Mary Jo Wick

The chronic inflammation of Crohn’s disease frequently leads to fibrosis and muscular hypertrophy of the intestinal wall. This often culminates in strictures, a serious condition lacking directed therapy. Severe pathological changes occur in the submucosa and muscularis propria intestinal wall layers of strictures, yet stricture-associated proteome changes in these layers is unexplored. We perform unbiased proteomics on submucosa and muscularis propria microdissected from transmural sections of strictured and nonstrictured ileum. Proteome changes in strictured submucosa reflected a transition from homeostasis to tissue remodeling, inflammation, and smooth muscle changes. Top submucosal features included reduced vascular components and lipid metabolism proteins accompanied by increased proteins with immune-, ECM-, or stress-related functions, including CTHRC1, TNC, IL-16, MZB1, and TXNDC5. In parallel, predominant changes in strictured muscularis propria included increased ECM (POSTN) and immune (mast cell CPA3) proteins alongside decreased proteins with lipid metabolic, mitochondrial, or key muscle functions. Finally, trends of differentially expressed proteins along nonstrictured submucosa suggest progressive profibrotic tissue remodeling and muscle expansion as proximity to strictures increases. The comprehensive proteome map presented here offers tissue-layer-resolved insight into the stricture microenvironment and potential drivers of fibrotic disease, providing a valuable resource to fuel biomarker and therapeutic target research.

Inflammasomes are intracellular protein complexes whose activation results in proinflammatory cytokines. Inflammasomes are implicated in Crohn´s disease (CD) pathogenesis, yet the contribution of inflammasomes in intestinal epithelial cells (IECs) versus lamina propria (LP) macrophages is poorly understood. Whether inflammasome expression in intestinal tissue reflects the serum inflammatory protein profile of patients is also not known. We aimed to determine the intestinal cell types where inflammasome expression is increased in CD and if they correlate with the serum protein profile. RT-PCR and NanoString nCounter technology were used to characterize inflammasome gene expression in CD patients and controls. The mucosa, LP and IEC cell fractions and FACS-sorted cells were analyzed. Proximity extension assay with a 92-protein panel was used to determine the serum inflammatory protein profile. Compositional analysis was used to correlate ileum inflammasome gene expression with intestinal mononuclear phagocyte populations. We show that NLRP3 and MEFV inflammasome sensors and downstream effector expression including IL-1β are increased in inflamed mucosa of IBD patients and correlate with disease activity. Inflammasome gene expression increased with the abundance of immature intestinal macrophages, and increased IL-1β released by CD LP cells correlated with immature macrophage frequency. Inflammasome gene expression was also increased in circulating monocytes, the precursors of immature intestinal macrophages. Finally, the serum inflammatory profile of CD patients correlates with ileal expression of genes related to NLRP3 and MEFV inflammasomes. Overall, we show that MEFV and NLRP3 inflammasome expression in CD intestine is attributed to the accumulation of immature macrophages and correlates with serum inflammatory proteins.

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract that leads to substantial suffering for millions of patients. In some patients, the chronic inflammation leads to remodelling of the extracellular matrix and fibrosis. Fibrosis, in combination with expansion of smooth muscle layers, leaves the bowel segment narrowed and stiff resulting in strictures, which often require urgent medical intervention. Although stricture development is associated with inflammation in the affected segment, anti‐inflammatory therapies fall far short of treating strictures. At best, current therapies might allow some patients to avoid surgery in a shorter perspective and no anti‐fibrotic therapy is yet available. This likely relates to our poor understanding of the mechanism underlying stricture development. Chronic inflammation is a prerequisite, but progression to strictures involves changes in fibroblasts, myofibroblasts and smooth muscle cells in a poorly understood interplay with immune cells and environmental cues. Much of the experimental evidence available is from animal models, cell lines or non‐strictured patient tissue. Accordingly, these limitations create the basis for many previously published reviews covering the topic. Although this information has contributed to the understanding of fibrotic mechanisms in general, in the end, data must be validated in strictured tissue from patients. As stricture formation is a serious complication of CD, we endeavoured to summarize findings exclusively performed using strictured tissue from patients. Here, we give an update of the mechanism driving this serious complication in patients, and how the strictured tissue differs from adjacent unaffected tissue and controls.

Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is a complex immune-mediated disease of the gastrointestinal tract that increases morbidity and negatively influences the quality of life. Intestinal mononuclear phagocytes (MNPs) have a crucial role in maintaining epithelial barrier integrity while controlling pathogen invasion by activating an appropriate immune response. However, in genetically predisposed individuals, uncontrolled immune activation to intestinal flora is thought to underlie the chronic mucosal inflammation that can ultimately result in IBD. Thus, MNPs are involved in fine-tuning mucosal immune system responsiveness and have a critical role in maintaining homeostasis or, potentially, the emergence of IBD. MNPs include monocytes, macrophages and dendritic cells, which are functionally diverse but highly complementary. Despite their crucial role in maintaining intestinal homeostasis, specific functions of human MNP subsets are poorly understood, especially during diseases such as IBD. Here we review the current understanding of MNP ontogeny, as well as the recently identified human intestinal MNP subsets, and discuss their role in health and IBD.

