in the supplementary materials and methods.

Macrophage culture protocols

Details of bone marrow derived and thioglycollate peritoneal macrophage culture protocols are available in the supplementary materials and methods.

In vitro cell stimulation assay

Cells were stimulated with E. coli 0111:B4 derived LPS (Sigma, L4130) from 10mg/ml stock, or 5mM Adenosine 5’-triphosphate disodium sate hydrate (ATP) from 90mM stock (Sigma, A22383-5G), stored at −20°C. Recombinant mouse IL-27 (NSO expressed, R&D systems, 2799-010/CF) was added to stimulation assay media as appropriate at 100ng/ml. Culture supernatant was collected and stored at −80°C for analysis.

Statistics

SigmaPlot 11.0 software was used for statistical analysis. Survival curves were analyzed according to the Kaplan-Meier estimator on GraphPad 6. Statistical differences were determined by ANOVA and non-parametric Rank Sum tests, with p values of ≤ 0.05 considered to represent a statistically significant difference between groups. If groups displayed normal variance, Student’s t-test was performed (as indicated in the figure legend text). Data is presented as mean +/− SEM.

Results

IL-27 attenuates acute severe colitis in vivo

Intra-rectal instillation of TNBS in SJL/J mice resulted in an acute severe left-sided colitis, with a rapid increase in disease activity index (DAI) within 24 hours. Mice treated with TNBS were included if their DAI was greater than 4 on day 1, reflecting appropriate response to TNBS. Mucosal delivery of LL-IL-27 attenuated TNBS colitis with a decreased DAI and significantly less weight loss from baseline starting weight, compared to L.lactis control (LL-C) (Figure 1a & 1b). LL-IL-27 was associated with a significantly lower colon weight and increased colon length (Figure 1c), resulting in a reduced colon weight:length ratio (p=0.005).

Figure 1. Oral delivery of IL-27 suppresses acute severe colitis.

Figure 1

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(a) LL-IL-27 evokes a decreased disease activity index (DAI) compared to LL-C; 5.8 vs. 8.2 on day 1, 5.3 vs. 8.9 on day 2 and 4.1 vs. 8.2, on day 3, maximum 12, *p=0.001. (b) LL-IL-27 led to significantly less weight loss than TNBS+LL-C or TNBS alone, p<0.01 and p<0.05 on days 2 and 3, respectively. Data in (a) and (b) combined from 3 separate experiments. n= 14, 21, 25, 25 for ethanol, TNBS, TNBS+LL-C and TNBS+LL-IL-27 groups, respectively. (c) Macroscopic colitis score represented by colon weight (mg) and length (cm). Colitis renders the colon shorter and heavier. Both parameters improved with LL-IL-27 vs. LL-C; colon length, p=0.001 and colon weight, p=0.017. Data combined from 2 separate experiments. n= 7, 16, 18, 19 for ethanol, TNBS, TNBS+LL-C and TNBS+LL-IL-27, respectively. (d) SJL/J colitic mice (2mg TNBS) receiving daily gavage of LL-IL27 for 4 days (n=10) showed serum IL-27 of 2.9pg/ml, indistinguishable from non-colitic untreated mice (n=8, 4.1 pg/ml), p=0.469 (t-test). (e) IL-27 provoked an improved systemic inflammatory response measured by serum CRP (day 2); LL-C vs. LL-IL-27, p=0.019 (t-test). Data from one experiment. n=5, 3, 10, 9, 10 for untreated, ethanol, TNBS, TNBS+LL-C and TNBS+LL-IL-27, respectively. All data (a–e) presented as mean+SEM.

Oral administration of LL-IL-27 is presumed to act locally since there was no evidence of systemic absorption, as determined by IL-27 p28 ELISA (Figure 1d). Although mucosally-delivered IL-27 was not measurable systemically, we observed a decrease in serum C-reactive protein (CRP) in LL-IL-27 treated mice (Figure 1e). This can be attributed to indirect effects on liver production of CRP, possibly downstream of inflammatory cytokines such as IL-6, as will be discussed.

