Double-stranded DNA coming from, for example, viruses, bacteria, or apoptotic cells is recognized by the cGAS-STING signaling pathway comprising the cyclic GMP-AMP synthase (cGAS) and the stimulator of interferon genes (STING) receptors. The pathway induces type I interferon response and activates transcription of interferon-stimulated genes and proinflammatory cytokines. Though the brain is an immune-privileged site, the blood-brain barrier (BBB) elicits inflammatory immune response in neurodegenerative diseases. Parkinson's disease is characterized by α-synuclein oligomer (αSO) aggregates, neurodegeneration, and mitophagy, which potential can activate the cGAS-STING pathway. Here, we studied the cGAS-STING pathway in a co-culture model of the rat BBB treated with and without α-synuclein monomers (αSM) or oligomers (αSO). Activation of the cGAS-STING pathway did not change barrier integrity and junctional protein staining, but it induced the transcription of the interferon-stimulated gene Viperin and the proinflammatory cytokine tumor necrosis factor-α in brain endothelial cells. Furthermore, STING activation increased the protein level of Viperin in astrocytes. The treatment with αSO, but not αSM, decreased barrier tightness and induced the transcription of Viperin and tumor necrosis factor-α in brain endothelial cells. In astrocytes, αSO treatment increased not only Viperin and tumor necrosis factor-α mRNA levels, but also interleukin-1β and interleukin-6. In conclusion, cGAS-STING pathway and downstream immune signaling pathways can be activated in the cells of a co-culture model of the BBB without influencing barrier integrity. However, αSO disrupts the BBB integrity and activates the cGAS-STING immune pathway in brain endothelial cells and astrocytes supporting the idea of using cGAS-STING as a therapeutic target in neuroinflammation.

The clinical outcome of SARS-CoV-2 infection spans from asymptomatic viral elimination to lethal COVID-19 pneumonia, which is due to type I interferon (IFN) deficiency in at least 15-20% of cases. We report two unrelated male patients with critical COVID-19 who are heterozygous for rare deleterious variants in RB1CC1, encoding the autophagy-related FIP200 protein. Airway epithelial cells genetically deprived of FIP200 or cell lines expressing the RB1CC1/FIP200 patient variants exhibit elevated SARS-CoV-2 replication and impaired autophagic flux. The antiviral function of FIP200 is independent of canonical autophagy and type I IFN, but involves the selective autophagy receptor NDP52. We identify a non-canonical function of FIP200 in a novel lysosomal degradation pathway, in which SARS-CoV-2 virions are targeted to single-membrane compartments for degradation of viral RNA in LC3B-positive acidified vesicles. This pathway is impaired in FIP200-deficient cells and in cells expressing FIP200 patient haplotypes. Collectively, we describe a cell-autonomous anti-SARS-CoV-2 restriction pathway, dependent on FIP200 and NDP52, and independent of canonical autophagy and type I IFN, which can underlie critical COVID-19 pneumonia.

OBJECTIVE:Pro-inflammatory T-cell responses dominate in Crohn's disease (CD). This may result from a dysbalanced expression of co-stimulatory and inhibitory T-cell receptors. The present study investigated if a dysbalanced co-stimulatory and inhibitory T-cell receptor expression are present in CD and can be rebalanced by anti-TNFα treatment. METHODS:Mucosal biopsies from 27 patients with active CD receiving anti-TNF treatment were examined for the mRNA levels of the co-stimulatory 4-1BB and inhibitory PD-1 T-cell receptor. Levels of mRNA were compared between inflamed and noninflamed tissue, and before and after treatment. Peripheral T cells from 12 healthy controls (HC) and 11 active CD patients were evaluated for their expression of 4-1BB and PD-1 by flow cytometry. RESULTS:The 4-1BB mRNA levels in inflamed mucosa were upregulated (> 2-fold) compared with uninflamed mucosa (p < 0.05). Anti-TNFα treatment reduced the 4-1BB and PD-1 mRNA levels in the inflamed gut tissue (p < 0.05). In in vitro activated T cells, the percentage of both 4-1BB and PD-1 positive CD4+ and CD8+ T cells increased more than 1.5 fold compared with HC (p < 0.05). The 4-1BB/PD-1 ratio on activated peripheral T cells was significantly reduced in CD after anti-TNF therapy (p < 0.05). CONCLUSIONS:A dysbalanced mucosal proinflammatory co-stimulatory T cell receptor expression was present in active CD and modified by anti-TNFα treatment. However, anti-TNFα treatment did not normalize the expression of 4-1BB or PD-1 on peripheral T cells although a modest increased immunoregulatory capacity could be demonstrated.

