The contribution of dendritic cell (DC) antigen cross-presentation to the activation of CD8 + T lymphocytes for immune defense against tumors, viruses, and intracellular pathogens has been recognized widely. Although originally thought to be an exclusive characteristic of DCs, recently also other immune cells, particularly macrophages, have been shown capable of cross-presentation. Here we provide an overview of in vitro and in vivo evidence on cross-presentation by macrophages. As we discuss, it is now firmly established that various types of tissue-resident macrophages are able to cross-present via similar cellular pathways as DCs. This is based on a wide range of antigens in macrophages from many different tissue origins such as blood, tumors, and lymphoid tissue. However, the physiological relevance of macrophage cross-presentation with potential contributions to activation of CD8 + T lymphocytes is still mostly unknown. While cross-presentation by various types of proinflammatory macrophages might be involved in cross-priming of naive CD8 + T lymphocytes, it might also be involved in local reactivation of memory and/or effector CD8 + T lymphocytes. Moreover, cross-presentation by anti-inflammatory macrophages could be related to immune tolerance. Because cross-presentation promotes the initiation and potentiation of antigen-specific CD8 + T lymphocyte responses, stimulating macrophages to crosspresent antigen might be a promising strategy for antitumor or antiviral therapies.

It is increasingly clear that inflammatory diseases and cancers are influenced by cleavage products of the pro-hormone chromogranin A (CgA), such as the 21-amino acids long catestatin (CST). The goal of this review is to provide an overview of the anti-inflammatory effects of CST and its mechanism of action. We discuss evidence proving that CST and its precursor CgA are crucial for maintaining metabolic and immune homeostasis. CST could reduce inflammation in various mouse models for diabetes, colitis and atherosclerosis. In these mouse models, CST treatment resulted in less infiltration of immune cells in affected tissues, although in vitro monocyte migration was increased by CST. Both in vivo and in vitro, CST can shift macrophage differentiation from a pro- to an anti-inflammatory phenotype. Thus, the concept is emerging that CST plays a role in tissue homeostasis by regulating immune cell infiltration and macrophage differentiation. These findings warrant studying the effects of CST in humans and make it an interesting therapeutic target for treatment and/or diagnosis of various metabolic and immune diseases.

Background and AimsA ‘leaky’ gut barrier has been implicated in the initiation and progression of a multitude of diseases, e.g., inflammatory bowel disease, irritable bowel syndrome, celiac disease, and colorectal cancers. Here we asked how Chromogranin A (CgA), a major hormone produced by the enteroendocrine cells, and Catestatin (CST), the most abundant CgA-derived proteolytic peptide, affect the gut barrier.Methods and ResultsUltrastructural studies on the colons from Catestatin (CST: hCgA352-372) knockout (CST-KO) mice revealed (i) altered morphology of tight (TJ) and adherens (AJ) junctions and desmosomes, indicative of junctional stress and (ii) an increased infiltration of immune cells compared to controls. Flow cytometry studies confirmed these cells to be macrophages and CD4+ T cells. Gene expression studies confirmed that multiple TJ-markers were reduced, with concomitant compensatory elevation of AJ and desmosome markers. Consistently, the levels of plasma FITC-dextran were elevated in the CST-KO mice, confirming leakiness’ of the gut. Leaky gut in CST-KO mice correlated with inflammation and a higher ratio of Firmicutes to Bacteroidetes, a dysbiotic pattern commonly encountered in a multitude of diseases. Supplementation of CST-KO mice with recombinant CST reversed this leakiness and key phenotypes. Supplementation of CgA-KO mice with either CST alone, or with the pro-inflammatory proteolytic CgA fragment pancreastatin (PST: CgA250-301) showed that gut permeability is regulated by the antagonistic roles of these two peptide hormones: CST reduces and PST increases leakiness.ConclusionWe conclude that the enteroendocrine cell-derived hormone, CgA regulates gut permeability. CST is both necessary and sufficient to reduce the leakiness. CST acts primarily via antagonizing the effects of PST.What you need to knowBackground and ContextThe intestinal barrier is disrupted in many intestinal diseases such as Crohn’s disease. Chromogranin A (CgA) is produced by enteroendocrine cells in the gut. CgA is proteolytically cleaved into bioactive peptides including catestatin (CST) and pancreastatin (PST). The role of CgA in the gut is unknown.New findingsCgA is efficiently processed to CST in the gut and this processing might be decreased during active Crohn’s disease. CST promotes epithelial barrier function and reduces inflammation by counteracting PST.LimitationsThe complete mechanism of intestinal barrier regulation by CST likely involves a complex interplay between the enteroendocrine system, metabolism, the epithelium, the immune system and the gut microbiota.ImpactOur findings indicate that CST is a key modulator of the intestinal barrier and immune functions that correlates with disease severity of Crohn’s disease. CST could be a target for therapeutic interventions in Crohn’s disease.

