Decoding spatiotemporal transcriptional dynamics and epithelial fibroblast crosstalk during gastroesophageal junction development through single cell analysis.

The gastroesophageal squamocolumnar junction (GE-SCJ) is a critical tissue interface between the esophagus and stomach, with significant relevance in the pathophysiology of gastrointestinal diseases. Despite this, the molecular mechanisms underlying GE-SCJ development remain unclear. Using single-cell transcriptomics, organoids, and spatial analysis, we examine the cellular heterogeneity and spatiotemporal dynamics of GE-SCJ development from embryonic to adult mice. We identify distinct transcriptional states and signaling pathways in the epithelial and mesenchymal compartments of the esophagus and stomach during development. Fibroblast-epithelial interactions are mediated by various signaling pathways, including WNT, BMP, TGF-ß, FGF, EGF, and PDGF. Our results suggest that fibroblasts predominantly send FGF and TGF-ß signals to the epithelia, while epithelial cells mainly send PDGF and EGF signals to fibroblasts. We observe differences in the ligands and receptors involved in cell-cell communication between the esophagus and stomach. Our findings provide insights into the molecular mechanisms underlying GE-SCJ development and fibroblast-epithelial crosstalk involved, paving the way to elucidate mechanisms during adaptive metaplasia development and carcinogenesis.

R-spondin/YAP axis promotes gastric oxyntic gland regeneration and Helicobacter pylori-associated metaplasia in mice.

The stomach corpus epithelium is organized into anatomical units that consist of glands and pits. Mechanisms that control the cellular organization of corpus glands and enable their recovery upon injury are not well understood. R-spondin 3 (RSPO3) is a WNT-signaling enhancer that regulates stem cell behavior in different organs. Here, we investigated the function of RSPO3 in the corpus during homeostasis, upon chief and/or parietal cell loss, and during chronic Helicobacter pylori infection. Using organoid culture and conditional mouse models, we demonstrate that RSPO3 is a critical driver of secretory cell differentiation in the corpus gland toward parietal and chief cells, while its absence promoted pit cell differentiation. Acute loss of chief and parietal cells induced by high dose tamoxifen - or merely the depletion of LGR5+ chief cells - caused an upregulation of RSPO3 expression, which was required for the initiation of a coordinated regenerative response via the activation of yes-associated protein (YAP) signaling. This response enabled a rapid recovery of the injured secretory gland cells. However, in the context of chronic H. pylori infection, the R-spondin-driven regeneration was maintained long term, promoting severe glandular hyperproliferation and the development of premalignant metaplasia.

BMP feed-forward loop promotes terminal differentiation in gastric glands and is interrupted by H. pylori-driven inflammation.

Helicobacter pylori causes gastric inflammation, gland hyperplasia and is linked to gastric cancer. Here, we studied the interplay between gastric epithelial stem cells and their stromal niche under homeostasis and upon H. pylori infection. We find that gastric epithelial stem cell differentiation is orchestrated by subsets of stromal cells that either produce BMP inhibitors in the gland base, or BMP ligands at the surface. Exposure to BMP ligands promotes a feed-forward loop by inducing Bmp2 expression in the epithelial cells themselves, enforcing rapid lineage commitment to terminally differentiated mucous pit cells. H. pylori leads to a loss of stromal and epithelial Bmp2 expression and increases expression of BMP inhibitors, promoting self-renewal of stem cells and accumulation of gland base cells, which we mechanistically link to IFN-γ signaling. Mice that lack IFN-γ signaling show no alterations of BMP gradient upon infection, while exposure to IFN-γ resembles H. pylori-driven mucosal responses.

Modelling Chlamydia and HPV co-infection in patient-derived ectocervix organoids reveals distinct cellular reprogramming.

