Intracellular invasion of Orientia tsutsugamushi activates inflammasome in asc-dependent manner

Abstract

Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular bacterium, which escapes from the endo/phagosome and replicates in the host cytoplasm. O. tsutsugamushi infection induces production of pro-inflammatory mediators including interleukin-1β (IL-1β), which is secreted mainly from macrophages upon cytosolic stimuli by activating cysteine protease caspase-1 within a complex called the inflammasome, and is a key player in initiating and maintaining the inflammatory response. However, the mechanism for IL-1β maturation upon O. tsutsugamushi infection has not been identified. In this study, we show that IL-1 receptor signaling is required for efficient host protection from O. tsutsugamushi infection. Live Orientia, but not heat- or UV-inactivated Orientia, activates the inflammasome through active bacterial uptake and endo/phagosomal maturation. Furthermore, Orientia-stimulated secretion of IL-1β and activation of caspase-1 are ASC- and caspase-1- dependent since IL-1β production was impaired in Asc- and caspase-1-deficient macrophages but not in Nlrp3-, Nlrc4- and Aim2-deficient macrophages. Therefore, live O. tsutsugamushi triggers ASC inflammasome activation leading to IL-1β production, which is a critical innate immune response for effective host defense.

Figure 1. IL-1 receptor signaling is required for effective host protection from O. tsutsugamushi infection.

(A) C57BL/6 mice (n=5) were i.p. inoculated with O. tsutsugamushi (OT) (5×10⁶ ICU) for the indicated time periods. Spleens were removed from infected mice and their weights were measured and normalized to body weight. (B) Serum IL-1β levels from infected mice were assessed by ELISA. A single circle represents an individual animal, and lines indicate the mean values. **p<0.01, ***p<0.001 versus uninfected mice. (C) Cytokine mRNA expression in the spleen from infected mice. At the indicated time periods after infection, total RNA was extracted and mRNA expression was determined by RNase protection assay. L32, a murine ribosomal protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Age and sex-matched wild-type (WT, n=7) and IL-1R-deficient mice (n=7) were infected with O. tsutsugamushi i.p. for 6 and 19 days. O. tsutsugamushi loads in blood and spleen were quantified by qPCR using primers specific for the O. tsutsugamushi tsa56 gene. Data were normalized by qPCR data for the GAPDH gene in host genomic DNA. A single circle represents an individual animal, and lines indicate the mean values. p<0.01, Wild-type vs. IL-1R−/−. Data are representative of three independent experiments in A-C.

Figure 2. Live O. tsutsugamushi infection activates caspase-1 and induces IL-1β secretion in LPS-primed BMDMs.

(A, B, D) BMDMs were primed with LPS (10 ng/ml) for 16 h and infected with OT. IL-1β release was assessed by ELISA. (A) LPS-primed BMDMs were treated with ATP (5 mM) for 3 h or infected with OT (ICU/cell=50) for the indicated time periods. (B) LPS-primed BMDMs were infected with OT of the indicated ICU/cell for 6 h. (C) BMDMs were primed with LPS (10 ng/ml), live OT, heat-inactivated (HOT) or UV-inactivated (UVOT) for 16 h and then treated with ATP (5 mM) for 3 h. (D) LPS-primed BMDMs were challenged with ATP, live OT, heat-inactivated OT (HOT), or UV-inactivated OT (UVOT) for 6 h. (A-D) Error bars represent SD of triplicate samples. N.D.; not detected. (E) LPS-primed BMDMs were challenged with the vehicle (-), ATP (5 mM, for 3 h), Salmonella enteritidis (Sal., MOI=25) or OT (ICU/cell=50) for the indicated time periods. The caspase-1 activation was analyzed by western blotting using rabbit polyclonal antibodies specific for the p10 subunits of caspase-1. Data are representative of three independent experiments in A-E.

Figure 3. Bacterial internalization and endosomal acidification are required for efficient IL-1β secretion upon O. tsutsugamushi infection.

LPS-primed BMDMs were pretreated with cytochalasin D (CD) (A) or NH₄Cl (B) at the indicated concentration for 1 h, and then challenged with OT (50 ICU/cell) for 6 h or ATP (5 mM) for 3 h. The production of IL-1β and TNF-α from infected cells was assessed by ELISA. Error bars represent SD of triplicate samples. *p<0.05, **p<0.01, ***p<0.001 versus vehicle-treated cells. Data are representative of three independent experiments in A and B.

