Plasmacytoid Dendritic Cells Are Not Major Producers of Type 1 IFN in Cutaneous Lupus: An In-Depth Immunoprofile of Subacute and Discoid Lupus.

The immunologic drivers of cutaneous lupus erythematosus (CLE) and its clinical subtypes remain poorly understood. We sought to characterize the immune landscape of discoid lupus erythematosus and subacute CLE using multiplexed immunophenotyping. We found no significant differences in immune cell percentages between discoid lupus erythematosus and subacute CLE (P > .05) with the exception of an increase in TBK1 in discoid lupus erythematosus (P < .05). Unbiased clustering grouped subjects into 2 major clusters without respect to clinical subtype. Subjects with a history of smoking had increased percentages of neutrophils, disease activity, and endothelial granzyme B compared with nonsmokers. Despite previous assumptions, plasmacytoid dendritic cells (pDCs) did not stain for IFN-1. Skin-eluted and circulating pDCs from subjects with CLE expressed significantly less IFNα than healthy control pDCs upon toll-like receptor 7 stimulation ex vivo (P < .0001). These data suggest that discoid lupus erythematosus and subacute CLE have similar immune microenvironments in a multiplexed investigation. Our aggregated analysis of CLE revealed that smoking may modulate disease activity in CLE through neutrophils and endothelial granzyme B. Notably, our data suggest that pDCs are not the major producers of IFN-1 in CLE. Future in vitro studies to investigate the role of pDCs in CLE are needed.

Fat grafts enriched with ex-vivo expanded adipose stem cells improve hyperpigmentation of face.

Facial contour deformities associated with pigmentary changes are of major concern for plastic surgeons being difficult to treat. No definite ideal treatment option is available to address simultaneously both the contour defects as well as related hyperpigmentation. The aim of the current study is to compare long-term effects of conventional adipose tissue grafting and ex-vivo expanded ASC-enriched adipose tissue grafting for the treatment of contour deformities related pigmentary changes on the face. In this study, 80 patients of contour deformities of face with hyperpigmentation were recruited after informed consent. Two techniques, that is, conventional fat grafting (C-FG) and fat grafts enriched with expanded adipose stem cells (FG-ASCs) were used to address the pigmentation. Both techniques were explained to patients and enrolled patients were divided into two groups, that is, C-FG and FG-ASCs as per patients' choice and satisfaction. Patients of the FG-ASCs group were treated with FG-ASCs while patients of C-FGs group were treated with C-FG (without expanded ASCs). Patients were followed for 12 months and improvement in face pigmentation was assessed clinically as well as measured objectively. Patients' satisfaction was also documented as highly satisfied, satisfied, and unsatisfied. This clinical trial was registered at www.clinicaltrials.gov with ID: NTC03564808. Mean age of patients was 24.42 (±4.49), 66 patients were females. Forehead was involved in 61.20% cases, cheek in 21.20% cases, chin in 11.20% cases, and nose in 6.20% cases. In GF-ASCs group, the integrated color density was decreased (1.08 × 106 ± 4.64 × 105 ) as compared with C-FG group (2.80 × 105 ± 1.69 × 105 ). Patients treated with fat grafts enriched with expanded ASCs were significantly more satisfied as compared with patients treated with C-FG only. In conclusion, ASC-enriched autologous fat grafting is preferred option for improving the contour deformities related increased pigmentation of face skin.

Safety and Efficacy of Lenabasum, a Cannabinoid Receptor Type 2 Agonist, in Patients with Dermatomyositis with Refractory Skin Disease: A Randomized Clinical Trial.

