Spleen tyrosine kinase inhibition restores myeloid homeostasis in COVID-19.

Spleen tyrosine kinase (SYK) is a previously unidentified therapeutic target that inhibits neutrophil and macrophage activation in coronavirus disease 2019 (COVID-19). Fostamatinib, a SYK inhibitor, was studied in a phase 2 placebo-controlled randomized clinical trial and was associated with improvements in many secondary end points related to efficacy. Here, we used a multiomic approach to evaluate cellular and soluble immune mediator responses of patients enrolled in this trial. We demonstrated that SYK inhibition was associated with reduced neutrophil activation, increased circulation of mature neutrophils (CD10+CD33-), and decreased circulation of low-density granulocytes and polymorphonuclear myeloid-derived suppressor cells (HLA-DR-CD33+CD11b-). SYK inhibition was also associated with normalization of transcriptional activity in circulating monocytes relative to healthy controls, an increase in frequency of circulating nonclassical and HLA-DRhi classical monocyte populations, and restoration of interferon responses. Together, these data suggest that SYK inhibition may mitigate proinflammatory myeloid cellular and soluble mediator responses thought to contribute to immunopathogenesis of severe COVID-19.

Small-molecule screening identifies Syk kinase inhibition and rutaecarpine as modulators of macrophage training and SARS-CoV-2 infection.

Biologically active small molecules can impart modulatory effects, in some cases providing extended long-term memory. In a screen of biologically active small molecules for regulators of tumor necrosis factor (TNF) induction, we identify several compounds with the ability to induce training effects on human macrophages. Rutaecarpine shows acute and long-term modulation, enhancing lipopolysaccharide (LPS)-induced pro-inflammatory cytokine secretion and relieving LPS tolerance in human macrophages. Rutaecarpine inhibits ß-glucan-induced H3K4Me3 marks at the promoters of several pro-inflammatory cytokines, highlighting the potential of this molecule to modulate chromosomal topology. Syk kinase inhibitor (SYKi IV), another screen hit, promotes an enhanced response to LPS similar to that previously reported for ß-glucan-induced training. Macrophages trained with SYKi IV show a high degree of resistance to influenza A, multiple variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and OC43 coronavirus infection, highlighting a potential application of this molecule and other SYKis as prophylactic treatments for viral susceptibility.

Spleen tyrosine kinase mediates innate and adaptive immune crosstalk in SARS-CoV-2 mRNA vaccination.

Durable cell-mediated immune responses require efficient innate immune signaling and the release of pro-inflammatory cytokines. How precisely mRNA vaccines trigger innate immune cells for shaping antigen specific adaptive immunity remains unknown. Here, we show that SARS-CoV-2 mRNA vaccination primes human monocyte-derived macrophages for activation of the NLRP3 inflammasome. Spike protein exposed macrophages undergo NLRP3-driven pyroptotic cell death and subsequently secrete mature interleukin-1ß. These effects depend on activation of spleen tyrosine kinase (SYK) coupled to C-type lectin receptors. Using autologous cocultures, we show that SYK and NLRP3 orchestrate macrophage-driven activation of effector memory T cells. Furthermore, vaccination-induced macrophage priming can be enhanced with repetitive antigen exposure providing a rationale for prime-boost concepts to augment innate immune signaling in SARS-CoV-2 vaccination. Collectively, these findings identify SYK as a regulatory node capable of differentiating between primed and unprimed macrophages, which modulate spike protein-specific T cell responses.

Markers of fungal translocation are elevated during post-acute sequelae of SARS-CoV-2 and induce NF-κB signaling.

