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BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming.

Bollampalli VP, Harumi Yamashiro L, Feng X, Bierschenk D, Gao Y, Blom H, Henriques-Normark B, Nylén S, Rothfuchs AG - PLoS Pathog. (2015)

Bottom Line: Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells.Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming.In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.

No MeSH data available.


Related in: MedlinePlus

Detection of BCG in the DLN correlates with the priming of P25 TCRTg cells.(A) 5% Evan’s blue was injected in the footpad of WT mice. At the indicated time points, animals were sacrificed, LNs isolated and the amount of dye per LN determined on a spectrophotometer against a standard curve. The concentration of Evan’s blue was normalized to the average cell number in each LN. (B) WT mice were inoculated with 1x106 CFUs of BCG in the footpad. CFUs of BCG in LNs and spleen were enumerated on 7H11 agar at the indicated time points after infection. (C) Frequency of P25 TCRTg cells was determined by flow cytometry in LNs and spleen of BCG-infected mice at the indicated time points after infection. Naïve P25 TCRTg cells were CFSE-labeled and transferred into CD45.1+ recipient mice inoculated 24hrs later with BCG as in (B). (D) Changes in MFI for CD69 (left panel), CD62L (center panel) and CD44 (right panel) on transferred P25 TCRTg cells (CD4+ CD45.2+) in the pLN at different time points after BCG infection. (E) Intracellular production of IFN-γ on transferred P25 TCRTg cells from pLN after in vitro recall with Ag85B240–254 peptide. Zebra plots depict intracellular IFN-γ in P25 TCRTg cells 9 days after BCG infection, following recall with peptide. Only baseline levels of IFN-γ were observed on non-transgenic (non-Tg) LN cells (CD45.2neg CD4+), shown here on day 9 after infection. Four to 8 animals per group were used in each experiment. Bars indicate standard error of the mean. One of two independent experiments shown.
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ppat.1005206.g001: Detection of BCG in the DLN correlates with the priming of P25 TCRTg cells.(A) 5% Evan’s blue was injected in the footpad of WT mice. At the indicated time points, animals were sacrificed, LNs isolated and the amount of dye per LN determined on a spectrophotometer against a standard curve. The concentration of Evan’s blue was normalized to the average cell number in each LN. (B) WT mice were inoculated with 1x106 CFUs of BCG in the footpad. CFUs of BCG in LNs and spleen were enumerated on 7H11 agar at the indicated time points after infection. (C) Frequency of P25 TCRTg cells was determined by flow cytometry in LNs and spleen of BCG-infected mice at the indicated time points after infection. Naïve P25 TCRTg cells were CFSE-labeled and transferred into CD45.1+ recipient mice inoculated 24hrs later with BCG as in (B). (D) Changes in MFI for CD69 (left panel), CD62L (center panel) and CD44 (right panel) on transferred P25 TCRTg cells (CD4+ CD45.2+) in the pLN at different time points after BCG infection. (E) Intracellular production of IFN-γ on transferred P25 TCRTg cells from pLN after in vitro recall with Ag85B240–254 peptide. Zebra plots depict intracellular IFN-γ in P25 TCRTg cells 9 days after BCG infection, following recall with peptide. Only baseline levels of IFN-γ were observed on non-transgenic (non-Tg) LN cells (CD45.2neg CD4+), shown here on day 9 after infection. Four to 8 animals per group were used in each experiment. Bars indicate standard error of the mean. One of two independent experiments shown.

