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 DUSP3−/− 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.

Sepsis and septic shock are complex clinical syndromes that arise when the local body response to pathogens becomes systemic and injures its own tissues and organs (1). When infection occurs, bacterial components, such as LPS, are recognized by the host, and inflammation is initiated. The TLR4 pathway is activated and triggers the release of cytokines, chemokines, and NO (2, 3). Systemic release of proinflammatory cytokines causes large-scale cellular and tissue injuries, leading to microvascular disruption, severe organ dysfunction, and, eventually, death (4). Sepsis occurrence and outcome depend on pathogen characteristics, as well as on risk factors, such as age or sex (1). Indeed, women are better protected against infection and sepsis compared with men. Women younger than 50 y show a lower incidence of severe sepsis and a better survival compared with age-matched men. This may be explained by the influence of female sex hormones on the immune system responses (5).

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 (6). DUSP3’s broader catalytic site allows the protein to dephosphorylate phospho-Tyr and phospho-Thr residues (7). The MAPK ERK1/2 and JNK were the first reported DUSP3 substrates (810). Other substrates, such as the EGFR and ErbB2 tyrosine receptors (11) and STAT5 transcription factor (12), were also reported. DUSP3’s physiological functions began to be elucidated as a result of the knockout (KO) mouse that we have generated. We have previously reported that DUSP3 plays an important role in platelets biology in monocytes, macrophages, and endothelial cells (1315). In platelets, DUSP3 plays an important role in arterial thrombosis and platelet activation through GPVI and CLEC-2 signaling pathways (14). DUSP3 also plays an important role in endothelial cells and angiogenesis and seems to act as a proangiogenic factor (16). Surprisingly, this function was not correlated with reduced tumor or metastatic growth. Indeed, in an experimental metastasis model using Lewis lung carcinoma cells, we found instead that DUSP3 plays an antitumor role, because DUSP3−/− mice were more sensitive to Lewis lung carcinoma cell metastatic growth compared with wild-type (WT) littermates. This enhanced tumor growth in DUSP3−/− mice was associated with greater recruitment of M2-like macrophages (M. Vandereyken, E. Van Overmeire, M. Amand, N. Rocks, C. Delierneux, P. Singh, M. Singh, C. Wathieu, T. Zurashvilli, N.E. Sounni, M. Moutschen, C. Gilles, C. Oury, D. Cataldo, J.A. Van Ginderachter, and S. Rahmouni, unpublished data). We and others showed that DUSP3 was downregulated in some human cancers and upregulated in others (reviewed in Refs. 16, 17). Further studies are required to better understand the role of this phosphatase in cancer biology.

DUSP3 also plays an important role in immune cell functions. In T cells, DUSP3 can be activated by ZAP-70 tyrosine kinase after TCR triggering (18). This activation, through tyrosine phosphorylation of DUSP3, allows the targeting of the MAPK ERK1/2 and the activation of its downstream signaling pathway. Moreover, in Jurkat leukemia T cells, DUSP3 targets ERK and JNK but not p38. Together, these data suggest that DUSP3 controls T cell physiological functions, at least in part, through the MAPKs ERK and JNK (8). In innate immune cells, we recently showed that DUSP3 is the most highly expressed atypical dual-specificity phosphatase in human monocytes. This was also true in mice (15). These findings suggested to us that DUSP3 could play an important role in innate immune responses. Indeed, using DUSP3−/− mice, we found that DUSP3 deletion conferred resistance to LPS-induced endotoxemia and polymicrobial infection–induced septic shock to female mice. This protection was macrophage dependent, and correlated with a higher percentage of M2-like macrophages in DUSP3−/− mice. Moreover, the resistance was associated with decreased phosphorylation of the tyrosine kinases ERK1/2 and a subsequent decrease in TNF-α production (15).

In this article, we report that DUSP3 deletion does not protect male mice from LPS-induced endotoxemia and cecal ligation and puncture (CLP)-induced septic shock and that this protection was dependent on female sex hormones. Furthermore, we report that sepsis resistance was associated with a higher percentage of M2-like macrophages in the peritoneal cavity of DUSP3−/− female mice but not with decreased production of proinflammatory cytokines. We also showed that sepsis resistance in females, but not in males or in ovariectomized (OVX) females, was associated with decreased ERK1/2, PI3K, and Akt activation.

C57BL/6-CD45.2 DUSP3−/− mice were generated by homologous recombination as previously reported (13). These mice were backcrossed with C57BL/6-CD45.2 mice (Charles River) to create heterozygotes that were mated to generate DUSP3+/+ and DUSP3−/− littermate colonies used for experimentation. Age-matched male and female DUSP3+/+ and DUSP3−/− mice were used in all experiments. Mice were kept in ventilated cages under a 12-h dark/light cycle in a specific pathogen–free animal facility and received food and water ad libitum. Health status was evaluated every 3 mo, and mice were always found to be free of specific pathogens.

All mouse experiments and procedures were approved by the animal ethics committees of the Universities of Ghent and Liege and were carried out according to their guidelines.

