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.

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.

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.

Noncoding RNA MaIL1 is an integral component of the TLR4-TRIF pathway.

RNA has been proposed as an important scaffolding factor in the nucleus, aiding protein complex assembly in the dense intracellular milieu. Architectural contributions of RNA to cytosolic signaling pathways, however, remain largely unknown. Here, we devised a multidimensional gradient approach, which systematically locates RNA components within cellular protein networks. Among a subset of noncoding RNAs (ncRNAs) cosedimenting with the ubiquitin-proteasome system, our approach unveiled ncRNA MaIL1 as a critical structural component of the Toll-like receptor 4 (TLR4) immune signal transduction pathway. RNA affinity antisense purification-mass spectrometry (RAP-MS) revealed MaIL1 binding to optineurin (OPTN), a ubiquitin-adapter platforming TBK1 kinase. MaIL1 binding stabilized OPTN, and consequently, loss of MaIL1 blunted OPTN aggregation, TBK1-dependent IRF3 phosphorylation, and type I interferon (IFN) gene transcription downstream of TLR4. MaIL1 expression was elevated in patients with active pulmonary infection and was highly correlated with IFN levels in bronchoalveolar lavage fluid. Our study uncovers MaIL1 as an integral RNA component of the TLR4-TRIF pathway and predicts further RNAs to be required for assembly and progression of cytosolic signaling networks in mammalian cells.

Dual RNA-seq unveils noncoding RNA functions in host-pathogen interactions.

Bacteria express many small RNAs for which the regulatory roles in pathogenesis have remained poorly understood due to a paucity of robust phenotypes in standard virulence assays. Here we use a generic 'dual RNA-seq' approach to profile RNA expression simultaneously in pathogen and host during Salmonella enterica serovar Typhimurium infection and reveal the molecular impact of bacterial riboregulators. We identify a PhoP-activated small RNA, PinT, which upon bacterial internalization temporally controls the expression of both invasion-associated effectors and virulence genes required for intracellular survival. This riboregulatory activity causes pervasive changes in coding and noncoding transcripts of the host. Interspecies correlation analysis links PinT to host cell JAK-STAT signalling, and we identify infection-specific alterations in multiple long noncoding RNAs. Our study provides a paradigm for a sensitive RNA-based analysis of intracellular bacterial pathogens and their hosts without physical separation, as well as a new discovery route for hidden functions of pathogen genes.

A ribosomal RNA fragment with 2',3'-cyclic phosphate and GTP-binding activity acts as RIG-I ligand.

The RNA helicase RIG-I plays a key role in sensing pathogen-derived RNA. Double-stranded RNA structures bearing 5'-tri- or diphosphates are commonly referred to as activating RIG-I ligands. However, endogenous RNA fragments generated during viral infection via RNase L also activate RIG-I. Of note, RNase-digested RNA fragments bear a 5'-hydroxyl group and a 2',3'-cyclic phosphate. How endogenous RNA fragments activate RIG-I despite the lack of 5'-phosphorylation has not been elucidated. Here we describe an endogenous RIG-I ligand (eRL) that is derived from the internal transcribed spacer 2 region (ITS2) of the 45S ribosomal RNA after partial RNase A digestion in vitro, RNase A protein transfection or RNase L activation. The immunostimulatory property of the eRL is dependent on 2',3'-cyclic phosphate and its sequence is characterized by a G-quadruplex containing sequence motif mediating guanosine-5'-triphosphate (GTP) binding. In summary, RNase generated self-RNA fragments with 2',3'-cyclic phosphate function as nucleotide-5'-triphosphate binding aptamers activating RIG-I.

Influenza virus-mediated suppression of bronchial Chitinase-3-like 1 secretion promotes secondary pneumococcal infection.

