Background: Pyroptosis is a type of proinflammatory regulated cell death (RCD) in which caspase-1 proteolytically cleaves gasdermin D (GSDMD) to yield a cytotoxic pore-forming protein. Recent studies have suggested that additional cell death pathways may interact with GSDMD under certain circumstances to execute pyroptosis. Microglia/macrophages in the central nervous system (CNS) undergo GSDMD-associated pyroptosis in multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE) but the contribution of other cell death pathways to this phenomenon is unknown. Herein, we tested the hypothesis that multiple RCD pathways underlie microglial pyroptosis in the context of neuroinflammation. Methods: A siRNA screen of genes with known RCD functions was performed in primary human microglia to evaluate their role in nigericin-induced pyroptosis using supernatant lactate dehydrogenase activity as a read-out of cell lysis. Activation of apoptotic executioner proteins and their contribution to pyroptosis was assessed using semiquantitative confocal microscopy, high-sensitivity ELISA, immunoblot, cell lysis assays, and activity-based fluorescent probes. Quantification of pyroptosis-related protein expression was performed in CNS lesions from patients with progressive MS and mice with MOG 35-55-induced EAE, and in matched controls. Results: Among progressive MS patients, activated caspase-3 was detected in GSDMD immunopositive pyroptotic microglia/macrophages within demyelinating lesions. In the siRNA screen, suppression of caspase-3/7, caspase-1, or GSDMD expression prevented plasma membrane rupture during pyroptosis. Upon exposure to pyroptotic stimuli (ATP or nigericin), human microglia displayed caspase-3/7 activation and cleavage of caspase-3/7-specific substrates (e.g., DFF45, ROCK1, and PARP), with accompanying features of pyroptosis including GSDMD immunopositive pyroptotic bodies, IL-1β release, and membrane rupture. Pyroptosis-associated nuclear condensation and pyroptotic body formation were suppressed by caspase-3/7 inhibition. Pharmacological and siRNA-mediated inhibition of caspase-1 diminished caspase-3/7 activation during pyroptosis. In mice with EAE-associated neurological deficits, activated caspase-3 colocalized with GSDMD immunopositivity in lesion-associated macrophages/microglia.

Multiple sclerosis (MS) is a progressive and inflammatory demyelinating disease of the CNS. Peroxisomes perform critical functions that contribute to CNS homeostasis. We investigated peroxisome injury and mitigating effects of peroxisome-restorative therapy on inflammatory demyelination in models of MS. Human autopsied CNS tissues (male and female), human cell cultures, and cuprizonemediated demyelination mice (female) were examined by RT-PCR, Western blotting, and immunolabeling. The therapeutic peroxisome proliferator, 4-phenylbutyrate (4-PBA) was investigated in vitro and in vivo. White matter from MS patients showed reduced peroxisomal transcript and protein levels, including PMP70, compared with non-MS controls. Cultured human neural cells revealed that human microglia contained abundant peroxisomal proteins. TNF-a-exposed microglia displayed reduced immunolabeling of peroxisomal proteins, PMP70 and PEX11b, which was prevented with 4-PBA. In human myeloid cells exposed to TNF-a or nigericin, suppression of PEX11b and catalase protein levels were observed to be dependent on NLRP3 expression. Hindbrains from cuprizoneexposed mice showed reduced Abcd1, Cat, and Pex5l transcript levels, with concurrent increased Nlrp3 and Il1b transcript levels, which was abrogated by 4-PBA. In the central corpus callosum, Iba-1 in CNS-associated macrophages and peroxisomal thiolase immunostaining after cuprizone exposure was increased by 4-PBA. 4-PBA prevented decreased myelin basic protein and neurofilament heavy chain immunoreactivity caused by cuprizone exposure. Cuprizone-induced neurobehavioral deficits were improved by 4-PBA treatment. Peroxisome injury in CNS-associated macrophages contributed to neuroinflammation and demyelination that was prevented by 4-PBA treatment. A peroxisome-targeted therapy might be valuable for treating inflammatory demyelination and neurodegeneration in MS.

Human pegivirus (HPgV) infects peripheral leukocytes but was recently shown to be a neurotropic virus associated with leukoencephalitis in humans. In the present study, we investigated the neural cell tropism of HPgV as well as its effects on host immune responses. HPgV wild type (WT) as well as a mutant virus with a deletion in the HPgV NS2 gene (ΔNS2) were able to productively infect human astrocytes and microglia but not neurons or an oligodendrocyte-derived cell line. Of note, the ΔNS2 virus replicated better than WT pegivirus in astrocytes with both viruses being able to subsequently infect and spread in fresh human astrocyte cultures. Infection of human glia by HPgV WT and ΔNS2 viruses resulted in suppression of peroxisome-associated genes including PEX11B, ABCD1, PEX7, ABCD3, PEX3 and PEX5L during peak viral production, which was accompanied by reduced expression of IFNB, IRF3, IRF1, and MAVS , particularly in ΔNS2-infected cells. These data were consistent with analyses of brain tissue from patients infected with HPgV in which we observed suppression of peroxisome and type I interferon gene transcripts including PEX11B, ABCD3, IRF1, and IRF3 with concurrent loss of PMP70 immunoreactivity in glia. Our data indicate that human astrocytes and microglia are permissive to HPgV infection resulting in peroxisome injury and suppressed antiviral signaling that is influenced by viral diversity. Importance: Human pegiviruses are detected in 1-5% of the general population, principally infecting leukocytes, although their effects on human health remain uncertain. Herein, we show that human pegivirus infects specific neural cell types in culture and human brain and like other neurotropic flaviviruses, it causes suppression of peroxisome and antiviral signalling pathways, which could favour ongoing viral infection and perhaps confer susceptibility to the development of neurological disease.