Oral interleukin-10 alleviates polyposis via neutralization of pathogenic T-regulatory cells

Abstract

Immune dysregulation drives the pathogenesis of chronic inflammatory, autoimmune and dysplastic disorders. While often intended to address localized pathology, most immune modulatory therapies are administered systemically and carry inherent risk of multi-organ toxicities. Here we demonstrate, in a murine model of spontaneous gastrointestinal polyposis, that site-specific uptake of orally-administered microparticles of the interleukin IL-10 ameliorates local and systemic disease to enhance survival. Mechanistic investigations showed that the therapeutic benefit of this treatment derived from neutralization of disease-promoting FoxP3+RoRγt+IL17+ pathogenic T-regulatory cells (pgTreg), with a concomitant restoration of FoxP3+RoRγt-IL17- conventional T regulatory cells (Treg). These findings provide a proof-of-principle for the ability of an oral biologic to restore immune homeostasis at the intestinal surface. Further, they implicate local manipulation of IL-10 as a tractable therapeutic strategy to address the inflammatory sequelae associated with mucosal premalignancy.

Introduction

IL-10 is critical for maintenance of immune homeostasis, particularly at the gastrointestinal interface¹. In both humans and mice, IL-10 deficiency is linked to the development of a wide range of immune-mediated pathologies^{2, 3} including cancer ⁴.

Pre-clinical studies in murine models of inflammatory disease have demonstrated therapeutic utility for recombinant IL-10^5,6. In clinical trials however, intravenous administration of IL-10 to patients with inflammatory bowel disease (IBD) resulted in dose-dependent paradoxical effects with only nominal therapeutic benefit⁷. Due to the lack of methods for administering IL-10 in a tissue-specific manner the clinical potential of IL-10 as a biological therapeutic remains undetermined.

Previous studies have revealed that bioerodible sustained-release microparticles prepared via phase inversion nanoencapsulation (PIN) can be used for oral delivery of biological molecules to the intestine⁸. When applied to models of intestinal inflammation and dysplasia, this strategy affords the ability to target physiological quantities of protein to the disease microenvironment with minimal escape into circulation. Whereas the utility of PIN for delivery of nucleic acids and peptides to the gut mucosa has been established, its effectiveness in targeting bioactive protein therapeutics to gut-associated lymphoid tissue (GALT) and mesenteric lymph nodes (MLN) remains undefined.

Based on the previous findings that adoptive transfer of CD4⁺CD25⁺ T-cells suppresses intestinal disease in the APC^min/+ mouse model of spontaneous gastrointestinal polyposis in an IL-10-dependent manner ⁹, we sought to determine whether IL-10-encapsulated PIN microparticles could directly modify the intestinal inflammatory landscape and alter the natural history of this genetically-driven pre-neoplastic disease. The results provide the first evidence for the therapeutic potential of an oral particulate cytokine formulation, in this case IL-10, to ameliorate both local and systemic disease. Furthermore, our findings identify pathogenic Tregs as a primary therapeutic target of IL-10 in the dysplastic intestine.

Materials and Methods

Animals

C57BL/6 (B6), B6.SJL-Ptprc^aPepc^b/BoyJ (CD45.1), APC^min/+, DEREG, and RAG1^−/− mice were purchased from Jackson Laboratory. BALB/c mice were purchased from Taconic. APC^min/+-DEREG mice were created in house. All experiments were conducted in accordance with guidelines set forth by the Institutional Animal Care and Use Committees at the University at Buffalo and Wadsworth Center.

Microparticle preparation and oral delivery

IL-10-loaded PIN particles were prepared using a modified PIN process¹⁰. Two formulations were produced: (a) control (no cytokine) and (b) recombinant murine IL-10 (Peprotech, Inc., Rocky Hill, > 98% purity, <1EU/μg) with a loading of 0.5 μg cytokine / mg of particles. Control or IL-10 microparticles were provided by oral gavage (1mg particles in 0.3 ml sterile water) 3 times per week for 4 weeks starting at 10 weeks of age. For experiments utilizing FITC-BSA microparticles, single 30mg bolus doses were given.

