Modulatory effects of vasoactive intestinal peptide on intestinal mucosal immunity and microbial community of weaned piglets challenged by an enterotoxigenic Escherichia coli (K88).

Toll-like receptors (TLRs) recognize microbial pathogens and trigger immune response, but their regulation by neuropeptide-vasoactive intestinal peptide (VIP) in weaned piglets infected by enterotoxigenic Escherichia coli (ETEC) K88 remains unexplored. Therefore, the study was conducted to investigate its role using a model of early weaned piglets infected by ETEC K88. Male Duroc × Landrace × Yorkshire piglets (n = 24) were randomly divided into control, ETEC K88, VIP, and ETEC K88+VIP groups. On the first three days, ETEC K88 and ETEC K88+VIP groups were orally administrated with ETEC K88, other two groups were given sterile medium. Then each piglet from VIP and ETEC K88+VIP group received 10 nmol VIP intraperitoneally (i.p.) once daily, on day four and six. On the seventh day, the piglets were sacrificed. The results indicated that administration of VIP improved the growth performance, reduced diarrhea incidence of ETEC K88 challenged pigs, and mitigated the histopathological changes of intestine. Serum levels of IL-2, IL-6, IL-12p40, IFN-γ and TNF-α in the ETEC K88+ VIP group were significantly reduced compared with those in the ETEC group. VIP significantly increased IL-4, IL-10, TGF-β and S-IgA production compared with the ETEC K88 group. Besides, VIP could inhibit the expression of TLR2, TLR4, MyD88, NF-κB p65 and the phosphorylation of IκB-α, p-ERK, p-JNK, and p-38 induced by ETEC K88. Moreover, VIP could upregulate the expression of occludin in the ileum mucosa compared with the ETEC K88 group. Colon and caecum content bacterial richness and diversity were lower for pigs in the ETEC group than the unchallenged groups. These results demonstrate that VIP is beneficial for the maturation of the intestinal mucosal immune system and elicited local immunomodulatory activities. The TLR2/4-MyD88 mediated NF-κB and MAPK signaling pathway may be critical to the mechanism underlying the modulatory effect of VIP on intestinal mucosal immune function and bacterial community

Design, Recombinant Fusion Expression and Biological Evaluation of Vasoactive Intestinal Peptide Analogue as Novel Antimicrobial Agent

Antimicrobial peptides represent an emerging category of therapeutic agents with remarkable structural and functional diversity. Modified vasoactive intestinal peptide (VIP) (VIP analogue 8 with amino acid sequence “FTANYTRLRRQLAVRRYLAAILGRR”) without haemolytic activity and cytotoxicity displayed enhanced antimicrobial activities against Staphylococcus aureus (S. aureus) ATCC 25923 and Escherichia coli (E. coli) ATCC 25922 than parent VIP even in the presence of 180 mM NaCl or 50 mM MgCl2, or in the range of pH 4–10. VIP analogue 8 was expressed as fusion protein thioredoxin (Trx)-VIP8 in E. coli BL21(DE) at a yield of 45.67 mg/L. The minimum inhibitory concentration (MIC) of the recombinant VIP analogue 8 against S. aureus ATCC 25923 and E. coli ATCC 25922 were 2 μM. These findings suggest that VIP analogue 8 is a promising candidate for application as a new and safe antimicrobial agent

Effect of VIP on morphology of the intestines in weaned piglets infected by ETEC K88.

<p>Note: ^#<i>P</i><0.05, ^##<i>P</i><0.01, ^###<i>P</i><0.001 compared to Control; *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001 compared to ETEC K88. Values are expressed as mean ± SEM (n = 6, each group).</p

Effect of VIP on growth performance and diarrhea incidence of piglets challenged with ETEC K88.

<p>Note: ^###<i>P</i><0.001 compared to Control; **<i>P</i><0.01, ***<i>P</i><0.001 compared to ETEC K88. Values are expressed as mean ± SEM. ADG, ADFI, G:F represents average daily gain, average daily feed intake, and feed conversion efficiency (G:F), respectively.</p

Effect of VIP on degradation and phosphorylation of IκBα, ERK1/2 (p44/p42) MAPK, p38 MAPK, and JNK/SAPK, and expression of TLRs, NF-κB p65, MyD88 and occludin in the ileum.

<p>Cytosolic occludin, IκB-α, p-IκB-α, p44/p42 (ERK1/2), p-p44/p42, p38, p-p38, JNK/SAPK, p-JNK/SAPK, p65, TLR4, TLR2, MyD88, nuclear NF-κB p65 proteins and actin were detected by western blot analysis. Each results is the mean (n = 6)±S.E.M. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Effect of VIP on expression of immune-related molecules in serum from piglets infected with ETEC K88. Each results is the mean (n = 6)±S.E.M. of cytokines levels determined in triplication.

<p>*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001.</p

Bacterial composition of the different communities.

<p>Relative read abundance of different bacterial genus within the different communities. Sequences that could not be classified into any known group were assigned as “unclassified bacteria”. The ETEC K88-challenged pigs and unchallenged pigs with same breed and age, and similar weight were not from the same litter and were assigned to the four treatments in a randomized complete block design. All pigs were housed in stainless steel pens (three piglets per pen) in the same unit.</p

Differentially Abundant Features of Colon Contents^*.

<p>*Bacterial community comparison between the groups in the level of genus; The genus listed in the table are significant between the groups. Among of them, shared genus of bacteria between groups is shown in italics bold.</p

Influence of VIP on morphology of intestine in piglets infected by ETEC K88 (H&E).

<p>Influence of VIP on morphology of intestine in piglets infected by ETEC K88 (H&E).</p