Profound Defect of Amphiregulin Secretion by Regulatory T Cells in the Gut of HIV-Treated Patients.

The persistence of a leaky gut in HIV-treated patients leads to chronic inflammation with increased rates of cardiovascular, liver, kidney, and neurological diseases. Tissue regulatory T (tTreg) cells are involved in the maintenance of intestinal homeostasis and wound repair through the IL-33 pathway. In this study, we investigated whether the persistence of gut mucosal injury during HIV infection might be explained in part by a flaw in the mechanisms involved in tissue repair. We observed an increased level of IL-33 in the gut of HIV-infected patients, which is associated with an increased level of fibrosis and a low peripheral reconstitution of CD4+ T cells. Our results showed that intestinal Treg cells from HIV-infected patients were enriched in tTreg cells prone to support tissue repair. However, we observed a functional defect in tTreg cells caused by the lack of amphiregulin secretion, which could contribute to the maintenance of intestinal damage. Our data suggest a mechanism by which the lack of amphiregulin secretion by tTreg may contribute to the lack of repair of the epithelial barrier.

Human lung fibroblasts may modulate dendritic cell phenotype and function: results from a pilot in vitro study.

In human lung fibrotic lesions, fibroblasts were shown to be closely associated with immature dendritic cell (DC) accumulation. The aim of the present pilot study was to characterize the role of pulmonary fibroblasts on DC phenotype and function, using co-culture of lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) and from control patients, with a DC cell line MUTZ-3. We observed that co-culture of lung control and IPF fibroblasts with DCs reduced the expression of specific DC markers and down-regulated their T-cell stimulatory activity. This suggests that pulmonary fibroblasts might sustain chronic inflammation in the fibrotic lung by maintaining in situ a pool of immature DCs.

Phenotypic and functional changes in dermal primary fibroblasts isolated from intrinsically aged human skin.

Dermal fibroblasts play a key role in maintaining skin homoeostasis by synthesizing and degrading extracellular matrix components. During ageing, they are subjected to changes, such as the loss of type I collagen expression and an increased synthesis of metalloproteinase I, leading to fragmentation of collagen fibrils with consequent reduction of the mechanical tension and defects of skin wound healing. Most information about fibroblast ageing was obtained from experiments performed on replicative-senescent dermal fibroblasts in vitro. However, the senescence status of fibroblasts isolated from intrinsically aged skins and its consequences on functionality need to be deeper investigated. Herein, we studied age-related phenotypic and functional alteration of fibroblasts from 'young' (<35 years) and 'old' (>50 years) donors. Our results brought evidence of the senescent status of 'old' fibroblasts by senescence associated ß-galactosidase (SA-ßgal) positive staining and p16 expression. A PCR array focusing on senescence highlighted a subset of downregulated genes including cell cycle progression and ECM genes in 'old' fibroblasts as well as a subset of upregulated genes involved in senescence features. In 'old' fibroblasts, we measured a downregulation of proliferative and contractile capacities of migratory potential under PDGF stimulation and activation into myofibroblasts under TGFß. Old fibroblasts were also more sensitive to oxidative stress than 'young' ones. Of interest, downregulation of p16 expression partially reversed the senescent phenotype of 'old' fibroblasts but failed to restore their functional properties. In conclusion, our data brought evidence of phenotypic and functional differences between fibroblasts from young and intrinsically aged skin that may contribute to the alterations observed with ageing.

Inducible expression and pathophysiologic functions of T-plastin in cutaneous T-cell lymphoma.

