Wnt signalling is required for hair follicle development and for the growth phase (anagen) of postnatal follicles. When the pathway is activated at high levels in adult mouse epidermis, ectopic follicles form from existing follicles, interfollicular epidermis (IFE) and sebaceous glands, revealing a remarkable ability of the tissue to be reprogrammed. To compare the competence of different epidermal cell populations to form ectopic follicles, we expressed a 4-hydroxy-tamoxifen (4OHT) inducible, stabilised beta-catenin transgene (DeltaNbeta-cateninER) under the control of two different promoters. We targeted the reservoir of stem cells in the hair follicle bulge via the keratin 15 (K15) promoter and targeted the sebaceous glands and base of the follicle (bulb) with a truncated K5 promoter (DeltaK5). No ectopic follicles formed in the IFE in either model, establishing the autonomy of the IFE stem cell compartment in undamaged epidermis. Activation of beta-catenin in the bulge stimulated proliferation and bulge expansion. Existing hair follicles entered anagen, but no ectopic follicles formed. DeltaK5DeltaNbeta-cateninER expressing hair follicles also entered anagen on 4OHT treatment. In addition, a subpopulation of cells at the base of the sebaceous gland readily formed ectopic follicles, resulting in complete and reversible conversion of sebaceous glands into hair follicles. Combined activation of beta-catenin and the vitamin D receptor enhanced differentiation of sebaceous gland-derived hair follicles and stimulated ectopic follicle formation in the hair follicle bulb, but not in the bulge. Our results suggest that the bulge and sebaceous gland are, respectively, non-permissive and permissive niches for Wnt induced hair follicle differentiation.

The nuclear vitamin D receptor (VDR) is generally recognized as a ligand-dependent transcription factor that mediates the actions of its natural ligand, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on multiple target genes involved in mineral homeostasis, bone development, as well as immune reactivity. As the VDR is widely distributed in nearly all cells of the body, it implies that the vitamin D endocrine system may regulate many cell types and functions. Experiments in VDR null mice established that the VDR has intrinsically critical roles in skin and keratinocyte biology but not in immune responses. Oppositely, absence of the VDR ligand is linked to susceptibility to autoimmunity, illustrating a potential role for the unliganded VDR in the immune system. This discrepancy stimulated us to further investigate the impact of the VDR on the phenotype and function of myeloid dendritic cells (DCs) generated ex vivo from bone marrow precursors of VDR null (with a truncated VDR) and VDR ΔAF2 mice (with a mutated C-terminal activation factor 2 domain thus rendering ligand-induced gene transcription impossible). Absent or unliganded VDR did not affect bone marrow-derived myeloid DC generation. DCs obtained from VDR null and VDR ΔAF2 bone marrow cells had comparable MHC-II, and costimulatory molecule CD86, CD80 and CD40 expression than DCs from wild-type bone marrow cells. Additionally, an unliganded VDR did not affect the cytokine production nor the antigen-specific T cell stimulatory capacity of bone marrow-derived DCs. In conclusion, we showed that although clear effects of 1α,25-dihydroxyvitamin D3 are described on DC generation, absence of VDR or presence of an unliganded VDR does not affect the profile and function of ex vivo generated bone marrow-derived DCs.

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12-20 or InsB13-2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function.

Chronic obstructive pulmonary disease (COPD) is characterized by a progressive and abnormal inflammatory response in the lungs, mainly caused by cigarette smoking. Animal models exposed to cigarette smoke (CS) are used to mimic human COPD but the use of different CS protocols makes it difficult to compare the immunological and structural consequences of using a nose-only or whole-body CS exposure system. We hypothesized that when using a standardized CS exposure protocol based on particle density and CO (carbon monoxide) levels, the whole-body CS exposure system would generate a more severe inflammatory response than the nose-only system, due to possible sensitization by uptake of CS-components through the skin or via grooming.

