Abstract Compared with skin wounds, oral mucosal wounds heal more quickly, with minimal scarring, faster re-epithelialization, and reduced inflammation. One differentiating factor may be the differential transcription factor-associated gene networks involved in tissue regeneration. One such transcription factor, BATF3, was recently shown by us to promote wound-healing responses in vitro and in vivo. Our prior analyses also suggest that CREB5 is a differentially regulated transcription factor in oral wounds and may be involved in early wound-healing gene expression programs. CREB5 expression was induced in immortalized skin keratinocytes (HaCaT) to examine its effect on in vitro wound healing relative to immortalized gingival keratinocytes (TIGK). CREB5 overexpression let to differential expression of predicted downstream genes and improved skin keratinocyte migration in vitro. This work suggests that examining transcription factors and gene networks that regulate wound-healing responses in the oral mucosa may lead to the discovery of novel targets to improve skin wound healing.

The response to injury involves a complex series of events, including cellular migration and proliferation, inflammatory processes, and tissue remodeling. The oral mucosa exhibits a more regenerative repair response that resolves with minimal scarring when compared to skin. To investigate mechanisms driving this differential healing response, we integrated gene expression data from adult human palate and skin wounds over seven days with transcription factor binding data and protein-protein interaction data to estimate sample-specific gene regulatory networks. Comparative analysis between unwounded palate and skin networks revealed tissue-specific transcription factor targeting and gene set co-expression. Upon injury, global network changes between tissues were divergent, indicating distinct gene regulatory programs. Gene regulatory analyses revealed that the acute response to injury is characterized by transcription factor mediated gene repression, followed by tissue-specific gene activation at later healing stages. Notably, a subset of palate-specific transcription factors previously linked to regeneration in model organisms correlated with gene targeting and expression during acute injury. Specifically, BATF3 promoted cell migration and re-epithelialization after injury, but not proliferation. In summary, these findings highlight the temporal dynamics of transcription factors in wound response between palate and skin and provide insights into the gene regulatory mechanisms governing regenerative and non-regenerative healing.

Streptococcus pyogenes, or Group A Streptococus (GAS), a significant human pathogen, employs quorum sensing (QS) systems to coordinate its behavior and genetic regulation in order to enhance survival. Our previous research established that one such QS system, the Rgg2/3 system, can suppress macrophage NFκB activity and production of pro-inflammatory cytokines. Yet, the scope of suppression and the mechanism by which it occurs remains unknown. In this study, we used transcriptomic and phosphoproteomic approaches to address these unanswered questions. We found QS-ON GAS broadly suppressed most inflammatory transcriptional pathways including those of NFκB, type I and type II interferon responses, and intracellular stress responses. Yet, we found no alternative transcriptional programs were activated after QS-ON GAS infection. Additionally, phosphoproteomics showed no disruption in typical inflammatory pathways such as those related to NFκB and MAPK activation, which was confirmed by western blotting and translocation assays. Instead, the proteomic data highlighted a potential role for epigenetic mechanisms of inflammatory regulation. To determine if epigenetic regulation was involved in QS-mediated immunomodulation, DNA methylation was measured and studies were performed inhibiting various histone and chromatin modifiers. These studies also showed no dijerence between QS-ON compared with QS-OFF infected macrophages. These findings expand our understanding of QS-mediated suppression and of GAS virulence strategies that appear to employ unusual methods of restricting inflammation. Uncovering this mechanism will ojer invaluable insight into GAS, itself, as well as understudied immunological pathways.

