Fig 2: Endogenous GBP Expression and Rate of Cell Death Are Controlled by a Narrow Window of Low-Dose IFN Signaling(A) Macrophages were challenged with L. pneumophila ⊿sdhA, and macrophage death was measured by PI incorporation. Left: representative kinetics of cell death. Right: average cell death at 6 hpi; N = 3.(B) lfnb−/− macrophages were treated for 20 hr with recombinant IFNβ. Cells were then challenged with L. pneumophila ⊿sdhA, and macrophage death was measured by PI incorporation.(C and D) Immunoblot (C) and qRT-PCR (D) after 20 hr of treatment of Ifnb-/ macrophages with low-dose IFNβ. The dotted black line indicates steady-state expression of genes of interest in B6 macrophages.(E and F) B6 and Ifnb−/−BMDMs were stimulated with recombinant IFNβ for 8 hr. Immunoblot shown in (E). Quantification of band intensity over GAPDH shown in (F). The dotted red line indicates the threshold of antibody detection on the LI-COR Biosciences. Representative of 3 experiments.See Figure S3.

Fig 3: Chromosome 3 GBPs Are Involved in Restriction of Legionella Bacterium during Pneumonia(A) Mice were infected with 1 × 107 L. pneumophila bacteria oropharyngeally. Rectal temperature of mice at various time points post-infection. Each dot represents an animal; results are pooled from 2 experiments.(B) Serum CXCL1 was measured by ELISA. Each dot represents an animal; results are pooled from 2 experiments in which tissues were harvested between 4 and 6 hr post-infection.(C) Lung IL-1a and IL-1β were measured by ELISA. Each dot represents an animal; results are pooled from 2 experiments in which tissues were harvested between 12 and 18 hr post-infection.(D and E) Legionella colony-forming units from whole-lung tissue; the geometric mean is shown. Each dot represents an animal; results are pooled from 2 experiments. Animals were infected with (D) WT or ⊿sdhA bacterium and (E) WT bacterium.(F) Whole-lung tissue was extracted from B6, ifnar−/−, and Ifnb−/− animals in the absence of bacterial challenge. Each symbol represents one mouse. mRNA transcripts for indicated Gbps were quantified by qRT-PCR.(G) Body temperature of mice infected with 1 × 107 L. pneumophila bacteria oropharyngeally. Each dot represents an animal; results are pooled from 2 experiments.(H and I) Lung IL-1 a and IL-1 β measured by ELISA. Each dot represents an animal; results are pooled from 2 experiments in which tissues were harvested between 4 and 6 hpi (H) or 12 and 18 hpi (I).(J) Legionella colony-forming units from whole-lung tissue of B6 and ifnar−/− animals.(K) Serum CXCL1 was measured by ELISA. Each dot represents an animal; results are pooled from 2 experiments in which tissues were harvested between 4 and 6 hpi.

Fig 4: RSV-derived neutrophils exacerbate allergic airway inflammation(A) Anti-Gr-1 antibody (Ab) or control IgG was administered every 24 h for 4 consecutive days starting 2 h before RSV infection.(B) Flow cytometric analysis of neutrophils in whole lungs. CD11b+Ly6Ghigh: neutrophil.(C) AHR in anti-Gr-1 Ab- or IgG-administered HDM or HDM/RSV mice.(D) HE staining of lung tissue from mice in the HDM + IgG, HDM + Gr-1 Ab, HDM/RSV + IgG, and HDM/RSV + Gr-1 Ab groups. Scale bars, 200 µm (upper), 50 µm (lower).(E) Number of total cells in the BAL fluid from mice in the HDM + IgG, HDM + Gr-1 Ab, HDM/RSV + IgG, and HDM/RSV + Gr-1 Ab groups.(F) mRNA levels of Mmp12, Ifnb, and Il17a in the whole lungs of mice in the HDM + IgG, HDM + Gr-1 Ab, HDM/RSV + IgG, and HDM/RSV + Gr-1 Ab groups. The data are expressed as the mean ± SEM (n = 5–6). *p < 0.05.

Fig 5: IL-4 receptor a (IL-4Ra)high M2-like alveolar macrophages highly produces MMP-12(A) Immunohistochemical staining of MMP-12 in lung tissue. Scale bars, 200 µm.(B) Flow cytometry of MMP-12 expression by alveolar macrophages (AMac), interstitial macrophages (IMac), and CD45- cells in HDM mice. CD45+Siglec-FhighCD64+: AMac, CD45+Siglec-F+CD64+: IMac.(C) Flow cytometric analysis and median fluorescence intensity of MMP-12 in M2-like alveolar macrophages (arginase-1+ macrophages gated on Siglec-FhighCD64+) in the whole lungs of mice in control (black), RSV (blue), HDM (green), and HDM/RSV groups (red). Gray line denotes isotype control.(D) mRNA levels of Arg1 and Mmp12 in RAW264.7 macrophages after stimulation with IL-4 and IL-13 in vitro.(E) Activation of STAT6 in RAW264.7 macrophages stimulated with IL-4 and IL-13 for 1 h. The ratios of phosphorylated (p)-STAT6 to total (t)-STAT6 are present.(F) Arg1 and Mmp12 mRNA levels in RAW264.7 macrophages treated with AS1517499 (STAT6 inhibitor).(G) mRNA levels of Ifnb in whole lungs from mice in the control, RSV, HDM, and HDM/RSV groups.(H) mRNA levels of Mmp12 and Il4ra in RAW264.7 macrophages after stimulation with IFN-ß in the presence of IL-4 (10 ng/mL) and IL-13 (10 ng/mL). (I) Flow cytometric analysis of IL-4Ra and the median fluorescence intensity of IL-4Ra and MMP-12 in alveolar macrophages in the whole lungs of mice in the control (black), RSV (blue), HDM (green), and HDM/RSV groups (red). Gray line denotes the isotype control. The data are expressed as the mean ± SEM (n = 5–6 [A, B, F, H], n = 3 [C, D, E, G]). *p < 0.05, **p < 0.05, ***p < 0.001, ****p < 0.0001.