Enhancing autophagy in CD11c+ antigen-presenting cells as a therapeutic strategy for acute respiratory distress syndrome

Abstract

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are severe clinical disorders that mainly develop from viral respiratory infections, sepsis, and chest injury. Antigen-presenting cells play a pivotal role in propagating uncontrolled inflammation and injury through the excess secretion of pro-inflammatory cytokines and recruitment of immune cells. Autophagy, a homeostatic process that involves the degradation of cellular components, is involved in many processes including lung inflammation. Here, we use a polyinosinic-polycytidylic acid (poly(I:C))-induced lung injury mouse model to mimic viral-induced ALI/ARDS and show that disruption of autophagy in macrophages exacerbates lung inflammation and injury, whereas autophagy induction attenuates this process. Therefore, induction of autophagy in macrophages can be a promising therapeutic strategy in ALI/ARDS.

Keywords: CD11c; CP: Immunology; acute respiratory distress syndrome (ARDS); antigen-presenting cells; autophagy; lung inflammation; lung injury; poly(I:C).

Figure 1.. Poly(I:C) stimulation induces lung inflammation and injury

(A) C57BL/6 mice were intranasally (i.n.) challenged with 40 μg poly(I:C) for 3 consecutive days, and on the fourth day, AHR was measured and BALF and lungs collected for analysis. (B and C) Lung resistance (B) and dynamic compliance (C) were measured in tracheostomized ventilated mice (n = 5–6 mice per group). (D) Hematoxylin and eosin (H&E) staining of lung sections (scale bar: 100 μm). (E) Average airway thickness. (F andG) Total number of immune cells in the (F) BALF and in the (G) lungs. (H) Levels of pro-inflammatory cytokines and chemokines in the BALF quantified by using BioLegend LEGENDplex bead-based immunoassay. (I) FITC-dextran fluorescence intensity. (J) Protein concentration in the BALF. Data are represented as means ± SEM (unpaired Student’s t test). ns, not significant, *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S1.

Figure 2.. Poly(I:C) stimulation downregulates autophagy in CD11c⁺ APCs

(A) Representative western blot of LC3-I and LC3-II and p62 levels in CD11c⁺ and CD11c⁻ cells sorted from the lungs of C57BL/6 mice (n = 7 mice per group) treated with 40 μg poly(I:C) or PBS. (B) Visual representation of the densiometric quantification of LC3-II/LC3-I ratio. (C) Visual representation of the densiometric quantification of p62/β-actin ratio in CD11c⁺ cells only. Data are represented as means ± SEM (unpaired Student’s t test). ns, not significant, *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. See also Figure S2.

Figure 3.. Lack of autophagy in pulmonary CD11c⁺ APCs exacerbates lung inflammation and injury

Atg5^flox/flox and Atg5^KO mice were i.n. challenged with 40 μg poly(I:C) or PBS for 3 consecutive days, and on the fourth day, AHR was measured and BALF and lungs collected for analysis (n = 3–4 mice per group). (A and B) Lung resistance (A) and dynamic compliance (B) were measured in tracheostomized ventilated mice (n = 3 mice per group). (C) H&E staining of lung sections (scale bar: 100 μm). (D) Average airway thickness. (E and F) Total number of immune cells in the (E) BALF and in the (F) lungs. (G) Levels of pro-inflammatory cytokines and chemokines in the BALF quantified by using BioLegend LEGENDplex bead-base immunoassay. (H) FITC-dextran fluorescence intensity. (I) Protein concentration in the BALF. Data are represented as means ± SEM (two-way ANOVA). ns, not significant, *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S3.

Figure 4.. Autophagy induction in pulmonary CD11c⁺ APCs attenuates lung inflammation and prevents lung injury

TFEB^flox/flox and Tfeb^TG mice were i.n. challenged with 40 μg poly(I:C) or PBS for 3 consecutive days, and on the fourth day, AHR was measured and BALF and lungs collected for analysis. (A and B) Lung resistance (A) and dynamic compliance (B) were measured in tracheostomized ventilated mice (n = 3–6 mice per group). (C) H&E staining of lung sections (scale bar: 100 μm). (D) Average airway thickness. (E and F) Total number of immune cells in the (E) BALF and in the (F) lungs. (G) Levels of pro-inflammatory cytokines and chemokines in the BALF quantified by using BioLegend LEGENDplex bead-base immunoassay. (H) FITC-dextran fluorescence intensity. (I) Protein concentration in the BALF. Data are represented as means ± SEM (two-way ANOVA). ns, not significant, *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S4.

Figure 5.. A lack of autophagy significantly impacts gene expression of pro-inflammatory cytokines

(A) Volcano plot of differentially expressed genes in poly(I:C)-treated Atg5^KO vs. Atg5^flox/flox mice. (B) Volcano plot of differentially expressed genes in poly(I:C)-treated Tfeb^TG vs. Tfeb^flox/flox mice. (C) Heatmap representation of cytokines significantly modulated in poly(I:C)-treated Atg5^KO mice. (D) Heatmap representation of cytokines significantly modulated in poly(I:C)-treated Tfeb^TG mice. n = 3 mice per group. (E and F) The regulation of genes encoding for relevant M1/M2 markers represented as fold change in poly(I:C)-treated Atg5^KO vs. Atg5^flox/flox control mice and in (F) Tfeb^TG vs. Tfeb^flox/flox control mice. (G) The number of M1 and M2 macrophages in the lungs of Atg5^flox/flox and Atg5^KO mice challenged with poly(I:C). (H–K) Frequency of CD11c⁺ CD11b⁺ CD64⁺ macrophages expressing (H) CD86, (I) CD206, (J) CD163, and (K) CD36. Data are represented as means ± SEM (two-way ANOVA). ns, not significant, *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S5.

Figure 6.. Use of an autophagy inducer, α4-vFLIP, ameliorates lung inflammation and injury

(A) C57BL/6 mice were i.n. challenged with 40 μg poly(I:C) for 3 consecutive days in the morning followed by intraperitoneal (i.p.) injection of vFLIP (300 mg per mouse) or control peptide, TAT, in the evening. On the fourth day, AHR was measured and BALF and lungs collected for analysis. (B) Western blot analysis of LC3-I and LC3-II and p62 levels in CD11c⁺ and CD11c⁻ cells sorted from the lungs of C57BL/6 mice treated with PBS, poly(I:C) only, poly(I:C) + α4-vFLIP, or poly(I:C) + TAT. (C and D) Lung resistance (C) and dynamic compliance (D) were measured in tracheostomized ventilated mice (n = 3–6 mice per group). (E) H&E staining of lung sections (scale bar: 100 μm). (F) Average airway thickness. (G and H) Total number of immune cells in the (G) BALF and in the (H) lungs. (I) Levels of pro-inflammatory cytokines and chemokines in the BALF quantified by using BioLegend LEGENDplex bead-base immunoassay. (J) FITC-dextran fluorescence intensity. (K) Protein concentration in the BALF. Data are represented as means ± SEM (one-way ANOVA). ns, not significant, *p < 0.05; **p < 0.01; ***p < 0.001. See also Figure S6.