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.

Molecular Determinants in Phagocyte-Bacteria Interactions.

Phagocytes are crucial for host defense against bacterial pathogens. As first demonstrated by Metchnikoff, neutrophils and mononuclear phagocytes share the capacity to engulf, kill, and digest microbial invaders. Generally, neutrophils focus on extracellular, and mononuclear phagocytes on intracellular, pathogens. Reciprocally, extracellular pathogens often capitalize on hindering phagocytosis and killing of phagocytes, whereas intracellular bacteria frequently allow their engulfment and then block intracellular killing. As foreseen by Metchnikoff, phagocytes become highly versatile by acquiring diverse phenotypes, but still retaining some plasticity. Further, phagocytes engage in active crosstalk with parenchymal and immune cells to promote adjunctive reactions, including inflammation, tissue healing, and remodeling. This dynamic network allows the host to cope with different types of microbial invaders. Here we present an update of molecular and cellular mechanisms underlying phagocyte functions in antibacterial defense. We focus on four exemplary bacteria ranging from an opportunistic extracellular to a persistent intracellular pathogen.

SARS-CoV-2 variants of concern elicit divergent early immune responses in hACE2 transgenic mice.

Knowledge about early immunity to SARS-CoV-2 variants of concern mainly comes from the analysis of human blood. Such data provide limited information about host responses at the site of infection and largely miss the initial events. To gain insights into compartmentalization and the early dynamics of host responses to different SARS-CoV-2 variants, we utilized human angiotensin converting enzyme 2 (hACE2) transgenic mice and tracked immune changes during the first days after infection by RNAseq, multiplex assays, and flow cytometry. Viral challenge infection led to divergent viral loads in the lungs, distinct inflammatory patterns, and innate immune cell accumulation in response to ancestral SARS-CoV-2, Beta (B.1.351) and Delta (B.1.617.2) variant of concern (VOC). Compared to other SARS-CoV-2 variants, infection with Beta (B.1.351) VOC spread promptly to the lungs, leading to increased inflammatory responses. SARS-CoV-2-specific antibodies and T cells developed within the first 7 days postinfection and were required to reduce viral spread and replication. Our studies show that VOCs differentially trigger transcriptional profiles and inflammation. This information contributes to the basic understanding of immune responses immediately postexposure to SARS-CoV-2 and is relevant for developing pan-VOC interventions including prophylactic vaccines.

The role of PB1-F2 in adaptation of high pathogenicity avian influenza virus H7N7 in chickens.

Avian influenza viruses (AIV) of the H7N7 subtype are enzootic in the wild bird reservoir in Europe, cause infections in poultry, and have sporadically infected humans. The non-structural protein PB1-F2 is encoded in a second open frame in the polymerase segment PB1 and its sequence varies with the host of origin. While mammalian isolates predominantly carry truncated forms, avian isolates typically express full-length PB1-F2. PB1-F2 is a virulence factor of influenza viruses in mammals. It modulates the host immune response, causing immunopathology and increases pro-inflammatory responses. The role of full-length PB1-F2 in IAV pathogenesis as well as its impact on virus adaptation and virulence in poultry remains enigmatic. Here, we characterised recombinant high pathogenicity AIV (HPAIV) H7N7 expressing or lacking PB1-F2 in vitro and in vivo in chickens. In vitro, full-length PB1-F2 modulated viability of infected chicken fibroblasts by limiting apoptosis. In chickens, PB1-F2 promoted gastrointestinal tropism, as demonstrated by enhanced viral replication in the gut and increased cloacal shedding. PB1-F2's effects on cellular immunity however were marginal. Overall, chickens infected with full-length PB1-F2 virus survived for shorter periods, indicating that PB1-F2 is also a virulence factor in bird-adapted viruses.

Nuclear cGAS suppresses DNA repair and promotes tumorigenesis.

