Elizabeth A. Grice, Ph.D. - US grants

Chronic non-healing wounds represent a major health care buriden especially in elderly, bed-ridden, and diabetic patient populations. Microbial bioburden and/or infection are generally acknowledged to deleteriously affect wound healing and are a significant source of medical complications. Little is known about the microbes that colonize chronic wounds and how they influence the host cutaneous defense response during wound healing. Most microbial surveys of chronic wounds have relied on culture-based methodologies, even though it is estimated that >99% of bacteria are estimated to resist isolation in pure culture. Sequencing bacterial 16S ribosomal RNA (rRNA) genes is a less-biased approach to characterizing bacterial diversity. 16s rRNA genes are present in every bacterial cell and can be used to identify bacteria. Using these methods, I have generated preliminary data in the Lepr^'' (db/db) mouse model of impaired wound healing that demonstrated a longitudinal selective shift in wound microbiota coinciding with impaired healing. Concurrent transcriptional profiling showed prolonged expression of cutaneous defense response genes, correlating with the selective shift observed in wound microbiota. Based on this preliminary data, the specific aims of this proposal test the hypothesis that wound microbiota integrates with an aberrant cutaneous host defense response to impair wound healing: (1) Characterize role for microbes in impaired wound healing by manipulating host skin and/or wound microbiota using gnotobiotic (germ-free) mice and antibiotic treatment regiments and (2) Investigate the impact of the cutaneous defense response on host microbiota and wound healing using db/db mice deficient for innate immune components or pro-inflammatory innate immune molecules. The completion of these aims will provide new insight into microbial interactions with the cutaneous defense response in impaired wound healing. The long-term goal is to enable the development of improved biomarkers and novel therapeutics by leveraging our understanding of host-microbe interactions in impaired wound healing. Furthermore, implementation of the proposed career development plan and completion of these aims will establish the foundation necessary for me to embark on a career as an independent investigator.

? DESCRIPTION (provided by applicant): The role of colonizing and infecting pathogens (wound bioburden) on diabetic foot ulcer (DFU) outcomes remains unclear. Our group has shown that the totality of microorganisms in DFUs measured using high throughput 16S ribosomal RNA (rRNA) gene sequencing, is directly related to: ulcer duration, ulcer depth, and poor glycemic control. However, changes in bioburden over time provided only limited insights into the role of these changes on DFU outcomes. Nonetheless, bioburden may be influencing DFU outcomes, but the specific dimensions of importance may not be discernable using 16S rRNA profiling. Major limitations of this molecular technique include the fact that it does not disclose functional potential of the microbiota and resolution to species or strain level is often impossible. To circumvent these limitations, we will employ whole genome shotgun (WGS) metagenomic sequencing and analysis, a powerful lens for deciphering the functional potential of microbial communities. Our overarching hypothesis is that specific dimensions of the dynamic DFU microbiome are predictive of outcome. Additionally, we hypothesize that different treatment regimens have different effects on the DFU microbiome. We have assembled a multi-disciplinary team of experts to execute the aims of this proposal, with basic science expertise in wound microbiome and metagenomics and clinical and research expertise in DFUs and infection. To test our hypothesis, we will complete the following aims: Aim 1: Determine if pathogenic species/strains and overall microbial diversity of the DFU metagenome are associated with specific DFU outcomes. We will characterize the changes in metagenomes over time for 107 subjects with DFUs recruited from our previous studies, from whom specimens were collected every 2 weeks from presentation to final outcome. We will determine if 1) the presence of specific species or strains of pathogens (e.g. beta-hemolytic Streptococcus); and 2) low microbial diversity are directly associated with lower rates of healing or infection-related complications (i.e., ulcer deterioration, osteomyelitis or amputation). Aim 2: Determine if pathogenicity genes in the DFU metagenome are associated with specific DFU outcomes. We will examine the presence and relative abundance of genes conferring 1) biofilm-formation potential, 2) antibiotic resistance and, 3) virulence factors, to determine if they are association with lower rates of healing and infection-related complications. Aim 3: Determine the effects of aggressive sharp debridement and antibiotic treatment on the DFU metagenome. This is the first longitudinal study of the role of the DFU microbiome on DFU outcomes using WGS metagenomic sequencing as well as the first to examine the impact of two commonly employed antimicrobial interventions on the DFU metagenome.

