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Dermal neutrophil, macrophage and dendritic cell responses to Yersinia pestis transmitted by fleas.

Shannon JG, Bosio CF, Hinnebusch BJ - PLoS Pathog. (2015)

Bottom Line: Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis.Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria.This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Plague Section, Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.

ABSTRACT
Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.

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Imaging and tracking dendritic cell movement in response to flea bites and flea-transmitted Y. pestis in vivo.CD11c-YFP mouse ears were fed upon by 1 uninfected flea for 10 min, Y. pestis pMcherry blocked fleas for 50 min (No Transmission = 5 fleas, +Transmission = 6 fleas), or no fleas (empty feeding chamber) for 10 min and imaged by confocal microscopy for 4 h. (A) Flea bite sites were identified by Sytox Blue staining (blue, upper panels only). Upper panels show t = 0 h and lower panels show t = 4 h. The full time series can be seen in S9–S12 Videos. Movement of YFP+ over the course of the experiment was tracked using the image-processing software Imaris. The lower panels also show the cell tracking data and displacement (arrows) for all displacement events ≥ 30 μm. YFP+ cells are dendritic cells (yellow), the red channel shows Y. pestis pMcherry, if present. All examples shown are representative of at least 3 independent experiments. Scale bar represents 100 μm. (B) The direction and length of net displacement were calculated and analysis was limited to displacement events ≥ 30 μm. The direction of cell displacement was manually scored as being “toward,” “neutral,” or “away” from the flea bite site (determined by the presence of bacteria and/or Sytox Blue staining). For the No Fleas condition, a spot at the center of the image field was arbitrarily chosen as the reference point for the scoring. The results shown are the mean of at 3 or 4 independent experiments. Error bars represent standard deviation. (C), The average numbers of displacement events ≥ 30 μm in length for each experimental condition were calculated. Error bars represent SEM. The results shown are a mean of 3 or 4 independent experiments. * = p≤0.05, ** = p≤0.01, and **** = p≤0.0001.
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ppat.1004734.g006: Imaging and tracking dendritic cell movement in response to flea bites and flea-transmitted Y. pestis in vivo.CD11c-YFP mouse ears were fed upon by 1 uninfected flea for 10 min, Y. pestis pMcherry blocked fleas for 50 min (No Transmission = 5 fleas, +Transmission = 6 fleas), or no fleas (empty feeding chamber) for 10 min and imaged by confocal microscopy for 4 h. (A) Flea bite sites were identified by Sytox Blue staining (blue, upper panels only). Upper panels show t = 0 h and lower panels show t = 4 h. The full time series can be seen in S9–S12 Videos. Movement of YFP+ over the course of the experiment was tracked using the image-processing software Imaris. The lower panels also show the cell tracking data and displacement (arrows) for all displacement events ≥ 30 μm. YFP+ cells are dendritic cells (yellow), the red channel shows Y. pestis pMcherry, if present. All examples shown are representative of at least 3 independent experiments. Scale bar represents 100 μm. (B) The direction and length of net displacement were calculated and analysis was limited to displacement events ≥ 30 μm. The direction of cell displacement was manually scored as being “toward,” “neutral,” or “away” from the flea bite site (determined by the presence of bacteria and/or Sytox Blue staining). For the No Fleas condition, a spot at the center of the image field was arbitrarily chosen as the reference point for the scoring. The results shown are the mean of at 3 or 4 independent experiments. Error bars represent standard deviation. (C), The average numbers of displacement events ≥ 30 μm in length for each experimental condition were calculated. Error bars represent SEM. The results shown are a mean of 3 or 4 independent experiments. * = p≤0.05, ** = p≤0.01, and **** = p≤0.0001.

Mentions: The bite sites of uninfected fleas, blocked fleas that did not transmit bacteria, and blocked fleas that had deposited Y. pestis in the dermis were imaged for at least 4 hours post-feeding (Fig. 6A). In response to uninfected flea bites DCs appear to randomly move through the dermis (Fig. 6A, S9 Video), similar to what is observed in a naïve mouse ear (Fig. 6A, S10 Video). Consistent with what was observed after needle inoculation of these mice [16], we did not observe any notable interaction between DCs and flea-transmitted bacteria (Fig. 6A, S11 Video). Interestingly, while there was no net influx of a large number of DCs like that seen with neutrophils, the cells that were present appeared to migrate towards the flea bite sites that contained Y. pestis. A similar phenomenon was seen at some blocked flea bites where no bacteria were deposited in the dermis, but was much more variable (Fig. 6A, S12 Video). To quantify this cellular movement, image analysis software was used to track the migration of these cells over the course of the experiment. Cell tracking and displacement are shown in the bottom panels of Fig. 6A. The direction of displacement of each cell track was scored as being “toward”, “away” from or “neutral” relative to the flea bite site (Fig. 6B). Additionally, the average number of cell tracks with displacement >30 μm was determined for each experiment (Fig. 6C). We conclude that DCs migrate towards flea-transmitted Y. pestis or blocked flea bites, but not to uninfected flea bites in the dermis and that there was overall more displacement of DCs when bacteria were present at the bite site.


Dermal neutrophil, macrophage and dendritic cell responses to Yersinia pestis transmitted by fleas.

