Manuel Koch

MicroRNA-155 Is Essential for the T Cell-Mediated Control of Helicobacter pylori Infection and for the Induction of Chronic Gastritis and Colitis

MicroRNAs govern immune responses to infectious agents, allergens, and autoantigens and function by posttranscriptional repression of their target genes. In this paper, we have addressed the role of microRNA-155 (miR-155) in the control of Helicobacter pylori infection of the gastrointestinal tract and the development of H. pylori-induced chronic gastritis and associated gastric preneoplastic pathology. We show that miR-155 is upregulated in the gastric mucosa of experimentally infected mice and that miR-155−/− mice fail to control H. pylori infection as a result of impaired pathogen-specific Th1 and Th17 responses. miR-155−/− mice are also less well protected against challenge infection after H. pylori-specific vaccination than their wild-type (wt) counterparts. As a consequence of their impaired T cell responses to H. pylori, miR-155−/− mice develop less severe infection-induced immunopathology manifesting as chronic atrophic gastritis, epithelial hyperplasia, and intestinal metaplasia. T cells from miR-155−/− mice that are activated by CD3/CD28 cross-linking expand less and produce less IFN-γ and IL-17 than wt T cells. Finally, we show in this paper using adoptive transfers that the phenotypes of miR-155−/− mice are likely due to T cell-intrinsic defects. In contrast to wt T cells, miR-155−/− T cells from infected donors do not control H. pylori infections in T cell-deficient recipients, do not differentiate into Th1 or Th17 cells, and do not cause immunopathology. In addition, naive miR-155−/− T cells fail to induce chronic Th17-driven colitis in an adoptive transfer model. In conclusion, miR-155 expression is required for the Th17/Th1 differentiation that underlies immunity to H. pylori infection on the one hand and infection-associated immunopathology on the other.

Helicobacter pylori Induces miR-155 in T Cells in a cAMP-Foxp3-Dependent Manner

Amongst the most severe clinical outcomes of life-long infections with Helicobacter pylori is the development of peptic ulcers and gastric adenocarcinoma - diseases often associated with an increase of regulatory T cells. Understanding H. pylori-driven regulation of T cells is therefore of crucial clinical importance. Several studies have defined mammalian microRNAs as key regulators of the immune system and of carcinogenic processes. Hence, we aimed here to identify H. pylori-regulated miRNAs, mainly in human T cells. MicroRNA profiling of non-infected and infected human T cells revealed H. pylori infection triggers miR-155 expression in vitro and in vivo. By using single and double H. pylori mutants and the corresponding purified enzymes, the bacterial vacuolating toxin A (VacA) and γ-glutamyl transpeptidase (GGT) plus lipopolysaccharide (LPS) tested positive for their ability to regulate miR-155 and Foxp3 expression in human lymphocytes; the latter being considered as the master regulator and marker of regulatory T cells. RNAi-mediated knockdown (KD) of the Foxp3 transcription factor in T cells abolished miR-155 expression. Using adenylate cyclase inhibitors, the miR-155 induction cascade was shown to be dependent on the second messenger cyclic adenosine monophosphate (cAMP). Furthermore, we found that miR-155 directly targets the protein kinase A inhibitor α (PKIα) mRNA in its 3′UTR, indicative of a positive feedback mechanism on the cAMP pathway. Taken together, our study describes, in the context of an H. pylori infection, a direct link between Foxp3 and miR-155 in human T cells and highlights the significance of cAMP in this miR-155 induction cascade.

Induction of microRNA-155 is TLR- and type IV secretion system-dependent in macrophages and inhibits DNA-damage induced apoptosis

Helicobacter pylori is a gastric pathogen responsible for a high disease burden worldwide. Deregulated inflammatory responses, possibly involving macrophages, are implicated in H. pylori-induced pathology, and microRNAs, such as miR-155, have recently emerged as crucial regulators of innate immunity and inflammatory responses. miR-155 is regulated by Toll-like receptor (TLR) ligands in monocyte-derived cells and has been shown to be induced in macrophages during H. pylori infection. Here, we investigated the regulation of miR-155 expression in primary murine bone marrow-derived macrophages (BMMs) during H. pylori infection and examined the downstream mRNA targets of this microRNA using microarray analysis. We report TLR2/4- and NOD1/2-independent up-regulation of miR-155, which was found to be dependent on the major H. pylori pathogenicity determinant, the type IV secretion system (T4SS). miR-155 expression was dependent on NF-κB signaling but was independent of CagA. Microarray analysis identified known gene targets of miR-155 in BMMs during H. pylori infection that are proapoptotic. We also identified and validated miR-155 binding sites in the 3′ UTRs of the targets, Tspan14, Lpin1, and Pmaip1. We observed that H. pylori-infected miR-155−/− BMMs were significantly more susceptible to cisplatin DNA damage-induced apoptosis than were wild-type BMMs. Thus, our data suggest a function for the prototypical H. pylori pathogenicity factor, the T4SS, in the up-regulation of miR-155 in BMMs. We propose the antiapoptotic effects of miR-155 could enhance macrophage resistance to apoptosis induced by DNA damage during H. pylori infection.

