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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

CFSE+ cells enter the paracortex, co-localize with BCG and are closely apposed to P25 TCRTg cells.P25 TCRTg cells were isolated from LNs of naïve P25 TCRTg RAG–1-/- ECFP mice using a CD4 negative selection procedure (Miltenyi Biotec), and transferred i.v. into WT recipients. Recipients were then inoculated with BCG-RFP and the CFSE-based migration assay performed. (A) Distribution of CFSE+ cells in the paracortex (T-cell area) of the pLN as determined by CD3 staining on pLN sections from BCG-injected mice. (B) Quantification of CFSE+ cells within the T-cell area of the pLN from BCG- and PBS-injected mice expressed as the number of CFSE+ cells per area of CD3 staining. (C) Micrograph showing distribution of BCG (red), CFSE+ cells (green), p25 TCRTg cells (blue) and HEVs (white) in the pLN. Boxed inserts in the micrograph were magnified to show x/y/z projections of two CFSE+ cells with intracellular BCG-RFP. BCG were overexposed in these magnified views to facilitate visualization. Infected cell in lower panel is also in contact with a p25TCRTg ECFP cell (blue). (D) Quantification of the number of CFSE+ cells containing BCG-RFP (left graph), found in close apposition with P25 TCRTg ECFP cells (center graph), and containing BCG-RFP and in close apposition with P25 TCRTg ECFP cells (right graph). Data samples represent average obtained from individual pLN from which multiple fields of maximum intensity projections were analyzed. Bars indicate standard error of the mean. White bar on micrographs depict 100 microns. *Denotes statistically significant difference between BCG-infected and PBS controls in (B) and between day 1 and 6 in (D).
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ppat.1005206.g004: CFSE+ cells enter the paracortex, co-localize with BCG and are closely apposed to P25 TCRTg cells.P25 TCRTg cells were isolated from LNs of naïve P25 TCRTg RAG–1-/- ECFP mice using a CD4 negative selection procedure (Miltenyi Biotec), and transferred i.v. into WT recipients. Recipients were then inoculated with BCG-RFP and the CFSE-based migration assay performed. (A) Distribution of CFSE+ cells in the paracortex (T-cell area) of the pLN as determined by CD3 staining on pLN sections from BCG-injected mice. (B) Quantification of CFSE+ cells within the T-cell area of the pLN from BCG- and PBS-injected mice expressed as the number of CFSE+ cells per area of CD3 staining. (C) Micrograph showing distribution of BCG (red), CFSE+ cells (green), p25 TCRTg cells (blue) and HEVs (white) in the pLN. Boxed inserts in the micrograph were magnified to show x/y/z projections of two CFSE+ cells with intracellular BCG-RFP. BCG were overexposed in these magnified views to facilitate visualization. Infected cell in lower panel is also in contact with a p25TCRTg ECFP cell (blue). (D) Quantification of the number of CFSE+ cells containing BCG-RFP (left graph), found in close apposition with P25 TCRTg ECFP cells (center graph), and containing BCG-RFP and in close apposition with P25 TCRTg ECFP cells (right graph). Data samples represent average obtained from individual pLN from which multiple fields of maximum intensity projections were analyzed. Bars indicate standard error of the mean. White bar on micrographs depict 100 microns. *Denotes statistically significant difference between BCG-infected and PBS controls in (B) and between day 1 and 6 in (D).

Mentions: Next, the distribution of these migratory skin DCs was assessed in the DLN. The CFSE-based migration assay was performed and sections of pLN from PBS- and BCG-infected animals were subjected to confocal microscopy. CFSE-labeled cells localized exclusively to the T-cell area of the LN (Fig 4A) and in increased numbers following BCG but not PBS injection (Fig 4B), the latter corroborating our flow cytometry data. The presence of BCG within this migratory cell-population was confirmed at all time-points analyzed by using DsRed-expressing bacilli (BCG-RFP) [15] (Fig 4C and 4D). To test whether migrating skin DCs were capable of engaging BCG-specific CD4+ T cells, naïve P25 TCRTg cells expressing ECFP were adoptively transferred into recipient mice. The latter were then infected with BCG-RFP in the footpad and the CFSE-based migration assay performed. Several CFSE+ cells were found in apposition to P25 TCRTg cells (Fig 4C and 4D), suggesting a possible role for this migratory DC sub-population in priming CD4+ T cells to BCG. Interestingly, such positioning was independent of whether or not CFSE+ cells were directly infected with BCG.