Accurate repair of DNA double-stranded breaks by homologous recombination preserves genome integrity and inhibits tumorigenesis. Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that activates innate immunity by initiating the STING-IRF3-type I IFN signalling cascade1,2. Recognition of ruptured micronuclei by cGAS links genome instability to the innate immune response3,4, but the potential involvement of cGAS in DNA repair remains unknown. Here we demonstrate that cGAS inhibits homologous recombination in mouse and human models. DNA damage induces nuclear translocation of cGAS in a manner that is dependent on importin-α, and the phosphorylation of cGAS at tyrosine 215-mediated by B-lymphoid tyrosine kinase-facilitates the cytosolic retention of cGAS. In the nucleus, cGAS is recruited to double-stranded breaks and interacts with PARP1 via poly(ADP-ribose). The cGAS-PARP1 interaction impedes the formation of the PARP1-Timeless complex, and thereby suppresses homologous recombination. We show that knockdown of cGAS suppresses DNA damage and inhibits tumour growth both in vitro and in vivo. We conclude that nuclear cGAS suppresses homologous-recombination-mediated repair and promotes tumour growth, and that cGAS therefore represents a potential target for cancer prevention and therapy.

Pharmacokinetics and Efficacy of the Benzothiazinone BTZ-043 against Tuberculous Mycobacteria inside Granulomas in the Guinea Pig Model.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is the world's leading cause of mortality from a single bacterial pathogen. With increasing frequency, emergence of drug-resistant mycobacteria leads to failures of standard TB treatment regimens. Therefore, new anti-TB drugs are urgently required. BTZ-043 belongs to a novel class of nitrobenzothiazinones, which inhibit mycobacterial cell wall formation by covalent binding of an essential cysteine in the catalytic pocket of decaprenylphosphoryl-ß-d-ribose oxidase (DprE1). Thus, the compound blocks the formation of decaprenylphosphoryl-ß-d-arabinose, a precursor for the synthesis of arabinans. An excellent in vitro efficacy against M. tuberculosis has been demonstrated. Guinea pigs are an important small-animal model to study anti-TB drugs, as they are naturally susceptible to M. tuberculosis and develop human-like granulomas after infection. In the current study, dose-finding experiments were conducted to establish the appropriate oral dose of BTZ-043 for the guinea pig. Subsequently, it could be shown that the active compound was present at high concentrations in Mycobacterium bovis BCG-induced granulomas. To evaluate its therapeutic effect, guinea pigs were subcutaneously infected with virulent M. tuberculosis and treated with BTZ-043 for 4 weeks. BTZ-043-treated guinea pigs had reduced and less necrotic granulomas than vehicle-treated controls. In comparison to the vehicle controls a highly significant reduction of the bacterial burden was observed after BTZ-043 treatment at the site of infection and in the draining lymph node and spleen. Together, these findings indicate that BTZ-043 holds great promise as a new antimycobacterial drug.

Sensing of mycobacterial arabinogalactan by galectin-9 exacerbates mycobacterial infection.

Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio-synthetical target for anti-tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin-9 with high affinity, and galectin-9 associates with transforming growth factor ß-activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal-regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin-9 or inhibition of MMPs blocks AG-induced pathological impairments in the lung, and the AG-galectin-9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin-9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.

Human GBP1 Is Involved in the Repair of Damaged Phagosomes/Endolysosomes.

