TLR9 promotes monocytic myeloid-derived suppressor cell induction during JEV infection.

Myeloid-derived suppressor cells (MDSCs) are a group of heterologous populations of immature bone marrow cells consisting of progenitor cells of macrophages, dendritic cells and granulocytes. Recent studies have revealed that the accumulation of MDSCs in the mouse spleen plays a pivotal role in suppressing the immune response following JEV infection. However, the mechanisms by which JEV induces MDSCs are poorly understood. Here, it was found that JEV infection induces mitochondrial damage and the release of mitochondrial DNA (mtDNA), which further leads to the activation of TLR9. TLR9 deficiency decreases the M-MDSCs population and their suppressive function both in vitro and in vivo. Moreover, the increase of MHCⅡ expression on antigen-presenting cells and CD28 expression on T cells in TLR9-/- mice was positively correlated with M-MDSCs reduction. Accordingly, the survival rate of TLR9-/- mice dramatically increased after JEV infection. These findings reveal the connections of mitochondrial damage and TLR9 activation to the induction of M-MDSCs during JEV infection.

A broadly applicable protein-polymer adjuvant system for antiviral vaccines.

Although protein subunit vaccines generally have acceptable safety profiles with precise antigenic content, limited immunogenicity can lead to unsatisfactory humoral and cellular immunity and the need for vaccine adjuvants and delivery system. Herein, we assess a vaccine adjuvant system comprising Quillaja Saponaria-21(QS-21) and cobalt porphyrin polymeric micelles that enabling the display of His-tagged antigen on its surface. The nanoscale micelles promote antigen uptake and dendritic cell activation to induce robust cytotoxic T lymphocyte response and germinal center formation. Using the recombinant protein antigens from influenza A and rabies virus, the micelle adjuvant system elicited robust antiviral responses and protected mice from lethal challenge. In addition, this system could be combined with other antigens to induce high titers of neutralizing antibodies in models of three highly pathogenic viral pathogens: Ebola virus, Marburg virus, and Nipah virus. Collectively, our results demonstrate this polymeric micelle adjuvant system can be used as a potent nanoplatform for developing antiviral vaccine countermeasures that promote humoral and cellular immunity.

The matrix protein of lyssavirus hijacks autophagosome for efficient egress by recruiting NEDD4 through its PPxY motif.

Lyssaviruses are well-known worldwide and often cause fatal encephalitis. Previous studies have shown that autophagy is beneficial for the replication of rabies virus (RABV), the representative lyssavirus, but the detailed mechanism remains obscure. In this study, we showed that the rabies virus matrix protein (RABV-M) used its PPxY motif to interact with the E3 ubiquitin-protein ligase NEDD4. NEDD4 then recruited MAP1LC3/LC3 via its LC3-interacting region (LIR). Interestingly, after binding to the ubiquitinated RABV-M, NEDD4 could bind more LC3 and enhance autophagosome accumulation, while NEDD4 knockdown significantly reduced M-induced autophagosome accumulation. Further study revealed that RABV-M prevented autophagosome-lysosome fusion and facilitated viral budding. Inhibition of RABV-M-induced autophagosome accumulation reduced the production of extracellular virus-like particles. We also found that M proteins of most lyssaviruses share the same mechanism to accumulate autophagosome by hijacking NEDD4. Collectively, this study revealed a novel strategy for lyssaviruses to achieve efficient viral replication by exploiting the host autophagy system.Abbreviations: ABLV: Australian bat lyssavirus; ATG5: autophagy related 5; Baf A1:bafilomycin A1;co-IP: co-immunoprecipitation; CQ: chloroquine; DAPI:4',6-diamidino-2'-phenylindole; DMSO: dimethyl sulfoxide; EBLV:European bat lyssavirus; GFP: green fluorescent protein; GST:glutathione S-transferase; hpi: hours post-infection; hpt: hourspost-transfection; LIR: LC3-interactingregion;MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; mCherry:red fluorescent protein; MOI: multiplicity of infection; NC: negativecontrol; MVB: multivesicular body; NEDD4: neural precursorcell-expressed developmentally down-regulated 4; RABV: rabies virus;SQSTM1/p62: sequestosome 1; VLP: virus-like particle; VPS4B: vacuolarprotein sorting 4B; TEM: transmission electron microscopy; WB:western blotting; WT: wild-type; µm: micrometer; µM: micromole.

