Ramirez‑Moral et al. Respir Res (2021) 22:230 https://doi.org/10.1186/s12931-021-01823-8

LETTER TO THE EDITOR Open Access Post‑treatment with the PPAR‑γ agonist pioglitazone inhibits infammation and bacterial growth during Klebsiella pneumonia Ivan Ramirez‑Moral1,2,5* , Bianca Lima Ferreira1,2,3, Alex F. de Vos1,2 and Tom van der Poll1,4

Abstract Agonists of peroxisome proliferator-activated receptor (PPAR)-γ have been suggested as potential adjuvant therapy in bacterial pneumonia because of their capacity to inhibit infammation and enhance bacterial clearance. Previous studies only assessed the efects of pretreatment with these compounds, thereby bearing less relevance for the clini‑ cal scenario. Moreover, PPAR-γ agonists have not been studied in pneumonia caused by Klebsiella pneumoniae, a com‑ mon human respiratory pathogen of which antibiotic treatment is hampered by increasing antimicrobial resistance. Here we show that administration of the PPAR-γ agonist pioglitazone 6 or 8 h after infection of mice with a highly virulent strain of Klebsiella pneumoniae via the airways results in reduced cytokine and myeloperoxidase levels in the lungs at 24 h after infection, as well as reduced bacterial growth in the lungs and decreased dissemination to distant organs at 42 h post-infection. These results suggest that pioglitazone may be an interesting agent in the treatment of Klebsiella pneumonia. Keywords: PPAR-γ, Pioglitazone, Pneumonia, Klebsiella

To the editor, studied as a potential adjunctive therapeutic strategy in Peroxisome proliferator-activated receptor (PPAR)-γ is bacterial infections [2]. a transcription factor belonging to the PPAR subfamily of Pneumonia is a major cause of morbidity and mortality nuclear hormone receptors [1]. While originally studied worldwide [3]. Treatment of pneumonia by antimicrobial for its role in lipid and glucose metabolism, by now the agents has become more difcult due to the emergence role of PPAR-γ in the regulation of immune responses of multidrug resistant pathogens. Antimicrobial resist- has been widely recognized [1, 2]. Endogenous ligands ance is especially problematic in infections caused by that can stimulate PPAR-γ activity include fatty acids gram-negative bacteria such as Pseudomonas (P.) aerugi- and eicosanoids. Considering the broad efects resulting nosa and microorganisms belonging to the Enterobacte- from PPAR-γ activation several synthetic agonists have riaceae family, such as Klebsiella (K.) pneumoniae [4, 5]. been developed, in particular thiazolidinediones such as Previous research on the potential of PPAR-γ agonists in pioglitazone, rosiglitazone, troglitazone, and ciglitazone gram-negative pneumonia has focused on P. aeruginosa [1]. Amongst others, these PPAR-γ agonists have been [6]. PPARγ activation enhanced phagocytosis and kill- ing of Pseudomonas by macrophages, and pretreatment with pioglitazone resulted in lower bacterial counts in *Correspondence: [email protected] lung homogenates of mice infected with this bacterium 5 Amsterdam UMC, Location Academic Medical Center, University via the airways [7]. With regard to gram-positive pneu- of Amsterdam, Meibergdreef 9, Room G2‑130, 1105 AZ Amsterdam, The monia, our group showed that pretreatment with ciglita- Netherlands Full list of author information is available at the end of the article zone lessens Streptococcus (S.) pneumoniae-induced lung

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infammation in mice by reducing bacterial outgrowth Prism 7.03. Te number of mice and the statistical tests and proinfammatory cytokine production [8]. Of note, used for each data set are described in the fgure legends. however, rosiglitazone treatment compromised bacte- A P value < 0.05 was considered statistically signifcant. rial clearance during post-infuenza super-infection by Two treatment strategies with pioglitazone (adminis- Staphylococcus aureus [9]. To the best of our knowledge tered by intraperitoneal injection) were evaluated. In a the efect of PPAR-γ stimulation during Klebsiella pneu- frst experiment mice were infected with K. pneumoniae monia in vivo has not been studied. In vitro, troglitazone via the airways and treated with a single dose of piogl- and rosiglitazone were shown to increase the ability of rat itazone (20 mg/g body weight; Cayman Chemical, Ann alveolar macrophages to phagocytose opsonized K. pneu- Arbor, MI, USA) or vehicle (10% dimethylsulfoxide in moniae [10]. In the present study we aimed to study the PBS) intraperitoneally 8 h later. At 24 h after infection efect of pioglitazone posttreatment, (i.e., administered all mice showed high bacterial loads in the lungs, which during an ongoing infection) in pneumonia caused by a were not diferent between treatment groups; likewise, highly virulent K. pneumoniae strain. bacterial burdens in distant body sites (blood, spleen and Pneumonia was induced in female C57BL/6 mice liver) were similar in pioglitazone and vehicle treated (Charles River, Maastricht, the Netherlands; 8–10 weeks mice (Fig. 1A). Pioglitazone strongly reduced TNF, IL-6, of age) by intranasal inoculation of K. pneumoniae IL-1β, CXCL2 and MPO concentrations in whole lung serotype 2 (43816; ATCC, Rockville, MD; 10­ 4 colony‐ homogenates (Fig. 1B). We next studied the longer-term forming units, CFU) as previously described [11–13]. consequences of pioglitazone treatment in an experiment Bacterial counts were determined in organ homogen- in which pioglitazone (10 mg/g) or vehicle was given ates and blood as described [11–13]. Tumor necrosis at 6 and 30 h after infection and efects were assessed factor (TNF)-α, interleukin (IL)-6, IL-1β, IL-10, C-X-C 42 h post-infection. In this setting, pioglitazone reduced motif ligand (CXCL)2 and myeloperoxidase (MPO) were bacterial loads in lungs and distant organs (Fig. 2A) measured in lung homogenates by ELISA according to and infammatory mediators and MPO levels in lungs, manufacturer’s instructions (R&D Systems, Minneapolis, although the diference between groups did not reach MN, USA). Statistical analysis was done using GraphPad statistical signifcance for IL-6 and IL-1β (Fig. 2B).

