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

• DOI: 10.15252/emmm.202012034
• Publication Date: 2020-06-29T08:27:07Z
• Authors: Fenghua Zhang; Ehab A Ayaub; Bingbing Wang; Estela Puchulu‐Ca...; Yen‐Hsing Li; Suraj U Hettiarac...; Spencer D Lindeman; Qian Luo; Sasmita Rout; Madduri Srinivasarao; Abigail Cox; Konstantin Tsoyi; Cheryl Nickerson‐...; Ivan O Rosas; Philip S Low
• Keywords: folate receptor β; 0301 basic medicine; Medicine (General); bleomycin; Macrophages; Pulmonary Fibrosis; Articles; QH426-470; Fibroblasts; idiopathic pulmonary fibrosis; macrophages; 3. Good health; Mice, Inbred C57BL; Bleomycin; Mice; 03 medical and health sciences; R5-920; Macrophages, Alveolar; Genetics; Animals; toll‐like receptor 7

Article29 June 2020Open Access Source DataTransparent process Reprogramming of profibrotic macrophages for treatment bleomycin-induced pulmonary fibrosis Fenghua Zhang orcid.org/0000-0002-7376-6029 Department Chemistry and Institute Drug Discovery, Purdue University, West Lafayette, IN, USA Search more papers by this author Ehab A Ayaub Division Pulmonary Critical Care Medicine, Brigham Women's Hospital, Harvard Medical School, Boston, MA, Bingbing Wang Estela Puchulu-Campanella orcid.org/0000-0002-3951-3925 Yen-Hsing Li Suraj U Hettiarachchi Spencer D Lindeman Qian Luo Sasmita Rout Madduri Srinivasarao Abigail Cox Comparative Pathobiology, College Veterinary Konstantin Tsoyi Cheryl Nickerson-Nutter Three Lakes Partners, Northbrook, IL, Ivan O Rosas Philip S Low Corresponding Author [email protected] orcid.org/0000-0001-9042-5528 Information Zhang1, Ayaub2, Wang1, Puchulu-Campanella1, Li1, Hettiarachchi1, Lindeman1, Luo1, Rout1, Srinivasarao1, Cox3, Tsoyi2, Nickerson-Nutter4, Rosas2 *,1 1Department 2Division 3Department 4Three *Corresponding author. Tel: [phone]; E-mail: EMBO Mol Med (2020)12:e12034https://doi.org/10.15252/emmm.202012034 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Fibrotic diseases cause organ failure that lead ~45% all deaths in United States. Activated stimulate secreting cytokines induce fibroblasts synthesize collagen extracellular matrix proteins. Although suppression macrophage-derived cytokine production can halt progression fibrosis, therapeutic agents prevent release these (e.g., TLR7 agonists) have proven too toxic administer systemically. Based on expression folate receptor β solely activated myeloid cells, we created folate-targeted agonist (FA-TLR7-54) selectively accumulates suppresses fibrosis-inducing production. We demonstrate FA-TLR7-54 reprograms M2-like into fibrosis-suppressing macrophages, resulting dramatic declines release, hydroxyproline biosynthesis, deposition, with concomitant increases alveolar airspaces. nontargeted TLR7-54 is lethal at doses, halts without evidence toxicity. Taken together, shown constitute novel potent approach treating causing dose-limiting systemic toxicities. Synopsis alleviate no detectable Achievement outcome enabled selective reprogramming antifibrotic suppressing consequent fibroblast activation biosynthesis. Folate (FRβ) over-expressed allowing targeting agonists specifically lungs fibrotic mice. Upon intravenous FA-TLR7-54, bleomycin-treated mice reprogram from phenotype, as evidenced changes many molecular markers. Treatment reduces collagen, hydroxyproline, alpha smooth muscle actin (Ashcroft) scores affected while improving available air space morphology. Therapeutic doses toxicities, causes prominent inflammation, animal weight loss, significantly increased mortality treated The paper explained Problem Medium survival idiopathic (IPF) patients following diagnosis only 2–3 years. While current therapies may mitigate symptoms or slow advancement disease, reverse existing nor disease progression. Given high probability mortality, there an urgent need development improved therapies. be achieved vitro toll-like 7 (TLR7) agonists, TLR7-based Results