Fig 2: Procedure for selecting inhibition antibody for IL-34-limitation treatment(A) Steps for selecting anti-human IL-34 antibody as a proper inhibitor in vivo experiment. The peripheral blood covers onto Histopaque®-1077 at first. After centrifugation, four layers (plasma, PBMCs, Histopaque®-1077, RBCs) are observed. PBMCs, peripheral blood mononuclear cells; RBCs, red blood cells.(B) For collecting the isolated PBMCs, aspirate the plasma layer. CD14+ cells from the PBMCs are filtered out using magnetic field and MACS column.(C) CD14+ cells are cultured with recombinant human IL-34 and anti-human IL-34 antibody for 5 days.(D) Representative images of the cultured cells at day 5. The right panel of each group is a magnified image of the left panel. Scale bar, 200 μm left panel, 50 μm right panel.

Fig 3: Assessment of dose for IL-34 treatment in immunotherapy using a mouse modelThe number of CTLA-4 injections in every group was once per week. Mice were divided into four groups depending upon the number of anti-mouse IL-34 antibody doses; Group 1, only CTLA-4 injection; Group 2, one time per week; Group 3, two times per week; Group 4, three times per week (n = 3 per group). Data represent mean ± SEM. Asterisks indicate significance of Group 4 compared to Groups 1, 2, and 3. ∗p < 0.05, Tukey’s multiple comparison test.

Fig 4: Smooth muscle cell‐specific IL34‐deficiency attenuates pulmonary hypertension in male mice with fibrosing interstitial lung disease. (A) Representative images of scratch assay in RAW264.7 cells with IL34 (100 ng/mL) for 24 h. Scale bar, 100 µm. The upper images (0 h) show the condition immediately before vehicle or IL34 stimulation. The lower images (24 h) show the condition just after vehicle or IL34 stimulation. The bar graph shows the relative migration rate (n = 3 per group). (B) A schematic diagram of experimental design. Male smooth muscle cell‐specific IL34‐deficient mice (IL34flox/flox; Tagln‐Cre+, IL34 CKO) were assigned to AAV‐Stuffer (n = 6) or AAV‐TGFβ (n = 6) group. IL34flox/flox; Tagln‐Cre− littermates (WT) were also assigned to AAV‐Stuffer (n = 6) or AAV‐TGFβ (n = 6) group. IL34 CKO and WT mice were intratracheally administered with AAV‐Stuffer or AAV‐TGFβ (2.5 × 1011 gc/mouse). Three weeks after administration, right heart catheterization was performed to measure right ventricular systolic pressure (RVSP), and the mice were sacrificed. (C) RVSP (n = 6 per group) and the weight ratio of right ventricle to left ventricle plus interventricular septum (RV/[LV + S], n = 6 per group) in mice 3 weeks after AAV‐Stuffer or AAV‐TGFβ administration. (D) Western blot analysis using the indicated antibodies of the lungs in mice 3 weeks after AAV‐Stuffer or AAV‐TGFβ administration. The graph presents the densitometric analysis (n = 6 per group). (E) Representative Elastica Masson's‐stained images of the lung section in mice 3 weeks after AAV‐Stuffer or AAV‐TGFβ administration. Scale bar, 100 µm. The graph presents the results of the Ashcroft score (n = 6 per group). Representative immunohistochemical images of the lung for αSMA in mice 3 weeks after AAV‐Stuffer or AAV‐TGFβ administration. Scale bar, 20 µm. The graph presents quantification of muscularized small pulmonary arteries (n = 6 per group). (F) Representative immunostained images of the lung for CD68, αSMA, and DAPI in mice 3 weeks after AAV‐Stuffer or AAV‐TGFβ administration. Scale bar, 20 µm. The graph presents the number of perivascular CD68‐positive cells in the lung of mice 3 weeks after AAV‐Stuffer or AAV‐TGFβ administration (n = 6 per group). Data are presented as mean ± SEM. Comparisons were performed using the one‐way ANOVA with Tukey's multiple comparisons test. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Fig 5: IL34 is upregulated in pulmonary arteries from patients with fibrosing interstitial lung disease (FILD) and pulmonary vascular remodeling (PVR). (A) Representative immunohistochemical stained images of IL34 and CD68 in lung sections from patients with FILD with (n = 5) and without PVR (n = 5), and control subjects (n = 5). The graph shows quantification of IL34‐positive areas and the number of perivascular CD68‐positive cells. Scale bar, 20 µm. (B) Representative Elastica Masson's‐stained images in lung sections from patients with FILD with and without PVR, and control subjects. Scale bar, 20 µm. Data are presented as mean ± SEM. Comparisons were performed using one‐way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.