**5. Infection and Immunity**

Pain sensation is a negative stress for animals, and CGRP release from nociceptors exacerbates symptoms. Conversely, we recently clarified that CGRP release dependent on TRPV1 and TRPA1 activity in DRG neurons is involved in bone protection during fungus infection. This phenomenon is supported by several physiological mechanisms, including ATP release from keratinocytes, neural excitation of sodium channel 1.8 (Nav1.8)-positive DRG neurons, and CGRP-dependent suppression of osteoclasts activated via TNF-. Thus, in this part, we comprehensively describe the physiological and pathological systems involved in cutaneous infection to bone inflammation (Figure 3).

**Figure 3.** Bone protection system from *Candida* infection. Bone disruption followed by inflammation is worsened by overactive osteoclasts. Nociceptors suppress osteoclast development through calcitonin gene-related peptide (CGRP) release. The process involves: (1) *Candida albicans* is activated in an optimal environment (e.g., body temperature); (2) β-glucan released from pseudohyphae bind to its receptor, dectin-1, on the plasma membrane of keratinocytes; (3) ATP release from keratinocytes is enhanced through the phospholipase C (PLC) pathway; (4) neuronal excitation in voltage-gated sodium channel 1.8 (Nav1.8)-positive dorsal root ganglion (DRG) neurons; and (5) CGRP release from DRG neurons. Jun dimerization protein 2 (Jdp2) is activated by CGRP through a cAMP cascade in myeloid cells. In turn, tumor necrosis factor-alpha (TNF-α release (which accelerates osteoclast development) is suppressed. TNF-α-dependent inflammation is induced by the direct effect of β-glucan on myeloid cells. Furthermore, the CGRP–cAMP axis in osteoclasts also inhibits over-development. Thus, these pathways from skin to bone induce bone protection and inhibit bone inflammation during fungus infection.

## *5.1. Nociception by C. albicans*

*Candida albicans* infections can cause skin or vulvar pain. Breast candidiasis is characterized by severe pain around the nipple [173]. *C. albicans* in the vagina causes itching and mechanical allodynia [174]. *C. albicans* can also enter skeletal tissue and induce painful bone infection [175]. Although *C. albicans* has algesic activity, the mechanisms by which this fungus triggers pain remains completely unknown.

TLR4 expressed on myeloid cells are involved in recognition of fungal mannan and cytokine production upon MyD88 and TIR-domain-containing adapter-inducing interferon-beta (TRIF) activation. The fungal cell wall contains β-glucan and mannan on the intracellular and extracellular face, respectively [176]. Surface exposure of β-glucan is sensed by dectin-1 [177]. Activated dectin-1 assembles as a multimeric complex and induces signaling via an ITAM-like motif, promoting formation of CARD-9–Bcl-10–Malt-1 trimers (CBM trimer) and activation of the NLRP3–ASC–ICE complex (NLRP3 inflammasome). CBM trimers and NLRP3 inflammasome activation are both required to induce cytokine production [178].

Candidalysin was recently discovered and is the first fungal cytolytic peptide [179]. This peptide may also contribute to the pathogenesis of fungal invasion. Pain induced by fungal infections is thought to be caused by inflammation, but a recent study suggested that both *Staphylococcus aureus*-derived N-formulated peptides and α-hemolysin can directly stimulate nociceptors [180]. Therefore, nociceptors may be able to sense pathogens, but the underlying molecular mechanisms behind fungal nociception remain unclear.

In the colonization phase, *C. albicans* forms yeast-like structures that are harmless because colony growth is suppressed by host immunity and the natural antagonistic effects of microbial flora. When the yeast form of *C. albicans* attaches to the skin of an immunocompromised host, budding growth is immediately induced and the soluble β-glucan form is secreted. Notably, β-glucan-induced allodynia is relatively severe compared with that induced by mannan and other pathological components such as Candidalysin. Furthermore, dectin-1-deficient mice are completely unresponsive to *C. albicans* or β-glucan-induced pain in a MyD88/TRIF/inflammasome-independent manner. Moreover, we discovered that *C. albicans* induces acute pain by stimulating Nav1.8-positive nociceptors in primary sensory neurons via the dectin-1-mediated PLCγ2–TRPV1/TRPA1 axis. β-Glucan also induces allodynia, which is dependent on dectin-1-mediated ATP secretion from keratinocytes. Notably, keratinocyte-derived extracellular ATP stimulates sensory neurons via P2X receptors. We also found that mice deficient in the ATP transporter, vesicular nucleotide transporter (VNUT), are unresponsive to β-glucan-induced allodynia, while the VNUT inhibitor clodronate has potent prophylactic potential to target fungal nociceptive symptoms. Together, these findings suggest that ATP- or VNUT-targeted therapies such as clodronate treatment may be a promising therapeutic option for treating pain or allodynia associated with fungal infections [181].

#### *5.2. Secondary Symptoms Following Nociception*

Nociceptor innervation is seen in skin and bone. Although the function of nociceptors in the osteo–immune system is unclear, ion channels in the DRG may be responsible for sensing noxious stimuli [182]. During inflammation, pro-algesic cytokines derived from immune cells gradually evoke allodynia, leading to production of neuropeptides such as CGRP [183], which in turn causes vasodilatation, impaired insulin release, and enhanced Th17 cell function [184–186]. Meanwhile, depletion of TRPV1-positive neurons or CGRP deficiency can lead to osteoporosis [187,188]. Thus, nociceptors may modulate osteo–immune system activity, but how they influence pathogen-induced inflammation and bone destruction in a physiological context remains unknown.

To investigate these questions, we injected LPS or β-glucan into the hind paw of *Nav1.8CreRosa26DTA* mice, a nociceptor-deficient line. Notably, LPS-induced osteo-inflammation was unaffected, suggesting that nociceptors do not affect TLR-induced osteo-inflammation. In contrast, *Nav1.8CreRosa26DTA* mice injected with β-glucan exhibit severe skin inflammation and bone

destruction, indicating that nociceptors are negative regulators of fungal osteo-inflammation. Similar to *Nav1.8CreRosa26DTA* mice, TRPV1/TRPA1 double-deficient mice exhibit severe osteo-inflammation in response to β-glucan, and this phenotype was rescued by CGRP administration. Notably, β-glucan injection into the hind paws of both *Nav1.8CreRosa26DTA* and TRPV1/TRPA1 double-deficient mice abolished serum CGRP, indicating that TRP channels acting as nociceptors are required for CGRP induction. To address how CGRP inhibits osteo-inflammation, we assessed the effects of CGRP on osteoclast formation and cytokine production. Intriguingly, we discovered that nerve-derived CGRP inhibits osteoclast actin polymerization via cAMP induction, leading to impaired osteoclast multinucleation. We also found that the CGRP-induced transcriptional repressor, Jun dimerization protein 2, selectively blocks dectin-1-mediated pro-inflammatory cytokine production in myeloid cells via direct inhibition of p65. These unexpected roles for β-glucan-stimulated nociceptors suggests the existence of novel sensocrine pathways that may play a role in fungal osteo-inflammation [181].
