γδ T Cells Mediate a Requisite Portion of a Wound Healing Response Triggered by Cutaneous Poxvirus Infection.

Infection at barrier sites, e.g., skin, activates local immune defenses that limit pathogen spread, while preserving tissue integrity. Phenotypically distinct γδ T cell populations reside in skin, where they shape immunity to cutaneous infection prior to onset of an adaptive immune response by conventional αß CD4+ (TCD4+) and CD8+ (TCD8+) T cells. To examine the mechanisms used by γδ T cells to control cutaneous virus replication and tissue pathology, we examined γδ T cells after infection with vaccinia virus (VACV). Resident γδ T cells expanded and combined with recruited γδ T cells to control pathology after VACV infection. However, γδ T cells did not play a role in control of local virus replication or blockade of systemic virus spread. We identified a unique wound healing signature that has features common to, but also features that antagonize, the sterile cutaneous wound healing response. Tissue repair generally occurs after clearance of a pathogen, but viral wound healing started prior to the peak of virus replication in the skin. γδ T cells contributed to wound healing through induction of multiple cytokines/growth factors required for efficient wound closure. Therefore, γδ T cells modulate the wound healing response following cutaneous virus infection, maintaining skin barrier function to prevent secondary bacterial infection.

Understanding Type 3 Innate Lymphoid Cells and Crosstalk with the Microbiota: A Skin Connection.

Innate lymphoid cells (ILCs) are a diverse population of lymphocytes classified into natural killer (NK) cells, ILC1s, ILC2s, ILC3s, and ILCregs, broadly following the cytokine secretion and transcription factor profiles of classical T cell subsets. Nonetheless, the ILC lineage does not have rearranged antigen-specific receptors and possesses distinct characteristics. ILCs are found in barrier tissues such as the skin, lungs, and intestines, where they play a role between acquired immune cells and myeloid cells. Within the skin, ILCs are activated by the microbiota and, in turn, may influence the microbiome composition and modulate immune function through cytokine secretion or direct cellular interactions. In particular, ILC3s provide epithelial protection against extracellular bacteria. However, the mechanism by which these cells modulate skin health and homeostasis in response to microbiome changes is unclear. To better understand how ILC3s function against microbiota perturbations in the skin, we propose a role for these cells in response to Cutibacterium acnes, a predominant commensal bacterium linked to the inflammatory skin condition, acne vulgaris. In this article, we review current evidence describing the role of ILC3s in the skin and suggest functional roles by drawing parallels with ILC3s from other organs. We emphasize the limited understanding and knowledge gaps of ILC3s in the skin and discuss the potential impact of ILC3-microbiota crosstalk in select skin diseases. Exploring the dialogue between the microbiota and ILC3s may lead to novel strategies to ameliorate skin immunity.

Treatment response to isotretinoin correlates with specific shifts in Cutibacterium acnes strain composition within the follicular microbiome.

There are no drugs as effective as isotretinoin for acne. Deciphering the changes in the microbiome induced by isotretinoin in the pilosebaceous follicle of successfully treated patients can pave the way to identify novel therapeutic alternatives. We determined how the follicular microbiome changes with isotretinoin and identified which alterations correlate with a successful treatment response. Whole genome sequencing was done on casts from facial follicles of acne patients sampled before, during and after isotretinoin treatment. Alterations in the microbiome were assessed and correlated with treatment response at 20 weeks as defined as a 2-grade improvement in global assessment score. We investigated the α-diversity, ß-diversity, relative abundance of individual taxa, Cutibacterium acnes strain composition and bacterial metabolic profiles with a computational approach. We found that increased ß-diversity of the microbiome coincides with a successful treatment response to isotretinoin at 20 weeks. Isotretinoin selectively altered C. acnes strain diversity in SLST A and D clusters, with increased diversity in D1 strains correlating with a successful clinical response. Isotretinoin significantly decreased the prevalence of KEGG Ontology (KO) terms associated with four distinct metabolic pathways inferring that follicular microbes may have limited capacity for growth or survival following treatment. Importantly, these alterations in microbial composition or metabolic profiles were not observed in patients that failed to achieve a successful response at 20 weeks. Alternative approaches to recapitulate this shift in the balance of C. acnes strains and microbiome metabolic function within the follicle may be beneficial in the future treatment of acne.

Human Regulatory T Cells: Understanding the Role of Tregs in Select Autoimmune Skin Diseases and Post-Transplant Nonmelanoma Skin Cancers.