NF-κB is central for activation of immune responses. Cytosolic DNA activates the cGAS–STING pathway to induce type I interferons (IFNs) and signaling through NF-κB, thus instigating host defenses and pathological inflammation. However, the mechanism underlying STING-induced NF-κB activation is unknown. Here we report that STING activates NF-κB in a delayed manner, following exit from the Golgi to endolysosomal compartments. Activation of NF-κB is dependent on the IFN-inducing transcription factor IRF3 but is independent of type I IFN signaling. This activation pattern is evolutionarily conserved in tetrapods. Mechanistically, the monomer IRF3 is recruited to STING pS358, with delayed kinetics relative to IRF3 recruitment to STING pS366, which promotes type I IFN responses. IRF3 engagement with STING pS358 induces trafficking to late endolysosomal compartments, supporting recruitment of TRAF6 and activation of NF-κB. We identify a TRAF6 binding motif in IRF3 that facilitates recruitment of TRAF6. This work defines a signaling surface on STING and a function for IRF3 as an adaptor in immune signaling. These findings indicate that STING signaling to NF-κB is enabled only within a short time window between exit from the Golgi and lysosomal degradation, possibly limiting inflammation under homeostatic and danger-sensing conditions. Here the authors show how the DNA-sensing cGAS–STING pathway activates NF-κB and inflammatory gene expression with delayed kinetics via post-Golgi endolysosomal signaling.

BACKGROUND:Herpes Simplex Virus 1 (HSV-1) is a neurotropic virus causing encephalitis and post-infectious complications. Infections can induce a range of acute, subacute, and progressing brain disease, and in recent years it has emerged that immune responses are involved in the pathogenesis of these diseases. METHODS:Mice were infected with HSV-1 through corneal infection, and the brain stem was analyzed using single-cell and GeoMx spatial transcriptomics. Through these technologies we profiled temporal transcriptomic changes in cell populations, pathways, and cell-cell communication associated with antiviral activity and inflammation-induced disturbance of physiological brain structures and activities. RESULTS:We found that microglia proportions increased early after HSV-1 infection, followed by monocyte influx and later by T cells. The blood-brain barrier was disrupted, and transcriptomic profiles associated with homeostatic brain transcriptional activities were altered. Early transcriptional responses were dominated by antiviral and inflammatory activities. A microglia subpopulation with high type I interferon and chemokine expression localized to infection sites, likely mediating antiviral defense and immune recruitment. Monocyte subpopulations displayed a broader activation profile than microglia and was a central mediator of crosstalk between immune cells. Cytokines from microglia, monocytes, and T cells reprogrammed brain cells, notably endothelial cells and oligodendrocytes, disrupting brain functions. Comparing datasets from various brain diseases revealed the identified microglia subpopulation as specific to viral infections. CONCLUSIONS:This study identifies a unique population of virus-activated microglia with antiviral and proinflammatory properties and reveals monocytes to be a key driver of interactions driving pathology in the virus-infected brain.

Herpes simplex encephalitis (HSE) is a devastating disease with high mortality and serious sequelae. Genetic defects in the IFN-I pathway predispose individuals to HSE, but underlying mechanisms remain unclear. Using transgenic mice with the IRF3 R278Q mutation, ortholog to HSE-associated IRF3 R285Q, and iPSC-derived CNS cells from a pediatric patient carrying the variant, we investigated mechanisms in HSE. IRF3 R278Q transgenic mice exhibited aggravated HSV-1 brain disease and elevated CNS viral loads. Accordingly, microglia from the IRF3 R278Q mice showed reduced HSV-1-induced IFN-I expression. Surprisingly, unaltered Ifnb levels along with elevated levels of inflammatory cytokines were detected in infected transgenic mouse brains, correlating with higher viral load. This was successfully modeled in patient microglia. Multiomics-based immune profiling revealed an inflammatory monocyte population in the infected IRF3 R278Q mouse brain, which was enriched for NF-κB activation. NF-κB inhibition improved disease outcomes, surpassing the effect of acyclovir. These findings suggest that IFN-I defects lead to elevated levels of HSV-1 replication in the brain, which subsequently enables NF-κB-driven immunopathology, offering insights with therapeutic potential.

