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 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

Increased levels of soluble forms of E-selectin and ICAM-1 adhesion molecules during human leptospirosis

<div><p>Leptospirosis is a multisystemic zoonotic disease with infiltration of visceral organs by <i>Leptospira</i>. The capacity of the vascular endothelium to grant immune cell recruitment and activation in target organs during the disease course remains poorly characterized. We ascertained the levels of expression of several soluble cell adhesion molecules (CAM) notably expressed by endothelial cells in human leptospirosis. We prospectively enrolled 20 hospitalized patients and compared them to 10 healthy controls. Disease severity was defined by one or more organ failures, or death. Plasmatic concentrations of soluble CAM were assessed by multiplex bead assay at the time of patient presentation (M0) and 1 month after hospital discharge. The levels of soluble E-selectin (sCD62E) and soluble intercellular adhesion molecule 1 (sICAM-1, sCD53) were significantly increased in patients compared to controls (p<0.0001) and at 1 month (p<0.0001) with median values at 978 ng/ml (interquartile ranges 787–1164; sCD62E) and 1021 ng/ml (690–1428; sCD53). At M0, Soluble P-selectin level (sCD62P) was found to be decreased with levels at 60 ng/ml (0–631) versus 711 ng/ml (343–1113) for healthy controls (p<0.05). Levels of sICAM-3 (sCD50), sVCAM-1 (vascular cell adhesion molecule, sCD106) and sPECAM-1 (platelet endothelial cell adhesion molecule, sCD31) were not different from healthy subjects at M0. This study shows that two adhesion molecules, shed as soluble forms, are elevated during the acute phase of leptospirosis: E-selectin and s-ICAM1. These molecules may interfere with the process of immune cell recruitment to clear <i>Leptospira</i> at tissue levels.</p></div

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

MTX treatment does not affect the expression of GM-CSF in HSF stimulated with PIC.

<p>HSF were exposed to PIC 100μg/mL and treated or not with MTX 1μM. <b>A)</b> GM-CSF mRNA levels from HSF stimulated by PIC100μg/mL in the absence and presence of MTX 1μM treatment were assessed by qRT-PCR. <b>B)</b> Levels of GM-CSF in HSF culture supernatants harvested after 6 hours, 24 hours, 48 hours and 72 hours were measured by ELISA assay. All 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 at 1 μM to 10 μM does not cause HSF cytotoxicity.

<p><b>A)</b> HSFs were treated with MTX at a concentration of 1μM or 10μM for 24 hours. Percentage of LDH released in culture supernatant was measured using the CytoTox 96 Non-Radioactive Cytotoxicity Assay. Results are from 3 independent experiments. <b>B)</b> HSF cell morphology after treatment with MTX 1μM and 10μM for 24 hours. Magnification is x100.</p

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

Characteristics of leptospirosis patient group at admittance and comparison to healthy subjects.

<p>Characteristics of leptospirosis patient group at admittance and comparison to healthy subjects.</p

Immunosuppressive MTX drug at micromolar concentrations does not affect the antiviral, inflammatory and pro-osteoclastogenic functions of HSF.

<p>Chikungunya virus (CHIKV, ✡) is a single strand RNA virus capable of replicating at high levels in human synovial fibroblasts (HSF) and to contribute to chronic arthritis through ill-characterized mechanisms. CHIKV will be recognized by RIG-I and MDA5 pattern recognition receptors to initiate the canonical interferon-type I dependent antiviral response and to control infection. Poly I:C (PIC) is a viral RNA mimetic. Upon stimulation by CHIKV (or PIC), HSF will express antiviral genes (e.g. ISG15) as well as a myriad of proinflammatory molecules (interleukins/IL1, chemokines/CCL2) and growth factors involved in bone repair (e.g. M-CSF, GM-CSF). CHIKV infection (but not PIC stimulation) also is known to upregulate the expression of RANKL involved in osteoclastogenic activities and bone destruction. We herein have shown that methotrexate (MTX) a well-known therapeutic agent (DHFR inhibitor) against arthritis does not interfere with the antiviral and bone repair mechanisms essential to regulate CHIKV-induced chronic joint diseases. Critically, MTX does not aggravate the level of CHIKV replication in HSF. Macrophages (latently infected by CHIKV) and recruited CD4 T lymphocytes will contribute to chronic arthritis post-CHIKV but a plausible role of MTX on these cells remains to be addressed.</p

List of primers used for qRT-PCR.

<p>List of primers used for qRT-PCR.</p