NLRP6 negatively regulates innate immunity and host defence against bacterial pathogens

Abstract

Members of the intracellular nucleotide-binding and oligomerization domain (NOD)-like receptor (NLR) family contribute to immune responses through activation of nuclear factor-κB (NF-κB), type I interferon and inflammasome signalling. Mice lacking the NLR family member NLRP6 were recently shown to be susceptible to colitis and colorectal tumorigenesis, but the role of NLRP6 in microbial infections and the nature of the inflammatory signalling pathways regulated by NLRP6 remain unclear. Here we show that Nlrp6-deficient mice are highly resistant to infection with the bacterial pathogens Listeria monocytogenes, Salmonella typhimurium and Escherichia coli. Infected Nlrp6-deficient mice had increased numbers of monocytes and neutrophils in circulation, and NLRP6 signalling in both haematopoietic and radioresistant cells contributed to increased susceptibility. Nlrp6 deficiency enhanced activation of mitogen-activated protein kinase (MAPK) and the canonical NF-κB pathway after Toll-like receptor ligation, but not cytosolic NOD1/2 ligation, in vitro. Consequently, infected Nlrp6-deficient cells produced increased levels of NF-κB- and MAPK-dependent cytokines and chemokines. Thus, our results reveal NLRP6 as a negative regulator of inflammatory signalling, and demonstrate a role for this NLR in impeding clearance of both Gram-positive and -negative bacterial pathogens.

Figure 1. Nlrp6^-/- mice are resistant to Listeria and Salmonella infection

a), WT and Nlrp6^-/- mice were infected i.p. with Listeria monocytogenes and survival was monitored daily for 20 days. b) Proportion of weight loss. c,d) Bacterial loads in the liver and spleen on day 1. e,f) Bacterial loads in the liver and spleen on day 3. g) Livers were collected on day 3 and immunohistochemistry was performed for L. monocytogenes using anti-Listeria antibody. Arrows indicate the presence of L. monocytogenes in the WT liver section. h) Livers from L. monocytogenes infected mice showing inflammatory lesions indicated by arrows. i,j) WT and Nlrp6^-/- mice were infected i.p. with Salmonella typhimurium. Bacterial loads were determined in the liver and spleen on day 1. k,l) Bacterial loads in the liver and spleen on day 3. Each point represents an individual mouse, and the line represents the mean ± s.e.m; p values were determined by an unpaired two-tailed test. Results show cumulative data from two different experiments.

Figure 2. Enhanced monocyte and neutrophil recruitment in infected Nlrp6^-/- mice

a,b) WT and Nlrp6^-/- mice were infected i.p. with L. monocytogenes. Blood was analyzed for different cell populations based on their morphology. c,d) At 6h post-infection, peritoneal lavage was performed on mice infected as above and the cells collected were stained for CD11b⁺Gr1^- (monocytes) and CD11b⁺Gr1⁺ (neutrophils) cells. e) Bone-marrow chimera mice were generated for Nlrp6 as described in methods. Bar graph shows the percent of weight loss in four groups of chimeric mice. f,g) Different groups of chimeric mice, as indicated in the figure panels, were infected with L. monocytogenes and bacterial loads were determined in the liver and spleen on day 3. Data show mean ± s.e.m. of a representative experiment.

Figure 3. NLRP6 negatively regulates NF-κB and ERK signaling

a,c,e) WT and Nlrp6^-/- macrophages were exposed to L. monocytogenes or Pam3 or LPS and samples were immunoblotted with indicated antibodies. b,d,f) Bar graphs showing semi-quantitation of pERK and pIκB levels relative to ERK and IκB levels. g) Liver lysates from WT and Nlrp6^-/- mice were examined for activation of NF-κB and MAP-kinase signaling by Western blot analysis. Each lane represents an individual mouse. h) Bar graphs showing semi-quantitation of pERK and pIκB levels relative to ERK and IκB levels, respectively.

Figure 4. NLRP6 negatively regulates canonical NF-κB activation pathway

a) Bone-marrow derived macrophages from WT and Nlrp6^-/- mice were infected with L. monocytogenes and phosphorylation of p105 and p100 was examined in total cell lysate and cytosolic fraction. Nuclear fraction was examined for RelA and RelB activation in WT and Nlrp6^-/- cells. b) Cells grown on coverslips were infected with GFP-labeled L. monocytogenes and examined for nuclear translocation of RelA using anti-RelA antibody (red). Scale bars, 10μm c) Quantitative analysis of RelA-positive nuclei is shown. d) WT and Nlrp6^-/- macrophages were stimulated with Pam3 and phosphorylation of p105 and p100 was examined in total cell lysate and cytosolic fraction. Nuclear fraction was examined for RelA and RelB activation in WT and Nlrp6^-/- cells. e) Cells grown on coverslips were stimulated with Pam3 and examined for nuclear translocation of RelA using anti-RelA antibody (red). Scale bars, 10μm f) Quantitative analysis of RelA-positive nuclei is shown. Data show mean ± s.e.m. from three different experiments.