Dual-Specificity Phosphatase 3 Deletion Protects Female, but Not

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 ovariectomized 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 phosphatase 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 León 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. Furthermore, we report that tubes; BD Biosciences) by puncturing the heart with a 26G needle. Cen- sepsis resistance was associated with a higher percentage of trifugation was performed twice at 800 3 g for 15 min at room temperature 2 2 M2-like macrophages in the peritoneal cavity of DUSP3 / female (RT). Plasma samples were separated in sterile Eppendorf tubes, aliquoted mice but not with decreased production of proinflammatory cy- in small volumes, and stored at 280˚C until used. tokines. We also showed that sepsis resistance in females, but not Meso Scale Discovery electrochemiluminescence assay in males or in ovariectomized (OVX) females, was associated with decreased ERK1/2, PI3K, and Akt activation. The Meso Scale Discovery (MSD) assay was performed according to manufacturer’s instructions (Meso Scale Discovery). Briefly, plasma was diluted 15 or 15,000 times for TNF or IL-6, respectively. For IL-10 and Materials and Methods IFN-g, samples were diluted twice. Samples were loaded onto 96-well Mice and ethics statement plates, incubated for 2 h at RT, and washed. Detection Abs were added for 2 h at RT. Signal detection was measured within 15 min after read buffer 2 2 C57BL/6-CD45.2 DUSP3 / mice were generated by homologous addition using an MSD instrument. recombination as previously reported (13). These mice were backcrossed with C57BL/6-CD45.2 mice (Charles River) to create heterozygotes that Isolation and stimulation of thioglycollate-elicited peritoneal 2 2 were mated to generate DUSP3+/+ and DUSP3 / littermate colonies macrophages used for experimentation. Age-matched male and female DUSP3+/+ and DUSP32/2 mice were used in all experiments. Mice were kept in ven- Peritoneal washes were performed 4 d after i.p. injection of 1 ml of 4% tilated cages under a 12-h dark/light cycle in a specific pathogen–free thioglycollate broth (Sigma). Five milliliters of 0.6 mM PBS-EDTA was animal facility and received food and water ad libitum. Health status was injected twice in the peritoneal cavity using an 18G needle and then col- evaluated every 3 mo, and mice were always found to be free of specific lected. Peritoneal macrophages (PMs) were selected by adherence to plastic pathogens. tissue culture dishes in complete RPMI 1640 medium. PMs were stimulated All mouse experiments and procedures were approved by the animal with 1 mg/ml LPS for 15, 30, or 60 min or for 8 or 24 h, depending on the ethics committees of the Universities of Ghent and Liege and were carried experiment performed out according to their guidelines. Phenotyping and flow cytometry CLP and in vivo LPS challenge Peritoneal washes were centrifuged for 10 min at 350 3 g, and the pellets were CLP was performed as previously described (19). For LPS challenge, resuspended in PBS. For cell surface staining, cells were incubated for 15 min mice were injected i.p. with 6 mg/kg LPS. Body temperature was with anti-CD16/CD32 (FcgIII/IIR) before labeling with specific Abs for 30 min monitored using a rectal thermometer at various times after LPS injection at 4˚C. Cells were washed, fixed with 1% paraformaldehyde solution. Flow and after CLP. Death of mice was recorded, and the data were analyzed cytometry was performed using BD FACSDiva software on FACSCanto II (BD for statistical significance of differences between the experimental Biosciences) and results were analyzed using FlowJo software (Tree Star). groups. Protein extraction and Western blot Mice irradiation and bone marrow transplantation For Western blot experiments, cells were stimulated for the indicated time Ten- to twelve-week-old C57BL/6-CD45.2 donor mice were killed by points and lysis was performed with RIPA buffer (50 mM Tris-HCl [pH 8], cervical dislocation. Tibiae and femurs were collected, and bone 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, marrow (BM) cells were flushed with PBS. BM cells (10 3 106)were 1 mM orthovanadate, complete protease inhibitor mixture tablets EDTA immediately injected i.v. into 6–8-wk-old lethally irradiated (866.3 cGy) free, and 1 mM PMSF) on ice for 20 min. Lysates were clarified by C57BL/6-CD45.1 recipient mice. Four weeks later, transplantation centrifugation at 19,000 3 g for 20 min at 4˚C. The resulting supernatants efficiency was evaluated based on the ratio of CD45.2/CD45.1 cells in were collected, and protein concentrations were determined using the the blood of transplanted mice. colorimetric Bradford reagent (Bio-Rad). Proteins were denatured at 95˚C The Journal of Immunology 3

FIGURE 1. Female sex hormones and BM cells are required for DUSP3 deletion-induced resistance to endotoxemia and septic shock. (A) DUSP3+/+ male (n = 12) and female (n = 17) mice and DUSP32/2 male (n = 13) and female (n = 19) mice were injected i.p. with 6 mg/kg LPS. Percentage survival was assessed twice a day for 10 d. (B) Body temperature of DUSP3+/+ and DUSP32/2 mice before, 6, and 24 h after LPS injection. (C) DUSP3+/+ male (n = 10) and female (n = 11) mice and DUSP32/2 male (n = 9) and female (n = 11) mice were subjected to CLP (one puncture with 21-gauge needle). Survival was documented twice a day for 7 d. (D) Body temperature of mice shown in panel (C) at basal (0), 6 h, and 24 h after LPS (Figure legend continues) 4 THE ROLE OF SEX-DEPENDENT DUSP3 IN SEPSIS in Laemmli buffer (40% glycerol, 8% SDS, 20% 2-ME, 20% Tris-HCl were still alive by the end of the experiment, whereas 70% of 0.5 M (pH 6.8), 0.05% bromophenol blue, and water) for 5 min. DUSP32/2 female mice survived (Fig. 1C). The body temperature