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Aberrant intestinal inflammation plays a critical role in the development of colitis-associated colorectal cancer (CAC), yet the mechanisms controlling tumor development by the myeloid immune compartment are not fully understood. Although altered microRNA expression is observed in CAC, it is also unclear how myeloid-specific microRNAs impact the inflammatory process that underpins the continuum from ulcerative colitis to tumorigenesis. In this study, we report that miR-223 acts to limit myeloid-driven inflammation in the azoxymethane (AOM)-dextran sodium sulfate (DSS) model of CAC in mice. In this model, miR-223-/y mice present with significantly larger tumors with an enhanced proliferative signature. Immunoprofiling showed that miR-223-/y mice have significantly increased colonic myeloid immune infiltrate (neutrophils, monocytes, and macrophages) following AOM-DSS. This was accompanied by an increased inflammatory chemokine and cytokine signature for monocytes and neutrophils. Bone marrow chimera studies demonstrate that myeloid-expressed miR-223 is responsible for the enhanced tumor proliferation and inflammatory response. RNA sequencing identified several pathways that could be contributing to the development of CAC in miR-223-/y mice, including the IL-6/IL-17a cytokine family and STAT3 signaling. Lastly, neutrophil depletion with an anti-GR1 Ab (Ly6G/Ly6C) during the initial phase of the AOM-DSS model reduced the tumor burden in miR-223-/y mice. Collectively, our data indicate that miR-223 is an important regulator of mucosal inflammation and acts to constrain the progression from ulcerative colitis to CAC by limiting myeloid-associated inflammation., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Background: Trained immunity results in long-term immunological memory, provoking a faster and greater immune response when innate immune cells encounter a secondary, often heterologous, stimulus. We have previously shown that house dust mite (HDM)-induced innate training is amplified in mice expressing the human macrophage migration inhibitory factor (MIF) CATT 7 functional polymorphism., Aim: This study investigated the ability of mesenchymal stromal cells (MSCs) to modulate MIF-driven trained immunity both in vitro and in vivo., Methods: Compared with wild-type mice, in vivo HDM-primed bone marrow-derived macrophages (BMDMs) from CATT 7 mice expressed significantly higher levels of M1-associated genes following lipopolysaccharide stimulation ex vivo. Co-cultures of CATT 7 BMDMs with MSCs suppressed this HDM-primed effect, with tumor necrosis factor alpha (TNF-α) being significantly decreased in a cyclooxygenase 2 (COX-2)-dependent manner. Interestingly, interleukin 6 (IL-6) was suppressed by MSCs independently of COX-2. In an in vitro training assay, MSCs significantly abrogated the enhanced production of pro-inflammatory cytokines by HDM-trained CATT 7 BMDMs when co-cultured at the time of HDM stimulus on day 0, displaying their therapeutic efficacy in modulating an overzealous human MIF-dependent immune response. Utilizing an in vivo model of HDM-induced trained immunity, MSCs administered systemically on day 10 and day 11 suppressed this trained phenomenon by significantly reducing TNF-α and reducing IL-6 and C-C motif chemokine ligand 17 (CCL17) production., Conclusions: This novel study elucidates how MSCs can attenuate an MIF-driven, HDM-trained response in CATT 7 mice in a model of allergic airway inflammation., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. All rights reserved.)

Atopic diseases such as Eosinophilic Esophagitis (EoE) often progress into fibrosis (FS-EoE), compromising organ function with limited targeted treatment options. Mechanistic understanding of FS-EoE progression is confounded by the lack of preclinical models and the heavy focus of research on eosinophils themselves. We found that macrophage accumulation precedes esophageal fibrosis in FS-EoE patients. We developed a FS-EoE model via chronic administration of oxazalone allergen, in a transgenic mouse over-expressing esophageal epithelial hIL-5 (L2-IL5 OXA ). These mice display striking histopathologic features congruent with that found in FS-EoE patients. Unbiased proteomic analysis, using a unique extracellular-matrix (ECM) focused technique, identified an inflammation-reactive provisional basal lamina membrane signature and this was validated in two independent EoE patient RNA-sequencing/proteomic cohorts, supporting model significance. A wound healing signature was also observed involving hemostasis-associated molecules previously unnoted in EoE. We further identified the ECM glycoprotein, Tenascin-C (TNC), and the stress-responsive keratin-16 (KRT16) as IL-4 and IL-13 responsive mediators, acting as biomarkers of FS-EoE. To mechanistically address how the immune infiltrate shapes FS-EoE progression, we phenotyped the major immune cell subsets that coalesce with fibrosis in both the L2-IL5 OXA mice and in FS-EoE patients. We found that macrophage are required for matrisome and cytoskeletal remodeling. Importantly, we show that macrophage accumulation precedes esophageal fibrosis and provide a novel therapeutic target in FS-EoE as their depletion with anti-CSF1 attenuated reactive matrisome and cytoskeletal changes. Thus, macrophage-based treatments and the exploration of TNC and KRT16 as biomarkers may provide novel therapeutic options for patients with fibrostenosis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metabolic imbalance leading to inflammatory hypoxia and stabilization of hypoxia-inducible transcription factors (HIFs) is a hallmark of inflammatory bowel diseases. We hypothesize that HIF could be stabilized in CD4 + T cells during intestinal inflammation and alter the functional responses of T cells via regulation of microRNAs. Our assays reveal markedly increased T cell-intrinsic hypoxia and stabilization of HIF protein during experimental colitis. microRNA screen in primary CD4 + T cells points us towards miR-29a and our subsequent studies identify a selective role for HIF-2α in CD4-cell-intrinsic induction of miR-29a during hypoxia. Mice with T cell-intrinsic HIF-2α deletion display elevated T-bet (target of miR-29a) levels and exacerbated intestinal inflammation. Mice with miR-29a deficiency in T cells show enhanced intestinal inflammation. T cell-intrinsic overexpression of HIF-2α or delivery of miR-29a mimetic dampen T H 1-driven colitis. In this work, we show a previously unrecognized function for hypoxia-dependent induction of miR-29a in attenuating T H 1-mediated inflammation., (© 2024. The Author(s).)

Background: Investigating the contributory role that epithelial cell metabolism plays in allergic inflammation is a key factor to understanding what influences dysfunction and the pathogenesis of the allergic disease eosinophilic esophagitis (EoE). We previously highlighted that the absence of hypoxia signaling through hypoxia-inducible factor (HIF)-1α in EoE contributes to esophageal epithelial dysfunction. However, metabolic regulation by HIF-1α has not been explored in esophageal allergy., Objectives: We sought to define the role of HIF-1α-mediated metabolic dysfunction in esophageal epithelial differentiation processes and barrier function in EoE., Methods: In RNA sequencing of EoE patient biopsy samples, we observed the expression pattern of key genes involved in mitochondrial metabolism/oxidative phosphorylation (OXPHOS) and glycolysis. Seahorse bioenergetics analysis was performed on EPC2-hTERT cells to decipher the metabolic processes involved in epithelial differentiation processes. In addition, air-liquid interface cultures were used to delineate metabolic dependency mechanisms required for epithelial differentiation., Results: Transcriptomic analysis identified an increase in genes associated with OXPHOS in patients with EoE. Epithelial origin of this signature was confirmed by complex V immunofluorescence of patient biopsy samples. Bioenergetic analysis in vitro revealed that differentiated epithelium was less reliant on OXPHOS compared with undifferentiated epithelium. Increased OXPHOS potential and reduced glycolytic capacity was mirrored in HIF1A-knockdown EPC2-hTERT cells that exhibited a significant absence of terminal markers of epithelial differentiation, including involucrin. Pharmacologic glucose transport inhibition phenocopied this, while rescue of the HIF-1α-deficient phenotype using the pan-prolyl hydroxylase inhibitor dimethyloxalylglycine resulted in restored expression of epithelial differentiation markers., Conclusions: An OXPHOS-dominated metabolic pattern in EoE patients, brought about largely by the absence of HIF-1α-mediated glycolysis, is linked with the deficit in esophageal epithelial differentiation., Competing Interests: Disclosure statement This publication emanated from research conducted with the financial support of Science Foundation Ireland under grants 18/FRL/6201 (E.N.M.) and 17/FRL/4863 (J.C.M.), and from the Health Research Board under grant ILP-POR-2022-026 (J.C.M.). Financial support for Attune NxT was provided to Maynooth University Department of Biology by Science Foundation Ireland (16/RI/3399). G.T.F. received support via appointment to the La Cache Chair in Gastrointestinal and Immunologic Diseases. The study sponsors played no role in the study design; in the collection, analysis, and interpretation of data; or in the preparation of the report. Contents do not necessarily represent the views of the official funder. Disclosure of potential conflict of interest: J. M. Espinosa reports providing consulting services to Eli Lilly, Gilead, Biohaven, and Perha Pharmaceuticals. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

