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. Author manuscript; available in PMC: 2023 Dec 2.
Published in final edited form as: Microbiol Res. 2023 Jul 14;275:127460. doi: 10.1016/j.micres.2023.127460

Inflammasome activation and pyroptosis mediate coagulopathy and inflammation in Salmonella systemic infection

Ankit Pandeya a,b, Yan Zhang a, Jian Cui c, Ling Yang a, Jeffery Li a, Guoying Zhang a, Congqing Wu c,d,e, Zhenyu Li a,*, Yinan Wei a,*
PMCID: PMC10693354  NIHMSID: NIHMS1941745  PMID: 37467711

Abstract

Inflammasome activation is a critical defense mechanism against bacterial infection. Previous studies suggest that inflammasome activation protects against Salmonella oral infection. Here we find inflammasome activation plays a critical role in the pathogenesis of Salmonella systemic infection. We show that in a systemic infection model by i.p. injection of Salmonella, deficiency of caspase-1 or gasdermin-D prolonged survival time, reduced plasma concentrations of the proinflammatory cytokines IL-1β, IL-6 and TNFα. These deficiencies also protected against coagulopathy during Salmonella infection as evidenced by diminished prolongation of prothrombin time and increase in plasma thrombin-antithrombin complex concentrations in the caspase-1 or gasdermin-D deficient mice. Activation of the NAIP/NLRC4 inflammasome by flagellin and/or the components of the SPI1 type 3 secretion system played a critical role in Salmonella-induced coagulopathy. In the absence of flagellin and SPI1, the Salmonella mutant strain still triggered coagulopathy through the caspase-11/NLRP3 pathway. Our results reveal a previously undisclosed role of the inflammasomes and pyroptosis in the pathogenesis of Salmonella systemic infection.

Keywords: Pyroptosis, Inflammasome, Macrophage, Salmonella, Coagulation, DIC

1. Introduction

Salmonella are enteric bacteria that enter the body through oral route because of contaminated food and water, and then can cross the intestinal epithelial barrier to reach the systemic sites (Franchi, 2011; Pham and McSorley, 2015; Velge et al., 2012). Several mouse models have been established to study Salmonella enterica serovar Typhimurium (referred to as Salmonella thereafter) infection where it causes lethal or chronic infection depending on the mouse strain (Brown et al., 2013; Kurtz et al., 2017; Monack et al., 2004; Roy and Malo, 2002; Søndberg and Jelsbak, 2016). Upon infection, Salmonella can cause inflammasome activation and pyroptosis in several different cell types ranging from epithelial cells in the intestinal barrier to immune cells (Brennan and Cookson, 2000; Crowley et al., 2020; Franchi, 2011; Rauch et al., 2017). Salmonella has several virulence factors that can activate the inflammasome and cause pyroptosis. For example, flagellin from Salmonella can activate the NLRC4 inflammasome (Miao et al., 2006; Zhao et al., 2011). Apart from this, Salmonella encodes two kinds of Type III Secretion System (T3SS), the Salmonella pathogenicity island (SPI) 1 and 2 (Shea et al., 1996). SPI1 T3SS plays an important role during host invasion and its components also activate NLRC4 inflammasome while SPI2 T3SS is essential during intracellular survival of Salmonella (Buckner et al., 2011; Lou et al., 2019).

The role of the NLRC4 inflammasome in Salmonella infection has been studied by several groups but most of them focused on the gastrointestinal infection model, which is the major route of Salmonella infection (Crowley et al., 2020; Knodler et al., 2014; Lara-Tejero et al., 2006; Rauch et al., 2017; Raupach et al., 2006). Extensive studies have been performed to find out the role played by inflammasome activation and pyroptosis during the intestinal infection and bacterial clearance. Inflammasome components and pyroptosis are shown to play protective roles during the early phase of gastrointestinal Salmonella infection, preventing systemic dissemination of Salmonella. Caspase-1 and caspase-11 have been shown to contribute to intestinal restriction of bacteria during Salmonella infection (Crowley et al., 2020; Knodler et al., 2014). NLRC4 inflammasome and caspase-8 activation in intestinal epithelial cells also caused expulsion of infected cells and restriction of the systemic dissemination of Salmonella (Rauch et al., 2017). Caspase-1 deficiency leads to increased lethality during oral Salmonella infection (Lara-Tejero et al., 2006; Raupach et al., 2006). Salmonella can also cause systemic infection and sepsis (Appiah et al., 2021; Chen et al., 2022; Kim et al., 2021; Nurnaningsih et al., 2022; Tamber et al., 2021), and ranks first among causes of bloodstream infection in children under five years old in the Democratic Republic of Congo with a fatality rate of 15 % (Tack et al., 2021). Unlike the gastrointestinal infection model, the contribution of inflammasome activation and pyroptosis to the pathogenesis of systemic infection of Salmonella has not been fully investigated. The role of inflammasome disruption has been found to be both protective and detrimental in the systemic infection of Salmonella (Broz et al., 2012; Miao et al., 2010; Raupach et al., 2006). Interleukin18 produced from caspase-1 activation has been found to be critical for host defense against Salmonella systemic infection (Raupach et al., 2006). However, septic shock induced by high dose of live attenuated Salmonella was protected by caspase-1 and IL-18 deficiency (Raupach et al., 2006). Salmonella downregulates the expression of flagellin and SPI1 genes during the systemic phase of infection, and NLRC4 inflammasome has been shown to have a minor role during systemic infection (Lara-Tejero et al., 2006; Miao et al., 2010). However, if the host was infected by Salmonella constantly expressing flagellin, NLRC4 inflammasome was found to play a protective role in the host defense and bacterial clearance (Miao et al., 2010). In addition, non-canonical caspase11 activation and pyroptosis were reported to have a detrimental role by allowing the intracellular bacteria escaping and spreading during Salmonella systemic infection when caspase-1 was absent and neutrophil mediated clearance was defective (Broz et al., 2012). These apparent paradoxes prompted us to re-examine the role of inflammasome activation and pyroptosis in the pathogenesis of systemic infection of Salmonella.

