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. 2013 Oct 15;191(8):3986-9.
doi: 10.4049/jimmunol.1301549. Epub 2013 Sep 16.

Cutting edge: Mouse NAIP1 detects the type III secretion system needle protein

Manira Rayamajhi  1 , Daniel E Zak, Joseph Chavarria-Smith, Russell E Vance, Edward A Miao
Affiliations

Cutting edge: Mouse NAIP1 detects the type III secretion system needle protein

Manira Rayamajhi et al. J Immunol. .

Abstract

The NAIP/NLRC4 inflammasomes activate caspase-1 in response to bacterial type III secretion systems (T3SSs). Inadvertent injection of the T3SS rod protein and flagellin into the cytosol is detected through murine NAIP2 and NAIP5/6, respectively. In this study, we identify the agonist for the orphan murine NAIP1 receptor as the T3SS needle protein. NAIP1 is poorly expressed in resting mouse bone marrow-derived macrophages; however, priming with polyinosinic-polycytidylic acid induces it and confers needle protein sensitivity. Further, overexpression of NAIP1 in immortalized bone marrow-derived macrophages by retroviral transduction enabled needle detection. In contrast, peritoneal cavity macrophages basally express NAIP1 and respond to needle protein robustly, independent of priming. Human macrophages are known to express only one NAIP gene, which detects the needle protein, but not rod or flagellin. Thus, murine NAIP1 is functionally analogous to human NAIP.

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Conflict of interest statement

Disclosures

The authors declare that they have no competing financial interests.

Figures

Figure 1
Figure 1. Human macrophages detect cytosolic PrgI while mouse macrophages show a weak response
Human monocytic cell lines U937 (A, B) and Thp1 (C, D) were differentiated into macrophages with phorbol myristate acetate (PMA), primed with 50 ng/ml LPS, and then transfected with purified PrgI or PrgJ. 2 h post transfection, cytotoxicity (A, C) and IL-1β secretion (B, D) were determined by LDH release and ELISA, respectively. BMMs primed with LPS (50ng/ml) for 4 hours were transfected with 125ng/well PrgI or PrgJ for 1 h and (E) cytotoxicity or (F) IL-1β secretion were determined. (G, H) Retroviral lethality screen was performed by transducing C57BL/6 and Nlrc4−/− BMMs with retroviruses expressing GFP alone (empty vector), prgJ-GFP, prgJΔLRRs-GFP, or prgI-GFP. Survival of GFP+ cells was determined by flow cytometry 2 days later by flow cytometry. Part of data in G and H (GFP alone and prgJ-GFP) were previously published in (8); prgJΔLRRs-GFP and prgI-GFP transduction data from the same experiment is now presented here. (H) Quantification of G. Graphs show mean and standard deviation of triplicate wells and are representative of at least 3 independent experiments.
Figure 2
Figure 2. Naip1 expression in BMMs with or without stimulation with LPS or PolyIC from the innate immune database
(A) BMMs were left untreated or primed with LPS or poly(I:C) 23 h and RQ-PCR was performed to assess Naip1 and Naip2 transcript levels relative to Rps17. (B) Cytotoxicity in response to cytosolic PrgI or PrgJ protein was determined at 2 h in unstimulated BMMs or BMMs primed with 6μg/ml Poly(I:C) for 2 days. Data representative of 2–3 independent experiments.
Figure 3
Figure 3. NAIP1 complementation confers robust PrgI sensitivity to BMMs
PrgI or PrgJ were transfected into the cytosol of iBMMs and (A) cytotoxicity and (B) IL-1β secretion were measured at 2 h. (C–D) iBMMs were transduced with retroviruses expressing either GFP alone (Empty vector) or Naip1-IRES-GFP and GFP+ cells were purified by FACS. (C) Cytotoxicity and (D) IL-1β secretion were determined 2 h after PrgI transfection. Data in (A–B) and (C–D) are representative of 2 and 3 independent experiments respectively.
Figure 4
Figure 4. Peritoneal cavity macrophages that express NAIP1 basally respond to T3SS needle
(A) Quantitative PCR was performed to assess the amounts of Naip1 (red bars), Naip2 (black bars), Naip5 (grey bars) and Naip6 (white bars) transcripts expressed in BMMs or the indicated organs and normalized to GAPDH. (B) Levels of Naip1 and Naip2 transcripts were determined in BMMs (white bars) or peritoneal cavity macrophages (PerC) by quantitative PCR.(C) PerC macrophages from C57BL/6 (black bars) or Nlrc4−/− mice (white bars) were primed with LPS (50ng/ml) before transfection with PrgI, PrgJ, or FlgE. IL-1β in the supernatants was measured at 2 h. Data representative of at least 2–3 independent observations.
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