Itaconate and derivatives reduce interferon responses and inflammation in influenza A virus infection.

Excessive inflammation is a major cause of morbidity and mortality in many viral infections including influenza. Therefore, there is a need for therapeutic interventions that dampen and redirect inflammatory responses and, ideally, exert antiviral effects. Itaconate is an immunomodulatory metabolite which also reprograms cell metabolism and inflammatory responses when applied exogenously. We evaluated effects of endogenous itaconate and exogenous application of itaconate and its variants dimethyl- and 4-octyl-itaconate (DI, 4OI) on host responses to influenza A virus (IAV). Infection induced expression of ACOD1, the enzyme catalyzing itaconate synthesis, in monocytes and macrophages, which correlated with viral replication and was abrogated by DI and 4OI treatment. In IAV-infected mice, pulmonary inflammation and weight loss were greater in Acod1-/- than in wild-type mice, and DI treatment reduced pulmonary inflammation and mortality. The compounds reversed infection-triggered interferon responses and modulated inflammation in human cells supporting non-productive and productive infection, in peripheral blood mononuclear cells, and in human lung tissue. All three itaconates reduced ROS levels and STAT1 phosphorylation, whereas AKT phosphorylation was reduced by 4OI and DI but increased by itaconate. Single-cell RNA sequencing identified monocytes as the main target of infection and the exclusive source of ACOD1 mRNA in peripheral blood. DI treatment silenced IFN-responses predominantly in monocytes, but also in lymphocytes and natural killer cells. Ectopic synthesis of itaconate in A549 cells, which do not physiologically express ACOD1, reduced infection-driven inflammation, and DI reduced IAV- and IFNγ-induced CXCL10 expression in murine macrophages independent of the presence of endogenous ACOD1. The compounds differed greatly in their effects on cellular gene homeostasis and released cytokines/chemokines, but all three markedly reduced release of the pro-inflammatory chemokines CXCL10 (IP-10) and CCL2 (MCP-1). Viral replication did not increase under treatment despite the dramatically repressed IFN responses. In fact, 4OI strongly inhibited viral transcription in peripheral blood mononuclear cells, and the compounds reduced viral titers (4OI>Ita>DI) in A549 cells whereas viral transcription was unaffected. Taken together, these results reveal itaconates as immunomodulatory and antiviral interventions for influenza virus infection.

SARS-CoV-2-free residual proteins mediated phenotypic and metabolic changes in peripheral blood monocytic-derived macrophages in support of viral pathogenesis.

The large-scale dissemination of coronavirus disease-2019 (COVID-19) and its serious complications have pledged the scientific research communities to uncover the pathogenesis mechanisms of its etiologic agent, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Methods of unveiling such mechanisms are rooted in understanding the viral agent's interactions with the immune system, including its ability to activate macrophages, due to their suggested role in prolonged inflammatory phases and adverse immune responses. The objective of this study is to test the effect of SARS-CoV-2-free proteins on the metabolic and immune responses of macrophages. We hypothesized that SARS-CoV-2 proteins shed during the infection cycle may dynamically induce metabolic and immunologic alterations with an inflammatory impact on the infected host cells. It is imperative to delineate such alterations in the context of macrophages to gain insight into the pathogenesis of these highly infectious viruses and their associated complications and thus, expedite the vaccine and drug therapy advent in combat of viral infections. Human monocyte-derived macrophages were treated with SARS-CoV-2-free proteins at different concentrations. The phenotypic and metabolic alterations in macrophages were investigated and the subsequent metabolic pathways were analyzed. The obtained results indicated that SARS-CoV-2-free proteins induced concentration-dependent alterations in the metabolic and phenotypic profiles of macrophages. Several metabolic pathways were enriched following treatment, including vitamin K, propanoate, and the Warburg effect. These results indicate significant adverse effects driven by residual viral proteins that may hence be considered determinants of viral pathogenesis. These findings provide important insight as to the impact of SARS-CoV-2-free residual proteins on the host cells and suggest a potential new method of management during the infection and prior to vaccination.

Highly restricted SARS-CoV-2 receptor expression and resistance to infection by primary human monocytes and monocyte-derived macrophages.

