Pathogenic Leptospira interrogans serovar Lai strain Lai (56601) was grown in liquid Ellinghausen–McCullough–Johnson–Harris (EMJH) medium at 29°C. The virulence of the leptospira was maintained by passage in hamster. Before use, bacterial concentration was determined using a Petroff–Hausser counting chamber. Leptospira was passaged less than three times in liquid EMJH for all infection studies. C57BL/6J wild-type (WT) mice were provided by the Model Animal Research Center of Nanjing University. C57BL/6J NLRP3-dificient (NLRP3^−/−) and NLRC4-dificient (NLRC4^−/−) mice were kindly provided by Dr. Feng Shao. Syrian golden hamsters (Mesocricetus auratus) were provided by the Animal Center of Jilin University. All animal experiments were performed according to regulations of the Administration of Affairs Concerning Experimental Animals in China. The protocol was approved by the Institutional Animal Care and Use Committee of Jilin University (20170318).

The extraction and culture of primary mouse macrophages were completed as previously described (14). In brief, WT, NLRP3^−/−, and NLRC4^−/− mice were injected with 2 ml of 3% thioglycolate medium (BD Biosciences, USA). Three days after the injection, peritoneal macrophages were isolated by washing the peritoneal cavity with PBS. The primary mouse macrophages and murine macrophage cell line J774A.1 were cultured in DMEM media (HyClone, USA) supplemented with 10% fetal bovine serum (FBS; HyClone, USA), 100 U/ml penicillin and 100 µg/ml streptomycin in 5% CO₂ atmosphere at 37°C. Before infection, cells were washed three times by DMEM media to dislodge the penicillin and streptomycin.

J774A.1 cells at a density of 10³ were incubated with Dox (0–20 µg/ml) for 24 and 48 h at 37°C in a 96-well cell culture plate, followed by the addition of 10 μl/well CCK-8 (TransGen Biotech, China). After incubated for 2 h at 37°C, the optical density of the samples at 450 nm was measured.

J774A.1 cells and primary mouse macrophages were seeded at 10⁶ cells in six-well culture plates and pre-incubated for 12 h in DMEM media supplemented with 10% FBS. In all experiments, unless otherwise noted, macrophages were incubated in the presence or absence of various concentrations of Dox (Sigma-Aldrich, USA) added 24 h prior to 10⁸ live or sonicated leptospira infection (MOI of 100:1) (7, 15). The cells were then washed by culture medium and incubated for another 24 h with live or sonicated leptospira alone or with added various concentrations of Dox (15). To activate NLRP3 inflammasome, J774A.1 cells were incubated in the presence or absence of Dox added 24 h prior to stimulate with LPS (1 µg/ml) from Escherichia coli 0111:B4 (Sigma-Aldrich, USA) for 4 h. The cells were then washed by culture medium and incubated for 2 h with ATP (5 mM) (Sigma-Aldrich, USA) alone or with added Dox. To distinguish priming from activating, J774A.1 cells were primed with LPS (1 µg/ml) for 4 h, and then incubated with Dox for 1 h before being pulsed with ATP (5 mM) for 2 h. To know how long these effects last once Dox has been removed, J774A.1 cells were incubated with Dox (20 µg/ml) for 24 h. After 0–4 h once Dox has been removed, the cells were stimulated with live leptospira for another 24 h without Dox.

Mice were injected intraperitoneally with Dox at the dose of 5 mg/kg before 24 h inoculated intraperitoneally with 2 × 10⁸ sonicated leptospira. Another 24 h later, mice were euthanized and their peritoneal cavities were lavaged with 5 ml of PBS and centrifuged at 2,000 rpm for 10 min as in a previous study (16). The cytokine expressions of the supernatants (Sups) were analyzed by enzyme-linked immunosorbent assay (ELISA). To verify the immunoregulatory activity of Dox against acute leptospirosis, hamsters were inoculated intraperitoneally with 10⁷ live leptospira, which induced 100% mortality in hamsters (17). Dox at the dose of 5 mg/kg was performed intraperitoneally at 48 and 72 h after infection (18). At 96 h after infection, hamsters were euthanized and their organs were collected.

Total RNA of cells and organs were extracted using TRIzol (Invitrogen, USA) following the manufacturer’s instructions. RNA was reverse transcribed into cDNA by using random primers from a TransScript One-Step gDNA Removal kit and cDNA Synthesis SuperMix (TransGen Biotech, China). The primers used in this study were listed in Table 1. The qPCR reaction was performed using an Applied Bioscience 7500 thermocycler and FastStart Universal SYBR Green Master (Roche Applied Science, Germany). PCR conditions were as follows: 50°C for 2 min, 95°C for 10 min, followed by 45 cycles of amplification 95°C for 15 s and 60°C for 60 s (14). The number of target gene was normalized to GAPDH using a 2^−ΔΔCT method.

