NLRP3 activation in neutrophils induces lethal autoinflammation, liver inflammation, and fibrosis

Abstract

Sterile inflammation is a central element in liver diseases. The immune response following injurious stimuli involves hepatic infiltration of neutrophils and monocytes. Neutrophils are major effectors of liver inflammation, rapidly recruited to sites of inflammation, and can augment the recruitment of other leukocytes. The NLRP3 inflammasome has been increasingly implicated in severe liver inflammation, fibrosis, and cell death. In this study, the role of NLRP3 activation in neutrophils during liver inflammation and fibrosis was investigated. Mouse models with neutrophil-specific expression of mutant NLRP3 were developed. Mutant mice develop severe liver inflammation and lethal autoinflammation phenocopying mice with a systemic expression of mutant NLRP3. NLRP3 activation in neutrophils leads to a pro-inflammatory cytokine and chemokine profile in the liver, infiltration by neutrophils and macrophages, and an increase in cell death. Furthermore, mutant mice develop liver fibrosis associated with increased expression of pro-fibrogenic genes. Taken together, the present work demonstrates how neutrophils, driven by the NLRP3 inflammasome, coordinate other inflammatory myeloid cells in the liver, and propagate the inflammatory response in the context of inflammation-driven fibrosis.

Keywords: NLR Family Pyrin Domain Containing 3; fibrosis; liver inflammation; macrophage; neutrophil.

Figure 1. Neutrophil‐specific mutant NLRP3 mice show impaired growth, weight, and survival

1. Schematic illustration of generation of both mutant neutrophil‐specific Nlrp3 ^D301NneoR and Nlrp3 ^A350VneoR mice.

2. Representative images of both Nlrp3 ^D301N‐MRP8 and Nlrp3 ^A350V‐MRP8 mutant mice.

3. Body weight of both Nlrp3 ^D301N‐MRP8 and Nlrp3 ^A350V‐MRP8 mutant mice at day 10 of age. n = 6–8.

4. Survival curve of both neutrophil‐ and myeloid‐specific Nlrp3 ^D301NneoR and Nlrp3 ^A350VneoR mutant mice. n ≥ 18.

5. Weight and survival curves of neutrophil‐specific Nlrp3 ^A350VneoR Gsdmd ^−/− mice. n ≥ 18.

Data information: Significance determined by unpaired t‐test (***P < 0.001), data are expressed as mean ± SD, and dots represent individual biological replicates.

Figure 2. Neutrophil‐specific NLRP3 activation induces systemic changes in hematopoiesis and accumulation of inflammatory neutrophils in the liver

1. Flow cytometry gating strategy used to identify mature leukocytes and HSPCs in the bone marrow, peripheral blood, and liver.

2. Frequency of LSKs, MPs, and leukocytes in bone marrow and peripheral blood of live CD45⁺ cells from WT (n = 6) and Nlrp3 ^D301N‐MRP8 (n = 6) neonatal mice at P8‐P9.

3. Frequency of LSKs, MPs, and leukocytes in liver of live CD45⁺ cells from WT (n = 6) and Nlrp3 ^D301N‐MRP8 (n = 6) neonatal mice at P8‐P9.

4. Markers of neutrophil activation FcγRIII/II and PSGL‐1 illustrated by geometric mean fluorescence intensity (gMFI) in neutrophils from the bone marrow (BM), blood (BLD), and liver (LIV) of WT (n = 3) and Nlrp3 ^D301N‐MRP8 (n = 4) neonatal mice at P8‐P9.

5. Loss of Ly6G expression in Nlrp3 ^D301N‐MRP8 neutrophils (n = 4) relative to WT littermate neutrophils (n = 3) by flow cytometry evaluation of gMFI.

Data information: Significance determined by unpaired t‐test (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001), data are expressed as mean ± SD, and dots represent individual biological replicates. CMP, common myeloid progenitor; HSPCs, hematopoietic stem and progenitor cells; LSKs, lineage⁻ ckit⁺ Sca‐1⁺; LT/ST‐HSC, long‐term/short‐term hematopoietic stem cell; Mac/DC, macrophage/dendritic cell; Mono, monocyte; MonP, monocyte progenitor; MPP, multipotent progenitor; MPs, myeloid progenitors; NeP, neutrophil progenitor; Neutro, neutrophil.

Figure EV1. Liver weight/body weight ratio of Nlrp3 mutant mice

Liver weight/body weight ratio of Nlrp3 ^A350V‐MRP8 and Nlrp3 ^D301N‐MRP8 mutant mice. Data information: n ≥ 6 mice per genotype, significance determined by unpaired t‐test (**P < 0.01), data are expressed as mean ± SD, and dots represent individual biological replicates.

Figure 3. Neutrophil‐specific NLRP3 activation leads to myeloid cell accumulation and elevated cell death in the liver

1. A, B

   MPO staining of liver issue used as a marker for myeloid cells including monocytes in both Nlrp3 ^D301N‐MRP8 and Nlrp3 ^A350V‐MRP8 mutant (n ≥ 3 mice per genotype, representative images shown, bar indicates 250 μM). H&E staining of liver tissue with histologic scoring of liver inflammation (bar indicates 100 μM, representative images are shown). TUNEL staining of liver tissue (Representative images shown, bar indicates 250 μM).

2. C

   Expression of mRNA level in whole liver tissue of MPO, MMP8, MMP9 LCN2, Chitinase‐3‐like proteins CHIL1 and CHIL3.

