Potential of Oral Nanoparticles Containing Cytokines as Intestinal Mucosal Immunostimulants in Pigs: A Pilot Study

Abstract

Antimicrobial resistance is a global threat that is worryingly rising in the livestock sector. Among the proposed strategies, immunostimulant development appears an interesting approach to increase animal resilience at critical production points. The use of nanoparticles based on cytokine aggregates, called inclusion bodies (IBs), has been demonstrated as a new source of immunostimulants in aquaculture. Aiming to go a step further, the objective of this study was to produce cytokine nanoparticles using a food-grade microorganism and to test their applicability to stimulate intestinal mucosa in swine. Four cytokines (IL-1β, IL-6, IL-8, and TNF-α) involved in inflammatory response were produced recombinantly in Lactococcus lactis in the form of protein nanoparticles (IBs). They were able to stimulate inflammatory responses in a porcine enterocyte cell line (IPEC-J2) and alveolar macrophages, maintaining high stability at low pH and high temperature. In addition, an in vivo assay was conducted involving 20 piglets housed individually as a preliminary exploration of the potential effects of IL-1β nanoparticles in piglet intestinal mucosa after a 7 d oral administration. The treated animals tended to have greater levels of TNF-α in the blood, indicating that the tested dose of nanoparticles tended to generate an inflammatory response in the animals. Whether this response is sufficient to increase animal resilience needs further evaluation.

Immunomodulation Evidence of Nanostructured Recombinant Proteins in Salmonid Cells

“…The possibility of modulating the immune system to enhance the response against pathogens seems very attractive, as it would reduce the negative impact of diseases and reduce the use of chemical compounds as treatments. The production of recombinant immune-related proteins, such as IBs, has been explored in animal models; for example, cytokines (IL1β, TNFα, IL6, IL8, and CCL4) in fish and pigs [ 15 , 16 , 31 ], and metalloproteinase-9 (MMP9) in cows [ 58 , 59 ]. Our results prompted us to explore antimicrobial peptides and cytokine IBs as possible immunomodulators in salmonids.…”

“…For the cultures with induction, an early induction strategy was defined using 1 mM IPTG (Merck, Darmstadt, Germany), a biomass of 0.5 OD 600nm , and a period of recombinant protein production that was extended for 3 h post-induction. IBs were isolated through enzymatic and mechanical disruption of the cells as described previously [ 31 ]. Briefly, bacterial cultures were incubated with lysozyme at 1 μg/mL (Winkler), phenylmethanesulfonyl fluoride (PMSF, Roche Diagnostic, Mannheim, Germany) at 0.4 mM was added, and the mixture was incubated at 37 °C for 2 h and 250 rpm.…”

“…In salmonids, hepcidin has shown antimicrobial activity against pathogens such as Piscirikettsia salmonis , A. salmonicida , Aeromonas hydrophila , and Pseudomonas aeruginosa [ 26 , 27 ]. Cytokines as IBs have been previously evaluated in fish, where TNFα was tested as an immunostimulant in rainbow trout and zebrafish ( Danio rerio ) [ 15 , 16 ], and in pigs, where IL1, IL6, IL8, and TNFα were tested as stimulators of the intestinal mucosa [ 31 ]. To the best of our knowledge, although AMP-based IBs have been described [ 18 ], no work on teleost IBs of AMPs has been published.…”

The future of recombinant host defense peptides

“…IBs are protein nanoparticles or aggregates whose formation has been widely described in E. coli [56,57], but also in other microbial expression systems such as lactic acid bacteria [66][67][68][69][70] and yeast [70,71]. These aggregates can be easily purified [72] and offer interesting features not available in a soluble form.…”