SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway.

Pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induces inflammatory cytokines and chemokines including IL-6, IL-1ß, TNFa, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and neucleocapsid (N) proteins. When stimulated with extracellular S protein, human lung epithelial cells A549 also produce inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly are non-inflammatory, but elicit an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-κB pathway in a MyD88-dependent manner. Further, such an activation of the NF-κB pathway is abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein induces IL-6, TNF-a, and IL-1 ß in wild-type, but not Tlr2-deficient mice. Together these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.

SARS-CoV-2 spike protein induces inflammation via TLR2-dependent activation of the NF-κB pathway.

The pathogenesis of COVID-19 is associated with a hyperinflammatory response; however, the precise mechanism of SARS-CoV-2-induced inflammation is poorly understood. Here, we investigated direct inflammatory functions of major structural proteins of SARS-CoV-2. We observed that spike (S) protein potently induced inflammatory cytokines and chemokines, including IL-6, IL-1ß, TNFα, CXCL1, CXCL2, and CCL2, but not IFNs in human and mouse macrophages. No such inflammatory response was observed in response to membrane (M), envelope (E), and nucleocapsid (N) proteins. When stimulated with extracellular S protein, human and mouse lung epithelial cells also produced inflammatory cytokines and chemokines. Interestingly, epithelial cells expressing S protein intracellularly were non-inflammatory, but elicited an inflammatory response in macrophages when co-cultured. Biochemical studies revealed that S protein triggers inflammation via activation of the NF-κB pathway in a MyD88-dependent manner. Further, such an activation of the NF-κB pathway was abrogated in Tlr2-deficient macrophages. Consistently, administration of S protein-induced IL-6, TNF-α, and IL-1ß in wild-type, but not Tlr2-deficient mice. Notably, upon recognition of S protein, TLR2 dimerizes with TLR1 or TLR6 to activate the NF-κB pathway. Taken together, these data reveal a mechanism for the cytokine storm during SARS-CoV-2 infection and suggest that TLR2 could be a potential therapeutic target for COVID-19.

The scavenger receptor class B type I adaptor protein PDZK1 maintains endothelial monolayer integrity.

Circulating levels of high-density lipoprotein (HDL) cholesterol are inversely related to the risk of cardiovascular disease, and HDL and the HDL receptor scavenger receptor class B type I (SR-BI) initiate signaling in endothelium through src that promotes endothelial NO synthase activity and cell migration. Such signaling requires the C-terminal PDZ-interacting domain of SR-BI. Here we show that the PDZ domain-containing protein PDZK1 is expressed in endothelium and required for HDL activation of endothelial NO synthase and cell migration; in contrast, endothelial cell responses to other stimuli, including vascular endothelial growth factor, are PDZK1-independent. Coimmunoprecipitation experiments reveal that Src interacts with SR-BI, and this process is PDZK1-independent. PDZK1 also does not regulate SR-BI abundance or plasma membrane localization in endothelium or HDL binding or cholesterol efflux. Alternatively, PDZK1 is required for HDL/SR-BI to induce Src phosphorylation. Paralleling the in vitro findings, carotid artery reendothelialization following perivascular electric injury is absent in PDZK1-/- mice, and this phenotype persists in PDZK1-/- mice with genetic reconstitution of PDZK1 expression in liver, where PDZK1 modifies SR-BI abundance. Thus, PDZK1 is uniquely required for HDL/SR-BI signaling in endothelium, and through these mechanisms, it is critically involved in the maintenance of endothelial monolayer integrity.

The Receptor for Hyaluronan-Mediated Motility (CD168) promotes inflammation and fibrosis after acute lung injury.

Acute lung injury results in early inflammation and respiratory distress, and later fibrosis. The glycosaminoglycan hyaluronan (HA) and the Receptor for Hyaluronan-Mediated Motility (RHAMM, CD168) have been implicated in the response to acute lung injury. We hypothesized that, compared to wild type (WT) mice, RHAMM knockout (KO) mice would be protected from, whereas mice with macrophage-specific transgenic overexpression of RHAMM (TG) would have worse inflammation, respiratory distress and fibrosis after intratracheal (IT) bleomycin. Compared to WT mice, 10 days after IT bleomycin, RHAMM KO mice had less weight loss, less increase in respiratory rate, and fewer CD45+ cells in the lung. At day 28, compared to injured WT animals, injured RHAMM KO mice had lower M1 macrophage content, as well as decreased fibrosis as determined by trichrome staining, Ashcroft scores and lung HPO content. Four lines of transgenic mice with selective overexpression of RHAMM in macrophages were generated using the Scavenger Receptor A promoter driving a myc-tagged full length RHAMM cDNA. Baseline expression of RHAMM and CD44 was the same in WT and TG mice. By flow cytometry, TG bone marrow-derived macrophages (BMDM) had increased cell surface RHAMM and myc, but equal CD44 expression. TG BMDM also had 2-fold increases in both chemotaxis to HA and proliferation in fetal bovine serum. In TG mice, increased inflammation after thioglycollate-induced peritonitis was restricted to macrophages and not neutrophils. For lung injury studies, non-transgenic mice given bleomycin had respiratory distress with increased respiratory rates from day 7 to 21. However, TG mice had higher respiratory rates from 4 days after bleomycin and continued to increase respiratory rates up to day 21. At 21 days after IT bleomycin, TG mice had increased lung macrophage accumulation. Lavage HA concentrations were 6-fold higher in injured WT mice, but 30-fold higher in injured TG mice. At 21 days after IT bleomycin, WT mice had developed fibrosis, but TG mice showed exaggerated fibrosis with increased Ashcroft scores and HPO content. We conclude that RHAMM is a critical component of the inflammatory response, respiratory distress and fibrosis after acute lung injury. We speculate that RHAMM is a potential therapeutic target to limit the consequences of acute lung injury.

