Interleukin-2-induced skin inflammation.

Recombinant human IL-2 has been used to treat inflammatory diseases and cancer; however, side effects like skin rashes limit the use of this therapeutic. To identify key molecules and cells inducing this side effect, we characterized IL-2-induced cutaneous immune reactions and investigated the relevance of CD25 (IL-2 receptor α) in the process. We injected IL-2 intradermally into WT mice and observed increases in immune cell subsets in the skin with preferential increases in frequencies of IL-4- and IL-13-producing group 2 innate lymphoid cells and IL-17-producing dermal γδ T cells. This overall led to a shift toward type 2/type 17 immune responses. In addition, using a novel topical genetic deletion approach, we reduced CD25 on skin, specifically on all cutaneous cells, and found that IL-2-dependent effects were reduced, hinting that CD25 - at least partly - induces this skin inflammation. Reduction of CD25 specifically on skin Tregs further augmented IL-2-induced immune cell infiltration, hinting that CD25 on skin Tregs is crucial to restrain IL-2-induced inflammation. Overall, our data support that innate lymphoid immune cells are key cells inducing side effects during IL-2 therapy and underline the significance of CD25 in this process.

Regulatory T cells in skin mediate immune privilege of the hair follicle stem cell niche.

Immune tolerance is maintained in lymphoid organs (LOs). Despite the presence of complex immune cell networks in non-LOs, it is unknown whether self-tolerance is maintained in these tissues. We developed a technique to restrict genetic recombination to regulatory T cells (Tregs) only in skin. Selective depletion of skin Tregs resulted in T cell-mediated inflammation of hair follicles (HFs). Suppression did not rely on CTLA-4, but instead on high-affinity interleukin-2 (IL-2) receptor expression by skin Tregs, functioning exclusively in a cell-extrinsic manner. In a novel model of HF stem cell (HFSC)-driven autoimmunity, we reveal that skin Tregs immunologically protect the HFSC niche. Finally, we used spatial transcriptomics to identify aberrant IL-2 signaling at stromal-HF interfaces in a rare form of human alopecia characterized by HFSC destruction and alopecia areata. Collectively, these results reveal the fundamental biology of Tregs in skin uncoupled from the systemic pool and elucidate a mechanism of self-tolerance.

Tertiary lymphoid structures sustain cutaneous B cell activity in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a chronic skin condition affecting approximately 1% of the US population. HS skin lesions are highly inflammatory and characterized by a large immune infiltrate. While B cells and plasma cells comprise a major component of this immune milieu, the biology and the contribution of these cells in HS pathogenesis are unclear. We aimed to investigate the dynamics and microenvironmental interactions of B cells within cutaneous HS lesions. Combining histological analysis, single-cell RNA sequencing, and spatial transcriptomics profiling of HS lesions, we defined the tissue microenvironment relative to B cell activity within this disease. Our findings identified tertiary lymphoid structures (TLSs) within HS lesions and described organized interactions among T cells, B cells, antigen-presenting cells, and skin stroma. We found evidence that B cells within HS TLSs actively underwent maturation, including participation in germinal center reactions and class switch recombination. Moreover, skin stroma and accumulating T cells were primed to support the formation of TLSs and facilitate B cell recruitment during HS. Our data definitively demonstrated the presence of TLSs in lesional HS skin and point to ongoing cutaneous B cell maturation through class switch recombination and affinity maturation during disease progression in this inflamed nonlymphoid tissue.

Genetic and Immunohistochemical Profiling of Mammary Hidradenoma and Comparison to Mucoepidermoid Carcinoma.

