Immunoregulatory Macrophages Modify Local Pulmonary Immunity and Ameliorate Hypoxic Pulmonary Hypertension

Abstract

Background: Macrophages play a significant role in the onset and progression of vascular disease in pulmonary hypertension, and cell-based immunotherapies aimed at treating vascular remodeling are lacking. We aimed to evaluate the effect of pulmonary administration of macrophages modified to have an anti-inflammatory/proresolving phenotype in attenuating early pulmonary inflammation and progression of experimentally induced pulmonary hypertension.

Methods: Mouse bone marrow-derived macrophages were polarized in vitro to a regulatory (M2_reg) phenotype. M2_reg profile and anti-inflammatory capacity were assessed in vitro upon lipopolysaccharide/IFNγ (interferon-γ) restimulation, before their administration to 8- to 12-week-old mice. M2_reg protective effect was evaluated at early (2-4 days) and late (4 weeks) time points during hypoxia (8.5% O₂) exposure. Levels of inflammatory markers were quantified in alveolar macrophages and whole lung, while pulmonary hypertension development was ascertained by right ventricular systolic pressure (RVSP) and right ventricular hypertrophy measurements. Bronchoalveolar lavage from M2_reg-transplanted hypoxic mice was collected and its inflammatory potential evaluated on naive bone marrow-derived macrophages.

Results: M2_reg macrophages expressing Tgfβ, Il10, and Cd206 demonstrated a stable anti-inflammatory phenotype in vitro, by downregulating the induction of proinflammatory cytokines and surface molecules (Cd86, Il6, and Tnfα) upon a subsequent proinflammatory stimulus. A single dose of M2_regs attenuated hypoxic monocytic recruitment and perivascular inflammation. Early hypoxic lung and alveolar macrophage inflammation leading to pulmonary hypertension development was significantly reduced, and, importantly, M2_regs attenuated right ventricular hypertrophy, right ventricular systolic pressure, and vascular remodeling at 4 weeks post-treatment.

Conclusions: Adoptive transfer of M2_regs halts the recruitment of monocytes and modifies the hypoxic lung microenvironment, potentially changing the immunoreactivity of recruited macrophages and restoring normal immune functionality of the lung. These findings provide new mechanistic insights into the diverse role of macrophage phenotype on lung vascular homeostasis that can be explored as novel therapeutic targets.

Keywords: adoptive transfer; hypoxia; macrophage activation; pulmonary arterial hypertension; vascular remodeling.

Figure 1.. Established M2regs show reduced pro-inflammatory phenotype after restimulation with LPS and IFNγ.

(A) M2_regs (Il4, Il10, TGFβ) showed induced mRNA expression of alternatively (M2) associated markers Tgfβ, Il10, Arg1, Cd206 (Mrc1) and Pdl2 and reduced mRNA levels of Cd86, a proinflammatory surface marker associated with macrophage activation and M1 activation compared to M0 (untreated) cells. (B) Flow cytometry was used to assess CD206, PDL2 and CD86 surface expression (median fluorescence intensity, MFI) in M2_reg and M1 (LPS/IFNγ) polarized BMDMs (used here as controls)_. Representative histograms for CD86, CD206 and PDL2 MFIs are shown. (C) Restimulated M2_regs retain part of their M2-associated phenotype (increased Il10 mRNA) and reduced expression of M1/classical markers (Cd86, Il6 and Tnf⍺) in response to LPSγ/IFNγ, compared with non-treated (N-LPS/IFNγ) cells. (D) CD206, PDL2 and CD86 surface expression (MFI) in restimulated M2_regs-LPS/IFNγ assessed by flow cytometry. Statistical significance was calculated using Mann-Whitney test (A, n=3–6; data represents mean ± SD) and one-way ANOVA with Tukey’s multiple comparison test (B, C and D, n=3; data represents mean ± SD).

Figure 2.. Adoptive transfer of M2_regs reduces vascular injury and PH progression.

