Gernot Sellge

Human Intestinal Mast Cells Are Capable of Producing Different Cytokine Profiles: Role of IgE Receptor Cross-Linking and IL-4

Mast cells are recognized as a new type of immunoregulatory cells capable of producing different cytokines. So far, little is known about the cytokine profile of mature human mast cells isolated from intestinal tissue and cultured in the presence of stem cell factor (SCF). We observed that these cells express the proinflammatory cytokines TNF-α, IL-1β, IL-6, IL-8, IL-16, and IL-18 without further stimulation. Both IgE-dependent and IgE-independent agonists (e.g., Gram-negative bacteria) enhanced expression of TNF-α. Another set of cytokines consisting of IL-3, IL-5, IL-9, and IL-13 was expressed following activation by IgE receptor cross-linking. If mast cells were cultured in the presence of IL-4 and SCF, the production and release of IL-3, IL-5, and IL-13 was increased up to 4-fold compared with mast cells cultured with SCF alone. By contrast, IL-6 expression was completely blocked in response to culture with IL-4. In summary, our data show that mature human mast cells produce proinflammatory cytokines that may be up-regulated following triggering with IgE-independent agonists such as bacteria, whereas activation by IgE receptor cross-linking results in the expression of Th2-type cytokines. IL-4 enhances the expression of Th2-type cytokines but does not affect or even down-regulates proinflammatory cytokines.

Nod2-Dependent Th2 Polarization of Antigen-Specific Immunity

While a number of microbial-associated molecular patterns have been known for decades to act as adjuvants, the mechanisms and the signaling pathways underlying their action have remained elusive. Here, we examined the unfolding of the adaptive immune response induced by Nod2 in vivo upon activation by its specific ligand, muramyl dipeptide, a component of peptidoglycan. Our findings demonstrate that this bacterial sensor triggers a potent Ag-specific immune response with a Th2-type polarization profile, characterized by the induction of IL-4 and IL-5 by T cells and IgG1 Ab responses. Nod2 was also found to be critical for the induction of both Th1- and Th2-type responses following costimulation with TLR agonists. Importantly, the synergistic responses to Nod2 and TLR agonists seen in vivo were recapitulated by dendritic cells in vitro, suggesting that these cells likely play a central role in the integration of Nod2- and TLR-dependent signals for driving the adaptive immune response. Taken together, our results identify Nod2 as a critical mediator of microbial-induced potentiation and polarization of Ag-dependent immunity. Moreover, these findings affect our understanding of Crohn’s diseases pathogenesis, where lack of Nod2-dependent Th2 signaling in a subset of these patients might explain heightened Th1-mediated inflammation at the level of the intestinal mucosa.

Selective expression of histamine receptors H1R, H2R, and H4R, but not H3R, in the human intestinal tract

Background and aims: Histamine is known as a regulator of gastrointestinal functions, such as gastric acid production, intestinal motility, and mucosal ion secretion. Most of this knowledge has been obtained from animal studies. In contrast, in humans, expression and distribution of histamine receptors (HR) within the human gastrointestinal tract are unclear. Methods: We analysed HR expression in human gastrointestinal tissue specimens by quantitative reverse transcription-polymerase chain reaction and immunostaining. Results: We found that H1R, H2R, and H4R mRNA were expressed throughout the gastrointestinal tract, while H3R mRNA was absent. No significant differences in the distribution of HR were found between different anatomical sites (duodenum, ileum, colon, sigma, and rectum). Immunostaining of neurones and nerve fibres revealed that H3R was absent in the human enteric nervous system; however, H1R and H2R were found on ganglion cells of the myenteric plexus. Epithelial cells also expressed H1R, H2R and, to some extent, H4R. Intestinal fibroblasts exclusively expressed H1R while the muscular layers of human intestine stained positive for both H1R and H2R. Immune cells expressed mRNA and protein for H1R, H2R, and low levels of H4R. Analysis of endoscopic biopsies from patients with food allergy and irritable bowel syndrome revealed significantly elevated H1R and H2R mRNA levels compared with controls. Conclusions: We have demonstrated that H1R, H2R and, to some extent, H4R, are expressed in the human gastrointestinal tract, while H3R is absent, and we found that HR expression was altered in patients with gastrointestinal diseases.

