Microbiota-derived butyrate restricts tuft cell differentiation via histone deacetylase 3 to modulate intestinal type 2 immunity.

Tuft cells in mucosal tissues are key regulators of type 2 immunity. Here, we examined the impact of the microbiota on tuft cell biology in the intestine. Succinate induction of tuft cells and type 2 innate lymphoid cells was elevated with loss of gut microbiota. Colonization with butyrate-producing bacteria or treatment with butyrate suppressed this effect and reduced intestinal histone deacetylase activity. Epithelial-intrinsic deletion of the epigenetic-modifying enzyme histone deacetylase 3 (HDAC3) inhibited tuft cell expansion in vivo and impaired type 2 immune responses during helminth infection. Butyrate restricted stem cell differentiation into tuft cells, and inhibition of HDAC3 in adult mice and human intestinal organoids blocked tuft cell expansion. Collectively, these data define a HDAC3 mechanism in stem cells for tuft cell differentiation that is dampened by a commensal metabolite, revealing a pathway whereby the microbiota calibrate intestinal type 2 immunity.

Microbiota-derived butyrate dampens linaclotide stimulation of the guanylate cyclase C pathway in patient-derived colonoids.

BACKGROUND & AIMS: Disorders of gut-brain interaction (DGBI) are complex conditions that result in decreased quality of life and a significant cost burden. Linaclotide, a guanylin cyclase C (GCC) receptor agonist, is approved as a DGBI treatment. However, its efficacy has been limited and variable across DGBI patients. Microbiota and metabolomic alterations are noted in DGBI patients, provoking the hypothesis that the microbiota may impact the GCC response to current therapeutics. METHODS: Human-derived intestinal organoids were grown from pediatric DGBI, non-IBD colon biopsies (colonoids). Colonoids were treated with 250 nM linaclotide and assayed for cGMP to develop a model of GCC activity. Butyrate was administered to human colonoids overnight at a concentration of 1 mM. Colonoid lysates were analyzed for cGMP levels by ELISA. For the swelling assay, colonoids were photographed pre- and post-treatment and volume was measured using ImageJ. Principal coordinate analyses (PCoA) were performed on the Bray-Curtis dissimilarity and Jaccard distance to assess differences in the community composition of short-chain fatty acid (SCFA) producing microbial species in the intestinal microbiota from pediatric patients with IBS and healthy control samples. KEY RESULTS: Linaclotide treatment induced a significant increase in [cGMP] and swelling of patient-derived colonoids, demonstrating a human in vitro model of linaclotide-induced GCC activation. Shotgun sequencing analysis of pediatric IBS patients and healthy controls showed differences in the composition of commensal SCFA-producing bacteria. Butyrate exposure significantly dampened linaclotide-induced cGMP levels and swelling in patient-derived colonoids. CONCLUSIONS & INFERENCES: Patient-derived colonoids demonstrate that microbiota-derived butyrate can dampen human colonic responses to linaclotide. This study supports incorporation of microbiota and metabolomic assessment to improve precision medicine for DGBI patients.

Deep Crypt Secretory Cell Differentiation in the Colonic Epithelium Is Regulated by Sprouty2 and Interleukin 13.

BACKGROUND & AIMS: Deep crypt secretory (DCS) cells are a critical component of the colonic stem cell niche. However, the regulatory mechanisms controlling DCS cell numbers and function are not well understood. Sprouty2 is an inflammation-responsive regulator of intracellular signaling that influences colonic secretory cell numbers in colitis via an epithelial-stromal interleukin (IL)33/IL13 signaling loop. Here, we tested the hypothesis that IL13, induced by epithelial Sprouty2 down-regulation, promotes DCS cell differentiation and function. METHODS: Distal colons from mice with an intestinal epithelial-specific Sprouty2 deletion (Spry2ΔIE) and littermate controls were analyzed by in situ hybridization for Reg4+ DCS cells. Single-cell RNA sequencing and immunostaining were used to identify DCS cell-derived host defense peptides (HDPs) and localization of IL13 and IL13 receptor; bulk RNA sequencing and quantitative polymerase chain reaction were used to quantify changes in expression of identified HDPs. Cytokine-treated colonoids were assessed for DCS cells. A requirement for an IL33/IL13 signaling loop in the regulation of DCS cells was assessed in vivo using IL13 null mice. RESULTS: Reg4+ DCS cell numbers were increased 2-fold in distal colons of Spry2ΔIE mice with a concomitant overall increase in DCS cell marker expression (Reg4, Spink4, and Agr2). Single-cell transcriptomics showed the HDP Retnlb/Resistin Like Beta (RELMß) is highly enriched in DCS cells. Retnlb/RELMß expression was increased in Spry2ΔIE colons. IL13, but not IL33, induced Reg4 and Retnlb expression in colonic epithelial organoids, and IL33-mediated expansion of the DCS cell population in vivo was dependent on IL13, which was expressed predominantly by type II innate lymphoid cells in the colonic mucosa. CONCLUSIONS: Sprouty2 limits colonic DCS cell differentiation through suppression of IL13 signaling. At homeostasis, DCS cells are marked by high levels of the HDP RELMß. Loss of epithelial Sprouty2 activates type II innate lymphoid cells to release IL13, promoting expansion of the DCS cell population and increased colonic RELMß levels.

