Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection disturbs the coagulation balance in the blood, triggering thrombosis and contributing to organ failure. The role of prothrombotic metabolites in COVID-19-associated coagulopathy remains elusive. Leveraging K18-hACE2 mice infected with SARS-CoV-2, we observed higher levels of the tryptophan metabolite, kynurenine, than in controls. SARS-CoV-2-infected mice showed a significant upregulation of enzymes controlling kynurenine biogenesis, such as indoleamine 2,3-dioxygenase 1 (IDO-1) and tryptophan 2,3-dioxygenase in the kidney and liver, respectively, as well as changes in the enzymes involved in kynurenine catabolism, including kynurenine monooxygenase and kynurinase. Consistent with the agonistic role of these metabolites in aryl hydrocarbon receptor (AHR) signaling, AHR activation and its downstream mediator, tissue factor (TF), a highly potent procoagulant factor, was observed in endothelial cells (ECs) of lungs and kidneys of infected mice. These findings were validated in humans. Compared with controls, sera of patients with COVID-19 showed increased levels of kynurenine, kynurenic acid, anthranilic acid, and quinolinic acid. Activation of the AHR-TF axis was noted in the kidneys and lungs of patients with COVID-19, and sera from patients infected with SARS-CoV-2 showed higher IDO-1 activity than controls. Kynurenine levels in patients with COVID-19 correlated strongly with the TF-inducing activity of sera from patients infected with SARS-CoV-2 on ECs. A specific IDO-1 inhibitor or AHR inhibitor separately or in combination suppressed sera from induced TF activity in ECs from patients with COVID-19. Together, we identified IDO-1 as upregulated by SARS-CoV-2 infection, resulting in augmented kynurenine and its prothrombotic catabolites, thereby suggesting the kynurenine-AHR-TF axis as a potential new diagnostic and therapeutic target.

Bacterial sepsis remains a devastating clinical problem. Here, we describe a protective role for the recently discovered acid-sensitive, proton-activated chloride channel, PACC1 (PAC/ASOR/TMEM206), during sepsis. Initially, we found PACC1 was enriched in healthy human and mouse mononuclear phagocytes, particularly macrophages, and differentially regulated by inflammatory stimuli, suggesting PACC1 involvement in innate immunity. To further investigate, we generated de novo Pacc1 knockout (-/-) mice, which presented without major immunologic abnormalities at baseline. Compared to wild-type (WT), Pacc1-/- myeloid cells showed normal phagocytic uptake of acid-insensitive Escherichia coli BioParticles, but impaired development of the acidifying phagolysosome using acid-sensitive E. coli BioParticles. Transcriptomic profiling of Pacc1-/- macrophages revealed dysregulated phagolysosomal and cytokine networks (e.g., interferons). Because phagolysosomal bacterial clearance is essential to resolve infection, we challenged Pacc1-/- mice with intraperitoneal gram-negative E. coli sepsis. Pacc1-/- mice displayed increased bacterial burden, immune cell infiltration, inflammation, and lethality. In contrast, phagocytosis-independent E. coli lipopolysaccharide (LPS)-induced endotoxemia yielded comparable WT and Pacc1-/- survival, as well as similar inflammatory responses. Finally, we engineered Pacc1-floxed (fl/fl) mice crossed with a myeloid lineage Cre-deleter strain to interrogate myeloid cell-intrinsic PACC1 in vivo. Consistent with a predominate role for PACC1 during phagocytosis and bacterial clearance in these cells, LysM-Cre/Pacc1fl/fl mice exhibited impaired E. coli sepsis survival but indifferent endotoxemia phenotypes. In conclusion, PACC1 links sterilizing phagolysosomal activity with immune networks in sepsis pathobiology.

Pneumonia remains a major global health burden, highlighting the need for host-directed therapies to complement antimicrobial treatment. Here, we identify Oncostatin M (OSM) as a critical regulator of pulmonary host responses during influenza and bacterial pneumonia. Loss of OSM shifted lung macrophages toward a pro-inflammatory phenotype during influenza infection and exacerbated lung injury during bacterial pneumonia, demonstrating an essential role for OSM in limiting immunopathology. Unexpectedly, OSM induced Signal Transducer and Activator of Transcription 3 (STAT3) activation in the absence of the canonical OSM receptor subunit OSMrβ, revealing previously unrecognized non-canonical OSM signaling in the mouse lungs. Consistent with this finding, loss of OSMrβ did not phenocopy the severe disease observed with loss of OSM. Together, these findings identify OSM as a key regulator of pulmonary immunity and reveal unexpected complexity in OSM signaling during pneumonia.