The suspensions of J774A.1 cells in six-well culture plates and the peritoneal cavities of mice were centrifuged at 2,000 rpm for 10 min at 4°C, then stored at −80°C. Cytokines were measured in Sups subsequently using mouse ELISA kits (eBioscience, USA) according to the instructions of the manufacturer. Three biological replicates were carried out.

J774A.1 cells at a density of 10⁴ were incubated in the presence or absence of Dox in 96-well culture plates for 24 h. Then cells were stimulated by live/dead leptospira and LPS/ATP with the presence or absence of Dox for another 24 h. The LDH release was tested by a Cytotoxicity Detection Kit (Roche Applied Science, Germany) according to the instructions of the manufacturer.

Total proteins of cells were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Scientific, USA). Protein concentrations were determined by Pierce BCA protein assay kit (Thermo Scientific, USA). Protein samples (50 μg/well) were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred onto the polyvinylidene difluoride membrane. After blocked 2 h at room temperature with 5% skim milk dissolved in Tris buffer solution containing 0.05% Tween-20 (TBS-T), and followed by washing with TBS-T, the membrane was incubated with primary antibodies overnight at 4°C. Subsequently, the membrane was washed and then incubated with secondary antibody at room temperature for 2 h. After washed in TBS-T three times, membranes were tested by ECL Plus Western Blotting Detection System (Amersham Life Science, UK). The primary antibodies used were rabbit monoclonal antibodies, and the secondary antibody used was HRP-conjugated goat anti-rabbit antibody (Cell Signaling Technology, Inc., USA). Antibodies p65, p-p65, p38, ERK, p-ERK, NLRP3, and IL-1β were purchased from Cell Signaling Technology, Inc. in USA. Antibody p-p38 was purchased from ABclonal in USA. Antibody β-actin was purchased from Bioworld in USA and caspase-1 was from Abcam in USA. The gray values of the western blotting bands were procured using Image-Pro Plus 6.0 software.

Data are presented as mean ± SD. Statistical analyses were performed by One-way ANOVA followed by the Newman–Keuls test. Differences were considered significant at p < 0.05.

To analyze the potential anti-inflammatory effects of Dox, IL-1β, and TNF-α, mRNA were analyzed by qRT-PCR, protein levels in Sup were tested by ELISA in leptospira-infected J774A.1 cells. Dox at the doses of 5, 10, and 20 µg/ml was not cytotoxic to J774A.1 cells for 24 and 48 h (Figure S1 in Supplementary Material). The live leptospira-induced high expressions and releases of IL-1β and TNF-α compared with the untreated control group (Figures 1A,B,E,F). Interestingly, after treatment with different concentrations of Dox, only the IL-1β expression and release were reduced dramatically in leptospira-infected J774A.1 cells (Figures 1A,B). In order to eliminate the influence of antibiotic sterilization function on live leptospira, this experiment was repeated using sonicated leptospira. The sonicated leptospira also induced high expressions and releases of IL-1β and TNF-α (Figures 1C,D,G,H). Compared with the live leptospira, sonicated leptospira-induced less IL-1β mRNA expression and protein release (Figures 1A–D). Similarly, when Dox was treated, only the IL-1β expression and release were reduced in sonicated leptospira-infected J774A.1 cells (Figures 1C,D). These results showed that Dox specifically inhibited leptospira-induced IL-1β but not TNF-α mRNA and protein level in macrophages of mice.

MAPK and NF-κB pathways play critical roles in the induction of proinflammatory gene expression. To confirm whether the inhibition of IL-1β by Dox is mediated through the MAPK and NF-κB pathways, activation of p38, ERK, and p65 was examined by western blot in sonicated leptospira-infected J774A.1 cells. In the MAPK pathway, Dox inhibited the activation of p38 and ERK in sonicated leptospira-infected J774A.1 cells (Figures 2A,B). In the NF-κB pathway, Dox inhibited the activation of p65 (Figures 2C,D). The activation of p38, ERK, and p65 was inhibited by Dox in a dose-dependent manner. These results indicated that the inhibition of the downregulation of MAPK and NF-κB pathways by Dox in leptospira-infected J774A.1 cells may be responsible for the decline of IL-1β.

Both NLRP3 and NLRC4 inflammasomes were responsible for the expression of mature IL-1β. To make clear, the role of NLRP3 and NLRC4 inflammasomes in leptospira-induced IL-1β release, peritoneal macrophages from WT, NLRP3^−/−, and NLRC4^−/− mice were used. The level of IL-1β release in Sup was decreased significantly in peritoneal macrophages of NLRP3^−/− and NLRC4^−/− mice after live leptospira infection compared with infected cells of WT mice (Figure 3A). Interestingly, the release of IL-1β was more seriously damaged in infected cells of NLRP3^−/− mice. Western blot analysis showed that the defect of NLRP3 damaged the activation of caspase-1 and the maturity of IL-1β (Figure 3B). These results indicated that leptospira-induced IL-1β release was mainly dependent on NLRP3 inflammasome activation.