IL-4-Induced Priming of Human Intestinal Mast Cells for Enhanced Survival and Th2 Cytokine Generation Is Reversible and Associated with Increased Activity of ERK1/2 and c-Fos

In synergy with stem cell factor (SCF), IL-4 strongly enhances mast cell proliferation and shifts IgE-dependent cytokine production in mature human mast cells toward an increased release of Th2 cytokines such as IL-3, IL-5, and IL-13 and a decreased IL-6 expression. In this study we analyzed the kinetics and the mechanisms of these IL-4 effects on mast cells purified from intestinal tissue. If the cells were first cultured with IL-4 for 14 days and then without IL-4 for another 14 days, mast cells lost the capacity of producing higher amounts of Th2 cytokines and regained the capacity of producing IL-6. The IL-4-induced up-regulation of mast cell proliferation and FcεRI expression was also reversible if IL-4 was withdrawn for 14 days. Interestingly, in contrast to IL-4, proliferation and phenotype of human intestinal mast cells were not affected by IL-13 although both cytokines were capable of inducing STAT6 activation. Instead, IL-4 treatment (but not IL-13 treatment) was associated with an increased activity of ERK1/2 and c-Fos, the downstream target of ERK1/2 and component of the transcription factor AP-1. Consistently, mast cell proliferation and cytokine expression in response to IL-4 was blocked by the MEK inhibitor PD98059. In summary, our data show that the IL-4 effects on human intestinal mast cell functions are reversible and accompanied by an increased activity of ERK1/2 and c-Fos.

Th17 Cells Are the Dominant T Cell Subtype Primed by Shigella flexneri Mediating Protective Immunity

The T cell response to Shigella, the causative agent of bacillary dysentery, remains poorly understood. Using a murine model of infection, we report that Shigella flexneri primes predominately IL-17A– and IL-22–producing Th17 cells. Shigella-specific Th1 cells are only significantly induced on secondary infection, whereas specific Th2 and CD8+ T cells are undetectable. Apart from Th17 cells that are primed in a MHC class II- and IL-6–dependent, but IL12/23p40-independent manner, we identified γδ T cells as an additional but minor source of IL-17A. Priming of IL-17A+ γδ T cells is dependent on IL12/23p40, but independent of MHC-class II and IL-6. Th17 cells have emerged as important players in inflammatory, autoimmune, and infectious diseases. Among the yet unresolved questions is their role in long-term immunity to pathogens. In this study, we show that the elicited S. flexneri-specific Th17 pool gives rise to an enhanced recall response up to 12 mo after priming, suggesting the presence of a long-term memory state. The clearance of primary infection is impaired in the absence of T cells, but independently of IL-17A. However, after reinfection, IL-17A produced by S. flexneri-specific Th17 cells becomes important to ultimately restrict bacterial growth. These findings bring new insights into the adaptive immune response to Shigella infection and highlight the importance of pathogen-specific Th17 cell immunity for secondary immune protection.

Cultured human intestinal mast cells express functional IL-3 receptors and respond to IL-3 by enhancing growth and IgE receptor-dependent mediator release

Mast cells are immunoregulatory effector cells capable of releasing different mediators and cytokines implicated in inflammatory tissue processes. Previous studies suggested that IL‐3 regulates growth and function of murine mast cells and human mast cell precursors, but does not affect mature human mast cells. In the present study, we found expression of IL‐3 receptors (IL‐3R) in freshlyisolated human intestinal mast cells by reverse transcriptase (RT)‐PCR and in mast cells cultured with stem cell factor (SCF) using RT‐PCR and flow cytometry. IL‐3R expression was enhanced when theculture medium was supplemented with IL‐4 in addition to SCF. In the presence of SCF, IL‐3 significantly enhanced mast cell growth in a dose‐dependent fashion (179±51% of control, p⩽0.004, n=9, ED50 ≈ 15 ng/ml) by decreasing mast cell apoptosis rather than inducing proliferation. Furthermore, IL‐3 selectively enhanced histamine (from 39.6±12.4 to 51.2±15.7% specific release, p<0.02, n=8) and leukotriene C4 (LTC4, 5.1±3.4 to 10.8±5.5 ng/106 mast cells, p<0.03, n=6) release triggered by IgE receptor cross‐linking without affecting prostaglandin D2 production. In conclusion, our data show that human intestinal mast cells express functional IL‐3R, indicating that IL‐3 not only regulates growth and function of immature, but also that of mature human mast cells.

