Lipopolysaccharide Enhances Microglia Antiviral Control of Betacoronavirus Infection through TLR4-Dependent Induction of IFNβ with Concomitant Modulation of dsRNA Sensors MDA5 and TLR3

doi:10.21203/rs.3.rs-4438872/v1

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Lipopolysaccharide Enhances Microglia Antiviral Control of Betacoronavirus Infection through TLR4-Dependent Induction of IFNβ with Concomitant Modulation of dsRNA Sensors MDA5 and TLR3

https://doi.org/10.21203/rs.3.rs-4438872/v1

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Microglia play a pivotal role in brain homeostasis and are essential to protective immunity within the central nervous system by coordinating the innate immune response to neuroinvasive pathogens. Bacterial endotoxin (lipopolysaccharide, LPS) is the most used pro-inflammatory stimulus for microglia, both in vitroand in vivo. However, the impact of LPS in microglia response to viral infection, including coronaviruses remains ill-defined. Murine coronaviruses productively infect microglia and are well established models of RNA virus-induced encephalitis and demyelination. In the present study, we investigated the effects of TLR4 stimulation with a TLR4-specific agonist [smooth (s)-form LPS (E. coli 0111:B4)] on murine coronavirus MHV-JHM and -A59 infection of microglia in vitro. Mechanistically, our data revealed that LPS stimulation of microglia restrict coronavirus replication through TLR4-dependent induction of interferon (IFN-b) with concomitant modulation of dsRNA sensors melanoma differentiation-associated gene 5 (MDA5) and toll-like receptor 3 (TLR3). Moreover, in the absence of coronavirus infection, LPS induces TLR4-dependent upregulation of MDA5 expression and activation of TLR3, as measured by phosphorylation of its residue Y759, a hallmark of TLR3 signaling initiation. Supporting LPS-induced TLR3 activation, we demonstrated that TLR3 selectively contributes to TLR4-dependent, LPS-induced neuroinflammation even in the absence of virus infection. Overall, the present study provides novel mechanistic insights regarding microglia responses to coronavirus infection and highlights a previously unrecognized crosstalk between bacterial TLR4 and dsRNA sensors in microglia with implications beyond viral infection.

Microglia

TLR4

TLR3

MDA5

Lipopolysaccharide

Coronavirus

Microglia, the innate immune cell within the central nervous system (CNS), contribute to the homeostatic control of brain function and plasticity in the developing and adult CNS [1–6]. As the only resident immune cell of the brain parenchyma, microglia are key orchestrators of immune mechanisms within the CNS in response to pathogen insult or sterile tissue damage, surveying the microenvironment for pathogen-associated molecular patterns (PAMPs) and host-derived damage associated molecular patterns (DAMPs) [7–10]. Detection of PAMPs and DAMPs by pattern recognition receptors (PRRs) causes resting microglia to transition into an activated state [11, 12]. This “activated” phenotype is defined, in part, by the production of innate immune effectors that facilitate inflammatory, antimicrobial, and chemotactic signaling pathways within the CNS [10].

Microglia express several types of PRRs to initiate innate immune responses early during infection, including Toll-like receptors (TLRs) and the cytosolic double stranded (ds) RNA sensors retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), RIG-I and melanoma differentiation-associated protein 5 (MDA5). Mammalian TLRs present at the plasma membrane include those that detect microbial cell surface components, such as TLR4, which detects Lipopolysaccharide (LPS) from Gram-negative bacteria, TLRs 1, 2 and 6 (bacterial lipoproteins), and TLR5 (flagellin). Nucleic acid sensing TLRs are present within endosomes and sense dsRNA (TLR3), single stranded (ss) RNA (TLR7, 8) and unmethylated CpG containing ssDNA (TLR9). The complexity of PAMP-PRR interactions allows multiple members of the TLR family to detect a plethora of PAMPs expressed by viruses, bacteria, and fungi, as well as host-derived DAMPS, such as alarmins, during infection and injury [13]. Interestingly, TLR4 is present in both plasma membrane and endosomes. The ectodomain of each TLR contain leucine-rich motifs that provide specificity for molecular recognition of PAMPs and DAMPs. The binding of these ligands to TLRs and RLRs activates specific intracellular signaling cascades that initiate host defense signaling, leading to production of pro-inflammatory cytokines and type 1 IFN (IFN-I). Though microglia-dependent responses aid in pathogen clearance, excessive immune activation through dysregulated PRRs signaling is detrimental to the host and exacerbates neuropathogenesis, providing strong rationale and scientific premise for considering microglia as a therapeutic target [14, 11, 15, 16]. Thus, it remains imperative that we advance research on the molecular mechanisms by which microglia sense and react to infectious agents to therapeutically modulate the balance between pathogen offense and host defense.

Viruses gain access to the CNS via multiple routes and depending on the route of entry and the type of virus, they can replicate within neurons, astrocytes, oligodendrocytes, and microglia, causing meningitis, and/or encephalitis during the acute phase, and long-term sequelae with or without virus persistence in the CNS [17]. DNA viruses such as herpes simplex type 1 and 2 remain the most common etiology for sporadic encephalitis worldwide [18]. Remarkably, neurotropic RNA viruses continue to emerge and are increasingly linked to chronic diseases of the CNS despite viral clearance [17, 19]. Viral infections of the CNS remain a mayor global public health concern because of high mortality and disability rates [20–22]. Therapeutic options to treat viral encephalitis (VE) remain very limited, and indeed, VE is still a cause for significant morbidity even with treatment [23]. Supportive measures are primarily used to treat arthropod-borne encephalitides, such as West Nile virus (WNV) encephalitis and Eastern equine encephalitis [24, 25]. In contrast, and unique to the herpesviridae family, herpes simplex encephalitis has been successfully treated with the antiviral acyclovir, although herpes virus infection remains persistent in infected individuals [26].

The development of in vivo models of CNS infection that recapitulate human disease is pivotal to identify mechanisms of neuroinvasion and neuropathogenesis by viruses. Emerging evidence from mouse models suggest a complex, double-edged sword for microglia function during virus infection of the CNS [27, 11, 28]. Microglia have been shown to contribute to survival during acute virus infection of the CNS. Pharmacological ablation of microglia with PLX5622, an antagonist of the colony stimulating factor 1 receptor (CSF1R), demonstrated increased mortality rates upon viral infections, including by pseudorabies virus, vesicular stomatitis virus (VSV), Theiler’s murine encephalomyelitis virus, Dengue virus (DENV), West Nile virus (WNV), Japanese encephalitis virus (JEV), herpes virus, and neurotropic betacoronavirus mouse hepatitis virus (MHV-JHM) [29–35]. The protective role of microglia during CNS viral infection can occur via multiple, non-redundant mechanisms including microglia-mediated by phagocytosis, T-cell recruitment, and production of innate antiviral effectors such as IFN-I and -IFN-II (recently reviewed in [28, 11]). However, it remains difficult to determine if increased morbidity after PLX5622 microglia depletion is a loss of a direct protective function or an exacerbation of detrimental pathways in response to PLX5622 experimental administration. While all the above-mentioned data suggest protective antiviral functions of microglia, detrimental contributions of microglia during JEV, Zika virus, and WNV infections in the mouse have also been demonstrated. Mechanistically, microglia detrimental effects in mice infected with JEV have been linked to exacerbated pro-inflammatory cytokines and chemokines including tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), IL-1β, IFN-β, type II IFN (IFN-γ), C-C motif ligand 2 (CCL2), CCL5, as well as complement factor 3 (C3) that can induce direct neurotoxic effects [36–39]. Microglia has been shown to facilitate virus entry into the CNS and exert neurotoxic effect in a mouse model of WNV infection [40, 41]. Sustained activation of microglia has been linked to long-term cognitive deficits in mouse models of JEV, WNV, and Zika virus, involving microglia in seizures, and elimination of synapses via upregulation of complement C3 and C1q subunit A or through microglia upregulation of IFN-γ [42, 43, 11].

Major questions remain about the mechanisms underlying microglia-specific immunity, including the intracellular pathways that sense and restrict coronavirus replication and/or promote neuroinflammation. Mouse hepatitis virus (MHV) is the prototype of betacoronaviruses, a group that includes severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [44]. Murine coronaviruses comprise several MHV strains that cause virus strain-specific pathogenesis in the mouse [45]. MHV-JHM and MHV-A59 induce acute and chronic demyelinating encephalomyelitis and are well-stablished models to study RNA virus-induced neuropathogenesis in C57BL/6J mice [46]. Interestingly, MHV-JHM and -A59 while productively infecting neurons, oligodendrocytes, and glial cells, including microglia, dramatically differ in their neurovirulence [45]. Following intranasal or intracranial infection with MHV-JHM mice develop acute fatal encephalitis with 100% mortality within 5–7 days post-infection (dpi). In contrast, the neuro-attenuated MHV-A59 causes acute, mild encephalitis and chronic demyelination in mice with only 5–10% mortality. Mice infected with MHV-A59 or a neuro-attenuated rJ2.2 variant of JHM develop immune-mediated demyelination peaking at 12–14 dpi, undergoing remyelination with pathological features resembling those of multiple sclerosis in humans [47]. Using reverse genetics to generate chimeric MHV-JHM /-A59 viruses, we have previously demonstrated that the Spike glycoprotein and the 21 kb replicase complex are major determinants of coronavirus pathogenesis [48–50] in C57BL/6J mice. Recent evidence demonstrated that while productively infected in vivo, microglial cells are required for early control of murine coronavirus acute infection of the brain, contributing to myelin repair in the spinal cord during chronic demyelination [32, 51]. Apart from coronavirus animal models, the COVID-19 pandemic has provided a unique opportunity to investigate the impact of an acute RNA virus respiratory infection on the human CNS. Remarkably, analysis of human brains using positron emission tomography (PET) and the ligand [18F]FEPPA for the translocator protein 18 kDa (TSPO), which is expressed by activated microglia, reveals that long-term neurocognitive impairment and depression in COVID-19 individuals correlate with microglia activation [52]. While the ability of SARS-CoV-2 to directly infect the CNS in COVID-19 individuals remains contradictory, there is solid clinical global data supporting the complexity of acute and long-term CNS complications associated with SARS-CoV-2 infection at the population level (recently reviewed in [53, 54]). However, the innate immune sensors, including TLRs, that orchestrate microglia response to coronavirus infection remain unknown.

The crosstalk between viruses and bacteria coinfections and their impact on microglia function remain unexplored. Lipopolysaccharide (LPS), a virulent factor and component of the outer membrane of gram-negative bacteria, is the most used pro-inflammatory stimulus for microglia, both in vitro and in vivo. In the absence of any infection, intraperitoneal injection of LPS, produces systemic inflammation with a spectrum of acute and long-term behavioral responses and neuroinflammation inducing profound microglia activation in animal models [55–58]. Although no potential correlation with neurocognitive impact was investigated, Teixeira et al., found increased microbial translocation (as measured by LPS concentration in plasma) and hyperinflammation in patients with severe COVID-19, leading to higher monocyte activation [59]. Moreover, TLR4, the classical sensor of LPS, induces the expression of both pro-inflammatory cytokines and IFN-I. Perhaps most intriguing, however, are recent reports demonstrating that LPS stimulation restricts replication of HIV-1, Influenza, and other RNA viruses within human and murine macrophages [60–64]. Collectively, these data provide evidence of an unexplored link between TLR4 and antiviral immunity. Despite the plethora of studies investigating microglia response to LPS using imaging, electrophysiology, and behavioral assays, the effects of LPS stimulation on microglia responses to viral infection, including coronaviruses, remain unexplored.