High level expression of the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) has been associated with severe asthma. The role of MIF and its functional promotor polymorphism in innate immune training is currently unknown. Using novel humanized CATT 7 MIF mice, this study is the first to investigate the effect of MIF on bone marrow-derived macrophage (BMDM) memory after house dust mite (HDM) challenge. CATT 7 BMDMs demonstrated a significant primed increase in M1 markers following HDM and LPS stimulation, compared to naive mice. This M1 signature was found to be MIF-dependent, as administration of a small molecule MIF inhibitor, SCD-19, blocked the induction of this pro-inflammatory M1-like phenotype in BMDMs from CATT 7 mice challenged with HDM. Training naive BMDMs in vitro with HDM for 24 h followed by a rest period and subsequent stimulation with LPS led to significantly increased production of the pro-inflammatory cytokine TNFα in BMDMs from CATT 7 mice but not WT mice. Addition of the pan methyltransferase inhibitor MTA before HDM training significantly abrogated this effect in BMDMs from CATT 7 mice, suggesting that HDM-induced training is associated with epigenetic remodelling. These findings suggest that trained immunity induced by HDM is under genetic control, playing an important role in asthma patients with the high MIF genotypes (CATT 6/7/8 )., (© 2024 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

MicroRNA (miRNA)-mediated mRNA regulation directs many homeostatic and pathological processes, but how miRNAs coordinate aberrant esophageal inflammation during eosinophilic esophagitis (EoE) is poorly understood. Here, we report a deregulatory axis where microRNA-155 (miR-155) regulates epithelial barrier dysfunction by selectively constraining tight junction CLDN7 (claudin-7). MiR-155 is elevated in the esophageal epithelium of biopsies from patients with active EoE and in cell culture models. MiR-155 localization using in situ hybridization (ISH) in patient biopsies and intra-epithelial compartmentalization of miR-155 show expression predominantly within the basal epithelia. Epithelial miR-155 activity was evident through diminished target gene expression in 3D organotypic cultures, particularly in relatively undifferentiated basal cell states. Mechanistically, generation of a novel cell line with enhanced epithelial miR-155 stable overexpression induced a functionally deficient epithelial barrier in 3D air-liquid interface epithelial cultures measured by transepithelial electrical resistance (TEER). Histological assessment of 3D esophageal organoid cultures overexpressing miR-155 showed notable dilated intra-epithelial spaces. Unbiased RNA-sequencing analysis and immunofluorescence determined a defect in epithelial barrier tight junctions and revealed a selective reduction in the expression of critical esophageal tight junction molecule, claudin-7. Together, our data reveal a previously unappreciated role for miR-155 in mediating epithelial barrier dysfunction in esophageal inflammation., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

MicroRNAs (miRNAs) are a class of small endogenous RNA molecules between 18 and 25 nucleotides long. The primary function of miRNAs is in the posttranscriptional regulation of mRNA targets through RNA interference culminating in mRNA degradation or translational repression. MiRNAs are fundamental in physiological and pathological processes such as cell proliferation, differentiation, apoptosis, and inflammation. Among this includes the uncovered potential of miRNAs in overall esophageal disease with a focus on the clinicopathologic allergic disease eosinophilic esophagitis (EoE), gastroesophageal reflux disease (GERD), and the tumorigenic continuum from Barrett's esophagus (BE) toward esophageal adenocarcinoma (EAC). Although these pathologies are distinct from one another, they share pathophysiological elements such as an intense inflammatory milieu, esophageal dysfunction, and as presented in this review, an overlap in miRNA expression which contributes to overall esophageal disease. The overlap in the dysregulated miRNA transcriptome of these pathologies highlights the key role miRNAs play in contributing to esophageal disease progression. Owing to this notable dysregulation, there is an attractive utility for miRNAs as diagnostic and prognostic biomarkers in esophageal diseases that already require invasive endoscopies and biopsy retrieval. In this review miRNAs within EoE, GERD, BE, EAC, and esophageal achalasia are discussed, as well as reviewing a core set of miRNAs shared in the disease progression among some of these pathologies, along with the potential utility of targeting miRNAs as therapeutic options in overall esophageal disease.

Background: The mucus gel layer (MGL) lining the colon is integral to exclusion of bacteria and maintaining intestinal homeostasis in health and disease. Some MGL defects allowing bacteria to directly contact the colonic surface are commonly observed in ulcerative colitis (UC). The major macromolecular component of the colonic MGL is the secreted gel-forming mucin MUC2, whose expression is essential for homeostasis in health. In UC, another gel-forming mucin, MUC5AC, is induced. In mice, Muc5ac is protective during intestinal helminth infection. Here we tested the expression and functional role of MUC5AC/Muc5ac in UC biopsies and murine colitis., Methods: We measured MUC5AC/Muc5ac expression in UC biopsies and in dextran sulfate sodium (DSS) colitis. We performed DSS colitis in mice deficient in Muc5ac (Muc5ac-/-) to model the potential functional role of Muc5ac in colitis. To assess MGL integrity, we quantified bacterial-epithelial interaction and translocation to mesenteric lymph nodes. Antibiotic treatment and 16S rRNA gene sequencing were performed to directly investigate the role of bacteria in murine colitis., Results: Colonic MUC5AC/Muc5ac mRNA expression increased significantly in active UC and murine colitis. Muc5ac-/- mice experienced worsened injury and inflammation in DSS colitis compared with control mice. This result was associated with increased bacterial-epithelial contact and translocation to the mesenteric lymph nodes. However, no change in microbial abundance or community composition was noted. Antibiotic treatment normalized colitis severity in Muc5ac-/- mice to that of antibiotic-treated control mice., Conclusions: MUC5AC/Muc5ac induction in the acutely inflamed colon controls injury by reducing bacterial breach of the MGL., (© 2020 Crohn’s & Colitis Foundation. Published by Oxford University Press on behalf of Crohn’s & Colitis Foundation.)

Epithelial barrier dysfunction is a significant factor in many allergic diseases, including eosinophilic esophagitis (EoE). Infiltrating leukocytes and tissue adaptations increase metabolic demands and decrease oxygen availability at barrier surfaces. Understanding of how these processes impact barrier is limited, particularly in allergy. Here, we identified a regulatory axis whereby the oxygen-sensing transcription factor HIF-1α orchestrated epithelial barrier integrity, selectively controlling tight junction CLDN1 (claudin-1). Prolonged experimental hypoxia or HIF1A knockdown suppressed HIF-1α-dependent claudin-1 expression and epithelial barrier function, as documented in 3D organotypic epithelial cultures. L2-IL5OXA mice with EoE-relevant allergic inflammation displayed localized eosinophil oxygen metabolism, tissue hypoxia, and impaired claudin-1 barrier via repression of HIF-1α/claudin-1 signaling, which was restored by transgenic expression of esophageal epithelial-targeted stabilized HIF-1α. EoE patient biopsy analysis identified a repressed HIF-1α/claudin-1 axis, which was restored via pharmacologic HIF-1α stabilization ex vivo. Collectively, these studies reveal HIF-1α's critical role in maintaining barrier and highlight the HIF-1α/claudin-1 axis as a potential therapeutic target for EoE.

A predominant feature of intestinal inflammation is the accumulation of neutrophils, which dictates a fine balance between epithelial repair or progression to chronic inflammation. While the processes of mucosal healing are well studied, how neutrophils advance an inflammatory insult towards epithelial neoplasia is less understood. In this issue of the JCI, Butin-Israeli et al. outline a mechanism whereby neutrophils control epithelial fitness and genomic instability via delivery of miR-23a-and miR-155-containing microparticles. Localized delivery of antisense oligonucleotides targeting miR-23a and miR-155 reversed this genomic instability and accelerated mucosal healing. This mechanism of neutrophil-derived microRNA shuttling opens up new therapeutic potential to enhance epithelial healing and limit mucosal injury.