Coinfections with pathogenic microbes continually confront cervical mucosa, yet their implications in pathogenesis remain unclear. Lack of in-vitro models recapitulating cervical epithelium has been a bottleneck to study coinfections. Using patient-derived ectocervical organoids, we systematically modeled individual and coinfection dynamics of Human papillomavirus (HPV)16 E6E7 and Chlamydia, associated with carcinogenesis. The ectocervical stem cells were genetically manipulated to introduce E6E7 oncogenes to mimic HPV16 integration. Organoids from these stem cells develop the characteristics of precancerous lesions while retaining the self-renewal capacity and organize into mature stratified epithelium similar to healthy organoids. HPV16 E6E7 interferes with Chlamydia development and induces persistence. Unique transcriptional and post-translational responses induced by Chlamydia and HPV lead to distinct reprogramming of host cell processes. Strikingly, Chlamydia impedes HPV-induced mechanisms that maintain cellular and genome integrity, including mismatch repair in the stem cells. Together, our study employing organoids demonstrates the hazard of multiple infections and the unique cellular microenvironment they create, potentially contributing to neoplastic progression.

Innate-like Gene Expression of Lung-Resident Memory CD8+ T Cells during Experimental Human Influenza: A Clinical Study.

Rationale: Suboptimal vaccine immunogenicity and antigenic mismatch, compounded by poor uptake, means that influenza remains a major global disease. T cells recognizing peptides derived from conserved viral proteins could enhance vaccine-induced cross-strain protection. Objectives: To investigate the kinetics, phenotypes, and function of influenza virus-specific CD8+ resident memory T (Trm) cells in the lower airway and infer the molecular pathways associated with their response to infection in vivo. Methods: Healthy volunteers, aged 18-55, were inoculated intranasally with influenza A/California/4/09(H1N1). Blood, upper airway, and (in a subgroup) lower airway samples were obtained throughout infection. Symptoms were assessed by using self-reported diaries, and the nasal viral load was assessed by using quantitative PCR. T-cell responses were analyzed by using a three-color FluoroSpot assay, flow cytometry with MHC I-peptide tetramers, and RNA sequencing, with candidate markers being confirmed by using the immunohistochemistry results for endobronchial biopsy specimens. Measurements and Main Results: After challenge, 57% of participants became infected. Preexisting influenza-specific CD8+ T cells in blood correlated strongly with a reduced viral load, which peaked at Day 3. Influenza-specific CD8+ T cells in BAL fluid were highly enriched and predominantly expressed the Trm markers CD69 and CD103. Comparison between preinfection CD8+ T cells in BAL fluid and blood by using RNA sequencing revealed 3,928 differentially expressed genes, including all major Trm-cell markers. However, gene set enrichment analysis of BAL-fluid CD8+ T cells showed primarily innate cell-related pathways and, during infection, included upregulation of innate chemokines (Cxcl1, Cxcl10, and Cxcl16) that were also expressed by CD8+ cells in bronchial tissues. Conclusions: CD8+ Trm cells in the human lung display innate-like gene and protein expression that demonstrates blurred divisions between innate and adaptive immunity. Clinical study registered with www.clinicaltrials.gov (NCT02755948).

Cellular stress promotes NOD1/2-dependent inflammation via the endogenous metabolite sphingosine-1-phosphate.

Cellular stress has been associated with inflammation, yet precise underlying mechanisms remain elusive. In this study, various unrelated stress inducers were employed to screen for sensors linking altered cellular homeostasis and inflammation. We identified the intracellular pattern recognition receptors NOD1/2, which sense bacterial peptidoglycans, as general stress sensors detecting perturbations of cellular homeostasis. NOD1/2 activation upon such perturbations required generation of the endogenous metabolite sphingosine-1-phosphate (S1P). Unlike peptidoglycan sensing via the leucine-rich repeats domain, cytosolic S1P directly bound to the nucleotide binding domains of NOD1/2, triggering NF-κB activation and inflammatory responses. In sum, we unveiled a hitherto unknown role of NOD1/2 in surveillance of cellular homeostasis through sensing of the cytosolic metabolite S1P. We propose S1P, an endogenous metabolite, as a novel NOD1/2 activator and NOD1/2 as molecular hubs integrating bacterial and metabolic cues.