BACKGROUND: Treatment options are limited for skin disease in dermatomyositis. Lenabasum is a cannabinoid receptor type 2 agonist that triggers the resolution of inflammation. OBJECTIVE: The objective of this study was to evaluate the safety and efficacy of lenabasum in patients with refractory cutaneous dermatomyositis. DESIGN: This study was a single-center, double-blind, randomized, placebo-controlled phase 2 study conducted from July 2015 to August 2017. POPULATION: The population included subjects aged ≥18 years with at least moderately active dermatomyositis skin activity by Cutaneous Dermatomyositis Disease Area and Severity Index activity ≥ 14 and failure or intolerance to hydroxychloroquine. INTERVENTION: Participants received 20 mg lenabasum daily for 28 days and then 20 mg twice per day for 56 days or placebo. MAIN OUTCOMES AND MEASURES: The primary outcome was a change in Cutaneous Dermatomyositis Disease Area and Severity Index activity. Safety and other secondary efficacy assessments were performed till day 113. RESULTS: A total of 22 subjects were randomized to lenabasum (n = 11) or placebo (n = 11). No serious or severe adverse events were related to lenabasum, and no participants discontinued the study. The adjusted least-squares mean for Cutaneous Dermatomyositis Disease Area and Severity Index activity decreased more for lenabasum, and the difference was significant on day 113 (least-squares mean [standard error] difference = ‒6.5 [3.1], P = 0.038). Numerically greater improvements were seen in multiple secondary efficacy outcomes and biomarkers with lenabasum. CONCLUSION: Lenabasum treatment was well tolerated and was associated with greater improvement in Cutaneous Dermatomyositis Disease Area and Severity Index activity and multiple efficacy outcomes. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov, NCT02466243.

Cannabinoid type 2 receptor (CB2R) distribution in dermatomyositis skin and peripheral blood mononuclear cells (PBMCs) and in vivo effects of LenabasumTM.

BACKGROUND: Lenabasum is a cannabinoid type 2 receptor (CB2R) reverse agonist that demonstrates anti-inflammatory effects in vivo and in vitro in dermatomyositis (DM) and is currently being investigated for therapeutic potential. The purpose of our study is to investigate CB2R distribution as well as the effects of lenabasum in DM. METHODS: Immunohistochemistry staining (IHC) was utilized to examine immune cell and cytokine production changes in lesional DM skin biopsies from lenabasum and placebo-treated patients. CB2R expression in various immune cell populations within DM skin was investigated with image mass cytometry (IMC), whereas flow cytometry elucidated CB2R expression in DM peripheral blood mononuclear cells (PBMCs) as well as cytokine production by CB2R-expressing cell populations. RESULTS: After 12 weeks of lenabasum treatment, IHC staining showed that CD4+ T cells, CB2R, IL-31, IFN-γ, and IFN-ß cytokines were downregulated. IFN-γ and IFN-ß mRNA decreased in lesional DM skin but not in PBMCs. IMC findings revealed that CB2R was upregulated in DM lesional skin compared to HC skin and DM PBMCs (p<0.05). In DM skin, CB2R was upregulated on dendritic cells, B cells, T cells, and macrophages while dendritic cells had the greatest expression in both DM skin and PBMCs (p<0.05). These CB2R+ cells in the skin produce IL-31, IL-4, IFN-γ, and IFN-ß. CONCLUSION: Our findings of differential CB2R expression based on location and cell type suggest modes by which lenabasum may exert anti-inflammatory effects in DM and highlights dendritic cells as potential therapeutic targets.

Plasma-derived DNA containing-extracellular vesicles induce STING-mediated proinflammatory responses in dermatomyositis.

Objectives: Extracellular vesicles (EVs) are lipid bilayer membrane vesicles that are present in various bodily fluids and have been implicated in autoimmune disease pathogenesis. Type I interferons (IFN), specifically IFN-ß, are uniquely elevated in dermatomyositis (DM). The stimulator of interferon genes (STING) works as a critical nucleic acid sensor and adaptor in type I IFN signaling with possible implications in autoimmune diseases such as DM. In the current study, we investigated whether circulating EVs contribute to proinflammatory effects in DM, whether these proinflammatory responses are mediated by the STING signaling pathway, and if so, by what mechanism STING is activated. Methods: We collected and characterized EVs from plasma of healthy controls (HC) and DM patients; analyzed their abilities to trigger proinflammatory cytokines release by ELISA, and explored STING signaling pathway activation using immunoblot and immunofluorescent staining. STING signaling pathway inhibitors and RNAi were used to further investigate whether STING was involved in EVs-triggered proinflammatory response. DNase/lipid destabilizing agent was utilized to digest EVs and their captured DNA contents to evaluate how EVs triggered STING-mediated proinflammatory response in DM. Results: EVs isolated from DM plasma triggered proinflammatory cytokines including type I IFN release with STING signaling pathway activation. The activated STING pathway was preferentially mediated by dsDNA captured by EVs. Suppression of STING or its downstream signaling proteins attenuated the EVs-mediated proinflammatory response. Conclusions: Plasma-derived, DNA containing-EVs induced STING-mediated proinflammatory effects in DM. Targeting the STING pathway may be a potential therapeutic approach for DM.