Mucoepidermoid carcinoma (MEC) is exceedingly rare in the breast, with <45 cases reported in the literature. Although estrogen receptor/progesterone receptor/human epidermal growth factor 2 triple-negative, MEC is characterized as a special subtype of breast carcinoma with significantly better prognosis than conventional basal-type tumors. Cutaneous hidradenoma (HA) is considered a benign adnexal neoplasm showing histomorphologic overlap with MEC. Rare cases of HA have also been reported in the breast, but these are relatively uncharacterized. In this study, we examined the clinicopathologic, immunohistochemical (IHC), and genetic features of 8 breast HAs, in comparison to 3 mammary MECs. All cases were positive for MAML2 break-apart fluorescence in situ hybridization. Eight cases demonstrated a CRTC1::MAML2 fusion, and one MEC harbored a CRTC3::MAML2 fusion; the latter is a novel finding in the breast. Mutational burden was very low, with only one HA exhibiting a MAP3K1 pathogenic alteration. By IHC, both MEC and HA demonstrated cell type-dependent expression of high- and low-molecular-weight keratins and p63, as well as negative to low-positive estrogen receptor and androgen receptor. Smooth muscle myosin and calponin highlighted an in situ component in the 3 cases of MEC; expression of these myoepithelial markers was negative in HAs. Additional distinguishing characteristics included the growth pattern and tumor architecture, the presence of glandular/luminal cells in HA, and overall higher IHC expression of SOX10, S100 protein, MUC4, and mammaglobin in MEC. Morphologic findings were also compared to a series of 27 cutaneous nonmammary HAs. Mucinous and glandular/luminal cells were identified in significantly more mammary HAs than nonmammary lesions. The findings provide insight into the pathogenesis of MAML2-rearranged neoplasms of the breast, underscore the overlapping genetic features of MEC and HA, and highlight similarities to their extramammary counterparts.

Tertiary Lymphoid Structures Sustain Cutaneous B cell Activity in Hidradenitis Suppurativa.

Background: Hidradenitis suppurativa (HS) skin lesions are highly inflammatory and characterized by a large immune infiltrate. While B cells and plasma cells comprise a major component of this immune milieu the biology and contribution of these cells in HS pathogenesis is unclear. Objective: We aimed to investigate the dynamics and microenvironmental interactions of B cells within cutaneous HS lesions. Methods: We combined histological analysis, single-cell RNA-sequencing (scRNAseq), and spatial transcriptomic profiling of HS lesions to define the tissue microenvironment relative to B cell activity within this disease. Results: Our findings identify tertiary lymphoid structures (TLS) within HS lesions and describe organized interactions between T cells, B cells, antigen presenting cells and skin stroma. We find evidence that B cells within HS TLS actively undergo maturation, including participation in germinal center reactions and class switch recombination. Moreover, skin stroma and accumulating T cells are primed to support the formation of TLS and facilitate B cell recruitment during HS. Conclusion: Our data definitively demonstrate the presence of TLS in lesional HS skin and point to ongoing cutaneous B cell maturation through class switch recombination and affinity maturation during disease progression in this inflamed non-lymphoid tissue.

Innate type 2 immunity controls hair follicle commensalism by Demodex mites.

Demodex mites are commensal parasites of hair follicles (HFs). Normally asymptomatic, inflammatory outgrowth of mites can accompany malnutrition, immune dysfunction, and aging, but mechanisms restricting Demodex outgrowth are not defined. Here, we show that control of mite HF colonization in mice required group 2 innate lymphoid cells (ILC2s), interleukin-13 (IL-13), and its receptor, IL-4Ra-IL-13Ra1. HF-associated ILC2s elaborated IL-13 that attenuated HFs and epithelial proliferation at anagen onset; in their absence, Demodex colonization led to increased epithelial proliferation and replacement of gene programs for repair by aberrant inflammation, leading to the loss of barrier function and HF exhaustion. Humans with rhinophymatous acne rosacea, an inflammatory condition associated with Demodex, had increased HF inflammation with decreased type 2 cytokines, consistent with the inverse relationship seen in mice. Our studies uncover a key role for skin ILC2s and IL-13, which comprise an immune checkpoint that sustains cutaneous integrity and restricts pathologic infestation by colonizing HF mites.

Xenograft Skin Model to Manipulate Human Immune Responses In Vivo.

