Sustained Remission Without Corticosteroids Among Patients With Pemphigus Who Had Rituximab as First-Line Therapy: Follow-Up of the Ritux 3 Trial.

Importance: The Ritux 3 trial demonstrated the short-term efficacy and safety of first-line treatment with rituximab compared with a standard corticosteroid regimen in pemphigus. No data on the long-term follow-up of patients who received rituximab as first line are available. Objective: To assess the long-term efficacy and safety of the Ritux 3 treatment regimen. Design, Setting, and Participants: This 7-year follow-up study of the Ritux 3 trial included patients with pemphigus from 25 dermatology departments in France from January 1, 2010, to December 31, 2015. Exposure: Patients were initially randomized in the rituximab plus prednisone group or prednisone-alone group. Main outcomes and measures: The primary outcome was the 5- and 7-year disease-free survival (DFS) without corticosteroids, assessed by Kaplan-Meier curves. Secondary outcomes were occurrence of relapse, occurrence of severe adverse events (SAEs), and evolution of antidesmoglein (Dsg) antibody enzyme-linked immunosorbent assay values to predict long-term relapse. Results: Of the 90 patients in the Ritux 3 trial, 83 were evaluated at the end of follow-up study visit (44 in the rituximab plus prednisone group; 39 in the prednisone-alone group) with a median (IQR) follow-up of 87.3 (79.1-97.5) months. Forty-three patients (93%) from the rituximab plus prednisone and 17 patients (39%) from the prednisone-alone group had achieved complete remission without corticosteroids at any time during the follow-up. Patients from the rituximab group had much longer 5- and 7-year DFS without corticosteroids than patients from the prednisone-alone group (76.7% and 72.1% vs 35.3% and 35.3%, respectively; P < .001), and had about half the relapses (42.2% vs 83.7%; P < .001). Patients who received rituximab as second-line treatment had shorter DFS than patients treated as first line (P = .007). Fewer SAEs were reported in the rituximab plus prednisone group compared with the prednisone-alone group, 31 vs 58 respectively, corresponding to 0.67 and 1.32 SAEs per patient, respectively (P = .003). The combination of anti-Dsg1 values of 20 or more IU/mL and/or anti-Dsg3 values of 48 or more IU/mL yielded 0.83 positive predictive value and 0.94 negative predictive value to predict long-term relapse. Conclusions and Relevance: In this secondary analysis of the Ritux 3 trail, first-line treatment of patients with pemphigus with the Ritux 3 regimen was associated with long-term sustained complete remission without corticosteroid therapy without any additional maintenance infusion of rituximab.

Efficacy of Methotrexate Alone vs Methotrexate Plus Low-Dose Prednisone in Patients With Alopecia Areata Totalis or Universalis: A 2-Step Double-Blind Randomized Clinical Trial.

Importance: Poor therapeutic results have been reported in patients with alopecia areata totalis (AT) or universalis (AU), the most severe and disabling types of alopecia areata (AA). Methotrexate, an inexpensive treatment, might be effective in AU and AT. Objective: To evaluate the efficacy and tolerance of methotrexate alone or combined with low-dose prednisone in patients with chronic and recalcitrant AT and AU. Design, Setting, and Participants: This academic, multicenter, double-blind, randomized clinical trial was conducted at 8 dermatology departments at university hospitals between March 2014 and December 2016 and included adult patients with AT or AU evolving for more than 6 months despite previous topical and systemic treatments. Data analysis was performed from October 2018 to June 2019. Interventions: Patients were randomized to receive methotrexate (25 mg/wk) or placebo for 6 months. Patients with greater than 25% hair regrowth (HR) at month 6 continued their treatment until month 12. Patients with less than 25% HR were rerandomized: methotrexate plus prednisone (20 mg/d for 3 months and 15 mg/d for 3 months) or methotrexate plus placebo of prednisone. Main Outcome and Measures: The primary end point assessed on photos by 4 international experts was complete or almost complete HR (Severity of Alopecia Tool [SALT] score <10) at month 12, while receiving methotrexate alone from the start of the study. Main secondary end points were the rate of major (greater than 50%) HR, quality of life, and treatment tolerance. Results: A total of 89 patients (50 female, 39 male; mean [SD] age, 38.6 [14.3] years) with AT (n = 1) or AU (n = 88) were randomized: methotrexate (n = 45) or placebo (n = 44). At month 12, complete or almost complete HR (SALT score <10) was observed in 1 patient and no patient who received methotrexate alone or placebo, respectively, in 7 of 35 (20.0%; 95% CI, 8.4%-37.0%) patients who received methotrexate (for 6 or 12 months) plus prednisone, including 5 of 16 (31.2%; 95% CI, 11.0%-58.7%) who received methotrexate for 12 months and prednisone for 6 months. A greater improvement in quality of life was observed in patients who achieved a complete response compared with nonresponder patients. Two patients in the methotrexate group discontinued the study because of fatigue and nausea, which were observed in 7 (6.9%) and 14 (13.7%) patients receiving methotrexate, respectively. No severe treatment adverse effect was observed. Conclusions and Relevance: In this randomized clinical trial, while methotrexate alone mainly allowed partial HR in patients with chronic AT or AU, its combination with low-dose prednisone allowed complete HR in up to 31% of patients. These results seem to be of the same order of magnitude as those recently reported with JAK inhibitors, with a much lower cost. Trial Registration: ClinicalTrials.gov Identifier: NCT02037191.