Neuroinvasive virus facilitates viral replication by employing lipid droplets to reduce arachidonic acid-induced ferroptosis.

Lipids have been previously implicated in the lifecycle of neuroinvasive viruses. However, the role of lipids in programmed cell death and the relationship between programmed cell death and lipid droplets (LDs) in neuroinvasive virus infection remains unclear. Here, we found that the infection of neuroinvasive virus, such as rabies virus and encephalomyocarditis virus could enhance the LD formation in N2a cells, and decreasing LDs production by targeting diacylglycerol acyltransferase could suppress viral replication. The lipidomics analysis revealed that arachidonic acid (AA) was significantly increased after reducing LD formation by restricting diacylglycerol acyltransferase, and AA was further demonstrated to induce ferroptosis to inhibit neuroinvasive virus replication. Moreover, lipid peroxidation and viral replication inhibition could be significantly alleviated by a ferroptosis inhibitor, ferrostatin-1, indicating that AA affected neuroinvasive virus replication mainly through inducing ferroptosis. Furthermore, AA was demonstrated to activate the acyl-CoA synthetase long-chain family member 4-lysophosphatidylcholine acyltransferase 3-cytochrome P450 oxidoreductase axis to induce ferroptosis. Our findings highlight novel cross-talks among viral infection, LDs, and ferroptosis for the first time, providing a potential target for antiviral drug development.

Lyssavirus M protein degrades neuronal microtubules by reprogramming mitochondrial metabolism.

Infection with neurotropic viruses may result in changes in host behavior, which are closely associated with degenerative changes in neurons. The lyssavirus genus comprises highly neurotropic viruses, including the rabies virus (RABV), which has been shown to induce degenerative changes in neurons, marked by the self-destruction of axons. The underlying mechanism by which the RABV degrades neuronal cytoskeletal proteins remains incomplete. In this study, we show that infection with RABV or overexpression of its M protein can disrupt mitochondrial metabolism by binding to Slc25a4. This leads to a reduction in NAD+ production and a subsequent influx of Ca2+ from the endoplasmic reticulum and mitochondria into the cytoplasm of neuronal cell lines, activating Ca2+-dependent proteinase calpains that degrade α-tubulin. We further screened the M proteins of different lyssaviruses and discovered that the M protein of the dog-derived RABV strain (DRV) does not degrade α-tubulin. Sequence analysis of the DRV M protein and that of the lab-attenuated RABV strain CVS revealed that the 57th amino acid is vital for M-induced microtubule degradation. We generated a recombinant RABV with a mutation at the 57th amino acid position in its M protein and showed that this mutation reduces α-tubulin degradation in vitro and axonal degeneration in vivo. This study elucidates the mechanism by which lyssavirus induces neuron degeneration.IMPORTANCEPrevious studies have suggested that RABV (rabies virus, the representative of lyssavirus) infection induces structural abnormalities in neurons. But there are few articles on the mechanism of lyssavirus' effect on neurons, and the mechanism of how RABV infection induces neurological dysfunction remains incomplete. The M protein of lyssavirus can downregulate cellular ATP levels by interacting with Slc25a4, and this decrease in ATP leads to a decrease in the level of NAD+ in the cytosol, which results in the release of Ca2+ from the intracellular calcium pool, the endoplasmic reticulum, and mitochondria. The presence of large amounts of Ca2+ in the cytoplasm activates Ca2+-dependent proteases and degrades microtubule proteins. The amino acid 57 of M protein is the key site determining its disruption of mitochondrial metabolism and subsequent neuron degeneration.

