Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity - PubMed (original) (raw)

. 2009 Jun 19;30(6):875-87.

doi: 10.1016/j.immuni.2009.05.005. Epub 2009 Jun 4.

James L Mueller, Matthew D McGeough, Carla A Pena, Amirhossein Misaghi, Chhavi Gandhi, Chris D Putnam, David L Boyle, Gary S Firestein, Anthony A Horner, Pejman Soroosh, Wendy T Watford, John J O'Shea, Daniel L Kastner, Hal M Hoffman

Affiliations

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

Susannah D Brydges et al. Immunity. 2009.

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.

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Figures

Figure 1

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_A350V_NeoR/+ or Nlrp3_L351P_NeoR/+ matings, with 50% of offspring expected to be mutant. Shown: total pups; total mutant pups days 1 and 2 post-birth.

Figure 2

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

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

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

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

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_A350V_neoR/+ 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.

Comment in

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