Unique lung has use target attached leading protection against fibrosis. This remarkable benefit anti-fibrotic macrophages. excessive toxicity rapid body premature demonstrated Impact results work inducing therefore constitutes agent worthy further examination nontoxic Introduction diseases, which normal tissue replaced scar ultimately failure, are reported responsible States (Nanchahal Hinz, 2016). Idiopathic interstitial deposition progressive stiffening loss ability mediate gas exchange (Plantier et al, 2018). Due constitutive decline vital capacity, median estimated years (Warheit-Niemi 2019), severity associated morbidities chronic hypoxia, fatigue, joint pain, persistent coughing, mobility) increasing continuously during later stages disease. With ~40,000 new cases IPF diagnosed per year most ending death, public awareness pathology finally (https://ghr.nlm.nih.gov/condition/idiopathic-pulmonary-fibrosis#statistics). Despite seriousness IPF, few options been developed nearly designed retard progression, but none capable curing (Somogyi 2019). Thus, oxygen therapy improve comfort lifestyle, little effect (Graney 2017). two FDA-approved drugs, pirfenidone nintedanib, neither (Dempsey clearly major strategies even Two cell types appear play essential roles (Prasse 2006; Byrne 2015; Pakshir First, derive peripheral blood fibrocytes (Darby 2014), epithelial cells undergo epithelial-to-mesenchymal transition (EMT) (Kage Borok, 2012), endogenous become form myofibroblasts turn secrete other proteins stiffen (Habiel Hogaboam, Second, tissue-resident monocytes, aforementioned via secretion CCL18, TGFβ, IL-1β, and/or PDGF, promoting their Wilson 2010; Braga 2016; Wynn Vannella, Misharin In thought cross-stimulate each other, vicious cycle assures propagation throughout study below, test hypothesis characteristics data demonstrating might disproportionately biased toward anti-inflammatory (M2-like) healing/repair phenotype 2015), screened immune modulators convert state. below successfully significant decrease markers (i.e., collagen), increase hallmarks activity CXCL10, IFNα, IFNγ, CD86) (Diaz Jiménez, 1997; Jiang Lutherer 2011; Murray, 2017), anticipated expansion Analysis macrophage As hypothesized introduction, one strategy halting M1-like phenotype. search surface targeted delivery noted highly expressed both human (Hu 2019) murine bleomycin (BLM)-induced (Nagai 2010), largely absent healthy tissues (Low 2007) (see also Fig EV1). Since Kularatne, 2009) others (Sudimack Lee, 2000) had already methods drugs receptor-expressing elected explore same deliver FRβ+ Click here expand figure. Figure EV1. FRβ (A&B) (C&D) A. Mice BLM-induced experimental were stained using monoclonal antibody mouse (F3). Representative FRβ-positive marked red arrows. H&E IHC staining performed days 7, 14, 21 post-BLM-induced injury. More than 90 × 106 quantified section Aperio Image Scope (Leica Biosystems). Scale bars, 100 μm. B. Quantification sections panel C, D. (C) (D) (m909). 200 online Download figure PowerPoint reports laboratories could induced (Rodell 2018; Mullins toxicities administration precluded such (Savage 1996; Harrison 2004; Geller Biffen 2012). if able any observations exploited decided investigate whether used initial strategy, examined THP-1 (a monocytic line adopt produce upon stimulation IL-4, IL-6 plus IL-13) (Fernando 2014; Genin 2015) reprogrammed vitro. For purpose, IL-13 mRNA levels M2 polarization marker (CD206) classic (CCL18, IL-1β) 2010) incubation presence absence TLR7-54; ref. Shukla see Appendix S2A). 