Fetal-placental crosstalk occurs through fetal cytokine synthesis and placental clearance.

BACKGROUND: Cytokines modulate fetal well-being and contribute to parturition. Their origin in fetal blood, whether maternal, placental or fetal, at the time of parturition remains unclear. OBJECTIVE: To determine fetal and placental contributions to circulating fetal cytokines by measuring umbilical arterial (UmA) and venous (UmV) concentration differences in uncomplicated term pregnancies in the absence and presence of labor. METHODS: Term uncomplicated pregnancies were assessed: Group 1 were not in labor and delivered by elective cesarean section (n = 20); Group 2 delivered vaginally following uncomplicated pregnancy and labor (n = 30). UmA and UmV blood was collected before delivery of the placenta to measure circulating cytokines. Placental tissue was collected for histology and to determine cytokine contents and localization. RESULTS: Group 1 UmA and UmV IL-10 concentrations were similar (504 ±â€¯15 and 468 ±â€¯16 pg/ml, respectively; P ≥ 0.1); other cytokines were below level of detection. During labor, IL-10 concentrations increased 15-34%, but placental contents decreased. Group 2 UmA IL-6 and IL-8 concentrations increased (P < 0.001) to 16.7 ±â€¯1.6 and 18.4 ±â€¯4.3 pg/ml, respectively, but were less (P < 0.001) in UmV, 0.29 ±â€¯0.2 and 0.74 ±â€¯0.3 pg/ml, respectively, demonstrating placental clearances ≥97%. This was associated with >6-fold increases in placental IL-6/IL-8 contents (P < 0.001) and chorioamniotic infiltration of activated maternal neutrophils. IL-6 and IL-10 were localized to villous syncytiotrophoblasts. CONCLUSIONS: In uncomplicated term pregnancies fetal circulating IL-10 is likely of placental origin, whereas IL-6/IL-8 are derived from the fetus, increase during parturition, and circulating levels are modulated by non-saturable placental clearance, revealing a novel pathway for fetal-placental crosstalk and signaling.

The NLRP3 inflammasome is critically involved in the development of bronchopulmonary dysplasia.

The pathogenesis of bronchopulmonary dysplasia (BPD), a devastating lung disease in preterm infants, includes inflammation, the mechanisms of which are not fully characterized. Here we report that the activation of the NLRP3 inflammasome is associated with the development of BPD. Hyperoxia-exposed neonatal mice have increased caspase-1 activation, IL1ß and inflammation, and decreased alveolarization. Nlrp3(-/-) mice have no caspase-1 activity, no IL1ß, no inflammatory response and undergo normal alveolarization. Treatment of hyperoxia-exposed mice with either IL1 receptor antagonist to block IL1ß or glyburide to block the Nlrp3 inflammasome results in decreased inflammation and increased alveolarization. Ventilated preterm baboons show activation of the NLRP3 inflammasome with increased IL1ß:IL1ra ratio. The IL1ß:IL1ra ratio in tracheal aspirates from preterm infants with respiratory failure is predictive of the development of BPD. We conclude that early activation of the NLRP3 inflammasome is a key mechanism in the development of BPD, and represents a novel therapeutic target for BPD.

Antioxidant protects against increases in low molecular weight hyaluronan and inflammation in asphyxiated newborn pigs resuscitated with 100% oxygen.

BACKGROUND: Newborn resuscitation with 100% oxygen is associated with oxidative-nitrative stresses and inflammation. The mechanisms are unclear. Hyaluronan (HA) is fragmented to low molecular weight (LMW) by oxidative-nitrative stresses and can promote inflammation. We examined the effects of 100% oxygen resuscitation and treatment with the antioxidant, N-acetylcysteine (NAC), on lung 3-nitrotyrosine (3-NT), LMW HA, inflammation, TNFα and IL1ß in a newborn pig model of resuscitation. METHODS & PRINCIPAL FINDINGS: Newborn pigs (n = 40) were subjected to severe asphyxia, followed by 30 min ventilation with either 21% or 100% oxygen, and were observed for the subsequent 150 minutes in 21% oxygen. One 100% oxygen group was treated with NAC. Serum, bronchoalveolar lavage (BAL), lung sections, and lung tissue were obtained. Asphyxia resulted in profound hypoxia, hypercarbia and metabolic acidosis. In controls, HA staining was in airway subepithelial matrix and no 3-NT staining was seen. At the end of asphyxia, lavage HA decreased, whereas serum HA increased. At 150 minutes after resuscitation, exposure to 100% oxygen was associated with significantly higher BAL HA, increased 3NT staining, and increased fragmentation of lung HA. Lung neutrophil and macrophage contents, and serum TNFα and IL1ß were higher in animals with LMW than those with HMW HA in the lung. Treatment of 100% oxygen animals with NAC blocked nitrative stress, preserved HMW HA, and decreased inflammation. In vitro, peroxynitrite was able to fragment HA, and macrophages stimulated with LMW HA increased TNFα and IL1ß expression. CONCLUSIONS & SIGNIFICANCE: Compared to 21%, resuscitation with 100% oxygen resulted in increased peroxynitrite, fragmentation of HA, inflammation, as well as TNFα and IL1ß expression. Antioxidant treatment prevented the expression of peroxynitrite, the degradation of HA, and also blocked increases in inflammation and inflammatory cytokines. These findings provide insight into potential mechanisms by which exposure to hyperoxia results in systemic inflammation.