Triangle of cytokine storm, central nervous system involvement, and viral infection in COVID-19: the role of sFasL and neuropilin-1

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) is identified as the cause of coronavirus disease 2019 (COVID-19), and is often linked to extreme inflammatory responses by over activation of neutrophil extracellular traps (NETs), cytokine storm, and sepsis. These are robust causes for multi-organ damage. In particular, potential routes of SARS-CoV2 entry, such as angiotensin-converting enzyme 2 (ACE2), have been linked to central nervous system (CNS) involvement. CNS has been recognized as one of the most susceptible compartments to cytokine storm, which can be affected by neuropilin-1 (NRP-1). ACE2 is widely-recognized as a SARS-CoV2 entry pathway; However, NRP-1 has been recently introduced as a novel path of viral entry. Apoptosis of cells invaded by this virus involves Fas receptor–Fas ligand (FasL) signaling; moreover, Fas receptor may function as a controller of inflammation. Furthermore, NRP-1 may influence FasL and modulate cytokine profile. The neuroimmunological insult by SARS-CoV2 infection may be inhibited by therapeutic approaches targeting soluble Fas ligand (sFasL), cytokine storm elements, or related viral entry pathways. In the current review, we explain pivotal players behind the activation of cytokine storm that are associated with vast CNS injury. We also hypothesize that sFasL may affect neuroinflammatory processes and trigger the cytokine storm in COVID-19.

A Putative Serine Protease is Required to Initiate the RIPK3-MLKL—Mediated Necroptotic Death Pathway in Neutrophils

Adhesion receptors, such as CD44, have been shown to activate receptor interacting protein kinase-3 (RIPK3)—mixed lineage kinase-like (MLKL) signaling, leading to a non-apoptotic cell death in human granulocyte/macrophage colony-stimulating factor (GM-CSF) – primed neutrophils. The signaling events of this necroptotic pathway, however, remain to be investigated. In the present study, we report the design, synthesis, and characterization of a series of novel serine protease inhibitors. Two of these inhibitors, compounds 1 and 3, were able to block CD44-triggered necroptosis in GM-CSF-primed neutrophils. Both inhibitors prevented the activation of MLKL, p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3’—kinase (PI3K), hence blocking the increased levels of reactive oxygen species (ROS) required for cell death. Although compounds one and three partially inhibited isolated human neutrophil elastase (HNE) activity, we obtained no pharmacological evidence that HNE is involved in the initiation of this death pathway within a cellular context. Interestingly, neither serine protease inhibitor had any effect on FAS receptor-mediated apoptosis. Taken together, these results suggest that a serine protease is involved in non-apoptotic CD44-triggered RIPK3-MLKL-dependent neutrophil cell death, but not FAS receptor-mediated caspase-dependent apoptosis. Thus, a pharmacological block on serine proteases might be beneficial for preventing exacerbation of disease in neutrophilic inflammatory responses.

IL-6-driven FasL promotes NF-κBp65/PUMA-mediated apoptosis in portal hypertensive gastropathy

Mucosal epithelial apoptosis with non-specific inflammation is an essential pathological characteristic in portal hypertensive gastropathy (PHG). However, whether a coordinated crosstalk between myeloid cells and epithelial cells involved in PHG remains unclear. IL-6, which is induced in the mucosa of PHG patients and mice, promotes FasL production via enhancing NF-κBp65 activation in myeloid cells, while blockage of IL-6 signaling by Tocilizumab or deletion of NF-κBp65 in myeloid cells attenuates the inflammatory response and Fas/FasL-mediated epithelial apoptosis in PHG. IL-6-driven FasL from myeloid cells combines with epithelial Fas receptor to encourage NF-κBp65/PUMA-mediated epithelial apoptosis in PHG, and inhibition of NF-κBp65 or knockout of PUMA alleviates Fas/FasL-mediated epithelial apoptosis in PHG. These results indicate that IL-6 drives FasL generation via NF-κBp65 in myeloid cells to promote Fas/NF-κBp65/PUMA-mediated epithelial apoptosis in PHG, and this coordinated crosstalk between myeloid cells and epithelial cells may provide a potential therapeutic target for PHG.

