A Subset of Type I Conventional Dendritic Cells Controls Cutaneous Bacterial Infections through VEGFα-Mediated Recruitment of Neutrophils.

Skin conventional dendritic cells (cDCs) exist as two distinct subsets, cDC1s and cDC2s, which maintain the balance of immunity to pathogens and tolerance to self and microbiota. Here, we examined the roles of dermal cDC1s and cDC2s during bacterial infection, notably Propionibacterium acnes (P. acnes). cDC1s, but not cDC2s, regulated the magnitude of the immune response to P. acnes in the murine dermis by controlling neutrophil recruitment to the inflamed site and survival and function therein. Single-cell mRNA sequencing revealed that this regulation relied on secretion of the cytokine vascular endothelial growth factor α (VEGF-α) by a minor subset of activated EpCAM+CD59+Ly-6D+ cDC1s. Neutrophil recruitment by dermal cDC1s was also observed during S. aureus, bacillus Calmette-Guérin (BCG), or E. coli infection, as well as in a model of bacterial insult in human skin. Thus, skin cDC1s are essential regulators of the innate response in cutaneous immunity and have roles beyond classical antigen presentation.

Modelling atopic dermatitis in healthy human skin for the characterization of topical compounds.

Suitable human models for the development and characterization of topical compounds for inflammatory skin diseases such as atopic dermatitis are not readily available to date. We describe here the development of a translational model involving healthy human skin mimicking major aspects of AD and its application for the characterization of topical Janus kinase inhibitors. Full thickness human abdominal skin obtained from plastic surgery stimulated in vitro with IL4 and IL13 shows molecular features of AD. This is evidenced by STAT6 phosphorylation assessed by immunohistochemistry and analysis of skin lysates. Broad transcriptome changes assessed by AmpliSeq followed by gene set variation analysis showed a consistent upregulation of gene signatures characterizing AD in this model. Topical application of experimental formulations of compounds targeting the JAK pathway to full thickness skin normalizes the molecular features of AD induced by IL4 and IL13 stimulation. The inhibitory effects of topical JAK inhibitors on molecular features of AD are supported by pharmacokinetic analysis. The model described here is suited for the characterization of topical compounds for AD and has the potential to be extended to other inflammatory skin diseases and pathophysiological pathways.

Discovery of a novel series of selective macrocyclic PKCTheta inhibitors.

A series of macrocyclic PKCθ inhibitors based on a 1,3-dihydro-2H-imidazo[4,5-b]pyridin-2-one hinge binder has been studied. Different aromatic and heteroaromatic substituents have been explored in order to optimize potency, isoform selectivity as well as DMPK properties. The importance of the length of the macrocyclic linker has also been analyzed. In particular, it has been found that methyl substitutions on the linker can have a profound influence on both potency and metabolic stability. Several compounds showing very good profiles, suitable for in vivo testing, are disclosed.

Transcriptomic characterization of prurigo nodularis and the therapeutic response to nemolizumab.

BACKGROUND: Prurigo nodularis (PN) is a debilitating, difficult-to-treat, intensely pruritic, chronic inflammatory skin disease characterized by hyperkeratotic skin nodules. The pathogenesis of PN is not well understood but is believed to involve cross talk between sensory nerve fibers, immune cells, and the epidermis. It is centered around the neuroimmune cytokine IL-31, driving an intractable itch-scratch cycle. OBJECTIVE: We sought to provide a comprehensive view of the transcriptomic changes in PN skin and characterize the mechanism of action of the anti-IL-31 receptor inhibitor nemolizumab. METHOD: RNA sequencing of biopsy samples obtained from a cohort of patients treated with the anti-IL-31 receptor inhibitor nemolizumab and taken at baseline and week 12. Generation and integration of patient data with RNA-Seq data generated from reconstructed human epidermis stimulated with IL-31 and other proinflammatory cytokines. RESULTS: Our results demonstrate that nemolizumab effectively decreases IL-31 responses in PN skin, leading to effective suppression of downstream inflammatory responses including TH2/IL-13 and TH17/IL-17 responses. This is accompanied by decreased keratinocyte proliferation and normalization of epidermal differentiation and function. Furthermore, our results demonstrate how transcriptomic changes associated with nemolizumab treatment correlate with improvement in lesions, pruritus, stabilization of extracellular matrix remodeling, and processes associated with cutaneous nerve function. CONCLUSION: These data demonstrate a broad response to IL-31 receptor inhibition with nemolizumab and confirm the critical upstream role of IL-31 in PN pathogenesis.

