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, 285 (34), 26223-32

A Role for the Human Nucleotide-Binding Domain, Leucine-Rich Repeat-Containing Family Member NLRC5 in Antiviral Responses

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A Role for the Human Nucleotide-Binding Domain, Leucine-Rich Repeat-Containing Family Member NLRC5 in Antiviral Responses

Andreas Neerincx et al. J Biol Chem.

Abstract

Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion. Here we report on the characterization of human NLRC5 and provide evidence that this NLR has a function in innate immune responses. We found that NLRC5 is a cytosolic protein expressed predominantly in hematopoetic cells. NLRC5 mRNA and protein expression was inducible by the double-stranded RNA analog poly(I.C) and Sendai virus. Overexpression of NLRC5 failed to trigger inflammatory responses such as the NF-kappaB or interferon pathways in HEK293T cells. However, knockdown of endogenous NLRC5 reduced Sendai virus- and poly(I.C)-mediated type I interferon pathway-dependent responses in THP-1 cells and human primary dermal fibroblasts. Taken together, this defines a function for NLRC5 in anti-viral innate immune responses.

Figures

FIGURE 1.
FIGURE 1.
NLRC5 structure and expression. A, model of the NLRC5 structure. The N-terminal effector and NACHT domain with a bound ATP molecule (blue) are based on the Apaf-1 effector domain structure (Protein Data Bank entry 1z6t). The C-terminal LRRs (red to yellow) are based on the TLR4 LRRs (Protein Data Bank entry 2z64). The figure was prepared with Pymol. The positions of the domains are indicated. B, quantitative PCR analysis of NLRC5 mRNA expression in the indicated human tissue and blood cells. The expression level of NLRC5 mRNA normalized to GAPDH expression relative to NLRC5 expression in CD4+ cells (set to 1) ± S.D. (n = 3) is shown. The order was determined by increasing expression levels. C, RT-PCR analysis of NLRC5 mRNA expression in the indicated cell culture lines. Amplification of GAPDH served as control. D, schematic representation of putative NLRC5 splice variants. Position of the DD, NACHT, and LRR domains are indicated by shaded boxes. The italic numbers refer to amino acid (aa) positions. E, RT-PCR analysis of the indicated tissue and cell culture lines using specific primer pairs to amplify NLRC5 full-length and isoform 3 expression and to detect the deletion in isoform 4 and 5 mRNA. Plasmids encoding NLRC5 full-length (fl), isoform 3, or isoform 4 cDNA served as controls. Amplification of GAPDH served as standard. WT, wild type.
FIGURE 2.
FIGURE 2.
NLRC5 protein expression. A, characterization of the 3H8 anti-NLRC5 monoclonal antibody. Western blot analysis of lysates of HEK293T (−) and HEK293T cells transfected with FLAG-NLRC5 (+) are shown. As control for expression of the NLRC5 plasmid, a similar blot was probed with anti-FLAG (M2) antibody (left panel). Running of a protein standard is indicated. B, THP-1 cells were treated for 48 h with a control (CTRL) or two NLRC5 specific siRNA duplexes. The cells were lysed in SDS-PAGE loading buffer, and NLRC5 was detected using the 3H8 antibody. Probing for α-tubulin served as a control for equal loading. Running of a protein standard is indicated. C, indirect immunofluorescence micrograph of HeLa cells, transiently transfected with FLAG-NLRC5 using anti-FLAG antibody. Co-staining with 4′,6-diamidino-2-phenylindole, dihydrochloride (DAPI, blue) is shown in the left panel. Bar, 10 μm.
FIGURE 3.
FIGURE 3.
Induction of NLRC5 expression by viral PAMPs. A, mRNA levels of NLRC5 after PAMP treatment in THP-1 cells. THP-1 cells were treated with 50 ng/ml tumor necrosis factor (TNF), 1 μm muramyl di-peptide (MDP), 50 ng/ml lipopolysaccharide (LPS), 0.5 μm phorbol 12-myristate 13-acetate (PMA), or 100 ng/ml Pam3CSK. 6 h after PAMP stimulation, RNA was prepared, and RT-PCRs were conducted amplifying NLRC5 and GAPDH cDNA as control. Interleukin-8 cDNA amplification served as an internal control for successful stimulation. B, quantitative PCR analysis of NLRC5 expression in HeLa cells treated for the indicated time with 100 μg/ml poly(I·C). The data are normalized to GAPDH expression (n = 3). C, Western blot analysis of HeLa, CaCo2, and THP-1 cells treated for 24 h with 100 μg/ml poly(I·C). Detection with the anti-NLRC5 antibody 3H8 is shown. Probing with a GAPDH-specific antibody served as loading control. BG indicates background bands. D, HeLa cells were treated for 48 h with either TAK1- or TBK1-specific siRNA or nontargeting siRNA (CTRL) as control. The cells were stimulated for an additional 24 h with 100 μg/ml poly(I·C) where indicated or left untreated (−). Probing with a GAPDH-specific antibody served as loading control. Knockdown of TBK1 is shown by probing with specific antibody (bottom panel). E, quantitative PCR analysis of NLRC5 expression in HeLa and THP-1 cells treated for the indicated time with Sendai virus. The data are normalized to GAPDH expression (n = 3). F, Western analysis of HeLa cells treated for 24 h with Sendai virus. Detection with the anti-NLRC5 antibody 3H8 is shown. Probing for α-tubulin served as a loading control.
FIGURE 4.
FIGURE 4.
NLRC5 signaling. A, co-immunoprecipitation of FLAG-tagged NLR proteins and GFP-NLRC5 transiently expressed in HEK293T cells. FLAG NLRs were precipitated with anti-FLAG antibody, and GFP-NLRC5 was detected in the lysate (Input) and the immunoprecipitations (IP). B, effect of overexpression of NLRC5 on the indicated pathways. HEK293T cells were transfected with increasing amounts (5, 10, and 30 ng) of FLAG-NLRC5 along with the indicated luciferase reporter constructs in 96-well plates. TBK1 overexpression, tumor necrosis factor treatment, or Sendai virus infection served as positive control for the reporter. Read-out was performed 16 h after transfection. Normalized relative light units (nRLU) of triplicate measurements ± S.D. representative of three independent experiments are shown. Upper right inset, Western blot showing expression of NLRC5 in the ISRE assay. Probing for GAPDH served as a loading control.
FIGURE 5.
FIGURE 5.
NLRC5 impacts on Sendai virus-mediated type I interferon responses. THP-1 cells were treated with the indicated siRNA duplexes. 72 h after transfection, the cells were stimulated with Sendai virus or left untreated as controls. A nontargeting siRNA (CTRL) was used as negative control. A, type I interferon secretion was assayed using a type I interferon secreted alkaline phosphatase reporter cell line (HEK-Blue IFN-α/β)-based bioassay. The data are shown relative to control siRNA set to 100%. B, efficient reduction of NLRC5 protein levels shown by Western analysis using the NLRC5-specific 3H8 antibody, probing with α-tubulin served as loading control. C, quantitative PCR analysis of IFN-β expression in the cells of A. The data are normalized to GAPDH expression (n = 2) and are presented relative to CTRL siRNA set to 100%. D, RANTES cytokine levels were measured in the supernatant of cells treated as in A after 16 h of SeV exposure (gray bars) or mock treatment (white bars). E, RT-PCR analysis of RANTES mRNA expression in THP-1 cells treated with control (ctrl) or NLRC5_4 siRNA for 72 h prior to SeV exposure for 24 h. Amplification of GAPDH served as a loading control.
FIGURE 6.
FIGURE 6.
Analysis of NLRC5 function in primary human dermal fibroblasts. A, quantitative PCR analysis of NLRC5 expression in cells treated for 24 h with 100 μg/ml poly(I·C). The data are normalized to GAPDH expression. CTRL, mock treated cells. B, cells were treated with the indicated NLRC5-specific siRNA duplexes (columns 1 and 4) for 72 h and subsequently stimulated with poly(I·C) (left panel) or Sendai virus (right panel). A nontargeting siRNA (CTRL) was used as a negative control. Type I interferon secretion was assayed using a type I interferon secreted alkaline phosphatase reporter cell line (HEK-Blue IFN-α/β)-based bioassay. The data are shown relative to CTRL siRNA set to 100%. C, RANTES cytokine levels measured in the supernatant of cells of B after 16 h of induction with poly(I·C) or SeV (gray bars) or mock treatment (white bars). D, RT-PCR analysis of NLRC5 mRNA expression in the cells shown in B and C treated with control (CTL) or NLRC5 siRNA for 72 h. Amplification of GAPDH served as a loading control.

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