? DESCRIPTION (provided by applicant): The skin harbors a diverse population of microbiota with important functions in cutaneous health and disease. The role of host-microbe interactions is well appreciated in multiple skin disorders. However, very little is known regarding host modulation of skin microbiota, and microbial regulation of host cutaneous immunity. We have previously shown that disrupting complement signaling alters the diversity and composition of skin microbiota in parallel with changing the cutaneous inflammatory milieu. Further, our RNA-seq studies indicate that genes related to complement signaling and activation are upregulated in conventionally raised mouse skin as compared to germ free mouse skin, suggesting that the commensal microbiota regulate their expression. Our overarching hypothesis is that there is a mechanistic link between the skin microbiota and complement. We posit that the skin microbiota are shaped and maintained by complement, and conversely, that the commensal microbiota modulate complement at the gene expression level. To test our hypothesis we propose the following aims: 1) Determine how commensals and pathogens differentially induce complement gene expression in skin. We will also determine if commensal microbiota reconstitution can tune complement gene expression and improve infection outcome in a model of skin and soft tissue infection. 2) Establish the complement effector pathways that impact cutaneous microbial composition, diversity, and quantity. Following up on preliminary data indicating that complement deficiency accelerates excisional wound healing in mouse models in parallel with shifting the colonizing microbiota, we will test the necessity and the sufficiency of the microbiot in this phenotype. Completion of the proposed studies will fill a gap in our fundamental knowledge of skin-microbe interactions, the functional consequences of disrupting these interactions, while facilitating future research questions regarding the role of skin microbiota in disease, wound healing, and infection.

Chronic, non-healing wounds are common and costly complications of diabetes. Microbial colonization and biofilm formation are hypothesized to impair wound healing and contribute to severe complications such as osteomyelitis and amputation. Although all chronic wounds are colonized with microbiota, its importance, in the absence of clinical infection, is currently unknown. In this competitive renewal, we hypothesize that host response, wound healing, and clinical DFU outcomes are determined by 1) genomic diversification of the wound pathogen Staphylococcus aureus; 2) commensal interactions with wound pathogens; and 3) commensal interactions with the host. In the previous cycle, we developed a shotgun metagenomic sequencing pipeline to analyze the microbiota colonizing neuropathic, non-infected DFU (n=100; the ?DFU100? cohort) in a longitudinal prospective cohort study. We observed that strain-level variation of the wound pathogen Staphylococcus aureus was associated with DFU outcomes. Therefore, in Aim 1, we will use a microbial genomic approach and matched clinical isolates from the DFU100 cohort to identify S. aureus genomic determinants of pathogenesis in DFU and their association with clinical outcomes. We also observed that species clinically regarded as ?bystanders? (e.g. skin commensals, environmental contaminants) influence the virulence of wound pathogens and tune host tissue repair responses to promote healing in vivo. Aim 2 will determine if a skin commensal, Corynebacterium striatum, tunes the virulence of S. aureus and improves wound healing in murine and porcine models of S. aureus wound infection. Aim 3 is based on our observation that Alcaligenes faecalis wound isolates promote keratinocyte migration, cytokine secretion, and accelerated wound closure in a murine model of diabetic wound healing. We will establish the mechanism and therapeutic potential of A. faecalis-mediated host responses that lead to accelerated wound healing. The proposed research will use cutting-edge, cross-disciplinary approaches to investigate interactions between wound pathogens, wound ?bystanders?, and the host; understanding these mechanisms will lead to improved DFU outcomes as our long-term objectives are to 1) develop novel microbiota-based interventions to improve healing that exploit microbial interactions with each other and the host; and 2) identify microbial biomarkers to classify patients at risk of complication.

The overarching goal of this well-established training program is to maintain and accelerate long term research progess and innovation in cutaneous biology, skin diseases, and dermatoepidemiology, leading to novel treatments for skin disease and improvements in overall human health. We aim to achieve this by identifying and training the next generation of leaders in this area. Our specific goals are to: (i) recruit diverse trainees at early stages in their careers by exposure to the excitement and impact of dermatology research; (ii) support the research and training of young scientists with established interests in the skin and its diseases; and (iii) provide cross-disciplinary training in skin biology and diverse fields such as genomics, epidemiology or bioengineering. To these ends we request support for: 4 long-term (1 year or more) predoctoral fellowships to support the thesis research of MD PhD students, PhD students engaging in innovative, interdisciplinary work relevant to cutaneous biology and skin disease, or students pursuing dual MD and Masters in Clinical Epidemiology (MSCE) degrees; and 4 postdoctoral fellowships for MD, PhD and MD PhD scientists committed to careers in cutaneous biology and skin disease. Our program involves 41 highly qualified trainers drawn from the Department of Dermatology, from other departments within the Perelman School of Medicine, and from other Schools at University of Pennsylvania including the School of Veterinary Medicine, the School of Engineering and Applied Sciences, and the School of Dental Medicine. To promote cutting-edge and interdisciplinary research, our trainers have expertise ranging from clinical epidemiology, genomics, bioinformatics, microbiology, immunology, to developmental biology, stem cells, and regeneration. A particular emphasis of our training program is to encourage and train physician scientists in Dermatology. In addition to training MD and MD PhD students in skin-related research, we established a four-year clinical/research dermatology residency program in which trainees pursue T32-supported postdoctoral research fellowships that provide a basis for obtaining independent funding. This program has already been extremely successful, attracting exceptionally qualified, diverse, and enthusiastic trainees, several of whom are now established as NIH-funded independent physician-scientists. Our training program includes opportunities for lab-based research, translational research, clinical trials, and epidemiology. In parallel with ?hands on? research training, our trainees attend seminars, journal clubs and technical and skills workshops both within and outside Dermatology. We provide networking lunches and research retreats for trainees, and trainees invite two seminar speakers per year to present their research in the Dermatology Seminar Series, followed by lunch with the speaker. Trainees receive formal instruction in the Responsible Conduct of Research, and extensive mentoring, career counseling, and assistance in identifying suitable positions in academia, industry, or for further training in clinical or research areas from the Co- Directors of the training program as well as from their mentors and, in the case of MD PhD and PhD thesis students, their home Graduate Programs.

