The commensal skin microbiota triggers type I IFN-dependent innate repair responses in injured skin.

Skin wounds heal by coordinated induction of inflammation and tissue repair, but the initiating events are poorly defined. Here we uncover a fundamental role of commensal skin microbiota in this process and show that it is mediated by the recruitment and the activation of type I interferon (IFN)-producing plasmacytoid DC (pDC). Commensal bacteria colonizing skin wounds trigger activation of neutrophils to express the chemokine CXCL10, which recruits pDC and acts as an antimicrobial protein to kill exposed microbiota, leading to the formation of CXCL10-bacterial DNA complexes. These complexes and not complexes with host-derived DNA activate pDC to produce type I IFNs, which accelerate wound closure by triggering skin inflammation and early T cell-independent wound repair responses, mediated by macrophages and fibroblasts that produce major growth factors required for healing. These findings identify a key function of commensal microbiota in driving a central innate wound healing response of the skin.

Diversification of human plasmacytoid predendritic cells in response to a single stimulus.

Innate immune cells adjust to microbial and inflammatory stimuli through a process termed environmental plasticity, which links a given individual stimulus to a unique activated state. Here, we report that activation of human plasmacytoid predendritic cells (pDCs) with a single microbial or cytokine stimulus triggers cell diversification into three stable subpopulations (P1-P3). P1-pDCs (PD-L1+CD80-) displayed a plasmacytoid morphology and specialization for type I interferon production. P3-pDCs (PD-L1-CD80+) adopted a dendritic morphology and adaptive immune functions. P2-pDCs (PD-L1+CD80+) displayed both innate and adaptive functions. Each subpopulation expressed a specific coding- and long-noncoding-RNA signature and was stable after secondary stimulation. P1-pDCs were detected in samples from patients with lupus or psoriasis. pDC diversification was independent of cell divisions or preexisting heterogeneity within steady-state pDCs but was controlled by a TNF autocrine and/or paracrine communication loop. Our findings reveal a novel mechanism for diversity and division of labor in innate immune cells.

T(H)17 cells promote microbial killing and innate immune sensing of DNA via interleukin 26.

Interleukin 17-producing helper T cells (T(H)17 cells) have a major role in protection against infections and in mediating autoimmune diseases, yet the mechanisms involved are incompletely understood. We found that interleukin 26 (IL-26), a human T(H)17 cell-derived cytokine, is a cationic amphipathic protein that kills extracellular bacteria via membrane-pore formation. Furthermore, T(H)17 cell-derived IL-26 formed complexes with bacterial DNA and self-DNA released by dying bacteria and host cells. The resulting IL-26-DNA complexes triggered the production of type I interferon by plasmacytoid dendritic cells via activation of Toll-like receptor 9, but independently of the IL-26 receptor. These findings provide insights into the potent antimicrobial and proinflammatory function of T(H)17 cells by showing that IL-26 is a natural human antimicrobial that promotes immune sensing of bacterial and host cell death.

Netting Neutrophils Activate Autoreactive B Cells in Lupus.

Lupus erythematosus (LE) patients develop autoantibodies that form circulating immune complexes (ICs) with extracellular self-nucleic acids. These ICs are deposited into peripheral tissues, where they trigger detrimental organ inflammation. Recent evidence suggests that ICs contain LL37-DNA complexes derived from neutrophil extracellular traps (NETs) and that LE patients develop pathogenic autoantibodies against these structures, including Abs to LL37. However, the mechanism that leads to the generation of these Abs is unknown. In this study, we show that NETs directly trigger Ab production by human memory B cells. This occurs via LL37-DNA complexes present in NETs, which have the unique ability to gain access to endosomal compartments of B cells and to trigger TLR9 activation. In LE patients, NET-derived LL37-DNA complexes trigger polyclonal B cell activation via TLR9, but also specifically expand self-reactive memory B cells producing anti-LL37 Abs in an Ag-dependent manner. These findings suggest a unique link between neutrophils and B cells in which NETs trigger a concerted activation of TLR9 and BCR leading to anti-NET autoantibody production in lupus.

CD28 Deficiency Enhances Type I IFN Production by Murine Plasmacytoid Dendritic Cells.