of Denatured samples were run on 10% SDS-PAGE gel and transferred onto each group dropped after surgery, and only DUSP32/2 female mice nitrocellulose membranes. To block the nonspecific binding sites, mem- recovered (Fig. 1D). These results indicate a sex-specific response to branes were incubated for 1 h at RT in TBS-Tween 20 containing 5% nonfat 2 2 milk or 3% BSA. Membranes were incubated overnight with primary Ab at septicshockinDUSP3 / mice. 4˚C, washed three times in TBS-Tween, and incubated with HRP- 2 2 conjugated secondary Ab for 1 h at RT. The blots were developed by OVX DUSP3 / mice are sensitive to LPS-induced death ECL (ECL kit; Amersham), according to the manufacturer’s instructions. Male and female sex hormone receptors have been identified on RNA purification, reverse transcription, and real-time PCR immune cells, suggesting direct effects of androgen and estrogen on RNAwas extracted from PMs using an miRNeasy Mini Kit (QIAGEN), and these cells (20). Sexual steroid hormones have been recognized to cDNA was synthesized using Expand reverse transcriptase (Roche), influence numerous immune pathophysiological processes (21). according to the recommendations of the manufacturer. cDNA was am- To elucidate the effect of female sex hormones, we subjected plified using SYBR Green PCR Master Mix (Roche) and 0.3 mM specific 4-wk-old DUSP3+/+ and DUSP32/2 mice to ovariectomy. As primers for Arginase 1 (Arg1), inducible NO synthase (iNOS), and b2- +/+ 2/2 microglobulin (b2M). All quantitative PCRs were performed on a Light- controls, another group of 4-wk-old DUSP3 and DUSP3 Cycler Real-Time PCR System (Roche). The ratio between the expression mice were sham operated. To assess the efficiency of ovariec- level of the gene of interest and b2M in the sample was defined as the tomy, we checked the presence and the size of the uterus. Suc- normalization factor. Relative mRNA quantities for Arg1 and iNOS were cessful OVX mice were deprived of normal uterus development, determined using the DCq method. All primers were from Eurogentec. The whereas sham-operated mice presented a normally developed following sequences were used: iNOS: forward, 59-GCTTCTGGTCG- ATGTCATGAG-39 and reverse, 59-TCCACCAGGAGATGTTGAAC-39, uterus (Fig. 1E). Six weeks after surgery, sham and OVX mice Arg1: forward, 59-CAGAAGAATGGAAGAGTCAG-39 and reverse, 59- were challenged with 6 mg/kg LPS, and survival and temperature AGATATGCAGGGAGTCACC-39,andb2M: forward, 59-CACCCCACTGA- were monitored (Fig. 1F, 1G). Ovariectomy impaired the observed GACTGATACA-39 and reverse, 59-TGATGCTTGATCACATGTCTCG-39. endotoxemia resistance of DUSP32/2 mice, whereas sham-operated 2/2 Statistical analysis DUSP3 mice were still fully protected from endotoxin-induced death. These data demonstrate that female sex hormones are in- The Student t test was used to assess statistical differences between groups. 2/2 Survival differences after LPS challenge and CLP were analyzed by volved in the observed resistance of DUSP3 female mice to Kaplan–Meier analysis with the log-rank test. Results were considered LPS-induced lethality. significant at p , 0.05. Results are presented as mean 6 SEM. Prism 2/2 +/+ software (GraphPad) was used to perform the statistical analysis. *p , DUSP3 female BM cells rescue DUSP3 female, but not 0.05, **p , 0.01, ***p , 0.001. male, mice from LPS-induced lethality We previously showed that adoptive transfer of DUSP32/2 female Results +/+ 2/2 BM cells or monocytes to DUSP3 female mice was sufficient to DUSP3 female, but not male, mice are resistant to LPS- transfer resistance to LPS-induced lethality (15). Therefore, we induced endotoxemia and to CLP-induced septic shock investigated whether this is also true when recipient mice are In a previous study, we showed that DUSP3 deletion protected mice males. To generate chimeric mice, 1 3 107 BM cells from female from LPS-induced endotoxemia and polymicrobial infection–in- DUSP32/2 C57BL/6-CD45.2 mice were injected i.v. into lethally duced septic shock (15). Only females were used in the first study. irradiated DUSP3+/+ C57BL/6-CD45.1 recipient male and female To investigate whether the protection observed is a general feature mice (DUSP32/2 → M-DUSP3+/+ and DUSP32/2 → F-DUSP3+/+, of DUSP3 deletion or is sex dependent, we challenged DUSP32/2 respectively). As a control, DUSP3+/+ female BM was trans- males with a lethal dose of LPS (i.p. injection of 6 mg/kg) and planted into lethally irradiated DUSP3+/+ male or female mice compared their survival with females and with WT control lit- (DUSP3+/+ → M-DUSP3+/+ and DUSP3+/+ → F-DUSP3+/+,re- termates of both sexes. Body temperature was also monitored. As spectively). Successful hemato-lymphoid reconstitution was veri- expected and previously reported, 90% of DUSP32/2 female mice fied by flow cytometry 3–4 wk after the transplantation. Ninety- were resistant to LPS, whereas only 5% of DUSP3+/+ female mice five percent of peripheral blood cells were CD45.2+ (Fig. 1H, 1I). survived the challenge (15). Interestingly, DUSP3+/+ and DUSP32/2 Moreover, in recipient mice, the expression of DUSP3 in PMs was male mice were equally sensitive to LPS-induced death (Fig. 1A). abolished in the recipient mice transplanted with DUSP32/2 BM Body temperature of all groups of mice, with the exception of cell suspension, as shown by DUSP3 immunoblotting (Fig. 1J). DUSP32/2 females, decreased after LPS injection. Twenty-four Four weeks after BM transplantation, 6 mg/kg LPS was injected i.p. hours later, almost all DUSP32/2 females recovered, whereas the into recipient mice, and survival was monitored for 8 d (Fig. 1K). other groups remained hypothermic (Fig. 1B). These results were Interestingly, .70% of the chimeric DUSP32/2 → F-DUSP3+/+ further confirmed in the CLP model performed on DUSP3+/+ and mice survived until the end of the experiment compared with 9% DUSP32/2 male and female mice. As expected, only 10% of of DUSP3+/+ → F-DUSP3+/+ mice. In contrast, all DUSP32/2 → DUSP3+/+ and DUSP32/2 male mice and DUSP3+/+ female mice M-DUSP3+/+ and DUSP3+/+ → M-DUSP3+/+ mice died within 4 d

