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Lupus miliaris disseminatus faciei (LMDF) is a chronic inflammatory dermatosis of unknown aetiology, most often seen in young adults. Although many treatments for LMDF exist, treatment guidelines have not been developed, and response to therapy is generally unpredictable. We present the results of transcriptomic analysis of LMDF lesional skin, which revealed a variety of differentially expressed genes linking LMDF to alterations in innate and adaptive T helper 1 immunity. Immunohistochemical analysis was also performed, identifying similar changes in T-cell immune responses. Given evidence for increased tumour necrosis factor (TNF) pathway activity, our patient, who had previously been refractory to multiple treatments, was initiated on TNF inhibitor therapy with excellent response. This characterization of the LMDF immune response may lead to improved treatment of this condition.

Porphyria cutanea tarda (PCT) is a phototoxic cutaneous disease linked to elevated liver iron levels and photosensitizing heme precursors. Here, we report a 65-year-old man with a history of hypertension and heavy alcohol and tobacco use who presented to clinic for evaluation of an intensely pruritic rash on his arms present for 6 years. A biopsy completed 3 years prior demonstrated a lichenoid drug eruption.

Fibrosis occurs when collagen deposition and fibroblast proliferation replace healthy tissue. Red light (RL) may improve skin fibrosis via photobiomodulation, the process by which photosensitive chromophores in cells absorb visible or near-infrared light and undergo photophysical reactions. Our previous research demonstrated that high fluence RL reduces fibroblast proliferation, collagen deposition, and migration. Despite the identification of several cellular mechanisms underpinning RL phototherapy, little is known about the transcriptional changes that lead to anti-fibrotic cellular responses. Herein, RNA sequencing was performed on human dermal fibroblasts treated with RL phototherapy. Pathway enrichment and transcription factor analysis revealed regulation of extracellular matrices, proliferation, and cellular responses to oxygen-containing compounds following RL phototherapy. Specifically, RL phototherapy increased the expression of MMP1, which codes for matrix metalloproteinase-1 (MMP-1) and is responsible for remodeling extracellular collagen. Differential regulation of MMP1 was confirmed with RT-qPCR and ELISA. Additionally, RL upregulated PRSS35, which has not been previously associated with skin activity, but has known anti-fibrotic functions. Our results suggest that RL may benefit patients by altering fibrotic gene expression.

IgE induced by type 2 immune responses in atopic dermatitis is implicated in the progression of atopic dermatitis to other allergic diseases, including food allergies, allergic rhinitis, and asthma. However, the keratinocyte-derived signals that promote IgE and ensuing allergic diseases remain unclear. Herein, in a mouse model of atopic dermatitis-like skin inflammation induced by epicutaneous Staphylococcus aureus exposure, keratinocyte release of IL‑36α along with IL-4 triggered B cell IgE class-switching, plasma cell differentiation, and increased serum IgE levels-all of which were abrogated in IL-36R-deficient mice or anti-IL‑36R-blocking antibody-treated mice. Moreover, skin allergen sensitization during S. aureus epicutaneous exposure-induced IL-36 responses was required for the development of allergen-specific lung inflammation. In translating these findings, elevated IL‑36 cytokines in human atopic dermatitis skin and in IL‑36 receptor antagonist-deficiency patients coincided with increased serum IgE levels. Collectively, keratinocyte-initiated IL‑36 responses represent a key mechanism and potential therapeutic target against allergic diseases.

Purpose: Prospective human data are lacking regarding safety, efficacy, and immunologic impacts of different radiation doses administered with combined PD-L1/CTLA-4 blockade. Patients and Methods: We performed a multicenter phase II study randomly assigning patients with metastatic microsatellite stable colorectal cancer to repeated low-dose fractionated radiation (LDFRT) or hypofractionated radiation (HFRT) with PD-L1/CTLA-4 inhibition. The primary endpoint was response outside the radiation field. Correlative samples were analyzed using multiplex immunofluorescence (IF), IHC, RNA/T-cell receptor (TCR) sequencing, cytometry by time-of-flight (CyTOF), and Olink. Results: Eighteen patients were evaluable for response. Median lines of prior therapy were four (range, 1–7). Sixteen patients demonstrated toxicity potentially related to treatment (84%), and 8 patients had grade 3–4 toxicity (42%). Best response was stable disease in 1 patient with out-of-field tumor shrinkage. Median overall survival was 3.8 months (90% confidence interval, 2.3–5.7 months). Correlative IF and RNA sequencing (RNA-seq) revealed increased infiltration of CD8+ and CD8+/PD-1+/Ki-67+ T cells in the radiation field after HFRT. LDFRT increased foci of micronuclei/primary nuclear rupture in two subjects. CyTOF and RNA-seq demonstrated significant declines in multiple circulating immune populations, particularly in patients receiving HFRT. TCR sequencing revealed treatment-associated changes in T-cell repertoire in the tumor and peripheral blood. Conclusions: We demonstrate the feasibility and safety of adding LDFRT and HFRT to PD-L1/CTLA-4 blockade. Although the best response of stable disease does not support the use of concurrent PD-L1/CTLA-4 inhibition with HFRT or LDFRT in this population, biomarkers provide support that both LDFRT and HFRT impact the local immune microenvironment and systemic immunogenicity that can help guide future studies.