Severe acute respiratory syndrome (SARS) coronavirus 2 (SARS-CoV2), which causes the disease COVID-19, has caused an unprecedented global pandemic. Angiotensin-converting enzyme 2 (ACE2) is the major cellular receptor for SARS-CoV2 entry, which is facilitated by viral Spike priming by cellular TMPRSS2. Macrophages play an important role in innate viral defense and are also involved in aberrant immune activation that occurs in COVID-19, and thus direct macrophage infection might contribute to severity of SARS-CoV2 infection. Here, we demonstrate that monocytes and monocyte-derived macrophages (MDM) under in vitro conditions express low-to-undetectable levels of ACE2 and TMPRSS2 and minimal coexpression. Expression of these receptors remained low in MDM induced to different subtypes such as unpolarized, M1 and M2 polarized. Untreated, unpolarized, M1 polarized, and M2 polarized MDM were all resistant to infection with SARS-CoV2 pseudotyped virions. These findings suggest that direct infection of myeloid cells is unlikely to be a major mechanism of SARS-CoV2 pathogenesis. Summary sentence: Monocytes and macrophages express minimal ACE2 and TMPRSS2 and resist SARS-CoV-2 Spike-mediated infection, suggesting direct myeloid cell infection is unlikely a major contributor to pathogenesis.

Inflammasome activation in infected macrophages drives COVID-19 pathology.

Severe COVID-19 is characterized by persistent lung inflammation, inflammatory cytokine production, viral RNA and a sustained interferon (IFN) response, all of which are recapitulated and required for pathology in the SARS-CoV-2-infected MISTRG6-hACE2 humanized mouse model of COVID-19, which has a human immune system1-20. Blocking either viral replication with remdesivir21-23 or the downstream IFN-stimulated cascade with anti-IFNAR2 antibodies in vivo in the chronic stages of disease attenuates the overactive immune inflammatory response, especially inflammatory macrophages. Here we show that SARS-CoV-2 infection and replication in lung-resident human macrophages is a critical driver of disease. In response to infection mediated by CD16 and ACE2 receptors, human macrophages activate inflammasomes, release interleukin 1 (IL-1) and IL-18, and undergo pyroptosis, thereby contributing to the hyperinflammatory state of the lungs. Inflammasome activation and the accompanying inflammatory response are necessary for lung inflammation, as inhibition of the NLRP3 inflammasome pathway reverses chronic lung pathology. Notably, this blockade of inflammasome activation leads to the release of infectious virus by the infected macrophages. Thus, inflammasomes oppose host infection by SARS-CoV-2 through the production of inflammatory cytokines and suicide by pyroptosis to prevent a productive viral cycle.

SARS-CoV-2 Spike protein is not pro-inflammatory in human primary macrophages: endotoxin contamination and lack of protein glycosylation as possible confounders.

INTRODUCTION: The inflammatory potential of SARS-CoV-2 Spike S1 (Spike) has never been tested in human primary macrophages (MΦ). Different recombinant Spikes might display different effects in vitro, according to protein length and glycosylation, and endotoxin (lipopolysaccharide, LPS) contamination. OBJECTIVES: To assess (1) the effects of different Spikes on human primary MΦ inflammation; (2) whether LPS contamination of recombinant Spike is (con)cause in vitro of increased MΦ inflammation. METHODS: Human primary MΦ were incubated in the presence/absence of several different Spikes (10 nM) or graded concentrations of LPS. Pro-inflammatory marker expression (qPCR and ELISA) and supernatant endotoxin contamination (LAL test) were the main readouts. RESULTS: LPS-free, glycosylated Spike (the form expressed in infected humans) caused no inflammation in human primary MΦ. Two (out of five) Spikes were contaminated with endotoxins ≥ 3 EU/ml and triggered inflammation. A non-contaminated non-glycosylated Spike produced in E. coli induced MΦ inflammation. CONCLUSIONS: Glycosylated Spike per se is not pro-inflammatory for human MΦ, a feature which may be crucial to evade the host innate immunity. In vitro studies with commercially available Spike should be conducted with excruciating attention to potential LPS contamination.

Specific inhibition of the NLRP3 inflammasome suppresses immune overactivation and alleviates COVID-19 like pathology in mice.