Multicenter Cohort Study of Infliximab Pharmacokinetics and Therapy Response in Pediatric Acute Severe Ulcerative Colitis.

BACKGROUND & AIMS: We aimed to model infliximab (IFX) pharmacokinetics (PK) in pediatric acute severe ulcerative colitis (ASUC) and assess the association between PK parameters, including drug exposure, and clinical response. METHODS: We studied a multicenter prospective cohort of hospitalized children initiating IFX for ASUC or IBD-unclassified. Serial IFX serum concentrations over 26 weeks were used to develop a PK model. We tested the association of PK parameter estimates with day 7 clinical response, week 8 clinical remission, week 26 corticosteroid-free clinical remission (CSF-CR) (using the Pediatric Ulcerative Colitis Activity Index), and colectomy-free survival. RESULTS: Thirty-eight participants received IFX (median initial dose, 9.9 mg/kg). Day 7 clinical response, week 8 clinical remission, and week 26 CSF-CR occurred in 71%, 55%, and 43%, respectively. Albumin, C-reactive protein, white blood cell count, platelets, weight, and antibodies to IFX were significant covariates incorporated into a PK model. Week 26 non-remitters exhibited faster IFX clearance than remitters (P = .013). However, cumulative IFX exposure did not differ between clinical response groups. One (2.7%) and 4 (10.8%) participants underwent colectomy by week 26 and 2 years, respectively. Day 3 IFX clearance >0.02 L/h was associated with colectomy (hazard ratio, 58.2; 95% confidence interval, 6.0-568.6; P < .001). CONCLUSIONS: At median higher-than-label IFX dosing for pediatric ASUC, baseline faster IFX CL was associated with colectomy and at week 26 with lack of CSF-CR. IFX exposure was not predictive of clinical outcomes. Higher IFX dosing may sufficiently optimize early outcomes in pediatric ASUC. Larger studies are warranted to determine whether sustained intensification can overcome rapid clearance and improve later outcomes. CLINICALTRIALS: gov identifier: NCT02799615.

IL-33 is produced by colon fibroblasts and differentially regulated in acute and chronic murine colitis.

IL-33 is upregulated in ulcerative colitis and has a protective role in chemically-induced acute murine colitis. We aimed to determine whether IL-33 influences Il10-/- chronic colitis and its cellular source in health and during colitis. Il10-/-Il33-/- and Il10-/-Il33+/+ littermates developed colitis of similar severity. Colon Il33 was induced in WT and Il10-/- mice exposed to DSS, but not in unchallenged Il10-/- mice with colitis. Il33-citrine reporter mice showed that Il33-citrine colocalized with α-smooth muscle actin+ myofibroblasts and vimentin+ fibroblasts in WT mice. Citrine+CD74+CD90hi inflammatory fibroblasts were increased with DSS treatment. IL-1ß induced Il33 expression in colon myofibroblasts, but colon Il33 expression did not differ between DSS-treated WT and Il1r1-/- mice. In conclusion, deficiency of IL-33 does not alter the severity of chronic colitis in Il10-/- mice. Induction of Il33 upon DSS exposure in WT and Il10-/- mice, but not in unchallenged Il10-/- mice, suggests epithelial injury induces colon IL-33. Fibroblasts are the primary colonic source of IL-33 and IL-33-expressing CD90hiCD74+ fibroblasts are increased during DSS-induced colitis. IL-1ß induces Il33 in colon myofibroblasts in vitro, but signaling through the IL-1R1 is not necessary for induction of IL-33 in DSS-induced colitis.

