Palmitoleate protects against lipopolysaccharide-induced inflammation and inflammasome activity

Figures

• 

  Fig 1. Palmitoleate prevents LPS-induced inflammation and inflammasome activation in macrophages. Mouse bone marrow–derived macrophages (BMDMs) and THP-1–derived macrophages (THP-1 DMs) were exposed with LPS (10 ng/ml) for 3 h to mimic inflammation, in vitro. LPS increased the expression of proinflammatory cytokines like IL-1β, IL-6, and TNF-α mRNA relative to control HPRT mRNA expression. Pretreatment of palmitoleate (PO), 200 μM, for 16 h significantly decreased the mRNA levels of proinflammatory cytokines in BMDMs, and THP-1 DM with LPS (A, B). BMDMs were exposed with LPS (10 ng/ml) for 3 h and ATP (5 mM) for 30 min showed increased protein expression of pro-IL-1β, IL-1β, and cleaved caspase 1 as measured by immunoblot. Pretreatment of PO for 16 h and then stimulation with LPS for 3 h followed by ATP for 30 min decreased the expression of pro-IL-1β, IL-1β, and active caspase 1 suggesting that PO prevents inflammasome activation (C). Increased IL-1β release into the culture supernatant with the exposure of LPS + ATP suggests inflammasome activation and treatment of PO significantly prevented the release of IL-1β into the culture supernatant, suggesting that palmitoleate has…

• 

  Fig 2. Palmitoleate (PO) prevents LPS-induced inflammation in trophoblasts. HTR-8 cells (A) or JEG-3 cells (B) with 1 μg/ml of LPS for 4 h to mimic maternal systemic inflammation in vitro. LPS increased the expression of proinflammatory cytokines IL-1β, IL-6, and TNF-α mRNA relative to control 18S rRNA expression in trophoblasts. Pretreatment of 200 μM PO for 16 h followed by LPS for 4 h prevents LPS alone induced inflammation as evidenced by decreased the mRNA levels of proinflammatory cytokine TNF-α in HTR-8 (A) and JEG-3 cells (B). The data represent the mean ± SEM for n = 3. ∗P < 0.05, ∗∗P < 0.01. IL, interleukin; LPS, lipopolysaccharide.

• 

  Fig 3. Palmitoleate decreases the expression of Pro-IL1β in trophoblasts. HTR-8 cells were exposed with LPS (1 μg/ml) for 4 h and ATP (5 mM) for 30 min showed increased protein expression of pro-IL-1β, and mature IL-1β measured by immunoblot. Pretreatment of PO for 16 h and then sequential treatment with LPS (4 h) + ATP (30 min), decreased the expression of Pro-IL-1β, and mature IL-1β. Actin levels were unchanged in all the treatment conditions (A). Levels of NLRP3, procaspase 1 and cleaved and active caspase 1 were also unchanged with the treatment of LPS + ATP, LPS + ATP + PO compared vehicle-treated cells (B). The images are representative of 3–4 independent experiments. IL, interleukin; LPS, lipopolysaccharide; NLRP3, NACHT, LRR, and PYD domain–containing protein 3.

• 

  Fig 4. Palmitoleate (PO) protects against LPS-induced inflammation by blocking NF-κB nuclear translocation or activation. BMDMs were stimulated with LPS (10 ng/ml) alone or in presence of PO (pretreatment for 16 h at 200 μM) for 5–10 min and analyzed for NF-κB signaling pathway activation. Immunoblot analysis showed decreased levels of IκBα after 10 min of LPS exposure (A). Pretreatment of PO however did not block the degradation of IκBα (A). LPS exposure (5–10 min) also increased phosphorylated forms of IKKα/β and PO did not alter the levels of phospho-IKKα/β after 5–10 min. Immunoblot analysis also showed that total IKK-α, IKK-β, and β-actin levels remained unchanged in all the treatment conditions tested (A). We also observed increased phosphorylation of JNK following LPS exposure, and treatment of palmitoleate did not change the levels of phosphorylated JNK (A). Total JNK level remained the same in all experimental conditions (A). NF-κB p65 nuclear translocation were tested by immunoblot following LPS exposure in BMDMs and observed to be increased in the nuclei, suggesting increased nuclear translocation of p65 subunit. Pretreatment of PO for 16 h followed by LPS exposure showed…