injection. (E–G) DUSP3+/+ and DUSP32/2 mice were sham operated (n = 9 for DUSP3+/+ and n = 8 for DUSP32/2) or OVX (n = 9 for DUSP3+/+ and n =11 for DUSP32/2) 4 wk after birth. (E) Representative macroscopic view of uterus after sham surgery or ovariectomy. Dashed lines denote the mice uterine lining axis. (F) Six weeks after surgery, mice were injected i.p. with 6 mg/kg LPS. Percentage survival was assessed twice a day for 5 d. (G) Body temperature of DUSP3+/+ and DUSP32/2 mice before and 8 and 24 h after LPS injection. (H–K)Atotalof103 106 BM cells from DUSP32/2 C57BL/6-CD45.2 female mice was injected i.v. into lethally irradiated DUSP3+/+ C57BL/6-CD45.1 recipient male and female mice (DUSP32/2 → M-DUSP3+/+ and DUSP32/2 → F-DUSP3+/+ mice, respectively). As control, DUSP3+/+ female BM was transplanted into lethally irradiated DUSP3+/+ male or female mice (DUSP3+/+ → M-DUSP3+/+ and DUSP3+/+ → F-DUSP3+/+mice, respectively). (H) Representative dot plot of CD45.1 and CD45.2 immune cells in BM-transplanted mice. (I) Percentage of CD45.1 and CD45.2 immune cells in all transplanted mice. (J) Western blot was performed on peritoneal cells from transplanted mice using anti- DUSP3 Ab. Anti-GAPDH was used as a loading control. Each lane corresponds to one mouse. Lane 1: lysate from peritoneal cavity cells of DUSP3+/+ mouse. Lanes 2-8: F-DUSP32/2 → M-DUSP3+/+. Lanes 9-14: F-DUSP32/2 into M-DUSP3+/+.(K) Transplanted mice survival after i.p. LPS injection (6 mg/ml). Data are presented as mean + SEM. Survival data were compared using the Kaplan–Meier test with the log-rank test. *p , 0.05, ***p , 0.001, ***p , 0.001. The Journal of Immunology 5

FIGURE 2. DUSP3-deletion-induced LPS shock resistance in female mice, but not in male WT or OVX mice, is associated with increased M2-like macrophages in the peritoneal cavity. (A) Peritoneal cells harvested from PBS and 24-h LPS-challenged DUSP3+/+ and DUSP32/2 mice were analyzed by flow cytometry to evaluate the percentage of T cell, B cell, NK cell, NKT cell, neutrophil, and macrophage populations. For lymphocyte and NK cell phenotyping, cells were stained using PE–anti-CD3, FITC–anti-CD4, PE–Cy7–anti-B220, and PerCP–Cy5–anti-NK1.1. Forward scatter and side scatter were used for gating on live cells and lymphocyte populations. CD4 T cells were B2202/NK1.12/CD3+/CD4+, CD8 T cells were B2202/NK1.12/CD3+/ CD8+, B cells were B220+/NK1.12/CD32, NK cells were B2202/CD32NK1.1+, and NKT cells were B2202/CD3+NK1.1+. For (Figure legend continues) 6 THE ROLE OF SEX-DEPENDENT DUSP3 IN SEPSIS after LPS injection (Fig. 1K). These data suggest that, in the ab- in DUSP3+/+ and DUSP32/2 OVX mice compared with DUSP3+/+ sence of DUSP3, female sex hormones and BM cells are required and DUSP32/2 sham mice at basal level. However, 24 h after LPS for resistance to LPS shock. challenge, the percentage of M2-like macrophages in the peritoneal cavity of OVX mice decreased, but it did not reach statistical DUSP3 deletion–induced LPS shock resistance in female mice, significance compared with DUSP32/2 sham mice (Fig. 2B, 2C). but not in male, OVX, or WT mice, is associated with increased These data suggest that M2-like macrophages could be involved in M2-like macrophages in the peritoneal cavity the resistance to LPS-induced endotoxemia. To further charac- We have previously reported that DUSP3 is expressed in several terize these cells, we measured the relative expression of genes immune cells where it plays an important role in macrophage and associated with M1-like and M2-like PMs (i.e., Nos2 and Arg1). T cell functions (15, 18). Because sepsis involves the participation At baseline, none of the transcript was detected (data not shown). of innate and adaptive immune cells (22), we investigated whether Two hours after LPS challenge, Arg1 expression increased sig- DUSP3 deletion–associated survival of shock in females was nificantly in DUSP32/2 sham mice compared with DUSP3+/+ linked to an unbalanced contribution of one cell type or another in sham mice (Fig. 2D). In males and OVX groups, Arg1 was de- LPS-resistant mice compared with LPS-sensitive mice. We found tected but at significantly lower levels compared with sham- that, at baseline, as well as after LPS injection, the percentages of operated female mice. Twenty-four hours after LPS injection, + + + 2 CD19 B cells, CD4 T cells, CD8 T cells, macrophages (Ly6G the level of Arg1 increased dramatically in the DUSP32/2 sham + + 2 + + CD11b F4/80 ), neutrophils (F4/80 /CD11b Ly6G ), NK cells group compared with all other groups (Fig. 2D). The Nos2 level 2 + + + (CD3 NK1.1 ), and NKT cells (CD3 NK1.1 ) were equal in was low 2 h after LPS injection, but it increased significantly 22 h males and females of both genotypes (Fig. 2A). LPS injection later in sham-operated female mice of both genotypes, although induced a significant reduction in T cells and macrophages, in- the increase was more significant in DUSP3+/+ female mice creased neutrophil infiltration of the peritoneal cavity, and had no (Fig. 2D). Altogether, these data suggest that M2-like macro- significant impact on the percentages of NK, NKT, and B cells phages and female hormones could be involved in DUSP3- (Fig. 2A). induced resistance to LPS-induced endotoxemia. We previously reported that increased survival of DUSP32/2 female mice after LPS and CLP was associated with a higher DUSP3-KO female mice survival against LPS is not due to a percentage of M2-like macrophages in the peritoneal cavity of modification in proinflammatory cytokine production these mice compared with DUSP3+/+ females (15). To