Background & Aims: Loss of leucine-rich repeat-containing G-protein-coupled receptor 5-positive crypt base columnar cells provides permissive conditions for different facultative stem cell populations to dedifferentiate and repopulate the stem cell compartment. In this study, we used a defensin α4-Cre recombinase (Defa4Cre) line to define the potential of Paneth cells to dedifferentiate and contribute to intestinal stem cell (ISC) maintenance during normal homeostasis and after intestinal injury., Methods: Small intestine and enteroids from Defa4 Cre ;Rosa26 tandem dimer Tomato (tdTomato), a red fluoresent protein, (or Rosa26 Enhanced Yellow Fluorescent Protein (EYFP)) reporter, Notch gain-of-function (Defa4 Cre ;Rosa26 Notch Intracellular Domain (NICD)-ires-nuclear Green Fluorescent Protein (nGFP) and Defa4 Cre ;Rosa26 reverse tetracycline transactivator-ires Enhanced Green Fluorescent Protein (EGFP);TetO NICD ), A Disintegrin and Metalloproteinase domain-containing protein 10 (ADAM10) loss-of-function (Defa4 Cre ;ADAM10 flox/flox ), and Adenomatous polyposis coli (APC) inactivation (Defa4 Cre ;APC flox/flox ) mice were analyzed. Doxorubicin treatment was used as an acute intestinal injury model. Lineage tracing, proliferation, and differentiation were assessed in vitro and in vivo., Results: Defa4 Cre -expressing cells are fated to become mature Paneth cells and do not contribute to ISC maintenance during normal homeostasis in vivo. However, spontaneous lineage tracing was observed in enteroids, and fluorescent-activated cell sorter-sorted Defa4 Cre -marked cells showed clonogenic enteroid growth. Notch activation in Defa4 Cre -expressing cells caused dedifferentiation to multipotent ISCs in vivo and was required for adenoma formation. ADAM10 deletion had no significant effect on crypt homeostasis. However, after acute doxorubicin-induced injury, Defa4 Cre -expressing cells contributed to regeneration in an ADAM10-Notch-dependent manner., Conclusions: Our studies have shown that Defa4 Cre -expressing Paneth cells possess cellular plasticity, can dedifferentiate into multipotent stem cells upon Notch activation, and can contribute to intestinal regeneration in an acute injury model., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Eosinophilic esophagitis (EoE) is a chronic antigen-mediated clinicopathologic disease of the esophagus characterized by an eosinophil-predominant inflammatory infiltrate. A clinical hallmark is extensive tissue remodeling including basal zone hyperplasia, fibrosis, and angiogenesis. However, the cellular mechanisms responsible for these processes are not fully defined. We hypothesized that targeting granulocyte-macrophage colony-stimulating factor (GM-CSF; an agonist cytokine linked with eosinophil survival and activation) would be protective in a preclinical model of EoE., Methods: Eosinophilic esophagitis-like esophageal inflammation was induced in the L2-IL5 OXA EoE mouse model, and GM-CSF production was assessed by mRNA and protein analyses. Granulocyte-macrophage colony-stimulating factor-receptor-alpha expression patterns were examined by flow cytometric and immunofluorescence analysis. L2-IL5 OXA EoE mice were treated with anti-GM-CSF neutralizing antibody or isotype control and assessed for histopathological indices of eosinophilia, epithelial hyperplasia, and angiogenesis by immunohistochemistry and RT-PCR., Results: Significantly increased levels of esophageal GM-CSF expression was detected in the L2-IL5 OXA mouse EoE model during active inflammation. Granulocyte-macrophage colony-stimulating factor-receptor-alpha was predominantly expressed on esophageal eosinophils during EoE, in addition to select cells within the lamina propria. Anti-GM-CSF neutralization in L2-IL5 OXA EoE mice resulted in a significant diminution of epithelial eosinophilia in addition to basal cell hyperplasia and vascular remodeling. This treatment response was independent of effects on esophageal eosinophil maturation or activation., Conclusion: Granulocyte-macrophage colony-stimulating factor is a potential therapeutic target to reduce esophageal eosinophilia and remodeling., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

MicroRNA (miRNA)-mediated RNA interference regulates many immune processes, but how miRNA circuits orchestrate aberrant intestinal inflammation during inflammatory bowel disease (IBD) is poorly defined. Here, we report that miR-223 limits intestinal inflammation by constraining the nlrp3 inflammasome. miR-223 was increased in intestinal biopsies from patients with active IBD and in preclinical models of intestinal inflammation. miR-223 -/y mice presented with exacerbated myeloid-driven experimental colitis with heightened clinical, histopathological, and cytokine readouts. Mechanistically, enhanced NLRP3 inflammasome expression with elevated IL-1β was a predominant feature during the initiation of colitis with miR-223 deficiency. Depletion of CCR2 + inflammatory monocytes and pharmacologic blockade of IL-1β or NLRP3 abrogated this phenotype. Generation of a novel mouse line, with deletion of the miR-223 binding site in the NLRP3 3' untranslated region, phenocopied the characteristics of miR-223 -/y mice. Finally, nanoparticle-mediated overexpression of miR-223 attenuated experimental colitis, NLRP3 levels, and IL-1β release. Collectively, our data reveal a previously unappreciated role for miR-223 in regulating the innate immune response during intestinal inflammation., (© 2017 Neudecker et al.)

A critical step in the pathogenesis of acute lung injury (ALI) is excessive recruitment of polymorphonuclear neutrophils (PMNs) into the lungs, causing significant collateral tissue damage. Defining the molecular and cellular steps that control neutrophil infiltration and activation during ALI is therefore of important therapeutic relevance. Based on previous findings implicating the transcription factor Tbet in mucosal Th1-inflammation, we hypothesized a detrimental role for Tbet during ALI. In line with our hypothesis, initial studies of endotoxin-induced lung injury revealed a marked protection of Tbet -/- mice, including attenuated neutrophilia compared to WT counterparts. Surprisingly, subsequent studies identified natural killer (NK) cells as the major source of pulmonary Tbet during ALI. In addition, a chemokine screen suggested that mature Tbet + NK-cells are critical for the production of pulmonary CXCL1 and -2, thereby contributing to pulmonary PMN recruitment. Indeed, both NK-cell Ab depletion and adoptive transfer studies provide evidence for NK cells in the orchestration of neutrophil recruitment during endotoxin-induced ALI. Taken together, these findings identify a novel role for Tbet + NK-cells in initiating the early events of noninfectious pulmonary inflammation., (© Society for Leukocyte Biology.)

Organized lymphoid tissues like the thymus first appeared in jawed vertebrates around 500 million years ago and have evolved to equip the host with a network of specialized sites, strategically located to orchestrate strict immune-surveillance and efficient immune responses autonomously. The gut-associated lymphoid tissues maintain a mostly tolerant environment to dampen our responses to daily dietary and microbial products in the intestine. However, when this homeostasis is perturbed by chronic inflammation, the intestine is able to develop florid organized tertiary lymphoid tissues (TLT), which heralds the onset of regional immune dysregulation. While TLT are a pathologic hallmark of Crohn's disease (CD), their role in the overall process remains largely enigmatic. A critical question remains; are intestinal TLT generated by the immune infiltrated intestine to modulate immune responses and rebuild tolerance to the microbiota or are they playing a more sinister role by generating dysregulated responses that perpetuate disease? Herein, we discuss the main theories of intestinal TLT neogenesis and focus on the most recent findings that open new perspectives to their role in inflammatory bowel disease.

Proinflammatory consequences have been described for lysophosphatidylcholine, a lipid product of cellular injury, signaling via the G protein-coupled receptor G2A on myeloid and lymphoid inflammatory cells. This prompted the hypothesis that genetic deletion of G2A would limit intestinal inflammation in a mouse model of colitis induced by dextran sodium sulfate. Surprisingly, G2A(-/-) mice exhibited significantly worsened colitis compared with wild-type mice, as demonstrated by disease activity, colon shortening, histology, and elevated IL-6 and IL-5 in colon tissues. Investigation of inflammatory cells recruited to inflamed G2A(-/-) colons showed significantly more TNF-α(+) and Ly6C(hi)MHCII(-) proinflammatory monocytes and eosinophils than in wild-type colons. Both monocytes and eosinophils were pathogenic as their depletion abolished the excess inflammation in G2A(-/-) mice. G2A(-/-) mice also had less IFN-γ in inflamed colon tissues than wild-type mice. Fewer CD4(+) lymphocytes were recruited to inflamed G2A(-/-) colons, and fewer colonic lymphocytes produced IFN-γ upon ex vivo stimulation. Administration of IFN-γ to G2A(-/-) mice during dextran sodium sulfate exposure abolished the excess colitic inflammation and reduced colonic IL-5 and eosinophil numbers to levels seen in wild-type mice. Furthermore, IFN-γ reduced the numbers of TNF-α(+) monocyte and enhanced their maturation from Ly6C(hi)MHCII(-) to Ly6C(int)MHCII(+) Taken together, the data suggest that G2A signaling serves to dampen intestinal inflammation via the production of IFN-γ, which, in turn, enhances monocyte maturation to a less inflammatory program and ultimately reduces eosinophil-induced injury of colonic tissues., (Copyright © 2016 by The American Association of Immunologists, Inc.)

