Deciphering the Role of Schwann Cells in Inflammatory Peripheral Neuropathies Post Alphavirus Infection

Old world alphaviruses (e.g., chikungunya) are known to cause severe acute and chronic debilitating arthralgia/arthritis. However, atypical neurological manifestations and, in particular, unexpected cases of acute inflammatory Guillain–Barre syndrome (GBS) have been associated with the arthritogenic alphaviruses. The pathogenesis of alphavirus-associated GBS remains unclear. We herein addressed for the first time the role of Schwann cells (SC) in peripheral neuropathy post-alphaviral infection using the prototypical ONNV alphavirus model. We demonstrated that human SC expressed the recently identified alphavirus receptor MxRA8 and granting viral entry and robust replication. A canonical innate immune response was engaged by ONNV-infected SC with elevated gene expression for RIG-I, MDA5, IFN-β, and ISG15 and inflammatory chemokine CCL5. Transcription levels of prostaglandin E2-metabolizing enzymes including cPLA2α, COX-2, and mPGES-1 were also upregulated in ONNV-infected SC. Counterintuitively, we found that ONNV failed to affect SC regenerative properties as indicated by elevated expression of the pro-myelinating genes MPZ and MBP1 as well as the major pro-myelin transcription factor Egr2. While ONNV infection led to decreased expression of CD55 and CD59, essential to control complement bystander cytotoxicity, it increased TRAIL expression, a major pro-apoptotic T cell signal. Anti-apoptotic Bcl2 transcription levels were also increased in infected SC. Hence, our study provides new insights regarding the remarkable immunomodulatory role of SC of potential importance in the pathogenesis of GBS following alphavirus infection.

A clinical and passage-limited isolate of ONNV was obtained from the National Reference Center (CNR arbovirus, Marseille, France) and titrated on Vero cells at 10 7 PFU/mL [27].

Cell Culture Treatment
SC were seeded in culture plates and incubated at 37 • C in a humid atmosphere with 5% CO 2 . When reaching 80-90% confluence, cells were infected with ONNV at different MOIs (1, 10 −1 , 10 −2 , and 10 −3 ) or stimulated with the double-stranded PIC (10 µg/mL), ATP (1 mM), anti-inflammatory TGF-β2 (10 ng/mL), or the proinflammatory cytokines IL-1β (10 ng/mL) and TNF-α (10 ng/mL) for 6 h and 24 h. SC were exposed to extracellular PIC stimulation via addition to the culture medium. The concentrations used for the different stimuli were chosen from data published in the literature [28].

Cytotoxicity Assay
Cell damages were evaluated by measuring lactate dehydrogenase (LDH) release using a colorimetric-based kit (CytoTox 96 non-radioactive cytotoxicity assay, Promega, Madison, WI, USA). Supernatants of infected or treated cells were recovered and cells were lysed according to the manufacturer's instructions. Cells lysed with 1% Triton X-100 were taken as the positive control and medium without cells as the negative control. Absorbance of converted dye was measured at 490 nm. Cytotoxicity was expressed relative to maximum LDH release with the formula: % cytotoxicity = 100 × experimental LDH release/maximum LDH release.

Quantitative Real-Time RT-PCR (qRT-PCR)
Total RNA was extracted from harvested cell culture using Zymo Research Quick-RNA Viral kit (Irvine, CA, USA, cat. no. R1035) according to the manufacturer's instructions. qRT-PCR experiments were performed using the One Step Prime Script Syber Green RT-PCR kit from TAKARA (Cat. No RR066A). qRT-PCR was performed in a final volume of 5 µL containing 1 µL of extracted total RNA per reaction, 2.7 µL of enzyme mix, and 1.3 µL of primer mix with a final primer concentration of 250 nM. Primer pairs were designed in-house using the NCBI Primer Designing Tool (http://www.ncbi.nlm.nih.gov/tools/ primer-blast/ accessed on 3 January 2020). The specific primers used are listed in Table 1. qRT-PCR was carried out using a Quant Studio 5 thermocycler from Applied Biosystems (Thermo Fisher Scientific) with the following steps: a reverse transcription at 42 • C for 5 min followed by 40 cycles of a denaturation step at 95 • C for 5 s, an annealing step at 58 • C for 15 s, and an extension step at 72 • C for 15 s. Expression of the target mRNA was normalized to the expression of GAPDH mRNA.