A modified recombinant adenovirus vector containing dual rabies virus G expression cassettes confers robust and long-lasting humoral immunity in mice, cats, and dogs.

During the COVID-19 epidemic, the incidence of rabies has increased in several countries, especially in remote and disadvantaged areas, due to inadequate surveillance and declining immunization coverage. Multiple vaccinations with inactivated rabies virus vaccines for pre- or post-exposure prophylaxis are considered inefficient, expensive and impractical in developing countries. Herein, three modified human recombinant adenoviruses type 5 designated Adv-RVG, Adv-E1-RVG, and Adv-RVDG, carrying rabies virus G (RVG) expression cassettes in various combinations within E1 or E3 genomic regions, were constructed to serve as rabies vaccine candidates. Adv-RVDG mediated greater RVG expression both in vitro and in vivo and induced a more robust and durable humoral immune response than the rabies vaccine strain SAD-L16, Adv-RVG, and Adv-E1-RVG by more effectively activating the dendritic cells (DCs) - follicular helper T (Tfh) cells - germinal centre (GC) / memory B cells (MBCs) - long-lived plasma cells (LLPCs) axis with 100% survival after a lethal RABV challenge in mice during the 24-week study period. Similarly, dogs and cats immunized with Adv-RVDG showed stronger and longer-lasting antibody responses than those vaccinated with a commercial inactivated rabies vaccine and showed good tolerance to Adv-RVDG. In conclusion, our study demonstrated that simultaneous insertion of protective antigens into the E1 and E3 genomic regions of adenovirus vector can significantly enhance the immunogenicity of adenoviral-vectored vaccines, providing a theoretical and practical basis for the subsequent development of multivalent and multi-conjugated vaccines using recombinant adenovirus platform. Meanwhile, our data suggest Adv-RVDG is a safe, efficient, and economical vaccine for mass-coverage immunization.

Circular RNA vaccines with long-term lymph node-targeting delivery stability after lyophilization induce potent and persistent immune responses.

IMPORTANCE: messenger RNA (mRNA) vaccines are a key technology in combating existing and emerging infectious diseases. However, the inherent instability of mRNA and the nonspecificity of lipid nanoparticle-encapsulated (LNP) delivery systems result in the need for cold storage and a relatively short-duration immune response to mRNA vaccines. Herein, we develop a novel vaccine in the form of circRNAs encapsulated in LNPs, and the circular structure of the circRNAs enhances their stability. Lyophilization is considered the most effective method for the long-term preservation of RNA vaccines. However, this process may result in irreversible damage to the nanoparticles, particularly the potential disruption of targeting modifications on LNPs. During the selection of lymph node-targeting ligands, we found that LNPs modified with mannose maintained their physical properties almost unchanged after lyophilization. Additionally, the targeting specificity and immunogenicity remained unaffected. In contrast, even with the addition of cryoprotectants such as sucrose, the physical properties of LNPs were impaired, leading to an obvious decrease in immunogenicity. This may be attributed to the protective role of mannose on the surface of LNPs during lyophilization. Freshly prepared and lyophilized mLNP-circRNA vaccines elicited comparable immune responses in both the rabies virus model and the SARS-CoV-2 model. Our data demonstrated that mLNP-circRNA vaccines elicit robust immune responses while improving stability after lyophilization, with no compromise in tissue targeting specificity. Therefore, mannose-modified LNP-circRNA vaccines represent a promising vaccine design strategy.

Cholesterol depletion inhibits rabies virus infection by restricting viral adsorption and fusion.

