Genetically engineered distal airway stem cell transplantation protects mice from pulmonary infection
2019; Springer Nature; Volume: 12; Issue: 1 Linguagem: Inglês
10.15252/emmm.201810233
ISSN1757-4684
AutoresYueqing Zhou, Yun Shi, Ling Yang, Yufen Sun, Yufei Han, Zixian Zhao, Yu‐jia Wang, Ying Liu, Yu Ma, Ting Zhang, Tao Ren, Tina P. Dale, Nicholas R. Forsyth, Faguang Jin, Jieming Qu, Wei Zuo, Jin‐Fu Xu,
Tópico(s)Respiratory Support and Mechanisms
ResumoArticle29 November 2019Open Access Source DataTransparent process Genetically engineered distal airway stem cell transplantation protects mice from pulmonary infection Yue-qing Zhou Yue-qing Zhou Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yun Shi Yun Shi Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Department of Respiratory and Critical Care Medicine, Tangdu Hospital, Fourth Military Medical University of PLA, Xi'an, China Search for more papers by this author Ling Yang Ling Yang Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yu-fen Sun Yu-fen Sun Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yu-fei Han Yu-fei Han Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Zi-xian Zhao Zi-xian Zhao Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yu-jia Wang Yu-jia Wang Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Ying Liu Ying Liu Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yu Ma Yu Ma Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Regend Therapeutics Co. Ltd, Zhejiang, China Search for more papers by this author Ting Zhang Ting Zhang Regend Therapeutics Co. Ltd, Zhejiang, China Search for more papers by this author Tao Ren Tao Ren Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Tina P Dale Tina P Dale Guy Hilton Research Center, School of Pharmacy and Bioengineering, Keele University, Staffordshire, UK Search for more papers by this author Nicholas R Forsyth Nicholas R Forsyth orcid.org/0000-0001-5156-4824 Guy Hilton Research Center, School of Pharmacy and Bioengineering, Keele University, Staffordshire, UK Search for more papers by this author Fa-guang Jin Fa-guang Jin Department of Respiratory and Critical Care Medicine, Tangdu Hospital, Fourth Military Medical University of PLA, Xi'an, China Search for more papers by this author Jie-ming Qu Corresponding Author Jie-ming Qu [email protected] orcid.org/0000-0003-4517-9473 Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Respiratory Diseases, Shanghai Jiaotong University School of Medicine, Shanghai, China Search for more papers by this author Wei Zuo Corresponding Author Wei Zuo [email protected] orcid.org/0000-0002-4460-0337 Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Regend Therapeutics Co. Ltd, Zhejiang, China Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China Ningxia Medical University, Yinchuan, China Search for more papers by this author Jin-fu Xu Corresponding Author Jin-fu Xu [email protected] orcid.org/0000-0002-8039-8973 Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yue-qing Zhou Yue-qing Zhou Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yun Shi Yun Shi Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Department of Respiratory and Critical Care Medicine, Tangdu Hospital, Fourth Military Medical University of PLA, Xi'an, China Search for more papers by this author Ling Yang Ling Yang Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yu-fen Sun Yu-fen Sun Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yu-fei Han Yu-fei Han Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Zi-xian Zhao Zi-xian Zhao Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yu-jia Wang Yu-jia Wang Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Ying Liu Ying Liu Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Yu Ma Yu Ma Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Regend Therapeutics Co. Ltd, Zhejiang, China Search for more papers by this author Ting Zhang Ting Zhang Regend Therapeutics Co. Ltd, Zhejiang, China Search for more papers by this author Tao Ren Tao Ren Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Tina P Dale Tina P Dale Guy Hilton Research Center, School of Pharmacy and Bioengineering, Keele University, Staffordshire, UK Search for more papers by this author Nicholas R Forsyth Nicholas R Forsyth orcid.org/0000-0001-5156-4824 Guy Hilton Research Center, School of Pharmacy and Bioengineering, Keele University, Staffordshire, UK