Most viruses have acquired mechanisms to suppress antiviral alpha/beta interferon (IFN-␣/␤) and stress responses. Enteroviruses (EVs) actively counteract the induction of IFN-␣/␤ gene transcription and stress granule (SG) formation, which are increasingly implicated as a platform for antiviral signaling, but the underlying mechanisms remain poorly understood. Both viral proteases (2A pro and 3C pro ) have been implicated in the suppression of these responses, but these conclusions predominantly rely on ectopic overexpression of viral proteases or addition of purified viral proteases to cell lysates. Here, we present a detailed and comprehensive comparison of the effect of individual enterovirus proteases on the formation of SGs and the induction of IFN-␣/␤ gene expression in infected cells for representative members of the enterovirus species EV-A to EV-D. First, we show that SG formation and IFN-␤ induction are suppressed in cells infected with EV-A71, coxsackie B3 virus (CV-B3), CV-A21, and EV-D68. By introducing genes encoding CV-B3 proteases in a recombinant encephalomyocarditis virus (EMCV) that was designed to efficiently activate antiviral responses, we show that CV-B3 2A pro , but not 3C pro , is the major antagonist that counters SG formation and IFN-␤ gene transcription and that 2A pro 's proteolytic activity is essential for both functions. 2A pro efficiently suppressed SG formation despite protein kinase R (PKR) activation and ␣ subunit of eukaryotic translation initiation factor 2 phosphorylation, suggesting that 2A pro antagonizes SG assembly or promotes its disassembly. Finally, we show that the ability to suppress SG formation and IFN-␤ gene transcription is conserved in the 2A pro of EV-A71, CV-A21, and EV-D68. Collectively, our results indicate that enterovirus 2A pro plays a key role in inhibiting innate antiviral cellular responses. IMPORTANCE Enteroviruses are important pathogens that can cause a variety of diseases in humans, including aseptic meningitis, myocarditis, hand-foot-and-mouth disease, conjunctivitis, and acute flaccid paralysis. Like many other viruses, enteroviruses must counteract antiviral cellular responses to establish an infection. It has been suggested that enterovirus proteases cleave cellular factors to perturb antiviral pathways, but the exact contribution of viral proteases 2A pro and 3C pro remains elusive. Here, we show that 2A pro , but not 3C pro , of all four human EV species (EV-A to EV-D) inhibits SG formation and IFN-␤ gene transcription. Our observations suggest that enterovirus 2A pro has a conserved function in counteracting antiviral host responses and thereby is the main enterovirus "security protein." Understanding the molecular mechanisms of enterovirus immune evasion strategies may help to develop countermeasures to control infections with these viruses. FIG 5 Enterovirus 2A pro suppresses the induction of type I IFN gene expression. (A)HeLa R19 cells were infected with the indicated viruses at an MOI of 10, and the cells were lysed at 8 hpi, total cell...