In the present study, we investigated the effects of TLR4 stimulation with a TLR4-specific agonist [smooth (s)-form LPS (E. coli 0111:B4)] on murine coronavirus MHV-JHM and -A59 infection of microglia in vitro. Our data revealed that LPS stimulation of microglia restrict coronavirus replication through TLR4-dependent induction of IFN-β with concomitant modulation of dsRNA sensors MDA5 and TLR3. Moreover, in the absence of coronavirus infection, LPS induces TLR4-dependent upregulation of MDA5 expression and activation of TLR3, as measured by phosphorylation of its residue Y759, a hallmark of TLR3 signaling initiation. Supporting LPS-induced TLR3 activation, we demonstrated that TLR3 selectively contributes to TLR4-dependent, LPS-induced neuroinflammation even in the absence of virus infection.

Here, we also stablished an in vitro model of coronavirus infection using immortalized microglia originally isolated from adult C57BL/6J mice (kindly provided by the NIH Repository). We demonstrated that the highly neurovirulent MHV-JHM and the neuro-attenuated MHV-A59 murine coronaviruses infect microglia in vitro to extents like those previously observed in whole brains of infected C57BL/6J mice, validating our in vitro microglia infection model. Importantly, in vitro infection of microglia with the neurovirulent MHV-JHM induces significant higher levels of IL-6, IL-1α, and IL-1β compared to the neuro-attenuated MHV-A59, mimicking previously reported data obtained from whole brains of MHV-infected C57BL/6J mice. The main cellular source of these pro-inflammatory cytokines in the brain of infected mice remains to be identified in vivo. Noteworthy, our data demonstrates the biological relevance of our in vitro model to investigate microglia response to murine coronavirus infection, suggesting a pivotal role of microglia in driving coronavirus neurovirulence.

Overall, the present study provides novel mechanistic insights regarding microglia responses to coronavirus infection and highlights a previously unrecognized crosstalk between bacterial and dsRNA sensors in microglia with implications beyond viral infection.

2.1 Cell culture

Immortalized murine microglia cell lines were previously established and characterized by Dr. Douglas Golenbock (The University of Massachusetts Medical School, Worcester, Massachusetts, USA) and obtained from the NIH, NIAID BEI Resources Repository (https://www.niaid.nih.gov/research/bei-resources-repository). The microglial cells were derived using brain tissue from wild type (WT) immunocompetent mice (NR-9460), TLR2 knockout (KO) mice (NR-15641), TLR4 KO mice (NR-9901), and TLR3 KO mice (NR-9900) all in the C57BL/6J background. The primary microglial cells were immortalized by infection with the ecotropic transforming replication-deficient retrovirus J2 using techniques described in the literature [65]. Characterization based on immunofluorescence, stimulation assays and flow cytometry demonstrated that the immortalized cell line retains its microglial-specific morphological, functional, and surface expression properties (Supplementary Fig. 1, and data not shown) [66]. Cells were used at passage 4–12 for different assays. All cell culture reagents were purchased from Corning Life Sciences (Corning, NY, USA) unless otherwise specified. Murine microglial cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with 4.5 g/L Glucose L-glutamine, and Sodium Pyruvate (Cat. # 10-013-CMR), 10 µg/mL ciprofloxacin (bioWORLD, Dublin, OH, USA; Cat. # 86483-48-9) and supplemented with 10% irradiated fetal bovine serum (FBS) (Cytiva, Marlborough, MA, USA; Cat. # SH30073), hereby referred to as Complete DMEM. L2 and 17Cl.1 murine fibroblasts cell lines (a gift from Dr. Susan R. Weiss, Perelman School of Medicine, University of Pennsylvania) were maintained in DMEM supplemented with 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, and 100 U/mL penicillin-streptomycin. All cells were cultured at 37°C in a humidified 5% CO₂ atmosphere and tested for potential Mycoplasma contamination using the MycoAlert kit (Lonza, Morristown, NJ, USA; Cat. #LT07-318).

2.2 Viruses, infections, and quantification of viral titers

Murine betacoronaviruses MHV-JHM and -A59 strains were propagated by infection of 17Cl.1 cells at low multiplicity of infection (MOI = 0.1) in complete DMEM supplemented with 2% FBS as previously described [67]. Viruses were aliquoted and stored at -80°C until further use. Titers of virus stocks and experimental samples were assessed on L2 monolayers using standard plaque assay [68]. Prior to infection, microglial cells were washed in Phosphate Buffered Saline with magnesium and calcium (PBS), and then inoculated with MHV-A59 or MHV-JHM at the indicated multiplicity of infection (MOI) for 1 hour at 37^oC in Complete DMEM containing 2% FBS. After 1 hour (h) of virus adsorption, the virus inoculum was removed, cells were gently washed with PBS, and complete DMEM containing 10% FBS was added to infected cells as indicated. The quantitation of released infectious virus particles into the supernatant of infected cells by plaque assay is a well-established method to measure infectious coronavirus production. Supernatants from infected cells were clarified by centrifugation at 200 xg for 10 minutes at 4°C to remove cell debris, aliquoted, and stored at -80°C until further analysis. MHV-JHM and -A59 titers of experimental samples were assessed on L2 monolayers using standard plaque assay [67, 66].

2.3 Microglia stimulation with TLR4-specific LPS:EB

To investigate the effect of LPS stimulation on microglia antiviral response to coronavirus infection, we used an ultra-pure commercial preparation of smooth (s)-form LPS purified from the Gram-negative E. coli 0111:B4, a pathogenic serotype of E. coli (Invivogen, San Diego, CA, USA; Cat. #tlrl-eblps). LPS:EB ultrapure is extracted by successive enzymatic hydrolysis steps and purified by the previously described phenol-TEA-DOC extraction protocol [69]. This process removes contaminating lipoproteins, and, as expected, LPS:EB ultrapure induces TLR4-dependent signaling (data shown in Supplementary Fig. 3). Four microglia experimental groups were investigated in the context of MHV-JHM and -A59 infections. The presence or absence of LPS stimulation in microglia is denoted as (+) or (-), respectively. A working concentration [1 µg/mL] of LPS:EB was used unless otherwise noted. 1) Infection only (-/-): cells were infected in the absence of LPS:EB treatment. 2) LPS:EB stimulation after virus adsorption (LPS-post, -/+): cells were treated with LPS:EB after virus adsorption for 20 h. 3) Priming with LPS for 3 h before virus adsorption (LPS-primed, +/-): cells were pretreated with LPS:EB for 3 hours prior to infection and left unstimulated after virus adsorption. 4) Cells were primed with LPS:EB prior to infection and then stimulated after virus adsorption (LPS-primed/post, +/+). Moreover, the following non-infected experimental groups were analyzed: i) untreated, non-infected cells (naïve control); ii) cells treated with LPS:EB for 3 hours, then left unstimulated, or iii) treated with LPS:EB; iv) cells treated with LPS:EB for 3 hours, followed by one hour of rest (to simulate virus adsorption time), then treated with LPS:EB for 20h. Supernatants and cell lysates of all experimental groups were collected 20 h after stimulation or infection unless otherwise specified.

2.4 Microglia stimulation with TLR3 agonist Poly(I:C) HMW

Microglial cells were stimulated with Poly(I:C) High Molecular Weight (HMW) which is comprised of long strands of inosine poly(I) homopolymer annealed to strands of cytidine poly(C) homopolymer. The average size of Poly(I:C) HMW is from 1.5 kb to 8 kb. Poly(I:C) HMW is functionally validated for TLR3 potency using HEK-Blue™ TLR3 Cells and the absence of contaminants (e.g. lipoproteins and endotoxin) is confirmed using TLR2 and TLR4 cellular assays, respectively (data not shown). Poly(I:C) HMW was purchased from Invivogen (Cat. #tlrl-pic), reconstituted as per the manufacturer’s instructions and stored at -80^oC. Cells were washed in PBS, then stimulated with 50µg/mL Poly(I:C) HMW in complete DMEM media. The negative control condition was comprised of complete DMEM alone. Cells were incubated at 37\(^\circ\)C and 5% CO₂ for the time indicated in the corresponding figure legends. Afterwards, supernatants and cell lysates were collected and stored at -80^oC until further analysis.

2.5 Pharmacological inhibition of TLR3 and TLR4

For specific pharmacological inhibition of TLR3 and TLR4 signaling, TLR3/dsRNA Complex Inhibitor and TLR4 Inhibitor TAK-242 were purchased from MilliporeSigma (EMD Millipore; Burlington, MA, USA, Cat. #614310-10MG, #614316-5MG, respectively). Inhibitors were first reconstituted in dimethylsulfoxide (DMSO, 4-X) (ATCC, Manassas, VA, USA) and then diluted in PBS to obtain final working concentrations in cells. Cells were then pre-treated with either 100 µM of iTLR3 or 10 µM of TAK-242 in Complete DMEM for 2 hours at 37^oC. Afterwards, cells were washed in PBS and infected/stimulated as described above. The media was then replaced with complete DMEM in the presence or absence of the respective inhibitor as described in corresponding figure legends.

2.6 Neutralization of mouse IFNβ

Anti-mouse IFNβ neutralizing antibody was purchased from PBL assay science (PBL Assay Science, Piscataway, NJ, USA; Cat. #32400-1; stock concentration of 3.59 x 10⁶ mammalian Neutralizing Units/mL, NU/mL). Microglial cells were pre-treated with 2000 Neutralizing Units (NU)/mL of anti-mouse IFNβ neutralizing antibody in complete DMEM at 37^oC for 2 hours, as previously described by our lab [70]. After incubation, cells were washed in PBS and the media was replaced with complete DMEM alone, 2000 NU/mL of anti-mouse IFNβ, or with conditional media described in corresponding figure legends.

2.7 Immunofluorescence analysis

Microglial cells were cultured at 37°C in 8-well slides (Nunc™ Lab-Tek II; Thermo Scientific, Waltham, MA, USA; Cat. #154461PK) containing 200 µl of Complete DMEM at a seed count of 50,000 cells per chamber for 24 h. After experimental conditions, cells were fixed in 3.7% paraformaldehyde for 15 min at room temperature. Fixed cells were permeabilized in 0.1% Triton X-100 (IBI Scientific, Dubuque, IA; Catalog # IB07100) in PBS for 10 min at room temperature. To prevent non-specific binding by primary antibodies, cells were treated with BlockAid (Invitrogen, Cat. #B10710) or 3% BSA in PBS with 10% horse serum (Vector Laboratories, Newark, CA) and rat IgG2a, λ anti-mouse CD16/32 Fc receptors blocking (Biolegend, San Diego, CA, USA) for 1 hour at room temperature. For indirect immunofluorescence, we used the following primary antibodies diluted in BlockAid reagent supplemented with 2.5% horse serum: anti-nucleocapsid MHV (anti-N) monoclonal antibody (MAb) (1.16.1 hybridoma was kindly provided by J. Leibowitz, Texas A&M University); monoclonal anti-dsRNA IgG2ak antibody [J2] (Kerafast, Boston, MA, USA; Catalog# Kf-Ab01299-2.0); goat anti-mouse/human IBA-1 (Novus, Centennial, CO, USA; Catalog# NB100-2833), followed by highly cross-adsorbed secondary antibodies donkey anti-rabbit, anti-mouse, or anti-goat IgG (H + L) Alexa Fluor™ 488, Alexa Fluor™ 594, and Alexa Fluor™ 647 (Invitrogen). Incubations of primary and secondary antibodies were performed at 4°C overnight and room temperature for one hour in the dark, respectively. The cells were mounted using ProLong Glass antifade with NucBlue (Invitrogen, Cat. #P36983). Fluorescence images were acquired using an Olympus IX-81 microscope (Olympus, Tokyo, Japan). Image acquisition was performed using cellSens Dimension software Ver2.0 (Olympus). IFA images were quantified using the ImageJ version 2.14.0/1.54f.