AbstractStreptococcus pyogenes, or Group A Streptococus (GAS), a significant human pathogen, employs quorum sensing (QS) systems to coordinate its behavior and genetic regulation in order to enhance survival. Our previous research established that one such QS system, the Rgg2/3 system, can suppress macrophage NFκB activity and production of pro-inflammatory cytokines. Yet, the scope of suppression and the mechanism by which it occurs remains unknown. In this study, we used transcriptomic and phosphoproteomic approaches to address these unanswered questions. We found QS-ON GAS broadly suppressed most inflammatory transcriptional pathways including those of NFκB, type I and type II interferon responses, and intracellular stress responses. Yet, we found no alternative transcriptional programs were activated after QS-ON GAS infection. Additionally, phosphoproteomics showed no disruption in typical inflammatory pathways such as those related to NFκB and MAPK activation, which was confirmed by western blotting and translocation assays. Instead, the proteomic data highlighted a potential role for epigenetic mechanisms of inflammatory regulation. To determine if epigenetic regulation was involved in QS-mediated immunomodulation, DNA methylation was measured and studies were performed inhibiting various histone and chromatin modifiers. These studies also showed no dijerence between QS-ON compared with QS-OFF infected macrophages. These findings expand our understanding of QS-mediated suppression and of GAS virulence strategies that appear to employ unusual methods of restricting inflammation. Uncovering this mechanism will ojer invaluable insight into GAS, itself, as well as understudied immunological pathways.ImportanceStreptococcus pyogenesis a ubiquitous pathogen that causes over 600 million infections every year and 500 thousand to 1 million fatalities. While in developed countries it is generally known to cause mild conditions such as pharyngitis, it can also manifest as severe infections such as necrotizing fasciitis, septic arthritis, and lead to post-infectious sequelae including rheumatic heart disease and glomerulonephritis. Elucidating new mechanisms of virulence in this organism, including how it evades and suppresses immune responses can be critical in understanding its pathogenicity, epidemiology, and identification of novel treatment avenues in this era of multi-drug-resistant bacteria. In this study, we characterize the broad spectrum by which GAS modulates the host innate immune response and begin to uncover host pathways that bacteria can use or inhibit for its survival.

An adequate wound healing response requires the coordination of intercellular signals between multiple cell types. After skin injury, endothelial cells and fibroblasts provisionally replace the site of injury with newly vascularized granulation tissue. Given the spatial and temporal overlap of these 2 cell types in the healing response, we hypothesize that endothelial cell-derived extracellular vesicles (ECEVs) could provide a communication pathway through which endothelial cells influence fibroblast behavior. In this study, we investigated the effects of ECEVs on fibroblast function both in vitro and in vivo through a murine skin wound healing model. Transcriptomic analysis showed that the uptake of ECEVs by fibroblasts altered functions and pathways relating to cell division, extracellular matrix organization, and fibrosis. This gene signature resulted from upregulation of the transcription factor ETV1, which enhanced fibroblast susceptibility to FGF2 found on ECEVs. This transcriptomic analysis was functionally corroborated through in vitro assays in which fibroblasts demonstrated enhanced proliferation, cell cycle progression, and altered extracellular matrix deposition after ECEV treatment. Administration of ECEVs to healing mouse wounds led to increased collagen density and fibroblast quantity in the wound bed of scar tissue. Our study highlights a previously undefined role for ECEVs in regulating fibroblast function during wound repair.