Using a systemic infection model by i.p. injection of Salmonella, we show that inflammasome activation and subsequent pyroptosis contribute to Salmonella-induced inflammation and lethality. Deficiency of NAIPs, caspase-1, or GSDMD reduced Salmonella-induced proinflammatory cytokine production and prolonged survival time. Salmonella-induced coagulopathy is dependent on caspase-1 activation and pyroptosis, which are probably mediated by flagellin and/or the components of the SPI1 T3SS. However, in the absence of flagellin and SPI1, LPS from the pathogen can trigger coagulopathy through the caspase11/NPRP3 pathway. Our findings identified a novel function of inflammasomes and pyroptosis in Salmonella systemic infection.

2. Results

2.1. Deficiency of caspase-1 prolongs mouse survival during Salmonella systemic infection

To investigate the role of inflammasome activation in Salmonella systemic infection, C57BL/6 J mice and caspase-1 deficient mice were injected with 5 × 106 CFU of Salmonella in 0.2 mL sterile saline by i.p. We observed that deficiency of caspase-1 significantly prolonged survival (Fig. 1A). Caspase-11 deficient mice were also more resistant than WT mice to Salmonella infection, but deficiency of caspase-11 delayed lethality significantly less than deficiency of caspase-1. These data suggest that unlike oral infection, in which caspase-1 is essential for bacterial clearance and protects against infection (Cai et al., 2022; Carvalho et al., 2012; Crowley et al., 2020; Lara-Tejero et al., 2006; Miao et al., 2010; Raupach et al., 2006), caspase-1 contributes to the pathogenesis of Salmonella systemic infection.

Fig. 1.

Fig. 1.

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Caspase 1 deficiency prolongs the mice survival upon Salmonella systemic infection. (A-C) C57BL/6J, Caspase-1−/−, Caspase-11−/−, Naip−/−, Gsdmd−/− mice, 10–14 weeks old sex matched (n = 8–10 per group), were intraperitoneally injected with 5 × 106 bacteria in 0.2 mL sterile saline and monitored for 4 days. Kaplan-Meier survival curves for (A) WT vs Caspase-1−/− vs Caspase-11−/− mice, (B) WT vs Naip−/− mice, (C) WT vs Gsdmd−/− mice were shown. ****p < 0.0001, Log-rank (Mantel-Cox) test.

Salmonella contains several virulence factors including flagellin and T3SS components, causing activation of the NLRC4 inflammasome and caspase-1. NAIP5 and 6 are intracellular receptors for flagellin, NAIP1 detects PrgI and NAIP2 detects PrgJ (Rauch et al., 2016). Binding of NAIPs with ligands triggers activation of NLRC4. We used mice deficient in Naip 1–6 to investigate the role of the NAIPs/NLRC4 inflammasome in Salmonella systemic infection. Consistent with a protective role of caspase-1 deficiency, the Naip1–6 deficient mice were less susceptible to Salmonella infection than the wild-type mice (Fig. 1B). Moreover, deficiency of GSDMD also prolonged survival upon Salmonella infection. These data indicate that pyroptosis following inflammasome activation contributes to the pathogenesis of Salmonella systemic infection (Fig. 1C). TLR5 is a membrane receptor of flagellin, which plays an important role in flagellin-mediated production of proinflammatory cytokines such as TNFα and IL-6 (Fitzgerald and Kagan, 2020; Hayashi et al., 2001). However, deficiency of Tlr5 did not protect against Salmonella systemic infection (Fig. S1).