Rabies is an ancient zoonotic disease caused by the rabies virus (RABV), and a sharp increase in rabies cases and deaths were observed following the COVID-19 pandemic, indicating that it still poses a severe public health threat in most countries in the world. Cholesterol is one of the major lipid components in cells, and the exact role of cholesterol in RABV infection remains unclear. In this study, we initially observed that cellular cholesterol levels were significantly elevated in RABV infected cells, while cholesterol depletion by using methyl-ß-cyclodextrin (MßCD) could restrict RABV entry. We further found that decreasing the cholesterol level of the viral envelope could change the bullet-shaped morphology of RABV and dislodge the glycoproteins on its surface to affect RABV entry. Moreover, the depletion of cholesterol could decrease lysosomal cholesterol accumulation to inhibit RABV fusion. Finally, it was found that the depletion of cholesterol by MßCD was due to the increase of oxygen sterol production in RABV-infected cells and the enhancement of cholesterol efflux by activating liver X receptor alpha (LXRα). Together, our study reveals a novel role of cholesterol in RABV infection, providing new insight into explore of effective therapeutics for rabies.

A single immunization with core-shell structured lipopolyplex mRNA vaccine against rabies induces potent humoral immunity in mice and dogs.

The persistence and clinical consequences of rabies virus (RABV) infection have prompted global efforts to develop a safe and effective vaccines against rabies. mRNA vaccines represent a promising option against emerging and re-emerging infectious diseases, gaining particular interest since the outbreak of COVID-19. Herein, we report the development of a highly efficacious rabies mRNA vaccine composed of sequence-modified mRNA encoding RABV glycoprotein (RABV-G) packaged in core-shell structured lipopolyplex (LPP) nanoparticles, named LPP-mRNA-G. The bilayer structure of LPP improves protection and delivery of RABV-G mRNA and allows gradual release of mRNA molecules as the polymer degrades. The unique core-shell structured nanoparticle of LPP-mRNA-G facilitates vaccine uptake and demonstrates a desirable biodistribution pattern with low liver targeting upon intramuscular immunization. Single administration of low-dose LPP-mRNA-G in mice elicited potent humoral immune response and provided complete protection against intracerebral challenge with lethal RABV. Similarly, single immunization of low-dose LPP-mRNA-G induced high levels of virus-neutralizing antibody titers in dogs. Collectively, our data demonstrate the potential of LPP-mRNA-G as a promising next-generation rabies vaccine used in human and companion animals.

Short-Chain Fatty Acids Alleviate Vancomycin-Caused Humoral Immunity Attenuation in Rabies-Vaccinated Mice by Promoting the Generation of Plasma Cells via Akt-mTOR Pathway.

Mounting evidence suggests that gut microbial composition and its metabolites, including short-chain fatty acids (SCFAs), have beneficial effects in regulating host immunogenicity to vaccines. However, it remains unknown whether and how SCFAs improve the immunogenicity of the rabies vaccine. In this study, we investigated the effect of SCFAs on the immune response to rabies vaccine in vancomycin (Vanco)-treated mice and found that oral gavage with butyrate-producing bacteria (C. butyricum) and butyrate supplementation elevated RABV-specific IgM, IgG, and virus-neutralizing antibodies (VNAs) in Vanco-treated mice. Supplementation with butyrate expanded antigen-specific CD4+ T cells and IFN-γ-secreting cells, augmented germinal center (GC) B cell recruitment, promoted plasma cells (PCs) and RABV-specific antibody-secreting cells (ASCs) generation in Vanco-treated mice. Mechanistically, butyrate enhanced mitochondrial function and activated the Akt-mTOR pathway in primary B cells isolated from Vanco-treated mice, ultimately promoting B lymphocyte-induced maturation protein-1 (Blimp-1) expression and CD138+ PCs generation. These results highlight the important role of butyrate in alleviating Vanco-caused humoral immunity attenuation in rabies-vaccinated mice and maintaining host immune homeostasis. IMPORTANCE The gut microbiome plays many crucial roles in the maintenance of immune homeostasis. Alteration of the gut microbiome and metabolites has been shown to impact vaccine efficacy. SCFAs can act as an energy source for B-cells, thereby promoting both mucosal and systemic immunity in the host by inhibiting HDACs and activation of GPR receptors. This study investigates the impact of orally administered butyrate, an SCFA, on the immunogenicity of rabies vaccines in Vanco-treated mice. The results showed that butyrate ameliorated humoral immunity by facilitating the generation of plasma cells via the Akt-mTOR in Vanco-treated mice. These findings unveil the impact of SCFAs on the immune response of the rabies vaccine and confirm the crucial role of butyrate in regulating immunogenicity to rabies vaccines in antibiotic-treated mice. This study provides a fresh insight into the relationship of microbial metabolites and rabies vaccination.