2.8 Near-Infrared (NIR) western blot analysis

Cells were lysed by adding Mammalian Protein Extraction Reagent (MPER) containing 1x EDTA and 1x HALT protease inhibitor (Invitrogen) for 20 min at 4°C with gentle rocking. After collection, lysates were centrifuged at 14,000xg for 15 min at 4°C. The pelleted non-soluble fraction was then discarded. The soluble protein content was quantified using the Pierce BCA Protein Assay Kit (ThermoFisher, Waltham, MA, USA; Cat. #23225) according to manufacturer’s directions. Cell lysates were prepared with 30 µg of total protein in 4X, non-reducing fluorescent compatible sample buffer (Invitrogen Cat. #LC2570) and 10X Bolt™ sample reducing agent (Invitrogen #B0009), heated at 70°C for 10 min, and immediately placed on ice. Samples were resolved in precast Bolt™ Bis-Tris Plus Mini Protein Gels, 4-12%, 1.0 mm, WedgeWell™ format (ThermoFisher, Cat. #NW04120BOX), and transferred using iBlot™ 2 Transfer Stacks, PVDF, mini (ThermoFisher, Cat. #IB24002) using the iBlot 2 Gel Transfer Device (ThermoFisher; Cat. #IB21001), as per the manufacturer’s protocol. Membranes were blocked using Intercept Tris-buffered saline (TBS) Blocking Buffer (LI-COR, Lincoln, NE, Cat. #927-60001) and stripped using Restore™ Fluorescent Western Blot Stripping Buffer (Invitrogen, Cat. #62300) diluted 1:5 in ultrapure H₂0 (Mili-Q, Millipore). Membranes were probed rocking overnight at 4°C with primary antibodies diluted in Intercept T20 antibody diluent preformulated with tween® 20 (LI-COR, Cat. #927-65001): anti-MDA5 (1:1000, Cell Signaling, Cat. #5321); anti-phospho-Y759-TLR3 (1:500 Novus Biologicals, Cat. # NBP2-24904); anti-TLR3 (1:1000, Novus Biologicals, Cat. #NB100-56571); anti-TLR3/CD283 (1:500, Novus Biologicals, Cat. #DDX0470P-100); anti-IFNβ (1:500, Bioss, Cat. #bs-0784R and Invitrogen, Cat. #PA5-203990); anti-nucleocapsid MHV (anti-N) MAb 1.16.1 (1:100, J. Leibowitz, Texas A&M University); and anti-β-actin (1:5,000, Cell Signaling, Danvers, MA, USA; Cat. #3700). Membranes were probed with 1:12,500-1:25,000 dilution of highly cross-adsorbed secondary antibodies (LI-COR) IRDye® 800CW Donkey anti-Mouse IgG (H + L) (Cat. #926-32212); IRDye® 800CW Donkey anti-Rabbit IgG (H + L) (Cat. #926-32213); IRDye® 680RD Donkey anti-Rabbit IgG (H + L) (Cat. #926-68073); IRDye® 680RD Donkey anti-mouse IgG (H + L) (Cat. #926-68072); and IRDye® 800CW Goat anti-Mouse IgM (µ chain specific) (Cat. #926-32280). Blots were imaged on the Odyssey® CLx Near-Infrared Fluorescence imaging system using 680 nm and 780 nm channels (LI-COR). Protein detection was performed using Image Studio Ver 5.2. (LI-COR) and data analyzed using ImageJ (https://imagej.nih.gov/ij/). Microsoft PowerPoint was used to prepare image panels and annotations.

2.9 Quantification of IL-6 and TNF-α in cell culture supernatants

Cell culture supernatants from naïve, LPS:EB stimulated, and/or MHV-infected microglia were analyzed for IL-6 and TNF-α by commercial enzyme-linked immunosorbent assays (ELISAs) [mouse IL-6 (Cat. #88-7064) and mouse TNF-α (Cat, #88-7124), Invitrogen]. Absorbance was measured at 450 nm (with a 540 nm reference wavelength) using the Azure Biosystems Ao microplate reader. Standard curves were generated by inputting obtained absorbance values directly into the online data analysis tool “Four Parameter Logistic Curve”, MyAssays Ltd, http://www.myassays.com/four-parameter-logistic-curve.assay. The concentration of secreted cytokines was determined by inputting obtained absorbance values directly into the “Four Parameter Logistic Curve” online data analysis tool as previously described.

2.10 Proteome profile analysis

Analysis of the innate immune secretome was performed via a membrane-based sandwich immunoassay Proteome Profiler Mouse Cytokine Array Kit, Panel A, as per manufacturer’s instructions (R&D Systems, Minneapolis, MN, USA; Cat. #ARY006). Briefly, samples were mixed with a cocktail of biotinylated detection antibodies and then incubated with the array membrane which is spotted in duplicate with capture antibodies to specific target proteins. To allow captured protein detection using Li-COR Odyssey CLx imaging system, membranes were incubated with 1:2000 dilution of IRDye 800CW Streptavidin (LI-COR, Cat. #926-32230) before washing and visualization. Membranes were imaged on the Odyssey® CLx Near-Infrared Fluorescence imaging system (LI-COR) using the 780 nm channel. Protein detection was performed using Image Studio Ver 5.2. (LI-COR) and quantification was performed using the image analysis program ImageJ NIH (https://imagej.nih.gov/ij/). Microsoft PowerPoint was used to prepare image panels and annotations. This kit detects 40 mouse cytokines, chemokines, and acute response proteins in duplicate simultaneously, including: CXCL13/BLC/BCA-1, C5a, G-CSF, GM-CSF, M-CSF, CCL1/I-309, CCL11/Eotaxin, CCL2/JE/MCP-1, CCL3/MIP-1 alpha, CCL4/MIP-1 beta, CCL5/RANTES, CCL12/MCP-5, CCL17/TARC, CXCL1/KC, CXCL2/MIP-2, CXCL9/MIG, CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1, TIMP-1, ICAM-1, TREM-1, IFN-gamma, IL-1 alpha/IL-1F1, IL-1 beta/IL-1F2, IL-1ra/IL-1F3, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 p70, IL-13, IL-16, IL-17, IL-23, IL-27, and TNF-alpha.

2.11 RNA silencing

Microglial cells were transfected with Silencer Select Pre-Designed siRNA MDA5 (Ambion; ID# s89789; Gene Symbol Ifih1; Locus ID 71586) or scrambled control (Ambion; Cat. # 4390843) using Lipofectamine RNAiMax Transfection Reagent (ThermoFisher; Cat. #13778075), as per manufacturer’s instructions. The oligonucleotide sequences of MDA5-silencing RNA were as follows: Sense strand – GCAAUACAACGACAAUCCAtt; Antisense strand – UGGAUUGUCGUUGUAUUGCtt. Briefly, siRNA and RNAiMax were diluted separately in Opti-MEM™ I Reduced Serum Medium (ThermoFisher, Cat. #31985062) and then combined at a 1:1 ratio for 20 minutes at room temperature. Microglia were then incubated with 25 pmols of this siRNA-lipid complex for 48 consecutive hours in complete DMEM. Following transfection, cells were washed with PBS and LPS:EB stimulated and/or infected with MHV as described. To determine the efficiency of the siRNA transfection protocol, microglia were similarly transfected with Silencer Cy3-Labeled negative control siRNA #1 (Invitrogen; Cat. #AM4621). Supplementary Fig. 4 shows transfection efficiency determined by confocal microscopy.

2.12 Statistical analysis

Data visualization and analyses were performed using build-in functions of GraphPad Prism (version 9.4.1; Graphpad Software, Inc., USA). Data were considered statistically significant if P values were < 0.05. All error bars represent SEM about the mean. The number of independent experiments and biological replicates per experimental groups is indicated in the respective figure legends. Ordinary one-way ANOVA and 2way-ANOVA, each with Tukey’s multiple comparison test, and Unpaired student t-test (two-tailed) were utilized as described in corresponding figure legends. Signified p values are as follows: ^*P ≤ 0.032, ^**P ≤ 0.002, ^***P ≤ 0.001, ^****P ≤ 0.0001. Datasets representing MHV-JHM are displayed in blue, while datasets representing MHV-A59 are displayed in pink.

2.13 Generation of diagrams and models

Schematics depicting the mechanism of action for each pharmacological TLR inhibitor, as well as those detailing our working model and summary, were created with BioRender scientific illustration software (Toronto, ON, Canada).

3.1 LPS:EB stimulation diminishes murine coronavirus replication in microglia

To investigate the potential effects of TLR4 activation on murine coronavirus infection of microglia, we designed an in vitro experimental system using immortalized microglial cells isolated from immunocompetent C57BL/6J mice. Murine coronaviruses MHV-JHM and -A59 were selected for our study based on their known ability to infect microglia in vivo. To activate TLR4, microglial cells were stimulated with 1 µg/mL of LPS:EB, a known TLR4 ligand, before (LPS-primed for 3 h), after (LPS-post for 20 h) or, before and after MHV infection (LPS-primed, LPS-post). No LPS:EB was present during virus adsorption for 1 h. Cells were infected with MHV-A59 or -JHM [MOI = 1] and virus inoculum was removed after virus adsorption. As depicted in Fig. 1A, infected microglial cells were subjected to four experimental groups with (+) or without (-) LPS:EB. Supernatants and cell lysates were collected at 20 hpi for quantification of viral titers by plaque assay and MHV-Nucleocapsid (N) expression by immunoblot. Group 1 (-/-): MHV infection; no LPS stimulation. Group 2 (-/+): MHV infection followed by LPS:EB stimulation for 20 h. Group 3 (+/+): LPS:EB priming for 3h before virus adsorption and then, LPS:EB stimulation for 20 hpi. Group 4 (+/-): LPS:EB priming for 3h before virus adsorption without further LPS:EB stimulation. We also included a negative control group of cells that were not stimulated, nor infected (Naïve microglia).

Viral titers in microglia infected with MHV-A59 were significantly higher than in MHV-JHM -infected cells (P < 0.001; one-Way ANOVA with Tukey’s multiple comparison’s test) (Fig. 1B). Importantly, viral titers in our in vitro model of microglia infection reproduced previous data from our lab and others using plaque assay analysis of whole mouse brains infected with MHV-A59 or -JHM [48, 71]. Stimulation with LPS:EB after virus infection significantly reduced MHV-A59 and -JHM viral titers. Similarly, LPS:EB priming before infection and priming and stimulation post-infection significantly reduced viral load in supernatants of microglia infected with MHV-A59 (P < 0.001) and MHV-JHM (P < 0.01) (Fig. 1B). As expected, immunoblot analysis of naïve microglia cell lysates demonstrated no expression of viral nucleocapsid in the absence of viral infection (Fig. 1C). Consistent with viral titers, the expression of MHV-Nucleocapsid (N) protein in microglia cell lysates was significantly inhibited irrespective of experimental group, although with a more pronounced effect in microglia stimulated with LPS:EB after viral infection [Group 2; (-/+)] (Fig. 1C, D). Due to this observation, and to avoid any potential effects of LPS:EB during virus entry in the cells, all further investigations were conducted in microglia infected with MHV and then stimulated with LPS:EB after infection [Group 2; (-/+)] .

Next, we performed immunofluorescence analysis of MHV-Nucleocapsid and viral dsRNA in microglia infected with MHV-A59, or -JHM in the absence of LPS:EB stimulation, or in cells stimulated with LPS:EB after infection (Group 2) (Fig. 2A, B). In agreement with data shown in Fig. 1, specific immunostaining for MHV-Nucleocapsid and viral dsRNA, a replicative intermediate produced during coronavirus replication, were significantly reduced in microglia stimulated with LPS:EB after virus infection (P < 0.001 for both MHV-A59 and -JHM infections) (Fig. 2C). Cell-to-cell fusion is a well-known cytopathic effect of many coronaviruses that is driven by the virus Spike glycoprotein and a cellular furin-like protease [72]. Immunofluorescence staining also showed that MHV-JHM induced bigger syncytia (multinucleated cells) than MHV-A59 infection. Importantly, our in vitro microglia model reproduced the ability of MHV-JHM and -A59 and other betacoronaviruses including SARS-CoV-2, to induce syncytia formation in vitro and in vivo [73–75, 72].