Search for more papers by this author Fa-guang Jin Fa-guang Jin Department of Respiratory and Critical Care Medicine, Tangdu Hospital, Fourth Military Medical University of PLA, Xi'an, China Search for more papers by this author Jie-ming Qu Corresponding Author Jie-ming Qu [email protected] orcid.org/0000-0003-4517-9473 Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China Institute of Respiratory Diseases, Shanghai Jiaotong University School of Medicine, Shanghai, China Search for more papers by this author Wei Zuo Corresponding Author Wei Zuo [email protected] orcid.org/0000-0002-4460-0337 Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China Regend Therapeutics Co. Ltd, Zhejiang, China Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China Ningxia Medical University, Yinchuan, China Search for more papers by this author Jin-fu Xu Corresponding Author Jin-fu Xu [email protected] orcid.org/0000-0002-8039-8973 Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China Search for more papers by this author Author Information Yue-qing Zhou1,‡, Yun Shi2,3,‡, Ling Yang1,‡, Yu-fen Sun1, Yu-fei Han1, Zi-xian Zhao1, Yu-jia Wang1, Ying Liu2, Yu Ma2,4, Ting Zhang4, Tao Ren2, Tina P Dale5, Nicholas R Forsyth5, Fa-guang Jin3, Jie-ming Qu *,6,7, Wei Zuo *,1,2,4,8,9 and Jin-fu Xu *,1 1Department of Respiratory and Critical Care Medicine, Clinical Translation Research Center, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, China 2Shanghai East Hospital, Tongji University School of Medicine, Shanghai, China 3Department of Respiratory and Critical Care Medicine, Tangdu Hospital, Fourth Military Medical University of PLA, Xi'an, China 4Regend Therapeutics Co. Ltd, Zhejiang, China 5Guy Hilton Research Center, School of Pharmacy and Bioengineering, Keele University, Staffordshire, UK 6Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China 7Institute of Respiratory Diseases, Shanghai Jiaotong University School of Medicine, Shanghai, China 8Guangzhou Institute of Respiratory Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China 9Ningxia Medical University, Yinchuan, China ‡These authors contribute equally to this work *Corresponding author. Tel: +8621 64370045-665852; E-mail: [email protected] *Corresponding author. Tel: +8621 65985082; E-mail: [email protected] *Corresponding author. Tel: +8621 65111298; E-mail: [email protected] EMBO Mol Med (2020)12:e10233https://doi.org/10.15252/emmm.201810233 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Severe pulmonary infection is a major threat to human health accompanied by substantial medical costs, prolonged inpatient requirements, and high mortality rates. New antimicrobial therapeutic strategies are urgently required to address the emergence of antibiotic resistance and persistent bacterial infections. In this study, we show that the constitutive expression of a native antimicrobial peptide LL-37 in transgenic mice aids in clearing Pseudomonas aeruginosa (PAO1), a major pathogen of clinical pulmonary infection. Orthotopic transplantation of adult mouse distal airway stem cells (DASCs), genetically engineered to express LL-37, into injured mouse lung foci enabled large-scale incorporation of cells and long-term release of the host defense peptide, protecting the mice from bacterial pneumonia and hypoxemia. Further, correlates of DASCs in adult humans were isolated, expanded, and genetically engineered to demonstrate successful construction of an anti-infective artificial lung. Together, our stem cell-based gene delivery therapeutic platform proposes a new strategy for addressing recurrent pulmonary infections with future translational opportunities. Synopsis Using adult distal airway stem cells (DASCs) as a natural vehicle to deliver the antimicrobial peptide LL-37 into damaged lung, this study reports a technology to restore epithelium barrier and pulmonary innate immunity, which could have applications for the treatment of infectious lung diseases. Bacterial clearance ability was greater in the lungs of LL-37 transgenic mice compared to those of wild-type mice. Genetically engineered DASCs expressing LL-37 peptide displayed normal stem cell properties and enhanced anti-microbial functions. Orthotopic transplantation of LL-37-expressing DASCs was successfully used for lung regeneration and enhanced host defense capability. Bioengineered artificial lungs