Monocyte-Mediated Thrombosis Linked to Circulating Tissue Factor and Immune Paralysis in COVID-19.

BACKGROUND: SARS-CoV-2 infections cause COVID-19 and are associated with inflammation, coagulopathy, and high incidence of thrombosis. Myeloid cells help coordinate the initial immune response in COVID-19. Although we appreciate that myeloid cells lie at the nexus of inflammation and thrombosis, the mechanisms that unite the two in COVID-19 remain largely unknown. METHODS: In this study, we used systems biology approaches including proteomics, transcriptomics, and mass cytometry to define the circulating proteome and circulating immune cell phenotypes in subjects with COVID-19. RESULTS: In a cohort of subjects with COVID-19 (n=35), circulating markers of inflammation (CCL23 [C-C motif chemokine ligand 23] and IL [interleukin]-6) and vascular dysfunction (ACE2 [angiotensin-converting enzyme 2] and TF [tissue factor]) were elevated in subjects with severe compared with mild COVID-19. Additionally, although the total white blood cell counts were similar between COVID-19 groups, CD14+ (cluster of differentiation) monocytes from subjects with severe COVID-19 expressed more TF. At baseline, transcriptomics demonstrated increased IL-6, CCL3, ACOD1 (aconitate decarboxylase 1), C5AR1 (complement component 5a receptor), C5AR2, and TF in subjects with severe COVID-19 compared with controls. Using stress transcriptomics, we found that circulating immune cells from subjects with severe COVID-19 had evidence of profound immune paralysis with greatly reduced transcriptional activation and release of inflammatory markers in response to TLR (Toll-like receptor) activation. Finally, sera from subjects with severe (but not mild) COVID-19 activated human monocytes and induced TF expression. CONCLUSIONS: Taken together, these observations further elucidate the pathological mechanisms that underlie immune dysfunction and coagulation abnormalities in COVID-19, contributing to our growing understanding of SARS-CoV-2 infections that could also be leveraged to develop novel diagnostic and therapeutic strategies.

Type-1 angiotensin receptor signaling in central nervous system myeloid cells is pathogenic during fatal alphavirus encephalitis in mice.

BACKGROUND: Alphaviruses can cause fatal encephalitis in humans. Natural infections occur via the bite of infected mosquitos, but aerosol transmissibility makes some of these viruses potential bioterrorism agents. Central nervous system (CNS) host responses contribute to alphavirus pathogenesis in experimental models and are logical therapeutic targets. We investigated whether reactive oxygen species (ROS) generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) activity within the CNS contributes to fatal alphavirus encephalitis in mice. METHODS: Infected animals were treated systemically with the angiotensin receptor-blocking drug, telmisartan, given its ability to cross the blood-brain barrier, selectively block type-1 angiotensin receptors (AT1R), and inhibit Nox-derived ROS production in vascular smooth muscle and other extraneural tissues. Clinical, virological, biochemical, and histopathological outcomes were followed over time. RESULTS: The importance of the angiotensin II (Ang II)/AT1R axis in disease pathogenesis was confirmed by demonstrating increased Ang II levels in the CNS following infection, enhanced disease survival when CNS Ang II production was suppressed, increased AT1R expression on microglia and tissue-infiltrating myeloid cells, and enhanced disease survival in AT1R-deficient mice compared to wild-type (WT) controls. Systemic administration of telmisartan protected WT mice from lethal encephalitis caused by two different alphaviruses in a dose-dependent manner without altering virus replication or exerting any anti-inflammatory effects in the CNS. Infection triggered up-regulation of multiple Nox subunits in the CNS, while drug treatment inhibited local Nox activity, ROS production, and oxidative neuronal damage. Telmisartan proved ineffective in Nox-deficient mice, demonstrating that this enzyme is its main target in this experimental setting. CONCLUSIONS: Nox-derived ROS, likely arising from CNS myeloid cells triggered by AT1R signaling, are pathogenic during fatal alphavirus encephalitis in mice. Systemically administered telmisartan at non-hypotensive doses targets Nox activity in the CNS to exert a neuroprotective effect. Disruption of this pathway may have broader implications for the treatment of related infections as well as for other CNS diseases driven by oxidative injury.

MicroRNA-34a Negatively Regulates Efferocytosis by Tissue Macrophages in Part via SIRT1.

