Published August 28, 2017, doi:10.4049/jimmunol.1602092 The Journal of Immunology Dual-Specificity Phosphatase 3 Deletion Protects Female, but Not Male, Mice from Endotoxemia-Induced and Polymicrobial-Induced Septic Shock Maud M. Vandereyken,*,1 Pratibha Singh,*,1 Caroline P. Wathieu,* Sophie Jacques,* Tinatin Zurashvilli,* Lien Dejager,†,‡ Mathieu Amand,* Lucia Musumeci,* Maneesh Singh,* Michel P. Moutschen,* Claude R. F. Libert,†,‡ and Souad Rahmouni* Dual-specificity phosphatase 3 (DUSP3) is a small phosphatase with poorly known physiological functions and for which only a few substrates are known. Using knockout mice, we recently reported that DUSP3 deficiency confers resistance to endotoxin- and polymicrobial-induced septic shock. We showed that this protection was macrophage dependent. In this study, we further investigated the role of DUSP3 in sepsis tolerance and showed that the resistance is sex dependent. Using adoptive-transfer experiments and ovariectomized mice, we highlighted the role of female sex hormones in the phenotype. Indeed, in ovariec- tomized females and in male mice, the dominance of M2-like macrophages observed in DUSP32/2 female mice was reduced, suggesting a role for this cell subset in sepsis tolerance. At the molecular level, DUSP3 deletion was associated with estrogen- dependent decreased phosphorylation of ERK1/2 and Akt in peritoneal macrophages stimulated ex vivo by LPS. Our results demonstrate that estrogens may modulate M2-like responses during endotoxemia in a DUSP3-dependent manner. The Journal of Immunology, 2017, 199: 000–000. epsis and septic shock are complex clinical syndromes that ally, death (4). Sepsis occurrence and outcome depend on arise when the local body response to pathogens becomes pathogen characteristics, as well as on risk factors, such as age S systemic and injures its own tissues and organs (1). When or sex (1). Indeed, women are better protected against infection infection occurs, bacterial components, such as LPS, are recog- and sepsis compared with men. Women younger than 50 y nized by the host, and inflammation is initiated. The TLR4 show a lower incidence of severe sepsis and a better survival pathway is activated and triggers the release of cytokines, che- compared with age-matched men. This may be explained by mokines, and NO (2, 3). Systemic release of proinflammatory the influence of female sex hormones on the immune system cytokines causes large-scale cellular and tissue injuries, leading to responses (5). microvascular disruption, severe organ dysfunction, and, eventu- Dual-specificity phosphatase 3 (DUSP3), or Vaccinia H1-related, is an atypical dual-specificity phosphatase of 21 kDa. The phos- phatase contains one catalytic domain but lacks a binding domain *Immunology and Infectious Disease Unit, GIGA-Research, University of Lie`ge, B-4000 Lie`ge, Belgium; †Inflammation Research Center, VIB, B-9052 Ghent, Belgium; (6). DUSP3’s broader catalytic site allows the protein to dephos- and ‡Department of Biomedical Molecular Biology, Ghent University, B-9000 Ghent, phorylate phospho-Tyr and phospho-Thr residues (7). The MAPK Belgium ERK1/2 and JNK were the first reported DUSP3 substrates (8–10). 1 M.M.V. and P.S. contributed equally to this work. Other substrates, such as the EGFR and ErbB2 tyrosine receptors ORCIDs: 0000-0003-1874-3657 (C.P.W.); 0000-0002-4488-2581 (T.Z.); 0000-0002- (11) and STAT5 transcription factor (12), were also reported. 6942-5299 (L.M.); 0000-0003-0956-0242 (S.R.). DUSP3’s physiological functions began to be elucidated as a result Received for publication December 13, 2016. Accepted for publication August 1, 2017. of the knockout (KO) mouse that we have generated. We have This work was supported by the Fonds Le´on Fredericq and Centre anticancereux pre`s previously reported that DUSP3 plays an important role in platelets de l’Universite´ de Lie`ge and by the Fond National de la Recherche Scientifique biology in monocytes, macrophages, and endothelial cells (13–15). (FNRS; to S.R.). This work was also supported by the Agency for Innovation of In platelets, DUSP3 plays an important role in arterial thrombosis Science and Technology in Flanders, the Research Council of Ghent University (Geconcerteerde Onderzoeksacties Program), the Research Foundation Flanders, Eu- and platelet activation through GPVI and CLEC-2 signaling path- ropean Commission 7th Framework Programme COST Action BM1402, and Belgian ways (14). DUSP3 also plays an important role in endothelial cells Science Policy Program Interuniversity Attraction Poles Grant IAP-VI-18 (to and angiogenesis and seems to act as a proangiogenic factor (16). C.R.F.L.). M.M.V. and M.A. are FNRS-Te´le´vie Ph.D. fellows. Surprisingly, this function was not correlated with reduced tumor or S.R. designed the research; M.M.V., C.P.W., P.S., S.J., T.Z., M.A., L.M., M.S., and L.D. performed experiments; S.R., M.P.M., and C.R.F.L. analyzed data; and S.R. and metastatic growth. Indeed, in an experimental