To further investigate the inhibitory effect of LPS:EB in murine coronavirus replication, we infected microglia with MHV-A59 or -JHM [MOI = 1] for 1 h and thereafter, the cells were cultured in the presence or absence of LPS:EB [1 µg/mL] for the indicated time points (experimental design is shown in Supplemental Fig. 2). Microglia supernatants and cell lysates were collected and analyzed for viral titers and MHV-Nucleocapsid expression by plaque assay and immunoblot, respectively. As shown in Fig. 3A, viral titers were significant reduced at 20 hpi, with no differences at earlier time points. We detected MHV-Nucleocapsid (N) protein expression as early as 6 hpi, and, as expected, the expression of N protein increased over time in cell lysates of MHV-infected microglia (Fig. 3B). Stimulation with LPS:EB after infection significantly reduced N expression at 20 hpi (P < 0.01) although the inhibitory effect was less pronounced at 6 and 14 hpi (Fig. 3C).

Overall, these data suggest that LPS:EB induced inhibition of MHV replication might require some degree of TLR4 activation that mediate the induction of molecular effectors of the antiviral response that interfere with viral replication late during infection.

3.2 LPS:EB triggers TLR4-dependent neuroinflammatory response

Extracellular LPS is bound by CD14 and transferred to a complex consisting of TLR4 and myeloid differentiation protein-2 (MD2) located on the cell surface [76, 77]. This TLR4/MD2 complex is considered the major receptor of the innate immune system to recognize lipopolysaccharides (LPSs). However, evidence has emerged that TLR2 can also sense some types of LPS depending on lipid A and core saccharide moiety structures composition in innate immune cells [78–80]. To exclude any potential cross-activation of TLR2 in the neuroinflammatory response induced by LPS:EB in microglia, we utilized an ultra-pure commercial preparation of smooth (s)-form LPS purified from the Gram-negative E. coli 0111:B4, a pathogenic serotype of E. coli. This was confirmed in our microglia in vitro model. First, pharmacological inhibition of TLR4 with TAK-242 [10 µM] in immunocompetent wild type (WT) microglia completely suppressed IL-6 and TNF-α production as measured by ELISAs (Supplemental Fig. 3A). Second, TLR4^−/− microglia did not produce IL-6 and TNF-α in response to LPS:EB stimulation (Supplemental Fig. 3B). Third, proteome array analysis of cytokines, acute response proteins, CXCL and CCL chemokines in WT, TLR2^−/−, and TLR4^−/− microglia revealed that LPS:EB stimulation does not trigger any effector in the absence of TLR4 signaling, demonstrating the requirement of TLR4 signaling for LPS:EB induced neuroinflammation (Supplemental Fig. 3C-F).

Upon stimulation with LPS:EB of WT microglia, we identified IL-6 and TNF-α as the highest upregulated cytokines followed by interleukin-1 receptor antagonist (IL-1RA), IL-1α, IL-27, IL-23, and IL-27. Moreover, CXCL2 (MIP-2), CXCL-10 (IP-10), CCL5 (RANTES), CCL12 (MCP-5), G-SSF, sICAM-1, and M-CSF were upregulated in supernatants of WT microglia in response to LPS:EB stimulation. All these effectors were abrogated in LPS:EB-stimulated TLR4^−/− microglia. As expected, analysis of the proinflammatory response in microglia WT and TLR2^−/− in response to stimulation with TLR2-specific ligands Pam3CSK4 or FSL-1 was abrogated in TLR2^−/− microglia (data not shown). Importantly, TLR2^−/− microglia were still as responsive to LPS:EB stimulation as WT counterparts, though we noted that the secretion CCL12, M-CSF, and GM-CSF were upregulated in TLR2^−/− microglia (Supplemental Fig. 3C-E). Therefore, although TLR2 signaling does not contribute to LPS:EB induced neuroinflammatory response, our data suggest that TLR2 might contribute to the negative regulation of CCL12, M-CSF, and GM-CSF secretion upon TLR4 activation in microglia.

Taken together, these results validate LPS:EB as a TLR4-dependent agonist, implying that TLR4 signaling facilitates LPS:EB-induced inhibition of MHV replication.

3.3 Analysis of the neuroinflammatory response in MHV-infected, or -infected and LPS:EB-stimulated microglia

Next we tested the hypothesis that MHV-A59 and -JHM infection of microglia induces virus strain-specific neuroinflammatory effectors, and that LPS:EB stimulation during infection contributes to exacerbated neuroinflammation. To test these hypotheses we characterized cytokines, chemokines, and acute response proteins in supernatants of WT microglia using proteome profile arrays. The following microglia experimental groups were analyzed: i) cells stimulated with LPS:EB in the absence of infection for 20 h; ii-iii) cells infected with MHV-A59 or -JHM [MOI = 1] for 1 h and then cultured in the absence or presence of LPS:EB for 20 hpi; iv) cells infected with MHV-A59 or -JHM [MOI = 1] for 1 h and then stimulated with LPS:EB in the presence of TAK-242 to pharmacologically inhibit TLR4 signaling during dual infection and stimulation (Fig. 4A-D).

Among 40 molecular effectors analyzed by proteome arrays, the highest upregulated cytokines in MHV-infected microglia were IL-6, TNF-α. Interestingly, the neurovirulent MHV-JHM induced stronger upregulation of both pro-inflammatory cytokines (IL-6: 5.07-fold; TNF-α: 4.07-fold) compared to the neuro-attenuated MHV-A59 (IL-6: 2.20-fold; TNF-α: 2.25-fold). Furthermore, IL-1α and IL-1β were upregulated by MHV-JHM infection in the absence of LPS stimulation. In contrast, MHV-A59 induced both IL-1α and IL-1β only in the presence of LPS:EB stimulation during infection (Fig. 4A). These results suggest a differential involvement of the inflammasome in microglia response to virulent vs. attenuated CNS-tropic murine coronaviruses demonstrating increased IL-1α and IL-1β production in microglia infected with the neurovirulent MHV-JHM. Noteworthy, CXCL-1, a chemokine previously known to activate the NLRP3 inflammasome in macrophages during Mycobacterium tuberculosis infection [81], was only upregulated by MHV-JHM (3.65-fold) (Fig. 4C). Therefore, the results obtained in our in vitro model of microglia infection suggest a correlation between the known MHV-JHM neurovirulence in vivo and our novel data in vitro, suggesting a detrimental contribution of microglia through exacerbated IL-1α, IL-1β, and CXCL1/inflammasome activation during MHV-JHM-induced fatal encephalitis. In contrast, both viruses induced similar upregulation of the expression of CXCL12 (MHV-A59: 4.01-fold; MHV-JHM: 3.09-fold) and CXCL2 (MHV-A59: 5.38-fold; MHV-JHM: 5.66-fold) (Fig. 4C) but did not induce CCL chemokine production (Fig. 4D).

Pharmacological inhibition of TLR4 signaling using TAK-242 in microglia stimulated with LPS:EB after MHV infection abrogated all molecular effectors except the expression of the chemokines CXCL12 and CXCL2 (Fig. 4C). Because these chemokines were abrogated in LPS:EB-stimulated TLR4^−/− microglia (Supplemental Fig. 3), it is tempting to speculate that MHV infection induces CXCL12 and CXCL2 chemokines independently of TLR4 signaling. Although the role of TLR4 in murine coronavirus neuroinflammatory response is not the focus of this study, our proteome array data suggest TLR4 involvement in the upregulation of the proinflammatory cytokines IL-6 and TNF-α in MHV-infected microglia (Fig. 4A).

Having demonstrated MHV strain-specific upregulation of neuroinflammatory effectors (IL-6, TNF-α, IL-1α, and IL-1β) and that LPS:EB stimulation during infection contributed to higher levels of IL-1α and IL-1β in a TLR4-dependent manner, we next interrogated the effect of LPS-EB on intracellular IFNβ protein expression in the absence of MHV infection and during MHV infection. In the absence of MHV infection, microglia were subjected to the following experimental groups: i) naïve cells; ii) cells stimulated with LPS:EB for 20 h; iii) cells primed with LPS:EB for 3 h, media removed, cells allowed to rest for 1 h, and cultured in the presence of LPS:EB for 20 h; and iv) cells stimulated with LPS:EB for 3 h, media removed and then cultured with fresh media without LPS:EB for 20 h. Furthermore, we analyzed the effect of MHV-A59 and -JHM infection in the absence or presence of LPS:EB stimulation (LPS-primed, and/or LPS-Post infection) on IFNβ intracellular protein expression at 20 hpi. Using immunoblot analysis of microglial cell lysates, we found that LPS:EB induced similar upregulation of IFNβ in LPS-Primed for 3 h, LPS-Post for 20h, or LPS-Primed/Post in the absence of MHV infection. Importantly, and somewhat expected based on the known ability of coronaviruses to counteract antiviral response, our data demonstrated that MHV-A59 and -JHM viruses significantly inhibited IFNβ expression (Fig. 4F). It is noteworthy, however, that MHV infection did not inhibit LPS-EB-induced IFNβ expression, suggesting an unforeseen inability of coronaviruses to efficiently counteract antiviral control in LPS-EB-stimulated microglia.

Taken together, these results demonstrate that LPS:EB activates TLR4-dependent signaling, resulting in IFNβ upregulation and secretion of IFN-inducible effectors. Our data reveal MHV strain-specific upregulation of neuroinflammatory effectors (IL-6, TNF-α, IL-1α, and IL-1β) in microglia. Furthermore, LPS:EB stimulation during infection contributed to higher levels of IL-1α, and IL-1β in a TLR4-dependent manner, and pharmacological inhibition of TLR4 signaling suppresses the neuroinflammatory response in MHV-infected, LPS-EB stimulated microglia. Moreover, our microglia in vitro model of MHV infection confirmed the known ability of betacoronaviruses to counteract IFNβ response in other cell types [82–86]. Paradoxically, MHV-A59 and -JHM were unable to inhibit IFN-β production induced by LPS:EB.

3.4 Neutralization of IFNβ in LPS:EB-stimulated microglia rescues MHV replication

The upregulation of IFNβ in response to LPS:EB stimulation prompted us to interrogate whether blocking IFNβ with a specific neutralizing Ab (nAb-IFNβ) would rescue MHV replication in microglia. Cells were infected with MHV-A59, or -JHM for 1 h following stimulation with LPS:EB in the presence or absence of nAb-IFNβ [2,000 NU/mL]. This concentration of Ab was selected based on previous data from our lab validating the neutralization activity against IFNβ in the antiviral response in Poly(I:C) stimulated, MHV-infected macrophages [70]. Microglial supernatants and cell lysates were collected at 20 hpi for quantification of viral titers and immunoblot analysis of MHV-Nucleocapsid protein expression, respectively.

Intriguingly, incubating MHV-A59 or -JHM -infected microglia with nAb-IFNβ did not increase MHV viral titers or nucleocapsid expression (Fig. 5A-C). This could be attributed to the IFNβ-suppressed state that is generated during MHV infection, as coronavirus have been shown to possess numerous mechanisms to antagonize IFN. As such, further inhibition of IFNβ may be inconsequential. Conversely, neutralization of IFNβ in MHV-infected, LPS:EB-stimulated microglia increased replication of both MHV-A59 and -JHM, restoring viral titers to the levels observed in unstimulated cells (Fig. 5A). Moreover, neutralization of IFNβ significantly upregulated MHV-A59 and -JHM Nucleocapsid expression (P < 0.001 and P < 0.01, respectively), although the levels of Nucleocapsid were not fully restored to those observed in infected microglia.

These data suggest that while LPS:EB stimulation restricts the production of infectious MHV via IFNβ-dependent mechanisms, IFNβ-independent pathways may work in concert to inhibit the synthesis of viral proteins.

Taken together, our results identified a role of IFNβ production in the inhibition of coronavirus infection during LPS:EB stimulation in microglia.

