DISCUSSION
Using in vivo and ex vivo methods, coupled with genetic
and pharmacological strategies, this work highlights the involvement of
Cav3.2 T-type channels in the development and
maintenance of edema and inflammatory hypersensitivity, with a
requirement of Cav3.2 channels being required in T
CD4+ cells, macrophages and the primary afferent
fibers C-LTMRs. From a clinical point of view, these results suggest
that T-type calcium channels could be an interesting target for the
treatment of inflammatory pain.
We first demonstrated that inhibition of T-type calcium channel by
TTA-A2, ABT-639 and Cav3.2 channels deletion
(Cav3.2 KO mice), reduced mechanical allodynia and
hyperalgesia in the carrageenan and CFA models. These results are
consistent with those of previous studies using the same models
(François et al., 2015; Kerckhove et al., 2019) and in other
inflammatory contexts (Choi et al., 2007; Kerckhove et al., 2014).
Interestingly, repeated administration of TTA-A2 accounts for a
maintained antihyperalgesic/anti-allodynic action. In parallel, we
reported for the first time the involvement of Cav3.2
channels in the inflammation process. We observed an anti-inflammatory
effect after genetic and pharmacological inhibition of
Cav3.2 channels, suggesting a pathophysiological role of
these channels not only in pain but also in the inflammatory process.
These results are consistent with others showing a contribution of
Cav3.2 to the development of bladder inflammation in
cyclophosphamide-induced cystitis (Matsunami et al., 2012). In the CFA
model, acute administration of TTA-A2 failed to reduce edema size,
suggesting that the dose or duration of treatment was not sufficient in
a setting of chronic inflammation. In contrast, their repeated
administration significantly reduced edema volume throughout the
experiment. The genetic and pharmacological inhibition of
Cav3.2 channels systematically induced an almost
complete reduction of allodynia/hyperalgesia but only partially
inhibited edema volume. These results confirm the well documented
dissociation between pain and edema (Lee and Jeong, 2002) and suggest a
greater involvement of Cav3.2 channels in the pain
process than in inflammation which is regulated by numerous various
mechanisms.
Given the marked effect of the inhibition of Cav3.2
channels on inflammatory pain, determination of their functional
location to modulate this pathophysiological process is of prime
interest. C-LTMRs are specialized subpopulations of cutaneous afferents
that modulate mechanical and chemical pain hypersensitivity and consist
of two third Cav3.2 channels containing primary afferent
neurons lumbar DRG in mice (François et al., 2015). We used
Cav3.2Nav1.8 cKO mice to delete
Cav3.2 channels solely in C-LTMRs. Strikingly, this
specific tool uncovers that pain-like symptoms were absent in
Cav3.2Nav1.8 cKO mice submitted to the
two inflammatory model studied, corroborating previous finding on
formalin pain (François et al., 2015). The same observation was also
reported in neuropathic (François et al., 2015) and visceral (Picard et
al., 2019) pain murine models further supporting that
Cav3.2 channels in C-LTMR are essential to build up
nociceptive symptoms. In the present study, the reduction of pain-like
inflammatory symptoms after treatment with systemic, intrathecal or
intraplantar administration of ABT-639 indicated that
Cav3.2 expressed at multiple subcellular levels from the
peripheral to the central terminal are involved in the antihyperalgesic
action induced by Cav3.2 inhibition. The study of Jarviset al . (2014) also evidenced the analgesic effect of ABT-639 in
different murine models of neuropathic pain. However, a clinical trial
with ABT-639 in patients with diabetic neuropathy failed to demonstrate
any efficacy of the antagonist, whose tolerability was nevertheless
good; the low doses used were considered as a possible explanation of
this negative result (Ziegler et al., 2015). Clinical evaluations of
local intradermal injection of TTA-A2 in a muscle pain model in healthy
volunteers and patients (three patients with chronic pain) showed a
decrease in mechanical and cold allodynia with no adverse effect (Samour
et al., 2015).