A molecular feature of Sézary syndrome (SS) is the abnormal expression of T-plastin by malignant T cells. Herein, we investigated the molecular mechanisms involved in T-plastin synthesis and the functions of this actin-binding protein, with a special interest in chemoresistance and migration. We confirm the specific expression of T-plastin in peripheral blood lymphocytes (PBLs) from SS patients and its total absence in PBLs from patients with mycosis fungoides, inflammatory cutaneous or hematologic diseases, and from healthy volunteers. Only 3 of 4 SS patients did constitutively express T-plastin. To assess whether T-plastin expression was inducible, T-plastin-negative PBLs were stimulated by phorbol 12-myristate 13-acetate and ionomycin. Our results demonstrate that T-plastin synthesis was induced in negative PBLs from SS patients, other studied patients, and healthy volunteers. Both constitutive and calcium-induced T-plastin expression was down-regulated by calcineurin inhibitors and involved nuclear factor of activated T cells transcription pathway. Constitutive T-plastin expression in SS was associated with resistance to etoposide-induced apoptosis and cell migration toward chemokines (TARC/CCL17, IP-10). In conclusion, T-plastin is a marker restricted to malignant lymphocytes from SS patients and plays a role for cell survival and migration. This opens new strategies for the treatment of SS advanced stages.

MHC class II engagement by its ligand LAG-3 (CD223) contributes to melanoma resistance to apoptosis.

Melanoma is the most aggressive skin cancer in humans that often expresses MHC class II (MHC II) molecules, which could make these tumors eliminable by the immune system. However, this MHC II expression has been associated with poor prognosis, and there is a lack of immune-mediated eradication. The lymphocyte activation gene-3 (LAG-3) is a natural ligand for MHC II that is substantially expressed on melanoma-infiltrating T cells including those endowed with potent immune-suppressive activity. Based on our previous data showing the signaling capacity of MHC II in melanoma cells, we hypothesized that LAG-3 could contribute to melanoma survival through its MHC II signaling capacity in melanoma cells. In this study, we demonstrate that both soluble LAG-3 and LAG-3-transfected cells can protect MHC II-positive melanoma cells, but not MHC II-negative cells, from FAS-mediated and drug-induced apoptosis. Interaction of LAG-3 with MHC II expressed on melanoma cells upregulates both MAPK/Erk and PI3K/Akt pathways, albeit with different kinetics. Inhibition studies using specific inhibitors of both pathways provided evidence of their involvement in the LAG-3-induced protection from apoptosis. Altogether, our data suggest that the LAG-3-MHC II interaction could be viewed as a bidirectional immune escape pathway in melanoma, with direct consequences shared by both melanoma and immune cells. In the future, compounds that efficiently hinder LAG-3-MHC II interaction might be used as an adjuvant to current therapy for MHC II-positive melanoma.

Hair follicle stem cell replication stress drives IFI16/STING-dependent inflammation in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic, relapsing, inflammatory skin disease. HS appears to be a primary abnormality in the pilosebaceous-apocrine unit. In this work, we characterized hair follicle stem cells (HFSCs) isolated from HS patients and more precisely the outer root sheath cells (ORSCs). We showed that hair follicle cells from HS patients had an increased number of proliferating progenitor cells and lost quiescent stem cells. Remarkably, we also showed that the progression of replication forks was altered in ORSCs from hair follicles of HS patients, leading to activation of the ATR/CHK1 pathway. These alterations were associated with an increased number of micronuclei and with the presence of cytoplasmic ssDNA, leading to the activation of the IFI16/STING pathway and the production of type I IFNs. This mechanistic analysis of the etiology of HS in the HFSC compartment establishes a formal link between genetic predisposition and skin inflammation observed in HS.

Inhibition of IkappaB kinase subunit 2 in cutaneous T-cell lymphoma down-regulates nuclear factor-kappaB constitutive activation, induces cell death, and potentiates the apoptotic response to antineoplastic chemotherapeutic agents.

