RNA-seq analysis provides insight into reprogramming of culm development in Zizania latifolia induced by Ustilago esculenta.

KEY MESSAGE: We report a transcriptome assembly and expression profiles from RNA-Seq data and identify genes responsible for culm gall formation in Zizania latifolia induced by Ustilago esculenta. The smut fungus Ustilago esculenta can induce culm gall in Zizania latifolia, which is used as a vegetable in Asian countries. However, the underlying molecular mechanism of culm gall formation is still unclear. To characterize the processes underlying this host-fungus association, we performed transcriptomic and expression profiling analyses of culms from Z. latifolia infected by the fungus U. esculenta. Transcriptomic analysis detected U. esculenta induced differential expression of 19,033 and 17,669 genes in Jiaobai (JB) and Huijiao (HJ) type of gall, respectively. Additionally, to detect the potential gall inducing genes, expression profiles of infected culms collected at -7, 1 and 10 DAS of culm gall development were  analyzed. Compared to control, we detected 8089 genes (4389 up-regulated, 3700 down-regulated) and 5251 genes (3121 up-regulated, 2130 down-regulated) were differentially expressed in JB and HJ, respectively. And we identified 376 host and 187 fungal candidate genes that showed stage-specific expression pattern, which are  possibly responsible for gall formation at the initial and later phases, respectively. Our results indicated that cytokinins play more prominent roles in regulating gall formation than do auxins. Together, our work provides general implications for the understanding of gene regulatory networks for culm gall development in Z. latifolia, and potential targets for genetic manipulation to improve the future yield   of  this crop.

Lack of microRNA miR-150 reduces the capacity of epidermal Langerhans cell cross-presentation.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that repress target genes at post-transcriptional level. Langerhans cells (LCs) are skin-residential dendritic cells (DCs) with a life cycle distinct from other types of DCs. miRNA deficiency interrupts the homoeostasis and function of epidermal LCs, suggesting the critical roles of miRNAs in LC development and function. However, the roles of individual miRNAs in regulating LC development and function remain completely unknown. MiRNA miR-150 is highly expressed in mature lymphocytes and regulates T- and B-cell development and function. Here, we reported that miR-150 is also expressed in epidermal LCs, and its expression is significantly down-regulated during in vitro LC maturation. Using a miR-150 knockout mouse model, we found that lack of miR-150 reduces the capacity of LCs to cross-present a soluble antigen to antigen-specific CD8(+) T cells, but does not disturb the development, maturation, migration and phagocytic capacity of LCs. Thus, our data indicate that miR-150 is required for LC cross-presentation.

FibROAD: a manually curated resource for multi-omics level evidence integration of fibrosis research.

Organ fibrosis represents a vital health threat that substantially contributes to yearly mortality rates. While a considerable amount of research has been conducted on fibrosis, these reports have only focused on specific organs as affected within distinct disorders. Accordingly, results from such studies have been unable to provide a comprehensive understanding of the pathological processes involved. Here, we describe the development of FibROAD, an open-access database that integrates evidence from fibrosis-associated disorders as obtained from both the literature and multi-omics data. This resource will greatly assist both researchers and clinicians in the comprehension and treatment of this condition. FibROAD currently involves an assembly of 232 strong evidence-based fibrosis-related genes (FRGs) as garnered from 909 PubMed publications and contains lists of multi-omics data from > 4000 samples including RNA-seq, single-cell RNA-seq, miRNA-seq, ChIP-seq, ATAC-seq MeDIP-seq and MBD-seq as obtained from 17 different organs in 5 species. Results from integrative analyses as obtained using FibROAD have demonstrated that FRGs can be indicators for a wide range of organ fibrosis and reveal potential pro-fibrotic candidate genes for fibrosis research. In conclusion, FibROAD serves as a convenient platform where researchers can acquire integrated evidence and a more comprehensive understanding of fibrosis-related disorders. Database URL  https://www.fibroad.org.

Single-cell RNA-seq reveals fibroblast heterogeneity and increased mesenchymal fibroblasts in human fibrotic skin diseases.

