Cutting edge: NLRP12 controls dendritic and myeloid cell migration to affect contact hypersensitivity

Abstract

Nucleotide-binding domain leucine-rich repeat (NLR) proteins are regulators of inflammation and immunity. Although first described 8 y ago, a physiologic role for NLRP12 has remained elusive until now. We find that murine Nlrp12, an NLR linked to atopic dermatitis and hereditary periodic fever in humans, is prominently expressed in dendritic cells (DCs) and neutrophils. Nlrp12-deficient mice exhibit attenuated inflammatory responses in two models of contact hypersensitivity that exhibit features of allergic dermatitis. This cannot be attributed to defective Ag processing/presentation, inflammasome activation, or measurable changes in other inflammatory cytokines. Rather, Nlrp12(-/-) DCs display a significantly reduced capacity to migrate to draining lymph nodes. Both DCs and neutrophils fail to respond to chemokines in vitro. These findings indicate that NLRP12 is important in maintaining neutrophils and peripheral DCs in a migration-competent state.

Fig. 1

(A) Targeted disruption of the Nlrp12 gene. (B) PCR genotyping from Nlrp12^+/− crosses. (C) Expression analysis of Nlrp12 by RT-PCR. BMD, bone marrow derived; iDC, immature DCs before maturation stimulus; mDC, DCs after TNFα maturation; mac, macrophage. (D) IL-1β production from BMDC. No tx, no treatment; pIC, polyinosine-polycytidylic acid; pLPS, phenol purified LPS; cLPS, commercial LPS.

Fig. 2

Nlrp12^−/− mice fail to mount a robust CHS response. (A–C) CHS-induced ear swelling in response to oxazalone (A–B, WT n=12, Nlrp12^−/− n=13, Ctrl n=6) and FITC (C), WT n=7, Nlrp12^−/− n=8, Ctrl n=3). (D) Cellularity of CHS ear tissue (WT n=8, Nlrp12^−/− n=7). (E) Quantification of MPO+ neutrophils in CHS ear tissue (WT n=8, Nlrp12^−/− n=8). (F) IL-1β and (G) TNFα in CHS ear tissue (WT n=5, Nlrp12^−/− n=5). All experiments were repeated 2–3 times, all data are presented as mean ± SEM. * P<0.05, ** P<0.01.

Fig. 3

DC migration to draining LNs is significantly impaired in Nlrp12^−/− mice (A–C) FITC⁺ CD11c⁺ cells in the draining LN 24 h (A–B) and 48 h (C) after topical application of FITC (WT n=5, Nlrp12^−/− n=5). (D) FITC⁺ CD11c⁺ cells in the draining LN of WT (n=5) and Nlrp3^−/− (n=5) DCs 24 h after topical application of FITC. (E) Quantification of I-Ab⁺ skin DCs in ear epidermal sheets, untreated (Untx) or FITC-treated (FITC) for 24 h (WT n=4, Nlrp12^−/− n=5). (F) Ova⁺ I-Ab⁺ CD11c⁺ cells in the draining LN 24 h following s.c. injection of fluorescent Ova in CFA. Nlrp12^−/− values were normalized to WT values set at 100% (WT n=6, Nlrp12^−/− n=5). All experiments were repeated 2–3 times, all data are presented as mean ± SEM. * P<0.05, ** P<0.01.

Fig. 4

Deletion of Nlrp12 impairs cell migration in vitro. Migration of (A–C, G) WT (○) and Nlrp12^−/− (◆) BMDCs, and of (D–F, H) WT (○) and Nlrp3^−/− () BMDCs to the indicated chemokine. Data are representative of 3–5 experiments and are presented as mean ± SEM of one experiment. Data from all experiments with associated pairwise comparison statistics are presented in table S4. (I) Migration of WT and Nlrp12^−/− neutrophils to CXCL1. Data are comprised of three independent experiments, presented as mean ± SEM, and pairwise comparisons were made using two-tailed Student’s t test, α = 0.05. * P<0.05.