Lipid Storage and Interferon Response Determine the Phenotype of Ground Glass Hepatocytes in Mice and Humans.

BACKGROUND AND AIMS: A histopathological hallmark of chronic hepatitis B virus (HBV) infection is the presence of ground glass hepatocytes (GGHs). GGHs are liver cells that exhibit eosinophilic, granular, glassy cytoplasm in light microscopy and are characterized by accumulation of HBV surface (HBs) proteins in the endoplasmic reticulum (ER). More important, GGHs have been accepted as a precursor of HCC and may represent preneoplastic lesions of the liver. METHODS: Here we show that the reason for ground glass phenotype of hepatocytes in patients with chronic hepatitis B (CHB) and in HBs transgenic mice is a complex formation between HBs proteins and lipid droplets (LDs) within the ER. RESULTS: As fat is a main component of LDs their presence reduces the protein density of HBs aggregates. Therefore, they adsorb less amount of eosin during hematoxylin-eosin staining and appear dull in light microscopy. However, after induction of interferon response in the liver LDs were not only co-localized with HBs but also distributed throughout the cytoplasm of hepatocytes. The uniform distribution of LDs weakens the contrast between HBs aggregates and the rest of the cytoplasm and complicates the identification of GGHs. Suppression of interferon response restored the ground glass phenotype of hepatocytes. CONCLUSIONS: Complex formation between HBs and LDs represents a very important feature of CHB that could affect LDs functions in hepatocytes. The strain specific activation of the interferon response in the liver of HBs/c mice prevented the development of GGHs. Thus, manipulation of LDs could provide a new treatment strategy in the prevention of liver cancer.

Pro- and Antitumorigenic Capacity of Immunoproteasomes in Shaping the Tumor Microenvironment.

Apart from the constitutive proteasome, the immunoproteasome that comprises the three proteolytic subunits LMP2, MECL-1, and LMP7 is expressed in most immune cells. In this study, we describe opposing roles for immunoproteasomes in regulating the tumor microenvironment (TME). During chronic inflammation, immunoproteasomes modulated the expression of protumorigenic cytokines and chemokines and enhanced infiltration of innate immune cells, thus triggering the onset of colitis-associated carcinogenesis (CAC) in wild-type mice. Consequently, immunoproteasome-deficient animals (LMP2/MECL-1/LMP7-null mice) were almost completely resistant to CAC development. In patients with ulcerative colitis with high risk for CAC, immunoproteasome-induced protumorigenic mediators were upregulated. In melanoma tumors, the role of immunoproteasomes is relatively unknown. We found that high expression of immunoproteasomes in human melanoma was associated with better prognosis. Similarly, our data revealed that the immunoproteasome has antitumorigenic activity in a mouse model of melanoma. The antitumor immunity against melanoma was compromised in immunoproteasome-deficient mice because of the impaired activity of CD8+ CTLs, CD4+ Th1 cells, and antigen-presenting cells. These findings show that immunoproteasomes may exert opposing roles with either pro- or antitumoral properties in a context-dependent manner.

Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia.

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages.

Stable expansion of high-grade serous ovarian cancer organoids requires a low-Wnt environment.

High-grade serous ovarian cancer (HGSOC) likely originates from the fallopian tube (FT) epithelium. Here, we established 15 organoid lines from HGSOC primary tumor deposits that closely match the mutational profile and phenotype of the parental tumor. We found that Wnt pathway activation leads to growth arrest of these cancer organoids. Moreover, active BMP signaling is almost always required for the generation of HGSOC organoids, while healthy fallopian tube organoids depend on BMP suppression by Noggin. Fallopian tube organoids modified by stable shRNA knockdown of p53, PTEN, and retinoblastoma protein (RB) also require a low-Wnt environment for long-term growth, while fallopian tube organoid medium triggers growth arrest. Thus, early changes in the stem cell niche environment are needed to support outgrowth of these genetically altered cells. Indeed, comparative analysis of gene expression pattern and phenotypes of normal vs. loss-of-function organoids confirmed that depletion of tumor suppressors triggers changes in the regulation of stemness and differentiation.