(A) Male mice received 0.6–1 × 10⁶ M2_regs or media (vehicle) by intrapulmonary administration followed by exposure to normal air (Nrmx) or hypoxia (8.5% oxygen, Hpx) for 4 weeks. (B) Fulton’s Index (RV/LV+S), right ventricular systolic pressure (RSVP) and Medial Thickness Index determined after long-term exposure. Line in the box plot (interquartile range) represents the median, and whiskers the min to max range of the data. (C) Representative images of lung vascular remodeling in small (50–150μm) pulmonary arterioles (PA) of vehicle (veh) and M2_regs-treated Nrmx or Hpx mice, as assessed by α-smooth muscle (αSMA) staining; scale bar = 50μm. (D) Peripheral muscularization of PAs in the lungs of Hpx mice and effect of M2_reg administration, analyzed by von Willebrand factor (vWf ) and αSMA double immunofluorescence; arrows indicate colocalization in small (20–100μm) PAs; scale bar = 50μm. (E) Quantification of muscularization of non-muscular (NM), partially muscular (PM), and fully muscular (FM) PAs is shown in D. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test (B, n=7–18 and E, n=4–6; data represents mean ± SEM).

Figure 3.. Chronic inflammation partially reverses the phenotype of M2_regs in vivo.

(A) CD86⁺, CD206⁺ and PDL2⁺ frequencies showing efficient M2_reg polarization before their transfer to mice and exposure to chronic hypoxia. Frequencies of LPS/IFNγ stimulated cells (M1) are shown for comparison. (B) Male mice received 0.6–1 × 10⁶ DiD-M2_regs or DiD-M0s_s by intrapulmonary administration followed by exposure to normal air (Nrmx) or hypoxia (8.5% oxygen, Hpx) for 2 or 4 weeks. Flow cytometry analysis of CD45⁺, DiD⁺, and CD11c⁺SiglecF⁺ cells recovered from cell suspensions of a representative mouse lung after adoptive transfer. (B) Quantification of CD86⁺, CD206⁺ and PDL2⁺ cell frequencies of CD45 DiD⁺ retrieved cells from M0 and M2_reg-treated mice after exposure to Nrmx or Hpx for 2 or 4 weeks. Statistical significance was calculated using one-way ANOVA with Tukey’s multiple comparison test (A, n=3–4; data represents mean ± SD) and two-way ANOVA with Tukey’s multiple comparison test (B, n=4–8; data represents mean ± SEM).

Figure 4.. M2_reg adoptive transfer decreases early Hpx-induced cytokine expression in lung and BAL.

(A) Male mice received 0.6–1 × 10⁶ M2_regs or media (vehicle) by intrapulmonary administration followed by exposure to normal air (Nrmx) or hypoxia (8.5% oxygen, Hpx) for 3 days. (B) Whole lung Il6, Ccl17, Fizz1 and Arg1 mRNA levels were analyzed by qPCR at this time point. Expression levels are shown relative to vehicle (veh)-Nrx mice (C) Representative images showing Fizz1 immunofluorescence (FITC) in macrophages and bronchial epithelial cells corresponding to lung sections of veh and M2_regs-treated Nrmx or Hpx mice; cell nuclei are stained with DAPI; arrows show Fizz1-stained macrophages in airspaces; scale bar = 50μm. (D) Quantification of Il6 and Ccl17 mRNA and Fizz1protein levels determined as mean fluorescence intensity (MFI) normalized for cell number (DAPI stain), in bronchoalveolar (BAL) cell pellet from mice housed in Nrmx or Hpx for 3 days. (E) Representative BAL cytospins showing Fizz1 immunofluorescence staining in veh- and M2_regs-treated Nrmx or Hpx mice; scale bar = 12.5μm. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test (B and D, n=3–5; data represents mean ± SEM).

Figure 5.. M2_regs attenuate monocytic recruitment and perivascular macrophage accumulation.