PROJECT SUMMARY Cutaneous leishmaniasis, brought about by infection with the intracellular parasite leishmania, exhibits a spectrum of clinical manifestations, ranging from single healing lesions to severe chronic lesions, and including both disseminated and mucosal lesions, all of which can be disfiguring and/or resistant to drug treatment. Lesion resolution requires parasite control, modulation of pathologic inflammatory responses, and initiation of wound healing responses. We recently discovered that leishmania infection, in murine models and in humans, results in specific alterations to the skin microbiome, or dysbiosis, and that these changes contribute to the immunopathologic response associated with infection. In mice, the alterations occurred in a severity-dependent manner, with non-healing cutaneous leishmaniasis lesions characterized by Streptococcus spp. and resolving lesions characterized by Staphylococcus spp. Leishmania-induced dysbiosis was transmissible to non-infected cage-mates, allowing us to show that pre-existing dysbiosis results in more severe disease following an insult. In humans, preliminary gene transcriptional studies show that cutaneous leishmaniasis lesions with measurable Streptococcus reads exhibited increased IL-1? expression and were enriched for differentially expressed genes associated with cytotoxicity. This is in line with previous work where we identified a pathway leading to severe inflammation that is initiated by exaggerated T cell cytotoxicity, leading to IL-1? production. These preliminary findings provide a platform for us to delineate the sufficiency and necessity of the skin microbiome in the development of cutaneous lesions and in the wound healing response that is essential for lesion resolution (Aim 1). Our studies indicate that dysbiotic mice exhibit changes in the immune response, and we propose to define the innate and adaptive responses associated with dysbiosis that contribute to lesion development and impair wound healing (Aim 2). Importantly, our approach is designed to maximize clinical relevance by evaluating the effect of patient bacterial isolates on lesion development and resolution in mice, and by investigating the predictive nature of the microbiome in cutaneous leishmaniasis patients (Aim 3). With these studies we will determine how the skin microbiome integrates with the immune response and the wound healing response to influence disease outcomes, which will allow us to identify potential therapies to block dysbiosis-associated pathology, promote increased wound healing, and lessen disease severity in cutaneous leishmaniasis patients.

PROJECT SUMMARY The objective of this project is to determine how early life exposures to persistent organic pollutants alter the skin microbiome, contribute to skin barrier defects and inflammation, and disrupt the establishment of immune tolerance to commensal microbes. The skin epidermal permeability barrier (EPB) forms late in development and is essential for life and health, preventing dehydration, and protecting against infection and physical and chemical insults. EPB dysfunction is a key feature of atopic dermatitis (AD; eczema), a common skin disorder characterized by chronic and relapsing, itchy, inflamed, skin lesions and dysbiotic microbiota. Recent dramatic increases in AD prevalence, especially in industrialized countries, suggest a strong environmental component to disease such as air pollution. We hypothesize that early in life exposures to environmental pollutants, such as 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD) or diesel exhaust particles (DEP), that persistently activate aryl hydrocarbon receptor (AhR) signaling, affect the development of the skin microbiome, the EPB, and the establishment of immune tolerance to the commensal skin microbiome, leading to AD-like phenotypes of skin barrier dysfunction, inflammation and dysbiosis. We propose three aims to examine a critical neonatal development period where the newly formed EPB is exposed to the ex utero environment, assembles its microbiota, and establishes immunotolerance to this commensal microbiome. In aim 1 we will determine the effect of AhR signaling and chemical exposures that activate AhR signaling on the assembly and function of the skin microbiome in concert with analyzing EPB function and inflammation. In aim 2 we will test the role of the microbiome in regulating epidermal AhR signaling, and contribution to AD-like phenotypes in AhR exposure models. In aim 3, we will investigate the role of AhR signaling in the establishment of tolerance to commensal microbes. Findings from the proposed studies should greatly enhance the understanding of how early life exposures to pollutants can disrupt the skin microbiome, its interactions with the host, and lead to skin disease.