Fibrotic skin disease represents a major global healthcare burden, characterized by fibroblast hyperproliferation and excessive accumulation of extracellular matrix. Fibroblasts are found to be heterogeneous in multiple fibrotic diseases, but fibroblast heterogeneity in fibrotic skin diseases is not well characterized. In this study, we explore fibroblast heterogeneity in keloid, a paradigm of fibrotic skin diseases, by using single-cell RNA-seq. Our results indicate that keloid fibroblasts can be divided into 4 subpopulations: secretory-papillary, secretory-reticular, mesenchymal and pro-inflammatory. Interestingly, the percentage of mesenchymal fibroblast subpopulation is significantly increased in keloid compared to normal scar. Functional studies indicate that mesenchymal fibroblasts are crucial for collagen overexpression in keloid. Increased mesenchymal fibroblast subpopulation is also found in another fibrotic skin disease, scleroderma, suggesting this is a broad mechanism for skin fibrosis. These findings will help us better understand skin fibrotic pathogenesis, and provide potential targets for fibrotic disease therapies.

TRAF4 Promotes Fibroblast Proliferation in Keloids by Destabilizing p53 via Interacting with the Deubiquitinase USP10.

Keloids represent one extreme of aberrant dermal wound healing. One of the important characteristics of keloids is uncontrolled fibroblasts proliferation. However, the mechanism of excessive proliferation of fibroblasts in keloids remains elusive. In this study, we demonstrated that TRAF4 was highly expressed in keloid fibroblasts and promoted fibroproliferation. We investigated the underlying molecular mechanism and found that TRAF4 suppressed the p53 pathway independent of its E3 ubiquitin ligase activity. Specifically, TRAF4 interacted with the deubiquitinase USP10 and blocked the access of p53 to USP10, resulting in p53 destabilization. Knockdown of p53 rescued cell proliferation in TRAF4-knockdown keloid fibroblasts, suggesting that the regulation of proliferation by TRAF4 in keloids relied on p53. Furthermore, in keloid patient samples, TRAF4 expression was inversely correlated with p53-p21 signaling activity. These findings help to elucidate the mechanisms underlying keloid development and indicate that blocking TRAF4 could represent a potential strategy for keloid therapy in the future.

BSNXD modulates mesenchymal stem cell differentiation into osteoblasts in a postmenopausal osteoporotic mouse model.

Mesenchymal stem cells (MSCs) are a type of stem cell that has multidirectional differentiation abilities. Under certain inducing factors, MSCs can differentiate into osteoblasts and adipocytes. Adipocytes and osteoblasts are derived from MSCs, and decreased osteoblastogenesis and increased adipocytes may be a primary cause of postmenopausal osteoporosis (PMO). The present study aimed to elucidate whether BuShen NingXin Decoction (BSNXD), a traditional Chinese medicinal compound, regulates MSC differentiation into both osteoblasts and adipocytes. The effects of BSNXD on bone morphometry were measured using micro-CT and its effects on the proportion of immune cells in the spleen were measured using flow cytometry (FCM). BSNXD-mediated regulation of MSC differentiation into osteoblasts and adipocytes was verified in vitro using ALP and Oil Red O staining. In addition, osteoblastogenesis-related genes and adipocyte transcription factors were measured using real-time PCR. We found that BSNXD increased bone volume, bone mineral density, and bone trabecular number, but decreased bone trabecular spacing. BSNXD treatment also increased regulatory T cell (Treg) function in vivo. In vitro, BSNXD serum increased ALP activity as well as collagen type I, osteocalcin, Runx2, and osterix mRNA expression. Moreover, BSNXD decreased adipocyte numbers and PPARγ mRNA expression, whereas in Tregs, BSNXD enhanced ALP activity. In conclusion, BSNXD promotes the differentiation of MSCs into osteoblasts and inhibits differentiation into adipocytes. BSNXD enhanced expression of osteoblastogenesis-related genes and decreased adipocyte transcription factor expression. We propose that BSNXD may be effective for the prevention of PMO.

Bu-Shen-Ning-Xin decoction: inhibition of osteoclastogenesis by abrogation of the RANKL-induced NFATc1 and NF-κB signaling pathways via selective estrogen receptor α.