The long-acting, highly lipophilic, β2-adrenoceptor agonist clenbuterol may represent a suitable therapeutic agent for the treatment of neuroinflammation as it drives an anti-inflammatory response within the CNS. However, clenbuterol is also known to increase the expression of IL-1β in the brain, a potent neuromodulator that plays a role in provoking sickness related symptoms including anxiety and depression-related behaviours. Here we demonstrate that, compared to the immunological stimulus lipopolysaccharide (LPS, 250μg/kg), clenbuterol (0.5mg/kg) selectively up-regulates expression of the central IL-1 system resulting in a mild stress-like response which is accompanied by a reduction in locomotor activity and food consumption in rats. We provide further evidence that clenbuterol-induced activation of the central IL-1 system occurs in a controlled and selective manner in tandem with its negative regulators IL-1ra and IL-1RII. Furthermore, we demonstrate that peripheral β2-adrenoceptors mediate the suppression of locomotor activity and food consumption induced by clenbuterol and that these effects are not linked to the central induction of IL-1β. Moreover, despite increasing central IL-1β expression, chronic administration of clenbuterol (0.03mg/kg; twice daily for 21days) fails to induce anxiety or depressive-like behaviour in rats in contrast to reports of the ability of exogenously administered IL-1 to induce these symptoms in rodents. Overall, our findings suggest that clenbuterol or other selective β2-adrenoceptor agonists could have the potential to combat neuroinflammatory or neurodegenerative disorders without inducing unwanted symptoms of depression and anxiety., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Migration and positioning of immune cells is fundamental for their differentiation and recruitment at sites of infection. Besides the fundamental role played by chemokines and their receptors, recent studies demonstrate that a complex network of purinergic signaling events plays a key role in these trafficking events. This process includes the release of nucleotides (such as ATP and ADP) and subsequent autocrine and paracrine signaling events through nucleotide receptors. At the same time, surface-expressed ectoapyrases and nucleotidases convert extracellular nucleotides to adenosine, and adenosine signaling events play additional functional roles in leucocyte trafficking. In this review we revisit classical paradigms of inflammatory cell trafficking in the context of recent studies implicating purinergic signaling events in this process., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In this issue of Science Signaling, Bullen et al demonstrate that protein kinase A (PKA) phosphorylates and stimulates the transcriptional activity of hypoxia-inducible transcription factor-1α (HIF-1α). This finding may have implications in diseases processes that occur at the interface of hypoxia and inflammation, where HIF-1α stabilization can function to dampen hypoxia-driven inflammation., (Copyright © 2016, American Association for the Advancement of Science.)

The incidence of cancer is higher in the elderly; however, many of the underlying mechanisms for this association remain unexplored. Here, we have shown that B cell progenitors in old mice exhibit marked signaling, gene expression, and metabolic defects. Moreover, B cell progenitors that developed from hematopoietic stem cells (HSCs) transferred from young mice into aged animals exhibited similar fitness defects. We further demonstrated that ectopic expression of the oncogenes BCR-ABL, NRAS(V12), or Myc restored B cell progenitor fitness, leading to selection for oncogenically initiated cells and leukemogenesis specifically in the context of an aged hematopoietic system. Aging was associated with increased inflammation in the BM microenvironment, and induction of inflammation in young mice phenocopied aging-associated B lymphopoiesis. Conversely, a reduction of inflammation in aged mice via transgenic expression of α-1-antitrypsin or IL-37 preserved the function of B cell progenitors and prevented NRAS(V12)-mediated oncogenesis. We conclude that chronic inflammatory microenvironments in old age lead to reductions in the fitness of B cell progenitor populations. This reduced progenitor pool fitness engenders selection for cells harboring oncogenic mutations, in part due to their ability to correct aging-associated functional defects. Thus, modulation of inflammation--a common feature of aging--has the potential to limit aging-associated oncogenesis.

NK cells are innate lymphoid cells important for immune surveillance, identifying and responding to stress, infection, and/or transformation. Whereas conventional NK (cNK) cells circulate systemically, many NK cells reside in tissues where they appear to be poised to locally regulate tissue function. In the present study, we tested the contribution of tissue-resident NK (trNK) cells to tissue homeostasis by studying ischemic injury in the mouse kidney. Parabiosis experiments demonstrate that the kidney contains a significant fraction of trNK cells under homeostatic conditions. Kidney trNK cells developed independent of NFIL3 and T-bet, and they expressed a distinct cell surface phenotype as compared with cNK cells. Among these, trNK cells had reduced asialo-GM1 (AsGM1) expression relative to cNK cells, a phenotype observed in trNK cells across multiple organs and mouse strains. Strikingly, anti-AsGM1 Ab treatment, commonly used as an NK cell-depleting regimen, resulted in a robust and selective depletion of cNKs, leaving trNKs largely intact. Using this differential depletion, we tested the relative contribution of cNK and trNK cells in ischemic kidney injury. Whereas anti-NK1.1 Ab effectively depleted both trNK and cNK cells and protected against ischemic/reperfusion injury, anti-AsGM1 Ab preferentially depleted cNK cells and failed to protect against injury. These data demonstrate unanticipated specificity of anti-AsGM1 Ab depletion on NK cell subsets and reveal a new approach to study the contributions of cNK and trNK cells in vivo. In total, these data demonstrate that trNK cells play a key role in modulating local responses to ischemic tissue injury in the kidney and potentially other organs., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Central to inflammatory bowel disease (IBD) pathogenesis is loss of mucosal barrier function. Emerging evidence implicates extracellular adenosine signaling in attenuating mucosal inflammation. We hypothesized that adenosine-mediated protection from intestinal barrier dysfunction involves tissue-specific signaling through the A2B adenosine receptor (Adora2b) at the intestinal mucosal surface. To address this hypothesis, we combined pharmacologic studies and studies in mice with global or tissue-specific deletion of the Adora2b receptor. Adora2b(-/-) mice experienced a significantly heightened severity of colitis, associated with a more acute onset of disease and loss of intestinal epithelial barrier function. Comparison of mice with Adora2b deletion on vascular endothelial cells (Adora2b(fl/fl)VeCadCre(+)) or intestinal epithelia (Adora2b(fl/fl)VillinCre(+)) revealed a selective role for epithelial Adora2b signaling in attenuating colonic inflammation. In vitro studies with Adora2b knockdown in intestinal epithelial cultures or pharmacologic studies highlighted Adora2b-driven phosphorylation of vasodilator-stimulated phosphoprotein (VASP) as a specific barrier repair response. Similarly, in vivo studies in genetic mouse models or treatment studies with an Adora2b agonist (BAY 60-6583) recapitulate these findings. Taken together, our results suggest that intestinal epithelial Adora2b signaling provides protection during intestinal inflammation via enhancing mucosal barrier responses.