Regulatory T cells (Tregs) play an important role in maintaining immune tolerance and homeostasis by modulating how the immune system is activated. Several studies have documented the critical role of Tregs in suppressing the functions of effector T cells and antigen-presenting cells. Under certain conditions, Tregs can lose their suppressive capability, leading to a compromised immune system. For example, mutations in the Treg transcription factor, Forkhead box P3 (FOXP3), can drive the development of autoimmune diseases in multiple organs within the body. Furthermore, mutations leading to a reduction in the numbers of Tregs or a change in their function facilitate autoimmunity, whereas an overabundance can inhibit anti-tumor and anti-pathogen immunity. This review discusses the characteristics of Tregs and their mechanism of action in select autoimmune skin diseases, transplantation, and skin cancer. We also examine the potential of Tregs-based cellular therapies in autoimmunity.

Keratinocytes and immune cells in the epidermis are key drivers of inflammation in hidradenitis suppurativa providing a rationale for novel topical therapies.

BACKGROUND: Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disease characterized by painful nodules, drainage and scarring in skin folds. Injectable adalimumab is the only drug approved by the US Food and Drug Administration for the treatment of HS. Although systemic Janus kinase (JAK) inhibitors show promise, serious side-effects have been reported. There are no highly effective topical treatments for HS; furthermore, the contribution of epidermal keratinocytes to the intense inflammation has largely been unexplored. OBJECTIVES: We investigated the role of keratinocytes and epidermal immune cells in HS inflammation at all Hurley stages of disease severity. We aimed to determine whether ruxolitinib can mitigate inflammation from keratinocytes and to develop a better understanding of how topical therapeutics might benefit patients with HS. METHODS: We used skin samples from 87 patients with HS (Hurley stages I-III) and 39 healthy controls to compare keratinocyte- and immune cell-driven epidermal inflammation, in addition to the response of lesional HS keratinocytes to treatment with interferon (IFN)-γ and ruxolitinib. We used haematoxylin and eosin staining, immunohistochemistry, immunoblotting and quantitative reverse-transcription polymerase chain reaction assessments in whole skin, isolated epidermis, and cultured keratinocytes from healthy controls and both nonlesional and lesional HS skin to identify and define epidermal and keratinocyte-mediated inflammation in HS and how this may be targeted by therapeutics. RESULTS: HS lesional keratinocytes autonomously secreted high levels of chemokines, such as CCL2, CCL3 and CXCL3, which recruited neutrophils, CD8 T cells, and natural killer cells to the epidermis. Keratinocytes were the dominant source of tumour necrosis factor-α and interleukin (IL)-6 in HS lesions with little to no contribution from underlying dermal immune cells. In the presence of IFN-γ, which is dependent on immune cell infiltrate in vivo, keratinocytes expressed increased levels of additional cytokines including IL-1ß, IL-12, IL-23 and IL-36γ. The JAK inhibitor ruxolitinib mitigated the expression of inflammatory cytokines and chemokines in HS lesional keratinocytes, thus providing a rationale for future study as a topical treatment for HS. CONCLUSIONS: This study demonstrates that keratinocytes actively recruit immune cells to HS epidermis and interactions between these cells drive a broad inflammatory profile in HS epidermis. Targeting epidermal inflammation in HS with novel topical formulations may be highly efficacious with reduced systemic side-effects.

Evolution of the facial skin microbiome during puberty in normal and acne skin.

BACKGROUND: The composition of the skin microbiome varies from infancy to adulthood and becomes most stable in adulthood. Adult acne patients harbour an 'acne microbiome' dominated by specific strains of Cutibacterium acnes. However, the precise timing of skin microbiome evolution, the development of the acne microbiome, and the shift to virulent C. acnes strain composition during puberty is unknown. OBJECTIVES: We performed a cross-sectional pilot study in a paediatric population to understand how and when the skin microbiome composition transitions during puberty and whether a distinct 'acne microbiome' emerges in paediatric subjects. METHODS: Forty-eight volunteers including males and females, ages 7-17 years, with and without acne were enrolled and evaluated for pubertal development using the Tanner staging criteria. Sebum levels were measured, and skin microbiota were collected by sterile swab on the subject's forehead. DNA was sequenced by whole genome shotgun sequencing. RESULTS: A significant shift in microbial diversity emerged between early (T1-T2) and late (T3-T5) stages of puberty, coinciding with increased sebum production on the face. The overall relative abundance of C. acnes in both normal and acne skin increased during puberty and individual C. acnes strains were uniquely affected by pubertal stage and the presence of acne. Further, an acne microbiome signature associated with unique C. acnes strain composition and metabolic activity emerges in late puberty in those with acne. This unique C. acnes strain composition is predicted to have increased porphyrin production, which may contribute to skin inflammation. CONCLUSIONS: Our data suggest that the stage of pubertal development influences skin microbiome composition. As children mature, a distinct acne microbiome composition emerges in those with acne. Understanding how both puberty and acne influence the microbiome may support novel therapeutic strategies to combat acne in the paediatric population.