Sprouty2 limits intestinal tuft and goblet cell numbers through GSK3ß-mediated restriction of epithelial IL-33.

Dynamic regulation of intestinal cell differentiation is crucial for both homeostasis and the response to injury or inflammation. Sprouty2, an intracellular signaling regulator, controls pathways including PI3K and MAPKs that are implicated in differentiation and are dysregulated in inflammatory bowel disease. Here, we ask whether Sprouty2 controls secretory cell differentiation and the response to colitis. We report that colonic epithelial Sprouty2 deletion leads to expanded tuft and goblet cell populations. Sprouty2 loss induces PI3K/Akt signaling, leading to GSK3ß inhibition and epithelial interleukin (IL)-33 expression. In vivo, this results in increased stromal IL-13+ cells. IL-13 in turn induces tuft and goblet cell expansion in vitro and in vivo. Sprouty2 is downregulated by acute inflammation; this appears to be a protective response, as VillinCre;Sprouty2F/F mice are resistant to DSS colitis. In contrast, Sprouty2 is elevated in chronic colitis and in colons of inflammatory bowel disease patients, suggesting that this protective epithelial-stromal signaling mechanism is lost in disease.

IL-33 Induces Murine Intestinal Goblet Cell Differentiation Indirectly via Innate Lymphoid Cell IL-13 Secretion.

Regulation of the intestinal mucus layer by goblet cells is important for preventing inflammation and controlling infection. IL-33, a cytokine upregulated in inflammatory bowel disease and helminth infection, induces intestinal goblet cells, but the mechanism remains unclear. Enteroids are three-dimensional structures of primary small intestinal epithelial cells that contain all differentiated intestinal epithelial cell types. We developed an enteroid-immune cell coculture model to determine the mechanism through which IL-33 affects intestinal goblet cell differentiation. We report that IL-33 does not directly induce goblet cell differentiation in murine enteroids; however, IL-13, a cytokine induced by IL-33, markedly induces goblet cells and gene expression consistent with goblet cell differentiation. When enteroids are cocultured with CD90+ mesenteric lymph node cells from IL-33-treated mice, IL-33 then induces IL-13 secretion by group 2 innate lymphoid cells and enteroid gene expression consistent with goblet cell differentiation. In cocultures, IL-33-induced Muc2 expression is dependent on enteroid Il4ra expression, demonstrating a requirement for IL-13 signaling in epithelial cells. In vivo, IL-33-induced intestinal goblet cell hyperplasia is dependent on IL-13. These studies demonstrate that IL-33 induces intestinal goblet cell differentiation not through direct action on epithelial cells but indirectly through IL-13 production by goup 2 innate lymphoid cells.

Mucosal Expression of Type 2 and Type 17 Immune Response Genes Distinguishes Ulcerative Colitis From Colon-Only Crohn's Disease in Treatment-Naive Pediatric Patients.

BACKGROUND & AIMS: There is controversy regarding the role of the type 2 immune response in the pathogenesis of ulcerative colitis (UC)-few data are available from treatment-naive patients. We investigated whether genes associated with a type 2 immune response in the intestinal mucosa are up-regulated in treatment-naive pediatric patients with UC compared with patients with Crohn's disease (CD)-associated colitis or without inflammatory bowel disease (IBD), and whether expression levels are associated with clinical outcomes. METHODS: We used a real-time reverse-transcription quantitative polymerase chain reaction array to analyze messenger RNA (mRNA) expression patterns in rectal mucosal samples from 138 treatment-naive pediatric patients with IBD and macroscopic rectal disease, as well as those from 49 children without IBD (controls), enrolled in a multicenter prospective observational study from 2008 to 2012. Results were validated in real-time reverse-transcription quantitative polymerase chain reaction analyses of rectal RNA from an independent cohort of 34 pediatric patients with IBD and macroscopic rectal disease and 17 controls from Cincinnati Children's Hospital Medical Center. RESULTS: We measured significant increases in mRNAs associated with a type 2 immune response (interleukin [IL]5 gene, IL13, and IL13RA2) and a type 17 immune response (IL17A and IL23) in mucosal samples from patients with UC compared with patients with colon-only CD. In a regression model, increased expression of IL5 and IL17A mRNAs distinguished patients with UC from patients with colon-only CD (P = .001; area under the receiver operating characteristic curve, 0.72). We identified a gene expression pattern in rectal tissues of patients with UC, characterized by detection of IL13 mRNA, that predicted clinical response to therapy after 6 months (odds ratio [OR], 6.469; 95% confidence interval [CI], 1.553-26.94), clinical response after 12 months (OR, 6.125; 95% CI, 1.330-28.22), and remission after 12 months (OR, 5.333; 95% CI, 1.132-25.12). CONCLUSIONS: In an analysis of rectal tissues from treatment-naive pediatric patients with IBD, we observed activation of a type 2 immune response during the early course of UC. We were able to distinguish patients with UC from those with colon-only CD based on increased mucosal expression of genes that mediate type 2 and type 17 immune responses. Increased expression at diagnosis of genes that mediate a type 2 immune response is associated with response to therapy and remission in pediatric patients with UC.