Systematic Evaluation of Kinetics and Distribution of Muscle and Lymph Node Activation Measured by 18F-FDG- and 11C-PBR28-PET/CT Imaging, and Whole Blood and Muscle Transcriptomics After Immunization of Healthy Humans With Adjuvanted and Unadjuvanted Vaccines.

Systems vaccinology has been applied to detect signatures of human vaccine induced immunity but its ability, together with high definition in vivo clinical imaging is not established to predict vaccine reactogenicity. Within two European Commission funded high impact programs, BIOVACSAFE and ADITEC, we applied high resolution positron emission tomography/computed tomography (PET/CT) scanning using tissue-specific and non-specific radioligands together with transcriptomic analysis of muscle biopsies in a clinical model systematically and prospectively comparing vaccine-induced immune/inflammatory responses. 109 male participants received a single immunization with licensed preparations of either AS04-adjuvanted hepatitis B virus vaccine (AHBVV); MF59C-adjuvanted (ATIV) or unadjuvanted seasonal trivalent influenza vaccine (STIV); or alum-OMV-meningococcal B protein vaccine (4CMenB), followed by a PET/CT scan (n = 54) or an injection site muscle biopsy (n = 45). Characteristic kinetics was observed with a localized intramuscular focus associated with increased tissue glycolysis at the site of immunization detected by 18F-fluorodeoxyglucose (FDG) PET/CT, peaking after 1-3 days and strongest and most prolonged after 4CMenB, which correlated with clinical experience. Draining lymph node activation peaked between days 3-5 and was most prominent after ATIV. Well defined uptake of the immune cell-binding radioligand 11C-PBR28 was observed in muscle lesions and draining lymph nodes. Kinetics of muscle gene expression module upregulation reflected those seen previously in preclinical models with a very early (~6hrs) upregulation of monocyte-, TLR- and cytokine/chemokine-associated modules after AHBVV, in contrast to a response on day 3 after ATIV, which was bracketed by whole blood responses on day 1 as antigen presenting, inflammatory and innate immune cells trafficked to the site of immunization, and on day 5 associated with activated CD4+ T cells. These observations confirm the use of PET/CT, including potentially tissue-, cell-, or cytokine/chemokine-specific radioligands, is a safe and ethical quantitative technique to compare candidate vaccine formulations and could be safely combined with biopsy to guide efficient collection of samples for integrated whole blood and tissue systems vaccinology in small-scale but intensive human clinical models of immunization and to accelerate clinical development and optimisation of vaccine candidates, adjuvants, and formulations.

Mycofactocin Is Associated with Ethanol Metabolism in Mycobacteria.

