The complement system: A key player in the host response to infections.

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.

Myeloid cell-derived interleukin-6 induces vascular dysfunction and vascular and systemic inflammation.

Aims: The cytokine interleukin-6 (IL-6) plays a central role in the inflammation cascade as well as cardiovascular disease progression. Since myeloid cells are a primary source of IL-6 formation, we aimed to generate a mouse model to study the role of myeloid cell-derived IL-6 in vascular disease. Methods and results: Interleukin-6-overexpressing (IL-6OE) mice were generated and crossed with LysM-Cre mice, to generate mice (LysM-IL-6OE mice) overexpressing the cytokine in myeloid cells. Eight- to 12-week-old LysM-IL-6OE mice spontaneously developed inflammatory colitis and significantly impaired endothelium-dependent aortic relaxation, increased aortic reactive oxygen species (ROS) formation, and vascular dysfunction in resistance vessels. The latter phenotype was associated with decreased survival. Vascular dysfunction was accompanied by a significant accumulation of neutrophils, monocytes, and macrophages in the aorta, increased myeloid cell reactivity (elevated ROS production), and vascular fibrosis associated with phenotypic changes in vascular smooth muscle cells. In addition to elevated Mcp1 and Cxcl1 mRNA levels, aortae from LysM-IL-6OE mice expressed higher levels of inducible NO synthase and endothelin-1, thus partially accounting for vascular dysfunction, whereas systemic blood pressure alterations were not observed. Bone marrow (BM) transplantation experiments revealed that vascular dysfunction and ROS formation were driven by BM cell-derived IL-6 in a dose-dependent manner. Conclusion: Mice with conditional overexpression of IL-6 in myeloid cells show systemic and vascular inflammation as well as endothelial dysfunction. A decrease in circulating IL-6 levels by replacing IL-6-producing myeloid cells in the BM improved vascular dysfunction in this model, underpinning the relevant role of IL-6 in vascular disease.

Aging is associated with an insufficient early inflammatory response of lung endothelial cells in SARS-CoV-2 infection.

Advanced age is associated with an increased susceptibility to Coronavirus Disease (COVID)-19 and more severe outcomes, although the underlying mechanisms are understudied. The lung endothelium is located next to infected epithelial cells and bystander inflammation may contribute to thromboinflammation and COVID-19-associated coagulopathy. Here, we investigated age-associated SARS-CoV-2 pathogenesis and endothelial inflammatory responses using humanized K18-hACE2 mice. Survival was reduced to 20% in aged mice (85-112 weeks) versus 50% in young mice (12-15 weeks) at 10 days post infection (dpi). Bulk RNA-sequencing of endothelial cells from mock and infected mice at 2dpi of both age groups (aged: 72-85 weeks; young: 15 weeks) showed substantially lower significant differentially regulated genes in infected aged mice than in young mice (712 versus 2294 genes). Viral recognition and anti-viral pathways such as RIG-I-like receptor signaling, NOD-like receptor signaling and interferon signaling were regulated in response to SARS-CoV-2. Young mice showed several fold higher interferon responses (Ifitm3, Ifit1, Isg15, Stat1) and interferon-induced chemokines (Cxcl10 and Cxcl11) than aged mice. Endothelial cells from infected young mice displayed elevated expression of chemokines (Cxcl9, Ccl2) and leukocyte adhesion markers (Icam1) underscoring that inflammation of lung endothelium during infection could facilitate leukocyte adhesion and thromboinflammation. TREM1 and acute phase response signaling were particularly prominent in endothelial cells from infected young mice. Immunohistochemistry was unable to detect viral protein in pulmonary endothelium. In conclusion, our data demonstrate that the early host response of the endothelium to SARS-CoV-2 infection declines with aging, which could be a potential contributor to disease severity.

Nucleic acid sensing promotes inflammatory monocyte migration through biased coagulation factor VIIa signaling.