LL-IL-27 resulted in a significant reduction in total histology colitis score compared to LL-C (Figure 2a & d), although this was surprisingly modest compared to clinical improvement. Assessment of the independent histological parameters of the composite score revealed significant protection against mucosal ulceration in those receiving LL-IL-27 (Figure 2b). We hypothesized that IL-27 may protect the epithelial barrier, shielding the innate immune system from contact with intestinal microbiota. IL-27 was previously shown to increase colonic epithelial cell proliferation, and to promote epithelial wound restitution in vitro 25. To explore this in vivo, we assessed epithelial cell proliferation by Ki67 immunohistochemistry. Eliciting colitis increased epithelial proliferation by 50% but there was no effect on proliferation index associated with LL-IL-27 (Figure 2c).

Figure 2. Oral delivery of IL-27 leads to improved histological colitis score and protection against ulceration in acute severe colitis.

Figure 2

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(a) Cumulative histological colitis score based on 5 parameters (degree of inflammatory cell infiltrate/ mucosal ulceration/ crypt hyperplasia/ goblet cell depletion, presence of granuloma) with a maximum of 14. TNBS+LL-C vs. TNBS+ LL-IL-27, p=0.035. (b) Degree of distal colonic mucosa ulceration score, graded on a scale of 0–4. 0=none, 1=erosion, 2=mild ulceration, 3=moderate ulceration, 4=severe ulceration. TNBS+LL-C vs. TNBS+ LL-IL-27, p=0.008. Data generated from randomly selected animals from 2 experiments. n=5,5,6,7 for ethanol, TNBS, TNBS+LL-C and TNBS+LL-IL27, respectively. Mean + SEM. (c) Epithelial proliferation index was assessed by Ki67 immunohistochemistry on paraffin embedded distal colon sections. n=5/group from 2 experiments. Index derived from number of Ki67 positive cells/total number epithelial cells/ HPF, expressed as percentage. Mean + SEM % from 5 HPF per sample used in analysis. Colitis evoked a significant increase in proliferation index across all groups compared to ethanol control; p=0.009, 0.001 & 0.002 for TNBS alone, +LL-C and +LL-IL-27, respectively. There was no significant difference between TNBS alone vs. +LL-C vs. +LL-IL-27 (p=0.243), t-test. (d) Representative distal colon photomicrographs of each experimental group. There is full-thickness necrosis of the mucosa and marked submucosal inflammation and edema in both the TNBS alone and TNBS+LL-C treated mice. LL-IL-27 treatment significantly reduced mucosal necrosis and decreased inflammatory infiltrates. Slides were optically scanned with an Aperio AT2 digital slide scanner. Hematoxylin and eosin staining.

Our in vivo data shows that oral IL-27 attenuates acute severe colitis when administered shortly after the time of colonic insult. An important question is whether pre-exposure to IL-27 could impact disease induction as a prophylactic effect. Mice were pre-treated with LL-IL-27 for 2 weeks prior to intra-rectal administration of TNBS. This did not affect acute colitis induction (Figure 3), suggesting that an active inflammatory microenvironment is required for IL-27 to suppress inflammation in the colonic mucosa.

Figure 3. An active inflammatory microenvironment is required for oral IL-27 to suppress inflammation in the colonic mucosa.

Figure 3

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Pre-exposure to LL-IL-27 (10 oral gavages over 2 week period) prior to intra-rectal instillation of 2mg TNBS (SJL/J 6–8 week old male mice) did not impact acute colitis induction as measured by (a) disease activity index, (b) weight loss from starting weight, or (c) macroscopic colitis score (colon length and weight). Data presented combined from 2 separate experiments, n=10/group, as mean + SEM; ANOVA on Ranks & Rank Sum test for pairwise comparisons.