BACKGROUND: The Coronavirus Disease 2019 (COVID-19) pandemic has been a great threat to global public health since 2020. Although the advance on vaccine development has been largely achieved, a strategy to alleviate immune overactivation in severe COVID-19 patients is still needed. The NLRP3 inflammasome is activated upon SARS-CoV-2 infection and associated with COVID-19 severity. However, the processes by which the NLRP3 inflammasome is involved in COVID-19 disease remain unclear. METHODS: We infected THP-1 derived macrophages, NLRP3 knockout mice, and human ACE2 transgenic mice with live SARS-CoV-2 in Biosafety Level 3 (BSL-3) laboratory. We performed quantitative real-time PCR for targeted viral or host genes from SARS-CoV-2 infected mouse tissues, conducted histological or immunofluorescence analysis in SARS-CoV-2 infected mouse tissues. We also injected intranasally AAV-hACE2 or intraperitoneally NLRP3 inflammasome inhibitor MCC950 before SARS-CoV-2 infection in mice as indicated. FINDINGS: We have provided multiple lines of evidence that the NLRP3 inflammasome plays an important role in the host immune response to SARS-CoV-2 invasion of the lungs. Inhibition of the NLRP3 inflammasome attenuated the release of COVID-19 related pro-inflammatory cytokines in cell cultures and mice. The severe pathology induced by SARS-CoV-2 in lung tissues was reduced in Nlrp3-/- mice compared to wild-type C57BL/6 mice. Finally, specific inhibition of the NLRP3 inflammasome by MCC950 alleviated excessive lung inflammation and thus COVID-19 like pathology in human ACE2 transgenic mice. INTERPRETATION: Inflammatory activation induced by SARS-CoV-2 is an important stimulator of COVID-19 related immunopathology. Targeting the NLRP3 inflammasome is a promising immune intervention against severe COVID-19 disease. FUNDING: This work was supported by grants from the Bureau of Frontier Sciences and Education, CAS (grant no. QYZDJ-SSW-SMC005 to Y.G.Y.), the key project of the CAS "Light of West China" Program (to D.Y.) and Yunnan Province (202001AS070023 to D.Y.).

SARS-CoV-2 infection triggers profibrotic macrophage responses and lung fibrosis.

COVID-19-induced "acute respiratory distress syndrome" (ARDS) is associated with prolonged respiratory failure and high mortality, but the mechanistic basis of lung injury remains incompletely understood. Here, we analyze pulmonary immune responses and lung pathology in two cohorts of patients with COVID-19 ARDS using functional single-cell genomics, immunohistology, and electron microscopy. We describe an accumulation of CD163-expressing monocyte-derived macrophages that acquired a profibrotic transcriptional phenotype during COVID-19 ARDS. Gene set enrichment and computational data integration revealed a significant similarity between COVID-19-associated macrophages and profibrotic macrophage populations identified in idiopathic pulmonary fibrosis. COVID-19 ARDS was associated with clinical, radiographic, histopathological, and ultrastructural hallmarks of pulmonary fibrosis. Exposure of human monocytes to SARS-CoV-2, but not influenza A virus or viral RNA analogs, was sufficient to induce a similar profibrotic phenotype in vitro. In conclusion, we demonstrate that SARS-CoV-2 triggers profibrotic macrophage responses and pronounced fibroproliferative ARDS.

Human nasal wash RNA-Seq reveals distinct cell-specific innate immune responses in influenza versus SARS-CoV-2.

BACKGROUNDInfluenza A virus (IAV) and SARS-CoV-2 are pandemic viruses causing millions of deaths, yet their clinical manifestations are distinctly different.METHODSWith the hypothesis that upper airway immune and epithelial cell responses are also distinct, we performed single-cell RNA sequencing (scRNA-Seq) on nasal wash cells freshly collected from adults with either acute COVID-19 or influenza or from healthy controls. We focused on major cell types and subtypes in a subset of donor samples.ResultsNasal wash cells were enriched for macrophages and neutrophils for both individuals with influenza and those with COVID-19 compared with healthy controls. Hillock-like epithelial cells, M2-like macrophages, and age-dependent B cells were enriched in COVID-19 samples. A global decrease in IFN-associated transcripts in neutrophils, macrophages, and epithelial cells was apparent in COVID-19 samples compared with influenza samples. The innate immune response to SARS-CoV-2 appears to be maintained in macrophages, despite evidence for limited epithelial cell immune sensing. Cell-to-cell interaction analyses revealed a decrease in epithelial cell interactions in COVID-19 and highlighted differences in macrophage-macrophage interactions for COVID-19 and influenza.ConclusionsOur study demonstrates that scRNA-Seq can define host and viral transcriptional activity at the site of infection and reveal distinct local epithelial and immune cell responses for COVID-19 and influenza that may contribute to their divergent disease courses.FundingMassachusetts Consortium on Pathogen Readiness, the Mathers Foundation, and the Department of Defense (W81XWH2110029) "COVID-19 Expansion for AIRe Program."