The Alphavirus Receptor, MxRA8, Is Highly Expressed in ONNV-Infected SC
Recently, the cell adhesion molecule MxRA8 has been identified as a receptor for multiple alphaviruses including CHIKV and ONNV [29]. We assessed by qRT-PCR the expression levels of MxRA8 in ONNV-infected SC as well as in response to different stimulatory conditions (Figure 1a,b). First, SC were infected with ONNV at a multiplicity of infection (MOI) 1, 10 −1 , 10 −2 , and 10 −3 or stimulated with the viral analog PIC 10 µg/mL, ATP 1 mM, TGF-β2 10 ng/mL, or the proinflammatory cytokines IL-1β 10 ng/mL and TNF-α 10 ng/mL. Experiments were carried out for 6 h and 24 h. We found that cultured mRNA levels from SC exposed to stimulatory treatments (IL-1β 10 ng/mL, TNF-α 10 ng/mL, TGF-β2 10 ng/mL, and ATP 1 mM) or (b) infected with ONNV (from MOI 10 −3 to 1) or stimulated with PIC 10 μg/mL for 6 h and 24 h as assessed by qRT-PCR. All experiments were done in triplicates. Results are expressed as mean ± SEM and presented as a normalized fold increase vs. control (CT).

Figure 1.
MxRA8 alphavirus entry receptor is upregulated in ONNV-infected SC. (a) MXRA8 mRNA levels from SC exposed to stimulatory treatments (IL-1β 10 ng/mL, TNF-α 10 ng/mL, TGF-β2 10 ng/mL, and ATP 1 mM) or (b) infected with ONNV (from MOI 10 −3 to 1) or stimulated with PIC 10 µg/mL for 6 h and 24 h as assessed by qRT-PCR. All experiments were done in triplicates. Results are expressed as mean ± SEM and presented as a normalized fold increase vs. control (CT). * p-values ≤ 0.05, ** p-values ≤ 0.01, and *** p-values ≤ 0.001 represent significant differences from controls by one-way ANOVA followed by the Bonferroni's test analysis.
ONNV infection strongly and significantly upregulated MxRA8 receptor mRNA levels at 24 h post infection, regardless of ONNV MOIs but with a five-fold increase with l ONNV MOI 1 (p < 0.01) compared to mock infected cells. We did not observe modulation of MxRA8 receptor expression under the following conditions: PIC, ATP, TGF-β2, IL-1β, and TNF-α.

SC Are Highly Susceptible to ONNV Infection
To address whether SC are able to be infected and to replicate ONNV, SC cultures were challenged with increasing MOIs of ONNV (MOI 10 −3 , 10 −2 , 10 −1 , and 1) and the levels of two different viral RNA (E2 and NSP2) were quantified by qRT-PCR at 6 h and 24 h post infection (Figure 2a,b). Expression levels of E2 and NSP2 genes increased in infected SC at 24 h with the different tested MOIs. At 24 h post infection with ONNV MOI 1, the levels of viral E2 and NSP2 RNA increased approximately 100-fold more than at 6 h post infection (4.3 ± 5.17 × 10 −1 and 1.58 × 10 −1 ± 1.14 × 10 −1 , respectively, at 24 h compared to 5.2 × 10 −2 ± 2.74 × 10 −2 and 1.09 × 10 −3 ± 7.47 × 10 −4 at 6 h). Of note, we confirmed the infection and expression of high levels of capsid viral protein following CHIKV infection of SC ( Figure S1).

