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. 2020 Aug 7;12(8):e12034.
doi: 10.15252/emmm.202012034. Epub 2020 Jun 29.

Reprogramming of profibrotic macrophages for treatment of bleomycin-induced pulmonary fibrosis

Fenghua Zhang  1 , Ehab A Ayaub  2 , Bingbing Wang  1 , Estela Puchulu-Campanella  1 , Yen-Hsing Li  1 , Suraj U Hettiarachchi  1 , Spencer D Lindeman  1 , Qian Luo  1 , Sasmita Rout  1 , Madduri Srinivasarao  1 , Abigail Cox  3 , Konstantin Tsoyi  2 , Cheryl Nickerson-Nutter  4 , Ivan O Rosas  2 , Philip S Low  1
Affiliations

Reprogramming of profibrotic macrophages for treatment of bleomycin-induced pulmonary fibrosis

Fenghua Zhang et al. EMBO Mol Med. .

Abstract

Fibrotic diseases cause organ failure that lead to ~45% of all deaths in the United States. Activated macrophages stimulate fibrosis by secreting cytokines that induce fibroblasts to synthesize collagen and extracellular matrix proteins. Although suppression of macrophage-derived cytokine production can halt progression of fibrosis, therapeutic agents that prevent release of these cytokines (e.g., TLR7 agonists) have proven too toxic to administer systemically. Based on the expression of folate receptor β solely on activated myeloid cells, we have created a folate-targeted TLR7 agonist (FA-TLR7-54) that selectively accumulates in profibrotic macrophages and suppresses fibrosis-inducing cytokine production. We demonstrate that FA-TLR7-54 reprograms M2-like fibrosis-inducing macrophages into fibrosis-suppressing macrophages, resulting in dramatic declines in profibrotic cytokine release, hydroxyproline biosynthesis, and collagen deposition, with concomitant increases in alveolar airspaces. Although nontargeted TLR7-54 is lethal at fibrosis-suppressing doses, FA-TLR7-54 halts fibrosis without evidence of toxicity. Taken together, FA-TLR7-54 is shown to constitute a novel and potent approach for treating fibrosis without causing dose-limiting systemic toxicities.

Keywords: bleomycin; folate receptor β; idiopathic pulmonary fibrosis; macrophages; toll-like receptor 7.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure EV1
Figure EV1. FRβ expression in murine (A&B) and human (C&D) lungs
  1. A

    Mice with BLM‐induced experimental fibrosis were stained using a monoclonal antibody to mouse FRβ (F3). Representative FRβ‐positive macrophages are marked with red arrows. H&E and FRβ IHC staining were performed on days 7, 14, and 21 post‐BLM‐induced lung injury. More than 90 × 106 cells were quantified per section using Aperio Image Scope (Leica Biosystems). Scale bars, 100 μm.

  2. B

    Quantification of FRβ staining in sections from panel A.

  3. C, D

    IHC staining of healthy (C) or IPF (D) human lung tissue with a monoclonal antibody to human FRβ (m909). Scale bars, 200 μm.

Source data are available online for this figure.
Figure 1
Figure 1. Nontargeted TLR7 agonist (TLR7‐54) and folate‐targeted TLR7 agonist (FA‐TLR7‐54) downregulate profibrotic macrophage markers
  1. A–C

    Human monocytic (THP‐1) cells were induced to acquire an M2‐like phenotype (see Materials and Methods) and treated with different concentrations of TLR7‐54 or FA‐TLR7‐54 for either 48 h (A–B) or 2 h (C). In the latter case, after the 2 h incubation, culture medium was replaced with drug‐free medium and incubation was continued for 46 h. All treatment groups were then analyzed by qPCR for gene expression, and cell supernatants were analyzed for secreted cytokines by ELISA. (A) Changes in mRNA levels of indicated profibrotic macrophage markers by induced by different concentrations of TLR7‐54 and FA‐TLR7‐54 (n = 3, technical replicates). (B‐C) Changes in CCL18 and IL‐1β in the culture media induced upon treatment with TLR7‐54 and FA‐TLR7‐54 for the treatment regimens (n = 3, technical replicates).