In some conditions, inflammation can be controlled by neuronal mediators
in a process called neurogenic inflammation (Xanthos and Sandkühler,
2014). To determine the involvement of Cav3.2 channels
in this process, two strategies were used. With regard to neuronal
Cav3.2 channels, we observed no change in edema size in
Cav3.2Nav1.8 cKO mice, which strongly
suggests that Cav3.2 channels expressed on C-LTMRs are
not involved in subacute or maintained inflammation. Experiments using
chimeric mice enabled us to demonstrate for the first time that the
absence of Cav3.2 channels in hematopoietic cells was
sufficient to reduce edema development and pro-inflammatory mediator
release. Interestingly, this reduction was close to that observed in
constitutive Cav3.2 KO mice, suggesting that
Cav3.2 channels expressed by hematopoietic cells were
involved in the control of inflammation. Accordingly, transplantation of
hematopoietic cells expressing Cav3.2 channels into
constitutive Cav3.2 KO animals completely restored edema
development and pro-inflammatory mediator release. Thus, these in
vivo experiments provide evidence that inflammation induced by
carrageenan and CFA involved Cav3.2 channels expressed
in hematopoietic cells. We then explored more specifically the potential
contribution of BMDM and CD4+ T cells, two actors that
play a crucial role in the inflammatory diseases (Weyand and Goronzy,
2021). Using immunocytochemistry, we demonstrated, for the first time,
that these cells expressed Cav3.2 channel protein
consistent with a previous demonstration of low level of
Cav3.2 transcript expression in murine
CD4+ T cells (Jarvis et al., 2014). Genetic deletion
of Cav3.2 channels in BMDM showed no morphological signs
of activation and lower production of pro-inflammatory mediators (IL-6
and TNF-α) in response to LPS stimulation. This result accounts for the
fact that Cav3.2 channels are involved in exocytose not
only in excitable cells but also in non-excitable cell (Carbone et al.,
2006). This could be related to a reduced calcium recruitment in
LPS-stimulated BMDM after Cav3.2 deletion, which lead to
a reduced level of pro-inflammatory mediators. A relationship between
the calcium signaling pathway and the production of pro-inflammatory
mediators has already been established in LPS-stimulated rat peritoneal
macrophages (Zhou et al., 2006). Cav3.2 channel deletion
was also associated with a lower activation status in spleen APC
suggesting an impaired ability of these cells to promote T cell
activation. Moreover, Cav3.2 gene invalidation strongly
affected T-cell proliferation upon CD3/CD28 stimulation, suggesting the
involvement of Cav3.2 channels in early events of
lymphocyte activation. In conclusion, Cav3.2 gene
deletion strongly impairs the function of APC and CD4+T cells, a process that could explain the involvement of
Cav3.2 in edema and inflammatory process observed in our
models.
Our study has certain limitations. First, although the spinal and
peripheral location of Cav3.2 channels was shown to be
involved in inflammatory pain, a supraspinal contribution cannot be
ruled out. Indeed, Cav3.2 channels are expressed in the
brain (Bernal Sierra et al., 2017) and intracerebroventricular injection
of TTA-A2 induced antinociception in the formalin test in mice
(Kerckhove et al., 2014). However, the fact that ablation of
Cav3.2 channels in C-LTMRs totally suppressed pain-like
symptoms in our inflammatory models detracts from this hypothesis. In
addition, we cannot definitively conclude that the reduced activation
and proliferation of macrophages and CD4+ T cells that
were proposed to explain the anti-edematous effect of
Cav3.2 inhibition are the only processes involved. It is
possible that a default in the fetal development of the hematopoietic
lineage in Cav3.2 KO mice or in the recruitment or
infiltration process in WT mice also contributes to the observed
phenotype. However, after analysis, we observed no significant
alteration in the number of the major populations of immune cells in
Cav3.2 KO mice. Furthermore, other subpopulations of
cells involved in inflammation such as monocytes and osteoblasts could
account for the action of Cav3.2 channels inhibition.
Elucidation of the mechanistic basis of these different questions will
require additional experimental approaches, possibly including
tissue‐specific manipulation of the expression of Cav3.2
channels.
In a clinical perspective, together with reports from the literature,
our study supports the need of a more thorough clinical evaluation of
T-type channels blockers in the treatment of chronic pain, in order to
draw firm conclusions on their potential efficacy, especially in
patients with inflammatory pain. The findings of our study would permit
a rapid translation in patients by performing a clinical study in
patients suffering from inflammatory pain.