Takeshi Yoshioka

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fatal lung disorder characterized by aberrant extracellular matrix deposition in the interstitium. Pirfenidone is an antifibrotic agent used to treat patients with IPF. Pirfenidone shows a pleiotropic mode of action, but its underlying antifibrotic mechanism is unclear. Transient receptor potential vanilloid 4 (TRPV4), which is a mechanosensitive calcium channel, was recently shown to be related to pulmonary fibrosis. To clarify the antifibrotic mechanisms of pirfenidone, we investigated whether TRPV4 blockade has a pharmacological effect in a murine model of pulmonary fibrosis and whether pirfenidone contributes to suppression of TRPV4. Our synthetic TRPV4 antagonist and pirfenidone treatment attenuated lung injury in the bleomycin mouse model. TRPV4-mediated increases in intracellular calcium were inhibited by pirfenidone. In addition, TRPV4-stimulated interleukin-8 release from cells was reduced and a delay in cell migration was abolished by pirfenidone. Furthermore, pirfenidone decreased TRPV4 endogenous ligands in bleomycin-administered mouse lungs and their production by microsomes of human lungs. We found TRPV4 expression in the bronchiolar and alveolar epithelium and activated fibroblasts of the lungs in patients with IPF. Finally, we showed that changes in forced vital capacity of patients with IPF treated with pirfenidone were significantly correlated with metabolite levels of TRPV4 endogenous ligands in bronchoalveolar lavage fluid. These results suggest that the antifibrotic action of pirfenidone is partly mediated by TRPV4 and that TRPV4 endogenous ligands in bronchoalveolar lavage fluid may be biomarkers for distinguishing responders to pirfenidone.

Pellagra is caused by an abnormal intake and/or use of niacin, but its phenotypes are diverse. The phenotypes of pellagra can also be atypical, such as nausea. We previously reported a mouse model of pellagra-related nausea. However, the mechanism of this model is unclear. In this study, we found that the gut microbiota, which is thought to be a source of niacin, played an important role in the development of pellagra-related nausea in germ-free mice. We also investigated the gut microbiome. We compared urinary niacin metabolite levels and the dermal response between mice fed a normal diet and those fed a low-niacin diet to investigate the putative trigger of pellagra. Epoxyeicosatrienoic and hydroxyeicosatetraenoic acid levels were higher in mice fed a low-niacin diet compared with those fed a normal diet. Furthermore, histological studies indicated a dermatological response to the low-niacin diet. Interestingly, higher levels of oxidised fatty acids in response to the germ-free state were also observed. These findings indicate successful establishment of our newly established mouse model of pellagra via the gut microbiota. We believe that this model could enable the discovery of the putative cause of pellagra and phenotypes of pellagra that have not been recognised yet.

Niacin deficiency causes pellagra, the symptoms of which include dermatitis, diarrhoea and dementia. Investigating the mechanism underlying these phenotypes has been challenging due to the lack of an appropriate animal model. Here, we report a mouse model of pellagra-related nausea induced by feeding mice a low-niacin diet and administering isoniazid (INH), which is thought to induce pellagra. Mice fed a normal or low-niacin diet received INH (0·3 or 1·0 mg/mg per animal, twice daily, 5 d), and nausea was evaluated based on pica behaviour, which considered the rodent equivalent of the emetic reflex. Furthermore, the effect of therapeutic niacin administration on nausea was evaluated in this model. Urinary and hepatic metabolite levels were analysed by LC coupled with MS. INH-induced pica was observed in mice fed a low-niacin diet but not in those fed a normal diet. Levels of urinary metabolites, such as 1-methyl-2-pyridone-5-carboxamide, kynurenic acid and xanthurenic acid, were significantly reduced in the mice treated with INH compared with those that did not receive INH. Furthermore, niacin supplementation prevented pica and restored the levels of some metabolites in this mouse model. Our findings suggest that INH-related nausea is pellagra-like. We also believe that our newly established method for quantifying pica is a useful tool for investigating the mechanisms of pellagra-related nausea.