Background: Fibrostenosis and stricture are well-recognized endpoints in Crohn's disease (CD). We hypothesized that stricturing CD is characterized by eosinophilia and epithelial IL-33. We proposed that eosinophil exposure to IL-33 would perpetuate inflammatory chronicity and subsequent fibrostenosis., Methods: We performed a retrospective study of 74 children with inflammatory and stricturing ileal CD comparing clinicopathological features to immunohistochemical measures of eosinophilia and IL-33. To scrutinize eosinophil patterns, we developed a novel eosinophil peroxidase score encompassing number, distribution, and degranulation. Human eosinophils and intestinal fibroblasts were cultured with IL-33 and IL-13, and inflammatory and remodeling parameters were assessed. Antieosinophil therapy was also administered to the Crohn's-like ileitis model (SAMP1/SkuSlc)., Results: Our novel eosinophil peroxidase score was more sensitive than H&E staining, revealing significant differences in eosinophil patterns, comparing inflammatory and stricturing pediatric CD. A significant relationship between ileal eosinophilia and complicated clinical/histopathological phenotype including fibrosis was determined. IL-33 induced significant eosinophil peroxidase secretion and IL-13 production. Exposure to eosinophils in the presence of IL-33, "primed" fibroblasts to increase proinflammatory cytokines (TNF-α, IL-1β, and IL-6), eosinophil-associated chemokines (CCL24 and CCL26), and IL-13Rα2 production. Production of fibrogenic molecules (collagen 1A2, fibronectin, and periostin) increased after exposure of "primed" fibroblasts to IL-13. Epithelial-IL-33 was increased in pediatric Crohn's ileitis and strongly associated with clinical and histopathological activity, ileal eosinophilia, and complicated fibrostenotic disease. SAMP1/SkuSlc eosinophil-targeted treatment resulted in significant improvements in inflammation and remodeling., Conclusions: Our study of specimens from pediatric patients with ileal CD linked eosinophil patterns and IL-33 to fibrosis and suggested that these may contribute to the perpetuation of inflammation and subsequent stricture in pediatric CD.

Objective: Eosinophils reside in the colonic mucosa and increase significantly during disease. Although a number of studies have suggested that eosinophils contribute to the pathogenesis of GI inflammation, the expanding scope of eosinophil-mediated activities indicate that they also regulate local immune responses and modulate tissue inflammation. We sought to define the impact of eosinophils that respond to acute phases of colitis in mice., Design: Acute colitis was induced in mice by administration of dextran sulfate sodium, 2,4,6-trinitrobenzenesulfonic acid or oxazolone to C57BL/6J (control) or eosinophil deficient (PHIL) mice. Eosinophils were also depleted from mice using antibodies against interleukin (IL)-5 or by grafting bone marrow from PHIL mice into control mice. Colon tissues were collected and analysed by immunohistochemistry, flow cytometry and reverse transcription PCR; lipids were analysed by mass spectroscopy., Results: Eosinophil-deficient mice developed significantly more severe colitis, and their colon tissues contained a greater number of neutrophils, than controls. This compensatory increase in neutrophils was accompanied by increased levels of the chemokines CXCL1 and CXCL2, which attract neutrophils. Lipidomic analyses of colonic tissue from eosinophil-deficient mice identified a deficiency in the docosahexaenoic acid-derived anti-inflammatory mediator 10, 17- dihydroxydocosahexaenoic acid (diHDoHE), namely protectin D1 (PD1). Administration of an exogenous PD1-isomer (10S, 17S-DiHDoHE) reduced the severity of colitis in eosinophil-deficient mice. The PD1-isomer also attenuated neutrophil infiltration and reduced levels of tumour necrosis factor-α, IL-1β, IL-6 and inducible NO-synthase in colons of mice. Finally, in vitro assays identified a direct inhibitory effect of PD1-isomer on neutrophil transepithelial migration., Conclusions: Eosinophils exert a protective effect in acute mouse colitis, via production of anti-inflammatory lipid mediators., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

The regulation of T cell and DC retention and lymphatic egress within and from the intestine is critical for intestinal immunosurveillance; however, the cellular processes that orchestrate this balance during IBD remain poorly defined. With the use of a mouse model of TNF-driven Crohn's-like ileitis (TNF(Δ) (ARE)), we examined the role of CCR7 in the control of intestinal T cell and DC retention/egress during experimental CD. We observed that the frequency of CCR7-expressing TH1/TH17 effector lymphocytes increased during active disease in TNF(Δ) (ARE) mice and that ΔARE/CCR7(-/-) mice developed exacerbated ileitis and multiorgan inflammation, with a marked polarization and ileal retention of TH1 effector CD4(+) T cells. Furthermore, adoptive transfer of ΔARE/CCR7(-/-) effector CD4(+) into lymphopenic hosts resulted in ileo-colitis, whereas those transferred with ΔARE/CCR7(+/+) CD4(+) T cells developed ileitis. ΔARE/CCR7(-/-) mice had an acellular draining MLN, decreased CD103(+) DC, and decreased expression of RALDH enzymes and of CD4(+)CD25(+)FoxP3(+) Tregs. Lastly, a mAb against CCR7 exacerbated ileitis in TNF(Δ) (ARE) mice, phenocopying the effects of congenital CCR7 deficiency. Our data underscore a critical role for the lymphoid chemokine receptor CCR7 in orchestrating immune cell traffic and TH1 versus TH17 bias during chronic murine ileitis., (© Society for Leukocyte Biology.)

Acute intestinal inflammation involves early accumulation of neutrophils (PMNs) followed by either resolution or progression to chronic inflammation. Based on recent evidence that mucosal metabolism influences disease outcomes, we hypothesized that transmigrating PMNs influence the transcriptional profile of the surrounding mucosa. Microarray studies revealed a cohort of hypoxia-responsive genes regulated by PMN-epithelial crosstalk. Transmigrating PMNs rapidly depleted microenvironmental O2 sufficiently to stabilize intestinal epithelial cell hypoxia-inducible factor (HIF). By utilizing HIF reporter mice in an acute colitis model, we investigated the relative contribution of PMNs and the respiratory burst to "inflammatory hypoxia" in vivo. CGD mice, lacking a respiratory burst, developed accentuated colitis compared to control, with exaggerated PMN infiltration and diminished inflammatory hypoxia. Finally, pharmacological HIF stabilization within the mucosa protected CGD mice from severe colitis. In conclusion, transcriptional imprinting by infiltrating neutrophils modulates the host response to inflammation, via localized O2 depletion, resulting in microenvironmental hypoxia and effective inflammatory resolution., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Eosinophilic oesophagitis (EoE) is a chronic inflammatory condition of the oesophagus with limited treatment options. No previous transgenic model has specifically targeted the oesophageal mucosa to induce oesophageal eosinophilia., Design: We developed a mouse model that closely resembles EoE by utilising oxazolone haptenation in mice with transgenic overexpression of an eosinophil poietic and survival factor (interleukin (IL)-5) in resident squamous oesophageal epithelia., Results: Overexpression of IL-5 in the healthy oesophagus was achieved in transgenic mice (L2-IL5) using the squamous epithelial promoter Epstein-Barr virus ED-L2. Oxazolone-challenged L2-IL5 mice developed dose-dependent pan-oesophageal eosinophilia, including eosinophil microabscess formation and degranulation as well as basal cell hyperplasia. Moreover, oesophagi expressed increased IL-13 and the eosinophil agonist chemokine eotaxin-1. Treatment of these mice with corticosteroids significantly reduced eosinophilia and epithelial inflammation., Conclusions: L2-IL5 mice provide a novel experimental model that can potentially be used in preclinical testing of EoE-related therapeutics and mechanistic studies identifying pathogenetic features associated with mucosal eosinophilia.

Pharmacological stabilization of hypoxia-inducible factor (HIF) through prolyl hydroxylase (PHD) inhibition limits mucosal damage associated with models of murine colitis. However, little is known about how PHD inhibitors (PHDi) influence systemic immune function during mucosal inflammation or the relative importance of immunological changes to mucosal protection. We hypothesized that PHDi enhances systemic innate immune responses to colitis-associated bacteremia. Mice with colitis induced by trinitrobenzene sulfonic acid were treated with AKB-4924, a new HIF-1 isoform-predominant PHDi, and clinical, immunological, and biochemical endpoints were assessed. Administration of AKB-4924 led to significantly reduced weight loss and disease activity compared with vehicle controls. Treated groups were pyrexic but did not become subsequently hypothermic. PHDi treatment augmented epithelial barrier function and led to an approximately 50-fold reduction in serum endotoxin during colitis. AKB-4924 also decreased cytokines involved in pyrogenesis and hypothermia, significantly reducing serum levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α while increasing IL-10. Treatment offered no protection against colitis in epithelial-specific HIF-1α-deficient mice, strongly implicating epithelial HIF-1α as the tissue target for AKB-4924-mediated protection. Taken together, these results indicate that inhibition of prolyl hydroxylase with AKB-4924 enhances innate immunity and identifies that the epithelium is a central site of inflammatory protection afforded by PHDi in murine colitis.

