Stimulatory effects of Lactobacillus casei derived extracellular vesicles on toll-like receptor 9 gene expression and cytokine profile in human intestinal epithelial cells

Abstract

Background A complex community of microorganisms in the gastrointestinal (GI) tract, known as the gut microbiota, exerts major effects on gene expression and cytokine profile. Extracellular vesicles (EVs) which are produced by bacteria could be sensed by Toll like receptors (TLRs). The interaction between gut microbiota and TLRs affects homeostasis and immune responses. In this study, we evaluated TLR9 gene expression and cytokines level in Caco-2 cell line treated with Lactobacillus casei as one of the gut microbiota and its EVs. Methods In the present study, L. casei derived EVs was extracted via ultracentrifugation. The quality control assessment included the evaluation of physicochemical characteristics of EVs. For the treatment of Caco-2 cell line, L. casei and its EVs (100 and 150 μg/mL) were used. In addition, qRT-PCR assay was carried out to evaluate the mRNA expression of TLR9 gene. ELISA assay was also performed to determine the levels of IFN γ , TNF-α, GM-CSF, IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-12, IL-17A, and IL-10 cytokines.Results The results showed that L. casei slightly increased TLR9 gene expression in the Caco-2 cell line. It was also found that EVs at concentrations of 100 and 150 μg/mL could significantly decrease TLR9 gene expression. Furthermore, L. casei significantly increased IL-10 and IFN γ levels. Based on the findings, the level of IL-17A, as a proinflammatory cytokine, decreased by L. casei. Both concentrations of EVs decreased the level of IFN γ , while increasing the concentrations of IL-4 and IL-10. EVs from L. casei could modulate immune responses in the Caco-2 cell line. Both EVs and L. casei activated the expression and secretion of several cytokines. Conclusions L. casei and its EVs have pivotal role in the cross talk between gut microbiota and the host especially in the modulation of the immune system. This study shows for the first time the increasing level of anti-inflammatory cytokines by EVs released by L. casei. Based on the last studies on immunomodulatory effect of EVs on immune cells and our results in cell line level, we postulate that L. casei derived EVs could be possible candidates for the reduction of immune responses.

Probiotics, Their Extracellular Vesicles and Infectious Diseases

“…On the other hand, EVs from E. coli and L. casei increased NF-κB levels per se in Caco-2 and HT-29 cell lines ( Cañas et al, 2018 ; Bäuerl et al, 2020 ). This opposite modulation of NFκB levels, in the presence or absence of LPS, was also observed for pro-inflammatory cytokines like IL-8 in both Caco-2 and HT-29 cell lines, and in ex vivo human colonic explant ( Fábrega et al, 2016 ; Choi et al, 2020 ; Vargoorani et al, 2020 ).…”

“…In vitro and in vivo experiments with intestinal epithelial cells have demonstrated that EVs released by E. coli , L. casei , L. paracasei , P. freudenreichii , and L. rhamnosus can modulate NFκB levels ( Cañas et al, 2018 ; Bäuerl et al, 2020 ; Choi et al, 2020 ; Vargoorani et al, 2020 ; Tong et al, 2021 ). NF-κB is a family of transcription factors and has an essential role in a variety of aspects related with human health including the development of both innate and adaptive immunity.…”

Extracellular Vesicles of Probiotics: Shedding Light on the Biological Activity and Future Applications

“…Dynamic light scattering (DLS) (9/60; 15%) was used less commonly for this purpose ( Figure 1 and Table S1 ). In 11 articles, more than one technique was applied to determine the size of EVs, including 6 with microscopy + NTA [ 29 , 30 , 31 , 32 , 33 , 34 ], 3 with microscopy + DLS [ 35 , 36 , 37 ], and 2 with microscopy + NTA + DLS [ 38 , 39 ] ( Figure 1 and Table S1 ). Among other parameters of EVs measured by researchers, the surface charge/zeta potential and the quantity of EVs can be distinguished.…”

“…As mentioned previously, the parameters of EVs other than dimensions—surface charge/zeta potential and quantity—are of little interest to scientists. In the studies in which this topic was addressed, the zeta potential of EVs had negative values between −0.5 mV [ 37 ] and −45 mV [ 42 ], with an average ranging from −10 mV to −20 mV ( Table S1 ). The phenomenon of negative charge of EVs is most often related to the presence of extracellular DNA on the surface of these structures, which translates into their important function in supporting adhesion, aggregation, and biofilm formation of microbes [ 43 , 44 ].…”

Metagenomic Profiling of Fecal-Derived Bacterial Membrane Vesicles in Crohn’s Disease Patients

“…This might describe one potential mechanism of the role of dysbiosis [15,16]. MVs isolated from different strains of Bifidobacterium and Lactobacillus may mediate the probiotic impacts of the MV-releasing microbes [20]. Studies investigating the immunoregulatory roles of MVs have unraveled pro-and anti-inflammatory effects [21][22][23].…”