3718 Diabetes Volume 65, December 2016 Naiara N. Dejani,1,2,3 Stephanie L. Brandt,1 Annie Piñeros,1,2 Nicole L. Glosson-Byers,1 Sue Wang,1 Young Min Son,1 Alexandra I. Medeiros,2,3 and C. Henrique Serezani1 Topical Prostaglandin E Analog Restores Defective Dendritic Cell– Mediated Th17 Host Defense Against Methicillin-Resistant Staphylococcus Aureus in the Skin of Diabetic Mice Diabetes 2016;65:3718–3729 | DOI: 10.2337/db16-0565 People with diabetes are more prone to Staphylococcus Diabetes is a clinical syndrome associated with deficiency in aureus skin infection than healthy individuals. Control of insulin secretion or action. As a consequence, diabetes causes S. aureus infection depends on dendritic cell (DC)–induced hyperglycemia, which has been associated with undermined T-helper 17 (Th17)–mediated neutrophil recruitment host defense and increased susceptibility to localized and and bacterial clearance. DC ingestion of infected apo- systemic infections (1). Skin and soft tissues are sites of prev- ptotic cells (IACs) drive prostaglandin E2 (PGE2) secretion alent infections primarily caused by Staphylococcus aureus in peo- to generate Th17 cells. We speculated that hyperglyce- ple with diabetes (2,3). Methicillin-resistant S. aureus (MRSA) is mia inhibits skin DC migration to the lymph nodes and a frequent causative pathogen of complicated skin and soft impairs the Th17 differentiation that accounts for poor tissue infections in patients with diabetes (2) that can be dif- skin host defense in diabetic mice. Diabetic mice showed ficult to treat and often require hospitalization and surgery (4). increased skin lesion size and bacterial load and de- COMPLICATIONS Innate immune recognition of S. aureus promotes proin- creased PGE secretion and Th17 cells compared with 2 flammatory signals that lead to neutrophil recruitment and nondiabetic mice after methicillin-resistant S. aureus abscess formation, a hallmark of S. aureus infection (5). Ex- (MRSA) infection. Bone marrow–derived DCs (BMDCs) tensive neutrophil apoptosis is also a hallmark of S. aureus cultured in high glucose (25 mmol/L) exhibited decreased infections (5). An additional complication is that neutrophils Ptges mRNA expression, PGE2 production, lower CCR7- dependent DC migration, and diminished maturation after from patients with diabetes have defective antimicrobial ef- recognition of MRSA-IACs than BMDCs cultured in low fector functions (6,7). glucose (5 mmol/L). Similar events were observed in DCs Clearance of apoptotic cells (efferocytosis) has long been fl from diabetic mice infected with MRSA. Topical treat- associated with resolution of in ammation (8). However, ment of diabetic mice with the PGE analog misoprostol efferocytosis of infected apoptotic cells (IACs) has been pro- improved host defense against MRSA skin infection by posed to be an innate antimicrobial mechanism that results restoring DC migration to draining lymph nodes, Th17 in pathogen destruction (9) and comprises a critical link to differentiation, and increased antimicrobial peptide ex- adaptive immune responses (10). Considering the increased pression. These findings identify a novel mechanism neutrophil recruitment and apoptosis at sites of MRSA in- involved in poor skin host defense in diabetes and pro- fection, we speculated that the uptake of infected apoptotic pose a targeted strategy to restore skin host defense in neutrophils by dendritic cells (DCs) dictate the type of host diabetes. defense produced against S. aureus in skin infections. 1Department of Microbiology and Immunology, Indiana University School of Med- This article contains Supplementary Data online at http://diabetes icine, Indianapolis, IN .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-0565/-/DC1. 2 University of São Paulo, Ribeirão Preto, Brazil C.H.S. is currently affiliated with the Department of Medicine, Vanderbilt 3 Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, University School of Medicine, Nashville, TN. Universidade Estadual Paulista “Júlio de Mesquita Filho,” Araraquara, Brazil © 2016 by the American Diabetes Association. Readers may use this article as Corresponding author: C. Henrique Serezani, [email protected]. long as the work is properly cited, the use is educational and not for profit, and the Received 6 May 2016 and accepted 30 August 2016. work is not altered. More information is available at http://www.diabetesjournals .org/content/license. diabetes.diabetesjournals.org Dejani and Associates 3719 In the skin, DCs are prompted to respond to intracellular RESEARCH DESIGN AND METHODS and extracellular modifications that induce a rapid change Animals in their function and phenotype accompanied by their Wild-type C57BL/6 mice (8–12 weeks old) were obtained migration