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Non-resolving pro-inflammatory macrophage polarization by super-low doses of bacterial endotoxin

Subclinical endotoxemia (low levels of circulating bacterial endotoxin) has been observed in patients suffering from chronic inflammatory diseases such as atherosclerosis, diabetes, and obesity. However, the link between this condition and chronic inflammation is poorly understood. Previous work from our lab has shown that chronic exposure to super-low doses of bacterial endotoxin (LPS) aggravates atherosclerosis resulting in increased plaque size and instability in a macrophage-dependent manner in a mouse model of atherosclerosis. Further, we showed that super-low dose LPS (SLD-LPS) treatment was able to inhibit lysosomal fusion in immortalized macrophages. However, this was done under more acute treatment conditions. The aim of this project was to examine the molecular mechanisms by which chronic SLD-LPS may polarize macrophages to a non-resolving pro-inflammatory state consistent with chronic inflammation. This was carried out in two projects, the first a more broad phenotypic paper showing the disruption in homeostasis by chronic SLD-LPS in immortalized macrophages, while the second uses primary bone marrow-derived mouse macrophages to identify specific molecular signaling pathways used by chronic SLD-LPS.

Here we show that chronic SLD-LPS led to the novel upregulation of pro-inflammatory mediators p62 and ccl2 with simultaneous downregulation of homeostatic mediators Nrf2 and slc40a1 in immortalized wild-type mouse macrophages. Further we showed this effect was reversed using the homeostatic restorative agent sodium phenylbutyrate (4-PBA), a newly reported activity for this reagent in mouse macrophages. This indicated that a disruption in homeostasis, possibly involving autophagy, may be responsible for the non-resolving pro-inflammatory polarization of macrophages. Therefore, in our second project, we further explored the effect of chronic SLD-LPS treatment on the homeostatic arm of the response by focusing on the Nrf2 inhibitor Keap1. Here we show that chronic SLD-LPS results in an accumulation of Keap1 in mouse bone marrow-derived macrophages, an effect specific to chronic SLD-LPS, as high doses of LPS failed to induce Keap1. We suggest that this effect may be related to a disruption in lysosomal fusion as evidenced by accumulation of autophagy flux markers MLKL and p62. Further, we show that these effects are dependent on the non-traditional TLR4 adaptor TRAM, suggesting an alternative dose-dependent signaling pathway for LPS.

Together this work identifies novel signaling mechanisms involved in non-resolving pro-inflammatory polarization of murine macrophages, providing new insight behind how chronic super-low dose LPS exposure may lead to chronic inflammation. / Doctor of Philosophy / Inflammation is the body's natural response to injury or insult and can be beneficial in certain contexts such as pathogen clearance. However, left un-checked, chronic inflammation can exacerbate or even lead to disease pathology, such as is the case with modern diseases such as atherosclerosis, obesity, diabetes, etc. Despite the high prevalence of these diseases, effective treatments and therapies are still lacking. Recently it was discovered that many patients suffering from chronic inflammatory diseases had low levels bacterial endotoxin (LPS) in their circulation, a condition referred to as subclinical endotoxemia. However, possible links between this condition and chronic inflammatory disease remain poorly understood. Using a mouse model of atherosclerosis, previous research from our lab showed that persistent exposure to super-low doses of bacterial endotoxin (similar to those observed in humans) lead to aggravated atherosclerosis with both increased plaque size and instability. Further, we showed that this effect was primarily mediated by pro-inflammatory polarized immune cells called macrophages, but the molecular mechanism behind this polarization is still unclear. Further research into these molecular mechanisms may provide better targets for the development of future chronic inflammatory disease treatments. Here using a combination of mouse cell line and primary cell cultures, we discuss how chronic exposure to super-low doses of bacterial endotoxin leads to the chronic non-resolving pro-inflammatory polarization of macrophage immune cells, with particular emphasis on the distinct molecular signaling mechanisms induced by chronic super-low dose LPS.

Identiferoai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/104112
Date10 January 2020
CreatorsRahtes, Allison Anne
ContributorsBiological Sciences, Li, Liwu, Lu, Chang, Jones, Caroline N., Cecere, Thomas E., Xu, Bin
PublisherVirginia Tech
Source SetsVirginia Tech Theses and Dissertation
Detected LanguageEnglish
TypeDissertation
FormatETD, application/pdf, application/vnd.openxmlformats-officedocument.wordprocessingml.document
RightsIn Copyright, http://rightsstatements.org/vocab/InC/1.0/

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