PURPOSE: A key molecular feature of cutaneous T-cell lymphomas (CTCL) is the constitutive activation of the nuclear factor-kappaB (NF-kappaB) transcription factor. We investigated in vitro the effects on CTCL survival and chemoresistance of a specific inhibition of IkappaB kinase subunit 2 (IKK2). EXPERIMENTAL DESIGN: Selective IKK2 inhibition was carried out by transfection of SeAx and MyLa CTCL lines with an inactive form of IKK2 and by exposing these lines and tumor cells from 10 patients with Sézary syndrome (SS) to AS602868, a new IKK2 inhibitor. The constitutive nuclear translocation of NF-kappaB was analyzed by electrophoretic mobility shift assay and confocal microscopy. Apoptosis was determined by Annexin V/propidium iodide-positive staining and mitochondrial transmembrane potential alterations as well as poly(ADP-ribose)polymerase cleavage. The expression of Bcl-2 family oncoproteins and survivin was studied by immunoblotting. RESULTS: Specific IKK2 inhibition resulting from transfection or from incubation with AS602868 allowed a down-regulation of NF-kappaB transcriptional activity. As shown by electrophoretic mobility shift assay and apoptosis assays, AS602868 down-regulated the nuclear translocation of NF-kappaB and induced a potent apoptotic response in CTCL lines and in tumor cells from patients with SS while preserving the viability of both peripheral blood lymphocytes from healthy donors and of nonmalignant T cells from SS patients. Moreover, CTCL death induction by conventional antineoplastic agents etoposide and vincristine was potentiated by AS602868. Finally, AS602868-induced apoptosis of CTCL cells was associated with an up-regulation of Bax dimers and a decrease of survivin. CONCLUSION: These results indicate that IKK2 inhibition represents a promising strategy for the treatment of advanced stages of CTCL.

Photobiomodulation therapy promotes in vitro wound healing in nicastrin KO HaCaT cells.

Mutations in NCSTN gene (encoding for nicastrin protein) are associated with hidradenitis suppurativa (HS), a chronic inflammatory disease involving hair follicles. HS is clinically handled with drugs but the most severe cases are treated with surgery. Photobiomodulation (PBM) therapy, already used in the treatment of skin diseases such as acne, herpes virus lesions, ultraviolet damage, vitiligo, hypertrophic scar, keloid, burn, psoriasis and diabetic chronic wounds, could be beneficial as an adjuvant supportive treatment to promote and foster the healing process after skin excision in HS. The effects of PBM therapy in promoting the wound closure are evaluated in a HaCaT cells NCSTN-/-, assessing cell metabolism, migration rate, proliferation and cell cycle progression. In our experimental model, PBM exerts a potent action on metabolism of mutated keratinocytes, incrementing adenosine triphosphate (ATP) production at 2 hours, while after 24 hours an increase of metabolism with a decrement of intracellular ATP levels were recorded. Moreover, PBM speeds up the wound closure, inducing cells' migration without affecting their proliferation.Based on our findings, we suggest the use of PBM in HS patients, who undergo major surgery with large skin excision.

Expression, subcellular localization and cytokinic modulation of Toll-like receptors (TLRs) in normal human keratinocytes: TLR2 up-regulation in psoriatic skin.

The aim of the research described here was to investigate the expression of Toll-like receptors (TLRs) in normal human keratinocytes, to study its modulation by proinflammatory cytokines, and to characterize the function of the latter within the epidermis. Our results demonstrate that normal human keratinocytes may present an intra-cytoplasmic expression of TLR2, TLR3, and TLR4. Exposure of keratinocytes to IFN-gamma and TNF-alpha increased intra-cytoplasmic expression and led to partial translocation at the cell surface. Keratinocyte activation by TLR2, TLR3, and TLR4 ligands led to the nuclear translocation of NF-kappab and the release of proinflammatory cytokines TNF-alpha and IL-8. In immunochemistry analysis, psoriatic skin showed a strong over-expression of TLR2 in the epidermis compared with normal skin. Our results thus demonstrate large TLR expression in keratinocytes and the functionality of TLRs 2, 3, and 4. TLR2 over-expression in psoriatic skin provides new insights into TLR implication in the pathogenesis of psoriasis, through inappropriate stimulation by infectious or endogen ligands.