Characteristic Pattern of IL-17RA, IL-17RB, and IL-17RC in Monocytes/Macrophages and Mast Cells From Patients With Bullous Pemphigoid.

Inflammation is largely implicated in bullous pemphigoid (BP), the most frequent skin auto-immune blistering disease. IL-17, essentially IL-17A/F, has been involved in blister formation through regulation of protease production, and its specific serum profile within BP was related to disease outcome. However, relationships between IL-17 family ligands and receptors are quite complex with six different IL-17 isoforms, and five different receptors. We here aimed at clarifying the contribution of the IL-17 axis in BP by characterizing not only the expression of IL-17 receptor (IL-17R) members within immune cells isolated from BP patients (PMNs, n = 9; T-lymphocytes, n = 10; and monocytes, n = 10) but also the expression of IL-17 isoforms in sera (n = 83), and blister fluid (n = 31) of BP patients. We showed that at diagnosis, IL-17RA and IL-17RC expression were significantly increased in monocytes isolated from BP patients as compared to those from control subjects (p = 0.006 and p = 0.016, respectively). Notably, both IL-17RA and IL-17RC mRNA expression remained elevated in BP monocytes at time of relapse. We further demonstrated a significant increase of all IL-17 isoforms tested in BP blister fluid compared with BP serum (IL-17A, p < 0.0001; IL-17A/F, p < 0.0001; IL-17B, p = 0.0023; IL-17C, p = 0.0022; IL-17E, p < 0.0001). Among all, IL-17B was the only cytokine for which a significant decreased concentration within blister fluid was observed in BP patients with severe disease compared to patients with moderate disease (p = 0.012). We further evidenced a significant negative correlation between IL-17B levels and blister/erosion BPDAI subscore (r = -0.52, p = 0.003). We finally identified mast cells as a potential target of IL-17B in lesional skin of BP patients. In conclusion, we showed here that IL-17RA and IL-17RC expression in monocyte was associated with disease activity and evidenced in situ a negative correlation between BP disease activity and IL-17B, whose effects could be mediated by IL-17RB expressed by mast cell in BP lesional skin.

IL-23/IL-17 Axis Activates IL-1ß-Associated Inflammasome in Macrophages and Generates an Auto-Inflammatory Response in a Subgroup of Patients With Bullous Pemphigoid.