Abstract Introduction We have recently identified the lung mesenchyme as a potent source of complement proteins during homeostasis. However, the changes in the complement transcriptomic profile in the mesenchyme in the context of infections has not yet been elucidated. Here, we show that mesenchymal cells express a defined pattern of complement genes during respiratory bacterial and viral infections. Methods Single cell RNA-sequencing data from selected published studies in murine experimental lung infection with Streptococcus pneumoniae, Pseudomonas aeruginosa, Klebsiella pneumoniae, influenza, and SARS-CoV-2 that included mesenchymal cells were analyzed. The raw counts data were retrieved from NCBI GEO and imported into RStudio/R (v4.3.1) and processed using Seurat (v5.0.1). Results Uninfected mesothelial cells exhibited high expression of classical pathway genes such as C1ra, C1s, C3, Serping1 (C1-INH), and Cfh, while fibroblasts highly expressed Serping1 and Cfh, consistent with our previous findings. During pneumococcal pneumonia, mesothelial cells showed downregulation of C2, C4b and Cfh expression, and upregulation of Cfb, whereas fibroblasts showed elevated expression of C3, Cfb and C4b, and repression of Cfh versus uninfected cells. Similar expression patterns in these cell types, though not always statistically significant, were observed in the other bacterial and viral infection models analyzed. As mesenchymal cells modulate inflammation and tissue repair through extracellular matrix (ECM) remodeling, we also explored ECM, collagen and cytokine expression. Across all infection models except Klebsiella, fibroblasts expressed higher levels of Timp1 (up to 100-fold), Col4a1, and Col4a2, while Stat1 was elevated in S. pneumoniae and viral infections. Conclusion Our study elucidates a cell-specific pattern of complement, ECM and cytokine/chemokine gene expression within lung mesenchymal cells during bacterial and viral infections and highlights a potential role for complement-driven ECM remodeling. Funding Source This work was funded by the National Institutes of Health (R01HL166588 to M.B.). Topic Categories Immune Response Regulation: Molecular Mechanisms (IRM)

Polyphosphates are evolutionarily conserved linear chains of phosphate residues present in all living cells. Bacteria accumulate polyphosphates under stress and starvation for energy and phosphate storage, protein folding, and stress adaptation. During infection, bacteria release polyphosphates that may impair host responses, although the exact mechanisms remain elusive. In this study, polyphosphates were found to be elevated in bronchoalveolar lavage fluids from patients with Legionnaires' disease, in Legionella pneumophila cultured alone, or during infection of bone marrow-derived macrophages (BMDMs) from C57BL/6J mice. We performed RNA-sequencing of infected BMDMs co-incubated with or without long-chain, bacterial-type polyphosphates. Among nearly 500 differentially expressed genes, Il12b (p40) showed the strongest suppression among highly expressed genes. IL-12p40 protein release was dose-dependently reduced by long-chain polyphosphates (~Pi700), but not by short-chain, mammalian-type polyphosphates (~Pi70), indicating a specific bacterial mechanism targeting innate immune signaling. In contrast, IL-18, processed by inflammasome activation and synergizing functionally with IL-12, was not consistently suppressed. Polyphosphates predominantly inhibited LPS/TLR4 signaling, with Legionella-induced IL-12 relying on the MyD88 pathway, but not TRIF. RAGE and P2Y1, previously implicated in polyphosphate biology, were not required for IL-12 suppression. However, PI3K/AKT signaling appeared to mediate polyphosphate effects, which were reversed by the PI3K inhibitors, LY294002, copanlisib, and eganelisib. Finally, long-chain polyphosphates suppressed IL-12 release also from human monocyte-derived macrophages exposed to L. pneumophila or LPS. In summary, our findings identify a selective inhibition of IL-12 by long-chain bacterial polyphosphates, suggesting that these molecules act as bacterial effectors capable of suppressing protective innate immune responses.