IL-27 reduces colonic neutrophil infiltrate associated with decreased CXC chemokine expression

Previous studies have shown that lack of IL-27 signaling increased neutrophil influx to the site of acute bacterial infection in the peritoneal cavity 26 and lung 24. This raised the question of whether mucosal IL-27 might suppress neutrophil infiltration. To explore this as a potential mechanism, neutrophil and macrophage infiltration was assessed by myeloperoxidase (MPO) and F4/80 immunohistochemistry, respectively. Induction of acute TNBS colitis led to a precipitous increase in mucosal neutrophils (p=0.001) and a modest increase in macrophages (p<0.05). LL-IL-27 led to a significant reduction in MPO+ neutrophil infiltration into the colonic mucosa (Figure 4a & Supplementary Figure 1a). F4/80+ macrophage infiltrate did not alter with LL-IL-27 treatment (Figure 4b & Supplementary Figure 1b).

Figure 4. Oral IL-27 reduces colonic neutrophil infiltrate associated with decreased CXC chemokine expression.

Figure 4

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Distal colon inflammatory cell phenotype assessed by immunohistochemistry using (a) myeloperoxidase (MPO) and (b) F4/80 positivity to identify neutrophils and macrophages, respectively. Data generated from randomly selected animals from 2 experiments. n=5,5,6,6,8 for untreated, ethanol, TNBS, TNBS+LL-C and TNBS+LL-IL-27, respectively. Mean + SEM. (a) Treatment with IL-27 led to a significant reduction in MPO+ neutrophil infiltration into the colonic mucosa (p=0.002, t-test). (b) F4/80+ macrophage infiltrate did not alter with LL-IL-27 treatment (p=0.250, t-test). (c) On day 2 post TNBS instillation, CXCL1 and CXCL2 but not CXCL5 protein (measured by ELISA and normalized to mg total protein concentration) were significantly reduced in distal colon homogenate in colitic mice treated with LL-IL-27 compared to LL-C (p=0.026, p=0.038 & p=0.427 (t-test), respectively). Data represents mean + SEM, generated from 2 separate experiments, n=10 /group. (d) This IL-27 associated reduction in CXCL2 expression persisted as measured later in the experimental protocol (day 4 or time of death), p=0.001 and p=0.003, compared to TNBS plus LL-C and TNBS alone, respectively. Mean + SEM, generated from one experiment, n=3, 3, 8, 8, 11 for untreated, ethanol, TNBS, TNBS+LL-C and TNBS+LL-IL27, respectively. (e) Ex-vivo derived colitis associated lamina propria F4/80+ macrophage cells secreted CXCL2 measured by ELISA after 24 hours culture at 37°C (105 cells/200µl), and this was significantly reduced in cells exposed in vivo to LL-IL-27, p=0.001 (t-test). Data generated from 5 pooled distal colon samples per group.

Neutrophils move to areas of acute inflammation via chemokine gradients, therefore expression of several candidate chemokines in the distal colon was assessed. On day 2 post-TNBS instillation, CXCL1 and CXCL2 but not CXCL5 protein was significantly reduced in colitic mice treated with LL-IL-27 compared to LL-C (Figure 4c). CXCL2, a potent neutrophil chemokine, was taken forward in further investigation and at the end of the treatment protocol (day 4 or time of death), the significant suppression persisted (Figure 4d).

Oral IL-27 reduces CXCL2 secretion in ex-vivo colon derived F4/80+macrophage

Macrophages are major producers of chemokines that induce a neutrophil influx 27. The cellular source of CXCL2 in our acute TNBS colitis model was assessed by selecting F4/80+ cells from distal colon lamina propria cell suspension, on day 2 post-TNBS instillation, cultured for 24 hours to assess CXCL2 response. These colitis derived F4/80+ macrophages secreted CXCL2, and this was significantly reduced in cells exposed to LL-IL-27 in vivo (Figure 4e).

IL-27 does not directly suppress macrophage CXCL2 expression ex vivo

Having shown that IL-27 suppressed chemokine expression by colonic macrophages in vivo, we next evaluated whether this was a direct effect by examining IL-27 responses of different macrophage preparations.