Bullous Pemphigoid (BP) is a skin autoimmune blistering disease characterized by immune-mediated degradation of the dermo-epidermal junction and release of a large number of inflammatory cytokines. Interleukin-1ß (IL-1ß) is a pleiotropic pro-inflammatory cytokine associated with inflammasome activation and known to be pivotal in several auto-immune and auto-inflammatory diseases. We sought to clarify the presence of inflammasome-dependent IL-1ß and to investigate its role in BP. Skin biopsy specimens (n = 13), serum (n = 60), blister fluid (n = 26), and primary inflammatory cells from patients with BP were used to investigate inflammasome activation and function. We here highlighted a differential occurrence of a functional in situ inflammasome in patients with BP, biologically distinguished by IL-1ß and NLRP3 expression. Clinically, elevated IL-1ß levels were associated with the presence of erythema and urticarial plaques reflecting the inflammatory phase preceding blister formation. We further identified IL-17 and IL-23 as important molecules favoring IL-1ß expression in monocyte-derived macrophages from BP patients. Finally, we demonstrated the ability of IL-1ß to stimulate the release of the matrix metalloproteinase-9 in those macrophages, reinforcing the role of IL-1ß in the auto-amplification loop of the inflammatory response associated to BP. However, whether this inflammasome is an epiphenomenon associated with BP disease or constitutes an amplification inflammatory step in certain patients still need to be determined. In the context of a precision medicine approach, our findings allowed us to delineate a subgroup of patients with BP that showed similarities with auto-inflammatory diseases. Subsequently, this opens up alternative therapeutic strategies targeting IL-1ß pathway in the aim to control the early, pre-blistering inflammatory phase. Ultimately, this could also help in reducing the detrimental effects associated with high doses of corticosteroids treatment.

Blister Fluid Induces MMP-9-Associated M2-Type Macrophages in Bullous Pemphigoid.

Bullous pemphigoid (BP) is a cutaneous autoimmune disease, characterized by an inflammatory cascade leading to blister formation. Although macrophages were shown to participate in BP pathophysiology, their role in the blister formation process still needs to be investigated. We here addressed the influence of serum and blister fluid (BF) from patients with BP on the polarization status of macrophages with regards to the metalloproteinase-9 (MMP-9) expression. We demonstrated that several markers related to the alternatively activated macrophage phenotype (M2) including IL-10, TARC, arginase, TNFα, and IL-1RA were meaningfully increased in BF of patients with BP. We further showed that BF, but not serum from patients with BP, significantly induced the expression of CD163, CD206, and IL-10 in BP monocyte-derived macrophages (MDMs). Notably IL-10 was the only cytokine to be correlated to the reference clinical score, BP disease activity index (BPDAI), especially to the inflammatory BPDAI subscore evaluating urticarial and erythematous skin lesions (r = 0.57, p = 0.0004). We also found elevated levels of MMP-9 to M2-type macrophages ex vivo and highlighted the presence of CD163+ MMP-9+ macrophages histologically, at skin lesional site. Finally, we showed that methylprednisolone reduced MMP-9 levels in MDMs without modifying the other M2 markers. All together these results strongly support the presence of M2-phenotype macrophages with pro-inflammatory properties susceptible to favor blister formation in BP.

NET Formation in Bullous Pemphigoid Patients With Relapse Is Modulated by IL-17 and IL-23 Interplay.

Background: DNA extracellular traps (ETs), released by neutrophils (NETs), or eosinophils (EETs), play a pathogenic role in several autoimmune disorders. However, to date, NETs have never been investigated in bullous pemphigoid (BP) with respect to clinical and immunological activities, both at baseline and at time of relapse which have been characterized with specific IL-17 and IL-23 patterns. Objective: We sought to assess whether ETs were associated with BP as well as the relative contribution of IL-17 axis cytokines to NET induction. Methods: Skin biopsy specimens were obtained from 11 patients with BP. Immuno-detection of neutrophils and eosinophils combined to DNA staining allowed us to investigate the in-situ presence of NETs and EETs using confocal scanning microscopy. NETs release was evaluated ex vivo by stimulating polymorphonuclear cells from BP patients with BP biological fluids in presence of IL-17A and IL-23 or of glucocorticoids. Results: At baseline, ETs were observed in BP lesions at the site of dermal-epidermal cleavage. Despite an important infiltrate of eosinophils, ETs were essentially associated with neutrophils in situ and were not related to BP clinical activity at diagnosis. In situ observation of NETs was associated in 6 among 8 patients with serum capacity of NET induction. Notably both blister fluid and sera from BP patients at diagnosis and at time of relapse could induce NET formation ex vivo. In contrast, a longitudinal investigation showed a decrease of NET formation with time of treatment in patients undergoing remission. Mimicking relapse, complementation of sera from BP patients with ongoing remission with either IL-17A or IL-23 increased NET formation. Conversely, IL-17A inhibited NET formation induced by serum from BP patients with relapse supplemented or not with IL-23. Finally, glucocorticoids also inhibited NET formation ex vivo in BP. Conclusion: NET formation is an associated phenomenon with BP. Furthermore, we showed that IL-23 favored NET formation, whereas the effects of IL-17A are environment dependent. Indeed, IL-17A displayed a protective effect on NET formation when associated with IL-23, showing for the first-time differential effects of these two cytokines in BP.

