VNAR domains are promissory antibody-based molecules because of their particular features such as low molecular weight and size, resistance to gastric pH and long CDR3 loop. VNAR domains are currently used to neutralize bioactive molecules and viruses [11, 14], as well as to perform in vitro diagnostic assays . Herein, we present the first study aimed to report an anti-TNF VNAR that improves survival in a murine model of endotoxic shock. Our results support a role for TNF bioactivity blockade in the treatment of sepsis, and suggest that different anti-TNF strategies may reach different survival rates through differentially attenuating inflammatory mechanisms.
In the present study, anti-TNF administration was not associated with early (3 h) depletion of serum TNF levels; on the contrary, TNF was detected in sera from animals in all groups. In contrast, serum TNF declined to almost undetectable levels at 24 h and 48 h after administration of either F(ab’)2 fragments or VNAR domains. These results are opposed to others studies showing that treatments with anti-TNF antibodies early reduce serum TNF concentration [15, 16]. A possible explanation is based on the molecular weight and bioavailability of each molecule. In this context, while conventional murine monoclonal IgG antibodies (159 kD) have a serum half-life of 25 days , mean serum half-life of smaller molecules is reduced, possibly as a result of improved clearance. Indeed, F(ab’)2 fragments (100 kD) show a mean serum half-life of 2 hrs , and VNAR (14 KD) single domains as little as 3 min to 1.5 hrs .
The liver plays key physiological roles including blood filtering of toxins as well as inactivation and clearing of bacterial antigens and products . Thus, the liver is a site in which early inflammatory changes in sepsis can be assessed by measuring the expression of several pro- and anti-inflammatory molecules . Due to the size of VNAR single domains, they may arrive to the liver and, possibly, deeply penetrate in hepatic tissue , which ultimately may result in the attenuation of inflammatory response. In our study, untreated animals with endotoxic shock showed an intense pro-inflammatory response featured by high expression of TNF and low expression of IL-10 in liver, while anti-TNF treated groups were characterized by an anti-inflammatory response featured by high expression of IL-10 and low expression of TNF.
The protective role of IL-10 in animal sepsis may be due to its antagonistic effect on the production and overall functioning of TNF [23–25]. In this way, it has been demonstrated that neutralization of IL-10 in septic animals and the induction of endotoxemic shock in IL-10 knockout animals are associated with increased tissue injury [26, 27] and higher serum levels of interferon-γ and TNF [28, 29]. In our study, a lower anti-inflammatory TNF/IL-10 ratio was associated with the administration of VNAR domains compared to F(ab’)2 fragments. This could be associated with the trend for higher survival observed in the group on VNAR therapy. In support to this notion, we have previously described that regulation in the IL-1β/IL-10 balance is associated with protection against lethality in a sepsis model of cecal ligation and puncture [7, 30]. The presence of similar results in different models of sepsis further supports that modulation of inflammation would tame tissue injury mechanisms, while full blockade of an inflammatory pathway would facilitate polarization of the immune response in either systemic inflammatory response syndrome or its counterpart, compensatory anti-inflammatory response syndrome .
Tumor necrosis factor directly influences the production of IL-6 and iNOS [31–33] and it is conceivable that its attenuation could have regulated both gene expression and serum concentration of IL-6 and nitric oxide (NO) in our experiments. For example, it has been reported that the administration of anti-IL-6 antibodies improves survival in sepsis [34, 35]; while TNF blockade inhibits hepatic expression of iNOS and nitrotyrosine in mice with endotoxic shock .
In addition, protection of liver seems to be critical to obtain beneficial outcomes in sepsis. In a recent study performed in rats with polymicrobial sepsis, treatment with hyperoncotic albumin attenuates hepatic injury in association with reduced plasma levels of IL-1β, IL-6, liver enzymes, and O2- concentrations . These results support anti-TNF VNAR domains as an alternative approach in the treatment of sepsis, due to its attenuating effects on the inflammatory response showed in liver; however these must be further studied in more aggressive models such as the polymicrobial sepsis induced by cecal ligation and puncture. Related to the latter study, a recent rat gut model of indomethacin-induced jejunoileitis showed that anti-TNF-α monoclonal antibody reduced iNOS expression and IL-1 beta, the latter two thought to be key mediators of inflammatory bowel disease .