were protected from bacterial infection by LL-37-expressing human DASC engraftment. Introduction Respiratory infection is amongst the leading causes of human death. These include lower respiratory tract infections by Gram-negative pathogens such as Pseudomonas aeruginosa which constitute the main reason for hospital-associated infections and are associated with high morbidity and mortality rates in hospitals. These continue to pose a therapeutic challenge due to the rapid development of resistance to standard antibiotic regimes during treatments. In the case of the opportunistic pathogen P. aeruginosa, broad antibiotic resistance has been observed, and 18–25% of clinical isolates demonstrated multidrug resistance (Souli et al, 2008). Unfortunately, relapse or re-infection is a frequent occurrence in patients with pulmonary infections. To this end, the development of new therapeutic strategies is needed to combat pulmonary bacterial infections. Antimicrobial peptides (AMPs) are substances produced by animals, bacteria, and plants that are regarded as naturally occurring broad-spectrum antibiotics. As an essential part of innate immunity, AMPs possess the ability to kill invading pathogens including bacteria, fungi, virus, and parasites (Zasloff, 2002; Fjell et al, 2011; Kovach et al, 2012). The peptide hCAP-18/LL-37 (LL-37) is the only human cathelicidin (CAMP) identified so far. The LL-37 peptide is cleaved from hCAP-18 by proteinase, which enables it to have a broad range of bactericidal activity against both Gram-negative and Gram-positive organisms, including P. aeruginosa. LL-37 has roles in multiple host defense processes by directly targeting microbial biofilm and activating innate immune cell function (Scott et al, 2002; Overhage et al, 2008; Yu et al, 2010; Bandurska et al, 2015). In the inflamed human lung, LL-37 was reported to be highly expressed and had potent anti-infective and anti-inflammatory potential (Nijnik & Hancock, 2009; Currie et al, 2016), suggesting that the LL-37 peptide can be used as an alternative medicine to conventional antibiotics for treating pulmonary infection. However, the degradation of the LL-37 peptide in vivo due to bacterial proteases may limit its clinical application (Vandamme et al, 2012). Furthermore, as a peptide with potential off-target toxicity, LL-37 requires topical delivery to infected foci, rather than systemically, with local concentration control (Johansson et al, 1998; Heilborn et al, 2005). Therefore, the development of a system to achieve local, long-term LL-37 release may help combat pulmonary infection. Viral systems have been utilized for exogenous gene expression; however, clinical applicability for this approach has multiple drawbacks including virus-induced tissue toxicity and inflammation post-virus infection, oncogenic risks and genotoxicity, and the off-target effects of the viral vector (i.e., liver). Here, we introduce a novel platform combining conventional viral-based gene engineering with intrapulmonary stem cell transplantation. We and others previously demonstrated that distal airway stem cells (DASCs) derived from p63+ lineage negative progenitors are the major regenerative cells following large-scale lung damage (Kumar et al, 2011; Vaughan et al, 2015; Zuo et al, 2015; Yang et al, 2018). DASCs have the capacity to rapidly restore epithelial barriers in vivo and differentiate into functional alveolar cells with accompanied Notch signaling (Vaughan et al, 2015; Xi et al, 2017). The feasibility for large-scale in vitro expansion and remarkable lung engraftment after transplantation (Zuo et al, 2015; Imai-Matsushima et al, 2018) make DASCs ideal candidates for cell therapy and gene engineering. In the current study, via a novel transgenic rodent model, we show that constitutive expression of human LL-37 peptide, in the lung, enhances the pulmonary host defense system. Introduction of LL-37 into mouse DASCs enable delivery of the antimicrobial peptide specifically into injured foci without distribution to other healthy lung regions, and endow the lung with enhanced bacterial clearance ability. An anti-infective human bioengineered lung is also constructed by engrafting LL-37-overexpressing human DASCs into decellularized lung scaffolds. Taken together, we demonstrate that genetically engineered DASCs can efficiently and specifically deliver the antimicrobial