Alterations in the human glycome have been associated with cancer and autoimmunity. Thus, constructing a site-specific map of the human glycome for biomarker research and discovery has been a highly sought-after objective. However, due to analytical barriers, comprehensive site-specific glycoprofiling is difficult to perform. To develop a platform to detect easily quantifiable, site-specific, disease-associated glycan alterations for clinical applications, we have adapted the multiple reaction monitoring mass spectrometry method for use in glycan biomarker research. The adaptations allow for highly precise site-specific glycan monitoring with minimum sample prep. Using this technique, we successfully mapped out the relative abundances of the most common 159 glycopeptides in the plasma of 97 healthy volunteers. This plasma glycome map revealed 796 significant (FDR 2 = 0.62 ± 0.12). The human plasma site-specific glycan map described herein has utility in applications ranging from glycan biomarker research and discovery to the development of novel glycan-altering interventions.

The mechanisms that mediate durable protection against Staphylococcus aureus skin reinfections are unclear, as recurrences are common despite high antibody titers and memory T cells. Here, we developed a mouse model of S. aureus skin reinfection to investigate protective memory responses. In contrast with WT mice, IL-1β-deficient mice exhibited poor neutrophil recruitment and bacterial clearance during primary infection that was rescued during secondary S. aureus challenge. The γδ T cells from skin-draining LNs utilized compensatory T cell-intrinsic TLR2/MyD88 signaling to mediate rescue by trafficking and producing TNF and IFN-γ, which restored neutrophil recruitment and promoted bacterial clearance. RNA-sequencing (RNA-seq) of the LNs revealed a clonotypic S. aureus-induced γδ T cell expansion with a complementarity-determining region 3 (CDR3) aa sequence identical to that of invariant Vγ5+ dendritic epidermal T cells. However, this T cell receptor γ (TRG) aa sequence of the dominant CDR3 sequence was generated from multiple gene rearrangements of TRGV5 and TRGV6, indicating clonotypic expansion. TNF- and IFN-γ-producing γδ T cells were also expanded in peripheral blood of IRAK4-deficient humans no longer predisposed to S. aureus skin infections. Thus, clonally expanded γδ T cells represent a mechanism for long-lasting immunity against recurrent S. aureus skin infections.

The present paradigm of psoriasis pathogenesis revolves around the IL-23/IL-17A axis. Dual-secreting Th17 T cells presumably are the predominant sources of the psoriasis phenotype-driving cytokines, IL-17A and IL-22. We thus conducted a meta-analysis of independently acquired RNA-seq psoriasis datasets to explore the relationship between the expression of IL17A and IL22. This analysis failed to support the existence of dual secreting IL-17A/IL-22 Th17 cells as a major source of these cytokines. However, variable relationships amongst the expression of psoriasis susceptibility genes and of IL17A, IL22, and IL23A were identified. Additionally, to shed light on gene expression relationships in psoriasis, we applied a machine learning nonlinear dimensionality reduction strategy (t-SNE) to display the entire psoriasis transcriptome as a 2-dimensonal image. This analysis revealed a variety of gene clusters, relevant to psoriasis pathophysiology but failed to support a relationship between IL17A and IL22. These results support existing theories on alternative sources of IL-17A and IL-22 in psoriasis such as a Th22 cells and non-T cell populations.

Psoriasis (PsO) and psoriatic arthritis (PsA) are chronic immune-mediated inflammatory diseases of multifactorial etiology. In addition to genetic and environmental factors, evidence supports involvement of a dysregulated human microbiome in the pathogenesis of psoriatic disease. In particular, alterations in the composition of the microbiome, termed dysbiosis, can result in downstream proinflammatory effects in the gut, skin, and joints. Both the cutaneous and intestinal microbial populations are implicated in the pathogenesis of psoriatic disease, although exact mechanisms are unclear. Herein, we review the relationship between the human microbiome and psoriatic disease. Further insight into the functions of the microbiome may allow for greater understanding of inflammatory disease processes and identification of additional therapeutic targets.