Intrinsic Defect in Keratinocyte Function Leads to Inflammation in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic, inflammatory, debilitating, follicular disease of the skin. Despite a high prevalence in the general population, the physiopathology of HS remains poorly understood. The use of antibiotics and immunosuppressive agents for therapy suggests a deregulated immune response to microflora. Using cellular and gene expression analyses, we found an increased number of infiltrating CD4(+) T cells secreting IL-17 and IFN-γ in perilesional and lesional skin of patients with HS. By contrast, IL-22-secreting CD4(+) T cells are not enriched in HS lesions contrasting with increased number of those cells in the blood of patients with HS. We showed that keratinocytes isolated from hair follicles of patients with HS secreted significantly more IL-1ß, IP-10, and chemokine (C-C motif) ligand 5 (RANTES) either constitutively or on pattern recognition receptor stimulations. In addition, they displayed a distinct pattern of antimicrobial peptide production. These findings point out a functional defect of keratinocytes in HS leading to a balance prone to inflammatory responses. This is likely to favor a permissive environment for bacterial infections and chronic inflammation characterizing clinical outcomes in patients with HS.

Transforming growth factor-ß increases interleukin-13 synthesis via GATA-3 transcription factor in T-lymphocytes from patients with systemic sclerosis.

INTRODUCTION: Transforming growth factor (TGF)-ß and interleukin (IL)-13 play a crucial role in the pathogenesis of systemic sclerosis (SSc), partly through activation of collagen production that leads to fibrosis. The aim of the present study was to determine whether TFG-ß alters IL-13 production in T lymphocytes from patients with SSc from that seen in those of healthy donors. METHODS: IL-13 mRNA and protein synthesis under TFG-ß exposure was measured in circulating T lymphocytes from healthy donors and patients with SSc and also in the Jurkat Th2 T-cell line, using quantitative real-time PCR and fluorescence-activated cell sorting analysis, respectively. The involvement of Smad and GATA-3 transcription factors was assessed by using specific inhibitors and small interfering RNA, and the binding capacity of GATA-3 to the IL-13 gene promoter was evaluated by chromatin immunoprecipitation assay. RESULTS: TGF-ß induced a significant decrease in IL-13 mRNA and protein levels in lymphocytes from healthy donors (mean [±SD] inhibition of 30% ± 10% and 20% ± 7%, respectively; p < 0.05). In contrast, TGF-ß promoted a significant increase in IL-13 mRNA levels and IL-13 synthesis by CD4(+) and CD8(+) T-cell subtypes from patients with SSc, with respective increases of 2.4 ± 0.3-fold, 1.6 ± 0.05-fold and 2.7 ± 0.02-fold. The involvement of the Smad signaling pathway and upregulation of GATA-3 binding capacity on the IL-13 promoter in lymphocytes from patients with SSc contributed to the effect of TGF-ß on IL-13 production. CONCLUSIONS: These results demonstrate that TGF-ß upregulates IL-13 synthesis through GATA-3 expression in the T lymphocytes of patients with SSc, confirming that the GATA-3 transcription factor can be regarded as a novel therapeutic target in patients with SSc.

Arsenic trioxide induces apoptosis of cutaneous T cell lymphoma cells: evidence for a partially caspase-independent pathway and potentiation by ascorbic acid (vitamin C).

Arsenic trioxide (As2O3) displays apoptogenic properties against various types of hematopoietic malignancies. We investigated the effects of As2O3 on the viability of the cutaneous T cell lymphoma cell lines HuT-78, SeAx, and Myla, and of peripheral blood mononuclear cells from patients with Sézary syndrome, by using propidium iodide and annexin-V staining, terminal deoxyuridine triphosphate nick end labeling (TUNEL), cell cycle analysis, mitochondrial transmembrane potential (delta psi(m)) alterations, cytochrome c release, and detection of processed caspase-3. We also report in vivo effects of As2O3 in two patients with cutaneous T cell lymphoma. The results show that As2O3 induces apoptosis of cutaneous T cell lymphoma lines and of Sézary cells from patients in a time- and concentration-dependent manner in vitro, as demonstrated by annexin-V staining, mitochondrial depolarization, and DNA fragmentation. Ascorbic acid 100 microM potentiated As2O3-induced Sézary cell death, whereas interferon-alpha had no synergistic effect. As2O3-induced Sézary cell death involves activation of caspase-3, cleavage of poly(ADP-ribose)polymerase, and cytochrome c release, but was only partially inhibited by the pancaspase inhibitor Z-VAD.fluoromethylketone. Finally, As2O3 was administered to two patients with cutaneous T cell lymphoma, allowing us to obtain a partial response in one case, whereas stability was observed in the second patient. These results demonstrate that As2O3 synergizes with ascorbic acid to induce Sézary cell death at clinically achievable concentrations, through a caspase-partially independent pathway, and provide a rationale for further in vivo studies addressing the therapeutic efficacy of As2O3 in cutaneous T cell lymphoma patients.