3.5 TLR4 signaling deficiency impairs MHV replication and increases IFNβ expression

Microglia deficient in TLR4 (TLR4^−/−) or pharmacological inhibition of TLR4 signaling with TAK-242 suppressed neuroinflammatory response to LPS:EB stimulation, demonstrating the specificity of LPS:EB as a TLR4 agonist (Supplemental Fig. 3, Fig. 4). These results prompted us to test the hypothesis that LPS:EB-induced restriction of MHV replication in microglia is dependent on TLR4 signaling. First, to determine a potential role of TLR4 signaling in MHV infection in resting microglia, WT or TLR4^−/− microglia were infected with either MHV-A59 or -JHM for 1 hour. The virus inoculum was removed, and cells were cultured for 20 hours in the presence of TAK-242 or left untreated. Unexpectedly, MHV-A59 and -JHM viral titers were

significantly reduced in TLR4^−/− or WT microglia supernatants treated with TAK-242 (P < 0.01), implying that loss of TLR4 signaling restricts MHV infection through mechanism(s) yet to be determined (Fig. 6A, B). Furthermore, inhibition of TLR4 in LPS-treated JHM-infected WT microglia failed to increase viral replication or nucleocapsid expression. Indeed, TAK-242 alone significantly reduced the expression of the Nucleocapsid protein to similar extent as LPS:EB stimulation (Fig. 5C-E), prompting us to interrogate whether pharmacological inhibition of TLR4 affects microglial IFNβ expression.

We investigated intracellular IFNβ expression in response to TAK-242, LPS:EB stimulation, or the combination of TLR4 inhibitor and agonist in the absence or the presence of viral infection (Fig. 6F-H). Surprisingly, TAK-242 induced an almost 2-fold induction in the expression of IFNβ, similar to the upregulation induced by LPS:EB alone. This upregulated expression was similarly observed in microglia stimulated with LPS:EB in the presence of TAK-242. IFNβ was also elevated in MHV-infected microglia treated with TAK-242, though the expression was slightly higher in MJV-JHM- than MHV-A59-infected cells (Fig. 5E-G). This may further reflect strain-specific differences between MHV-A59 and MHV-JHM to antagonize IFNβ-inducing pathways.

Taken together, these novel results demonstrate that coronavirus replication is restricted in TLR4^−/− or WT microglia treated with TLR4 inhibitor TAK-242, suggesting a complexity beyond potential non-specific, off-target effects of TAK-242. TLR4 inhibitor TAK-242 did not inhibit LPS:EB-induced IFNβ expression (Fig. 6). In contrast, TAK-242 abrogated the neuroinflammatory response in LPS:EB stimulated microglia (as shown by ELISA and proteome arrays, Fig. 5). Mechanistically, we identified a previously unforeseen effect of the TLR4 inhibitor TAK-242 on IFNβ expression upregulation that correlates with decreased MHV replication (viral titers and Nucleocapsid expression) in TAK-242-treated, infected microglia. This paradoxical effect of LPS:EB (TLR4 agonist) and TAK-242 (TLR4 inhibitor) on the upregulation of IFNβ expression is puzzling. It's possible that while activation of TLR4 induces antiviral control, inhibition of TLR4 signaling might enhance certain antiviral pathways in microglia. This hypothesis will be further investigated in experiments shown Fig. 11.

3.6 Contrasting effects of MHV infection and LPS:EB stimulation on MDA5 expression

Type I IFNs induce hundreds of genes with antiviral functions, including the interferon induced with helicase C domain 1 (IFIH1) gene encoding MDA5, a cytosolic dsRNA sensor with critical roles in some viral infections, including coronaviruses [87–90]. Furthermore, in vitro LPS stimulation of murine macrophages and U373MG human astrocytoma cells induces upregulation of MDA5 expression [91, 92]. Although LPS is the most potent stimuli that induces microglial activation, the effects of TLR4 stimulation on dsRNA sensors including MDA5 remain to be elucidated. Taken together, we tested the hypothesis that LPS:EB stimulation of TLR4 induces MDA5 expression in microglia.

Microglial cells were i) left untreated; ii) stimulated with the TLR4 agonist LPS:EB for 20 h without priming (-/+); iii) LPS:EB primed for 3 h and, rested for 1 h with additional agonist stimulation for 20 h (+/+); and iv) LPS:EB primed for 3 h, rested for 1 h, and cultured without the agonist for 20 h. Immunoblot analysis of microglial cell lysates at 20 h demonstrated that MDA5 protein expression is low in resting microglia, and that irrespective of experimental stimulation condition, LPS:EB induced significant upregulation of MDA5 expression (P < 0.001) (Fig. 7A, B). Second, we used the same experimental set up of LPS:EB stimulation in the context of MHV-A59 and -JHM infections (Fig. 7C-F). MHV infection did not inhibit LPS:EB-induced upregulation of MDA5 (Fig. 7C, D). In contrast, both viruses significantly reduced MDA5 expression (P < 0.001) (Fig. 7F). These results imply distinct capabilities of coronavirus infection to counteract/antagonize MDA5 during agonist-driven activation of TLR4 in microglia. Third, we investigated whether pharmacological inhibition of TLR4 with TAK-242 inhibited MDA5 expression. As shown in Fig. 7E, TAK-242 inhibited MDA5 expression in resting and in LPS:EB stimulated cells, suggesting a regulatory role of TLR4 in basal expression of microglial MDA5. Importantly, these results demonstrate that LPS:EB-induced MDA5 upregulation is dependent on TLR4 signaling.

Next we investigated the temporal expression of MDA5 in naïve, LPS:EB-stimulated, and MHV-infected microglia cultured without or with LPS:EB stimulation at the indicated times (0, 1.5, 3, 6, and 14 h) (Fig. 8A, B). Immunoblot analysis of cell lysates showed that MDA5 expression started to be induced at 3 h after LPS:EB treatment with increasing concentrations thereafter. Interestingly, MHV-A59 and -JHM infection slightly decreased expression of MDA5 in LPS-EB-stimulated cells at 3 hpi, a time within the coronavirus replication cycle with undetectable viral protein expression; however, both viruses did not show inhibitory effect on MDA5 expression at 14 hpi. Interestingly, in contrast to MHV-JHM, MHV-A59 reduced LPS:EB-induced MDA5 expression at 6 hpi, a time in the coronavirus replication cycle known for the start of viral proteins expression.

Overall, these results demonstrate that LPS:EB induced MDA5 expression is fully dependent on TLR4 signaling. Interestingly, we found that while MHV infection of microglia reduced the expression of MDA5, MHV did not inhibit the induction of MDA5 expression by LPS:EB, suggesting distinct mechanisms regulating MDA5 expression upon agonist activation of TLR4 signaling that MHV infection is not effective at counteracting. Interestingly, our findings align with previous data in murine fibroblasts showing that MHV-A59 and -JHM viruses do not induce IFNβ and do not inhibit its induction by ligand activation of TLR3, MDA5, or RIG-I antiviral pathways, consistent with the notion that dsRNA, produced during MHV infection, is not accessible to cellular PRRs [83].

3.7 MDA5 siRNA silencing significantly inhibits LPS:EB-induced MDA5 protein expression

MDA5 contributes to the antiviral response of fibroblast, macrophages, and lung epithelial cells to coronavirus infection, including MHV and SARS-CoV-2 [87, 88, 93]. Our results demonstrate that LPS:EB stimulation inhibit MHV replication with concomitant TLR4-dependent upregulation of the antiviral dsRNA sensor MDA5 in microglia. To further investigate a potential mechanistic link between these two findings, we tested the hypothesis that LPS:EB inhibits MHV replication via upregulation of MDA5 expression in microglia.

Microglia are known for their difficulty to achieve successful transfection, including siRNA silencing. First, we demonstrated siRNA transfection efficiency in microglia using a commercially available fluorescent siRNA control of transfection (Supplemental Fig. 5). After having demonstrated some level of successful transfection, we further investigated the effect of silencing with a siRNA MDA5 or a scramble siRNA control on the expression of MDA5 protein (Fig. 9A-C). Microglial cells were left non-transfected, or transfected with siRNA-scramble or siRNA-MDA5 for 48 h, MHV-infected for 1 h, and then cultured in the presence or absence of LPS:EB for 20 hpi. Immunoblot analysis of MDA5 expression demonstrated that as expected, the scramble siRNA exhibited no suppressive effect on MDA5 expression. However, somewhat surprisingly, the scramble siRNA induced significant upregulation of MDA5, implying non-specific effects of RNAi on the activation of innate sensors such as MDA5 in microglia. Importantly, specific siRNA silencing of MDA5 significantly abrogated MDA5 protein upregulation in LPS:EB stimulated, and in MHV-infected and LPS:EB stimulated microglia. In LPS-treated naïve and MHV-infected microglia, transfection with MDA5-silencing siRNA resulted in a 50–80% reduction in MDA5. The magnitude and specificity of this silencing confirms that our transfection procedure efficiently downregulates LPS-enhanced expression of MDA5.

Overall, these results demonstrate the feasibility of siRNA MDA5 silencing to significantly inhibit the LPS:EB-induced upregulation of MDA5 in microglia prompting us to interrogate the effects of siRNA MDA5 silencing on MHV replication in infected, and LPS:EB stimulated microglia.

3.8 siRNA MDA5 silencing does not rescue MHV replication in LPS:EB stimulated microglia

After confirming we could effectively silence MDA5 expression in vitro, we sought to test the hypothesis that LPS:EB-induced MDA5 upregulation is involved in the reduction of MHV viral titers and Nucleocapsid expression in stimulated microglia. Microglial cells were transfected with siRNA scramble or siRNA MDA5 for 48 h, then infected with MHV for 1 h, and cultured in the absence or presence of LPS:EB for 20 hpi. An infection control group was included, consisting of cells infected with MHV-A59 or -JHM and cultured in the presence or absence of LPS:EB for 20 hpi. We quantified viral titers and MHV-Nucleocapsid expression in microglia supernatants and cells lysates using plaque assay and immunoblot, respectively.

Compared to infection control, siRNA scramble or specific siRNA MDA5 silencing slightly reduced MHV-A59 production while inducing a more pronounced inhibition of MHV-JHM viral titers, although without reaching statistical significance (Fig. 10A). In alignment with viral loads, both siRNA scramble and siRNA MDA5 significantly inhibited MHV-JHM Nucleocapsid (P < 0.001) with no effects on MHV-A59 Nucleocapsid expression (Fig. 10B, C). LPS:EB stimulation, or transfection with siRNA scramble control or siRNA MDA5 reduced viral loads to the same extent. Therefore, siRNA MDA5 silencing did not rescue the reduced viral titers observed upon LPS:EB stimulation of infected microglia (Fig. 10A). Moreover, in microglia infected and stimulated with LPS:EB, only the expression of MHV-A59 Nucleocapsid protein was significantly increased in cells transfected with either siRNA scramble or specific siRNA MDA5 (P < 0.01 and P < 0.001, respectively).

Taken together, though effective at silencing MDA5 (Fig. 9), it is evident that transfection of siRNA can induce off-target effects that may impact host responses to coronavirus infection. Moreover, our results suggest potential differences between MHV-A59 and -JHM in counteracting/evading antiviral pathways, including RNAi machinery in microglia. While these findings don’t argue against potential MDA5 sensing of MHV dsRNA, these data suggest that i) MDA5 may play a redundant role if any, in antiviral control of coronavirus infection in microglia; ii) LPS:EB induced MDA5 upregulation is not required, or perhaps, it is redundant with concomitant activation of yet to be identified antiviral sensors mediating IFN-dependent control during LPS stimulation of MHV-infected microglia.

3.9 LPS:EB induces TLR4-dependent activation of TLR3 signaling in microglia

Our data demonstrates that TLR4 activation with LPS:EB induced IFNβ-dependent restriction of coronavirus replication in microglia. While we observed that LPS:EB stimulation results in TLR4-dependent MDA5 upregulation, our siRNA silencing approach failed to demonstrate its causative role in LPS:EB-induced antiviral response. Results shown in Fig. 6 demonstrate that paradoxically, microglial IFNβ expression is upregulated in response to TLR4 activation with LPS:EB or pharmacological TLR4 inhibition with TAK-242. We speculated that while agonist activation of TLR4 induces antiviral control, inhibition of TLR4 signaling might enhance certain antiviral pathways in microglia. Here we test the hypothesis that LPS:EB stimulation of microglia induces TLR4-dependent phosphorylation of residue Y759-TLR3, a critical activation required for TLR3 signaling in response to dsRNA [94, 95].

