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Th2 immunity plays important roles in both protective and allergic responses. Nevertheless, the nature of antigen-presenting cells responsible for Th2 cell differentiation remains ill-defined compared with the nature of the cells responsible for Th1 and Th17 cell differentiation. Basophils have attracted attention as a producer of Th2-inducing cytokine IL-4, whereas their MHC class II (MHC-II) expression and function as antigen-presenting cells are matters of considerable controversy. Here we revisited the MHC-II expression on basophils and explored its functional relevance in Th2 cell differentiation. Basophils generated in vitro from bone marrow cells in culture with IL-3 plus GM-CSF displayed MHC-II on the cell surface, whereas those generated in culture with IL-3 alone did not. Of note, these MHC-II–expressing basophils showed little or no transcription of the corresponding MHC-II gene. The GM-CSF addition to culture expanded dendritic cells (DCs) other than basophils. Coculture of basophils and DCs revealed that basophils acquired peptide–MHC-II complexes from DCs via cell contact-dependent trogocytosis. The acquired complexes, together with CD86, enabled basophils to stimulate peptide-specific T cells, leading to their proliferation and IL-4 production, indicating that basophils can function as antigen-presenting cells for Th2 cell differentiation. Transfer of MHC-II from DCs to basophils was also detected in draining lymph nodes of mice with atopic dermatitis-like skin inflammation. Thus, the present study defined the mechanism by which basophils display MHC-II on the cell surface and appears to reconcile some discrepancies observed in previous studies.

Atopic dermatitis (AD) is a complex, often lifelong allergic disease with severe pruritus affecting around 10% of both humans and dogs. To investigate the role of mast cells (MCs) and MC-specific proteases on the immunopathogenesis of AD, a vitamin D3-analog (MC903) was used to induce clinical AD-like symptoms in c-kit-dependent MC-deficient Wsh−/− and the MC protease-deficient mMCP-4−/−, mMCP-6−/−, and CPA3−/− mouse strains. MC903-treatment on the ear lobe increased clinical scores and ear-thickening, along with increased MC and granulocyte infiltration and activity, as well as increased levels of interleukin 33 (IL-33) locally and thymic stromal lymphopoietin (TSLP) both locally and systemically. The MC-deficient Wsh−/− mice showed significantly increased clinical score and ear thickening albeit having lower ear tissue levels of IL-33 and TSLP as well as lower serum levels of TSLP as compared to the WT mice. In contrast, although having significantly increased IL-33 ear tissue levels the chymase-deficient mMCP-4−/− mice showed similar clinical score, ear thickening, and TSLP levels in ear tissue and serum as the WT mice, whereas mMCP-6 and CPA3 -deficient mice showed a slightly reduced ear thickening and granulocyte infiltration. Our results suggest that MCs promote and control the level of MC903-induced AD-like inflammation.

Vitamin D is known to play an important role in cancer-prevention. One of the features associated with the onset of malignancy is the elevation of Cu (II) levels. The mode of cancer-prevention mediated by calcitriol, the biologically active form of vitamin D, remain largely unknown. Using exogenously added Cu (II) to stimulate a malignancy like condition in a novel cellular system of rabbit calcitriol overloaded lymphocytes, we assessed lipid peroxidation, protein carbonylation, DNA damage and consequent apoptosis. Free radical mediators were identified using free radical scavengers and the role of Cu (II) in the reaction was elucidated using chelators of redox active cellular metal ions. Lipid peroxidation and protein carbonylation (markers of oxidative stress), consequent DNA fragmentation and apoptosis were observed due to calcitriol-Cu (II) interaction. Hydroxyl radicals, hydrogen peroxide and superoxide anions mediate oxidative stress produced during this interaction. Amongst cellular redox active metals, copper was found to be responsible for this reaction. This is the first report implicating Cu (II) and calcitriol interaction as the cause of selective cytotoxic action of calcitriol against malignant cells. We show that this interaction leads to the production of oxidative stress due to free radical production and consequent DNA fragmentation, which leads to apoptosis. A putative mechanism is presented to explain this biological effect.

