Hair Follicle Dermal Sheath Derived Cells Improve Islet Allograft Survival without Systemic Immunosuppression

Immunosuppressive drugs successfully prevent rejection of islet allografts in the treatment of type I diabetes. However, the drugs also suppress systemic immunity increasing the risk of opportunistic infection and cancer development in allograft recipients. In this study, we investigated a new treatment for autoimmune diabetes using naturally immune privileged, hair follicle derived, autologous cells to provide localized immune protection of islet allotransplants. Islets from Balb/c mouse donors were cotransplanted with syngeneic hair follicle dermal sheath cup cells (DSCC, group 1) or fibroblasts (FB, group 2) under the kidney capsule of immune-competent, streptozotocin induced, diabetic C57BL/6 recipients. Group 1 allografts survived significantly longer than group 2 (32.2 ± 12.2 versus 14.1 ± 3.3 days, P<0.001) without administration of any systemic immunosuppressive agents. DSCC reduced T cell activation in the renal lymph node, prevented graft infiltrates, modulated inflammatory chemokine and cytokine profiles, and preserved better beta cell function in the islet allografts, but no systemic immunosuppression was observed. In summary, DSCC prolong islet allograft survival without systemic immunosuppression by local modulation of alloimmune responses, enhancing of beta cell survival, and promoting of graft revascularization. This novel finding demonstrates the capacity of easily accessible hair follicle cells to be used as local immunosuppression agents in islet transplantation

Hair Follicle Mesenchyme-Associated PD-L1 Regulates T-Cell Activation Induced Apoptosis: A Potential Mechanism of Immune Privilege

The immune privilege (IP) of hair follicles (HFs) has been well established in previous studies. However, whether cultured HF cells still exhibit IP properties, the individual factors involved in this process, and the detailed mechanisms that drive and maintain IP, are largely unidentified. We found preferential expression of IP-associated genes in cultured HF dermal papilla and dermal sheath cup cells (DSCCs) compared with non-follicular fibroblasts (FBs) at passage 4, suggesting a potential for functional IP. Notably, programmed cell death 1 ligand 1 (PD-L1) was significantly upregulated in DSCCs and dermal papilla cells relative to FBs. IFNγ secretion from peripheral blood mononuclear cells (PBMCs) co-cultured with histoincompatible DSCCs was significantly lower than with FB and higher percentages of early apoptotic, Annexin V+ cells were observed in PBMC co-cultured with DSCCs. Knockdown of PD-L1 translation by silencing interfering RNA in DSCCs enabled increased IFNγ secretion by PBMCs, whereas transfection of pCMV6-XL4/hPD-L1 in FB significantly reduced IFNγ secretion and increased apoptosis in co-cultured PBMCs. We also found that apoptosis in allogeneic T cells induced by DSCCs was largely dependent on the mitochondrial pathway. Our study suggests IP properties are exhibited in cultured DSCCs in part through expression of negative co-signaling molecule PD-L1

Host Response of Human Epidermis to Methicillin-Resistant Staphylococcus aureus Biofilm Infection and Synthetic Antibiofilm Peptide Treatment

Bacterial biofilm infections associated with wounded skin are prevalent, recalcitrant, and in urgent need of treatments. Additionally, host responses in the skin to biofilm infections are not well understood. Here we employed a human organoid skin model to explore the transcriptomic changes of thermally-injured epidermis to methicillin-resistant Staphylococcus aureus (MRSA) biofilm colonization. MRSA biofilm impaired skin barrier function, enhanced extracellular matrix remodelling, elicited inflammatory responses including IL-17, IL-12 family and IL-6 family interleukin signalling, and modulated skin metabolism. Synthetic antibiofilm peptide DJK-5 effectively diminished MRSA biofilm and associated skin inflammation in wounded human ex vivo skin. In the epidermis, DJK-5 shifted the overall skin transcriptome towards homeostasis including modulating the biofilm induced inflammatory response, promoting the skin DNA repair function, and downregulating MRSA invasion of thermally damaged skin. These data clarified the underlying immunopathogenesis of biofilm infections and revealed the intrinsic promise of synthetic peptides in reducing inflammation and biofilm infections

Hair Follicle Dermal Sheath Derived Cells Improve Islet Allograft Survival without Systemic Immunosuppression