Streptococcus pyogenes or Group A Streptococcus (GAS), a significant human pathogen, employs quorum sensing (QS) systems to coordinate its behavior and genetic regulation to enhance survival. Our previous research established that one such QS system, the Rgg2/3 system, can suppress macrophage NFκB activity and production of pro-inflammatory cytokines. Yet, the scope of suppression and the mechanism by which it occurs remain unknown. In this study, transcriptomic and phosphoproteomic approaches were used to address these unanswered questions. It was found that QS-ON GAS broadly suppressed most inflammatory transcriptional pathways, including those of NFκB, type I and type II interferon responses, and intracellular stress responses. Yet, we found that no alternative transcriptional programs were activated after QS-ON GAS infection. Additionally, phosphoproteomics showed no disruption in typical inflammatory pathways such as those related to NFκB and MAPK activation, which was confirmed by western blotting and translocation assays. Instead, the proteomic data highlighted a potential role for epigenetic mechanisms of inflammatory regulation. A role for epigenetic regulation in QS-mediated immunomodulation was validated using pharmacological inhibitors of histone and chromatin modifiers. These findings expand our understanding of QS-mediated suppression and of GAS virulence strategies that appear to employ atypical methods of restricting inflammation. Uncovering this mechanism will offer invaluable insight into GAS itself, as well as into understudied immunological pathways.IMPORTANCEStreptococcus pyogenes is a ubiquitous pathogen that causes over 600 million infections every year and 500,000 to 1 million fatalities. While in developed countries, it is generally known to cause mild conditions such as pharyngitis, it can also manifest as severe infections, such as necrotizing fasciitis and septic arthritis, and lead to post-infectious sequelae including rheumatic heart disease and glomerulonephritis. Elucidating new mechanisms of virulence in this organism, including how it evades and suppresses immune responses, is critical in understanding its pathogenicity and epidemiology, as well as identifying novel treatment avenues in this era of multidrug-resistant bacteria. In this study, we characterize the broad spectrum by which GAS modulates the host innate immune response and begin to uncover host pathways that the bacteria can use or inhibit for its survival.

Prostate cancer is the second leading cause of malignancy-related deaths among American men. Active surveillance is a safe option for many men with less aggressive disease, yet definitively determining low-risk cancer is challenging with biopsy alone. Herein, we sought to identify prostate-derived microRNAs in patient sera and serum extracellular vesicles, and determine if those microRNAs improve upon the current clinical risk calculators for prostate cancer prognosis before and after biopsy. Prostate-derived intracellular and extracellular vesicle-contained microRNAs were identified by small RNA sequencing of prostate cancer patient explants and primary cells. Abundant microRNAs were included in a custom microRNA PCR panel that was queried in whole serum and serum extracellular vesicles from a diverse cohort of men diagnosed with prostate cancer. The levels of these circulating microRNAs significantly differed between indolent and aggressive disease and improved the area under the curve for pretreatment nomograms of prostate cancer disease risk. The microRNAs within the extracellular vesicles had improved prognostic value compared to the microRNAs in the whole serum. In summary, quantifying microRNAs circulating in extracellular vesicles is a clinically feasible assay that may provide additional information for assessing prostate cancer risk stratification.

Introduction: Microscopic colitis (MC) is a subset of the inflammatory bowel disease (IBD) family of diseases. Its pathogenesis is multifactorial, and risk factors include older age, woman sex, medication use, and autoimmune disease. Symptoms include chronic, persistent watery diarrhea, and patients are not thought to be at an increased risk of colon cancer. We present a rare case of MC with adjacent colonic metastatic serous carcinoma and focal intramucosal carcinoma. Case Description/Methods: A 66-year-old woman with a history of stage IV serous endometrial carcinoma who had chemotherapy and surgical debulking 2 years prior presented with >15 episodes of watery diarrhea with daily abdominal cramping for 3 months. Computed tomography (CT) of the abdomen and pelvis obtained 2 weeks prior for evaluation of diarrhea demonstrated new rectosigmoid wall thickening. Laboratory investigations for infectious diarrhea, IBD, anemia, and celiac disease were negative. Stool osmolar gap was consistent with secretory diarrhea. The patient then underwent a colonoscopy which revealed malignant appearing mass-like lesions in the ascending and transverse colon, and a nodular rectum. Biopsies from the ascending colon lesion revealed tubulovillous adenoma with high-grade dysplasia and focal intramucosal carcinoma. Biopsies from the transverse colon lesion and nodular rectum showed metastatic endometrial serous carcinoma. Random colon biopsies were consistent with MC. Patient was discharged with instructions to complete a tapered dose of budesonide, to follow up with her gynecologic oncologist, and to undergo repeat colonoscopy to rule out primary colon cancer at the ascending colon mass-like lesion. On follow-up 2 weeks later, patient reported significant improvement in diarrhea with 2-3 formed stools daily (Figure 1). Discussion: Patients with MC usually do not experience progression of the disease. Unlike Crohn’s disease or ulcerative colitis, MC is not associated with an increased risk of colon cancer. On endoscopy, colon is grossly normal, and colonic biopsies are required to confirm the diagnosis. Interestingly, our patient’s colon had multiple distinct pathologies present which is a very rare presentation. Further studies are needed to evaluate the association between MC, primary colon cancer, endometrial cancer, and other malignancies. Treatment of MC varies based on severity, but medical therapy (e.g., budesonide) remains first-line with surgical intervention reserved for refractory cases.Figure 1.: Focal intramucosal carcinoma arising from a tubulovillous adenoma in the ascending colon is shown in A. Cells in the transverse colon with abundant cytoplasm, marked atypia, and prominent nucleoli consistent with metastatic serous carcinoma are shown in B. Colonic mucosa with moderate chronic inflammation with lymphocytosis and lymphoid aggregates compatible with microscopic/lymphocytic colitis is shown in C. Nodular rectum is shown in D, the transverse colon mass-like lesion is shown in E, and the ascending colon mass-like lesion is shown in F.

