Alveolar macrophage‐derived extracellular vesicles inhibit endosomal fusion of influenza virus
2020; Springer Nature; Volume: 39; Issue: 16 Linguagem: Inglês
10.15252/embj.2020105057
ISSN1460-2075
AutoresDaniel J. Schneider, Katherine Smith, Catrina E. Latuszek, Carol A. Wilke, D. Lyons, Loka R. Penke, Jennifer M. Speth, Matangi Marthi, Joel A. Swanson, Bethany B. Moore, Adam S. Lauring, Marc Peters‐Golden,
Tópico(s)interferon and immune responses
ResumoArticle9 July 2020free access Source DataTransparent process Alveolar macrophage-derived extracellular vesicles inhibit endosomal fusion of influenza virus Daniel J Schneider Corresponding Author Daniel J Schneider [email protected] orcid.org/0000-0002-0344-2811 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Katherine A Smith Katherine A Smith Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Catrina E Latuszek Catrina E Latuszek Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Carol A Wilke Carol A Wilke Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Danny M Lyons Danny M Lyons Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Division of Infectious Disease, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Loka R Penke Loka R Penke Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Jennifer M Speth Jennifer M Speth Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Matangi Marthi Matangi Marthi orcid.org/0000-0002-1622-5701 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Joel A Swanson Joel A Swanson Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Bethany B Moore Bethany B Moore orcid.org/0000-0003-3051-745X Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Adam S Lauring Adam S Lauring Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Division of Infectious Disease, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Marc Peters-Golden Marc Peters-Golden Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Daniel J Schneider Corresponding Author Daniel J Schneider [email protected] orcid.org/0000-0002-0344-2811 Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Katherine A Smith Katherine A Smith Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Catrina E Latuszek Catrina E Latuszek Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Carol A Wilke Carol A Wilke Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Danny M Lyons Danny M Lyons Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Division of Infectious Disease, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Loka R Penke Loka R Penke Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Jennifer M Speth Jennifer M Speth Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Matangi Marthi Matangi Marthi orcid.org/0000-0002-1622-5701 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Joel A Swanson Joel A Swanson Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Bethany B Moore Bethany B Moore orcid.org/0000-0003-3051-745X Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Adam S Lauring Adam S Lauring Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Division of Infectious Disease, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Marc Peters-Golden Marc Peters-Golden Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI, USA Search for more papers by this author Author Information Daniel J Schneider *,1, Katherine A Smith1, Catrina E Latuszek1, Carol A Wilke1,2, Danny M Lyons2,3, Loka R Penke1, Jennifer M Speth1, Matangi Marthi2, Joel A Swanson2, Bethany B Moore1,2,4, Adam S Lauring2,3,4 and Marc Peters-Golden1,4 1Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA 2Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI, USA 3Division of Infectious Disease, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA 4Graduate Program in Immunology, University of Michigan Medical School, Ann Arbor, MI, USA *Corresponding author (lead contact). Tel: +1(734) 615-2319; E-mail: [email protected] The EMBO Journal (2020)39:e105057https://doi.org/10.15252/embj.2020105057 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 Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM-EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes. Synopsis Extracellular vesicles are emerging as homeostatic vectors, but poorly understood in influenza infection. Here, alveolar macrophage-derived extracellular vesicles inhibit influenza-endosome fusion in a strain-specific, and pH-dependent manner. Following initial infection of epithelial cells, the influenza virus traffics within host cell endosomes which undergo progressive acidification. Prior to gaining entry into the nucleus for its replication, influenza virus must fuse with endosome membranes—an event initiated at a strain-specific pH. Alveolar macrophages secrete extracellular vesicles which, when internalized by epithelial cells, lead to accelerated acidification of endosomes. Infection of epithelial cells by influenza strains which preferentially fuse with endosome membranes at high