metastasis model M.M.V. wrote the manuscript. using Lewis lung carcinoma cells, we found instead that DUSP3 2/2 Address correspondence and reprint requests to Dr. Souad Rahmouni, University of plays an antitumor role, because DUSP3 mice were more sen- Lie`ge, Immunology and Infectious Diseases Research Unit, GIGA B34, 1, Avenue de sitive to Lewis lung carcinoma cell metastatic growth compared l’Hoˆpital, Lie`ge B-4000, Belgium. E-mail address: [email protected] with wild-type (WT) littermates. This enhanced tumor growth in Abbreviations used in this article: Arg1, arginase 1; BM, bone marrow; CLP, cecal 2/2 ligation and puncture; DUSP3, dual-specificity phosphatase 3; iNOS, inducible NO DUSP3 mice was associated with greater recruitment of M2-like synthase; KO, knockout; b2M, b2-microglobulin; MSD, Meso Scale Discovery; macrophages (M. Vandereyken, E. Van Overmeire, M. Amand, OVX, ovariectomized; PM, peritoneal macrophage; RT, room temperature; TSC, N. Rocks, C. Delierneux, P. Singh, M. Singh, C. Wathieu, T. Zurashvilli, tuberous sclerosis protein; WT, wild-type. N.E. Sounni, M. Moutschen, C. Gilles, C. Oury, D. Cataldo, J.A. Van Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 Ginderachter, and S. Rahmouni, unpublished data). We and others www.jimmunol.org/cgi/doi/10.4049/jimmunol.1602092 2 THE ROLE OF SEX-DEPENDENT DUSP3 IN SEPSIS showed that DUSP3 was downregulated in some human cancers Female ovariectomy and in vivo estrogen complementation and upregulated in others (reviewed in Refs. 16, 17). Further studies Four-week-old females were anesthetized using ketamine/xylazine (150 and are required to better understand the role of this phosphatase in 20 mg/kg). A vertical incision of 2–3 cm was performed in the middle of cancer biology. the back. One centimeter lateral of the midline, another incision of 2– DUSP3 also plays an important role in immune cell functions. 3 mm was performed in the fascia. Adipose tissue surrounding the ovary In T cells, DUSP3 can be activated by ZAP-70 tyrosine kinase was pulled out, and the ovary was removed after clamping. The same operation was performed for the contralateral ovary. The incision in fascia after TCR triggering (18). This activation, through tyrosine was closed with stitches, and the skin incision was closed with clips. phosphorylation of DUSP3, allows the targeting of the MAPK Sham-operated mice were used as a control. All of the above procedures ERK1/2 and the activation of its downstream signaling pathway. were applied to these mice, with the exception of the removal of ovaries. Moreover, in Jurkat leukemia T cells, DUSP3 targets ERK and For in vivo estrogen complementation, s.c. implants for controlled release of 17b-estradiol (1.5 mg/d; Belma Technologies) were applied to OVX JNK but not p38. Together, these data suggest that DUSP3 mice 2 wk after surgery, and mice were sacrificed 3 wk later. controls T cell physiological functions, at least in part, through the MAPKs ERK and JNK (8). In innate immune cells, we re- Abs and reagents cently showed that DUSP3 is the most highly expressed atypical The following materials were from Cell Signaling Technology: anti– dual-specificity phosphatase in human monocytes. This was also phospho-Akt (Ser473), anti-Akt, anti–phospho-ERK1/2 (Thr202/Tyr204), true in mice (15). These findings suggested to us that DUSP3 anti-ERK, anti–phospho-PI3K p85 (Tyr458)/p55 (Tyr199), anti-PI3K p85, 21/9 could play an important role in innate immune responses. Indeed, and anti–phospho-GSK3a/b (Ser ). Anti-GSK3a/b was from Santa 2/2 Cruz Biotechnology, anti-GAPDH Ab was from Sigma, HRP-conjugated using DUSP3 mice, we found that DUSP3 deletion conferred anti-goat Ab was from Dako, HRP-conjugated anti-mouse Ab was from resistance to LPS-induced endotoxemia and polymicrobial in- GE Healthcare, and HRP-conjugated anti-rabbit Ab was from Merck fection–induced septic shock to female mice. This protection Millipore. Allophycocyanin–anti-CD45.1 (A20), PerCP–Cy5.5–anti-CD45.2 was macrophage dependent, and correlated with a higher per- (104), FITC-anti-CD11b, allophycocyanin–Cy7–anti-Ly6G, PE–anti-CD3, 2/2 PerCP–anti-CD8, FITC–anti-CD4, biotin–anti-B220, and streptavidin– centage of M2-like macrophages in DUSP3 mice. Moreover, PE–Cy7 were from BD Biosciences. Allophycocyanin–anti-F4/80, PerCP– the resistance was associated with decreased phosphorylation of Cy5–anti-NK1.1, and PerCP–Cy5.5–anti-CD11b were from eBio- the tyrosine kinases ERK1/2 and a subsequent decrease in TNF-a science, and PE–Cy–anti-Ly6G Ab was from BioLegend. LPS from production (15). Escherichia coli serotype O111:B4 was from Sigma and was diluted in In this article, we report that DUSP3 deletion does not protect pyrogen-free PBS. male mice from LPS-induced endotoxemia and cecal ligation and Animal blood sampling and plasma preparation puncture (CLP)-induced septic shock and that this protection was Peripheral blood was drawn into EDTA-coated tubes (BD Microtainer K2E dependent on female sex hormones.