Evidence for a crucial role of a host non-coding RNA in influenza A virus replication

A growing body of evidence suggests the non-protein coding human genome is of vital importance for human cell function. Besides small RNAs, the diverse class of long non-coding RNAs (lncRNAs) recently came into focus. However, their relevance for infection, a major evolutionary driving force, remains elusive. Using two commercially available microarray systems, namely NCode™ and Sureprint™ G3, we identified differential expression of 42 ncRNAs during influenza A virus (IAV) infection in human lung epithelial cells. This included several classes of lncRNAs, including large intergenic ncRNAs (lincRNAs). As analyzed by qRT-PCR, expression of one lincRNA, which we termed virus inducible lincRNA (VIN), is induced by several IAV strains (H1N1, H3N2, H7N7) as well as vesicular stomatitis virus. However, we did not observe an induction of VIN by influenza B virus, treatment with RNA mimics, or IFNβ. Thus, VIN expression seems to be a specific response to certain viral infections. RNA fractionation and RNA-FISH experiments revealed that VIN is localized to the host cell nucleus. Most importantly, we show that abolition of VIN by RNA interference restricts IAV replication and viral protein synthesis, highlighting the relevance of this lincRNA for productive IAV infection. Our observations suggest that viral pathogens interfere with the non-coding portion of the human genome, thereby guaranteeing their successful propagation, and that the expression of VIN correlates with their virulence. Consequently, our study provides a novel approach for understanding virus pathogenesis in greater detail, which will enable future design of new antiviral strategies targeting the host’s non-protein coding genome.

Inflammation, Immunity, Vaccines for Helicobacter pylori infection

Over the last decades, it has become evident that chronic infection by Helicobacter pylori is achieved by colonizing an almost exclusive niche and hiding from many of the hosts cellular immune defense mechanisms. Although recent years have seen progress in our understanding of the innate and adaptive immune response against H. pylori, it is still uncertain how to promote the development of immunity with the final goal of a successful vaccine. Research published in the last year revealed an intriguing mutual regulation of innate response mechanisms of mucosal epithelial cells by the host and H. pylori, respectively. A further focus was put on the interaction between H. pylori and dendritic cells, with some emphasis on the inflammasome and the resulting T‐cell responses. Moreover, the function of microRNAs in macrophages and gastric MALT lymphoma development has been studied in more detail. Several novel antigens and adjuvants have been tested as vaccination strategies, primarily in mice. In this review, we present a concise summary of advances in the area of inflammation, immunity, and vaccines during the last twelve months.

Lipidomic profiling before and after Roux-en-Y gastric bypass in obese patients with diabetes

Bariatric surgery is a well-established approach to improve metabolic disease in morbidly obese patients with high cardiovascular risk. The post-operative normalization of lipid metabolism has a central role in the prevention of future cardiovascular events. The aim of the present study therefore was to characterize changes of plasma lipidomic patterns, consisting of 229 lipid species of 13 lipid classes, 3 months after Roux-en-Y gastric bypass (RYGB) in morbidly obese patients with and without diabetes. RYGB resulted in a 15–32% decrease of body mass index, which was associated with a significant reduction of total cholesterol (TC, −28.3%; P=0.02), LDL-cholesterol (LDL-C, −26.8%; P=0.03) and triglycerides (TGs, −63.0%; P=0.05) measured by routine clinical chemistry. HDL-cholesterol remained unchanged. The effect of RYGB on the plasma lipidomic profile was characterized by significant decreases of 87 lipid species from triacylglycerides (TAGs), cholesterol esters (CholEs), lysophosphatidylcholines (LPCs), phosphatidylcholines (PCs), phosphatidylethanolamine ethers (PEOs), phosphatidylinositols (PIs) and ceramides (Cers). The total of plasma lipid components exhibited a substantial decline of 32.6% and 66 lipid species showed a decrease by over 50%. A direct correlation with HbA1C values could be demonstrated for 24 individual lipid species (10 TAG, three CholE, two LPC, one lysophosphatidylcholine ethers (LPCO) (LPC ether), one PC, two phosphatidylcholine ethers (PCO) and five Cer). Notably, two lipid species (TAG 58:5 and PEO 40:5) were inversely correlated with HbA1C. LPCO, as single whole lipid class, was directly related to HbA1C. These data indicate that RYGB-induced modulation of lipidomic profiles provides important information about post-operative metabolic adaptations and might substantially contribute to improvements of glycemic control. These striking changes in the human plasma lipidome may explain acute, weight independent and long-term effects of RYGB on the cardiovascular system, mental status and immune regulation.

Macrophages recognize the Helicobacter pylori type IV secretion system in the absence of toll-like receptor signalling.