CLP was performed as previously described (19). For LPS challenge, mice were injected i.p. with 6 mg/kg LPS. Body temperature was monitored using a rectal thermometer at various times after LPS injection and after CLP. Death of mice was recorded, and the data were analyzed for statistical significance of differences between the experimental groups.

Ten- to twelve-week-old C57BL/6-CD45.2 donor mice were killed by cervical dislocation. Tibiae and femurs were collected, and bone marrow (BM) cells were flushed with PBS. BM cells (10 × 106) were immediately injected i.v. into 6–8-wk-old lethally irradiated (866.3 cGy) C57BL/6-CD45.1 recipient mice. Four weeks later, transplantation efficiency was evaluated based on the ratio of CD45.2/CD45.1 cells in the blood of transplanted mice.

Four-week-old females were anesthetized using ketamine/xylazine (150 and 20 mg/kg). A vertical incision of 2–3 cm was performed in the middle of the back. One centimeter lateral of the midline, another incision of 2–3 mm was performed in the fascia. Adipose tissue surrounding the ovary was pulled out, and the ovary was removed after clamping. The same operation was performed for the contralateral ovary. The incision in fascia was closed with stitches, and the skin incision was closed with clips. Sham-operated mice were used as a control. All of the above procedures were applied to these mice, with the exception of the removal of ovaries. For in vivo estrogen complementation, s.c. implants for controlled release of 17β-estradiol (1.5 μg/d; Belma Technologies) were applied to OVX mice 2 wk after surgery, and mice were sacrificed 3 wk later.

The following materials were from Cell Signaling Technology: anti–phospho-Akt (Ser473), anti-Akt, anti–phospho-ERK1/2 (Thr202/Tyr204), anti-ERK, anti–phospho-PI3K p85 (Tyr458)/p55 (Tyr199), anti-PI3K p85, and anti–phospho-GSK3α/β (Ser21/9). Anti-GSK3α/β was from Santa Cruz Biotechnology, anti-GAPDH Ab was from Sigma, HRP-conjugated anti-goat Ab was from Dako, HRP-conjugated anti-mouse Ab was from GE Healthcare, and HRP-conjugated anti-rabbit Ab was from Merck Millipore. Allophycocyanin–anti-CD45.1 (A20), PerCP–Cy5.5–anti-CD45.2 (104), FITC-anti-CD11b, allophycocyanin–Cy7–anti-Ly6G, PE–anti-CD3, PerCP–anti-CD8, FITC–anti-CD4, biotin–anti-B220, and streptavidin–PE–Cy7 were from BD Biosciences. Allophycocyanin–anti-F4/80, PerCP–Cy5–anti-NK1.1, and PerCP–Cy5.5–anti-CD11b were from eBioscience, and PE–Cy–anti-Ly6G Ab was from BioLegend. LPS from Escherichia coli serotype O111:B4 was from Sigma and was diluted in pyrogen-free PBS.

Peripheral blood was drawn into EDTA-coated tubes (BD Microtainer K2E tubes; BD Biosciences) by puncturing the heart with a 26G needle. Centrifugation was performed twice at 800 × g for 15 min at room temperature (RT). Plasma samples were separated in sterile Eppendorf tubes, aliquoted in small volumes, and stored at −80°C until used.

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 IFN-γ, samples were diluted twice. Samples were loaded onto 96-well 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 addition using an MSD instrument.

Peritoneal washes were performed 4 d after i.p. injection of 1 ml of 4% thioglycollate broth (Sigma). Five milliliters of 0.6 mM PBS-EDTA was injected twice in the peritoneal cavity using an 18G needle and then collected. Peritoneal macrophages (PMs) were selected by adherence to plastic tissue culture dishes in complete RPMI 1640 medium. PMs were stimulated with 1 μg/ml LPS for 15, 30, or 60 min or for 8 or 24 h, depending on the experiment performed

Peritoneal washes were centrifuged for 10 min at 350 × g, and the pellets were resuspended in PBS. For cell surface staining, cells were incubated for 15 min with anti-CD16/CD32 (FcγIII/IIR) before labeling with specific Abs for 30 min at 4°C. Cells were washed, fixed with 1% paraformaldehyde solution. Flow cytometry was performed using BD FACSDiva software on FACSCanto II (BD Biosciences) and results were analyzed using FlowJo software (Tree Star).

For Western blot experiments, cells were stimulated for the indicated time points and lysis was performed with RIPA buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM orthovanadate, complete protease inhibitor mixture tablets EDTA free, and 1 mM PMSF) on ice for 20 min. Lysates were clarified by centrifugation at 19,000 × g for 20 min at 4°C. The resulting supernatants were collected, and protein concentrations were determined using the colorimetric Bradford reagent (Bio-Rad). Proteins were denatured at 95°C in Laemmli buffer (40% glycerol, 8% SDS, 20% 2-ME, 20% Tris-HCl 0.5 M (pH 6.8), 0.05% bromophenol blue, and water) for 5 min.