Antibodies

The following antibodies were used for immunofluorescence and immunohistochemistry: CD45R/B220 (RA3-6B2, BD Pharmingen), CD11c (N418, eBioscience), Fc receptor block;anti-CD16/CD32 (2.4G2, BD Pharmingen), CD3 (F7.2.38, Agilent Technologies), von Willebrand factor (vWF) (A0082, Dako). The following antibodies were used for flow cytometry: CD45.1 (A20, eBioscience), CD4 (RM4-5, eBioscience), IL-17A (TC11-18H10, BD Pharmingen), FoxP3 (FJK-16s, eBioscience), RoRγt (Q31-378, BD Pharmingen), CD44 (IM7, eBioscience), IFNγ (XMG1.2, BD Pharmingen), CD8α (53-6.7, eBioscience), γδ-TCR (eBioGL3, eBioscience). Anti-mCD4 (GK1.5, BioXCell) was used for in vivo CD4⁺ T-cell depletion.

Immunofluorescence tissue collection and staining

Tissues were embedded in OCT compound (Sakura Finetek) and snap frozen in liquid nitrogen. 20μm serial cryosections were immunostained as indicated previously¹¹. Scanning laser confocal images were collected using a Leica SP5 ABOS microscope and processed using Fiji Software (http://fiji.sc/).

Gross intestinal preparation and polyp quantification

Formalin-fixed intestines were opened longitudinally and polyp burdens were quantified using a dissecting microscope.

Flow Cytometry

Spleens and mesenteric lymph nodes (MLN) were processed into single cell suspensions. Intestines were digested and fractionated into lamina propria mononuclear cells (LPMCs) and intraepithelial lymphocytes (IELs) as described previously¹². For experiments requiring detection of intracellular antigens (FoxP3, RoRγt, IL-17A and IFNγ), cell suspensions were cultured for 5h in the presence of Golgistop (5μl/ml; BD), phorbol myristate acetate (PMA) (50ng/ml; Sigma) and ionomycin (1μg/ml; Sigma). Cells were then permeabilized and fixed using an intracellular staining kit (eBioscience) overnight at 4°C.

Histological and immunohistochemical preparation

Paraffin-embedded tissue sections (5μm) were stained with Hematoxylin and Eosin. For immunohistochemistry (IHC), slides were subjected to antigen retrieval prior to application of the indicated primary antibodies. Biotinylated secondary antibodies (anti-rat IgG, BD Pharmingen), HRP-SA conjugate (Invitrogen), and diaminobenzidine (DAB) chromogen (Dako) were used for visualization. Images were taken at 400X magnification.

Histological Scoring

For intestines, histological scores were assigned in a blinded fashion using criteria established in our laboratory (see supplementary data). For spleens, histological scores were assigned using modified criteria from a previously published report¹³ (see supplementary data).

Hematological indices

Red blood cell (RBC), hemoglobin (HgB) and hematocrit (HCT) levels were determined using a Vetscan HM5 automated analyzer (Abaxis Veterinary Diagnostics).

Mortality Tracking

Mortality criteria for APC^min/+ mice were developed and applied to survival studies. A combined scoring system, taking into account the degree of weight loss and the severity of change in activity was selected (see supplementary data).

Microarray data processing

RNA was isolated from nonpolyp (for WT and APC^min/+ mice) and polyp sections (for APC ^min/+ mice) of terminal ileum using the RNeasy minikit (Qiagen) and subjected to gene expression profiling. Briefly, RNA was processed into cRNA using the Affymetrix Genechip 3’ IVT Expression Protocol and applied to Affymetrix Mouse Genome 430A 2.0 arrays. Interference from bacterial RNA was selected against using a probe-set enriched in oligos extending into 3’-poly-A tails. Details with respect to sample processing and preparation can be accessed from the GEO (NIH) under accession number GSE49970.

Subtotal Treg depletion

DEREG or APC^min/+-DEREG mice received 1μg of Diphtheria toxin (DT, Sigma) dissolved in 100μl of Dulbecco’s Phosphate Buffered Saline (DPBS) by intraperitoneal (i.p.) injection on days 0, 1 and 14.

In vivo Treg Suppression Assay

MLN CD45.2⁺CD4⁺CD25⁺ cells and total lymph node CD45.1⁺CD4⁺CD25⁻ cells were isolated by magnetic selection from either APC^min/+ or BoyJ mice, respectively. 1x10⁶ CD45.1⁺CD4⁺CD25⁻ cells were CFSE-labeled using the Vybrant CFDA SE cell tracer kit and adoptively transferred with 4x10⁵ MLN CD45.2⁺CD4⁺CD25⁺ as indicated, into 8 week old female RAG1^−/− recipients.

In vivo adoptive cell transfer

1x10⁶ magnetically selected MLN CD4⁺CD25⁺ T-cells from 14 week old APC^min/+ mice were adoptively transferred into sex-matched 10 week old APC^min/+ recipients. 4 days before transfer, recipient mice were transiently depleted of their CD4⁺ cells using a single 300μg i.p. dose of anti-CD4 antibody.

