Fig 1.
Alveolar macrophage deficient CBFβΔLysM mice exhibit enhanced mortality after influenza infection.
WT and CBFβΔLysM mice were infected i.n. with a 0.1LD50 of A/PR/8. a) Survival (left) and weight loss (right) (with surviving CBFβΔLysM mice removed) out to day 20 PI. b) Representative flow plots and total numbers of AlvMΦs (left) and CD11b- AlvMΦs (right) in the BAL fluid at day 0 PI. c) Total protein detected in the BAL at the indicated days PI. d) Total number of AlvMΦs in the BAL and lungs at the indicated days PI. e) Total number of neutrophils in the lung and their f) percent with cell surface CD107a (first panel) and CD11b MFI (second panel) at the indicated days PI. g) Total numbers of lung interstitial macrophages and h) respiratory dendritic cells at day 0 PI. i) Total numbers of inflammatory mononuclear cells and j) percentage that are Ly6C+ in the lungs at the indicated days PI. Data were pooled from a minimum of 3 experiments with a total of 5–12 infected mice per genotype at each indicate time point. Error bars are standard error mean. Statistical analysis is a two- tailed non-paired students t test for single time points or 2-way ANOVA when multiple time points are present. * indicates P< .05, ** for P < .001 and *** for P < .001.
Table 1.
Pulmonary YFP expression in LysM-Cre x ROSA26 reporter mice prior to and during IAV infection.
Fig 2.
Virus clearance and adaptive immune responses in CBFβΔLysM mice.
WT and CBFβΔLysM mice were infected i.n. with a 0.1LD50 dose of A/PR/8. Kinetics of virus replication and clearance as determined a) in BAL fluid by TCID50 units (dashed line is the lower limit of detection) and b) by qRT-PCR for the spliced IAV M2 gene at the indicated days PI. c) Representative levels of IAV specific IgG antibodies in the BAL fluid at day 11 PI. d) Total number of CD4 T cells and e) CD8 T cells positive for the IAV PA antigen tetramer in the lungs at the indicated days PI. Kinetics of BAL f)I FNγ, IL-10 and TNF g) IL-4 and IL-17. For IAV specific antibodies, data is representative of 2 experiments for a total of 5 WT and 6 CBFβΔLysM mice. For all other data, data were pooled from a minimum of 3 experiments with a total of 4–9 mice per genotype at each indicate time point. Error bars are standard error mean. A 2-way ANOVA was used for statistical analysis. * indicates P< .05, ** for P < .001 and *** for P < .001. L.D. is limit of detection, NS is not significant, N.D. is not detectable.
Fig 3.
AlvMΦs regulate the susceptibility of type 1 alveolar epithelial cells to IAV infection.
WT and CBFβΔLysM mice were infected i.n. with a 0.1LD50 dose of a-c) 0.1LD50 of A/PR/8 or d-g) NS1-GFP A/PR/8. a) At the indicated days PI Evans Blue dye leak into the airspace was quantified. b) Representative H&E section images from day 12 PI IAV infected WT and CBFβΔLysM. c) Percent blood oxygen saturation at the indicated days PI. d) Day 7 PI blood oxygen saturation after i.p. injection of CD4 and CD8 depleting antibodies at day 3 PI. Percent of (left) and total numbers of (right) infected e) T1AECs and f) conducting airway epithelial cells at day 4 & 7 PI. g) AlvMΦs were transferred i.n. at day -1PI and (left) the percent of T1AECs that were infected at day 4PI was determined, as well as, (right) CBFβΔLysM survival out to day 20PI (n = 4 CBFβΔLysM mice treated with AlvMΦs). h-j) WT mice were irradiated and given either congenic CD45.1 WT bone marrow, CD45.2 CBFβΔLysM bone marrow, or a mixture of 90% CBFβΔLysM and 10% WT bone marrow (Mix). h) Seven weeks after reconstitution, the origin of the pulmonary myeloid cells in the mixed bone marrow chimera and i) the total number of AlvMΦs in the BAL was quantified. j) The percent of T1AECs that were infected (GFP+) at day 4 and 7 PI. Data were pooled from a minimum of 3 experiments with a total of 4–11 mice per genotype at each indicate time point. Error bars are standard error mean. For statistical analysis a two-tailed non-paired students t test, 1-way ANOVA or 2-way ANOVA was used where appropriate. * indicates P< .05, ** for P < .001 and *** for P < .001; NS is not significant.
