Membrane microdomains regulate NLRP10- and NLRP12-dependent signalling in A549 cells challenged with cigarette smoke extract

  • Dhirendra P. Singh
  • Gagandeep Kaur
  • Prathyusha Bagam
  • Rakeysha Pinkston
  • Sanjay Batra
In vitro systems
  • 103 Downloads

Abstract

Chronic obstructive pulmonary disease (COPD) is predicted to become the third leading cause of death and disability worldwide by 2030; with cigarette smoking (active or passive) being one of the chief cause of its occurrence. Cigarette smoke exposure has been found to result in excessive inflammation and tissue injury, which might lead to COPD, although the exact pathophysiology of the disease remains elusive. While previous studies have demonstrated the role of membrane-bound Toll-like receptors (TLRs) in cigarette smoke (CS)-induced inflammation, scant information is available about the role of cytosolic NOD-like receptors (NLRs) in regulating CS-mediated inflammatory responses. Thus, we investigated the role of NLRP10 and NLRP12 in regulating inflammatory responses in human alveolar type II epithelial cells (A549) and human monocytic cells (THP-1) in response to a challenge with cigarette smoke extract (CSE). We observed CSE-mediated increase in caspase-1 activity; production of IL-1β and IL-18; and expression of NLRP10 and NLRP12 in A549 and THP-1 cells. Interestingly, immunofluorescence imaging results demonstrated an increase in the membrane recruitment of NLRP10 and NLRP12 proteins in CSE-challenged A549 cells. We also observed an increase in the expression of lipid raft proteins (caveolin-1, caveolin-2, and flotillin-1) and an induction of lipid raft assembly following CSE-exposure in A549 cells. Lipid rafts are cholesterol-rich membrane microdomains well known to act as harbours for signalling molecules. Here we demonstrate  the recruitment of NLRP10 and NLRP12 in lipid raft entities as well as the interaction of NLRP12 with the lipid raft protein caveolin-1 in CSE-challenged A549 cells. Furthermore, enrichment of lipid raft entities with poly-unsaturated fatty acids (PUFA) rescued A549 cells from CSE-mediated membrane recruitment of NLRP10 and NLRP12, and also from inflammatory responses and inflammasome activation. Enrichment of membrane microdomains with PUFA was able to reverse filipin (chemical agent used for disrupting lipid rafts)-mediated enhanced inflammation in CSE-challenged A549 cells. Overall, our findings unveil a novel mechanism by identifying an important role of membrane microdomains (lipid rafts) in regulating CSE-induced inflammation and NLRP10/NLRP12-dependent signalling in A549 cells.

Keywords

Cigarette smoke extract (CSE) NLRP10 NLRP12 Lipid Rafts And COPD 

Notes

Acknowledgements

This work was supported by Young Clinical Scientist Award from the Flight Attendant Medical Research Institute (FAMRI-123253_YCSA_Faculty); NIH R15 (7 R15 ES023151 02); Southern University Foundation Grant (FY2017-017/FY2018-020); and LBRN Startup Grant (2P20GM103424-14 -Subaward No. 100011) to SB. Authors would like to acknowledge the VCU Lipidomics/Metabolomics Core, the NIH-NCI Cancer Center Support Grant P30 CA016059, as well as a shared resource grant (S10RR031535) from the National Institutes of Health to the VCU Massey Cancer Center. Authors would also like to sincerely thank Ms. Karen McDonough, AgCenter Cell Culture Facility, Louisiana State University, Baton Rouge, Louisiana for maintaining the cell culture for experiments.

Compliance with ethical standards

Conflict of interest

Authors declare no conflict of interest.

Supplementary material

204_2018_2185_MOESM1_ESM.jpg (292 kb)
PUFA or DHA enriched A549 cells show differential membrane recruitment of NLRP10 and NLRP12 in response to CSE-challenge. Immunoblotting and densitometry results show differential recruitment of NLRP10 and NLRP12 proteins in the membrane fractions isolated from CSE-challenged control and PUFA/DHA enriched-A549 cells. (n = 3–4/group). Results are a representation of single set from three independent experiments. Here, CSE-0.25 represents 0.25 µg/ml concentration (JPEG 293 KB)
204_2018_2185_MOESM2_ESM.jpg (1.4 mb)
Enrichment with saturated fatty acid does not rescue A549 cells from CSE-induced inflammatory responses. a Cytokine/chemokine (IL-1β, CCL-2 and CXCL-8) production in arachidic acid (saturated fatty acid) enriched CSE-challenged A549 cells. (n = 3–4/group). Results are a representation of three independent experiments. Error bars represent SEM, * (p <= 0.05), ** (p <= 0.01), and *** (p <= 0.001), as per one-way ANOVA for multiple comparisons. b PUFA mediated decrease in membrane cholesterol in CSE-challenged A549 cells. Results demonstrating cellular localization of cholesterol in CSE-challenged control and PUFA-enriched A549 cells. (n = 3–4/group). Results are a representation of three independent experiments (JPEG 1484 KB)
204_2018_2185_MOESM3_ESM.doc (85 kb)
Supplementary material 3 (85 KB)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Dhirendra P. Singh
    • 1
  • Gagandeep Kaur
    • 1
  • Prathyusha Bagam
    • 1
  • Rakeysha Pinkston
    • 1
  • Sanjay Batra
    • 1
    • 2
  1. 1.Laboratory of Pulmonary Immuno-toxicology, Environmental Toxicology DepartmentSouthern University and A&M CollegeBaton RougeUSA
  2. 2.Department of Pathobiological Sciences, School of Veterinary MedicineLouisiana State UniversityBaton RougeUSA

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