Quantitative RT-PCR (qPCR)

Steady-state mRNA levels were detected with SYBR Green PCR Master Mix (Applied Biosystems) using the Mx3000p qPCR system (Agilent Technologies). Results were normalized to the expression of β-actin. The expression level was scaled using the 2^−ΔΔCT method, with the average levels obtained for wild type intestines set arbitrarily to 1. Primer sequences utilized were: β-actin forward 5’-TCACCCACACTGGCCCATCTACGA-3’, reverse 5’-TGGTGAAGCTGTAGCCACGCT-3’; IL-1β forward 5’-GCCCATCCTCTGTGACTCAT-3’, reverse 5’-AGGCCACAGGTATTTTGTCG-3’; IL-6 forward 5’-CCATCCAGTTGCCTTCTTGG-3’, reverse 5’-TTTCTGCAAGTGCATCATCG-3’; IL-17A forward 5’-CTGAGCTTCCCAGATCACAGAG-3’, reverse 5’-CGCAAAAGTGAGCTCCAGAAAG-3’; GM-CSF forward 5’-CACGTTGAATGAAGAGGTAGAAG-3’, reverse 5’-CATGTTCAAGGCGCCCTTGAG-3’; TNFα forward 5’-GAACTGGCAGAAGAGGCACT-3’, reverse 5’-AGGGTCTGGGCCATAGAACT-3’.

Statistical Analysis

Statistical calculations were performed using Student’s t-Test in pairwise comparisons of groups. Log-rank tests were utilized for survival studies. P-values of 0.05 or less considered statistically significant.

Results and Discussion

Oral PIN microparticles loaded with FITC-BSA were taken up and retained in the Peyer’s patches (PPs) and MLNs of both APC^min/+ and wild type mice (Fig. 1A, Supplementary Fig. 1A, B). Microparticles were not detected in the colon, liver or spleen at any time point after feeding (Supplementary Fig. 1C). Moreover, when IL-10 loaded microparticles were delivered in bolus doses, IL-10 could not be detected in serum (data not shown). These results suggest that uptake of microparticles and release of IL-10 was effectively localized to intestinal immune structures.

Figure 1. Chronic oral IL-10 microparticle therapy alleviates polyposis and systemic disease symptoms.

A. Particle uptake. Six hours after oral gavage, FITC-BSA microparticles (areas circled) were localized from the PPs and MLNs of treated APC^min/+ mice. B. Effect of treatment on polyp burden. Mice were gavaged with 1 mg of control or IL-10 microspheres. Numbers indicate the mean. Boxes have lines at the median plus lower and upper quartiles, with whiskers extending to show the remaining data (n = 5, 9, 9, for wt, control, IL-10 respectively). Increasing the IL-10 dose did not improve therapeutic efficacy (data not shown). C. Anemia. RBC levels (n = 10, 15, 13 for wt, control, IL-10, respectively). D. Splenic pathology. Representative H&E and anti-vWF-stained sections are shown. Megakaryocytes were visualized directly (inset panels). Splenic pathology scores (n is identical to panel A) and megakaryocytosis (over 10-12 high power fields, n = 3 per group) were quantified. E. Body weight. Statistical comparison is made between Control and IL10-treated groups (n = 4, 6, 6 for wt, control, IL-10, respectively). F. Survival. Mortality was determined for APC^min/+ mice receiving either no treatment (control), or chronic IL-10 microsphere treatment (n = 13 and 11, respectively). Therapy was initiated on day 70. *, **, ***, **** = p<0.05, p<0.01,<0.001, and <0.0001, respectively. Error bars, s.e.m.

We directly tested whether oral administration of IL-10 microparticles could suppress established polyposis in APC^min/+ mice. Therapy resulted in a greater than 2-fold reduction in polyp burden (Fig. 1B) and a decreased severity of disease pathology in the intestine (Supplementary Fig. 2A, B). In APC^min/+ mice, intestinal disease is accompanied by predictable systemic abnormalities, namely anemia, splenomegaly and weight loss¹⁴. To determine whether amelioration of local disease would result in improvement of systemic symptoms, we monitored experimental animals over the course of therapy. In all cases, treatment with IL-10 resulted in significant benefit (Fig. 1C-E, Supplementary Fig. 2C). Relief of systemic symptoms was likely secondary to the suppression of chronic intestinal inflammation, as such activity has been shown to cause anemia¹⁵ and to promote the establishment of extramedullary hematopoiesis¹⁶. Accordingly, analysis of APC^min/+ mouse spleens revealed significant megakaryocytosis, which was abrogated following treatment (Fig. 1D). Chronic treatment resulted in a 15% extension of both median and maximal lifespan, suggesting that IL-10 could suppress but not arrest disease (Fig. 1F, Supplementary Fig. 3A).