Stimulator of interferon genes (STING) is a cytosolic DNA sensor that activates type I interferon (IFN) signaling, which plays a key role in neuroinflammation. Although the role of STING in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS), remains debated, its involvement in the development of CNS lesions, particularly within localized pathology, modeled here by targeting the corpus callosum, has yet to be explored. Using a focal EAE model, we compared the induction of lesions in wild-type and STING-deficient (STINGgt/gt) mice. Lesions were analyzed by immunohistochemistry, flow cytometry, and transcriptomics. STING-deficient mice had significantly larger demyelinated lesions, reduced ISG expression, and modified immune cell infiltration. STING signaling limits lesion severity in focal EAE by promoting IFN responses and regulating immune infiltration. These findings position STING as a potential target for MS therapy.

Neurotropic viruses affecting the central nervous system cause significant short- and long-term morbidity and mortality. Sequelae are extremely prevalent, with up to 50% of survivors experiencing neurological, neuropsychiatric, and behavioral issues. However, the immunopathological and virological mechanisms and factors influencing these outcomes remain poorly understood. In this review, we outline the available knowledge on the long-term outcomes of brain infections of several widespread viruses and highlight key current research gaps.

Microglia response is proposed to be relevant in the neurogenerative process associated with alpha-synuclein (α-syn) pathology in Parkinson’s disease (PD). STING is a protein related to the immune sensing of DNA and autophagy, and it has been proposed to be involved in PD neurodegeneration. To investigate this, we injected 10 µg of murine pre-formed fibrils (PFFs) of α-syn (or monomeric and PBS as controls) into the striatum of wild-type (WT) and STINGgt/gt mice, which lack functional STING. We examined motor behavior and brain pathology at 1- and 6-month post-injection. STINGgt/gt mice showed more motor changes associated with PFF injection than WT mice. STINGgt/gt mice had a differential immune response to PFF with early and sustained increased microglia numbers and higher macrophagic CD68 response, but milder changes in the expression of immune-relevant markers such as TLR2, TLR4, IL1ß, and TREM2. However, the lack of STING did not induce changes in the extent of α-syn pathology nor the p62 accumulation seen in the model. Altogether, this resulted in a faster but similar degree of nigrostriatal dopaminergic degeneration after 6 months. Therefore, the data do not support a necessary role for STING in the α-syn-induced nigral neuronal loss in the PFF-PD mouse model used here. However, the results suggest a functional relevance for STING in the brain response to the excess and aggregation of amyloidogenic proteins such as α-syn that can contribute to symptomatic changes.

Herpes Simplex Virus 1 (HSV-1) is a common human neurotropic virus with the majority of adults harboring latent-recurrent infections. In rare cases, HSV-1 infection can access the central nervous system through the neuronal route and develop into life-threatening encephalitis. Here, we used a mouse model for HSV-1 infection to describe the transcriptomic profile of the infected brain stem at the single-cell level and with temporal resolution. Among resident brain cells, microglia increased in proportion during the course of infection, while astrocytes, pericytes, and endothelial cell levels decrease. At the levels of peripheral immune cells, we found notably monocytes to strongly influx the infected brain. Large dynamic changes were found in the abundance of subpopulations of the different cell types following virus infection. For instance, we identify one subpopulation of microglia exhibiting very high type I interferon and chemokine expression early during infection. This population was also enriched for viral transcripts, suggesting localization at foci of infection, and orchestrating recruitment of other immune cells. In contrast, for the infiltrating monocytes, we identified a larger panel of unique subpopulations with antiviral and inflammatory phenotypes, and found not all of these being highly positive for viral transcripts, thus indicating monocyte activities beyond the infected brain areas. Finally, investigation of endothelial cell cross-talk with other cell types revealed that cytokines derived from microglia and monocyte, but also T cells, contribute to disturbance of the blood brain barrier. Our work thus reveals for the first time the complex nature of the cellular response in the virus-infected brain, which seeks to eliminate infection but can also prime for pathological changes. ### Competing Interest Statement The authors have declared no competing interest.

Pattern recognition receptors (PRRs) induce host defense but can also induce exacerbated inflammatory responses. This raises the question of whether other mechanisms are also involved in early host defense. Using transcriptome analysis of disrupted transcripts in herpes simplex virus (HSV)-infected cells, we find that HSV infection disrupts the hypoxia-inducible factor (HIF) transcription network in neurons and epithelial cells. Importantly, HIF activation leads to control of HSV replication. Mechanistically, HIF activation induces autophagy, which is essential for antiviral activity. HSV-2 infection in vivo leads to hypoxia in CNS neurons, and mice with neuron-specific HIF1/2α deficiency exhibit elevated viral load and augmented PRR signaling and inflammatory gene expression in the CNS after HSV-2 infection. Data from human stem cell-derived neuron and microglia cultures show that HIF also exerts antiviral and inflammation-restricting activity in human CNS cells. Collectively, the HIF transcription factor system senses virus-induced hypoxic stress to induce cell-intrinsic antiviral responses and limit inflammation.

