Cycloadduits 1,3-N,X précurseurs de vecteurs de principe actif (mécanisme de cyclisation, effet anomère, élaboration de synthons polymérisables)

MONTPELLIER-BU Sciences (341722106) / SudocSudocFranceF

Immunomodulatory drug methotrexate used to treat patients with chronic inflammatory rheumatisms post-chikungunya does not impair the synovial antiviral and bone repair responses

International audienceChikungunya virus (CHIKV) is a mosquito-transmitted RNA alphavirus causing major outbreaks of infectious chronic inflammatory rheumatisms (CIR). Recently, methotrexate (MTX), a disease modifying anti-rheumatic drug has been used successfully to treat patients suffering from rheumatoid-like arthritis post-CHIK but its immunomodulatory activity in the context of viral persistence has been a matter of concerns. We herein used a model of primary human synovial fibroblasts (HSF) and the synthetic molecule polyriboinosinic:polyribocytidylic acid (PIC) to mimic chronic infectious settings in the joints of CHIKV infected patients. The innate antiviral immune and inflammatory responses were investigated in response to MTX used at the therapeutic concentration of 1 μM. We found that MTX did not affect cellular viability as indicated by the LDH release assay. By quantitative RT-PCR, we observed that HSF responded robustly to PIC by increasing ISG15 and IFNβ mRNA levels. Furthermore, PIC upregulated the mRNA expression of two of the major pattern recognition receptors, RIG-I and MDA5 involved in the innate immune detection of viral RNA. MTX did not impact the antiviral response of PIC on ISG15, IFNβ, RIG-I and MDA5 mRNA expressions. MTX alone or combined with PIC did not affect the expression of proinflammatory CCL2 and CXCL8 chemokines. PIC strongly upregulated the mRNA and protein expression of osteoclastogenic factors (IL-6, GM-CSF but not RANKL). Critically, MTX treatment alone or combined with PIC did not affect the expression of all three tested osteoclastogenic cytokines. We found that MTX alone did not increase the capacity of CHIKV to infect and replicate in HSF. In conclusion, our study argues for a beneficial effect of MTX to treat CIR post-CHIKV given that it does not critically impact the antiviral, the proinflammatory and the bone tissue remodeling responses of synovial cells

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

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

Robust COX-2-mediated prostaglandin response may drive arthralgia and bone destruction in patients with chronic inflammation post-chikungunya

International audiencePatients following infection by chikungunya virus (CHIKV) can suffer for months to years from arthralgia and arthritis. Interestingly, methotrexate (MTX) a major immune-regulatory drug has proved to be of clinical benefit. We have previously shown that CHIKV can persist in the joint of one patient 18 months post-infection and plausibly driving chronic joint inflammation but through ill-characterized mechanisms. We have pursued our investigations and report novel histological and in vitro data arguing for a plausible role of a COX-2-mediated inflammatory response post-CHIKV. In the joint, we found a robust COX-2 staining on endothelial cells, synovial fibroblasts and more prominently on multinucleated giant cells identified as CD11c+ osteoclasts known to be involved in bone destruction. The joint tissue was also strongly stained for CD3, CD8, CD45, CD14, CD68, CD31, CD34, MMP2, and VEGF (but not for NO synthase and two B cell markers). Dendritic cells were rarely detected. Primary human synovial fibroblasts were infected with CHIKV or stimulated either by the synthetic molecule polyriboinosinic:polyribocytidylic acid (PIC) to mimic chronic viral infection or cytokines. First, we found that PIC and CHIKV enhanced mRNA expression of COX-2. We further found that PIC but not CHIKV increased the mRNA levels of cPLA2α and of mPGES-1, two other central enzymes in PGE2 production. IFNβ upregulated cPLA2α and COX-2 transcription levels but failed to modulated mPGES-1 mRNA expression. Moreover, PIC, CHIKV and IFNβ decreased mRNA expression of the PGE2 degrading enzyme 15-PGDH. Interestingly, MTX failed to control the expression of all these enzymes. In sharp contrast, dexamethasone was able to control the capacity of pro-inflammatory cytokines, IL-1β as well as TNFα, to stimulate mRNA levels of cPLA2α, COX-2 and mPGES-1. These original data argue for a concerted action of CHIKV (including viral RNA) and cytokines plausibly released from recruited leukocytes to drive a major COX-2-mediated PGE2 proinflammatory responses to induce viral arthritis