2.2. Salmonella induces severe inflammation in vivo, which depends largely on inflammasome activation and pyroptosis

To further evaluate the role of inflammasome activation and pyroptosis in Salmonella systemic infection, we measured plasma concentrations of the proinflammatory cytokines, IL-1β, IL-6 and TNFα. Plasma TNFα concentration reached a peak at 90 min post infection, whereas the levels of IL-1β and IL-6 kept on increasing to a plateau 4–8 h post-infection (Fig. 2A). As expected, Salmonella-induced IL-1β production was abolished in mice deficient in caspase-1 (Fig. 2A). TNFα and IL-6 concentrations were also significantly less in the caspase-1 deficient mice compared to WT mice. Deletion of the Naip1–6 genes had a similar effect on the cytokine productions (Fig. 2B). These data indicate that the NLRC4 inflammasome plays an important role in Salmonella-induced inflammation. We also studied the level of inflammation in GSDMD deficient mice and found that the plasma concentrations of these cytokines reduced as compared to WT mice (Fig. 2A). GSDMD is known to induce pore formation, which is required for cytokine release and its deficiency leads to reduced plasma cytokine levels (Evavold et al., 2018). Thus, reduced plasma concentrations of the proinflammatory cytokines in the GSDMD deficient mice may be due to reduced production, release of the cytokines, or both.

Fig. 2.

Fig. 2.

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Salmonella causes systemic inflammation depending on NAIP, caspase-1, and GSDMD. (A and B) WT, Caspase-1−/−, Gsdmd−/− (A) and Naip−/− mice (B) were intraperitoneally injected with 1 × 108 CFU bacteria, and blood was collected over a period of 8 h through retroorbital bleeding. Plasma levels of IL-1β, TNFα and IL-6 were measured using ELISA. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA with Holm-Sidak multiple comparison test.

Gram-negative bacteria can activate the caspase-11 non-canonical inflammasome through LPS. We tested whether LPS/caspase-11 also contributes to inflammation during Salmonella systemic infection. Plasma concentrations of IL-1β, TNFα, and IL-6 in Caspase-11 deficient mice were very similar to WT mice (Fig. S2A). Plasma concentrations of these cytokines in the Tlr5 deficient mice were also similar to the values for WT mice (Fig. S2B). These data suggest that the LPS/caspase-11 pathway or the flagellin/TLR5 pathway plays a minimal role in inflammation upon Salmonella infection (Fig. S2A–B), which are consistent with the survival data showing that Salmonella-induced lethality was partially protected by deficiency of caspase-11 and not protected by deficiency of TLR5 (Fig. 1A, S1).

2.3. Salmonella induces DIC during systemic infection in a caspase-1-dependent manner

DIC is a common and fatal complication of bacteria systemic infection. The mortality rate is dramatically increased in septic patients with DIC (Fujishima et al., 2014; Gando et al., 2013; Rangel-Frausto et al., 1995; Venugopal, 2014). Recently, our lab and Yang et al. have shown that inflammasome activation and pyroptosis play a major role in DIC during sepsis (Wu et al., 2019; Yang et al., 2019). Here we investigated whether they contribute to DIC during the Salmonella pathogenesis (Fig. 3A and B). Prothrombin time (PT) was significantly prolonged after Salmonella infection. Similarly, the plasma concentrations of thrombin-antithrombin (TAT) complex were also significantly increased upon Salmonella infection. These two indicators of coagulopathy were diminished in caspase-1 deficient mice compared to the WT mice, highlighting a role of caspase-1 in the process. Similarly, mice deficient in GSDMD had shorter PT and lower plasma TAT levels compared with WT mice in response to Salmonella challenge, which is consistent with our previous findings that GSDMD-dependent pyroptosis is required for the release of the tissue factor, a key trigger of coagulopathy in sepsis (Wu et al., 2019). GSDMD pores are also involved in the release of extracellular traps from neutrophils (Sollberger et al., 2018). Hence, GSDMD deficient mice are likely less able to release extracellular traps, which may also contribute to coagulation (Massberg et al., 2010) as well as defense against various pathogens including salmonella (Brinkmann et al., 2004; Clark et al., 2007; Ma and Kubes, 2008; Monaco et al., 2021).

Fig. 3.

Fig. 3.

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Salmonella causes coagulopathy upon systemic infection through inflammasome activation and pyroptosis. (A-B) Measurement of PT and plasma TAT concentration in the indicated mice strain (n = 5–10, 10–14 weeks old) 8 h after i.p. injection of Salmonella at a dose of 1 × 108 CFU. Blood was collected from control and infected mice through cardiac puncture into sodium citrate tube. Plasma was isolated, and PT and TAT were measured. (C-D) LDH release determined in the supernatant of different genotypes of BMDMs after incubation with 25 MOI Salmonella for 90 min. (E) Western blot for caspase-1 and IL-1β in supernatant of different genotypes BMDMs after incubation with 25 MOI Salmonella for 90 min. (F) Western blot for caspase-1 and IL-1β in the supernatant (S/N) and cell lysate samples of BMDMs after incubation with different strains of Salmonella at 25 MOI for 90 min. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way ANOVA with Holm-Sidak multiple comparison test.