Denatured samples were run on 10% SDS-PAGE gel and transferred onto nitrocellulose membranes. To block the nonspecific binding sites, membranes were incubated for 1 h at RT in TBS-Tween 20 containing 5% nonfat milk or 3% BSA. Membranes were incubated overnight with primary Ab at 4°C, washed three times in TBS-Tween, and incubated with HRP-conjugated secondary Ab for 1 h at RT. The blots were developed by ECL (ECL kit; Amersham), according to the manufacturer’s instructions.

RNA was extracted from PMs using an miRNeasy Mini Kit (QIAGEN), and cDNA was synthesized using Expand reverse transcriptase (Roche), according to the recommendations of the manufacturer. cDNA was amplified using SYBR Green PCR Master Mix (Roche) and 0.3 mM specific primers for Arginase 1 (Arg1), inducible NO synthase (iNOS), and β2-microglobulin (β2M). All quantitative PCRs were performed on a LightCycler Real-Time PCR System (Roche). The ratio between the expression level of the gene of interest and β2M in the sample was defined as the normalization factor. Relative mRNA quantities for Arg1 and iNOS were determined using the ΔCq method. All primers were from Eurogentec. The following sequences were used: iNOS: forward, 5′-GCTTCTGGTCGATGTCATGAG-3′ and reverse, 5′-TCCACCAGGAGATGTTGAAC-3′, Arg1: forward, 5′-CAGAAGAATGGAAGAGTCAG-3′ and reverse, 5′-AGATATGCAGGGAGTCACC-3′, and β2M: forward, 5′-CACCCCACTGAGACTGATACA-3′ and reverse, 5′-TGATGCTTGATCACATGTCTCG-3′.

The Student t test was used to assess statistical differences between groups. Survival differences after LPS challenge and CLP were analyzed by Kaplan–Meier analysis with the log-rank test. Results were considered significant at p < 0.05. Results are presented as mean ± SEM. Prism software (GraphPad) was used to perform the statistical analysis. *p < 0.05, **p < 0.01, ***p < 0.001.

In a previous study, we showed that DUSP3 deletion protected mice from LPS-induced endotoxemia and polymicrobial infection–induced septic shock (15). Only females were used in the first study. To investigate whether the protection observed is a general feature of DUSP3 deletion or is sex dependent, we challenged DUSP3−/− males with a lethal dose of LPS (i.p. injection of 6 mg/kg) and compared their survival with females and with WT control littermates of both sexes. Body temperature was also monitored. As expected and previously reported, 90% of DUSP3−/− female mice were resistant to LPS, whereas only 5% of DUSP3+/+ female mice survived the challenge (15). Interestingly, DUSP3+/+ and DUSP3−/− male mice were equally sensitive to LPS-induced death (Fig. 1A). Body temperature of all groups of mice, with the exception of DUSP3−/− females, decreased after LPS injection. Twenty-four hours later, almost all DUSP3−/− females recovered, whereas the other groups remained hypothermic (Fig. 1B). These results were further confirmed in the CLP model performed on DUSP3+/+ and DUSP3−/− male and female mice. As expected, only 10% of DUSP3+/+ and DUSP3−/− male mice and DUSP3+/+ female mice were still alive by the end of the experiment, whereas 70% of DUSP3−/− female mice survived (Fig. 1C). The body temperature of each group dropped after surgery, and only DUSP3−/− female mice recovered (Fig. 1D). These results indicate a sex-specific response to septic shock in DUSP3−/− mice.

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 DUSP3−/− 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 DUSP3−/− mice before, 6, and 24 h after LPS injection. (C) DUSP3+/+ male (n = 10) and female (n = 11) mice and DUSP3−/− 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 injection. (EG) DUSP3+/+ and DUSP3−/− mice were sham operated (n = 9 for DUSP3+/+ and n = 8 for DUSP3−/−) or OVX (n = 9 for DUSP3+/+ and n = 11 for DUSP3−/−) 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 DUSP3−/− mice before and 8 and 24 h after LPS injection. (HK) A total of 10 × 106 BM cells from DUSP3−/− C57BL/6-CD45.2 female mice was injected i.v. into lethally irradiated DUSP3+/+ C57BL/6-CD45.1 recipient male and female mice (DUSP3−/− → M-DUSP3+/+ and DUSP3−/− → 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-DUSP3−/− → M-DUSP3+/+. Lanes 9-14: F-DUSP3−/− 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.

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 DUSP3−/− 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 DUSP3−/− mice before, 6, and 24 h after LPS injection. (C) DUSP3+/+ male (n = 10) and female (n = 11) mice and DUSP3−/− 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 injection. (EG) DUSP3+/+ and DUSP3−/− mice were sham operated (n = 9 for DUSP3+/+ and n = 8 for DUSP3−/−) or OVX (n = 9 for DUSP3+/+ and n = 11 for DUSP3−/−) 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 DUSP3−/− mice before and 8 and 24 h after LPS injection. (HK) A total of 10 × 106 BM cells from DUSP3−/− C57BL/6-CD45.2 female mice was injected i.v. into lethally irradiated DUSP3+/+ C57BL/6-CD45.1 recipient male and female mice (DUSP3−/− → M-DUSP3+/+ and DUSP3−/− → 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-DUSP3−/− → M-DUSP3+/+. Lanes 9-14: F-DUSP3−/− 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.

