Helicobacter pylori Inhibits Dendritic Cell Maturation via Interleukin-10-Mediated Activation of the Signal Transducer and Activator of Transcription 3 Pathway.

Helicobacter pylori infects the human gastric mucosa causing a chronic infection that is the primary risk factor for gastric cancer development. Recent studies demonstrate that H. pylori promotes tolerogenic dendritic cell (DC) development indicating that this bacterium evades the host immune response. However, the signaling pathways involved in modulating DC activation during infection remain unclear. Here, we report that H. pylori infection activated the signal transducer and activator of transcription 3 (STAT3) pathway in murine bone marrow-derived DCs (BMDCs) and splenic DCs isolated ex vivo. Isogenic cagA-, cagE-, vacA- and urease-mutants exhibited levels of phosphoSTAT3 that were comparable to in the wild-type (WT) parent strain. H. pylori-infected BMDCs produced increased immunosuppressive IL-10, which activated STAT3 in an autocrine/paracrine fashion. Neutralization of IL-10 prevented H. pylori-mediated STAT3 activation in both BMDCs and splenic DCs. In addition, anti-IL-10 treatment of infected H. pylori-BMDCs was associated with increased CD86 and MHC II expression and enhanced proinflammatory IL-1β cytokine secretion. Finally, increased CD86 and MHC II expression was detected in H. pylori-infected STAT3 knockout DCs when compared to WT controls. Together, these results demonstrate that H. pylori infection induces IL-10 secretion in DCs, which activates STAT3, thereby modulating DC maturation and reducing IL-1β secretion. These findings identify a host molecular mechanism by which H. pylori can manipulate the innate immune response to potentially favor chronic infection and promote carcinogenesis. © 2014 S. Karger AG, Basel.

Pestiviruses.

Pestiviruses cause economically important diseases among domestic ruminants and pigs, but they may also infect a wide spectrum of wild species of even-toed ungulates (Artiodactyla). Bovine viral diarrhea virus (BVDV) and Border disease virus of sheep infect their hosts either transiently or persistently. Cellular and humoral immunotolerance to the infecting strain is a unique feature of persistent infection (PI) by ruminant pestiviruses. Persistence, caused by transplacental infection early in fetal development, depends on virally encoded interferon antagonists that inactivate the host's innate immune response to the virus without globally interfering with its function against other viruses. At epidemiological equilibrium, approximately 1-2% of animals are PI. Successful BVDV control programs show that removal of PI animals results in viral extinction in the host population. The nucleotide sequences of ruminant pestiviruses change little during persistent infection. Nevertheless, they display large heterogeneity, pointing to a long history of virus-host coevolution in which avirulent strains are more successful.

Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein Npro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE-, Npro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE- vaccine virus in pigs, the IRF3-degrading function of Npro was not recovered. Therefore, we examined whether restoring the ability of Npro to block IFN-α/β induction of both the avirulent and moderately virulent GPE--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in Npro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional Npro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional Npro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.

Prolonged activity of the pestiviral RNase Erns as an interferon antagonist after uptake by clathrin-mediated endocytosis