Matrix remodelling and MMP expression/activation are associated with hidradenitis suppurativa skin inflammation.

Hidradenitis suppurativa/acne inversa (HS) is a chronic, inflammatory, recurrent, debilitating skin disease of the hair follicle, associated with considerable tissue remodelling. Although abnormal cytokine expression was detected both in perilesional and in uninvolved skin, up to now there is no model allowing a better understanding of the implicit inflammatory mechanisms in HS. The aim of this study was to investigate the inflammatory response in HS skin by mean of an ex vivo model culture. To that purpose, nine skin biopsy specimens from patients suffering from HS and controls were cultured up to 4 days. Microscopy imaging investigations showed variations of collagen I and III organization, and an increase in elastin fibres fragmentation in HS skin after 4 days of culture. The HS matrix structure remodelling was associated with high level of MMP-2 and MMP-9 in HS lesional skin. After 4 days of culture, the MMP expression in HS perilesional skin reached the level observed in HS lesional skin. Concomitantly, an increase in IL-1ß concentration was observed in all skin samples after 4 days of culture, although IL-1ß concentrations remained significantly higher in HS lesional skin as compared with control skin. Meanwhile, neither IL-17 concentrations nor the inflammasome components NLRP3 and caspase-1 varied. Thus, our HS skin model culture showed that MMP-induced matrix alteration could participate in HS inflammation by releasing biological active peptides and inflammatory factors from the extracellular matrix (ECM), and open new opportunities to investigate the regulation of the inflammatory mechanism associated with HS.

B-cell depletion induces a shift in self antigen specific B-cell repertoire and cytokine pattern in patients with bullous pemphigoid.

Bullous Pemphigoid is the most common auto-immune bullous skin disease. It is characterized by the production of auto-antibodies directed against 2 proteins of the hemi-desmosome (BP180 and BP230). We assessed the efficacy and mechanisms of action of rituximab, an anti-CD20 monoclonal antibody, in 17 patients with severe and relapsing type of bullous pemphigoid. The phenotype, cytokine gene expression, and rearrangement of BP180-specific B-cell receptor genes were performed over 2 years following treatment. At the end of the study, 5 patients had died, 3 had withdrawn from the study, and 9 patients were in complete remission. The one- and two-year relapse rates were 44.1% (95% Confidence Interval (CI): 21.0-76.0%) and 66.5%, (95% CI: 38.4-91.4%), respectively. Phenotypic analyses confirmed dramatic B-cell depletion, which lasted for 9 to 12 months. The ELISA values of serum anti-BP180 antibodies and the frequency of BP180-specific circulating B cells decreased dramatically following treatment, which paralleled the improvement of skin lesions. During B-cell reconstitution, a polyclonal IgM repertoire appeared and a shift in the rearrangement of the B-cell receptor genes of BP180-specific circulating B cells was observed. Concurrently, we observed a decrease of IL-15, IL-6 and TNFα expressing BP180-specific B cells, and the emergence of IL-10 and IL-1RA-expressing BP180-specific IgM+ B cells in patients in complete remission off therapy, suggesting the functional plasticity of BP180-specific auto-immune B cells after rituximab treatment.

Ustekinumab for skin reactions associated with anti-tumor necrosis factor-α agents in patients with inflammatory bowel diseases: A single-center retrospective study.