The human skin xenograft model, in which human donor skin is transplanted onto an immunodeficient mouse host, is an important option for translational research in skin immunology. Murine and human skin differ substantially in anatomy and immune cell composition. Therefore, traditional mouse models have limitations for dermatological research and drug discovery. However, successful xenotransplants are technically challenging and require optimal specimen and mouse graft site preparation for graft and host survival. The present protocol provides an optimized technique for transplanting human skin onto mice and discusses necessary considerations for downstream experimental aims. This report describes the appropriate preparation of a human donor skin sample, assembly of a surgical setup, mouse and surgical site preparation, skin transplantation, and post-surgical monitoring. Adherence to these methods allows for maintenance of xenografts for over 6 weeks post-surgery. The techniques outlined below allow maximum grafting efficiency due to the development of engineering controls, sterile technique, and pre- and post-surgical conditioning. Appropriate performance of the xenograft model results in long-lived human skin graft samples for experimental characterization of human skin and preclinical testing of compounds in vivo.

Intestinal inflammation alters the antigen-specific immune response to a skin commensal.

Resident microbes in skin and gut predominantly impact local immune cell function during homeostasis. However, colitis-associated neutrophilic skin disorders suggest possible breakdown of this compartmentalization with disease. Using a model wherein neonatal skin colonization by Staphylococcus epidermidis facilitates generation of commensal-specific tolerance and CD4+ regulatory T cells (Tregs), we ask whether this response is perturbed by gut inflammation. Chemically induced colitis is accompanied by intestinal expansion of S. epidermidis and reduces gut-draining lymph node (dLN) commensal-specific Tregs. It also results in reduced commensal-specific Tregs in skin and skin-dLNs and increased skin neutrophils. Increased CD4+ circulation between gut and skin dLN suggests that the altered cutaneous response is initiated in the colon, and resistance to colitis-induced effects in Cd4creIl1r1fl/fl mice implicate interleukin (IL)-1 in mediating the altered commensal-specific response. These findings provide mechanistic insight into observed connections between inflammatory skin and intestinal diseases.

Obesity alters pathology and treatment response in inflammatory disease.

Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation. We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.

Cicatricial pemphigoid Brunsting-Perry variant masquerading as neutrophil-mediated cicatricial alopecia.

A 72-year-old male presented with scarring alopecia on the scalp vertex, multiple crusted plaques on the hairline, and a history of vesicular eruption on the face. The scalp showed crusted plaques with loss of follicular ostia. No follicular pustules or compound follicles were present. An initial transverse scalp biopsy showed perifollicular neutrophils, lymphocytes, and plasma cells along with dermal fibrosis. Focal epidermal/dermal and follicular/adventitial dermal clefts were apparent but were thought to be secondary to fibrosis, and the biopsy result was interpreted to represent a neutrophil-mediated cicatricial alopecia. Concurrently, direct immunofluorescence (DIF) analysis showed linear junctional deposition of IgG and C3. A repeat scalp biopsy revealed more prominent epidermal/dermal clefts, fibrosis, mixed infiltrate with neutrophils, lymphocytes, histiocytes, and plasma cells, as well as prominent follicular/adventitial dermal clefts with perifollicular neutrophils. Given the combination of clefts, perijunctional neutrophils, and positive DIF findings, it became clear that this eruption represented the Brunsting-Perry variant of cicatricial pemphigoid. Here, we illustrated that a neutrophil-rich form of cicatricial pemphigoid can masquerade as a neutrophil-mediated scarring alopecia. In evaluating a specimen suspected to be a neutrophil-mediated scarring alopecia, one should be alert to the presence of subepidermal and perifollicular clefting, and consider cicatricial pemphigoid.

Superficial Angiomyxomas Frequently Demonstrate Loss of Protein Kinase A Regulatory Subunit 1 Alpha Expression: Immunohistochemical Analysis of 29 Cases and Cutaneous Myxoid Neoplasms With Histopathologic Overlap.