The RNase activity of the envelope glycoprotein E(rns) of the pestivirus bovine viral diarrhea virus (BVDV) is required to block type I interferon (IFN) synthesis induced by single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA) in bovine cells. Due to the presence of an unusual membrane anchor at its C terminus, a significant portion of E(rns) is also secreted. In addition, a binding site for cell surface glycosaminoglycans is located within the C-terminal region of E(rns). Here, we show that the activity of soluble E(rns) as an IFN antagonist is not restricted to bovine cells. Extracellularly applied E(rns) protein bound to cell surface glycosaminoglycans and was internalized into the cells within 1 h of incubation by an energy-dependent mechanism that could be blocked by inhibitors of clathrin-dependent endocytosis. E(rns) mutants that lacked the C-terminal membrane anchor retained RNase activity but lost most of their intracellular activity as an IFN antagonist. Surprisingly, once taken up into the cells, E(rns) remained active and blocked dsRNA-induced IFN synthesis for several days. Thus, we propose that E(rns) acts as an enzymatically active decoy receptor that degrades extracellularly added viral RNA mainly in endolysosomal compartments that might otherwise activate intracellular pattern recognition receptors (PRRs) in order to maintain a state of innate immunotolerance. IMPORTANCE The pestiviral RNase E(rns) was previously shown to inhibit viral ssRNA- and dsRNA-induced interferon (IFN) synthesis. However, the localization of E(rns) at or inside the cells, its species specificity, and its mechanism of interaction with cell membranes in order to block the host's innate immune response are still largely unknown. Here, we provide strong evidence that the pestiviral RNase E(rns) is taken up within minutes by clathrin-mediated endocytosis and that this uptake is mostly dependent on the glycosaminoglycan binding site located within the C-terminal end of the protein. Remarkably, the inhibitory activity of E(rns) remains for several days, indicating the very potent and prolonged effect of a viral IFN antagonist. This novel mechanism of an enzymatically active decoy receptor that degrades a major viral pathogen-associated molecular pattern (PAMP) might be required to efficiently maintain innate and, thus, also adaptive immunotolerance, and it might well be relevant beyond the bovine species.

Microbe sampling by mucosal dendritic cells is a discrete, MyD88-independent step in DeltainvG S. Typhimurium colitis.

Intestinal dendritic cells (DCs) are believed to sample and present commensal bacteria to the gut-associated immune system to maintain immune homeostasis. How antigen sampling pathways handle intestinal pathogens remains elusive. We present a murine colitogenic Salmonella infection model that is highly dependent on DCs. Conditional DC depletion experiments revealed that intestinal virulence of S. Typhimurium SL1344 DeltainvG mutant lacking a functional type 3 secretion system-1 (DeltainvG)critically required DCs for invasion across the epithelium. The DC-dependency was limited to the early phase of infection when bacteria colocalized with CD11c(+)CX3CR1(+) mucosal DCs. At later stages, the bacteria became associated with other (CD11c(-)CX3CR1(-)) lamina propria cells, DC depletion no longer attenuated the pathology, and a MyD88-dependent mucosal inflammation was initiated. Using bone marrow chimeric mice, we showed that the MyD88 signaling within hematopoietic cells, which are distinct from DCs, was required and sufficient for induction of the colitis. Moreover, MyD88-deficient DCs supported transepithelial uptake of the bacteria and the induction of MyD88-dependent colitis. These results establish that pathogen sampling by DCs is a discrete, and MyD88-independent, step during the initiation of a mucosal innate immune response to bacterial infection in vivo.

Listeria monocytogenes spreads within the brain by actin-based intra-axonal migration

Listeria monocytogenes rhombencephalitis is a severe progressive disease despite a swift intrathecal immune response. Based on previous observations, we hypothesized that the disease progresses by intra-axonal spread within the central nervous system. To test this hypothesis, neuroanatomical mapping of lesions, immunofluorescence analysis, and electron microscopy were performed on brains of ruminants with naturally occurring rhombencephalitis. In addition, infection assays were performed in bovine brain cell cultures. Mapping of lesions revealed a consistent pattern with a preferential affection of certain nuclear areas and white matter tracts, indicating that Listeria monocytogenes spreads intra-axonally within the brain along interneuronal connections. These results were supported by immunofluorescence and ultrastructural data localizing Listeria monocytogenes inside axons and dendrites associated with networks of fibrillary structures consistent with actin tails. In vitro infection assays confirmed that bacteria were moving within axon-like processes by employing their actin tail machinery. Remarkably, in vivo, neutrophils invaded the axonal space and the axon itself, apparently by moving between split myelin lamellae of intact myelin sheaths. This intra-axonal invasion of neutrophils was associated with various stages of axonal degeneration and bacterial phagocytosis. Paradoxically, the ensuing adaxonal microabscesses appeared to provide new bacterial replication sites, thus supporting further bacterial spread. In conclusion, intra-axonal bacterial migration and possibly also the innate immune response play an important role in the intracerebral spread of the agent and hence the progression of listeric rhombencephalitis.

