Fig 2: Gating scheme explaining the identification of the Ly6G-, F4/80+, CD11b+, CD11c+, CD206- and Ly6G-, F4/80+, CD11b+, CD11c-, CD206+ BMDMs1. gating of live cells, 2. removal of doublets, 3. selection of viable cells, 4. Selection of myeloid cells (CD64+) 5. exclusion of neutrophils, 6. gating of mononuclear macrophages, 7. Gating on CD11c and CD206.

Fig 3: Deletion of Hif2a in LysM+ cells attenuates hypoxia-induced Pdgfb expression, distal muscularization, and PH.(A and B) BALF cells were isolated from WT mice exposed to normoxia or hypoxia (10% FiO2) for up to 21 days. Western blot was used to assess HIF2-a and ß-actin protein levels (A) with densitometry of HIF2-a relative to ß-actin (B). n = 3 mice per time point. (C–I) Hif2afl/fl mice also carrying no Cre or LysM-Cre were exposed to hypoxia for 3 or 21 days. At hypoxia day 3, BALF cells were isolated, with Pdgfb mRNA levels determined by qRT-PCR (C), and vibratome sections of the lung were stained for SMA, CD64, and nuclei (DAPI) with arterioles indicated by “a” and with close-ups of the boxed regions shown below (D). The numbers of macrophages (asterisks) and alveolar myofibroblasts (arrowheads) were quantified per 100 alveoli (D–F). n = 3–5 mice; qRT-PCR was done in triplicate. More than 700 alveoli were quantified per mouse. At hypoxia day 21, vibratome sections with distal arterioles in the L.L1.A1.L1 area were stained for SMA and MECA-32 (G), and RVSP and the Fulton index were measured (H and I). n = 3 mice. One-way ANOVA with Tukey’s multiple-comparison test was used in B, H, and I (* vs. normoxia, P < 0.05), and Student’s t test was used in C, E, and F. Scale bars: 50 µm (D) and 25 µm (G).

Fig 4: Lung macrophages accumulate with hypoxia and are critical for hypoxia-induced pulmonary vascular remodeling and PH.WT mice were exposed to hypoxia (10% FiO2) for up to 21 days or maintained in normoxia as indicated. (A) Vibratome sections including distal arterioles of the L.L1.A1 regions of left lung were stained for markers of SMCs (a–smooth muscle actin [SMA]), macrophages (CD68), and ECs (CD31). The boxed region is shown as close-ups below. n = 6 mice. (B and C) BALF and residual lung were harvested, and single-cell suspensions were subjected to flow cytometric analysis. The percentage of total cells in the given compartment that are CD64+Ly6G– macrophages was determined. n = 3 mice per time point. (D–J) Liposomes containing PBS (vehicle) or clodronate were administered orotracheally at the onset of hypoxia (or normoxia as a control) and two times per week thereafter during the 21-day treatment. (D) Lung vibratome sections of the L.L1.A1.M1 region were stained for SMA, CD64, and CD31 with boxed regions magnified below. n = 4–5 mice. RVSP (E) and Fulton index (F; weight ratio of the right ventricle [RV] to sum of the left ventricle [LV] and septum [S]) are shown. n = 3 mice. (G and H) The percent of CD64+Ly6G– macrophages in total cells of the BALF and residual lung was determined. n = 3 mice. (I and J) Alveolar regions were stained for SMA and nuclei (DAPI), and the number of alveolar myofibroblasts (arrowheads) per 100 alveoli was determined. Arterioles are indicated by “a.” n = 3 mice. More than 500 alveoli were quantified per mouse. One-way ANOVA with Tukey’s multiple-comparison test (*, **, ***, # vs. normoxia, P < 0.05, < 0.01, < 0.001, < 0.0001, respectively) was used in B, C, and E–H, and Student’s t test was used in J. Scale bars: 25 µm.

Fig 5: Characterization of vaccine proteins. (A) Schematic illustration of a dimeric vaccine protein. The targeting units, either an scFv specific for mouse I-Ed (aMHCII), the chemokine MIP1a (Mip1a), or a scFv specific for the hapten NIP (aNip; nontargeted control), are connected to HA via a dimerization unit containing the hinge and CH3 domain from human IgG3. (B) Western blot of supernatants from 293E cells transfected with the indicated vaccine plasmids. Molecular sizes and corresponding structures are indicated. (C) Vaccine proteins in supernatants from transiently transfected 293E cells were detected in an ELISA specific for HA from A/Shanghai/1/2013 (H7N9). (D) Binding of vaccine proteins to B cells (CD19+), macrophages (F4/80+ CD64+), DCs (Lin- CD11chi) divided into conventional DC1 (CD24+) and conventional DC2 (CD11b+), and T cells (CD3+) from BALB/c splenocytes.