First we investigated the temporal expression and activation of TLR3 in WT, TLR3^−/−, and TLR4^−/− microglial cells stimulated with LPS:EB for 0.5, 1.5, 3, 6, and 18 h. Immunoblot analysis of microglial cell lysates at the indicated times demonstrated that WT microglia responded to LPS:EB stimulation with early (30 minutes) and late (6, 18 h) phosphorylation of pY759-TLR3. In contrast, expression of TLR3 was not significantly affected by LPS:EB stimulation (Fig. 11A, B). Importantly, activation of pY759-TLR3 was abrogated in microglia deficient in TLR3 or TLR4 signaling, demonstrating that LPS:EB-induced activation of TLR3 is dependent of TLR4, and arguing against a potential direct stimulation effect of LPS:EB on TLR3.

Next we investigated the effects of pharmacological inhibition of TLR4 with TAK-242 in WT microglia stimulated with LPS:EB, infected with MHV, or infected and stimulated for 20 h. Microglial cells were left untreated or pretreated with TAK-242 for 2 h before infection and/or LPS:EB stimulation and cultured in the presence of TAK-242 after virus adsorption for 20 h. TAK-242 treatment alone did not show any effect on TLR3. Importantly, and in agreement with results obtained in TLR4 deficient microglia (Fig. 11A), TAK-242 suppressed LPS:EB phosphorylation of pY759-TLR3 (Fig. 11C). MHV-JHM infection did not induced pY759-TLR3 at least at the time analyzed (20 hpi). However, pharmacological inhibition of TLR4 induced 2.45-fold increase of pY759-TLR3 in MHV-JHM infected cells, suggesting that MHV-JHM infection may hijack TLR4 signaling to counteract TLR3 sensing and activation. Similar levels of increased phosphorylation of pY759-TLR3 were observed in LPS:EB-stimulated microglia compared to JHM-infected and LPS:EB (2.65- and 2.64-fold, respectively), implying that MHV-JHM does not counteract LPS:EB-induced activation of TLR3 (Fig. 11C). Finally, only a slight reduction in pY759-TLR3 was observed in TAK-242-treated, infected and stimulated microglia, mimicking our data on IFNβ expression under these experimental conditions (Fig. 6).

Taken together, these results demonstrate that i) LPS:EB induces biphasic TLR4-dependent activation of pY759-TLR3 with no significant effects in TLR3 expression; ii) MHV-JHM infection does not induce phosphorylation of Y759-TLR3, and it does not counteract its activation by LPS:EB; iii) while pharmacological inhibition of TLR4 signaling with TAK-242 suppresses LPS:EB-induced pY759-TLR3, intriguingly, this suppression turns into activation of pY759-TLR3 during MHV-JHM infection. Overall, these findings further support the existence of a TLR4-TLR3 crosstalk axis that is activated upon LPS stimulation and that may fine-tune microglial antiviral and inflammatory response to coronavirus infection.

3.10 TLR3 selectively contributes to LPS:EB-induced pro-inflammatory milieu

Considering the results above, we next investigated if TLR3 signaling contributes to neuroinflammation in response to LPS:EB stimulation of microglia. WT, TLR3^−/−, and TLR4^−/− microglia were stimulated with LPS:EB for 20 hours, after which the supernatants were collected for proteome array analysis (Fig. 11D-G). As expected, cytokines, chemokines and acute response proteins were suppressed in LPS:EB-stimulated TLR4-deficient (TLR4^−/−) compared to WT microglia. Noteworthy, several effectors were downregulated in LPS:EB stimulated TLR3^−/− microglia, including the cytokines IL-1RA, IL-23, IL-6, IL-16, IL-1α, IL-27, and TNF-α, the chemokine CXCL10 (IP-10), and the grow factors G-CSF and GM-CSF. Interestingly, TLR3-deficiency did not inhibit LPS:EB-induced production of CXCL2, CCL5, CCL12 chemokines, neither the soluble intercellular adhesion molecule-1 (sICAM-1), suggesting no involvement of TLR3 signaling in the production of these effectors. Importantly, and as expected, in response to Poly(I:C) HMW, a synthetic dsRNA ligand of TLR3, IL-6 and TNF-α were inhibited in TLR3^−/− compared to WT microglia, demonstrating lack of signaling in response to dsRNA in TLR3-deficient microglia (ELISA data, Supplemental Fig. 6). Overall, the proteome array analysis demonstrated that TLR3 selectively contribute to proinflammatory cytokine milieu during LPS:EB stimulation of microglia, implying a TLR4-TLR3 crosstalk that fine-tunes neuroinflammation in response to bacterial LPS.

We were intrigued by TLR3’s influence on the secretion of IL-6 and TNF-α, key inflammatory mediators of the cytokine storm seen in cases of severe sepsis and certain viral infections, including fatal encephalitis induced by MHV-JHM infection in the mouse, and severe COVID-19 by SARS-CoV-2 infection in human lungs. To examine this relationship using a more sensitive approach, we analyzed LPS-mediated secretion of these two cytokines by ELISA (Fig. 12A). In response to LPS:EB stimulation, both IL-6 and TNF-α were significantly reduced (P < 0.0001) in supernatants of TLR3^−/− compared to WT microglia (Fig. 12A). These results further confirmed the contribution of TLR3 signaling to TLR4-dependent, LPS:EB-induced proinflammatory milieu. To further investigate the contribution of TLR3-TLR4 crosstalk in IL-6 and TNF-α secretion, we inhibited TLR3 signaling with the previously characterized TLR3/dsRNA complex inhibitor that acts as a direct, competitive, and high affinity inhibitor of dsRNA binding to TLR3 (iTLR3) (Fig. 12B). WT microglial cells were pretreated with iTLR3 for 2 h before, and during LPS:EB or Poly(I:C) stimulation for 20 h and cytokines were measured by ELISA. TLR3 inhibition significantly reduced LPS:EB-induced IL-6 and TNF-α and as expected, suppressed Poly (I:C)-induced cytokines (Fig. 12B). Temporal analysis of IL-6 and TNF-α in response to LPS:EB in the presence or absence of iTLR3 demonstrated the involvement of TLR3 in early (3, 6 h, P < 0.001) and late (18 h, P < 0.001) IL-6 production (Fig. 12C). While production of TNF-α was independent of TLR3 at 3 h post-stimulation, TLR3 was involved in LPS:EB-induction of TNF-α at 6 and 18 h (P < 0.001) (Fig. 12C). Overall, these findings demonstrate that TLR3 differentially contributes to LPS:EB-induced pro-inflammatory cytokines.

Lastly, we further investigated the potential contribution of TLR3-dependent signaling to upregulated MDA5 expression in microglia upon LPS:EB stimulation, MHV infection, or infection and stimulation. Immunoblot analysis revealed that pharmacological inhibition of TLR3 (iTLR3) did not affect upregulation of MDA5 expression LPS:EB stimulated cells (Fig. 13A, B). Conversely, iTLR3 significantly inhibited MDA5 expression in microglia infected with MHV-JHM or -A59 and then stimulated microglia (P < 0.001, P < 0.01, respectively) (Fig. 13A, C, D). In agreement with results shown in Fig. 7E, MHV infection did not inhibit LPS:EB-induced MDA5 expression. Taken together, these findings demonstrate that LPS:EB-induced MDA5 upregulation is not dependent on TLR3. However, our data suggest that TLR3 signaling contributes to MDA5 upregulation in MHV-infected, LPS:EB-stimulated microglia. Considering the observed inability of MHV infection to inhibit MDA5 upregulation in response to LPS:EB, our findings may also imply that during MHV infection and LPS:EB stimulation, a TLR4-TLR3 crosstalk may fine-tune MDA5 expression in microglia. Future work will be directed at further understanding cross regulatory pathways between bacterial sensor TLR4 and dsRNA sensors, including TLR3 and MDA5, in microglia response to infection as well as sterile inflammation.

There were several motivations for the present study. First, microglia responses to coronavirus infection remains ill-defined. Second, the potential crosstalk between bacteria and virus innate immune sensors within the central nervous system remains poorly understood. Although the bacterial endotoxin LPS is the most used pro-inflammatory stimulus for microglia, both in vitro and in vivo, little is known on the potential effects of LPS in promoting or restricting viral infections of microglia, including coronaviruses. Third, microglia are emerging as critical regulators of neuronal function with implications in brain homeostasis, as well as key players influencing viral neuropathogenesis through mechanisms that are not well characterized. It is pivotal that efforts to develop microglia-targeted therapeutic strategies are based on a profound molecular knowledge of microglia function in health and disease. Fourth, there are multiple challenges to study microglia in vivo and in vitro, including the realization that there is no in vitro method available yet that fully recapitulates microglia function and heterogeneity within the CNS environment.

Here we tested the overall hypothesis that stimulation of microglia with the TLR4-specific agonist smooth (s)-form LPS isolated from pathogenic E. coli 0111:B4, impact microglia response to murine coronavirus infection. Fig. 14 summarizes our findings in the context of (A) LPS:EB stimulated, uninfected microglia and (B) during MHV infection in the presence of LPS stimulation.

A) In the context of uninfected microglia, LPS:EB stimulation induced TLR4-dependent upregulation of MDA5 (Fig. 7E) with increasing protein expression after the first 3 h post-stimulation (Fig. 8). Unexpectedly, LPS:EB stimulation induced activation of TLR3 signaling as measured by phosphorylation of the TLR3 residue Y759 (Fig. 11A). Importantly, LPS:EB-driven activation of pTLR3-Y759 was fully dependent on TLR4 expression (Fig. 11A) and meditated by the MyD88, with an early (but not late) involvement of the TRIF axis of the TLR4 signaling (data not shown). Pharmacological inhibition of TLR4 with TAK-242 suppressed LPS:EB-induced pTLR3-Y759 (Fig. 11C), suggesting that phosphorylation of TLR3-Y759 is secondary to initial TLR4 sensing of LPS:EB and recruitment of TLR4 adaptors TIRAP/MyD88 and TRAM/TRIF. LPS:EB-induced upregulation of MDA5 expression was independent of TLR3 (Fig. 13A, B). Intriguingly, using TLR3 deficient microglia (TLR3^-/-), our data demonstrated that TLR3 contributed to IL-6, and TNFα production in LPS:EB stimulated microglia (Fig. 11, 12). These results linked the observed activation of pTLR3-Y759 residue with functional activation of the TLR3 pathway in LPS:EB stimulated microglia. Moreover, our data revealed a previously unrecognized effect of the TLR4 inhibitor TAK-242 on IFNβ expression upregulation in microglia. TAK-242 induced upregulation of IFNβ in the absence of LPS stimulation and did not inhibit LPS-EB-induced upregulation of IFNβ (Fig. 6F). Mechanistically, TAK-242-induced IFNβ was likely independent of MDA5 expression as the inhibitor reduces MDA5 expression in the absence of LPS (Fig. 7E). Further studies will characterize the molecular mechanisms that induce IFNβ expression upon TAK-242 treatment in microglia.