pH is inhibited by extracellular vesicles. Infection by influenza strains which fuse at low pH is unaffected by extracellular vesicles. Introduction Influenza respiratory infection is a global health problem affecting people of all ages. Patients who smoke or have chronic lung disease have enhanced susceptibility and more severe infection (Arcavi & Benowitz, 2004). Influenza is transmitted person-to-person to the proximal airways where it infects and replicates within epithelial cells (ECs). As the replication cycle generates infectious virions, these travel distally to infect ECs in the delicate alveolar spaces responsible for gas exchange. The subsequent occurrence of diffuse alveolar damage is a major determinant of the morbidity and mortality associated with influenza (Korteweg & Gu, 2008; Taubenberger & Morens, 2008). Current pharmacologic treatments and vaccination strategies for influenza have limited applications and efficacy (Dobson et al, 2015; CDC, 2020), and improved approaches are needed. Naïve individuals require up to 5 days for the development of specific T- and B-cell responses to influenza (Hermans et al, 2018). Therefore, the resident immune cells of the lung—alveolar macrophages (AMs) and the alveolar ECs (AECs) that comprise the alveolar surface—have an indispensable role in host defense at the early stages of infection. An improved understanding of these early innate immune protective mechanisms and how they become dysregulated is required to inform future therapeutic approaches. The AM is uniquely adapted to cope with the distinct and numerous challenges of this microenvironment. While resident AMs are now considered to be protective against influenza (Kim et al, 2008, 2013; Purnama et al, 2014; Schneider et al, 2014; Cardani et al, 2017), the mechanisms responsible remain to be elucidated. Electron microscopic morphometric analysis of human (Fehrenbach et al, 1994) and mammalian (Hyde et al, 2004) lungs as well as recent live microscopic studies of the mouse lung (Westphalen et al, 2014) demonstrate that there are fewer than one AM per each pulmonary alveolus. Moreover, and contrary to traditional notions, live microscopy has suggested that AMs may remain relatively stationary in vivo (Westphalen et al, 2014). Taken together, these observations strongly favor an important role for paracrine communication in the ability of AMs to protect AECs from influenza infection. Extracellular vesicles (EVs) represent one mode of paracrine intercellular communication whose importance is increasingly appreciated over the last decade. EVs are small (< 1 μm) membrane-delimited structures emanating from endosomal or plasma membranes of a wide range of cell types and organisms. Surface proteins on EVs can neutralize extracellular antigens or engage surface receptors on target cells (Lima et al, 2009; Atay et al, 2011). Additionally, biologically active vesicular cargo (lipids, nucleic acids, proteins) can exert intracellular actions in recipient cells upon internalization of EVs (Pitt et al, 2016; Robbins et al, 2016). The packaging of cargo molecules within EVs serves to protect them from degradation in the extracellular space (Zhang et al, 2015). Recent work from our laboratory has revealed that cues from neighboring AECs and external stimuli [e.g., soluble mediators, cigarette smoke (CS), and lipopolysaccharide] can rapidly modify the number of secreted AM-derived EVs, their cargo, and their uptake, with resulting modulation of inflammatory responses within target AECs (Bourdonnay et al, 2015; Speth et al, 2016; Schneider et al, 2017). These features position EVs as nimble vectors for signaling within the dynamic lung environment. Despite the public health impact of influenza, little is known about cell communication via EVs in the host response to this infection, and EVs secreted from AMs in influenza have not been considered. Using in vitro and in vivo models of alveolar communication, we investigated the ability of EVs secreted from AMs to inhibit the replication within AECs of a panel of patient-derived influenza viruses. We report here that AM-derived EVs are capable of inhibiting only a subset of these influenza strains. Only those strains susceptible to inhibition by EVs exhibited defects in endosomal fusion and egress. This differential strain susceptibility facilitated the determination that AM-EVs inhibit influenza replication in AECs by enhancing endosomal acidification. These studies provide new insights into the mechanisms by which AMs promote anti-viral defense within the lung and reveal important characteristics of influenza virus that facilitate evasion of the early innate immune response. In addition, these studies reveal an important consequence of EV uptake within endosomes which has potential relevance to numerous endocytosis-dependent pathophysiologic processes beyond influenza. Results Paracrine inhibitory activity of AMs against laboratory