Title : Investigation of the immunomodulatory effects of cecropin A on a primary hepatic coculture of chicken origin
The indiscriminate use of conventional antibiotics has contributed to the global spread of antibiotic resistance, therefore, there is an urgent demand to search for alternatives that can provide a novel antimicrobial mode of action. Finding a potential replacement is crucial for livestock farming, where animals are largely exposed to pathogens, while production efficiency, as well as animal health and well-being, have to be maintained. In this field, antimicrobial peptides (AMPs) have recently aroused great interest. Besides owning the ability to directly attack microbes, it has attracted increasing attention that AMPs are able to influence the host immune response, thereby being promising candidates for designing new antimicrobial agents. Despite being a thoroughly investigated molecule, only a few studies are available concerning the effects of the AMP cecropin A on cellular level in production animals, and to date, none of them were carried out on the liver. However, the liver plays a key role in maintaining local and systemic homeostasis by regulating loads of inflammatory processes. In our research, the effects of cecropin A were investigated on cell viability and inflammatory response in a chicken primary hepatocyte-non-parenchymal cell co-culture.The peptide was used alone and in combination with polyinosynic-polycytidylic acid (poly I:C, 50 µg/ml)- induced inflammation at concentrations of 1, 3.125, 6.25, 12.5 and 25 µg/ml. Cell viability was determined by colorimetric measurement of extracellular (EC) lactate dehydrogenase (LDH) activity, which indicates the extent of cell membrane damage. To monitor the inflammatory state, the level of transforming growth factor (TGF)-ß1 was measured by ELISA, and the concentrations of interleukin (IL)-6, IL-10, and interferon (IFN)-γ were assayed by Luminex method. In our study, we found that the three lowest concentrations of cecropin A did not affect extracellular LDH activity alone or in poly I:C-induced inflammation, however, the solely applied 12.5 and 25 µg/ml concentrations contributed to significantly increased membrane leakage. Based on these results, inflammatory markers continued to be examined only in treatment groups below these latter concentrations. When measuring TGF-ß1, we found that the sole dose of cecropin A at 6.25 µg/ml could decrease the level of the cytokine. In the case of IL-6, IL-10 and IFN-γ, cecropin A significantly reduced their production when applied alone at 1 µg/ml, furthermore at 1 and 6.25 µg/ml – and also at 3.125 µg/ml when assaying IL-6 – in poly I:C-induced inflammation, respectively. According to our results, the treatment of cells with cecropin A at lower concentrations did not result in a change in cell viability, suggesting its safe application. However, from a hepatic perspective, avoiding its higher concentrations might be advisable to consider. In addition, we found that the peptide might display immunomodulatory activity, as when used solely it could affect the production of TGF-ß1, IL-6, IL-10 and IFN-γ. Furthermore, it could also affect the levels of IL-6, IL-10 and IFN-γ during poly I:C-induced inflammation. Thus, our results suggest that cecropin A might be a promising molecule for the development of new antibiotic-substitutive agents, however, there is still a lot to clarify regarding its cellular effects.
Audience take away:
• The spread of antibiotic resistance contributed to the global aspiration for the reduction of the use of conventional antibiotics. Therefore, finding new agents that might serve as potential replacers for them became increasingly important, and AMPs are especially worth to investigate in this regard. Still, for the future application of AMPs, it is crucial to thoroughly investigate their cellular effects on various cell types. Our study provides useful results regarding the potential cytotoxic effect and role of cecropin A in inflammatory processes to consider for the subsequent design of cecropin A-based drugs.
• Determining an optimal dose for a future drug candidate is of great importance, which might be also supported by our research, where different concentrations of cecropin A were tested.
• In in vitro experiments on cell cultures, evoking inflammation is a common part of various types of research. The results of our study might provide additional useful information about poly I:C, which has been of great interest in recently developed inflammatory models.
• To date, limited data are available on poultry or chicken cell cultures. However, they might provide novel and valuable information about physiological and pathological processes on cellular level, that could be taken into account from a veterinary aspect.