Migratory DC subsets are reduced in the dLN in C3^−/− mice after influenza infection.

<p>(<b>A</b>) Kinetics of CD103⁺ DCs and CD11b⁺ DCs in the lungs during influenza infection in WT and C3^−/− mice. Populations were gated as show in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003115#ppat.1003115.s002" target="_blank">Fig. S2</a>. Numbers within the dot plot represent average percentage of cells for the group. (<b>B</b> and <b>C</b>) Absolute numbers of CD103⁺ DC (<b>B</b>) and CD11b⁺ DC (<b>C</b>) subsets in lungs during steady state and during influenza infection. (<b>D</b>) Kinetics of CD103⁺ DCs and CD11b⁺ DCs in the dLN during influenza infection in WT and C3^−/− mice. Populations were gated as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003115#ppat.1003115.s001" target="_blank">Fig. S1</a>. Numbers within the dot plot represent average percentage of cells for the group. (<b>E</b> and <b>F</b>) Absolute numbers of CD103⁺ DC (<b>E</b>) and CD11b⁺ DC (<b>F</b>) subsets in dLN during steady state and during influenza infection. The values are expressed as mean ± SEM. The data are representative of three different experiments with similar results. *, P<0.05, and **P<0.01.</p

C3^−/− mice show greater weight loss and mortality during influenza infection.

<p>(<b>A</b>) Percentage of body weight loss after influenza infection. A weight loss of <20% and recovery represents a sub-lethal infection. (<b>B</b>) Survival curve comparing WT and C3^−/− mice during influenza infection (n = 6–7 in each group). (<b>C</b>) Plots represent <i>ex vivo</i> analysis of CFSE-labeled OT-I CD8⁺ T cell proliferation in the dLN 3 days after infection with PR8 and PR8-OT-I (bottom). The graph on the right shows the respective division index for the proliferating OT-I CD8⁺ T cells for each group of mice. (<b>D</b>) Plots represent <i>ex vivo</i> analysis of CFSE-labeled OT-II CD4⁺ T cell proliferation in the dLN 3 days after infection with PR8 and PR8-OT-II (bottom). The graph on the right shows the respective division index for the proliferating OT-II CD4⁺ T cells for each group of mice. (<b>E</b>) Histogram showing expression of costimulatory molecules CD86, CD80 and CD40 on CD103⁺ DCs and CD11b⁺ DCs in the dLN on day 2 post infection. Filled histogram: Isotype control, dashed histogram: Naïve, open histogram: infected. (<b>F</b>) Bar graphs show the mean fluorescent intensity (MFI) of CD86, CD80 and CD40 expression on CD103⁺DC and CD11b⁺ DC subsets in the dLN of naïve and influenza infected (Day 2) WT or C3^−/− mice. (<b>G</b>) Plots represent CFSE-labeled OT-I CD8⁺ T cell proliferation 3 days after co-culture with sorted CD103⁺ DCs and CD11b⁺ DCs obtained from pooled dLN of PR8-OT-I influenza infected mice at a ratio of 1∶10 (DC:T cells). Results shown are representative of four experiments with similar results. The values are expressed as mean ± SEM.</p

Tracking of mDC migration from the lung to the dLN demonstrates defective mDC migration in C3^−/− mice during influenza infection and intratracheal administration of LPS does not recover the defective mDC migration during influenza infection.

<p>WT and C3^−/− mice were infected with influenza virus; 16 hours before sacrificing, cells in the lungs were labeled with 8 mM CFSE by intranasal instillation. dLN was harvested and analysed for CFSE⁺ mDCs using gating strategy shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003115#ppat.1003115.s001" target="_blank">Fig. S1</a>. (<b>A</b>) Dot plots show CFSE⁺ mDCs (first panel) in the dLN and subsequent panels show CD103⁺ DCs and CD11b⁺ DCs after gating on CFSE⁺ mDCs at indicated times after influenza infection. Numbers within the dot plot represent cell number. (<b>B</b>) Kinetics of frequency of CFSE⁺ mDCs in the dLN of influenza infected mice. Absolute number of CFSE⁺ CD103⁺ DCs (<b>C</b>) and CFSE⁺ CD11b⁺ DCs (<b>D</b>) in the dLN at indicated time points after influenza infection. LPS (2 µg in PBS) was administered intratracheally after 6 hours to either uninfected or flu infected C3^−/− mice and the migration of mDCs from the lung to the dLN were followed as described earlier using CFSE on day 2 post infection. (E) Number of CFSE⁺ CD103⁺ (left) and CD11b⁺ (right) DCs in LPS administered C3^−/− mice in comparison with WT mice without LPS administration. The values are expressed as mean ± SEM. The data are representative of three different experiments with similar results. *, P<0.05, ** P<0.01 and *** P<0.001.</p

Lung C3a and C5a levels are up regulated during influenza infection by exclusive contribution from CD103⁺ DCs.

