Gasdermin D-dependent neutrophil pyroptosis exacerbates Coxsackievirus A6-induced neuroinflammation through NET formation

Figures

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  The proposed mechanism of CVA6-induced neuroinflammation: Following the invasion of the central nervous system by CVA6, neutrophil aggregation is induced, which triggers the activation of the classical pyroptosis pathway. During this process, Caspase-1 cleaves GSDMD, pro-IL-1β, and pro-IL-18 to produce active N-GSDMD, mature IL-1β, and IL-18, respectively. The pyroptosis mediated by N-GSDMD leads to the release of neutrophil extracellular traps (NETs) and is accompanied by a robust secretion of inflammatory cytokines. This cascade ultimately results in a cytokine storm and severe neuroinflammatory responses.

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  Fig. 1. The activation of the Caspase-1/GSDMD pathway and NETs formation in neutrophils are involved in CVA6-induced neuroinflammation. Ten-day-old ICR mice were i.p. inoculated with a lethal dose of the CVA6 strain (104 TCID50 /mouse) or saline, and sacrificed at 3 dpi and 5 dpi. (A): The percentage of neutrophils in brain tissues and blood samples from Mock- or CVA6-infected mice by using FACS (n = 4 per group); (B): The concentrations of MPO-DNA in brain lysis from Mock- or CVA6-infected mice at 3 dpi and 5 dpi (n = 4 per group); (C): BMDNs infected by CVA6 or stimulated by PMA to release NETs under bright field microscopy, and the black arrows represent the NETs, bar = 20 μm; (D): Ties with NETs (CitH3/DAPI) in BMDNs infected by CVA6 or stimulated by PMA (n = 12 per group), and the white arrows represent the NETs, bar = 50 μm; (E): NETs formation after CVA6 infection in BMDNs under TEM; (F): Expression profiles of key pyroptosis-associated proteins, namely pro-Casp1, cleaved Casp1 (p20), full-length GSDMD, and GSDMD-N, alongside the mature forms of IL-1β (17 kD) and IL-18 (18 kD) in CVA6-infected BMDNs. (G): The expression profiles of key pyroptosis-associated proteins, namely…

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  Fig. 2. Blocking Caspase-1 suppressed GSDMD expression and NETs release in brain of mice infected with CVA6. Ten-day-old C57BL/6J and Casp1-/- mice were i.p. inoculated with a lethal dose of the CVA6 (104 TCID50 /mouse) or saline. Mean clinical scores (A) and survival rates (B) were monitored until 15 dpi (n = 9–12 per group). Brain tissues were harvested for analysis at 5 dpi. (C): Expression profiles of key pyroptosis-associated proteins—including full-length GSDMD, GSDMD-N, and the mature forms of IL-1β (17 kDa) and IL-18 (18 kDa)—in brain tissues from WT or Casp1-/- mice following CVA6 infection (n = 3 per group). (D): Co-localization between neutrophils (MPO) and pyroptosis (GSDMD-N) in brain slices from WT or Casp1-/- mice following CVA6 infection, marked by white arrows. Bar = 20 μm. (E): Co-localization between neutrophils (MPO) and IL-1β, and marked by white arrows. Bar = 20 μm. (F): Co-localization between neutrophils (MPO) and IL-18, and marked by white arrows. Bar = 20 μm. (G): Quantification of IL-1β and IL-18 in brain slices (n = 3 per group). (H): Expression profiles of key pyroptosis-associated proteins in brain tissues of mice infected with CVA6 and treated with either…

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  Fig. 3. Gasdermin D deficiency inhibits the release of NETs and alleviates neuroinflammation induced by CVA6. Ten-day-old C57BL/6J and Gsdmd-/- mice were i.p. inoculated with a lethal dose of the CVA6 strain (104 TCID50 /mouse) or saline (A). A daily record was kept on the body weight (B), mean clinical score (C) and percent survival (D) of mice (n = 9–12 per group). Tissue analysis was performed at 5 dpi. (E): Expression profiles of key pyroptosis-associated proteins, namely full-length GSDMD, and GSDMD-N, alongside the mature forms of IL-1β (17 kD) and IL-18 (18 kD) in the brains tissues from Mock- or CVA6-infected mice (WT, Gsdmd-/-) (n = 3 per group). (F): Pathological damage and viral replication of brain were measure by H&E staining, Nissl staining and IHC (CVA6 VP1) of brain from Mock- or CVA6-infected mice (WT, Gsdmd-/-) (n = 3 per group), and black arrows indicate inflammatory cell infiltration in H&E, viral replication in IHC, and neuronal damage in Nissl staining, bar = 20 μm. (G): MPO concentrations in brain lysates and serum, MPO–DNA complex levels, and relative mRNA expression of TNFA and IL-6 in brain lysates from mock- or CVA6-infected mice (WT, Gsdmd-/-) (n = 4 per…

