CLEC5A mediates Zika virus-induced testicular damage.

BACKGROUND: Zika virus (ZIKV) infection is clinically known to induce testicular swelling, termed orchitis, and potentially impact male sterility, but the underlying mechanisms remain unclear. Previous reports suggested that C-type lectins play important roles in mediating virus-induced inflammatory reactions and pathogenesis. We thus investigated whether C-type lectins modulate ZIKV-induced testicular damage. METHODS: C-type lectin domain family 5 member A (CLEC5A) knockout mice were generated in a STAT1-deficient immunocompromised background (denoted clec5a-/-stat1-/-) to enable testing of the role played by CLEC5A after ZIKV infection in a mosquito-to-mouse disease model. Following ZIKV infection, mice were subjected to an array of analyses to evaluate testicular damage, including ZIKV infectivity and neutrophil infiltration estimation via quantitative RT-PCR or histology and immunohistochemistry, inflammatory cytokine and testosterone detection, and spermatozoon counting. Furthermore, DNAX-activating proteins for 12 kDa (DAP12) knockout mice (dap12-/-stat1-/-) were generated and used to evaluate ZIKV infectivity, inflammation, and spermatozoa function in order to investigate the potential mechanisms engaged by CLEC5A. RESULTS: Compared to experiments conducted in ZIKV-infected stat1-/- mice, infected clec5a-/-stat1-/- mice showed reductions in testicular ZIKV titer, local inflammation and apoptosis in testis and epididymis, neutrophil invasion, and sperm count and motility. CLEC5A, a myeloid pattern recognition receptor, therefore appears involved in the pathogenesis of ZIKV-induced orchitis and oligospermia. Furthermore, DAP12 expression was found to be decreased in the testis and epididymis tissues of clec5a-/-stat1-/- mice. As for CLEC5A deficient mice, ZIKV-infected DAP12-deficient mice also showed reductions in testicular ZIKV titer and local inflammation, as well as improved spermatozoa function, as compared to controls. CLEC5A-associated DAP12 signaling appears to in part regulate ZIKV-induced testicular damage. CONCLUSIONS: Our analyses reveal a critical role for CLEC5A in ZIKV-induced proinflammatory responses, as CLEC5A enables leukocytes to infiltrate past the blood-testis barrier and induce testicular and epididymal tissue damage. CLEC5A is thus a potential therapeutic target for the prevention of injuries to male reproductive organs in ZIKV patients.

CLEC5A and TLR2 are critical in SARS-CoV-2-induced NET formation and lung inflammation.

BACKGROUND: Coronavirus-induced disease 19 (COVID-19) infects more than three hundred and sixty million patients worldwide, and people with severe symptoms frequently die of acute respiratory distress syndrome (ARDS). Recent studies indicated that excessive neutrophil extracellular traps (NETs) contributed to immunothrombosis, thereby leading to extensive intravascular coagulopathy and multiple organ dysfunction. Thus, understanding the mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-induced NET formation would be helpful to reduce thrombosis and prevent ARDS in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. METHODS: We incubated SARS-CoV-2 with neutrophils in the presence or absence of platelets to observe NET formation. We further isolated extracellular vesicles from COVID-19 patients' sera (COVID-19-EVs) to examine their ability to induce NET formation. RESULTS: We demonstrated that antagonistic mAbs against anti-CLEC5A mAb and anti-TLR2 mAb can inhibit COVID-19-EVs-induced NET formation, and generated clec5a-/-/tlr2-/- mice to confirm the critical roles of CLEC5A and TLR2 in SARS-CoV-2-induced lung inflammation in vivo. We found that virus-free extracellular COVID-19 EVs induced robust NET formation via Syk-coupled C-type lectin member 5A (CLEC5A) and TLR2. Blockade of CLEC5A inhibited COVID-19 EVs-induced NETosis, and simultaneous blockade of CLEC5A and TLR2 further suppressed SARS-CoV-2-induced NETosis in vitro. Moreover, thromboinflammation was attenuated dramatically in clec5a-/-/tlr2-/- mice. CONCLUSIONS: This study demonstrates that SARS-CoV-2-activated platelets produce EVs to enhance thromboinflammation via CLEC5A and TLR2, and highlight the importance of CLEC5A and TLR2 as therapeutic targets to reduce the risk of ARDS in COVID-19 patients.

