Clinically used broad-spectrum antibiotics compromise inflammatory monocyte-dependent antibacterial defense in the lung.

Hospital-acquired pneumonia (HAP) is associated with high mortality and costs, and frequently caused by multidrug-resistant (MDR) bacteria. Although prior antimicrobial therapy is a major risk factor for HAP, the underlying mechanism remains incompletely understood. Here, we demonstrate that antibiotic therapy in hospitalized patients is associated with decreased diversity of the gut microbiome and depletion of short-chain fatty acid (SCFA) producers. Infection experiments with mice transplanted with patient fecal material reveal that these antibiotic-induced microbiota perturbations impair pulmonary defense against MDR Klebsiella pneumoniae. This is dependent on inflammatory monocytes (IMs), whose fatty acid receptor (FFAR)2/3-controlled and phagolysosome-dependent antibacterial activity is compromized in mice transplanted with antibiotic-associated patient microbiota. Collectively, we characterize how clinically relevant antibiotics affect antimicrobial defense in the context of human microbiota, and reveal a critical impairment of IM´s antimicrobial activity. Our study provides additional arguments for the rational use of antibiotics and offers mechanistic insights for the development of novel prophylactic strategies to protect high-risk patients from HAP.

EGF and BMPs Govern Differentiation and Patterning in Human Gastric Glands.

BACKGROUND & AIMS: The homeostasis of the gastrointestinal epithelium relies on cell regeneration and differentiation into distinct lineages organized inside glands and crypts. Regeneration depends on Wnt/ß-catenin pathway activation, but to understand homeostasis and its dysregulation in disease, we need to identify the signaling microenvironment governing cell differentiation. By using gastric glands as a model, we have identified the signals inducing differentiation of surface mucus-, zymogen-, and gastric acid-producing cells. METHODS: We generated mucosoid cultures from the human stomach and exposed them to different growth factors to obtain cells with features of differentiated foveolar, chief, and parietal cells. We localized the source of the growth factors in the tissue of origin. RESULTS: We show that epidermal growth factor is the major fate determinant distinguishing the surface and inner part of human gastric glands. In combination with bone morphogenetic factor/Noggin signals, epidermal growth factor controls the differentiation of foveolar cells vs parietal or chief cells. We also show that epidermal growth factor is likely to underlie alteration of the gastric mucosa in the precancerous condition atrophic gastritis. CONCLUSIONS: Use of our recently established mucosoid cultures in combination with analysis of the tissue of origin provided a robust strategy to understand differentiation and patterning of human tissue and allowed us to draw a new, detailed map of the signaling microenvironment in the human gastric glands.

The ALPK1/TIFA/NF-κB axis links a bacterial carcinogen to R-loop-induced replication stress.

Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate ß-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of ß-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.

ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.

The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP-glycero-ß-D-manno-heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the ß-form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori-induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.

Helicobacter pylori Depletes Cholesterol in Gastric Glands to Prevent Interferon Gamma Signaling and Escape the Inflammatory Response.

BACKGROUND & AIMS: Despite inducing an inflammatory response, Helicobacter pylori can persist in the gastric mucosa for decades. H pylori expression of cholesterol-α-glucosyltransferase (encoded by cgt) is required for gastric colonization and T-cell activation. We investigated how cgt affects gastric epithelial cells and the host immune response. METHODS: MKN45 gastric epithelial cells, AGS cells, and human primary gastric epithelial cells (obtained from patients undergoing gastrectomy or sleeve resection or gastric antral organoids) were incubated with interferon gamma (IFNG) or interferon beta (IFNB) and exposed to H pylori, including cagPAI and cgt mutant strains. Some cells were incubated with methyl-ß-cyclodextrin (to deplete cholesterol from membranes) or myriocin and zaragozic acid to prevent biosynthesis of sphingolipids and cholesterol and analyzed by immunoblot, immunofluorescence, and reverse transcription quantitative polymerase chain reaction analyses. We compared gene expression patterns among primary human gastric cells, uninfected or infected with H pylori P12 wt or P12Δcgt, using microarray analysis. Mice with disruption of the IFNG receptor 1 (Ifngr1-/- mice) and C57BL6 (control) mice were infected with PMSS1 (wild-type) or PMSS1Δcgt H pylori; gastric tissues were collected and analyzed by reverse transcription quantitative polymerase chain reaction or confocal microscopy. RESULTS: In primary gastric cells and cell lines, infection with H pylori, but not cgt mutants, blocked IFNG-induced signaling via JAK and STAT. Cells infected with H pylori were depleted of cholesterol, which reduced IFNG signaling by disrupting lipid rafts, leading to reduced phosphorylation (activation) of JAK and STAT1. H pylori infection of cells also blocked signaling by IFNB, interleukin 6 (IL6), and IL22 and reduced activation of genes regulated by these signaling pathways, including cytokines that regulate T-cell function (MIG and IP10) and anti-microbial peptides such as human ß-defensin 3 (hBD3). We found that this mechanism allows H pylori to persist in proximity to infected cells while inducing inflammation only in the neighboring, non-infected epithelium. Stomach tissues from mice infected with PMSS1 had increased levels of IFNG, but did not express higher levels of interferon-response genes. Expression of the IFNG-response gene IRF1 was substantially higher in PMSS1Δcgt-infected mice than PMSS1-infected mice. Ifngr1-/- mice were colonized by PMSS1 to a greater extent than control mice. CONCLUSIONS: H pylori expression of cgt reduces cholesterol levels in infected gastric epithelial cells and thereby blocks IFNG signaling, allowing the bacteria to escape the host inflammatory response. These findings provide insight into the mechanisms by which H pylori might promote gastric carcinogenesis (persisting despite constant inflammation) and ineffectiveness of T-cell-based vaccines against H pylori.

ALPK1- and TIFA-Dependent Innate Immune Response Triggered by the Helicobacter pylori Type IV Secretion System.

Activation of transcription factor NF-κB is a hallmark of infection with the gastric pathogen Helicobacter pylori, associated with inflammation and carcinogenesis. Genome-wide RNAi screening revealed numerous host factors involved in H. pylori-, but not IL-1ß- and TNF-α-dependent NF-κB regulation. Pathway analysis including CRISPR/Cas9-knockout and recombinant protein technology, immunofluorescence microscopy, immunoblotting, mass spectrometry, and mutant H. pylori strains identified the H. pylori metabolite D-glycero-ß-D-manno-heptose 1,7-bisphosphate (ßHBP) as a cagPAI type IV secretion system (T4SS)-dependent effector of NF-κB activation in infected cells. Upon pathogen-host cell contact, TIFA forms large complexes (TIFAsomes) including interacting host factors, such as TRAF2. NF-κB activation, TIFA phosphorylation, and TIFAsome formation depend on a functional ALPK1 kinase, highlighting the ALPK1-TIFA axis as a core innate immune pathway. ALPK1-TIFA-mediated NF-κB activation was independent of CagA protein translocation, indicating that CagA translocation and HBP delivery to host cells are distinct features of the pathogen's T4SS.