Type I IFNs (IFN-I) are key innate mediators that create a profound antiviral state and orchestrate the activation of almost all immune cells. Plasmacytoid dendritic cells (pDCs) are the most powerful IFN-I-producing cells and play important roles during viral infections, cancer, and autoimmune diseases. By comparing gene expression profiles of murine pDCs and conventional DCs, we found that CD28, a prototypic T cell stimulatory receptor, was highly expressed in pDCs. Strikingly, CD28 acted as a negative regulator of pDC IFN-I production upon TLR stimulation but did not affect pDC survival or maturation. Importantly, cell-intrinsic CD28 expression restrained pDC (and systemic) IFN-I production during in vivo RNA and DNA viral infections, limiting antiviral responses and enhancing viral growth early after exposure. Finally, CD28 also downregulated IFN-I response upon skin injury. Our study identified a new pDC regulatory mechanism by which the same CD28 molecule that promotes stimulation in most cells that express it is co-opted to negatively regulate pDC IFN-I production and limit innate responses.

STING activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity.

Spontaneous CD8 T-cell responses occur in growing tumors but are usually poorly effective. Understanding the molecular and cellular mechanisms that drive these responses is of major interest as they could be exploited to generate a more efficacious antitumor immunity. As such, stimulator of IFN genes (STING), an adaptor molecule involved in cytosolic DNA sensing, is required for the induction of antitumor CD8 T responses in mouse models of cancer. Here, we find that enforced activation of STING by intratumoral injection of cyclic dinucleotide GMP-AMP (cGAMP), potently enhanced antitumor CD8 T responses leading to growth control of injected and contralateral tumors in mouse models of melanoma and colon cancer. The ability of cGAMP to trigger antitumor immunity was further enhanced by the blockade of both PD1 and CTLA4. The STING-dependent antitumor immunity, either induced spontaneously in growing tumors or induced by intratumoral cGAMP injection was dependent on type I IFNs produced in the tumor microenvironment. In response to cGAMP injection, both in the mouse melanoma model and an ex vivo model of cultured human melanoma explants, the principal source of type I IFN was not dendritic cells, but instead endothelial cells. Similarly, endothelial cells but not dendritic cells were found to be the principal source of spontaneously induced type I IFNs in growing tumors. These data identify an unexpected role of the tumor vasculature in the initiation of CD8 T-cell antitumor immunity and demonstrate that tumor endothelial cells can be targeted for immunotherapy of melanoma.

[The skin microbiota: a colossus steps into the spotlight]. / Le microbiote cutané: le poids lourd sort de l'ombre.

The skin contains many commensal bacteria. For years, these microbes have been considered to be exploiters of the human host for nutrients. However, recent findings indicates that the skin microbiota is also used by the human host to protect himself against invading pathogens as the commensal bacteria have direct antimicrobial capacity and provide factors required to mount a protective immune responses in the skin. While the healthy skin microbiome functions as guardians of host defense, increased or decreased bacterial composition of the skin microbiome (called dysbiosis) leads to skin inflammation and disease. Here we will review the emerging data on the role of distinct types of dysbiosis in the pathogenesis skin diseases and illustrate how the new understanding of the role of the skin microbiome has implications in the clinical management of skin diseases.

Nucleic acid-containing amyloid fibrils potently induce type I interferon and stimulate systemic autoimmunity.

The immunopathophysiologic development of systemic autoimmunity involves numerous factors through complex mechanisms that are not fully understood. In systemic lupus erythematosus, type I IFN (IFN-I) produced by plasmacytoid dendritic cells (pDCs) critically promotes the autoimmunity through its pleiotropic effects on immune cells. However, the host-derived factors that enable abnormal IFN-I production and initial immune tolerance breakdown are largely unknown. Previously, we found that amyloid precursor proteins form amyloid fibrils in the presence of nucleic acids. Here we report that nucleic acid-containing amyloid fibrils can potently activate pDCs and enable IFN-I production in response to self-DNA, self-RNA, and dead cell debris. pDCs can take up DNA-containing amyloid fibrils, which are retained in the early endosomes to activate TLR9, leading to high IFNα/ß production. In mice treated with DNA-containing amyloid fibrils, a rapid IFN response correlated with pDC infiltration and activation. Immunization of nonautoimmune mice with DNA-containing amyloid fibrils induced antinuclear serology against a panel of self-antigens. The mice exhibited positive proteinuria and deposited antibodies in their kidneys. Intriguingly, pDC depletion obstructed IFN-I response and selectively abolished autoantibody generation. Our study reveals an innate immune function of nucleic acid-containing amyloid fibrils and provides a potential link between compromised protein homeostasis and autoimmunity via a pDC-IFN axis.

Human neutrophils drive skin autoinflammation by releasing interleukin (IL)-26.

