Monogenic causes of inflammatory bowel diseases (IBD) are increasingly being discovered. To date, much attention has been placed in those resulting from inborn errors of immunity. Therapeutic efforts have been largely focused on offering personalized immune modulation or curative bone marrow transplant for patients with IBD and underlying immune disorders. To date, less emphasis has been placed on monogenic causes of IBD that pertain to impairment of the intestinal epithelial barrier. Here, we provide a comprehensive review of monogenic causes of IBD that result in impaired intestinal epithelial barrier that are categorized into 6 important functions: (1) epithelial cell organization, (2) epithelial cell intrinsic functions, (3) epithelial cell apoptosis and necroptosis, (4) complement activation, (5) epithelial cell signaling, and (6) control of RNA degradation products. We illustrate how impairment of any of these categories can result in IBD. This work reviews the current understanding of the genes involved in maintaining the intestinal barrier, the inheritance patterns that result in dysfunction, features of IBD resulting from these disorders, and pertinent translational work in this field.

Afzali et al. described 3 patients with heterozygous point mutations in the transcription factor BACH2, characterized by lymphocyte maturation defects, immunoglobulin deficiency and intestinal inflammation secondary to BACH2-related immunodeficiency and autoimmunity (BRIDA). Molecular testing suggested these mutations lead to disruption of protein stability. Mice with heterozygous Bach2 deficiency display similar lymphocyte defects, supporting that the disease-mechanism was BACH2 haploinsufficiency. We present a patient with recurrent infections and hypogammaglobulinemia with heterozygous chromosome 6 deletion encompassing the BACH2 gene, in absence of gastrointestinal or rheumatologic manifestations.