Bacterial sepsis remains a devastating clinical problem. Here, we describe a protective role for the recently discovered acid-sensitive, proton-activated chloride channel, PACC1 (PAC/ASOR/TMEM206), during sepsis. Initially, we found PACC1 was enriched in healthy human and mouse mononuclear phagocytes, particularly macrophages, and differentially regulated by inflammatory stimuli, suggesting PACC1 involvement in innate immunity. To further investigate, we generated de novo Pacc1 knockout ( -/- ) mice, which presented without major immunologic abnormalities at baseline. Compared to wildtype (WT), Pacc1 -/- myeloid cells showed normal phagocytic uptake of acid-insensitive Escherichia coli BioParticles , but impaired development of the acidifying phagolysosome using acid-sensitive E. coli BioParticles. Transcriptomic profiling of Pacc1 -/- macrophages revealed dysregulated phagolysosomal and cytokine networks (e.g., interferons). Because phagolysosomal bacterial clearance is essential to resolve infection, we challenged Pacc1 -/- mice with intraperitoneal gram-negative E. coli sepsis. Pacc1 -/- mice displayed increased bacterial burden, immune cell infiltration, inflammation, and lethality. In contrast, phagocytosis-independent E. coli lipopolysaccharide (LPS)-induced endotoxemia yielded comparable WT and Pacc1 -/- survival, as well as similar inflammatory responses. Finally, we engineered Pacc1 -floxed ( fl/fl ) mice crossed with a myeloid lineage Cre-deleter strain to interrogate myeloid cell-intrinsic PACC1 in vivo . Consistent with a predominate role for PACC1 during phagocytosis and bacterial clearance in these cells, LysM-Cre/Pacc1 fl/fl mice exhibited impaired E. coli sepsis survival but indifferent endotoxemia phenotypes. In conclusion, PACC1 links sterilizing phagolysosomal activity with immune networks in sepsis pathobiology. Significance Statement:Bacterial sepsis remains a major global health burden. Here, we report an essential role for the recently discovered acid-sensitive chloride channel, PACC1 (PAC/ASOR/TMEM206), in protective host defense during bacterial infection and sepsis. PACC1 is highly expressed in human and mouse phagocytic myeloid cells, particularly macrophages, where it regulates phagocytic bacterial clearance and inflammatory responses. Using de novo generated mice, we show that global or myeloid cell-targeted deletion of PACC1 impairs development of phagolysosomal acidification, confers susceptibility to bacterial infection and excessive inflammation, and undermines host defense. These findings warrant further investigation of PACC1 in sepsis pathobiology.

Legionnaires’ disease (LD) is a severe respiratory illness caused by Legionella spp., with a 7-10% fatality rate in the US and limited knowledge of its pathobiology. Interleukin (IL)-27 is an immunomodulatory cytokine that signals through the IL-27RA and gp130 receptors. Here, we show that IL-27 modulates lymphocyte-specific responses in the lung during Legionella pneumonia. IL-27 was highly elevated in the bronchoalveolar lavage fluid (BALF) of patients with LD compared to healthy controls (p < 0.001). Comparable results were noted in C57BL/6 mice, with rapid IL-27 induction 24h after L. pneumophila infection. Il-27p28 mRNA was highest in alveolar macrophages, and recruited monocytes in the BALF while IL-27RA was detected in NK and T cells. Global IL-27RA-/- mice showed less severe disease, increased NK and T cell activity, and higher protective IFN-γ levels. These findings were concordant with the highly virulent L. longbeachae. IL-27 chiefly affected immunopathology but not bacterial burden. Single-cell CITE-seq using BALF from L. pneumophila-infected IL27RA-/- mice revealed enhanced expression of the activation markers CD25 and CD69 in NK and T cells, and co-stimulatory over co-inhibitory receptors in T cells versus wild type mice. NK cell-specific IL-27RA deletion worsened disease, while CD8+ T cell-specific deletion had no effect. In conclusion, cell type-specific targeting of IL-27RA activity could be a novel therapeutic approach in treating Legionnaires’ disease. This work was funded by the National Institutes of Health (R01HL166588 to M.B.), the Federal Ministry of Education and Research (01EO1003, 01EO1503 to M.B.), and the Deutsche Forschungsgemeinschaft (BO3482/3-3, BO3482/4-1 to M.B.). Cytokines and Chemokines and Their Receptors (CCR)

Sepsis affects ∼50 million people annually, with mortality rates of 20–50%. Host-pathogen interactions are implicated in its etiology but are understudied. Bacterial polyphosphates are metabolites with pleiotropic functions in stress response, virulence and host evasion. Here, we reveal polyphosphate-mediated changes during sepsis. In cecal ligation and puncture (CLP)-induced sepsis, gnotobiotic mice colonized with wild type E. coli showed reduced survival versus mice with polyphosphate kinase-deficient E. coli (38% vs 75%), implying polyphosphates mediate lethality. Moreover, polyphosphates impaired local innate immune responses, altered basal and maximal metabolism (OXPHOS, glycolysis), and dysregulated transcriptional programs of metabolic genes (Ldha, Hk2, Slc2a1, Atp5h) as studied by snRNA-Seq in macrophages. Polyphosphates bound to lactate dehydrogenase and affected its activity. In the serum from patients with sepsis (n = 140) polyphosphate accumulation was highest compared to systemic inflammatory response syndrome (n = 114) and healthy controls (n = 143), and higher in non-survivors of sepsis than survivors. Polyphosphate amounts positively correlated with lactate, an established prognostic marker for sepsis. Notably, in murine CLP sepsis, recombinant exopolyphosphate improved survival and lactate by neutralizing polyphosphates. Our findings elucidate how polyphosphates disrupt macrophage functioning, reprogramming innate immune responses and influencing sepsis outcomes. This work was funded by the National Institutes of Health (R01AI153613 to M.B.) and Deutsche Forschungsgemeinschaft (BO3482/3-3, BO3482/4-1 to M.B.). Microbial, Parasitic, and Fungal Immunology (MPF)