Figure 4. IL-1β secretion induced by O. tsutsugamushi infection in LPS-primed BMDMs was blocked by pan-caspase inhibitor.

LPS-primed BMDMs were pretreated with pan-caspase inhibitor Z-VAD-FMK (VAD) at the indicated concentration for 1 h and then challenged with ATP (5 mM) for 3 h or OT (A. ICU/cell=10; B,C. ICU/cell=50) for the indicated period (A) or 6 h (B, C). (A) Cells were fixed and stained with human antiserum against OT and a FITC-conjugated goat anti-human IgG antibody, and examined using a fluorescence microscope. Green spots indicate OT and red areas indicate the host cell. Scale bar, 50 µm. (B) Cell viability was measured by MTT assay. (C) The production of IL-1β and IL-6 was assessed by ELISA. Error bars represent SD of triplicate samples. **p<0.01, ***p<0.001 versus vehicle-treated cells. Data are representative of three independent experiments in A-C.

Figure 5. O. tsutsugamushi infection did not induce pyroptosis.

LPS-primed BMDMs were challenged with ATP (5 mM) or OT with the indicated ICU/cell for the indicated time periods. Pyroptosis was assessed by LDH release. Error bars represent SD of triplicate samples. Data are representative of three independent experiments.

Figure 6. O. tsutsugamushi activates caspase-1 in LPS-primed BMDMs in Nlrp3-, Nlrc4- and AIM2-independent manners.

Wild-type (WT), MyD88-, Rip2-, Nlrp3-, Nlrc4- or Aim2-deficient BMDMs were primed with LPS (10 ng/ml) for 16 h and then treated with ATP (5 mM) for 3 h or infected with OT (ICU/cell=50) for 6 h or the indicated time periods. (A, B) IL-1β and IL-6 production from the cells was assessed by ELISA. (C) The cleaved caspase-1 and procaspase-1 were analyzed by western blotting using rabbit polyclonal antibodies specific for the p10 subunits of caspase-1. Error bars represent SD of triplicate samples. N.D.; not detected. -; untreated. ***p<0.001 versus wild-type. Data are representative of three independent experiments in A-C.

Figure 7. Live O. tsutsugamushi activates caspase-1 in LPS-primed BMDMs in caspase-1- and ASC-dependent manners.

(A) Wild-type (WT) or caspase-1-deficient BMDMs were primed with LPS (10 ng/ml) for 16 h and then treated with ATP (5 mM) for 3 h or infected with OT (ICU/cell=50) for 6 h. IL-1β and IL-6 production from infected cells was assessed by ELISA. (B) Wild-type (WT) or Asc-deficient BMDMs (left panel) or BMDCs (right panel) were primed with LPS and treated as in panel A. IL-1β and IL-6 production from infected cells was assessed by ELISA. (C) Wild-type (WT) or Asc-deficient BMDMs were primed with LPS (10 ng/ml) for 16 h and then infected with OT (ICU/cell=50) for the indicated time periods. The cleaved caspase-1 and procaspase-1 were analyzed by western blotting using rabbit polyclonal antibodies specific for the p10 subunits of caspase-1. (D) Caspase-1 activation in LPS-primed wild-type (WT) or Asc-deficient BMDMs after infection with O. tsutsugamushi. Numbers above bracketed lines indicate the percent of cells positive for FAM–YVAD staining. Error bars represent SD of triplicate samples. N.D.; not detected. ***p<0.001 versus wild-type. Data are representative of three independent experiments in A-D.

Figure 8. Model of inflammasome activation by O. tsutsugamushi.

OT infection requires attachment to the host cell surface (binding), followed by uptake of the bacteria by clathrin-mediated endo/phagocytosis (internalization). After maturation of the endo/phagosome, OT can be released into the cytoplasm (escape), where OT multiplies (replication). OT in the mature endo/phagosome or cytoplasm activates ASC inflammasome, which induce activation of caspase-1. Active caspase-1 processes pro-IL-1β cleavage, which results in the maturation and secretion of IL-1β.