Infections of the lung are among the leading causes of death worldwide. Despite the preactivation of innate defense programs during viral infection, secondary bacterial infection substantially elevates morbidity and mortality rates. Particularly problematic are co-infections with influenza A virus (IAV) and the major bacterial pathogen Streptococcus pneumoniae. However, the molecular processes underlying the severe course of such co-infections are not fully understood. Previously, the absence of secreted glycoprotein Chitinase-3-like 1 (CHI3L1) was shown to increase pneumococcal replication in mice. We therefore hypothesized that an IAV preinfection decreases CHI3L1 levels to promote pneumococcal infection. Indeed, in an air-liquid interface model of primary human bronchial epithelial cells (hBECs), IAV preinfection interfered with apical but not basolateral CHI3L1 release. Confocal time-lapse microscopy revealed that the gradual loss of apical CHI3L1 localization during co-infection with influenza and S. pneumoniae coincided with the disappearance of goblet as well as ciliated cells and increased S. pneumoniae replication. Importantly, extracellular restoration of CHI3L1 levels using recombinant protein significantly reduced bacterial load in influenza preinfected bronchial models. Thus, recombinant CHI3L1 may provide a novel therapeutic means to lower morbidity and mortality associated with post-influenza pneumococcal infections.

The role of lncRNAs in innate immunity and inflammation.

The innate immune system relies on a germ-line-encoded repertoire of pattern recognition receptors (PRRs), activated by deeply conserved pathogen signatures, such as bacterial cell wall components or foreign nucleic acids. To enable effective defence against invading pathogens and prevent from deleterious inflammation, PRR-driven immune responses are tightly controlled by a dense network of nuclear and cytoplasmic regulators. Long non-coding RNAs (lncRNAs) are increasingly recognized as important components of these regulatory circuitries, providing positive and negative control of PRR-induced innate immune responses. The present review provides an overview of the presently known roles of lncRNAs in human and murine innate antiviral and antibacterial immunity. The emerging roles in host defence and inflammation suggest that further mechanistic insights into the cellular functions of lncRNAs will decisively advance our molecular understanding of immune-associated diseases and open new avenues for therapeutic intervention.

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.

Proviral MicroRNAs Detected in Extracellular Vesicles From Bronchoalveolar Lavage Fluid of Patients With Influenza Virus-Induced Acute Respiratory Distress Syndrome.

Influenza A virus (IAV) causes severe respiratory infections and alveolar epithelial damage resulting in acute respiratory distress syndrome (ARDS). Extracellular vesicles (EVs) have been shown to mediate cellular crosstalk in inflammation by transfer of microRNAs (miRNAs). In this study, we found significant changes in the miRNA composition of EVs in the bronchoalveolar lavage fluid from patients with IAV-induced ARDS. Among the 9 significantly deregulated microRNAs, miR-17-5p was upregulated in patients' BALF and in EVs of IAV-infected lung epithelial cells (A549). In these cells, transfer of miR-17-5p strongly downregulated expression of the antiviral factor Mx1 and significantly enhanced IAV replication.

A Far-Red Fluorescent DNA Binder for Interaction Studies of Live Multidrug-Resistant Pathogens and Host Cells.

Transgene expression of green fluorescent protein (GFP) has facilitated the spatiotemporal investigation of host-pathogen interactions; however, introduction of the GFP gene remains challenging in drug-resistant bacteria. Herein, we report a novel far-red fluorescent nucleic acid stain, 6-TramTO-3, which efficiently labels bacteria through a DNA binding mode without affecting growth and viability. Exemplarily, we stained Klebsiella pneumoniae, a major threat to hospitalized patients, and deciphered divergent interaction strategies of antibiotic-resistant and antibiotic-sensitive Klebsiella strains with immune cells. 6-TramTO-3 constitutes an off-the-shelf reagent for real-time analysis of bacterial infection, including strains for which the use of genetically encoded reporters is not feasible. Eventually, our approach may aid the development of strategies to combat a major worldwide health threat: multidrug-resistant bacteria.

Analysis of the host microRNA response to Salmonella uncovers the control of major cytokines by the let-7 family.