Apoptotic cell (AC) clearance (efferocytosis) is an evolutionarily conserved process essential for immune health, particularly to maintain self-tolerance. Despite identification of many recognition receptors and intracellular signaling components of efferocytosis, its negative regulation remains incompletely understood and has not previously been known to involve microRNAs (miRs). In this article, we show that miR-34a (gene ID 407040), well recognized as a p53-dependent tumor suppressor, mediates coordinated negative regulation of efferocytosis by resident murine and human tissue macrophages (Mø). The miR-34a expression varied greatly between Mø from different tissues, correlating inversely with their capacity for AC uptake. Transient or genetic knockdown of miR-34a increased efferocytosis, whereas miR-34a overexpression decreased efferocytosis, without altering recognition of live, necrotic, or Ig-opsonized cells. The inhibitory effect of miR-34a was mediated both by reduced expression of Axl, a receptor tyrosine kinase known to recognize AC, and of the deacetylase silent information regulator T1, which had not previously been linked to efferocytosis by tissue Mø. Exposure to AC downregulated Mø miR-34a expression, resulting in a positive feedback loop that increased subsequent capacity to engulf AC. These findings demonstrate that miR-34a both specifically regulates and is regulated by efferocytosis. Given the ability of efferocytosis to polarize ingesting Mø uniquely and to reduce their host-defense functions, dynamic negative regulation by miR-34a provides one means of fine-tuning Mø behavior toward AC in specific tissue environments with differing potentials for microbial exposure.

Astrocyte matricellular proteins that control excitatory synaptogenesis are regulated by inflammatory cytokines and correlate with paralysis severity during experimental autoimmune encephalomyelitis.

The matricellular proteins, secreted protein acidic and rich in cysteine (SPARC) and SPARC-like 1 (SPARCL1), are produced by astrocytes and control excitatory synaptogenesis in the central nervous system. While SPARCL1 directly promotes excitatory synapse formation in vitro and in the developing nervous system in vivo, SPARC specifically antagonizes the synaptogenic actions of SPARCL1. We hypothesized these proteins also help maintain existing excitatory synapses in adult hosts, and that local inflammation in the spinal cord alters their production in a way that dynamically modulates motor synapses and impacts the severity of paralysis during experimental autoimmune encephalomyelitis (EAE) in mice. Using a spontaneously remitting EAE model, paralysis severity correlated inversely with both expression of synaptic proteins and the number of synapses in direct contact with the perikarya of motor neurons in spinal gray matter. In both remitting and non-remitting EAE models, paralysis severity also correlated inversely with sparcl1:sparc transcript and SPARCL1:SPARC protein ratios directly in lumbar spinal cord tissue. In vitro, astrocyte production of both SPARCL1 and SPARC was regulated by T cell-derived cytokines, causing dynamic modulation of the SPARCL1:SPARC expression ratio. Taken together, these data support a model whereby proinflammatory cytokines inhibit SPARCL1 and/or augment SPARC expression by astrocytes in spinal gray matter that, in turn, cause either transient or sustained synaptic retraction from lumbar spinal motor neurons thereby regulating hind limb paralysis during EAE. Ongoing studies seek ways to alter this SPARCL1:SPARC expression ratio in favor of synapse reformation/maintenance and thus help to modulate neurologic deficits during times of inflammation. This could identify new astrocyte-targeted therapies for diseases such as multiple sclerosis.

Manipulation of host factors optimizes the pathogenesis of western equine encephalitis virus infections in mice for antiviral drug development.

While alphaviruses spread naturally via mosquito vectors, some can also be transmitted as aerosols making them potential bioterrorism agents. One such pathogen, western equine encephalitis virus (WEEV), causes fatal human encephalitis via multiple routes of infection and thus presumably via multiple mechanisms. Although WEEV also produces acute encephalitis in non-human primates, a small animal model that recapitulates features of human disease would be useful for both pathogenesis studies and to evaluate candidate antiviral therapies. We have optimized conditions to infect mice with a low passage isolate of WEEV, thereby allowing detailed investigation of virus tropism, replication, neuroinvasion, and neurovirulence. We find that host factors strongly influence disease outcome, and in particular, that age, gender, and genetic background all have significant effects on disease susceptibility independent of virus tropism or replication within the central nervous system. Our data show that experimental variables can be adjusted in mice to recapitulate disease features known to occur in both non-human primates and humans, thus aiding further study of WEEV pathogenesis and providing a realistic therapeutic window for antiviral drug delivery.

Novel indole-2-carboxamide compounds are potent broad-spectrum antivirals active against western equine encephalitis virus in vivo.