SC Upregulate Antiviral Innate Immune-Related Genes in Response to ONNV Infection
We evaluated the capacity of ONNV to modulate the expression of several genes coding for proteins having a key antiviral role. We thus carried out qRT-PCR to screen the expression of RLRs (RIG-I and MDA5) as well as IFN-β and ISG15 in SC at 6 h an h post infection. First, in response to PIC stimulation (Figure 4a,b), the relative mR expression of the virus sensors RIG-I and MDA5 were significantly increased in SC at and 24 h. At 6 h post PIC treatments, the levels of expression were (3.5 × 10 −2 ± 1.48 × p < 0.01) for RIG-I with a fold increase of 20 and (2.26 × 10 −3 ± 2.23 × 10 −3 , p < 0.05) for MD with a fold increase of 80 compared to control conditions (1.75 × 10 −3 ± 5.44 × 10 −4 for R I and 2.71 × 10 −5 ± 1.67 × 10 −5 for MDA5). The mRNA levels of IFN-β and ISG15 were Figure 3. ONNV does not cause SC cytotoxicity at 24 h. SC were infected with ONNV (from MOI 10 −3 to 1) or exposed to stimulatory treatments (PIC 10 µg/mL, IL-1β 10 ng/mL, TNF-α 10 ng/mL, TGF-β2 10 ng/mL, and ATP 1 mM) for 24 h. The percentage of LDH released in the culture supernatant was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay. Results are from 4 independent experiments. * p-values ≤ 0.05 represent significant differences from controls (CT) by one-way ANOVA followed by the Bonferroni's test analysis.

SC Induce a Robust Chemokine Response Following ONNV Infection and after IL-1β or TNF-α Stimulation
Chemokines are important mediators of inflammation known to be involved in chemotaxis and the activation of phagocytes and lymphocytes [30]. Inflammatory chemokines and infiltration of activated monocytes and lymphocytes are described as key events in the pathogenesis of GBS [17,31]. In particular, CCL2, CXCL8, and CCL5 chemokines are known to be principally involved in the recruitment of macrophages, neutrophils, and T cells, respectively.
We next evaluated the stimulatory effects of the pro-inflammatory cytokines IL-1β and TNF-α and the anti-inflammatory growth factor TGF-β2 and ATP (DAMP) on CCL2, CXCL8, and CCL5 expressions (Figure 6a-c). All experiments were done in triplicates. Results are expressed as mean ± SEM and presented as a normalized fold increase vs. control (CT). * p-values ≤ 0.05 and ** p-values ≤ 0.01 represent significant differences from controls by one-way ANOVA followed by the Bonferroni's test analysis.
When exposed to IL-1β and TNF-α, SC significantly increased the expression of all tested chemokines at 24 h. CCL2 mRNA levels were increased by about 13-fold post-IL-1β stimulation (p < 0.01) and 21-fold post-TNF-α stimulation (p < 0.01). Up to a 133-fold increase was detected on CXCL8 mRNA levels after IL-1β stimulation (p < 0.01) and up to a 95-fold increase in response to TNF-α stimulation (p < 0.01). CCL5 mRNA levels were increased by about 260-fold post-IL-1β stimulation (p < 0.05) and 100-fold post-TNF-α stimulation (p < 0.05). However, TGF-β2 and ATP failed to modulate CCL2, CXCL8, and CCL5 expressions in SC at 6 h and 24 h. All experiments were done in triplicates. Results are expressed as mean ± SEM and presented as a normalized fold increase vs. control (CT). * p-values ≤ 0.05 and ** p-values ≤ 0.01 represent significant differences from controls by one-way ANOVA followed by the Bonferroni's test analysis.