Sex differences are increasingly recognized to play an important role in the epidemiology, treatment and outcomes of many diseases. This study aims to describe differences between sexes in patient characteristics, ulcer severity and outcome after 6 months in individuals with a diabetic foot ulcer (DFU).

Active vitamin D3, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], and its synthetically derived analogs possess potent anticancer properties. In breast cancer (BC) cells, 1,25(OH)2D3 blocks cell proliferation and induces apoptosis through different cell-type specific mechanisms. In this study, we evaluated if the combination of the potent vitamin D3 analog, inecalcitol, with a selective CDK4/6 inhibitor, palbociclib, enhanced the antiproliferative effects of both single compounds in hormone-sensitive (ER+) BC, for which palbociclib treatment is already approved, but also in triple-negative BC (TNBC). Inecalcitol and palbociclib combination treatment decreased cell proliferation in both ER+ (T47D-MCF7) and TNBC (BT20-HCC1143-Hs578T) cells, with a more pronounced antiproliferative effect in the former. In ER+ BC cells, the combination therapy downregulated cell cycle regulatory proteins (p)-Rb and (p)-CDK2 and blocked G1-S phase transition of the cell cycle. Combination treatment upregulated p-mTOR and p-4E-BP1 protein expression in MCF7 cells, whereas it suppressed expression of these proteins in BT20 cells. Cell survival was decreased after inecalcitol treatment either alone or combined in MCF7 cells. Interestingly, the combination therapy upregulated mitochondrial ROS and mitotracker staining in both cell lines. Furthermore, in vivo validation in a MCF7 cell line-derived xenograft mouse model decreased tumor growth and cell cycle progression after combination therapy, but not in a TNBC BT20 cell line-derived xenograft model. In conclusion, we show that addition of a potent vitamin D3 analog to selective CDK4/6 inhibitor treatment results in increased antiproliferative effects in ER+ BC both in vitro and in vivo.

We investigated the functions of critical adhesion molecules ICAM-1 and VCAM-1 in a keratin-14 IL-4-transgenic (Tg) mouse model of atopic dermatitis, the skin lesions of which are characterized by prominent inflammatory cell infiltration, significantly increased mRNAs and proteins of ICAM-1, VCAM-1, E-selectin, P-selectin, L-selectin, and PSGL-1, and significantly increased numbers of dermal vessels expressing these adhesion molecules. We tested the hypotheses that deletion or blockade of these molecules may impede the inflammation by examining the disease progresses in the Tg mice crossed with ICAM-1-knockout mice and Tg mice received anti-VCAM-1-neutralizing antibody. Although the findings of the ICAM-1-knockout Tg mice (Tg/ICAM-1(-/-)) developed skin lesions similar to wide-type ICAM-1 Tg mice (Tg/ICAM-1(+/+)) were surprising, a compensatory mechanism may account for it: the frequency of VCAM-1 ligand, CD49d, on CD3(+) T cells in the lesional skin significantly increased in the Tg/ICAM-1(-/-) mouse, compared with the Tg/ICAM-1(+/+) mice. In contrast, anti-VCAM-1-treated Tg/ICAM-1(-/-) or Tg/ICAM-1(+/+) mice had significantly delayed onset of skin inflammation compared with isotype antibody-treated groups. Moreover, anti-VCAM-1 significantly reduced the skin inflammation severity in Tg/ICAM-1(+/+) mice, accompanied with reduction of mast cell, eosinophil, and CD3(+) T cell infiltration. VCAM-1 is more critical in developing skin inflammation in this model.