Numerous studies of relatively few patients have linked T cell receptor (TCR) genes to psoriasis but have yielded dramatically conflicting results. To resolve these discrepancies, we have chosen to mine RNA-Seq datasets for patterns of TCR gene segment usage in psoriasis. A meta-analysis of 3 existing and 1 unpublished datasets revealed a statistically significant link between the relative expression of TRAJ23 and psoriasis and the psoriasis-associated cytokine IL-17A. TRGV5, a TCR-γ segment, was also associated with psoriasis but correlated instead with IL-36A, other IL-36 family members, and IL-17C (not IL-17A). In contrast, TRAJ39 was strongly associated with healthy skin. T cell diversity measurements and analysis of CDR3 sequences were also conducted, revealing no psoriasis-associated public CDR3 sequences. Finally, in comparison with the expression of TCR-αβ genes, the expression of TCR-γδ genes was relatively low but mildly elevated in psoriatic skin. These results have implications for the development of targeted therapies for psoriasis and other autoimmune diseases. Also, the techniques employed in this study have applications in other fields, such as cancer immunology and infectious disease.

Therapeutic applications for mesenchymal stem/stromal cells (MSCs) are growing; however, the successful implementation of these therapies requires the development of appropriate MSC delivery systems. Hydrogels are ideally suited to cultivate MSCs but tuning hydrogel properties to match their specific in vivo applications remains a challenge. Thus, further characterization of how hydrogel-based delivery vehicles broadly influence MSC function and fate will help lead to the next generation of more intelligently designed delivery vehicles. To date, few attempts have been made to comprehensively characterize hydrogel impact on the MSC transcriptome. Herein, we have synthesized cell-degradable hydrogels based on bio-inert poly(ethylene glycol) tethered with specific integrin-binding small molecules and have characterized their resulting effect on the MSC transcriptome when compared with 2D cultured and untethered 3D hydrogel cultured MSCs. The 3D culture systems resulted in alterations in the MSC transcriptome, as is evident by the differential expression of genes related to extracellular matrix production, glycosylation, metabolism, signal transduction, gene epigenetic regulation, and development. For example, genes important for osteogenic differentiation were upregulated in 3D hydrogel cultures, and the expression of these genes could be partially suppressed by tethering an integrin-binding RGD peptide within the hydrogel. Highlighting the utility of tunable hydrogels, when applied to ex vivo human wounds the RGD-tethered hydrogel was able to support wound re-epithelialization, possibly due to its ability to increase PDGF expression and decrease IL-6 expression. These results will aid in future hydrogel design for a broad range of applications.

TO THE EDITOR Transforming growth factor-β (TGF-β) and platelet-derived growth factor (PDGF) are central mediators of fibrosis, and their overexpression contributes to the pathophysiology of scleroderma, chiefly by inducing the overproduction of extracellular matrix proteins (ECM) by dermal fibroblasts (Gay et al., 1989; Sargent et al., 2010; Bhattacharyya et al., 2012). TGF-β also promotes the differentiation of dermal fibroblasts into myofibroblasts, which are key mediators of scleroderma (Abraham et al., 2007). Thus, targeting this pathway is a reasonable strategy to treat a variety of fibrotic diseases including scleroderma, for which current treatment options are limited. Herein we explore the potential of novel mTOR inhibition as a means to block the pro-fibrotic effects of TGF-β. Recent studies have suggested a functional role of mTOR in fibrotic diseases and autoimmunity (Ong et al., 2007; Fried et al., 2008; Su et al., 2009; Raychaudhuri and Raychaudhuri, 2014). This pathway is initiated by cytokines and growth factors that induce phosphorylation of Akt(Thr308) and its downstream mediators mTORC1, p70S6K1, and 4E-BP1. In addition to mTORC1, mTOR is now known to comprise a second multi-protein complex, mTORC2, which positively regulates the activity of mTOR through phosphorylation of AktSer473 (Bhagwat et al., 2011). Blockade of mTORC1 has already been attempted as a treatment for scleroderma without much understanding about its molecular mechanism (Fried et al., 2008; Su et al., 2009; Yoshizaki et al., 2010). Such strategies focused only on mTORC1 and did not account for the contributions of mTORC2 (Bhagwat et al., 2011). To increase the effectiveness of mTOR blockade, dual inhibitors targeting both mTORC1 and mTORC2 have now been developed (Bhagwat et al., 2011). Herein, we demonstrate that dual mTOR blockade can more effectively inhibit the pro-fibrotic effects of TGF-β and PDGF. These have therapeutic implications for scleroderma and other TGF-β dominant fibrotic diseases.

