SKIN November 2022 Volume 6 Issue 6 (c) 2022 THE AUTHORS. Published by the National Society for Cutaneous Medicine. 534 BRIEF ARTICLE Identifying Inflammatory Gene Expression Signatures for Skin and Soft Tissue Infections Jennifer E. Yeh, MD, PhD1,2, Marcelo Pereira da Silva, BS3, Adam B. Raff, MD, PhD4, Ruth K. Foreman, MD, PhD5 1 Harvard Combined Dermatology Residency Training Program, Boston, MA 2 Stanford University School of Medicine, Department of Dermatology, Palo Alto, CA 3 Massachusetts General Hospital, Cutaneous Biology Research Center, Boston, MA 4 Beth Israel Lahey Health, Department of Dermatology, Boston, MA 5 Massachusetts General Hospital, Department of Pathology, Boston, MA Skin and soft tissue infections (SSTIs) are one of the most common reasons to seek medical care in the United States. Cellulitis accounts for ~60% of SSTIs, with 14.5 million cases annually in the United States accounting for $3.7 billion in healthcare costs.1 SSTIs are frequently misdiagnosed ABSTRACT Background: Misdiagnosis of skin and soft tissue infections (SSTIs) due to clinical mimics can result in delay of care, unnecessary antibiotic exposure, and inappropriate hospitalization. Comprehensive screening of inflammatory genes in SSTIs could identify biomarkers to distinguish SSTIs from mimics. Methods: We performed a search of the MGH James Homer Wright Pathology Laboratories database from 2008-2018 for diagnoses of necrotizing fasciitis, cellulitis, and stasis dermatitis, yielding 103 cases. Diagnoses were verified by chart review and categorized by discharge diagnosis. Three samples from each category, along with three controls from location-matched skin were selected for further study. mRNA isolated from paraffin- embedded skin biopsies was analyzed by Nanostring, with 594 inflammatory genes profiled. Results: We identified differentially expressed genes between necrotizing fasciitis, cellulitis, and infectious cases (necrotizing fasciitis and cellulitis) compared to non-infectious stasis dermatitis. Differentially upregulated genes in SSTIs included those with known roles in inflammation (CXCR2, IL6, IFI16, TNFRSF1B) and transcriptional regulation (BCL3, MBP). We also identified differential upregulation of genes not previously associated with SSTIs including S100A8, S100A9, MCL1, CD14, and LTF. Conclusion: We characterized transcriptomic signatures of severe and moderate SSTIs compared to stasis dermatitis and normal skin from the lower extremities. Though limited by small sample size, these data support the utility of a prospective study analyzing outcomes of patients diagnosed with SSTIs based on gene expression signatures. Identifying SSTI- specific gene expression signatures could help differentiate true skin infections from non- infectious inflammatory skin conditions, facilitating more accurate diagnoses and improving patient care. INTRODUCTION SKIN November 2022 Volume 6 Issue 6 (c) 2022 THE AUTHORS. Published by the National Society for Cutaneous Medicine. 535 due to clinical overlap with inflammatory dermatoses (e.g., stasis dermatitis), resulting in delay of effective care, unnecessary antibiotic exposure, and inappropriate hospitalization.2 Misdiagnosis is partially due to an incomplete understanding of SSTI biology leading to ineffective diagnostic strategies. SSTIs are thought to be mediated by toxin-induced inflammatory pathways3; however, published literature lacks comprehensive screening of inflammatory genes in SSTIs. Identifying SSTI-specific gene expression signatures could help differentiate true skin infections from inflammatory mimics, thereby reducing misdiagnosis and broadening understanding of the pathophysiology to identify potential therapeutic targets. We performed a search of the MGH James Homer Wright Pathology Laboratories database from 2008-2018 for diagnoses of necrotizing fasciitis, cellulitis, and stasis dermatitis, yielding 103 cases. Diagnoses were verified by chart review and categorized by discharge diagnosis. Three samples from each category, along with three controls from location-matched skin were selected for further study. mRNA isolated from paraffin- embedded skin biopsies was analyzed by Nanostring, with 594 inflammatory genes profiled. One hundred thirty-four cases were identified and verified by dermatologist chart review of discharge diagnosis and dermatopathologist review of skin biopsies, yielding 103 cases which were filtered by lower extremity because this location is common for SSTI mimics, resulting in 71 cases (Figure 1). We confirmed necrotizing fasciitis cases with imaging evidence of subcutaneous air and cellulitis cases with positive bacterial data by culture or Gram stain. Cases with concomitant clinical scenarios such as osteomyelitis, immunosuppression, implanted hardware, and neutrophilic dermatoses were excluded. For this pilot study, three samples from each diagnosis were chosen for further cytokine interrogation. Cultured bacteria from these three cases included methicillin-sensitive Staphylococcus aureus and Klebsiella oxytoca. Additionally, three normal skin controls from location-matched skin and age- matched cases (+/- 10 years) were selected to account for