investigate We previously reported that DUSP32/2 female survival to LPS whether this is associated with DUSP3-deficient female survival, was associated with a decreased systemic TNF level compared we phenotyped DUSP3+/+ and DUSP32/2 PMs from male and with DUSP3+/+ mice (15). Therefore, we wanted to know whether female mice (sham operated and OVX) challenged with LPS, the susceptibility of DUSP32/2 male and OVX mice to LPS- based on the characterization previously reported by Ghosn et al. induced death could be linked to differential expression of TNF (23). M1 macrophages are F4/80intCD11bintLy6G2, whereas M2- or to other proinflammatory cytokines, such as IL-6, IFN-g, and like macrophages are F4/80hiCD11bhiLy6G2 (Fig. 2B, 2C). We IL-10. We measured and compared plasma levels of these four confirmed previous findings showing that the percentage of M2- cytokines at basal levels and at 2 and 24 h after LPS challenge in like macrophages was higher in the peritoneal cavity of DUSP32/2 all groups of mice using an MSD assay. For TNF, there was no female mice compared with littermate controls at basal and 24 h difference between DUSP3+/+ and DUSP32/2 male mice. How- after LPS injection (Fig. 2B, 2C). Interestingly, we observed that ever, and as previously reported (15), there was a significant de- the percentage of M2-like macrophages in male mice was slightly crease in this cytokine in DUSP32/2 female mice compared with lower compared with DUSP32/2 female mice at basal level; this DUSP3+/+ female mice 2 and 24 h after LPS challenge (Fig. 3A). difference was exacerbated at 24 h after LPS injection. There was Compared with DUSP3+/+ mice, DUSP32/2 mice of both sexes not a significant difference for the percentage of M2-like macro- had a slight, but nonsignificant, decrease in IL-6 2 h after LPS phages between DUSP3+/+ and DUSP32/2 male mice. Similarly, injection (Fig. 3B). These differences were maintained in OVX there was no difference in the percentage of M1-like macrophages mice groups (Fig. 3A, 3B). For IFN-g, secretion was equal in all at basal level and 24 h after LPS injection between DUSP3+/+ and groups of mice 2 h after LPS challenge. At 24 h after LPS in- DUSP32/2 female mice. However, we noticed a slight increase in jection, IFN-g levels were lower in sham and OVX DUSP32/2 the percentage of M1-like macrophages in male mice compared females compared with sham and OVX DUSP3+/+ females. with female mice at basal level. This difference was accentuated, However, there was a 10-fold decrease in IFN-g in all OVX mice, although not significantly, at 24 h (Fig. 2B, 2C). For OVX mice, regardless of their genotype. In males, the level of IFN-g was the percentage of M1-like macrophages (F4/80intCD11bint)was significantly higher in DUSP32/2 mice than in the littermate higher in DUSP3+/+ and DUSP32/2 OVX mice compared with controls at 24 h, but not at 2 h, after LPS injection (Fig. 3C). DUSP32/2 sham mice at basal level. The difference was main- Finally, the level of IL-10 was lower in DUSP32/2 mice compared tained at 24 h after LPS injection, although not significantly with controls, regardless of sex or type of surgery (Fig. 3D). Al- (Fig. 2B, 2C). The percentage of M2-like macrophages was equal together, these data strongly suggest that DUSP3 deletion–induced

neutrophils and macrophages, phenotyping was performed using PerCP–Cy5.5–anti-CD11b, allophycocyanin–Cy7–anti-Ly6G, and allophycocyanin–anti- F4/80. Neutrophils were F4/802/CD11b+/Ly6G+, and macrophages were considered Ly6G2/F4/80+/CD11b+. Percentage of the indicated cell population of live cells (total live cells for macrophage and neutrophil analysis and leukocyte gate for the analysis of lymphocytes and neutrophils) are presented as bar graphs of means (n = 3 in each group) + SEM. (B) Peritoneal cells from PBS or LPS (24 h)–injected DUSP3+/+ and DUSP32/2 male mice and DUSP3+/+ and DUSP32/2 sham-operated or OVX female mice were analyzed to discriminate between M1-like macrophages (F4/80intCD11bint) and M2-like macrophages (F4/80hiCD11bhi). Analyses were performed on the Ly6G2 live cell gate. A representative dot plot from each group of mice is shown. (C) Quantiﬁcation of M1-like and M2-like macrophages out of total live Ly6G2 cells. Results are presented as mean + SEM (n = 6–10 mice per group). (D) Quantitative RT-PCR analysis of the expression of Arg1 and Nos2 transcripts in PMs harvested from the indicated groups of mice at 2 h and 24 h after LPS injection. The expression of genes of interest was relative to b2M. n = 4 mice in each group. Results are presented as mean + SEM. *p , 0.5, **p , 0.01. The Journal of Immunology 7

FIGURE 3. Survival of DUSP3-KO female mice against LPS is not due to a modification in proinflammatory cytokine production. Plasma levels of TNF (A), IL-6 (B), IFN-g (C), and IL-10 (D) in DUSP3+/+ and DUSP32/2 male mice and sham-operated or OVX female mice before and 2 and 24 h after LPS challenge (6 mg/ml). Cytokine levels were determined using MSD assays. Results are presented as mean + SEM (n = 5 mice per group). The same mice were used at all time points. *p , 0.05, **p , 0.01. 