Autoinflammation is a sterile inflammatory process resulting from increased neutrophil infiltration and overexpression of IL-1 cytokines. The factors that trigger these events are, however, poorly understood. By investigating pustular forms of psoriasis, we show that human neutrophils constitutively express IL-26 and abundantly release it from granular stores upon activation. In pustular psoriasis, neutrophil-derived IL-26 drives the pathogenic autoinflammation process by inducing the expression of IL-1 cytokines and chemokines that further recruit neutrophils. This occurs via activation of IL-26R in keratinocytes and via the formation of complexes between IL-26 and microbiota DNA, which trigger TLR9 activation of neutrophils. Thus our findings identify neutrophils as an important source of IL-26 and point to IL-26 as the key link between neutrophils and a self-sustaining autoinflammation loop in pustular psoriasis.

Cross-Comparison of Inflammatory Skin Disease Transcriptomics Identifies PTEN as a Pathogenic Disease Classifier in Cutaneous Lupus Erythematosus.

Tissue transcriptomics is used to uncover molecular dysregulations underlying diseases. However, the majority of transcriptomics studies focus on single diseases with limited relevance for understanding the molecular relationship between diseases or for identifying disease-specific markers. In this study, we used a normalization approach to compare gene expression across nine inflammatory skin diseases. The normalized datasets were found to retain differential expression signals that allowed unsupervised disease clustering and identification of disease-specific gene signatures. Using the NS-Forest algorithm, we identified a minimal set of biomarkers and validated their use as diagnostic disease classifier. Among them, PTEN was identified as being a specific marker for cutaneous lupus erythematosus and found to be strongly expressed by lesional keratinocytes in association with pathogenic type I IFNs. In fact, PTEN facilitated the expression of IFN-ß and IFN-κ in keratinocytes by promoting activation and nuclear translocation of IRF3. Thus, cross-comparison of tissue transcriptomics is a valid strategy to establish a molecular disease classification and to identify pathogenic disease biomarkers.

Binding with nucleic acids or glycosaminoglycans converts soluble protein oligomers to amyloid.

Ample evidence suggests that almost all polypeptides can either adopt a native structure (folded or intrinsically disordered) or form misfolded amyloid fibrils. Soluble protein oligomers exist as an intermediate between these two states, and their cytotoxicity has been implicated in the pathology of multiple human diseases. However, the mechanism by which soluble protein oligomers develop into insoluble amyloid fibrils is not clear, and investigation of this important issue is hindered by the unavailability of stable protein oligomers. Here, we have obtained stabilized protein oligomers generated from common native proteins. These oligomers exert strong cytotoxicity and display a common conformational structure shared with known protein oligomers. They are soluble and remain stable in solution. Intriguingly, the stabilized protein oligomers interact preferentially with both nucleic acids and glycosaminoglycans (GAG), which facilitates their rapid conversion into insoluble amyloid. Concomitantly, binding with nucleic acids or GAG strongly diminished the cytotoxicity of the protein oligomers. EGCG, a small molecule that was previously shown to directly bind to protein oligomers, effectively inhibits the conversion to amyloid. These results indicate that stabilized oligomers of common proteins display characteristics similar to those of disease-associated protein oligomers and represent immediate precursors of less toxic amyloid fibrils. Amyloid conversion is potently expedited by certain physiological factors, such as nucleic acids and GAGs. These findings concur with reports of cofactor involvement with disease-associated amyloid and shed light on potential means to interfere with the pathogenic properties of misfolded proteins.

Activin A-Mediated Polarization of Cancer-Associated Fibroblasts and Macrophages Confers Resistance to Checkpoint Immunotherapy in Skin Cancer.

PURPOSE: Cemiplimab is approved for the treatment of locally advanced basal cell carcinomas (BCC), although with mitigated results. We sought to interrogate the cellular and molecular transcriptional reprogramming underlying BCC resistance to immunotherapy. EXPERIMENTAL DESIGN: Here, we combined spatial and single-cell transcriptomics to deconvolute the spatial heterogeneity of the tumor microenvironment in regard with response to immunotherapy, in a cohort of both naïve and resistant BCCs. RESULTS: We identified subsets of intermingled cancer-associated fibroblasts (CAF) and macrophages contributing the most to CD8 T-cell exclusion and immunosuppression. Within this spatially resolved peritumoral immunosuppressive niche, CAFs and adjacent macrophages were found to display Activin A-mediated transcriptional reprogramming towards extracellular matrix remodeling, suggesting active participation to CD8 T-cell exclusion. In independent datasets of human skin cancers, Activin A-conditioned CAFs and macrophages were associated with resistance to immune checkpoint inhibitors (ICI). CONCLUSIONS: Altogether, our data identify the cellular and molecular plasticity of tumor microenvironment (TME) and the pivotal role of Activin A in polarizing the TME towards immune suppression and ICI resistance.