Aim A “leaky” gut barrier has been implicated in the initiation and progression of a multitude of diseases, for example, inflammatory bowel disease (IBD), irritable bowel syndrome and celiac disease. Here we show how pro‐hormone Chromogranin A (CgA), produced by the enteroendocrine cells, and Catestatin (CST: hCgA352‐372), the most abundant CgA‐derived proteolytic peptide, affect the gut barrier. Methods Colon tissues from region‐specific CST‐knockout (CST‐KO) mice, CgA‐knockout (CgA‐KO) and WT mice were analysed by immunohistochemistry, western blot, ultrastructural and flowcytometry studies. FITC‐dextran assays were used to measure intestinal barrier function. Mice were supplemented with CST or CgA fragment pancreastatin (PST: CgA250‐301). The microbial composition of cecum was determined. CgA and CST levels were measured in blood of IBD patients. Results Plasma levels of CST were elevated in IBD patients. CST‐KO mice displayed (a) elongated tight, adherens junctions and desmosomes similar to IBD patients, (b) elevated expression of Claudin 2, and (c) gut inflammation. Plasma FITC‐dextran measurements showed increased intestinal paracellular permeability in the CST‐KO mice. This correlated with a higher ratio of Firmicutes to Bacteroidetes, a dysbiotic pattern commonly encountered in various diseases. Supplementation of CST‐KO mice with recombinant CST restored paracellular permeability and reversed inflammation, whereas CgA‐KO mice supplementation with CST and/or PST in CgA‐KO mice showed that intestinal paracellular permeability is regulated by the antagonistic roles of these two peptides: CST reduces and PST increases permeability. Conclusion The pro‐hormone CgA regulates the intestinal paracellular permeability. CST is both necessary and sufficient to reduce permeability and primarily acts by antagonizing PST.

SummaryAntigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal “clickable” antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.

Major histocompatibility complex (MHC) molecules are well-known for their role in antigen (cross-) presentation, thereby functioning as key players in the communication between immune cells, for example dendritic cells (DCs) and T cells, or immune cells and their targets, such as T cells and virus-infected or tumor cells. However, much less appreciated is the fact that MHC molecules can also act as signaling receptors. In this process, here referred to as reverse MHC class I (MHC-I) signaling, ligation of MHC molecules can lead to signal-transduction and cell regulatory effects in the antigen presenting cell. In the case of MHC-I, reverse signaling can have several outcomes, including apoptosis, migration, induced or reduced proliferation and cytotoxicity towards target cells. Here, we provide an overview of studies showing the signaling pathways and cell outcomes upon MHC-I stimulation in various immune and non-immune cells. Signaling molecules like RAC-alpha serine/threonine-protein kinase (Akt1), extracellular signal-regulated kinases 1/2 (ERK1/2), and nuclear factor-κB (NF-κB) were common signaling molecules activated upon MHC-I ligation in multiple cell types. For endothelial and smooth muscle cells, the in vivo relevance of reverse MHC-I signaling has been established, namely in the context of adverse effects after tissue transplantation. For other cell types, the role of reverse MHC-I signaling is less clear, since aspects like the in vivo relevance, natural MHC-I ligands and the extended downstream pathways are not fully known.The existing evidence, however, suggests that reverse MHC-I signaling is involved in the regulation of the defense against bacterial and viral infections and against malignancies. Thereby, reverse MHC-I signaling is a potential target for therapies against viral and bacterial infections, cancer immunotherapies and management of organ transplantation outcomes.

Background: The pro-hormone chromogranin A (CgA) and its bioactive cleavage product catestatin (CST) are both associated with inflammatory bowel disease (IBD) and dysregulated barrier functions, but their exact role has remained elusive. Here, we demonstrate that CST regulates the colonic mucus layer. Methods: CST levels were measured in feces of IBD patients. The mucus layer, goblet cells, and immune cell infiltration were analyzed by histology and electron microscopy in colon tissue from IBD patients and mice with selective deletion of the CST-coding region of the CgA gene. Results: CST levels were elevated in feces of IBD patients compared to healthy controls. The thickness of the mucus layer was increased in non-affected, but not in inflamed, regions of the colon in IBD patients. The thickness of the mucus layer and concomitant mucus production were also increased in the CST-KO mouse. This mucus phenotype in CST-KO mice could be reversed by bone marrow transplantation from wildtype mice. Conclusions: CST produced by bone-marrow derived immune cells reduces production of the mucus layer in the intestine. This might contribute to the reduced mucus layer in inflamed colon regions of IBD patients. Additionally, CST feces levels might be a biomarker for IBD.