The CH24H metabolite, 24HC, blocks viral entry by disrupting intracellular cholesterol homeostasis.

Cholesterol-24-hydroxylase (CH24H or Cyp46a1) is a reticulum-associated membrane protein that plays an irreplaceable role in cholesterol metabolism in the brain and has been well-studied in several neuro-associated diseases in recent years. In the present study, we found that CH24H expression can be induced by several neuroinvasive viruses, including vesicular stomatitis virus (VSV), rabies virus (RABV), Semliki Forest virus (SFV) and murine hepatitis virus (MHV). The CH24H metabolite, 24-hydroxycholesterol (24HC), also shows competence in inhibiting the replication of multiple viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 24HC can increase the cholesterol concentration in multivesicular body (MVB)/late endosome (LE) by disrupting the interaction between OSBP and VAPA, resulting in viral particles being trapped in MVB/LE, ultimately compromising VSV and RABV entry into host cells. These findings provide the first evidence that brain cholesterol oxidation products may play a critical role in viral infection.

Inhibition of miR-200b-3p confers broad-spectrum resistance to viral infection by targeting TBK1.

The host innate immune system's defense against viral infections depends heavily on type I interferon (IFN-I) production. Research into the mechanisms of virus-host interactions is essential for developing novel antiviral therapies. In this study, we compared the effect of the five members of the microRNA-200 (miR-200) family on IFN-I production during viral infection and found that miR-200b-3p displayed the most pronounced regulatory effect. During viral infection, we discovered that the transcriptional level of microRNA-200b-3p (miR-200b-3p) increased with the infection of influenza virus (IAV) and vesicular stomatitis virus (VSV), and miR-200b-3p production was modulated by the activation of the ERK and p38 pathways. We identified cAMP response element binding protein (CREB) as a novel transcription factor that binds to the miR-200b-3p promoter. MiR-200b-3p reduces NF-κB and IRF3-mediated IFN-I production by targeting the 3' untranslated region (3' UTR) of TBK1 mRNA. Applying miR-200b-3p inhibitor enhances IFN-I production in IAV and VSV-infected mouse models, thus inhibiting viral replication and improving mouse survival ratio. Importantly, in addition to IAV and VSV, miR-200b-3p inhibitors exhibited potent antiviral effects against multiple pathogenic viruses threatening human health worldwide. Overall, our study suggests that miR-200b-3p might be a potential therapeutic target for broad-spectrum antiviral therapy. IMPORTANCE The innate immune response mediated by type I interferon (IFN-I) is essential for controlling viral replication. MicroRNAs (miRNAs) have been found to regulate the IFN signaling pathway. In this study, we describe a novel function of miRNA-200b-3p in negatively regulating IFN-I production during viral infection. miRNA-200b-3p was upregulated by the MAPK pathway activated by IAV and VSV infection. The binding of miRNA-200b-3p to the 3' UTR of TBK1 mRNA reduced IFN-I activation mediated by IRF3 and NF-κB. Application of miR-200b-3p inhibitors exhibited potent antiviral effects against multiple RNA and DNA viruses. These results provide fresh insight into understanding the impact of miRNAs on host-virus interactions and reveal a potential therapeutic target for common antiviral intervention.