Inflammatory bowel disease (IBD) is associated with immune dysregulation triggered by environmental factors, microbial dysbiosis, and genetical susceptibility. Regulatory T cells (Tregs) are critical in controlling intestinal immune homeostasis and Treg deficiencies trigger intestinal inflammation.1Brunkow M.E. et al.Nat Genet. 2001; 27: 68-73Crossref PubMed Scopus (2023) Google Scholar, 2Bennett C.L. et al.Nat Genet. 2001; 27: 20-21Crossref PubMed Scopus (2639) Google Scholar Interleukin (IL)-2 is a key cytokine controlling differentiation, survival, and function of Tregs.3Klatzmann D. et al.Nat Rev Immunol. 2015; 15: 283-294Crossref PubMed Scopus (399) Google Scholar In contrast to conventional T cells (Tcon), Tregs exhibit higher sensitivity to IL-2 due to constitutive expression of CD25, the high-affinity subunit of the IL-2 receptor.3Klatzmann D. et al.Nat Rev Immunol. 2015; 15: 283-294Crossref PubMed Scopus (399) Google Scholar Low-dose (LD) IL-2 has been reported to selectively expand Tregs and used as a therapeutic strategy in chronic graft-versus-host disease, hepatitis C virus–induced vasculitis, systemic lupus erythematosus, and Wiskott-Aldrich syndrome.4He J. et al.Nat Med. 2016; 22: 991-993Crossref PubMed Scopus (365) Google Scholar, 5Koreth J. et al.N Engl J Med. 2011; 365: 2055-2066Crossref PubMed Scopus (826) Google Scholar, 6Saadoun D. et al.N Engl J Med. 2011; 365: 2067-2077Crossref PubMed Scopus (588) Google Scholar, 7Jyonouchi S. et al.Clin Immunol. 2017; 179: 47-53Crossref PubMed Scopus (24) Google Scholar Thus, we sought to investigate LD IL-2 as an IBD therapeutic using humanized mice. Similar to other disease settings,4He J. et al.Nat Med. 2016; 22: 991-993Crossref PubMed Scopus (365) Google Scholar, 5Koreth J. et al.N Engl J Med. 2011; 365: 2055-2066Crossref PubMed Scopus (826) Google Scholar, 6Saadoun D. et al.N Engl J Med. 2011; 365: 2067-2077Crossref PubMed Scopus (588) Google Scholar, 7Jyonouchi S. et al.Clin Immunol. 2017; 179: 47-53Crossref PubMed Scopus (24) Google Scholar LD IL-2 specifically activated peripheral blood and colonic lamina propria Tregs from patients with IBD in vitro (Supplementary Figure 1). To study the efficacy of LD IL-2 in a preclinical setting, we treated NSG mice reconstituted with healthy donor peripheral blood mononuclear cells with phosphate-buffered saline (PBS) or LD IL-2 (1.0 × 104 IU/day [10K]; 5.0 × 104 IU/day [50K]) (Figure 1A). On day 5 following immune reconstitution, colitis was induced by rectal application of 2,4-dinitrobenzene sulfonic acid. Mice receiving 10K IL-2 had reduced weight loss and reduced histology scores compared with mice treated with PBS or 50K IL-2 (Figure 1B and C). Phospho-flow analysis of STAT5 confirmed that 10K IL-2 specifically activated Tregs, whereas STAT5 phosphorylation was also detected in Tcons from mice receiving 50K IL-2 (Figure 1D). Expansion of Tregs was observed in blood, spleen, and colon of mice treated with either dose of IL-2 (Figure 1E). However, reduced body weight loss upon treatment with 10K IL-2 associated with Treg expansion in the absence of significant Tcon activation suggest that a therapeutic range of LD IL-2 is critical (Figure 1C–E). To evaluate the efficacy of LD IL-2 in a fully reconstituted humanized murine system, we developed NSG mice that lack murine MHCII but express human HLA-DQ8 (NSGIIDQ8 mice). Sixteen-week-old mice reconstituted with human healthy donor CD34+ hematopoietic stem cells at birth were sensitized with 2,4,6-trinitrobenzenesulfonic acid (TNBS) and treated with 10K IL-2 daily followed by induction of colitis with TNBS rectal challenge (Figure 2A). In contrast to mice treated with PBS, mice receiving LD IL-2 exhibited significant improvement in histological disease activity with a trend in reduced weight loss (Figure 2B and C). LD IL-2 was associated with significant expansion of human Tregs in the blood and spleen but not in the mesenteric lymph nodes or colon (Figure 2D and E). Correspondingly, no difference in the frequency of FOXP3-expressing T cells was detected using RNAscope analysis of paraffin-embedded colonic lamina propria sections (Supplementary Figure 2). The expansion of peripheral Tregs and improvement in disease activity in LD IL-2-treated mice was not attributed to alterations in the human immune reconstitution between groups (Figure 2F) or frequency of effector T or natural killer cells (Supplementary Figure 3). Tregs have been reported to suppress pathogenic effector T cell function and autoimmunity through both contact-dependent and contact-independent mechanisms.8Sakaguchi S. et al.Nat Rev Immunol. 2010; 10: 490-500Crossref PubMed Scopus (1794) Google Scholar CyTOF analysis of splenic CD25+ cells showed an increased frequency of FOXP3+ T cells in mice receiving LD IL-2 with majority of expanded cells falling within the CD45RO+FOXP3+ Treg cluster (Figure 2G). The majority of FOXP3+ Tregs that expanded after LD IL-2-treatment exhibited increased expression of molecules associated with Treg activation or function (HLA-DR, CD45RO, and CTLA4) or chemokines important for trafficking and migration to sites of inflammation (CCR4 and CCR6) (Figure 2H). Our data suggest that expansion and activation of memory Tregs might be critical clinical determinants. Taken together, our study demonstrates that LD IL-2 expands Tregs and ameliorates experimental colitis in humanized mice. While these data support a rationale for LD IL-2 in IBD therapy, the safety of long-term drug administration needs further investigation to ensure continued selective activation of Tregs over Tcons. Based on these promising results, we have initiated a phase 1b/2a clinical trial investigating the safety and therapeutic efficacy of LD IL-2 in patients with moderate to severe ulcerative colitis (NCT02200445). The authors thank the Harvard Digestive Disease Center for their core support services. Download .pdf (2.13 MB) Help with pdf files Supplemental Methods Download .pdf (.12 MB) Help with pdf files Supplemental Figures 1–4