SARS-CoV-2 infection disturbs the coagulation balance in the blood, triggering thrombosis and contributing to organ failure. The role of prothrombotic metabolites in COVID-19-associated coagulopathy remains elusive. Leveraging K18-hACE2 mice infected with SARS-CoV-2, we observed higher levels of the tryptophan metabolite, kynurenine, compared to controls. SARS CoV-2 infected mice showed a significant upregulation of enzymes controlling Kynurenine biogenesis, such as indoleamine 2,3-dioxygenase (IDO-1) and tryptophan 2,3-dioxygenase levels in kidneys and liver, respectively, as well as changes in the enzymes involved in kynurenine catabolism, including kynurenine monooxygenase and kynurinase. Consistent with the agonistic role of these metabolites in Aryl Hydrocarbon Receptor (AHR) signaling, AHR activation and its downstream mediator, tissue factor (TF), a highly potent procoagulant factor, was observed in endothelial cells (ECs) of lungs and kidneys of infected mice. These findings were validated in humans, where compared to controls, sera of COVID-19 patients showed increased levels of Kynurenine, kynurenic acid, anthranilic acid, and quinolinic acid. Activation of the AHR-TF axis was noted in the kidneys and lungs of COVID-19 patients, and COVID-19 sera showed higher IDO-1 activity than controls. Levels of Kyn in COVID-19 patients correlated strongly with the TF inducing activity of COVID-19 sera on ECs. A specific IDO-1 inhibitor or AHR inhibitor separately or in combination suppressed COVID-19 sera-induced TF activity in ECs. Together, we identified IDO-1 as upregulated by SARS-CoV-2 infection, resulting in augmented Kyn and its prothrombotic catabolites, thereby suggesting the Kyn AHR-TF axis as possibly a new diagnostic and/or therapeutic target.

The role of mesenchymal cells during respiratory infection is not well defined, including whether, which, and how the different types of mesenchymal cells respond. We collected all mesenchymal cells from lung single-cell suspensions of mice that were naive (after receiving only saline vehicle), pneumonic (after intratracheal instillation of pneumococcus 24 hours previously), or resolved from infection (after nonlethal pneumococcal infections 6 weeks previously) and performed single-cell RNA sequencing. Cells clustered into 5 well-separated groups based on their transcriptomes: matrix fibroblasts, myofibroblasts, pericytes, smooth muscle cells, and mesothelial cells. Fibroblasts were the most abundant and could be further segregated into Pdgfra+Npnt+Ces1d+Col13a1+ alveolar fibroblasts and Cd9+Pi16+Sca1+Col14a1+ adventitial fibroblasts. The cells from naive and resolved groups overlapped in dimension reduction plots, suggesting the mesenchymal cells returned to baseline transcriptomes after resolution. During pneumonia, all mesenchymal cells responded with altered transcriptomes, revealing a core response that had been conserved across cell types as well as distinct mesenchymal cell type-specific responses. The different subsets of fibroblasts induced similargene sets, but the alveolar fibroblasts responded more strongly than the adventitial fibroblasts. These data demonstrated diverse and specialized immune activities of lung mesenchymal cells during pneumonia.

Streptococcus pneumoniae (Spn) is the leading cause of pneumonia-associated mortality in adults. Complement activation is critical for encapsulated Spn clearance but also augments ARDS pathogenesis. C5a binding to its two receptors, C5aR1 and C5aR2, activates inflammatory cells. While C5a neutralization is protective in sepsis and inflammation models, its role in Spn infection remains unclear. To address this gap, we generated a novel C5aR1/2-/- double-knockout mouse strain using CRISPR/Cas9. Alveolar leukocytes were profiled at the single-cell level using CITE-sequencing containing a ∼200 antibody panel. Despite the lack of C5a-induced neutrophil influx, C5aR1/2-/- mice exhibited paradoxical exacerbation of alveolar inflammation after Spn infection, with increased neutrophil infiltration, elevated cytokine/chemokine release, and enhanced lung vascular permeability. The recruited neutrophils in C5aR1/2-/- mice showed greater NETosis but impaired bactericidal function evidenced by higher CFUs in bronchoalveolar lavage fluid. CITE-seq revealed distinct subclusters of alveolar macrophages and neutrophils after Spn infection, with heterogeneous C5aR1 and C5aR2 expression in WT mice. C5aR1/2-/- neutrophils showed increased apoptosis and signs of exhaustion (elevated PD-L1/ICAM-1 and reduced CXCR2/CD62L). In conclusion, C5aR1/2-/- neutrophils fail to control bacterial growth, fostering a potent chemotactic local environment for sustained inflammation, thereby promoting lung damage. Supported by NIH RO1HL139641 and the Federal Ministry of Education and Research (01EO1503) Microbial, Parasitic, and Fungal Immunology (MPF)

IntroductionCoronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is a public health emergency with phenotypes ranging from asymptomatic to severe sequelae that can lead to multiple organ failure and death (1, 2). SARS-CoV-2 efficiently infects airway epithelial cells and alveolar pneumocytes, causing in high viral loads and inflammatory responses, including the interferon response (3). In hospitalized patients, COVID-19 increases the risk of venous and arterial thromboembolic events due to vascular barrier failure, edema, endotheliitis, thrombosis, and inflammatory cell infiltration (4, 5). Hypercoagulation and micro- and macro-circulatory thrombosis are major causes of multiple organ failure in COVID-19 (6). Although many people have survived COVID-19 without long-term symptoms, a considerable portion of COVID-19 survivors reportedly have continuing cardiovascular issues such as coagulopathy or bleeding disorders (7). This suggests that, in addition to the respiratory epithelium, the endothelium lining of blood vessels may also be impacted by SARS-CoV-2 infection. The pathophysiology of COVID-19 has been explored in recent reviews (reviewed in (8, 9)). Due to the conflicting data, there is ongoing controversy about the endothelial tropism (refers to the ability of SARS-CoV-2 to interact with endothelial cells) and productive endothelial infection (viral replication within the ECs) of SARS-CoV-2. Here, we share our perspective on challenging the notion of endothelial tropism and productive endothelial infection, drawing insights from the current scientific evidence.Endothelial dysfunction and hypercoagulation in COVID-19The endothelium, which lines the inside of arteries, is crucial for controlling vascular tone and preserving vascular homeostasis (10). Disseminated intravascular coagulation (DIC), vasculitis, and thrombosis can all be attributable to endothelial damage (11, 12). Numerous prevalent viruses and bacteria have been found to directly infect ECs, causing necrosis, apoptosis and/or damage to the vessel wall (13-15). Upon infection by viruses such as Dengue, Hantaan, Marburg, Lassa, and Ebola, both immune and non-immune cells (including endothelial cells, monocytes, and macrophages) express tissue factor (TF), leading to hypercoagulation and often culminating in disseminated intravascular coagulation (DIC) (16-20). Studies have demonstrated that Dengue, Ebola, and Marburg viruses can directly infect endothelial cells (ECs) and replicate within them, as reviewed in detail elsewhere (13, 21, 22). However, it remains unclear whether SARS-CoV-2 exhibits a similar phenomenon due to conflicting observations. SARS-CoV-2 infection of endothelium is less studied than airway epithelium and alveolar pneumocytes (10, 23-25). Despite thromboprophylaxis, 31-49% of COVID-19 emergency care patients had arterial and venous thromboembolism (26-30). This shows that endothelial impairment must be addressed aggressively to prevent thrombosis. However, it is unclear whether the hypercoagulation is driven by lung-induced systemic inflammation upon infection, or by endothelial injury or dysfunction due to direct SARS-CoV-2 infection. Several pro-inflammatory cytokines including TNF-α, IL-1α, IL-1β, IL-6, IL-8, MCP-1, IFN- that are responsible for the cytokine storm in COVID-19 (31, 32) may induce COVID-19-associated coagulopathy (CAC) via expression of TF on ECs, monocytes, macrophages and T cells (33-40). The IL-6 signaling complex damages liver sinusoidal ECs and produces liver injury, suggesting that endothelial dysfunction and hypercoagulation may cause severe COVID-19 (41). SARS-CoV-2 infection in the Syrian hamster model showed inflammation and type I interferon dysregulation in respiratory and non-respiratory tissues like the heart and kidney, shedding light on COVID-19 as a multiorgan disease and possible post-acute sequelae (42). SARS-CoV-2 Spike and Nucleocapsid protein directly activate ECs, inducing mitochondrial dysfunction, vasculopathy, and coagulopathy (43, 44) (Figure 1). Furthermore, our recent study showed that the early host response of the endothelium to SARS-CoV-2 infection declines with aging, potentially contributing to increased disease severity (45).Proposed novel endothelial (co)-receptors for SARS-CoV-2 entry SARS-CoV-2 and SARS-CoV utilize human ACE2 as an entry receptor and TMPRSS2, primarily expressed by ECs in the respiratory and digestive tracts, as a co-factor to degrade extracellular matrix proteins for viral entry (46). ACE2 is variably expressed on arterial and venous ECs, smooth muscle cells, and pericytes across organs, facilitating systemic viral dissemination upon entry into the circulatory system. However, studies, including in-house immunohistochemistry, showed that humanized ACE2 mice express hACE2 in brain blood vessels but not in lung, gastrointestinal, or renal vessels, suggesting SARS-CoV-2 may employ both hACE2-dependent and independent entry mechanisms (47) (Figure 1B). Low expression of ACE2 and TMPRSS2 in human ECs limits the ability of SARS-CoV-2 to infect ECs (48). Thus, the variations in ACE2 expression across different microvascular beds, or alternative receptors on ECs may facilitate the entry of