3. The role of cytohesins in the innate immune response to Legionella
3.2 Results
3.2.7 Phagocytosis of L. pneumophila by different myeloid cells
Figure 3.14: Enumeration of L. pneumophila+ neutrophils in the lung of cy-tohesin knockout mice after infection. C57BL/6 (WT), cytohesin-1 (Cyth1-/-) and cytohesin-3 (Cyth3-/-) knockout mice, as well as cytohesin-2 conditional knockout (Cyth2fl/fl LysM/Cre), floxed cytohesin-2 (Cyth2fl/fl) and LysM/Cre
eutrophils
L pneumophila poi i e
Days
eutrophils
Percentage ofL pn neutrophils of total neutrophils in lung
Days
B C
uninfected infected
A
Days
L pneumophila poi i e
Days Percentage ofL pn neutrophils of total neutrophils in lung
WT h1 h3
h fl fl
h e h fl fl e
(Cyth2+/+ LysM/Cre) control mice were challenged with L. pneumophila flaA over a 7 day time course. Murine lung was collected to different time points and L.
pneumophila+(L.pn+) neutrophils were identified and quantified via flow cytometry analysis as in Figure 3.10A with the use of an antibody that stains L.pn LPS. A.
Simplified gating for L.pn+ neutrophils in murine lung 3 days after infection and not infected mice (control). B. Enumeration of L.pn+ neutrophils presented lung tissue. C. Percentage of L.pn+ neutrophils of all neutrophils in the lung tissue.
Graphs present the mean with SEM. n≥8 pooled from n≥2 independent experi-ments (*, p<0.05; two-tailed Mann-Whitney U-test).
After infection, MCs engulf L. pneumophila and the number of L.pn+ MCs de-creases slowly over time Figure 3.15B. Notably, the percentage of L.pn+ MC in relation to all lung tissue MCs increased from ~50% to ~80% (Figure 3.15C).
Cyth1-/- mice displayed a lower number of L.pn+ MCs on day 7 p.i. (Figure 3.15B), although proportionally they did not alter compared to WT L.pn+ MCs (Figure 3.15C).
Moreover, Cyth3-/- L.pn+ MCs were numerically increased versus WT L.pn+ MCs in the early stage (Figure 3.15B). However, these differences were not significant when calculated as a percentage of total lung infiltrated MCs in these mice (Figure 3.15C).
In Cyth2 experiments the Cyth2+/+ LysM/Cre control mice showed elevation of L.pn+ MCs compared to myeloid-specific Cyth2 KO mice (Figure 3.15B), which was not significantly different percentage-wise (Figure 3.15C). However, similar to L.pn+ neutrophils, L.pn+ MCs in myeloid-specific Cyth2 KO were proportionally lower compared to the controls on day 7 (Figure 3.15C).
Therefore, deletion of either Cyth1, Cyth2 or Cyth3 did not remarkably modify the phagocytosis of L. pneumophila by MCs.
Figure 3.15: Enumeration of L. pneumophila+ monocyte-derived cells in the lung of cytohesin knockout mice after infection. C57BL/6 (WT), cytohesin-1 (Cyth1-/-) and cytohesin-3 (Cyth3-/-) knockout mice, as well as cytohesin-2 condi-tional knockout (Cyth2fl/fl LysM/Cre), floxed cytohesin-2 (Cyth2fl/fl) and LysM/Cre
MCs
Days
MCs
WT h1 h3
Days
B C
uninfected infected
A
Days
L pneumophila poi i e
h fl fl
h e h fl fl e
L pneumophila poi i e
Percentage ofL pn MCs of total MCs in lung
Days Percentage ofL pn MCs of total MCs in lung
(Cyth2+/+ LysM/Cre) control mice were challenged with L. pneumophila flaA over a 7 day time course. Murine lung was collected to different time points and L. pneumophila+ MCs were identified and quantified via flow cytometry analysis as in Figure 3.11A with the use of an antibody that stains L.pn LPS. A. Simplified gating for L.pn+ MCs in murine lung 3 days after infection and not infected mice (control). B. Enumeration of L.pn+ MCs presented lung tissue. C. Percentage of L.pn+ MCs of all MCs in the lung tissue. Graphs present the mean with SEM. n≥8 pooled from n≥2 independent experiments (*, p<0.05; **, p<0.01; two-tailed Mann-Whitney U-test).
The number of cDCs which engulfed L. pneumophila in the course of infection was lower than other phagocytes, with only 4-8% of all lung located cDCs staining positive for L. pneumophila (Figure 3.16).
Following infection, Cyth1-/- mice displayed similar absolute counts (Figure 3.16B) and percentage (Figure 3.16C) of L.pn+ cDC compared to WT mice. Sim-ilar to neutrophils, the absolute count of L.pn+ cDC was increased in Cyth3-/- mice (Figure 3.16B), but was not different as a proportion of total cDCs (Figure 3.16C).
Decreased numbers of L.pn+ cDC were observed in myeloid-specific Cyth2 KO mice on day 3 (Figure 3.16B). However, this was in line with generally lower numbers of cDCs (see Figure 3.12) and was not significantly different as a pro-portion of total cDC (Figure 3.16C). The percentage of L.pn+ cDC in these mice was inconsistent and showed a higher percentage of L.pn+ cDCs on day 5 and lower on day 7 p.i. (Figure 3.16C). It therefore seems unlikely that phagocytosis by cDCs is altered overall in myeloid-specific Cyth2 KO mice.