Bone marrow derived macrophage (BMDM) expressed IL-27Rα (Figure 5a) demonstrating their capability to respond to IL-27. LPS stimulation provoked robust CXCL2 secretion. However, addition of recombinant IL-27 (rIL-27) for the 5 hour duration of LPS stimulation did not suppress this response (Figure 5b). To investigate whether the macrophage chemokine response was suppressed by priming with IL-27 prior to LPS challenge, BMDM were cultured in media containing rIL-27 for 48 hours prior to LPS stimulation. Again, exposure to LPS led to CXCL2 secretion and this was not reduced by rIL-27 pre-exposure (Figure 5c).

Figure 5. IL-27 does not directly suppress macrophage CXCL2 expression.

Figure 5

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To investigate the mechanism of IL-27 mediated immunosuppression, chemokine response in bone marrow derived macrophage (BMDM) (a–c), thioglycollate induced peritoneal macrophage (TPM) (d & e) and colonic lamina propria F4/80+ macrophage (f) were assessed. (a) >90% of M-CSF cultured bone marrow derived cells expressed F4/80 and IL-27Rα. (b) BMDM were stimulated for 5 hours with various concentrations of LPS as labelled +/− rIL27 (100ng/ml) and CXCL2 measured in supernatant by ELISA. Combined data from several experiments, generated from >3 wells/experimental condition. LPS resulted in a robust CXCL2 response but there was no differential response in association with IL-27, t-test. (c) Exposure for >48 hours prior to LPS stimulation did not impact CXCL2 response, t-test. (d) Peritoneal cell phenotype day 4 post IP thioglycollate injection. Cells expressed IL-27Rα. (e) Adherent cells were stimulated with 50ng/ml of LPS +/− 100ng/ml rIL27, for 5 or 72 hours. Again, LPS resulted in a robust CXCL2 response but there was no differential response in association with IL-27. (f) This was also seen in ex vivo colonic lamina propria F4/80+ cells stimulated for 24 hours with 50ng/ml of LPS +/− 100ng/ml rIL27, t-test. Results in C, E & F presented from one experiment, with the same pattern of expression seen in repeat experiment. All data presented as mean + SEM.

To evaluate a different macrophage population, thioglycollate-induced peritoneal cells were examined, perhaps better representing the acute inflammatory macrophages in this in vivo colitis model. At day 4 post-thioglycollate injection, the majority of cells were F4/80+ macrophages, representing F4/80intermediate inflammatory and F4/80high resident macrophages, co-existing with Ly6G+ neutrophils (Figure 5d). Adherent cells were used as a macrophage enriched population, and expressed IL-27Rα. As in BMDM, LPS induced robust CXCL2 expression and this was not altered by presence of rIL-27 (Figure 5e).

Overall, LPS induced CXCL2 protein expression in macrophages was not inhibited by rIL-27 in two independent in vitro cell culture models, suggesting this is not the direct mechanism for CXCL2 suppression in vivo.

However, it remained possible that macrophage responses would be site specific to the colon. To explore this, F4/80+ cells isolated from murine colon lamina propria were LPS stimulated for 24 hours, with or without rIL-27. As seen in the other macrophage culture models, LPS induced a striking increase in CXCL2 compared to unstimulated cells (Figure 5f). Once again, there was no significant IL-27 suppression of CXCL2 production in response to LPS. This supports the hypothesis that mucosal delivery of IL-27 does not directly interfere with the ability of colonic lamina propria macrophages to secrete CXCL2 after an inflammatory stimulus.

IL-27 suppression of acute severe colitis is T cell-independent

Since IL-27 inhibits several T cell subsets, we assessed whether T cell mediated immunosuppression could be central to IL-27 effect in acute colitis.

TNBS colitis was evoked in Rag1-deficient (Rag1−/−) mice that lack mature T and B cells, and responses to LL-C or LL-IL-27 assessed. There was reduced disease severity evoked in the C57BL/6 strain of mice due to relative resistance to TNBS (compared to SJL/J strain) 28. LL-IL-27 led to a significantly improved DAI (Figure 6a). LL-IL-27 treated mice lost significantly less weight initially, though the difference between the groups narrowed during the protocol with a strong trend for improvement in the LL-IL-27 treated group (Figure 6b). CXCL2 protein in distal colon homogenates was significantly inhibited by LL-IL-27 (Figure 6c), and there was a significant reduction in histological colitis score (Figure 6d). Taken together, these data show that IL-27 mediated inhibition of acute colitis persists in Rag1-deficient mice, implying the mechanism is T cell-independent.