Introduction: Recent clinical trials show Lactobacillus rhamnosus GG (LGG) administration in critical illness has the potential to reduce nosocomial infections and improve clinical outcome. However, the mechanism(s) of LGG-mediated benefit following illness and injury remain elusive., Objective: The aim of this study was to determine the effect of LGG treatment on survival and lung injury in a mouse model of Pseudomonas aeruginosa-induced pneumonia. As increased T regulatory (Treg) cell numbers have been shown to improve outcome in experimental pneumonia, we examined the potential role of Treg cells in probiotic-mediated benefit., Methods: FVB/N mice were subjected to intratracheal injection of either P. aeruginosa or saline and received LGG or vehicle immediately before procedure. T regulatory cell responses in the lung were evaluated by polymerase chain reaction, Western blotting, and flow cytometry., Results: Mice treated with LGG had significantly improved 7-day survival (P < 0.01) compared with saline-treated control pneumonia mice (55% LGG vs. 14% control). The survival advantage was associated with reduced bacterial counts in bronchoalveolar lavage and with decreased markers of the systemic inflammatory response and improved lung pathology in the probiotic group. Probiotic treatment influenced immune response in the lungs of mice with pneumonia as demonstrated by increased levels of Treg cell marker Foxp3., Conclusions: These data demonstrate that early administration of LGG improves outcome following P. aeruginosa-induced pneumonia. An effect of LGG on Treg cells may play a role in this protection.

In this study we examined the impact of systemic treatment with the long-acting brain penetrant β2-adrenoceptor agonist clenbuterol on NFκB activity and IκB expression in rat brain. Clenbuterol decreased NFκB activity (p65 DNA binding) in nuclear extracts prepared from rat cortex and hippocampus for up to 8h following a single treatment. This was accompanied by increased expression of IκBα mRNA and protein. The temporal increase in IκB protein expression paralleled the suppression of NFκB activity, suggesting that IκBα mediates the suppression NFκB activity observed. These actions of clenbuterol were prevented by pre-treatment with the non-selective β-adrenoceptor antagonist propranolol, the β2-adrenoceptor antagonist ICI-118,551, but not the β1-adrenoceptor antagonist metoprolol, suggesting that the effects of clenbuterol on IκBα expression and NFκB activity are mediated specifically by the β2-adrenoceptor. In addition, the actions of clenbuterol were mimicked by systemic administration of another highly selective long-acting β2-adrenoceptor agonist formoterol. As neurodegenerative diseases are associated with inflammation we determined if clenbuterol could suppress NFκB activation that occurs in response to an inflammatory stimulus. In this regard we demonstrate that clenbuterol inhibited IκB phosphorylation and IκB degradation and inhibited NFκB activity in hippocampus and cortex of rats following a central injection of the inflammagen bacterial lipopolysaccharide (LPS). In tandem, clenbuterol blocked expression of the NFκB-inducible genes TNF-α and ICAM-1 following LPS administration. Our finding that clenbuterol and formoterol inhibit NFκB activity in the CNS further supports the idea that β2-adrenoceptors may be an attractive target for treating neuroinflammation and combating inflammation-related neurodegeneration., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Fecal alpha-1-antitrypsin (AAT) clearance has been a marker of clinical disease severity in inflammatory bowel diseases (IBDs) for many years. Although AAT deficiency is more often associated with lung and liver pathologies, AAT-deficient patients with concomitant IBD have been shown to develop more aggressive disease and rapid progression to surgery. Although recent studies have highlighted the pleiotropic anti-inflammatory functions of AAT, including reducing proinflammatory cytokine production and suppressing immune cell activation, its potential therapeutic role in IBD has not been described., Methods: The therapeutic potential of human AAT administration was assessed in murine models of IBD including new-onset and established chemically induced colitis and spontaneous chronic murine ileitis. Histological assessment of inflammation, cytokine secretion profiling, and flow cytometric evaluation of inflammatory infiltrate were performed in each model. The effect of AAT on intestinal barrier function was also examined both in vitro and in vivo., Results: AAT attenuated inflammation in small and large intestinal IBD models through reduced secretion of proinflammatory cytokines, inflammatory cell infiltration, and reduced tissue injury. AAT also increased intestinal restitution after chemically induced colitis. AAT significantly decreased intestinal permeability in vitro and in vivo as part of a protective mechanism for both acute and chronic models of IBD., Conclusions: Our findings describe a beneficial role for AAT in IBD models through suppression of cytokine production and enhanced intestinal barrier function. This raises the possibility that AAT supplementation, which has a long history of proven safety, may have a therapeutic effect in human IBD.

Acute lung injury (ALI) is associated with high morbidity and mortality in critically ill patients. At present, the functional contribution of airway mucins to ALI is unknown. We hypothesized that excessive mucus production could be detrimental during lung injury. Initial transcriptional profiling of airway mucins revealed a selective and robust induction of MUC5AC upon cyclic mechanical stretch exposure of pulmonary epithelia (Calu-3). Additional studies confirmed time- and stretch-dose-dependent induction of MUC5AC transcript or protein during cyclic mechanical stretch exposure in vitro or during ventilator-induced lung injury in vivo. Patients suffering from ALI showed a 58-fold increase in MUC5AC protein in their bronchoalveolar lavage. Studies of the MUC5AC promoter implicated nuclear factor κB in Muc5ac induction during ALI. Moreover, mice with gene-targeted deletion of Muc5ac⁻/⁻ experience attenuated lung inflammation and pulmonary edema during injurious ventilation. We observed that neutrophil trafficking into the lungs of Muc5ac⁻/⁻ mice was selectively attenuated. This implicates that endogenous Muc5ac production enhances pulmonary neutrophil trafficking during lung injury. Together, these studies reveal a detrimental role for endogenous Muc5ac production during ALI and suggest pharmacological strategies to dampen mucin production in the treatment of lung injury.

Acute lung injury (ALI) is characterized by alveolar injury and uncontrolled inflammation. Since most cases of ALI resolve spontaneously, understanding the endogenous mechanisms that promote ALI resolution is important to developing effective therapies. Previous studies have implicated extracellular adenosine signaling in tissue adaptation and wound healing. Therefore, we hypothesized a functional contribution for the endogenous production of adenosine during ALI resolution. As a model, we administered intratracheal LPS and observed peak lung injury at 3 d, with resolution by d 14. Treatment with pegylated adenosine-deaminase to enhance extracellular adenosine breakdown revealed impaired ALI resolution. Similarly, genetic deletion of cd73, the pacemaker for extracellular adenosine generation, was associated with increased mortality (0% wild-type and 40% in cd73(-/-) mice; P<0.05) and failure to resolve ALI adequately. Studies of inflammatory cell trafficking into the lungs during ALI resolution revealed that regulatory T cells (Tregs) express the highest levels of CD73. While Treg numbers in cd73(-/-) mice were similar to controls, cd73-deficient Tregs had attenuated immunosuppressive functions. Moreover, adoptive transfer of cd73-deficient Tregs into Rag(-/-) mice emulated the observed phenotype in cd73(-/-) mice, while transfer of wild-type Tregs was associated with normal ALI resolution. Together, these studies implicate CD73-dependent adenosine generation in Tregs in promoting ALI resolution.

Oxygen is a molecule that is central to cellular respiration and viability, yet there are multiple physiologic and pathological contexts in which cells experience conditions of insufficient oxygen availability, a state known as hypoxia. Given the metabolic challenges of a low oxygen environment, hypoxia elicits a range of adaptive responses at the cellular, tissue, and systemic level to promote continued survival and function. Within this context, T lymphocytes are a highly migratory cell type of the adaptive immune system that frequently encounters a wide range of oxygen tensions in both health and disease. It is now clear that oxygen availability regulates T cell differentiation and function, a response orchestrated in large part by the hypoxia-inducible factor transcription factors. Here, we discuss the physiologic scope of hypoxia and hypoxic signaling, the contribution of these pathways in regulating T cell biology, and current gaps in our understanding. Finally, we discuss how emerging therapies that modulate the hypoxic response may offer new modalities to alter T cell function and the outcome of acute and chronic pathologies.