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Male and female sex hormone receptors have been identified on immune cells, suggesting direct effects of androgen and estrogen on these cells (20). Sexual steroid hormones have been recognized to influence numerous immune pathophysiological processes (21). To elucidate the effect of female sex hormones, we subjected 4-wk-old DUSP3+/+ and DUSP3−/− mice to ovariectomy. As controls, another group of 4-wk-old DUSP3+/+ and DUSP3−/− mice were sham operated. To assess the efficiency of ovariectomy, we checked the presence and the size of the uterus. Successful OVX mice were deprived of normal uterus development, whereas sham-operated mice presented a normally developed uterus (Fig. 1E). Six weeks after surgery, sham and OVX mice were challenged with 6 mg/kg LPS, and survival and temperature were monitored (Fig. 1F, 1G). Ovariectomy impaired the observed endotoxemia resistance of DUSP3−/− mice, whereas sham-operated DUSP3−/− mice were still fully protected from endotoxin-induced death. These data demonstrate that female sex hormones are involved in the observed resistance of DUSP3−/− female mice to LPS-induced lethality.

We previously showed that adoptive transfer of DUSP3−/− female BM cells or monocytes to DUSP3+/+ female mice was sufficient to transfer resistance to LPS-induced lethality (15). Therefore, we investigated whether this is also true when recipient mice are males. To generate chimeric mice, 1 × 107 BM cells from female DUSP3−/− C57BL/6-CD45.2 mice were injected i.v. into lethally irradiated DUSP3+/+ C57BL/6-CD45.1 recipient male and female mice (DUSP3−/− → M-DUSP3+/+ and DUSP3−/− → F-DUSP3+/+, respectively). As a control, DUSP3+/+ female BM was transplanted into lethally irradiated DUSP3+/+ male or female mice (DUSP3+/+ → M-DUSP3+/+ and DUSP3+/+ → F-DUSP3+/+, respectively). Successful hemato-lymphoid reconstitution was verified by flow cytometry 3–4 wk after the transplantation. Ninety-five percent of peripheral blood cells were CD45.2+ (Fig. 1H, 1I). Moreover, in recipient mice, the expression of DUSP3 in PMs was abolished in the recipient mice transplanted with DUSP3−/− BM cell suspension, as shown by DUSP3 immunoblotting (Fig. 1J). Four weeks after BM transplantation, 6 mg/kg LPS was injected i.p. into recipient mice, and survival was monitored for 8 d (Fig. 1K). Interestingly, >70% of the chimeric DUSP3−/− → F-DUSP3+/+ mice survived until the end of the experiment compared with 9% of DUSP3+/+ → F-DUSP3+/+ mice. In contrast, all DUSP3−/− → M-DUSP3+/+ and DUSP3+/+ → M-DUSP3+/+ mice died within 4 d after LPS injection (Fig. 1K). These data suggest that, in the absence of DUSP3, female sex hormones and BM cells are required for resistance to LPS shock.

We have previously reported that DUSP3 is expressed in several immune cells where it plays an important role in macrophage and T cell functions (15, 18). Because sepsis involves the participation of innate and adaptive immune cells (22), we investigated whether DUSP3 deletion–associated survival of shock in females was linked to an unbalanced contribution of one cell type or another in LPS-resistant mice compared with LPS-sensitive mice. We found that, at baseline, as well as after LPS injection, the percentages of CD19+ B cells, CD4+ T cells, CD8+ T cells, macrophages (Ly6GCD11b+F4/80+), neutrophils (F4/80/CD11b+Ly6G+), NK cells (CD3NK1.1+), and NKT cells (CD3+NK1.1+) were equal in males and females of both genotypes (Fig. 2A). LPS injection induced a significant reduction in T cells and macrophages, increased neutrophil infiltration of the peritoneal cavity, and had no significant impact on the percentages of NK, NKT, and B cells (Fig. 2A).

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 DUSP3−/− 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 B220/NK1.1/CD3+/CD4+, CD8 T cells were B220/NK1.1/CD3+/CD8+, B cells were B220+/NK1.1/CD3, NK cells were B220/CD3NK1.1+, and NKT cells were B220/CD3+NK1.1+. For neutrophils and macrophages, phenotyping was performed using PerCP–Cy5.5–anti-CD11b, allophycocyanin–Cy7–anti-Ly6G, and allophycocyanin–anti-F4/80. Neutrophils were F4/80/CD11b+/Ly6G+, and macrophages were considered Ly6G/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 DUSP3−/− male mice and DUSP3+/+ and DUSP3−/− 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 Ly6G 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 Ly6G 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 β2M. n = 4 mice in each group. Results are presented as mean + SEM. *p < 0.5, **p < 0.01.