Myeloid-derived NF-κB negative regulation of PU.1 and c/EBP-ß-driven pro-inflammatory cytokine production restrains LPS-induced shock.

Sepsis is a life-threatening event predominantly caused by Gram-negative bacteria. Bacterial infection causes a pronounced macrophage (MΦ) and dendritic cell activation that leads to excessive pro-inflammatory cytokine IL-1ß, IL-6 and TNF-α production (cytokine storm), resulting in endotoxic shock. Previous experimental studies have revealed that inhibiting NF-κB signaling ameliorates disease symptoms; however, the contribution of myeloid p65 in endotoxic shock remains elusive. In this study, we demonstrate increased mortality in mice lacking p65 in the myeloid lineage (p65Δmye) compared with wild type mice upon ultra-pure LPS challenge. We show that increased susceptibility to LPS-induced shock was associated with elevated serum level of IL-1ß and IL-6. Mechanistic analyses revealed that LPS-induced pro-inflammatory cytokine production was ameliorated in p65-deficient bone marrow-derived MΦs; however, p65-deficient 'activated' peritoneal MΦs exhibited elevated IL-1ß and IL-6. We show that the elevated pro-inflammatory cytokine secretion was due, in part, to increased accumulation of IL-1ß mRNA and protein in activated inflammatory MΦs. The increased IL-1ß was linked with heightened binding of PU.1 and CCAAT/enhancer binding protein-ß to Il1b and Il6 promoters in activated inflammatory MΦs. Our data provide insight into a role for NF-κB in the negative regulation of pro-inflammatory cytokines in myeloid cells.

IL-33 Signaling Protects from Murine Oxazolone Colitis by Supporting Intestinal Epithelial Function.

BACKGROUND: IL-33, a member of the IL-1 cytokine family that signals through ST2, is upregulated in ulcerative colitis (UC); however, the role of IL-33 in colitis remains unclear. IL-33 augments type 2 immune responses, which have been implicated in UC pathogenesis. We sought to determine the role of IL-33 signaling in oxazolone (OXA) colitis, a type 2 cytokine-mediated murine model of UC. METHODS: Colon mucosal IL-33 expression was compared between pediatric and adult UC and non-IBD patients using immunohistochemistry and real-time PCR. OXA colitis was induced in WT, IL-33, and ST2 mice, and histopathology, cytokine levels, and goblet cells were assessed. Transepithelial resistance was measured across IL-33-treated T84 cell monolayers. RESULTS: Colon mucosal IL-33 was increased in pediatric patients with active UC and in OXA colitis. IL-33 and ST2 OXA mice exhibited increased disease severity compared with WT OXA mice. OXA induced a mixed mucosal cytokine response, but few differences were observed between OXA WT and IL-33 or ST2 mice. Goblet cells were significantly decreased in IL-33 and ST2 OXA compared with WT OXA mice. IL-33 augmented transepithelial resistance in T84 cells, and this effect was blocked by the ERK1/2 inhibitor PD98,059. CONCLUSIONS: OXA colitis is exacerbated in IL-33 and ST2 mice. Increased mucosal IL-33 in human UC and murine colitis may be a homeostatic response to limit inflammation, potentially through effects on epithelial barrier function. Further investigation of IL-33 protective mechanisms would inform the development of novel therapeutic approaches.

NKT cells can help mediate the protective effects of 1,25-dihydroxyvitamin D3 in experimental autoimmune encephalomyelitis in mice.