Mice deficient in NAIP 1–6 showed slightly reduced PT and plasma TAT levels (Figs. 3A and 3B). While the TAT levels were significantly less in caspase-1 deficient mice compared to NAIPs deficient mice, the difference in PT time between the two strains is not statistically significant. Mice deficient in caspase-11 displayed modestly reduced PT time but similar TAT level compared to the WT mice. These results are consistent with the mouse survival data upon Salmonella infection, showing most significantly prolonged survival of the caspase-1 deficient strain.

2.4. Salmonella induces rapid inflammasome activation and pyroptosis through the NAIPs/NLRC4/Caspase-1 pathway

Salmonella causes rapid and robust inflammasome activation and pyroptosis in mouse BMDMs upon infection. More than 50 % of the cells died as measured by lactate dehydrogenase (LDH) release within 90 min of infection, and cleaved caspase-1 and IL-1β indicated robust inflammasome activation (Fig. 3C, E). As expected, BMDMs derived from caspase-1 deficient mice did not undergo lytic cell death as indicated by the lack of LDH release in the supernatant (Fig. 3C). Similarly, BMDMs from the NAIPs and GSDMD deficient mice did not undergo lytic cell death. In contrast, deficiency in caspase-11 or NLRP3 had no impact on cell death (Fig. 3D). These data demonstrate that Salmonella induces pyroptosis mainly through the NAIPs/NLRC4/Caspase-1/GSDMD axis. In support of this conclusion, incubation of BMDMs from WT or NLRP3 deficient mice, but not the NAIPs deficient mice, with Salmonella resulted in cleavage of caspase-1 and IL-1β (Fig. 3E).

2.5. Flagellin and T3SS components are responsible for rapid inflammasome activation and pyroptosis caused by Salmonella

Rapid inflammasome activation and pyroptosis in BMDM caused by Salmonella were abolished when flagellin was deleted (Fig. 3F and Fig. S3), suggesting that flagellin is the key component for Salmonella-induced inflammasome activation, which agrees with the previous findings (Reyes Ruiz et al., 2017). Apart from flagellin, Salmonella can activate the NAIPs/NLRC4 inflammasome through the T3SS components rod protein PrgJ and needle protein PrgI (Rauch et al., 2016; Rayamajhi et al., 2013; Zhao and Shao, 2015; Zhao et al., 2011). Flagellin can be secreted into the cytoplasm of host cells by Salmonella using the SPI1 T3SS (Sun et al., 2007). We used SPI1 deficient Salmonella to investigate its role in inflammasome activation and pyroptosis. SPI1 deficient strain failed to induce rapid cell death in macrophages in vitro (Fig. S3). Correspondingly, SPI1 deficient strain did not cause activation of caspase-1 and IL-1β as shown by Western blot (Fig. 3F).

2.6. Deficiency of flagellin or SPI1 is not sufficient to prevent inflammation in vivo

To investigate the contribution of flagellin to inflammation during Salmonella infection, we compared the ability of WT and the flagellin deficient strain in the induction of proinflammatory cytokines. Plasma concentration of IL-1β in mice challenged with the flagellin deficient strain was about half of that in the mice challenged with WT strain of Salmonella (Fig. 4A). Plasma concentrations of TNFα and IL-6 were not significantly affected by flagellin deficiency. Because Salmonella-induced proinflammatory cytokines, including IL-1β, TNFα and IL-6, was diminished in mice deficient in caspase-1 or NAIPs (Fig. 2A–B), these data suggest that virulent factors other than flagellin can activate the NLRC4 inflammasome, leading to caspase-1 activation. Next, we used a strain lacking the SPI1 gene cluster to determine whether PrgJ and PrgI are involved in the activation of the NLRC4 inflammasome by Salmonella. Similar as the flagellin deficient strain, the strain deficient in the SPI1 genes reduced the secretion of IL-1β, but not TNFα and IL-6 (Fig. 4A).

Fig. 4.

Fig. 4.

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Deficiency of flagellin and SPI1 do not protect against Salmonella mediated lethality. (A) Mice were injected intraperitoneally with 1×108 CFU bacteria and blood was collected through retroorbital bleeding. Plasma concentrations of IL-1β, IL-6 and TNFα were determined using ELISA. (B) Kaplan Meier survival curve obtained after mice were injected intraperitoneally with the indicated strains of bacteria at a dose of 5 × 106 CFU in 0.2 mL sterile saline and monitored over time. Log-Rank (Mantel-Cox) test was done to evaluate the significance between curves. (C-D) PT and Plasma TAT determined for uninfected and infected mice. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way ANOVA with Holm-Sidak multiple comparison test for A, one way ANOVA test for C and D.