into secondary lymphoid tissue (11). During from The Jackson Laboratory. Mice were maintained ac- this process, DCs require maturation, a process associ- cording to National Institutes of Health guidelines for the ated with upregulation of class II MHC and costimulatory use of experimental animals in the Laboratory Animal molecules (CD80, CD86, CD40) (12) and consequent Resource Center at Indiana University School of Medicine migration to lymph nodes in a CC-chemokine receptor 7 (Indianapolis, IN). Experiments were performed under a – (CCR7) dependent response to its ligands CCL19 and protocol approved by the Indiana University School of CCL21 (13). In addition, the lipid mediator prostaglan- Medicine Animal Care and Use Committee. din E2 (PGE2) has been shown to be a key factor for DC maturation and migration through CCR7 (14). More- Induction of Diabetes fi over, PGE2 is required to initiate skin immunity by in- Diabetes was induced by ve daily intraperitoneal injec- creasing maturation and migration of Langerhans cells, tions of low-dose (40 mg/kg) streptozotocin as previously a subtype of skin-resident DCs (14). described (21). Mice with blood glucose .300 mg/dL were PGE2 synthesis involves activation of phospholipase considered diabetic. Experiments were performed using A2 family members that mobilize arachidonic acid from mice at 30 days after diabetes onset. cellular membranes. Cyclooxygenase (COX) 1 and 2 then MRSA Skin Infection and Misoprostol Topical convert arachidonic acid into prostaglandin H2 (PGH2), Treatment fi which is further metabolized by speci c synthases to gen- Control and diabetic mice were anesthetized (ketamine/ fi erate speci c prostanoids. Microsomal PGE synthase 1 is xylazine), their backs were shaved, and MRSA USA300 fl induced during the in ammatory response that acts mainly LAC strain (5 3 106 colony-forming units [cfu]) in 50 mL downstream of COX-2 to convert PGH2 to PGE2. The levels PBS was injected subcutaneously. Lesion and abscess size of PGE2 are regulated by the local balance between the were monitored daily and determined by affected area – COX-2 driven synthesis and 15-hydroxyprostaglandin calculated by the standard equation for area [area = – dehydrogenase mediated degradation of PGE2 or the actions (p/2) 3 length 3 width] (22). Mice were topically treated of prostaglandin transporters (15). PGE2 acts through four at the site of infection twice a day for 7 days with 0.03% – – G-protein coupled receptors, designated EP1 EP4, which misoprostol (prepared by emulsifying 150 mg misoprostol display different tissue distributions and deliver distinct in 0.5 g petroleum gel or vehicle control). intracellular signals (16). Moreover, efferocytosis has been In Vivo DC Migration shown to trigger PGE2 production (17), although the role of PGE2 on DC functions during efferocytosis remains to Control and diabetic mice were injected subcutaneously 6 be determined. with 5 3 10 cfu of MRSA plus 50 mg of CellTrace FarRed Although efferocytosis leads to secretion of anti- DDAO-SE (Invitrogen) as suggested by the manufacturer. inflammatory mediators that promote expansion of regula- Brachial lymph nodes were harvested 36 or 48 h postin- + + tory Foxp3 T cells, efferocytosis of Escherichia coli–infected fection. Migrating DCs were identified as FarRed CD11c neutrophils by DCs induces interleukin-6 (IL-6), IL-23, and cells detected by flow cytometry. b b transforming growth factor- (TGF- ) production, which Skin Biopsy Specimens and Bacterial Load triggers T-helper 17 (Th17) differentiation (10,18). Punch biopsy specimens (8 mm) from noninfected or in- Th17 cells are fundamental players in host defense fected skin were harvested at different time points and against S. aureus infection (19) by activating keratinocytes used for determining bacterial counts, cytokine production, and resulting in the induction of neutrophil chemoattrac- RNA extraction, and cell isolation (23). For bacterial counts, tants and antimicrobial peptides (5). Patients with defec- skin biopsy specimens were collected at day 7 postinfection, tive Th17 responses exhibit increased susceptibility to processed, and homogenized in tryptic soy broth media, and S. aureus skin infections (5); however, whether patients serial dilutions were plated on tryptic soy broth agar. Colo- with diabetes also exhibit defective Th17 cells during nies were counted after incubation overnight at 37°C. S. aureus skin infection is unknown. Reduced frequencies of IL-17–producing memory CD4+ T cells associated with MRSA-IACs elevated glucose and increased glycated hemoglobin A1c DMSO-differentiated neutrophil-like cells were incubated have been observed in response to Streptococcus pneumo- with green fluorescent protein-MRSA (multiplicity of in-