C-type lectins and extracellular vesicles in virus-induced NETosis.

Dysregulated formation of neutrophil extracellular traps (NETs) is observed in acute viral infections. Moreover, NETs contribute to the pathogenesis of acute viral infections, including those caused by the dengue virus (DV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Furthermore, excessive NET formation (NETosis) is associated with disease severity in patients suffering from SARS-CoV-2-induced multiple organ injuries. Dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) and other members of C-type lectin family (L-SIGN, LSECtin, CLEC10A) have been reported to interact with viral glycans to facilitate virus spreading and exacerbates inflammatory reactions. Moreover, spleen tyrosine kinase (Syk)-coupled C-type lectin member 5A (CLEC5A) has been shown as the pattern recognition receptor for members of flaviviruses, and is responsible for DV-induced cytokine storm and Japanese encephalomyelitis virus (JEV)-induced neuronal inflammation. Moreover, DV activates platelets via CLEC2 to release extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs). The DV-activated EXOs (DV-EXOs) and MVs (DV-MVs) stimulate CLEC5A and Toll-like receptor 2 (TLR2), respectively, to enhance NET formation and inflammatory reactions. Thus, EVs from virus-activated platelets (PLT-EVs) are potent endogenous danger signals, and blockade of C-type lectins is a promising strategy to attenuate virus-induced NETosis and intravascular coagulopathy.

CLEC5A: A Promiscuous Pattern Recognition Receptor to Microbes and Beyond.

CLEC5A is a spleen tyrosine kinase (Syk)-coupled C-type lectin that is highly expressed by monocytes, macrophages, neutrophils, and dendritic cells and interacts with virions directly, via terminal fucose and mannose moieties of viral glycans. CLEC5A also binds to N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc) disaccharides of bacterial cell walls. Compared to other C-type lectins (DC-SIGN and DC-SIGNR) and TLRs, CLEC5A binds its ligands with relatively low affinities. However, CLEC5A forms a multivalent hetero-complex with DC-SIGN and other C-type lectins upon engagement with ligands, and thereby mediates microbe-induced inflammatory responses via activation of Syk. For example, in vivo studies in mouse models have demonstrated that CLEC5A is responsible for flaviviruses-induced hemorrhagic shock and neuroinflammation, and a CLEC5A polymorphism in humans is associated with disease severity following infection with dengue virus. In addition, CLEC5A is co-activated with TLR2 by Listeria and Staphylococcus. Furthermore, CLEC5A-postive myeloid cells are responsible for Concanavilin A-induced aseptic inflammatory reactions. Thus, CLEC5A is a promiscuous pattern recognition receptor in myeloid cells and is a potential therapeutic target for attenuation of both septic and aseptic inflammatory reactions.

NanoBioAnalytical characterization of extracellular vesicles in 75-nm nanofiltered human plasma for transfusion: A tool to improve transfusion safety.

Plasma transfusion induces some transfusion related acute lung injury (TRALI) mediated through neutrophil extracellular traps (NETs). We investigated whether extracellular vesicles (EVs) present in plasma or obtained from resting (N-PEVs) or thrombin activated platelets (T-PEVs) can trigger NETs, and whether 75 nm-nanofiltration, to partially remove EVs, prohibits NETs formation. EVs size and concentration were determined by conventional biophysical approaches and by an original NanoBioAnalytical (NBA) platform based on EV immunocapture biochip, combining Surface Plasmon Resonance Imaging (SPRi) and Atomic Force Microscopy (AFM) exploration. EVs effective diameter was in the 25-1000 nm range, with a majority (≈ 90%) ≤ 100 nm. Both T-PEVs in buffer (but not N-PEVs) and non-nanofiltered plasma containing T-PEVs triggered NETs formation. Nanofiltration depleted large EVs (> 70 nm) and decreased NETs formation. The NBA platform was found to be a suitable tool to investigate the safety of plasma for transfusion.

Inhibition of SARS-CoV-2-mediated thromboinflammation by CLEC2.Fc.