The intestinal commensal microbiota is essential for many host physiological processes, but its impact on infectious diseases is poorly understood. Here we investigate the influence of the gut microbiota during oral Salmonella infection. We report a higher bacterial burden in mesenteric lymph nodes (MLN) of intragastrically infected germ-free (GF) mice compared to conventionally-raised (CONV-R) animals, despite similar inflammatory phagocyte recruitment. Salmonella penetration into the lamina propria of the small intestine and splenic bacterial burden were not altered in the absence of the microbiota. Intragastrically infected GF mice also displayed a higher frequency of IFN-γ-producing NK, NKT, CD4+ and CD8+ T cells in the MLN despite IL-12 levels similar to infected CONV-R mice. However, infecting mice intraperitoneally abrogated the difference in MLN bacterial load and IFN-γ-producing cells observed in intragastrically-infected animals. Moreover, mice treated with antibiotics (ABX) and intragastrically infected with Salmonella had a greater bacterial burden and frequency of IFN-γ-producing cells in the MLN. In ABX mice the number of Salmonella correlated with the frequency of IFN-γ-producing lymphocytes in the MLN, while no such correlation was observed in the MLN of infected GF mice. Overall, the data show that the lack of the microbiota influences pathogen colonization of the MLN, and the increased IFN-γ in the MLN of infected GF mice is not only due to the absence of commensals at the time of infection but the lack of immune signals provided by the microbiota from birth.

Previous studies using purified TLR ligands plus agonistic anti-CD40 antibodies showed that TLRs and CD40 can act synergistically on dendritic cells (DCs) to optimize T cell activation and Th1 differentiation. However, a synergistic effect of TLRs and CD40 during bacterial infection is not known. Here we show that mice lacking the TLR adaptor MyD88 alone, or lacking both MyD88 and CD40 (DKO mice), are compromised in survival to Salmonella infection but have intact recruitment of neutrophils and inflammatory monocytes as well as unaltered abundance of DC subsets and DC activation in infected tissues. In contrast to infected wildtype and CD40-/- mice, both MyD88-/- mice and DKO mice lack detectable serum IFN-γ and have elevated IL-10. A synergistic effect of TLRs and CD40 was revealed in co-culture experiments where OT-II T cell proliferation was compromised when DKO DCs were pulsed with OVA protein and OVA323-339 peptide, but not with heat-killed Salmonella expressing OVA (HKSOVA), relative to MyD88-/- DCs. In contrast, MyD88-/- or DKO DCs pulsed with any of the antigens had a similar ability to induce IFN-γ that was lower than WT or CD40-/- DCs. DKO DCs pulsed with HKSOVA, but not with OVA or OVA323-339, had increased IL-10 relative to MyD88-/- DCs. Finally, HKSOVA-pulsed MyD88-/- and DKO DCs had similar and low induction of NFκB- dependent and -independent genes upon co-culture with OT-II cells. Overall, our data revealed that synergistic effects of CD40 and MyD88 do not influence host survival to Salmonella infection or serum levels of IFN-γ or IL-10. However, synergistic effects of MyD88 and CD40 may be apparent on some (IL-10 production) but not all (OT-II proliferation and IFN-γ production) DC functions and depend on the complexity of the antigen. Indeed, synergistic effects observed using purified ligands and well-defined antigens may not necessarily apply when complex antigens, such as live bacteria, challenge the immune system.

Mucus alterations are a feature of ulcerative colitis (UC) and can drive inflammation by compromising the mucosal barrier to luminal bacteria. The exact pathogenesis of UC remains unclear, but CD4⁺ T cells reacting to commensal antigens appear to contribute to pathology. Given the unique capacity of dendritic cells (DCs) to activate naive T cells, colon DCs may activate pathogenic T cells and contribute to disease. Using Muc2^-/- mice, which lack a functional mucus barrier and develop spontaneous colitis, we show that colitic animals have reduced colon CD103⁺CD11b^- DCs and increased CD103^-CD11b⁺ phagocytes. Moreover, changes in colonic DC subsets and distinct cytokine patterns distinguish mice with distally localized colitis from mice with colitis spread proximally. Specifically, mice with proximally spread, but not distally contained, colitis have increased IL-1β, IL-6, IL-17, TNFα, and IFNγ combined with decreased IL-10 in the distal colon. These individuals also have increased numbers of CD103⁺CD11b⁺ DCs in the distal colon. CD103⁺CD11b⁺ DCs isolated from colitic but not noncolitic mice induced robust differentiation of Th17 cells but not Th1 cells ex vivo. In contrast, CD103^-CD11b⁺ DCs from colitic Muc2^-/- mice induced Th17 as well as Th1 differentiation. Thus, the local environment influences the capacity of intestinal DC subsets to induce T cell proliferation and differentiation, with CD103⁺CD11b⁺ DCs inducing IL-17-producing T cells being a key feature of extensively spread colitis.