Since deficiency of flagellin or SPI1 alone was unable to abolish systemic inflammation in vivo, we constructed a Salmonella strain lacking both SPI1 and flagellin (ΔSPI1ΔfliCfljB). When mice were challenged with this strain, the severity of inflammation, as indicated by the plasma cytokine concentrations, was further reduced compared to the SPI1 or flagellin single deficient strains (Fig. 4A), suggesting that both flagellin and SPI1 T3SS contribute to Salmonella-induced inflammation in vivo.

2.7. Deficiency of SPI1 and flagellin does not prolong survival of mice

Next, we observed survival of mice challenged with WT or the 2206SPI1ΔfliCfljB strain. Surprisingly, although inflammation was reduced, removal of both SPI1 and flagellin had minimal impact on mouse survival (Fig. 4B). These data suggest that inflammation may not be the major cause of lethality during Salmonella systemic infection.

We then tested the occurrence of DIC upon infection by these knockout strains. The PT of mice infected with the mutant Salmonella strains were longer than the untreated control, but significantly shorter than those infected with the WT Salmonella. Plasma TAT concentrations in mice treated with the mutant Salmonella strains were similar as those treated with the WT strain (Fig. 4C and D), indicating that in the absence of flagellin and SPI1, other mechanisms induced DIC and lethality.

2.8. Flagellin and SPI1 deficient strain causes caspase-11 and NLRP3 inflammasome activation during longer incubation in vitro

To determine whether LPS is responsible for Salmonella-induced DIC and lethality in the absence of flagellin and SPI1, we investigated whether caspase-11 can mediate macrophage pyroptosis by Salmonella. BMDMs from C57BL/6 J mice were incubated with 25 MOI bacteria for 90 mins before gentamicin was added to a final concentration of 100 μg/mL to kill the extracellular bacteria. Cells were incubated for an additional 6 or 16.5 h. The Salmonella mutant strains lacking SPI1, flagellin, or both induced cell death and cleavage of caspase-1 and IL-1β in a time-dependent manner (Fig. 5). After 18 h of infection, there was significant amount of LDH release as well as caspase-1 and IL-1β cleavage in BMDMs treated with all Salmonella mutant strains (Fig. 5C and D).

Fig. 5.

Fig. 5.

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Flagellin and/or SPI1 deficient Salmonella causes inflammasome activation and pyroptosis in macrophages. (A-B) Caspase-1 and IL-1β Western blot and LDH release after 8 h of inoculation at 25 MOI. (C-D) Caspase-1 and IL-1β western blot and LDH release after 18 h of inoculation at 25 MOI. In all cases, 100 μg/mL gentamicin was added 90 min post infection. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, One-way ANOVA with Holm-Sidak multiple comparison test.

We then used the Salmonella mutant strains to investigate the mechanism of flagellin/SPI1-independent inflammasome activation. When theΔSPI1ΔfliCfljB strain was used, cleavage of caspase-1 and IL-1β was completely abolished in the casapase-11 deficient BMDMs (Fig. 6A). Since LPS-induced caspase-1 activation requires the NLRP3 inflammasome (Kayagaki et al., 2011; Rühl and Broz, 2015), we further investigated the role of NLRP3 in caspase-1 activation by the ΔSPI1ΔfliCfljB strain. Cleavage of caspase-1 and IL-1β induced by the ΔSPI1ΔfliCfljB strain was abolished in the NLRP3 deficient cells (Fig. 6A). In contrast, WT strain induced similar level of inflammasome activation in the NLRP3 deficient cells (Fig. 6A). Consistent with the caspase-1 activation, cell death induced by the mutants deficient in SPI1 and flagellin was protected by deficiency of caspase-11 (Fig. 6B).

Fig. 6.

Fig. 6.

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Caspase-11/NLRP3 mediates flagellin- and SPI1-independent inflammasome activation, pyroptosis and coagulopathy. (A) Western blot of caspase-1 and IL-1β after 18 h of incubation with 25 MOI wild-type or ΔSPI1ΔfliCfljB Salmonella. (B) LDH release from BMDMs after incubated with 25 MOI of the indicated strains of Salmonella for 18 h. (C-D) Prothrombin time and Plasma TAT concentration of mice 8 h after inoculated with 1 × 108 CFU ΔSPI1ΔfliCfljB Salmonella intraperitoneally. (E) Pathogenesis of Salmonella through inflammasome activation during severe systemic infection. All data are presented as mean ± SEM (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Two-way ANOVA with Holm-Sidak multiple comparison test.

2.9. LPS mediates DIC induced by ΔSPI1ΔfliCfljB strain through caspase-11/NLRP3

To determine whether LPS is responsible for coagulopathy induced by strainΔSPI1ΔfliCfljB, we tested whether deficiency of caspase-11 or NLRP3 protect from DIC in vivo upon infection by the strain. Prolongation of PT and increase in plasma TAT concentrations were abolished by deficiency of ether casaspe-11 or NLRP3 (Fig. 6C and D). These results indicate that in the absence of flagellin and SPI1, LPS mediates DIC through the caspase-11 and NLRP3 pathway.