Figure 6. IL-27 immunosuppression in acute TNBS colitis is T cell-independent and IL-10-dependent, evoking early IL-10 secretion in the colonic mucosa.

Figure 6

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(a–d) 4mg TNBS instilled intra-rectally into Rag−/− mice. Data combined from 2 separate experiments. n=10/group. There was a significant difference in (a) DAI in mice treated with LL-IL-27 compared to LL-C on day 1 and 2, p=0.013 & p=0.040 respectively, t-test, and (b) degree of weight loss from baseline on day 1 (p=0.019), then a trend just under significance on days 2&3 (p=0.06), t-test. (c) Distal colon homogenate CXCL2 protein was significantly reduced with LL-IL-27, p=0.028, (d) as was distal colon histology score, p=0.028, t-test. (e–h) 6mg TNBS instilled intra-rectally into IL-10−/− mice. Data combined from 2 separate experiments. n=8/group. (e) DAI, p=0.237, 0.903, 0.681, respectively on day 1, 2 & 3, LL-IL-27 vs. LL-C, t-test, (f) weight loss from baseline, p=0.727, 0.751, 0.716, respectively on day 1, 2 and 3, LL-IL-27 vs. LL-C, t-test. (g) CXCL2 protein in distal colon homogenate, p=0.959 and (h) distal colon histology score, p=0.608, t-test, were not significantly different in mice treated with LL-IL-27 compared to LL-C. (i) On day 2 post TNBS instillation in SJL/J mice, there was a significant increase in distal colon IL-10 protein associated with IL-27, p=0.01, t-test. (j) Later in the treatment protocol, at day 4 or time of death, this difference in IL-10 protein expression was not apparent, p=0.653. At both time points, n=10/group, combined data from 2 experiments.

IL-27 immunosuppression in acute severe colitis is IL-10-dependent

It is known that IL-27 can induce the immunosuppressive cytokine, IL-10 29, 30 from Tr1 T regulatory cells 31, and other cell types, such as macrophages 17. Our previous data revealed that LL-IL-27 evoked a significant colonic IL-10 response, from CD4+CD8α intra-epithelial T cells, and overall clinical improvement mediated by IL-27 was dependent on production of this cytokine in a chronic enterocolitis model 11. To evaluate whether IL-10 played a major role in the immunosuppressive function of IL-27 in the context of acute innate cell driven colitis, we employed an IL-10 deficient (C57BL/6 IL-10−/−) mouse model in our experimental colitis protocol. There was no significant difference in DAI (Figure 6e) or weight loss (Figure 6f) between mice treated with LL-C versus LL-IL-27. There was no reduction in CXCL2 (Figure 6g) nor total histology score (Figure 6h) associated with LL-IL-27. This suggests IL-27 mediated immunosuppression in acute colitis is IL-10-dependent and CXCL2 reduction is downstream of this IL-10 dependency.

Next, IL-10 protein expression was assessed in distal colon homogenate on either day 2 (Figure 6i) or day 4/time of death post-TNBS instillation (Figure 6j). On day 2, distal colonic IL-10 was increased by 25% in mice exposed to IL-27. Later in the treatment protocol there was no significant difference in mucosal IL-10 detected between colitic mice who received LL-C or LL-IL-27. This suggests that early in disease pathogenesis, L.lactis delivered IL-27 hastens the immunoregulatory IL-10 response, increasing IL-10 in the colonic mucosal microenvironment, impacting clinical disease course and outcome.

IL-27 induces phosphorylated STAT-1 in mucosal epithelial cells

To identify the cell type directly impacted by IL-27 treatment, immunohistochemistry was employed to identify STAT-1 phosphorylation in response to orally administered LL-IL-27. Multifocal mucosal epithelial cells and scattered inflammatory cells within the lamina propria and submucosa showed strong positive nuclear labeling for phosphorylated STAT 1 (Figure 7a). This suggests efficacy of LL-IL-27 is a consequence of both immune and non-immune cell mediated effects. In keeping with this, IL-27 was previously shown to promote epithelial wound restitution in vitro 25. As expected, gavage of LL-IL-27 in healthy non colitic mice did not result in STAT-1 phosphorylation response, again demonstrating that an inflammatory microenvironment is required for LL-IL-27 immunosuppression.