T-plastin expression downstream to the calcineurin/NFAT pathway is involved in keratinocyte migration.

Cutaneous wound healing requires keratinocyte proliferation, migration and differentiation to restore the barrier function of the skin. The calcineurin/nuclear factor of activated-T-cell (NFAT) signaling pathway has been recently shown to be involved in keratinocyte growth, differentiation and migration. It is induced by an increased intracellular calcium rate and its inhibition results in decreased capacities of keratinocytes to migrate. Nevertheless, the link between calcineurin activation and keratinocyte migration remains unknown. Recently, Orai1, a pore subunit of a store-operated calcium channel that favors calcium influx, was shown to play a critical role to control proliferation and migration of basal keratinocytes. Of interest, the actin-bundling T-plastin is crucial in cell motility through cross-linking to actin filament and its synthesis was shown to be induced by calcium influx and regulated by the calcineurin/NFAT pathway in tumor Sezary cells. We investigated herein the role of the calcineurin/NFAT pathway-dependent T-plastin in keratinocyte migration, by quantifying T-plastin expression in keratinocytes and by analyzing their migration under calcineurin inhibition or knockdown of NFAT2 or T-plastin. We did confirm the role of the calcineurin/NFAT pathway in keratinocyte migration as shown by their decreased capacities to migrate after FK506 treatment or siNFAT2 transfection in both scratching and Boyden assays. The expression of NFAT2 and T-plastin in keratinocytes was decreased under FK506 treatment, suggesting that T-plastin plays a role in keratinocyte migration downstream to the calcineurin/NFAT pathway. Accordingly, siRNA knockdown of T-plastin expression also decreased their migration capacities. Actin lamellipodia formation as well as FAK and ß6-integrin expression were also significantly decreased after treatment with FK506 or siRNA, reinforcing that NFAT2-dependent T-plastin expression plays a role in keratinocyte migration. These results indicate that T-plastin might be considered as a major actor in the mechanisms underlying calcineurin/NFAT-dependent keratinocyte migration and may explain wound-healing defects observed in patients under calcineurin inhibitor long-term treatment.

Expression of transforming growth factor ß receptor II in mesenchymal stem cells from systemic sclerosis patients.

OBJECTIVES: The present work aimed to evaluate the expression of transforming growth factor-ß (TGF-ß) receptors on bone marrow-derived multipotent mesenchymal stromal cells (MSCs) in patients with systemic sclerosis (SSc) and the consequences of TGF-ß activation in these cells, since MSC have potential therapeutic interest for SSc patients and knowing that TGF-ß plays a critical role during the development of fibrosis in SSc. DESIGN: This is a prospective research study using MSC samples obtained from SSc patients and compared with MSC from healthy donors. SETTING: One medical hospital involving collaboration between an internal medicine department for initial patient recruitment, a clinical biotherapeutic unit for MSC preparation and an academic laboratory for research. PARTICIPANTS: 9 patients with diffuse SSc for which bone marrow (BM) aspiration was prescribed by sternum aspiration before haematopoietic stem cell transplantation, versus nine healthy donors for normal BM. PRIMARY AND SECONDARY OUTCOME MEASURES: TGF-ß, TGF-ß receptor types I (TBRI) and II (TBRII) mRNA and protein expression were assessed by quantitative PCR and flow cytometry, respectively, in MSC from both SSc patients and healthy donors. MSC were exposed to TGF-ß and assessed for collagen 1α2 synthesis and Smad expression. As positive controls, primary cultures of dermal fibroblasts were also analysed. RESULTS: Compared with nine controls, MSC from nine SSc patients showed significant increase in mRNA levels (p<0.002) and in membrane expression (p<0.0001) of TBRII. In response to TGF-ß activation, a significant increase in collagen 1α synthesis (p<0.05) and Smad-3 phosphorylation was upregulated in SSc MSC. Similar results were obtained on eight SSc-derived dermal fibroblasts compared to six healthy controls. CONCLUSIONS: TBRII gene and protein expression defect in MSC derived from SSc patients may have pathological significance. These findings should be taken into account when considering the use of MSC-based therapies in an autologous setting.

