The mesenchymal stromal cell secretome promotes tissue regeneration and increases macrophage infiltration in acute and methicillin-resistant Staphylococcus aureus-infected skin wounds in vivo.

BACKGROUND AIMS: The prevalence of chronic wounds continues to be a burden in human medicine. Methicillin-resistant Staphylococcus aureus (MRSA) is commonly isolated from infected wounds. MRSA infections primarily delay healing by impairing local immune cell functions. This study aimed to investigate the potential of mesenchymal stromal cell (MSC)-secreted bioactive factors, defined as the secretome, to improve innate immune responses in vivo. MSCs were isolated from the bone marrow of horses, which serve as valuable translational models for wound healing. The MSC secretome, collected as conditioned medium (CM), was evaluated in vivo using mouse models of acute and MRSA-infected skin wounds. METHODS: Punch biopsies were used to create two full-thickness skin wounds on the back of each mouse. Acute wounds were treated daily with control medium or bone marrow-derived MSC (BM-MSC) CM. The antibiotic mupirocin was administered as a positive control for the MRSA-infected wound experiments. Wounds were photographed daily, and wound images were measured to determine the rate of closure. Trichrome staining was carried out to examine wound tissue histologically, and immunofluorescence antibody binding was used to assess immune cell infiltration. Wounds in the MRSA-infected model were swabbed for quantification of bacterial load. RESULTS: Acute wounds treated with BM-MSC CM showed accelerated wound closure compared with controls, as illustrated by enhanced granulation tissue formation and resolution, increased vasculature and regeneration of hair follicles. This treatment also led to increased neutrophil and macrophage infiltration. Chronic MRSA-infected wounds treated with BM-MSC CM showed reduced bacterial load accompanied by better resolution of granulation tissue formation and increased infiltration of pro-healing M2 macrophages compared with control-treated infected wounds. CONCLUSIONS: Collectively, our findings indicate that BM-MSC CM exerts pro-healing, immunomodulatory and anti-bacterial effects on wound healing in vivo, validating further exploration of the MSC secretome as a novel treatment option to improve healing of both acute and chronic wounds, especially those infected with antibiotic-resistant bacteria.

The High-Risk Human Papillomavirus Type Influences the Tissue Microenvironment in Cervical Intraepithelial Neoplasia Grade 2.

High-risk, cancer-causing human papillomavirus (HPV) types are associated with cervical precancer and cancer. A high proportion of high-risk HPV precancer lesions undergo immune-mediated regression. The purpose of this study was to determine if the tissue microenvironment of HPV16 and 18 (HPV16/18) cervical intraepithelial neoplasia grade 2 lesions differed from other high-risk types (HPV 'other'). Consistent with other studies, we found that progression to higher-grade disease was more frequent in HPV16/18 lesions when compared with HPV 'other' lesions. HPV16/18 lesions were significantly more likely to be indoleamine 2,3,-dioxygenase 1 (IDO1)-positive and were associated with reduced CD8 and FoxP3 T cells in the lesion. In the stroma, reduced Tbet- and CD32-positive cells and increased Blimp1-positive cells were significantly associated with HPV16/18 lesions when compared with HPV 'other' types. On analysis of the IDO1-positive tissues, lesional IDO1 was associated with significantly decreased numbers of CD4-, CD8-, and FoxP3-positive cells in the stroma compared with IDO1-negative tissues. These data suggest that IDO1 expression may impair infiltration of CD4, CD8, and FoxP3 cells into the stroma beneath the precancer lesion. Increased expression of IDO1 may contribute to immune avoidance and an increased frequency of disease progression in HPV16- and 18-positive lesions.

Use of Biologics and Stem Cells for Wound Healing in the Horse.

Treatment of skin wounds is a high priority in veterinary medicine because healthy uncompromised skin is essential for the well-being of horses. Stem cells and other biologic therapies offer benefits by reducing the need for surgical procedures and conventional antibiotics. Evidence from in vitro studies and small in vivo trials supports the use of equine stem cells and biologics for the treatment of acute and chronic cutaneous wounds. Larger clinical trials are warranted to better evaluate the regenerative and immunological responses to these treatments. Additionally, delivery methods and treatment schedules should be optimized to improve efficacy of these novel therapies.

Characterisation of the immune microenvironment of cutaneous squamous cell carcinoma in immunosuppression.

Cutaneous squamous cell carcinoma (cSCC) is a common cancer. Systemic immunosuppression with drugs such as Prednisone results in more aggressive disease. We hypothesise that more aggressive disease in immunosuppression is the result of immune changes in the tumor microenvironment. We characterised T cell, phagocytic and antigen presenting cell subsets in cSCC and determined if these infiltrates were altered by immunosuppressive therapy. We found a dominant "CD8 profile" in the centre of cSCC lesions, with CD8 cells correlating with Tbet, FoxP3, OX40 and "M2-like" macrophages, whereas a "Tbet and granulocyte profile" with associated inflammation predominated at the margin of the tumor. Individuals on systemic immunosuppressive therapy had lesions that were comparable in size, stage and number of vessels to immune competent individuals; however, the number of CD11c positive cells in the lesion centre was significantly reduced. We conclude that cSCC lesions are immunologically heterogeneous across the lesion and that systemically immunosuppressed individuals have reduced CD11c positive cells in the centre of the lesion. The role and detailed phenotype of CD11c positive cells in cSCC lesions warrant further investigation.