Figure 7. IL-27 is associated with phosphorylated STAT-1 in colonic epithelial cells and significantly reduces pro-inflammatory cytokine profile in vivo.

Figure 7

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(a) Immunohistochemistry for phosphorylated STAT-1 (Tyr701). Multifocal mucosal epithelial cells and scattered inflammatory cells within the lamina propria and submucosa show strong positive nuclear labeling for phosphorylated STAT-1 in response to oral LL-IL-27. Occasional cells show both strong nuclear and variable cytoplasmic labeling. There is no phosphorylated STAT-1 immunopositivity in colitic mice who received no treatment or treatment with LL-C. Diaminobenzidine chromogen and hematoxylin counterstain. This pattern of expression was seen in all mice per group (n=3). (b) Cytokine gene expression profile in distal colon homogenate was assessed by Taqman RT-PCR assays (Applied Biosystems) and analyzed with ddCt method of relative quantification; normalization to endogenous GAPDH control and level of expression compared to ethanol control group. Data, combined from 2 experiments, presented as mean + SEM. Il6 (p=0.003), Il1b (p=0.001) and Tnf (p=<0.01) were significantly increased in TNBS treated mice. Expression of Il6 was significantly reduced with LL-IL-27 compared to LL-C (p=0.036), whereas no such effect was seen for Il1b and Tnf. n=7, 10–13, 10–15, 10–15, for ethanol, TNBS alone, TNBS+LL-C and TNBS+LL-IL-27, respectively. (c) Cytokine protein expression in distal colon homogenate measured by ELISA and normalized to mg total protein. Combined data presented from 2 experiments as mean + SEM. n=5, 8–10, 7–10, 10, for ethanol, TNBS alone, TNBS+LL-C and TNBS+LL-IL-27, respectively. Protein expression of IL-6 (p=0.002), IL-1β (p=0.003) and TNF (p=0.031) were significantly reduced in the distal colon of colitic mice receiving LL-IL-27 treatment compared to LL-C.

IL-27 significantly reduces pro-inflammatory cytokine profile in vivo

Gene expression of pro-inflammatory cytokines were assessed in distal colon homogenate, namely Il6, Il1β, Tnf, Il13, Il12, Ifng, Il17a, and Il22, along with the regulatory T cell associated transcription factor Foxp3. The majority of these genes were expressed at low abundance or undetectable and not investigated further. Il6, Il1β, and Tnf were significantly increased in all TNBS treated mice compared to ethanol controls (Figure 7b). Il6 expression was significantly lower in mice treated with LL-IL-27 compared to LL-C. No differential expression of Il1β or Tnf was seen with LL-IL-27 treatment.

In contrast, distal colon protein expression of IL6, IL1β and TNF were significantly reduced in colitic mice receiving LL-IL-27 treatment (Figure 7c), highlighting a discrepancy between gene and protein expression for the latter two cytokines. This suggests potential influences on translational or post-translational regulation. Thus we hypothesized that IL-27 may inhibit the inflammasome as a mechanism of immunosuppression. However, there was no impact on expression of distal colon il-18 or inflammasome components (Nlrp3, Caspase-1 and Asc), nor LPS induced IL1β secretion from in vitro macrophage preparations (Supplementary Figure 2), suggesting IL-27 does not directly influence acute colonic inflammasome activation in vivo.

Discussion

Our data show a novel immunosuppressive activity of the cytokine IL-27 in acute severe colitis, directed toward gut mucosal innate immune responses. Mucosal delivery of IL-27 reduced clinical parameters of colonic disease activity, repressed systemic inflammatory response, and improved colitis histology score. LL-IL-27 improved mucosal ulceration. This is important because mucosal healing is defined by the absence of ulcerated lesions in the gut mucosa during endoscopy, and this represents a primary endpoint in assessing effectiveness of IBD therapy.