Interleukin-1ß suppresses rabies virus infection by activating cGAS-STING pathway and compromising the blood-brain barrier integrity in mice.

Rabies, caused by rabies virus (RABV), is an ancient zoonotic disease that severely threatens the public health throughout the world. Previous study indicated that interleukin-1ß (IL-1ß) plays an important role in RABV infection. However, the mechanism how IL-1ß affects RABV pathogenicity is still unknown yet. In this study, we confirmed that IL-1ß was able to reduce viral titers of RABV in different cells, and the recombinant RABV expressing IL-1ß, designated as rCVS-IL1ß, could be suppressed in different cells due to the expression of IL-1ß. Furthermore, the survival rates of mice infected with rCVS-IL1ß by intramuscular route was significantly higher than those of mice infected with parent virus rCVS, which is associated with the less viral loads for entry into the central nervous system (CNS). We further characterized that the cGAS-STING pathway was activated in rCVS-IL1ß infected bone marrow derived dendritic cells (BMDC), which could contribute to the decreased viral loads of RABV after intramuscular infection. Moreover, we also observed that the expression of IL-1ß by rCVS-IL1ß could compromise the blood-brain barrier (BBB) integrity by degrading the tight junction proteins, which allowing peripheral inflammatory cytokines, chemokines, and CD4+T cells to enter into the brain for the clearance of RABV in the CNS. Together, our study suggests that IL-1ß could attenuate RABV pathogenicity through activating cGAS-STING pathway in to decrease the viral entry into the CNS and enhance the BBB permeability to promote RABV clearance in the CNS as well, which provides new insight into developing effective therapeutics for rabies.

Substitution of S179P in the Lyssavirus Phosphoprotein Impairs Its Interferon Antagonistic Function.

Lyssaviruses cause rabies, which is an acute neurological disease responsible for more than 59,000 human deaths annually and has no available effective treatments. The phosphoprotein (P) of lyssaviruses (lyssavirus-P) plays multiple roles in virus replication and immune evasion. Lyssavirus-P has been identified as the major type I interferon (IFN-I) antagonist, while the precise site and precise molecular mechanism remain unclear. Herein, we found that substitution of site 179 of lyssavirus-P from serine (Ser) to proline (Pro) impairs its antagonism function of IFN-I by sequence alignment and site mutations. Subsequent studies demonstrated that lyssavirus-P containing S179 specifically interacted with I-kappa B kinase ε (IKKε). Specifically, lyssavirus-P containing S179 interacted simultaneously with the kinase domain (KD) and scaffold dimerization domain (SDD) of IKKε, competing with TNF receptor-associated factor 3 (TRAF3) and IFN regulatory factor 3 (IRF3) for binding with IKKε, leading to the inhibition of IFN production. Furthermore, S179 was involved in the viral pathogenicity of the typical lyssavirus rabies virus in a mouse model. Interestingly, we found that S179 is conserved among most lyssavirus-P and functional for IFN antagonism. Collectively, we identified S179 of lyssavirus-P is essential for IFN-I inhibition, which provides deep insight into the immune evasion strategies of lyssaviruses. IMPORTANCE Interferon (IFN) and the IFN-induced cellular antiviral response constitute the first line of defense against viral invasion. Evading host innate immunity, especially IFN signaling, is the key step required for lyssaviruses to establish infection. In this study, S179 of lyssavirus phosphoprotein (lyssavirus-P) was identified as the key site for antagonizing IFN-I production. Mechanistically, lyssavirus-P containing S179 specifically targets the key kinase IKKε and disrupts its interaction with TRAF3 and IRF3. S179P mutation in the P protein of the typical lyssavirus rabies virus (RABV) attenuated its pathogenicity in a mouse model. Our findings provide deep insight into the immune evasion strategies of lyssaviruses, which is helpful for the development of effective antiviral therapeutics.