INTRODUCTION: Bu-Shen-Ning-Xin decoction (BSNXD) is a traditional Chinese medicinal composition that has been used as a remedy for postmenopausal osteoporosis, but the mechanisms affecting bone metabolism are not fully understood. PURPOSE: We investigated the molecular mechanism and signaling pathway underlying the effect of BSNXD on osteoclastogenesis. MATERIALS AND METHODS: A postmenopausal osteoporosis animal model generated by ovariectomy was administered BSNXD and drug-derived serum was prepared. An enzyme immunoassay was conducted to measure the 17-ß-estradiol (E2) concentration in the drug-derived serum. Bone marrow-derived monocyte/macrophage precursor cells were treated with drug-derived serum, and tartrate-resistance acid phosphatase staining was conducted to observe osteoclastogenesis. A bone resorption assay was performed to analyze the effect on osteoclastic resorptive function. Real-time PCR, flow cytometry, Western blotting, transfection, and luciferase assays were conducted to explore the related mechanism. RESULTS: E2 was not elevated in BSNXD-derived serum. BSNXD-derived serum suppressed receptor activation of nuclear factor κB ligand (RANKL)-activated osteoclastogenesis in a dose-dependent manner; this effect could be reversed by estrogen receptor α antagonist methyl-piperidino-pyrazole. The serum suppressed RANKL-induced NF-κB transcription and inhibited the accumulation of nuclear factor of activated T-cells, cytoplasmic 1 in osteoclast precursor cells; the inhibitory effect was abolished by methyl-piperidino-pyrazole but not the estrogen receptor ß antagonist or androgen receptor antagonist. CONCLUSION: These results collectively suggest that administration of BSNXD presents inhibitory effects on osteoclast differentiation by abrogating the RANKL-induced nuclear factor of activated T-cells, cytoplasmic 1 and NF-κB signaling pathways downstream of estrogen receptor α, thereby contributing to the inhibitory effect on bone resorption.

Bu-Shen-Ning-Xin Decoction ameliorated the osteoporotic phenotype of ovariectomized mice without affecting the serum estrogen concentration or uterus.

INTRODUCTION: Bu-Shen-Ning-Xin Decoction (BSNXD), a traditional Chinese medicinal composition, has been used as a remedy for postmenopausal osteoporosis, but its effects on bone metabolism and the uterus have not been reported. PURPOSE: We aimed to determine the respective effects of BSNXD on the bones and the uterus of ovariectomized (OVX) mice to evaluate the efficacy and safety of this herbal formula. MATERIALS AND METHODS: Postmenopausal osteoporosis animal models that were generated by ovariectomy were treated with BSNXD. Dual-energy X-ray absorptiometry was performed to analyze the bone mineral density, and histomorphometric analysis was performed to measure the parameters related to bone metabolism. Calcein labeling was performed to detect bone formation. The uteruses from the mice were weighed, and the histomorphometry was analyzed. Drug-derived serum was prepared to assess the 17-ß-estradiol concentration via enzyme immunoassay. RESULTS: BSNXD administration ameliorated the osteoporotic phenotype of OVX mice, as evidenced by an increase in the bone mineral density and bone volume; these effects could not be abolished by the administration of the aromatase inhibitor letrozole. Moreover, BSNXD had no effect on the serum estrogen concentration or uterus. CONCLUSION: These results suggest that BSNXD has ameliorating effects on bone loss due to estrogen deprivation without affecting the peripheral blood estrogen concentration or the uterus in OVX mice.

[Anti-tumor effects of 10-hydroxycamptothecinc-treated DC-Hepa1-6 fusion vaccines].

OBJECTIVE: To investigate the induction of antitumor immune responses and therapeutic effects of 10-hydroxycamptothecinc-treated (HCPT) DC-Hepa fusion vaccines by DC fused with hepal-6 cell from hepatoma. METHODS: The fused cells were isolated by magnetic cell sorting and adherent culture. Cell apoptosis was detected by Rhodamine123/PI double-labeled assay, CTL activity by 4 h (51)Cr releasing assay. Protective and therapeutic effects of the fusion vaccine to the tumor-bearing mice was also observed. RESULTS: The apoptosis rate was 29.7%+/-4.1% when DC-Hepa fusion vaccine was treated with 50 microg/ml HCPT for 24 h. After treatment with the HCPT-DC-Hepa fusion vaccine, the tumor grew obviously slowly, survival period of the mice was prolonged, induced more potent CTL cytotoxicity, and resisted against the rechallenge of Hepal-6 cells. CONCLUSION: Vaccination with HCPT-DC-Hepa fusion vaccine could elicit potent antitumor responses, which will provide a new approach to the DC-mediated therapeutic antitumor immunity.

