TRPA1 activation and Hsp90 inhibition synergistically downregulate macrophage activation and inflammatory responses in vitro.

BACKGROUND: Transient receptor potential ankyrin 1 (TRPA1) channels are known to be actively involved in various pathophysiological conditions, including neuronal inflammation, neuropathic pain, and various immunological responses. Heat shock protein 90 (Hsp90), a cytoplasmic molecular chaperone, is well-reported for various cellular and physiological processes. Hsp90 inhibition by various molecules has garnered importance for its therapeutic significance in the downregulation of inflammation and are proposed as anti-cancer drugs. However, the possible role of TRPA1 in the Hsp90-associated modulation of immune responses remains scanty. RESULTS: Here, we have investigated the role of TRPA1 in regulating the anti-inflammatory effect of Hsp90 inhibition via 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) in lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA) stimulation in RAW 264.7, a mouse macrophage cell lines and PMA differentiated THP-1, a human monocytic cell line similar to macrophages. Activation of TRPA1 with Allyl isothiocyanate (AITC) is observed to execute an anti-inflammatory role via augmenting Hsp90 inhibition-mediated anti-inflammatory responses towards LPS or PMA stimulation in macrophages, whereas inhibition of TRPA1 by 1,2,3,6-Tetrahydro-1,3-dimethyl-N-[4-(1-methylethyl)phenyl]-2,6-dioxo-7 H-purine-7-acetamide,2-(1,3-Dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7 H-purin-7-yl)-N-(4-isopropylphenyl)acetamide (HC-030031) downregulates these developments. LPS or PMA-induced macrophage activation was found to be regulated by TRPA1. The same was confirmed by studying the levels of activation markers (major histocompatibility complex II (MHCII), cluster of differentiation (CD) 80 (CD80), and CD86, pro-inflammatory cytokines (tumor necrosis factor (TNF) and interleukin 6 (IL-6)), NO (nitric oxide) production, differential expression of mitogen-activated protein kinase (MAPK) signaling pathways (p-p38 MAPK, phospho-extracellular signal-regulated kinase 1/2 (p-ERK 1/2), and phosphor-stress-activated protein kinase/c-Jun N-terminal kinase (p-SAPK/JNK)), and induction of apoptosis. Additionally, TRPA1 has been found to be an important contributor to intracellular calcium levels toward Hsp90 inhibition in LPS or PMA-stimulated macrophages. CONCLUSION: This study indicates a significant role of TRPA1 in Hsp90 inhibition-mediated anti-inflammatory developments in LPS or PMA-stimulated macrophages. Activation of TRPA1 and inhibition of Hsp90 has synergistic roles towards regulating inflammatory responses associated with macrophages. The role of TRPA1 in Hsp90 inhibition-mediated modulation of macrophage responses may provide insights towards designing future novel therapeutic approaches to regulate various inflammatory responses.

Elevation of TRPV1 expression on T-cells during experimental immunosuppression.

The transient receptor potential vanilloid 1 (TRPV1) channel is a thermo-sensitive, polymodal cation channel. An increase in intracellular calcium (Ca2+) is essential for T-cell responses. Similarly, various immunosuppressive agents are also reported to induce Ca2+ influx. However, the possible involvement of TRPV1 during immunosuppression has not been studied yet. Here, we investigated the possible functional role of TRPV1 in FK506 or B16F10-culture supernatant (B16F10-CS)-driven experimental immunosuppression in T-cells. Intriguingly, it was found that TRPV1 surface expression was further significantly elevated during immunosuppression compared with concanavalin A (ConA) or TCR-activated T-cells. Moreover, in B16F10 tumor-bearing mice, TRPV1 expression was upregulated on splenic T-cells as compared with T-cells derived from control mice. We also observed an immediate increase in intracellular Ca2+ levels in FK506 (marked increase) and B16F10-CS treatment (modest increase) or in combination with T-cell activation as compared with resting and activated T-cells. Likewise, in B16F10 tumor-bearing mice, the basal intracellular calcium level was upregulated in T-cells as compared with controls. The elevated Ca2+ level(s) were found to be significantly downregulated by 5'-iodoresiniferatoxin (50-IRTX) (a TRPV1-specific inhibitor), suggesting an important role of TRPV1 during immune activation and immunosuppression. The current study may have implications for immunosuppressive diseases along with inflammatory disorders associated with the coordinating role of TRPV1 and Ca2+ influx.

Inhibition of transient receptor potential vanilloid 1 (TRPV1) channel regulates chikungunya virus infection in macrophages.

Chikungunya virus (CHIKV), a virus that induces pathogenic inflammatory host immune responses, is re-emerging worldwide, and there are currently no established antiviral control measures. Transient receptor potential vanilloid 1 (TRPV1), a non-selective Ca2+-permeable ion channel, has been found to regulate various host inflammatory responses including several viral infections. Immune responses to CHIKV infection in host macrophages have been reported recently. However, the possible involvement of TRPV1 during CHIKV infection in host macrophages has not been studied. Here, we investigated the possible role of TRPV1 in CHIKV infection of the macrophage cell line RAW 264.7. It was found that CHIKV infection upregulates TRPV1 expression in macrophages. To confirm this observation, the TRPV1-specific modulators 5'-iodoresiniferatoxin (5'-IRTX, a TRPV1 antagonist) and resiniferatoxin (RTX, a TRPV1 agonist) were used. Our results indicated that TRPV1 inhibition leads to a reduction in CHIKV infection, whereas TRPV1 activation significantly enhances CHIKV infection. Using a plaque assay and a time-of-addition assay, it was observed that functional modulation of TRPV1 affects the early stages of the viral lifecycle in RAW 264.7 cells. Moreover, CHIKV infection was found to induce of pNF-κB (p65) expression and nuclear localization. However, both activation and inhibition of TRPV1 were found to enhance the expression and nuclear localization of pNF-κB (p65) and production of pro-inflammatory TNF and IL-6 during CHIKV infection. In addition, it was demonstrated by Ca2+ imaging that TRPV1 regulates Ca2+ influx during CHIKV infection. Hence, the current findings highlight a potentially important regulatory role of TRPV1 during CHIKV infection in macrophages. This study might also have broad implications in the context of other viral infections as well.