8 THE ROLE OF SEX-DEPENDENT DUSP3 IN SEPSIS resistance to LPS-induced shock in female mice is not a conse- levels of ERK1/2 and Akt in DUSP32/2, but not in DUSP3+/+, quence of the observed modifications of the measured cytokines. PMs (Fig. 6). These data clearly suggest that the DUSP3- dependent reduced phosphorylation of ERK1/2 and Akt is estro- DUSP3 deletion alters ERK1/2 and PI3K/Akt phosphorylation gen dependent. magnitudes and kinetics in an estrogen-dependent manner We have previously reported that, although DUSP3 is a ubiqui- Discussion tously expressed protein, the level of expression varies signifi- It is well recognized that immune responses to infection are sex cantly between cell types (14, 15) and during cell cycle progression dependent. Indeed, stronger immune responses confer protection (24). Therefore, we investigated whether its expression varies be- against infections and sepsis to women (26). Several epidemio- tween males and females and changes in response to LPS or after logical studies have been performed and showed a greater inci- ovariectomy. As shown in Fig. 4A, DUSP3 expression level was dence of sepsis in males compared with females (27). Consequently, similar in males and females and was not influenced by LPS or compared with males, there are less female hospitalizations as- OVX (Fig. 4A). sociated with infections. In addition, male sex and the presence We have previously reported that DUSP3 deletion in female mice of comorbidities were commonly reported independent predictors of macrophages was associated with decreased ERK1/2 phosphory- postacute mortality in sepsis survivors (28). Interestingly, many of lation levels after ex vivo LPS stimulation (15). To investigate the differences between males and females in response to infections whether this alteration was also associated with the sex-specific become apparent at puberty (29). In line with this, women younger resistance to septic shock, DUSP3+/+ and DUSP32/2 PMs from than 50 y of age show a lower incidence of severe sepsis and better sham or OVX mice were stimulated ex vivo with LPS (1 mg/ml) at survival compared with age-matched men (30). Altogether, these different time points, and cell lysates were probed with phospho- observations suggest a role for sexual hormones in the protection specific ERK1/2 Abs. As expected, ERK1/2 phosphorylation was from severe infections and sepsis. This hypothesis has been sup- significantly lower in DUSP32/2 sham PMs at all time points ported by the finding that receptors for reproductive hormones are compared with DUSP3+/+ macrophages. Interestingly, in OVX present in a variety of immune cell types (31). In contrast, estrogen mice, LPS stimulation led to equal ERK1/2 activation in DUSP32/2 has been demonstrated to increase resistance to several bacterial and DUSP3+/+ PMs, as demonstrated by the observed phosphor- infections, whereas the removal of endogenous estrogens have been ylation levels. There was no difference in ERK1/2 phosphoryla- shown, for example, to markedly increase the severity of Myco- tion in male mice from both genotypes (Fig. 4B, 4C). bacterium avium infections, an effect that can be reversed after 17b- The observed reduced phosphorylation of ERK1/2 in DUSP32/2 estradiol replacement (32, 33). However, the role of female repro- sham mice suggests that DUSP3 could be targeting ERK1/2 up- ductive hormones in the susceptibility to acute infection and sepsis stream kinase or one of the ERK1/2 phosphatases. Therefore, we remains poorly understood. analyzed MAPKK MEK1/2 activation following ex vivo LPS In this article, we report that DUSP3 deletion confers resistance stimulation (1 mg/ml) of PMs. MEK1/2 kinetic phosphorylation to LPS-induced lethality and to polymicrobial-induced septic shock was equal between DUSP3+/+ and DUSP32/2 sham mice of both in female mice but not in male mice. We demonstrated that this sexes (Fig. 4D, 4E), suggesting that MEK1/2 is not targeted by protection is dependent on female sexual hormones and monocytes/ DUSP3. macrophages. Indeed, ovariectomy induced a loss of resistance. In The PI3K/Akt pathway is another important pathway that is contrast, DUSP32/2 monocyte transfer to WT females was suf- activated after TLR4 triggering (25). Therefore, we investigated ficient to transfer the resistance to WT recipient mice (15); how- whether DUSP3 deletion could impact this pathway after activa- ever, this protection was not due to decreased TNF production, as tion with LPS and whether the kinetics and magnitude of this suggested by our previous study (15). To our knowledge, this is activation could be sex dependent. PI3K and Akt activation was the first report demonstrating a signaling molecule–induced syn- evaluated using phospho-specific Abs and Western blot after ex ergistic immunoprotective effect of monocytes/macrophages and vivo LPS stimulation (1 mg/ml) of PMs at different time points. female sexual hormones against sepsis. Interestingly, PI3K and Akt activation decreased in DUSP32/2 The observed resistance to LPS-induced septic shock in sham PMs compared with DUSP3+/+ PMs at all time points. This DUSP32/2 female mice was associated with a modest increase in difference was abolished in OVX mice, because the phosphory- M2-like macrophages in the peritoneal cavity of mice. This ob- lation level of PI3K and Akt remained equal between DUSP3+/+ servation was strengthened by the increase in Arg1 gene expres- and DUSP32/2 PMs. However, activation of GSK3 downstream sion in DUSP32/2 female mice but not in male or OVX mice. target of Akt was not affected by DUSP3 deficiency in sham or Arg1 is a known marker for M2-like macrophages (34). Ovari- OVX mice (Fig. 5). There was no difference in PI3K or Akt ac- ectomy of DUSP3-deficient mice induced a loss of resistance to tivation in male PMs after LPS stimulation. PI3K and Akt were LPS-induced death, with no difference in the percentage of M2- equally activated at all time points in DUSP3+/+ and DUSP32/2 like macrophages between control groups and OVX DUSP32/2 LPS- stimulated PMs, and GSK3 activation was not affected by mice. Together with the fact that the percentage of M2-like DUSP3 deficiency (Fig. 5). macrophages was also equal in DUSP3+/+ and DUSP32/2 male These data suggest that DUSP3 affects ERK1/2, PI3K, and Akt mice, it suggests that female sex hormones may influence the activation, probably in concert with estrogens. To investigate this alternative activation of macrophages. Our observations are in line hypothesis, DUSP32/2 and DUSP3+/+ female mice underwent with studies