Human intestinal dendritic cells decrease cytokine release against Salmonella infection in the presence of Lactobacillus paracasei upon TLR activation

Abstract

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs) are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs) and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells) harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants, Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS) decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-β2 secretion, whereas the supernatant increased the secretion of TGF-β1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR) signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods.

Conflict of interest statement

Competing Interests: Esther Matencio, María J. Bernal and Fernando Romero are members of the Hero Institute for Infant Nutrition, Hero Spain S. A. The sponsor had no role in the biological sample analysis, statistical analysis or data interpretation. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1. Pro-inflammatory cytokine production in DCs after exposure to L. paracasei, Salmonella or both.

Dendritic cells (DCs) were incubated with the probiotic L. paracasei CNCM I-4034 (Prob) or its cell-free supernatant (CFS), Salmonella (S.typhi), or both. E. coli lipopolysaccharide (LPS, 20 ng/ml) was used as a positive control. Negative-control cultures contained unstimulated DCs. IL-1β, IL-6, IL-8, IL-12(p40) and IL-12(p70) production was measured. The data shown are the mean values and SEM of three independent experiments. *p<0.05 compared to controls; #p<0.05 compared to S.typhi.

Figure 2. Measure of anti-inflammatory cytokines and TNF-α in DCs after exposure to L. paracasei, Salmonella or both.

DCs were incubated with the probiotic L. paracasei CNCM I-4034 (Prob) or its cell-free supernatant (CFS), Salmonella (S.typhi), or both. IL-10, TNF-α, TGF-β1 and TGF-β2 production was measured. LPS, 20 ng/ml, was used as a positive control. Negative-control cultures contained unstimulated DCs. The data shown are the mean values and SEM of three independent experiments. *p<0.05 compared to controls; #p<0.05 compared to S.typhi; N.D indicate no detected.

Figure 3. Measure of chemokines and IFNγ in DCs after exposure to L. paracasei, Salmonella or both.

DCs were incubated with the probiotic L. paracasei CNCM I-4034 (Prob) or its cell-free supernatant (CFS), Salmonella (S.typhi), or both. The production of IFNγ and the chemokines MCP-1/CCL2, MIP-1α/CCL3, RANTES/CCL5 and MDC/CCL22 was measured. LPS, 20 ng/ml, was used as a positive control. Negative-control cultures contained unstimulated DCs. The data shown are the mean values and SEM of three independent experiments. *p<0.05 compared to controls; #p<0.05 compared to S.typhi.

Figure 4. Expression of TLRs genes in DCs in the presence of L. paracasei, Salmonella or both.

Comparison of the expression of TLR1, TLR2, TLR3, TLR4 and TLR5 in DCs in the presence of the probiotic (Prob), its supernatant (CFS), Salmonella (S.typhi) or a combination. LPS, 20 ng/ml, was used as a positive control. The data shown are the mean values and SEM of three independent experiments. The fold change (Fc) represents the ratio of the expression in treated DCs to the expression in control cells. *p<0.05 compared to controls; #p<0.05 compared to S.typhi. N.D indicate no detected.

Figure 5. Expression levels of TLR signalling pathway in DCs treated with L. paracasei, Salmonella or both.

Comparison of the expression of TLR9, MYD88, IRAK-1, IRAK-4 and TOLLIP in DCs in the presence of the probiotic (Prob), its supernatant (CFS), Salmonella (S.typhi) or a combination. LPS, 20 ng/ml, was used as a positive control. The fold change (Fc) represents the ratio of the expression in treated DCs to the expression in control cells. The data shown are the mean values and SEM of three independent experiments. *p<0.05 compared to controls; #p<0.05 compared to S.typhi.

Figure 6. Expression levels of TLR signalling pathway in DCs treated with L. paracasei, Salmonella or both.

Comparison of the expression of CASP8, TAK-1, JNK, IRF-3 and MAPK14 in DCs in the presence of the probiotic (Prob), its supernatant (CFS), Salmonella (S.typhi) or a combination. LPS, 20 ng/ml, was used as a positive control. The fold change (Fc) represents the ratio of the expression in treated DCs to the expression in control cells. The data shown are the mean values and SEM of three independent experiments. *p<0.05 compared to controls; #p<0.05 compared to S.typhi.

Figure 7. Expression levels of TLR signalling pathway in DCs treated with L. paracasei, Salmonella or both.

Comparison of the expression of NFKBIA, NFKB-1, TBK-1, IL-10 and TNF-α in DCs in the presence of the probiotic (Prob), its supernatant (CFS), Salmonella (S.typhi) or a combination. LPS, 20 ng/ml, was used as a positive control. The fold change (Fc) represents the ratio of the expression in treated DCs to the expression in control cells. The data shown are the mean values and SEM of three independent experiments. *p<0.05 compared to controls; #p<0.05 compared to S.typhi. N.D indicate no detected.