The vitamin D endocrine system is essential for calcium metabolism and skeletal integrity. 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] regulates bone mineral homeostasis and acts directly on osteoblasts. In the present study we characterized the transcriptional regulation of the class 3 semaphorin (Sema3) gene family by 1,25(OH)2D3 in osteoblastic cells. Class 3 semaphorins are secreted proteins that regulate cell growth, morphology and migration, and were recently shown to be involved in bone homeostasis. In ST2, MC3T3-E1 and primary calvarial osteoblast cell cultures we found that all members of the Sema3 gene family were expressed, and that Sema3e and Sema3f were the most strongly induced 1,25(OH)2D3 target genes among the studied cell types. In addition, transcription of Sema3b and Sema3c was upregulated, whereas Sema3d and Sema3g was downregulated by 1,25(OH)2D3 in different osteoblastic cells. Chromatin immunoprecipitation analysis linked to DNA sequencing (ChIP-seq analysis) revealed the presence of the vitamin D receptor at multiple genomic loci in the proximity of Sema3 genes, demonstrating that the genes are primary 1,25(OH)2D3 targets. Furthermore, we showed that recombinant SEMA3E and SEMA3F protein were able to inhibit osteoblast proliferation. However, recombinant SEMA3s did not affect ST2 cell migration. The expression of class 3 semaphorins in osteoblasts together with their regulation by 1,25(OH)2D3 suggests that these genes, involved in the regulation of bone homeostasis, are additional mediators for 1,25(OH)2D3 signaling in osteoblasts.

Inflammation, including reactive oxygen species and inflammatory cytokines in tissues amplify various post-translational modifications of self-proteins. A number of post-translational modifications have been identified as autoimmune biomarkers in the initiation and progression of Type 1 diabetes. Here we show the citrullination of pancreatic glucokinase as a result of inflammation, triggering autoimmunity and affecting glucokinase biological functions. Glucokinase is expressed in hepatocytes to regulate glycogen synthesis, and in pancreatic beta cells as a glucose sensor to initiate glycolysis and insulin signaling. We identify autoantibodies and autoreactive CD4+ T cells to glucokinase epitopes in the circulation of Type 1 diabetes patients and NOD mice. Finally, citrullination alters glucokinase biologic activity and suppresses glucose-stimulated insulin secretion. Our study define glucokinase as a Type 1 diabetes biomarker, providing new insights of how inflammation drives post-translational modifications to create both neoautoantigens and affect beta cell metabolism.

Increasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2) is a necessary step for SARS-CoV-2 infection permissiveness. In light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyze ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing β-cells. ACE2 is also highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human β-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increase ACE2 expression in the β-cell line EndoC-βH1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct β-cell virus tropism.

In type 1 diabetes, autoimmune β-cell destruction may be favored by neoantigens harboring posttranslational modifications (PTMs) such as citrullination. We studied the recognition of native and citrullinated glucose-regulated protein (GRP)78 peptides by CD8+ T cells. Citrullination modulated T-cell recognition and, to a lesser extent, HLA-A2 binding. GRP78-reactive CD8+ T cells circulated at similar frequencies in healthy donors and donors with type 1 diabetes and preferentially recognized either native or citrullinated versions, without cross-reactivity. Rather, the preference for native GRP78 epitopes was associated with CD8+ T cells cross-reactive with bacterial mimotopes. In the pancreas, a dominant GRP78 peptide was instead preferentially recognized when citrullinated. To further clarify these recognition patterns, we considered the possibility of citrullination in the thymus. Citrullinating peptidylarginine deiminase (Padi) enzymes were expressed in murine and human medullary epithelial cells (mTECs), with citrullinated proteins detected in murine mTECs. However, Padi2 and Padi4 expression was diminished in mature mTECs from NOD mice versus C57BL/6 mice. We conclude that, on one hand, the CD8+ T cell preference for native GRP78 peptides may be shaped by cross-reactivity with bacterial mimotopes. On the other hand, PTMs may not invariably favor loss of tolerance because thymic citrullination, although impaired in NOD mice, may drive deletion of citrulline-reactive T cells.

Achieving weight loss is the cornerstone of the treatment of the metabolic consequences of obesity, in particular of glucose intolerance.