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 DUSP3−/− 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 B220/NK1.1/CD3+/CD4+, CD8 T cells were B220/NK1.1/CD3+/CD8+, B cells were B220+/NK1.1/CD3, NK cells were B220/CD3NK1.1+, and NKT cells were B220/CD3+NK1.1+. For neutrophils and macrophages, phenotyping was performed using PerCP–Cy5.5–anti-CD11b, allophycocyanin–Cy7–anti-Ly6G, and allophycocyanin–anti-F4/80. Neutrophils were F4/80/CD11b+/Ly6G+, and macrophages were considered Ly6G/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 DUSP3−/− male mice and DUSP3+/+ and DUSP3−/− 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 Ly6G 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 Ly6G 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 β2M. n = 4 mice in each group. Results are presented as mean + SEM. *p < 0.5, **p < 0.01.

Close modal

We previously reported that increased survival of DUSP3−/− female mice after LPS and CLP was associated with a higher percentage of M2-like macrophages in the peritoneal cavity of these mice compared with DUSP3+/+ females (15). To investigate whether this is associated with DUSP3-deficient female survival, we phenotyped DUSP3+/+ and DUSP3−/− PMs from male and female mice (sham operated and OVX) challenged with LPS, based on the characterization previously reported by Ghosn et al. (23). M1 macrophages are F4/80intCD11bintLy6G, whereas M2-like macrophages are F4/80hiCD11bhiLy6G (Fig. 2B, 2C). We confirmed previous findings showing that the percentage of M2-like macrophages was higher in the peritoneal cavity of DUSP3−/− female mice compared with littermate controls at basal and 24 h after LPS injection (Fig. 2B, 2C). Interestingly, we observed that the percentage of M2-like macrophages in male mice was slightly lower compared with DUSP3−/− female mice at basal level; this difference was exacerbated at 24 h after LPS injection. There was not a significant difference for the percentage of M2-like macrophages between DUSP3+/+ and DUSP3−/− male mice. Similarly, there was no difference in the percentage of M1-like macrophages at basal level and 24 h after LPS injection between DUSP3+/+ and DUSP3−/− female mice. However, we noticed a slight increase in the percentage of M1-like macrophages in male mice compared with female mice at basal level. This difference was accentuated, although not significantly, at 24 h (Fig. 2B, 2C). For OVX mice, the percentage of M1-like macrophages (F4/80intCD11bint) was higher in DUSP3+/+ and DUSP3−/− OVX mice compared with DUSP3−/− sham mice at basal level. The difference was maintained at 24 h after LPS injection, although not significantly (Fig. 2B, 2C). The percentage of M2-like macrophages was equal in DUSP3+/+ and DUSP3−/− OVX mice compared with DUSP3+/+ and DUSP3−/− sham mice at basal level. However, 24 h after LPS challenge, the percentage of M2-like macrophages in the peritoneal cavity of OVX mice decreased, but it did not reach statistical significance compared with DUSP3−/− sham mice (Fig. 2B, 2C). These data suggest that M2-like macrophages could be involved in the resistance to LPS-induced endotoxemia. To further characterize these cells, we measured the relative expression of genes associated with M1-like and M2-like PMs (i.e., Nos2 and Arg1). At baseline, none of the transcript was detected (data not shown). Two hours after LPS challenge, Arg1 expression increased significantly in DUSP3−/− sham mice compared with DUSP3+/+ sham mice (Fig. 2D). In males and OVX groups, Arg1 was detected but at significantly lower levels compared with sham-operated female mice. Twenty-four hours after LPS injection, the level of Arg1 increased dramatically in the DUSP3−/− sham group compared with all other groups (Fig. 2D). The Nos2 level was low 2 h after LPS injection, but it increased significantly 22 h later in sham-operated female mice of both genotypes, although the increase was more significant in DUSP3+/+ female mice (Fig. 2D). Altogether, these data suggest that M2-like macrophages and female hormones could be involved in DUSP3-induced resistance to LPS-induced endotoxemia.

We previously reported that DUSP3−/− female survival to LPS was associated with a decreased systemic TNF level compared with DUSP3+/+ mice (15). Therefore, we wanted to know whether the susceptibility of DUSP3−/− male and OVX mice to LPS-induced death could be linked to differential expression of TNF or to other proinflammatory cytokines, such as IL-6, IFN-γ, and IL-10. We measured and compared plasma levels of these four cytokines at basal levels and at 2 and 24 h after LPS challenge in all groups of mice using an MSD assay. For TNF, there was no difference between DUSP3+/+ and DUSP3−/− male mice. However, and as previously reported (15), there was a significant decrease in this cytokine in DUSP3−/− female mice compared with DUSP3+/+ female mice 2 and 24 h after LPS challenge (Fig. 3A). Compared with DUSP3+/+ mice, DUSP3−/− mice of both sexes had a slight, but nonsignificant, decrease in IL-6 2 h after LPS injection (Fig. 3B). These differences were maintained in OVX mice groups (Fig. 3A, 3B). For IFN-γ, secretion was equal in all groups of mice 2 h after LPS challenge. At 24 h after LPS injection, IFN-γ levels were lower in sham and OVX DUSP3−/− females compared with sham and OVX DUSP3+/+ females. However, there was a 10-fold decrease in IFN-γ in all OVX mice, regardless of their genotype. In males, the level of IFN-γ was significantly higher in DUSP3−/− mice than in the littermate controls at 24 h, but not at 2 h, after LPS injection (Fig. 3C). Finally, the level of IL-10 was lower in DUSP3−/− mice compared with controls, regardless of sex or type of surgery (Fig. 3D). Altogether, these data strongly suggest that DUSP3 deletion–induced resistance to LPS-induced shock in female mice is not a consequence of the observed modifications of the measured cytokines.