ABSTRACT: Protease activated receptors (PARs) are cleaved by coagulation proteases and thereby connect hemostasis with innate immune responses. Signaling of the tissue factor (TF) complex with factor VIIa (FVIIa) via PAR2 stimulates extracellular signal-regulated kinase (ERK) activation and cancer cell migration, but functions of cell autonomous TF-FVIIa signaling in immune cells are unknown. Here, we show that myeloid cell expression of FVII but not of FX is crucial for inflammatory cell recruitment to the alveolar space after challenge with the double-stranded viral RNA mimic polyinosinic:polycytidylic acid [Poly(I:C)]. In line with these data, genetically modified mice completely resistant to PAR2 cleavage but not FXa-resistant PAR2-mutant mice are protected from lung inflammation. Poly(I:C)-stimulated migration of monocytes/macrophages is dependent on ERK activation and mitochondrial antiviral signaling (MAVS) but independent of toll-like receptor 3 (TLR3). Monocyte/macrophage-synthesized FVIIa cleaving PAR2 is required for integrin αMß2-dependent migration on fibrinogen but not for integrin ß1-dependent migration on fibronectin. To further dissect the downstream signaling pathway, we generated PAR2S365/T368A-mutant mice deficient in ß-arrestin recruitment and ERK scaffolding. This mutation reduces cytosolic, but not nuclear ERK phosphorylation by Poly(I:C) stimulation, and prevents macrophage migration on fibrinogen but not fibronectin after stimulation with Poly(I:C) or CpG-B, a single-stranded DNA TLR9 agonist. In addition, PAR2S365/T368A-mutant mice display markedly reduced immune cell recruitment to the alveolar space after Poly(I:C) challenge. These results identify TF-FVIIa-PAR2-ß-arrestin-biased signaling as a driver for lung infiltration in response to viral nucleic acids and suggest potential therapeutic interventions specifically targeting TF-VIIa signaling in thrombo-inflammation.

Sterility testing of germ-free mouse colonies.

In biomedical research, germ-free and gnotobiotic mouse models enable the mechanistic investigation of microbiota-host interactions and their role on (patho)physiology. Throughout any gnotobiotic experiment, standardized and periodic microbiological testing of defined gnotobiotic housing conditions is a key requirement. Here, we review basic principles of germ-free isolator technology, the suitability of various sterilization methods, and the use of sterility testing methods to monitor germ-free mouse colonies. We also discuss their effectiveness and limitations, and share the experience with protocols used in our facility. In addition, possible sources of isolator contamination are discussed and an overview of reported contaminants is provided.

Externalized histones fuel pulmonary fibrosis via a platelet-macrophage circuit of TGFß1 and IL-27.

Externalized histones erupt from the nucleus as extracellular traps, are associated with several acute and chronic lung disorders, but their implications in the molecular pathogenesis of interstitial lung disease are incompletely defined. To investigate the role and molecular mechanisms of externalized histones within the immunologic networks of pulmonary fibrosis, we studied externalized histones in human and animal bronchoalveolar lavage (BAL) samples of lung fibrosis. Neutralizing anti-histone antibodies were administered in bleomycin-induced fibrosis of C57BL/6 J mice, and subsequent studies used conditional/constitutive knockout mouse strains for TGFß and IL-27 signaling along with isolated platelets and cultured macrophages. We found that externalized histones (citH3) were significantly (P < 0.01) increased in cell-free BAL fluids of patients with idiopathic pulmonary fibrosis (IPF; n = 29) as compared to healthy controls (n = 10). The pulmonary sources of externalized histones were Ly6G+CD11b+ neutrophils and nonhematopoietic cells after bleomycin in mice. Neutralizing monoclonal anti-histone H2A/H4 antibodies reduced the pulmonary collagen accumulation and hydroxyproline concentration. Histones activated platelets to release TGFß1, which signaled through the TGFbRI/TGFbRII receptor complex on LysM+ cells to antagonize macrophage-derived IL-27 production. TGFß1 evoked multiple downstream mechanisms in macrophages, including p38 MAPK, tristetraprolin, IL-10, and binding of SMAD3 to the IL-27 promotor regions. IL-27RA-deficient mice displayed more severe collagen depositions suggesting that intact IL-27 signaling limits fibrosis. In conclusion, externalized histones inactivate a safety switch of antifibrotic, macrophage-derived IL-27 by boosting platelet-derived TGFß1. Externalized histones are accessible to neutralizing antibodies for improving the severity of experimental pulmonary fibrosis.

Commensal bacteria weaken the intestinal barrier by suppressing epithelial neuropilin-1 and Hedgehog signaling.

The gut microbiota influences intestinal barrier integrity through mechanisms that are incompletely understood. Here we show that the commensal microbiota weakens the intestinal barrier by suppressing epithelial neuropilin-1 (NRP1) and Hedgehog (Hh) signaling. Microbial colonization of germ-free mice dampens signaling of the intestinal Hh pathway through epithelial Toll-like receptor (TLR)-2, resulting in decreased epithelial NRP1 protein levels. Following activation via TLR2/TLR6, epithelial NRP1, a positive-feedback regulator of Hh signaling, is lysosomally degraded. Conversely, elevated epithelial NRP1 levels in germ-free mice are associated with a strengthened gut barrier. Functionally, intestinal epithelial cell-specific Nrp1 deficiency (Nrp1ΔIEC) results in decreased Hh pathway activity and a weakened gut barrier. In addition, Nrp1ΔIEC mice have a reduced density of capillary networks in their small intestinal villus structures. Collectively, our results reveal a role for the commensal microbiota and epithelial NRP1 signaling in the regulation of intestinal barrier function through postnatal control of Hh signaling.