Immunomodulatory drug methotrexate used to treat patients with chronic inflammatory rheumatisms post-chikungunya does not impair the synovial antiviral and bone repair responses

<div><p>Chikungunya virus (CHIKV) is a mosquito-transmitted RNA alphavirus causing major outbreaks of infectious chronic inflammatory rheumatisms (CIR). Recently, methotrexate (MTX), a disease modifying anti-rheumatic drug has been used successfully to treat patients suffering from rheumatoid-like arthritis post-CHIK but its immunomodulatory activity in the context of viral persistence has been a matter of concerns. We herein used a model of primary human synovial fibroblasts (HSF) and the synthetic molecule polyriboinosinic:polyribocytidylic acid (PIC) to mimic chronic infectious settings in the joints of CHIKV infected patients. The innate antiviral immune and inflammatory responses were investigated in response to MTX used at the therapeutic concentration of 1 μM. We found that MTX did not affect cellular viability as indicated by the LDH release assay. By quantitative RT-PCR, we observed that HSF responded robustly to PIC by increasing ISG15 and IFNβ mRNA levels. Furthermore, PIC upregulated the mRNA expression of two of the major pattern recognition receptors, RIG-I and MDA5 involved in the innate immune detection of viral RNA. MTX did not impact the antiviral response of PIC on ISG15, IFNβ, RIG-I and MDA5 mRNA expressions. MTX alone or combined with PIC did not affect the expression of proinflammatory CCL2 and CXCL8 chemokines. PIC strongly upregulated the mRNA and protein expression of osteoclastogenic factors (IL-6, GM-CSF but not RANKL). Critically, MTX treatment alone or combined with PIC did not affect the expression of all three tested osteoclastogenic cytokines. We found that MTX alone did not increase the capacity of CHIKV to infect and replicate in HSF. In conclusion, our study argues for a beneficial effect of MTX to treat CIR post-CHIKV given that it does not critically impact the antiviral, the proinflammatory and the bone tissue remodeling responses of synovial cells.</p></div

MTX effects on the expression of proinflammatory chemokines in response to PIC stimulation.

<p><b>A)</b> Relative expression of CCL2 and CXCL8 from HSF stimulated by PIC100μg/mL for 6 hours and 24 hours in the absence and presence of MTX 1μM treatment was assessed by qRT-PCR. <b>B)</b> HSF were exposed to PIC 100μg/mL and treated or not with MTX 1μM. Supernatants were harvested after 6 hours and 24 hours and levels of CCL2 and CXCL8 were quantitated by ELISA assay. All experiments were done in quadruplicates and results are expressed as mean ± standard error. *: p-values ≤ 0.05, **: p-values ≤ 0.01, ***: p-values ≤ 0.001 and ****: p-values ≤ 0.0001.</p

MTX treatment does not affect the expression of IFN β and ISG15 antiviral innate immune genes in response to CHIKV infection.

<p>Relative expression IFN β (A) and ISG15 (B) mRNA from HSF infected with CHIKV at MOI1 for 24 hours after exposure or not to MTX 1μM as assessed by qRT-PCR. Experiments were done in triplicates and results are expressed as mean ± standard error. *: p-values ≤ 0.05, **: p-values ≤ 0.01, ***: p-values ≤ 0.001 and ****: p-values ≤ 0.0001.</p

MTX treatment has not effect on the expression of IL-6 cytokine in HSF exposed to PIC.

<p>HSF were stimulated by PIC 100μg/mL and treated or not with MTX 1μM. <b>A)</b> IL-6 mRNA levels from HSF stimulated by PIC100μg/mL for 6 hours and 24 hours in the absence and presence of MTX 1μM treatment were evaluated by qRT-PCR. <b>B)</b> Supernatants were harvested after 6 hours and 24 hours and levels of IL-6 were measured by ELISA assay. All experiments were done in quadruplicates and results are expressed as mean ± standard error. *: p-values ≤ 0.05, **: p-values ≤ 0.01, ***: p-values ≤ 0.001 and ****: p-values ≤ 0.0001.</p

MTX treatment does not affect CHIKV replication in HSF.

<p>Relative expression of viral NSP1 (A) and E2 (B) mRNA from HSF infected with different MOIs of CHIKV for 24 hours after exposure or not to MTX 1μM treatment (qRT-PCR data). Experiments were done in triplicates and results are expressed as mean ± standard error. *: p-values ≤ 0.05, **: p-values ≤ 0.01, ***: p-values ≤ 0.001 and ****: p-values ≤ 0.0001.</p