Intestinal dendritic cell licensing through Toll-like receptor 4 is required for oral tolerance in allergic contact dermatitis.

BACKGROUND: Induction of oral tolerance to haptens is an efficient way to prevent allergic contact dermatitis (ACD) in mice. Toll-like receptor (TLR)-mediated sensing of the microbiota contributes to gut homeostasis, yet whether it contributes to induction of oral tolerance has not been documented. OBJECTIVE: We examined whether oral tolerance to the contact sensitizer 2,4-dinitro-fluorobenzene (DNFB) depends on microbiota/TLRs and evaluated the role of TLR4 on the tolerogenic function of intestinal dendritic cells (DCs). METHODS: Oral tolerance was induced by DNFB gavage in germ-free and mice deficient in several TLRs. Tolerance was assessed by means of suppression of contact hypersensitivity and hapten-specific IFN-γ-producing effector T cells. The tolerogenic function of intestinal DCs was tested by adoptive transfer experiments, ex vivo hapten presentation, and forkhead box p3 regulatory T-cell conversion. RESULTS: Oral tolerance induced by DNFB gavage was impaired in germ-free mice and TLR4-deficient mice. Bone marrow chimeras revealed that TLR4 expression on hematopoietic cells was necessary for oral tolerance induction. TLR4 appeared to be essential for the ability of intestinal dendritic cells from DNFB-fed mice to inhibit ACD on adoptive transfer. Indeed, TLR4 conditioned the in vivo mobilization to mesenteric lymph nodes of intestinal migratory CD103+ DCs carrying oral DNFB, especially the CD103+CD11b+ DC subset expressing the vitamin A-converting enzyme retinaldehyde dehydrogenase and specialized in forkhead box p3-positive regulatory T-cell conversion. CONCLUSIONS: Our data demonstrate that TLR4 conditions induction of oral tolerance to DNFB through licensing tolerogenic gut DCs. Oral biotherapy with TLR4 ligands might be useful to potentiate oral tolerance to haptens and alleviate ACD in human subjects.

Sulfoximines as potent RORγ inverse agonists.

Progress in the identification of suitable RORγ inverse agonists as clinical candidates has been hampered by the high lipophilicity that seems required for high potency on this nuclear receptor. In this context, we decided to focus on the replacement of the hydroxymethyl group found on known modulators to determine if more polarity could be tolerated in this position. SAR of the replacement of this moiety is presented in this article leading to the identification of sulfoximine derivatives as potent modulators with pharmacological activity in the in vivo mouse Imiquimod psoriasis model.

Discovery and process development of a novel TACE inhibitor for the topical treatment of psoriasis.

Targeting the TNFα pathway is a validated approach to the treatment of psoriasis. In this pathway, TACE stands out as a druggable target and has been the focus of in-house research programs. In this article, we present the discovery of clinical candidate 26a. Starting from hits plagued with poor solubility or genotoxicity, 26a was identified through thorough multiparameter optimisation. Showing robust in vivo activity in an oxazolone-mediated inflammation model, the compound was selected for development. Following a polymorph screen, the hydrochloride salt was selected and the synthesis was efficiently developed to yield the API in 47% overall yield.

Identification of novel TACE inhibitors compatible with topical application.