Immunomodulation of neutrophil granulocyte functions by bacterial polyphosphates.

Polyphosphates are highly conserved, linear polymers of monophosphates that reside in all living cells. Bacteria produce long chains containing hundreds to thousands of phosphate units, which can interfere with host defense to infection. Here, we report that intratracheal long-chain polyphosphate administration to C57BL/6J mice resulted in the release of proinflammatory cytokines and influx of Ly6G+ polymorphonuclear neutrophils in the bronchoalveolar lavage fluid causing a disruption of the physiologic endothelial-epithelial small airway barrier and histologic signs of lung injury. Polyphosphate-induced effects were attenuated after neutrophil depletion in mice. In isolated murine neutrophils, long-chain polyphosphates modulated cytokine release induced by lipopolysaccharides (LPS) from Gram-negative bacteria or lipoteichoic acid from Gram-positive bacteria. In addition, long-chain polyphosphates induced immune evasive effects in human neutrophils. In detail, long-chain polyphosphates downregulated CD11b and curtailed the phagocytosis of Escherichia coli particles by neutrophils. Polyphosphates modulated the migration capacity by inducing CD62L shedding resulting in CD62Llow and CD11blow neutrophils. The release of IL-8 induced by LPS was also significantly reduced. Pharmacologic blockade of PI3K with wortmannin antagonized long-chain polyphosphate-induced effects on LPS-induced IL-8 release. In conclusion, polyphosphates govern immunomodulation in murine and human neutrophils, suggesting polyphosphates as a therapeutic target for bacterial infections to restore innate immune defense.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron.

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

Bacterial polyphosphates induce CXCL4 and synergize with complement anaphylatoxin C5a in lung injury.

Polyphosphates are linear polymers of inorganic phosphates that exist in all living cells and serve pleiotropic functions. Bacteria produce long-chain polyphosphates, which can interfere with host defense to infection. In contrast, short-chain polyphosphates are released from platelet dense granules and bind to the chemokine CXCL4. Here, we report that long-chain polyphosphates induced the release of CXCL4 from mouse bone marrow-derived macrophages and peritoneal macrophages in a dose-/time-dependent fashion resulting from an induction of CXCL4 mRNA. This polyphosphate effect was lost after pre-incubation with recombinant exopolyphosphatase (PPX) Fc fusion protein, demonstrating the potency of long chains over monophosphates and ambient cations. In detail, polyphosphate chains >70 inorganic phosphate residues were required to reliably induce CXCL4. Polyphosphates acted independently of the purinergic P2Y1 receptor and the MyD88/TRIF adaptors of Toll-like receptors. On the other hand, polyphosphates augmented LPS/MyD88-induced CXCL4 release, which was explained by intracellular signaling convergence on PI3K/Akt. Polyphosphates induced Akt phosphorylation at threonine-308. Pharmacologic blockade of PI3K (wortmannin, LY294002) antagonized polyphosphate-induced CXCL4 release from macrophages. Intratracheal polyphosphate administration to C57BL/6J mice caused histologic signs of lung injury, disruption of the endothelial-epithelial barrier, influx of Ly6G+ polymorphonuclear neutrophils, depletion of CD11c+SiglecF+ alveolar macrophages, and release of CXCL4. Long-chain polyphosphates synergized with the complement anaphylatoxin, C5a, which was partly explained by upregulation of C5aR1 on myeloid cells. C5aR1-/- mice were protected from polyphosphate-induced lung injury. C5a generation occurred in the lungs and bronchoalveolar lavage fluid (BALF) of polyphosphate-treated C57BL/6J mice. In conclusion, we demonstrate that polyphosphates govern immunomodulation in macrophages and promote acute lung injury.

Tissue factor in COVID-19-associated coagulopathy.

