Potential probiotic Lactiplantibacillus plantarum strains alleviate TNF-α by regulating ADAM17 protein and ameliorate gut integrity through tight junction protein expression in in vitro model

Abstract

Background: Lactiplantibacillus species are extensively studied for their ability to regulate host immune responses and functional therapeutic potentials. Nevertheless, there is a lack of understanding on the mechanisms of interactions with the hosts during immunoregulatory activities.

Methods: Two Lactiplantibacillus plantarum strains MKMB01 and MKMB02 were tested for probiotic potential following Indian Council of Medical Research (ICMR) guidelines. Human colorectal adenocarcinoma cells such as HT-29, caco-2, and human monocytic cell THP-1 were also used to study the potential of MKMB01 and MKMB02 in regulating the host immune response when challenged with enteric pathogen Salmonella enterica typhimurium. Cells were pre-treated with MKMB01 and MKMB02 for 4 h and then stimulated with Salmonella. qRT-PCR and ELISA were used to analyze the genes and protein expression. Confocal microscopy and field emission scanning electron microscopy (FESEM) were used to visualize the effects. An Agilent Seahorse XF analyzer was used to determine real-time mitochondrial functioning.

Results: Both probiotic strains could defend against Salmonella by maintaining gut integrity via expressing tight junction proteins (TJPs), MUC-2, and toll-like receptors (TLRs) negative regulators such as single Ig IL-1-related receptor (SIGIRR), toll-interacting protein (Tollip), interleukin-1 receptor-associated kinase (IRAK)-M, A20, and anti-inflammatory transforming growth factor-β and interleukin-10. Both strains also downregulated the expression of pro-inflammatory cytokines/chemokines interleukin-1β, monocyte chemoattractant protein (MCP)-1, tumor necrosis factor-alpha (TNF-α), interleukin 6, and nitric oxide (NO). Moreover, TNF-α sheddase protein, a disintegrin and metalloproteinase domain 17 (ADAM17), and its regulator iRhom2 were downregulated by both strains. Moreover, the bacteria also ameliorated Salmonella-induced mitochondrial dysfunction by restoring bioenergetic profiles, such as non-mitochondrial respiration, spare respiratory capacity (SRC), basal respiration, adenosine triphosphate (ATP) production, and maximal respiration.

Conclusions: MKMB01 and MKMB02 can reduce pathogen-induced gut-associated disorders and therefore should be further explored for their probiotic potential.

Keywords: ADAM17 and iRhom2; Anti-inflammatory; Gut integrity; Immunomodulatory; Oxidative stress; Probiotics.

Fig. 1

Effect of L. plantarum stains MKMB01 and MKMB02 on gut integrity. Representative confocal images of tight junction protein (TJP) expression with Salmonella-infected caco-2 cells are shown as (A) claudin-1, (B) ZO-1, and (C) occludin; a, b, c, and d denote control, negative control, MKMB01, and MKMB02, respectively for each group. Graphical representation of TJP expression is represented by (e). (D) RT-PCR result of MUC-2 gene expression and (E) TEER assay. Changes in TEER with time are compared with negative control. Values are shown as mean (± SEM) from three replicates. Significant differences are marked by */# = p ≤ 0.05, **/## = p ≤ 0.01, and ***/### = p ≤ 0.001 (*between Salmonella and probiotic treated groups whereas #between Salmonella treated and control groups). Scale bar: 25 μm

Fig. 2

Immunoregulatory effect of L. plantarum MKMB01 and MKMB02. Graphical representation of the genes (A) NO, (B) IL-1β, (C) MCP-1, (D) SIGIRR, (E) TOLLIP, (F) IRAK-M, (G) A20, (H) IL-10, (I) TGF-β, (J) IL-6,(K) TNF-α, (L)), and (M)). TNF-α release by THP-1 and HT-29 cells in pg/mlmL; (N) and (O) IL-6 release by THP-1 and HT-29 cells in pg/mlmL; (P) ADAM10, (Q) ADAM17, (R) iRhom-1, (S) iRhom-2, and (T) ADAM17 expression in THP-1 cells (a, b, c, and d denote control, negative control, MKMB01, and MKMB02, respectively for each group), ); and (U) graphical representation of ADAM17 expression. Changes in gene/protein expression were compared with untreated control and Salmonella treatment. Values are shown as mean (± SEM) from three replicates. Significant differences are marked by */# = p ≤ 0.05, **/## = p ≤ 0.01, and ***/### = p ≤ 0.001 (*between Salmonella and probiotic treated groups, whereas #between Salmonella treated and control groups). Scale bar: 25 μm

Fig. 3

Effect of L. plantarum MKMB01 and MKMB02 on apoptosis, ROS generation, and mitochondrial function. (A-E) denote apoptosis [Upper left quadrants = necrotic cells (P1-1), upper right quadrants = apoptotic cells (P1-2), lower left quadrants = viable cells (P1-3), and lower right quadrants = pre-apoptotic cells (P1-4)]; (F-J) denotes ROS generation (P2 = ROS negative and P3 = ROS positive); (K) Mitochondrial respiration; (L) Non-mitochondrial respiration; (M) Basal respiration; (N) Maximal respiration; (O) Spare respiratory capacity (SRC) and (P) ATP Production. Values are expressed as means (± SEM) for three replicates. Significant differences are marked by */# = p ≤ 0.05, **/## = p ≤ 0.01, and ***/### = p ≤ 0.001 (*between Salmonella and probiotic treated groups, whereas #between Salmonella treated and control groups)

Fig. 4

The whole genome sequences of the MKMB01 and MKMB02 probiotic strains. The genomic sequences of MKMB01 (A) and MKMB02 (B) reveal their functional capabilities (C & D). Putative bacteriocin ORFs (antimicrobial peptides) were detected (E & F)