Anti-tumor necrosis factor (TNF)-α agents may induce skin reactions, in particular in patients with inflammatory bowel diseases (IBD). The objective of this study was to determine the efficacy of ustekinumab in these patients. IBD patients facing therapeutic issues because of cutaneous reactions or tolerance issues, consequently treated with ustekinumab in our department, were included. Retrospective review of case records and clinical photographs was carried out. Twenty-six patients were included. Twenty-three patients were treated for Crohn's disease and three for ulcerative colitis. Fourteen patients presented psoriasiform lesions, nine eczematiform lesions, four anaphylactoid reactions, two alopecia areata-like lesions, one injection-site reaction, one cutaneous polyarteritis nodosa and five other skin reactions. Most of them resolved under ustekinumab. In detail, eczematiform lesions completely resolved in all cases, psoriasiform lesions completely resolved in 12 cases (85.7%) and had partial response in two cases (14.3%). Two cases of alopecia areata showed complete response (complete hair regrowth). Fourteen patients showed complete digestive response, 10 patients partial digestive response (seven of which needed IBD treatment optimization) and only two failure. In conclusion, ustekinumab is a drug of choice in patients with IBD who cannot tolerate TNF blockers.

Association of Transient Palmoplantar Keratoderma With Clinical and Immunologic Characteristics of Bullous Pemphigoid.

Importance: Development of transient palmoplantar keratoderma (PPK) with bullous pemphigoid (BP) has only been described in 2 isolated case reports. The clinical significance and the pathophysiologic mechanisms of this association are unknown. Objective: To examine the clinical characteristics and immunological profile of patients with BP who develop transient PPK and analyze therapeutic options and outcomes. Design, Setting, and Participants: In this case series, patients with BP who developed acquired, transient PPK, and were treated at a single institution from January 1, 2015, through December 31, 2017, were studied. Main Outcomes and Measures: Clinical and immunological activity of BP, treatment administrated before and after PPK appearance, and patient outcomes. Results: Six patients with BP and transient PPK were identified and included in the study. There were 5 women and 1 man with a mean age of 72 years. At baseline, all patients had a generalized, multibullous BP and high serum anti-BP180 antibodies (mean, 130 U/mL; range, 73-150), whereas anti-BP230 antibodies were elevated in only 1 case. The PPK appeared a mean 6.2 (range, 2-12) months after BP diagnosis, following a prolonged period of disease activity with recurrent flares. When the PPK occurred, BP was uncontrolled on therapy (mean Bullous Pemphigoid Disease Activity Index [BPDAI] score, 57; range, 34-105; mean anti-BP180 antibodies titer, 122 U/mL; range, 81-150). On administration of additional systemic immunosuppressive therapies, the PPK healed progressively in a mean 4.3 months (range, 2-9), along with BP clinical remission in 4 of 6 patients. No relationship was found between PPK occurrence and anti-BP180/230 antibodies profiles. In contrast, blister fluids collected at the time of PPK displayed a much higher level of interleukin 1ß (IL-1ß) compared with those collected in the absence of PKK. Expression of IL-17A, IL-17F, and IL-22 was also enhanced in the blister fluid of patients with BP who had PPK. Conclusions and Relevance: To our knowledge, this is the first report of 6 cases of BP with transient PPK with extensive immunological investigation. The PPK appeared after a prolonged period of clinical BP activity punctuated with recurrent relapses, was transient, and healed after BP control with additional immunosuppressive therapy. Enhanced expression of a particular cytokine panel in the blister fluid at time of PPK could support keratinocyte proliferation as described in patients with psoriasis. Transient PPK could represent a clinical marker of severe, treatment-resistant BP.

Variation of the epidermal expression of glucocorticoid receptor-beta as potential predictive marker of bullous pemphigoid outcome.

Bullous pemphigoid (BP) is the most common autoimmune subepidermal blistering disease in Western countries. Although topical and/or systemic glucocorticoids treatment efficacy is widely recognized, up to 30% of patients with BP may undergo a relapse during the first year of treatment. We investigated the protein expression of the total glucocorticoid receptor and GRß isoform in the skin biopsy specimens from patients with BP and wondered whether such investigation at baseline provided a tool to predict disease outcome. Total GR and GRß protein expressions were detected by immunohistochemistry at baseline on 12 patients who later relapse and 11 patients who remained on remission in comparison with 14 control patients. The expression of GRß in the epidermis of patients with BP who later relapse was significantly higher than that in the epidermis of patients with BP controlled upon corticosteroid treatment, which was also higher than control patients. Thus, our results suggest that increased protein expression of GRß in skin epithelial cells is predictive of reduced steroid treatment efficacy, and therefore of increased risk of disease relapse in patients with BP.