Signal signature of aboveground-induced resistance upon belowground herbivory in maize

Plants activate local and systemic defence mechanisms upon exposure to stress. This innate immune response is partially regulated by plant hormones, and involves the accumulation of defensive metabolites. Although local defence reactions to herbivores are well studied, less is known about the impact of root herbivory on shoot defence. Here, we examined the effects of belowground infestation by the western corn rootworm Diabrotica virgifera virgifera on aboveground resistance in maize. Belowground herbivory by D. v. virgifera induced aboveground resistance against the generalist herbivore Spodoptera littoralis, and the necrotrophic pathogen Setosphaeria turcica. Furthermore, D. v. virgifera increased shoot levels of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and primed the induction of chlorogenic acid upon subsequent infestation by S. littoralis. To gain insight into the signalling network behind this below- and aboveground defence interaction, we compiled a set of 32 defence-related genes, which can be used as transcriptional marker systems to detect activities of different hormone-response pathways. Belowground attack by D. v. virgifera triggered an ABA-inducible transcription pattern in the shoot. The quantification of defence hormones showed a local increase in the production of oxylipins after root and shoot infestation by D. v. virgifera and S. littoralis, respectively. On the other hand, ABA accumulated locally and systemically upon belowground attack by D. v. virgifera. Furthermore, D. v. virgifera reduced the aboveground water content, whereas the removal of similar quantities of root biomass had no effect. Our study shows that root herbivory by D. v. virgifera specifically alters the aboveground defence status of a maize, and suggests that ABA plays a role in the signalling network mediating this interaction.

The long pentraxin PTX3 at the crossroads between innate immunity and tissue remodelling

Innate immunity represents the first line of defence against pathogens and plays key roles in the activation and orientation of the adaptive immune response. The innate immune system comprises both a cellular and a humoral arm. Components of the humoral arm include soluble pattern recognition molecules that recognize pathogen-associated molecular patterns and initiate the immune response in coordination with the cellular arm, therefore acting as functional ancestors of antibodies. Pentraxins are essential constituents of the humoral arm of innate immunity and represent a superfamily of highly conserved acute phase proteins, traditionally classified into short and long pentraxins. Pentraxin 3 (PTX3) is the prototypic member of the long pentraxins subfamily. As opposed to C-reactive protein, whose sequence and regulation have not been conserved during evolution from mouse to man, the evolutionary conservation of sequence, gene organization and regulation of PTX3 has allowed addressing its pathophysiological roles in genetically modified mice, in diverse conditions, ranging from infections to sterile inflammation, angiogenesis and female fertility. Despite this conservation, a number of predominantly non-coding polymorphisms have been identified in the PTX3 gene which, when associated in particular haplotypes, have been shown to be relevant in clinical conditions including infection and fertility. Here we review the studies on PTX3, with emphasis on pathogen recognition, tissue remodelling and crosstalk with other components of the innate immune system.

