Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity

Abstract

NLRP3 nucleates the inflammasome, a protein complex responsible for cleavage of prointerleukin-1beta (IL-1beta) to its active form. Mutations in the NLRP3 gene cause the autoinflammatory disease spectrum cryopyrin-associated periodic syndromes (CAPS). The central role of IL-1beta in CAPS is supported by the response to IL-1-targeted therapy. We developed two Nlrp3 mutant knockin mouse strains to model CAPS to examine the role of other inflammatory mediators and adaptive immune responses in an innate immune-driven disease. These mice had systemic inflammation and poor growth, similar to some human CAPS patients, and demonstrated early mortality, primarily mediated by myeloid cells. Mating these mutant mice to various gene mutant backgrounds showed that the mouse disease phenotype required an intact inflammasome, was only partially dependent on IL-1beta, and was independent of T cells. These data suggest that CAPS are true inflammasome-mediated diseases and provide insight for more common inflammatory disorders.

Figure 1. Expression of L351P is lethal in the perinatal period and expression of A350V causes a severe inflammatory phenotype with death by day fourteen post-birth

(A) Gross phenotype of Nlrp3^A350V/+/CreL, representative pictures. Mutants are depicted at the top, days 2 and 4, and on the left, days 6 and 8. Black arrows denote an abscess (day 2) and scaling erythema (day 8). (B) Survival (top) and growth (bottom) curves for WT (n=86, CreZ shown only), Nlrp3^A350V/+/CreZ (n=50), and Nlrp3^A350V/+/CreL, (n=40). A log-rank (Mantel-Cox) test comparing Nlrp3^A350V/+/CreZ and Nlrp3^A350V/+/CreL survival curves yielded a P value <0.0001. Error bars shown for mean daily weights on growth curves are SDev (n=1–17). (C) Nlrp3^A350V/+/CreL, day 8, tissue sections from synovium, liver, meninges, and conjunctiva, stained with haematoxylin and eosin at 10X and 40X (insets) magnification. (D) Birth table of pups resulting from CreL^+/+ x Nlrp3^A350VNeoR/+ or Nlrp3^L351PNeoR/+ matings, with 50% of offspring expected to be mutant. Shown: total pups; total mutant pups days 1 and 2 post-birth.

Figure 2. Multiple cytokines are upregulated in the serum and skin of Nlrp3^A350V/+/CreL mice

(A) Luminex analysis of serum obtained at days 6–8 from WT and Nlrp3^A350V/+/CreL pups, n=10–12 mice, each graph point represents one mouse, mean and SEM are shown. (B) Immunohistochemistry on skin from WT and Nlrp3^A350V/+/CreL pups for IL-1β or IL-6 (red stain), and an isotype control followed by haematoxylin staining. Similar staining was observed on sections from Nlrp3^A350V/+/CreZ (not shown).

Figure 3. Myeloid cells from Nlrp3^A350V/+/CreT and Nlrp3^L351p/+/CreT mice and CAPS patients react similarly to innate stimuli

Tamoxifen-treated Nlrp3^A350V/+/CreT, Nlrp3^L351P/+/CreT, and littermate WT BMDC were incubated with varying amounts of crude LPS (A) or pure LPS (100 ng/ml) with and without ATP (5mM) (B). (C) Tamoxifen-treated peritoneal macrophages (PM) were incubated with pure LPS with and without ATP. IL-1β in the supernatants was measured by ELISA. (A–C) n=2–3 mice with 3 wells each / genotype. (D) Western blotting for IL-1β or actin loading control, Nlrp3^A350V/+/CreT BMDC lysates (top), and supernatants (bottom). (E) In vitro stimulation of monocytes from 3 CAPS patients and 3 normal human controls with pure LPS with and without ATP. (F) In vitro cold stimulation of WT, Nlrp3^A350V/+/CreT, and Nlrp3^L351P/+/CreT BMDC, n=2–3. Cells were incubated at 32°C overnight and supernatants were analyzed by ELISA for IL-1β. (G) In vitro cold stimulation of monocytes from 5 FCAS patients and 2 normal human controls, 4 hrs and overnight. Also shown, incubation at 37°C.

Figure 4. IL-1β is required for murine disease

(A) Nlrp3^A350V/+/CreL and wildtype littermates were treated with subcutaneously injected mIL-1 Trap every other day beginning day 1–2 post-birth. Survival (left) (n=9) and growth (right) (n=2–9 mice), P=0.003 by log-rank (Mantel-Cox) comparison. (B) Survival (left) and growth (right) of Nlrp3^A350V/+/CreL/Il-1r^+/− and Nlrp3^A350V/+/CreL/Il-lr^−/−, (n=13–18 for survival and 1–9 for growth). (C) Survival (left) and growth (right) of Nlrp3^L351P/+/CreL/Il-1r^−/−, (n=33 for survival and 2–12 for growth). Error bars shown for mean daily weights on growth curves are SDev. (D) Luminex analysis of serum from Nlrp3^A350V/+/CreL/Il-lr^−/−, n=5, compared with Luminex data from WT and Nlrp3^A350V/+/CreL in figure 2 (averages and SEM shown only).

Figure 5. Nlrp3^A350V/+/creT BMDC drive an inflammatory T cell phenotype

(A) Tamoxifen-treated BMDC from Nlrp3^A350V/+/creT and WT mice were treated with LPS, and stained for CD40, OX40L, and MHC class II receptor. (B–I) BMDC were plated with OT II naïve T cells and OVA antigen under Th1, Th2, or Thl7 cell polarizing conditions, or left unpolarized. (B–D) FACS analysis of intracellular IFNγ and IL-17. (E–I) Luminex analysis of culture supernatants for IFNγ, IL-17, IL-5, and IL-4.

Figure 6. An intact inflammasome is required for pathology mediated by cryopyrin knock-in mutations, but T and B cells are not. Mutant phenotype does not require wildtype cryopyrin

Nlrp3^A350VneoR/+ were bred with Asc^−/−, Rag^−/−, and Nlrp3^−/−. (A) Survival (top) and growth (bottom) of Nlrp3^A350V/+/CreL/Rag^−/− (n=l–3 for growth, 6 for survival), Nlrp3^A350V/+/CreL/Asc^−/− (n=1–3 for growth, 7 for survival), and Nlrp3^A350V/−/CreL (n=l–3 for growth, 7 for survival). (B) Survival (top) and growth (bottom) of Nlrp3^L351P/+/CreL/Asc^−/− (n=13 for survival, 2–3 for growth). Error bars shown for mean daily weights on growth curves are SDev.