Superficial angiomyxomas (SAMs) are benign cutaneous tumors that arise de novo and in the setting of the Carney complex (CC), an autosomal dominant disease with several cutaneous manifestations including lentigines and pigmented epithelioid melanocytomas. Although most SAM do not pose a diagnostic challenge, a subset can demonstrate histopathologic overlap with other myxoid tumors that arise in the skin and subcutis. Traditional immunohistochemical markers are of limited utility when discriminating SAM from histopathologic mimics. Since protein kinase A regulatory subunit 1 alpha (PRKAR1A) genetic alterations underlie most CC cases, we investigated whether SAM demonstrate loss of PRKAR1A protein expression by immunohistochemistry. In our series, 29 SAM, 26 myxofibrosarcoma, 5 myxoid dermatofibrosarcoma protuberans, 11 superficial acral fibromyxomas, and 18 digital mucous cysts were characterized. Of the 29 SAM examined in this study, 1 was associated with documented CC in a 5-year-old girl. SAM tended to arise in adults (mean 49.7 y; range: 5 to 87 y). Loss of PRKAR1A was seen in 55.2% of cases (16/29) and had a male predilection (87.5%, 12/16). PRKAR1A-inactivated SAM demonstrated significant nuclear enlargement (100%, 16/16 vs. 23.1%, 3/13), multinucleation (81.3%, 13/16 vs. 23.1%, 3/13), and presence of neutrophils (43.8%, 7/16 vs. 0%, 0/13). In contrast, PRKAR1A was retained in all cases of myxofibrosarcoma (100%, 26/26), myxoid dermatofibrosarcoma protuberans (100%, 5/5), superficial acral fibromyxomas (100%, 11/11), and digital mucous cyst (100%, 18/18). Taken together, PRKAR1A loss by immunohistochemistry can be used as an adjunctive assay to support the diagnosis of SAM given the high specificity of this staining pattern compared with histopathologic mimics.

Early-life inflammation primes a T helper 2 cell-fibroblast niche in skin.

Inflammation early in life can prime the local immune milieu of peripheral tissues, which can cause lasting changes in immunological tone that confer disease protection or susceptibility1. The cellular and molecular mechanisms that prompt changes in immune tone in many nonlymphoid tissues remain largely unknown. Here we find that time-limited neonatal inflammation induced by a transient reduction in neonatal regulatory T cells causes a dysregulation of subcutaneous tissue in mouse skin. This is accompanied by the selective accumulation of type 2 helper T (TH2) cells within a distinct microanatomical niche. TH2 cells are maintained into adulthood through interactions with a fibroblast population in skin fascia that we refer to as TH2-interacting fascial fibroblasts (TIFFs), which expand in response to TH2 cytokines to form subcutaneous fibrous bands. Activation of the TH2-TIFF niche due to neonatal inflammation primes the skin for altered reparative responses to wounding. Furthermore, we identify fibroblasts in healthy human skin that express the TIFF transcriptional signature and detect these cells at high levels in eosinophilic fasciitis, an orphan disease characterized by inflammation and fibrosis of the skin fascia. Taken together, these data define a previously unidentified TH2 cell niche in skin and functionally characterize a disease-associated fibroblast population. The results also suggest a mechanism of immunological priming whereby inflammation early in life creates networks between adaptive immune cells and stromal cells to establish an immunological set-point in tissues that is maintained throughout life.

Regulatory T cells promote innate inflammation after skin barrier breach via TGF-ß activation.

Regulatory T cells (Tregs) use multiple mechanisms to attenuate inflammation and prevent autoimmunity. Tregs residing in peripheral (i.e., nonlymphoid) tissues have specialized functions; specifically, skin Tregs promote wound healing, suppress dermal fibrosis, facilitate epidermal regeneration, and augment hair follicle cycling. Here, we demonstrated that skin Tregs were transcriptionally attuned to interact with their tissue environment through increased expression of integrin and TGF-ß pathway genes that influence epithelial cell biology. We identified a molecular pathway where skin Tregs license keratinocytes to promote innate inflammation after skin barrier breach. Using a single-cell discovery approach, we identified preferential expression of the integrin αvß8 on skin Tregs Upon skin injury, Tregs used this integrin to activate latent TGF-ß, which acted directly on epithelial cells to promote CXCL5 production and neutrophil recruitment. Induction of this circuit delayed epidermal regeneration but provided protection from Staphylococcus aureus infection across a compromised barrier. Thus, αvß8-expressing Tregs in the skin, somewhat paradoxical to their canonical immunosuppressive functions, facilitated inflammation acutely after loss of barrier integrity to promote host defense against infection.