Thromboinflammation is the major cause of morbidity and mortality in COVID-19 patients, and post-mortem examination demonstrates the presence of platelet-rich thrombi and microangiopathy in visceral organs. Moreover, persistent microclots were detected in both acute COVID-19 and long COVID plasma samples. However, the molecular mechanism of SARS-CoV-2-induced thromboinflammation is still unclear. We found that the spleen tyrosine kinase (Syk)-coupled C-type lectin member 2 (CLEC2), which was highly expressed in platelets and alveolar macrophages, interacted with the receptor-binding domain (RBD) of SARS-CoV-2 spike protein (SARS-CoV-2 RBD) directly. Unlike the thread-like NETs, SARS-CoV-2-induced aggregated NET formation in the presence of wild-type (WT), but not CLEC2-deficient platelets. Furthermore, SARS-CoV-2 spike pseudotyped lentivirus was able to induce NET formation via CLEC2, indicating SARS-CoV-2 RBD engaged CLEC2 to activate platelets to enhance NET formation. Administration of CLEC2.Fc inhibited SARS-CoV-2-induced NET formation and thromboinflammation in AAV-ACE2-infected mice. Thus, CLEC2 is a novel pattern recognition receptor for SARS-CoV-2, and CLEC2.Fc and may become a promising therapeutic agent to inhibit SARS-CoV-2-induced thromboinflammation and reduced the risk of post-acute sequelae of COVID-19 (PASC) in the future.

CLEC5A is critical in Pseudomonas aeruginosa-induced NET formation and acute lung injury.

Pseudomonas aeruginosa is one of the most common nosocomial infections worldwide, and it frequently causes ventilator-associated acute pneumonia in immunocompromised patients. Abundant neutrophil extracellular traps (NETs) contribute to acute lung injury, thereby aggravating ventilator-induced lung damage. While pattern recognition receptors (PRRs) TLR4 and TLR5 are required for host defense against P. aeruginosa invasion, the PRR responsible for P. aeruginosa-induced NET formation, proinflammatory cytokine release, and acute lung injury remains unclear. We found that myeloid C-type lectin domain family 5 member A (CLEC5A) interacts with LPS of P. aeruginosa and is responsible for P. aeruginosa-induced NET formation and lung inflammation. P. aeruginosa activates CLEC5A to induce caspase-1-dependent NET formation, but it neither causes gasdermin D (GSDMD) cleavage nor contributes to P. aeruginosa-induced neutrophil death. Blockade of CLEC5A attenuates P. aeruginosa-induced NETosis and lung injury, and simultaneous administration of anti-CLEC5A mAb with ciprofloxacin increases survival rate and decreases collagen deposition in the lungs of mice challenged with a lethal dose of P. aeruginosa. Thus, CLEC5A is a promising therapeutic target to reduce ventilator-associated lung injury and fibrosis in P. aeruginosa-induced pneumonia.

Endosomal TLR3 co-receptor CLEC18A enhances host immune response to viral infection.

Human C-type lectin member 18A (CLEC18A) is ubiquitously expressed in human, and highest expression levels are found in human myeloid cells and liver. In contrast, mouse CLEC18A (mCLEC18A) is only expressed in brain, kidney and heart. However, the biological functions of CLEC18A are still unclear. We have shown that a single amino acid change (S339 →R339) in CTLD domain has profound effect in their binding to polysaccharides and house dust mite allergens. In this study, we further demonstrate that CLEC18A and its mutant CLEC18A(S339R) associate with TLR3 in endosome and bind poly (I:C) specifically. Compared to TLR3 alone, binding affinity to poly (I:C) is further increased in TLR3-CLEC18A and TLR3-CLEC18A(S339R) complexes. Moreover, CLEC18A and CLEC18A(S339R) enhance the production of type I and type III interferons (IFNs), but not proinflammatory cytokines, in response to poly (I:C) or H5N1 influenza A virus (IAV) infection. Compared to wild type (WT) mice, ROSA-CLEC18A and ROSA-CLEC18A(S339R) mice generate higher amounts of interferons and are more resistant to H5N1 IAV infection. Thus, CLEC18A is a TLR3 co-receptor, and may contribute to the differential immune responses to poly (I:C) and IAV infection between human and mouse.

SIGLEC-3 (CD33) serves as an immune checkpoint receptor for HBV infection.