Background: The earliest endoscopically-evident lesion in Crohn's disease is the aphthous ulcer, which develops over ectopic lymphoid tissues (ie, inducible lymphoid follicles (ILF), tertiary lymphoid tissue (TLT)) in the chronically inflamed intestine. ILF/TLT are induced within effector sites by homeostatic lymphoid chemokines, but their role in the development of intestinal ILF/TLT and in the pathogenesis of Crohn's disease is poorly understood., Design: Using a mouse model of Crohn's-like ileitis (TNFARE) which develops florid induction of ILF/TLT within its terminal ileum, the contribution of the CCR7/CCL19/CCL21 chemokine axis during the development of TLT and its role in disease pathogenesis were assessed., Results: Both CCL19 and CCL21 were increased within the inflamed ileum of TNFARE mice, which resulted in CCR7 internalisation and impaired T cell chemotaxis. ILF/TLT were a major source of CCL19 and CCL21 and increased local synthesis, augmented recruitment/retention of effector, naïve and central memory T cell subsets within the inflamed ileum. Immunoblockade of CCR7 resulted in further effector T cell retention and exacerbation of ileitis., Conclusions: Induction of ILF/TLT in the chronically inflamed intestine alters the homeostatic CCL19-CCL21 lymphoid-chemokine gradient and increases recruitment/retention of effector CCR7+ T cell subsets within the terminal ileum, contributing to the perpetuation of chronic inflammation. Thus, blockade of CCR7 or its ligands might result in deleterious consequences for subjects with chronic inflammatory diseases.

Recent studies have demonstrated dramatic shifts in metabolic supply-and-demand ratios during inflammation, a process resulting in localized tissue hypoxia within inflammatory lesions ("inflammatory hypoxia"). As part of the adaptive immune response, T cells are recruited to sites of inflammatory hypoxia. Given the profound effects of hypoxia on gene regulation, we hypothesized that T-cell differentiation is controlled by hypoxia. To pursue this hypothesis, we analyzed the transcriptional consequences of ambient hypoxia (1% oxygen) on a broad panel of T-cell differentiation factors. Surprisingly, these studies revealed selective, robust induction of FoxP3, a key transcriptional regulator for regulatory T cells (Tregs). Studies of promoter binding or loss- and gain-of-function implicated hypoxia-inducible factor (HIF)-1α in inducing FoxP3. Similarly, hypoxia enhanced Treg abundance in vitro and in vivo. Finally, Treg-intrinsic HIF-1α was required for optimal Treg function and Hif1a-deficient Tregs failed to control T-cell-mediated colitis. These studies demonstrate that hypoxia is an intrinsic molecular cue that promotes FoxP3 expression, in turn eliciting potent anti-inflammatory mechanisms to limit tissue damage in conditions of reduced oxygen availability.

Unlabelled: BACKGROUND; Imprinting an effector or regulatory phenotype on naïve T cells requires education at induction sites by dendritic cells (DC). Objectives To analyse the effect of inflammation on the frequency of mononuclear phagocytes (MP) and the effect of altering their frequency by administration of Flt3-L in chronic ileitis., Methods: Using a tumour necrosis factor (TNF) driven model of ileitis (ie, TNFΔARE) that recapitulates many features of Crohn's disease (CD), dynamic changes in the frequency and functional state of MP within the inflamed ileum were assessed by flow cytometry, immunofluorescence and real-time reverse-transcription PCR and by generating CX(3)CR1 GFP-reporter TNFΔARE mice. The effect of Flt3-L supplementation on the severity of ileitis, and the frequency of CD103(+) DC and of FoxP3(+) regulatory T cells was also studied in TNFΔARE mice., Results: CD11c(Hi)/MHCII(+) MP accumulated in inflamed ilea, predominantly mediated by expansion of the CX(3)CR1(+) MP subpopulation. This coincided with a decreased pro-regulatory CD103(+) DC. The phenotype of these MP was that of activated cells, as they expressed increased CD80 and CD86 on their surface. Flt3-ligand administration resulted in a preferential expansion of CD103(+) DC that attenuated the severity of ileitis in 20-week-old TNFΔARE mice, mediated by increased CD4(+)/CD25(+)/FoxP3(+) regulatory T cells., Conclusions: Results support a role for Flt3-L as a potential therapeutic agent in Crohn's-like ileitis.

A complex biologic network regulates kidney perfusion under physiologic conditions. This system is profoundly perturbed following renal ischemia, a leading cause of acute kidney injury (AKI) - a life-threatening condition that frequently complicates the care of hospitalized patients. Therapeutic approaches to prevent and treat AKI are extremely limited. Better understanding of the molecular pathways promoting postischemic reflow could provide new candidate targets for AKI therapeutics. Due to its role in adapting tissues to hypoxia, we hypothesized that extracellular adenosine has a regulatory function in the postischemic control of renal perfusion. Consistent with the notion that equilibrative nucleoside transporters (ENTs) terminate adenosine signaling, we observed that pharmacologic ENT inhibition in mice elevated renal adenosine levels and dampened AKI. Deletion of the ENTs resulted in selective protection in Ent1-/- mice. Comprehensive examination of adenosine receptor-knockout mice exposed to AKI demonstrated that renal protection by ENT inhibitors involves the A2B adenosine receptor. Indeed, crosstalk between renal Ent1 and Adora2b expressed on vascular endothelia effectively prevented a postischemic no-reflow phenomenon. These studies identify ENT1 and adenosine receptors as key to the process of reestablishing renal perfusion following ischemic AKI. If translatable from mice to humans, these data have important therapeutic implications.

Intestinal remodeling and stricture formation is a complication of inflammatory bowel disease (IBD) that often requires surgical intervention. Although eosinophils are associated with mucosal remodeling in other organs and are increased in IBD tissues, their role in IBD-associated remodeling is unclear. Histological and molecular features of ileitis and remodeling were assessed using immunohistochemical, histomorphometric, flow cytometric, and molecular analysis (real-time RT-PCR) techniques in a murine model of chronic eosinophilic ileitis. Collagen protein was assessed by Sircol assay. Using a spontaneous eosinophilic Crohn's-like mouse model SAMP1/SkuSlc, we demonstrate an association between ileitis progression and remodeling over the course of 40 weeks. Mucosal and submucosal eosinophilia increased over the time course and correlated with increased histological inflammatory indices. Ileitis and remodeling increased over the 40 weeks, as did expression of fibronectin. CCR3-specific antibody-mediated reduction of eosinophils resulted in significant decrease in goblet cell hyperplasia, muscularis propria hypertrophy, villus blunting, and expression of inflammatory and remodeling genes, including fibronectin. Cellularity of local mesenteric lymph nodes, including T- and B-lymphocytes, was also significantly reduced. Thus, eosinophils participate in intestinal remodeling, supporting eosinophils as a novel therapeutic target., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background & Aims: Retinoic acid (RA), produced by intestinal epithelial cells (IECs) and dendritic cells (DCs) promotes the induction of regulatory T cells (Tregs) and decreases the induction of T-helper (Th)17 cells., Methods: We studied the roles of RA in mice that overproduce tumor necrosis factor (TNF) and develop chronic ileitis (TNF&#95;ARE mice). We assessed the frequency and function of CD103+ DCs, Th17 cells, and Tregs by flow cytometry, and we measured expression of cytokines and retinaldehyde dehydrogenase (RALDH) enzymes in ileum samples, DCs, and IECs by real-time polymerase chain reaction. We quantified RA by electrochemical analysis and examined the effect of RA supplementation on TNF-induced ileitis using histologic, coculture, and suppression assays and flow cytometry., Results: Numbers of CD103+ DCs decreased in the inflamed ilea of mice with chronic disease; RA synthetic machinery (RALDH1,2) was down-regulated. Nevertheless, the proportion of CD4+, CD25+, FoxP3+ Tregs increased, indicating an alternate source for RA. IECs responded to reduced levels of RA by up-regulating RALDH3 in vivo and in vitro. Net tissue levels of RA remained lower in TNF+ARE than wild-type mice, indicating that epithelial up-regulation of RALDH3 could not maintain adequate concentrations of RA, probably because of loss of IEC mass. RA supplementation significantly attenuated disease by increasing the number and function of CD103+ DCs and Tregs and reducing Th17 cells., Conclusions: Reduced levels of RA appear to induce IECs to up-regulate synthesis of RA. RA supplementation attenuates ileitis through its effects on CD103+ DCs, Tregs, and Th17 cells. RA supplementation might offer therapeutic benefit in Crohn's disease., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

IL-37, a newly described member of the IL-1 family, functions as a fundamental inhibitor of innate inflammation and immunity. In the present study, we examined a role for IL-37 during experimental colitis. A transgenic mouse strain was generated to express human IL-37 (hIL-37tg), and these mice were subjected to dextran sulfate sodium (DSS)-induced colitis. Despite the presence of a CMV promoter to drive expression of IL-37, mRNA transcripts were not present in colons at the resting state. Expression was observed only upon disruption of the epithelial barrier, with a six- to sevenfold increase (P = 0.02) on days 3 and 5 after continuous exposure to DSS. During the development of colitis, clinical disease scores were reduced by 50% (P < 0.001), and histological indices of colitis were one-third less in hIL-37tg mice compared with WT counterparts (P < 0.001). Reduced inflammation was associated with decreased leukocyte recruitment into the colonic lamina propria. In addition, release of IL-1β and TNFα from ex vivo colonic explant tissue was decreased 5- and 13-fold, respectively, compared with WT (P ≤ 0.005), whereas IL-10 was increased sixfold (P < 0.001). However, IL-10 was not required for the anti-inflammatory effects of IL-37 because IL-10-receptor antibody blockade did not reverse IL-37-mediated protection. Mechanistically, IL-37 originating from hematopoietic cells was sufficient to exert anti-inflammatory effects because WT mice reconstituted with hIL-37tg bone marrow were protected from colitis. Thus, IL-37 emerges as key modulator of intestinal inflammation.