4-Aminopyridine Induces Nerve Growth Factor to Improve Skin Wound Healing and Tissue Regeneration.

The discovery of ways to enhance skin wound healing is of great importance due to the frequency of skin lesions. We discovered that 4-aminopyridine (4-AP), a potassium channel blocker approved by the FDA for improving walking ability in multiple sclerosis, greatly enhances skin wound healing. Benefits included faster wound closure, restoration of normal-appearing skin architecture, and reinnervation. Hair follicle neogenesis within the healed wounds was increased, both histologically and by analysis of K15 and K17 expression. 4-AP increased levels of vimentin (fibroblasts) and alpha-smooth muscle actin (α-SMA, collagen-producing myofibroblasts) in the healed dermis. 4-AP also increased neuronal regeneration with increased numbers of axons and S100+ Schwann cells (SCs), and increased expression of SRY-Box Transcription Factor 10 (SOX10). Treatment also increased levels of transforming growth factor-ß (TGF-ß), substance P, and nerve growth factor (NGF), important promoters of wound healing. In vitro studies demonstrated that 4-AP induced nerve growth factor and enhanced proliferation and migration of human keratinocytes. Thus, 4-AP enhanced many of the key attributes of successful wound healing and offers a promising new approach to enhance skin wound healing and tissue regeneration.

T Cell Extracellular Traps: Tipping the Balance Between Skin Health and Disease.

The role of extracellular traps (ETs) in the innate immune response against pathogens is well established. ETs were first identified in neutrophils and have since been identified in several other immune cells. Although the mechanistic details are not yet fully understood, recent reports have described antigen-specific T cells producing T cell extracellular traps (TETs). Depending on their location within the cutaneous environment, TETs may be beneficial to the host by their ability to limit the spread of pathogens and provide protection against damage to body tissues, and promote early wound healing and degradation of inflammatory mediators, leading to the resolution of inflammatory responses within the skin. However, ETs have also been associated with worse disease outcomes. Here, we consider host-microbe ET interactions by highlighting how cutaneous T cell-derived ETs aid in orchestrating host immune responses against Cutibacterium acnes (C. acnes), a commensal skin bacterium that contributes to skin health, but is also associated with acne vulgaris and surgical infections following joint-replacement procedures. Insights on the role of the skin microbes in regulating T cell ET formation have broad implications not only in novel probiotic design for acne treatment, but also in the treatment for other chronic inflammatory skin disorders and autoimmune diseases.

Biomarkers of Tretinoin Precursors and Tretinoin Efficacy in Patients With Moderate to Severe Facial Photodamage: A Randomized Clinical Trial.

Importance: Topical formulations of tretinoin precursors (retinol and its ester derivatives) are widely available over the counter and may offer similar clinical benefits to those of tretinoin for treatment of photoaging. However, which of the many purported molecular effects of retinoids most strongly drives clinical improvements in tretinoin-treated skin remains unclear. Objectives: To evaluate the clinical efficacy of topical tretinoin precursors (TTP) vs tretinoin (RA) in treating moderate to severe facial photodamage and to identify potential biomarkers that correlate with clinical efficacy. Design, Setting, and Participants: This randomized, double-blind, single-center, parallel-arm study of 24 patients with moderate to severe facial photodamage was conducted at an academic referral center from November 2010 to December 2011, with data analysis performed from January 2012 to December 2021. Interventions: Daily topical application of 0.02% RA or 1.1% TTP formulation containing retinol, retinyl acetate, and retinyl palmitate for 24 weeks. Main Outcomes and Measures: Photoaging and tolerability were assessed by dermatologist evaluations and patient-reported outcomes. Target gene expression was assessed by real-time quantitative polymerase chain reaction of biopsied tissue from treated areas. Results: A total of 20 White women were ultimately analyzed (9 randomized to TTP, 11 randomized to RA). At week 24, there was no significant difference in Griffiths photoaging scores among patients receiving TTP vs RA (median, 4 vs 5) (TTP - RA difference: -1; 95% CI, -2 to 1; P = .27). Treatment with TTP was associated with erythema 6 times less frequently than RA (11% vs 64%) (TTP - RA difference: -0.53; 95% CI, -0.88 to -0.17; P = .01). Target gene analysis showed significant CRABP2 messenger RNA (mRNA) induction (confirming retinoic acid receptor signaling) but no significant changes in procollagen I or MMP1/3/9 mRNA in TTP-treated samples. Instead, MMP2 mRNA, which encodes a type IV collagenase, was significantly reduced in TTP-treated samples (week 24 - baseline mRNA difference: -5; 96% CI, -33 to 1.6; P = .02), and changes in MMP2 were strongly correlated with changes in fine wrinkles (r = 0.54; 95% CI, 0.12 to 0.80; P = .01). Interestingly, patients with severe baseline wrinkles exhibited greater improvements (r = -0.74; 95% CI, -0.89 to -0.43; P < .001). This trend was mirrored in MMP2 mRNA, with initial expression strongly predicting subsequent changes (r = -0.78; 95% CI, -0.89 to -0.43; P < .001). Conclusions and Relevance: In this randomized clinical trial, there was no significant difference in efficacy between this particular formulation of TTP and tretinoin 0.02%. However, the results of these mechanistic studies highlight MMP2 as a possible mediator of retinoid efficacy in photoaging. Trial Registration: ClinicalTrials.gov Identifier: NCT01283464.

