Peptidylarginine deiminase 4 (PAD4) catalyzes citrullination of histones, an important step for neutrophil extracellular trap (NET) formation. We aimed to determine the role of PAD4 during pneumonia. Markers of NET formation were measured in lavage fluid from airways of critically ill patients. NET formation and host defense were studied during pneumonia-derived sepsis caused by in PAD4 and PAD4 mice. Patients with pneumosepsis, compared with those with nonpulmonary disease, showed increased citrullinated histone 3 (CitH3) levels in their airways and a trend toward elevated levels of NET markers cell-free DNA and nucleosomes. During murine pneumosepsis, CitH3 levels were increased in the lungs of PAD4 but not of PAD4 mice. Combined light and electron microscopy showed NET-like structures surrounding in areas of CitH3 staining in the lung; however, these were also seen in PAD4 mice with absent CitH3 lung staining. Moreover, cell-free DNA and nucleosome levels were mostly similar in both groups. Moreover, and LPS could still induce NETosis in PAD4 neutrophils. Both groups showed largely similar bacterial growth, lung inflammation, and organ injury. In conclusion, these data argue against a major role for PAD4 in NET formation, host defense, or organ injury during pneumonia-derived sepsis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease characterized by strong induction of inflammatory cytokines, progressive lung inflammation, and potentially multiorgan dysfunction. It remains unclear how SARS-CoV-2 infection leads to immune activation. The Spike (S) protein of SARS-CoV-2 has been suggested to trigger TLR4 and thereby activate immunity. Here, we have investigated the role of TLR4 in SARS-CoV-2 infection and immunity. Neither exposure of isolated S protein, SARS-CoV-2 pseudovirus nor primary SARS-CoV-2 isolate induced TLR4 activation in a TLR4-expressing cell line. Human monocyte-derived DCs express TLR4 but not angiotensin converting enzyme 2 (ACE2), and DCs were not infected by SARS-CoV-2. Notably, neither S protein nor SARS-CoV-2 induced DC maturation or cytokines, indicating that both S protein and SARS-CoV-2 virus particles do not trigger extracellular TLRs including TLR4. Ectopic expression of ACE2 in DCs led to efficient infection by SARS-CoV-2 and, strikingly, efficient type I IFN and cytokine responses. These data strongly suggest that not extracellular TLRs but intracellular viral sensors are key players in sensing SARS-CoV-2. These data imply that SARS-CoV-2 escapes direct sensing by TLRs, which might underlie the lack of efficient immunity to SARS-CoV-2 early during infection.

Platelet Bruton's tyrosine kinase (Btk) is an essential signalling protein for the collagen receptor glycoprotein VI (GPVI) and podoplanin receptor C-type-lectin-like receptor-2, which are platelet receptors implicated in the maintenance of vascular integrity during inflammation. Moreover, platelets, platelet GPVI and Btk are important for host defence during murine bacterial pneumosepsis. The aim of this study was to determine the role of platelet Btk in vascular integrity and host defence during murine pneumosepsis caused by the common human pathogens Streptococcus pneumoniae and Klebsiella pneumoniae. Using the Cre-loxP system, male platelet-specific Btk-deficient mice (PF4creBtkfl/Y) were created. Similar to platelets from total Btk-deficient mice, platelets from PF4creBtkfl/Y mice showed abrogated aggregation and P-selectin expression when stimulated with the GPVI ligand cross-linked collagen-related peptide. Upon infection with S. pneumoniae, PF4creBtkfl/Y mice showed increased lung bleeding, but unimpaired anti-bacterial defence. During pneumosepsis evoked by K. pneumoniae, platelet Btk deficiency was not associated with lung bleeding and did not impact on host defence, even when platelet function was further compromised by blocking secondary platelet activation by the P2Y12 receptor antagonist clopidogrel. Together, these data indicate that, while platelet Btk is not important for anti-bacterial defence in pneumosepsis, its role in maintaining vascular integrity in the lung depends on the causative pathogen.