Long COVID, a type of post-acute sequelae of SARS-CoV-2 (PASC), has been associated with sustained elevated levels of immune activation and inflammation. However, the mechanisms that drive this inflammation remain unknown. Inflammation during acute coronavirus disease 2019 could be exacerbated by microbial translocation (from the gut and/or lung) to blood. Whether microbial translocation contributes to inflammation during PASC is unknown. We did not observe a significant elevation in plasma markers of bacterial translocation during PASC. However, we observed higher levels of fungal translocation - measured as ß-glucan, a fungal cell wall polysaccharide - in the plasma of individuals experiencing PASC compared with those without PASC or SARS-CoV-2-negative controls. The higher ß-glucan correlated with higher inflammation and elevated levels of host metabolites involved in activating N-methyl-d-aspartate receptors (such as metabolites within the tryptophan catabolism pathway) with established neurotoxic properties. Mechanistically, ß-glucan can directly induce inflammation by binding to myeloid cells (via Dectin-1) and activating Syk/NF-κB signaling. Using a Dectin-1/NF-κB reporter model, we found that plasma from individuals experiencing PASC induced higher NF-κB signaling compared with plasma from negative controls. This higher NF-κB signaling was abrogated by piceatannol (Syk inhibitor). These data suggest a potential targetable mechanism linking fungal translocation and inflammation during PASC.

Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

Listeria toxin promotes phosphorylation of the inflammasome adaptor ASC through Lyn and Syk to exacerbate pathogen expansion.

Inflammasome activation exacerbates infectious disease caused by pathogens such as Listeria monocytogenes, Staphylococcus aureus, and severe acute respiratory syndrome coronavirus 2. Although these pathogens activate host inflammasomes to regulate pathogen expansion, the mechanisms by which pathogen toxins contribute to inflammasome activation remain poorly understood. Here we show that activation of inflammasomes by Listeria infection is promoted by amino acid residue T223 of listeriolysin O (LLO) independently of its pore-forming activity. LLO T223 is critical for phosphorylation of the inflammasome adaptor ASC at amino acid residue Y144 through Lyn-Syk signaling, which is essential for ASC oligomerization. Notably, a Listeria mutant expressing LLO T223A is impaired in inducing ASC phosphorylation and inflammasome activation. Furthermore, the virulence of LLO T223A mutant is markedly attenuated in vivo due to impaired ability to activate the inflammasome. Our results reveal a function of a pathogen toxin that exacerbates infection by promoting phosphorylation of ASC.

Immune thrombocytopenia: options and new perspectives.

Immune thrombocytopenia is a haematological, autoimmune disorder characterized by elevated platelet demolition due to the presence of antiplatelet autoantibodies derived from B cells and to an irregular, deficient process of platelets production in bone marrow. In this review, after a brief presentation of 'old' strategies used nowadays yet, we focused on new drugs used in the treatment of immune thrombocytopenia and their mechanism of action and posology, basing on the last scientific literature. The observation that CoViD-19 can be associated with immune thrombocytopenia is also put in evidence. Particular attention will be dedicated on the concept that the ideal treatment should represent a solution not only for the failure of normal processes of production and survival of platelets, but also it should improve quality of life of patients, with minimum adverse events. Anyway, despite enormous advances of the last years, further investigations are necessary in order to define scrupulously long-term efficacy of new molecules proposed.

Complement activation and increased expression of Syk, mucin-1 and CaMK4 in kidneys of patients with COVID-19.

Acute and chronic kidney failure is common in hospitalized patients with COVID-19, yet the mechanism of injury and predisposing factors remain poorly understood. We investigated the role of complement activation by determining the levels of deposited complement components (C1q, C3, FH, C5b-9) and immunoglobulin along with the expression levels of the injury-associated molecules spleen tyrosine kinase (Syk), mucin-1 (MUC1) and calcium/calmodulin-dependent protein kinase IV (CaMK4) in the kidney tissues of people who succumbed to COVID-19. We report increased deposition of C1q, C3, C5b-9, total immunoglobulin, and high expression levels of Syk, MUC1 and CaMK4 in the kidneys of COVID-19 patients. Our study provides strong rationale for the expansion of trials involving the use of inhibitors of these molecules, in particular C1q, C3, Syk, MUC1 and CaMK4 to treat patients with COVID-19.

Repurposing Fostamatinib to Combat SARS-CoV-2-Induced Acute Lung Injury.

A screen by Kost-Alimova et al.1 suggests that the FDA-approved SYK inhibitor fostamatinib inhibits MUC1 in the respiratory tract and has the potential to treat serious outcomes of coronavirus COVID-19, including acute respiratory distress syndrome (ARDS) and acute lung injury (ALI).