infectious particles. Supporting this concept, numerous additional receptors have been identified over the past three years as potentially relevant to viral particle entry in ECs. Endosomal cysteine peptidases like cathepsins B and L activate the spike (S) protein, enhancing viral entry (49-51). SARS-CoV-2 also binds heparan sulfate, sialic acid-containing glycoproteins, and gangliosides on ECs (52, 53). Proteolytic cleavage at furin-type cleavage sites in the S protein exposes a conserved motif that interacts with Neuropilin-1/2 receptors, significantly increasing infectivity (54, 55). Vimentin, CD147, and TMEM106B have been identified as co-receptors or alternative receptors, though role of TMEM106B in COVID-19 pathology lacks experimental validation (56-59). Further research is needed to confirm these mechanisms and in vivo relevance.Controversies in COVID-19 and direct infection of ECs Endotheliitis is regarded as a host immune-inflammatory response of the endothelium forming the inner surface of blood vessels in association with a direct consequence of infectious pathogen invasion. Systemic endotheliitis causes organ damage (60). Human autopsies, non-human primates (NHPs) and mice models showed sporadic endothelial infection and consistently observed in Syrian hamsters (61, 62). Compared to healthy individuals, circulating markers of endothelial and platelet activation are elevated in severe COVID-19 (63). Evidence of myeloid polarization, such as elevated levels of shed CD16 and CD163, have been linked to the expression of TF by proinflammatory macrophages and are related with poor clinical outcomes (64). Elevated D-dimer and thrombocytopenia in severe COVID-19 could be explained by dysregulated inflammation and microthrombus formation that are complicated by endothelial dysfunction (65). Consecutively, in patients with severe COVID-19, hypoxia due to pulmonary microvascular dysfunction might cause the classic acute respiratory distress syndrome (ARDS) (66). Furthermore, compared to controls, human pulmonary microvascular ECs isolated from human lungs challenged with lipopolysaccharide and tumor necrosis factor alpha showed increased pro-coagulant activity and PAI-1, and decreased fibrinolytic potential, emphasizing the pro-coagulant features of the pulmonary endothelium in ARDS (67).Earlier studies support that SARS-CoV-2 viral particles were detected in highly vascularized organs in which endothelial dysfunction plays a fundamental role (24, 68-76). Initial transmission electron microscopy (TEM) studies revealed the presence of viral particles in kidney ECs, venous ECs, and liver sinusoidal ECs in autopsy samples from COVID-19 patients (76-78). However, owing to the challenges of interpreting TEM and the high variability in experience of those interpreting images, the presence of viral particle in the endothelium remains debatable (76, 79, 80). Irrespective of these controversies, there has been increasing evidence to indicate that coated vesicles and multivesicular bodies closely mimic viral particles even in the lung epithelium by TEM and are not uncommonly misinterpreted (81). Despite the thrombo-inflammatory phenotype, no definitive animal models or human biopsies have yet shown direct SARS-CoV-2 infection of ECs or the presence of viral particles (82-85). Human micro- and macrovascular ECs are resistant to SARS-CoV-2 infection, and ACE2 overexpression is necessary for endothelial infection (86-89). Montezano et al. demonstrated that recombinant Spike protein-1 induced endothelial inflammation via ACE2 independent of ACE2 enzymatic activity and viral replication in vitro (90). On ex vivo lung cultures from a patient who had SARS-CoV-2 infection found no signs of the virus in the vascular endothelium following immunohistochemical labeling of SARS-CoV-2 Spike protein (91). ECs primed with (IL-1) produced more pro-inflammatory cytokines, such as IL-6 and IL-8, and were resistant to direct SARS-CoV-2 infection (92). Furthermore, two separate investigations found that human pulmonary microvascular ECs were resistant to SARS-CoV-2 infection (MOI=0.5-3 after 2 hours of adsorption) (93, 94).Based on these findings, it is possible to hypothesize that prior reports of endothelial viral presence in patients are not a universal hallmark of illness and may be restricted to certain patient groups or isolated episodes. In light of this, we carefully evaluated the immunoreactivity of the SARS-CoV-2 Nucleocapsid (N) Protein in lung slices from translational preclinical animal models (transgenic K18-hACE2 mice (expression of hACE2 in lung epithelial cells), hACE2-KI (global hACE2 knock-in by replacing mouse ACE2), Syrian hamsters, and African green monkeys (AGM) and human postmortem lung samples. A board-certified veterinary pathologist (N.A.C.) immunohistochemically analyzed hundreds of organ slices from previous studies in each species (45, 61, 62, 69, 95-99). A PCR-positive human autopsy samples with clear hyaline membrane formation and AGMs at 7 days post-infection (dpi) showed no N Protein, suggesting viral antigen is only present during the acute phase of disease (Figure 2A and 2B). The lung epithelium of K18-hACE2, Syrian hamsters, NHPs, and ACE2-KI mice showed varying and decreasing levels of SARS-CoV-2 N protein, with airway tropism only present in ACE2-KI mice and Syrian hamsters. No tissues had viral antigens in ECs (Figure 2C-E). To further support the absence of direct endothelial infection, we performed duplex fluorescent IHC targeting the pulmonary endothelium (CD34 or CD31) and SARS-CoV-2 N protein. In both the AGM and K18-hACE2 mouse, luminal alveolar pneumocytes exclusively displayed SARS-CoV-2 N protein as evidenced by absence of colocalization with vascular endothelium at 4DPI (Figure 2F-G). EC and inflammatory mediators in COVID-19Collectively, findings from our group and others question the universality of SARS- CoV-2's direct endothelial infectivity. Because active viral infection of lung epithelial cells is associated with a systemic pro-inflammation, it is possible that inflammatory mediators can facilitate endothelial injury by activating immunothrombosis mechanisms such as complement activation, antiphospholipid antibodies, and so on. ECs treated with human sera from COVID-19 hospitalized patients (n=118) demonstrated anti-cardiolipin IgG/IgM and anti-phosphatidlyserine/prothrombin (anti-PS/PT) IgG/IgM-driven elevation of surface adhesion markers E-selectin, VCAM-1, and ICAM-1 (100-102). Infection-induced proinflammatory cytokines, such as IL1β, TNFα, can stimulate coagulation, which may influence thrombin generation, fibrin formation, and TF-dependent thrombo-inflammatory responses via protease-activated receptors (PARs) (6, 103-107) (Figure 2H). SARS-CoV-2 infection induces increased production of superoxide anion and release of mitochondrial DNA (mtDNA), activating Toll-like receptor 9 (TLR9) and NFκ-B. Consequently, this activation orchestrates the expression of inflammatory genes, contributing to the pathological processes associated with COVID-19 (108). Numerous studies have highlighted changes in lipid profiles linked with COVID-19. Among the most commonly observed changes are reductions in serum cholesterol and ApoA1 levels, coupled with elevated triglycerides (109). Lipidomic analysis in COVID-19 patients showed high levels of eicosanoids in the lungs which might be a potential contributor for endothelial dysfunction. A systemic inflammatory response and aberrant expression of the extracellular matrix (ECM) during COVID-19 controls the balance and repair of ECs (110). A study on human lung autopsy confirmed that Hyaluronan is an important compound ECM of all vital organ systems (111). COVID-19 is characterized by significantly increased levels of MMP-1 and vascular endothelial growth factor (VEGF)-A, which are directly correlated with the severity of the disease (112). More than 50% of patients who had experienced moderate or severe cases of COVID-19 had reduced pulmonary diffusion and early fibrotic changes, which were correlated with elevated levels of MMP-1 (113). A detailed dysregulation of the ECM in COVID-19 reviewed elsewhere (114).The endothelial glycocalyx (EG) is crucial for maintaining vascular homeostasis, and it is linked to vascular endothelial dysfunction (115). Numerous studies indicate that severe COVID-19 patients experience EG damage on the endothelial cell surface, evidenced by elevated plasma levels of glycocalyx components like syndecan-1, heparan sulfate, and hyaluronan. These biomarkers, along with high levels of IL-1β, IL-6, TNF-α, hsCRP, and procalcitonin, are associated with increased severity and mortality in COVID-19 cases (116-118). However, despite numerous reports of EG injury in COVID-19, the underlying mechanisms are still not fully understood (118-122). Furthermore, several drugs, including heparin and tocilizumab, are already being used in COVID-19 treatment to protect the endothelial glycocalyx damage (123-126). Marine algae extracts, including fucoidan and rhamnan sulfate (RS) have been shown to restore the EG in both in vivo and in vitro studies (127, 128). Specifically, fucoidan, a heparan sulfate (HS) mimetic, demonstrated the ability to reduce endothelial activation and facilitate EG restoration in endothelial cells treated with COVID-19 serum(125). Vascular EG damage and potential targeted therapy in COVID-19 has been explored in recent reviews (reviewed in (84, 129, 130). The drugs used for COVID-19 treatment often exhibit multifunctional properties; however, no agents specifically aimed at directly improving the glycocalyx structure or integrity have been reported to date. Nevertheless, several treatment regimens targeting EG repair and protection have been applied in clinical practice for COVID-19 patients. These approaches were comprehensively reviewed elsewhere (129, 131). Most extracellularvesicles (EVs) found in the blood originate from platelets and erythrocytes (132). Under physiological conditions, the proportion of circulating EVs secreted by ECs is relatively low, but notably increases in pathological conditions marked by endothelial dysfunction. EVs released by ECs contain numerous endothelial markers, including endoglin/CD105, E-selectinCD62E, S-endo/CD146, vascular endothelial cadherin/CD144, platelet endothelial cell adhesion molecule 1/ CD31, and intercellular adhesion molecule 1 /CD54 (133). SARS-CoV-2 infection associated with the release of EVs carrying TF into the bloodstream which activate platelets and ECs, thereby contributing to COVID-19-related thrombosis in patients (134, 135). Given the critical role of ECs in vascular homeostasis and the prospective association of COVID-19 with endothelial injury or dysfunction, it appears that patients with