Data information: Mean ± SD. Statistical significance between TLR7‐54‐ or FA‐TLR7‐54‐treated groups versus M2‐untreated group compared using Dunnett's multiple comparison test (# P < 0.05, ## P < 0.01 ### P < 0.001, #### P < 0.0001).
Figure EV2
Figure EV2. TLR7‐54 specifically targets TLR7
NF‐κB‐luc‐transduced or NF‐κB‐luc/hTLR7‐transduced THP‐1 cells were incubated with 100 nM TLR7‐54 or 10 ng/ml TNFα (positive control) for 6 h. NF‐κB‐induced luciferase activity was then quantitated by ONE‐Gloucodep™ Luciferase Assay (n = 3, technical replicates). Mean ± SD. Statistical significance between groups was determined using unpaired two‐tailed t‐test (**P < 0.01, ***P < 0.001).
Figure 2
Figure 2. Both targeted and nontargeted TLR7 agonists reprogram human monocyte‐derived profibrotic macrophages to an anti‐fibrotic phenotype
  1. A–F

    M2‐induced human monocyte‐derived macrophages were treated with 100 nM of the indicated drug either continuously for 48 h, or initially for 2 h in the presence or absence of FA‐glucosamine (competition) followed by 46 h in the absence of drug (2+46 h), as described in Fig 1. mRNA levels of profibrotic markers, Arg1 (A), CD206 (B), and CD163 (C), and protein levels of secreted profibrotic CCL18 (D) and anti‐fibrotic cytokines, CXCL10 (E) and IL‐6 (F) (n = 3, technical replicates), were then determined. Changes in both sets of cytokines were inhibited by blockade of unoccupied folate receptors with excess FA‐glucosamine (2+46 h, competition).

Data information: Mean ± SD. Statistical significance between groups was determined using unpaired two‐tailed t‐test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
Figure 3
Figure 3. Evaluation of folate‐dye conjugate targeting to FRβ+ macrophages in lungs of mice with pulmonary fibrosis
  1. A

    Healthy mice or BLM‐induced mice were tail vein injected on day 10 with 10 nmol OTL38 in the absence or presence of 200‐fold excess FA‐glucosamine to block all folate receptors. After 2 h, mice were euthanized, resected, and imaged for fluorescence intensity (n = 5).

  2. B

    Alternatively, lungs from mice injected with 100 nmol OTL38 in the presence or absence of 200‐fold excess FA‐glucosamine were collagenase digested and stained with 7‐AAD plus antibodies to CD11b and F4/80 prior to FACS analysis (n = 3). Representative plots showing the gating strategy yielding live macrophages (7‐AAD− CD11b+ F4/80+) and OTL38‐positive macrophages are shown.

  3. C

    Percentages of live macrophages in BLM‐induced mice (n = 3).

  4. D

    Percentages of lung macrophages that accumulated OTL38 in vivo (n = 3).

Data information: Mean ± SD. Statistical significance between groups was compared using unpaired two‐tailed t‐test (***P < 0.001, ****P < 0.0001). Source data are available online for this figure.
Figure EV3
Figure EV3. Monocyte‐derived alveolar macrophages constitute the predominant macrophage subpopulation that expresses FRβ following BLM‐induced lung injury
Ten days following intratracheal instillation of bleomycin (0.75 mg/kg), mice were sacrificed and lungs were processed for flow cytometric staining and analysis.
  1. A

    Representative plots showing the gating strategy leading to various macrophage subpopulations.

  2. B

    Representative plots showing FRβ expression on interstitial macrophages (IMs), monocyte‐derived alveolar macrophages (Mono‐AMs), and tissue‐resident alveolar macrophages (TR‐AMs).

  3. C

    Percentages of IMs, Mono‐AMs, and TR‐AMs present in the total macrophage pool (Ly6C‐ gate) (n = 5–7).

  4. D

    Proportion of FRβ‐expressing IMs, FRβ‐expressing Mono‐AMs, and FRβ‐expressing TR‐AMs in the corresponding parent populations (n = 5–7). All samples were derived at the same time and processed in parallel.

Data information: Mean ± SEM. Significance was compared with one‐way ANOVA using Tukey's multiple comparison test (**P < 0.01, ****P < 0.0001).
Figure 4
Figure 4. Effect of a single dose of targeted or nontargeted TLR7‐54 on phenotypic markers of pulmonary fibrosis macrophages in vivo
  1. A–C

    Healthy mice or BLM‐induced mice were injected intravenously on day 10 with either vehicle (3% DMSO in PBS), or 10 nmol TLR7‐54 or FA‐TLR7‐54 dissolved in vehicle, and 1 or 4 h later sacrificed to collect both lungs and bronchoalveolar lavage fluid (BALF). (A) Lungs were digested, and macrophages were isolated by flow cytometry prior to analysis for expression of the indicated mRNAs by qPCR (n = 3). (B) BALF cells were pelleted and similarly analyzed for the indicated mRNAs (n = 3). (C) BALF supernatant was also collected and analyzed by ELISA for IL‐6, IFNα, and TNFα (n = 3).