B) In the context of MHV infection and LPS-EB-stimulation, LPS significantly reduced MHV replication, as measured by viral titers, MHV Nucleocapsid protein expression, and formation of dsRNA in infected and stimulated microglia (Fig. 1-3). Mechanistically, neutralization of IFNβ demonstrated its critical role in LPS:EB-induced antiviral control (Fig. 5). MHV-JHM and -A59 inhibited basal expression of MDA5 and IFNβ in non-stimulated microglia, but MHVs were not able to counteract LPS:EB-induced upregulation of MDA5 or IFNβ expression in LPS-stimulated cells (Fig. 4E, 7E). Although LPS induced significant MDA5 expression, MDA5 siRNA silencing did not rescue MHV replication in LPS-stimulated cells (Fig. 10), suggesting the involvement of sensor(s) other than MDA5 in LPS-induced IFNβ antiviral response in microglia. Paradoxically, we found that TLR4 activation with LPS:EB or suppression of TLR4 signaling by genetic deficiency (TLR4^-/-) or pharmacological inhibition (TAK-242) significantly inhibited MHV replication (Fig. 6). TAK-242 inhibition of TLR4 suppressed pTLR3-Y759 activation in LPS:EB-stimulated microglia, demonstrating that TLR4 signaling is required for LPS:EB-induced activation of pTLR3-Y759 (Fig. 11C). MHV infection alone did not induce phosphorylation of TLR3-Y759 at least at the time analyzed (20 hpi) (Fig. 11C). However, TAK-242 activated pTLR3-Y759 in MHV-infected microglia and it did not fully suppress LPS-induced pTLR3-Y759 in infected, and stimulated microglia (Fig. 11C). Considering that our data demonstrates that TAK-242 suppresses activation of pTLR3-Y759 in response to LPS:EB stimulation, taken together, these results suggest the existence of a previously unrecognized TLR4-3 crosstalk in microglia response to coronavirus infection.

LPS:EB is a TLR4-specific agonist. Liposaccharides are constituents of the Gram-negative bacterial outer membrane and play pivotal roles in bacteria integrity and resistance to outside stressors [96]. The host response to Gram-negative bacteria is determined at least in part, by the complexity of the biochemical composition of the LPS. Structural modifications of bacterial LPS can facilitate Gram-negative bacteria evasion of host innate immunity. It is therefore not surprising that through evolution, Gram-negative bacteria have evolved fascinating complexity in LPSs. The LPS structure comprises a conserved phosphoglycolipid (the lipid A), a short oligosaccharide chain (core OS region), and a surface-exposed O-polysaccharide (O-antigen) [97]. Based on this biochemical composition, LPSs are categorized into two main types, smooth and rough LPS, which express or lack the O-antigen, respectively. Chemical modifications of these three constituents, and specially, modifications on lipid A dramatically influence the overall bioactivity of an LPS. In 1998, the discovery of the TLR4 gene and the identification of TLR4 as the innate immune sensor of LPS marked the beginning of pivotal research in the characterization of the host immunological response to Gram-negative bacteria [98,99]. The canonical view of LPS sensing includes its binding by soluble LPS-binding protein (LBP) following transportation to membrane-bound CD14 (mCD14) and subsequent presentation to myeloid differentiation protein-2 (MD-2)/TLR4 complex [77]. LPS lipid A recognition by MD-2 triggers MD-2/TLR4 complex dimerization, resulting in the activation of two signaling transduction pathways, the myeloid differentiation primary response 88 (Myd88)-dependent axis and the Toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent axis. Among all TLRs, TLR4 is unique in its ability to mediate both MyD88 and TRIF pathways. Importantly, the adaptors Toll-interleukin-1 Receptor (TIR) domain-containing adaptor protein (TIRAP) and the TRIF-related adaptor molecule (TRAM) bridge MyD88 and TRIF to the TIR domain of TLR4 to initiate signaling from the cell membrane (TIRAP/MyD88) and from the endosome (TRAM/TRIF). In response to LPS, activation of the MyD88 pathway induces pro-inflammatory mediators, whereas the TRIF pathway mediates IFN responses [100]. Noteworthy, this canonical view of TLR4 is being challenged by emerging evidence demonstrating the existence of cellular responses to LPS that do not involve TLR4, including sensing of extracellular LPS by transient receptor potential (TRP) cation channels, as well as sensing of intracellular LPS by CD14-dependent signaling and Caspases 11 (human) and 4/5 (mouse) noncanonical inflammasome [101,102].

Therefore, pivotal to our studies, here we demonstrated that the smooth (s)-form LPS isolated from pathogenic E. coli 0111:B4 (LPS:EB) is dependent on TLR4 for the induction neuroinflammatory mediators in microglia. Importantly, cytokines, chemokines, and acute response proteins were fully abrogated in response to LPS:EB stimulation of microglia deficient in TLR4 (TLR4^-/-), or in immunocompetent WT microglia treated with TAK-242, a cell-permeable compound that selectively binds to Cys747 of TLR4 and selectively disrupts its interaction with adaptors TIRAP/MyD88 and TRAM/TRIF to block TLR4 signaling [103-105]. In contrast, proteome arrays demonstrated that TLR2-deficient microglia (TLR2^-/-) were responsive to LPS:EB, excluding the possibility of TLR2 involvement in microglia response to LPS:EB. This was an important finding as some types of LPS can induce TLR2-mediated inflammatory responses and functional heterodimers of TLR4/TLR2 have been found in immune cells [106-108,80].

Murine coronaviruses MHV-JHM and -A59 productively infect microglia in vitro inducing distinct neuroinflammatory response. The CNS tropic murine coronaviruses MHV-A59 and -JHM are well characterized models of acute, mild to fatal encephalomyelitis with chronic demyelination observed in mice that survive infection. The molecular mechanisms that drive neuro-attenuation/survival (MHV-A59) or neurovirulence/death (MHV-JHM) in infected mice remain ill-defined. However, we and others have previously demonstrated that MHV viral loads as measured by plaque assays of whole brains of infected mice do not correlate with CNS disease outcome [71,50,109]. These data suggest that immune-mediated mechanisms drive coronavirus neuropathogenesis. Recent evidence demonstrated that while productively infected in vivo, microglial cells are required for early control of murine coronavirus acute infection of the brain, contributing to myelin repair in the spinal cord during chronic demyelination [32,51], recently reviewed in [110]. However, the innate immune sensors, including TLRs, that orchestrate microglia response to coronavirus infection remain unknown.

Here we established an in vitro microglia model system using immortalized microglia previously isolated from brains of adult C57BL/6J mice. The biological significance of this in vitro system is supported by the following findings. First, our results demonstrate that MHV-JHM replicates to significantly lower titers compared to MHV-A59 in in vitro infected microglia, recapitulating viral loads previously observed in whole brains from infected mice. While MHV-JHM is highly neurovirulent and induces fatal encephalitis in infected C57BL/6J mice, viral titers in brains are significantly lower compared to neuro-attenuated MHV-A59 infected mice [48,71,109,50]. Second, proinflammatory cytokines IL-6, TNFa, IL-1a, and IL-1b are more elevated in the brain of MHV-JHM-infected mice compared to neuro-attenuated MHV-A59 [111].

Using our in vitro model of microglia infection, we demonstrate that the neurovirulent MHV-JHM induced stronger upregulation of pro-inflammatory cytokines IL-6 and TNFα compared to the neuro-attenuated MHV-A59. Furthermore, IL-1α and IL-1β were upregulated by MHV-JHM infection in the absence of LPS stimulation. In contrast, MHV-A59 induced both IL-1α and IL-1β only in the presence of LPS:EB stimulation during infection. Therefore, our data suggest a differential involvement of the inflammasome in microglia response to virulent vs. attenuated CNS-tropic. Noteworthy, CXCL-1, a chemokine previously known to activate the NLRP3 inflammasome in macrophages during Mycobacterium tuberculosis infection [81], was upregulated only by MHV-JHM. Therefore, our results identify an important association between the previously known MHV-JHM neurovirulence in vivo and our novel data in vitro, suggesting a detrimental contribution of microglia through exacerbated IL-1α, IL-1β, and CXCL1/inflammasome activation during MHV-JHM-induced fatal encephalitis in vivo. The main cellular source of these pro-inflammatory cytokines in the brain of infected mice remains to be identified in vivo. Importantly, our in vitro data suggest a pivotal role of microglia in driving coronavirus neurovirulence. A systematic investigation on the TLRs that mediate antiviral control and/or neuroinflammation in response to MHV-A59 and -JHM was beyond the goal of the present study. However, it is tempting to hypothesize that a TLR4 / inflammasome crosstalk in microglia may contribute to neurovirulence and inflammation in response to coronavirus infection in the brain. Future studies will test this hypothesis in experimentally infected mice.

LPS:EB stimulation restricts murine coronavirus replication in microglia through IFNβ. The E. coli LPS is the most common model of neuroinflammation induction in vivo and in vitro. A plethora of studies investigating microglia response to LPS using imaging, electrophysiology, and behavioral assays have demonstrated diverse effects of LPS in microglia cellular functions with implications in the development of neurocognitive impairment and depression-like behavior [56,112,113]. However, the effects of LPS stimulation on microglia responses to viral infection, including coronaviruses, remain unexplored. Here we demonstrate that LPS:EB stimulation before or during infection significantly restricts MHV-JHM and -A59 replication. We did not observed differences in the reduction of viral titers among the three LPS:EB-stimulated microglia experimental groups (3 h before infection; 20 h after virus adsorption; or the combination of both pre- and post-stimulation). Interestingly, despite just a 3-hour pre-treatment, LPS-primed cells were as refractory to MHV as cells incubated with LPS throughout infection. The addition of LPS after priming did not further impair MHV replication. These results suggest a lack of synergistic effect between LPS pre- and post-stimulation on virus restriction. There are multiple steps in the coronavirus replication cycle that LPS:EB may inhibit, including attachment, entry, uncoating, replication, maturation, and/or virus release. In the present study, we further mechanistically investigated the effects of LPS:EB stimulation after viral infection. Importantly, the reduction in viral titers associated with significant inhibition of dsRNA formation -a known intermediate of virus replication-, and Nucleocapsid expression reduction, suggesting that LPS:EB restricts coronavirus protein synthesis and replication. Interestingly, temporal analysis of Nucleocapsid expression and viral titers revealed no significant differences between LPS:EB-stimulated compared to unstimulated microglia until 20 hpi. These results suggest that the production of antiviral molecular effectors such as IFN are required to restrict coronavirus replication. Indeed, our data demonstrates that neutralization of IFNb in LPS:EB-stimulated microglia restores MHV viral titers to the levels observed in unstimulated cells.

LPS is known to induce IFNs in vitro in human and murine cells, as well as in vivo [114-117]. Moreover, IFNb has an essential role in the induction of IFN-stimulated gene expression by LPS in macrophages [118]. In agreement, our findings demonstrate that LPS:EB induce IFNb in microglia. Coronavirus’ severe pathogenesis associates with suboptimal antiviral but a simultaneously excessive inflammatory response in both humans and animal models [86]. Results from in vitro studies in other cell types previously showed that MHV significantly reduced levels of IFNs compared to other viruses such as Sendai virus or Newcastle disease virus. Moreover, suppression of MHV required high concentrations of recombinant IFNs, suggesting that CoVs elicit robust anti-IFN mechanisms at least in mouse fibroblasts [84]. Not surprisingly, our data revealed that MHV-JHM and -A59 inhibit IFNb expression, as measured by intracellular protein levels in infected microglia. In contrast, MHVs did not reduce IFNb expression in the presence of LPS:EB stimulation. Our findings agree with previous data showing that MHV infection did not inhibit IFNb production mediated by PRRs dsRNA sensors RIG-I, MDA-5, and TLR3 in macrophages [83]. Positive single-strand RNA (+ RNA) viruses, including coronaviruses, remodel host cell membranes to induce double-membrane vesicles (DMVs) as a replication organelle to isolate the replication of their genome from innate immunity mechanisms [119]. It has been previously hypothesized that dsRNA, produced during MHV infection, is not accessible to cellular PRRs. However, there is a battery of coronavirus-encoded proteins with emerging roles in hijacking and/or antagonizing the host antiviral response [120]. Adding to the complexity of coronavirus infection and IFN response, lack of type I IFN receptor (IFNAR) signaling resulted in dramatically high virus replication in vitro, and particularly in vivo, such that MHV spread to multiple organs with exacerbated mortality within 3 days post-infection [121,122]. These results showed that IFNAR signaling is critical to suppress initial virus replication and prevent the systemic spread of MHV in vivo.