1A, three M2/profibrotic CD206), suggesting indeed promote shift profibrotically polarized less Because studies IL-6-stimulated bone marrow-derived S3, similarly To characterize specificity TLR7-54, next incubated 6 h either THP-1-NF-κB-luc transduced express TLR7. EV2, failed activate NF-κB former consistent Eng 2018), strongly latter. These requires 1. Nontargeted (TLR7-54) downregulate A–C. Human (THP-1) acquire Materials Methods) different concentrations 48 (A–B) 2 (C). latter case, after incubation, culture medium was drug-free continued 46 h. All groups then analyzed qPCR gene expression, supernatants secreted ELISA. (A) Changes indicated (n = 3, technical replicates). (B-C) CCL18 IL-1β media regimens Data information: Mean ± SD. Statistical significance between TLR7-54- FA-TLR7-54-treated versus M2-untreated group compared Dunnett's multiple comparison (#P < 0.05, ##P 0.01 ###P 0.001, ####P 0.0001). EV2. targets TLR7NF-κB-luc-transduced NF-κB-luc/hTLR7-transduced nM 10 ng/ml TNFα (positive control) NF-κB-induced luciferase quantitated ONE-Gloucodep™ Luciferase Assay determined unpaired two-tailed t-test (**P 0.01, ***P 0.001). determine receptor-targeted enable similar FRβ-expressing THP-1-induced S4), prepared folate-conjugated version (FA-TLR7-54; Figs S1 S2A) linker connecting constructed disulfide bond would allow unmodified its internalization reducing environment intracellular endosomes (Yang 2006). 1A (gray bars), yielded those seen impact drug somewhat because enter receptor-mediated endocytosis (Varghese 2014). Finally, ensure above analyses accurately reflect cytokines, polypeptides 1B, found reductions when importantly, exposure limited mimic time rapidly excreted conjugates vivo; superior (Fig 1C), likely it captured internalized receptors, i.e., preventing removal Next, confirm properties physiologically relevant model, isolated monocytes differentiated them before exposing FA-TLR7-54. 2A–D, suppress (Arg1, CD206, CD163) cytokine, CCL18. Moreover, revealed panels 2E 2F, elevate CXCL10 IL-6, shifts sets inhibited blockade unoccupied receptors excess folate-glucosamine (FA-glucosamine, competitive inhibitor FRβ-binding) (Gent 2013), confirming uptake receptor. comprised predominantly M2-polarized contribute prominently suggest 2. Both monocyte-derived A–F. M2-induced h, initially FA-glucosamine (competition) followed (2+46 h), described markers, Arg1 (A), CD206 (B), CD163 (C), protein (E) (F) replicates), determined. competition). (*P **P ****P vivo Encouraged results, undertook folate-linked drugs. After testing induction mice, selected protocol where 0.75 mg/kg BLM instilled C57BL/6 allowed progress through inflammatory prior initiation therapy. inadequacies still exist, methodology Official American Thoracic Society Workshop Report "the best-characterized model preclinical testing" (Jenkins 2017; Tashiro EV1, display indications day post-BLM nascent develops severe 14. Progression continues additional 2–5 begins spontaneously resolve 21. evaluate injected fluorescent dye (OTL38) (Mahalingam 2018) tail veins BLM-treated organs. 3A, OTL38 fluorescence observed kidneys site excretion), tissues. contrast, injecting not kidneys, pronounced accumulation lungs. That mediated simultaneously 200-fold FA-glucosamine. molecule will bind accumulating body. 3. Evaluation folate-dye conjugate Healthy vein nmol block receptors. euthanized, resected, imaged intensity 5). Alternatively, collagenase digested 7-AAD antibodies CD11b F4/80 FACS analysis 3). plots showing gating yielding live (7-AAD− CD11b+ F4/80+) OTL38-positive shown. C. Percentages accumulated (***P 3 [emmm202012034-sup-0003-SDataFig3.pdf] what type capturing animals flow cytometry cell-specific uptake. 3B–D, showed percentages displayed OTL38. ~20% OTL38-injected retention. observation concurrent injection blocked considerable retention, required subpopulations subsequently segregated (Mono-AMS), considerably small population barely EV3). localization Mono-AMs informative, (Misharin Significantly, conclusion supported essentially nondetectable 3A) dramatically B). EV1D), individuals EV1C). EV3. Monocyte-derived predominant subpopulation expresses injuryTen intratracheal instillation (0.75 mg/kg), sacrificed processed cytometric