showing that estrogens influence numerous immu- ovariectomy at the age of 4 wk. Two weeks later, half of the mice nological processes, including the physiological functions of from each group received estrogen via s.c. implant for controlled monocytes and macrophages (35). Indeed, ovarian sex hormones release of 17b-estradiol (1.5 mg/d). Mice were kept for 3 wk modulate monocyte adhesion and chemotaxis, TLR expression, before sacrifice. PMs were stimulated ex vivo with LPS (1 mg/ml) cytokine production, and phagocytosis activity (36). Moreover, at different time points, and cell lysates were probed with anti– several lines of evidence suggest that estrogens also influence phospho-ERK1/2, anti-ERK, anti–phospho-PI3K, anti-PI3K, anti– macrophage polarization. ER-a–KO mice undergo a decrease in phospho-Akt, and anti-Akt Abs. As shown in Fig. 6, estrogen alternative activated macrophages (36). ER-a–deficient macro- complementation significantly reduced the phosphorylation phages are indeed refractory to IL-4–induced alternative activation, The Journal of Immunology 9

FIGURE 4. DUSP3 deficiency affects ERK1/2 phosphorylation in macrophages from female mice but not from male mice. PMs isolated from 12-wk-old DUSP3+/+ and DUSP32/2 female, male, and OVX mice were stimulated ex vivo with 1 mg/ml LPS for the indicated times. (A) Immunoblot of DUSP3 expression in resting or LPS activated peritoneal macrophages from DUSP3+/+ male and female mice and from OVX female mice. (B) Western blots were performed using anti–phospho-ERK1/2 (Thr202/Tyr204) and anti-ERK1/2 as a loading control. Representative blots are shown for each detected (phospho) protein. (C) Densitometry quantifications of phospho-ERK1/2 and ERK1/2 were performed. (D) Anti–phospho-MEK1/2 (Ser217/221) and anti-MEK1/2, as loading control and (E) densitometry quantification of phospho-MEK and MEK. Results are presented as the ratio of phospho-ERK/ERK and phospho- MEK/MEK from four independent experiments. For each experiment, peritoneal cells from two or three individual mice were pooled prior to stimulation with LPS and lysis. Data are shown as mean + SEM. *p , 0.05. as demonstrated by a decrease in IL-4R and STAT6 phosphory- LPS challenge (data not shown). In contrast, TNF production does lation in these cells (37). Estrogens have also been reported to not seem to play a role in the observed phenotype, because increase the expression of the transcription factor IFN regulatory ovariectomy of DUSP32/2 mice did not influence the level of this factor-4, which is involved in the alternative activation of mac- proinflammatory cytokine, although mice did succumb to endo- rophages (38). Using a transcriptomic assay, we did not observe toxemia. These data were rather surprising because sex steroids are differences in IL-4, IL-4R, or IFN regulatory factor-4 expression known to regulate pro- and anti-inflammatory cytokine levels re- levels between DUSP3-KO males and females at baseline or after leased by macrophages. In contrast, female sex hormones are 10 THE ROLE OF SEX-DEPENDENT DUSP3 IN SEPSIS

FIGURE 5. DUSP3 deficiency affects the PI3K/Akt pathway in macrophages from female, but not male, mice. PMs isolated from 12-wk-old DUSP3+/+ and DUSP32/2 female, male, and OVX mice were stimulated ex vivo with 1 mg/ml LPS for the indicated times. (A) Western blots were performed on peritoneal cell lysates using anti–phospho-PI3K (p85 Tyr458/p55 Tyr199), anti–phospho-Akt (Ser473), anti–phospho-GSK3a/b (Ser21/9) and anti-PI3K, anti-Akt, and anti- GSK3a/b as loading controls. (B) Densitometry quantifications of phospho-PI3K, phospho-Akt, phospho-GSK3a/b, PI3K, Akt, and GSK3a/b. Results are presented as the ratio of phospho-PI3K/PI3K, phospho-Akt/Akt, and phospho-GSK3a/b/GSK3a/b from four independent experiments. For each experiment, peritoneal cells from two or three individual mice were pooled prior to stimulation with LPS and lysis. Data are shown as mean + SEM. *p = 0.05. known to negatively regulate the production of TNF (39), one of linked to the observed decrease in ERK1/2 and Akt/PI3K acti- the most important cytokines in sepsis (40, 41). Therefore, the vation. Upon ex vivo LPS stimulation, DUSP32/2 female PMs change in TNF production, as well as the observed changes in showed reduced phosphorylation of ERK1/2 and Akt compared IFN-g, IL-6, IL-10, and, perhaps other cytokines, upon DUSP3 with DUSP3+/+ female macrophages. These differences were not deletion, should be considered an independent phenomenon that observed in macrophages from OVX DUSP32/2 mice, but they is not related to DUSP32/2 female mice survival to sepsis. were maintained in DUSP32/2 OVX mice receiving estrogen How DUSP3 regulates macrophage alternative activation in a complementation. Together, these data suggest that, under inflam- female sexual hormone–dependent manner is a complex question matory conditions, estrogen controls macrophage polarization to answer. The molecular mechanisms involved are probably through the DUSP3–ERK1/2–Akt signaling pathway axis. The Journal of Immunology 11

FIGURE 6. Alteration of ERK1/2 and Akt phosphorylation in DUSP32/2 female macrophages is estrogen dependent. PMs isolated from OVX DUSP3+/+ and DUSP32/2 mice and from OVX DUSP3+/+ and DUSP32/2 mice receiving estrogen complementation (3 wk, 1.5 mg/d) were stimulated ex vivo with 1 mg/ml LPS for the indicated times. (A) Western blots were performed using anti–phospho-ERK1/2 (Thr202/Tyr204), anti–phospho-PI3K (p85 Tyr458/p55 Tyr199), anti–phospho-Akt (Ser473), anti-PI3K, anti-ERK1/2, and anti-Akt as loading controls. (B) Densitometry quantiﬁcations of phospho-ERK, phospho- PI3K, phospho-Akt, ERK1/2, PI3K, and Akt. Results are presented as the ratio of phospho-ERK1/2/ERK1/2, phospho-PI3K/PI3K, and phospho-Akt/Akt from three independent experiments. For each experiment, peritoneal cells from three individual mice were pooled prior to stimulation with LPS and lysis. Data are shown as mean + SEM. *p , 0.05, **p , 0.01. 12 THE ROLE OF SEX-DEPENDENT DUSP3 IN SEPSIS