Mycofactocin (MFT) belongs to the class of ribosomally synthesized and posttranslationally modified peptides conserved in many ActinobacteriaMycobacterium tuberculosis assimilates cholesterol during chronic infection, and its in vitro growth in the presence of cholesterol requires most of the MFT biosynthesis genes (mftA, mftB, mftC, mftD, mftE, and mftF), although the reasons for this requirement remain unclear. To identify the function of MFT, we characterized MFT biosynthesis mutants constructed in Mycobacterium smegmatis, M. marinum, and M. tuberculosis We found that the growth deficit of mft deletion mutants in medium containing cholesterol-a phenotypic basis for gene essentiality prediction-depends on ethanol, a solvent used to solubilize cholesterol. Furthermore, functionality of MFT was strictly required for growth of free-living mycobacteria in ethanol and other primary alcohols. Among other genes encoding predicted MFT-associated dehydrogenases, MSMEG_6242 was indispensable for M. smegmatis ethanol assimilation, suggesting that it is a candidate catalytic interactor with MFT. Despite being a poor growth substrate, ethanol treatment resulted in a reductive cellular state with NADH accumulation in M. tuberculosis During ethanol treatment, mftC mutant expressed the transcriptional signatures that are characteristic of respirational dysfunction and a redox-imbalanced cellular state. Counterintuitively, there were no differences in cellular bioenergetics and redox parameters in mftC mutant cells treated with ethanol. Therefore, further understanding of the function of MFT in ethanol metabolism is required to identify the cause of growth retardation of MFT mutants in cholesterol. Nevertheless, our results establish the physiological role of MFT and also provide new insights into the specific functions of MFT homologs in other actinobacterial systems.IMPORTANCE Tuberculosis is caused by Mycobacterium tuberculosis, and the increasing emergence of multidrug-resistant strains renders current treatment options ineffective. Although new antimycobacterial drugs are urgently required, their successful development often relies on complete understanding of the metabolic pathways-e.g., cholesterol assimilation-that are critical for persistence and for pathogenesis of M. tuberculosis In this regard, mycofactocin (MFT) function appears to be important because its biosynthesis genes are predicted to be essential for M. tuberculosisin vitro growth in cholesterol. In determining the metabolic basis of this genetic requirement, our results unexpectedly revealed the essential function of MFT in ethanol metabolism. The metabolic dysfunction thereof was found to affect the mycobacterial growth in cholesterol which is solubilized by ethanol. This knowledge is fundamental in recognizing the bona fide function of MFT, which likely resembles the pyrroloquinoline quinone-dependent ethanol oxidation in acetic acid bacteria exploited for industrial production of vinegar.

Chronic Chlamydia infection in human organoids increases stemness and promotes age-dependent CpG methylation.

Chronic infections of the fallopian tubes with Chlamydia trachomatis (Ctr) cause scarring and can lead to infertility. Here we use human fallopian tube organoids and genital Ctr serovars D, K and E for long-term in vitro analysis. The epithelial monolayer responds with active expulsion of the bacteria into the lumen and with compensatory cellular proliferation-demonstrating a role of epithelial homeostasis in the defense against this pathogen. In addition, Ctr infection activates LIF signaling, which we find to be an essential regulator of stemness in the organoids. Infected organoids exhibit a less differentiated phenotype with higher stemness potential, as confirmed by increased organoid forming efficiency. Moreover, Ctr increases hypermethylation of DNA, which is an indicator of accelerated molecular aging. Thus, the chronic organoid infection model suggests that Ctr has a long-term impact on the epithelium. These heritable changes might be a contributing factor in the development of tubal pathologies, including the initiation of high grade serous ovarian cancer.

cGAS facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity.

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

Long-Term Culture of Distal Airway Epithelial Cells Allows Differentiation Towards Alveolar Epithelial Cells Suited for Influenza Virus Studies.

As the target organ for numerous pathogens, the lung epithelium exerts critical functions in health and disease. However, research in this area has been hampered by the quiescence of the alveolar epithelium under standard culture conditions. Here, we used human distal airway epithelial cells (DAECs) to generate alveolar epithelial cells. Long-term, robust growth of human DAECs was achieved using co-culture with feeder cells and supplementation with epidermal growth factor (EGF), Rho-associated protein kinase inhibitor Y27632, and the Notch pathway inhibitor dibenzazepine (DBZ). Removal of feeders and priming with DBZ and a cocktail of lung maturation factors prevented the spontaneous differentiation into airway club cells and instead induced differentiation to alveolar epithelial cells. We successfully transferred this approach to chicken distal airway cells, thus generating a zoonotic infection model that enables studies on influenza A virus replication. These cells are also amenable for gene knockdown using RNAi technology, indicating the suitability of the model for mechanistic studies into lung function and disease.