Regulated Expression of Prostaglandin E2-Metabolic Pathway in Cytokine-Stimulated and ONNV-Infected SC
We assessed the capacity of SC to modulate the expression of several enzymes of the PGE2 biosynthetic pathway, including cPLA2α, mPGES-1, COX-2, and 15-PGDH. Cellular responses were evaluated after ONNV infection and PIC, IL-1β, TNF-α, TGF-β2, and ATP exposure.
To support qRT-PCR data, we performed immunofluorescence to detect and evaluate the expression level of COX-2 in ONNV-infected SC (Figure 8e). ONNV-infected cells visually led to enhanced COX-2 detection in neighboring cells. ONNV infection also increased mPGES-1 detection in SC compared to mock infected cells ( Figure S4).
To support qRT-PCR data, we performed immunofluorescence to detect MPZ expression in ONNV-infected SC (Figure 9e). By immunofluorescence assay, we showed a slightly more intense fluorescence for the structural protein MPZ in ONNV-infected SC compared to mock infected cells. Collectively and counterintuitively, we can conclude that ONNV infection upregulated pro-myelinating MPZ, MBP1, and Egr2 expression in SC.

Regulated Expression of Complement Regulatory Proteins CD55 and CD59 in ONNV-Infected SC
The complement system is an essential component of the innate immune response. It is involved in pathogen recognition and immunoregulation [22,23]. Aberrant complement cascade activation on "self" membranes can be prevented by several regulators such as: decay-accelerating factor (CD55), which inhibits C3 and C5 convertase formation, and CD59, which prevents terminal cytolytic membrane attack complex formation. Some viruses were reported to avoid complement destruction by modulating the expression of complement regulatory proteins [33].

SC Modulate the Expression of the Proapoptotic Ligand TRAIL and the Apoptotic Regulator Bcl 2 in Response to ONNV Infection
Apoptosis is a tightly regulated form of controlled cellular self-destruction. The induction of apoptosis or programmed cell death in virus-infected cells is an important antiviral defense mechanism [34]. We assessed the expression of the proapoptotic factor TNF-related, apoptosis-inducing ligand (TRAIL) and the anti-apoptotic protein B-cell lymphoma 2 (Bcl 2) in ONNV-infected and PIC-stimulated SC (Figure 11a,b). Our results showed that PIC strongly upregulated TRAIL mRNA levels up to 75-fold at 6 h (1.77 × 10 −2 ± 1.17 × 10 −2 , p < 0.0001) compared to control cells (2.36 × 10 −4 ± 1.22 × 10 −4 ). However, Bcl 2 gene expression was not modulated by PIC at 6 h and 24 h.

