LDN diminished LPS induced cytokine storm.
Cytokine storm is a very commonly observed factor in most severe COVI19 patients and also one of the leading causes of mortality5–7. Peripheral blood of severe COVID-19 patients has also shown a high level of cytokine storm14. Keeping in mind the ability of lipopolysaccharide (LPS) to cause sepsis and triggers an uncontrolled systemic inflammatory response in murine macrophage cells15, we treated macrophage cells with LPS (1μg/ml) in presence and absence of LDN (5 μM). Results demonstrated that LPS treatment significantly induced expression on pro-inflammatory cytokines (IL-1β, IL-6and TNF-a) whereas, LDN significantly inhibited LPS expression of IL-1β, IL-6and TNF-a in macrophage cells (Fig. 1A ). Adipose tissue macrophage (ATM) is closely linked to this inflammatory condition which leads to numbers of diseases and the ability of HFD feeding on increased LPS uptake and trafficking to macrophages and other targets are well known16. Hence in this study, we investigated the effect of High Fat Diet (HFD) on cytokines expression in purified ATMs. Expression of pro-inflammatory markers such as IL-1β , TNF-a, and IL6 was induced (Fig-1B ) whereas LDN attenuates HFD induced expression profile of pro-inflammatory cytokine. LDN shows a protective (by reducing elevated M1 cytokines) from HFD mediated inflammation. Next, we tested the possible involvement of High Fat Diet (HFD) in inducing the release of proinflammatory cytokines, and we also determine the effects of LDN on release of proinflammatory mediators in HFD mice model (Fig 1C ). Serum from HFD mice show a significantly enhanced release proinflammatory mediators (IL-1Β, TNF-α and IL-6) and interestingly we found that LDN treatment attenuated HFD induced serum IL-1Β, TNF-α and IL-6 level (Fig-1C ). Cell viability was determined by utilizing MTT assay in macrophage cell line. The assay revealed that the selected range of dose is safe and did not affect cell viability at the different doses exposed (Fig S1). These data suggest that HFD diet induced mice persuaded the release of various pro-inflammatory mediators, LDN treatment can significantly abrogate HFD induced release of proinflammatory mediators in serum.