Myeloid Dendritic Cells Are Major Producers of IFN-ß in Dermatomyositis and May Contribute to Hydroxychloroquine Refractoriness.

Dermatomyositis pathogenesis remains incompletely understood; however, recent work suggests a predominant IFN-1 response. We explored dermatomyositis pathogenesis by quantifying the inflammatory cells in the skin, comparing myeloid with plasmacytoid dendritic cell release of IFN-ß, and assessing myeloid dendritic cell (mDC) contribution to hydroxychloroquine refractoriness. Immunohistochemistry was performed to assess cell-type expression in lesional skin biopsies from 12 patients with moderate-to-severe cutaneous dermatomyositis. Immunofluorescence, laser-capture microdissection, and flow cytometry were used to assess mDC release of IFN-ß in lesional skin biopsies and blood of patients with dermatomyositis. Immunohistochemistry was utilized to determine whether myeloid or plasmacytoid dendritic cells were increased in hydroxychloroquine nonresponders. CD4+, CD11c+, and CD69+ cells were more populous in lesional skin of patients with dermatomyositis. mDCs colocalized with IFN-ß by immunofluorescence and laser-capture microdissection revealed increased IFN-ß mRNA expression by mDCs in lesional skin of patients with dermatomyositis. In blood, both mDCs and plasmacytoid dendritic cells were major producers of IFN-ß in patients with dermatomyositis, whereas plasmacytoid dendritic cells predominately released IFN-ß in healthy controls (P < 0.01). mDCs were significantly increased in the skin of hydroxychloroquine nonresponders compared with that in the skin of responders (P < 0.05). mDCs cells appear to play an important role in dermatomyositis pathogenesis and IFN-ß production.

Preenrichment with Adipose Tissue-Derived Stem Cells Improves Fat Graft Retention in Patients with Contour Deformities of the Face.

Quick absorption of adipose tissue grafts makes the outcomes less satisfactory for clinical applications. In the current study, adipose tissue grafts were mixed with adipose tissue-derived stem cells (ASCs) to improve retention of adipose tissue grafts and to make the clinical outcomes of fat grafting more reliable. Adipose tissue was either injected alone (conventional group) or mixed with ASCs (stem cell group) before injection. In both groups, adipose tissue was injected at the site of contour throughout layers of tissues till visual clinical symmetry with the opposite side was achieved. The volume of injected fat graft was measured after 72 hours and 6 months using a B-mode ultrasound device connected with a 12 MH frequency probe. The percentage reduction in the volume of injected fat, physician satisfaction scores (Ph-SCs), and patient satisfaction scores (P-SCs) were also recorded. After 6 months, there was significantly lower fat absorption in the stem cell group as compared to the conventional group. Mean physician and patient satisfaction scores were significantly improved in the stem cell group. No significant adverse effects were noted in any patient. Significantly lower absorption of graft due to the use of ASCs improves the clinical outcomes of conventional fat grafting for contour deformities of the face. The current preenrichment strategy is noninvasive, safe and can be applied to other diseases that require major tissue augmentation such as breast surgery. This trial is registered with NCT02494752.

Quinacrine Suppresses Tumor Necrosis Factor-α and IFN-α in Dermatomyositis and Cutaneous Lupus Erythematosus.