PROJECT SUMMARY Towards the overall theme of translating basic research into new treatments for skin disease, the goals of the Penn SBDRC are to promote multi-disciplinary, collaborative research among skin investigators; draw new investigators with diverse perspectives to skin research; foster an interactive, multi-disciplinary environment that cultivates the next generation of skin researchers; increase access to state-of-the-art approaches and resources for conducting rigorous skin research, and accelerate translation of research findings into innovative therapies for skin disease. In addition to providing state-of-the-art service offerings through 3 Resource Cores, the Penn SBDRC includes an Administrative Core, which unifies skin researchers across the University, the Philadelphia region and beyond. The Aims are: 1) To provide oversight and evaluation of the Penn SBDRC; 2): To coordinate, integrate, and communicate the activities of the Penn SBDRC. The Administrative Core provides administrative, logistical, regulatory, and financial support to the overall Center, the Resource Cores, and the Enrichment Programs. The SBDRC website and its underlying REDCap databases serve as the major platform for interacting with members, including ordering of Core services, tracking membership data, evaluating services and programs, tracking outcomes, and relaying announcements such as seminar and meeting notices. A quarterly SBDRC Newsletter and Twitter feed are additional tools for disseminating information and highlighting the research and achievements of SBDRC members. 3) To implement an Enrichment Program that enhances the scientific environment and supports the next generation of skin researchers. The SBDRC Enrichment Program is divided into four Sub-Cores representing individual objectives: (a) The Scientific Enrichment Sub-Core sponsors an Annual Scientific Symposium, seminars, and workshops. (b) The Community Outreach Sub-Core introduces underserved Philadelphia public high school students to the excitement of scientific research and dermatology through the Penn Academy of Skin Health (PASH), a Saturday academy program and summer internship program. (c) The Mentoring Sub-Core promotes the career success and training of a diverse pool of future skin researchers through structured mentoring for senior trainees and junior faculty, and specialized mentoring programs for women and minorities underrepresented in medicine. (d) The Seed Funding Sub-Core offers 3 different funding mechanisms that attract new investigators and foster the independence of junior investigators.

PROJECT SUMMARY The Penn Skin Biology and Diseases Resource-based Center (Penn SBDRC) will continue to support and accelerate skin disease research and its translation by providing critical infrastructure, resources, and expertise to skin investigators. The overall goals of the Penn SBDRC are to promote collaboration among skin investigators, especially across disciplines; to draw new investigators with diverse perspectives to skin research; to foster an environment that supports and cultivates the next generation of skin researchers; to increase access to technology and resources for conducting rigorous, impactful skin research; and to accelerate translation of research findings into innovative therapies for skin disease. To accomplish these goals, the aims of the Center are: Aim 1) To provide cutting-edge approaches and related expertise centered around 3 multi-disciplinary Resource Cores: Cutaneous Phenomics and Transcriptomics (CPAT) Core, Skin Translational Research (STaR) Core, and Data Science and Informatics (DSI) Core. These Cores are highly synergistic, providing high-demand and/or specialized services, valuable tissue/cellular resources, and the expertise to ensure rigorous experimental design, analytical strategies, and accurate interpretations. All Cores take advantage of the deep expertise of the Directors and Co-Directors and their research groups, to continuously improve and innovate upon techniques/approaches offered by the Center. Aim 2) To establish an Administrative Core that unifies skin investigators and promotes the goals of the SBDRC. The Administrative Core provides oversight and implements the activities of the center. This includes an Enrichment Program that is subdivided into 4 Sub-Cores: The Community Outreach Sub-Core introduces underserved Philadelphia public high school students to the excitement of biomedical research and dermatology through the Penn Academy of Skin Health (PASH), a Saturday academy and summer internship program. These programs also provide volunteer and mentorship opportunities for SBDRC members; The Mentoring Sub-Core promotes career success and training through structured mentorship for trainees and junior faculty. Through a partnership with the Dermatology Department?s Committee on Diversity and Inclusion, specialized mentoring programs for women and minorities underrepresented in medicine will be provided, as well as training for SBDRC mentors. The Scientific Enrichment Sub-Core sponsors a seminar series, workshops, and a yearly scientific symposium to promote interactions, collaboration, and innovative approaches among skin researchers. The Seed Funding Sub-Core offers 3 different funding mechanisms that attract new investigators and support the independence of junior investigators. The Penn SBDRC leverages the excellence of Penn Dermatology?s research and clinical programs as well as that of the entire Penn community to enhance collaboration, bringing new technology to skin research, and providing access to critical intellectual and technical resources that greatly enhance innovation and rigor in the study of skin disease. The Center impacts skin research well beyond Pennsylvania?s borders, and ultimately advances the treatment, prevention, and diagnosis of skin diseases.