Type I IFNs link skin-associated dysbiotic commensal bacteria to pathogenic inflammation and angiogenesis in rosacea.

Rosacea is a common chronic inflammatory skin disease with a fluctuating course of excessive inflammation and apparent neovascularization. Microbial dysbiosis with a high density of Bacillus oleronius and increased activity of kallikrein 5, which cleaves cathelicidin antimicrobial peptide, are key pathogenic triggers in rosacea. However, how these events are linked to the disease remains unknown. Here, we show that type I IFNs produced by plasmacytoid DCs represent the pivotal link between dysbiosis, the aberrant immune response, and neovascularization. Compared with other commensal bacteria, B. oleronius is highly susceptible and preferentially killed by cathelicidin antimicrobial peptides, leading to enhanced generation of complexes with bacterial DNA. These bacterial DNA complexes but not DNA complexes derived from host cells are required for cathelicidin-induced activation of plasmacytoid DCs and type I IFN production. Moreover, kallikrein 5 cleaves cathelicidin into peptides with heightened DNA binding and type I IFN-inducing capacities. In turn, excessive type I IFN expression drives neoangiogenesis via IL-22 induction and upregulation of the IL-22 receptor on endothelial cells. These findings unravel a potentially novel pathomechanism that directly links hallmarks of rosacea to the killing of dysbiotic commensal bacteria with induction of a pathogenic type I IFN-driven and IL-22-mediated angiogenesis.

Differentiation of IL-26+ TH17 intermediates into IL-17A producers via epithelial crosstalk in psoriasis.

Interleukin (IL)-26 is a TH17 cytokine with known antimicrobial and pro-inflammatory functions. However, the precise role of IL-26 in the context of pathogenic TH17 responses is unknown. Here we identify a population of blood TH17 intermediates that produce high levels of IL-26 and differentiate into IL-17A-producing TH17 cells upon TGF-ß1 exposure. By combining single cell RNA sequencing, TCR sequencing and spatial transcriptomics we show that this process occurs in psoriatic skin. In fact, IL-26+ TH17 intermediates infiltrating psoriatic skin induce TGF-ß1 expression in basal keratinocytes and thereby promote their own differentiation into IL-17A-producing cells. Thus, our study identifies IL-26-producing cells as an early differentiation stage of TH17 cells that infiltrates psoriatic skin and controls its own maturation into IL17A-producing TH17 cells, via epithelial crosstalk involving paracrine production of TGF-ß1.

Single cell and spatial sequencing define processes by which keratinocytes and fibroblasts amplify inflammatory responses in psoriasis.

The immunopathogenesis of psoriasis, a common chronic inflammatory disease of the skin, is incompletely understood. Here we demonstrate, using a combination of single cell and spatial RNA sequencing, IL-36 dependent amplification of IL-17A and TNF inflammatory responses in the absence of neutrophil proteases, which primarily occur within the supraspinous layer of the psoriatic epidermis. We further show that a subset of SFRP2+ fibroblasts in psoriasis contribute to amplification of the immune network through transition to a pro-inflammatory state. The SFRP2+ fibroblast communication network involves production of CCL13, CCL19 and CXCL12, connected by ligand-receptor interactions to other spatially proximate cell types: CCR2+ myeloid cells, CCR7+ LAMP3+ dendritic cells, and CXCR4 expressed on both CD8+ Tc17 cells and keratinocytes, respectively. The SFRP2+ fibroblasts also express cathepsin S, further amplifying inflammatory responses by activating IL-36G in keratinocytes. These data provide an in-depth view of psoriasis pathogenesis, which expands our understanding of the critical cellular participants to include inflammatory fibroblasts and their cellular interactions.

Integrated multi-omics reveals cellular and molecular interactions governing the invasive niche of basal cell carcinoma.

Tumors invade the surrounding tissues to progress, but the heterogeneity of cell types at the tumor-stroma interface and the complexity of their potential interactions hampered mechanistic insight required for efficient therapeutic targeting. Here, combining single-cell and spatial transcriptomics on human basal cell carcinomas, we define the cellular contributors of tumor progression. In the invasive niche, tumor cells exhibit a collective migration phenotype, characterized by the expression of cell-cell junction complexes. In physical proximity, we identify cancer-associated fibroblasts with extracellular matrix-remodeling features. Tumor cells strongly express the cytokine Activin A, and increased Activin A-induced gene signature is found in adjacent cancer-associated fibroblast subpopulations. Altogether, our data identify the cell populations and their transcriptional reprogramming contributing to the spatial organization of the basal cell carcinoma invasive niche. They also demonstrate the power of integrated spatial and single-cell multi-omics to decipher cancer-specific invasive properties and develop targeted therapies.