Down-regulating constitutive activation of the NF-kappaB canonical pathway overcomes the resistance of cutaneous T-cell lymphoma to apoptosis.

Constitutive activation of the nuclear factor-kappaB (NF-kappaB) pathway has been shown to be involved in the resistance of tumor cells to apoptosis in several human malignancies of the hematopoietic lineage. By using electrophoretic mobility shift assay (EMSA) and confocal microscopic analysis, we demonstrate that NF-kappaB is constitutively activated in cutaneous T-cell lymphoma (CTCL) cell lines HuT-78, MyLa, and SeAx and in peripheral blood lymphocytes (PBLs) from patients with Sézary syndrome (SS) presenting a high ratio of tumor cells, with evidence of p50 and RelA/p65 in DNA-linked complexes. Transfection of SeAx line with a kappaB/luciferase reporter plasmid showed that translocated NF-kappaB complexes were functional. Selective inhibition of NF-kappaB, by transfecting CTCL cell lines with a super-repressor form of IkappaB alpha, led to apoptosis. We evidenced down-regulation of NF-kappaB activation and induction of CTCL cell apoptosis in the presence of proteasome 26S inhibitors ALLN, MG132, and bortezomib. Bortezomib at nanomolar concentrations inhibited constitutive activation of NF-kappaB and induced apoptosis of CTCL cells, with evidence of an upregulation of Bax expression. These results demonstrate the key role played by NF-kappaB in the resistance of CTCL to apoptosis and suggest that bortezomib might be useful for the treatment of patients with advanced stages of CTCL refractory to standard antineoplastic chemotherapy.

Expression and activity of IL-17 in cutaneous T-cell lymphomas (mycosis fungoides and Sezary syndrome).

Interleukin-17 (IL-17) is a proinflammatory cytokine mainly produced by activated CD4+ CD45RO T cells. In mice, we have demonstrated that, depending on the model, IL-17 may act as a tumor growth-promoting or -inhibiting factor. In order to address the relevance of these models in human tumors, we look for the natural expression and activity of IL-17 in mycosis fungoides (MF) and Sezary syndrome (SS). These cutaneous T-cell lymphomas were selected because they are usually CD3+ CD4+ CD45RO+, a phenotype similar to nontransformed T cells producing IL-17. We show that in vitro activated malignant T cells derived from MF or SS patients express IL-17 mRNA and secrete this cytokine. However, IL-17 does not act in vitro as a growth factor for MF or SS cell lines. In addition, 5 out of 10 MF/SS biopsies expressed IL-17 mRNA, while this cytokine was not detected in normal skin. IL-17 was not observed in the biopsies derived from 2 patients initially identified as MF, whereas an upregulation of this cytokine was clearly demonstrated during progression of the disease in these patients. An association between IL-17 expression and polymorphonuclear neutrophil infiltration was also recorded in this group of MF/SS patients. A more detailed analysis of 2 patients with a pustular form of MF and SS revealed that IL-17 may participate in the recruitment of polymorphonuclear neutrophils via a paracrine mechanism involving keratinocyte-released IL-8. This study is the first report demonstrating that some human tumor cells could express IL-17, a cytokine that represents an early event in the development of the inflammatory reaction within the tumor microenvironment, a process that may influence tumor phenotype and growth.