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.

Stromal R-spondin orchestrates gastric epithelial stem cells and gland homeostasis.

The constant regeneration of stomach epithelium is driven by long-lived stem cells, but the mechanism that regulates their turnover is not well understood. We have recently found that the gastric pathogen Helicobacter pylori can activate gastric stem cells and increase epithelial turnover, while Wnt signalling is known to be important for stem cell identity and epithelial regeneration in several tissues. Here we find that antral Wnt signalling, marked by the classic Wnt target gene Axin2, is limited to the base and lower isthmus of gastric glands, where the stem cells reside. Axin2 is expressed by Lgr5+ cells, as well as adjacent, highly proliferative Lgr5- cells that are able to repopulate entire glands, including the base, upon depletion of the Lgr5+ population. Expression of both Axin2 and Lgr5 requires stroma-derived R-spondin 3 produced by gastric myofibroblasts proximal to the stem cell compartment. Exogenous R-spondin administration expands and accelerates proliferation of Axin2+/Lgr5- but not Lgr5+ cells. Consistent with these observations, H. pylori infection increases stromal R-spondin 3 expression and expands the Axin2+ cell pool to cause hyperproliferation and gland hyperplasia. The ability of stromal niche cells to control and adapt epithelial stem cell dynamics constitutes a sophisticated mechanism that orchestrates epithelial regeneration and maintenance of tissue integrity.

Inhibitors of Apoptosis Protein Antagonists (Smac Mimetic Compounds) Control Polarization of Macrophages during Microbial Challenge and Sterile Inflammatory Responses.

Apoptosis is a physiological cell death process essential for development, tissue homeostasis, and for immune defense of multicellular animals. Inhibitors of apoptosis proteins (IAPs) regulate apoptosis in response to various cellular assaults. Using both genetic and pharmacological approaches we demonstrate here that the IAPs not only support opportunistic survival of intracellular human pathogens like Chlamydia pneumoniae but also control plasticity of iNOS+ M1 macrophage during the course of infection and render them refractory for immune stimulation. Treatment of Th1 primed macrophages with birinapant (IAP-specific antagonist) inhibited NO generation and relevant proteins involved in innate immune signaling. Accordingly, birinapant promoted hypoxia, angiogenesis, and tumor-induced M2 polarization of iNOS+ M1 macrophages. Interestingly, birinapant-driven changes in immune signaling were accompanied with changes in the expression of various proteins involved in the metabolism, and thus revealing the new role of IAPs in immune metabolic reprogramming in committed macrophages. Taken together, our study reveals the significance of IAP targeting approaches (Smac mimetic compounds) for the management of infectious and inflammatory diseases relying on macrophage plasticity.

Propionibacterium acnes inhibits FOXM1 and induces cell cycle alterations in human primary prostate cells.

Propionibacterium acnes has been detected in diseased human prostate tissue, and cell culture experiments suggest that the bacterium can establish a low-grade inflammation. Here, we investigated its impact on human primary prostate epithelial cells. Microarray analysis confirmed the inflammation-inducing capability of P. acnes but also showed deregulation of genes involved in the cell cycle. qPCR experiments showed that viable P. acnes downregulates a master regulator of cell cycle progression, FOXM1. Flow cytometry experiments revealed that P. acnes increases the number of cells in S-phase. We tested the hypothesis that a P. acnes-produced berninamycin-like thiopeptide is responsible for this effect, since it is related to the FOXM1 inhibitor siomycin. The thiopeptide biosynthesis gene cluster was strongly expressed; it is present in subtype IB of P. acnes, but absent from type IA, which is most abundant on human skin. A knock-out mutant lacking the gene encoding the berninamycin-like peptide precursor was unable to downregulate FOXM1 and to halt the cell cycle. Our study reveals a novel host cell-interacting activity of P. acnes.

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-1ß (IL-1ß) 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.