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  Fig. 4. Pharmacologic inhibition of gasdermin D prevents NET release and neuroinflammation induced by CVA6. Ten-day-old ICR mice treated with PBS or Dissulfiram (10 mg/kg) were i.p. inoculated with a lethal dose of the CVA6 strain (104 TCID50 /mouse) or saline (A). A daily record was kept on the body weight (B), mean clinical score (C) and percent survival (D) of mice (n = 9–12 per group). Tissue analysis was performed at 5 dpi. Tissue analysis was performed at 5 dpi. (E): Expression profiles of key pyroptosis-associated proteins, namely full-length GSDMD, and GSDMD-N, alongside the mature forms of IL-1β (17 kD) and IL-18 (18 kD) in the brains tissues from Mock- or CVA6-infected mice treated with PBS or Dissulfiram (n = 3 per group). (F): Pathological damage and viral replication of brain were measure by H&E staining, Nissl staining and IHC (CVA6 VP1) of brain from Mock- or CVA6-infected mice treated with PBS or Dissulfiram (n = 3 per group), and black arrows indicate inflammatory cell infiltration in H&E, viral replication in IHC, and neuronal damage in Nissl staining, bar = 20 μm. (G): MPO concentrations in brain lysates and serum, MPO–DNA complex levels, and relative mRNA expression…

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  Fig. 5. An inducible mouse model for specific neutrophil depletion was reproduced. A: Strategy for hDTR gene knock-in using the CRISPR/Cas9 system. B: Macroscopic observations of Ly6G-DTR knock-in and wild-type C57BL/6 mice at 6 weeks old. C: Agarose gel electrophoresis images for mouse genotyping; red labels indicate heterozygous mice with bands at 364 bp and 558 bp. D-G: Flow cytometry plots show CD3ε-CD19-CD11b + Ly6G+ neutrophils in peripheral blood (D) and lung tissues (F), collected 24 and 48 h post-DT treatment (n = 3 per group). Neutrophil percentages in blood (E) and lung tissues (G)

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  Fig. 6. Effects of complete specific depletion of neutrophils after CVA6 infection. Ten-day-old WT or Ly6G-DTR KI mice were administered i.p. with either a lethal dose of the CVA6 strain (104 TCID50 /mouse) or PBS. DT was administered to mice at the indicated time points. A: The CVA6 infection and DT intervention were performed. Body weight (B), clinical scores (C), and survival rates (D) were monitored until 15 dpi (n = 6–8 per group). E: H&E staining of brain and lung tissues showed characteristic lesions, marked by black arrows or circles, bar = 20 μm. Histopathological scores for the brain (F) and lung (G) were evaluated with n = 3/group. Viral loads in the brain (H), lung (I), and muscle (J) were measured, with n = 8/group. no significance; *P < 0.05, ***P < 0.001, ****P < 0.0001

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  Fig. 7. The effects of anti-Ly6G-mediated neutrophil depletion and genetic NETs ablation on CVA6-induced mortality in mice. Ten-day-old ICR mice were administered i.p. with either the anti-Ly6G neutralizing antibody (clone 1A8; 12.5 mg/kg) or a rat IgG2a isotype control antibody (12.5 mg/kg), beginning 48 h prior to CVA6 infection and repeated once daily for two consecutive days (i.e., at 48 and 24 h before infection). A daily record was kept on the mean clinical score (A) and percent survival (B) of mice (n = 9–12 per group). Ten-day-old PAD4fl/fl and PAD4 Ne-KO were administered i.p. with either a lethal dose of the CVA6 strain (104 TCID50 /mouse) or saline. A daily record was kept on the mean clinical score (A) and percent survival (B) of mice (n = 9–12 per group). ns, no significance; * P < 0.05; ** P < 0.01; *** P < 0.001

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  Fig. 8. Gasdermin D is associated with NET release in patients with HFMD. A: The concentration of MPO in mild (n = 83) and severe cases (n = 83) with CVA6 infection was determined by MPO kit; B-D: The relative mRNA level of GSDMD (B), IL-1β (C) and IL-18 (D) in mild (n = 79) and severe cases (n = 79) with CVA6 infection was determined by qPCR; E-F: Correlation analysis between GSDMD mRNA level and IL-1β (E) / IL-18 (F) among all CVA6 infected cases (n = 158); * P < 0.05