preexisting endothelial dysfunction in various disease states (e.g., diabetes, atherosclerosis, and hypertension) are vulnerable to a more severe disease course (136). For instance, among the comorbidities, diabetes mellitus (DM) was the most frequently reported (10.9% of cases) condition (137). Studies from China, Europe, UK and the US have also found that when people with DM acquire COVID-19, they are more likely to develop COVID-19-related complications, require ICU hospitalization, or die from the disease (138-140). The possible root cause might be chronic endothelial dysfunction due to DM together with the direct damage of ECs by SARS-CoV-2-mediated inflammatory responses result in further impairment of the microcirculation contributing to pathophysiology of acute respiratory syndrome and multi-organ failure.ConclusionExperimental and clinical evidence from our group and others suggests that SARS-CoV-2 is unlikely to productively infect endothelial cells. Instead, elevated circulatory mediators, such as cytokines, extracellular matrix components, extracellular vesicles, lipids/lipoproteins, and thrombin, are likely the primary drivers of endothelial dysfunction during SARS-CoV-2 infection. Additionally, accumulating evidence indicates that SARS-CoV-2-mediated endothelial glycocalyx damage disrupts vascular homeostasis by altering vascular permeability, cell adhesion, mechanosensing, and antithrombotic and anti-inflammatory functions.Given the mixed findings on endothelial infectivity and the involvement of multiple (co)-receptors, further research is needed. First, developing better animal models that demonstrate endothelial infectivity of SARS-CoV-2 could help identify potential co-receptors and validate direct infection. Second, there is a possibility that SARS-CoV-2 enters endothelial cells without effective replication. Initial entry may trigger significant interferon and inflammatory signaling, but rapid RNA degradation could render the infection undetectable. These hypotheses warrant further investigation.Besides, the variation in detection of SARS-CoV-2 or viral particles might be due to the methodological differences, such as tissue sampling techniques, sensitivity of detection methods, and variations in experimental models. In addition, biological variability, including differences in patient population and disease severity, could lay a role in the observed variability across studies. Thus, further systematic review is required to identify the methodological approaches contributes to discrepancies. Although many models tested by the researchers to dissect out the endothelial involvement and insights into pathogen-host interactions during SARS-CoV-2 infection, the effects of interventions, differences in immune responses, disease progression, and tissue pathology between animals and humans can influence the relevance of the findings.Alternatively, infected ECs can be cleared from the system by various immune mechanisms, such as phagocytosis by immune cells like macrophages, or through apoptosis induced by immune responses. This can make it challenging to detect infections or inflammation in the endothelial layer, as the infected cells may be removed before they can be adequately studied or identified. However, considering that studies typically sample at different time points, the likelihood of this occurring is low. However, it is important to carefully investigate this possibility to draw a well-informed conclusion, ensuring that immune clearance mechanisms are accounted for in the analysis.Nonetheless, current clinical and experimental evidence confirms that endothelial dysfunction is a hallmark of COVID-19, making it a critical therapeutic target. Addressing this dysfunction may help protect vulnerable individuals from severe hyperinflammation and hypercoagulation. To mitigate pro-thrombotic complications, the International Society on Thrombosis and Haemostasis (ISTH) recommends universal standard thromboprophylaxis with low molecular weight heparin or unfractionated heparin (LMWH/UFH) for all hospitalized patients, unless contraindicated (141).

Infections are one of the most significant healthcare and economic burdens across the world as underscored by the recent coronavirus pandemic. Moreover, with the increasing incidence of antimicrobial resistance, there is an urgent need to better understand host-pathogen interactions to design effective treatment strategies. The complement system is a key arsenal of the host defense response to pathogens and bridges both innate and adaptive immunity. However, in the contest between pathogens and host defense mechanisms, the host is not always victorious. Pathogens have evolved several approaches, including co-opting the host complement regulators to evade complement-mediated killing. Furthermore, deficiencies in the complement proteins, both genetic and therapeutic, can lead to an inefficient complement-mediated pathogen eradication, rendering the host more susceptible to certain infections. On the other hand, overwhelming infection can provoke fulminant complement activation with uncontrolled inflammation and potentially fatal tissue and organ damage. This review presents an overview of critical aspects of the complement-pathogen interactions during infection and discusses perspectives on designing therapies to mitigate complement dysfunction and limit tissue injury.