Developing Human Skin Contains Lymphocytes Demonstrating a Memory Signature.

Lymphocytes in barrier tissues play critical roles in host defense and homeostasis. These cells take up residence in tissues during defined developmental windows, when they may demonstrate distinct phenotypes and functions. Here, we utilized mass and flow cytometry to elucidate early features of human skin immunity. Although most conventional αß T (Tconv) cells in fetal skin have a naive, proliferative phenotype, a subset of CD4+ Tconv and CD8+ cells demonstrate memory-like features and a propensity for interferon (IFN)γ production. Skin regulatory T cells dynamically accumulate over the second trimester in temporal and regional association with hair follicle development. These fetal skin regulatory T cells (Tregs) demonstrate an effector memory phenotype while differing from their adult counterparts in expression of key effector molecules. Thus, we identify features of prenatal skin lymphocytes that may have key implications for understanding antigen and allergen encounters in utero and in infancy.

Regulatory T cells in skin injury: At the crossroads of tolerance and tissue repair.

Skin injury is a highly inflammatory process that is carefully regulated to mitigate tissue damage and allow for proper barrier repair. Regulatory T cells (Tregs) are crucial coordinators of the immune response to injury in several organs. Here, we review the emerging role of Tregs in facilitating skin repair after injury. We focus on recently discovered interactions between lymphocytes and nonhematopoietic cells during wound healing and discuss how these interactions are regulated both by "classical" suppressive mechanisms of Tregs and by "nonclassical" reparative Treg functions.

Genomic and Clinicopathologic Characteristics of PRKAR1A-inactivated Melanomas: Toward Genetic Distinctions of Animal-type Melanoma/Pigment Synthesizing Melanoma.

Melanocytic tumors with inactivation of protein kinase A regulatory subunit-α (PRKAR1A) have large oval nuclei and intense pigmentation. Historically, these tumors have been categorized under various names, including epithelioid blue nevus, pigmented epithelioid melanocytoma (PEM) and animal-type melanoma. Although a subset of PEM harbor BRAF activating mutations and biallelic inactivation of PRKAR1A, there are only a few reports of melanomas, or of tumors with genomic alterations beyond those of PEMs. Herein, we describe the clinicopathologic and genetic features of 8 melanomas and tumors that lack PRKAR1α expression by immunohistochemistry but do not fit with conventional PRKAR1A-inactivated melanocytomas. These tumors tended to affect younger patients than conventional melanomas (median age=38 y) and presented as dark brown/black papules and nodules. Histopathologically, they demonstrated nodularity, sometimes in a background of conventional melanoma, and large vesicular nuclei with prominent nucleoli. With the exception of 1 case, the mitotic index was not significantly elevated. Immunohistochemically, all cases showed loss of PRKAR1α and of p16 expression. Seven tumors underwent massively parallel short read (next-generation) sequencing of a panel of 480 cancer-associated genes. Five tumors demonstrated truncating mutations of PRKAR1A and the 2 in which such mutations were not identified demonstrated loss of heterozygosity of the PRKAR1A locus. Four of the tumors harbored BRAF V600E mutations, and 1 harbored a FAM39B-BRAF gene fusion. Another harbored a GNA11 activating mutation. A MAP kinase activating mutation was not identified in the remaining case. Four tumors displayed TERT promoter mutations and chromosomal copy number changes supporting the diagnosis of melanoma. Two cases without these alterations and were classified as "high-grade PRKAR1A-inactivated melanocytomas". The 1 case with widespread metastases demonstrated mutations in TP53 and RB1. Overall, we provide the first genetic characterization of PRKAR1A-inactivated melanomas, discuss the differential diagnosis of heavily pigmented epithelioid melanocytic neoplasms, and propose a new nomenclature for such tumors.