We sought to define the potential mechanisms underlying the therapeutic activity of oral IL-10. Since IL-17 has recently been implicated as an essential driver of polyp growth in APC^min/+ mice¹⁷, and IL-10 has been shown to suppress IL-17 production by T-cells¹⁸, we hypothesized that the observed therapeutic effects of IL-10 microparticles may partially be attributed to modification of T-cell activity. APC^min/+ nonpolyp and polyp areas demonstrated increasing levels of T-cell activation, with a progressive shift from a balanced T-helper (Th) 1/2/17 profile to an IL-1β/IL-6/IL-23-enriched Th17-promoting profile during early-stage disease (Fig. 2A). Importantly, quantification of select Th17-related inducer/effector cytokines confirmed that IL-10 microparticles were highly effective at suppressing the expression of these genes in mice with established disease (Fig. 2B). In contrast, treatment did not result in significant changes in IFNγ or IL-4 levels (data not shown). Taken together, these results demonstrate that IL-10 microparticle therapy strongly antagonizes IL-17-mediated inflammation.

Figure 2. IL-10-mediated neutralization of Th-17-like intestinal inflammatory profile is associated with suppression of pgTreg IL-17 production.

A. Pre-therapy inflammatory landscape. HeatMap assessment of select T-cell related functional genes between 12 week old APC^min/+-nonpolyp and polyp intestine ranked by average fold-change over WT nonpolyp conditions. B. Post-therapy inflammatory landscape. Messenger RNA expression levels for Th17-associated cytokines from 14 week old Control or IL-10 microparticle treated mice. C. Post-therapy T-cell IL-17 production. LPMCs from 14 week old Control or IL-10 microparticle treated mice were analyzed (n= 4-6 per group). *, ** = p<0.05 and p<0.01, respectively. Error bars, s.e.m.

T-cells are thought to be the primary producers of IL-17 during chronic intestinal inflammation¹⁹. Accordingly, we examined the IL-17 expression profile in various types of intestinal T-cells from our APC^min/+ mice. Although intraepithelial γδ-T cells did not produce significant levels of IL-17 during disease (Supplementary Fig. 3B), there was an increase in both the total number of CD4⁺ T-cells and in their ability to produce IL-17 (Fig. 2C). IL-10 treatment did not impact the absolute numbers of CD4⁺ T-cells, but did act to dramatically reduce the percentage of IL-17-producing cells within this population (Fig. 2C). Since both FoxP3⁻RORγt⁺IL-17⁺ Th17 cells and FoxP3⁺RoRγt⁺IL-17⁺ pgTregs are known to be a major contributor to polyposis in this model^19–21, we examined the impact of IL-10 on IL-17 expression by these two subsets. IL-10 treatment resulted in a dramatic reduction in IL-17 production by pgTregs, with only a modest decline in IL-17 production by Th17 cells (Fig. 2C). Collectively, these results suggest that of the IL-17-producing T-cell subtypes present during disease, pgTregs are uniquely sensitive to modulation by IL-10.

Next we examined whether modulation of pgTregs was the primary mechanism underlying IL-10 microparticle therapeutic effect. We crossed APC^min/+ mice with the DEREG murine model of inducible Treg depletion and developed a subtotal Treg depletion protocol appropriate for our desire to assess the contribution of the IL-10-pgTreg axis to our model system. Subtotal Treg depletion resulted in a greater than 70% reduction in FoxP3⁺ cells over 28 days, yet avoided the catastrophic myelo- and lympho-proliferative disorder typically seen in mice undergoing total Treg ablation²² (Supplementary Fig. 4, 5A). Subtotal Treg depletion reduced polyp burden, anemia and splenic pathology (Fig. 3A, Supplementary Fig 5B, C), supporting the notion that pgTregs are the major contributors to both local and systemic disease. Administration of IL-10 microparticles further enhanced depletion-induced therapeutic benefits, suggesting that IL-10 was acting, in part, through neutralization of residual pgTregs. Consistent with this hypothesis, analysis of MLNs revealed that subtotal Treg depletion reduced the prevalence of pgTregs, while IL-10 both diminished the pgTreg presence and promoted an increase in conventional Tregs; MLN Th17 cell numbers, however, remained unaffected (Fig. 3B, Supplementary Fig 5D, E). Analysis of the lamina propria mononuclear cells (LPMCs) revealed a similar pattern, i.e. both subtotal Treg depletion and IL-10 therapy diminished the proportion of IL-17-positive cells, but the most significant reduction was obtained in the combination treatment group (Fig. 3C). Whether the superior effect that was obtained in this group was due to the appearance of a qualitatively unique Treg population is currently under investigation.

