Leukotriene B4 licenses inflammasome activation to enhance skin host defense Ana Carolina Guerta Salinaa,b,c,1, Stephanie L. Brandta,d,1, Nathan Klopfensteina,e, Amondrea Blackmana, Júlia Miranda Ribeiro Bazzanoa, Anderson Sá-Nunesa,f, Nicole Byers-Glossond, Claudia Brodskyng, Natalia Machado Tavaresg, Icaro Bonyek Santos Da Silvag, Alexandra I. Medeirosb, and C. Henrique Serezania,h,e,i,2 aDepartment of Medicine, Division of Infectious Diseases, Vanderbilt University Medical Center, Nashville, TN 37232; bDepartment of Biological Sciences, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Araraquara, São Paulo 14800-903, Brazil; cDepartment of Biochemistry and Immunology, Faculty of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, São Paulo 14049-900, Brazil; dDepartment of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202-3082; eVanderbilt Institute of Infection, Immunology and Inflammation, Vanderbilt University Medical Center, Nashville, TN 37232; fDepartment of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo 05508-000, SP, Brazil; gOswaldo Cruz Foundation, Gonçalo Moniz Institute, FIOCRUZ, Salvador 40296-710, Brazil; hDepartment of Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN 37232; and iVanderbilt Center for Immunobiology, Vanderbilt University Medical Center, Nashville, TN 37232 Edited by Jenny P.-Y. Ting, University of North Carolina at Chapel Hill, Chapel Hill, NC, and accepted by Editorial Board Member Carl F. Nathan October 15, 2020 (received for review February 12, 2020) The initial production of inflammatory mediators dictates host de- DAMPs and further amplifies the inflammatory response. In- fense as well as tissue injury. Inflammasome activation is a constit- creased production of IL-1β and IL-18, along with DAMPs gener- uent of the inflammatory response by recognizing pathogen and ation, have been heavily implicated in the pathogenesis of a myriad host-derived products and eliciting the production of IL-1β and IL-18 in of inflammatory diseases and facilitates antimicrobial activities “ addition to inducing a type of inflammatory cell death termed pyrop- (1, 6). ” tosis. Leukotriene B4 (LTB4) is a lipid mediator produced quickly (sec- Staphylococcus aureus skin infection is controlled by the syn- onds to minutes) by phagocytes and induces chemotaxis, increases chronized actions of structural cells (keratinocytes) and skin cytokine/chemokine production, and enhances antimicrobial effector β phagocytes. Inflammasome-dependent IL-1 production is re- IMMUNOLOGY AND INFLAMMATION functions. Whether LTB directly activates the inflammasome remains 4 quired for neutrophil recruitment, abscess formation, and bacte- to be determined. Our data show that endogenously produced LTB4 is required for the expression of pro-IL-1β and enhances inflammasome rial clearance (7, 8). The mechanisms underlying inflammasome activation and IL-1β production during skin infection is not well assembly in vivo and in vitro. Furthermore, LTB4-mediated Bruton’s tyrosine kinase (BTK) activation is required for inflammasome assem- understood. blyinvivoaswellforIL-1β–enhanced skin host defense. Together, LTB4 is a bioactive lipid mediator that is quickly produced by these data unveil a new role for LTB4 in enhancing the expression and phagocytes, such as macrophages and neutrophils. LTB4 syn- assembly of inflammasome components and suggest that while thesis involves several rate-limiting steps that include activation blocking LTB4 actions could be a promising therapeutic strategy to of phospholipase A2 (PLA2) and arachidonic acid (AA) release prevent inflammasome-mediated diseases, exogenous LTB4 can be used as an adjuvant to boost inflammasome-dependent host defense. Significance inflammasome | leukotriene | skin | innate immunity Production of IL-1β is an essential component of the inflam- matory response and host defense. IL-1β secretion is depen- pon infection, a fast and highly synchronized inflammatory dent on the activation of an intracellular platform termed Uresponse is mounted to restrict microbial growth and eventu- inflammasome. The initial inflammatory signals that drive ally eliminate the pathogen. Therefore, studies focusing on early inflammasome activation remains elusive. Here, we show that events could unmask new players involved in the generation and the bioactive lipid leukotriene B4 enhances both transcriptional magnitude of host defense. Inflammasomes are multiprotein in- and posttranscriptional programs that activate the inflamma- tracellular platforms that detect both pathogen and host-derived some in vivo and in vitro. We identified critical signaling pro- products and induce the inflammatory response. The proteins that grams required for inflammasome assembly, IL-1β secretion, form the inflammasome consist of upstream sensors that belong to and its consequences in skin host defense. Our data also sug- the nucleotide-binding oligomerization domain-like receptor (NLR) gest that the prevention of LTB4 actions might be an important family, the adaptor protein apoptosis-associated speck-like protein therapeutic strategy to prevent IL-1β–dependent inflammatory containing CARD (ASC), and the downstream effector caspase-1. diseases by inhibiting both first and second signals necessary There are different NLR proteins, including NLRP1, 2, 3, 6, 7, and for inflammasome activation. NLRC4, and absent in melanoma 2 (AIM2) (1, 2). The different NLRs recognize both distinct and overlapping stressors to elicit the Author contributions: A.C.G.S., S.L.B., A.S.