thapsigargin and Sepsis

Immune cell chemotaxis to the sites of pathogen invasion is critical for fighting infection, but in life-threatening conditions such as sepsis and Covid-19, excess activation of the innate immune system is thought to cause a damaging invasion of immune cells into tissues and a consequent excessive release of cytokines, chemokines and neutrophil extracellular traps (NETs). In these circumstances, tempering excessive activation of the innate immune system may, paradoxically, promote recovery. Here we identify the antimalarial compound artemisinin as a potent and selective inhibitor of neutrophil and macrophage chemotaxis induced by a range of chemotactic agents. Artemisinin released calcium from intracellular stores in a similar way to thapsigargin, a known inhibitor of the Sarco/Endoplasmic Reticulum Calcium ATPase pump (SERCA), but unlike thapsigargin, artemisinin blocks only the SERCA3 isoform. Inhibition of SERCA3 by artemisinin was irreversible and was inhibited by iron chelation, suggesting iron-catalysed alkylation of a specific cysteine residue in SERCA3 as the mechanism by which artemisinin inhibits neutrophil motility. In murine infection models, artemisinin potently suppressed neutrophil invasion into both peritoneum and lung in vivo and inhibited the release of cytokines/chemokines and NETs. This work suggests that artemisinin may have value as a therapy in conditions such as sepsis and Covid-19 in which over-activation of the innate immune system causes tissue injury that can lead to death.

Topics: Animals; Artemisinins; Calcium; Calcium-Transporting ATPases; Chemotaxis; COVID-19 Drug Treatment; Cytokines; Extracellular Traps; Macrophages; Mice; Neutrophils; Sepsis; Thapsigargin

Previous studies have shown that pretreatment with thapsigargin (TG), a cellular stress inducer, produced potent protective actions against various pathologic injuries. So far there is no information on the effects of TG on the development of bacterial sepsis. Using lipopolysaccharides- and cecal ligation/puncture-induced sepsis models in mice, we demonstrated that preconditioning with a single bolus administration of TG conferred significant improvements in survival. The beneficial effects of TG were not mediated by ER stress induction or changes in Toll-like receptor 4 signaling. In vivo and in cultured macrophages, we identified that TG reduced the protein production of pro-inflammatory cytokines, but exhibited no significant effects on steady state levels of their transcriptions. Direct measurement on the fraction of polysome-bound mRNAs revealed that TG reduced the translational efficiency of pro-inflammatory cytokines in macrophages. Moreover, we provided evidence suggesting that repression of the mTOR (the mammalian target of rapamycin) signaling pathway, but not activation of the PERK (protein kinase R-like endoplasmic reticulum kinase)-eIF2α (eukaryotic initiation factor 2α) pathway, might be involved in mediating the TG effects on cytokine production. In summary, our results support that pharmacological preconditioning with TG may represent a novel strategy to prevent sepsis-induced mortality and organ injuries.

Topics: Animals; Anti-Inflammatory Agents; Cytokines; Disease Models, Animal; Humans; Male; Mice; Mice, Inbred C57BL; Oxidative Stress; Pre-Exposure Prophylaxis; Protective Agents; RAW 264.7 Cells; Sepsis; Thapsigargin; Toll-Like Receptor 4

Sepsis is a systemic inflammatory response syndrome induced by infection. T Lymphocytes play an important role in this disease. Transient receptor potential (TRP) channels and calcium-sensing receptors (CaSR) are expressed in lymphocytes to promote intracellular Ca(2+) release. However, data about the link between CaSR and TRP channels in septic T lymphocytes are few. In this study, by Ca(2+) imaging and Western blotting, we found that in septic rat peripheral blood T lymphocytes expressions of TRPC3 and TRPC6 proteins are higher. The SR/ER Ca(2+) ATPase inhibitor thapsigargin (TG) and CaSR agonist NPS R-568 also increased expressions of TRPC3 and TRPC6 proteins, which were reversed by PLC-IP3 channel blocker U73122 and TRPC channels inhibitor SKF96365. By Ca(2+) imaging, we found that the depletion of ER Ca(2+) stores by TG elicited a transient rise in cytoplasmic Ca(2+), followed by sustained increase depending on extracellular Ca(2+). But, SKF96365, not Verapamil (L-type channels inhibitor) and NiCl2 (Na(+)/Ca(2+) exchanger inhibitor), inhibited the relatively high [Ca(2+)]i. NPS R-568 also resulted in the same effect, and the duration of [Ca(2+)]i increase was eliminated completely by U73122 and was reduced in the absence of [Ca(2+)]o. NPS R-568 and TG increased the apoptotic ratio of septic T lymphocytes, which can be suppressed by SKF96365 and U73122. These results suggested that CaSR activation promoted the expression of TRPC3 and TRPC6 and enhanced T lymphocytes apoptosis through PLC-IP3 signaling pathway in sepsis.

Topics: Animals; Apoptosis; Calcium Signaling; Extracellular Space; Flow Cytometry; Inositol 1,4,5-Trisphosphate; Intracellular Space; Ion Channel Gating; Rats, Wistar; Receptors, Calcium-Sensing; Sepsis; T-Lymphocytes; Thapsigargin; TRPC Cation Channels; Type C Phospholipases

The endoplasmic reticulum (ER) is responsible for the synthesis and folding of secretory, transmembrane and ER-resident proteins. Conditions that impair protein folding or overwhelm its protein folding capacity disrupt ER homeostasis, thereby causing ER stress. ER stress-induced apoptosis and inflammation are involved in the pathogenesis of inflammatory diseases. Activated protein C (APC) inhibits inflammation and apoptosis in monocytes, and this may partly explain the protective effects of APC treatment in severe sepsis. However, the precise molecular pathways by which APC modulates these effects remain unknown.. To investigate whether APC modulates the ER stress response in human monocytes.. We treated monocytes with ER stress-inducing agents in the presence or absence of APC to determine the effect on this response. Protein and mRNA levels were determined by immunoblotting and real-time PCR, respectively. Enzyme assays and flow cytometry were used to determine the role of APC in this model.. In thapsigargin (Tg)-treated cells, APC dampened unfolded protein response activation, as indicated by reduced levels of the 78-kDa glucose-regulated protein (GRP78), in an endothelial protein C receptor-independent and protease-activated receptor-1-independent manner. Consistent with this, APC decreased phosphorylated eukaryotic translational initiation factor 2α and C/EBP homologous protein levels induced by Tg. APC inhibited Tg-induced ER Ca(2+) flux and reactive oxygen species generation. Functionally, APC diminished Tg-induced caspase-3 activity and degradation of the nuclear factor kappaB inhibitor IκBα. Furthermore, APC dampened the induction of tissue factor procoagulant activity facilitated by Tg.. These studies suggest that APC modulates the adverse effects of ER Ca(2+) depletion in human monocytes.

Topics: Anti-Inflammatory Agents; Apoptosis; Base Sequence; Calcium; Calcium Signaling; Caspase 3; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Gene Expression; Heat-Shock Proteins; Humans; In Vitro Techniques; Monocytes; NF-kappa B; Protein C; Reactive Oxygen Species; Recombinant Proteins; RNA, Messenger; Sepsis; Stress, Physiological; Thapsigargin; Thromboplastin