Background： Tetracycline （TET） has been found to have both antibiotic and anti-inflammatory properties. The anti-inflammatory effect of topical TET on atopic dermatitis （AD） has not been reported. The purpose of this study was to explore the potential role of topical TET and its anti-inflammatory effects in a mouse model of AD. Methods： The 2% TET was applied topically to ears of MC903-induced AD-like BALB/c mice once a day. AD-like symptoms and severity were evaluated by assessing skin scoring of dermatitis, ear thickness, and frequency of scratching. Serum lgE and thymic stromal lymphopoietin （TSLP） levels were measured by enzyme-linked immunosorbent assay. Western blot was used for analyzing the expressions of TSLP, protease-activated receptor 2 （PAR2）, and nuclear factor-kappa B （NF-~：B） in skin lesions. Real-time polymerase chain reaction was performed to assess the mRNA levels of TSLP and inflammatory cytokines including interleukin （IL）-4, IL-13, tumor necrosis factor （TNF）-α, and IL-1β in skin lesions. Results： Scoring of dermatitis （9.00 ± 0.63 vs. 6.67± 1.03, P = 0.001）, ear thickness （0.44± 0.02 mm vs. 0.40±0.03 mm, P = 0.018）, and serum IgE level （421.06 ± 212.13 pg/ml vs. 244.15±121.39 pg/ml, P 0.047） were all improved in the 2% TET treatment group compared with AD group. Topical TET significantly reduced the serum level of TSLP （119.04 ±38.92 pg/ml vs. 65.95± 54.61 μg/ml, P = 0.011） and both mRNA and protein expressions of TSLP in skin lesions compared with AD group （P = 0.003 and 0.011, respectively）, and NF-κB and PAR2 expression in skin lesions were also suppressed （P = 0.016 and 0.040, respectively）. Furthermore, expressions of inflammatory cytokines IL-4, IL- 13, and TNF-α in skin lesions were down-regulated in 2% TET group compared with AD group （P = 0.035, 0.008, and 0.044, respectively）. Conclusions： Topical TET exerted anti-inflammatory effects through suppression of TSLP and inflammatory cytokines in AD mouse model, suggesting TET as a potential agent for the topical treatment of AD in the future.

The cytokines IL-1 beta, IL-6 and the chemokine IL-8 are key mediators of host inflammation. 1 alpha,25-Dihydroxyvitamin D3 (VD3) has been shown to regulate host immune responses in vivo and in vitro. The purpose of this study was to investigate whether the addition of VD3 to human corneal epithelial cells colonized with Pseudomonas aeruginosa altered the expression of IL-1 beta, IL-6 and IL-8. An immortalized human corneal epithelial (HCE) cell line was used in this study. After growth to confluency, HCE cells were challenged with P. aeruginosa strain 6294 in the presence or absence of 10-6 mol/L VD3 for 4 h, 8 h and 12 h. Gene expression of IL-1 beta, IL-6 and IL-8 was detected by reverse transcription-PCR (RT-PCR) from total RNA extracted from HCE cells. Protein concentrations of IL-1 beta, IL-6 and IL-8 in culture supernatants were measured by ELISA. Addition of VD3 to HCE cells colonized with P. aeruginosa significantly inhibited the expression of IL-1 beta and IL-8 mRNA and protein (P < 0.05). Although the expression of IL-6 mRNA was stimulated at 12 h post-challenge (P < 0.05), the expression of IL-6 protein was inhibited at all time points after the addition of VD3. In conclusion, this study demonstrated that VD3 inhibited the P. aeruginosa-induced expression of IL-1 beta, IL-6 and IL-8 in HCE cells, suggesting that this vitamin may have the potential to become a novel anti-inflammatory agent in ocular disease.

Promotion of apoptosis (which is frequently dependent on functional p53) is thought to be critical for the effectiveness of chemotherapy or radiotherapy. Studies in this as well as other laboratories have demonstrated that breast tumor cells are relatively refractory to apoptosis in response to modalities that induce DNA damage. This report describes our efforts to understand the basis for the absence of an apoptotic response to adriamycin and ionizing radiation in the breast tumor cell based on alterations in cell-cycle and apoptotic regulatory proteins. We also report on the permissive effects of Vitamin D3 and the Vitamin D3 analog EB 1089 in the promotion of apoptosis in p53-wild-type cells. Our studies suggest that regulation of apoptosis in the breast tumor cell may require modulation of signaling events other than or in addition to the p53-dependent DNA damage response.

Hypercalciuria was induced in female Sprague-Dawley rats, aged 40+/-2 days, by 7-day administration (mean+/-SEM) of calcitriol (5.4+/-0.1 ng/100 g per day, intraperitoneal), furosemide (14.9+/-1.9 mg/100 g per day, oral), or ammonium chloride (3.8+/-0.1 mEq/100 g per day, oral). Calciuria increased from 1.9+/-0.2, 1.6+/-0.2, and 1.9+/-0.3 to 5.4+/-0.5, 4.0+/-0.9, and 5.4+/-0.5 mg/100 g per day in the calcitriol (VD, n = 9), furosemide (F, n = 6), and ammonium chloride (AC, n = 10) groups, respectively. Calciuria did not change (1.9+/-0.3 vs. 1.6+/-0.1 mg/100 g per day) in control rats (n = 8). Ninety-six percent of treated rats became hypercalciuric as assessed by urine calcium excretion above the 90th percentile of normal values. Hypercalciuria was of similar degree in the three groups of rats and was not associated with hypercalcemia, metabolic acidosis, severe serum electrolyte imbalance, or growth impairment. VD rats had low serum parathyroid hormone (PTH) concentrations (3.0+/-0.5 pg/ml vs. 15.8+/-1.3 pg/ml in controls, P <0.05), whereas serum PTH was not significantly elevated in F rats (16.2+/-1.8 pg/ml). Thus, the protocol caused three forms of hypercalciuria that mimicked the clinical conditions of idiopathic hypercalciuria in humans and may clearly be differentiated according to their mechanism of production. This experimental model of normocalcemic hypercalciuria may be useful to clarify unknown aspects of pathogenesis and pathophysiology of idiopathic hypercalciuria in children.