(A) Representative flow cytometry contour plots showing the gating strategy used to identify monocytes and macrophages in lung mononuclear cell suspensions from 2-day housed normoxia (Nrmx) or hypoxia (Hpx) male mice having received media (vehicle) or M2_regs. (B) Histograms showing frequencies (as percentages of the parent cell) of alveolar (CD11b^low, MHCII⁺, CD64⁺, CD11c⁺) and interstitial (CD11b⁺, MHCII⁺, CD64⁺, CDl1c^low) macrophages, and non-classical (CD11b⁺, MHCII⁻, CD64^+/−, Ly6c^low) and classical (CD11b⁺, MHCII⁻, CD64^+/−, Ly6c^high) monocytes in each experimental group. (C, upper panel) Representative images of lung complement C5a receptor 1 (C5ar1) perivascular immunostaining in vehicle and M2_reg-treated mice after 3 days of Hpx exposure. Arrows show increased C5ar1staining in areas surrounding airway (AW)-associated pulmonary arteries (PA) from vehicle-treated tissue sections, compared to M2_reg-treated lungs; Scale bar = 50μm. (C, lower panel) Scatter plot showing quantification of C5ar1 immunostaining as percentage of C5ar1 immunofluorescence per high power field (hpf) area. (D, upper panel) Immunolocalization of CD68⁺ macrophages are shown in PAs (arrows) of vehicle treated murine lungs exposed to 3-day Hpx and absent in lung sections from M2_reg-treated counterparts. Scale bar = 100μm. (D, lower panel) Scatter plot showing quantification of CD68 immunostaining as percentage of CD68 immunofluorescence per high power field (hpf) area. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test (B, n=4; C and D, n=3–4; data represents mean ± SEM).

Figure 6.. Delayed M2_reg administration fails to reduce perivascular inflammation and PH development.

(A) Scheme showing delayed M2_reg administration and analysis of PH progression in 4-week housed normoxic (Nrmx) or hypoxic (Hpx) male mice. (B) Hpx-induced lung tissue CD68⁺ macrophages accumulation was ameliorated in Hpx mice that received M2_regs at the beginning (day 0, d0) of the 4-week exposure period but not in mice treated with M2_regs at day 14 (d14) after the initiation of Hpx. Arrowheads showing perivascular CD68 immunofluorescence (red); cell nuclei are stained with DAPI. Scale bar = 50μm. (C) Scatter plot showing quantification of CD68 immunostaining as percentage of CD68 immunofluorescence per high power field (hpf) area. (D) Fulton’s Index (RV/LV+S) determined in mice exposed to 4 weeks of Nrmx or Hpx that were left untreated (vehicle) or treated with early (d0) or late (d14) M2_regs. Line in the box plot (interquartile range) represents the median and whiskers the min to max range of the data. Statistical significance was calculated using two-way ANOVA with Tukey’s multiple comparison test (C, n=4 and D, n=4–6; data represents mean ± SEM).

Figure 7.. M2_regs modulate the lung milieu to suppress pro-inflammatory signals.

(A) Scheme showing treatment of bone marrow derived macrophages (BMDMs) with bronchoalveolar (BAL) supernatants obtained from male mice receiving media (vehicle) or M2_regs and housed in Nrmx or Hpx for three consecutive days. (B) BMDM expression level of cytokines and inflammatory mediators, determined by qPCR, showing normalization (Il6, Ccl17 and Fizz1) or augmentation (Chi3l3) by BAL supernatant derived from Hpx M2_regs-treated mice, compared to supernatants from vehicle-treated counterparts. Expression levels are shown relative to (vehicle) veh-Nrx BAL-treated BMDMs. (C) Scheme showing treatment of reporter BMDMs with BAL supernatants obtained from wild type (Hmox1^+/+) or Hmox1 deficient (Hmox1^−/−) mice treated with vehicle or M2_regs. (D) Hmox1^−/− BAL supernatants induced the expression of Il6 and Ccl17 mRNA in BMDMs compared to Hmox1^+/+ BAL, with negligible effect on Fizz1 and Arg1 gene expression. (E) Il6 and Ccl17 mRNA expression in cell pellets from BAL fluid from the same experimental groups. Statistical significance was calculated using Kruskal-Wallis with Dunn’s multiple comparison test (B, D and E, n=3–6; data represents mean ± SEM).