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin.

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

Yin and Yang: A disrupted skin microbiome and an aberrant host immune response in hidradenitis suppurativa.

The skin microbiome plays an important role in maintaining skin homeostasis by controlling inflammation, providing immune education and maintaining host defense. However, in many inflammatory skin disorders the skin microbiome is disrupted. This dysbiotic community may contribute to disease initiation or exacerbation through the induction of aberrant immune responses in the absence of infection. Hidradenitis suppurativa (HS) is a complex, multifaceted disease involving the skin, innate and adaptive immunity, microbiota and environmental stimuli. Herein, we discuss the current state of HS skin microbiome research and how microbiome components may activate pattern recognition receptor (PRR) pathways, metabolite sensing pathways and antigenic receptors to drive antimicrobial peptide, cytokine, miRNA and adaptive immune cell responses in HS. We highlight the major open questions that remain to be addressed and how antibiotic therapies for HS likely influence both microbial burden and inflammation. Ultimately, we hypothesize that the two-way communication between the skin microbiome and host immune response in HS skin generates a chronic positive feed-forward loop that perpetuates chronic inflammation, tissue destruction and disease exacerbation.

Keeping the peace: commensal Cutibacterium acnes trains CD4+ TH17 cells to trap and kill.

Commensal or pathogenic bacterial communities of the skin interact with the host immune system to preserve homeostasis or sustain disease. In this issue of the JCI, Agak et al. substantially advance our conceptual understanding of TH17 cell biology. The researchers identified IL-26-independent mechanisms by which CD4+ TH17 clones directly kill bacteria. These CD4+ TH17 clones share antimicrobial properties with cytotoxic T cells and granulocytes as evidenced by secretion of granulysin, granzyme B, and histone-laden DNA extracellular traps. Interestingly, these clones emerged following monocyte education by Cutibacterium acnes strains associated with healthy skin, but not those associated with acne. Overall, the antimicrobial mechanisms employed by these TH17 subsets suggest a unique link between innate and adaptive immune responses.

Runoff water quantity and quality data from native tallgrass prairie and crop-livestock systems in Oklahoma between 1977 and 1999.

Erosion and sedimentation pose serious threats to soil and water quality worldwide, including in the U.S. southern Great Plains. To better understand these processes in agricultural landscapes, eight 1.6-ha watersheds were established and instrumented in 1976 at the USDA-ARS Grazinglands Research Laboratory, ∼50 km west of Oklahoma City near El Reno, OK, to measure precipitation and surface runoff quantity and quality. Prior to construction, all watersheds were in native grass, primarily big bluestem (Andropogon gerardii Vitman.), little bluestem [Schizachyrium scoparium (Michx.) Nash], and Indiangrass [Sorghastrum nutans (L.) Nash]; afterwards, four of the eight watersheds were cropped initially into winter wheat (Triticum aestivum L.) (two conventionally tilled and two minimally or no-till). Although there have been many peer-reviewed papers from the Water Resources and Erosion (WRE) watersheds, none included all the datasets collected during the period 1977-1999. The objectives of this paper were (a) to present and discuss all archived historical data, including methods of collection and analysis, (b) to provide summary analyses of the variability in each dataset, and (c) to provide details about how to access these datasets. These datasets are valuable resources to improve modeling in relation to land use and management changes, climate variability, and other environmental factors and may be useful in developing strategies to mitigate environmental impacts of agricultural systems. They are available at https://doi.org/10.15482/USDA.ADC/1518421.