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-γ (C), and IL-10 (D) in DUSP3+/+ and DUSP3−/− 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.

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-γ (C), and IL-10 (D) in DUSP3+/+ and DUSP3−/− 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.

Close modal

We have previously reported that, although DUSP3 is a ubiquitously expressed protein, the level of expression varies significantly between cell types (14, 15) and during cell cycle progression (24). Therefore, we investigated whether its expression varies between males and females and changes in response to LPS or after ovariectomy. As shown in Fig. 4A, DUSP3 expression level was similar in males and females and was not influenced by LPS or OVX (Fig. 4A).

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 DUSP3−/− 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.

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 DUSP3−/− 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.

Close modal

We have previously reported that DUSP3 deletion in female mice macrophages was associated with decreased ERK1/2 phosphorylation levels after ex vivo LPS stimulation (15). To investigate whether this alteration was also associated with the sex-specific resistance to septic shock, DUSP3+/+ and DUSP3−/− PMs from sham or OVX mice were stimulated ex vivo with LPS (1 μg/ml) at different time points, and cell lysates were probed with phospho-specific ERK1/2 Abs. As expected, ERK1/2 phosphorylation was significantly lower in DUSP3−/− sham PMs at all time points compared with DUSP3+/+ macrophages. Interestingly, in OVX mice, LPS stimulation led to equal ERK1/2 activation in DUSP3−/− and DUSP3+/+ PMs, as demonstrated by the observed phosphorylation levels. There was no difference in ERK1/2 phosphorylation in male mice from both genotypes (Fig. 4B, 4C).

The observed reduced phosphorylation of ERK1/2 in DUSP3−/− sham mice suggests that DUSP3 could be targeting ERK1/2 upstream kinase or one of the ERK1/2 phosphatases. Therefore, we analyzed MAPKK MEK1/2 activation following ex vivo LPS stimulation (1 μg/ml) of PMs. MEK1/2 kinetic phosphorylation was equal between DUSP3+/+ and DUSP3−/− sham mice of both sexes (Fig. 4D, 4E), suggesting that MEK1/2 is not targeted by DUSP3.

The PI3K/Akt pathway is another important pathway that is activated after TLR4 triggering (25). Therefore, we investigated whether DUSP3 deletion could impact this pathway after activation with LPS and whether the kinetics and magnitude of this activation could be sex dependent. PI3K and Akt activation was evaluated using phospho-specific Abs and Western blot after ex vivo LPS stimulation (1 μg/ml) of PMs at different time points. Interestingly, PI3K and Akt activation decreased in DUSP3−/− sham PMs compared with DUSP3+/+ PMs at all time points. This difference was abolished in OVX mice, because the phosphorylation level of PI3K and Akt remained equal between DUSP3+/+ and DUSP3−/− PMs. However, activation of GSK3 downstream target of Akt was not affected by DUSP3 deficiency in sham or OVX mice (Fig. 5). There was no difference in PI3K or Akt activation in male PMs after LPS stimulation. PI3K and Akt were equally activated at all time points in DUSP3+/+ and DUSP3−/− LPS- stimulated PMs, and GSK3 activation was not affected by DUSP3 deficiency (Fig. 5).

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 DUSP3−/− female, male, and OVX mice were stimulated ex vivo with 1 μg/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-GSK3α/β (Ser21/9) and anti-PI3K, anti-Akt, and anti-GSK3α/β as loading controls. (B) Densitometry quantifications of phospho-PI3K, phospho-Akt, phospho-GSK3α/β, PI3K, Akt, and GSK3α/β. Results are presented as the ratio of phospho-PI3K/PI3K, phospho-Akt/Akt, and phospho-GSK3α/β/GSK3α/β 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.

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 DUSP3−/− female, male, and OVX mice were stimulated ex vivo with 1 μg/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-GSK3α/β (Ser21/9) and anti-PI3K, anti-Akt, and anti-GSK3α/β as loading controls. (B) Densitometry quantifications of phospho-PI3K, phospho-Akt, phospho-GSK3α/β, PI3K, Akt, and GSK3α/β. Results are presented as the ratio of phospho-PI3K/PI3K, phospho-Akt/Akt, and phospho-GSK3α/β/GSK3α/β 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.

