Development of a skin microbiome diagnostic method to assess skin condition in healthy individuals: Application of research on skin microbiomes and skin condition.

OBJECTIVE: Skin microbiomes vary across individuals. They are known to play essential roles in maintaining homeostasis and preventing infectious pathogens. In recent years, cosmetic product development has begun to focus on the relationship between skin microbiomes and skin conditions. However, the statistical methods used in many studies include the standard t-test and small-scale correlation analysis, which do not take into account the internal correlation structure in data on skin microbiomes and skin features. In this study, we aimed to understand the relationship between skin microbiomes and skin features by analysing complex microbiomes and skin data. METHODS: We obtained data on 19 skin characteristics and skin microbiomes based on 16s ribosomal RNA (16S rRNA) gene analysis of 276 healthy Japanese women. We then performed the principal component analysis (PCA), a method that takes into account the internal correlation structure, on 234 panels of them that did not contain outliers or missing values. We confirmed the relationship between skin microbiomes and skin features with principal component regression analysis and hierarchical clustering analysis (HCA). RESULTS: The principal component regression analysis showed strong relationships between skin microbiomes and sebum-related skin characteristics and skin pH. In the HCA, the female panel was classified into two major groups based on the skin microbiome. Furthermore, there were significant differences in sebum-related skin characteristics and the way skin condition changes with ageing between those groups, suggesting the possibility of measuring skin condition and age-related skin risk based on microbiome data. In addition, sebum-related characteristics differed significantly among middle-aged participants, suggesting a strong relationship between skin microbiomes and sebum-related characteristics. CONCLUSION: Analysis of skin condition and skin microbiome in Japanese women, taking into account the correlation between variables, showed that skin microbiome was significantly related to the number of pores and the amount of sebum. Furthermore, it was suggested that the skin condition and the way the skin condition changes with ageing may differ depending on the type of skin microbiome. The finding of a relationship between skin condition and skin microbiome suggests the possibility of proposing a new beauty method focusing on the skin microbiome in the future.

OBJECTIF: Les microbiomes de la peau varient selon les individus. Ils sont connus pour jouer des rôles essentiels dans le maintien de l'homéostasie et la prévention des agents pathogènes infectieux. Ces dernières années, le développement de produits cosmétiques a commencé à se concentrer sur la relation entre les microbiomes cutanés et les conditions de la peau. Cependant, les méthodes statistiques utilisées dans de nombreuses études comprennent le t-test standard et l'analyse de corrélation à petite échelle, qui ne tiennent pas compte de la structure de corrélation interne dans les données sur les microbiomes cutanés et les caractéristiques de la peau. Dans cette étude, nous avons cherché à comprendre la relation entre les microbiomes cutanés et les caractéristiques de la peau en analysant des données complexes sur les microbiomes et la peau. MÉTHODES: Nous avons obtenu des données sur 19 caractéristiques de la peau et sur les microbiomes cutanés à partir de l'analyse du gène de l'ARNr 16S (16S rRNA) de 276 femmes japonaises en bonne santé. Nous avons ensuite effectué l'analyse en composantes principales (PCA: principal component analysis), une méthode qui prend en compte la structure de corrélation interne, sur 234 d'entre elles qui ne contenaient pas de valeurs aberrantes ou manquantes. Nous avons confirmé la relation entre les microbiomes cutanés et les caractéristiques de la peau à l'aide d'une analyse de régression en composantes principales et d'une analyse de regroupement hiérarchique (HCA: hierarchical clustering analysis). RÉSULTATS: L'analyse de régression en composantes principales a montré des relations fortes entre les microbiomes cutanés et les caractéristiques de la peau liées au sébum et au pH de la peau. Dans l'étude HCA, le panel de femmes a été classé en deux grands groupes sur la base du microbiome cutané. En outre, il y avait des différences significatives dans les caractéristiques de la peau liées au sébum et dans la façon dont l'état de la peau change avec l'âge entre ces groupes, ce qui suggère la possibilité de mesurer l'état de la peau et le risque cutané lié à l'âge à partir des données du microbiome. En outre, les caractéristiques liées au sébum différaient de manière significative chez les participants d'âge moyen, ce qui suggère une forte relation entre les microbiomes cutanés et les caractéristiques liées au sébum. CONCLUSION: L'analyse de l'état de la peau et du microbiome cutané chez les femmes japonaises, en tenant compte de la corrélation entre les variables, a montré que le microbiome cutané était significativement lié au nombre de pores et à la quantité de sébum. En outre, il a été suggéré que l'état de la peau et la façon dont l'état de la peau évolue avec le vieillissement peuvent différer en fonction du type de microbiome cutané. La découverte d'une relation entre l'état de la peau et le microbiome cutané suggère la possibilité de proposer à l'avenir une nouvelle méthode de beauté axée sur le microbiome cutané.