Mentions: To begin dissecting the early events following BCG infection, we established a model where C57BL/6 mice are inoculated with BCG in the footpad skin and immune responses assessed in the DLN. The popliteal LN (pLN) was established as the DLN after injection of Evan’s Blue in the footpad, with secondary drainage to lumbar aortic and sciatic LNs (Fig 1A). In accordance with the above, inoculation of BCG in the footpad lead to a major detection of bacilli in the draining, pLN (Fig 1B). To examine the outcome of mycobacterial antigen-specific CD4+ T-cell responses in this setting, the fate of naïve P25 TCRTg cells was followed in vivo. These CD4+ T cells have a transgenic T-cell receptor specific for peptide 25 of mycobacterial antigen 85B (Ag85B240–254) [11]. The expansion of P25 TCRTg cells was first evident in the pLN, which was also the dominant site for P25 TCRTg cell expansion (Fig 1C). Up-regulation of CD69 and down-regulation of CD62L were clearly evident on P25 TCRTg cells 1 day after BCG infection, prior to T-cell expansion (Fig 1D). The expansion of P25 TCRTg cells correlated with sequential dilution of CFSE labeling in this population and was corroborated by an increase in the absolute number of activated P25 TCRTg cells in the DLN (S1A and S1C Fig). As reported by others, [12], surface expression of CD69 on transgenic cells was progressively down-regulated with cell division while cells that had divided expressed more CD44 compared to undivided cells (S1B Fig). Moreover, P25 TCRTg cells were found to produce IFN-γ upon recall with Ag85B240–254. IFN-γ+ cells were CFSElow and CD44+ (Fig 1E). Overall, our observations suggest that the priming of P25 TCRTg cells is concentrated to the DLN where also the majority of culturable bacilli are found.


BCG Skin Infection Triggers IL-1R-MyD88-Dependent Migration of EpCAMlow CD11bhigh Skin Dendritic cells to Draining Lymph Node During CD4+ T-Cell Priming.

Bollampalli VP, Harumi Yamashiro L, Feng X, Bierschenk D, Gao Y, Blom H, Henriques-Normark B, Nylén S, Rothfuchs AG - PLoS Pathog. (2015)

Detection of BCG in the DLN correlates with the priming of P25 TCRTg cells.(A) 5% Evan’s blue was injected in the footpad of WT mice. At the indicated time points, animals were sacrificed, LNs isolated and the amount of dye per LN determined on a spectrophotometer against a standard curve. The concentration of Evan’s blue was normalized to the average cell number in each LN. (B) WT mice were inoculated with 1x106 CFUs of BCG in the footpad. CFUs of BCG in LNs and spleen were enumerated on 7H11 agar at the indicated time points after infection. (C) Frequency of P25 TCRTg cells was determined by flow cytometry in LNs and spleen of BCG-infected mice at the indicated time points after infection. Naïve P25 TCRTg cells were CFSE-labeled and transferred into CD45.1+ recipient mice inoculated 24hrs later with BCG as in (B). (D) Changes in MFI for CD69 (left panel), CD62L (center panel) and CD44 (right panel) on transferred P25 TCRTg cells (CD4+ CD45.2+) in the pLN at different time points after BCG infection. (E) Intracellular production of IFN-γ on transferred P25 TCRTg cells from pLN after in vitro recall with Ag85B240–254 peptide. Zebra plots depict intracellular IFN-γ in P25 TCRTg cells 9 days after BCG infection, following recall with peptide. Only baseline levels of IFN-γ were observed on non-transgenic (non-Tg) LN cells (CD45.2neg CD4+), shown here on day 9 after infection. Four to 8 animals per group were used in each experiment. Bars indicate standard error of the mean. One of two independent experiments shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4594926&req=5

