Dissecting the metabolic signaling pathways by which microbial molecules drive the differentiation of regulatory B cells.

The host-microbiome axis has been implicated in promoting anti-inflammatory immune responses. Yet, the underlying molecular mechanisms of commensal-mediated IL-10 production by regulatory B cells (Bregs) are not fully elucidated. Here, we demonstrate that bacterial CpG motifs trigger the signaling downstream of TLR9 promoting IκBNS-mediated expression of Blimp-1, a transcription regulator of IL-10. Surprisingly, this effect was counteracted by the NF-κB transcription factor c-Rel. A functional screen for intestinal bacterial species identified the commensal Clostridium sporogenes, secreting high amounts of short-chain fatty acids (SCFAs) and branched-chain fatty acids (BCFAs), as an amplifier of IL-10 production by promoting sustained mTOR signaling in B cells. Consequently, enhanced Breg functionality was achieved by combining CpG with the SCFA butyrate or the BCFA isovalerate thereby synergizing TLR- and mTOR-mediated pathways. Collectively, Bregs required two bacterial signals (butyrate and CpG) to elicit their full suppressive capacity and ameliorate T cell-mediated intestinal inflammation. Our study has dissected the molecular pathways induced by bacterial factors, which might contribute not only to better understanding of host-microbiome interactions, but also to exploration of new strategies for improvement of anti-inflammatory cellular therapy.

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.

Biofilm formation on human immune cells is a multicellular predation strategy of Vibrio cholerae.

Biofilm formation is generally recognized as a bacterial defense mechanism against environmental threats, including antibiotics, bacteriophages, and leukocytes of the human immune system. Here, we show that for the human pathogen Vibrio cholerae, biofilm formation is not only a protective trait but also an aggressive trait to collectively predate different immune cells. We find that V. cholerae forms biofilms on the eukaryotic cell surface using an extracellular matrix comprising primarily mannose-sensitive hemagglutinin pili, toxin-coregulated pili, and the secreted colonization factor TcpF, which differs from the matrix composition of biofilms on other surfaces. These biofilms encase immune cells and establish a high local concentration of a secreted hemolysin to kill the immune cells before the biofilms disperse in a c-di-GMP-dependent manner. Together, these results uncover how bacteria employ biofilm formation as a multicellular strategy to invert the typical relationship between human immune cells as the hunters and bacteria as the hunted.

Bacterial vesicles block viral replication in macrophages via TLR4-TRIF-axis.

Gram-negative bacteria naturally secrete nano-sized outer membrane vesicles (OMVs), which are important mediators of communication and pathogenesis. OMV uptake by host cells activates TLR signalling via transported PAMPs. As important resident immune cells, alveolar macrophages are located at the air-tissue interface where they comprise the first line of defence against inhaled microorganisms and particles. To date, little is known about the interplay between alveolar macrophages and OMVs from pathogenic bacteria. The immune response to OMVs and underlying mechanisms are still elusive. Here, we investigated the response of primary human macrophages to bacterial vesicles (Legionella pneumophila, Klebsiella pneumoniae, Escherichia coli, Salmonella enterica, Streptococcus pneumoniae) and observed comparable NF-κB activation across all tested vesicles. In contrast, we describe differential type I IFN signalling with prolonged STAT1 phosphorylation and strong Mx1 induction, blocking influenza A virus replication only for Klebsiella, E.coli and Salmonella OMVs. OMV-induced antiviral effects were less pronounced for endotoxin-free Clear coli OMVs and Polymyxin-treated OMVs. LPS stimulation could not mimic this antiviral status, while TRIF knockout abrogated it. Importantly, supernatant from OMV-treated macrophages induced an antiviral response in alveolar epithelial cells (AEC), suggesting OMV-induced intercellular communication. Finally, results were validated in an ex vivo infection model with primary human lung tissue. In conclusion, Klebsiella, E.coli and Salmonella OMVs induce antiviral immunity in macrophages via TLR4-TRIF-signaling to reduce viral replication in macrophages, AECs and lung tissue. These gram-negative bacteria induce antiviral immunity in the lung through OMVs, with a potential decisive and tremendous impact on bacterial and viral coinfection outcome. Video Abstract.

