Anti-tumor necrosis factor VNAR single domains reduce lethality and regulate underlying inflammatory response in a murine model of endotoxic shock

Abstract

Background: In sepsis, tumor necrosis factor (TNF) is the key factor triggering respiratory burst, tissue injury and disseminated coagulation. Anti-TNF strategies based on monoclonal antibodies or F(ab')₂ fragments have been used in sepsis with contradictory results. Immunoglobulin new antigen receptors (IgNAR) are a unique subset of antibodies consisting of five constant (CNAR) and one variable domains (VNAR). VNAR domains are the smallest, naturally occurring, antibody-based immune recognition units, having potential use as therapy. Our aim was to explore the impact of an anti-TNF VNAR on survival in an experimental model of endotoxic shock. Also, mRNA expression and serum protein of several inflammatory molecules were measured.

Results: Endotoxic shock was induced by lipopolysaccharide (LPS) in male Balb/c mice. Animals were treated with anti-TNF VNAR domains, F(ab')₂ antibody fragments, or saline solution 15 minutes before, 2 h and 24 h after lethal dose₁₀₀ (LD₁₀₀) LPS administration. TNF blockade with either VNAR domains or F(ab')₂ fragments were associated with lower mortality (60% and 75%, respectively) compared to LD₁₀₀. Challenge with LPS induced significant production of serum TNF and interleukins -10 and -6 at 3 h. After that, significant reduction of IL-6 at 24 h (vs 3 h) was shown only in the VNAR group. Nitrites level also increased in response to LPS. In liver, TNF and IL-10 mRNA expression showed a pro-inflammatory imbalance in response to LPS. Blocking TNF was associated with a shift towards an anti-inflammatory status; however, polarization was more pronounced in animals receiving F(ab')₂ fragments than in those with VNAR therapy. With regard to IL-6, gene expression was increased at 3 h in all groups. TNF blockade was associated with rapid and sustained suppression of IL-6 expression, even more evident in the VNAR group. Finally, expression of inducible-nitric oxide synthase (iNOS) increased in response to LPS at 3 h, but this was decreased at 24 h only in the anti-TNF VNAR group.

Conclusions: Anti-TNF VNAR single domains improved survival in a murine model of endotoxic shock. Protection was associated with regulation in the TNF/IL-10 balance, attenuation of IL-6 and iNOS gene expression in the liver as well as decreased serum IL-6 concentration.

Figure 1

Survival of animals treated with LD₁₀₀ of LPS. The figure is a compilation of two independent assays with animals treated with _VNAR single domains Anti-TNF or Fragments F(ab)² anti-TNF. Each group included 20 animals. Survival comparisons were analyzed with the Mantel-Haenzel Log Rank test, differences were considered significant when * p < 0.05, ***p < 0.001 vs control.

Figure 2

Production and expression of TNF, IL-6 and IL-10. Panels A, B, and C show levels of cytokines in serum of groups LD₁₀₀, LD₁₀₀ + _VNAR or LD₁₀₀ + F(ab)² at different times; while panels D, E, and F show the expression in liver of the same cytokines at same groups and times; data represent mean ± standard error. The differences among groups were determined using a Kruskal-Wallis test and post hoc analysis using a Dunn’s multiple comparison test; differences were considered significant when *p < 0.05, **p < 0.01, ***p < 0.001. N = 10 animals for each group and time point.

Figure 3

Production of NO₂^- and expression of iNOS. Panel A shows levels of NO₂^- in serum of groups LD₁₀₀, LD₁₀₀ + _VNAR or LD₁₀₀ + F(ab)² at different times; panel B shows expression of iNOS in the same groups and times; data represent mean ± standard error. Differences among groups were determined using a Kruskal-Wallis test and post hoc using a Dunn’s multiple comparison test; differences were considered significant when *p < 0.05, **p < 0.01, **p < 0.001. N = 10 animals for each group and time point.