Palmitoleate protects against lipopolysaccharide-induced inflammation and inflammasome activity

Abstract

Inflammation is part of natural immune defense mechanism against any form of infection or injury. However, prolonged inflammation could perturb cell homeostasis and contribute to the development of metabolic and inflammatory diseases, including maternal obesity, diabetes, cardiovascular diseases, and metabolic dysfunction-associated steatotic liver diseases (MASLD). Polyunsaturated fatty acids have been shown to mitigate inflammatory response by generating specialized proresolving lipid mediators, which take part in resolution of inflammation. Similarly here, we show that palmitoleate, an omega-7 monounsaturated fatty acid exerts anti-inflammatory properties in response to lipopolysaccharide (LPS)-mediated inflammation. Exposure of bone marrow-derived macrophages (BMDMs) to LPS or TNFα induces robust increase in the expression of proinflammatory cytokines and supplementation of palmitoleate inhibited LPS-mediated upregulation of proinflammatory cytokines. We also observed that palmitoleate was able to block LPS + ATP-induced inflammasome activation mediated cleavage of procaspase 1 and prointerleukin-1β. Further, treatment of palmitoleate protects against LPS-induced inflammation in human THP-1-derived macrophages and trophoblasts. Coexposure of LPS and palmitate (saturated free fatty acid) induces inflammasome and cell death in BMDMs, however, treatment of palmitoleate blocked LPS and palmitate-induced cell death in BMDMs. Further, LPS and palmitate together results in the activation of mitogen-activated protein kinases and pretreatment of palmitoleate inhibited the activation of mitogen-activated protein kinases and nuclear translocation of nuclear factor kappa B in BMDMs. In conclusion, palmitoleate shows anti-inflammatory properties against LPS-induced inflammation and LPS + palmitate/ATP-induced inflammasome activity and cell death.

Keywords: macrophages; mitogen-activated protein kinase; monounsaturated fatty acids; obesity; placenta; pregnancy; trophoblasts.

Graphical abstract

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 anti-inflammatory and anti-inflammasome properties in macrophages (D). BMDMs were exposed with TNFα (10 ng/ml) for 30 min to mimic inflammation, in vitro. TNFα increased the expression of proinflammatory cytokines like IL-1β, IL-6, andTNF-α mRNA relative to control HPRT mRNA expression. Pretreatment of palmitoleate (PO), 200 μM, for 16 h followed by TNFα for 30 min significantly decreased the mRNA levels of proinflammatory cytokines in BMDMs (E).The data represent the mean ± SEM for n = 3. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001. IL, interleukin; LPS, lipopolysaccharide.

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 decreased nuclear levels of p65 subunit in BMDMs (B). Histone deacetylase 1 was used as nuclear loading control and showed no change under any treatment conditions (B). BMDM, bone marrow–derived macrophage; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide.

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.