Investigation of Immunostimulatory Effects of Heat-Treated Lactiplantibacillus plantarum LM1004 and Its Underlying Molecular Mechanism

Abstract

Postbiotics are defined as probiotics inactivated by heat, ultraviolet radiation, sonication, and other physical or chemical stresses. Postbiotics are more stable than probiotics, and these properties are advantageous for food additives and pharmacological agents. This study investigated the immunostimulatory effects of heat-treated Lactiplantibacillus plantarum LM1004 (HT-LM1004). Cellular fatty acid composition of L. plantarum LM1004 isolated form kimchi was analyzed by gas chromatography-mass spectrometry detection system. The nitric oxide (NO) content was estimated using Griess reagent. Immunostimulatory cytokines were evaluated using enzyme-linked immunosorbent assay. Relative protein expressions were evaluated by western blotting. Phagocytosis was measured using enzyme-labelled Escherichia coli particles. L. plantarum LM1004 showed 7 kinds of cellular fatty acids including palmitic acid (C16:0). The HT-LM1004 induced release of NO and upregulated the inducible NO synthase in RAW 264.7 macrophage cells. Tumor necrosis factor-α and interleukin-6 levels were also increased compared to control (non-treated macrophages). Furthermore, HT-LM1004 modulated mitogen-activated protein kinase (MAPK) subfamilies including p38 MAPK, extracellular signal-regulated kinase 1/2, and c-Jun N-terminal kinase. Therefore, these immunostimulatory effects were attributed to the production of transcriptional factors, such as nuclear factor kappa B (NF-κB) and the activator protein 1 family (AP-1). However, HT-LM1004 did not showed significant phagocytosis of RAW 264.7 macrophage cells. Overall, HT-LM1004 stimulated MAPK/AP-1 and NF-κB expression, resulting in the release of NO and cytokines. These results will contribute to the development of diverse types of food and pharmacological products for immunostimulatory agents with postbiotics.

Keywords: Lactiplantibacillus plantarum; immunostimulatory effect; nuclear factor kappa B; postbiotics.

Fig. 1.. Circular genome map of Lactiplantibacillus LM1004 and cellular membrane fatty acid analysis of heat-treated Lactiplantibacillus plantarum LM1004.

(A) Circular genome map of Lactiplantibacillus plantarum LM1004. Each circle from outside to inside indicates protein-coding sequences (CDS) on forward strand, CDS on reverse strand, tRNA, rRNA, GC content, and GC skew. (B) Cellular fatty acid composition of heat-treated Lactiplantibacillus plantarum LM1004.

Fig. 2.. Nitric oxide production and relative protein expression of iNOS in heat-treated Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macrophage cells.

(A) Cell viability of Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macrophage cells, (B) release of nitric oxide in Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macrophage cells, (C) relative protein expression in Lactiplantibacillus plantarum LM1004 treated RAW macrophage 264.7 cells. Data are shown as the means±SDs of three independent experiments. ^** p<0.01 and ^*** p<0.001, compared to the control. LPS, lipopolysaccharides; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 3.. Cytokine production and COX-2 level in heat-treated Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macrophage cells.

(A, B) Concentration of TNF-α and IL-6; (C) COX-2 expression in Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macrophage cells. Data are shown as the means±SDs of three independent experiments. ^* p<0.05, ^** p<0.01, and ^*** p<0.001, compared to the control. TNF-α, tumor necrosis factor-α; LPS, lipopolysaccharides; IL-6, interleukin-6; COX-2, cyclooxygenase-2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 4.. MAPK activation by heat-treated Lactiplantibacillus plantarum LM1004.

Data are shown as the means±SDs of three independent experiments. ^* p<0.05, ^** p<0.01, and ^*** p<0.001, compared to the control. LPS, lipopolysaccharides; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase.

Fig. 5.. Change of transcription factor protein level in heat-treated Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macrophage cells.

Data are shown as the means±SDs of three independent experiments. ^* p<0.05, ^** p<0.01, and ^*** p<0.001, compared to the control. LPS, lipopolysaccharides; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 6.. Modulation of nitric oxide and iNOS protein expression level in APDC treated RAW 264.7 macrophage cells. APDC inhibited NF-κB as pharmacological inhibitor.

(A) Cell viability of RAW 264.7 macrophage cells, (B) production of nitric oxide in NF-κB inhibited cells by heat-treated Lactiplantibacillus plantarum LM1004, (C) overexpression of iNOS protein level in NF-κB inhibited cells by heat-treated Lactiplantibacillus plantarum LM1004. Data are shown as the means±SDs of three independent experiments. ^** p<0.01 and ^*** p<0.001, compared to the control (non-treated RAW 264.7 macrophage cells). LPS, lipopolysaccharides; APDC, ammonium pyrrolidine dithiocarbamate; iNOS, inducible nitric oxide synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NF-κB, nuclear factor kappa B.

Fig. 7.. Phagocytosis of heat-treated Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macro-phage cells.

(A) Phagocytosis effect of heat-treated Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macrophage cells, (B, C) AMPK and ACC protein expression level in heat-treated Lactiplantibacillus plantarum LM1004 treated RAW 264.7 macrophage cells. Data are shown as the means±SDs of three independent experiments. LPS, lipopolysaccharides; AMPK, AMP-activated protein kinase; ACC, acetyl-CoA carboxylase.