Blimp-1 is a prognostic indicator for progression of cervical intraepithelial neoplasia grade 2.

BACKGROUND: Progression of cervical intraepithelial neoplasia (CIN) to higher grade disease is associated with persistent human papillomavirus (HPV) infection and an absence of immune-mediated regression. However, the immune microenvironment that distinguishes progression from persistent or regressing lesions has not been well defined. METHODS: A total of 69 patients under the age of 25 with high-risk HPV-positive cytology and biopsy-confirmed p16-positive CIN2 were included in the study. Biopsies were stained using 20 antibodies to a range of immune markers. Based on a 2-year follow-up, samples were analysed in "progressor" (CIN3 +) or "persister/regressor" (CIN1, 2 or normal) groups. RESULTS: Progression was most strongly associated with Blimp-1 positive cell staining in the lesion (P = 0.0019) and with low numbers of infiltrating CD4 cells in the dermal region beneath the lesion (P = 0.0022). The presence of CD4, CD8 and T bet-positive cells in the dermal region most strongly correlated with CD11c cells in the persister/regressor but not the progressor group. CONCLUSION: High numbers of Blimp-1 + cells in CIN2 lesions may predict progression to more severe disease. Measurement of Blimp-1 may have diagnostic utility for the determination of the need to treat women with cervical pre-cancer. HIGHLIGHTS: CIN2 progression is associated with high numbers of Blimp-1 positive cells in the lesion. Detection of Blimp-1 in the lesion may have utility as a prognostic test to inform the need to treat CIN2.

Skin antigen-presenting cells and wound healing: New knowledge gained and challenges encountered using mouse depletion models.

The role of antigen-presenting cells in the skin immune system, in particular Langerhans cells and dendritic cells, has not been well defined. We recently published a study in 'Immunology' where we reported that the loss of langerin-positive cells in the skin accelerated wound repair in the Lang-DTR mouse. The study published here by Li, et al. reports delayed wound closure following depletion of CD11c-positive cells in the CD11c-DTR mouse. In this commentary, we attribute the differences between these results to several factors that differ between the studies including the depletion of different cell populations; differences in the age and the sex of mice; differences in antibiotic use between the studies; and differences in the location of the biopsies that were taken. Here, we describe the impact of these differences on wound healing and conclude that further standardization of the wound model, and further characterization of the specific cells that are depleted in these mice, is necessary to better understand how antigen-presenting cells contribute to wound healing.

Novel Concepts: Langerhans Cells in the Tumour Microenvironment.

Langerhans cells (LCs) are immune cells that reside in the stratified epithelium of the skin and mucosal membranes. They play a range of roles in the skin, including antigen presentation and maintenance of peripheral tolerance. Reports of LC numbers have been variable in different cancer types, with the majority of studies indicating a reduction in their number. Changes in the cytokine profile and other secreted molecules, downregulation of surface molecules on cells and hypoxia all contribute to the regulation of LCs in the tumour microenvironment. Functionally, LCs have been reported to regulate immunity and carcinogenesis in different cancer types. An improved understanding of the function and biology of LCs in tumours is essential knowledge that underpins the development of new cancer immunotherapies.

Depletion of langerin+ cells enhances cutaneous wound healing.

Langerin is a C-type lectin receptor that is expressed on Langerhans cells and langerin-positive dermal dendritic cells in the skin. Little is known about the function of langerin+ cells in wound healing. In this study, the effects of ablation of langerin+ cells on healing of a full-thickness excision wound were investigated using the langerin-DTR depletable mouse. Strikingly, depletion of langerin+ cells resulted in more rapid reduction in wound area. Accelerated wound healing in the langerin+ -cell-depleted group was characterized by enhanced neo-epidermis and granulation tissue formation, and increased cellular proliferation within the newly formed tissues. Accelerated healing in the absence of langerin+ cells was associated with increased levels of granulocyte-macrophage colony-stimulating factor, F4/80+ cells and blood vessels within the granulation tissue. These data support an inhibitory role for langerin+ cells during wound healing. Therapies that suppress langerin+ cells or their function may therefore have utility in progressing the healing of wounds in humans.

The role of Langerhans cells in pathologies of the skin.

Langerhans cells (LCs) are epidermal immune cells of myeloid origin. Although these cells were primarily thought to play a defensive role in the skin, evidence now indicates a diverse range of LC-mediated effects including the relay of viral antigens in herpes simplex infection, recruitment of eosinophils in atopic dermatitis and promotion of a Th17 response in Candida infection. LCs may have a protective or suppressive function in pathologies of the skin, with differing functions being driven by the skin milieu. Understanding LC function will help guide the development of interventions that modulate these cells for therapeutic benefit.