Involvement of immune cells in the anti-inflammatory action of Cav3.2 channels inhibition
We then studied the mechanisms underlying the involvement of Cav3.2 channels in edema development. One possible explanation for the decreased edema observed in mice after Cav3.2 channel inhibition was a reduction of the neurogenic inflammation process (Xanthos and Sandkühler, 2014). Indeed, inhibition of Cav3.2 located in C-LTMRs could reduce the release of peripheral pro‐inflammatory neuropeptides in response to antidromic nerve stimulation. To test this hypothesis, we assessed edema development in Cav3.2Nav1.8 cKO mice. No significant difference was observed in edema volume between Cav3.2Nav1.8cKO mice and their control Cav3.2GFP-flox KI littermates after carrageenan injection (Figure 5A ) thereby excluding the hypothesis of the neuronal nature of the anti-edematous effect of Cav3.2 channel inhibition at least originating from C-LTMR afferents. The other explanation was based on the involvement of Cav3.2 channels expressed in immune cells in edema formation. To test this, we designed chimeric mice with genetic deletion of Cav3.2 only in hematopoietic cells (progenitor of immune cells) or only in non-hematopoietic cells (Figure 5B ). The absence of Cav3.2 channels specifically in the immune system (WT recipient mice transplanted with KO immune cells) reduced edema development, as in constitutive Cav3.2 KO mice (KO recipient mice transplanted with KO immune cells) (Figure 5C ). In contrast, edemas were restored in Cav3.2 KO recipient mice receiving WT hematopoietic cells (Figure 5C ). Consistently, the absence of Cav3.2 channels only in hematopoietic cells significantly reduced the production of IL-6, as in constitutive Cav3.2 KO mice (Figure 5D ). Our results showed that Cav3.2 channels expressed in immune cells are strongly involved in edema development and IL-6 production.
To identify the nature of the immune cells involved in Cav3.2-dependent edema development, Cav3.2 channel expression was investigated in macrophages and T cells. We used Cav3.2GFP-flox KI mice to detect Cav3.2 channel protein. Thus, bone marrow derived macrophages (BMDM) and CD4 positive T cells (CD4+ T cells) were obtained from the femurs and spleen, respectively, of Cav3.2GFP-flox KI mice or control WT mice and were labelled with an anti-eGFP antibody. Immunocytochemistry revealed the expression of Cav3.2 channels in both BMDM and lymphocyte CD4+ T cells (Figure 5E ).