Background: Transcriptional profiling has been performed on biopsies from ulcerative colitis patients. Limitations in prior studies include the variability introduced by inflammation, anatomic site of biopsy, extent of disease, and medications. We sought to more globally understand the variability of gene expression from patients with ulcerative colitis to advance our understanding of its pathogenesis and to guide clinical study design. Methods: We performed transcriptional profiling on 13 subjects, including pediatric and adult patients from 2 hospital sites. For each patient, we collected 6 biopsies from macroscopically inflamed tissue and 4 biopsies from macroscopically healthy-appearing tissue. Isolated RNA was used for microarray gene expression analysis utilizing Affymetrix Human Primeview microarrays. Ingenuity pathway analysis was used to assess over-representation of gene ontology and biological pathways. RNAseq was also performed, and differential analysis was assessed to compare affected vs unaffected samples. Finally, we modeled the minimum number of biopsies required to reliably detect gene expression across different subject numbers. Results: Transcriptional profiles co-clustered independently of the hospital collection site, patient age, sex, and colonic location, which parallels prior gene expression findings. A small set of genes not previously described was identified. Our modeling analysis reveals the number of biopsies and patients per cohort to yield reliable results in clinical studies. Conclusions: Key findings include concordance, including some expansion, of previously published gene expression studies and similarity among different age groups. We also established a reliable statistical model for biopsy collection for future clinical studies.

Mutations in Wiskott-Aldrich syndrome protein (WASP) cause autoimmune sequelae including colitis. Yet, how WASP mediates mucosal homeostasis is not fully understood. Here we show that WASP-mediated regulation of anti-inflammatory macrophages is critical for mucosal homeostasis and immune tolerance. The generation and function of anti-inflammatory macrophages are defective in both human and mice in the absence of WASP. Expression of WASP specifically in macrophages, but not in dendritic cells, is critical for regulation of colitis development. Importantly, transfer of WT anti-inflammatory macrophages prevents the development of colitis. DOCK8-deficient macrophages phenocopy the altered macrophage properties associated with WASP deficiency. Mechanistically, we show that both WASP and DOCK8 regulates macrophage function by modulating IL-10-dependent STAT3 phosphorylation. Overall, our study indicates that anti-inflammatory macrophage function and mucosal immune tolerance require both WASP and DOCK8, and that IL-10 signalling modulates a WASP-DOCK8 complex.

Background: IL10 receptor (IL10R) deficiency causes severe infantile-onset inflammatory bowel disease. Intact IL10R-dependent signals have been shown to be important for innate and adaptive immune cell functions in mice. We have previously reported a key role of IL10 in the generation and function of human anti-inflammatory macrophages. Independent of innate immune cell defects, the aim of the current study was to determine the role of IL10R signaling in regulating human CD4+ T-cell function. Methods: Peripheral blood mononuclear cells and intestinal biopsies cells were collected from IL10/IL10R-deficient patients and controls. Frequencies of CD4+ T-cell subsets, naive T-cell proliferation, regulatory T cell (Treg)-mediated suppression, and Treg and TH17 generation were determined by flow cytometry. Transcriptional profiling was performed by NanoString and quantitative real-time polymerase chain reaction. RNA in situ hybridization was used to determine the quantities of various transcripts in intestinal mucosa. Results: Analysis of 16 IL10- and IL10R-deficient patients demonstrated similar frequencies of peripheral blood and intestinal Tregs, compared with control subjects. In addition, in vitro Treg suppression of CD4+ T-cell proliferation and generation of Treg were not dependent on IL10R signaling. However, IL10R-deficient T naive cells exhibited higher proliferative capacity, a strong TH17 signature, and an increase in polarization toward TH17 cells, compared with controls. Moreover, the frequency of TH17 cells was increased in the colon and ileum of IL10R-deficient patients. Finally, we show that stimulation of IL10R-deficient Tregs in the presence of IL1&bgr; leads to enhanced production of IL17A. Conclusions: IL10R signaling regulates TH17 polarization and T-cell proliferation in humans but is not required for the generation and in vitro suppression of Tregs. Therapies targeting the TH17 axis might be beneficial for IL10- and IL10R-deficient patients as a bridge to allogeneic hematopoietic stem cell transplantation.