Multiple Death Pathways of Neutrophils Regulate Alveolar Macrophage Proliferation.

Alveolar macrophage (AM) proliferation and self-renewal play an important role in the lung tissue microenvironment. However, the impact of immune cells, especially the neutrophils, on AM homeostasis or function is not well characterized. In this study, we induced in vivo migration of neutrophils into bronchoalveolar lavage (BAL) fluid and lung using CXCL1, and then co-cultured these with AMs in vitro. Neutrophils in the BAL (BAL-neutrophils), rather than neutrophils of bone marrow (BM-neutrophils), were found to inhibit AM proliferation. Analysis of publicly available data showed high heterogeneity of lung neutrophils with distinct molecular signatures of BM- and blood-neutrophils. Unexpectedly, BAL-neutrophils from influenza virus PR8-infected mice (PR8-neutrophils) did not inhibit the proliferation of AMs. Bulk RNA sequencing further revealed that co-culture of AMs with PR8-neutrophils induced IFN-α and -γ responses and inflammatory response, and AMs co-cultured with BAL-neutrophils showed higher expression of metabolism- and ROS-associated genes; in addition, BAL-neutrophils from PR8-infected mice modulated AM polarization and phagocytosis. BAL-neutrophil-mediated suppression of AM proliferation was abrogated by a combination of inhibitors of different neutrophil death pathways. Collectively, our findings suggest that multiple cell death pathways of neutrophils regulate the proliferation of AMs. Targeting neutrophil death may represent a potential therapeutic strategy for improving AM homeostasis during respiratory diseases.

Decoding the spatial chromatin organization and dynamic epigenetic landscapes of macrophage cells during differentiation and immune activation.

Immunocytes dynamically reprogram their gene expression profiles during differentiation and immunoresponse. However, the underlying mechanism remains elusive. Here, we develop a single-cell Hi-C method and systematically delineate the 3D genome and dynamic epigenetic atlas of macrophages during these processes. We propose "degree of disorder" to measure genome organizational patterns inside topologically-associated domains, which is correlated with the chromatin epigenetic states, gene expression, and chromatin structure variability in individual cells. Furthermore, we identify that NF-κB initiates systematic chromatin conformation reorganization upon Mycobacterium tuberculosis infection. The integrated Hi-C, eQTL, and GWAS analysis depicts the atlas of the long-range target genes of mycobacterial disease susceptible loci. Among these, the SNP rs1873613 is located in the anchor of a dynamic chromatin loop with LRRK2, whose inhibitor AdoCbl could be an anti-tuberculosis drug candidate. Our study provides comprehensive resources for the 3D genome structure of immunocytes and sheds insights into the order of genome organization and the coordinated gene transcription during immunoresponse.

Lab-Attenuated Rabies Virus Facilitates Opening of the Blood-Brain Barrier by Inducing Matrix Metallopeptidase 8.