Data information: n ≥ 6 mice per genotype, significance determined by unpaired t‐test (*P < 0.05; **P < 0.01; ***P < 0.001), data are expressed as mean ± SD, and dots represent individual biological replicates.

Figure EV2. Nlrp3 ^A350V‐MRP8 mice display a near absence of Ly6G expression in bone marrow and blood

1. Frequency of leukocytes and HSPC in bone marrow and blood of live CD45+ cells from WT (n = 4) and Nlrp3 ^A350V‐MRP8 (n = 2) neonatal mice at P0.

2. Loss of Ly6G expression in Nlrp3 ^A350V‐MRP8 neutrophils (n = 2) relative to WT littermate neutrophils (n = 4) at P0 by flow cytometry evaluation of gMFI.

Data information: Data are expressed as mean, and dots represent individual biological replicates. CMP, common myeloid progenitor; HSPCs, hematopoietic stem and progenitor cells; LSKs, lineage⁻ ckit⁺ Sca‐1⁺; LT/ST‐HSC, long‐term/short‐term hematopoietic stem cell; Mac/DC, macrophage/dendritic cell; Mono, monocyte; MonP, monocyte progenitor; MPP, multipotent progenitor; MPs, myeloid progenitors; NeP, neutrophil progenitor; Neutro, neutrophil.

Figure 4. Neutrophil‐specific NLRP3 activation leads to increased cytokine production and acute phase proteins in the liver

1. A

   Expression of NLRP3, IL‐1β, and TNF‐α on mRNA level in whole liver lysate.

2. B, C

   Protein expression in liver tissue of cleaved and mature forms of Casp1 (p20), IL‐1β and TNF‐α (17 kDa; n = 3 mice per genotype for Western blots).

3. D

   Expression of mRNA of SAA 1 and 3, the chemoattractants CXCL1, CXCL2, its receptor CXCR2, and CCL2.

Data information: n ≥ 6 mice per genotype, significance determined by unpaired t‐test (*P < 0.05; **P < 0.01; ***P < 0.001), data are expressed as mean ± SD, and dots represent individual biological replicates.

Figure 5. NLRP3 activation in neutrophils alters macrophage population in the liver

1. Immunohistochemical staining of F4/80, a marker for resident macrophages, and mRNA expression of F4/80 in liver tissue.

2. Immunofluorescence staining of CLEC4F, a specific marker of Kupffer cells, and gene expression of CLEC4F in liver tissue.

3. Immunohistochemical staining of Ly6C, a marker for pro‐inflammatory macrophages, in liver tissue.

4. Gene expression of the pro‐inflammatory marker of macrophages iNOS as well as gene expression of CD11b.

Data information: Representative images shown, bar indicates 250 μM for F4/80 and Ly6C, bar indicates 100 μM for CLEC4F, n ≥ 6 mice per genotype, significance determined by unpaired t‐test (**P < 0.01; ***P < 0.001), data are expressed as mean ± SD, and dots represent individual biological replicates.

Figure 6. Neutrophil‐specific NLRP3 activation impairs collagen deposition and drives liver fibrosis

1. Collagen deposition assessed by Sirius red staining.

2. Gene expression of collagen 1a and collagen 3a in whole liver lysate analyzed by qPCR.

3. Protein expression of collagen 1a in whole liver lysate. n = 3.

4. Collagen deposition and fibrosis assessed by Sirius red staining in WT and Nlrp3 ^A350V‐MRP8 mutant mice to illustrate vascular as well as bile duct collagen deposition and foci of fibrosis (black arrows) in areas with severe liver inflammation.

5. Fibrotic foci in Nlrp3 ^D301N‐MRP8 mice over time. n ≥ 3.

6. Gene expression in liver tissue of the profibrotic genes CTGF and TIMP1 as well as the metalloproteinases 10 and 13.

Data information: Representative images shown, bar indicates 250 μM, n ≥ 6 per genotype, significance determined by unpaired t‐test (*P < 0.05; **P < 0.01; ***P < 0.001), data are expressed as mean ± SD, and dots represent individual biological replicates.

Figure EV3. Illustration of the crosstalk between cells in neutrophil‐specific NLRP3 inflammasome‐driven liver inflammation

The activation of the NLRP3 inflammasome in neutrophils leads to the secretion of active IL‐1β, IL‐18, and TNF‐α. These cytokines trigger a pro‐inflammatory cell response by promoting the secretion of additional cytokines such as CXCL1, CXCL2, and CCL2 that attract more neutrophils and BMDMs to the liver. Once recruited, BMDMs mature to pro‐inflammatory macrophages. Upregulation of LCN2 in neutrophils leads to an increase in CXCR2, which enhances leukocyte recruitment to the liver through CXCL1/CXCL2‐CXCR2 binding. In addition, increased CXCR2 expression enhances the secretion of cytokines. MMP8 and MMP9 have a chemotactic effect on neutrophils via the proline–glycine–proline tripeptide (PGP) that binds to CXCR2. Chitinase‐3‐like protein contributes to the recruitment of leukocytes to the site of inflammation by augmenting the secretion of cytokines such as CXCL2 and CCL2. Inflammation in the liver induces the secretion of the acute phase protein SAA in hepatocytes. SAA forms part of a feed‐forward loop and increases the release of IL‐1β and TNF‐α. While inflammation in the liver increases total leukocyte population, a decrease in the number of Kupffer cells is seen. The inflammatory milieu in the liver leads to the upregulation of profibrotic genes and the activation of stellate cells, which together promote liver fibrosis.