Discussion
Alphavirus infection has been reported to trigger the development of GBS in some patients through an ill-characterized mechanism. Acute and rapidly progressive flaccid peripheral paralysis may be driven through a localized inflammatory response orchestrated by recruited immune cells and/or compromised SC [35]. However, the direct or indirect mechanisms involved in peripheral neuropathology remain largely unknown. Indeed, alphavirus may induce GBS by damaging SC either directly or through the robust innate and adaptive immune inflammatory responses against the virus. We used the alphavirus ONNV to better investigate SC innate immune antiviral and inflammatory responses potentially protecting the nerve or contributing further to the injury. We demonstrated for the first time that SC express the recently discovered alphavirus receptor, MxRA8 [29], whose expression was significantly increased in response to ONNV infection. It has been shown that the deletion of MxRA8 gene or blocking of the surface protein in human and murine cells reduced alphaviral infection. MxRA8 was found to bind directly to CHIKV E2 protein and enhance virus attachment and internalization into the cells [29,36]. MxRA8, initially known as limitrin (also called DICAM or ASP3), is an adhesion molecule expressed on epithelial, myeloid, and mesenchymal cells [37][38][39]. The role of MxRA8 in the peripheral nerve is yet to be identified. In the CNS, MxRA8 was found to be highly expressed in astrocyte endfeet (glia limitans) where it was suggested to regulate the maturation and efficacy of the blood brain barrier [39]. Of note, astrocytes are highly susceptible to CHIKV infection and link to nervous tissue invasion [40]. Whether MxRA8 has a role at the level of the blood peripheral nerve barrier is unknown. Further studies using specific MXRA8 antagonists will need to address the role of the alphavirus receptor MxRA8 in downstream signaling events in Schwann cells. Studying these different post infectious signaling cascades also opens the way to new therapies, targeting in particular MxRA8 receptor of intracellular pathways.
We showed that primary SC are susceptible to ONNV infection associated with elevated expression of E2 and NSP2 viral genes between 6 h and 24 h post infection. CHIKV was reported to be able to infect a wide variety of neuroglial cell types including neurons, astrocytes, and oligodendrocytes in the CNS, but the infection of SC remained to be tested [40,41]. Murine in vivo infection studies showed that CHIKV can efficiently replicate in the brain of newborn mice, reaching high viral titers of 10 6 -10 7 PFU/mL [40,42]. Here, we demonstrated for the first time the ability of alphaviruses (ONNV but also CHIKV, data not shown) to infect the PNS-related glia cells, the SC.
Following ONNV infection, SC were able to initiate a robust antiviral response by upregulating the mRNA expression of the two major pattern recognition receptors RIG-I and MDA5 but also mRNA levels of antiviral IFN-β and the downstream IFN-stimulated antiviral gene ISG15. SC are known as immune competent cells and can express a broad range of pattern recognition receptors that allows them to recognize exogenous as well as endogenous danger signals [18]. In a previous in vitro study, SC were found to be able to initiate a robust antiviral response to infection with flaviviruses Zika virus (ZIKV) and yellow fever virus (YFV) [43].
In addition to their ability to activate an antiviral response, we showed that SC upregulated chemokines production including CXCL8, CCL5, and CCL2 under proinflammatory conditions. Cytokines and chemokines have been described to play a pivotal role in the pathogenesis of GBS. Chemokines are involved in chemotaxis and activation of phagocytes and lymphocytes. Macrophage and T cell infiltration into peripheral nerves and spinal nerve roots has been associated with GBS [19]. Several chemokines, such as CCL2 and CCL5, were found to be upregulated in experimental model of GBS [44]. CCL2 chemokine allows the recruitment of macrophages that can actively participate to the destruction of myelin [45]. Using an in vivo model of GBS, Orlikowski and colleagues observed a significant infiltration of macrophages in peripheral nerves of affected mice. Similarly, they detected a strong macrophages infiltration in nerve biopsies of GBS patients with elevated CCL2 concentrations in the serum of patients [30]. Elevated levels of both CCL2 and CCL5 were also detected in a mice experimental model of GBS at expected peak clinical severity [46]. Using immunohistochemical techniques, Xia et al. demonstrated that CCL2 primarily colocalized to SC and CCL5 to axons and suggested that CCL2 may be required for macrophage-dependent demyelination or SC injury, whereas CCL5 may be required for axonal degeneration [46]. We showed that ONNV infection increased CCL5 but not CCL2 and CXCL8 production, suggesting that ONNV-induced inflammatory response in SC can potentially lead to a T-dependent rather than a macrophage-or neutrophil-dependent response.
