Objective: Induction of secondary necrosis/pyroptosis contributes to the toxicity of chemotherapeutic drugs, in which gasdermin E (GSDME) plays critical roles. This study aimed to explore whether GSDME is involved in mediating the cytotoxic effects of cisplatin and doxorubicin on mouse macrophages.Methods: RAW 264.7 cells and bone marrow-derived macrophages (BMDMs) were treated with cisplatin or doxorubicin. Propidium iodide staining was used to assay necrosis, and immunoblotting was performed to detect protein expression. GSDME was knocked down by using small interfering RNA. Mice were injected intraperitoneally to evaluate toxicity to macrophages in vivo. Flow cytometry and immunofluorescence microscopy were adopted to analyse phenotypes of peritoneal cells.Cytokine levels were assayed by cytometric bead array. Results: Both cisplatin and doxorubicin dose-dependently induced necrosis in mouseRAW 264.7 macrophages and BMDMs. Accompanying this, multiple caspases were activated, concomitant with the cleavage of poly (ADP-ribose) polymerase. Consistent with caspase-3 activation, GSDME was cleaved to generate its N-terminal fragment (GSDME-NT), thus leading to secondary necrosis/pyroptosis. Inhibition of caspase-3 significantly attenuated the generation of GSDME-NT concurrently with decreased necrosis in macrophages. GSDME knockdown also evidently decreased the necrosis in RAW 264.7 and BMDMs. Besides, cisplatin administration depleted peritoneal macrophages in mice, which was associated with caspase-3 activation and GSDME-NT generation. Consistent with the macrophage depletion, cisplatin administration significantly decreased survival of mice with bacterial infection. Conclusion:Chemotherapeutic cisplatin and doxorubicin exerted their cytotoxicity on macrophages partly by inducing caspase-3/GSDME-mediated secondary necrosis.

Inflammatory caspase-11 senses and is activated by intracellular lipopolysaccharide (LPS) leading to pyroptosis that has critical role in defensing against bacterial infection, whereas its excess activation under pathogenic circumstances may cause various inflammatory diseases. However, there are few known drugs that can control caspase-11 activation. We report here that scutellarin, a flavonoid from Erigeron breviscapus , acted as an inhibitor for caspase-11 activation in macrophages. Scutellarin dose-dependently inhibited intracellular LPS-induced release of caspase-11p26 (indicative of caspase-11 activation) and generation of N-terminal fragment of gasdermin D (GSDMD-NT), leading to reduced pyroptosis. It also suppressed the activation of non-canonical nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome as evidenced by reduced apoptosis-associated speck-like protein containing a CARD (ASC) speck formation and decreased interleukin-1 beta (IL-1 β ) and caspase-1p10 secretion, whereas the NLRP3-specific inhibitor MCC950 only inhibited IL-1 β and caspase-1p10 release and ASC speck formation but not pyroptosis. Scutellarin also suppressed LPS-induced caspase-11 activation and pyroptosis in RAW 264.7 cells lacking ASC expression. Moreover, scutellarin treatment increased Ser/Thr phosphorylation of caspase-11 at protein kinase A (PKA)-specific sites, and its inhibitory action on caspase-11 activation was largely abrogated by PKA inhibitor H89 or by adenylyl cyclase inhibitor MDL12330A. Collectively, our data indicate that scutellarin inhibited caspase-11 activation and pyroptosis in macrophages at least partly via regulating the PKA signaling pathway.

Aging is a critical risk factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine efficacy. The immune responses to inactivated vaccine for older adults, and the underlying mechanisms of potential differences to young adults, are still unclear. Here we show that neutralizing antibody production by older adults took a longer time to reach similar levels in young adults after inactivated SARS-CoV-2 vaccination. We screened SARS-CoV-2 variant strains for epitopes that stimulate specific CD8 T cell response, and older adults exhibited weaker CD8 T-cell-mediated responses to these epitopes. Comparison of lymphocyte transcriptomes from pre-vaccinated and post-vaccinated donors suggested that the older adults had impaired antigen processing and presentation capability. Single-cell sequencing revealed that older adults had less T cell clone expansion specific to SARS-CoV-2, likely due to inadequate immune receptor repertoire size and diversity. Our study provides mechanistic insights for weaker response to inactivated vaccine by older adults and suggests the need for further vaccination optimization for the old population.

Here, we describe the use of the artificial antigen presenting cell (aAPC) system for the verification of T cell epitopes. We purify and activate CD8 + T cells from blood samples from HLA-A2 that are negative for SARS-CoV-2. CD8 + T cells are combined with peptide-loaded T2-A2 cells, which are then stained with a SARS-CoV-2 specific MHC-1 tetramer to identify specific HLA-A2 restricted T cell epitopes. The use of aAPC and healthy donors means that only BSL2 lab conditions are needed.