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)

CFSE+ cells enter the paracortex, co-localize with BCG and are closely apposed to P25 TCRTg cells.P25 TCRTg cells were isolated from LNs of naïve P25 TCRTg RAG–1-/- ECFP mice using a CD4 negative selection procedure (Miltenyi Biotec), and transferred i.v. into WT recipients. Recipients were then inoculated with BCG-RFP and the CFSE-based migration assay performed. (A) Distribution of CFSE+ cells in the paracortex (T-cell area) of the pLN as determined by CD3 staining on pLN sections from BCG-injected mice. (B) Quantification of CFSE+ cells within the T-cell area of the pLN from BCG- and PBS-injected mice expressed as the number of CFSE+ cells per area of CD3 staining. (C) Micrograph showing distribution of BCG (red), CFSE+ cells (green), p25 TCRTg cells (blue) and HEVs (white) in the pLN. Boxed inserts in the micrograph were magnified to show x/y/z projections of two CFSE+ cells with intracellular BCG-RFP. BCG were overexposed in these magnified views to facilitate visualization. Infected cell in lower panel is also in contact with a p25TCRTg ECFP cell (blue). (D) Quantification of the number of CFSE+ cells containing BCG-RFP (left graph), found in close apposition with P25 TCRTg ECFP cells (center graph), and containing BCG-RFP and in close apposition with P25 TCRTg ECFP cells (right graph). Data samples represent average obtained from individual pLN from which multiple fields of maximum intensity projections were analyzed. Bars indicate standard error of the mean. White bar on micrographs depict 100 microns. *Denotes statistically significant difference between BCG-infected and PBS controls in (B) and between day 1 and 6 in (D).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4594926&req=5

ppat.1005206.g004: CFSE+ cells enter the paracortex, co-localize with BCG and are closely apposed to P25 TCRTg cells.P25 TCRTg cells were isolated from LNs of naïve P25 TCRTg RAG–1-/- ECFP mice using a CD4 negative selection procedure (Miltenyi Biotec), and transferred i.v. into WT recipients. Recipients were then inoculated with BCG-RFP and the CFSE-based migration assay performed. (A) Distribution of CFSE+ cells in the paracortex (T-cell area) of the pLN as determined by CD3 staining on pLN sections from BCG-injected mice. (B) Quantification of CFSE+ cells within the T-cell area of the pLN from BCG- and PBS-injected mice expressed as the number of CFSE+ cells per area of CD3 staining. (C) Micrograph showing distribution of BCG (red), CFSE+ cells (green), p25 TCRTg cells (blue) and HEVs (white) in the pLN. Boxed inserts in the micrograph were magnified to show x/y/z projections of two CFSE+ cells with intracellular BCG-RFP. BCG were overexposed in these magnified views to facilitate visualization. Infected cell in lower panel is also in contact with a p25TCRTg ECFP cell (blue). (D) Quantification of the number of CFSE+ cells containing BCG-RFP (left graph), found in close apposition with P25 TCRTg ECFP cells (center graph), and containing BCG-RFP and in close apposition with P25 TCRTg ECFP cells (right graph). Data samples represent average obtained from individual pLN from which multiple fields of maximum intensity projections were analyzed. Bars indicate standard error of the mean. White bar on micrographs depict 100 microns. *Denotes statistically significant difference between BCG-infected and PBS controls in (B) and between day 1 and 6 in (D).
Mentions: Next, the distribution of these migratory skin DCs was assessed in the DLN. The CFSE-based migration assay was performed and sections of pLN from PBS- and BCG-infected animals were subjected to confocal microscopy. CFSE-labeled cells localized exclusively to the T-cell area of the LN (Fig 4A) and in increased numbers following BCG but not PBS injection (Fig 4B), the latter corroborating our flow cytometry data. The presence of BCG within this migratory cell-population was confirmed at all time-points analyzed by using DsRed-expressing bacilli (BCG-RFP) [15] (Fig 4C and 4D). To test whether migrating skin DCs were capable of engaging BCG-specific CD4+ T cells, naïve P25 TCRTg cells expressing ECFP were adoptively transferred into recipient mice. The latter were then infected with BCG-RFP in the footpad and the CFSE-based migration assay performed. Several CFSE+ cells were found in apposition to P25 TCRTg cells (Fig 4C and 4D), suggesting a possible role for this migratory DC sub-population in priming CD4+ T cells to BCG. Interestingly, such positioning was independent of whether or not CFSE+ cells were directly infected with BCG.

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