Neurotropic alphaviruses, including western, eastern, and Venezuelan equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication, using a cell-based phenotypic assay (W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275), and subsequently developed second- and third-generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). In this report, we describe the antiviral activity of the most promising third-generation lead compound, CCG205432, and closely related analogs CCG206381 and CCG209023. These compounds have half-maximal inhibitory concentrations of ∼1 µM and selectivity indices of >100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism-of-action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses and, potentially, of other RNA viruses. IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). We have now produced third-generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced-generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.

Type-I interferons suppress microglial production of the lymphoid chemokine, CXCL13.

Lymphoid chemokines are crucial for the development and maintenance of lymphoid organs, but their ectopic expression in non-lymphoid tissues is implicated in both local response to infection and chronic organ-specific autoimmunity. Production of one such chemokine, C-X-C motif ligand 13 (CXCL13), within the central nervous system (CNS) has been linked to the pathogenesis of multiple sclerosis (MS), although little is known about factors controlling its expression in different neural cell types and across a range of disease states. We provoked acute neuroinflammation in experimental animals without causing any associated demyelination using neuroadapted Sindbis virus (NSV) to better understand the sources and regulators of this chemokine in the CNS. We found that mice genetically deficient in the transcription factor, interferon (IFN) regulatory factor-7 (IRF7), made significantly higher CXCL13 protein levels in the CNS compared with wild-type (WT) controls. Microglia proved to be the main producer of CXCL13 in the brain during infection of both WT and IRF7(-/-) mice, and primary microglia cultured in vitro generated CXCL13 following stimulation with either virus particles or synthetic Toll-like receptor (TLR) ligands. Microglia cultured from IRF7(-/-) mice selectively overproduced CXCL13, and manipulation of extracellular type-I IFN levels demonstrated the existence of a negative feedback loop whereby type-I IFN receptor signaling specifically suppressed microglial CXCL13 release. Since IFN-ß is used to treat patients with relapsing-remitting MS and yet acts through unknown mechanisms, we speculate that suppressed lymphoid chemokine production by microglia could contribute to its therapeutic effects.

Discovery of potent broad spectrum antivirals derived from marine actinobacteria.

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds.

Optimization of novel indole-2-carboxamide inhibitors of neurotropic alphavirus replication.

Neurotropic alphaviruses, which include western equine encephalitis virus (WEEV) and Fort Morgan virus, are mosquito-borne pathogens that infect the central nervous system causing acute and potentially fatal encephalitis. We previously reported a novel series of indole-2-carboxamides as alphavirus replication inhibitors, one of which conferred protection against neuroadapted Sindbis virus infection in mice. We describe here further development of this series, resulting in 10-fold improvement in potency in a WEEV replicon assay and up to 40-fold increases in half-lives in mouse liver microsomes. Using a rhodamine123 uptake assay in MDR1-MDCKII cells, we were able to identify structural modifications that markedly reduce recognition by P-glycoprotein, the key efflux transporter at the blood-brain barrier. In a preliminary mouse PK study, we were able to demonstrate that two new analogues could achieve higher and/or longer plasma drug exposures than our previous lead and that one compound achieved measurable drug levels in the brain.

Virus-induced CD8+ T cells accelerate the onset of experimental autoimmune encephalomyelitis: implications for how viral infections might trigger multiple sclerosis exacerbations.

Viral infections can exacerbate multiple sclerosis (MS) through poorly defined mechanisms. We developed an experimental system whereby infection with an asymptomatic neurotropic alphavirus caused a transient acceleration of experimental autoimmune encephalomyelitis (EAE) without altering the expansion or differentiation of autoreactive CD4+ T cells. Instead, this effect on the clinical course of EAE depended on CD8+ T cells that neither participate in viral clearance nor induce neuropathology in infected mice without EAE. Our system should be useful to further unravel how certain viral infections trigger MS exacerbations and to understand how CD8+ T cells can exert pathogenic effects within active demyelinating lesions.

Complexity of the microglial activation pathways that drive innate host responses during lethal alphavirus encephalitis in mice.

