Treponema pallidum promotes macrophage polarization and activates the NLRP3 inflammasome pathway to induce interleukin-1β production

Abstract

Background: The involvement of inflammasome activation and macrophage polarization during the process of syphilis infection remains unknown. In this study, A series of experiments were performed using human macrophages to research the role of NLRP3 inflammasome regulation in interleukin (IL)-1β production and its influence on macrophage polarization triggered by T. pallidum.

Results: The results showed that in M0 macrophages treated with T. pallidum, the M1-associated markers inducible nitric oxide synthase (iNOS), IL-1β and TNF-α were upregulated, and the M2-associated markers CD206 and IL-10 were downregulated. In addition, we observed NLRP3 inflammasome activation and IL-1β secretion in T. pallidum-treated macrophages, and the observed production of IL-1β occurred in a dose- and time-dependent manner. Moreover, the secretion of IL-1β by macrophages after T. pallidum treatment was notably reduced by anti-NLRP3 siRNA and caspase-1 inhibitor treatment. NAC, KCl, and CA074-ME treatment also suppressed IL-1β release from T. pallidum-treated macrophages.

Conclusions: These findings showed that T. pallidum induces M0 macrophages to undergo M1 macrophage polarization and elevate IL-1β secretion through NLRP3. Moreover, the process of NLRP3 inflammasome activation and IL-1β production in macrophages in response to T. pallidum infection involves K⁺ efflux, mitochondrial ROS production and cathepsin release. This study provides a new insight into the innate immune response to T. pallidum infection.

Keywords: IL-1β; Macrophage; NLRP3; Polarization; Treponema pallidum.

Fig. 1

T. pallidum promotes macrophage polarization. a, THP-1 cells differentiated to M0 macrophages, and the morphological changes were observed by confocal microscopy (1000×). Nuclei were stained with DAPI (blue), and actin was stained with Alexa Fluor 633 phalloidin (red). Confocal laser scanning microscopy was conducted using an LSM700 confocal microscope (Zeiss, Oberkochen, Germany). b, Morphological changes in the macrophages treated with T. pallidum for 12 h (1000×). The image shows the typical morphological changes in the macrophages. The percentage of long orbicular macrophage cells among the macrophages after T. pallidum infection for 12 h was approximately 60% (data not shown). c, Cytokine mRNA levels in M0 macrophages treated with T. pallidum for 12 h. d, Cytokine protein levels in M0 macrophages treated with T. pallidum for 12 h. The results shown are from one experiment that is representative of three independent experiments and are expressed as the means±SDs. Student’s t-test was applied to compare the means between two groups. *** P < 0.001

Fig. 2

T. pallidum (at different MOIs) promotes NLRP3 inflammasome activation and IL-1β expression in macrophages. a, mRNA levels of NLRP3. b, mRNA levels of caspase-1. c, mRNA levels of IL-1β. d, Protein levels of IL-1β. e and f, Protein levels of NLRP3, active caspase-1, and IL-1β determined through a western blotting analysis. M0 macrophages treated with PBS served as the control group. The values represent the means±SDs of triplicate trials and are representative of three independent experiments. The western blotting results are representative of three independent experiments. Student’s t-test was used to compare the different MOI treatments with the control group. ** P < 0.01; ***P < 0.001

Fig. 3

T. pallidum infection for different times promotes NLRP3 inflammasome activation and IL-1β expression in macrophages. a, mRNA levels of NLRP3. b, mRNA levels of caspase-1. c, mRNA levels of IL-1β. d, Protein levels of IL-1β. e and f, Protein levels of NLRP3, active caspase-1, and IL-1β determined by western blotting analysis. The values represent the means±SDs of triplicate trials and are representative of three independent experiments. The western blotting results are representative of three independent experiments. Student’s t-test was applied to compare the cells infected for different times with the cells infected for 0 h. ** P < 0.01; *** P < 0.001

Fig. 4

Impact of NLRP3-targeting siRNA on IL-1β expression. a, Inhibition rate of NLRP3, caspase-1 and IL-1β mRNA and IL-1β protein. The mRNA levels were measured by real-time PCR, and the proteins levels were measured via ELISA. b and c, Inhibitory rates of NLRP3, IL-1β and active caspase-1 proteins measured by western blotting analysis. The values represent the means±SDs of triplicate trials and are representative of three independent experiments. The western blotting results are representative of three independent experiments. One-way analysis of variance (ANOVA) was employed to examine differences between groups, and Dunnett’s post-comparison test was used to conduct multiple comparisons. NS, non-significant difference; *** P < 0.001

Fig. 5

Inhibition of cathepsin B, ROS, K+ and caspase-1 impairs NLRP3 inflammasome activation and IL-1β expression in T. pallidum-infected macrophages. a, mRNA level of NLRP3. b, mRNA level of caspase-1. c, mRNA level of IL-1β. d, Protein level of IL-1β determined by ELISA. e and f, Protein levels of NLRP3, active caspase-1, and IL-1β determined by western blotting analysis. The values represent the means±SDs of triplicate trials and are representative of three independent experiments. The western blotting results are representative of three independent experiments. Student’s t-test was applied to compare the treatment groups with the control group. NS, non-significant difference, **P < 0.01; *** P < 0.001. NAC, N-acetylcysteine (an ROS inhibitor); CA074-ME, a cathepsin B inhibitor; KCl, a potassium channel inhibitor; Z-VAD-FMK, N-benzyloxycarbonyl-Val-Ala-Asp (O-methyl)-fluoromethyl ketone (a caspase-1 inhibitor)