strains of influenza infection localizes to their secreted EVs Investigation of the paracrine anti-viral activity of AMs requires a sensitive assay of influenza replication. A previously constructed luciferase reporter was inserted downstream of the PA segment in the genome of the commonly studied laboratory strain of influenza A/WSN/33 (Tran et al, 2013). The resulting Luc-WSN/33 generates luminescence with high specific activity from infected cells which correlates with virus replication. Luc-WSN/33 infectivity is similar to that of its unmodified wild-type (WT) counterpart. This permits a sensitive, time-integrated, high-throughput assay of viral replication. We set out to determine the capacity of substances constitutively secreted by AMs to protect ECs against influenza infection. We therefore collected conditioned medium (CM) from the mouse MH-S AM cell line which we have previously demonstrated readily sheds EVs with functional and structural characteristics similar to those of primary AMs (Bourdonnay et al, 2015; Schneider et al, 2017). Luc-WSN/33 infection was initially assayed in MDCK-SIAT1 cells derived from the standard (Green, 1962) MDCK cells, and which were engineered to overexpress α2,6-sialic acid for improved infectivity of recent circulating influenza strains (Oh et al, 2008). Indeed, CM from naïve AMs inhibited Luc-WSN/33 replication in these ECs (Fig 1A and B). This inhibitory effect of CM was localized to ultracentrifugation-purified EVs contained within CM (Fig 1A and B) and was not shared by the remaining EV-free CM. This inhibition of EC influenza replication increased with the dose of AM-EVs provided (Fig 1C) and was not the result of decreased EC viability (Appendix Fig S1). This inhibition of influenza replication in ECs by MH-S cell EVs was confirmed by using EVs isolated from primary mouse AMs (Fig 1D) or peritoneal macrophages (Fig EV1) both obtained by lavage from naïve C57BL/6 mice and then subjected to retroviral immortalization. Finally, EVs isolated from primary rat AMs isolated by lung lavage (Fig 1E) and human macrophages differentiated from the THP-1 monocyte cell line (Fig 1F) also demonstrated inhibition of influenza replication. These data demonstrate that the inhibitory activity against influenza constitutively secreted by AMs localizes to EVs. Figure 1. Paracrine inhibitory activity of AMs against laboratory strains of influenza infection localizes to their secreted EVs A, B. MDCK-SIAT1 cells co-incubated with Luc-WSN/33 and MH-S CM fractions. Replication was quantified by luminescence. (A) Replication curves display average luminescence reads from 5 wells per condition (measured at 7.5 min intervals—displayed here at 30 min intervals) from 1 experiment representative of 7 independent experiments. (B) Mean calculated area under the luminescence curves (AUC) for individual wells normalized to the corresponding mean of the control condition from these 7 experiments. C. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with flow cytometry-quantified MH-S AM-EVs at specified EV:cell ratios. D. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with CM fractions of J2 retrovirus-immortalized primary mouse AMs. E. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with primary rat AM-derived EVs. F. Luc-WSN/33 replication in MDCK-SIAT1 cells incubated with human THP-1 macrophage-derived EVs. Data information: Luc-WSN/33 replication in each condition represented by mean luminescence AUC for individual wells normalized to the mean of the control condition from 3 (C–E) and 4 (F) independent experiments. Error bars = SD. One-way ANOVA (B–D, F) and unpaired Student's t-test (E) (*P value of < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Source data are available online for this figure. Source Data for Figure 1 [embj2020105057-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. EVs isolated from immortalized mouse primary peritoneal macrophages inhibit influenza replication MDCK-Siat1 cells were co-incubated with Luc-WSN/33 and EVs isolated from immortalized primary mouse peritoneal macrophages. Luc-WSN/33 replication in each condition represented by mean luminescence AUC for individual wells normalized to the mean of the control condition from 4 independent experiments.Data information: Error bars = SD. Unpaired Student's t-test (****P value of < 0.0001).Source data are available online for this figure. Download figure Download PowerPoint AM-EVs inhibit influenza infection of AECs in vitro and in vivo To confirm that inhibition of influenza replication observed in MDCK-SIAT1 ECs (Fig 1) applies to AECs, we tested the effects of AM-EVs on the human A549 carcinoma (Fig 2A) and mouse MLE-12 (Fig 2B) AEC lines infected with Luc-WSN/33. A dose-dependent effect of AM-EVs against influenza replication was also observed in these AEC lines. In separate experiments, we validated the inhibitory effect of AM-EVs on infection of MLE-12 AECs with an additional WT laboratory strain PR/8 by more conventionally assessing replication using RT–qPCR analysis of the influenza M1 gene (Fig 2C). Figure 2. AM-EVs inhibit influenza replication in AECs in vitro and in vivo A, B. Luc-WSN/33 replication co-incubated with MH-S EVs was assessed in (A) human A549 and (B) mouse MLE-12 AECs. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments. C. MLE-12 AECs incubated with MH-S EVs infected with PR/8 influenza. Replication was quantified by RT–qPCR at 12 h post-infection. Data represent mean M gene transcripts normalized to β-actin from 3 independent experiments. D. Schematic outline for AM depletion in lungs of mice with o.p. Clo followed by intrapulmonary delivery of AM-EVs PR/8 infection. E. Total lung RNA was isolated from mice on day 1 post-infection and virus was quantified by RT–qPCR. Data represent mean M gene transcripts from individual mice normalized to β-actin from 3 independent experiments (4–5 mice per group). Data information: Error bars = SD. One-way ANOVA (A, B) and unpaired Student's t-test (C) (*P value of < 0.05, ***P < 0.001, ****P < 0.0001). For (E), the comparison between conditions was performed with one-way ANOVA (multiple comparisons) on values obtained from individual mice across all experiments. Source data are available online for this figure. Source Data for Figure 2 [embj2020105057-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint Our findings that AM-EVs protect AECs against influenza offers a possible mechanistic explanation for previous in vivo studies in multiple species in which depletion of AMs leads to exacerbation of influenza-related pathology (Kim et al, 2008, 2013; Purnama et al, 2014). We therefore assessed whether intrapulmonary delivery of MH-S cell EVs could rescue the impaired host defense against influenza exhibited by mice depleted of AMs following intrapulmonary instillation of clodronate-loaded liposomes (Clo) (Fig 2D). As we have reported previously (Zaslona et al, 2014), Clo resulted in 80% depletion of AMs at day 2 compared to empty liposomes (Emp) (Appendix Fig S2). At this time point, AM-depleted mice were infected oropharyngeally (o.p.) with the PR/8 influenza strain with and without co-administration of MH-S AM-EVs. Consistent with previous reports (Tate et al, 2010), Clo depletion of AMs resulted in higher viral transcript levels in the lungs of influenza-infected mice compared to mice treated with Emp (Fig 2E). Indeed, o.p. treatment with AM-EVs significantly lowered influenza transcript levels in lungs from AM-depleted influenza-infected mice, confirming that AM-EVs inhibit influenza replication in vivo. Cell-type specificity, modulation, and characteristics of the AM-EV activity against influenza That AECs were protected against influenza infection by AM-EVs suggests that these EVs contain bioactive constituents not elaborated by AECs themselves. To test this hypothesis, EVs were isolated in parallel from MH-S AMs and A549 cells, quantified by flow cytometry, and incubated at equal concentrations with Luc-WSN/33-infected MDCK-SIAT1 cells (Fig 3A). In contrast to the effects of MH-S-EVs (Fig 3A, middle bar), the same number of A549-EVs had no significant inhibitory action in MDCK-SIAT1 cells (Fig 3A, right bar), demonstrating that the inhibitory capacity of AM-EVs against influenza is not a property shared by all alveolar cell-derived EVs. Figure 3. Cell-type specificity, modulation, and characteristics of the AM-EV activity against influenza EVs isolated from MH-S AMs and A549 AECs were co-incubated in parallel with Luc-WSN/33 (MOI = 0.5) in MDCK-SIAT1 cells. Data represent mean luminescence AUC from individual wells normalized to the mean of the corresponding control condition from 3 independent experiments. EVs isolated from MH-S cells treated with increasing concentrations of CSE were co-incubated with Luc-WSN/33 in MLE-12 cells. Data represent mean luminescence at 12 h post-infection from individual wells normalized to the mean of untreated AM-EVs from 3 independent experiments per condition. Data information: Error bars = SD. One-way ANOVA (A, B) (*P value of < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Source data are available online for this figure. Source Data for Figure 3 [embj2020105057-sup-0005-SDataFig3.xlsx] Download figure Download PowerPoint Both experimental and epidemiologic studies have concluded that cigarette smoking (CS) and exposure both increase susceptibility to various infections, including influenza (Arcavi & Benowitz, 2004). This vulnerability occurs despite intact humoral immunity (Robbins et al, 2006), suggesting that CS dysregulates important innate immune responses to influenza. Therefore, we tested the effect of CS exposure of AMs on the anti-viral function of their secreted EVs using an established in vitro model of CS exposure (Phipps et al, 2010), aqueous CS extract (CSE). EVs were isolated from MH-S cells incubated