Targeting the Tumor Necrosis Factor α signalling with antibodies has led to a revolution in the treatment of psoriasis. Locally inhibiting Tumor Necrosis Factor α Converting Enzyme (TACE or ADAM17) could potentially mimic those effects and help treat mild to moderate psoriasis, without the reported side effect of systemic TACE inhibitors. Efforts to identify new TACE inhibitors are presented here. Enzymatic SAR as well as ADME and physico-chemistry data are presented. This study culminated in the identification of potent enzymatic inhibitors. Suboptimal cellular activity of this series is discussed in the context of previously published results.

Low-allergenic hydrolyzed egg induces oral tolerance in mice.

BACKGROUND: Egg allergy is one of the most common food allergies in children. The standard therapy for egg allergy is strict avoidance. Yet, there is considerable clinical and scientific interest in primary or secondary prevention. A major drawback of oral tolerance (OT) induction protocols, however, is the possibility of severe side effects; thus, we have formulated a hypoallergenic egg product and demonstrate its in vivo capacity to modulate the immune system in the current study. METHODS: Hydrolyzed egg (HE) was produced using a combination of moderate heat treatment and enzymatic hydrolysis. The capacity of HE to induce OT was tested in experimental models and compared to whole egg (WE). Delayed-type hypersensitivity (DTH) responses, immune markers and potential early markers of OT were analyzed. RESULTS: Allergic responses, assessed by both DTH responses upon OVA challenge and serum OVA-specific IgE and IgG1, were decreased after treatment with HE and WE compared to the control group. Additionally, feeding WE and HE significantly decreased Th2 cytokine induction and cell proliferation, induced the activation of effector CD4+ T cells and increased numbers and percentages of ICOS+CD4+CD25+Foxp3+ cells. Furthermore, DO11.10 mouse experiments showed that HE contains other peptides than the OVA323-339 peptide that are able to induce tolerance to OVA. CONCLUSIONS: Altogether, results showed that HE induces OT in mice in a dose-dependent manner. Due to its low allergenicity compared to WE, it may represent a safer alternative for OT induction in at-risk subjects or oral immunotherapy in allergic patients.

Design of a Supersoft Topical JAK Inhibitor, Which Is Effective in Human Skin but Rapidly Deactivated in Blood.

We describe the discovery and characterization of the supersoft topical JAK inhibitor 3(R), which is potent in biochemical and cellular assays as well as in human skin models. In blood, the neutral ester 3(R) is rapidly hydrolyzed (t1/2 ∼ 6 min) to the corresponding charged carboxylic acid 4 exhibiting >30-fold reduced permeability. Consequently, acid 4 does not reach the intracellular JAK kinases and is inactive in cellular assays and in blood. Thus, hydrolysis by blood esterases leads to the rapid deactivation of topically active ester 3(R) at a rate beyond the maximal hepatic clearance.

CD4+ T cells and Lactobacillus casei control relapsing colitis mediated by CD8+ T cells.

Evidence that CD4(+) regulatory T cells can control Ag-specific CD8(+) T cell-mediated colitis in immunocompetent mice is poorly documented. To examine the potential of CD4(+) T cells to control colitis, we used our model of CD8(+) T cell-mediated colitis induced by intracolonic sensitization followed by challenge with the hapten 2,4-dinitrobenzene sulfonic acid. The defect of CD4(+) T cells in MHC class II-deficient (Abeta(degrees/degrees)) mice allowed priming of 2,4-dinitrobenzene sulfonic acid-specific IFN-gamma-producing CD8 colitogenic effectors and development of colitis in the otherwise resistant C57BL/6 strain. Cotransfer experiments in RAG2(degrees/degrees) mice and ex vivo studies showed that CD4(+)CD25(+) T cells completely prevented CD8(+) T cell-mediated colitis and controlled CD8(+) T cell activation, respectively. In the susceptible BALB/c strain, Ab depletion revealed that lack of CD4(+) regulatory T cells resulted in 1) acute colitis elicited by a suboptimal dose of hapten challenge and 2) more severe relapsing episodes of colitis induced by effector/memory CD8(+) T cell-mediated colitis at an optimal dose of hapten challenge, even when CD4 depletion was performed just before the second challenge. Oral administration of the probiotic strain Lactobacillus casei DN-114 001 alleviated colitis and increased the suppressive function of Foxp3(+)CD4(+) regulatory T cells of colon lamina propria. These data demonstrate that CD4(+) regulatory T cells exert a protective effect on colitis by controlling colitogenic effector/memory CD8(+) T cells during the effector (symptomatic) phase of acute and relapsing colitis, respectively. Probiotics with natural adjuvant effects on mucosal regulatory T cells may represent a valuable approach to alleviate the colitogenic effect of Tc1-type CD8(+) effectors.