Interestingly, we also demonstrated that ONNV-infected SC showed significant activation of the prostaglandin pathway by upregulating the expression of cPLA2α, mPGES-1, and COX-2 mRNA. PGE2 is known as an important lipid mediator that is tied closely with inflammatory processes [20]. COX-2, a key enzyme of the prostaglandin biosynthetic pathway, was found to be highly expressed in SC and activated macrophages in a nerve injury in vivo model [47]. Interestingly, increased COX-2 protein levels were detected in serum and cerebrospinal fluid of GBS patients. Furthermore, increased levels of PGE2 were measured in sera from patients with GBS [21]. Targeting inflammatory pathways with anti-inflammatory drugs would therefore be beneficial for both the acute (GBS) and chronic (arthritis) phases of alphavirus infections [48].
Another essential component of the innate immune response is the complement system. The complement system is involved in the host defense against pathogens and the removal of immune complexes or apoptotic cells [22]. The complement system has been demonstrated to have both an antiviral and pathogenic role during alphavirus infection. For instance, the complement system has been shown to limit viremia and reduce the brain amount of Sindbis virus in infected mice while shortening mouse survival time after infection [49]. Some viruses were reported to avoid complement destruction by modulating the expression of complement regulatory proteins [33]. Hepatitis C virus was shown to upregulate the expression of CD55 onto the hepatocyte [50]. Complement regulatory proteins CD55 and CD59 were found to be expressed in SC and oligodendrocytes [51]. CD59 was detected on SC membranes, satellite cells in spinal ganglia, and endothelial cells in GBS and control patients' tissues [52]. CD59 expression was found to be upregulated at sites of myelin disruption on the human material, supporting a protective role of CD59 in inflammatory diseases of the PNS [52]. We showed that ONNV reduced CD55 and CD59 expression in infected SC. Decreased CD55 and CD59 expression in ONNV-infected SC may potentially contribute to bystander complement activation, myelin opsonization C3b opsonins, and phagocytosis by macrophages through complement surface receptor CR3 and possibly contribute to SC complement-mediated lysis.
Apoptosis or programmed cell death is a tightly regulated form of controlled cellular self-destruction. The induction of apoptosis in virus-infected cells is an important antiviral defense mechanism [34]. Some viruses have therefore evolved strategies to modulate host cell apoptosis in order to enhance their survival and dissemination [53]. CHIKV was shown to mobilize the apoptotic machinery of cultured fibroblasts to invade cell defenses [54]. We showed that ONNV strongly upregulated the proapoptotic factor TRAIL mRNA levels but also the anti-apoptotic Bcl 2 gene expression in infected SC. The capacity of ONNV to upregulate Bcl 2 gene expression may possibly prevent TRAIL-induced apoptosis in SC. Further studies are needed to investigate the mechanisms of apoptosis regulation in alphavirus-infected SC and their potential involvement in the pathogenesis of alphavirusassociated GBS.
Myelination is a process by which SC in the PNS produce the myelin sheath around the axonal membrane [12]. In peripheral nerve injury, SC play a major role in several aspects of nerve repair such as degeneration and remyelination [55]. SC originate from the embryonic neural crest. After birth, the SC retain their inherent plasticity which allows them to differentiate into non-myelinating and myelinating cells [56]. SC differentiation into mature myelinating cells is critical for remyelination and is under the control of several transcription factors including Egr2 [57]. Pathogen infection will potentially disturb these pathways, pre-venting SC from differentiating into cells able to support remyelination [14]. Mycobacterium leprae (M. leprae), a species of bacterium responsible for leprosy neuropathy in humans, can lead to PNS pathologies characterized by a process of nerve demyelination [58]. M. leprae was shown to infect and persist in SC and subsequent infection promoted dedifferentiation of host SC into progenitor stem-like cells with a marked downregulation of myelin genes (MPZ, MBP, and Erg2) and a reduced remyelination capacity in infected SC [58,59]. Myelin breakdown was shown to favor M. leprae viability and persistence inside SC [59].
Acute inflammatory demyelinating neuropathy may occur with CHIKV [60]. CHIKV can induce demyelination either directly by altering SC regenerative properties or through the immune inflammatory response to the virus and immune cells infiltration. In contrast to the capacity of M. leprae to downregulate myelin genes, we found that ONNV infection upregulated the expression of the pro-myelinating genes MPZ, MBP1, and the major pro-myelin transcription factor Egr2 in SC.