Most human tissue injuries lead to the formation of a fibrous scar and result in the loss of functional tissue. One adult tissue that exhibits a more regenerative response to injury with minimal scarring is the oral mucosa. We generated a microarray gene expression dataset to examine the response to injury from human palate and skin excisional biopsies spanning the first seven days after wounding. Differential expression analyses were performed in each tissue to identify genes overexpressed or underexpressed over time compared to baseline unwounded tissue. To attribute biological processes of interest to these gene expression changes, gene set enrichment analysis was used to identify core gene sets that are enriched over the time-course of the wound healing process with respect to unwounded tissue. This analysis identified gene sets uniquely enriched in either palate or skin wounds and gene sets that are enriched in both tissues in at least one time point after injury. Finally, a cell-deconvolution analysis was performed to better understand the cell type distribution in these tissues and how it changes over the time course of wound healing. This work provides a source of human wound gene expression data that includes two tissue types with distinct regenerative and scarring phenotypes.

Many factors regulate scar formation, which yields a modified extracellular matrix (ECM). Among ECM components, microfibril-associated proteins have been minimally explored in the context of skin wound repair. Microfibril-associated protein 5 (MFAP5), a small 25 kD serine and threonine rich microfibril-associated protein, influences microfibril function and modulates major extracellular signaling pathways. Though known to be associated with fibrosis and angiogenesis in certain pathologies, MFAP5’s role in wound healing is unknown. Using a murine model of skin wound repair, we found that MFAP5 is significantly expressed during the proliferative and remodeling phases of healing. Analysis of existing single-cell RNA-sequencing data from mouse skin wounds identified two fibroblast subpopulations as the main expressors of MFAP5 during wound healing. Furthermore, neutralization of MFAP5 in healing mouse wounds decreased collagen deposition and refined angiogenesis without altering wound closure. In vitro, recombinant MFAP5 significantly enhanced dermal fibroblast migration, collagen contractility, and expression of pro-fibrotic genes. Additionally, TGF-ß1 increased MFAP5 expression and production in dermal fibroblasts. Our findings suggest that MFAP5 regulates fibroblast function and influences scar formation in healing wounds. Our work demonstrates a previously undescribed role for MFAP5 and suggests that microfibril-associated proteins may be significant modulators of wound healing outcomes and scarring.