Fig. 1 Short-term efect of pioglitazone in Klebsiella-induced pneumonia. Mice were infected with K. pneumoniae via the airways and treated with a single dose of pioglitazone (20 mg/g body weight) or vehicle intraperitoneally (n 8 per group) 8 h later; measurements were done 24 h after = infection. A Bacterial loads in lungs, blood, spleen and liver. B TNF, IL-6, IL-1β, CXCL2 and MPO levels in lung homogenates. Graphs show median and every dot represents one individual mouse. P values were calculated using Mann–Whitney U tests. *P < 0.05, **P < 0.01, ***P < 0.001, ns not signifcant Ramirez‑Moral et al. Respir Res (2021) 22:230 Page 3 of 4

Fig. 2 Infuence of pioglitazone administration 42 h after induction of pneumonia. Mice were infected with K. pneumoniae via the airways and treated with pioglitazone (10 mg/g) or vehicle at 6 and 30 h after infection (n 8 per group); efects were assessed 42 h post-infection. A Bacterial = loads in lungs, blood, spleen and liver. B TNF, IL-6, IL-1β, CXCL2 and MPO levels in lung homogenates. Graphs show median and every dot represents one individual mouse. P values were calculated using Mann–Whitney U tests. *P < 0.05, **P < 0.01, ***P < 0.001, ns not signifcant

Te present results of pioglitazone mediated inhibition studies have reported on the effects of pre-treatment of infammatory responses in the lungs during Klebsiella with a thiazolidinedione in mouse models of Pseu- pneumonia is in agreement with the previously described domonas and pneumococcal pneumonia [6–8], and efects of ciglitazone during pneumonia caused by S. bacterial superinfection following influenza [9]. The pneumoniae-induced pneumonia [8]. PPAR-γ agonists present study is the first to describe the effect of a can exert anti-infammatory efects on macrophages, PPAR-γ agonist during pneumonia caused by K. pneu- including inhibition of proinfammatory cytokine pro- moniae, a common human respiratory pathogen for duction, via multiple mechanisms, encompassing inhibi- which adjunctive therapies are urgently needed [5]. tion of nuclear factor-ĸB and mitogen-activated protein Moreover, this investigation is the first to investi- kinases [2, 6]. Te role of PPAR-γ in the anti-infamma- gate the effect of administration of a PPAR-γ agonist tory potential of macrophages is further supported by the in mice with an ongoing lung infection, thereby more fnding that disruption of endogenous PPAR-γ in myeloid closely mimicking a clinical scenario. Our results cells impairs alternative macrophage activation [14], and argue for clinical evaluation of PPAR-γ agonists as is associated with a spontaneous low-grade infammatory immune modulating agents in the treatment of bacte- response in lungs of mice [15]. In pneumonia caused by rial pneumonia. S. pneumoniae mice with macrophage-specifc PPAR-γ Acknowledgements defciency displayed impaired antibacterial defense [15]. The authors thank Marieke ten Brink and Joost Daalhuisen (Center for Experi‑ Together these data suggest that the inhibitory efect of mental and Molecular Medicine, Amsterdam-UMC) for their technical support carrying the animal experiments. both endogenous PPAR-γ and synthetic PPAR-γ ago- nists like thiazolidinediones on infammation in the Authors’ contributions lungs drives its bacterial growth limiting efect during Conceptualization: IR-M and TvdP. Methodology: IR-M and BLF, Validation: IR-M. Formal analysis: IR-M. Investigation: IR-M, BLF and AFdV. Resources: TvdP. pneumonia. Writing—original draft: TvdP. Writing—review and editing: IR-M, BLF, AFdV PPAR-γ agonists have been extensively studied in and TvdP. Visualization: IR-M. Funding acquisition: TvdP. All authors read and the context of lung inflammation [6, 16]. Previous approved the fnal manuscript. Ramirez‑Moral et al. Respir Res (2021) 22:230 Page 4 of 4