Introduction T‐cell antigen receptor (TCR) interaction with cognate peptide:MHC complexes trigger clustering of TCR:CD3 complexes and signal transduction. Triggered TCR:CD3 complexes are rapidly internalized and degraded in a process called ligand‐induced TCR downregulation. Classic studies in immortalized T‐cell lines have revealed a major role for the Src family kinase Lck in TCR downregulation. However, to what extent a similar mechanism operates in primary human T cells remains unclear. Methods Here, we developed an anti‐CD3‐mediated TCR downregulation assay, in which T‐cell gene expression in primary human T cells can be knocked down by microRNA constructs. In parallel, we used CRISPR/Cas9‐mediated knockout in Jurkat cells for validation experiments. Results We efficiently knocked down the expression of tyrosine kinases Lck, Fyn, and ZAP70, and found that, whereas this impaired T cell activation and effector function, TCR downregulation was not affected. Although TCR downregulation was marginally inhibited by the simultaneous knockdown of Lck and Fyn, its full abrogation required broad‐acting tyrosine kinase inhibitors. Conclusions These data suggest that there is substantial redundancy in the contribution of individual tyrosine kinases to TCR downregulation in primary human T cells. Our results highlight that TCR downregulation and T cell activation are controlled by different signaling events and illustrate the need for further research to untangle these processes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), an infectious disease characterized by strong induction of inflammatory cytokines, progressive lung inflammation and potentially multi-organ dysfunction. It remains unclear whether SARS-CoV-2 is sensed by pattern recognition receptors (PRRs) leading to immune activation. Several studies suggest that the Spike (S) protein of SARS-CoV-2 might interact with Toll-like receptor 4 (TLR4) and thereby activate immunity. Here we have investigated the role of TLR4 in SARS-CoV-2 infection and immunity. Neither exposure of isolated S protein, SARS-CoV-2 pseudovirus nor a primary SARS-CoV-2 isolate induced TLR4 activation in a TLR4-expressing cell line. Human monocyte-derived dendritic cells (DCs) express TLR4 but not ACE2, and DCs were not infected by a primary SARS-CoV-2 isolate. Notably, neither S protein nor the primary SARS-CoV-2 isolate induced DC maturation or cytokines, indicating that both S protein and SARS-CoV-2 virus particles do not trigger extracellular TLRs, including TLR4. Ectopic expression of ACE2 in DCs led to efficient infection by SARS-CoV-2. Strikingly, infection of ACE2-positive DCs induced type I IFN and cytokine responses, which was inhibited by antibodies against ACE2. These data strongly suggest that not extracellular TLRs but intracellular viral sensors are key players in sensing SARS-CoV-2. These data imply that SARS-CoV-2 escapes direct sensing by TLRs, which might underlie the lack of efficient immunity to SARS-CoV-2 early during infection.

BackgroundInterferon (IFN)-β induction via activation of the stimulator of interferon genes (STING) pathway has shown promising results in tumor models. STING is activated by cyclic dinucleotides such as cyclic GMP–AMP dinucleotides with phosphodiester linkages 2′–5′ and 3′–5′ (cGAMPs), that are produced by cyclic GMP–AMP synthetase (cGAS). However, delivery of STING pathway agonists to the tumor site is a challenge. Bacterial vaccine strains have the ability to specifically colonize hypoxic tumor tissues and could therefore be modified to overcome this challenge. Combining high STING-mediated IFN-β levels with the immunostimulatory properties ofSalmonella typhimuriumcould have potential to overcome the immune suppressive tumor microenvironment.MethodsWe have engineeredS. typhimuriumto produce cGAMP by expression of cGAS. The ability of cGAMP to induce IFN-β and its IFN-stimulating genes was addressed in infection assays of THP-I macrophages and human primary dendritic cells (DCs). Expression of catalytically inactive cGAS is used as a control. DC maturation and cytotoxic T-cell cytokine and cytotoxicity assays were conducted to assess the potential antitumor response in vitro. Finally, by making use of differentS. typhimuriumtype III secretion (T3S) mutants, the mode of cGAMP transport was elucidated.ResultsExpression of cGAS inS. typhimuriumresults in a 87-fold stronger IFN-β response in THP-I macrophages. This effect was mediated by cGAMP production and is STING dependent. Interestingly, the needle-like structure of the T3S system was necessary for IFN-β induction in epithelial cells. DC activation included upregulation of maturation markers and induction of type I IFN response. Coculture of challenged DCs with cytotoxic T cells revealed an improved cGAMP-mediated IFN-γ response. In addition, coculture of cytotoxic T cells with challenged DCs led to improved immune-mediated tumor B-cell killing.ConclusionS. typhimuriumcan be engineered to produce cGAMPs that activate the STING pathway in vitro. Furthermore, they enhanced the cytotoxic T-cell response by improving IFN-γ release and tumor cell killing. Thus, the immune response triggered byS. typhimuriumcan be enhanced by ectopic cGAS expression. These data show the potential ofS. typhimurium–cGAS in vitro and provides rationale for further research in vivo.

Dysregulated immune responses contribute to pathogenesis of COVID-19 leading to uncontrolled and exaggerated inflammation observed during severe COVID-19. However, it remains unclear how immunity to SARS-CoV-2 is induced and subsequently controlled. Notably, here we have uncovered an important role for complement in the induction of innate and adaptive immunity to SARS-CoV-2. Complement rapidly opsonized SARS-CoV-2 via the lectin pathway. Complement-opsonized SARS-CoV-2 efficiently interacted with dendritic cells (DCs), inducing type I IFN and pro-inflammatory cytokine responses, which were inhibited by antibodies against the complement receptors (CR)3 and CR4. These data suggest that complement is important in inducing immunity via DCs in the acute phase against SARS-CoV-2. Strikingly, serum from COVID-19 patients as well as monoclonal antibodies against SARS-CoV-2 attenuated innate and adaptive immunity induced by complement-opsonized SARS-CoV-2. Blocking the FcyRII, CD32, restored complement-induced immunity. These data strongly suggest that complement opsonization of SARS-CoV-2 is important for inducing innate and adaptive immunity to SARS-CoV-2. Subsequent induction of antibody responses is important to limit the immune responses and restore immune homeostasis. These data suggest that dysregulation in complement and FcyRII signalling might underlie mechanisms causing severe COVID-19.

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