Helicobacter pylori strains carrying the cag pathogenicity island (cagPAI) provoke an increased inflammatory response, conferring an increased risk of ulcer formation and carcinogenesis. How the immune system recognizes the presence of cagPAI positive strains is yet unclear. By comparing the transcriptional response of wild type and MyD88/Trif–/– bone marrow macrophages to infection with H. pylori, we found that the majority of regulated genes were dependent on toll‐like receptor (TLR) signalling. To determine the role of TLR‐independent responses, we analysed the transcriptome of MyD88/Trif–/– bone marrow macrophages at different time points after infection with cagPAI positive versus negative strains. We identified a group of genes that exhibited different kinetic behaviour depending on whether cagPAI was present. Analysis of their gene expression kinetics demonstrated that this responsiveness to cagPAI was observed only in MyD88/Trif–/– macrophages. This group of cagPAI‐sensing genes was enriched for AU‐rich element containing early response genes involved in immune regulation, including interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α). Recognition of cagPAI positive strains was found to be mediated by the type IV secretion system (cagT4SS), rather than its effector protein CagA. We hypothesize that anergic macrophages of the gastric mucosa initiate an innate immune response following detection of the T4SS of H. pylori.

Macrophages recognize the Helicobacter pylori type IV secretion system in the absence of TLR signaling

Helicobacter pylori strains carrying the cag pathogenicity island (cagPAI) provoke an increased inflammatory response, conferring an increased risk of ulcer formation and carcinogenesis. How the immune system recognizes the presence of cagPAI positive strains is yet unclear. By comparing the transcriptional response of wild type and MyD88/Trif–/– bone marrow macrophages to infection with H. pylori, we found that the majority of regulated genes were dependent on toll‐like receptor (TLR) signalling. To determine the role of TLR‐independent responses, we analysed the transcriptome of MyD88/Trif–/– bone marrow macrophages at different time points after infection with cagPAI positive versus negative strains. We identified a group of genes that exhibited different kinetic behaviour depending on whether cagPAI was present. Analysis of their gene expression kinetics demonstrated that this responsiveness to cagPAI was observed only in MyD88/Trif–/– macrophages. This group of cagPAI‐sensing genes was enriched for AU‐rich element containing early response genes involved in immune regulation, including interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNF‐α). Recognition of cagPAI positive strains was found to be mediated by the type IV secretion system (cagT4SS), rather than its effector protein CagA. We hypothesize that anergic macrophages of the gastric mucosa initiate an innate immune response following detection of the T4SS of H. pylori.

Sensing the enemy: New role for a bacterial secretion system in activation of an innate immunity-associated microRNA

Both pathogenic and non-pathogenic microbes express common microbe-associated molecular patterns (MAMPs) that can activate innate pro-inflammatory responses. There is evidence to suggest that host immune systems are adapted to minimize unwarranted, potentially damaging pro-inflammatory responses, e.g., to commensals at muscosal surfaces where very high microbe loads are found, while maintaining the ability to mount aggressive responses to invading pathogens. The recognition of bacterial secretion systems by host innate immune pathways may represent one mechanism used by host immune cells to discriminate between pathogen and non-pathogen, or strains with reduced virulence potential. Most recently, it has been shown that the type IV secretion system (T4SS) encoded by virulent cagPAI-positive strains of the pro-carcinogenic pathogen Helicobacter pylori induces the expression of microRNA-155, a known mediator of innate immunity that is also implicated in oncogenesis. The T4SS-specific activation of miR-155 occurred independently of TLR and NOD1/2 pattern-recognition receptor (PRR) signaling. We discuss the potential role of the T4SS-dependent activation of miR-155 as a pathogen-specific immune response and the possible implications of this in the context of gastrointestinal macrophage inflammatory anergy, a phenomenon in which PRR signaling is inhibited in gastrointestinal resident phagocytes. We also touch on the observed anti-apoptotic role of miR-155 during H. pylori infection, and speculate as to its possible pathological consequences.

Genomic features of the Helicobacter pylori strain PMSS1 and its virulence attributes as deduced from its in vivo colonisation patterns: Virulence and genome of H. pylori PMSS1

The human gastric pathogen Helicobacter pylori occurs in two basic variants, either exhibiting a functional cagPAI‐encoded type‐4‐secretion‐system (T4SS) or not. Only a few cagPAI‐positive strains have been successfully adapted for long‐term infection of mice, including the pre‐mouse Sydney strain 1 (PMSS1). Here we confirm that PMSS1 induces gastric inflammation and neutrophil infiltration in mice, progressing to intestinal metaplasia. Complete genome analysis of PMSS1 revealed 1,423 coding sequences, encompassing the cagPAI gene cluster and, unusually, the location of the cytotoxin‐associated gene A (cagA) approximately 15 kb downstream of the island. PMSS1 harbours three genetically exchangeable loci that are occupied by the hopQ coding sequences. HopQ represents a critical co‐factor required for the translocation of CagA into the host cell and activation of NF‐κB via the T4SS. Long‐term colonisation of mice led to an impairment of cagPAI functionality. One of the bacterial clones re‐isolated at four months post‐infection revealed a mutation in the cagPAI gene cagW, resulting in a frame shift mutation, which prevented CagA translocation, possibly due to an impairment of T4SS function. Rescue of the mutant cagW re‐established CagA translocation. Our data reveal intriguing insights into the adaptive abilities of PMSS1, suggesting functional modulation of the H. pylori cagPAI virulence attribute.