A small peptide antagonist of the Fas receptor inhibits neuroinflammation and prevents axon degeneration and retinal ganglion cell death in an inducible mouse model of glaucoma

Background Glaucoma is a complex, multifactorial disease where apoptosis, microglia activation, and inflammation have been linked to the death of retinal ganglion cells (RGCs) and axon degeneration. We demonstrated previously that FasL-Fas signaling was required for axon degeneration and death of RGCs in chronic and inducible mouse models of glaucoma and that Fas activation triggered RGC apoptosis, glial activation, and inflammation. Here, we investigated whether targeting the Fas receptor with a small peptide antagonist, ONL1204, has anti-inflammatory and neuroprotective effects in a microbead-induced mouse model of glaucoma. Methods Intracameral injection of microbeads was used to elevate intraocular pressure (IOP) in Fas-deficient (Faslpr) mice and WT C57BL/6J mice that received an intravitreal injection of the Fas inhibitor, ONL1204 (2 μg/1 μl) (or vehicle only), on day 0 or day 7 after microbead injection. The IOP was monitored by rebound tonometry, and at 28 days post-microbead injection, Brn3a-stained RGCs and paraphenylenediamine (PPD)-stained axons were analyzed. The effects of ONL1204 on retinal microglia activation and the expression of inflammatory genes were analyzed by immunostaining of retinal flatmounts and quantitative PCR (qPCR). Results Rebound tonometry showed equivalent elevation of IOP in all groups of microbead-injected mice. At 28 days post-microbead injection, the RGC and axon counts from microbead-injected Faslpr mice were equivalent to saline-injected (no IOP elevation) controls. Treatment with ONL1204 also significantly reduced RGC death and loss of axons in microbead-injected WT mice when compared to vehicle-treated controls, even when administered after IOP elevation. Confocal analysis of Iba1-stained retinal flatmounts and qPCR demonstrated that ONL1204 also abrogated microglia activation and inhibited the induction of multiple genes implicated in glaucoma, including cytokines and chemokines (GFAP, Caspase-8, TNFα, IL-1β, IL-6, IL-18, MIP-1α, MIP-1β, MIP-2, MCPI, and IP10), components of the complement cascade (C3, C1Q), Toll-like receptor pathway (TLR4), and inflammasome pathway (NLRP3). Conclusions These results serve as proof-of-principal that the small peptide inhibitor of the Fas receptor, ONL1204, can provide robust neuroprotection in an inducible mouse model of glaucoma, even when administered after IOP elevation. Moreover, Fas signaling contributes to the pathogenesis of glaucoma through activation of both apoptotic and inflammatory pathways.

Antiapoptotic and mitochondrial biogenetic effects of exercise training on ovariectomized hypertensive rat hearts

This study was to investigate the effects of exercise training on antiapoptotic pathways and mitochondrial biogenesis in ovariectomized hypertensive rats. Histopathological analysis, TUNEL assay, and Western blotting were performed on the excised hearts from female spontaneously hypertensive rats (SHR), which were divided into a sham-operated sedentary hypertensive (SHR-S), a sedentary hypertensive ovariectomized (SHR-O), and hypertensive ovariectomized rats that underwent treadmill exercise training (SHR-OT; 60 min/day, 5 days/wk) for 8 wk, along with normotensive Wistar Kyoto rats (WKY). When compared with the WKY group, the SHR-S group exhibited decreased protein levels of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial OPA-1 (mitochondrial biogenesis) and decreased further in the SHR-O group. The protein levels of p-PI3K, p-Akt, Bcl-2, Bcl-xL (prosurvival pathways), and the protein levels of PGC-1α and mitochondrial OPA1 (mitochondrial biogenesis) were increased in the SHR-OT group, but estrogen receptor (ER)α and ERβ were not changed when compared with the SHR-O group. The protein levels of t-Bid, Bad, Bax, cytosolic cytochrome c, activated caspase 9, and activated caspase 3 (mitochondria-dependent apoptotic pathways), as well as Fas ligand, TNF-α, Fas receptors, Fas-associated death domain, activated caspase 8 (Fas receptor-dependent apoptotic pathways) were decreased in the SHR-OT group, when compared with the SHR-O group. Exercise training protection on the coexistence of hypertension and ovariectomy-induced cardiac mitochondria-dependent and Fas receptor-dependent apoptotic pathways by enhancing the Bcl2-related and mitochondrial biogenetic prosurvival pathways might provide a new therapeutic effect on cardiac protection in oophorectomized early postmenopausal hypertensive women. NEW & NOTEWORTHY Widely dispersed cardiac apoptosis was found in the coexistence of hypertension and ovariectomy. Exercise training on a treadmill could prevent ovariectomized hypertension-induced widely dispersed cardiac apoptosis via mitochondria-dependent apoptotic pathway (t-Bid, Bad, Bax, cytosolic cytochrome c, activated caspase 9, and activated caspase 3) and Fas receptor-dependent apoptotic pathway (Fas ligand, tumor necrosis factor-α, Fas receptors, Fas-associated death domain, activated caspase 8, and activated caspase 3) through enhancing the Bcl2-related (p-PI3K, p-Akt, Bcl-2, Bcl-xL) and mitochondrial biogenetic (PGC-1α and mitochondrial optic atrophy 1) prosurvival pathways.