First-line rituximab combined with short-term prednisone versus prednisone alone for the treatment of pemphigus (Ritux 3): a prospective, multicentre, parallel-group, open-label randomised trial.

BACKGROUND: High doses of corticosteroids are considered the standard treatment for pemphigus. Because long-term corticosteroid treatment can cause severe and even life-threatening side-effects in patients with this disease, we assessed whether first-line use of rituximab as adjuvant therapy could improve the proportion of patients achieving complete remission off-therapy, compared with corticosteroid treatment alone, while decreasing treatment side-effects of corticosteroids. METHODS: We did a prospective, multicentre, parallel-group, open-label, randomised trial in 25 dermatology hospital departments in France (Ritux 3). Eligible participants were patients with newly diagnosed pemphigus aged 18-80 years being treated for the first time (not at the time of a relapse). We randomly assigned participants (1:1) to receive either oral prednisone alone, 1·0 or 1·5 mg/kg per day tapered over 12 or 18 months (prednisone alone group), or 1000 mg of intravenous rituximab on days 0 and 14, and 500 mg at months 12 and 18, combined with a short-term prednisone regimen, 0·5 or 1·0 mg/kg per day tapered over 3 or 6 months (rituximab plus short-term prednisone group). Follow-up was for 3 years (study visits were scheduled weekly during the first month of the study, then monthly until month 24, then an additional visit at month 36). Treatment was assigned through central computer-generated randomisation, with stratification according to disease-severity (severe or moderate, based on Harman's criteria). The primary endpoint was the proportion of patients who achieved complete remission off-therapy at month 24 (intention-to-treat analysis). This study is registered with ClinicalTrials.gov, number NCT00784589. FINDINGS: Between May 10, 2010, and Dec 7, 2012, we enrolled 91 patients and randomly assigned 90 to treatment (90 were analysed; 1 patient withdrew consent before the random assignment). At month 24, 41 (89%) of 46 patients assigned to rituximab plus short-term prednisone were in complete remission off-therapy versus 15 (34%) of 44 assigned to prednisone alone (absolute difference 55 percentage points, 95% CI 38·4-71·7; p<0·0001. This difference corresponded to a relative risk of success of 2·61 (95% CI 1·71-3·99, p<0·0001), corresponding to 1·82 patients (95% CI 1·39-2·60) who would need to be treated with rituximab plus prednisone (rather than prednisone alone) for one additional success. No patient died during the study. More severe adverse events of grade 3-4 were reported in the prednisone-alone group (53 events in 29 patients; mean 1·20 [SD 1·25]) than in the rituximab plus prednisone group (27 events in 16 patients; mean 0·59 [1·15]; p=0·0021). The most common of these events in both groups were diabetes and endocrine disorder (11 [21%] with prednisone alone vs six [22%] with rituximab plus prednisone), myopathy (ten [19%] vs three [11%]), and bone disorders (five [9%] vs five [19%]). INTERPRETATION: Data from our trial suggest that first-line use of rituximab plus short-term prednisone for patients with pemphigus is more effective than using prednisone alone, with fewer adverse events. FUNDING: French Ministry of Health, French Society of Dermatology, Roche.

Bullous pemphigoid outcome is associated with CXCL10-induced matrix metalloproteinase 9 secretion from monocytes and neutrophils but not lymphocytes.