Melanotic Schwannoma of the Vulva: A Case Report and Review of the Literature.

Melanotic schwannoma is a pigmented tumor of peripheral nerve differentiation. Primary cutaneous presentations are extremely rare, as the bulk of melanotic schwannomas tend to develop in paraspinal and axial sites. Tumors arise sporadically and in the setting of the Carney complex. Alterations in the gene encoding protein kinase A regulatory subunit-α (PRKAR1A) underlie most patients with the Carney complex and mediate melanotic schwannoma tumorigenesis. Melanotic schwannomas from noncutaneous sites can locally recur and metastasize widely, leading to a recent proposal to change the nomenclature to "malignant melanotic schwannian tumor." However, the clinicopathologic features of primary cutaneous melanotic schwannomas are relatively unexplored. We present a case of a nodule arising on the vulva of a 34-year-old woman. Microscopically, a dermal-based, heavily pigmented proliferation of plump spindled and epithelioid cells arrayed in nodules and fascicles was seen. Lesional cells stained positively for S100, Melan-A, and BAP1 but were negative for Prkar1α. Next-generation sequencing of a panel of 480 cancer-associated genes revealed that the tumor harbored a PRKAR1A p.S299fs truncating mutation and copy neutral loss of heterozygosity of chromosome 17q, the locus at which PRKAR1A resides. Importantly, no other genetic abnormalities or chromosomal copy number changes were identified. On the basis of combined histopathologic, immunohistochemical, and genetic features, a diagnosis of melanotic schwannoma was rendered. Overall, we present the first clinicopathologic description of a vulvar melanotic schwannoma, review the literature concerning cutaneous presentations of melanotic schwannoma, and propose that melanotic schwannian tumors native to skin may behave more indolently than their noncutaneous counterparts.

Clinicopathologic overlap of psoriasis, eczema, and psoriasiform dermatoses: A retrospective study of T helper type 2 and 17 subsets, interleukin 36, and ß-defensin 2 in spongiotic psoriasiform dermatitis, sebopsoriasis, and tumor necrosis factor α inhibitor-associated dermatitis.

BACKGROUND: T helper (Th) type 17 and Th2 cells mediate psoriasis and eczema, respectively. Some dermatoses exhibit overlapping clinicopathologic features, and their immunopathology is relatively unexplored. OBJECTIVE: To determine whether Th17 and Th2 subsets and interleukin (IL) 36 and ß-defensin 2 (BD-2) markers of IL-17 signaling expression can discriminate between biopsy samples of psoriasis and eczematous/spongiotic dermatitis and to use those markers to immunophenotype cases with clinicopathologic overlap. METHODS: A retrospective study was performed on biopsy samples of psoriasis, eczema/spongiotic dermatitis, sebopsoriasis, tumor necrosis factor α inhibitor-associated psoriasiform dermatitis, and ambiguous cases diagnosed as spongiotic psoriasiform dermatitis. Dual CD4/GATA3 and CD4/RORC, IL-36, and BD-2 immunohistochemistry was performed. RESULTS: IL-36 and BD-2 were strongly expressed in biopsy samples of psoriasis compared with eczema/spongiotic dermatitis. No significant differences were observed in the percentages of Th2 and Th17 cells between disease types. Strong expression of IL-36 and BD-2 was observed in a subset of spongiotic psoriasiform dermatitis, sebopsoriasis, and tumor necrosis factor α inhibitor-associated psoriasiform dermatitis biopsy samples. LIMITATIONS: This was an exploratory study with a small sample size. No multiple testing adjustment was done. Clinical follow-up was limited. CONCLUSIONS: In cases with clinicopathologic overlap between psoriasis and spongiotic dermatitis, IL-36, and to a lesser extent BD-2, may be used to assess for a psoriasis-like/IL-17 phenotype, which could inform therapeutic clinical decisions.