Evidence of micro- and macro-thrombi in the arteries and veins of critically ill COVID-19 patients and in autopsies highlight the occurrence of COVID-19-associated coagulopathy (CAC). Clinical findings of critically ill COVID-19 patients point to various mechanisms for CAC; however, the definitive underlying cause is unclear. Multiple factors may contribute to the prothrombotic state in patients with COVID-19. Aberrant expression of tissue factor (TF), an initiator of the extrinsic coagulation pathway, leads to thrombotic complications during injury, inflammation, and infections. Clinical evidence suggests that TF-dependent coagulation activation likely plays a role in CAC. Multiple factors could trigger abnormal TF expression and coagulation activation in patients with severe COVID-19 infection. Proinflammatory cytokines that are highly elevated in COVID-19 (IL-1ß, IL-6 and TNF-α) are known induce TF expression on leukocytes (e.g. monocytes, macrophages) and non-immune cells (e.g. endothelium, epithelium) in other conditions. Antiphospholipid antibodies, TF-positive extracellular vesicles, pattern recognition receptor (PRR) pathways and complement activation are all candidate factors that could trigger TF-dependent procoagulant activity. In addition, coagulation factors, such as thrombin, may further potentiate the induction of TF via protease-activated receptors on cells. In this systematic review, with other viral infections, we discuss potential mechanisms and cell-type-specific expressions of TF during SARS-CoV-2 infection and its role in the development of CAC.

A single-cell lung atlas of complement genes identifies the mesothelium and epithelium as prominent sources of extrahepatic complement proteins.

To understand functional duality of the complement system in host defense and lung injury, a more comprehensive view of its localized production in the lung, and the impact of age on complement production are essential. Here, we explored the expression of complement genes through computational analysis of preexisting single cell RNA sequencing data from lung transcriptomes of healthy young (3 months) and old C57BL/6 mice (24 months), and humans. We characterized the distribution of 48 complement genes. Across 28 distinct immune and non-immune cell types in mice, mesothelial cells expressed the greatest number of complement genes (e.g., C1ra, C2, C3), and regulators (e.g., Serping1, Cfh). C5 was abundant in type II alveolar epithelial cells and C1q in interstitial lung macrophages. There were only moderate differences in gene expression between young and old mice. Among 57 human lung cell types, mesothelial cells showed abundant complement expression. A few differences in gene  expression (e.g., FCN1, CFI, C6, C7) were also evident between mice and human lung cells. Our findings present a novel perspective on the expression patterns of complement genes in normal lungs. These findings highlight the potential functions of complement in tissue-specific homeostasis and immunity and may foster a mechanistic understanding of its role in lung health and disease.

Humanized mice reveal a macrophage-enriched gene signature defining human lung tissue protection during SARS-CoV-2 infection.

The human immunological mechanisms defining the clinical outcome of SARS-CoV-2 infection remain elusive. This knowledge gap is mostly driven by the lack of appropriate experimental platforms recapitulating human immune responses in a controlled human lung environment. Here, we report a mouse model (i.e., HNFL mice) co-engrafted with human fetal lung xenografts (fLX) and a myeloid-enhanced human immune system to identify cellular and molecular correlates of lung protection during SARS-CoV-2 infection. Unlike mice solely engrafted with human fLX, HNFL mice are protected against infection, severe inflammation, and histopathological phenotypes. Lung tissue protection from infection and severe histopathology associates with macrophage infiltration and differentiation and the upregulation of a macrophage-enriched signature composed of 11 specific genes mainly associated with the type I interferon signaling pathway. Our work highlights the HNFL model as a transformative platform to investigate, in controlled experimental settings, human myeloid immune mechanisms governing lung tissue protection during SARS-CoV-2 infection.

Fatal Neurodissemination and SARS-CoV-2 Tropism in K18-hACE2 Mice Is Only Partially Dependent on hACE2 Expression.

Animal models recapitulating COVID-19 are critical to enhance our understanding of SARS-CoV-2 pathogenesis. Intranasally inoculated transgenic mice expressing human angiotensin-converting enzyme 2 under the cytokeratin 18 promoter (K18-hACE2) represent a lethal model of SARS-CoV-2 infection. We evaluated the clinical and virological dynamics of SARS-CoV-2 using two intranasal doses (104 and 106 PFUs), with a detailed spatiotemporal pathologic analysis of the 106 dose cohort. Despite generally mild-to-moderate pneumonia, clinical decline resulting in euthanasia or death was commonly associated with hypothermia and viral neurodissemination independent of inoculation dose. Neuroinvasion was first observed at 4 days post-infection, initially restricted to the olfactory bulb suggesting axonal transport via the olfactory neuroepithelium as the earliest portal of entry. Absence of viremia suggests neuroinvasion occurs independently of transport across the blood-brain barrier. SARS-CoV-2 tropism was neither restricted to ACE2-expressing cells (e.g., AT1 pneumocytes), nor inclusive of some ACE2-positive cell lineages (e.g., bronchiolar epithelium and brain vasculature). Absence of detectable ACE2 protein expression in neurons but overexpression in neuroepithelium suggest this as the most likely portal of neuroinvasion, with subsequent ACE2 independent lethal neurodissemination. A paucity of epidemiological data and contradicting evidence for neuroinvasion and neurodissemination in humans call into question the translational relevance of this model.