Role of macrophage-mediated Toll-like receptor 4-interleukin-1R signaling in ectopic tongue pain associated with tooth pulp inflammation.

BACKGROUND: The existence of referred pain and ectopic paresthesia caused by tooth pulp inflammation may make definitive diagnosis difficult and cause misdiagnosis or mistreatment; thus, elucidation of that molecular mechanism is urgent. In the present study, we investigated the mechanisms underlying ectopic pain, especially tongue hyperalgesia, after tooth pulp inflammation. METHODS: A rat model with mandibular first molar tooth pulp exposure was employed. Tooth pulp exposure-induced heat and mechanical-evoked tongue hypersensitivity was measured, and immunohistochemical staining for Iba1, a marker of active macrophages, IL-1ß, IL-1 type I receptor (IL-1RΙ), and toll-like receptor 4 in the trigeminal ganglion was performed. In addition, we investigated the effects of injections of liposomal clodronate Clophosome-A (LCCA), a selective macrophage depletion agent, lipopolysaccharide from Rhodobacter sphaeroides (LPS-RS, a toll-like receptor 4 antagonist), IL-1ß, or heat shock protein 70 (Hsp70, a selective agonist of toll-like receptor 4), to examine changes in tongue hypersensitivity and in the regulation of IL-1RΙ, toll-like receptor 4, and transient receptor potential vanilloid 1 (TRPV1) biosynthesis. RESULTS: At day 1 after tooth pulp exposure, obvious tooth pulp inflammation was observed. Tooth pulp exposure-induced heat and mechanical tongue hypersensitivity was observed from days 1 to 3 after tooth pulp exposure. The production of IL-1ß in activated macrophages and toll-like receptor 4 and IL-1RΙ expression were significantly increased in trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of LCCA significantly suppressed tongue hypersensitivity; however, toll-like receptor 4 and IL-1RΙ expression in trigeminal ganglion neurons innervating the tongue was not significantly altered. Intra-trigeminal ganglion injection of LPS-RS significantly suppressed tongue hypersensitivity and reduced IL-1RΙ expression in the trigeminal ganglion neurons innervating the tongue following tooth pulp exposure. Intra-trigeminal ganglion injection of recombinant Hsp70 significantly promoted tongue hypersensitivity and increased IL-1RI expression in trigeminal ganglion neurons innervating the tongue in naive rats. Furthermore, intra-trigeminal ganglion injection of recombinant IL-1ß led to tongue hypersensitivity and enhanced TRPV1 expression in trigeminal ganglion neurons innervating the tongue in naive rats. CONCLUSIONS: The present findings suggest that the neuron-macrophage interaction mediated by toll-like receptor 4 and IL-1RI activation in trigeminal ganglion neurons affects the pathogenesis of abnormal tongue pain following tooth pulp inflammation via IL-1RI and TRPV1 signaling in the trigeminal ganglion. Further research may contribute to the establishment of new therapeutic and diagnostic methods.

Involvement of Satellite Cell Activation via Nitric Oxide Signaling in Ectopic Orofacial Hypersensitivity.

: The mechanical head-withdrawal threshold (MHWT) was significantly reduced following inferior alveolar nerve transection (IANX) in rats. Nitrate and nitrite synthesis was dramatically increased in the trigeminal ganglion (TG) at 6 h after the IANX. The relative number of neuronal nitric oxide synthase (nNOS)-immunoreactive (IR) cells was significantly higher in IANX rats compared to sham-operated and N-propyl-L-arginine (NPLA)-treated IANX rats. On day 3 after NPLA administration, the MHWT recovered considerably in IANX rats. Following L-arginine injection into the TG, the MHWT was significantly reduced within 15 min, and the mean number of TG cells encircled by glial fibrillary acidic protein (GFAP)-IR cells was substantially higher. The relative number of nNOS-IR cells encircled by GFAP-IR cells was significantly increased in IANX rats. In contrast, after NPLA injection into the TG, the relative number of GFAP-IR cells was considerably reduced in IANX rats. Fluorocitrate administration into the TG significantly reduced the number of GFAP-IR cells and prevented the MHWT reduction in IANX rats. The present findings suggest that following IANX, satellite glial cells are activated via nitric oxide (NO) signaling from TG neurons. The spreading satellite glial cell activation within the TG results in mechanical hypersensitivity of face regions not directly associated with the trigeminal nerve injury.