The intestinal mucosal surface is continuously exposed to microbial communities and intestinal homeostasis is dependent on host barrier function and the establishment and persistence of well-regulated immune responses. Innate immune cells play a critical role in the maintenance of mucosal homeostasis by facilitating immune tolerance to luminal antigens. A recent study identified an inflammatory bowel disease (IBD) causal sub-network of genes (e.g., IL10, ARC, NOD2, HCK, WAS), which were highly enriched in bone marrow derived macrophages (Mφ) and predicted to have a role in anti-inflammatory macrophage function. The Wiskott-Aldrich syndrome (WAS) gene was identified within this causal sub-network along with HCK, a protein that interacts with Wiskott-Aldrich syndrome protein (WASP). WASP-deficient mice and up to 10% of WAS patients develop IBD. We have shown that WASP-deficiency in mice, when limited to innate immune cells, is sufficient to render normal wild-type CD4+ T cells colitogenic. We hypothesized that WASP-mediated regulation of anti-inflammatory Mφ function is critical for the maintenance of intestinal homeostasis. We analyzed the expression of Ly6c and MHCII in mucosal macrophages (CD45+CD11b+CD11c−CD64+CD103−) to distinguish them into pro- and anti-inflammatory Mφ populations. Bone marrow derived Mφ (BMDM) in mice and monocyte derived Mφ in human was polarized into anti-inflammatory M2 Mφ in presence of IL4, TGFb and IL10. Analysis of gut resident Mφ population in 5-weeks-old pre-colitic Was−/− mice showed an increase in the percentage of pro-inflammatory Mφ (Ly6chigh MHCIIhigh) and a concomitant decrease in the percentage of anti-inflammatory Mφ (Ly6clow MHCIIhigh). As predicted, in a bone-marrow chimera experiment, where lethally irradiated Was−/− mice were transferred with a mixture of WT (CD45.1) and Was−/− (CD45.2) bone-marrow in a 1:1 ratio, the Was−/− compartment, harbored an increased percentage of pro-inflammatory Mφ and decreased anti-inflammatory Mφ compared to the WT compartment. Next we examined the role of Mφ–intrinsic WASP in intestinal homeostasis by transferring CD4+ T cell into Rag1−/− mice lacking WASP only in Mφ (Rag1−/−Wasfl/flLysMCre). Rag1−/−Wasfl/flLysMCre recipient mice, when compared to Rag1−/− mice, developed more severe weight loss and colonic inflammation with increased pro-inflammatory cytokine expression. In addition, in vitro generation of BMDM M2 Mφ from Was−/− mice expressing classical M2 markers Arg1 and Fizz1, was also markedly reduced when compared with M2 Mφ derived from WT mice. LPS induced a much higher expression of pro-inflammatory cytokines in Was−/− M2 Mφ compared to WT M2 Mφ. Moreover, Was−/− M2 Mφ induced greater proliferation of CD4+ T cells, cytokine production, and decreased generation of Tregs compared to WT Mφ. Importantly, the colitis that results from the transfer of CD4+ T cells into Rag1−/−Was−/− mice was rescued by the transfer of WT M2 Mφ but not Was−/− M2 Mφ. Similarly, we found that the M2 Mφ generation was also severely impaired in patients with Wiskott-Aldrich syndrome. M2 Mφ from patients expressed higher levels of pro-inflammatory cytokines and was less efficient in the generation Tregs in an in vitro co-culture system. Collectively, these data implicates a critical Mφ-intrinsic functional role for WASP for the generation and maintenance of tolerance in the intestinal mucosa.

Existing therapies for inflammatory bowel disease that are based on broad suppression of inflammation result in variable clinical benefit and unwanted side effects. A potential therapeutic approach for promoting immune tolerance is the in vivo induction of regulatory T cells (Tregs). Here we report that activation of the aryl hydrocarbon receptor using the nontoxic agonist 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) induces human Tregs in vitro that suppress effector T cells through a mechanism mediated by CD39 and Granzyme B. We then developed a humanized murine system whereby human CD4(+) T cells drive colitis upon exposure to 2,4,6-trinitrobenzenesulfonic acid and assessed ITE as a potential therapeutic. ITE administration ameliorated colitis in humanized mice with increased CD39, Granzyme B, and IL10-secreting human Tregs. These results develop an experimental model to investigate human CD4(+) T responses in vivo and identify the non-toxic AHR agonist ITE as a potential therapy for promoting immune tolerance in the intestine.