As the NLRP3 inflammasome is vital to the release of mature IL-1β, we wanted to make clear whether the inhibition of IL-1β by Dox is also mediated through the NLRP3 inflammasome. The NLRP3 mRNA and protein were analyzed by qRT-PCR and western blot, respectively, in sonicated leptospira-infected J774A.1 cells. Leptospira induced the high expression of NLRP3 in both mRNA and protein levels. After treatment with the Dox at a dose of 20 µg/ml, the expression of leptospira-induced NLRP3 was declining (Figures 4A,B). The activation of caspase-1 and the release of mature IL-1β were also analyzed by western blot. As shown in Figures 4C,D, Dox inhibited the leptospira-induced activation of caspase-1 and the release of mature IL-1β. These results indicated that Dox suppressed NLRP3 inflammasome priming during leptospira infection.

Leptospira can activate the NLRP3 inflammasome by downregulation of the Na/K-ATPase pump. The Na/K-ATPase pump is composed of Na/Kα and Na/Kβ1 subunits (10). It is reported that leptospira activated the NLRP3 inflammasome by downregulating the expression of the Na/Kβ1 subunit (10). To make clear whether Dox upregulated the expression of the Na/Kβ1 subunit in leptospira-infected macrophages, the Na/Kβ1 subunit mRNA was analyzed by qRT-PCR. After infection of sonicated leptospira, the expression of the Na/Kβ1 subunit was not downregulated (Figure 4E). Then we used live leptospira to repeat this experiment and the markedly downregulated Na/Kβ1 subunit was found (Figure 4F). Dox upregulated the expression of the Na/Kβ1 subunit in both sonicated and live leptospira-infected macrophages (Figures 4E,F). These results indicated that Dox suppressed NLRP3 inflammasome possibly by upregulating the expression of the Na/Kβ1 subunit in leptospira-infected macrophages.

To determine whether Dox-mediated NLRP3 inflammasome inhibition is specific to leptospira stimulation, the effects of Dox on LPS and ATP stimulation, a conventional NLRP3 inflammasome agonist (16), was assessed. The LPS and ATP stimulation induced massive expression of IL-1β, which was stronger than sonicated leptospira infection in macrophages. Interestingly, treatment with Dox inhibited the expression of IL-1β in both leptospira and agonist stimulation (Figure 5A). As expected, Dox suppressed the activation of caspase-1 and the release of mature IL-1β in LPS and ATP stimulated macrophages by western blot analysis (Figure 5B). These results indicated that Dox-mediated NLRP3 inflammasome inhibition was not limited to leptospira stimulation. To distinguish priming from activating, LPS-primed J774A.1 cells were incubated with Dox (20 µg/ml) for 1 h, followed by treatment with ATP for 2 h. The releases of IL-1β in Sup were tested by ELISA. The level of IL-1β release was still high after Dox treated (Figure 5C). So Dox is only affecting priming of NLRP3 inflammasome. The effect of Dox on leptospira- and agonist-induced cell death was studied by LDH release. As expected, pretreatment with Dox dramatically reduced the level of cell death induced by leptospira and agonist (Figure 5D). To study how long these effects last once Dox has been removed, J774A.1 cells were incubated with Dox (20 µg/ml) for 24 h. After 0–4 h once Dox has been removed, the cells were stimulated with live leptospira for another 24 h without Dox. The expression and release of IL-1β was tested by qRT-PCR and western blot, respectively. The data showed that the inhibitory effect of Dox persisted at least 4 h after Dox had been removed (Figures 5E,F).

To verify the suppression effect of Dox on leptospira-induced IL-1β in vivo, mice were injected intraperitoneally with Dox before 24 h inoculated intraperitoneally with 2 × 10⁸ sonicated leptospira. Another 24 h later, the peritoneal cavities were lavaged and the cytokines were analyzed by ELISA. The expression of IL-1β and TNF-α was improved with leptospira stimulation compared with untreated controls. After treatment with Dox, only IL-1β levels were downregulated (Figure 6A). This was consistent to the results in vitro. To make clear whether Dox exerted the immunoregulatory activity against acute leptospirosis, hamsters were infected leptospira and treated with Dox. At 96 h after infection, the mRNA expression of IL-1β was downregulated in all the organs (liver, kidney, and lung) (Figure 6B), while TNF-α was downregulated only in the kidney (Figure 6C). These results indicated that Dox suppressed leptospira-induced IL-1β in vivo.