MicroRNAs have well-established roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. In contrast, microRNA responses to invasive bacteria have remained unknown. Here, we report cell type-dependent microRNA regulations upon infection of mammalian cells with the enteroinvasive pathogen, Salmonella Typhimurium. Murine macrophages strongly upregulate NF-κB associated microRNAs; strikingly, these regulations which are induced by bacterial lipopolysaccharide (LPS) occur and persist regardless of successful host invasion and/or replication, or whether an inflammatory response is mounted, suggesting that microRNAs belong to the first line of anti-bacterial defence. However, a suppression of the global immune regulator miR-155 in endotoxin-tolerant macrophages revealed that microRNA responses also depend on the status of infected cells. This study identifies the let-7 family as the common denominator of Salmonella-regulated microRNAs in macrophages and epithelial cells, and suggests that repression of let-7 relieves cytokine IL-6 and IL-10 mRNAs from negative post-transcriptional control. Our results establish a paradigm of microRNA-mediated feed-forward activation of inflammatory factors when mammalian cells are targeted by bacterial pathogens.

Differential activation and functional specialization of miR-146 and miR-155 in innate immune sensing.

Many microRNAs (miRNAs) are co-regulated during the same physiological process but the underlying cellular logic is often little understood. The conserved, immunomodulatory miRNAs miR-146 and miR-155, for instance, are co-induced in many cell types in response to microbial lipopolysaccharide (LPS) to feedback-repress LPS signalling through Toll-like receptor TLR4. Here, we report that these seemingly co-induced regulatory RNAs dramatically differ in their induction behaviour under various stimuli strengths and act non-redundantly through functional specialization; although miR-146 expression saturates at sub-inflammatory doses of LPS that do not trigger the messengers of inflammation markers, miR-155 remains tightly associated with the pro-inflammatory transcriptional programmes. Consequently, we found that both miRNAs control distinct mRNA target profiles; although miR-146 targets the messengers of LPS signal transduction components and thus downregulates cellular LPS sensitivity, miR-155 targets the mRNAs of genes pervasively involved in pro-inflammatory transcriptional programmes. Thus, miR-155 acts as a broad limiter of pro-inflammatory gene expression once the miR-146 dependent barrier to LPS triggered inflammation has been breached. Importantly, we also report alternative miR-155 activation by the sensing of bacterial peptidoglycan through cytoplasmic NOD-like receptor, NOD2. We predict that dose-dependent responses to environmental stimuli may involve functional specialization of seemingly co-induced miRNAs in other cellular circuitries as well.

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.

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.

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.

Emerging Roles of Long Noncoding RNAs in the Cytoplasmic Milieu.

While the important functions of long noncoding RNAs (lncRNAs) in nuclear organization are well documented, their orchestrating and architectural roles in the cytoplasmic environment have long been underestimated. However, recently developed fractionation and proximity labelling approaches have shown that a considerable proportion of cellular lncRNAs is exported into the cytoplasm and associates nonrandomly with proteins in the cytosol and organelles. The functions of these lncRNAs range from the control of translation and mitochondrial metabolism to the anchoring of cellular components on the cytoskeleton and regulation of protein degradation at the proteasome. In the present review, we provide an overview of the functions of lncRNAs in cytoplasmic structures and machineries und discuss their emerging roles in the coordination of the dense intracellular milieu. It is becoming apparent that further research into the functions of these lncRNAs will lead to an improved understanding of the spatiotemporal organization of cytoplasmic processes during homeostasis and disease.

Disease-Causing Mutations and Rearrangements in Long Non-coding RNA Gene Loci.

The classic understanding of molecular disease-mechanisms is largely based on protein-centric models. During the past decade however, genetic studies have identified numerous disease-loci in the human genome that do not encode proteins. Such non-coding DNA variants increasingly gain attention in diagnostics and personalized medicine. Of particular interest are long non-coding RNA (lncRNA) genes, which generate transcripts longer than 200 nucleotides that are not translated into proteins. While most of the estimated ~20,000 lncRNAs currently remain of unknown function, a growing number of genetic studies link lncRNA gene aberrations with the development of human diseases, including diabetes, AIDS, inflammatory bowel disease, or cancer. This suggests that the protein-centric view of human diseases does not capture the full complexity of molecular patho-mechanisms, with important consequences for molecular diagnostics and therapy. This review illustrates well-documented lncRNA gene aberrations causatively linked to human diseases and discusses potential lessons for molecular disease models, diagnostics, and therapy.

An Advanced Human Intestinal Coculture Model Reveals Compartmentalized Host and Pathogen Strategies during Salmonella Infection.

A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.