background gene expression. Slides were reviewed to confirm the histopathological diagnoses in selected cases (RKF). mRNA from FFPE skin biopsy samples was analyzed by Nanostring, and 594 inflammatory and immunologic signaling genes were profiled. Raw data were normalized to GUSB expression. T-tests were performed between infectious cases (necrotizing fasciitis and cellulitis) and non- infectious stasis dermatitis to identify candidate genes (p < 0.05). A heatmap was plotted with the resulting genes (Figure 2). Differentially upregulated genes in both necrotizing fasciitis and cellulitis included those with known roles in inflammation (CXCR2, IL6, IFI16, TNFRSF1B), transcription regulation (BCL3, MBP, SOCS3), and complement activation (C1QB, C1QA, CR1). Neutrophil recruitment by cytokines (IL1a, IL1b, IL6, TNF) and chemokines (CXCL1, CXCL2, CXCL5, CXCL8) is thought to be a key component of the immune response in SSTIs.4 We also identified differential upregulation of genes not previously associated with SSTIs METHODS RESULTS SKIN November 2022 Volume 6 Issue 6 (c) 2022 THE AUTHORS. Published by the National Society for Cutaneous Medicine. 536 Figure 1. Flowchart of case selection for small-scale pilot study. SKIN November 2022 Volume 6 Issue 6 (c) 2022 THE AUTHORS. Published by the National Society for Cutaneous Medicine. 534 Figure 2. A) Heatmap of differentially expressed genes between infectious (necrotizing fasciitis and cellulitis) vs non-infectious (stasis dermatitis) cases. B) List of differentially expressed genes with associated p-values. including S100A8, S100A9, MCL1, CD14, and LTF. S100A8 and S100A9 are calcium- binding proteins which form an antimicrobial complex secreted by neutrophils during inflammation.5 MCL1 is upregulated during phagocytosis to protect macrophages from apoptosis.6,7 CD14 recognizes lipopolysaccharides in bacterial membranes.8 LTF helps in antimicrobial defense by competing with microbes for iron.9 Here, we characterized transcriptomic signatures of severe and moderate SSTIs compared to a common clinical mimic, stasis DISCUSSION SKIN November 2022 Volume 6 Issue 6 (c) 2022 THE AUTHORS. Published by the National Society for Cutaneous Medicine. 535 dermatitis, identifying known and novel genes involved in inflammatory processes and infection. Although limited by its small scale, our study identified additional genes beyond what was previously identified in another transcriptomics study comparing bacterial cellulitis to normal-skin controls.10 Though limited in sample size, these data represent a pilot study in the understanding if SSTI gene regulation and support the utility of a prospective study analyzing patient immune signatures in conjunction with histology to differentiate SSTIs from clinical mimics. This work could inform innovative strategies that would fundamentally change SSTI management by providing rapid, point- of-care, objective diagnostics. Identification of tissue-specific and systemic biomarkers in patients with SSTIs may allow prognostic stratification to guide choice of therapeutics. Overall, improved diagnosis for SSTIs will help reduce unnecessary hospitalizations, antibiotic overuse, and healthcare costs and complications. Conflict of Interest Disclosures: None Funding: MGH Wellman Center for Photomedicine Early Discovery Fund Grant Corresponding Author: Ruth K. Foreman, MD, PhD Department of Pathology, Dermatopathology 55 Fruit St, Warren 8 Boston MA 02114 Phone: 617-726-8490 Email: rkforeman@partners.org References: 1. Hersh AL, Chambers HF, Maselli JH, Gonzales R. National trends in ambulatory visits and antibiotic prescribing for skin and soft-tissue infections. Arch Intern Med. 2008;168(14):1585-91. 2. Weng QY, Raff AB, Cohen JM, et al. Costs and Consequences Associated With Misdiagnosed Lower Extremity Cellulitis. JAMA Dermatol. 2017;153(2):141-6. 3. Hansen MB, Rasmussen LS, Svensson M, et al. Association between cytokine response, the LRINEC score and outcome in patients with necrotising soft tissue infection: a multicentre, prospective study. Sci Rep. 2017;7:42179. 4. Ley K, Laudanna C, Cybulsky MI, Nourshargh S. Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol. 2007;7(9):678-89. 5. Striz I, Trebichavsky I. Calprotectin - a pleiotropic molecule in acute and chronic inflammation. Physiol Res. 2004;53(3):245- 53. 6. Steimer DA, Boyd K, Takeuchi O, et al. Selective roles for antiapoptotic MCL-1 during granulocyte development and macrophage effector function. Blood. 2009;113(12):2805-15. 7. Koziel J, Maciag-Gudowska A, Mikolajczyk T, et al. Phagocytosis of Staphylococcus aureus by macrophages exerts cytoprotective effects manifested by the upregulation of antiapoptotic factors. PLoS One. 2009;4(4):e5210. 8. Zanoni I, Granucci F. Role of CD14 in host protection against infections and in metabolism regulation. Front Cell Infect Microbiol. 2013;3:32. 9. Jacobsen M, Repsilber D, Gutschmidt A, et al. Candidate biomarkers for discrimination between infection and disease caused by Mycobacterium tuberculosis. J Mol Med (Berl). 2007;85(6):613-21. 10. Pallin DJ, Bry L, Dwyer RC, et al. Toward an Objective Diagnostic Test for Bacterial Cellulitis. PLoS One. 2016;11(9):e0162947.