The emergence of regulatory T cells (Tregs) as central mediators of peripheral tolerance in the immune system has led to an important area of clinical investigation to target these cells for the treatment of autoimmune diseases such as type 1 diabetes. We have demonstrated earlier that in vitro treatment of T cells from healthy individuals with TX527, a low-calcemic analog of bioactive vitamin D, can promote a CD4+ CD25high CD127low regulatory profile and imprint a migratory signature specific for homing to sites of inflammation. Towards clinical application of vitamin D-induced Tregs in autologous adoptive immunotherapy for type 1 diabetes, we show here that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and TX527 similarly imprint T cells from type 1 diabetes patients with a CD4+ CD25high CD127low regulatory profile, modulate surface expression of skin- and inflammation-homing receptors, and increase expression of CTLA-4 and OX-40. Also, 1,25(OH)2D3 and TX527 treatment inhibit the production of effector cytokines IFN-γ, IL-9, and IL-17. Importantly, 1,25(OH)2D3 and TX527 promote the induction of IL-10-producing CD4+ CD25high CD127low T cells with a stable phenotype and the functional capacity to suppress proliferation of autologous responder T cells in vitro. These findings warrant additional validation of vitamin D-induced Tregs in view of future autologous adoptive immunotherapy in type 1 diabetes.

The detection of cis-regulatory modules (CRMs) that mediate transcriptional responses in eukaryotes remains a key challenge in the postgenomic era. A CRM is characterized by a set of co-occurring transcription factor binding sites (TFBS). In silico methods have been developed to search for CRMs by determining the combination of TFBS that are statistically overrepresented in a certain geneset. Most of these methods solve this combinatorial problem by relying on computational intensive optimization methods. As a result their usage is limited to finding CRMs in small datasets (containing a few genes only) and using binding sites for a restricted number of transcription factors (TFs) out of which the optimal module will be selected.

Post-translational modifications can lead to a break in immune tolerance in autoimmune diseases such as type 1 diabetes (T1D). Deamidation, the conversion of glutamine to glutamic acid by transglutaminase (TGM) enzymes, is a post-translational modification of interest, with deamidated peptides being reported as autoantigens in T1D. However, little is known about how Tgm2, the most ubiquitously expressed Tgm isoform, is regulated and how tolerance against deamidated peptides is lost. Here, we report on the aberrant expression and regulation of Tgm2 in the pancreas and thymus of NOD mice. We demonstrate that Tgm2 expression is induced by the inflammatory cytokines IL1β and IFNγ in a synergistic manner and that murine pancreatic islets of NOD mice have higher Tgm2 levels, while Tgm2 levels in medullary thymic epithelial cells are reduced. We thus provide the first direct evidence to our knowledge that central tolerance establishment against deamidated peptides might be impaired due to lower Tgm2 expression in NOD medullary thymic epithelial cells, which together with the aberrantly high levels of deamidated peptides in NOD β-cells underscores the role of deamidation in amplifying T-cell reactivity.

The hormonally-active form of vitamin D, 1,25-dihydroxyvitamin D3, can modulate both innate and adaptive immunity, through binding to the nuclear vitamin D receptor expressed in most immune cells. A high dose of regular vitamin D protected non-obese diabetic (NOD) mice against type 1 diabetes (T1D), when initiated at birth and given lifelong. However, considerable controversy exists on the level of circulating vitamin D (25-hydroxyvitamin D3, 25(OH)D3) needed to modulate the immune system in autoimmune-prone subjects and protect against T1D onset. Here, we evaluated the impact of two doses of dietary vitamin D supplementation (400 and 800 IU/day), given to female NOD mice from 3 until 25 weeks of age, on disease development, peripheral and gut immune system, gut epithelial barrier function, and gut bacterial taxonomy. Whereas serum 25(OH)D3 concentrations were 2.6- (400 IU/day) and 3.9-fold (800 IU/day) higher with dietary vitamin D supplementation compared to normal chow (NC), only the 800 IU/day vitamin D-supplemented diet delayed and reduced T1D incidence compared to NC. Flow cytometry analyses revealed an increased frequency of FoxP3+ Treg cells in the spleen of mice receiving the 800 IU/day vitamin D-supplemented diet. This vitamin D-induced increase in FoxP3+ Treg cells, also expressing the ecto-5'-nucleotidase CD73, only persisted in the spleen of mice at 25 weeks of age. At this time point, the frequency of IL-10-secreting CD4+ T cells was also increased in all studied immune organs. High-dose vitamin D supplementation was unable to correct gut leakiness nor did it significantly modify the increased gut microbial diversity and richness over time observed in NOD mice receiving NC. Intriguingly, the rise in alpha-diversity during maturation occurred especially in mice not progressing to hyperglycaemia. Principal coordinates analysis identified that both diet and disease status significantly influenced the inter-individual microbiota variation at the genus level. The abundance of the genera Ruminoclostridium_9 and Marvinbryantia gradually increased or decreased, respectively in faecal samples of mice on the 800 IU/day vitamin D-supplemented diet compared to mice on the 400 IU/day vitamin D-supplemented diet or NC, irrespective of disease outcome. In summary, dietary vitamin D reduced T1D incidence in female NOD mice at a dose of 800, but not of 400, IU/day, and was accompanied by an expansion of Treg cells in various lymphoid organs and an altered intestinal microbiota signature.

