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Crohn's disease (CD) is a complex and highly heterogeneous chronic inflammatory disorder, primarily affecting the gastrointestinal tract. Genetic and functional studies have highlighted a key role for innate immunity in its pathogenesis. Profound systemic defects in innate immunity and acute inflammation are understood to result in markedly delayed clearance of bacteria from the tissues, leading to local chronic granulomatous inflammation and compensatory adaptive immunological changes. Macrophages, key orchestrators of acute inflammation, are likely to play an important role in the initial impaired innate immune response. Monocyte-derived macrophages from CD patients stimulated with Escherichia coli were shown to release attenuated levels of tumour necrosis factor and interferon-γ with normal secretion of interleukin-8 (IL-8), IL-10 and IL-6. In controls, the secretion of these cytokines was strongly positively correlated, which was not seen with CD macrophages. The transcriptomes of CD and control macrophages were examined in an attempt to understand the molecular basis of this defect. There were no differentially expressed genes identified between the two groups, consistent with genetic heterogeneity; however, a number of molecules were found to be under-expressed in subgroups of CD patients. The most common of these was optineurin (OPTN) which was under-expressed in approximately 10% of the CD patients. Reduced OPTN expression coincided with lower intracellular protein levels and diminished cytokine secretion after bacterial stimulation both in the patients and with small interfering RNA knockdown in THP-1 cells. Identifying and studying subgroups of patients with shared defective gene expression could aid our understanding of the mechanisms underlying highly heterogeneous diseases such as CD., (© 2014 The Authors. Immunology published by John Wiley & Sons Ltd., Immunology.)

The epoxygenase CYP2J2 has an emerging role in inflammation and vascular biology. The role of CYP2J2 in phagocytosis is not known and its regulation in human inflammatory diseases is poorly understood. Here we investigated the role of CYP2J2 in bacterial phagocytosis and its expression in monocytes from healthy controls and Crohns disease patients. CYP2J2 is anti-inflammatory in human peripheral blood monocytes. Bacterial LPS induced CYP2J2 mRNA and protein. The CYP2J2 arachidonic acid products 11,12-EET and 14,15-EET inhibited LPS induced TNFα release. THP-1 monocytes were transformed into macrophages by 48h incubation with phorbol 12-myristate 13-acetate. Epoxygenase inhibition using a non-selective inhibitor SKF525A or a selective CYP2J2 inhibitor Compound 4, inhibited E. coli particle phagocytosis, which could be specifically reversed by 11,12-EET. Moreover, epoxygenase inhibition reduced the expression of phagocytosis receptors CD11b and CD68. CD11b also mediates L. monocytogenes phagocytosis. Similar, to E. coli bioparticle phagocytosis, epoxygenase inhibition also reduced intracellular levels of L. monocytogenes, which could be reversed by co-incubation with 11,12-EET. Disrupted bacterial clearance is a hallmark of Crohn's disease. Unlike macrophages from control donors, macrophages from Crohn's disease patients showed no induction of CYP2J2 in response to E. coli. These results demonstrate that CYP2J2 mediates bacterial phagocytosis in macrophages, and implicates a defect in the CYP2J2 pathway may regulate bacterial clearance in Crohn's disease.

Background: Recent work provides evidence of a failure of acute inflammation in Crohn's disease (CD), and suggests that the primary defect operates at the level of the macrophage and cytokine release. Here we extend the characterization of the innate immune defect in CD by investigating the macrophage response to Toll-like receptor (TLR) agonists and assess potential links between genome-wide association study (GWAS) susceptibility loci, disease phenotype, and therapeutic regimens on tumor necrosis factor α (TNF) release., Methods: Peripheral blood-derived macrophages were cultured from control subjects and patients with CD, stimulated with TLR ligands, and the release of TNF measured. Genomic DNA was purified from blood and genotyped for 34 single nucleotide polymorphisms (SNPs) identified as being associated with CD by GWAS., Results: All stimuli resulted in a reduction (32%-48%) in TNF release from macrophages derived from CD patients (n = 28-101) compared to those from healthy control (HC) subjects. All phenotypes demonstrated impaired TNF release, with the greatest defect in patients with colonic disease. There was no detectable relationship between the level of TNF released and the presence of GWAS susceptibility loci in CD patients. Reduced TNF levels were not influenced by age, gender, or use of aminosalicylate (5-ASA) medication., Conclusions: This study supports the hypothesis of defective proinflammatory cytokine secretion and an innate immunodeficiency in CD. Abnormal TNF secretion is evident downstream of multiple TLRs, affects all disease phenotypes, and is unrelated to 34 polymorphisms associated with CD by GWAS., (Copyright © 2012 Crohn's & Colitis Foundation of America, Inc.)