Antimalarials are used to treat dermatomyositis (DM) and cutaneous lupus erythematosus (CLE). Although hydroxychloroquine (HCQ) is frequently used, addition of quinacrine (QC) has shown additional clinical effects when combined with HCQ. To quantify the effects of HCQ versus QC in suppressing secretion of tumor necrosis factor-α (TNF-α) and IFN-α from the peripheral blood mononuclear cells of DM and CLE patients, lipopolysaccharide-stimulated and control peripheral blood mononuclear cells from DM and CLE patients and control subjects were analyzed for the effect of HCQ and QC on TNF-α and IFN-α production using ELISA testing. Flow cytometry showed the effects of these therapies on intracellular TNF-α in myeloid dendritic cells and monocytes of DM patients and control subjects. QC significantly suppressed TNF-α relative to HCQ from unstimulated and lipopolysaccharide-stimulated peripheral blood mononuclear cells of DM and CLE patients (P < 0.0001). It suppressed IFN-α as significantly as HCQ from cytosine phosphodiester guanine-stimulated peripheral blood mononuclear cells of DM and CLE patients (P < 0.0001). Flow cytometry showed that QC significantly suppressed intracellular expression of TNF-α from the lipopolysaccharide-stimulated myeloid dendritic cells and monocytes of DM patients (P-values ≤ 0.0008). In conclusion, QC likely has a different mechanism of action than HCQ, given the broader inhibition of proinflammatory cytokines, including both TNF-α and IFN-α.

Gottron's papules exhibit dermal accumulation of CD44 variant 7 (CD44v7) and its binding partner osteopontin: a unique molecular signature.

The accumulated mucin in non-Gottron's dermatomyositis (DM) lesions is primarily chondroitin-4-sulfate (C4S), which is immunomodulatory in vitro. Gottron's papules are a particularly resistant manifestation of DM that often persist after other lesions have resolved with therapy. We examined non-Gottron's DM lesions and Gottron's papule skin biopsies for C4S, CD44 variant 7 (CD44v7), a chondroitin sulfate-binding isoform causally implicated in autoimmunity, and osteopontin (OPN), a CD44v7 ligand implicated in chronic inflammation. Gottron's papule dermis contained more C4S and CD44v7 than non-Gottron's lesions. Normal skin showed less CD44v7 over joints relative to Gottron's lesions. All DM dermis had increased OPN compared with healthy skin. Mechanically stretching cultured fibroblasts for 6 hours induced CD44v7 mRNA and protein, whereas IFN-γ treatment induced OPN mRNA and protein. OPN alone did not induce CD44v7, but stretching dermal fibroblasts in the presence of OPN increased human acute monocytic leukemia cell line (THP-1) monocyte binding, which is blunted by anti-CD44v7 blocking antibody. C4S, CD44v7, and OPN are three molecules uniquely present in Gottron's papules that contribute to inflammation individually and in association with one another. We propose that stretch-induced CD44v7 over joints, in concert with dysregulated OPN levels in the skin of DM patients, increases local inflammatory cell recruitment and contributes to the pathogenesis and resistance of Gottron's papules.

Tumor necrosis factor α release in peripheral blood mononuclear cells of cutaneous lupus and dermatomyositis patients.

INTRODUCTION: Several studies have reported that TNFα is substantially increased within skin lesions of patients with discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE) and dermatomyositis (DM) compared to controls. Elevated TNFα has been reported in the sera of some patients with systemic lupus erythematosus, DLE and SCLE, but not in the sera of patients with DM. Because of the key pathogenic role of autoimmunity in these diseases, in this study we sought to evaluate TNFα production by a readily available source of immune cells (namely, peripheral blood mononuclear cells (PBMCs)) taken from controls and from patients with cutaneous lupus or DM. METHODS: Freshly isolated PBMCs were cultured overnight, and TNFα protein accumulation in conditioned medium was determined. In addition, flow cytometry using cell-type-specific markers was performed to determine the sources of TNFα. One-way analysis of variance and Dunnett's multiple comparisons test were performed for statistical comparisons. RESULTS: Accumulation of TNFα protein in conditioned medium containing PBMCs from DLE patients, but not from SCLE, TLE or DM patients, was significantly greater (19-fold) than that from controls (P < 0.001). In DLE PBMCs, increased TNFα was produced by circulating monocytes and myeloid dendritic cells (mDCs). The mean TNFα fluorescence intensity, but not the total number, of both monocytes and mDCs (P < 0.01) from DLE patients was significantly greater (2.3-fold) than that of controls. There were significantly more (13.3-fold) mDCs with intracellular TNFα in blood from DLE patients (P < 0.001) and DM patients (P < 0.001) compared to controls. Most importantly, a positive correlation was seen in DLE patients between their disease activity measured using the Cutaneous Lupus Erythematosus Disease Area and Severity Index and TNFα protein secretion (r = 0.61, P < 0.08). CONCLUSIONS: TNFα protein production by PBMCs is greater in DLE patients than in patients with other cutaneous forms of lupus and DM or in controls. Flow cytometric studies demonstrated that circulating monocytes and mDCs contributed to this increased TNFα production. Monocytes and mDCs are present in lesional skin, and the increased TNFα production by these cells and other PBMCs likely increase the number of inflammatory cells seen in DLE skin relative to other subsets of cutaneous lupus erythematosus and DM. These results provide a possible biological explanation for the denser infiltrate seen in DLE relative to DM.

