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Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

El-Awady AR, Miles B, Scisci E, Kurago ZB, Palani CD, Arce RM, Waller JL, Genco CA, Slocum C, Manning M, Schoenlein PV, Cutler CW - PLoS Pathog. (2015)

Bottom Line: Survival was decreased by activation of TLR2 and/or autophagy.Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1.These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Periodontics, Georgia Regents University, Augusta, Georgia, United States of America.

ABSTRACT
Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

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Mfa1+Pg up-regulate the expression of DC-SIGN in human MoDCs.A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001). B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons 3 different experiments (* p<0.01).
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ppat.1004647.g002: Mfa1+Pg up-regulate the expression of DC-SIGN in human MoDCs.A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001). B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons 3 different experiments (* p<0.01).

Mentions: To determine whether expression of DC-SIGN [14] was altered by P. gingivalis infection, MoDCs were infected with all the strains at different multiplicities of infection (MOIs) and gene expression of DC-SIGN was quantified at 2, 6, 12 and 24 hours (Fig. 2A) (S2 Fig.). At 12 hours, a distinct pattern of DC-SIGN expression was detected in MoDCs infected with Mfa1+Pg compared to Pg381 and FimA+Pg. Infection with Mfa1+Pg up-regulated DC-SIGN mRNA at 1 and 10 MOIs in a dose dependent manner (p<0.01) (Fig. 2A) (Table 2). In contrast, we observed decreased expression of DC-SIGN when MoDCs were incubated with Pg381 (MOI-10) down-regulated DC-SIGN mRNA expression significantly (p<0.05) at 12 hours (-4.55 fold). Fold regulations were calculated relative to un-infected MoDCs (Fig. 2A) (Table 2).


Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

El-Awady AR, Miles B, Scisci E, Kurago ZB, Palani CD, Arce RM, Waller JL, Genco CA, Slocum C, Manning M, Schoenlein PV, Cutler CW - PLoS Pathog. (2015)

Mfa1+Pg up-regulate the expression of DC-SIGN in human MoDCs.A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001). B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons 3 different experiments (* p<0.01).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352937&req=5

ppat.1004647.g002: Mfa1+Pg up-regulate the expression of DC-SIGN in human MoDCs.A) DC-SIGN mRNA expression in P. gingivalis-infected MoDCs at 0.1, 1 and 10 MOIs. The figure shows the gene expression after 12 hours of Pg381 and mutant strains infections. The target gene (DC-SIGN) was normalized using the endogenous control GAPDH (ΔCt) and fold regulations were calculated using 2-(ΔΔCt) method. The statistical analysis was performed using the t-test, which accounts for the clustering of infected and un-infected controls within 3 different experiments (* p<0.001). B) Immuno-electron microscopy of un-infected MoDCs (Cont.) (upper panel), MoDCs infected with Pg381 (middle panel) and Mfa1+Pg mutants (lower panel). Gold particles (marked with red rings) for positive DC-SIGN were detected in the cell membrane and cytoplasm of cells infected with Mfa1+Pg strains. Minimal positive staining for DC-SIGN was detected in the membranes of MoDCs infected with Pg381, while no cytoplasmic gold labeling was detected in these cells. C) Flow cytometry analysis of surface DC-SIGN in human MoDCs after infection with Pg381, Mfa1+Pg and FimA+Pg. The analysis of the intensity used Kruskal-Wallis test analysis of different groups and Dunn’s test for multiple comparisons 3 different experiments (* p<0.01).
Mentions: To determine whether expression of DC-SIGN [14] was altered by P. gingivalis infection, MoDCs were infected with all the strains at different multiplicities of infection (MOIs) and gene expression of DC-SIGN was quantified at 2, 6, 12 and 24 hours (Fig. 2A) (S2 Fig.). At 12 hours, a distinct pattern of DC-SIGN expression was detected in MoDCs infected with Mfa1+Pg compared to Pg381 and FimA+Pg. Infection with Mfa1+Pg up-regulated DC-SIGN mRNA at 1 and 10 MOIs in a dose dependent manner (p<0.01) (Fig. 2A) (Table 2). In contrast, we observed decreased expression of DC-SIGN when MoDCs were incubated with Pg381 (MOI-10) down-regulated DC-SIGN mRNA expression significantly (p<0.05) at 12 hours (-4.55 fold). Fold regulations were calculated relative to un-infected MoDCs (Fig. 2A) (Table 2).

Bottom Line: Survival was decreased by activation of TLR2 and/or autophagy.Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1.These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Periodontics, Georgia Regents University, Augusta, Georgia, United States of America.

ABSTRACT
Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

Show MeSH
Related in: MedlinePlus