Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin

Abstract

Murine NLR family, apoptosis inhibitory protein (Naip)1, Naip2, and Naip5/6 are host sensors that detect the cytosolic presence of needle and rod proteins from bacterial type III secretion systems and flagellin, respectively. Previous studies using human-derived macrophage-like cell lines indicate that human macrophages sense the cytosolic needle protein, but not bacterial flagellin. In this study, we show that primary human macrophages readily sense cytosolic flagellin. Infection of primary human macrophages with Salmonella elicits robust cell death and IL-1β secretion that is dependent on flagellin. We show that flagellin detection requires a full-length isoform of human Naip. This full-length Naip isoform is robustly expressed in primary macrophages from healthy human donors, but it is drastically reduced in monocytic tumor cells, THP-1, and U937, rendering them insensitive to cytosolic flagellin. However, ectopic expression of full-length Naip rescues the ability of U937 cells to sense flagellin. In conclusion, human Naip functions to activate the inflammasome in response to flagellin, similar to murine Naip5/6.

FIGURE 1.

Flagellin stimulates inflammasome activation in primary human macrophages. Human MDM were primed with 100 ng/ml LPS and infected with log phase WT Serovars S. Typhimurium (Stm) and S. Typhi (St) or the respective nonflagellated (ΔFla) variant (multiplicity of infection of 50). IL-1β secretion (A) and cytotoxicity (B) were determined 1 h after infection. (C) Activation of pyroptotic caspase-1 (Casp1) processing was visualized by immunoblotting (kDa). (D and E) IL-1β secretion and cell death caused by lethal factor–coupled protein variants were tested as described above for (A) and (B). Graphs show mean and SD of six replicate wells and are representative of at least three independent experiments. Significance was calculated using an unpaired Student t test. ****p < 0.0001. ns, p = 0.1–0.9999.

FIGURE 2.

Flagellin-dependent inflammasome activation correlates with the expression level of a human Naip isoform. (A) Quantitative RT-PCR was performed to assess the amounts of transcript isoform NAIP* expressed in primary macrophages (MDM) and differentiated U937 cells and normalized to GAPDH. (B and C) MDM and differentiated U937 cells were tested for their ability to undergo Salmonella-induced cell death and secretion of IL-1β (as described). Results are representative of at least two or more independent experiments. Significance was determined using an unpaired Student t test. **p < 0.05, ***p < 0.001, ****p < 0.0001.

FIGURE 3.

Expression of human Naip isoform that responds to flagellin in U937 monocytes increases cell death during Salmonella infection. Undifferentiated U937 cells were transfected with either an empty vehicle (V) or a construct containing the Naip* isoform (Naip*) under control of a CMV promoter. Twenty-four hours later the cells were infected with log phase S. Typhimurium (Stm) variants as indicated and the amount of cell death was determined by lactate dehydrogenase assay. Statistics were done using an unpaired Student t test. ***p < 0.001, ****p < 0.0001.

FIGURE 4.

Depletion of the human Naip sensor decreases inflammasome activation in response to flagellin. Fully differentiated U937 cells stably expressing either a scrambled control shRNA or Naip-specific shRNA were treated with either PA alone (−) or in combination with LFn-fusion proteins as indicated. IL-1β secretion (A) and cell death (B) were determined following each stimulus. Results are representative of at least two or more independent experiments. Statistics were done using an unpaired Student t test. ****p < 0.0001. C, control shRNA; N, Naip-specific shRNA.