RNA vaccines elicit protective immunity against SARS-CoV-2, but the use of mRNA as an antiviral immunotherapeutic is unexplored. Here, we investigate the activity of lipidoid nanoparticle (LNP)-formulated mRNA encoding human IFNλ1 (ETH47), which is a critical driver of innate immunity at mucosal surfaces protecting from viral infections. IFNλ1 mRNA administration promotes dose-dependent protein translation, induction of interferon-stimulated genes without relevant signs of unspecific immune stimulation, and dose-dependent inhibition of SARS-CoV-2 replication in vitro. Pulmonary administration of IFNλ1 mRNA in mice results in a potent reduction of virus load, virus-induced body weight loss and significantly increased survival. These data support the development of inhaled administration of IFNλ1 mRNA as a potential prophylactic option for individuals exposed to SARS-CoV-2 or at risk suffering from COVID-19. Based on the broad antiviral activity of IFNλ1 regardless of virus or variant, this approach might also be utilized for other respiratory viral infections or pandemic preparedness.

The inhibition of heat shock protein 90 (HSP90), a molecular chaperone, has been proposed to be a potential novel treatment strategy for Coronavirus disease 2019 (COVID-19). In contrast to other studies, our data demonstrated that RGRN-305, a HSP90 inhibitor, exacerbated the cytopathic effect and did not reduce the viral shedding in VeroE6-hTMPRSS2 cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Likewise in a murine model of SARS-CoV-2, transgenic mice treated orally with RGRN-305 exhibited reduced survival by the end of the experiment (day 12) as 14% (1/7) survived compared to 63% (5/8) of those treated with drug-vehicle. Animal weight was not reduced by the RGRN-305 treatment. Interestingly, we demonstrated that inhibition of HSP90 by RGRN-305 significantly dampened the inflammatory response induced by SARS-CoV-2 spike protein in human macrophage-like cells (U937) and human lung epithelial cells (A549). Measured by quantitative real-time PCR, the mRNA expression of the proinflammatory cytokines TNF, IL1B and IL6 were significantly reduced. Together, these data suggest that HSP90 inhibition by RGRN-305 exacerbates the SARS-CoV-2 infection in vitro and reduces the survival of mice infected with SARS-CoV-2, but exhibits strong anti-inflammatory properties. This data shows that while RGRN-305 may be helpful in a ‘cytokine storm’, it has no beneficial impact on viral replication or survival in animals as a monotherapy. Further animal studies with HSP90 inhibitors in combination with an anti-viral drug may provide additional insights into its utility in viral infections and whether HSP90 inhibition may continue to be a potential treatment strategy for COVID-19 disease.

The brain is highly sensitive to damage caused by infection and inflammation1,2. Herpes simplex virus 1 (HSV-1) is a neurotropic virus and the cause of herpes simplex encephalitis3. It is unknown whether neuron-specific antiviral factors control virus replication to prevent infection and excessive inflammatory responses, hence protecting the brain. Here we identify TMEFF1 as an HSV-1 restriction factor using genome-wide CRISPR screening. TMEFF1 is expressed specifically in neurons of the central nervous system and is not regulated by type I interferon, the best-known innate antiviral system controlling virus infections. Depletion of TMEFF1 in stem-cell-derived human neurons led to elevated viral replication and neuronal death following HSV-1 infection. TMEFF1 blocked the HSV-1 replication cycle at the level of viral entry through interactions with nectin-1 and non-muscle myosin heavy chains IIA and IIB, which are core proteins in virus-cell binding and virus-cell fusion, respectively4-6. Notably, Tmeff1-/- mice exhibited increased susceptibility to HSV-1 infection in the brain but not in the periphery. Within the brain, elevated viral load was observed specifically in neurons. Our study identifies TMEFF1 as a neuron-specific restriction factor essential for prevention of HSV-1 replication in the central nervous system.

Neurotropic viruses, including herpes simplex virus (HSV) types 1 and 2, have the capacity to infect neurons and can cause severe diseases. This is associated with neuronal cell death, which may contribute to morbidity or even mortality if the infection is not controlled. However, the mechanistic details of HSV‐induced neuronal cell death remain enigmatic. Here, we report that lytic HSV‐2 infection of human neuron‐like SH‐SY5Y cells and primary human and murine brain cells leads to cell death mediated by gasdermin E (GSDME). HSV‐2‐induced GSDME‐mediated cell death occurs downstream of replication‐induced endoplasmic reticulum stress driven by inositol‐requiring kinase 1α (IRE1α), leading to activation of caspase‐2, cleavage of the pro‐apoptotic protein BH3‐interacting domain death agonist (BID), and mitochondria‐dependent activation of caspase‐3. Finally, necrotic neurons released alarmins, which activated inflammatory responses in human iPSC‐derived microglia. In conclusion, lytic HSV infection in neurons activates an ER stress‐driven pathway to execute GSDME‐mediated cell death and promote inflammation.