Immunosuppressive drugs successfully prevent rejection of islet allografts in the treatment of type I diabetes. However, the drugs also suppress systemic immunity increasing the risk of opportunistic infection and cancer development in allograft recipients. In this study, we investigated a new treatment for autoimmune diabetes using naturally immune privileged, hair follicle derived, autologous cells to provide localized immune protection of islet allotransplants. Islets from Balb/c mouse donors were cotransplanted with syngeneic hair follicle dermal sheath cup cells (DSCC, group 1) or fibroblasts (FB, group 2) under the kidney capsule of immune-competent, streptozotocin induced, diabetic C57BL/6 recipients. Group 1 allografts survived significantly longer than group 2 (32.2 ± 12.2 versus 14.1 ± 3.3 days, P<0.001) without administration of any systemic immunosuppressive agents. DSCC reduced T cell activation in the renal lymph node, prevented graft infiltrates, modulated inflammatory chemokine and cytokine profiles, and preserved better beta cell function in the islet allografts, but no systemic immunosuppression was observed. In summary, DSCC prolong islet allograft survival without systemic immunosuppression by local modulation of alloimmune responses, enhancing of beta cell survival, and promoting of graft revascularization. This novel finding demonstrates the capacity of easily accessible hair follicle cells to be used as local immunosuppression agents in islet transplantation

Hair follicle dermal sheath derived cells improve islet allograft survival without systemic immunosuppression.

Immunosuppressive drugs successfully prevent rejection of islet allografts in the treatment of type I diabetes. However, the drugs also suppress systemic immunity increasing the risk of opportunistic infection and cancer development in allograft recipients. In this study, we investigated a new treatment for autoimmune diabetes using naturally immune privileged, hair follicle derived, autologous cells to provide localized immune protection of islet allotransplants. Islets from Balb/c mouse donors were cotransplanted with syngeneic hair follicle dermal sheath cup cells (DSCC, group 1) or fibroblasts (FB, group 2) under the kidney capsule of immune-competent, streptozotocin induced, diabetic C57BL/6 recipients. Group 1 allografts survived significantly longer than group 2 (32.2 ± 12.2 versus 14.1 ± 3.3 days, P &lt; 0.001) without administration of any systemic immunosuppressive agents. DSCC reduced T cell activation in the renal lymph node, prevented graft infiltrates, modulated inflammatory chemokine and cytokine profiles, and preserved better beta cell function in the islet allografts, but no systemic immunosuppression was observed. In summary, DSCC prolong islet allograft survival without systemic immunosuppression by local modulation of alloimmune responses, enhancing of beta cell survival, and promoting of graft revascularization. This novel finding demonstrates the capacity of easily accessible hair follicle cells to be used as local immunosuppression agents in islet transplantation

Development of Autoimmune Hair Loss Disease Alopecia Areata Is Associated with Cardiac Dysfunction in C3H/HeJ Mice

<div><p>Alopecia areata (AA) is a chronic autoimmune hair loss disease that affects several million men, women and children worldwide. Previous studies have suggested a link between autoimmunity, stress hormones, and increased cardiovascular disease risk. In the current study, histology, immunohistology, quantitative PCR (qPCR) and ELISAs were used to assess heart health in the C3H/HeJ mouse model for AA and heart tissue response to adrenocorticotropic hormone (ACTH) exposure. Mice with AA exhibited both atrial and ventricular hypertrophy, and increased collagen deposition compared to normal-haired littermates. QPCR revealed significant increases in <i>Il18</i> (4.6-fold), IL18 receptor-1 (<i>Il18r1</i>; 2.8-fold) and IL18 binding protein (<i>Il18bp</i>; 5.2-fold) in AA hearts. Time course studies revealed a trend towards decreased <i>Il18</i> in acute AA compared to controls while <i>Il18r1</i>, <i>Il18bp</i> and <i>Casp1</i> showed similar trends to those of chronic AA affected mice. Immunohistochemistry showed localization of IL18 in chronic AA mouse atria. ELISA indicated cardiac troponin-I (cTnI) was elevated in the serum and significantly increased in AA heart tissue. Cultures of heart atria revealed differential gene expression between AA and control mice in response to ACTH. ACTH treatment induced significant increase in cTnI release into the culture medium in a dose-dependent manner for both AA and control mice. In conclusion, murine AA is associated with structural, biochemical, and gene expression changes consistent with cardiac hypertrophy in response to ACTH exposure.</p></div

Notch signaling is significantly suppressed in basal cell carcinomas and activation induces basal cell carcinoma cell apoptosis.