• 

  Fig 5. Palmitoleate protects against LPS and palmitate mediated cell death and IL-1β release in BMDMs. We exposed BMDMs with LPS for 3 h and then treated with palmitate (PA, 200–400 μM) for 16 h and percent cell death were measured. LPS alone did not increase percent cell death, and treatment of PA post LPS exposure resulted in a significant increase in percent cell death compared to vehicle (Veh) treated BMDMs (A, B). Pretreatment of palmitoleate (PO, 200 μM) followed by LPS and PA treatment, significantly prevented LPS + PA-induced increase in percent cell death in BMDMs (A, B). Release of IL-1β in cell culture supernatant showed that LPS and palmitate exposure to BMDMs results in a significant increase in mature IL-1β release to culture supernatant compared to Veh cells. Pretreatment of PO prevented IL-1β release in response to LPS and palmitate coexposure (C). The image shown is representative images from n = 3. Data represents means ± SEM, ∗∗∗∗P < 0.0001. BMDM, bone marrow–derived macrophage; IL, interleukin; LPS, lipopolysaccharide.

• 

  Fig 6. Palmitoleate inhibits inflammasome-mediated caspase 1 and pro-IL-1β cleavage. Immunoblot analysis of BMDMs exposed with LPS and palmitate (PA) resulted in the cleavage of caspase 1 to cleaved caspase 1 p20 subunit (A, lane 3) and pretreatment of PO blocked cleavage of caspase 1 (A, lane 6). BMDM exposed to LPS and PA, showed an increase in both the pro and mature form of IL-1β compared to vehicle-treated cells (B, lane 1 and 3). Pretreatment of PO followed by LPS and PA resulted in a decreased levels of both pro and mature IL-1β protein compared to LPS and palmitate-treated cells (B, lane 3 and 6). NLRP3 and β-actin levels remains unchanged with different treatment conditions tested. BMDM, bone marrow–derived macrophage; IL, interleukin; LPS, lipopolysaccharide.

• 

  Fig 7. LPS and palmitate (PA) activates MAPKs in BMDMs. Immunoblot analysis of BMDMs exposed with LPS (10 ng/ml) for 3 h and then palmitate (PA, 400 μM) for 16 h showed MAPKs activation as evidenced by robust increase in the levels of phospho-JNK, phospho-p38, and phospho-ERK1/2 (lane 3) compared LPS alone or vehicle-treated cells (lane 1 and 2). Pretreatment of palmitoleate (200 μM, PO) followed by LPS and PA sequential exposure blocked phosphorylation status of all three MAPKs (p-JNK, p-p38, and p-ERK levels) compared to LPS and palmitate treated BMDMs (lane 6). Total JNK, p38, ERK1/2, and β-actin levels remained similar in all experimental conditions tested. BMDM, bone marrow–derived macrophage; IL, interleukin; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase.

• 

  Fig 8. Schematic diagram on the protective role of palmitoleate against inflammation and inflammasome activity. Palmitoleate blocks NF-κB p65 subunit nuclear translocation, thereby blocking LPS-mediated activation of NF-κB pathway and proinflammatory cytokine gene upregulation. Palmitoleate also blocks NLRP3 inflammasome-mediated cleavage of procaspase 1 and pro-IL-1β to active caspase 1 p10/p20 subunits and IL-1β, respectively, thereby preventing LPS and saturated free fatty acid–induced cellular death and inflammation. Further, palmitoleate also prevents LPS + palmitate (PA)-induced cellular MAPK and inflammatory responses. Illustration was made using BioRender. IL, interleukin; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; NLRP3, NACHT, LRR, and PYD domain–containing protein 3.