Deletion of microRNA miR-223 increases Langerhans cell cross-presentation.

Langerhans cells (LCs) are skin-residential dendritic cells that regulate skin immunity. MicroRNAs (miRNAs) are key regulators in the control of biological functions in a variety of cell types. Deletion of all miRNAs interrupts the homeostasis and function of epidermal LCs. However, the roles of individual miRNAs in regulating LC development and function are still completely unknown. MiRNA miR-223 is especially expressed in the myeloid compartment. Here, we reported that miR-223 is highly expressed in freshly isolated epidermal LCs, and tested whether miR-223 regulates LC development and function using miR-223 knockout (KO) mice. We found that the number, maturation, migration and phagocytic capacity of LCs were comparable between miR-223KO and wild-type mice. However, lack of miR-223 significantly increases LCs-mediated antigen-specific CD8(+) T cell proliferation in vivo and in vitro, while LCs from KO and WT mice showed comparable stimulation for antigen-specific CD4(+) T cells. Our data suggest that miR-223 negatively regulates LC cross-presentation, but may not be required for normal LC homeostasis and development.

Histone deacetylases inhibitor Trichostatin A ameliorates DNFB-induced allergic contact dermatitis and reduces epidermal Langerhans cells in mice.

BACKGROUND: Histone deacetylases (HDACs) influence chromatin organization, representing a key epigenetic regulatory mechanism in cells. Trichostatin A (TSA), a potent HDAC inhibitor, has anti-tumor and anti-inflammatory effects. Allergic contact dermatitis (ACD) is a T-cell-mediated inflammatory reaction in skin and is regulated by epidermal Langerhans cells (LCs). OBJECTIVE: The aim of this study was to investigate if TSA treatment prevents 2,4-dinitrofluorobenzene (DNFB)-induced ACD in mice and regulates epidermal LCs and other immune cells during ACD development. METHODS: ACD was induced by sensitizing and challenging with DNFB topically. Mice were treated intraperitoneally with TSA or vehicle DMSO as a control every other day before and during induction of ACD. The ear swelling response was measured and skin biopsies from sensitized skin areas were obtained for histology. Epidermal cells, thymus, spleen and skin draining lymph nodes were collected for immune staining. RESULTS: TSA treatment ameliorated skin lesion severity of DNFB-induced ACD. The percentages of epidermal LCs and splenic DCs as well as LC maturation were significantly reduced in TSA-treated mice. However, TSA treatment did not significantly affect the homeostasis of conventional CD4(+) and CD8(+) T cells, Foxp3(+)CD4(+) regulatory T cells, iNKT cells, and γδ T cells in thymus, spleen and draining lymph nodes (dLNs). Furthermore, there were no significant differences in IL-4 and IFN-γ-producing T cells and iNKT cells between TSA- and DMSO-treated mice. CONCLUSION: Our findings suggest that TSA may ameliorate ACD through the regulation of epidermal LCs and HDACs could serve as potential therapeutic targets for ACD and other LCs-related skin diseases.

Aging affects epidermal Langerhans cell development and function and alters their miRNA gene expression profile.

Immunosenescence is a result of progressive decline in immune system function with advancing age. Epidermal Langerhans cells (LCs), belonging to the dendritic cell (DC) family, act as sentinels to play key roles in the skin immune responses. However, it has not been fully elucidated how aging affects development and function of LCs. Here, we systemically analyzed LC development and function during the aging process in C57BL/6J mice, and performed global microRNA (miRNA) gene expression profiles in aged and young LCs. We found that the frequency and maturation of epidermal LCs were significantly reduced in aged mice starting at 12 months of age, while the Langerin expression and ability to phagocytose Dextran in aged LCs were increased compared to LCs from < 6 month old mice. The migration of LCs to draining lymph nodes was comparable between aged and young mice. Functionally, aged LCs were impaired in their capacity to induce OVA-specific CD4+ and CD8+ T cell proliferation. Furthermore, the expression of miRNAs in aged epidermal LCs showed a distinct profile compared to young LCs. Most interestingly, aging-regulated miRNAs potentially target TGF-ß-dependent and non- TGF-ß-dependent signal pathways related to LCs. Overall, our data suggests that aging affects LCs development and function, and that age-regulated miRNAs may contribute to the LC developmental and functional changes in aging.