UVB and proinflammatory cytokines synergistically activate TNF-alpha production in keratinocytes through enhanced gene transcription.

UVB irradiation potently induces cytokines in the skin, including IL-1alpha and tumor necrosis factor-alpha (TNF-alpha). The mechanism for TNF-alpha induction in UVB-irradiated keratinocytes is not clear. In this study, we explored the effects of UVB and cytokines, alone or in combination in human keratinocytes. Keratinocytes were sham- or UVB-irradiated with 30 mJ cm(-2), and then incubated in the absence or presence of IFN-alpha2b, TNF-alpha, or IL-1alpha. UVB and IL-1alpha treatment synergistically enhanced TNF-alpha secretion and mRNA levels in human keratinocytes, similar to the findings reported previously in human fibroblasts. Exogenous recombinant TNF-alpha up-regulates its own mRNA level. However, addition of IFN-alpha2b did not show any additive effect on TNF-alpha mRNA induction. To understand the regulation of TNF-alpha mRNA by UVB, with or without IL-1alpha, we examined the transcription rate and half-life of TNF-alpha mRNA. Treatment of keratinocytes with IL-1alpha or UVB alone increased TNF-alpha gene transcription 4- to 5-fold over sham treatment, and TNF-alpha gene transcription increased 11-fold in cells treated with UVB plus IL-1alpha over sham. UVB with IL-1alpha did not enhance the half-life of TNF-alpha mRNA over that seen with UVB alone. In conclusion, TNF-alpha expression in primary keratinocytes is upregulated transcriptionally by UVB and IL-1alpha.

IL-12 completely blocks ultraviolet-induced secretion of tumor necrosis factor alpha from cultured skin fibroblasts and keratinocytes.

Interleukin-12 is an important regulator of other cytokines. Although interleukin-12 is considered to act primarily on lymphocytes, provoking a shift from T helper 2 to T helper 1 cells and an increase in lymphocyte-derived tumor necrosis factor alpha, we hypothesized that interleukin-12 might also affect tumor necrosis factor alpha secretion from skin cells. In this study, keratinocytes were treated with ultraviolet-B, ultraviolet-A, or sham irradiation, without or with exogenous interleukin-12. Remarkably, the exogenous interleukin-12 totally blocked ultraviolet-B-induced tumor necrosis factor alpha production. Both ultraviolet-A and ultraviolet-B were capable of inducing interleukin-12 production. To determine the molecular mechanism of this effect, we used a chloramphenicol acetyl transferase reporter under the control of a 1.2 kb fragment of the wild-type (-308G) human tumor necrosis factor alpha promoter and found significant suppression of promoter activity with interleukin-12. Studies using the -308A variant of the human tumor necrosis factor alpha promoter showed much higher promoter activity overall, but also a greater sensitivity to suppression by interleukin-12. The mechanism did not involve blockage of the interleukin-1 receptor, because interleukin-12 did not suppress interleukin-1-mediated induction of collagenase mRNA. To determine the role of endogenous interleukin-12, we found that anti-interleukin-12 antibodies enhanced ultraviolet-B-induced tumor necrosis factor alpha secretion. Thus, interleukin-12 strongly inhibits tumor necrosis factor alpha production by noninflammatory skin cells, mostly or entirely through inhibition of gene transcription via an element within the first 1.2 kb of the tumor necrosis factor alpha promoter. The result is a shift in tumor necrosis factor alpha production from noninflammatory cells to T helper 1 cells. Because tumor necrosis factor alpha is central to the pathogenesis of several photosensitive skin diseases and certain forms of immune suppression, interleukin-12 may have important physiologic, pathophysiologic, and therapeutic roles.