Mouse guanylate-binding proteins (mGBPs) are recruited to various invasive pathogens, thereby conferring cell-autonomous immunity against these pathogens. However, whether and how human GBPs (hGBPs) target M. tuberculosis (Mtb) and L. monocytogenes (Lm) remains unclear. Here, we describe hGBPs association with intracellular Mtb and Lm, which was dependent on the ability of bacteria to induce disruption of phagosomal membranes. hGBP1 formed puncta structures which were recruited to ruptured endolysosomes. Furthermore, both GTP-binding and isoprenylation of hGBP1 were required for its puncta formation. hGBP1 was required for the recovery of endolysosomal integrity. In vitro lipid-binding assays demonstrated direct binding of hGBP1 to PI4P. Upon endolysosomal damage, hGBP1 was targeted to PI4P and PI(3,4)P2-positive endolysosomes in cells. Finally, live-cell imaging demonstrated that hGBP1 was recruited to damaged endolysosomes, and consequently mediated endolysosomal repair. In summary, we uncover a novel interferon-inducible mechanism in which hGBP1 contributes to the repair of damaged phagosomes/endolysosomes.

Targeting of myeloid-derived suppressor cells by all-trans retinoic acid as host-directed therapy for human tuberculosis.

Conventional anti-tuberculosis (TB) therapies comprise lengthy antibiotic treatment regimens, exacerbated by multi-drug resistant and extensively drug resistant mycobacterial strains. We assessed the ability of all-trans retinoic acid (ATRA), as repurposed compound serving as host-directed therapy (HDT), to counteract the suppressive effects of myeloid-derived suppressor cells (MDSCs) obtained from active TB cases (untreated or during week one of treatment) on T-cell responsiveness. We show for the first time that MDSCs suppress non-specific T-cell activation and production of interleukin (IL)-2, IL-4, IL-13 and GM-CSF via contact-dependent mechanisms. ATRA treatment decreases MDSC frequency, but fails to mature MDSCs to non-suppressive, terminally differentiated myeloid cells and does not restore T-cell function or cytokine production in the presence of MDSCs. The impact of ATRA treatment on improved immunity, using the concentration tested here, is likely to be minimal, but further identification and development of MDSC-targeting TB host-directed therapies are warranted.

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.

Platelets Restrict the Oxidative Burst in Phagocytes and Facilitate Primary Progressive Tuberculosis.

Rationale: Platelets are generated in the capillaries of the lung, control hemostasis, and display immunological functions. Tuberculosis primarily affects the lung, and patients show platelet changes and hemoptysis. A role of platelets in immunopathology of pulmonary tuberculosis requires careful assessment.Objectives: To identify the dynamics and interaction partners of platelets in the respiratory tissue and establish their impact on the outcome of pulmonary tuberculosis.Methods: Investigations were primarily performed in murine models of primary progressive pulmonary tuberculosis, by analysis of mouse strains with variable susceptibility to Mycobacterium tuberculosis infection using platelet depletion and delivery of antiplatelet drugs.Measurements and Main Results: Platelets were present at the site of infection and formed aggregates with different myeloid subsets during experimental tuberculosis. Such aggregates were also detected in patients with tuberculosis. Platelets were detrimental during the early phase of infection, and this effect was uncoupled from their canonical activation. Platelets left lung cell dynamics and patterns of antimycobacterial T-cell responses unchanged but hampered antimicrobial defense by restricting production of reactive oxygen species in lung-residing myeloid cells.Conclusions: Platelets are detrimental in primary progressive pulmonary tuberculosis, orchestrate lung immunity by modulating innate immune responsiveness, and may be amenable to new interventions for this deadly disease.

NOD-Like Receptors: Guards of Cellular Homeostasis Perturbation during Infection.

The innate immune system relies on families of pattern recognition receptors (PRRs) that detect distinct conserved molecular motifs from microbes to initiate antimicrobial responses. Activation of PRRs triggers a series of signaling cascades, leading to the release of pro-inflammatory cytokines, chemokines and antimicrobials, thereby contributing to the early host defense against microbes and regulating adaptive immunity. Additionally, PRRs can detect perturbation of cellular homeostasis caused by pathogens and fine-tune the immune responses. Among PRRs, nucleotide binding oligomerization domain (NOD)-like receptors (NLRs) have attracted particular interest in the context of cellular stress-induced inflammation during infection. Recently, mechanistic insights into the monitoring of cellular homeostasis perturbation by NLRs have been provided. We summarize the current knowledge about the disruption of cellular homeostasis by pathogens and focus on NLRs as innate immune sensors for its detection. We highlight the mechanisms employed by various pathogens to elicit cytoskeleton disruption, organelle stress as well as protein translation block, point out exemplary NLRs that guard cellular homeostasis during infection and introduce the concept of stress-associated molecular patterns (SAMPs). We postulate that integration of information about microbial patterns, danger signals, and SAMPs enables the innate immune system with adequate plasticity and precision in elaborating responses to microbes of variable virulence.