Active vitamin D [1,25-dihydroxyvitamin D3 (1,25D3)] blocks the development of experimental autoimmune diseases. However, the molecular and immunobiological mechanisms underlying 1,25D3's anti-inflammatory properties are not fully understood. We employed a murine model of experimental autoimmune encephalomyelitis (EAE) in order to determine the role of NKT cells in 1,25D3-mediated protection from EAE. Wild-type (WT) mice or mice lacking all NKT cells (CD1d(-/-)) or invariant NKT cells (Jα18(-/-)) were fed control or 1,25D3-supplemented diets. All mice fed with the control diet developed severe EAE. 1,25D3 treatment of WT mice protected them from developing EAE. CD1d(-/-) and Jα18(-/-) mice treated with 1,25D3 were not protected to the same extent as WT mice. Myelin oligodendrocyte glycoprotein-specific IL-17 and IFN-γ production was significantly reduced in 1,25D3 WT mice compared with WT but was not decreased in 1,25D3 CD1d(-/-) mice compared with CD1d(-/-) mice. IL-4(-/-) mice were utilized to determine how IL-4 deficiency affects susceptibility to EAE. IL-4(-/-) mice were not protected from developing EAE by α-galactosylceramide (α-GalCer) or 1,25D3 treatment. Furthermore, 1,25D3 treatment of splenocytes in vitro decreased α-GalCer-induced IL-17 and increased IL-4, IL-5 and IL-10 production. 1,25D3 alters the cytokine profile of invariant NKT cells in vitro. These studies demonstrate that NKT cells are important mediators of 1,25D3-induced protection from EAE in mice and NKT cell-derived IL-4 may be an important factor in providing this protection.

The vitamin D receptor turns off chronically activated T cells.

T cell proliferation and T helper (TH ) cells that make IL-17 (TH 17 cells) and IFN-γ (TH 1 cells) have been shown to be inhibited by 1,25(OH)2 D3 . Previous work has shown that immune-mediated diseases, where TH 1 and TH 17 cells are pathogenic, are ameliorated with 1,25(OH)2 D3 treatment. Paradoxically, infectious diseases that require TH 1 and TH 17 responses for host resistance are unaffected by 1,25(OH)2 D3 treatment. Resting T cells are not responsive to vitamin D because they do not express the vitamin D receptor (VDR) until late after activation. T cells activated following an infection help clear the infection, and since the antigen is eliminated, vitamin D is not needed to dampen the immune response. Conversely, in immune-mediated disease, there is chronic T cell activation, and in this scenario, vitamin D and 1,25(OH)2 D3 are critical for inhibiting T cell proliferation and cytokine production. Vitamin D is a late regulator of T cell function and acts to turn off T cells. This paper will review these data.

Dominant effects of the diet on the microbiome and the local and systemic immune response in mice.

Outside the nutrition community the effects of diet on immune-mediated diseases and experimental outcomes have not been appreciated. Investigators that study immune-mediated diseases and/or the microbiome have overlooked the potential of diet to impact disease phenotype. We aimed to determine the effects of diet on the bacterial microbiota and immune-mediated diseases. Three different laboratory diets were fed to wild-type mice for 2 weeks and resulted in three distinct susceptibilities to dextran sodium sulfate (DSS)-induced colitis. Examination of the fecal microbiota demonstrated a diet-mediated effect on the bacteria found there. Broad-spectrum antibiotics disturbed the gut microbiome and partially eliminated the diet-mediated changes in DSS susceptibility. Dietary changes 2 days after DSS treatment were protective and suggested that the diet-mediated effect occurred quickly. There were no diet-mediated effects on DSS susceptibility in germ-free mice. In addition, the diet-mediated effects were evident in a gastrointestinal infection model (Citrobacter rodentium) and in experimental autoimmune encephalomyelitis. Taken together, our study demonstrates a dominant effect of diet on immune-mediated diseases that act rapidly by changing the microbiota. These findings highlight the potential of using dietary manipulation to control the microbiome and prevent/treat immune-mediated disease.

CD14+CD33+ myeloid cell-CCL11-eosinophil signature in ulcerative colitis.