ppat.1005206.g001: Detection of BCG in the DLN correlates with the priming of P25 TCRTg cells.(A) 5% Evan’s blue was injected in the footpad of WT mice. At the indicated time points, animals were sacrificed, LNs isolated and the amount of dye per LN determined on a spectrophotometer against a standard curve. The concentration of Evan’s blue was normalized to the average cell number in each LN. (B) WT mice were inoculated with 1x106 CFUs of BCG in the footpad. CFUs of BCG in LNs and spleen were enumerated on 7H11 agar at the indicated time points after infection. (C) Frequency of P25 TCRTg cells was determined by flow cytometry in LNs and spleen of BCG-infected mice at the indicated time points after infection. Naïve P25 TCRTg cells were CFSE-labeled and transferred into CD45.1+ recipient mice inoculated 24hrs later with BCG as in (B). (D) Changes in MFI for CD69 (left panel), CD62L (center panel) and CD44 (right panel) on transferred P25 TCRTg cells (CD4+ CD45.2+) in the pLN at different time points after BCG infection. (E) Intracellular production of IFN-γ on transferred P25 TCRTg cells from pLN after in vitro recall with Ag85B240–254 peptide. Zebra plots depict intracellular IFN-γ in P25 TCRTg cells 9 days after BCG infection, following recall with peptide. Only baseline levels of IFN-γ were observed on non-transgenic (non-Tg) LN cells (CD45.2neg CD4+), shown here on day 9 after infection. Four to 8 animals per group were used in each experiment. Bars indicate standard error of the mean. One of two independent experiments shown.
Mentions: To begin dissecting the early events following BCG infection, we established a model where C57BL/6 mice are inoculated with BCG in the footpad skin and immune responses assessed in the DLN. The popliteal LN (pLN) was established as the DLN after injection of Evan’s Blue in the footpad, with secondary drainage to lumbar aortic and sciatic LNs (Fig 1A). In accordance with the above, inoculation of BCG in the footpad lead to a major detection of bacilli in the draining, pLN (Fig 1B). To examine the outcome of mycobacterial antigen-specific CD4+ T-cell responses in this setting, the fate of naïve P25 TCRTg cells was followed in vivo. These CD4+ T cells have a transgenic T-cell receptor specific for peptide 25 of mycobacterial antigen 85B (Ag85B240–254) [11]. The expansion of P25 TCRTg cells was first evident in the pLN, which was also the dominant site for P25 TCRTg cell expansion (Fig 1C). Up-regulation of CD69 and down-regulation of CD62L were clearly evident on P25 TCRTg cells 1 day after BCG infection, prior to T-cell expansion (Fig 1D). The expansion of P25 TCRTg cells correlated with sequential dilution of CFSE labeling in this population and was corroborated by an increase in the absolute number of activated P25 TCRTg cells in the DLN (S1A and S1C Fig). As reported by others, [12], surface expression of CD69 on transgenic cells was progressively down-regulated with cell division while cells that had divided expressed more CD44 compared to undivided cells (S1B Fig). Moreover, P25 TCRTg cells were found to produce IFN-γ upon recall with Ag85B240–254. IFN-γ+ cells were CFSElow and CD44+ (Fig 1E). Overall, our observations suggest that the priming of P25 TCRTg cells is concentrated to the DLN where also the majority of culturable bacilli are found.

Bottom Line: Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells.Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming.In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden.

ABSTRACT
The transport of antigen from the periphery to the draining lymph node (DLN) is critical for T-cell priming but remains poorly studied during infection with Mycobacterium bovis Bacille Calmette-Guérin (BCG). To address this we employed a mouse model to track the traffic of Dendritic cells (DCs) and mycobacteria from the BCG inoculation site in the skin to the DLN. Detection of BCG in the DLN was concomitant with the priming of antigen-specific CD4+ T cells at that site. We found EpCAMlow CD11bhigh migratory skin DCs to be mobilized during the transport of BCG to the DLN. Migratory skin DCs distributed to the T-cell area of the LN, co-localized with BCG and were found in close apposition to antigen-specific CD4+ T cells. Consequently, blockade of skin DC traffic into DLN dramatically reduced mycobacterial entry into DLN and muted T-cell priming. Interestingly, DC and mycobacterial entry into the DLN was dependent on IL-1R-I, MyD88, TNFR-I and IL-12p40. In addition, we found using DC adoptive transfers that the requirement for MyD88 in BCG-triggered migration was not restricted to the migrating DC itself and that hematopoietic expression of MyD88 was needed in part for full-fledged migration. Our observations thus identify a population of DCs that contribute towards the priming of CD4+ T cells to BCG infection by transporting bacilli into the DLN in an IL-1R-MyD88-dependent manner and reveal both DC-intrinsic and -extrinsic requirements for MyD88 in DC migration.

No MeSH data available.


Related in: MedlinePlus