Role of glutathione metabolism in host defense PNAS PLUS against Borrelia burgdorferi infection Mariska Kerstholta,b, Hedwig Vrijmoetha,b, Ekta Lachmandasa,b, Marije Oostinga,b, Mihaela Lupsec, Mirela Flontac, Charles A. Dinarelloa,b,d,1, Mihai G. Neteaa,b,e, and Leo A. B. Joostena,b,1 aDepartment of Internal Medicine, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands; bRadboud Center for Infectious Diseases, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands; cDepartment of Infectious Diseases, University of Medicine and Pharmacy “Iuliu Hatieganu,” 400349 Cluj-Napoca, Romania; dDepartment of Medicine, University of Colorado Denver, Aurora, CO 80045; and eHuman Genomics Laboratory, Craiova University of Medicine and Pharmacy, 200349 Craiova, Romania Contributed by Charles A. Dinarello, January 18, 2018 (sent for review December 7, 2017; reviewed by Pietro Ghezzi and Georg Schett) Pathogen-induced changes in host cell metabolism are known to central glucose metabolism in host mononuclear cells which was be important for the immune response. In this study, we in- crucial for cytokine production (13). vestigated how infection with the Lyme disease-causing bacterium In the present study, we aimed to further explore the metabolic Borrelia burgdorferi (Bb) affects host metabolic pathways and pathways induced by Bb and analyze their role in immune cell how these metabolic pathways may impact host defense. First, function. To achieve this, we performed metabolomic analysis metabolome analysis was performed on human primary mono- of primary human monocytes stimulated with Bb or other in- cytes from healthy volunteers, stimulated for 24 h with Bb at flammatory stimuli. Identified pathways were then further validated low multiplicity of infection (MOI). Pathway analysis indicated that using in vitro intervention experiments to elucidate their role in glutathione (GSH) metabolism was the pathway most significantly the inflammatory response. Lastly, we examined the relevant affected by Bb. Specifically, intracellular levels of GSH increased on metabolites in serum samples from acute Lyme disease patients. average 10-fold in response to Bb exposure. Furthermore, these changes were found to be specific, as they were not seen during Results stimulation with other pathogens. Next, metabolome analysis was Primary Human Monocytes Exposed to Bb Display Altered Glutathione performed on serum samples from patients with early-onset Lyme Metabolism. To determine which metabolic pathways were affected INFLAMMATION IMMUNOLOGY AND disease in comparison with patients with other infections. Sup- by Bb infection, metabolome analysis was performed on primary porting the in vitro analysis, we identified a cluster of GSH-related human monocytes stimulated with Bb or medium control for 24 h. metabolites, the γ-glutamyl amino acids, specifically altered in pa- Pathway analysis was performed to identify specific metabolic tients with Lyme disease, and not in other infections. Lastly, we performed in vitro experiments to validate the role for GSH metab- pathways altered by Bb exposure (Table S1). As seen in Fig. 1A,the olism in host response against Bb. We found that the GSH pathway pathways most significantly affected by Bb were glutathione (GSH) is essential for Bb-induced cytokine production and identified glu- metabolism, arachidonic acid metabolism, and pyrimidine metab- tathionylation as a potential mediating mechanism. Taken together, olism. When analyzing individual metabolites, eight compounds these data indicate a central role for the GSH pathway in the host related to GSH metabolism were found among the top 25 most response to Bb. GSH metabolism and glutathionylation may there- fore be important factors in the pathogenesis of Lyme disease and Significance potentially other inflammatory diseases as well. Inflammation plays a crucial role in the pathogenesis of Lyme Lyme disease | B. burgdorferi | cell metabolism | glutathione disease, caused by the spirochete Borrelia burgdorferi. In- tracellular metabolism is increasingly being recognized as a yme disease, caused by Borrelia burgdorferi (Bb) sensu lato,is major determinant of inflammation. In this study, we in- Lthe most common vector-borne disease in the Northern vestigated how B. burgdorferi affects host cell metabolism by hemisphere (1, 2), transmitted by ticks. Lyme disease most often analyzing the intracellular metabolome in vitro, as well as the presents locally with a migrating skin rash called erythema circulating metabolome in patients with early-onset Lyme dis- migrans (EM) but, if left untreated, can give rise to inflammatory ease. We identify glutathione metabolism as the most impor- complications in the joints (3), heart (4), or