Neutrophils in Tuberculosis: Cell Biology, Cellular Networking and Multitasking in Host Defense.

Neutrophils readily infiltrate infection foci, phagocytose and usually destroy microbes. In tuberculosis (TB), a chronic pulmonary infection caused by Mycobacterium tuberculosis (Mtb), neutrophils harbor bacilli, are abundant in tissue lesions, and their abundances in blood correlate with poor disease outcomes in patients. The biology of these innate immune cells in TB is complex. Neutrophils have been assigned host-beneficial as well as deleterious roles. The short lifespan of neutrophils purified from blood poses challenges to cell biology studies, leaving intracellular biological processes and the precise consequences of Mtb-neutrophil interactions ill-defined. The phenotypic heterogeneity of neutrophils, and their propensity to engage in cellular cross-talk and to exert various functions during homeostasis and disease, have recently been reported, and such observations are newly emerging in TB. Here, we review the interactions of neutrophils with Mtb, including subcellular events and cell fate upon infection, and summarize the cross-talks between neutrophils and lung-residing and -recruited cells. We highlight the roles of neutrophils in TB pathophysiology, discussing recent findings from distinct models of pulmonary TB, and emphasize technical advances that could facilitate the discovery of novel neutrophil-related disease mechanisms and enrich our knowledge of TB pathogenesis.

Susceptibility of Raccoon Dogs for Experimental SARS-CoV-2 Infection.

Raccoon dogs might have been intermediate hosts for severe acute respiratory syndrome-associated coronavirus in 2002-2004. We demonstrated susceptibility of raccoon dogs to severe acute respiratory syndrome coronavirus 2 infection and transmission to in-contact animals. Infected animals had no signs of illness. Virus replication and tissue lesions occurred in the nasal conchae.

AhR sensing of bacterial pigments regulates antibacterial defence.

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

How to measure the immunosuppressive activity of MDSC: assays, problems and potential solutions.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of mononuclear and polymorphonuclear myeloid cells, which are present at very low numbers in healthy subjects, but can expand substantially under disease conditions. Depending on disease type and stage, MDSC comprise varying amounts of immature and mature differentiation stages of myeloid cells. Validated unique phenotypic markers for MDSC are still lacking. Therefore, the functional analysis of these cells is of central importance for their identification and characterization. Various disease-promoting and immunosuppressive functions of MDSC are reported in the literature. Among those, the capacity to modulate the activity of T cells is by far the most often used and best-established read-out system. In this review, we critically evaluate the assays available for the functional analysis of human and murine MDSC under in vitro and in vivo conditions. We also discuss critical issues and controls associated with those assays. We aim at providing suggestions and recommendations useful for the contemporary biological characterization of MDSC.

Deciphering myeloid-derived suppressor cells: isolation and markers in humans, mice and non-human primates.