Background: Crohn's Disease (CD) is a chronic relapsing disorder characterized by granulomatous inflammation of the gastrointestinal tract. Although its pathogenesis is complex, we have recently shown that CD patients have a systemic defect in macrophage function, which results in the defective clearance of bacteria from inflammatory sites., Methodology/principal Findings: Here we have identified a number of additional macrophage defects in CD following diacylglycerol (DAG) homolog phorbol-12-myristate-13-acetate (PMA) activation. We provide evidence for decreased DNA fragmentation, reduced mitochondrial membrane depolarization, impaired reactive oxygen species production, diminished cytochrome c release and increased IL-6 production compared to healthy subjects after PMA exposure. The observed macrophage defects in CD were stimulus-specific, as normal responses were observed following p53 activation and endoplasmic reticulum stress., Conclusion: These findings add to a growing body of evidence highlighting disordered macrophage function in CD and, given their pivotal role in orchestrating inflammatory responses, defective apoptosis could potentially contribute to the pathogenesis of CD.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is critical for phagocyte anti-microbial activity and plays a major role in innate immunity. Defects in genes coding for components of the NADPH oxidase enzyme system are responsible for chronic granulomatous disease (CGD), a rare primary neutrophil immunodeficiency associated with recurrent, life-threatening bacterial and fungal infections. Microbial killing and digestion within the neutrophil phagosomal compartment are defective in these patients. NADPH oxidase activity is also crucial for optimal macrophage and dendritic cell function and has recently been implicated in both cross-presentation and T-cell priming. We present evidence of impaired macrophage function in CGD, with attenuated pro-inflammatory cytokine and increased interleukin-10 secretion following bacterial stimulation. These results highlight additional abnormalities in macrophage function associated with CGD and the importance of NADPH oxidase activity in immunity.

The cause of Crohn's disease (CD) remains poorly understood. Counterintuitively, these patients possess an impaired acute inflammatory response, which could result in delayed clearance of bacteria penetrating the lining of the bowel and predispose to granuloma formation and chronicity. We tested this hypothesis in human subjects by monitoring responses to killed Escherichia coli injected subcutaneously into the forearm. Accumulation of (111)In-labeled neutrophils at these sites and clearance of (32)P-labeled bacteria from them were markedly impaired in CD. Locally increased blood flow and bacterial clearance were dependent on the numbers of bacteria injected. Secretion of proinflammatory cytokines by CD macrophages was grossly impaired in response to E. coli or specific Toll-like receptor agonists. Despite normal levels and stability of cytokine messenger RNA, intracellular levels of tumor necrosis factor (TNF) were abnormally low in CD macrophages. Coupled with reduced secretion, these findings indicate accelerated intracellular breakdown. Differential transcription profiles identified disease-specific genes, notably including those encoding proteins involved in vesicle trafficking. Intracellular destruction of TNF was decreased by inhibitors of lysosomal function. Together, our findings suggest that in CD macrophages, an abnormal proportion of cytokines are routed to lysosomes and degraded rather than being released through the normal secretory pathway.

The pathogenesis of Crohn's disease (CD) has widely been regarded as the consequence of a dysregulated T-cell-mediated response to intestinal microbes, and the majority of the worldwide research effort has focused on characterizing and treating the chronic inflammatory phase of the disease. However, recent molecular biological and clinical investigations indicate that CD is actually a primary immunodeficiency. At first counter-intuitive, the apparent paradox of a pathogenic innate immune defect can be linked mechanistically to the granulomatous chronic inflammation characteristic of the disease. Genome-wide association studies have corroborated the involvement of innate immune dysfunction in the pathogenesis of CD, but less than 20% of the heritable risk is accounted for. By contrast, in vitro and in vivo stimulation of the immune system has highlighted novel areas of interest that may lead to the development of targeted therapeutic and diagnostic tools. INSET: Key issues. [ABSTRACT FROM AUTHOR]

Background: Ulcerative colitis (UC) is widely viewed as a leukocyte-mediated disorder. Although strong evidence implicates an exuberant response to microbial components in its pathogenesis, no intrinsic immune defect has been identified and the underlying pathogenic mechanisms remain obscure. Methodology/Principal Findings: The acute immune response to bacterial injection was determined in UC patients with quiescent disease and directly compared to healthy control subjects. Monocyte-derived macrophages were used to investigate bacterial recognition mechanisms in vitro. An exuberant and protracted acute inflammatory response to bacteria was evident in patients with UC, which coincides with increased systemic levels of CXCL10. Macrophages stimulated with bacteria and Toll-like receptor (TLR) ligands revealed a specific defect in the TLR4 response in UC. The defect resulted in the over-expression of a number of pro-inflammatory molecules under transcriptional control of the adaptor TIR-domain containing adaptor inducing interferon-β (TRIF). Conclusion: These findings highlight a dysregulated innate immune response with over-expression of molecules associated with leukocyte recruitment and activation that may eventuate in the hallmark chronic immune-mediated inflammation of UC. [ABSTRACT FROM AUTHOR]