Altered and allele-specific open chromatin landscape reveals epigenetic and genetic regulators of innate immunity in COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes severe COVID-19 in some patients and mild COVID-19 in others. Dysfunctional innate immune responses have been identified to contribute to COVID-19 severity, but the key regulators are still unknown. Here, we present an integrative single-cell multi-omics analysis of peripheral blood mononuclear cells from hospitalized and convalescent COVID-19 patients. In classical monocytes, we identified genes that were potentially regulated by differential chromatin accessibility. Then, sub-clustering and motif-enrichment analyses revealed disease condition-specific regulation by transcription factors and their targets, including an interaction between C/EBPs and a long-noncoding RNA LUCAT1, which we validated through loss-of-function experiments. Finally, we investigated genetic risk variants that exhibit allele-specific open chromatin (ASoC) in COVID-19 patients and identified a SNP rs6800484-C, which is associated with lower expression of CCR2 and may contribute to higher viral loads and higher risk of COVID-19 hospitalization. Altogether, our study highlights the diverse genetic and epigenetic regulators that contribute to COVID-19.

Single-cell RNA sequencing uncovers the nuclear decoy lincRNA PIRAT as a regulator of systemic monocyte immunity during COVID-19.

The systemic immune response to viral infection is shaped by master transcription factors, such as NF-κB, STAT1, or PU.1. Although long noncoding RNAs (lncRNAs) have been suggested as important regulators of transcription factor activity, their contributions to the systemic immunopathologies observed during SARS-CoV-2 infection have remained unknown. Here, we employed a targeted single-cell RNA sequencing approach to reveal lncRNAs differentially expressed in blood leukocytes during severe COVID-19. Our results uncover the lncRNA PIRAT (PU.1-induced regulator of alarmin transcription) as a major PU.1 feedback-regulator in monocytes, governing the production of the alarmins S100A8/A9, key drivers of COVID-19 pathogenesis. Knockout and transgene expression, combined with chromatin-occupancy profiling, characterized PIRAT as a nuclear decoy RNA, keeping PU.1 from binding to alarmin promoters and promoting its binding to pseudogenes in naïve monocytes. NF-κB-dependent PIRAT down-regulation during COVID-19 consequently releases a transcriptional brake, fueling alarmin production. Alarmin expression is additionally enhanced by the up-regulation of the lncRNA LUCAT1, which promotes NF-κB-dependent gene expression at the expense of targets of the JAK-STAT pathway. Our results suggest a major role of nuclear noncoding RNA networks in systemic antiviral responses to SARS-CoV-2 in humans.

Long noncoding RNAs in bacterial infection.

Infectious and inflammatory diseases remain major causes of mortality and morbidity worldwide. To combat bacterial infections, the mammalian immune system employs a myriad of regulators, which secure the effective initiation of inflammatory responses while preventing pathologies due to overshooting immunity. Recently, the human genome has been shown to be pervasively transcribed and to generate thousands of still poorly characterized long noncoding RNAs (lncRNAs). A growing body of literature suggests that lncRNAs play important roles in the regulatory circuitries controlling innate and adaptive immune responses to bacterial pathogens. This review provides an overview of the roles of lncRNAs in the interaction of human and rodent host cells with bacterial pathogens. Further decoding of the lncRNA networks that underlie pathological inflammation and immune subversion could provide new insights into the host cell mechanisms and microbial strategies that determine the outcome of bacterial infections. This article is categorized under: RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

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.

Efficient antisense inhibition reveals microRNA-155 to restrain a late-myeloid inflammatory programme in primary human phagocytes.

A persisting obstacle in human immunology is that blood-derived leukocytes are notoriously difficult to manipulate at the RNA level. Therefore, our knowledge about immune-regulatory RNA-networks is largely based on tumour cell-line and rodent knockout models, which do not fully mimic human leukocyte biology. Here, we exploit straightforward cell penetrating peptide (CPP) chemistry to enable efficient loss-of-function phenotyping of regulatory RNAs in primary human blood-derived cells. The classical CPP octaarginine (R8) enabled antisense peptide-nucleic-acid (PNA) oligomer delivery into nearly 100% of human blood-derived macrophages without apparent cytotoxicity even up to micromolar concentrations. In a proof-of-principle experiment, we successfully de-repressed the global microRNA-155 regulome in primary human macrophages using a PNA-R8 oligomer, which phenocopies a CRISPR-Cas9 induced gene knockout. Interestingly, although it is often believed that fairly high concentrations (µM) are needed to achieve antisense activity, our PNA-R8 was effective at 200 nM. RNA-seq characterized microRNA-155 as a broad-acting riboregulator, feedback restraining a late myeloid differentiation-induced pro-inflammatory network, comprising MyD88-signalling and ubiquitin-proteasome components. Our results highlight the important role of the microRNA machinery in fine-control of blood-derived human phagocyte immunity and open the door for further studies on regulatory RNAs in difficult-to-transfect primary human immune cells.