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. 2024 Apr 4:15:1309160.
doi: 10.3389/fmicb.2024.1309160. eCollection 2024.

Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice

Juliana Guimarães Laguna  1 , Andria Dos Santos Freitas  1 , Fernanda Alvarenga Lima Barroso  1 , Luís Cláudio Lima De Jesus  1 , Octávio Augusto Greco Gomes De Vasconcelos  1 , Ludmila Silva Quaresma  1 , Monique Ferrary Américo  1 , Gabriela Munis Campos  1 , Rafael de Assis Glória  1 , Joyce da Cruz Ferraz Dutra  1 , Tales Fernando Da Silva  1 , Kátia Duarte Vital  2 , Simone O Fernandes  2 , Ramon O Souza  3 , Flaviano Dos Santos Martins  3 , Enio Ferreira  4 , Túlio Marcos Santos  1 , Alexander Birbrair  4   5 , Marcos Felipe Andrade De Oliveira  6 , Ana Maria Caetano Faria  6 , Rodrigo Dias de Oliveira Carvalho  1 , Franco Maria Venanzi  7 , Yves Le Loir  8 , Gwénaël Jan  8 , Éric Guédon  8 , Vasco Ariston de Carvalho Azevedo  1
Affiliations

Recombinant probiotic Lactococcus lactis delivering P62 mitigates moderate colitis in mice

Juliana Guimarães Laguna et al. Front Microbiol. .

Abstract

Introduction and objective: p62 is a human multifunctional adaptor protein involved in key cellular processes such as tissue homeostasis, inflammation, and cancer. It acts as a negative regulator of inflammasome complexes. It may thus be considered a good candidate for therapeutic use in inflammatory bowel diseases (IBD), such as colitis. Probiotics, including recombinant probiotic strains producing or delivering therapeutic biomolecules to the host mucosal surfaces, could help prevent and mitigate chronic intestinal inflammation. The objective of the present study was to combine the intrinsic immunomodulatory properties of the probiotic Lactococcus lactis NCDO2118 with its ability to deliver health-promoting molecules to enhance its protective and preventive effects in the context of ulcerative colitis (UC).

Material and methods: This study was realized in vivo in which mice were supplemented with the recombinant strain. The intestinal barrier function was analyzed by monitoring permeability, secretory IgA total levels, mucin expression, and tight junction genes. Its integrity was evaluated by histological analyses. Regarding inflammation, colonic cytokine levels, myeloperoxidase (MPO), and expression of key genes were monitored. The intestinal microbiota composition was investigated using 16S rRNA Gene Sequencing.

Results and discussion: No protective effect of L. lactis NCDO2118 pExu:p62 was observed regarding mice clinical parameters compared to the L. lactis NCDO2118 pExu: empty. However, the recombinant strain, expressing p62, increased the goblet cell counts, upregulated Muc2 gene expression in the colon, and downregulated pro-inflammatory cytokines Tnf and Ifng when compared to L. lactis NCDO2118 pExu: empty and inflamed groups. This recombinant strain also decreased colonic MPO activity. No difference in the intestinal microbiota was observed between all treatments. Altogether, our results show that recombinant L. lactis NCDO2118 delivering p62 protein protected the intestinal mucosa and mitigated inflammatory damages caused by dextran sodium sulfate (DSS). We thus suggest that p62 may constitute part of a therapeutic approach targeting inflammation.

Keywords: epithelial barrier; gut microbiota; immunomodulation; inflammatory bowel disease; probiotic bacteria; recombinant protein.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

Figures

Figure 1
Figure 1
The experimental design. For 5 days, mice received treatments via gavage with recombinant Lactococcus lactis (pExu:empty or pExu:p62). After 10 days of intermission, mice were continuously inflamed by DSS 2% in drinking water and euthanized on the 22nd.
Figure 2
Figure 2
Effect of p62 protein on mice clinical parameters and colon length. Analysis of food (A) and liquid intake (B), body weight loss (C), and colon length (D) in mice inflamed with DSS 2%. Means ± SE followed by different lowercase letters indicate a statistically significant difference (p < 0.05) assessed by one-way ANOVA to multiple comparisons among treatments, followed by Tukey’s test.
Figure 3
Figure 3
p62 protein Does not improve intestinal mucosa damage induced by DSS 2%. (A) Colon mucosa histopathology (B) histopathological score; and (C) crypt depth. Means ± SE followed by different lowercase letters indicate a statistically significant difference (p < 0.05) assessed by one-way ANOVA to multiple comparisons among treatments, followed by Tukey’s test.
Figure 4
Figure 4
Protective effect of recombinant p62 protein on epithelial barrier. Intestinal permeability (A); The number of goblet cells/field (B); Representative photomicrographs from colon section stained with Periodic Acid-Schiff (PAS) (C); and level of mRNA transcript levels of mucin 2 (Muc2) (D), Zonulin (Hp) (E), Occludin (Ocln) (F). Means ± SE followed by different lowercase letters indicate a statistically significant difference (p < 0.05) assessed by one-way ANOVA followed by Tukey’s test.
Figure 5
Figure 5
Effect of recombinant p62 protein on inflammatory markers. (A) Myeloperoxidase activity; (B) sIgA levels and total protein pg./mL of IL10 (C), IL6 (D), and TGFβ (E). Means ± SE followed by different lowercase letters indicate a statistically significant difference (p < 0.05) assessed by one-way ANOVA to multiple comparisons among treatments, followed by Tukey’s test.
Figure 6
Figure 6
Effect of recombinant p62 protein on inflammatory markers. (A) Tnf, (B) Ifng, (C) Il17a, (D) Tgfb1, and (E) Il1b. Means ± SE followed by different lowercase letters indicate a statistically significant difference (p < 0.05) assessed by one-way ANOVA to multiple comparisons among treatments, followed by Tukey’s test.
Figure 7
Figure 7
Effect of recombinant p62 protein on intestinal microbiota. (A) Shannon index, (B) Principal component analysis (PCA), and (C) Heatmap of bacterial taxon relative abundance. Data in the graph represent the proportion of reads assigned to a given phylum-level taxon. The dendrogram represents a hierarchical clustering based on the Unweighted Pair Group Method using the Arithmetic averages method.
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