Introduction: Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease characterized by extracellular matrix deposition in lung interstitium. Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel activated by physical stimuli and arachidonic acid metabolites. In a recent study, TRPV4-deficient mice did not develop bleomycin-induced lung fibrosis. Pirfenidone is an antifibrotic agent used to treat patients with IPF, although its underlying antifibrotic mechanism is poorly understood. Aims and Objectives: To investigate the effect of TRPV4 blockade by pirfenidone and the role of TRPV4 in lung fibrosis. Methods: We measured the agonist-induced Ca²⁺ influx of TRPV4 to determine whether its channel activation was inhibited by pirfenidone. A TRPV4 antagonist and pirfenidone were also evaluated in a mouse model of bleomycin-induced lung fibrosis. Arachidonic acid metabolites were determined using a targeted lipidomics approach in fibrotic lung from mice and bronchoalveolar lavage fluid (BALF) from patients with IPF prior to pirfenidone treatment. Results: TRPV4-mediated Ca²⁺ influx was inhibited by pirfenidone. The TRPV4 antagonist and pirfenidone significantly improved lung fibrosis. Arachidonic acid metabolite levels increased in fibrotic lung and were reduced by pirfenidone. Change in forced vital capacity of patients with IPF treated with pirfenidone significantly correlated with arachidonic acid metabolite levels in BALF (n = 8, r = 0.79, p = 0.02). Conclusions: The antifibrotic action of pirfenidone appears to be partly mediated by TRPV4. Levels of arachidonic acid metabolites in BALF may predict the antifibrotic efficacy of pirfenidone.

Arachidonic acid (AA) plays a pivotal role in the development of edema via its oxidized metabolites derived from cyclooxygenase (COX) and lipoxygenase (LOX), and is recently recognized as an activator of TRPV3. However, it is not clear whether AA plays some TRPV3-mediated pathological roles in the development of edema. Pharmacological and histological studies using ICRTRPV3+/+ and ICRTRPV3-/- mice indicated that higher ear edema responses to topical application of AA were observed in ICRTRPV3+/+ mice compared with ICRTRPV3-/- mice. However, there was no difference in the ear edema response to 12-O-tetradecanoylphorbol 13-acetate, skin histology, and skin barrier function between these mouse strains. Furthermore, oxidized fatty acids from the lesional site were analyzed to elucidate the TRPV3-mediated pathological roles of AA, and the results revealed that there were no differences in the level of COX or LOX metabolites derived from AA between both mouse strains. We concluded that AA plays a role in the development of TRPV3-mediated ear edema and that this result may contribute to better understanding of the pathophysiological mechanisms involved in the development of a certain type of edema.

Rationale: Targeted oxidized fatty acid analysis has been widely used to understand the roles of fatty acids in the development of diseases. However, because of the extensive structural diversity of fatty acids, it is considered that unknown lipid metabolites will remain undetected. Here, to discover and identify unknown lipid metabolites in biological samples, a global analytical system and a method of synthesizing lipid standards were investigated. Methods: Oxidized fatty acids in mouse lung tissues were extracted using mixed‐mode spin columns. Separation was achieved via ultra‐high‐performance liquid chromatography, mass spectrometric (MS) analysis was conducted in full scan mode using a Q Exactive Plus instrument equipped with an electrospray ionization probe, and structure analysis was carried out by high‐resolution data‐dependent tandem mass spectrometry (dd‐MS ² ). In addition, lipid standards, which are not commercially available, were synthesized by bioconversion using Bacillus circulans . Results: Oxidized fatty acids in mouse lung tissues were analyzed by high‐resolution accurate‐mass analysis, and multiple unknown molecules were discovered and tentatively identified using high‐resolution dd‐MS ² . Among these molecules, 21‐hydroxydocosahexaenoic acid (21‐HDoHE) and 22‐HDoHE, which are not commercially available, were synthesized by bioconversion. By comparing the exact masses, retention times, and characteristic fragment ions of the synthesized standards, 21‐HDoHE and 22‐HDoHE were definitively identified in the mouse lung tissue. Conclusions: Our strategy of global analysis and bioconversion can be used for the discovery and identification of unknown lipid molecules. Copyright © 2016 John Wiley & Sons, Ltd.