peptide LL-37 in vivo and protect the lung against pathogen infection. Results Constitutive LL-37 expression clears pulmonary infection in vivo Clinical observations have indicated the elevation of LL-37 expression in lung disease exacerbation (Schaller-Bals et al, 2002; Pouwels et al, 2015), suggesting that pulmonary infection and inflammation could activate the LL-37-based protective mechanism. Here, to understand whether the elevated expression of LL-37 in vivo is beneficial, we constructed a novel transgenic mouse strain that constitutively expressed the human hCAP-18 gene driven by the EF1a promoter (Fig 1A). The expressed 18-kD hCAP-18 precursor required additional processing to produce 4-kD LL-37 and acquired biological functionality. To detect LL-37 expression, we collected the lysate from wild-type FVB and LL-37 transgenic mice and concentrated low-molecular-weight proteins by passing through a 10-kD centrifugal filter device. The expression of 4-kD LL-37, but not larger proteins (i.e., GAPDH), was detected in the low-molecular-weight ultrafiltrate in the transgenic mice (Fig 1B). Figure 1. Constitutive expression of LL-37 protected mouse lung from bacterial infection A. The schematic of human LL-37(CAMP) transgenic mouse strain. B. LL-37(4-kD) detection by Western blotting. Prior to loading, samples were centrifuged through 10-kD ultrafiltration membranes and an equal amount of ultrafiltrate (19 μg/lane) was subjected to immunoblotting. High-molecular-weight proteins (GAPDH) were not detected in ultrafiltrate. C. The CFU of PAO1 was measured by culturing in ultrafiltrate (upper panel) or retentate (lower panel) of mouse BALF samples from indicated mice. Initial additions of PAO1 were 1 × 103 CFU. Co-culture duration, 6 h. n = 3. Error bars, SEM. D. The bacterial CFU (per gram) in lungs of indicated mice with and without PAO1 infection (5 × 106 CFU). n = 3. Error bars, SEM. E. Representative histological sections of indicated lungs with PAO1 infection (5 × 106 CFU) for 6 h. H&E staining. Scale bar, 1,000 μm (upper panel) and 50 μm (lower panel). F. Histopathological injury score of indicated mouse lungs with PAO1 infection (5 × 106 CFU) based on blinded expert judgment. n = 3. Error bars, SEM. G. Gene expression level of IL-1β and IL-6 of indicated mouse lung with PAO1 infection (5 × 106 CFU). n ≥ 3. Error bars, SEM. Data information: Statistics for graphs: unpaired two-tailed t-test (C) and two-way ANOVA followed by Sidak's test (D, F, G). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 1 [emmm201810233-sup-0004-SDataFig1.zip] Download figure Download PowerPoint Bronchoalveolar lavage fluid (BALF) ultrafiltrate from LL-37 transgenic mice demonstrated a moderately enhanced bacteria inhibitory function than wild-type FVB BALF when used to culture PAO1 in vitro. In contrast, high-molecular-weight retentates displayed little difference in their bacterial inhibitory effect (Fig 1C). We also challenged mouse lung with equal amounts of PAO1 (5 × 106 CFU of PAO1) and analyzed the lung homogenate at different time points after infection. The results showed that the LL-37 transgenic mice had significantly enhanced bacterial clearance compared to wild-type FVB mice, leaving less residual infection (Fig 1D). Histological analysis indicated that LL-37 transgenic mice lung tissue displayed alleviated alveolar tissue damage at 6 h after PAO1 infection, potentially due to reduced bacterial burden (Fig 1D–F). Furthermore, the mRNA levels of major pro-inflammatory cytokines including IL-6 and IL-1β decreased in the lungs of LL-37 transgenic mice (Fig 1G). To further characterize the LL-37 transgenic mouse lung before and after bacterial infection, we performed RNA-Seq on lung tissues to analyze their whole transcriptomic profiles. As expected, FVB lungs had distinct transcriptomic profiles before and after PAO1 infection (PCC = 0.672). In contrast, LL-37+/+ lungs shared highly similar whole transcriptomic profiles before and after PAO1 infection (PCC = 0.977), suggesting that the LL-37+/+ lungs were protected from PAO1 challenge-induced alterations (Fig 2A). Interestingly, we found that overexpression of LL-37 gave rise to upregulation of multiple immune response-related genes even prior to infection (Fig 2B). Further analysis on gene ontology revealed that LL-37 expression enhanced normal mice development, including