Open Access Research article

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

Rafael Bojalil12, María Teresa Mata-González3, Fausto Sánchez-Muñoz1, Yepci Yee1, Iván Argueta1, Lucía Bolaños1, Luis Manuel Amezcua-Guerra1, Tanya Amanda Camacho-Villegas45, Edna Sánchez-Castrejón5, Walter Jakob García-Ubbelohde3, Alexei Fedorovish Licea-Navarro5, Ricardo Márquez-Velasco16* and Jorge Fernando Paniagua-Solís37

Author Affiliations

1 Department of Immunology, Instituto Nacional de Cardiología Ignacio Chávez, Mexico City, Mexico

2 Department of Health Care, Universidad Autónoma Metropolitana-Xochimilco, Mexico City, Mexico

3 Laboratorios Silanes, S. A. de C. V. Amores 1304, Col del Valle, Mexico City, 03100, Mexico

4 Marine Science Faculty, Universidad Autónoma de Baja California, Km. 103 Carretera Tijuana-Ensenada, Ensenada, Mexico

5 Marine Biotechnology Department, Centro de Investigación Científica y de Educación Superior de Ensenada (CICESE), Ensenada, Mexico

6 Department of Health Sciences, Universidad del Valle de México, Mexico City, Mexico

7 Current Address (JFP-S): Teraclon IDF; Parque Científico de Madrid, Tres Cantos, Madrid, España

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BMC Immunology 2013, 14:17  doi:10.1186/1471-2172-14-17

Published: 2 April 2013

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.

Keywords:
Endotoxic shock; Sepsis; Anti-TNF; VNAR; Inflammation