Tolerogenic dendritic cells (tolDCs) instruct regulatory T cells (Tregs) to dampen autoimmunity. Active vitamin D3 (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) imprints human monocyte-derived DCs with tolerogenic properties by reprogramming their glucose metabolism. Here we identify the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) as a critical checkpoint and direct transcriptional target of 1α,25(OH)2D3 in determining the tolDC profile. Using tracer metabolomics, we show that PFKFB4 activity is essential for glucose metabolism, especially for glucose oxidation, which is elevated upon 1α,25(OH)2D3 exposure. Pharmacological inhibition of PFKFB4 reversed the 1α,25(OH)2D3-mediated shift in metabolism, DC profile and function, as determined by expression of inhibitory surface markers and secretion of regulatory cytokines and factors. Moreover, PFKFB4 inhibition in 1α,25(OH)2D3-treated DCs blocked their hallmark capacity to induce suppressive Tregs. This work demonstrates that alterations in the bioenergetic metabolism of immune cells are central to the immunomodulatory effects induced by 1α,25(OH)2D3.

In chronic obstructive pulmonary disease (COPD), the bronchial epithelium is the first immune barrier that is triggered by cigarette smoke. Although vitamin D (vitD) has proven anti-inflammatory and antimicrobial effects in alveolar macrophages, little is known about the direct role of vitD on cigarette smoke-exposed bronchial epithelial cells. We examined the effects of vitD on a human bronchial epithelial cell line (16HBE) and on air-liquid culture of primary bronchial epithelial cells (PBEC) of COPD patients and controls exposed for 24 h to cigarette smoke extract (CSE). VitD decreased CSE-induced IL-8 secretion by 16HBE cells, but not by PBEC. VitD significantly increased the expression of the antimicrobial peptide cathelicidin in 16HBE and PBEC of both COPD subjects and controls. VitD did not affect epithelial to mesenchymal transition or epithelial MMP-9 expression and was not able to restore impaired wound healing by CSE in 16HBE cells. VitD increased the expression of its own catabolic enzyme CYP24A1 thereby maintaining its negative feedback. In conclusion, vitD supplementation may potentially reduce infectious exacerbations in COPD by the upregulation of cathelicidin in the bronchial epithelium.

The measurement of 1α,25(OH)2D3 in human serum poses a true challenge as concentrations are very low and structurally similar metabolites can interfere.

Neuropilin 2 (NRP2) mediates the effects of class 3 semaphorins and vascular endothelial growth factor and is implicated in axonal guidance and angiogenesis. Moreover, NRP2 expression is suggested to be involved in the regulation of bone homeostasis. Indeed, osteoblasts and osteoclasts express NRP2 and male and female global Nrp2 knockout mice have a reduced bone mass accompanied by reduced osteoblast and increased osteoclast counts.