Close modal

These data suggest that DUSP3 affects ERK1/2, PI3K, and Akt activation, probably in concert with estrogens. To investigate this hypothesis, DUSP3−/− and DUSP3+/+ female mice underwent ovariectomy at the age of 4 wk. Two weeks later, half of the mice from each group received estrogen via s.c. implant for controlled release of 17β-estradiol (1.5 μg/d). Mice were kept for 3 wk before sacrifice. PMs were stimulated ex vivo with LPS (1 μg/ml) at different time points, and cell lysates were probed with anti–phospho-ERK1/2, anti-ERK, anti–phospho-PI3K, anti-PI3K, anti–phospho-Akt, and anti-Akt Abs. As shown in Fig. 6, estrogen complementation significantly reduced the phosphorylation levels of ERK1/2 and Akt in DUSP3−/−, but not in DUSP3+/+, PMs (Fig. 6). These data clearly suggest that the DUSP3-dependent reduced phosphorylation of ERK1/2 and Akt is estrogen dependent.

FIGURE 6.

Alteration of ERK1/2 and Akt phosphorylation in DUSP3−/− female macrophages is estrogen dependent. PMs isolated from OVX DUSP3+/+ and DUSP3−/− mice and from OVX DUSP3+/+ and DUSP3−/− mice receiving estrogen complementation (3 wk, 1.5 μg/d) were stimulated ex vivo with 1 μg/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.

FIGURE 6.

Alteration of ERK1/2 and Akt phosphorylation in DUSP3−/− female macrophages is estrogen dependent. PMs isolated from OVX DUSP3+/+ and DUSP3−/− mice and from OVX DUSP3+/+ and DUSP3−/− mice receiving estrogen complementation (3 wk, 1.5 μg/d) were stimulated ex vivo with 1 μg/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.

Close modal

It is well recognized that immune responses to infection are sex dependent. Indeed, stronger immune responses confer protection against infections and sepsis to women (26). Several epidemiological studies have been performed and showed a greater incidence of sepsis in males compared with females (27). Consequently, compared with males, there are less female hospitalizations associated with infections. In addition, male sex and the presence of comorbidities were commonly reported independent predictors of postacute mortality in sepsis survivors (28). Interestingly, many of the differences between males and females in response to infections become apparent at puberty (29). In line with this, women younger than 50 y of age show a lower incidence of severe sepsis and better survival compared with age-matched men (30). Altogether, these observations suggest a role for sexual hormones in the protection from severe infections and sepsis. This hypothesis has been supported by the finding that receptors for reproductive hormones are present in a variety of immune cell types (31). In contrast, estrogen has been demonstrated to increase resistance to several bacterial infections, whereas the removal of endogenous estrogens have been shown, for example, to markedly increase the severity of Mycobacterium avium infections, an effect that can be reversed after 17β-estradiol replacement (32, 33). However, the role of female reproductive hormones in the susceptibility to acute infection and sepsis remains poorly understood.

In this article, we report that DUSP3 deletion confers resistance to LPS-induced lethality and to polymicrobial-induced septic shock in female mice but not in male mice. We demonstrated that this protection is dependent on female sexual hormones and monocytes/macrophages. Indeed, ovariectomy induced a loss of resistance. In contrast, DUSP3−/− monocyte transfer to WT females was sufficient to transfer the resistance to WT recipient mice (15); however, this protection was not due to decreased TNF production, as suggested by our previous study (15). To our knowledge, this is the first report demonstrating a signaling molecule–induced synergistic immunoprotective effect of monocytes/macrophages and female sexual hormones against sepsis.

The observed resistance to LPS-induced septic shock in DUSP3−/− female mice was associated with a modest increase in M2-like macrophages in the peritoneal cavity of mice. This observation was strengthened by the increase in Arg1 gene expression in DUSP3−/− female mice but not in male or OVX mice. Arg1 is a known marker for M2-like macrophages (34). Ovariectomy of DUSP3-deficient mice induced a loss of resistance to LPS-induced death, with no difference in the percentage of M2-like macrophages between control groups and OVX DUSP3−/− mice. Together with the fact that the percentage of M2-like macrophages was also equal in DUSP3+/+ and DUSP3−/− male mice, it suggests that female sex hormones may influence the alternative activation of macrophages. Our observations are in line with studies showing that estrogens influence numerous immunological processes, including the physiological functions of monocytes and macrophages (35). Indeed, ovarian sex hormones modulate monocyte adhesion and chemotaxis, TLR expression, cytokine production, and phagocytosis activity (36). Moreover, several lines of evidence suggest that estrogens also influence macrophage polarization. ER-α–KO mice undergo a decrease in alternative activated macrophages (36). ER-α–deficient macrophages are indeed refractory to IL-4–induced alternative activation, as demonstrated by a decrease in IL-4R and STAT6 phosphorylation in these cells (37). Estrogens have also been reported to increase the expression of the transcription factor IFN regulatory factor-4, which is involved in the alternative activation of macrophages (38). Using a transcriptomic assay, we did not observe differences in IL-4, IL-4R, or IFN regulatory factor-4 expression levels between DUSP3-KO males and females at baseline or after LPS challenge (data not shown). In contrast, TNF production does not seem to play a role in the observed phenotype, because ovariectomy of DUSP3−/− mice did not influence the level of this proinflammatory cytokine, although mice did succumb to endotoxemia. These data were rather surprising because sex steroids are known to regulate pro- and anti-inflammatory cytokine levels released by macrophages. In contrast, female sex hormones are known to negatively regulate the production of TNF (39), one of the most important cytokines in sepsis (40, 41). Therefore, the change in TNF production, as well as the observed changes in IFN-γ, IL-6, IL-10, and, perhaps other cytokines, upon DUSP3 deletion, should be considered an independent phenomenon that is not related to DUSP3−/− female mice survival to sepsis.