muscle, and blood circulation, and augmented mucosal immune response and organ-specific immune responses (Fig 2C). This finding indicated that LL-37 could stimulate lung immunity to protect infection, which was consistent with previous reports on other cathelicidin or cathelicidin-related peptides (Kovach et al, 2012; Beaumont et al, 2014). Figure 2. The altered transcriptomic profiles of transgenic mouse lungs before and after PAO1 infection A. Heatmap showing transcriptome profile correlation values of indicated lung tissue samples before and after PAO1 infection. B. Histogram of selected differentially expressed genes of LL-37+/+ mouse lung versus wild-type FVB mouse lung prior to infection. Blue bars indicated genes upregulated in wild-type FVB mouse lungs, while red bars indicated genes upregulated in LL-37+/+ mouse lungs. C, D. Enriched Gene Ontology classes of uninfected (C) and PAO1-infected (D) lungs. Red bar, GO class of upregulated gene in LL-37+/+ mice. Blue bar, GO class of upregulated gene in wild-type FVB mouse lung. GO terms were ranked by the enrichment P-value. E. Protein–protein interaction network of selected genes with high expression level in PAO1-infected wild-type lung (blue) and PAO1-infected LL-37+/+ lung (red), respectively. Source data are available online for this figure. Source Data for Figure 2 [emmm201810233-sup-0005-SDataFig2.zip] Download figure Download PowerPoint Next, we analyzed the lung transcriptomic profiles after PAO1 infection. The infected FVB mouse lungs were characterized by elevated infection and immune-related processes, such as “defense response” and “leukocyte activation”; in contrast, the infected LL-37+/+ lungs were characterized by lung tissue homeostasis/development-related processes, such as “lung epithelium development,” “respiratory system development,” and “cilium” (Fig 2D and Table EV1). Protein–protein interaction network analysis of overexpressed genes identified an inflammation-related molecular network in infected WT lungs, and in contrast, a lung development-related molecular network in infected LL-37+/+ lungs (Fig 2E). These data indicate that constitutive LL-37 expression in mouse lung can protect the lung from bacterial infection and inflammation. Genetically engineered mDASCs express functional LL-37 peptide LL-37 aids bacterial clearance in the mouse lung. We next determined if this peptide could be used in combination with DASCs to protect damaged lung from infection. P63+/Krt5+ DASCs (mDASCs) were isolated from normal adult mouse lung and expanded on 3T3 feeder cells as stem cell clones. The LL-37 gene was introduced into mDASCs by lentiviral transduction. Constitutive LL-37 expression was detected at both RNA and protein levels in LL-37-mDASCs but not in their wild-type counterpart (Fig 3A–C). WT-mDASCs and LL-37-mDASCs expressed similar levels of stem cell markers P63 and Krt5 (Fig 3D). mDASCs, irrespective of types, were able to be passaged indefinitely in our system. Elevated LL-37 expression was previously reported to affect cell viability and proliferation (Heilborn et al, 2005), while in this study we did not detect significant differences in either proliferation rate (Fig EV1A) or clonogenic ability (Fig 3E). In a three-dimensional organoid culture system, both mDASC cell lines formed alveolar-like sphere structures consisting of differentiated cells expressing AQP5 and PDPN, type I alveolar cell markers (Fig 3F). To confirm that genetically modified mDASCs were not tumorigenic, we assessed the anchorage-independent growth potential of these cells. mDASCs after LL-37 lentiviral transduction were unable to grow in soft agar medium, while mouse melanoma cells (B16) exhibited robust colony-forming efficiency under identical conditions (Fig EV1B). This indicated the successful generation of a LL-37-expressing mDASC cell line with unaltered self-renewal and differentiation properties. Figure 3. Engineered mDASCs possessed normal stem cell properties and enhanced antimicrobial potency A–C. Detection of LL-37 expression in the engineered mDASCs by immunofluorescence (A), real-time quantitative PCR (B), and Western blot (C). Scale bar, 50 μm. BF, bright field. n = 10. Error bars, SEM. D. Anti-Krt5 (red) and anti-P63 (green) immunostaining of WT- and LL-37-mDASC colonies. Scale bar, 70 μm. E. Stem cell colony-forming efficiency of WT- and LL-37-mDASCs during