Macrophages and Monocytes: "Trojan Horses" in COVID-19.

We aimed to explore whether variants of SARS-CoV-2 (Chinese-derived strain (D614, lineage A), Italian strain PV10734 (D614G, lineage B.1.1) and Alpha strain (lineage B.1.1.7)) were able to infect monocytes (MN) and monocyte-derived macrophages (MDM) and whether these infected cells may, in turn, be vectors of infection. For this purpose, we designed an in vitro study following the evolution of MN and MDM infection at different time points in order to confirm whether these cells were permissive for SARS-CoV-2 replication. Finally, we investigated whether, regardless of viral replication, the persistent virus can be transferred to non-infected cells permissive for viral replication. Thus, we co-cultured the infected MN/MDM with permissive VERO E6 cells verifying the viral transmission. This is a further in vitro demonstration of the important role of MN and MDM in the dissemination of SARS-CoV-2 and evolution of the COVID-19 disease.

Hofbauer Cells and COVID-19 in Pregnancy.

CONTEXT.­: SARS-CoV-2 can undergo maternal-fetal transmission, heightening interest in the placental pathology findings from this infection. Transplacental SARS-CoV-2 transmission is typically accompanied by chronic histiocytic intervillositis together with necrosis and positivity of syncytiotrophoblast for SARS-CoV-2. Hofbauer cells are placental macrophages that have been involved in viral diseases, including HIV and Zika virus, but their involvement in SARS-CoV-2 is unknown. OBJECTIVE.­: To determine whether SARS-CoV-2 can extend beyond the syncytiotrophoblast to enter Hofbauer cells, endothelium, and other villous stromal cells in infected placentas of liveborn and stillborn infants. DESIGN.­: Case-based retrospective analysis by 29 perinatal and molecular pathology specialists of placental findings from a preselected cohort of 22 SARS-CoV-2-infected placentas delivered to pregnant women testing positive for SARS-CoV-2 from 7 countries. Molecular pathology methods were used to investigate viral involvement of Hofbauer cells, villous capillary endothelium, syncytiotrophoblast, and other fetal-derived cells. RESULTS.­: Chronic histiocytic intervillositis and trophoblast necrosis were present in all 22 placentas (100%). SARS-CoV-2 was identified in Hofbauer cells from 4 of 22 placentas (18.2%). Villous capillary endothelial staining was positive in 2 of 22 cases (9.1%), both of which also had viral positivity in Hofbauer cells. Syncytiotrophoblast staining occurred in 21 of 22 placentas (95.5%). Hofbauer cell hyperplasia was present in 3 of 22 placentas (13.6%). In the 7 cases having documented transplacental infection of the fetus, 2 (28.6%) occurred in placentas with Hofbauer cell staining positive for SARS-CoV-2. CONCLUSIONS.­: SARS-CoV-2 can extend beyond the trophoblast into the villous stroma, involving Hofbauer cells and capillary endothelial cells, in a small number of infected placentas. Most cases of SARS-CoV-2 transplacental fetal infection occur without Hofbauer cell involvement.

Defining the Innate Immune Responses for SARS-CoV-2-Human Macrophage Interactions.