High glucose inhibits oral keratinocyte proliferation. Diabetes can lead to delayed oral wound healing and periodontal disease. L-Arginine, one of the most versatile amino acids, plays an important role in wound healing, organ maturation, and development. In this study, L-Arginine was found to enhance oral keratinocyte proliferation under high-glucose conditions. RNA sequencing analysis discovered a significant number of genes differentially upregulated following L-Arginine treatment under high-glucose conditions. Cytochrome P450 family 1 subfamily A member 1 (CYP1A1) was the most significantly upregulated gene at 24 and 48 h after L-Arginine treatment. Gene Ontology enrichment analysis found that cell proliferation- and mitosis-related biological processes, such as mitotic nuclear division, mRNA processing, and positive regulation of cell cycle processes, were significantly upregulated. Pathway enrichment analysis found that S-phase kinase-associated protein 2 (SKP2) and serine- and arginine-rich splicing factor 5 (SRSF5) were the top upregulated genes in cell cycle and spliceosome pathways, respectively. Indirect immunofluorescent cytochemistry confirmed increased protein levels of CYP1A1, SKP2, and SRSF5 after L-Arginine treatment. Knockdown of CYP1A1, SKP2, and SRSF5 abolished the enhanced proliferative effect of L-Arginine on oral keratinocytes under high-glucose conditions. In conclusion, L-Arginine enhances oral keratinocyte proliferation under high-glucose conditions via upregulation of CYP1A1, SKP2, and SRSF5, suggesting that supplemental L-Arginine in oral care products may be beneficial for oral tissue repair and regeneration.

The oral cavity is a highly regenerative epithelial tissue that results in minimal scarring after injury. This protocol describes the preparation of a mouse palate wound model. The protocol includes steps to place an excisional wound on the mouse palate, followed by harvesting of wound tissue and bone decalcification. We detail how to overcome the technical challenge of limited anatomical space, avoid damaging the nasal cavity, manage bleeding, and collect tissue for downstream genomic or immunohistochemical analysis.

The effective clearance of apoptotic cells is an essential step in the resolution of healing wounds. In particular, blood vessel regression during wound resolution produces a significant number of apoptotic endothelial cells (ApoEC) that must be cleared. In considering the fate of ApoEC and the presence of fibroblasts during wound resolution, we hypothesized that fibroblasts might serve as phagocytes involved in endothelial cell removal. The current study investigated whether dermal fibroblasts engulf ApoEC, whether this uptake alters the phenotype of dermal fibroblasts, and the biological molecules involved. In both in vitro and in vivo studies, following ApoEC engulfment, fibroblasts acquired a pro-healing phenotype (increased cell migration, contractility, α-smooth muscle actin expression, and collagen deposition). In addition, fibroblast uptake of ApoEC was shown to be mediated in part by the milk fat globule-EGF factor 8 protein/integrin αv β5 pathway. Our study demonstrates a novel function of fibroblasts in the clearance of ApoEC and suggests that this capability has significant implications for tissue repair and fibrosis.

Bicellular tight junctions are multiprotein complexes that are required for maintenance of barrier function and fence function in epithelial tissues. Wound healing in the oral cavity leads to minimal scar formation compared to the skin, and the precise mechanisms for this regenerative response remain to be elucidated. We hypothesized that oral and skin tissues express a different tight junction repertoire both at baseline and during the wound healing response, and that these molecules may be critical to the differential repair between the two tissues. We re-analyzed a mouse skin and palate epithelium microarray dataset to identify the tight junction repertoire of these tissue types. We then re-analyzed a skin and tongue wound healing microarray dataset to see how expression levels of tight junction genes change over time in response to injury. We performed in vitro scratch assays on human oral and skin keratinocyte cell lines to assay for tight junction expression over time, tight junction expression in response to lipopolysaccharide and histamine treatment, and the effects of siRNA knockdown of claudin 1 or occludin on migration and proliferation. Our data showed that oral and skin epithelium expressed different tight junction genes at baseline and during the wound healing response. Knockdown of claudin 1 or occludin led to changes in proliferation and migration in human skin keratinocytes but not oral keratinocytes. Furthermore, we also showed that skin keratinocytes were more permeable than oral keratinocytes upon histamine treatment. In conclusion, this study highlights a specific subset of functional tight junction genes that are differentially expressed between the oral and skin tissues, which may contribute to the mechanisms leading to distinct healing phenotypes in response to injury in the two tissues.