IL-27 Enhances γδ T Cell-Mediated Innate Resistance to Primary Hookworm Infection in the Lungs.

IL-27 is a heterodimeric IL-12 family cytokine formed by noncovalent association of the promiscuous EBI3 subunit and selective p28 subunit. IL-27 is produced by mononuclear phagocytes and unfolds pleiotropic immune-modulatory functions through ligation to IL-27 receptor α (IL-27RA). Although IL-27 is known to contribute to immunity and to limit inflammation after various infections, its relevance for host defense against multicellular parasites is still poorly defined. Here, we investigated the role of IL-27 during infection with the soil-transmitted hookworm, Nippostrongylus brasiliensis, in its early host intrapulmonary life cycle. IL-27(p28) was detectable in bronchoalveolar lavage fluid of C57BL/6J wild-type mice on day 1 after s.c. inoculation. IL-27RA expression was most abundant on lung-invading γδ T cells. Il27ra-/- mice showed increased lung parasite burden together with aggravated pulmonary hemorrhage and higher alveolar total protein leakage as a surrogate for epithelial-vascular barrier disruption. Conversely, injections of recombinant mouse (rm)IL-27 into wild-type mice reduced lung injury and parasite burden. In multiplex screens, higher airway accumulations of IL-6, TNF-α, and MCP-3 (CCL7) were observed in Il27ra-/- mice, whereas rmIL-27 treatment showed a reciprocal effect. Importantly, γδ T cell numbers in airways were enhanced by endogenous or administered IL-27. Further analysis revealed a direct antihelminthic function of IL-27 on γδ T cells as adoptive intratracheal transfer of rmIL-27-treated γδ T cells during primary N. brasiliensis lung infection conferred protection in mice. In summary, this report demonstrates protective functions of IL-27 to control the early lung larval stage of hookworm infection.

Neutrophil Extracellular Traps as an Exacerbating Factor in Bacterial Pneumonia.

Neutrophils are capable of extruding neutrophil extracellular traps (NETs), a network of granule proteins and chromatin material, upon activation. NETs provide defense against extracellular microbes, but histones in NETs can also induce cytotoxicity and activate inflammatory responses. The relevance of NETs to bacterial pneumonias is beginning to be defined. In the present study, we found that the extracellular concentration of citrullinated histone H3, a component of NETs, was elevated in bronchoalveolar lavage fluid recovered from mice with diverse bacterial pneumonias and correlated with neutrophil infiltration and cell death in the lungs as well as levels of H4. Because the histone H4 component of NETs is sufficient to stimulate inflammation, we tested its effects in the air spaces of the lungs. Recombinant histone H4 in the noninflamed lung produced only modest effects, but in the setting of neutrophilic inflammation, H4 substantially increased pulmonary neutrophils, NETs, necrosis, and edema. However, blockade of histone H4 with a monoclonal antibody during pneumonia did not significantly alter measures of lung damage. Taken together, these results implicate NETs and extracellular histone H4 in exacerbating the lung injury resulting from bacterial pneumonia.

Advocacy of targeting protease-activated receptors in severe coronavirus disease 2019.

Identifying drug targets mitigating vascular dysfunction, thrombo-inflammation and thromboembolic complications in COVID-19 is essential. COVID-19 coagulopathy differs from sepsis coagulopathy. Factors that drive severe lung pathology and coagulation abnormalities in COVID-19 are not understood. Protein-protein interaction studies indicate that the tagged viral bait protein ORF9c directly interacts with PAR2, which modulates host cell IFN and inflammatory cytokines. In addition to direct interaction of SARS-CoV-2 viral protein with PARs, we speculate that activation of PAR by proteases plays a role in COVID-19-induced hyperinflammation. In COVID-19-associated coagulopathy elevated levels of activated coagulation proteases may cleave PARs in association with TMPRSS2. PARs activation enhances the release of cytokines, chemokines and tissue factor expression to propagate IFN-dependent inflammation, leukocyte-endothelial interaction, vascular permeability and coagulation responses. This hypothesis, corroborated by in vitro findings and emerging clinical evidence, will focus targeted studies of PAR1/2 blockers as adjuvant drugs against cytokine release syndrome and COVID-19-associated coagulopathy. LINKED ARTICLES: This article is part of a themed issue on The second wave: are we any closer to efficacious pharmacotherapy for COVID 19? (BJP 75th Anniversary). To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.10/issuetoc.