TLR7 stimulation in human plasmacytoid dendritic cells leads to the induction of early IFN-inducible genes in the absence of type I IFN.

On recognition of influenza virus (Flu) by TLR7, plasmacytoid dendritic cells (pDCs) produce type I IFN in significant amounts. Synthetic TLR7 ligands induce the maturation of pDCs, as evidenced by the expression of costimulatory molecules and the production of proinflammatory cytokines; however, they induce only low-level production of IFN-alpha. To dissect the TLR7 signaling in pDCs and how these different profiles are induced, we studied the effects of 2 TLR7 ligands (Flu and CL097) on the activation of blood-isolated pDCs and the human GEN2.2 pDC cell line. Type I IFN production by pDCs correlates with differential interferon regulatory factor 7 (IRF7) translocation into the nucleus induced by the 2 TLR7 ligands. Surprisingly, with both activators we nevertheless observed the rapid expression of the IFN-inducible genes mxa, cxcl10, and trail within 4 hours of stimulation. This expression, controlled by STAT1 phosphorylation, was independent of type I IFN. STAT1 activation was found to be strictly dependent on the PI3K-p38MAPK pathway, showing a new signaling pathway leading to rapid expression of IFN-inducible genes after TLR7 triggering. Thus, pDCs, through this unusual TLR7 signaling, have the capacity to promptly respond to viral infection during the early phases of the innate immune response.

Plasmacytoid dendritic cells and dermatological disorders: focus on their role in autoimmunity and cancer.

Dendritic cells (DC), considered as immunological sentinels of the organism since they are antigen presenting cells, create the link between innate and adaptive immunity. DC include myeloid dendritic cells (MDC) and plasmacytoid dendritic cells (PDC). The presence of PDC, cells capable of producing large quantities of interferon alpha (IFN-alpha) in response to pathogenic agents or danger signals, seems to be closely related to pathological conditions. PDC have been observed in inflammatory immunoallergic dermatological disorders, in malignant cutaneous tumours and in cutaneous lesions of infectious origin. They seem to play a crucial role in the initiation of the pathological processes of autoimmune diseases such as lupus or psoriasis. Their function within a tumour context is not as well known and is controversial. They could have a tolerogenic role towards tumour cells in the absence of an activator but they also have the capacity to become activated in response to Toll-like receptor (TLR) ligands and could therefore be useful for therapeutic purposes.

Interleukin-26 activates macrophages and facilitates killing of Mycobacterium tuberculosis.

Tuberculosis-causing Mycobacterium tuberculosis (Mtb) is transmitted via airborne droplets followed by a primary infection of macrophages and dendritic cells. During the activation of host defence mechanisms also neutrophils and T helper 1 (TH1) and TH17 cells are recruited to the site of infection. The TH17 cell-derived interleukin (IL)-17 in turn induces the cathelicidin LL37 which shows direct antimycobacterial effects. Here, we investigated the role of IL-26, a TH1- and TH17-associated cytokine that exhibits antimicrobial activity. We found that both IL-26 mRNA and protein are strongly increased in tuberculous lymph nodes. Furthermore, IL-26 is able to directly kill Mtb and decrease the infection rate in macrophages. Binding of IL-26 to lipoarabinomannan might be one important mechanism in extracellular killing of Mtb. Macrophages and dendritic cells respond to IL-26 with secretion of tumor necrosis factor (TNF)-α and chemokines such as CCL20, CXCL2 and CXCL8. In dendritic cells but not in macrophages cytokine induction by IL-26 is partly mediated via Toll like receptor (TLR) 2. Taken together, IL-26 strengthens the defense against Mtb in two ways: firstly, directly due to its antimycobacterial properties and secondly indirectly by activating innate immune mechanisms.

Psoriasis Caught in the NET.

A report in the June 2019 issue of the Journal of Investigative Dermatology reveals a role of neutrophil extracellular traps (NETs) in the induction of T helper type 17 cell responses and shows the relevance of this pathway in patients with psoriasis carrying a common risk variant in the TRAF3IP2 gene (Lambert et al., 2019). This work provides a new piece to the puzzle that links neutrophils to the T helper type 17-mediated pathogenesis of psoriasis.