Host innate immune response follows severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and it is the driver of the acute respiratory distress syndrome (ARDS) amongst other inflammatory end-organ morbidities. Such life-threatening coronavirus disease 2019 (COVID-19) is heralded by virus-induced activation of mononuclear phagocytes (MPs; monocytes, macrophages, and dendritic cells). MPs play substantial roles in aberrant immune secretory activities affecting profound systemic inflammation and end-organ malfunctions. All follow the presence of persistent viral components and virions without evidence of viral replication. To elucidate SARS-CoV-2-MP interactions we investigated transcriptomic and proteomic profiles of human monocyte-derived macrophages. While expression of the SARS-CoV-2 receptor, the angiotensin-converting enzyme 2, paralleled monocyte-macrophage differentiation, it failed to affect productive viral infection. In contrast, simple macrophage viral exposure led to robust pro-inflammatory cytokine and chemokine expression but attenuated type I interferon (IFN) activity. Both paralleled dysregulation of innate immune signaling pathways, specifically those linked to IFN. We conclude that the SARS-CoV-2-infected host mounts a robust innate immune response characterized by a pro-inflammatory storm heralding end-organ tissue damage.

Can molecular mimicry explain the cytokine storm of SARS-CoV-2?: An in silico approach.

PARP14 and PARP9 play a key role in macrophage immune regulation. SARS-CoV-2 is an emerging viral disease that triggers hyper-inflammation known as a cytokine storm. In this study, using in silico tools, we hypothesize about the immunological phenomena of molecular mimicry between SARS-CoV-2 Nsp3 and the human PARP14 and PARP9. The results showed an epitope of SARS-CoV-2 Nsp3 protein that contains consensus sequences for both human PARP14 and PARP9 that are antigens for MHC Classes 1 and 2, which can potentially induce an immune response against human PARP14 and PARP9; while its depletion causes a hyper-inflammatory state in SARS-CoV-2 patients.

Spike protein of SARS-CoV-2 activates macrophages and contributes to induction of acute lung inflammation in male mice.

The spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in mediating viral entry into host cells. However, whether it contributes to pulmonary hyperinflammation in patients with coronavirus disease 2019 is not well known. In this study, we developed a spike protein-pseudotyped (Spp) lentivirus with the proper tropism of the SARS-CoV-2 spike protein on the surface and determined the distribution of the Spp lentivirus in wild-type C57BL/6J male mice that received an intravenous injection of the virus. Lentiviruses with vesicular stomatitis virus glycoprotein (VSV-G) or with a deletion of the receptor-binding domain (RBD) in the spike protein [Spp (∆RBD)] were used as controls. Two hours postinfection (hpi), there were 27-75 times more viral burden from Spp lentivirus in the lungs than in other organs; there were also about 3-5 times more viral burden from Spp lentivirus than from VSV-G lentivirus in the lungs, liver, kidney, and spleen. Deletion of RBD diminished viral loads in the lungs but not in the heart. Acute pneumonia was observed in animals 24 hpi. Spp lentivirus was mainly found in SPC+ and LDLR+ pneumocytes and macrophages in the lungs. IL6, IL10, CD80, and PPAR-γ were quickly upregulated in response to infection in the lungs as well as in macrophage-like RAW264.7 cells. Furthermore, forced expression of the spike protein in RAW264.7 cells significantly increased the mRNA levels of the same panel of inflammatory factors. Our results demonstrated that the spike protein of SARS-CoV-2 confers the main point of viral entry into the lungs and can induce cellular pathology. Our data also indicate that an alternative ACE2-independent viral entry pathway may be recruited in the heart and aorta.

Monocytes and Macrophages, Targets of Severe Acute Respiratory Syndrome Coronavirus 2: The Clue for Coronavirus Disease 2019 Immunoparalysis.