Background & Aims: A balance between effector and regulatory T-cell (Treg) responses is required to maintain intestinal homeostasis. To regulate immunity, T cells migrate to the intestine using a combination of adhesion molecules and chemokine receptors. However, it is not known whether the migration pathways of effector cells and Tregs are distinct or shared. We sought to determine whether interaction between the chemokine receptor 9 (CCR9) and its ligand, chemokine ligand 25 (CCL25), allows effectors or Tregs to localize to chronically inflamed small intestine., Methods: By using a mouse model that develops Crohn's-like ileitis (tumor necrosis factor Δadenosine uracyl-rich element [TNFΔARE] mice) we examined the role of CCL25-CCR9 interactions for effector and Treg traffic using flow cytometry, quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, immunoneutralization, and proliferation analyses., Results: In TNFΔARE mice, expression of CCL25 and the frequency of CCR9-expressing lymphocytes increased during late-stage disease. In the absence of CCR9, TNFΔARE mice developed exacerbated disease, compared with their CCR9-sufficient counterparts, which coincided with a deficiency of CD4(+)/CD25(+)/forkhead box P3(+) and CD8(+)/CD103(+) Tregs within the intestinal lamina propria and mesenteric lymph nodes. Furthermore, the CD8(+)/CCR9(+) subset decreased the proliferation of CD4(+) T cells in vitro. Administration of a monoclonal antibody against CCR9 to TNFΔARE mice exacerbated ileitis in vivo, confirming the regulatory role of CD8(+)/CCR9(+) cells., Conclusions: Signaling of the chemokine CCL25 through its receptor CCR9 induces Tregs to migrate to the intestine. These findings raise concerns about the development of reagents to disrupt this pathway for the treatment of patients with Crohn's disease., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Evidence indicates that noradrenaline elicits anti-inflammatory actions in the central nervous system (CNS), and plays a neuroprotective role where inflammatory events contribute to pathology. Here we examined the ability of pharmacological enhancement of central noradrenergic tone to impact upon activation of the IL-1 system in rat brain. Treatment with the noradrenaline reuptake inhibitor reboxetine combined with the alpha(2)-adrenoceptor antagonist idazoxan induced expression of IL-1beta as well as its negative regulators, IL-1 receptor antagonist (IL-1ra) and IL-1 type II receptor (IL-1RII) in rat cortex. The ability of reboxetine/idazoxan treatment to activate the IL-1 system was mediated by beta-adrenoceptors, as the aforementioned effects were blocked by the beta-adrenoceptor antagonist propranolol. Moreover, administration of the brain penetrant beta(2)-adrenoceptor agonist clenbuterol induced expression of IL-1beta, IL-1ra and IL-1RII in rat brain. This action was selective to the IL-1 system, as other inflammatory cytokines including TNF-alpha, IL-6 or IFN-gamma were not induced by clenbuterol. Induction of IL-1beta was accompanied by activation of NFkappaB and of the MAP kinase ERK, and clenbuterol also induced expression of the IL-1beta-inducible gene CINC-1. The ability of clenbuterol to activate the IL-1 system was blocked by propranolol, and was mimicked by the highly selective beta(2)-adrenoceptor agonist formoterol. Despite the ability of clenbuterol to activate the central IL-1 system, it largely combated the neuroinflammatory response induced by systemic inflammatory stimulus (bacterial lipopolysaccharide; LPS). Specifically, whilst the ability of clenbuterol to induce expression of IL-1RII and IL-1Ra was maintained following the inflammatory challenge, its ability to induce IL-1beta was reduced. In addition, clenbuterol suppressed LPS-induced expression of the inflammatory cytokines TNF-alpha and IL-6, the inflammatory chemokines RANTES and IP-10, the co-stimulatory molecules CD40 and ICAM-1. Thus overall, clenbuterol suppresses the innate inflammatory response in rat brain., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Background: SAMP1/Yit mice develop spontaneous, segmental, transmural ileitis recapitulating many features of Crohn's disease (CD). The ileitic phenotype may have arisen during crosses of SAMP1 mice selected for the presence of skin lesions. We hereby describe that the original SAMP1 strain similarly develops ileitis. Our aim was to characterize the histopathological and immunological features of this model and assess its responsiveness to standard inflammatory bowel disease (IBD) therapy., Methods: The time course of histopathological features of ileitis was assessed. Immune compartments were characterized by flow cytometry. Ileal cytokine profiles and transcription factors were determined by real-time reverse-transcription polymerase chain reaction (RT-PCR). Finally, response to corticosteroid therapy and its effect on immune compartments and cellularity was evaluated., Results: Histological features and time course of disease were conserved, compared to those reported in SAMP1/Yit strains, with similar expansion of CD19+, CD4+, and CD8+ effector (CD44(high) CD62L(low)), and central memory lymphocytes (CD44(high)CD62L(high)). However, different from SAMP1/YitFc mice, analysis of ileal cytokine profiles revealed initial T(H)1 polarization followed by T(H)2-polarized profile accompanied by prominent eosinophilia during late disease. Lastly, corticosteroids attenuated ileitis, resulting in decreased lymphocyte subsets and cellularity of compartments., Conclusions: Here we report that the ileitic phenotype of SAMP1-related strains was already present in the original SAMP1 strain. By contrast, the cytokine profile within the terminal ilea of SAMP1 is distinct from the mixed T(H)1/T(H)2 profile of SAMP1/YitFc mice during late disease, as it shows predominant T(H)2 polarization. Dissemination of these strains may advance our understanding of CD pathogenesis, which in 60% of patients involves the terminal ileum.

Evidence indicates that the monoamine neurotransmitter noradrenaline elicits anti-inflammatory actions in the central nervous system (CNS), and consequently may play a neuroprotective role where inflammatory events contribute to CNS pathology. Here we examined the ability of pharmacologically enhancing central noradrenergic tone to induce expression of anti-inflammatory cytokines in rat brain. Administration of the noradrenaline reuptake inhibitor reboxetine (15mg/kg; ip) combined with the alpha(2)-adrenoceptor antagonist idazoxan (1mg/kg; ip) induced interleukin-10 (IL-10) expression in rat cortex and hippocampus. In addition, these drug treatments induced IL-10 signaling as indicated by increased STAT3 phosphorylation and suppressor of cytokine signaling-3 (SOCS-3) mRNA expression. In contrast to the profound increase in IL-10 induced by the reboxetine/idazoxan combination, the other two broad spectrum anti-inflammatory cytokines IL-4 and TGF-beta were not induced by this treatment. The ability of combined treatment with reboxetine and idazoxan to induce IL-10 and SOCS3 expression was mediated by beta-adrenoceptor activation, as their induction was blocked by pre-treatment with the beta-adrenoceptor antagonist propranolol. Moreover, administration of the brain penetrant beta(2)-adrenoceptor agonist clenbuterol induced a time- and dose-dependent increase in central IL-10 and SOCS3 expression, and the ability of clenbuterol to induce IL-10 and SOCS-3 expression was blocked by the centrally acting beta-adrenoceptor antagonist, propranolol, and was mimicked by the highly selective beta(2)-adrenoceptor agonist formoterol. In all, these data indicate that increasing central noradrenergic tone induces IL-10 production and signaling in the CNS, which may protect against neurodegeneration., (Copyright 2010 Elsevier Inc. All rights reserved.)