In conclusion, this data suggests that Cyth1, Cyth2 or Cyth3 did not heavily im-pact the uptake of L. pneumophila in cDCs.
Figure 3.16: Enumeration of L. pneumophila+ cDCs in the lung of cytohesin knockout mice after infection. C57BL/6 (WT), cytohesin-1 (Cyth1-/-) and cyto-hesin-3 (Cyth3-/-) knockout mice, as well as cytohesin-2 conditional knockout (Cyth2fl/fl LysM/Cre), floxed cytohesin-2 (Cyth2fl/fl) and LysM/Cre (Cyth2+/+
cDCs
Days
cDCs
B C
uninfected infected
A
Days Days
L pneumophila poi i e
Percentage ofL pn cDCs of total cDCs in lung
Percentage ofL pn cDCs of total cDCs in lung
L pneumophila poi i e
WT h1 h3
h fl fl
h e h fl fl e
LysM/Cre) control mice were challenged with L. pneumophila flaA over a 7 day time course. Murine lung was collected to different time points and L. pneumoph-ila+ cDCs were identified and quantified via flow cytometry analysis as in Figure 3.12A with the use of an antibody that stains L.pn LPS. A. Simplified gating for L.pn+ cDCs in murine lung 3 days after infection and not infected mice (control).
B. Enumeration of L.pn+ cDCs presented lung tissue. C. Percentage of L.pn+ cDCs of all cDCs in the lung tissue. Graphs present the mean with SEM. n≥8 pooled from n≥2 independent experiments (*, p<0.05; **, p<0.01; two-tailed Mann-Whitney U-test).
Although the AM population decreased in the early stages of infection, the num-ber of L.pn+ AM consistently increased, possibly because of the uptake of bacte-rial debris and Legionella antigens (Figure 3.17B). Approximately 50-85% of AM were stained positive for L.pn and therefore, represented the phagocyte with the highest L. pneumophila content (Figure 3.17C).
During the course of infection, no significant differences were found in the abso-lute (Figure 3.17B) and relative number (Figure 3.17C) for L.pn+ AM among Cyth1-/-, Cyth3-/- and WT mice.
Similarly, in Cyth2 experiments the absolute values for L.pn+ AM were equal to the controls (Figure 3.17B). However, on day 7 p.i. the percentage of L.pn+ AM in myeloid-specific Cyth2 KO mice was much lower than in the controls (Figure 3.17C) which could indicate that there were less L.pn antigens present in these mice by this time point.
In summary, Cyth1, Cyth2 or Cyth3 were not required in the phagocytosis of L. pneumophila in AM upon infection in mice.
Figure 3.17: Enumeration of L. pneumophila+ alveolar macrophages in the lung of cytohesin knockout mice after infection. C57BL/6 (WT), cytohesin-1 (Cyth1-/-) and cytohesin-3 (Cyth3-/-) knockout mice, as well as cytohesin-2
condi-AM AM
B C
uninfected infected
A
Days
Percentage ofL pn AMs of total AM in lung
Days
L pneumophila poi i e
Days
Days
L pneumophila poi i e
Percentage ofL pn AM of total AM in lung
WT h1 h3
h fl fl
h e h fl fl e
tional knockout (Cyth2fl/fl LysM/Cre), floxed cytohesin-2 (Cyth2fl/fl) and LysM/Cre (Cyth2+/+ LysM/Cre) control mice were challenged with L. pneumophila flaA over a 7 day time course. Murine lung was collected to different time points and L. pneumophila+ alveolar macrophages (AM) were identified and quantified via flow cytometry analysis as in Figure 3.13A with the use of an antibody that stains L.pn LPS. A. Simplified gating for L.pn+ AM in murine lung 3 days after infection and not infected mice (control). B. Enumeration of L.pn+ AM presented lung tis-sue. C. Percentage of L.pn+ AM of all AM in the lung tissue. Graphs present the mean with SEM. n≥8 pooled from n≥2 independent experiments (*, p<0.05; two-tailed Mann-Whitney U-test).
In summary, deletion of Cyth1-/- led to a reduction in the absolute count of L.pn+ neutrophils and L.pn+ MCs, while Cyth3-/- deficiency caused an increase in the absolute count of L.pn+ neutrophils, L.pn+ MCs and L.pn+ cDCs at certain time points during L. pneumophila infection. Nonetheless, these alterations were not present when calculated as a percentage of the total number of the respective myeloid population present in the lung tissue, indicating that phagocytosis in these cell populations was not altered in the absence of Cyth1 of Cyth3, and any difference was potentially due to differences in cell numbers present.
Myeloid-specific Cyth2 KO mice appeared to have a reduced percentage of phag-ocytes positive for L. pneumophila when compared to the cell amount in the lung tissue in the late stage of infection. However, these alterations were not dramatic and did not lead to changes in the overall bacterial burden in the lung of myeloid-specific Cyth2 KO mice. This suggested that Cyth2 does not play an essential role in the phagocytosis of L. pneumophila in mice.