The transient receptor potential channel vanilloid subfamily V member 3 (TRPV3), which functions as a thermosensor in keratinocytes, plays an important role in the development of allergic and itchy dermatitis in rodents. Although real‐time PCR analysis using lesional and non‐lesional skin samples from patients with atopic dermatitis showed that TRPV3 was expressed in lesional skin, the role that TRPV3 plays in patients with dermatitis is still relatively obscure. Here, we determined whether TRPV3 was a dendritic cell (DC) modulator using DS‐Nh mice with a gain‐of‐function mutation in TRPV3 (TRPV3Gly573Ser), because increasing skin temperature is associated with the modulation of dermal dendritic cells (DCs). Interestingly, increased responses to haptens by skin and DCs were observed in DS‐Nh mice compared with those from DS mice with wild‐type TRPV3. Increased thymic stromal lymphopoietin (TSLP) responses were also observed in keratinocytes from DS‐Nh mice compared with those from DS mice. Taken together, we propose that the DS‐Nh mouse is a good model to use in order to better understand the role of this orphan channel and that TRPV3 may represent a new therapeutic target in certain types of dermatitis through the control of DCs.

Background: Recently, it has been reported that the Gly573Ser substitution of transient receptor potential V3 (TRPV3) leads to increased ion-channel activity in keratinocytes. Our previous studies have indicated that the spontaneous hairless and dermatitis phenotypes of DS-Nh mice, which were newly established as an animal model of atopic dermatitis (AD), are caused by TRPV3^Gly573Ser. Although this substitution causes hairlessness in several kinds of rodents, in our investigations, dermatitis developed in only a few animals. Here, we generated NC/Nga-Nh mice to elucidate the role of TRPV3^Gly573Ser in NC/Nga mice, which is one of the most studied animal models of AD. Methods: To establish and validate the new AD animal model, NC/Nga-Nh mice were generated using NC/Nga and DS-Nh mice, and their clinical features were compared. Next, T-cell receptor (TCR) Vβ usage in splenocytes, evaluation of bacterial colonization, and serological and histological analyses were carried out. Finally, repeated-hapten-application dermatitis was induced in these mice. Results: NC/Nga-Nh mice did not develop spontaneous dermatitis, whereas DS-Nh mice displayed this phenotype when maintained under the same conditions. Serological analysis indicated that there really was a phenotypic difference between these mice, and TCR repertoire analysis indicated that TCRVβ haplotypes played an important role in the development of dermatitis. Artificial dermatitis developed in DS and NC/Nga-Nh mice, but not in DS-Nh and NC/Nga mice. Histological and serological analyses indicated that mouse strains were listed in descending order of number of skin mast cells: DS-Nh > DS ≈ NC/Nga-Nh > NC/Nga, and serum IgE levels were increased after 2,4,6 trinitrochlorobenzene application in these mice. Serum IgE level in DS-Nh mice was lower than that mesured in other strains. Conclusion: Our results confirm the contribution of the TRPV3^Gly573Ser gene to the development of repeated hapten dermatitis, but not spontaneous dermatitis in NC/Nga mice.

Background. Although dry skin and T cell‐dependent disease exacerbation are characteristic features of atopic dermatitis (AD), the involvement of T cells in the development of dry skin remains unclear. Aims. We aimed to elucidate the role of T cells in the development of dry skin in DS‐Nh mice as a model for AD, and to evaluate this skin condition pharmacologically. Methods. We prepared DS‐Nh mice harbouring a T‐cell receptor (TCR)Vβ^a haplotype with a central deletion in the TCRBV gene segments, and mice harbouring a TCRVβ^b haplotype without any deletion. We analysed the TCRVβ chain usage and cytokine response to antimouse CD3 monoclonal antibodies in the splenocytes from the two mouse substrains. Transepidermal water loss (TEWL) was measured, and histochemical examination of these mice was carried out. Finally, a pharmacological analysis using loratadine was also performed to evaluate the features of spontaneous dry skin in DS‐Nh mice as a model of AD. Results. Although the deletion of TCRBV gene segments in the TCRVβ^a haplotype yielded different representations of each TCRVβ mRNA, this deletion did not evoke distinct cytokine profiles in the splenocytes compared with those of mice with the TCRVβ^b haplotype. Furthermore, our results indicated that the onset of dry skin occurred earlier in mice with TCRVβ^b than in those with TCRVβ^a. Pharmacologically, AD‐like dry skin in DS‐Nh with TCRVβ^b mice is susceptible to an H1 blocker. Conclusions. A specific lymphocyte subpopulation bearing T‐cell receptors may be responsible for loratadine‐responsive dermatitis in DS‐Nh mice.