BACKGROUND: The outcome of bullous pemphigoid (BP), the most frequent autoimmune skin-blistering disease, involves matrix metalloproteinase 9 (MMP-9), IL-17, and IL-23 release from infiltrated inflammatory cells. The chemokine CXCL10 has been associated with several autoimmune diseases, but its participation in BP pathophysiology still needs to be clarified. OBJECTIVE: We sought to assess whether BP outcome was associated with different CXCL10 levels and to evaluate the contribution of CXCL10 to the described cytokine/protease inflammatory loop associated with disease outcome. METHODS: Skin biopsy specimens (n = 16), serum (n = 114), blister fluid (n = 23), and primary inflammatory cells from patients with BP were used to investigate CXCL10 expression and function. RESULTS: At baseline, both resident cells, such as keratinocytes and fibroblasts, and infiltrating immune cells expressed CXCL10 at lesional sites in skin of patients with BP. CXCL10 levels were higher in blister fluid (P < .0001) and serum (P < .005) from patients with BP than in serum from age- and sex-matched control subjects (n = 34). Furthermore, CXCL10 serum levels increased at day 60 only in patients who relapsed within the first year of treatment (n = 33, P < .005). Interestingly, CXCL10 expression could be upregulated by itself and IL-17 in inflammatory cells. Notably, neutrophils and monocytes from patients with BP, but not lymphocytes, responded to CXCL10 by increasing MMP-9 secretion through the activation of extracellular signal-regulated kinase 1/2, p38, phosphoinositide-3 kinase signaling pathways. Finally, CXCL10-increased MMP-9 secretion was inhibited by methylprednisolone and also by compound A, a novel nonsteroidal glucocorticoid receptor ligand. CONCLUSION: We showed that increased levels of inflammatory biomarkers in patients with BP, such as CXCL10, favor neutrophil- and monocyte-associated MMP-9 release and disease relapse and opened new therapeutic horizons in patients with this autoimmune disease.

Histone deacetylases meet microRNA-associated MMP-9 expression regulation in glucocorticoid-sensitive and -resistant cell lines.

Glucocorticoids are largely used in the treatment of inflammatory pathologies and/or hematological malignancies and regulate the expression of a variety of genes involved in inflammation or metastasis such as matrix metalloproteinases (MMP). Long-term exposure to glucocorticoids can result in failure of responsiveness, which is often associated with an unwanted gene expression. Epigenetic mechanisms are involved in gene expression modulated after development of glucocorticoid resistance but how these mechanisms take place must be further studied. The effects of HDAC inhibitors (HDACi) in a context of glucocorticoid resistance are still not well understood and need to be further investigated. We hypothesized that acquired glucocorticoid resistance associated to HDACi could disturbs epigenetic landscape, especially miR expression, leading to a modulation of MMP-9 gene expression and/or protein secretion, described as largely involved in bone remodeling and tumor invasion in multiple myeloma. To this aim, we used sensitive RPMI-8226 cell line and its dexamethasone- and methylprednisolone-resistant derivatives. The resistant cell lines displayed an 'open chromatin' and an MMP-9 overexpression comparatively to the sensitive cell line. HDACi treatment with MS-275 increased even more MMP-9 overexpression not only at an mRNA level but also at the protein level. We showed that MMP-9 expression regulation was not directly linked with HAT/HDAC balance alterations but rather with the deregulation of MMP-9-targeting miRs. Then, we first demonstrated that miR­149 downregulation was directly involved in the MMP-9 overexpression following a chronic glucocorticoid exposure and that MS-275 could amplify this overexpression by inhibition of miR­149 expression and miR­520c overexpression. Taken together, these results indicate that the use of HDACi in a context of acquired glucocorticoid resistance could modify the epigenetic landscape, highlighting the importance of taking the glucocorticoid response status into consideration in treatment with HDACi.

The insect repellent N,N-diethyl-m-toluamide (DEET) induces angiogenesis via allosteric modulation of the M3 muscarinic receptor in endothelial cells.

The insect repellent N,N-diethyl-m-toluamide (DEET) has been reported to inhibit AChE (acetylcholinesterase) and to possess potential carcinogenic properties with excessive vascularization. In the present paper, we demonstrate that DEET specifically stimulates endothelial cells that promote angiogenesis which increases tumor growth. DEET activates cellular processes that lead to angiogenesis including proliferation, migration and adhesion. This is associated with an enhancement of NO production and VEGF expression in endothelial cells. M3 silencing or the use of a pharmacological M3 inhibitor abrogates all of these effects which reveals that DEET-induced angiogenesis is M3 sensitive. The experiments involving calcium signals in both endothelial and HEK cells overexpressing M3 receptors, as well as binding and docking studies demonstrate that DEET acts as an allosteric modulator of the M3 receptor. In addition, DEET inhibited AChE which increased acetylcholine bioavailability and binding to M3 receptors and also strengthened proangiogenic effects by an allosteric modulation.

The Combination of Marketed Antagonists of α1b-Adrenergic and 5-HT2A Receptors Inhibits Behavioral Sensitization and Preference to Alcohol in Mice: A Promising Approach for the Treatment of Alcohol Dependence.