3. Discussion

Inflammasome activation and subsequent pyroptosis is a double-edged sword of innate immune system. While activation of inflammatory caspases is a critical immune response against bacterial infections, excessive activation of the inflammatory caspases and pyroptosis lead to coagulopathy, multiple organ damage, and host lethality (Kayagaki et al., 2011; Wu et al., 2019; Zhao et al., 2016). Previous studies show that inflammasome activation protects against oral Salmonella infection (Crowley et al., 2020; Knodler et al., 2014; Lara-Tejero et al., 2006; Rauch et al., 2017; Raupach et al., 2006). Here, we demonstrate a novel function of inflammasome activation and pyroptosis in the pathogenesis of Salmonella systemic infection. Using an i.p. injection model, we show that Salmonella-induced inflammation and coagulopathy are largely dependent on caspase-1 activation and GSDMD-mediated processes. We identified that flagellin and SPI1 T3SS are the major virulent factors causing NLRC4 inflammasome activation in Salmonella infection under the current experimental condition. However, in the absence of these virulent factors, LPS can also trigger coagulopathy through the caspase-11 and NLRP3 pathway (Fig. 6E).

We observed that inflammasome activation and pyroptosis play a key role in proinflammatory cytokine production during Salmonella systemic infection. Plasma concentrations of all three cytokines tested, including IL-1β, IL-6 and TNFα, are dramatically reduced in the caspase-1 or Gsdmd deficient mice challenged with Salmonella by i.p. IL-1β and IL-18 are proinflammatory cytokines generated directly from inflammasome activation. Therefore, it is not surprising that IL-1β production is dependent on inflammasome activation. IL-6 and TNFα are produced through the NF-κb pathway. It is interesting that Salmonella-induced TNFα and IL-6 production is also significantly affected by the lack of caspase-1 and GSDMD (Fig. 2A). The mechanism by which inflammasome activation and pyroptosis regulate TNFα and IL-6 production is not yet well established. In a recent study de Lima Buzzo et al. reported a role of NLRC4/caspase-1 axis acting downstream of NF-κB activation (Buzzo et al., 2017). Activated caspase-1 cleaved the chromatin regulator PARP1 (also known as ARTD1) and enhanced accessibility of the NF-κB binding sites located at the Nos2 promoter. This could potentially be the mechanism leading to upregulated TNFα and IL-6 production as well. Previous studies show that secretion of TNFα does not require GSDMD-mediated pore formation (Evavold et al., 2018). IL-1β has been shown to induce the generation of IL-6 in vivo (Skelly et al., 2013). However, generation of TNFα is unlikely to be secondary to IL-1β (Skelly et al., 2013), and the serum TNFα concentration reached a peak at 90 min after challenged with Salmonella, when there was minimal IL-1β production (Fig. 2). It appears that Salmonella-induced inflammation is largely dependent on the canonical inflammasome pathway, but not the LPS/caspase-11 pathway. This conclusion is supported by the data that Salmonella-induced cytokine production was abolished by deficiency of NAIPs or caspase-1, but not caspase-11. In consistent with these findings, Salmonella mutants deficient in flagellin and/or T3SS induced less cytokine production than the WT strain. Thus, flagellin and/or T3SS play an important role in Salmonella induced inflammation through activating the NAIPs/NLRC4/caspase-1 pathway.

Cytokine storm is suggested as one of the major lethal complications during sepsis (Chousterman et al., 2017; Fajgenbaum and June, 2020). Surprisingly, survival time was very similar when mice were challenged with the ΔSPI1ΔfliCfljB strain or WT strain of Salmonella (Fig. 4B), although theΔSPI1ΔfliCfljB strain induced much less cytokine production (Fig. 4A). These data suggest that cytokine storm or inflammation is not a major contributor to Salmonella-induced lethality during systemic infection. We recently reported that inflammasome activation and pyroptosis triggers DIC during sepsis (Wu et al., 2019), another major lethal complication of sepsis. Salmonella-induced prolongation of prothrombin time and increase in plasma TAT concentrations are diminished in the caspase-1 or GSDMD deficient mice, indicating an important role of the canonical inflammasome pathway and pyroptosis in Salmonella-induced coagulopathy. It was unexpected that theΔSPI1ΔfliCfljB strain induced coagulopathy to a similar extent as the WT strain (Fig. 4C–D). Thus, in the absence of flagellin and SPI1 T3SS, an alternative mechanism could activate caspase-1 and pyroptosis, which is responsible of coagulopathy. In support of this hypothesis, we found that the ΔSPI1ΔfliCfljB strain induced caspase-1 activation and IL-1β cleavage in BMDMs. Since theΔSPI1ΔfliCfljB strain failed to induce caspase-1 activation and IL-1β cleavage in caspase-11 deficient BMDMs, we speculate that theΔSPI1ΔfliCfljB strain-induced caspase-1 activation is mediated by LPS through the caspase-11 pathway. It is known that activation of caspase-1 by LPS requires NLRP3 (Kayagaki et al., 2011; Rühl and Broz, 2015). Accordingly, ΔSPI1ΔfliCfljB strain-induced caspase-1 activation and IL-1β cleavage was abolished in the NLRP3 deficient BMDMs. In consistent with the in vitro findings, the ΔSPI1ΔfliCfljB strain-induced coagulopathy in mice deficient in either caspase-11 or NLRP3 was diminished. Taken together, our data indicate that Salmonella-induced coagulopathy mainly depends on the caspase-1 and GSDMD pathway, which is likely triggered by flagellin and the SPI1 T3SS components. However, in the absence of these proteins, LPS can also induce coagulopathy through the caspase-11/NLRP3 pathway (Figs. 4C–D, 6C–D).