In conclusion, our study demonstrated that SC, major PNS glial cells, are susceptible to alphavirus infection and that they are yet able to initiate robust antiviral and inflammatory responses following infection (summary Figure 12). This virus-mediated inflammation, with notably an important production of chemokines such as CCL5 by infected SC, can be potentially involved in the pathogenesis of PNS neurological complications, such as GBS. Our study provides new insights regarding the susceptibility and the remarkable immunomodulatory responses of SC to alphavirus infection of potential importance in the pathogenesis of GBS following alphavirus infection. . Canonically, it is well established that a pathogen can infiltrate a damaged blood peripheral nerve barrier and to cause bystander (indirect) injury to Schwann cells, demyelination, and axonal injury. Host cell debris (damage-associated molecular patterns DAMPs/alarmins such as ATP, HMGB1) may contribute further to the local inflammatory process. Indeed, the recruitment by chemokines (CCL2, CCL5, CXCL8) of innate (neutrophils, monocytes, and differentiating into macrophages) and adaptive (T lymphocytes) immune cells to the nerve will lead to the release of cytotoxic proinflammatory cytokines (IL1-β, TNF-α), outweighing those with anti-inflammatory activities (IL10, TGF-β). Other components such as prostaglandins (PGE2), complement, will stimulate altogether macrophages to phagocytose complement-or antibody-opsonized cell debris, including degraded myelin and axons. Critically, our data highlight herein, for the first time, a pos- . Canonically, it is well established that a pathogen can infiltrate a damaged blood peripheral nerve barrier and to cause bystander (indirect) injury to Schwann cells, demyelination, a-nd axonal injury. Host cell debris (damage-associated molecular patterns DAMPs/alarmins such as ATP, HMGB1) may contribute further to the local inflammatory process. Indeed, the recruitment by chemokines (CCL2, CCL5, CXCL8) of innate (neutrophils, monocytes, and differentiating into macrophages) and adaptive (T lymphocytes) immune cells to the nerve will lead to the release of cytotoxic proinflammatory cytokines (IL1-β, TNF-α), outweighing those with anti-inflammatory activities (IL10, TGF-β). Other components such as prostaglandins (PGE2), complement, will stimulate altogether macrophages to phagocytose complement-or antibody-opsonized cell debris, including degraded myelin and axons. Critically, our data highlight herein, for the first time, a possible more direct mechanism of nerve injury after alphavirus infection. First, we describe that, in fact, SC expressing the MxRA8 alphavirus receptor 1 can be infected directly by the prototype ONNV and yet capable of mounting a robust anti-viral response (IFN type I and ISG, e.g., ISG15) and as verified using the PIC viral analogue molecule 2 . ONNV-infected SC were also mobilized in the robust production of all major inflammatory molecules listed above. We further observed that the infection led to upregulated expression of three key enzymes involved in the biosynthesis of PGE2 (cPLA2α, mPGES-1, and COX-2). The pro-inflammatory effects of ONNV infection (as tested at different MOI) were comparable (or even stronger) to that observed when SC were stimulated with recombinant cytokines or ATP 3 to 6 . Interestingly, we also observed that ONNV-infected SC were able to upregulate several key factors associated with immunoregulation and tissue homeostasis. In response to infection, SC upregulated the expression of molecules involved in the control of apoptosis (e.g., Bcl 2) and, unexpectedly, the expression of myelin (transcription factor Egr2, Myelin P zero (MPZ), and MBP). Collectively, our original data suggest that GBS could potentially be mediated by a direct viral effect on SC. Alphaviruses can replicate within SC and induce a robust immunomodulatory response with potential importance in the GBS-like neuropathology. Figure created with Biorender.com (accessed on 5 May 2022).

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/cells12010100/s1, Figure S1: The infection and expression of high levels of capsid viral protein following CHIKV infection and comparing Schwann cells together with fibroblast-like synoviocytes; Figure S2: ONNV induces SC cytotoxicity at 48 h; Figure S3: ONNV does not increase CCL2 and CXCL8 proinflammatory chemokine production at 30 h post-infection; Figure S4: ONNV infection enhances the PGE2-synthesizing enzyme PGES detection in SC; Figure S5: SC modulate prostaglandin E2-biosynthesis pathway in response to the proinflammatory cytokines IL-1β and TNF-α stimulations.