Alcohol-dependence is a chronic disease with a dramatic and expensive social impact. Previous studies have indicated that the blockade of two monoaminergic receptors, α1b-adrenergic and 5-HT2A, could inhibit the development of behavioral sensitization to drugs of abuse, a hallmark of drug-seeking and drug-taking behaviors in rodents. Here, in order to develop a potential therapeutic treatment of alcohol dependence in humans, we have blocked these two monoaminergic receptors by a combination of antagonists already approved by Health Agencies. We show that the association of ifenprodil (1 mg/kg) and cyproheptadine (1 mg/kg) (α1-adrenergic and 5-HT2 receptor antagonists marketed as Vadilex ® and Periactine ® in France, respectively) blocks behavioral sensitization to amphetamine in C57Bl6 mice and to alcohol in DBA2 mice. Moreover, this combination of antagonists inhibits alcohol intake in mice habituated to alcohol (10% v/v) and reverses their alcohol preference. Finally, in order to verify that the effect of ifenprodil was not due to its anti-NMDA receptors property, we have shown that a combination of prazosin (0.5 mg/kg, an α1b-adrenergic antagonist, Mini-Press ® in France) and cyproheptadine (1 mg/kg) could also reverse alcohol preference. Altogether these findings strongly suggest that combined prazosin and cyproheptadine could be efficient as a therapy to treat alcoholism in humans. Finally, because α1b-adrenergic and 5-HT2A receptors blockade also inhibits behavioral sensitization to psychostimulants, opioids and tobacco, it cannot be excluded that this combination will exhibit some efficacy in the treatment of addiction to other abused drugs.

Ethanolic Extract of Algerian Propolis and Galangin Decreased Murine Melanoma Tumor Progression in Mice

Melanoma is the more dangerous skin cancer, and metastatic melanoma still carries poor prognosis. Despite recent therapeutic advances, prolonged survival remains rare and research is still required. Propolis extracts from many countries have attracted a great deal of attention for their biological properties. We here investigated the ability of an ethanolic extract of Algerian propolis (EEP) to control melanoma tumour growth when given to mice bearing B16F1melanoma tumour either as preventive or as therapeutic treatment. EEP given after tumour occurrence increased mice survival (+30%) and reduced tumour growth (-75%). This was associated with a decrease of the Mitotic Index (-75%) and of Ki-67 (-50%) expression. When given either before or both before and after tumour occurrence, EEP reduced tumour growth but without prolonging mice life. Isolation of B16F1 melanoma cells from resected tumour showed that preventive and curative EEP treatments reduced invasiveness by 55% and 40% respectively compared to control. Galangin, one of the most abundant flavonoids in propolis, significantly reduced the number of melanoma cells in vitro and induced autophagy/apoptosis dose dependently. In conclusion, we showed that EEP reduced melanoma tumour progression/dissemination and could extend mice lifespan when used as therapeutic treatment. Then, EEP may help patients with melanoma when used as a complementary therapy to classical treatment for which autophagy is not contraindicated.

A strategy to discover decoy chemokine ligands with an anti-inflammatory activity.

Excessive signaling by chemokines has been associated with chronic inflammation or cancer, thus attracting substantial attention as promising therapeutic targets. Inspired by chemokine-clearing molecules shaped by pathogens to escape the immune system, we designed a generic screening assay to discover chemokine neutralizing molecules (neutraligands) and unambiguously distinguish them from molecules that block the receptor (receptor antagonists). This assay, called TRIC-r, combines time-resolved intracellular calcium recordings with pre-incubation of bioactive compounds either with the chemokine or the receptor-expressing cells. We describe here the identification of high affinity neutraligands of CCL17 and CCL22, two chemokines involved in the Th2-type of lung inflammation. The decoy molecules inhibit in vitro CCL17- or CCL22-induced intracellular calcium responses, CCR4 endocytosis and human T cell migration. In vivo, they inhibit inflammation in a murine model of asthma, in particular the recruitment of eosinophils, dendritic cells and CD4(+)T cells. Altogether, we developed a successful strategy to discover as new class of pharmacological tools to potently control cell chemotaxis in vitro and in vivo.