with increasing concentrations of CSE, quantified by flow cytometry, and delivered at equal concentrations to Luc-WSN/33-infected MLE-12 cells (Fig 3B). CSE treatment of AMs dose-dependently attenuated the inhibitory actions of their EVs on Luc-WSN/33 replication in AECs. With exposure of MH-S cells to 0.8% CSE, their secreted EVs lost all activity against Luc-WSN/33 replication. The role of coding and non-coding RNA species in mediating the biological actions of EVs has gained substantial interest (Mateescu et al, 2017). We therefore developed a means to selectively deplete RNA within AM-EVs to examine the impact of this macromolecule class on the anti-viral actions of EVs. Subjecting plasma membranes to multiple freeze-thaw cycles is known to result in their permeabilization (Mardones & Gonzalez, 2003) and has been shown to provide access to constituents contained within EVs (Genschmer et al, 2019). This permeabilization method was used to facilitate incorporation of RNase A (Fig EV2) to permit the selective degradation of RNA within EVs. This protocol yielded the same number of EVs by flow cytometry, depleted ~ 90% of total RNA within EVs (Fig EV2, right column vs. middle column), and yet failed to abrogate the inhibitory effect of AM-EVs on Luc-WSN/33 replication in ECs. These data thus implicate non-RNA cargo within EVs as the predominant source of activity against influenza. Of note, the vast majority of characterized endogenous inhibitors of influenza infection are proteins (Iwasaki & Pillai, 2014). Moreover, we previously demonstrated that CS disrupts the packaging of selected proteins into AM-EVs (Bourdonnay et al, 2015). We therefore speculated that protein cargo may mediate the "anti-viral" actions of constitutively released AM-EVs and that CSE might result in a reduction in the incorporation of this protein cargo within AM-EVs. Click here to expand this figure. Figure EV2. AM-EV inhibitory activity against influenza is not sensitive to RNase A Schematic outline of the strategy to deplete RNA within EVs. Three groups of EVs were isolated from equal volumes of MH-S CM, subsequently treated with either RNase A, and ultimately subjected to gentle permeabilization by repeated freeze-thaw cycles at −80°C to permit RNase A entry into EVs. This permeabilization occurred before (right column) or after (left and middle columns) addition of an RNAse inhibitor. Analysis of EVs in each condition included RNA quantification with Agilent Bioanalyzer 2100 and TapeStation Analysis Software 3.1.1 to quantify the RNA present within EVs (n = 2) or quantified with flow cytometry (n = 2). These EVs were co-incubated with Luc-WSN/33 in MDCK-SIAT1 cells, and replication was assessed using luminescence. Data represent mean luminescence at 12 h post-infection from individual wells normalized to control from 4 independent experiments.Data information: Error bars = SD. One-way ANOVA (****P value of < 0.0001).Source data are available online for this figure. Download figure Download PowerPoint To identify candidate proteins within AM-EVs that might mediate the inhibitory activity against influenza replication within AECs and whose quantity was reduced by CSE, we assessed the differential abundance of proteins from control-treated and 0.8% CSE-treated AM-EVs using tandem mass tag mass spectrometry (TMT-MS) (McAlister et al, 2014). A schematic outline of the TMT workflow is shown in Fig EV3. Of the ~ 4,500 EV proteins identified with high confidence, only eight proteins exhibiting statistically significant differential abundance (−log10 q value ≥ 2, Fig EV3B, y-axis) and > 25% absolute difference in abundance ratio (Fig EV3B, x-axis), and all were upregulated with CSE. Therefore, this method failed to identify any EV protein candidates that could account for the activity of naïve AM-derived EVs against influenza and whose abundance is reduced by CSE. Click here to expand this figure. Figure EV3. Differential abundance of proteins within AM-EVs in response to CSE Flowchart demonstrating the number proteins within each EV condition compared using TMT-MS and each of the listed criteria. Volcano plot of all 4,497 proteins identified in both EV conditions. Orange boxes collectively denote the proteins with significantly different relative abundance (a −log10 q value ≥ 2) and with > 25% difference in abundance ratio. List of the 8 proteins identified that exhibited > log2 0.33 in abundance in EVs isolated from 0.8% CSE-treated AMs compared to 0% CSE-treated AM-EVs. Download figure Download PowerPoint Patient-derived influenza isolates exhibit strain-dependent sensitivity to the inhibitory effects of AM-EVs The WSN/33 and PR/8 influenza strains utilized thus far have been subjected to decades of adaptation to mice and laboratory cell lines through serial passaging. Consequently, these strains no longer cause disease in wild mice or humans (Sta
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