A subset of basal cell carcinomas (BCCs) are directly derived from hair follicles (HFs). In some respects, HFs can be defined as 'ordered' skin appendage growths, while BCCs can be regarded as 'disordered' skin appendage growths. The aim of the present study was to examine HFs and BCCs to define the expression of common and unique signaling pathways in each skin appendage. Human nodular BCCs, along with HFs and non‑follicular skin epithelium from normal individuals, were examined using microarrays, qPCR, and immunohistochemistry. Subsequently, BCC cells and root sheath keratinocyte cells from HFs were cultured and treated with Notch signaling peptide Jagged1 (JAG1). Gene expression, protein levels, and cell apoptosis susceptibility were assessed using qPCR, immunoblotting, and flow cytometry, respectively. Specific molecular mechanisms were found to be involved in the process of cell self‑renewal in the HFs and BCCs, including Notch and Hedgehog signaling pathways. However, several key Notch signaling factors showed significant differential expression in BCCs compared with HFs. Stimulating Notch signaling with JAG1 induced apoptosis of BCC cells by increasing Fas ligand expression and downstream caspase-8 activation. The present study showed that Notch signaling pathway activity is suppressed in BCCs, and is highly expressed in HFs. Elements of the Notch pathway could, therefore, represent targets for the treatment of BCCs and potentially in hair follicle engineering

Identification of Autoantigen Epitopes in&nbsp;Alopecia Areata.

Alopecia areata (AA) is believed to be a cell-mediated autoimmune hair loss disease. Both CD4 and cytotoxic CD8 T cells (CTLs) are important for the onset and progression of AA. Hair follicle (HF) keratinocyte and/or melanocyte antigen epitopes are suspected potential targets of autoreactive CTLs, but the specific epitopes have not yet been identified. We investigated the potential for a panel of known epitopes, expressed by HF keratinocytes and melanocytes, to induce activation of CTL populations in peripheral blood mononuclear cells. Specific synthetic epitopes derived from HF antigens trichohyalin and tyrosinase-related protein-2 induced significantly higher frequencies of response in AA CTLs compared with healthy controls (IFN-gamma secretion). Apoptosis assays revealed conditioned media from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides elevated the expression of apoptosis markers in primary HF keratinocytes. A cytokine array revealed higher expression of IL-13 and chemokine ligand 5 (CCL5, RANTES) from AA peripheral blood mononuclear cells stimulated with trichohyalin peptides compared with controls. The data indicate that AA affected subjects present with an increased frequency of CTLs responsive to epitopes originating from keratinocytes and melanocytes; the activated CTLs secreted soluble factors that induced apoptosis in HF keratinocytes. Potentially, CTL response to self-antigen epitopes, particularly trichohyalin epitopes, could be a prognostic marker for human AA

Evaluation of heart collagen deposition and cardiac troponin-I (cTnI) in AA and sham-grafted mice.

<p>Areas encompassed by collagen, lumen, blood vessels, and blood vessels plus surrounding collagens, were measured with ImageJ (A, C, D). There was a higher amount of collagen infiltration into the blood vessels within the hearts of AA mice (B, AA left; Control right). AA mice had significantly larger regions of collagen deposition around blood vessels but had significantly thinner blood vessel wall thickness compared to normal controls. Three random readings performed from each of 3 different non-consecutive slides per mouse for AA (n = 4) and control (n = 4) mice. ELISA revealed a higher amount of serum cTnI associated with AA (n = 5 per group) compared to control mice, though not statistically significant (E). The concentration of cTnI in the heart tissue of AA mice was significantly higher than the control mice (F). Statistical significance was determined with Student’s <i>t</i>-test where *denotes p<0.05. Bar = 50 µm.</p

qPCR and ELISA analysis of <i>Il18</i> and related genes and protein expression in AA and sham-grafted mice during the onset of AA.

<p>Comparing AA mice to sham grafted mice (n = 3 per time point), <i>Il18</i> gene expression fold change in mouse hearts showed an increase in the expression pattern before the onset of AA, but decreased expression around the time of AA onset. <i>Il18r1</i>, <i>Il18bp</i> and <i>Casp1</i> (A–D) showed mostly significant elevation both before and after onset of AA. At the protein level, the expression of IL18, IL18R1 and IL18BP displayed similar trends as with qPCR (E–G). However, <i>Casp1</i> expression was significantly higher in sham grafted mice at 10 and 12 weeks (H). QPCR analyses for gene expression levels were calculated as fold change by using the 2^−ΔΔCt, average fold change presented. Error bars represent the range factor difference (2^{−ΔΔCt±ΔCtSD}). Statistical significance determined with Student’s <i>t</i>-test; *denotes p<0.05.</p