Background Pyoderma gangrenosum (PG) is a debilitating ulcerative skin disease that is one of the most common associated diseases seen in patients with inflammatory bowel disease and rheumatoid arthritis. Although PG is classified as a neutrophilic dermatosis, its pathophysiology is poorly understood. Objective Use data obtained from patient-reported histories, immunohistochemistry, and gene expression analysis to formulate a hypothesis on PG pathophysiology. Methods Ten PG patients participated and answered questions about new ulcer formation. Skin biopsies of healed prior ulcers and adjacent normal skin were obtained from four patients for immunohistochemistry. Scars from healthy patients and patients with discoid lupus were used as additional controls. New onset PG papules were analyzed using immunohistochemistry and gene expression analysis via quantitative real-time PCR. Results All PG patients reported that healed sites of previous ulceration are refractory to re-ulceration. Simultaneous biopsies of healed and uninvolved skin triggered ulceration only in the latter. On immunohistochemistry, healed PG scars showed complete loss of pilosebaceous units, which were present in normal skin, and to a lesser extent in control scars, and discoid scars. Early PG papules showed perivascular and peripilosebaceous T cell infiltrates, rather than neutrophils. These early inflammatory events were dominated by increased gene expression of CXCL9, CXCL10, CXCL11, IL-8, IL-17, IFNG, and IL-36G and transcription factors consistent with Th1 phenotype. Limitations Small sample size was the main limitation. Conclusion We put forth the hypothesis that PG is a T cell response resulting in the destruction of pilosebaceous units.

It is widely accepted that central and effector memory CD4+ T cells originate from naïve T cells after they have encountered their cognate antigen in the setting of appropriate co-stimulation. However, if this were true the diversity of T cell receptor (TCR) sequences within the naïve T cell compartment should be far greater than that of the memory T cell compartment, which is not supported by TCR sequencing data. Here we demonstrate that aged mice with far fewer naïve T cells, respond to the model antigen, hen eggwhite lysozyme (HEL), by utilizing the same TCR sequence as their younger counterparts. CD4+ T cell repertoire analysis of highly purified T cell populations from naive animals revealed that the HEL-specific clones displayed effector and central “memory” cell surface phenotypes even prior to having encountered their cognate antigen. Furthermore, HEL-inexperienced CD4+ T cells were found to reside within the naïve, regulatory, central memory, and effector memory T cell populations at similar frequencies and the majority of the CD4+ T cells within the regulatory and memory populations were unexpanded. These findings support a new paradigm for CD4+ T cell maturation in which a specific clone can undergo a differentiation process to exhibit a “memory” or regulatory phenotype without having undergone a clonal expansion event. It also demonstrates that a foreign-specific T cell is just as likely to reside within the regulatory T cell compartment as it would the naïve compartment, arguing against the specificity of the regulatory T cell compartment being skewed towards self-reactive T cell clones. Finally, we demonstrate that the same set of foreign and autoreactive CD4+ T cell clones are repetitively generated throughout adulthood. The latter observation argues against T cell-depleting strategies or autologous stem cell transplantation as therapies for autoimmunity-as the immune system has the ability to regenerate pathogenic clones.

Purpose To examine the association between phosphodiesterase type 5 (PDE5) inhibitor use and melanoma by 1) conducting a systematic review of observational studies; and 2) determining if low PDE5A gene expression in human melanoma correlated with decreased overall survival. Materials and methods A systematic search of observational studies examining the association between PDE5 inhibitor use and melanoma was performed through ClinicalTrials.gov, the Cochrane Library, EMBASE, PubMed, and Web of Science databases, and seven eligible studies were identified. PDE5A gene expression was analyzed with RNA sequencing data from 471 human melanoma samples obtained from The Cancer Genome Atlas. Results Four studies reported a positive association between PDE5 inhibitor use and melanoma, and three studies found no correlation. RNA sequencing data analysis revealed that under-expression of the PDE5A gene did not impact clinical outcomes in melanoma. Conclusions There is currently no evidence to suggest that PDE5 inhibition in patients causes increased risk for melanoma. The few observational studies that demonstrated a positive association between PDE5 inhibitor use and melanoma often failed to account for major confounders. Nonetheless, the substantial evidence implicating PDE5 inhibition in the cyclic guanosine monophosphate (cGMP)-mediated melanoma pathway warrants further investigation in the clinical setting.