Nucleotide oligomerization domain-containing proteins instruct T cell helper type 2 immunity through stromal activation

Although a number of studies have examined the development of T-helper cell type 2 (Th2) immunity in different settings, the mechanisms underlying the initiation of this arm of adaptive immunity are not well understood. We exploited the fact that immunization with antigen plus either nucleotide-binding oligomerization domain-containing proteins 1 (Nod1) or 2 (Nod2) agonists drives Th2 induction to understand how these pattern-recognition receptors mediate the development of systemic Th2 immune responses. Here, we show in bone-marrow chimeric mice that Nod1 and Nod2 expression within the stromal compartment is necessary for priming of effector CD4+ Th2 responses and specific IgG1 antibodies. In contrast, sensing of these ligands by dendritic cells was not sufficient to induce Th2 immunity, although these cells contribute to the response. Moreover, we determined that CD11c+ cells were the critical antigen-presenting cells, whereas basophils and B cells did not affect the capacity of Nod ligands to induce CD4+ Th2 effector function. Finally, we found that full Th2 induction upon Nod1 and Nod2 activation was dependent on both thymic stromal lymphopoietin production by the stromal cells and the up-regulation of the costimulatory molecule, OX40 ligand, on dendritic cells. This study provides in vivo evidence of how systemic Th2 immunity is induced in the context of Nod stimulation. Such understanding will influence the rational design of therapeutics that could reprogram the immune system during an active Th1–mediated disease, such as Crohns disease.

TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohns disease. We found that TRIM27 expression is increased in Crohns disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus. We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.

Mast Cell Hyperplasia: Role of Cytokines

Mast cell hyperplasia is found in different pathologies such as chronic inflammatory processes, fibrotic disorders, wound healing or neoplastic tissue transformation. The functional significance of the accumulation of mast cells in these processes is largely unknown. It is now established that bone marrow-derived mast cell progenitors circulate in peripheral blood and subsequently migrate into the tissue where they undergo final maturation under the influence of local microenvironmental factors. Cytokines are of particular importance for mast cell recruitment, development, and function. Stem cell factor (SCF) is a unique mast cell growth factor, since mast cells disappear completely in the absence of SCF. However, several other cytokines such as IL-3 and IL-4 have been shown to influence mast cell proliferation and function also. This review focuses on the role of cytokines in the regulation of mast cell hyperplasia.

Hepatocyte caspase‐8 is an essential modulator of steatohepatitis in rodents

In human and murine models of nonalcoholic steatohepatitis (NASH), increased hepatocyte apoptosis is a critical mechanism contributing to inflammation and fibrogenesis. Caspase 8 (Casp8) is essential for death‐receptor‐mediated apoptosis activity and therefore its modulation might be critical for the pathogenesis of NASH. The aim was to dissect the role of hepatocyte Casp8 in a murine model of steatohepatitis. We generated hepatocyte‐specific Casp8 knockout (Casp8Δhep) mice. Animals were fed with a methionine‐choline‐deficient (MCD) diet. Liver injury was assessed by histopathological analysis, apoptotic death, serum alanine aminotransferase (ALT), fluorescent‐activated cell sorter (FACS), analysis of liver infiltration and inflammation, reactive oxygen species (ROS), and liver fibrosis. MCD feeding triggered steatosis, hepatic lipid storage, and accumulation of free fatty acid (FFA) in wildtype (WT) livers, which were significantly reduced in Casp8Δhep animals. Additionally, lack of Casp8 expression in hepatocytes reduced the MCD‐dependent increase in apoptosis and decreased expression of proinflammatory cytokines as well as hepatic infiltration. As a consequence, ROS production was lower, leading to a reduction in the progression of liver fibrosis in Casp8Δhep livers. Conclusion: Selective ablation of Casp8 in hepatocytes ameliorates development of NASH by modulating liver injury. Casp8‐directed therapy might be a plausible treatment for patients with steatohepatitis. (HEPATOLOGY 2013;57:2189–2201)