Although the complex response of coronaviruses to IFNs within microglia remains to be further analyzed, the present study is focused on mechanistically understand LPS:EB-induced restriction of MHV in microglia. As a logical next step, we investigated the effects of LPS:EB on MDA5 expression, a dsRNA sensor that has been shown to mediate antiviral response during MHV in vivo and in SARS-CoV-2 infected human lung epithelial cells in vitro [93,87,123].

Contrasting effects of LPS:EB stimulation and coronavirus infection on MDA5 expression. Our findings demonstrate that LPS:EB induced robust MDA5 expression starting at 6 h post-stimulation. Pharmacological inhibition of TLR4 with TAK-242 demonstrated that TLR4 is required for MDA5 upregulation in response to LPS:EB. In contrast, MHV-A59 and -JHM significantly reduced basal MDA5 expression. Similarly to our results on IFNb expression discussed above, MHVs did not inhibit LPS:EB-induced MDA5 upregulation, suggesting that coronaviruses are not effective at counteracting agonist-activation of PRPPs.

MDA-5 is an early response gene inducible by IFN and tumor necrosis factor-α, responding predominantly to IFN-β [124]. TLR4 signaling, induced by LPS, has been previously shown to increase the expression of MDA5 in U373MG human astrocytoma cells [91]. Moreover, LPS induced MDA5 expression in primary murine macrophages [92]. Therefore, our study expands previous knowledge on other cell types and demonstrates that LPS:EB induces upregulation of the dsRNA sensor MDA5 in murine microglia.

To mechanistically investigate whether MDA5 contributed to LPS:EB induced restriction of MHV replication, we used siRNA silencing approach. A caveat of our studies is that despite efficient specific silencing of MDA5, transfection of microglia with siRNA scramble control upregulated MDA5 expression, implying non-specific effects of RNAi machinery on the activation of innate sensors such as MDA5 in microglia. In LPS-treated naïve and MHV-infected microglia, transfection with MDA5-silencing siRNA resulted in 50-80% reduction in MDA5. The magnitude and specificity of this silencing confirms that our transfection procedure efficiently downregulates LPS-enhanced expression of MDA5. Although specific siRNA silencing of MDA5 did not restore viral titers in microglia infected with MHVs and stimulated with LPS:EB, it did slightly increase Nucleocapsid expression. Taken together, our results suggest a minor contribution, if any, of MDA5 in LPS:EB-induced restriction of coronavirus replication in microglia.

Paradoxical effects of TLR4 activation and inhibition on coronavirus replication.

The role of TLR4 in coronavirus infection and/or pathogenesis remains poorly understood. Using microglia deficient in TLR4 (TLR4^-/-) and pharmacological inhibition of TLR4 using TAK-242 in immunocompetent WT microglia, our results demonstrate that both MHV-JHM and -A59 replicate to significantly lower titers compared to WT microglia. Moreover, expression of Nucleocapsid protein was also significant reduced. These results, although surprising, suggest a positive role of TLR4 signaling in coronavirus life cycle. Although the identification of the precise role of TLR4 in coronavirus replication is beyond the goal of the present study, we speculate that TLR4 signaling could be beneficial for productive viral infection by inducing specific host factors that promote viral replication or repress those with antiviral activity. In this regard, TLR4 activation during Ebola virus infection increases the expression of suppressor of cytokine signaling 3 (SOCS3), which has been shown to enhance viral particle release [125].

TAK-242 is a cell-permeable compound that selectively binds to Cys747 of TLR4 and selectively disrupts its interaction with adaptors TRAM/TRIF and TYRAP/MyD88 [105,104,103]. Data in the present study demonstrates that TAK-242 suppresses LPS:EB-induced neuroinflammatory response and abolishes LPS:EB-induced upregulation of MDA5, adding to its known TLR4-specific inhibition. However, we have identified that TAK-242 increases intracellular IFNb protein expression in microglia, and does not inhibit IFNb induced by LPS:EB; indeed, a tendency to synergy in IFN b expression was observed. Our data suggest an off-target effect or perhaps highlights a potential role of TLR4 as negative regulator of an antiviral pathway. Interestingly, here we identify that TAK-242 treatment induces TLR3-Y759 activation only during MHV infection, suggesting a potential role of TLR3 in antiviral response. Future studies will test this hypothesis. Based on our surprising results on TAK-242 restriction of coronavirus replication, we searched the literature on potential effects of TAK-242 on viruses’ life cycle. In agreement with TAK-242 induced restriction of coronavirus replication, Cai et al., have demonstrated that TAK-242 restricts hepatitis B virus (a DNA virus) inhibiting HBsAg, HBV DNA, HBV RNAs, and cccDNA [126]. Mechanistically, TAK-242 increased the expression of elongation factor Tu GTP-binding domain containing 2 (EFTUD2), an innate immune regulator. Moreover, inhibition of the TLR4 signaling pathway with TAK-242 reduces respiratory syncytial virus (RSV) infection and cytokine release in primary airway epithelial cells [127].

An important consideration from our studies is that both TLR4^-/- microglia or pharmacological inhibition with TAK-242 restrict murine coronavirus replication. Ongoing studies in our lab are directed to investigate the role of TLR4 and its inhibition in coronaviruses life cycle.

LPS:EB induces TLR4-dependent activation of TLR3, while TLR3 selectively contributes to LPS:EB neuroinflammatory response in microglia.

Previously, LPS was shown to upregulate the expression of the endosomal dsRNA sensor, TLR3 in murine and human macrophages [128,129]. To the best of our knowledge, the present study is the first to demonstrate that LPS:EB induces phosphorylation of TLR3-Y759, a hallmark of TLR3 signaling initiation [94]. Importantly, LPS:EB-induced phosphorylation of TLR3 was abrogated in TLR4^-/- microglia or pharmacologically treated with the TLR4 inhibitor TAK-242. These results suggest that activation of TLR3 in response to LPS:EB is secondary to TLR4 sensing. LPS:EB induced biphasic activation of TLR3 at 30 minutes after stimulation and peaking at 18 h post-stimulation. Importantly, using TLR3^-/- microglia and pharmacological inhibition of TLR3, we demonstrate that TLR3 selectively contributes to TLR4-dependent, LPS:EB-induced proinflammatory milieu in microglia, including the cytokines IL-1RA, IL-23, IL-6, IL-16, IL-1α, IL-27, and TNF-α, the chemokine CXCL10 (IP-10), and the grow factors G-CSF and GM-CSF, implying a TLR4-TLR3 crosstalk that fine-tunes neuroinflammation in response to bacterial LPS. This TLR4/TLR3 complexity may impact brain function beyond acute response to infection. Based on our data, it is tempting to speculate that microglia exposure to different types of LPS, a marker of microbial translocation that drives chronic immune activation, may selectively dysregulate dsRNA sensing PRRs, including TLR3, even in the absence of viral infection, contributing to long-term neuroinflammation and/or neurodegeneration in the brain.

Major questions remain about the mechanisms underlying microglia-specific immunity, including the intracellular pathways that sense and restrict coronavirus replication and/or promote neuroinflammation. The present study highlights the complexity of microglia response to bacterial endotoxin LPS:EB and coronavirus infection.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Author contributions

Conceived and designed the experiments: EHD, AMG, SNM. Performed the experiments: EHD, AMG, BAL, AAT, AAM, VBP. Analyzed the data: EHD, AMG, BAL, SNM. Figure preparation: EHD, AMG. Writing the manuscript: EHD, SNM. Editing the manuscript: EHD, AMG, BAL, AAT, VBP, AAM, SNM. Supervision: SNM. Funding acquisition: SNM

Funding

This work was supported in part by the National Institute of Health, National Institute of Neurological Disease and Stroke grant number 1R01 NDS104365 and by funds from Drexel University. The funders have no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability The datasets generated during and/or analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

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No competing interests reported.

SupplementalFigure1Davisetal.tif
Supplemental Fig. 1 Iba-1 expression in mouse microglial cells. Immortalized microglial cells from wild type (WT) immunocompetent mice (NR-9460, NIAID BEI Resources Repository) were incubated with goat anti-mouse/human IBA-1 followed by highly cross-adsorbed secondary antibodies donkey anti-goat IgG (H+L) Alexa Fluor™ 594 and mounted using ProLong Glass antifade with NucBlue as detailed in Methods. Fluorescence images were acquired using an Olympus IX-81 microscope (Olympus, Tokyo, Japan) and captured at 400X. Image acquisition was performed using cellSens Dimension software Ver2.0 (Olympus)
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Supplemental Fig 2 Experimental design of the temporal analysis of LPS:EB-induced impairment of MHV replication and nucleocapsid expression. Microglia were infected with MHV-JHM or MHV-A59 (MOI=1) for 1 hour; then, virus inoculum was removed, and after gently washing, cells were cultured in the presence or absence of LPS:EB [1ug/mL] for 20 hpi. No LPS:EB was present during virus adsorption. At indicated time points, cell lysates and supernatants were collected for quantification of infectious virions in supernatants and analysis of MHV-Nucleocapsid expression in cell lysates by plaque assays and immunoblot, respectively. Results are shown in Figure 3
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Supplemental Fig. 3 LPS:EB induces TLR4-dependent secretion of pro-inflammatory mediators in microglia. (A, B) Quantification of IL-6 and TNF-α secretion by ELISA in (A) supernatants of microglia wild type (immunocompetent) treated with the TLR4 inhibitor TAK-242 (iTLR4) and (B) TLR4 deficient (TLR4^-/-) after overnight stimulation with LPS:EB [1μg/mL]. Graphs are representative of 2 independent experiments with 4-6 biological replicates each. (C, E) Characterization of the innate immune secretome of immunocompetent (WT), TLR2 deficient (TLR2^-/-), and TLR4^-/- microglia stimulated with LPS:EB [1μg/mL] for 20 hours. (C) Cytokines, (D) acute response proteins, (E) CXCL, and CCL chemokines were analyzed by proteome profiler array as described in Methods. Heatmaps shown indicate log2-fold increases/decreases in innate immune effector secretion as compared to resting cells. Data is representative of two independent experiments. Statistical Analysis (A; C) One-Way ANOVA with Tukey’s multiple comparison’s test; α < 0.05
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Supplemental Fig. 4 Pharmacological inhibition of TLR3 and TLR4 signaling schematics. (A) Diagram detailing the mechanism of action of TAK-242 TLR4 inhibitor. TAK-242 is a cell-permeable compound that selectively binds to Cys747 of the TIR domain of TLR4 and selectively disrupts its interaction with adaptor molecules TIRAP and TRAM, thereby inhibiting TLR4 signal transduction and its downstream signaling events. (B) Diagram detailing the mechanism of action for the TLR3/dsRNA complex inhibitor (iTLR3). TLR3/dsRNA Complex Inhibitor blocks dsRNA binding to TLR3 and suppress TLR3 signaling
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Supplemental Fig. 5 Confocal microscopy of microglia transfected for 48hrs with a fluorescent non-specific scramble control siRNA [25 pmol] or transfection reagent alone as negative control. Images representative of 2 independent experiments with each condition performed in duplicate. Fluorescence images were acquired using an Olympus IX-81 microscope (Olympus, Tokyo, Japan) and captured at 100X. Image acquisition was performed using cellSens Dimension software Ver2.0 (Olympus)
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Supplemental Fig. 6 Synthetic dsRNA Poly (I:C) high molecular weight (HMW) -induced IL-6 and TNF-α secretion is abrogated in TLR3-deficient microglia. Immunocompetent wild type, and TLR3-deficient (TLR3^-/-) microglia were stimulated with 25 μg/ml Poly (I:C) HMW for 20 h and supernatants collected for further measurements. (A) IL-6 and (B) TNF-α were quantified by ELISA. Graphs are representative of 2-3 independent experiments with 4-9 biological replicates each