Chronic hepatitis B (CHB) infection is rarely eradicated by current antiviral nucleos(t)ide analogues. We found that α2,6-biantennary sialoglycans of HBV surface antigen (HBsAg) bound human SIGLEC-3 (CD33) by IP and ELISA, and the binding affinity between SIGLEC-3 and α2,6-biantennary sialoglycans was determined by biolayer interferometry (equilibrium dissociation constant [KD]: 1.95 × 10-10 ± 0.21 × 10-10 M). Moreover, HBV activated SIGLEC-3 on myeloid cells and induced immunosuppression by stimulating immunoreceptor tyrosine-based inhibitory motif phosphorylation and SHP-1/-2 recruitment via α2,6-biantennary sialoglycans on HBsAg. An antagonistic anti-SIGLEC-3 mAb reversed this effect and enhanced cytokine production in response to TLR-7 agonist GS-9620 in PBMCs from CHB patients. Moreover, anti-SIGLEC-3 mAb alone was able to upregulate the expression of molecules involved in antigen presentation, such as CD80, CD86, CD40, MHC-I, MHC-II, and PD-L1 in CD14+ cells. Furthermore, SIGLEC-3 SNP rs12459419 C, which expressed a higher amount of SIGLEC-3, was associated with increased risk of hepatocellular carcinoma (HCC) in CHB patients (HR: 1.256, 95% CI: 1.027-1.535, P = 0.0266). Thus, blockade of SIGLEC-3 is a promising strategy to reactivate host immunity to HBV and lower the incidence of HCC in the CHB patient population.

CLEC2 and CLEC5A: Pathogenic Host Factors in Acute Viral Infections.

The protective roles of endosomal toll-like receptors (TLRs) and cytosolic nucleic acid sensors are well elucidated, but the pathogenic host factors during viral infections remain unclear. Spleen tyrosine kinase (Syk)-coupled C-type lectins (CLECs) CLEC2 and CLEC5A are highly expressed on platelets and myeloid cells, respectively. CLEC2 has been shown to recognize snake venom aggretin and the endogenous ligand podoplanin and acts as a critical regulator in the development and immunothrombosis. Although CLEC2 has been reported to interact with type I immunodeficiency virus (HIV-1), its role in viral infections is still unclear. CLEC5A binds to fucose and mannose moieties of dengue virus membrane glycans, as well as to N-acetylglucosamine (GlcNAc)/N-acetylmuramic acid (MurNAc) disaccharides that form the backbone of L. monocytogenes peptidoglycans. Recently, we demonstrated that both CLEC2 and CLEC5A are critical in microbe-induced "neutrophil extracellular trap" (NET) formation and proinflammatory cytokine production. Moreover, activation of CLEC2 by dengue virus (DV) and H5N1 influenza virus (IAV) induces the release of extracellular vesicles (EVs), which further enhance NETosis and proinflammatory cytokine production via CLEC5A and Toll-like receptor 2 (TLR2). These findings not only illustrate the immunomodulatory effects of EVs during platelet-leukocyte interactions, but also demonstrate the critical roles of CLEC2 and CLEC5A in acute viral infections.

Extracellular vesicles from CLEC2-activated platelets enhance dengue virus-induced lethality via CLEC5A/TLR2.

Platelet-leukocyte interactions amplify inflammatory reactions, but the underlying mechanism is still unclear. CLEC5A and CLEC2 are spleen tyrosine kinase (Syk)-coupled C-type lectin receptors, abundantly expressed by leukocytes and platelets, respectively. Whereas CLEC5A is a pattern recognition receptor (PRR) to flaviviruses and bacteria, CLEC2 is the receptor for platelet-activating snake venom aggretin. Here we show that dengue virus (DV) activates platelets via CLEC2 to release extracellular vesicles (EVs), including exosomes (EXOs) and microvesicles (MVs). DV-induced EXOs (DV-EXOs) and MVs (DV-MVs) further activate CLEC5A and TLR2 on neutrophils and macrophages, thereby induce neutrophil extracellular trap (NET) formation and proinflammatory cytokine release. Compared to  stat1-/- mice, simultaneous blockade of CLEC5A and TLR2 effectively attenuates DV-induced inflammatory response and increases survival rate from 30 to 90%. The identification of critical roles of CLEC2 and CLEC5A/TLR2 in platelet-leukocyte interactions will support the development of novel strategies to treat acute viral infection in the future.