The mechanism of IL-27 directed immunosuppression was multifactorial. This data represents first in field evidence of IL-27 evoked reduction in colonic mucosal neutrophil infiltrate, associated with a decreased CXC chemokine gradient. This was T cell independent and IL-10 dependent, with early enhanced mucosal IL-10. IL-27 also significantly attenuated pro-inflammatory cytokine protein expression. Although there was no evidence of direct impact on epithelial cell proliferation, IL-27 provoked an epithelial phosphorylated STAT-1 response. For the first time, we have revealed that IL-27 attenuates acute immune responses in the colon in vivo in a non-infective, genetically intact host.

IL-27 evoked reduction in myeloid cell infiltrate has been reported in different body sites, identified through genetic manipulation of IL-27 receptor signaling. Atherosclerotic prone Ldlr−/− mice had attenuated disease with hematopoietic cell IL-27Rα deficiency, mediated partly via reduced plaque chemokine gradient and infiltration of myeloid cells 32. Ebi3−/− mice demonstrated reduced granulocyte flux to acute peritonitis 26. When IL-27 receptor signaling was interrupted by genetic deficiency or antibody blocking, mice with systemic sepsis secondary to cecal puncture ligation and exposed to a secondary pneumonic bacterial insult, exhibited enhanced neutrophil infiltrate to the lungs This was associated with increased chemokine expression including CXCL2 and pro-inflammatory mediators such as IL-6, TNF, and IL1β 24. This IL-27 mediated blunting of granulocyte response was deleterious overall and associated with inability to resolve the secondary bacterial infection.

Neutrophils are necessary for efficient resolution of acute microbial attack through a variety of mechanisms 33. The suppression of neutrophil influx by IL-27 complicates IL-27 therapy for application to human disease. However, LL-IL-27 represents a localized rather than systemic therapy, delivered orally to act directly on the inflamed colonic mucosa, with no evidence of systemic IL-27 absorption as reported here and in our previous publication 11. The balance of beneficial to deleterious IL-27 inhibition of innate immunity appears site specific. Thus, IL-27 is beneficial in the circumstance of acute non-infective colitis, by limiting granulocyte directed tissue damage, release of pro-inflammatory mediators and activation of downstream adaptive immune responses.

CXCL2 is a potent neutrophil chemokine, secreted mainly by monocytes and macrophages, in response to LPS stimulation 27. There is existing evidence that IL-27 can modulate the functional capacity of macrophages and other innate cells. It is unclear whether IL-27 effects on innate cells are pro-inflammatory or anti-inflammatory and it appears that the outcome is dependent on stimulus, cell type and surrounding or experimental microenvironment 14, akin to its pleiotropic effect on T cell biology. For example, IL-27 can enhance pro-inflammatory cytokine and chemokine expression from blood derived human monocytes cultured and stimulated in vitro 34,35. In the gut, host or recipient IL-27R knockout mice, with or without genetic silencing of TCR revealed a role of IL-27-induced, APC-derived, cytokine mediated Th17 responses in T cell driven colitis 36. Conversely, lung macrophages from IL-27Rα knockout mice exert enhanced pro-inflammatory cytokine and chemokine secretion, along with increased anti-microbial killing capacity 24 exemplifying a suppressive activity of IL-27 on innate immune cells. Iyer 17 revealed macrophage IL-10 secretion in response to LPS or type I interferons is dependent on endogenous IL-27 signaling. Therefore, it was reasonable to speculate that our orally delivered IL-27 may suppress the proinflammatory function of the colonic macrophage population. Indeed, our data suggest that colonic macrophages are a potent source of CXCL2 during acute colitis and this was blunted by in vivo exposure to IL-27. We did not find evidence to support a direct immunosuppressive effect of IL-27 on macrophage chemokine response, despite utilizing three independent ex vivo macrophage culture models including colonic macrophages.