BACKGROUND: Coronavirus disease 2019 (COVID-19) clinical expression is pleiomorphic, severity is related to age and comorbidities such as diabetes and hypertension, and pathophysiology involves aberrant immune activation and lymphopenia. We wondered if the myeloid compartment was affected during COVID-19 and if monocytes and macrophages could be infected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: Monocytes and monocyte-derived macrophages (MDMs) from COVID-19 patients and controls were infected with SARS-CoV-2 and extensively investigated with immunofluorescence, viral RNA extraction and quantification, and total RNA extraction followed by reverse-transcription quantitative polymerase chain reaction using specific primers, supernatant cytokines (interleukins 6, 10, and 1ß; interferon-ß; transforming growth factor-ß1, and tumor necrosis factor-α), and flow cytometry. The effect of M1- vs M2-type or no polarization prior to infection was assessed. RESULTS: SARS-CoV-2 efficiently infected monocytes and MDMs, but their infection is abortive. Infection was associated with immunoregulatory cytokines secretion and the induction of a macrophagic specific transcriptional program characterized by the upregulation of M2-type molecules. In vitro polarization did not account for permissivity to SARS-CoV-2, since M1- and M2-type MDMs were similarly infected. In COVID-19 patients, monocytes exhibited lower counts affecting all subsets, decreased expression of HLA-DR, and increased expression of CD163, irrespective of severity. CONCLUSIONS: SARS-CoV-2 drives monocytes and macrophages to induce host immunoparalysis for the benefit of COVID-19 progression.SARS-CoV-2 infection of macrophages induces a specific M2 transcriptional program. In Covid-19 patients, monocyte subsets were decreased associated with up-expression of the immunoregulatory molecule CD163 suggesting that SARS-CoV-2 drives immune system for the benefit of Covid-19 disease progression.

Pathogenesis of Coronaviruses Through Human Monocytes and Tissue Macrophages.

Coronaviruses (CoVs) contribute significantly to the burden of respiratory diseases, frequently as upper respiratory tract infections. Recent emergence of novel coronaviruses in the last few decades has highlighted the potential transmission, disease, and mortality related to these viruses. In this literature review, we shall explore the disease-causing mechanism of the virus through human monocytes and macrophages. Common strains will be discussed; however, this review will center around coronaviruses responsible for epidemics, namely severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and -2 and the Middle East Respiratory Syndrome Coronavirus (MERS-CoV). Macrophages are key players in the immune system and have been found to play a role in the pathogenesis of lethal coronaviruses. In physiology, they are white blood cells that engulf and digest cellular debris, foreign substances, and microbes. They play a critical role in innate immunity and help initiate adaptive immunity. Human coronaviruses utilize various mechanisms to undermine the innate immune response through its interaction with macrophages and monocytes. It is capable of entering immune cells through DPP4 (dipeptidyl-peptidase 4) receptors and antibody-dependent enhancement, delaying initial interferon response which supports robust viral replication. Pathogenesis includes triggering the production of overwhelming pro-inflammatory cytokines that attract other immune cells to the site of infection, which propagate prolonged pro-inflammatory response. The virus has also been found to suppress the release of anti-inflammatory mediators such as IL-10, leading to an aberrant inflammatory response. Elevated serum cytokines are also believed to contribute to pathological features seen in severe disease such as coagulopathy, acute lung injury, and multiorgan failure.

SARS-CoV-2/ACE2 Interaction Suppresses IRAK-M Expression and Promotes Pro-Inflammatory Cytokine Production in Macrophages.

The major cause of death in SARS-CoV-2 infected patients is due to de-regulation of the innate immune system and development of cytokine storm. SARS-CoV-2 infects multiple cell types in the lung, including macrophages, by engagement of its spike (S) protein on angiotensin converting enzyme 2 (ACE2) receptor. ACE2 receptor initiates signals in macrophages that modulate their activation, including production of cytokines and chemokines. IL-1R-associated kinase (IRAK)-M is a central regulator of inflammatory responses regulating the magnitude of TLR responsiveness. Aim of the work was to investigate whether SARS-CoV-2 S protein-initiated signals modulate pro-inflammatory cytokine production in macrophages. For this purpose, we treated PMA-differentiated THP-1 human macrophages with SARS-CoV-2 S protein and measured the induction of inflammatory mediators including IL6, TNFα, IL8, CXCL5, and MIP1a. The results showed that SARS-CoV-2 S protein induced IL6, MIP1a and TNFα mRNA expression, while it had no effect on IL8 and CXCL5 mRNA levels. We further examined whether SARS-CoV-2 S protein altered the responsiveness of macrophages to TLR signals. Treatment of LPS-activated macrophages with SARS-CoV-2 S protein augmented IL6 and MIP1a mRNA, an effect that was evident at the protein level only for IL6. Similarly, treatment of PAM3csk4 stimulated macrophages with SARS-CoV-2 S protein resulted in increased mRNA of IL6, while TNFα and MIP1a were unaffected. The results were confirmed in primary human peripheral monocytic cells (PBMCs) and isolated CD14+ monocytes. Macrophage responsiveness to TLR ligands is regulated by IRAK-M, an inactive IRAK kinase isoform. Indeed, we found that SARS-CoV-2 S protein suppressed IRAK-M mRNA and protein expression both in THP1 macrophages and primary human PBMCs and CD14+ monocytes. Engagement of SARS-CoV-2 S protein with ACE2 results in internalization of ACE2 and suppression of its activity. Activation of ACE2 has been previously shown to induce anti-inflammatory responses in macrophages. Treatment of macrophages with the ACE2 activator DIZE suppressed the pro-inflammatory action of SARS-CoV-2. Our results demonstrated that SARS-CoV-2/ACE2 interaction rendered macrophages hyper-responsive to TLR signals, suppressed IRAK-M and promoted pro-inflammatory cytokine expression. Thus, activation of ACE2 may be a potential anti-inflammatory therapeutic strategy to eliminate the development of cytokine storm observed in COVID-19 patients.

