Fig 2: NLRP6 is involved in the expression of NLRP3 in P. multocida infected macrophages. Macrophages were infected with the P. multocida at a MOI of 1 for 9 h. Then 100 μg/mL ciprofloxacin was added for an additional 15 h incubation. After 3, 6, 9 and 24 h incubation, cell lysates were collected for RT-PCR and western blot assays. nlrp3 mRNA expression was determined by RT-PCR (A). NLRP3 protein expression was detected by western blot (B) and the ratio of NLRP3 level against GAPDH level was quantified (C). P. multocida-infection after 3, 6, 9 and 24 h detected relative mRNA expression of cxcr2 (D), cxcl1 (E), and cxcl2 (F) were also determined using RT-PCR. The results are representative of three independent experiments. Student t-test was used to analyze the statistical differences for comparisons between two groups. The statistical significance was determined as the p value, *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance.

Fig 3: NLRP6 mediates activation of caspase-1 and ASC in macrophages infected with P. multocida. The peritoneal macrophages of WT mice and Nlrp6−/− mice were infected with P. multocida (1.3 × 105 CFU) for 3 h. NLRP6 protein (red) and ASC specks (green) were observed through fluorescence microscopy (A). The white arrow indicates the co-localization of NLRP6 and ASC. The number of ASC specks in P. multocida-infected WT and Nlrp6−/− mice macrophages was quantified (B). Macrophages from WT or Nlrp6−/− mice were infected with P. multocida at an MOI of 1 for 9 h. The supernatants (Sup) and cell lysates (Lys) of WT and Nlrp6−/− mice peritoneal macrophages infected with P. multocida were collected after 24 h infection. Western blot analysis was used to detect the ASC oligomerization (C) and secretion of IL-1β (p17: subunit; p31: precursor), caspase-1 (p20: subunit; p45: precursor) (D) as well as GSDMD (G). Image J was used to quantify the ratio of IL-1β (E), caspase-1 (F), and GSDMD-N (H) to GAPDH. The results are representative of three independent experiments. Student’s t-test was used to analyze the statistical differences for comparisons between two groups. Statistical significance was determined as p value, *P < 0.05, **P < 0.01, ns: no significance.

Fig 4: NLRP6 protects the lung from P. multocida infection. WT and Nlrp6−/− mice were intranasally infected with 20 µL P. multocida (1000 CFU) and the same volume of sterilized PBS were used as control. Lungs were collected after 48 h infection for hematoxylin and eosin staining (original magnification × 100 and × 400). A Pictures are representative of three mice from each group of mice (n = 3). B The lungs were homogenized and colony counting was performed after 48 h infection (n = 5). Flow cytometry was used to analyze the number of neutrophils (C) and macrophages after 24 h infection (D) (n = 5–8). Lung homogenates were collected after 3 h, 6 h, 9 h, 24 h infection, and tissue RNA was extracted to detect mRNA expression of cxcl1 (E) and cxcl2 (F) mRNA (n = 5 in each time point). The student t-test was used to analyze the statistical differences for comparisons between two groups. Statistical significance was determined as p value, *P < 0.05, **P < 0.01, ***P < 0.001, ns: no significance.

Fig 5: NLRP6 mediates expression of inflammatory cytokines in macrophages infected with P. multocida. WT and Nlrp6−/− mice were intranasally infected with 20 µL P. multocida (1000 CFU) and the same volume of sterilized PBS were used as the control. Bronchoalveolar lavage fluid (BALF) and peripheral serum were collected after 48 h infection (n = 5–8). ELISA was used to detect the levels of IL-1β (A), IL-6 (B), TNF-α (C) in BALF and IL-1β (D), IL-6 (E) and TNF-α (F) in peripheral serum (n = 5–8). Macrophages from WT or Nlrp6−/− mice were infected with P. multocida at an MOI of 1 for 9 h. Then 100 μg/mL ciprofloxacin was added for an additional 15 h incubation. After 24 h infection, the supernatants were collected, and IL-1β (G), IL-6 (H) and TNF-α (I) were detected by ELISA. The student’s t-test was used to analyze the statistical differences for comparisons between two groups. Statistical significance was determined as the p value, *P < 0.05, **P < 0.01, ns: no significance.