We then assessed the involvement of T cells in the IL-27-mediated immunosuppression in this acute colitis model. IL-27 inhibits Th2 and Th17 responses 13,14, although this was not likely to be prominent in our acute model given the rapid onset of pathology. T cells present in the normal colonic mucosa are part of the ‘physiological immunity’ that evokes tolerance to luminal antigens and appropriate response to pathogenic insult. IL-27 is known to promote T regulatory cell subsets 31, 37. However in our model, IL-27 immunosuppression of innate cell driven colonic pathology was T cell independent.

We acknowledge that the direct cell target of orally delivered IL-27 in acute colitis remains elusive. We predict that IL-27 in acute severe colitis acts on epithelial cells and inflammatory cells of unknown phenotype. There are several immune cell types that may, independently or in conjunction, act in response to exogenous IL-27, such as neutrophils, dendritic cells or innate lymphoid cells and although outside the scope of this current study, warrants further investigation to reveal novel biological functions of IL-27. Neutrophils can secrete chemokines and attract an increasing neutrophil influx 38. IL-27 can inhibit neutrophil ROS production 16, 19, reduce their expression of the surface integrin Mac-1 16, 19 and provoke short lived IL1β secretion either independently 16 or in response to bacterial challenge 19, 24. IL-27 receptor deficiency led to enhanced neutrophil intracellular bacterial killing 24. Neither neutrophil survival nor apoptosis is known to be affected by IL-27 16. Another potential cell candidate is dendritic cells (DCs). Human immature DCs exposed to IL-27 displayed reduced antigen presenting capacity 22. IL-27 provokes DC derived IL-10, and suppresses downstream Th1 and Th17 response 20, 21. This mechanism impacts disease outcome in CNS autoimmune pathology, with vaccination of IL-27 primed DCs attenuating murine EAE 21. Another potential cell candidate is colonic epithelial cells, as this cell compartment can secrete chemokines 39, 40 and our results indicate that these cells strongly express phosphorylated STAT1 in response to mucosally delivered LL-IL-27 in vivo.

This data confirms the potential for oral L. lactis IL-27 to be a potential rescue medical therapy in acute severe colitis. Lactococcus lactis is a non-pathogenic, non-colonizing and non-transmissible food grade bacterium. The use of this bacteria as a mucosal delivery system for therapeutic proteins and vaccines has been reviewed elsewhere 41–43. We showed previously that orally delivered IL-27 attenuates chronic T cell derived colonic inflammation 11 and now present novel evidence to show efficacy towards acute innate cell derived colonic pathology, highlighting translational impact as a new or adjunct medical treatment strategy in acute severe colitis.

Supplementary Material

Supplementary Fig 1
Supplementary Fig 2

Acknowledgments

We thank Kelli Czarra (Laboratory Animal Sciences Program, Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, Maryland, USA) for her assistance with animal studies, and Kathleen Noer and Roberta Matthai of the Flow cytometry core facility, NCI for assistance with cell sorting.

Source of Funding

Lothar Steidler is a shareholder of Intrexon Corporation and employee of Intrexon Actobiotics N.V.

This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, as well as a fellowship funded by Crohn’s and Colitis Foundation of America (MHM) and a grant from the Broad Medical Research Foundation. This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. HHSN261200800001E (MRA). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.

Abbreviations

IL-27

interleukin 27

LL-IL-27

Lactococcus lactis expressing IL-27

LL-C

Lactococcus lactis empty vector control

DAI

Disease activity index

Footnotes

Conflicts of Interest

The other authors have declared that no conflict of interest exists.

Author contributions

MHM designed and executed experiments, analyzed data, and wrote the manuscript. LS provided L. lactis strains. WQL designed the IL-27 construct and assisted with experiments. MRA provided pathology reporting. All other authors assisted with design, execution and analysis of experiments. Specific input with macrophage in vitro culture and flow cytometry (WAB & LCD), preventative assessment (CS), ex vivo colonic cell isolation, pathology, and digital photography (CA) is noted. SKD designed experiments, wrote the manuscript and provided overall direction. All authors edited and agreed on the final manuscript.

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Supplementary Materials

Supplementary Fig 1
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Supplementary Fig 2
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