Microglia express multiple TLRs (Toll-like receptors) and provide important host defence against viruses that invade the CNS (central nervous system). Although prior studies show these cells become activated during experimental alphavirus encephalitis in mice to generate cytokines and chemokines that influence virus replication, tissue inflammation and neuronal survival, the specific PRRs (pattern recognition receptors) and signalling intermediates controlling microglial activation in this setting remain unknown. To investigate these questions directly in vivo, mice ablated of specific TLR signalling molecules were challenged with NSV (neuroadapted Sindbis virus) and CNS viral titres, inflammatory responses and clinical outcomes followed over time. To approach this problem specifically in microglia, the effects of NSV on primary cells derived from the brains of wild-type and mutant animals were characterized in vitro. From the standpoint of the virus, microglial activation required viral uncoating and an intact viral genome; inactivated virus particles did not elicit measurable microglial responses. At the level of the target cell, NSV triggered multiple PRRs in microglia to produce a broad range of inflammatory mediators via non-overlapping signalling pathways. In vivo, disease survival was surprisingly independent of TLR-driven responses, but still required production of type-I IFN (interferon) to control CNS virus replication. Interestingly, the ER (endoplasmic reticulum) protein UNC93b1 facilitated host survival independent of its known effects on endosomal TLR signalling. Taken together, these data show that alphaviruses activate microglia via multiple PRRs, highlighting the complexity of the signalling networks by which CNS host responses are elicited by these infections.

Novel inhibitors of neurotropic alphavirus replication that improve host survival in a mouse model of acute viral encephalitis.

Arboviral encephalitis is a potentially devastating human disease with no approved therapies that target virus replication. We previously discovered a novel class of thieno[3,2-b]pyrrole-based inhibitors active against neurotropic alphaviruses such as western equine encephalitis virus (WEEV) in cultured cells. In this report, we describe initial development of these novel antiviral compounds, including bioisosteric replacement of the 4H-thieno[3,2-b]pyrrole core with indole to improve metabolic stability and the introduction of chirality to assess target enantioselectivity. Selected modifications enhanced antiviral activity while maintaining low cytotoxicity, increased stability to microsomal metabolism, and also revealed striking enantiospecific activity in cultured cells. Furthermore, we demonstrate improved outcomes (both symptoms and survival) following treatment with indole analogue 9h (CCG-203926) in an in vivo mouse model of alphaviral encephalitis that closely correlate with the enantiospecific in vitro antiviral activity. These results represent a substantial advancement in the early preclinical development of a promising class of novel antiviral drugs against virulent neurotropic alphaviruses.

Disrupted glutamate transporter expression in the spinal cord with acute flaccid paralysis caused by West Nile virus infection.

Neuroinvasive West Nile virus (WNV) infections may cause acute flaccid paralysis (AFP); in fatal cases, anterior horn cell loss is presumed to be caused by direct viral infection. In related animal models, however, glutamate excitotoxicity mediates bystander injury of uninfected anterior horn cells, suggesting additional pathogenic mechanisms. We examined expression of the principal excitatory amino acid transporter (EAAT) of astrocytes (i.e. EAAT-2 in humans, glutamate transporter 1 in hamsters) in the spinal cord of human WNV-induced AFP patients and in hamsters with WNV-induced AFP by immunohistochemistry. Glial fibrillary acidic protein, synaptic and dendritic markers (i.e. synaptophysin, microtubule-associated protein 2), immune activation (HLA-DR), and viral antigens were also evaluated. Humans and hamsters with WNV-induced AFP had decreased spinal gray matter EAAT expression despite greater numbers of glial fibrillary acidic protein-positive astrocytes compared with controls. Areas of diminished EAAT expression showed reduced synaptic and dendritic protein expression and prominent local inflammation but few infected neurons. These findings suggest that WNV infection results in local immune activation within the spinal cord that in turn causes a failure of astrocyte glutamate reuptake even as the number of astrocytes increases; rising extracellular glutamate levels may then drive excitotoxic injury of both infected and uninfected anterior horn cells. The pathogenesis of this increasingly common disorder likely involves immune response and excitotoxicity mechanisms that are potential therapeutic targets.

A novel trafficking signal within the HLA-C cytoplasmic tail allows regulated expression upon differentiation of macrophages.

MHC class I molecules (MHC-I) present peptides to CTLs. In addition, HLA-C allotypes are recognized by killer cell Ig-like receptors (KIR) found on NK cells and effector CTLs. Compared with other classical MHC-I allotypes, HLA-C has low cell surface expression and an altered intracellular trafficking pattern. We present evidence that this results from effects of both the extracellular domain and the cytoplasmic tail. Notably, we demonstrate that the cytoplasmic tail contains a dihydrophobic (LI) internalization and lysosomal targeting signal that is partially attenuated by an aspartic acid residue (DXSLI). In addition, we provide evidence that this signal is specifically inhibited by hypophosphorylation of the adjacent serine residue upon macrophage differentiation and that this allows high HLA-C expression in this cell type. We propose that tightly regulated HLA-C surface expression facilitates immune surveillance and allows HLA-C to serve a specialized role in macrophages.