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Highly Efficient Transfection of Human THP-1 Macrophages by Nucleofection
Published on: September 2, 2014
Cutting Edge: Inflammasome Activation in Primary Human Macrophages Is Dependent on Flagellin
Jens Kortmann1, Sky W Brubaker1, Denise M Monack2
1Department of Microbiology and Immunology, Stanford School of Medicine, Stanford University, Stanford, CA 94305.
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.
Insights
Primary human macrophages detect bacterial flagellin using NLR family, apoptosis inhibitory protein (Naip), activating the inflammasome. This sensing mechanism is impaired in certain cancer cell lines but can be restored by Naip expression.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Murine NLR family, apoptosis inhibitory proteins (Naips) sense bacterial components like flagellin.
- Previous studies suggested human macrophages do not sense flagellin, unlike needle proteins.
Purpose of the Study:
- To investigate flagellin sensing by primary human macrophages.
- To determine the role of human Naip in detecting cytosolic flagellin and activating the inflammasome.
Main Methods:
- Infection of primary human macrophages with Salmonella.
- Assessing cell death and IL-1β secretion.
- Analyzing Naip isoform expression in different cell types.
- Ectopic expression of Naip in U937 cells.
Main Results:
- Primary human macrophages effectively sense cytosolic flagellin, leading to cell death and IL-1β secretion.
- Flagellin detection is dependent on a full-length human Naip isoform.
- This Naip isoform is highly expressed in primary macrophages but reduced in THP-1 and U937 cells.
- Restoring Naip expression in U937 cells rescued flagellin sensing.
Conclusions:
- Human Naip acts as a cytosolic flagellin sensor, activating the inflammasome.
- This function is analogous to murine Naip5/6.
- Naip expression levels influence inflammasome activation in human macrophages.
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