In cancer, infection and inflammation, the immune system's function can be dysregulated. Instead of fighting disease, immune cells may increase pathology and suppress host-protective immune responses. Myeloid cells show high plasticity and adapt to changing conditions and pathological challenges. Despite their relevance in disease pathophysiology, the identity, heterogeneity and biology of myeloid cells is still poorly understood. We will focus on phenotypical and functional markers of one of the key myeloid regulatory subtypes, the myeloid derived suppressor cells (MDSC), in humans, mice and non-human primates. Technical issues regarding the isolation of the cells from tissues and blood, timing and sample handling of MDSC will be detailed. Localization of MDSC in a tissue context is of crucial importance and immunohistochemistry approaches for this purpose are discussed. A minimal antibody panel for MDSC research is provided as part of the Mye-EUNITER COST action. Strategies for the identification of additional markers applying state of the art technologies such as mass cytometry will be highlighted. Such marker sets can be used to study MDSC phenotypes across tissues, diseases as well as species and will be crucial to accelerate MDSC research in health and disease.

MDSCs in infectious diseases: regulation, roles, and readjustment.

Many pathogens, ranging from viruses to multicellular parasites, promote expansion of MDSCs, which are myeloid cells that exhibit immunosuppressive features. The roles of MDSCs in infection depend on the class and virulence mechanisms of the pathogen, the stage of the disease, and the pathology associated with the infection. This work compiles evidence supported by functional assays on the roles of different subsets of MDSCs in acute and chronic infections, including pathogen-associated malignancies, and discusses strategies to modulate MDSC dynamics to benefit the host.

Mycobacterium tuberculosis infection modulates adipose tissue biology.

Mycobacterium tuberculosis (Mtb) primarily resides in the lung but can also persist in extrapulmonary sites. Macrophages are considered the prime cellular habitat in all tissues. Here we demonstrate that Mtb resides inside adipocytes of fat tissue where it expresses stress-related genes. Moreover, perigonadal fat of Mtb-infected mice disseminated the infection when transferred to uninfected animals. Adipose tissue harbors leukocytes in addition to adipocytes and other cell types and we observed that Mtb infection induces changes in adipose tissue biology depending on stage of infection. Mice infected via aerosol showed infiltration of inducible nitric oxide synthase (iNOS) or arginase 1 (Arg1)-negative F4/80+ cells, despite recruitment of CD3+, CD4+ and CD8+ T cells. Gene expression analysis of adipose tissue of aerosol Mtb-infected mice provided evidence for upregulated expression of genes associated with T cells and NK cells at 28 days post-infection. Strikingly, IFN-γ-producing NK cells and Mtb-specific CD8+ T cells were identified in perigonadal fat, specifically CD8+CD44-CD69+ and CD8+CD44-CD103+ subpopulations. Gene expression analysis of these cells revealed that they expressed IFN-γ and the lectin-like receptor Klrg1 and down-regulated CD27 and CD62L, consistent with an effector phenotype of Mtb-specific CD8+ T cells. Sorted NK cells expressed higher abundance of Klrg1 upon infection, as well. Our results reveal the ability of Mtb to persist in adipose tissue in a stressed state, and that NK cells and Mtb-specific CD8+ T cells infiltrate infected adipose tissue where they produce IFN-γ and assume an effector phenotype. We conclude that adipose tissue is a potential niche for Mtb and that due to infection CD8+ T cells and NK cells are attracted to this tissue.

The Mycobacterium tuberculosis regulatory network and hypoxia.

We have taken the first steps towards a complete reconstruction of the Mycobacterium tuberculosis regulatory network based on ChIP-Seq and combined this reconstruction with system-wide profiling of messenger RNAs, proteins, metabolites and lipids during hypoxia and re-aeration. Adaptations to hypoxia are thought to have a prominent role in M. tuberculosis pathogenesis. Using ChIP-Seq combined with expression data from the induction of the same factors, we have reconstructed a draft regulatory network based on 50 transcription factors. This network model revealed a direct interconnection between the hypoxic response, lipid catabolism, lipid anabolism and the production of cell wall lipids. As a validation of this model, in response to oxygen availability we observe substantial alterations in lipid content and changes in gene expression and metabolites in corresponding metabolic pathways. The regulatory network reveals transcription factors underlying these changes, allows us to computationally predict expression changes, and indicates that Rv0081 is a regulatory hub.