Involvement of TNFα in the enhancement of hypersensitivity in the adulthood-injured face associated with facial injury in infancy.

To evaluate the mechanisms underlying acceleration of hypersensitivity in the adulthood-injured face following facial injury in infants, we developed the rats model with facial skin injury in infants and adulthoods (incision + incision), and facial skin suture in infants and facial skin injury in adulthoods (sham + incision), and analyzed the mechanical head-withdrawal threshold (MHWT) of the facial skin, immunohistochemical analysis of trigeminal ganglion (TG) and the effects of intra-ganglionic administration of neutralizing ant-TNFα antibody and recombinant TNFα on nocifensive behavior. The MHWT became considerably lower in incision + incision rats than in sham + incision rats at 10-14 days after the surgery. We observed many TG neurons encircled by glial fibrillary acidic protein-immunoreactive (GFAP-IR) cells and those exhibited TNFα immunoreactivity. TNFα was also expressed in GFAP-IR cells in incision + inicision TG. TNFα protein levels and the relative number of TNFα-IR cells were significantly higher in incision + incision rats than in sham + incision rats. The MHWT was significantly recovered during the intra-ganglionic administration of neutralizing anti-TNFα antibody 4-14 days after the incision. Furthermore, the MHWT was significantly decreased in sham + incision rats following the intra-ganglionic administration of recombinant TNFα. The present findings suggest that the neuron-satellite glial cell communication via TNFα is a critical mechanism in the enhancement of mechanical hypersensitivity in the adulthood-injured face following facial injury in infants.

Connexin 43 expression in satellite glial cells contributes to ectopic tooth-pulp pain.

Pulpitis often causes referred pain in opposing teeth. However, the precise mechanism underlying ectopic pain associated with tooth-pulp inflammation remains unclear. We performed the present study to test the hypothesis that functional interactions between satellite glial cells (SGCs) and trigeminal ganglion (TG) neurons are involved in ectopic orofacial pain associated with tooth-pulp inflammation. Digastric muscle electromyograph (D-EMG) activity elicited by administration of capsaicin into the upper second molar pulp (U2) was analyzed to evaluate noxious reflex responses. D-EMG activity was significantly increased in rats with lower first molar (L1) inflammation relative to saline-treated rats. Significantly increased expression of glial fibrillary acid protein (GFAP), a marker of activated glial cells, and connexin 43 (Cx43), a gap-junction protein, was observed in activated SGCs surrounding U2-innervating TG-neurons after L1-pulp inflammation. Daily administration of Gap26, a Cx43-inhibiting mimetic peptide, into the TG significantly suppressed capsaicin-induced D-EMG activity enhancement and reduced the percentage of fluorogold-labeled (U2-innervated) cells that were surrounded by GFAP-immunoreactive (IR) and Cx43-IR cells after L1-pulp inflammation. These findings indicate that tooth-pulp inflammation induces SGC activation and subsequent spread of SGC activation in the TG via Cx43-containing gap junctions. Thus, remote neuron excitability becomes enhanced in the TG following tooth-pulp inflammation, resulting in ectopic tooth-pulp pain in the contralateral tooth.

Role of medullary astroglial glutamine synthesis in tooth pulp hypersensitivity associated with frequent masseter muscle contraction.