<p>(<b>A</b>) Bar graph shows the amounts of C3a and C5a per 100 µg protein in BAL fluid from naïve and PR8 infected WT mice on day 2, 4 and 7 post infection. (<b>B</b> and <b>C</b>) Bar graphs showing the relative mRNA expression levels of C3 and C5 in FACS sorted lung CD103⁺ DC and CD11b⁺ DC subsets in naïve or PR8 infected WT mice on day 1 and 3 post infection. Highest expression level in the naïve mice is taken as 100%. (<b>D</b> and <b>E</b>) Bar graphs show the amount of C3a and C5a per 100 µg protein in BAL fluid from naïve and PR8 infected langerin DTR mice with or without the depletion of CD103⁺ DCs. The data are representative of three different experiments with similar results. The values are expressed as mean ± SEM.</p

C3^−/− mDCs display similar capacity for virus uptake and maturation.

<p>(<b>A</b>) Dot plots show the percentage of DiD flu⁺ uptake by CD103⁺ DC (left panel) and CD11b⁺ DC (right panel) in WT and C3^−/− mice 16 hrs after DiD flu administration on day 1 post infection with PR8. (<b>B</b>) Bar graph shows the average numbers of DiD flu⁺ CD103⁺ DCs and DiD flu⁺ CD11b⁺ DCs in the lungs of WT and C3^−/− mice. (<b>C</b>)Single cell preparations from the lungs were enriched for DCs by a density gradient method. Enriched DCs were infected with influenza virus under <i>ex vivo</i> culture conditions and six hours later the CD103⁺ and CD11b⁺ DCs were flow sorted. Q-RT-PCR for the indicated cytokines were performed on the RNA obtained from the CD103⁺ and CD11b⁺ DC populations. Bar graph shows % relative expression in comparison to un-infected control. (<b>D</b>) WT and C3^−/− mice were infected with flu and 24 hours later the CD103⁺ and CD11b⁺ DCs from the lungs were flow sorted. Q-RT-PCR for the indicated cytokines were performed on the RNA obtained from the CD103⁺ and CD11b⁺ DC populations. Bar graph shows % relative expression in comparison to un-infected control. (<b>E</b>) Bar graphs shows the mean fluorescent intensity of CD86, CD40 and CD80 expression on CD103⁺DC and CD11b⁺ DC subsets in the lungs of naïve and PR8 infected WT or C3^−/− mice. The data are representative of three different experiments with similar results. The values are expressed as mean ± SEM.</p

Langerin-DTR mice show effector T cells response and survival characteristics similar to those of C3^−/− mice upon influenza infection and has defective CD11b⁺ DC migration.

<p>(<b>A</b>) Plots represent <i>ex vivo</i> analysis of CFSE-labeled OT-I CD8⁺ T cells proliferation in the dLN 3 days after infection with PR8 and PR8-OT-I (bottom) in WT and CD103⁺ DCs depleted langerin-DTR mice. (<b>B</b>) Percentage of body weight loss after influenza infection. (<b>C</b>) Survival curve comparing WT, C3^−/− and Langerin –DTR mice during influenza infection (n = 6 per group). (<b>D</b>) Graph shows the relative mRNA expression level of influenza M protein in lung tissues of WT and CD103⁺ DCs depleted langerin-DTR mice infected with 4 PFU of PR8. (<b>E</b>) Graph shows the frequency of IFNγ secreting CD4⁺ T cells in lungs by <i>ex vivo</i> overnight stimulation with MHC-II flu peptide on day 7 post infection. (<b>F</b>) Bar graph shows the absolute numbers of IFNγ secreting CD4⁺ T cells in lungs on day 7 post infection (<b>G</b>) Graph shows the frequency of Flu specific CTL response in lung as measured by Flu _peptide (ASNENMETM (NP _366–374)/H-2D^b tetramer staining on day 7 post infection. (<b>H</b>) Bar graph shows the absolute numbers of flu specific CD8⁺ T cells in lungs by tetramer staining on day 7 post infection. DT treated WT and Langerin-DTR mice were flu infected and the migration of CD103⁺ and CD11b⁺ DCs were evaluated as described before on day 3 post infection. (<b>I</b>) Dot plots show CD103⁺ and CD11b⁺ DCs mDCs in the dLN of DT treated and untreated WT and Langerin-DTR mice. Numbers within the dot plot represent cell number. (<b>J</b>) Total number of CD103⁺(top) and CD11b⁺(bottom) DCs in the dLN of flu infected mice. Results shown are representative of at least three different experiments with similar results. The values are expressed as mean ± SEM.</p