Several studies reported a protective role of the intestinal epithelial inflammasome against Salmonella infection (Crowley et al., 2020; Lara-Tejero et al., 2006; Rauch et al., 2017). We also found that the mortality rate was higher in mice deficient in NAIP than the WT controls in a Salmonella oral infection model (Fig. S4A), which is consistent with previous findings. These data suggest that inflammasome activation and pyroptosis may play distinct roles under different conditions, and probably also in different stages, of infections. In this regard, Miao et al. reported a protective role of caspase-1 during WT Salmonella systemic infection (Miao et al., 2010). They show that inflammasome activation successfully clear the flagellin-expressing strain from mice. In these studies, a low dose (100 CFUs) of bacteria was used. In our study a higher dose was used to mimic severe sepsis, which causes severe inflammatory response and DIC. When used a low dose (100 CFUs) of Salmonella, we also observed that the mice mortality was not much affected by the presence or absence of inflammasome components (Fig. S4B). Therefore, the inflammasome pathway is a double-edged sword in the infectious disease, could be both beneficial and detrimental, depending on the stage of disease and/or the path of infections. Our data indicate that in systemic infections, inflammasome activation and subsequent pyroptosis contribute to the onset of cytokine storm and coagulopathy, leading to death of the host. Our findings not only advance the understanding of the role of inflammasome and pyroptosis in bacterial infections, but also suggest that therapeutics targeting inflammasome and pyroptosis could be effective in preventing cytokine storm and coagulopathy, the lethal complications of severe infectious conditions during septic shock.

4. Methods

4.1. Mice

C57BL/6J (WT), Naip−/−, Caspase-1−/−, Caspase-11−/−, Caspase-1&11−/−, Tlr5−/−, Gsdmd−/− and Nlrp3−/− mice were housed in the Animal Care Facility at University of Kentucky as well as Texas A&M University, School of Pharmacy, following institutional and National Institutes of Health guidelines after approval by the Institutional Animal Care and Use Committee. Male mice of age 10–14 weeks were used in all experiments.

4.2. Bacteria

Salmonella enterica serovar Typhimurium (ATCC 14028) and its mutant ΔfliCfljB was obtained from Dr. Edward Miao at Duke University. ΔSPI1 was obtained from Dr. James M Slauch at the University of Illinois. Using the obtained ΔSPI1 strain, we created ΔSPI1ΔfliCfljB knockout using the Quick and easy gene deletion kit (Gene bridges GmbH, Heidelberg, Germany) following the manufacturer’s protocol. The target gene fliC was first replaced with FRT-flanked kanamycin resistance cassette which was then removed and again inserted in place of fljB. Colony PCR was performed to confirm the gene replacement and later confirmed through sequencing. All bacterial strains were grown in LB with appropriate antibiotics for selection and used for infection after their number counted by spreading onto LB-agar dishes.

4.3. BMDMs isolation and culture

BMDMs were isolated and cultured as described in published protocols (Davies and Gordon, 2005; Wu, 2019). Briefly, mouse femurs were isolated and bone marrow was flushed out and cultured in the BMDM medium (RPMI-1640 supplemented with 15 % L929-cell conditioned medium, 10 % FBS, 1 % HEPES, 1 % L-Glutamine and 1 % Penicillin/Streptomycin) for 5–6 days. Then the BMDMs were harvested and seeded in 100 μL in 96 well plate or 1 mL in 12 well plate at a density of 1 × 106 cells/mL.

4.4. In vitro infection

For in vitro infection assays, isolated BMDMs were plated in either 96 well plate or 12 well plate at a density of 1 × 106 cells/mL. The cells were then allowed to attach to the plates by incubating at 37 °C and 5 % CO2 for a few hours to overnight. After the cells properly attached, the medium was changed into Opti-MEM low serum medium. For infection, the designated strain of bacteria resuspended in sterile PBS were added to the cells at 25 MOI. The plates were briefly centrifuged at 500 g for 5 mins and incubated at 37 °C and 5 % CO2. At 90 mins post infection, either samples were collected and analyzed, or gentamicin was added to a concentration of 100 μg/mL and further incubated to a total of 18 h before samples were collected and analyzed.