Background The mechanisms underlying tooth pulp hypersensitivity associated with masseter muscle hyperalgesia remain largely underinvestigated. In the present study, we aimed to determine whether masseter muscle contraction induced by daily electrical stimulation influences the mechanical head-withdrawal threshold and genioglossus electromyography activity caused by the application of capsaicin to the upper first molar tooth pulp. We further investigated whether astroglial glutamine synthesis is involved in first molar tooth pulp hypersensitivity associated with masseter muscle contraction. Methods The first molar tooth pulp was treated with capsaicin or vehicle in masseter muscle contraction or sham rats, following which the astroglial glutamine synthetase inhibitor methionine sulfoximine or Phosphate buffered saline (PBS) was applied. Astroglial activation was assessed via immunohistochemistry. Results The mechanical head-withdrawal threshold of the ipsilateral masseter muscle was significantly decreased in masseter muscle contraction rats than in sham rats. Genioglossus electromyography activity was significantly higher in masseter muscle contraction rats than sham rats. Glial fibrillary acidic protein-immunoreactive cell density was significantly higher in masseter muscle contraction rats than in sham rats. Administration of methionine sulfoximine induced no significant changes in the density of glial fibrillary acidic protein-immunoreactive cells relative to PBS treatment. However, mechanical head-withdrawal threshold was significantly higher in masseter muscle contraction rats than PBS-treated rats after methionine sulfoximine administration. Genioglossus electromyography activity following first molar tooth pulp capsaicin treatment was significantly lower in methionine sulfoximine-treated rats than in PBS-treated rats. In the ipsilateral region, the total number of phosphorylated extracellular signal-regulated protein kinase immunoreactive cells in the medullary dorsal horn was significantly smaller upon first molar tooth pulp capsaicin application in methionine sulfoximine-treated rats than in PBS-treated rats. Conclusions Our results suggest that masseter muscle contraction induces astroglial activation, and that this activation spreads from caudal to the obex in the medullary dorsal horn, resulting in enhanced neuronal excitability associated with astroglial glutamine synthesis in medullary dorsal horn neurons receiving inputs from the tooth pulp. These findings provide significant insight into the mechanisms underlying tooth pulp hypersensitivity associated with masseter muscle contraction.

Interaction between calcitonin gene-related peptide-immunoreactive neurons and satellite cells via P2Y12 R in the trigeminal ganglion is involved in neuropathic tongue pain in rats.

The P2Y12 receptor expressed in satellite cells of the trigeminal ganglion is thought to contribute to neuropathic pain. The functional interaction between neurons and satellite cells via P2Y12 receptors and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) underlying neuropathic pain in the tongue was evaluated in this study. Expression of P2Y12 receptor was enhanced in pERK1/2-immunoreactive cells encircling trigeminal ganglion neurons after lingual nerve crush. The administration to lingual nerve crush rats of a selective P2Y12 receptor antagonist, MRS2395, attenuated tongue hypersensitivity to mechanical and heat stimulation and suppressed the increase in the relative numbers of calcitonin gene-related peptide (CGRP)-immunoreactive neurons and neurons encircled by pERK1/2-immunoreactive cells. Administration of the P2Y1,12,13 receptor agonist, 2-(methylthio)adenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), to naïve rats induced neuropathic pain in the tongue, as in lingual nerve crush rats. Co-administration of 2-MeSADP + MRS2395 to naïve rats did not result in hypersensitivity of the tongue. The relative number of CGRP-immunoreactive neurons increased following this co-administration, but to a lesser degree than observed in 2-MeSADP-administrated naïve rats, and the relative number of neurons encircled by pERK1/2-immunoreactive cells did not change. These results suggest that the interaction between activated satellite cells and CGRP-immunoreactive neurons via P2Y12 receptors contributes to neuropathic pain in the tongue associated with lingual nerve injury.

PHD1 interacts with ATF4 and negatively regulates its transcriptional activity without prolyl hydroxylation.

Cellular response to hypoxia plays an important role in both circulatory and pulmonary diseases and cancer. Hypoxia-inducible factors (HIFs) are major transcription factors regulating the response to hypoxia. The α-subunits of HIFs are hydroxylated by members of the prolyl-4-hydroxylase domain (PHD) family, PHD1, PHD2, and PHD3, in an oxygen-dependent manner. Here, we report on the identification of ATF4 as a protein interacting with PHD1 as well as PHD3, but not with PHD2. The central region of ATF4 including the Zipper II domain, ODD domain and ß-TrCP recognition motif were involved in the interaction with PHD1. Coexistence of PHD1 stabilized ATF4, as opposed to the destabilization of ATF4 by PHD3. Moreover, coexpression of ATF4 destabilized PHD3, whereas PHD1 stability was not affected by the presence of ATF4. Mutations to alanine of proline residues in ATF4 that satisfied hydroxylation consensus by PHDs did not affect binding activity of ATF4 to PHD1 and PHD3. Furthermore, in vitro prolyl hydroxylation assay clearly indicated that ATF4 did not serve as a substrate of both PHD1 and PHD3. Coexpression of PHD1 or PHD3 with ATF4 repressed the transcriptional activity of ATF4. These results suggest that PHD1 and PHD3 control the transactivation activity of ATF4.