Immunological control of viral infections in the brain exerts immediate protection and also long-term maintenance of brain integrity. Microglia are important for antiviral defense in the brain. Here, we report that herpes simplex virus type 1 (HSV1) infection of human induced pluripotent stem cell (hiPSC)–derived microglia down-regulates expression of genes in the TREM2 pathway. TREM2 was found to be important for virus-induced IFNB induction through the DNA-sensing cGAS-STING pathway in microglia and for phagocytosis of HSV1-infected neurons. Consequently, TREM2 depletion increased susceptibility to HSV1 infection in human microglia–neuron cocultures and in the mouse brain. TREM2 augmented STING signaling and activation of downstream targets TBK1 and IRF3. Thus, TREM2 is important for the antiviral immune response in microglia. Since TREM2 loss-of-function mutations and HSV1 serological status are both linked to Alzheimer’s disease, this work poses the question whether genetic or virus-induced alterations of TREM2 activity predispose to post-infection neurological pathologies.

Supplementary Figure Legend and Table 1 from MCT-1 Protein Interacts with the Cap Complex and Modulates Messenger RNA Translational Profiles

Glioblastoma (GBM) is an aggressive brain tumor with a median survival of 15 months and has limited treatment options. Immunotherapy with checkpoint inhibitors has shown minimal efficacy in combating GBM, and large clinical trials have failed. New immunotherapy approaches and a deeper understanding of immune surveillance of GBM are needed to advance treatment options for this devastating disease. In this study, we used two preclinical models of GBM: orthotopically delivering either GBM stem cells or employing CRISPR-mediated tumorigenesis by adeno-associated virus, to establish immunologically proficient and non-inflamed tumors, respectively. After tumor development, the innate immune system was activated through long-term STING activation by a pharmacological agonist, which reduced tumor progression and prolonged survival. Recruitment and activation of cytotoxic T-cells were detected in the tumors, and T-cell specificity towards the cancer cells was observed. Interestingly, prolonged STING activation altered the tumor vasculature, inducing hypoxia and activation of VEGFR, as measured by a kinome array and VEGF expression. Combination treatment with anti-PD1 did not provide a synergistic effect, indicating that STING activation alone is sufficient to activate immune surveillance and hinder tumor development through vascular disruption. These results guide future studies to refine innate immune activation as a treatment approach for GBM, in combination with anti-VEGF to impede tumor progression and induce an immunological response against the tumor.

Immunological control of viral infection in the brain is essential for immediate protection, but also for long-term maintenance of brain integrity. As the primary resident immune cell of the brain, microglia protect against viral infections through key macrophage functions, including release of the antiviral type I interferons (IFN-I) and clearance of infected cells. Microglia express the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS), which can bind viral DNA leading to signaling through stimulator of interferon genes (STING), and downstream immune activation. Here we report that herpes simplex virus (HSV) 1 infection of microglia leads to activation of IFN-I genes and pro-inflammatory cytokines. However, HSV1 also down-regulated expression of a subset of genes, including genes in the pathway engaged by the microglial receptor triggering receptor expressed on myeloid cells-2 (TREM2). Knockdown experiments revealed that TREM2 is important for viral activation of cGAS-STING signaling in microglia, induction of IFN-I, and phagocytosis of HSV1 infected neurons. Consequently, TREM2 depletion increased susceptibility to HSV1 infection in human microglia-neuron co-cultures and mice in vivo . Mechanistically, we show that TREM2 is essential for phosphorylation of STING, and downstream activation of the IFN-inducing transcription factor IRF3. We conclude that TREM2 is a novel component of the antiviral immune response in microglia, crucial for immediate host defense against HSV1 in the brain. Since both TREM2 loss-of-function mutations and HSV1 serological status are linked to development of Alzheimeŕs disease (AD), this work opens the question whether defects in TREM2 could predispose to impaired viral clearance and post-infection pathological neurological changes.

Supplementary Figure Legend and Table 1 from MCT-1 Protein Interacts with the Cap Complex and Modulates Messenger RNA Translational Profiles