nervous system (5). tant target of B. burgdorferi infection and discover that this In most cases, Lyme disease can be effectively treated by antibi- pathway is essential for cytokine production, likely through otics, yet a small percentage of patients experience persisting glutathionylation. These findings not only provide more in- sight into the pathogenesis of Lyme disease but also underline symptoms even after extensive antibiotic treatment (6, 7). how host–pathogen interactions in metabolism can play crucial Interestingly, Bb is not known to produce toxic factors (8). The roles in host defense against pathogens. majority of Lyme disease symptoms are therefore attributed to ’ the host s immune response against the pathogen. In addition, it Author contributions: M.K., C.A.D., M.G.N., and L.A.B.J. designed research; M.K. per- is hypothesized that persistent symptoms after treatment are not formed research; H.V., E.L., M.O., M.L., M.F., M.G.N., and L.A.B.J. contributed new re- due to continuous infection, but rather due to an aberrant in- agents/analytic tools; M.K., H.V., E.L., M.O., and C.A.D. analyzed data; M.K. and C.A.D. flammatory response (7, 9, 10). Together, this suggests a crucial wrote the paper; and M.G.N. supervised research. role for the host immune response in the initiation and outcome Reviewers: P.G., Brighton and Sussex Medical School; and G.S., University of Erlangen– Nuremberg. of the infection. The authors declare no conflict of interest. An upcoming topic in the study of the immune system is immunometabolism, which investigates the impact of cellular Published under the PNAS license. metabolism on immune cell function. This is of particular in- Data deposition: The metabolome data reported in this paper have been deposited in the MetaboLights database, https://www.ebi.ac.uk/metabolights/ (accession no. MTBLS625). terest in the case of Bb as the spirochete is known to have very 1To whom correspondence may be addressed. Email: [email protected] or limited metabolic capabilities (11, 12). This might cause the [email protected]. spirochete to induce specific changes in host cell metabolism. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Supporting this, we recently showed that Bb induces a switch in 1073/pnas.1720833115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1720833115 PNAS Latest Articles | 1of9 Downloaded by guest on September 24, 2021 AC Glutathione metabolism GSH 6 s 10 el ** Arachidonic acid ev Pyrimidine metabolism l 1 metabolism H 5 S 0.1 eG iv 4 0.01 lat Re 0.001 3 Bb -log(p) D RPMI GSSG s 10 * 12 SG level S 1 0.0 0.2 0.4 0.6 ative G Pathway impact Rel 0.1 b B B Bb RPMI RPMI N6,N6,N6-Trimethyl-L-lysine Cysteineglutathione disulfide # N-Acetylputrescine Orotidine Eicosapentaenoic acid Gamma Glutamylglutamic acid 11 Homocysteine 4 Cysteinylglycine 12 Putrescine L-Lactic acid Maltotriose Maltotetraose Erythronic acid L-Glutamic acid Pyridoxal 5'-phosphate 13 Phosphorylcholine PE(16:0/18:2(9Z,12Z)) Gamma-Glutamylcysteine 8 Uridine diphosphategalactose Niacinamide Glutathione 9 Acetylcysteine 6 Quinolinic acid 4-Guanidinobutanoic acid Adenosine monophosphate Fig. 1. Metabolome analysis of primary monocytes stimulated with Bb versus RPMI. (A) Scatter plot of KEGG metabolic pathways in primary human monocytes (n = 5) affected by Bb stimulation, showing log P value of the enrichment analysis (y axis and visualized by node color) and pathway impact, taking into account the importance of the affected metabolites within a pathway (x axis and visualized by node radius; range 0 to 1, where 1 is maximal impact). (B) Heat map depicting the top 25 most significantly affected metabolites after Bb stimulation, where red indicates an increase and blue indicates a decrease. Numbers represent references to Fig. S1. #, GSH derivative, formed upon oxidative stress of GSH. (C and D)Rawdataof(C) reduced glutathione (GSH) and (D)oxidized glutathione (GSSG) levels in primary monocytes stimulated with Bb or control (RPMI). Box plot indicates median ± min/max values. Max *P > 0.05, **P > 0.01. significantly affected metabolites (Fig. 1B, with references to Fig. lism in Bb infection (13), lactate was among the most significantly S1). Most noteworthy, Bb stimulation induced a dramatic increase affected metabolites. in reduced GSH levels (Fig. 1C), while only modestly increasing Next, we compared fold changes (FCs) relative to medium oxidized glutathione (GSSG) (Fig. 1D). This indicates a shift in the control (RPMI) in metabolite levels in monocytes exposed to Bb GSH/GSSG ratio, suggesting a more antioxidative state. Other with cells exposed to the TLR4 ligand LPS and the TLR2 ligand metabolites significantly increased by Bb stimulation included Pam3Cys. Fig. 2A