Shannon JG, Bosio CF, Hinnebusch BJ - PLoS Pathog. (2015)

Imaging and tracking dendritic cell movement in response to flea bites and flea-transmitted Y. pestis in vivo.CD11c-YFP mouse ears were fed upon by 1 uninfected flea for 10 min, Y. pestis pMcherry blocked fleas for 50 min (No Transmission = 5 fleas, +Transmission = 6 fleas), or no fleas (empty feeding chamber) for 10 min and imaged by confocal microscopy for 4 h. (A) Flea bite sites were identified by Sytox Blue staining (blue, upper panels only). Upper panels show t = 0 h and lower panels show t = 4 h. The full time series can be seen in S9–S12 Videos. Movement of YFP+ over the course of the experiment was tracked using the image-processing software Imaris. The lower panels also show the cell tracking data and displacement (arrows) for all displacement events ≥ 30 μm. YFP+ cells are dendritic cells (yellow), the red channel shows Y. pestis pMcherry, if present. All examples shown are representative of at least 3 independent experiments. Scale bar represents 100 μm. (B) The direction and length of net displacement were calculated and analysis was limited to displacement events ≥ 30 μm. The direction of cell displacement was manually scored as being “toward,” “neutral,” or “away” from the flea bite site (determined by the presence of bacteria and/or Sytox Blue staining). For the No Fleas condition, a spot at the center of the image field was arbitrarily chosen as the reference point for the scoring. The results shown are the mean of at 3 or 4 independent experiments. Error bars represent standard deviation. (C), The average numbers of displacement events ≥ 30 μm in length for each experimental condition were calculated. Error bars represent SEM. The results shown are a mean of 3 or 4 independent experiments. * = p≤0.05, ** = p≤0.01, and **** = p≤0.0001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4363629&req=5

ppat.1004734.g006: Imaging and tracking dendritic cell movement in response to flea bites and flea-transmitted Y. pestis in vivo.CD11c-YFP mouse ears were fed upon by 1 uninfected flea for 10 min, Y. pestis pMcherry blocked fleas for 50 min (No Transmission = 5 fleas, +Transmission = 6 fleas), or no fleas (empty feeding chamber) for 10 min and imaged by confocal microscopy for 4 h. (A) Flea bite sites were identified by Sytox Blue staining (blue, upper panels only). Upper panels show t = 0 h and lower panels show t = 4 h. The full time series can be seen in S9–S12 Videos. Movement of YFP+ over the course of the experiment was tracked using the image-processing software Imaris. The lower panels also show the cell tracking data and displacement (arrows) for all displacement events ≥ 30 μm. YFP+ cells are dendritic cells (yellow), the red channel shows Y. pestis pMcherry, if present. All examples shown are representative of at least 3 independent experiments. Scale bar represents 100 μm. (B) The direction and length of net displacement were calculated and analysis was limited to displacement events ≥ 30 μm. The direction of cell displacement was manually scored as being “toward,” “neutral,” or “away” from the flea bite site (determined by the presence of bacteria and/or Sytox Blue staining). For the No Fleas condition, a spot at the center of the image field was arbitrarily chosen as the reference point for the scoring. The results shown are the mean of at 3 or 4 independent experiments. Error bars represent standard deviation. (C), The average numbers of displacement events ≥ 30 μm in length for each experimental condition were calculated. Error bars represent SEM. The results shown are a mean of 3 or 4 independent experiments. * = p≤0.05, ** = p≤0.01, and **** = p≤0.0001.
Mentions: The bite sites of uninfected fleas, blocked fleas that did not transmit bacteria, and blocked fleas that had deposited Y. pestis in the dermis were imaged for at least 4 hours post-feeding (Fig. 6A). In response to uninfected flea bites DCs appear to randomly move through the dermis (Fig. 6A, S9 Video), similar to what is observed in a naïve mouse ear (Fig. 6A, S10 Video). Consistent with what was observed after needle inoculation of these mice [16], we did not observe any notable interaction between DCs and flea-transmitted bacteria (Fig. 6A, S11 Video). Interestingly, while there was no net influx of a large number of DCs like that seen with neutrophils, the cells that were present appeared to migrate towards the flea bite sites that contained Y. pestis. A similar phenomenon was seen at some blocked flea bites where no bacteria were deposited in the dermis, but was much more variable (Fig. 6A, S12 Video). To quantify this cellular movement, image analysis software was used to track the migration of these cells over the course of the experiment. Cell tracking and displacement are shown in the bottom panels of Fig. 6A. The direction of displacement of each cell track was scored as being “toward”, “away” from or “neutral” relative to the flea bite site (Fig. 6B). Additionally, the average number of cell tracks with displacement >30 μm was determined for each experiment (Fig. 6C). We conclude that DCs migrate towards flea-transmitted Y. pestis or blocked flea bites, but not to uninfected flea bites in the dermis and that there was overall more displacement of DCs when bacteria were present at the bite site.

Bottom Line: Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis.Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria.This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Plague Section, Laboratory of Zoonotic Pathogens, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, United States of America.

ABSTRACT
Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.

Show MeSH
Related in: MedlinePlus