NLRP10 Enhances CD4+ T-Cell-Mediated IFNγ Response via Regulation of Dendritic Cell-Derived IL-12 Release

NLRP10 is a nucleotide-binding oligomerization domain-like receptor that functions as an intracellular pattern recognition receptor for microbial products. Here, we generated a Nlrp10−/− mouse to delineate the role of NLRP10 in the host immune response and found that Nlrp10−/− dendritic cells (DCs) elicited sub-optimal IFNγ production by antigen-specific CD4+ T cells compared to wild-type (WT) DCs. In response to T-cell encounter, CD40 ligation or Toll-like receptor 9 stimulation, Nlrp10−/− DCs produced low levels of IL-12, due to a substantial decrease in NF-κB activation. Defective IL-12 production was also evident in vivo and affected IFNγ production by CD4+ T cells. Upon Mycobacterium tuberculosis (Mtb) infection, Nlrp10−/− mice displayed diminished T helper 1-cell responses and increased bacterial growth compared to WT mice. These data indicate that NLRP10-mediated IL-12 production by DCs is critical for IFNγ induction in T cells and contributes to promote the host defense against Mtb.


Generation of Nlrp10 -/mouse model
Sixteen ZNF pairs targeting Nlrp10 exon 2 were assembled by PCR and sub-cloned into a pZNF plasmid.
All pairs were tested for efficiency of generating double-strand breaks using the Surveyor Mutation assay in cultured mouse Neuro2A cells. The best performing pair sequence (5'GACCATCATGGCCTTGGCACGGGCCAACAGCCCCCAGGAGGCA-3') was microinjected into one-cell stage mouse embryos harvested from C57/BL6J females. Heterozygous mutant mice were screened by PCR and sequencing, and the targeted mutation was found in intron 2 by PCR and sequencing ( Figure   S1A-S1D in the Supplementary Material). The mutant founder mouse #25 carrying a 31 bp deletion was bred to C57/BL6J mice in order to generate F1 mutants. These animals were intercrossed to generate homozygous knockouts. Congenic wild-type littermate controls (WT) were used in all experiments.

Flow cytometry and cell sorting
The following antibody clones from BioLegend, BD Biosciences and eBioscience were used: anti-CD3

RNA extraction and semi-quantitative RT-PCR
Total RNA was isolated using Trizol ® (Invitrogen) and cleaned using an RNeasy Total RNA Clean-up kit (Qiagen). RNA from heart tissue was isolated using a Qiagen RNeasy Kit. Genomic DNA was removed using a Turbo DNA-free kit (Ambion) before reverse transcription using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qRT-PCR was performed in triplicates using iQ SYBR Green Supermix (BioRad) and the following primers: Nlrp10 forward 5'-CAACAGCCCCCAGGAG-3', Nlrp10 Amplification was performed on an Applied Biosystems 7500 Real-Time PCR System. The relative expression level of each gene was evaluated by DDCt method, comparing to the Gapdh housekeeping gene expression in the untreated condition or the WT control, as indicated.

Confocal microscopy
BMDCs ( Finally, the cells were washed and resuspended in 100 µl PBS and kept in the dark at 4˚C. Images were acquired with an Olympus FY1000 system at 40X magnification.

Histological analysis
Formalin-fixed lung tissues of Mtb-infected mice were paraffin-embedded, sectioned and stained with hematoxylin and eosin (H&E) or acid fast staining by Ziehl-Neelsen (ZN) method (IDEXX-RADIL Laboratories Inc, MO) as described previously (Singhal, A. et al. 2014, Sci Transl Med). Stained sections were photographed in a Nikon Microphot-FX photomicrographic system and analyzed with NIS-Elements F3.0 software (Nikon Instruments Inc, NY).

NF-kB Phospho Antibody Array
The cell lysates from WT and Nlrp10 -/-BMDCs stimulated or not stimulated with CpG DNA (1 µM) for 45 min were carried out a high-throughput proteomic screen of potential Nlrp10 targets using NF-kB Phospho Antibody Array (FullMoon BioSystems). The Full Moon arrays contain 215 highly specific antibodies against phospho and total proteins involved in the NF-kB pathway. Arrays were prepared following manufacturer's instructions. The raw fluorescence signals were background subtracted and averaged amongst the replicate spots. The averaged background subtracted signals were then logarithmically transformed and then normalized using the house keeping gene b-actin to account for slide to slide variations. Heat maps were generated using the log2 fold changes of the averaged signals and the proteins clustered using hierarchical clustering with Euclidean distance in R 3.3.1.
Lysates were prepared by mixing ( Millipore). Densitometry was performed using ImageJ and data from cytoplasmic and nuclear extracts were normalized to GAPDH, and lamin B, respectively.