Our preliminary work has revealed that vitamin D receptor (VDR) activation is protective against cisplatin induced acute kidney injury (AKI). Ferroptosis was recently reported to be involved in AKI. Here in this study, we investigated the internal relation between ferroptosis and the protective effect of VDR in cisplatin induced AKI. By using ferroptosis inhibitor ferrostatin-1 and measurement of ferroptotic cell death phenotype in both in vivo and in vitro cisplatin induced AKI model, we observed the decreased blood urea nitrogen, creatinine, and tissue injury by ferrostatin-1, hence validated the essential involvement of ferroptosis in cisplatin induced AKI. VDR agonist paricalcitol could both functionally and histologically attenuate cisplatin induced AKI by decreasing lipid peroxidation (featured phenotype of ferroptosis), biomarker 4-hydroxynonenal (4HNE), and malondialdehyde (MDA), while reversing glutathione peroxidase 4 (GPX4, key regulator of ferroptosis) downregulation. VDR knockout mouse exhibited much more ferroptotic cell death and worsen kidney injury than wild type mice. And VDR deficiency remarkably decreased the expression of GPX4 under cisplatin stress in both in vivo and in vitro, further luciferase reporter gene assay showed that GPX4 were target gene of transcription factor VDR. In addition, in vitro study showed that GPX4 inhibition by siRNA largely abolished the protective effect of paricalcitol against cisplatin induced tubular cell injury. Besides, pretreatment of paricalcitol could also alleviated Erastin (an inducer of ferroptosis) induced cell death in HK-2 cell. These data suggested that ferroptosis plays an important role in cisplatin induced AKI. VDR activation can protect against cisplatin induced renal injury by inhibiting ferroptosis partly via trans-regulation of GPX4.

Environmental exposure to per- and polyfluoroalkyl substances (PFASs) has been increasingly associated with skin disorders, including atopic dermatitis (AD); however, the underlying molecular mechanisms remain unclear. This study aimed to evaluate the effects of perfluorononanoic acid (PFNA) and perfluorooctanoic acid (PFOA)—two widely detected PFASs—on epidermal function and gene expression in Human Epithelial Keratinocyte, neonatal (HEKn). We assessed cell viability, morphology, and transcriptomic changes using in vitro assays and RNA-seq analysis from a neonatal cohort. PFASs induced dose-dependent cytotoxicity and downregulation of barrier-related genes. Ingenuity pathway analysis identified calcitriol as a suppressed upstream regulator. Functional validation revealed that calcitriol partially reversed the PFAS-induced suppression of antimicrobial peptide genes. These findings support the hypothesis that PFASs may contribute to AD-like skin pathology by impairing vitamin D receptor signaling and antimicrobial defense, and calcitriol demonstrates potential as a protective modulator. This study provides mechanistic insights into the impact of environmental toxicants on skin homeostasis and suggests a potential protective role for calcitriol in PFAS-induced skin barrier damage.

Recent studies indicated that intramammary administration of active vitamin D3 hormone (1,25D3) inhibits the inflammatory process associated with mastitis. We hypothesized that attenuation of endoplasmic reticulum (ER) stress by 1,25D3 in mammary epithelial cells (MECs) is an important cellular mechanism contributing to this beneficial effect of intramammary treatment with 1,25D3. To test this hypothesis, the effect of 1,25D3 was studied on induction of ER stress in a transformed human MEC line, MCF-7 cells. Treatment with two different ER stress inducers, thapsigargin (TG) and tunicamycin (TM), caused a dose-dependent induction of ER stress as evident from up-regulation of protein kinase RNA-like ER kinase (PERK), heat shock protein family A (Hsp70) member 5 (HSPA5), activating transcription factor (ATF4), ATF6, DNA damage inducible transcript 3 (DDIT3) and spliced X-box binding protein 1 (sXBP1) and impaired cell viability and decreased expression of vitamin D receptor (VDR) in MCF-7 cells (P < 0.05). Treatment with 1,25D3 (100 nM) inhibited TG (10 nM)- and TM (1 μg/mL)-induced mRNA and/or protein levels of ATF4, ATF6, DDIT3 and HSPA5 in MCF-7 cells (P < 0.05). In addition, 1,25D3 (100 nM) antagonized the effect of TG (10 nM) and TM (1 μg/mL) on mRNA and protein levels of VDR and mRNA levels of genes involved in production and degradation of 1,25D3 in MCF-7 cells (P < 0.05). Moreover, 1,25D3 (100 nM) inhibited nuclear factor-κB (NF-κB) activation in response to TM (10 nM) and TG (1 μg/mL) in MCF-7 cells. In conclusion, the present findings show that 1,25D3 is effective in attenuating ER stress and the NF-κB-driven inflammatory response in MCF-7 cells. This indicates that attenuation of ER stress by 1,25D3 in MECs may contribute to the recently observed inhibitory effect of intramammary treatment of dairy cows with 1,25D3 on the inflammatory process associated with mastitis.