Data information: Mean ± SD. Statistical significance between groups was compared using unpaired two‐tailed t‐test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Statistical significance between groups at 1 h or 4 h time point was compared using Dunnett's multiple comparison test (# P < 0.05, ## P < 0.01, #### P < 0.0001).
Figure 5
Figure 5. Effect of alternate‐day dosing with FA‐TLR7‐54 on fibrotic markers in BLM‐induced mice
  1. A

    BLM‐induced mice were treated every other day beginning on day 10 with 10 nmol/dose FA‐TLR7‐54 or TLR7‐54.

  2. B

    Survival analysis of the treatment groups (healthy, n = 5; others, n = 10).

  3. C–H

    BALF was collected on day 21 and centrifuged to isolate cell pellets. Cells (primarily macrophages) were analyzed by qPCR for Arg1 (C), MMP9 (D), TIMP3 (E), CD86 (F), and IRAK4 (G) (n = 5). BALF supernatant was analyzed by ELISA for IFNγ (H) (n = 5).

  4. I–K

    Lungs were resected and subjected to hematoxylin–eosin (H&E), trichome (collagen), and α‐SMA IHC staining (scale bars, 200 μm), (J) or hydrolyzed and analyzed for hydroxyproline content as a measure of collagen content (healthy, n = 5; others, n = 10), (K) Ashcroft score quantitation of fibrosis (n = 5).

Data information: Mean ± SD. Statistical significance between groups was compared using unpaired two‐tailed t‐test (*P < 0.05, **P < 0.01, ***P < 0.001). Source data are available online for this figure.
Figure EV4
Figure EV4. Treatment with FA‐TLR7‐54 reduces CD206‐positive macrophages in fibrotic lungs
Sections from the same healthy and fibrotic lungs described in Fig 5 were stained with DAPI (nuclei; blue), anti‐F4/80 (macrophages; red), and anti‐CD206 (M2 macrophage marker; green), and images were obtained with a Leica Versa 8 whole‐slide scanner as described in Materials and Methods (n = 2). Scale bars, 100 μm. Source data are available online for this figure.
Figure 6
Figure 6. Concentration dependence of FA‐TLR7‐54 suppression of BLM‐induced fibrosis
Healthy control (filled circles) or BLM‐induced mice were treated with vehicle (filled squares), 1 nmol (empty circles), 3 nmol (empty squares), or 10 nmol (filled triangles) FA‐TLR7‐54 and then sacrificed on day 21 for analysis.
  1. A

    Analysis of body weight change versus time (n = 10).

  2. B

    Analysis of the number of cells per milliliter of BALF (n = 5).

  3. C

    Quantitation of total hydroxyproline content per right lung (healthy control, n = 5; others, n = 7–9).

  4. D

    H&E staining and trichrome staining of lung tissue (scale bars, 200 μm).

Data information: Mean ± SD. Statistical significance between groups was determined using unpaired two‐tailed t‐test (*P < 0.05, **P < 0.01). Source data are available online for this figure.
Figure 7
Figure 7. Comparison of plasma cytokine levels in healthy mice following treatment with nontargeted versus folate‐targeted TLR7‐54
Healthy mice were tail vein injected with 10 nmol TLR7‐54 (circles) or FA‐TLR7‐54 (squares), and peripheral blood was collected at indicated time points after drug injection.
  1. A–C

    Measurement of plasma IL‐6 (A), IFNα (B), and TNFα (C) (n = 3).

  2. D–F

    Effect of drug concentration on plasma levels of IL‐6 (D), IFNα (E), and TNFα (F) at 1.5 h, 1 h, or 1 h after treatment, respectively (n = 2).

  3. G

    Change in body weight as a measure of systemic toxicity during alternate‐day dosing (n = 2).

Data information: Mean ± SD. Statistical significance between groups was compared using unpaired two‐tailed t‐test (*P < 0.05, **P < 0.01, ***P < 0.001).
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