Immune pathomechanism of drug hypersensitivity reactions

Drug hypersensitivity research has progressed enormously in recent years, and a greater understanding of mechanisms has contributed to improved drug safety. Progress has been made in genetics, enabling personalized medicine for certain drugs, and in understanding drug interactions with the immune system. In a recent meeting in Rome, the clinical, chemical, pharmacologic, immunologic, and genetic aspects of drug hypersensitivity were discussed, and certain aspects are briefly summarized here. Small chemicals, including drugs, can induce immune reactions by binding as a hapten to a carrier protein. Park (Liverpool, England) demonstrated (1) that drug haptens bind to protein in patients in a highly restricted manner and (2) that irreversibly modified carrier proteins are able to stimulate CD4(+) and CD8(+) T cells from hypersensitive patients. Drug haptens might also stimulate cells of the innate immune system, in particular dendritic cells, and thus give rise to a complex and complete immune reaction. Many drugs do not have hapten-like characteristics but might gain them on metabolism (so-called prohaptens). The group of Naisbitt found that the stimulation of dendritic cells and T cells can occur as a consequence of the transformation of a prohapten to a hapten in antigen-presenting cells and as such explain the immune-stimulatory capacity of prohaptens. The striking association between HLA-B alleles and the development of certain drug reactions was discussed in detail. Mallal (Perth, Australia) elegantly described a highly restricted HLA-B∗5701-specific T-cell response in abacavir-hypersensitive patients and healthy volunteers expressing HLA-B∗5701 but not closely related alleles. Expression of HLA-B∗1502 is a marker known to be necessary but not sufficient to predict carbamazepine-induced Stevens-Johnson syndrome/toxic epidermal necrolysis in Han Chinese. The group of Chen and Hong (Taiwan) described the possible "missing link" because they showed that the presence of certain T-cell receptor (TCR) clonotypes was necessary to elicit T-cell responses to carbamazepine. The role of TCRs in drug binding was also emphasized by Pichler (Bern, Switzerland). Following up on their "pharmacological interactions of drugs with immune receptors" concept (p-i concept), namely that drugs can bind directly to TCRs, MHC molecules, or both and thereby stimulate T cells, they looked for drug-binding sites for the drug sulfamethoxazole in drug-specific TCRs: modeling revealed up to 7 binding sites on the CDR3 and CDR2 regions of TCR Vα and Vβ. Among many other presentations, the important role of regulatory T cells in drug hypersensitivity was addressed.

The mucosal immune system at the gastrointestinal barrier

The immune system faces a considerable challenge in its efforts to maintain tissue homeostasis in the intestinal mucosa. It is constantly confronted with a large array of antigens, and has to prevent the dissemination and proliferation of potentially harmful agents while sparing the vital structures of the intestine from immune-mediated destruction. Complex interactions between the highly adapted effector cells and mechanisms of the innate and adaptive immune system generally prevent the luminal microflora from penetrating the intestinal mucosa and from spreading systemically. Non-haematopoietic cells critically contribute to the maintenance of local tissue homeostasis in an antigen-rich environment by producing protective factors (e.g. production of mucus by goblet cells, or secretion of microbicidal defensins by Paneth cells) and also through interactions with the adaptive and innate immune system (such as the production of chemotactic factors that lead to the selective recruitment of immune cell subsets). The complexity of the regulatory mechanisms that control the local immune response to luminal antigens is also reflected in the observation that mutations in immunologically relevant genes often lead to the development of uncontrolled inflammatory reactions in the microbially colonized intestine of experimental animals.

Early gene expression analysis in 9L orthotopic tumor-bearing rats identifies immune modulation in molecular response to synchrotron microbeam radiation therapy

Synchrotron Microbeam Radiation Therapy (MRT) relies on the spatial fractionation of the synchrotron photon beam into parallel micro-beams applying several hundred of grays in their paths. Several works have reported the therapeutic interest of the radiotherapy modality at preclinical level, but biological mechanisms responsible for the described efficacy are not fully understood to date. The aim of this study was to identify the early transcriptomic responses of normal brain and glioma tissue in rats after MRT irradiation (400Gy). The transcriptomic analysis of similarly irradiated normal brain and tumor tissues was performed 6 hours after irradiation of 9 L orthotopically tumor-bearing rats. Pangenomic analysis revealed 1012 overexpressed and 497 repressed genes in the irradiated contralateral normal tissue and 344 induced and 210 repressed genes in tumor tissue. These genes were grouped in a total of 135 canonical pathways. More than half were common to both tissues with a predominance for immunity or inflammation (64 and 67% of genes for normal and tumor tissues, respectively). Several pathways involving HMGB1, toll-like receptors, C-type lectins and CD36 may serve as a link between biochemical changes triggered by irradiation and inflammation and immunological challenge. Most immune cell populations were involved: macrophages, dendritic cells, natural killer, T and B lymphocytes. Among them, our results highlighted the involvement of Th17 cell population, recently described in tumor. The immune response was regulated by a large network of mediators comprising growth factors, cytokines, lymphokines. In conclusion, early response to MRT is mainly based on inflammation and immunity which appear therefore as major contributors to MRT efficacy.