Figure 3. Subtotal depletion of Tregs in APC^min/+ mice ameliorates disease and boosts oral IL-10 microparticle therapeutic effect.

Ten week-old APC ^min/+-DEREG mice received either mock (PBS) or subtotal Treg depletion (DT), concomitant with either Control or IL-10 microparticle therapy. Disease markers were quantified at the end of the therapeutic period; A. Polyp burdens, RBC levels and splenic pathology scores. B. MLN Tregs, pgTregs and Th17 cells. Cells were defined within the CD4⁺ population as FoxP3⁺RoRγt⁻ (Treg) and FoxP3⁻RoRγt⁺ (Th17) or within the FoxP3⁺ population as RoRγt⁺ (pgTregs). C. LPMC IL-17 expression profiles. Plots are representative of experimental groups and display events within a size-excluded subgate. For A and B, n = 7-8. For C, n = 3-4. *, **, ***, **** = p<0.05, p<0.01, <0.001, p<0.0001, respectively. Error bars, s.e.m.

To determine whether the dual activity of IL-10 on pgTregs and Tregs resulted in altered functional capacity within the total Treg population, we tested the ability of post-therapy MLN CD4⁺CD25⁺ T-cells to suppress naïve T-cell proliferation and activation. In comparison to cells isolated from control treated APC^min/+ mice, the cells from IL-10 treated APC^min/+ mice were significantly more effective at suppressing these processes both in vivo and in vitro (Fig. 4A, Supplementary Fig. 6C).

Figure 4. IL-10-mediated neutralization of pgTregs enhances Treg-mediated suppression of polyposis.

A. Suppression of cellular proliferation and activation. Isolated CD45.2⁺CD4⁺CD25⁺ Treg were mixed with CFSE-labeled CD45.1⁺ responder cells and applied to an in vivo Treg suppression assay. Responder cells were assessed for generation count, expression of CD44 and total number. The prevalences of IFNγ⁺CD45.1⁺ CD4⁺ cells in recipient lymph nodes were also determined. B. Suppression of disease. MLN CD45.2⁺CD4⁺CD25⁺ cells were adoptively transferred into 10 week old untreated CD45.2⁺ APC^min/+ mice. Polyp burdens, RBC levels and prevalences of MLN Th17 cells were assessed in recipients 4 weeks after transfer. For A plots are representative of each experimental group, (n=4-5); for B, (n=4) for the Mock Transfer group, and (n=8) for each of the transfer groups. *, **, *** = p<0.05, p<0.01, and <0.001, respectively. Error bars, s.e.m.

The above results demonstrated that the suppressive capacity of APC^min/+ MLN CD4⁺CD25⁺ T-cells was restored by IL-10 microparticle therapy. We predicted that functional rescue of APC^min/+ intestinal Treg population via reversal of the pgTreg/Treg ratio was ultimately responsible for disease amelioration. To confirm this notion, we adoptively transferred MLN CD4⁺CD25⁺ T-cells from control or IL-10-treated APC^min/+ mice into 10 week old APC^min/+ recipients. IL-10-conditioned CD4⁺CD25⁺ T-cells reduced polyposis, corrected anemia and decreased the prevalence of Th17 cells in recipient MLNs (Fig. 4B, Supplementary Fig. 7A-C). Splenic disease, however, was not alleviated after transfer (Supplementary Fig. 7D, E). Whether the inability to restore full therapeutic effect was due to the limitations of the experimental approach, i.e. accumulation/persistence of transferred cells in the intestine; or to the absence of Treg-independent effects of IL-10 microparticle therapy, i.e. impacts on the innate immune system, was not determined.

Our findings demonstrate that tissue-specific delivery of a particulate cytokine formulation can change the natural history of a highly penetrant genetic condition. We propose that tissue-localized IL-10 serves as a molecular rheostat, specifically modulating pgTreg function during intestinal inflammatory and dysplastic disease. The overall concepts tested here are expected to extend to other inflammation-driven diseases, especially those manifesting at mucosal surfaces, including IBD, asthma and genital tract infection.