4.5. LDH release assay

LDH CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega, Cat#G1780) was used to determine cell viability. Each well contained 50 μL of collected samples and 50 μL of CytoTox 96 reagent in a 96-well plate. The plate was incubated for 10–15 min at room temperature or until a sufficient colorimetric change was visible. The reaction was terminated using the stop solution, and the absorbance was read at 490 nm. Percentage LDH release was calculated using the maximum LDH release prepared using lysis buffer.

4.6. Western blots

For western blot, the collected cell supernatants were subjected to TCA precipitation to pellet proteins, whereas cell lysates were prepared in SDS sample buffer. Both pro-caspase-1 and caspase-1-p20 were detected using anti-caspase-1(p20) (Adipogen, Cat#AG-20B-0042) at 1:1000 dilution. Pro-IL-1β and IL-1β (p17) were detected using anti-IL-1β (GeneTex Cat#GTX74034) at 1:1000 dilution. Blots were imaged using the BIO-RAD ChemiDoc MP imaging system as well as the Azure imaging system.

4.7. In vivo inflammation study

For in vivo studies C57BL/6J and the corresponding gene deficient mice Naip−/−, Caspase 1−/−, Caspase-11−/−, Caspase-1&11−/−, Tlr5−/− and Gsdmd−/− (8–14 weeks old) were intraperitoneally injected with 1 × 108 CFU Salmonella in 0.2 mL sterile PBS. Blood samples were collected via retro-orbital bleeding in an EDTA tube before or at various time (1.5 h, 4 h, and 8 h) following injection. Blood samples were centrifuged at 10,000g for 1 min at room temperature to obtain plasma. IL-1β, IL-6, and TNFα were measured using ELISA kits (Invitrogen) following manufacturer’s instructions.

4.8. Coagulopathy

For the determination of coagulopathy, mice were infected with 1×108 CFU bacteria through intraperitoneal injection in 0.2 mL sterile saline as described. After 7.5–8 h of infection, blood was collected, and PT and plasma TAT concentrations were measures as indicators of coagulopathy.

4.9. Prothrombin time

To measure the PT, mice were subjected to tribromoethanol (avertin) anesthesia and blood was collected by the cardiac puncture method. Blood was collected using a 23-gauge needle in a syringe containing 3.8 % sodium citrate (final citrate to blood ratio 1:7). Plasma was obtained after centrifugation at 1500g for 15 min at 4 °C. PT was determined with Thromboplastin-D (Pacific Hemostasis, Cat#100357) in a manual setting according to manufacturer’s instruction, using CHRONO-LOG #367 plastic cuvette.

4.10. Plasma TAT concentration

Using the plasma obtained while determining the PT, plasma TAT concentrations were determined using a mouse TAT ELISA kit (Abcam, Cat#ab137994) at 1:50 dilution according to manufacturer’s instruction.

4.11. Survival assays

For survival assays, mice were infected with the indicated strain of Salmonella through intraperitoneal injection of 5 × 106 CFU bacteria in 0.2 mL sterile saline. For gastrointestinal infection, 1 × 107 CFU bacteria in 0.2 mL sterile saline were subjected through oral gavage. For low dose systemic infection, 100 CFU bacteria were injected intraperitoneally in 0.2 mL sterile saline. Mice were then provided with ad libitum food and water and observed over time to monitor their activity and health deterioration symptoms.

4.12. Statistical analysis

All the data are presented in mean±SEM. Depending upon the case, either two-way ANOVA with Holm-Sidak multiple comparisons or one-way ANOVA with Holm-Sidak multiple comparisons were used to determine the significance. P < 0.05 was considered statistically significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. All statistical analyses were conducted on biological replicates in GraphPad Prism 6.

Supplementary Material

supplementary figures

Acknowledgements

We sincerely thank Dr. Dr. Edward Miao (Duke University) for providing the Salmonella enterica serovar Typhimurium (ATCC 14028) and the ΔfliCfljB mutant strain, and Dr. James M. Slauch (University of Illinois Urbana Champaign) for the ΔSPI1 mutant strain. The authors declare no competing financial interests. This work was supported by: This work was supported by the National Institutes of Health (R00HL145117 to C.W., R01 HL142640 and GM132443 to Y.W. and Z. L., R01HL146744 to Z.L).

Footnotes

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Zhenyu Li : Conceptualization, Methodology, Writing – review & editing. Yinan Wei : Conceptualization, Methodology, Writing – review & editing.

Appendix A. Supporting information

Supplementary data associated with this article can be found in the online version at doi:10.1016/j.micres.2023.127460.

Supporting information

Supplemental Figs. S1 to S4 are available online.

Data Availability

Data will be made available on request.

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Supplementary Materials

supplementary figures
NIHMS1941745-supplement-supplementary_figures.pdf (382.7KB, pdf)

Data Availability Statement

Data will be made available on request.

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