five serial passages. n = 6. Error bars, SD. F. Representative 3D organoid culture of mDASCs with expression of type I alveolar cell markers (Aqp5 and Pdpn). Left panels, bright-field imaging of 3D organoids. Right panels, immunofluorescence of organoid sections. Scale bar, 20 μm. G. Co-culture of bacteria with DASCs shows antimicrobial effects in dose-dependent manner. Initial additions of PAO1 were 0.1 × , 0.5 × and 1 × 104 CFU, respectively. Co-culture duration, 6 h. n = 4. Error bars, SEM. MOI, multiplicity of infection. H. Co-culture of bacteria with DASCs shows antimicrobial effects in time-dependent manner. Initial concentration of PAO1 was 1 × 104 CFU. MOI = 1. n = 3. Error bars, SEM. I, J. Preincubation of cells with anti-LL-37 antibody, but not IgG control, significantly reduced anti-PAO1 (I) and anti-Escherichia coli (J) effects of LL-37-mDASCs. Initial dose of bacteria was 103 CFU. Co-culture duration, 18 h. n = 4 in (I) and n = 3 in (J). Error bars, SEM. Data information: Statistics for graphs: unpaired two-tailed t-test (B), two-way ANOVA followed by Sidak's test (G, H) and one-way ANOVA followed by Tukey's test (I, J). *P < 0.05; **P < 0.01; ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 3 [emmm201810233-sup-0006-SDataFig3.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The antimicrobial effect of LL-37-mDASCs in vitro A. Cell growth curve of WT- and LL-37-mDASCs was measured by MTT assay. n = 3–5. Error bars, SD. B. Soft agar assay of WT- and LL-37-mDASCs. Mouse melanoma cell line was included as a positive control. C. Histogram showed that LL-37-mDASCs conditioned medium (CM) had potent growth inhibitory effect on PAO1. Initial addition of PAO1 was 1 × 104 CFU. n = 5. Error bars, SEM. D. Histogram shows that LL-37-mDASCs CM had potent growth inhibitory effect on Escherichia coli. Initial addition of E. coli was 1 × 104 CFU. n = 3. Error bars, SEM. E. Clone formation unit assay of E. coli following incubation with indicated cellular CM. F. Preincubation of CM with anti-LL-37 antibody, but not mouse IgG, reduced the antimicrobial effect of LL-37-mDASCs against E. coli. n = 3. Error bars, SEM. Data information: Statistics for graphs: one-way ANOVA followed by Tukey's test. *P < 0.05; ***P < 0.001. Download figure Download PowerPoint To test whether functional LL-37 peptide could be produced and secreted by LL-37-mDASCs, we assessed the bacterial clearance ability of their culture-conditioned medium (CM). PAO1 growth was significantly inhibited by the CM from LL-37-mDASCs but not from WT-mDASCs (Fig EV1C). In a series of cell/bacteria co-culture assays, LL-37-mDASCs, when compared to WT-mDASCs, showed impaired bacterial growth of PAO1 at different infection doses and time points, although substantial bacterial proliferation was observed under both conditions (Fig 3G–I). A similar bacterial growth inhibitory effect of LL-37-mDASCs was also detected with Gram-negative pathogen, Escherichia coli (Figs 3J and EV1D and E). To confirm that the inhibitory effect of engineered cells was attributed to LL-37 peptide production, we used anti-LL-37 antibody to neutralize the secreted peptide. Compared with IgG control, anti-LL-37 antibody compromised the inhibitory effect of LL-37-mDASCs (Fig 3I and J) and their cellular CM (Fig EV1F). Collectively, the above data demonstrate successful engineering of LL-37-mDASCs with normal stem cell properties and antimicrobial functions. Regeneration of LL-37-Lung by transplantation of genetically engineered mDASCs To investigate the potential therapeutic effect of engineered DASCs in vivo, we transplanted the LL-37-mDASCs into the damaged lung of syngeneic animals. The chemotherapeutic drug bleomycin was intratracheally instilled into the mouse lung to induce acute pulmonary inflammation and alveolar tissue damage. Seven days after bleomycin administration, 106 GFP-labeled WT-mDASCs or LL-37-mDASCs were intratracheally delivered into injured mouse lungs. We named the lungs with WT-mDASCs or LL-37-mDASCs engraftment as WT-Lung or LL-37-Lung, respectively. The lung tissues were harvested for analysis on different days after transplantation. Substantial incorporation of mDASCs into mouse lung was detected without observing significant rejection of cells (Fig 4A). An equal engraft ratio was observed for the two cell types, w
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