ERK1/2 was previously reported to play a role in macrophage 9. Todd, J. L., J. D. Rigas, L. A. Rafty, and J. M. Denu. 2002. Dual-specificity protein tyrosine phosphatase VHR down-regulates c-Jun N-terminal kinase polarization through the mTOR signaling pathway (42). Indeed, (JNK). Oncogene 21: 2573–2583. ERK1/2 phosphorylates and dissociates the tuberous sclerosis 10. Todd, J. L., K. G. Tanner, and J. M. Denu. 1999. Extracellular regulated kinases protein (TSC) complex, leading to its inactivation and the subse- (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein- tyrosine phosphatase VHR. A novel role in down-regulating the ERK path- quent activation of mTOR (42), which, in turn, leads to decreased way. J. Biol. Chem. 274: 13271–13280. IL-4–induced M2 polarization in TSC-deficient mice (42, 43). The 11. Wang, J. Y., C. L. Yeh, H. C. Chou, C. H. Yang, Y. N. Fu, Y. T. Chen, role of sex hormones has not been investigated in these studies. In H. W. Cheng, C. Y. Huang, H. P. Liu, S. F. Huang, and Y. R. Chen. 2011. Vaccinia H1-related phosphatase is a phosphatase of ErbB receptors and is our model, it would be interesting to investigate whether the ob- J. Biol. Chem. 2 2 down-regulated in non-small cell lung cancer. 286: 10177–10184. served decreased phosphorylation of ERK1/2 in DUSP3 / fe- 12. Hoyt, R., W. Zhu, F. Cerignoli, A. Alonso, T. Mustelin, and M. David. 2007. male PMs could lead to TSC activation and, consequently, to M2 Cutting edge: selective tyrosine dephosphorylation of interferon-activated nu- clear STAT5 by the VHR phosphatase. J. Immunol. 179: 3402–3406. polarization. In contrast, it has been reported that, upon TLR4 13. Amand, M., C. Erpicum, K. Bajou, F. Cerignoli, S. Blacher, M. Martin, stimulation, PI3K engagement is followed by Akt and mTORC1 F. Dequiedt, P. Drion, P. Singh, T. Zurashvili, et al. 2014. DUSP3/VHR is a pro- activation that is due to TSC inactivation by Akt (44); this may angiogenic atypical dual-specificity phosphatase. Mol. Cancer 13: 108. 14. Musumeci, L., M. J. Kuijpers, K. Gilio, A. Hego, E. Theáˆtre, L. Maurissen, lead to the polarization of M1 macrophages (44, 45). Similarly to M. Vandereyken, C. V. Diogo, C. Lecut, W. Guilmain, et al. 2015. Dual- decreased ERK phosphorylation, decreased PI3K/Akt activation specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis. Circulation 131: 656–668. may lead to TSC activation and shift macrophage polarization 15. Singh, P., L. Dejager, M. Amand, E. Theatre, M. Vandereyken, T. Zurashvili, toward the M2 phenotype. M. Singh, M. Mack, S. Timmermans, L. Musumeci, et al. 2015. DUSP3 genetic Another important question raised by our study is how does deletion confers M2-like macrophage-dependent tolerance to septic shock. J. Immunol. 194: 4951–4962. DUSP3 deletion lead to decreased activation of ERK1/2 and Akt 16. Amand, M., C. Erpicum, C. Gilles, A. Noel, and S. Rahmouni. 2016. Functional signaling molecules under the control of estrogen? Decreased analysis of dual specificity phosphatases in angiogenesis. Methods Mol Biol phosphorylation of these kinases clearly suggests that they are not 1447: 331–349. 17. Pavic, K., G. Duan, and M. Ko¨hn. 2015. VHR/DUSP3 phosphatase: structure, directly targeted by DUSP3. The observed decreased phosphory- function and regulation. FEBS J. 282: 1871–1890. lation of ERK1/2 and Akt could be due to reduced activation of 18. Alonso, A., S. Rahmouni, S. Williams, M. van Stipdonk, L. Jaroszewski, specific ERK1/2 and PI3K/Akt, yet unknown, phosphatases. In- A. Godzik, R. T. Abraham, S. P. Schoenberger, and T. Mustelin. 2003. Tyrosine phosphorylation of VHR phosphatase by ZAP-70. Nat. Immunol. 4: 44–48. deed, preliminary data show that pervanadate (nonspecific protein 19. Rittirsch, D., M. S. Huber-Lang, M. A. Flierl, and P. A. Ward. 2009. Immunodesign tyrosine phosphatases inhibitor) treatment of LPS-stimulated PMs of experimental sepsis by cecal ligation and puncture. Nat. Protoc. 4: 31–36. 20. Klein, S. L. 2012. Immune cells have sex and so should journal articles. En- restores ERK1/2 phosphorylation, whereas okadaic acid (inhibitor docrinology 153: 2544–2550. of Ser/Thr PP1/PP2A), at low and high concentrations, did not 21. Verthelyi, D. 2001. Sex hormones as immunomodulators in health and disease. (data not shown). Further investigations using phosphoproteomic Int. Immunopharmacol. 1: 983–993. 22. Hotchkiss, R. S., G. Monneret, and D. Payen. 2013. Sepsis-induced immuno- approaches, among others, are required to confirm this hypothesis, suppression: from cellular dysfunctions to immunotherapy. Nat. Rev. Immunol. identify the specific substrate(s) for DUSP3, and assess the exact 13: 862–874. role of this phosphatase in TLR4 signaling under the influence of 23. Ghosn, E. E., A. A. Cassado, G. R. Govoni, T. Fukuhara, Y. Yang, D. M. Monack, K. R. Bortoluci, S. R. Almeida, L. A. Herzenberg, and L. A. Herzenberg. 2010. female sex hormones. Two physically, functionally, and developmentally distinct peritoneal macrophage In summary, we identified DUSP3 as a new key signaling subsets. Proc. Natl. Acad. Sci. USA 107: 2568–2573. molecule that plays an important role in macrophage alternative 24. Rahmouni, S., F. Cerignoli, A. Alonso, T. Tsutji, R. Henkens, C. Zhu, C. Louis- dit-Sully, M. Moutschen, W. Jiang, and T. Mustelin. 2006. Loss of the VHR activation and sexual dimorphism in the innate immune response dual-specific phosphatase causes cell-cycle arrest and senescence. Nat. Cell to infection. Our data suggest that DUSP3 inhibition, combined Biol. 8: 524–531. 25. Laird, M. H., S. H. Rhee, D. J. Perkins, A. E. Medvedev, W. Piao, M. J. Fenton, with estrogen administration, may lead to protection from sepsis and S. N. Vogel. 