Fig 4.
AlvMΦs function early in infection to confer resistance of type 1 alveolar epithelial cell to IAV infection.
a) CD11c-DTr+ and WT control littermates were given 40ng of DTx i.n. at the indicated time points pre or post infection with NS1-GFP A/PR/8. The percentage of infected T1AECs was quantified at day 4PI. b) WT mice were infected i.n. with a 0.1LD50 of A/PR/8 and BAL AlvMΦs were isolated and stained for cell surface IAV HA antigen and intracellular IAV NP antigen at the indicated time points. Data were pooled from a minimum of 3 experiments with a total of 4–6 mice per genotype at each indicate time point. Flow plots are representative from 5 mice. Error bars are standard error mean. For statistical analysis 2-way ANOVA test were used. * indicates P< .05, ** for P < .001 and *** for P < .001.
Fig 5.
AlvMΦs suppress T1AEC expression of arachidonic acid metabolism pathway genes.
a-e) WT and CBFβΔLysM mice were infected i.n. with a 0.1LD50 of A/PR/8. a) IFNα and IFNλ protein in the BAL fluid prior to and during IAV infection. Representative interferon stimulated genes b) detected by qRT-PCR of whole lung homogenates c) and detected by RNAseq on sorted T1AECs at day 2 PI. d) Genes identified as over expressed in T1AECs from CBFβΔLysM mice at day 2PI were grouped by pathway analysis. e) Arachidonic acid metabolism genes differentially expressed as determined by RNAseq. f) Percent of infected (GFP+) LET1 cells at 24hours post infection when cultured with media vehicle, AlvMΦs directly, AlvMΦs in transwell inserts or directly with splenic CD11c+ cells. g) Expression of the corresponding arachidonic acid metabolism pathway genes by qRT-PCR at 8 hours post infection in LET1 cells cultured alone or with AlvMΦs. a) BAL fluid was isolated from 4–10 mice per genotype at each indicate time point. c-d) 2–3 samples of pooled T1AECs from day 2 PI mice were used for RNAseq. For in vitro analyses, data were pooled from or is representative of a minimum of 3 experiments. Error bars are standard error mean. Statistical analysis is a) 2-way ANOVA, c) a linear regression analysis or f) 1-way ANOVA. * indicates P< .05, ** for P < .001 and *** for P < .001. N.S. is not significant.
Fig 6.
Inhibition of the 5-LOX pathway or blockade CysLT1 renders T1AECs resistant to IAV infection.
a) Infectivity of LET1 cells infected with NS1-GFP A/PR/8 in the presence of the specified treatment. b) Lentivirus shRNA knockdown of GGT1 (left panel) and the impact LET1 cell infectivity (right). c) WT and CBFβΔLysM mice were infected i.n. with NS1-GFP A/PR/8 and treated i.n. with 2.5mg/kg of Acivicin or vehicle at 5 and 29hours post infection. Infection of T1AECs (left) and conducting airway epithelial cells (right) was analyzed at day 4 PI. d) Expression of the CysLT1 and CysLT2 receptors in sorted T1AECs from WT and CBFβΔLysM at day 2PI as determined by RNAseq. e) Day 4 T1AEC infectivity in WT and CBFβΔLysM mice that were infected i.n. with NS1-GFP A/PR/8 and. f) CBFβΔLysM mice that were infected i.n. with 0.1LD50 of A/PR/8 and treated i.p. with 10mg/kg of Zafirlukast or vehicle every 24 hours starting at 5 hours PI until day 3 PI. g) Relative fluorescence of pHrodo Red labeled transferrin that has been taken up via the endocytic route by LET1 cells in the presence of different treatments. For in vitro analyses, data were pooled from, or is representative of, a minimum of 3 experiments with each dot representing 2-pooled wells from a 24well plate. c and e) data was pooled 3 experiments for a total of 4–6 mice for each treatment and genotype. Error bars are standard error mean. Statistical analysis is a either a 2-way ANOVA, 1-way ANOVA, or a two-tailed non-paired students t test. * indicates P< .05, ** for P < .001 and *** for P < .001.