Interleukin 10 receptor (IL10R)-deficient mice develop spontaneous colitis and, similarly, patients with loss-of-function mutations in IL10R develop severe infant-onset inflammatory bowel disease. Loss of IL10R signaling in mouse and human macrophages is associated with increased production of interleukin 1β. We demonstrated that innate immune production of IL1β mediates colitis in IL10R-deficient mice. Transfer of Il1r1-/- CD4+ T cells into Rag1-/-/Il10rb-/- mice reduced the severity of their colitis (compared to mice that received CD4+ T cells that express IL1R), accompanied by decreased production of interferon gamma, tumor necrosis factor-α, and IL17A. In macrophages from mice without disruption of IL10R signaling or from healthy humans (controls), incubation with IL10 reduced canonical activation of the inflammasome and production of IL1β through transcriptional and post-translational regulation of NLRP3. Lipopolysaccharide and adenosine triphosphate stimulation of macrophages from Il10rb-/- mice or IL10R-deficient patients resulted in increased production of IL1β. Moreover, in human IL10R-deficient macrophages, lipopolysaccharide stimulation alone triggered IL1β secretion via non-canonical, caspase 8-dependent activation of the inflammasome. We treated 2 IL10R-deficient patients with severe and treatment-refractory infant-onset inflammatory bowel disease with the IL1-receptor antagonist anakinra. Both patients had marked clinical, endoscopic, and histologic responses after 4-7 weeks. This treatment served as successful bridge to allogeneic hematopoietic stem cell transplantation in 1 patient. Our findings indicate that loss of IL10 signaling leads to intestinal inflammation, at least in part, through increased production of IL1 by innate immune cells, leading to activation of CD4+ T cells. Agents that block IL1 signaling might be used to treat patients with inflammatory bowel disease resulting from IL10R deficiency.

Background Although deleterious mutations in interleukin-10 and its receptor molecules cause severe infantile-onset inflammatory bowel disease, there are no reports of mutations affecting this signaling pathway in Japanese patients. Here we report a novel exonic mutation in the IL10RA gene that caused unique splicing aberrations in a Japanese patient with infantile-onset of inflammatory bowel disease in association with immune thrombocytopenic purpura and a transient clinical syndrome mimicking juvenile myelomonocytic leukemia. Case presentation A Japanese boy, who was the first child of non-consanguineous healthy parents, developed bloody diarrhea, perianal fistula, and folliculitis in early infancy and was diagnosed with inflammatory bowel disease. He also developed immune thrombocytopenic purpura and transient features mimicking juvenile myelomonocytic leukemia. The patient failed to respond to various treatments, including elemental diet, salazosulfapyridine, metronidazole, corticosteroid, infliximab, and adalimumab. We identified a novel mutation (c.537G > A, p.T179T) in exon 4 of the IL10RA gene causing unique splicing aberrations and resulting in lack of signaling through the interleukin-10 receptor. At 21 months of age, the patient underwent allogeneic hematopoietic stem cell transplantation and achieved clinical remission. Conclusions We describe a novel exonic mutation in the IL10RA gene resulting in infantile-onset inflammatory bowel disease. This mutation might also be involved in his early-onset hematologic disorders. Physicians should be familiar with the clinical phenotype of IL-10 signaling defects in order to enable prompt diagnosis at an early age and referral for allogeneic hematopoietic stem cell transplantation.

data.RESULTS.Overall, HD ADA-treated pts with abnormal laboratory values at BL achieved normal albumin levels (75%), platelet counts (53%), and CRP levels (64%) at wk 52 (Table ).HD ADA also improved mean hemoglobin levels at wk 52 (mean change from BL LD -0.96 v. HD 3.43 g/L, p<0.001).CONCLUSION.ADA led to normalization of laboratory values in clinically meaningful proportions of pts who had abnormal albumin levels, platelet counts, and CRP levels at BL in IMAgINE 1. Improvements in hemoglobin levels were also observed at wk 52.Reference: 1.