Salmonella exploits NLRP12-dependent innate immune signaling to suppress host defenses during infection

Abstract

The nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain containing 12 (NLRP12) plays a protective role in intestinal inflammation and carcinogenesis, but the physiological function of this NLR during microbial infection is largely unexplored. Salmonella enterica serovar Typhimurium (S. typhimurium) is a leading cause of food poisoning worldwide. Here, we show that NLRP12-deficient mice were highly resistant to S. typhimurium infection. Salmonella-infected macrophages induced NLRP12-dependent inhibition of NF-κB and ERK activation by suppressing phosphorylation of IκBα and ERK. NLRP12-mediated down-regulation of proinflammatory and antimicrobial molecules prevented efficient clearance of bacterial burden, highlighting a role for NLRP12 as a negative regulator of innate immune signaling during salmonellosis. These results underscore a signaling pathway defined by NLRP12-mediated dampening of host immune defenses that could be exploited by S. typhimurium to persist and survive in the host.

Fig. 1.

Absence of NLRP12 protects mice from exacerbated Salmonella infection. WT and Nlrp12^−/− mice were infected with 1 × 10⁵ (oral route) or 5 × 10³ (i.p. route) cfu of S. typhimurium. (A) Bacterial count in liver and spleen of WT and Nlrp12^−/− mice was measured by colony-forming unit (CFU) assay after 5 d (i.p.) or 7 d (oral) postinfection. Gross anatomy of Salmonella-infected spleen (B) and liver (C) collected at day 7 after oral infection. The arrow in C indicates microabscesses in the liver. (D) H&E staining of Salmonella-infected liver collected at day 7 after oral infection. The arrows indicate microabscesses and a necrotic area in the liver. (Magnifications: 4× and 10×.) (E) Histological scores of Salmonella-infected liver at day 7. (F) WT and Nlrp12^−/− mice were treated with a combination of broad-spectrum antibiotics for 3 wk or left untreated before oral infection with S. typhimurium (1 × 10⁵ cfu per mouse). Survival of the mice was monitored. d, day. Experiments in A–E were performed without the use of antibiotics. Data are representative of at least three independent experiments (n = 8–10). Data represent the mean ± SEM. *P < 0.05.

Fig. 2.

NLRP12 attenuates antimicrobial killing of Salmonella in macrophages. (A and B) BMDMs from WT and Nlrp12^−/− mice were infected with S. typhimurium [multiplicity of infection (MOI) of 5]. (A) Numbers of intracellular bacteria in Salmonella-infected cells were enumerated by colony-forming unit (CFU) assay. (B) Culture supernatants collected at 2, 8, and 24 h were analyzed for LDH release. (C) mRNA was isolated from Salmonella-infected BMDMs, and real-time quantitative PCR (qPCR) analysis of Il-6, Kc, and Tnf-α was performed. (D) Culture supernatant collected at 4 h after Salmonella infection was analyzed for IL-6, KC, and TNF-α by ELISA. (E) Real-time qPCR analysis of inducible nitric oxide synthase (iNOS) in BMDMs infected with S. typhimurium. (F) Cell lysates collected from BMDMs infected with S. typhimurium for 4 h were analyzed for caspase-1 (Casp-1) activation by Western blotting. (G) Culture supernatant collected from BMDMs infected with S. typhimurium for 4 h was analyzed for IL-1β by ELISA. Data represent the mean ± SD of triplicate wells. Data are representative of at least three independent experiments. *P < 0.05; **P < 0.01.

Fig. 3.

NLRP12 suppresses NF-κB and ERK activation during Salmonella infection. (A) Liver tissue samples collected from WT and Nlrp12^−/− mice at day 7 after i.p. infection with S. typhimurium were homogenized, and lysates were analyzed for phosphorylated (P)-IκBα, IκBα, P-ERK, and ERK using Western blotting. Densitometric analysis of P-IκBα and P-ERK relative to IκBα and ERK, respectively, was performed. (B) BMDMs from WT and Nlrp12^−/− mice were infected with S. typhimurium (MOI of 3), and cell lysates were used to analyze for P-IκBα, IκBα, P-ERK, and ERK by Western blotting. Densitometric analysis of P-IκBα relative to IκBα (C) and P-ERK relative to ERK (D) was performed. BMDMs were stimulated with S. typhimurium (E, MOI of 3) or LPS (F, 1 μg/mL), and cell lysates were analyzed for P-p105, P-p100, and P-IKKα/β by Western blotting. β-actin was used as a loading control. Data are representative of at least three independent experiments. Data represent the mean ± SEM. *P < 0.05; **P < 0.01.

Fig. 4.

NLRP12-mediated inhibition of NF-κB and ERK dampens cytokine production and clearance of Salmonella in macrophages. (A) BMDMs from WT and Nlrp12^−/− mice were treated with Salmonella LPS. Nlrp12^−/− BMDMs were also treated with either the ERK inhibitor U0126 or the IKK2 inhibitor SC-514 before and during LPS stimulation. Cell lysates were collected and analyzed for P-ERK, ERK, P-IκBα, and IκBα by Western blotting. (B–D) WT and Nlrp12^−/− BMDMs were treated with SC-514 during S. typhimurium infection. (B) Cell lysates collected after 2 and 24 h of infection were plated for bacterial colony-forming units (CFU). (C) Culture supernatant from BMDMs collected after 16 h of Salmonella infection was analyzed for IL-6 and KC by ELISA. (D) Nitrite in the culture supernatant collected after 24 h of infection was measured with Griess reagents. Data represent the mean ± SD of triplicate wells. Data are representative of at least three independent experiments. *P < 0.05.

Fig. 5.

NLRP12 suppresses NF-κB and ERK activation in response to Salmonella LPS but not to flagellin or the SPI-1 or SPI-2 type III secretion system. BMDMs from WT and Nlrp12^−/− mice were infected with S. typhimurium ΔsipB (A), Δspi-2 (B), and ΔfliCΔfljB (C) (MOI of 5) or with Salmonella LPS (D). Cell lysates were analyzed for P-ERK, ERK, P-IκBα, and IκBα by Western blotting. Data are representative of at least three independent experiments.

Fig. 6.

Model for NLRP12-mediated inhibition of canonical NF-κB and ERK signaling pathways in response to Salmonella infection. Salmonella infection of host macrophages leads to the activation of TLRs, which induces signal transduction via MyD88 and/or Toll or interleukin-1 receptor domain-containing adaptor inducing interferon-β (Trif) to activate NF-κB and ERK. Phosphorylation of IκBα and subsequent degradation of IκBα (an inhibitor of NF-κB) enables the translocation of NF-κB into the nucleus. ERK, as part of the MAPK pathway, is also phosphorylated. Both NF-κB and ERK activation lead to transcription of genes encoding proinflammatory cytokines and antimicrobial molecules, including iNOS, TNF-α, IL-6, and KC. During S. typhimurium infection, NLRP12 responds to an unknown activator and mediates the inhibition of IκBα and ERK phosphorylation, which results in a reduction in the levels of proinflammatory cytokines and antimicrobial molecules and a decreased capacity to control the replication and spread of S. typhimurium in the host cell. LRRs, leucine-rich repeats; NBD, nucleotide-binding domain; PYD, pyrin domain; SCV, Salmonella-containing vacuole.