Journal: Immunity
Article Title: Inhibition of transcription factor NFAT activity in activated platelets enhances their aggregation and exacerbates gram-negative bacterial septicemia
doi: 10.1016/j.immuni.2021.12.002
Figure Lengend Snippet: (A) Schematic of the septic shock model. Mice were intraperitoneally (i.p.) injected, or not, with 25 mg/kg of GSK484 (−24 h and −6 h) and then were i.p. administered with two doses of LPS (−4 h, 1 mg/kg and 0 h, 2 mg/kg) to induce septic shock. Peritoneal lavage and blood were collected 2 h after the LPS challenge.
(B) Schematic of gram-negative bacteria-induced sepsis. Mice were i.p. injected with 1 × 108 CFU of E. coli. Peritoneal lavage and blood were collected 6 h after the E. coli challenge.
(C) Littermates or iNFATuation-Pf4-cre mice were challenged with two doses of LPS (−4 h, 1 mg/kg and 0 h, 2 mg/kg). Temperatures were recorded at the indicated time points. n = 4 mice per group. Data show mean ± SEM.
(D–I) Littermates or iNFATuation-Pf4-cre mice were pretreated, or not, with GSK484 and then challenged with two doses of LPS. 2 h after LPS challenge blood was collected, platelet number (D) was assessed by cytofluorimetry, and plasma fibrinogen content (E) and thrombin-antithrombin complexes (F) were measured by ELISA. Neutrophils positive for platelet marker CD41 (G) and for citrullinated histone H3 (H) were quantified in the peritoneal lavage by cytofluorimetry. Plasma IL-6 content was measured by ELISA 18 h post challenge (I). Each dot represents a mouse. “Saline” are WT mice not challenged with LPS used as control.
(J) Littermates or iNFATuation-Pf4-cre mice were challenge with 1 × 108 CFU of E. coli. 6 h post infection, plasma IL-6 was quantified by ELISA. Each dot represents a mouse. “Saline” are WT mice not challenged with E. coli used as control.
(K–M) Platelet-depleted mice reconstituted with WT or Nfat1−/− platelets were challenged with 2 doses of LPS. 2 h after LPS challenge temperatures were recorded (K), percentage of neutrophils positive for citrullinated histone H3 were analyzed from the peritoneum by cytofluorimetry (L), and plasma IL-6 concentration was measured by ELISA (M). Each dot represents a mouse. “Saline” are WT mice not challenged with LPS used as control. Two-way ANOVA (A), and one-way ANOVA (D–M) were used for statistics. n.s., not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. In (D–M) gray stars represent statistics compared with saline group. See also Figure S7.
Article Snippet: Escherichia coli (O111) , ATCC , Cat# BAA-2440.
Techniques: Injection, Bacteria, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Marker, Saline, Control, Infection, Concentration Assay
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