-N., C.B., A.I.M., and C.H.S. designed research; A.C.G.S., S.L.B., N.K., A.B., J.M.R.B., A.S.-N., N.B.-G., N.M.T., and I.B.S.D.S. performed re- maturation and secretion of the inflammatory cytokines IL-1β and search; A.C.G.S., S.L.B., A.S.-N., C.B., A.I.M., and C.H.S. analyzed data; and A.C.G.S. and IL-18. Upon cell stimulation with either pathogen- or damage- C.H.S. wrote the paper. associated molecular patterns (PAMPs and DAMPs, respectively), The authors declare no competing interest. the inflammasome is activated in two sequential steps: 1) tran- This article is a PNAS Direct Submission. J.P.-Y.T. is a guest editor invited by the scription of the long forms (procytokines) of IL-1β and IL-18; and Editorial Board. 2) the assembly of the inflammasome complex ASC/NLRP and Published under the PNAS license. procaspase-1, followed by autocatalytic cleavage of caspase-1, and 1A.C.G.S. and S.L.B. contributed equally to this work. processing and secretion of mature IL-1β and IL-18, as well as the 2To whom correspondence may be addressed. Email: [email protected]. production of lipid mediators, such as prostaglandins and leuko- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ triene B4 (LTB4)(3–5). The secretion of these cytokines is followed doi:10.1073/pnas.2002732117/-/DCSupplemental. by a form of programmed cell death called pyroptosis that releases www.pnas.org/cgi/doi/10.1073/pnas.2002732117 PNAS Latest Articles | 1of9 Downloaded by guest on September 29, 2021 from phospholipids in cellular membranes. Activation of L-glutamine, 100 U/mL of penicillin G, and 100 mg/mL streptomycin (all from 5-lipoxygenase (5-LO) and 5-LO activation protein together ThermoFisher Scientific) at 37 °C and 5% CO2. PMNs were cultured with 100 ng/mL lipopolysaccharide (LPS) (Sigma-Aldrich, St. Louis, MO) for 2 h, metabolize AA to LTA4, which is converted to LTB4 by LTA4 washed with PBS, and challenged with 10 nM LTB4. Culture supernatants were hydrolase (LTA4H) (9). LTB4 binds to two different G protein- harvested to measure IL-1β release and active caspase-1 by enzyme-linked coupled receptors (GPCR): a high-affinity receptor (BLT1) and immunosorbent assay (ELISA) as described below. a low-affinity receptor (BLT2) (10). LTB4 via BLT1 enhances inflammatory response by increasing phagocyte chemotaxis and Inflammasome Activation. Macrophages and neutrophils were challenged activating transcription factors required to produce inflamma- with 100 ng/mL LPS for 3 h, followed by treatment with nigericin 1 μM, μ μ tory cytokines. We and others have shown that LTB4 enhances monosodium urate crystal (MSU; 10 M), flagellin 20 g/mL, and Poly μ pattern recognition response (PRR)-mediated responses by in- (dA:dT)/LyoVec 2 g/mL for 1 h. To assess the role of LTB4 in amplifying μ creasing the expression of the TIR adaptor MyD88 and the inflammasome activity, cells were pretreated with 10 nM LTB4 or 10 M BLT1 transcription factors NF-κB, AP-1, and PU.1 (11–14). Also, antagonist U-75302 5 min before the addition of the inflammasome acti- LTB is required for the control of a myriad of pathogens once vators. The direct effect of LTB4 in inflammasome activation was studied in 4 cells treated with LPS as above, followed by LTB for 3 h. this mediator enhances microbial clearance by controlling the 4 generation of reactive oxygen and nitrogen species and antimi- S. aureus – Methicillin-Resistant Skin Infection and Topical Ointment Treatment. crobial peptides in vivo (15 18). We have also shown that the Mice were infected with methicillin-resistant S. aureus (MRSA) USA300 LAC treatment of mice with a topical ointment containing LTB4 en- strain (∼5 × 106 colony-forming units [cfu]) subcutaneously (s.c.) in 50 μLof hances S. aureus clearance from the skin, production of inflam- PBS as previously shown (25). Lesion and abscess sizes were monitored and matory mediators, and abscess formation (19). Whether LTB4 determined by affected areas calculated using a standard formula for the enhances skin host defense directly or indirectly increases the area: (A = [π/2] × l × w) (26). The final concentrations of the ointments per – × −6 production of proinflammatory mediators remains to be deter- animal were as follows: LTB4 (33.7 ng 3.37 10 %), U-75302 (0.001%), all in 1 g of petroleum jelly (vehicle control). The ointment treatments were mined. Also, whether LTB4 influences inflammasome activation applied to the infected area with a clean cotton swab. Mice were treated is poorly understood.