Funding 5. Paczosa MK, Mecsas J. Klebsiella pneumoniae: going on the ofense with a Ivan Ramirez-Moral was funded by the Era-Net JPIAMR/ZonMW (Grant strong defense. Microbiol Mol Biol Rev. 2016;80:629–61. 50-52900-98-201); and Bianca Lima Ferreira was supported by FAPESP (Grant 6. de Carvalho MV, Gonçalves-De-albuquerque CF, Silva AR. PPAR gamma: 2019/02224-0). from defnition to molecular targets and therapy of lung diseases. Int J Mol Sci. 2021;22:805. Availability of data and materials 7. Bedi B, Yuan Z, Joo M, Zughaier SM, Goldberg JB, Arbiser JL, et al. Not applicable. Enhanced clearance of Pseudomonas aeruginosa by peroxisome proliferator-activated receptor gamma. Infect Immun. 2016;84:1975–85. 8. Stegenga ME, Florquin S, De Vos AF, Van Der Poll T. The thiazolidinedi‑ Declarations one ciglitazone reduces bacterial outgrowth and early infammation during Streptococcus pneumoniae pneumonia in mice. Crit Care Med. Ethics approval and consent to participate 2009;37:614–8. The Institutional Animal Care and Use Committee of the Academic Medical 9. Gopal R, Mendy A, Marinelli MA, Richwalls LJ, Seger PJ, Patel S, et al. Center approved all experiments. Peroxisome proliferator-activated receptor gamma (PPARγ) suppresses infammation and bacterial clearance during infuenza-bacterial super- Consent of publication infection. Viruses. 2019;11:505. Not applicable. 10. Aronof DM, Serezani CH, Carstens JK, Marshall T, Gangireddy SR, Peters- Golden M, et al. Stimulatory efects of peroxisome proliferator-activated Competing interests receptor-γ on Fcγ receptor-mediated phagocytosis by alveolar mac‑ The authors declare no commercial of fnancial confict of interest. rophages. PPAR Res. 2007;2017:52546. 11. Claushuis TAM, de Vos AF, Nieswandt B, Boon L, Roelofs JJTH, de Author details 1 Boer OJ, et al. Platelet glycoprotein VI aids in local immunity during Center of Experimental and Molecular Medicine, Amsterdam UMC, University pneumonia-derived sepsis caused by gram-negative bacteria. Blood. of Amsterdam, Amsterdam, The Netherlands. 2 Amsterdam Infection & Immu‑ 3 2018;131:864–76. nity Institute, Amsterdam, The Netherlands. Division of Infectious Diseases, 12. Ding C, Scicluna BP, Stroo I, Yang J, Roelofs JJ, de Boer OJ, et al. Prekal‑ Department of Medicine, Escola Paulista de Medicina, Universidade Federal de likrein inhibits innate immune signaling in the lung and impairs host Sao Paulo, Sao Paulo, Brazil. 4 Division of Infectious Diseases, Amsterdam UMC, 5 defense during pneumosepsis in mice. J Pathol. 2020;250:95–106. University of Amsterdam, Amsterdam, The Netherlands. Amsterdam UMC, 13. Ramirez-Moral I, Blok DC, Bernink JH, Garcia-Laorden MI, Florquin S, Boon Location Academic Medical Center, University of Amsterdam, Meibergdreef 9, L, et al. Interleukin-33 improves local immunity during gram-negative Room G2‑130, 1105 AZ Amsterdam, The Netherlands. pneumonia by a combined efect on neutrophils and infammatory monocytes. J Pathol. 2020;253:374–83. Received: 10 July 2021 Accepted: 9 August 2021 14. Odegaard JI, Ricardo-Gonzalez RR, Goforth MH, Morel CR, Subramanian V, Mukundan L, et al. Macrophage-specifc PPARγ controls alternative activation and improves insulin resistance. Nature. 2007;447:1116–20. 15. Gautier EL, Chow A, Spanbroek R, Marcelin G, Greter M, Jakubzick C, et al. Systemic analysis of PPARγ in mouse macrophage populations reveals References marked diversity in expression with critical roles in resolution of infam‑ 1. Ahmadian M, Suh JM, Hah N, Liddle C, Atkins AR, Downes M, et al. Pparγ mation and airway immunity. J Immunol. 2012;189:2614–24. signaling and metabolism: the good, the bad and the future. Nat Med 16. Nobs SP, Kopf M. PPAR-γ in innate and adaptive lung immunity. J Leukoc Nat Med. 2013;19:557–66. Biol. 2018;104:737–41. 2. Reddy AT, Lakshmi SP, Reddy RC. PPAR γ in bacterial infections: a friend or foe? PPAR Res. 2016;2016:7963540. 3. Torres A, Cilloniz C, Niederman MS, Menéndez R, Chalmers JD, Wunderink Publisher’s Note RG, et al. Pneumonia. Nat Rev Dis Prim. 2021;7:25. Springer Nature remains neutral with regard to jurisdictional claims in pub‑ 4. Enne VI, Personne Y, Grgic L, Gant V, Zumla A. Aetiology of hospital- lished maps and institutional afliations. acquired pneumonia and trends in antimicrobial resistance. Curr Opin Pulm Med. 2014;20:252–8.

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