The Immune System of Spiders

Spiders, as all other arthropods, have an open circulatory system, and their body fluid, the hemolymph, freely moves between lymphatic vessels and the body cavities (see Wirkner and Huckstorf 2013). The hemolymph can be considered as a multifunctional organ, central for locomotion (Kropf 2013), respiration (Burmester 2013) and nutrition, and it amounts to approximately 20 % of a spider’s body weight. Any injury includes not only immediate hemolymph loss but also pathogen attacks and subsequent infections. Therefore spiders have to react to injuries in a combined manner to stop fluid loss and to defend against microbial invaders. This is achieved by an innate immune system which involves several host defence systems such as hemolymph coagulation and the production of a variety of defensive substances (Fukuzawa et al.2008). In spiders, the immune system is localised in hemocytes which are derived from the myocardium cells of the heart wall where they are produced as prohemocytes and from where they are released as different cell types into the hemolymph (Seitz 1972). They contribute to the defence against pathogens by phagocytosis, nodulation and encapsulation of invaders. The humoral response includes mechanisms which induce melanin production to destroy pathogens, a clotting cascade to stop hemolymph loss and the constitutive production of several types of antimicrobial peptides, which are stored in hemocyte granules and released into the hemolymph (Fukuzawa et al.2008) (Fig.7.1). The immune system of spiders is an innate immune system. It is hemolymph-based and characterised by a broad but not very particular specificity. Its advantage is a fast response within minutes to a few hours. This is in contrast to the adaptive immune system of vertebrates which can react to very specific pathogens, thus resulting in much more specific responses. Moreover, it creates an immunological memory during the lifetime of the species. The disadvantage is that it needs more time to react with antibody production, usually many hours to a few days, and needs to be built up during early ontogenesis.

Fish Immune Responses to Myxozoa

Myxozoans evoke important economic losses in aquaculture production, but there is almost a total lack of disease control methods as no vaccines or commercial treatments are currently available. Knowledge of the immune responses that lead to myxozoan elimination and subsequent disease resistance is vital for shaping the future development of disease control measures. Different fish immune factors triggered by myxozoan parasites are reviewed in this chapter. Detailed information on the phenotypic and underlying molecular aspects of innate and adaptive responses, at both cellular and humoral levels, is provided for some well-studied fishmyxozoan systems. The importance of the local immune response, mainly at mucosal sites, is also highlighted. Myxozoan tactics to disable or avoid immune responses, such as modulation of immune gene transcription and immune evasion, are also reviewed. The existence of innate and acquired resistance to some myxozoan species suggest promising possibilities for controlling myxozooses through immune-based strategies, such as genetic selection for host resistance, vaccination, immune therapies and administration of immunostimulants.

Deletion of CD39 on natural killer cells attenuates hepatic ischemia/reperfusion injury in mice

Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.

Effects of TLR agonists on the hypoxia-regulated transcription factor HIF-1alpha and dendritic cell maturation under normoxic conditions

Dendritic cells (DC) are professional antigen presenting cells that represent an important link between innate and adaptive immunity. Danger signals such as toll-like receptor (TLR) agonists induce maturation of DC leading to a T-cell mediated adaptive immune response. In this study, we show that exogenous as well as endogenous inflammatory stimuli for TLR4 and TLR2 induce the expression of HIF-1alpha in human monocyte-derived DC under normoxic conditions. On the functional level, inhibition of HIF-1alpha using chetomin (CTM), YC-1 and digoxin lead to no consistent effect on MoDC maturation, or cytokine secretion despite having the common effect of blocking HIF-1alpha stabilization or activity through different mechanisms. Stabilization of HIF-1alpha protein by hypoxia or CoCl(2) did not result in maturation of human DC. In addition, we could show that TLR stimulation resulted in an increase of HIF-1alpha controlled VEGF secretion. These results show that stimulation of human MoDC with exogenous as well as endogenous TLR agonists induces the expression of HIF-1alpha in a time-dependent manner. Hypoxia alone does not induce maturation of DC, but is able to augment maturation after TLR ligation. Current evidence suggests that different target genes may be affected by HIF-1alpha under normoxic conditions with physiological roles that differ from those induced by hypoxia.