Single-cell analysis of human skin identifies CD14+ type 3 dendritic cells co-producing IL1B and IL23A in psoriasis.

Inflammatory skin diseases including atopic dermatitis (AD) and psoriasis (PSO) are underpinned by dendritic cell (DC)-mediated T cell responses. Currently, the heterogeneous human cutaneous DC population is incompletely characterized, and its contribution to these diseases remains unclear. Here, we performed index-sorted single-cell flow cytometry and RNA sequencing of lesional and nonlesional AD and PSO skin to identify macrophages and all DC subsets, including the newly described mature LAMP3+BIRC3+ DCs enriched in immunoregulatory molecules (mregDC) and CD14+ DC3. By integrating our indexed data with published skin datasets, we generated a myeloid cell universe of DC and macrophage subsets in healthy and diseased skin. Importantly, we found that CD14+ DC3s increased in PSO lesional skin and co-produced IL1B and IL23A, which are pathological in PSO. Our study comprehensively describes the molecular characteristics of macrophages and DC subsets in AD and PSO at single-cell resolution, and identifies CD14+ DC3s as potential promoters of inflammation in PSO.

Discovery and Characterization of CD12681, a Potent RORγ Inverse Agonist, Preclinical Candidate for the Topical Treatment of Psoriasis.

With possible implications in multiple autoimmune diseases, the retinoic acid receptor-related orphan receptor RORγ has become a sought-after target in the pharmaceutical industry. Herein are described the efforts to identify a potent RORγ inverse agonist compatible with topical application for the treatment of skin diseases. These efforts culminated in the discovery of N-(2,4-dimethylphenyl)-N-isobutyl-2-oxo-1-[(tetrahydro-2H-pyran-4-yl)methyl]-2,3-dihydro-1H-benzo[d]imidazole-5-sulfonamide (CD12681), a potent inverse agonist with in vivo activity in an IL-23-induced mouse skin inflammation model.

Topical Ivermectin 10 mg/g and Oral Doxycycline 40 mg Modified-Release: Current Evidence on the Complementary Use of Anti-Inflammatory Rosacea Treatments.

Rosacea is a common, chronic inflammatory skin disease that can present with a variety of signs and symptoms. The potentially simultaneous occurrence of different signs and symptoms is due to different underlying inflammatory pathways, emphasizing the need for complementary treatment approaches. Topical ivermectin cream (10 mg/g) and systemic, oral anti-inflammatory doxycycline (40 mg modified-release) are both approved for the treatment of papulopustular rosacea (PPR). Whether or not a combined therapeutic approach may be more beneficial than monotherapy for patients with PPR remains to be tested. Here, we summarize underlying inflammatory pathways implicated in rosacea and clarify the impact of these two agents on selective pathways during inflammation, due to specific characteristics of their individual mechanisms of action (MoA). Based on the complementary MoA of doxycycline modified-release and ivermectin, a scientific rationale for a combined therapy targeting inflammatory lesions in rosacea is given. We propose that topical ivermectin cream is a promising new candidate as first-line treatment to target the inflammatory lesions of rosacea, which can be used in combination with systemic doxycycline modified-release to provide an optimal treatment approach considering all inflammatory pathways involved in PPR. Funding Galderma.

Langerhans cells protect from allergic contact dermatitis in mice by tolerizing CD8(+) T cells and activating Foxp3(+) regulatory T cells.