Infection with laboratory-attenuated rabies virus (RABV), but not wild-type (wt) RABV, can enhance the permeability of the blood-brain barrier (BBB), which is considered a key determinant for RABV pathogenicity. A previous study showed that the enhancement of BBB permeability is directly due not to RABV infection but to virus-induced inflammatory molecules. In this study, the effect of the matrix metallopeptidase (MMP) family on the permeability of the BBB during RABV infection was evaluated. We found that the expression level of MMP8 was upregulated in mice infected with lab-attenuated RABV but not with wt RABV. Lab-attenuated RABV rather than wt RABV activates inflammatory signaling pathways mediated by the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Activated NF-κB (p65) and AP-1 (c-Fos) bind to the MMP8 promoter, resulting in upregulation of its transcription. Analysis of mouse brains infected with the recombinant RABV expressing MMP8 indicated that MMP8 enhanced BBB permeability, leading to infiltration of inflammatory cells into the central nervous system (CNS). In brain-derived endothelial cells, treatment with MMP8 recombinant protein caused the degradation of tight junction (TJ) proteins, and the application of an MMP8 inhibitor inhibited the degradation of TJ proteins after RABV infection. Furthermore, an in vivo experiment using an MMP8 inhibitor during RABV infection demonstrated that BBB opening was diminished. In summary, our data suggest that the infection of lab-attenuated RABV enhances the BBB opening by upregulating MMP8. IMPORTANCE The ability to change BBB permeability was associated with the pathogenicity of RABV. BBB permeability was enhanced by infection with lab-attenuated RABV instead of wt RABV, allowing immune cells to infiltrate into the CNS. We found that MMP8 plays an important role in enhancing BBB permeability by degradation of TJ proteins during RABV infection. Using an MMP8 selective inhibitor restores the reduction of TJ proteins. We reveal that MMP8 is upregulated via the MAPK and NF-κB inflammatory pathways, activated by lab-attenuated RABV infection but not wt RABV. Our findings suggest that MMP8 has a critical role in modulating the opening of the BBB during RABV infection, which provides fresh insight into developing effective therapeutics for rabies and infection with other neurotropic viruses.

JEV Infection Induces M-MDSC Differentiation Into CD3+ Macrophages in the Brain.

Japanese encephalitis virus (JEV) is one of the most important members of the flavivirus family. It is a typical zoonotic pathogen that has caused substantial social and economic losses worldwide. The relation between JEV-induced immunosuppression and inflammatory responses has not been thoroughly investigated. In this study, cells infiltrating the brain tissue of JEV-infected mice were mainly identified as monocytic myeloid-derived suppressor cells (M-MDSCs), which subsequently differentiated into CD3+ macrophages. Co-culture with T cells showed that both splenic M-MDSCs and brain infiltrated M-MDSCs isolated from JEV-infected mice inhibited T cell proliferation through ARG1 and iNOS. The splenectomy model revealed that JEV-induced M-MDSCs were mainly derived from bone marrow and migrated to the spleen and central nervous system (CNS). The results of the transcriptome analysis and IRF7-deficient mice indicated that the ZBP1-IRF7 signaling pathway stimulated by JEV RNA played a central role in the induction of M-MDSCs. M-MDSCs migrated into the CNS through the chemokine CCL2/N-CCL2 derived from astrocytes and brain infiltrated M-MDSCs differentiated into CD3+ macrophages through a mechanism mediated by M-CSF, IL-6 and IFN-γ in the brain microenvironment. These findings provide evidence for the mechanism that JEV regulates the differentiation of M-MDSCs and thereby exacerbates pathogenicity, which represents a potential therapeutic target for Japanese encephalitis (JE).

lncRNA EDAL restricts rabies lyssavirus replication in a cell-specific and infection route-dependent manner.

Rabies, caused by rabies lyssavirus (RABV), is a fatal disease among humans and almost all warm-blooded animals. Our previous study showed that the long non-coding RNA (lncRNA) EZH2 degradation-associated lncRNA (EDAL) effectively inhibits RABV infection both in vitro and in vivo by degrading EZH2 and promoting the transcription of an antiviral gene, Pcp4l1. Herein, we found that recombinant RABV expressing EDAL (rRABV-EDAL) restricts RABV replication in primary granule neurons but not in primary cortical neurons or astrocytes. Further study revealed that EDAL induced EZH2 protein degradation and thereby decreased trimethylation of lysine 27 on the histone 3 (H3K27me3) level in granule neuron cells but not in cortical neurons or astrocytes. Furthermore, rRABV-EDAL infection induces more Pcp4l1 mRNA transcription in granule neurons, while there are almost no obvious changes in cortical neurons or astrocytes. Consistently, compared with the parent virus RABV, reduced pathogenicity of rRABV-EDAL was observed in mice post-intranasal infection but not intramuscular infection. These results suggest that the lncRNA EDAL restricts RABV replication in a cell-specific and infection route-dependent manner.