Oral delivery of pancreatitis-associated protein by Lactococcus lactis displays protective effects in dinitro-benzenesulfonic-acid-induced colitis model and is able to modulate the composition of the microbiota

Abstract

Antimicrobial peptides secreted by intestinal immune and epithelial cells are important effectors of innate immunity. They play an essential role in the maintenance of intestinal homeostasis by limiting microbial epithelium interactions and preventing unnecessary microbe-driven inflammation. Pancreatitis-associated protein (PAP) belongs to Regenerating islet-derived III proteins family and is a C-type (Ca⁺² dependent) lectin. PAP protein plays a protective effect presenting anti-inflammatory properties able to reduce the severity of colitis, preserving gut barrier and epithelial inflammation. Here, we sought to determine whether PAP delivered at intestinal lumen by recombinant Lactococcus lactis strain (LL-PAP) before and after chemically induced colitis is able to reduce the severity in two models of colitis. After construction and characterization of our recombinant strains, we tested their effects in dinitro-benzenesulfonic-acid (DNBS) and Dextran sulfate sodium (DSS) colitis model. After the DNBS challenge, mice treated with LL-PAP presented less severe colitis compared with PBS and LL-empty-treated mice groups. After the DSS challenge, no protective effects of LL-PAP could be detected. We determined that after 5 days administration, LL-PAP increase butyrate producer's bacteria, especially Eubacterium plexicaudatum. Based on our findings, we hypothesize that a treatment with LL-PAP shifts the microbiota preventing the severity of colon inflammation in DNBS colitis model. These protective roles of LL-PAP in DNBS colitis model might be through intestinal microbiota modulation.

Figure 1

Characterization of human PAP production by Lactococcus lactis. PAP was identified in the pellet and supernatant of nisin‐induced recombinant L. lactis PAP culture by ELISA. S NI = supernatant from non‐induced culture; S I = supernatant from induced culture; P NI = pellet from non‐induced culture; and P I = pellet from induced culture. NZ9000: L. lactis control strain, containing the plasmid pNIS empty; pSECPAP: L. lactis strain secreting PAP; and pCYTPAP: L. lactis strain expressing PAP into the cytoplasm.

Figure 2

Effect of LL‐PAP on DNBS‐induced colitis. Mice were orally administered with LL‐empty or LL‐PAP during 5 days before and 4 days after intra‐rectal injection of DNBS. Mice were sacrificed 4 days after DNBS injection.A. Percentage of weight loss among the groups. B. Macroscopic score. C. Microscopic score.

Figure 3

Effect of LL‐PAP on DSS‐induced colitis. Mice were orally administered with LL‐empty or LL‐PAP during 5 days before and 12 days after DSS administration. Mice were sacrificed 12 days after DSS administration.A. Percentage of weight loss among the groups. B. Disease Activity Index.

Figure 4

Cytokine production in mesenteric lymph nodes. Mice were orally administered with LL or LL‐PAP during 5 days before and 4 days after intra‐rectal injection of DNBS. Mice were sacrificed 4 days after DNBS injection. Cells were isolated from MLN and re‐stimulated in vitro by anti‐CD3 and anti‐CD28 during 48 h. Supernatants were recovered and cytokine measured using ELISA kits.

Figure 5

Cytokine production in colon.Mice were orally administered with LL‐empty or LL‐PAP during 5 days before and 4 days after intra‐rectal injection of DNBS. Mice were sacrificed 4 days after DNBS injection. Colon from each mouse was mashed in 1 ml of PBS using Gentle Max and cytokines were measured using ELISA kits.

Figure 6

Percentage of FoxP3⁺ cells population from intestinal lamina propria. Mice were orally administered with LL‐empty or LL‐PAP during 5 days before and 4 days after intra‐rectal injection of DNBS. Mice were sacrificed 4 days after DNBS injection. Cells isolated from intestinal lamina propria from PBS, DNBS, LL‐empty and LL‐PAP treated mice were stained with anti‐CD4⁺ and anti‐FoxP3⁺ and analysed by Flow cytometer. The percentage of cells obtained is represented in the graph.

Figure 7

Intestinal microbiota analysis after oral gavage with LL‐PAP. A. Principal coordinate analysis. B. The α‐diversity into the LL‐PAP treated mice microbiota population compared with LL‐empty‐treated mice. C. Comparison of the relative abundance of OTUs in LL‐empty and LL‐PAP groups. [Color figure can be viewed at http://wileyonlinelibrary.com]

Figure 8

Intestinal microbiota analysis after DNBS administration.PCoA of microbiota from mice orally administered with LL‐empty or LL‐PAP during 5 days before and 4 days after intra‐rectal injection of DNBS. [Color figure can be viewed at http://wileyonlinelibrary.com]