2009. TLR4/MyD88/PI3K interactions regulate TLR4 signal- and septic shock. ing. J. Leukoc. Biol. 85: 966–977. 26. Straub, R. H. 2007. The complex role of estrogens in inflammation. Endocr. Rev. 28: 521–574. Acknowledgments 27. Suarez De La Rica, A., F. Gilsanz, and E. Maseda. 2016. Epidemiologic trends We thank the GIGA-animal, GIGA-imaging, and GIGA-immunohistochemistry of sepsis in western countries. Ann. Transl. Med. 4: 325. core facilities for technical assistance and help. 28. Shankar-Hari, M., M. Ambler, V. Mahalingasivam, A. Jones, K. Rowan, and G. D. Rubenfeld. 2016. Evidence for a causal link between sepsis and long-term mortality: a systematic review of epidemiologic studies. Crit. Care 20: 101. Disclosures 29. Beery, T. A. 2003. Sex differences in infection and sepsis. Crit. Care Nurs. Clin. The authors have no financial conflicts of interest. North Am. 15: 55–62. 30. Wichmann, M. W., D. Inthorn, H. J. Andress, and F. W. Schildberg. 2000. In- cidence and mortality of severe sepsis in surgical intensive care patients: the influence of patient gender on disease process and outcome. Intensive Care Med. References 26: 167–172. 1. Singer, M., C. S. Deutschman, C. W. Seymour, M. Shankar-Hari, D. Annane, 31. Angele, M. K., M. G. Schwacha, A. Ayala, and I. H. Chaudry. 2000. Effect of M. Bauer, R. Bellomo, G. R. Bernard, J. D. Chiche, C. M. Coopersmith, et al. gender and sex hormones on immune responses following shock. Shock 14: 81–90. 2016. The third international consensus definitions for sepsis and septic shock 32. Tsuyuguchi, K., K. Suzuki, H. Matsumoto, E. Tanaka, R. Amitani, and F. Kuze. (sepsis-3). JAMA 315: 801–810. 2001. Effect of oestrogen on Mycobacterium avium complex pulmonary infec- 2. Rittirsch, D., M. A. Flierl, and P. A. Ward. 2008. Harmful molecular mechanisms tion in mice. Clin. Exp. Immunol. 123: 428–434. in sepsis. Nat. Rev. Immunol. 8: 776–787. 33. Leone, M., J. Textoris, C. Capo, and J. Mege. 2012. Sex hormones and bacterial 3. Cohen, J. 2002. The immunopathogenesis of sepsis. Nature 420: 885–891. infections. In Sex Hormones. R. Dubey, ed. InTech, Rijeka, Croatia. Available at: 4. Seeley, E. J., M. A. Matthay, and P. J. Wolters. 2012. Inflection points in sepsis https://www.intechopen.com/books/sex-hormones/sex-hormones-and-bacterial- biology: from local defense to systemic organ injury. Am. J. Physiol. Lung Cell. infections. Mol. Physiol. 303: L355–L363. 34. Murray, P. J., and T. A. Wynn. 2011. Protective and pathogenic functions of 5. Angele, M. K., S. Pratschke, W. J. Hubbard, and I. H. Chaudry. 2014. Gender macrophage subsets. Nat. Rev. Immunol. 11: 723–737. differences in sepsis: cardiovascular and immunological aspects. Virulence 5: 35. Fairweather, D., and D. Cihakova. 2009. Alternatively activated macrophages in 12–19. infection and autoimmunity. J. Autoimmun. 33: 222–230. 6. Ishibashi, T., D. P. Bottaro, A. Chan, T. Miki, and S. A. Aaronson. 1992. Ex- 36. Bolego, C., A. Cignarella, B. Staels, and G. Chinetti-Gbaguidi. 2013. Macro- pression cloning of a human dual-specificity phosphatase. Proc. Natl. Acad. Sci. phage function and polarization in cardiovascular disease: a role of estrogen USA 89: 12170–12174. signaling? Arterioscler. Thromb. Vasc. Biol. 33: 1127–1134. 7. Yuvaniyama, J., J. M. Denu, J. E. Dixon, and M. A. Saper. 1996. Crystal structure 37. Ribas, V., B. G. Drew, A. Le, T. Soleymani, P. Daraei, D. Sitz, D. C. Henstridge, of the dual specificity protein phosphatase VHR. Science. 272: 1328–1331. M. A. Febbraio, S. C. Hewitt, K. S. Korach, et al 2011. Myeloid-specific estrogen 8. Alonso, A., M. Saxena, S. Williams, and T. Mustelin. 2001. Inhibitory role for receptor alpha deficiency impairs metabolic homeostasis and accelerates ath- dual specificity phosphatase VHR in T cell antigen receptor and CD28-induced erosclerotic lesion development. [Published erratum appears in 2012 Proc. Natl. Erk and Jnk activation. J. Biol. Chem. 276: 4766–4771. Acad. Sci. USA 109: 645.] Proc. Natl. Acad. Sci. USA 108: 16457–16462. The Journal of Immunology 13

38. Carreras, E., S. Turner, M. B. Frank, N. Knowlton, J. Osban, M. Centola, the transcription factors NF-IL6 and NF-kappa B by the estrogen receptor. FEBS C. G. Park, A. Simmons, J. Alberola-Ila, and S. Kovats. 2010. Estrogen receptor Lett. 409: 79–85. signaling promotes dendritic cell differentiation by increasing expression of the 42. Ma, L., Z. Chen, H. Erdjument-Bromage, P. Tempst, and P. P. Pandolfi. 2005. transcription factor IRF4. Blood 115: 238–246. Phosphorylation and functional inactivation of TSC2 by Erk implications for 39. Angele, M. K., M. W. Kno¨ferl, M. G. Schwacha, A. Ayala, W. G. Cioffi, tuberous sclerosis and cancer pathogenesis. Cell 121: 179–193. K. I. Bland, and I. H. Chaudry. 1999. Sex steroids regulate pro- and anti- 43. Byles, V., A. J. Covarrubias, I. Ben-Sahra, D. W. Lamming, D. M. Sabatini, inflammatory cytokine release by macrophages after trauma-hemorrhage. Am. B. D. Manning, and T. Horng. 2013. The TSC-mTOR pathway regulates mac- J. Physiol. 277: C35–C42. rophage polarization. Nat. Commun. 4: 2834. 40. Srivastava, S., M. N. Weitzmann, S. Cenci, F. P. Ross, S. Adler, and R. Pacifici. 44. Inoki, K., Y. Li, T. Zhu, J. Wu, and K. L. Guan. 2002. TSC2 is phosphorylated and 1999. Estrogen decreases TNF gene expression by blocking JNK activity and the inhibited by Akt and suppresses mTOR signalling. Nat. Cell Biol. 4: 648–657. resulting production of c-Jun and JunD. J. Clin. Invest. 104: 503–513. 45. Covarrubias, A. J., H. I. Aksoylar, and T. Horng. 2015. Control of macrophage 41. Ray, P., S. K. Ghosh, D. H. Zhang, and A. Ray. 1997. Repression of interleukin-6 metabolism and activation by mTOR and Akt signaling. Semin. Immunol. 27: gene expression by 17 beta-estradiol: inhibition of the DNA-binding activity of 286–296.