How DUSP3 regulates macrophage alternative activation in a female sexual hormone–dependent manner is a complex question to answer. The molecular mechanisms involved are probably linked to the observed decrease in ERK1/2 and Akt/PI3K activation. Upon ex vivo LPS stimulation, DUSP3−/− female PMs showed reduced phosphorylation of ERK1/2 and Akt compared with DUSP3+/+ female macrophages. These differences were not observed in macrophages from OVX DUSP3−/− mice, but they were maintained in DUSP3−/− OVX mice receiving estrogen complementation. Together, these data suggest that, under inflammatory conditions, estrogen controls macrophage polarization through the DUSP3–ERK1/2–Akt signaling pathway axis.

ERK1/2 was previously reported to play a role in macrophage polarization through the mTOR signaling pathway (42). Indeed, ERK1/2 phosphorylates and dissociates the tuberous sclerosis protein (TSC) complex, leading to its inactivation and the subsequent activation of mTOR (42), which, in turn, leads to decreased IL-4–induced M2 polarization in TSC-deficient mice (42, 43). The role of sex hormones has not been investigated in these studies. In our model, it would be interesting to investigate whether the observed decreased phosphorylation of ERK1/2 in DUSP3−/− female PMs could lead to TSC activation and, consequently, to M2 polarization. In contrast, it has been reported that, upon TLR4 stimulation, PI3K engagement is followed by Akt and mTORC1 activation that is due to TSC inactivation by Akt (44); this may lead to the polarization of M1 macrophages (44, 45). Similarly to decreased ERK phosphorylation, decreased PI3K/Akt activation may lead to TSC activation and shift macrophage polarization toward the M2 phenotype.

Another important question raised by our study is how does DUSP3 deletion lead to decreased activation of ERK1/2 and Akt signaling molecules under the control of estrogen? Decreased phosphorylation of these kinases clearly suggests that they are not directly targeted by DUSP3. The observed decreased phosphorylation of ERK1/2 and Akt could be due to reduced activation of specific ERK1/2 and PI3K/Akt, yet unknown, phosphatases. Indeed, preliminary data show that pervanadate (nonspecific protein tyrosine phosphatases inhibitor) treatment of LPS-stimulated PMs restores ERK1/2 phosphorylation, whereas okadaic acid (inhibitor of Ser/Thr PP1/PP2A), at low and high concentrations, did not (data not shown). Further investigations using phosphoproteomic approaches, among others, are required to confirm this hypothesis, identify the specific substrate(s) for DUSP3, and assess the exact role of this phosphatase in TLR4 signaling under the influence of female sex hormones.

In summary, we identified DUSP3 as a new key signaling molecule that plays an important role in macrophage alternative activation and sexual dimorphism in the innate immune response to infection. Our data suggest that DUSP3 inhibition, combined with estrogen administration, may lead to protection from sepsis and septic shock.

We thank the GIGA-animal, GIGA-imaging, and GIGA-immunohistochemistry core facilities for technical assistance and help.

This work was supported by the Fonds Léon Fredericq and Centre anticancereux près de l'Université de Liège and by the Fond National de la Recherche Scientifique (FNRS; to S.R.). This work was also supported by the Agency for Innovation of Science and Technology in Flanders, the Research Council of Ghent University (Geconcerteerde Onderzoeksacties Program), the Research Foundation Flanders, European Commission 7th Framework Programme COST Action BM1402, and Belgian Science Policy Program Interuniversity Attraction Poles Grant IAP-VI-18 (to C.R.F.L.). M.M.V. and M.A. are FNRS-Télévie Ph.D. fellows.

Abbreviations used in this article:

     
  • Arg1

    arginase 1

  •  
  • BM

    bone marrow

  •  
  • CLP

    cecal ligation and puncture

  •  
  • DUSP3

    dual-specificity phosphatase 3

  •  
  • iNOS

    inducible NO synthase

  •  
  • KO

    knockout

  •  
  • β2M

    β2-microglobulin

  •  
  • MSD

    Meso Scale Discovery

  •  
  • OVX

    ovariectomized

  •  
  • PM

    peritoneal macrophage

  •  
  • RT

    room temperature

  •  
  • TSC

    tuberous sclerosis protein

  •  
  • WT

    wild-type.

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The authors have no financial conflicts of interest.