Allergic contact dermatitis is the most frequent occupational disease in industrialized countries. It is caused by CD8(+) T cell-mediated contact hypersensitivity (CHS) reactions triggered at the site of contact by a variety of chemicals, also known as weak haptens, present in fragrances, dyes, metals, preservatives, and drugs. Despite the myriad of potentially allergenic substances that can penetrate the skin, sensitization is relatively rare and immune tolerance to the substance is often induced by as yet poorly understood mechanisms. Here we show, using the innocuous chemical 2,4-dinitrothiocyanobenzene (DNTB), that cutaneous immune tolerance in mice critically depends on epidermal Langerhans cells (LCs), which capture DNTB and migrate to lymph nodes for direct presentation to CD8(+) T cells. Depletion and adoptive transfer experiments revealed that LCs conferred protection from development of CHS by a mechanism involving both anergy and deletion of allergen-specific CD8(+) T cells and activation of a population of T cells identified as ICOS(+)CD4(+)Foxp3(+) Tregs. Our findings highlight the critical role of LCs in tolerance induction in mice to the prototype innocuous hapten DNTB and suggest that strategies targeting LCs might be valuable for prevention of cutaneous allergy.

Infant gut microbiota is protective against cow's milk allergy in mice despite immature ileal T-cell response.

Faecal microbiota of healthy infant displays a large abundance of Bifidobacterium spp. and Bacteroides spp. Although some studies have reported an association between these two genera and allergy, these findings remain a subject of debate. Using a gnotobiotic mouse model of cow's milk allergy, we investigated the impact of an infant gut microbiota ­ mainly composed of Bifidobacterium and Bacteroides spp. ­ on immune activation and allergic manifestations. The transplanted microbiota failed to restore an ileal T-cell response similar to the one observed in conventional mice. This may be due to the low bacterial translocation into Peyer's patches in gnotobiotic mice. The allergic response was then monitored in germ-free, gnotobiotic, and conventional mice after repeated oral sensitization with whey proteins and cholera toxin. Colonized mice displayed a lower drop of rectal temperature upon oral challenge with b-lactoglobulin, lower plasma mMCP-1, and lower anti-BLG IgG1 than germ-free mice. The foxp3 gene was highly expressed in the ileum of both colonized mice that were protected against allergy. This study is the first demonstration that a transplanted healthy infant microbiota mainly composed of Bifidobacterium and Bacteroides had a protective impact on sensitization and food allergy in mice despite altered T-cell response in the ileum.

Oral probiotic control skin inflammation by acting on both effector and regulatory T cells.

Probiotics are believed to alleviate allergic and inflammatory skin disorders, but their impact on pathogenic effector T cells remains poorly documented. Here we show that oral treatment with the probiotic bacteria L. casei (DN-114 001) alone alleviates antigen-specific skin inflammation mediated by either protein-specific CD4(+) T cells or hapten-specific CD8(+) T cells. In the model of CD8(+) T cell-mediated skin inflammation, which reproduces allergic contact dermatitis in human, inhibition of skin inflammation by L. casei is not due to impaired priming of hapten-specific IFNgamma-producing cytolytic CD8(+) effector T cells. Alternatively, L. casei treatment reduces the recruitment of CD8(+) effector T cells into the skin during the elicitation (i.e. symptomatic) phase of CHS. Inhibition of skin inflammation by L. casei requires MHC class II-restricted CD4(+) T cells but not CD1d-restricted NK-T cells. L casei treatment enhanced the frequency of FoxP3(+) Treg in the skin and increased the production of IL-10 by CD4(+)CD25(+) regulatory T cells in skin draining lymph nodes of hapten-sensitized mice. These data demonstrate that orally administered L. casei (DN-114 001) efficiently alleviate T cell-mediated skin inflammation without causing immune suppression, via mechanisms that include control of CD8(+) effector T cells and involve regulatory CD4(+) T cells. L. casei (DN-114 001) may thus represent a probiotic of potential interest for immunomodulation of T cell-mediated allergic skin diseases in human.