Unique Mutations in the Murine Hepatitis Virus Macrodomain Differentially Attenuate Virus Replication, Indicating Multiple Roles for the Macrodomain in Coronavirus Replication.

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in nonstructural protein 3 (nsp3) that binds and hydrolyzes mono-ADP-ribose (MAR) covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutation of a highly conserved asparagine residue, which makes contact with the distal ribose of ADP-ribose. To determine if additional Mac1 activities contribute to CoV replication, we compared the replication of murine hepatitis virus (MHV) Mac1 mutants, D1329A and N1465A, to the previously mentioned asparagine mutant, N1347A. These residues contact the adenine and proximal ribose in ADP-ribose, respectively. N1465A had no effect on MHV replication or pathogenesis, while D1329A and N1347A both replicated poorly in bone marrow-derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. Interestingly, D1329A was also significantly more attenuated than N1347A in all cell lines tested. Conversely, D1329A retained some ability to block beta interferon (IFN-ß) transcript accumulation compared to N1347A, indicating that these mutations have different effects on Mac1 functions. Combining these two mutations resulted in a virus that was unrecoverable, suggesting that the combined activities of Mac1 are essential for MHV replication. We conclude that Mac1 has multiple functions that promote the replication of MHV, and that these results provide further evidence that Mac1 is a prominent target for anti-CoV therapeutics. IMPORTANCE In the wake of the COVID-19 epidemic, there has been a surge to better understand how CoVs replicate and to identify potential therapeutic targets that could mitigate disease caused by SARS-CoV-2 and other prominent CoVs. The highly conserved macrodomain, also termed Mac1, is a small domain within nonstructural protein 3. It has received significant attention as a potential drug target, as previous studies demonstrated that it is essential for CoV pathogenesis in multiple animal models of infection. However, the functions of Mac1 during infection remain largely unknown. Here, using targeted mutations in different regions of Mac1, we found that Mac1 has multiple functions that promote the replication of MHV, a model CoV, and, therefore, is more important for MHV replication than previously appreciated. These results will help guide the discovery of these novel functions of Mac1 and the development of inhibitory compounds targeting this domain.

COVID-19 myocarditis: quantitative analysis of the inflammatory infiltrate and a proposed mechanism.

COVID-19 has a significant effect upon the cardiovascular system. While a number of different cardiovascular histopathologies have been described at post-mortem examination, the incidence of typical viral myocarditis in COVID-19 positive patients appears very low [1-3]. In this study, we further characterize and quantify the inflammatory cell infiltrate in a COVID-19 study cohort and compare the findings to both an age and disease matched control cohort and a cohort of patients diagnosed with typical inflammatory myocarditis. All study and control cohorts had 1 or more of the comorbidities most commonly associated with severe disease (hypertension, type II diabetes, obesity, or known cardiovascular disease). The results demonstrate a skewed distribution of the number of CD68+ cells in COVID-19 hearts, with upper quantiles showing a significant increase as compared to both matched control hearts, and those with myocarditis. In contrast, hearts from typical inflammatory myocarditis contained increased numbers of CD4+, and CD8+ cells compared to both COVID-19 and control cohorts. In conclusion, the presence of an increased number of CD68+ cells suggests that COVID-19 may incite a form of myocarditis different from typical viral myocarditis, and associated with diffusely infiltrative cells of monocytes/macrophage lineage.