This study tested the hypothesis that eotaxins (CCL11, CCL24, and CCL26) and IL-5 contribute to eosinophil recruitment to the intestine in UC and that intestinal macrophages are important producers of CCL11 in this disease. Peripheral blood and rectal biopsy samples were obtained from patients with active (n=18) and quiescent UC (n=9), and control patients (n=7). Eosinophil and macrophage levels and activation were analyzed by flow cytometry. Rectal mRNA levels of CCL11, CCL24, CCL26, and IL-5 were determined by qRT-PCR. The cellular source of CCL11 was visualized by immunofluorescence analyses. Eosinophil numbers were elevated in the blood and rectum of active and quiescent UC patients compared with controls. Levels of activated eosinophils (CD66b(high)) correlated with disease severity. Rectal CCL11, CCL24, and CCL26 mRNA levels were increased in active UC, whereas only CCL11 was elevated in quiescent UC. Levels of CCL11, but not CCL24 and CCL26, positively correlated with eosinophil numbers. Numbers of CD14(+)CD33(+) cells correlated with CCL11 and eosinophil levels. Immunofluorescence analyses revealed the presence of CD14(+)CCL11(+) mononuclear cells in colonic biopsies in UC. These results support the hypothesis that CCL11 contributes to eosinophil recruitment in UC and that intestinal myeloid cells are a source of CCL11. Interestingly, rectal levels of CCL24, CCL26, and IL-5 only increase during active UC, coinciding with further elevation of eosinophil numbers and with the activation of rectal eosinophils. In conclusion, there is a link among CD14(+)CD33(+) myeloid cells, CCL11, and eosinophils in adult UC.

Intestinal CCL11 and eosinophilic inflammation is regulated by myeloid cell-specific RelA/p65 in mice.

In inflammatory bowel diseases (IBDs), particularly ulcerative colitis, intestinal macrophages (MΦs), eosinophils, and the eosinophil-selective chemokine CCL11, have been associated with disease pathogenesis. MΦs, a source of CCL11, have been reported to be of a mixed classical (NF-κB-mediated) and alternatively activated (STAT-6-mediated) phenotype. The importance of NF-κB and STAT-6 pathways to the intestinal MΦ/CCL11 response and eosinophilic inflammation in the histopathology of experimental colitis is not yet understood. Our gene array analyses demonstrated elevated STAT-6- and NF-κB-dependent genes in pediatric ulcerative colitis colonic biopsies. Dextran sodium sulfate (DSS) exposure induced STAT-6 and NF-κB activation in mouse intestinal F4/80(+)CD11b(+)Ly6C(hi) (inflammatory) MΦs. DSS-induced CCL11 expression, eosinophilic inflammation, and histopathology were attenuated in RelA/p65(Δmye) mice, but not in the absence of STAT-6. Deletion of p65 in myeloid cells did not affect inflammatory MΦ recruitment or alter apoptosis, but did attenuate LPS-induced cytokine production (IL-6) and Ccl11 expression in purified F4/80(+)CD11b(+)Ly6C(hi) inflammatory MΦs. Molecular and cellular analyses revealed a link between expression of calprotectin (S100a8/S100a9), Ccl11 expression, and eosinophil numbers in the DSS-treated colon. In vitro studies of bone marrow-derived MΦs showed calprotectin-induced CCL11 production via a p65-dependent mechanism. Our results indicate that myeloid cell-specific NF-κB-dependent pathways play an unexpected role in CCL11 expression and maintenance of eosinophilic inflammation in experimental colitis. These data indicate that targeting myeloid cells and NF-κB-dependent pathways may be of therapeutic benefit for the treatment of eosinophilic inflammation and histopathology in IBD.

Eosinophils in infection and intestinal immunity.

PURPOSE OF REVIEW: Inflammatory bowel diseases (IBDs, e.g., Crohn's disease and ulcerative colitis) are thought to be a consequence of an uncontrolled inflammatory response against luminal antigens, including commensal bacteria. The observed link between eosinophil levels and severity and remission rates in IBD has led to speculation that eosinophils may contribute to the antimicrobial inflammatory response in IBD. RECENT FINDINGS: Eosinophils express the necessary cellular machinery (innate immune receptors, proinflammatory cytokines, antibacterial proteins, and DNA traps) to mount an efficient antibacterial response; however, the rapid decline in eosinophil numbers following acute systemic bacterial infection suggests a very limited role for eosinophils in bacterial responses. SUMMARY: We describe the clinical evidence of eosinophil involvement in IBD, summarize the in-vitro and in-vivo evidence of eosinophil antibacterial activity and the biology of eosinophils focusing on eosinophil-mediated bactericidal mechanisms and the involvement of eosinophil-derived granule proteins in this response, and conceptualize the contribution of eosinophils to a response against commensal bacteria in IBD.

Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11.

Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate (DSS) treatment promotes the recruitment of F4/80(+)CD11b(+)CCR2(+)Ly6C(high) inflammatory monocytes into the colon. F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6C(high) intestinal inflammatory macrophages revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocyte recruitment to the colon in CCR2(-/-) mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6C(high)CCR2(+) inflammatory monocyte/macrophage-derived CCL11.

Resistin-like molecule alpha enhances myeloid cell activation and promotes colitis.

BACKGROUND: Resistin-like molecule (Relm) alpha is a secreted protein and a hallmark signature gene for alternatively activated macrophages. Relm-alpha is highly induced by allergic inflammatory triggers and perceived to promote tissue repair. Yet the function of Relm-alpha remains unknown. OBJECTIVE: We sough to determine the role of Relm-alpha in dextran sodium sulfate (DSS)-induced colonic injury. METHODS: The cellular source of Relm-alpha was determined after oral DSS-induced colitis. Retnla(-/-) mice were generated, subjected to DSS treatment, and monitored for disease progression (clinical and histopathologic features). Cytokine production in the supernatants of ex vivo colon cultures, and of LPS-stimulated macrophages incubated with Relm-alpha was assessed. Relm-alpha was administered intraperitoneally, and the cellular recruitment to the peritoneum was assessed. RESULTS: After innate intestinal stimulation with DSS, Relm-alpha was highly expressed by eosinophils and epithelial cells. Retnla gene-targeted mice were protected from DSS-induced colitis (eg, decreased diarrhea, rectal bleeding, colon shortening, disease score, and histopathologic changes). Relm-alpha coactivated IL-6 and TNF-alpha release and inhibited IL-10 release from LPS-activated bone marrow-derived macrophages. Consistent with these finding, colon cultures of DSS-treated Retnla(-/-) mice produced decreased IL-6 and increased IL-10 ex vivo. Furthermore, Retnla(-/-) mice had substantially decreased c-Jun N-terminal kinase phosphorylation in vivo. In vivo administration of Relm-alpha initiated cellular recruitment to the peritoneum, and Relm-alpha was able to induce eosinophil chemotaxis in vitro. CONCLUSIONS: These findings demonstrate a central proinflammatory role for Relm-alpha in colonic innate immune responses, identifying a novel pathway for regulation of macrophage activation.

Intestinal macrophage/epithelial cell-derived CCL11/eotaxin-1 mediates eosinophil recruitment and function in pediatric ulcerative colitis.

Clinical studies have demonstrated a link between the eosinophil-selective chemokines, eotaxins (eotaxin-1/CCL11 and eotaxin-2/CCL24), eosinophils, and the inflammatory bowel diseases, Crohn's disease and ulcerative colitis (UC). However, the cellular source and individual contribution of the eotaxins to colonic eosinophilic accumulation in inflammatory bowel diseases remain unclear. In this study we demonstrate, by gene array and quantitative PCR, elevated levels of eotaxin-1 mRNA in the rectosigmoid colon of pediatric UC patients. We show that elevated levels of eotaxin-1 mRNA positively correlated with rectosigmoid eosinophil numbers. Further, colonic eosinophils appeared to be degranulating, and the levels positively correlated with disease severity. Using the dextran sodium sulfate (DSS)-induced intestinal epithelial injury model, we show that DSS treatment of mice strongly induced colonic eotaxin-1 and eotaxin-2 expression and eosinophil levels. Analysis of eosinophil-deficient mice defined an effector role for eosinophils in disease pathology. DSS treatment of eotaxin-2(-/-) and eotaxin-1/2(-/-) mice demonstrated that eosinophil recruitment was dependent on eotaxin-1. In situ and immunofluorescence analysis-identified eotaxin-1 expression was restricted to intestinal F4/80(+)CD11b(+) macrophages in DSS-induced epithelial injury and to CD68(+) intestinal macrophages and the basolateral compartment of intestinal epithelial cells in pediatric UC. These data demonstrate that intestinal macrophage and epithelial cell-derived eotaxin-1 plays a critical role in the regulation of eosinophil recruitment in colonic eosinophilic disease such as pediatric UC and provides a basis for targeting the eosinophil/eotaxin-1 axis in UC.