Control of Overly Secreted Tryptophanyl tRNA Synthetase Attenuates Sepsis Severity in a Porcine Model

Abstract

Sepsis is a leading cause of mortality in hospitals with a lack of reliable biomarkers and specialized therapeutics. Recently, highly secreted tryptophanyl-tRNA synthetase 1 (WARS1), an endogenous ligand for Toll-like receptor (TLR) 2 and TLR4, was found to be a potential theranostic target for hypercytokinemic severe sepsis. In this study, using the minipig sepsis model inoculated with cecum slurry, we demonstrated that increases in WARS1 levels were associated with severity of sepsis and showed strong correlations with RBC count and the levels of HGB, HCT, EPO, lactate, and PLT count in the acute phase of sepsis. Further, administration of the WARS1 neutralizing antibody to the septic minipigs inhibited the increase in the overall SOFA score with a significantly lower P/F ratio, which was accompanied by the suppression of proinflammatory cytokine and chemokine expressions as well as EPO production, a decrease in AST and ALT levels, and inflammatory immune cell infiltration in the lung. Taken together, these findings provide a novel insight into the pathophysiology of acute phase of sepsis and suggest the clinical application of WARS1 neutralizing therapeutics in the treatment of sepsis.

Keywords: Anti-WARS1 neutralizing antibody; Organ dysfunction; Sepsis; Severity biomarker; Tryptophanyl-tRNA synthetase (WARS1).

Fig. 1

Correlation between plasma levels of WARS1 and sepsis severity-related parameters in the CS-inoculated minipigs. (A) Survival plot of minipigs inoculated with different CS concentrations. (B) Plasma WARS1 levels in minipigs at indicated time after inoculation with different CS concentrations. (C-I) Correlation between WARS1 levels and various clinical and laboratory parameters. Data are presented as mean ± SEM (B). Statistical analysis is performed with a log-rank test (A), an ANOVA with Dunnett corrections (B), and Pearson’s correlation coefficient test (C-I). ns, not significant; *p<0.05, **p<0.01.

Fig. 2

Interaction of anti-WARS1 MAb with porcine WARS1. (A) Similarity of protein sequence between human and porcine WARS1. (B) Purified recombinant porcine WARS1 protein. (C) Immunoblot for recombinant porcine WARS1 protein (RP) with anti-WARS1 MAb and tissue homogenates (TH). (D) Binding affinity of anti-WARS1 MAb to recombinant porcine WARS1 protein.

Fig. 3

Effect of anti-WARS1 MAb administration on the elimination of secreted WARS1 in the CS-inoculated minipigs. (A) Experimental scheme of CS-inoculated septic minipig model. (B) WARS1 levels in plasma at indicated time-point. Naïve vs. CS control, ^##p<0.01, ^###p<0.001; CS control vs. CS+anti-WARS1 MAb, ***p<0.001. (C, D) WARS1 levels in PLF (C) and BALF (D). **p<0.01. Data are presented as mean ± SEM (B-D). Statistical analysis is performed with an ANOVA with Dunnett corrections (B), and a Mann-Whitney U-test (C, D).

Fig. 4

Effect of anti-WARS1 MAb on organ damage in the CS-inoculated minipigs. (A) Total mSOFA scores at the indicated time point. (B) Representative images of H&E-stained lung sections at 20 h (magnification, 200×; scale bars, 100 μm). (C-E) Levels of ALT (C), AST (D), and lactate (E) in plasma at indicated time-point. Data are presented as mean ± SEM (A, C-E). Statistical analysis is performed with ANOVA with Dunnett corrections (A, C-E). *p<0.05.

Fig. 5

Effect of anti-WARS1 MAb on various cytokines in the CS-inoculated minipigs. (A, B) Levels of inflammatory cytokines (A) and EPO (B) at the indicated time point in plasma. (C, D) Gene expression of inflammatory cytokines in lung (C) and liver (D) homogenates at 20 h. Data are presented as mean ± SEM (A-D). Statistical analysis is performed with ANOVA with Dunnett corrections (A-D). CS control vs. CS+anti-WARS1 MAb, ns, not significant; *p<0.05, **p<0.01.

Fig. 6

Schematic presentation of putative mechanisms for anti-WARS1 MAb treatment in minipigs with sepsis. Anti-WARS1 MAb treatment lowers the production of proinflammatory cytokines and chemokines, reduces EPO levels, decreases RBC count, and prevents related organ dysfunction. WARS1, tryptophanyl-tRNA synthetase 1; EPO, erythropoietin; RBC, red blood cell; P/F ratio, PaO₂, partial pressure of oxygen/FiO₂, fraction of inspired oxygen; AST, aspartate aminotransferase; ALT, alanine aminotransferase; SOFA, sequential organ failure assessment; TLR, Toll-like receptor; IL, interleukin; TNF, tumor necrosis factor.