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The TORC1-activated Proteins, p70S6K and GRB10, Regulate IL-4 Signaling and M2 Macrophage Polarization by Modulating Phosphorylation of Insulin Receptor Substrate-2*

  • Kristi J. Warren
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Xi Fang
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Nagaraj M. Gowda
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Joshua J. Thompson
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Nicola M. Heller
    Correspondence
    To whom correspondence should be addressed: Johns Hopkins University School of Medicine, Dept. of ACCM, Division of Allergy and Clinical Immunology, 720 Rutland Ave., Ross 367, Baltimore, MD 21205. Tel.: 410-955-1743; Fax: 410-614-0083;
    Affiliations
    From Johns Hopkins University, School of Medicine, Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland 21205
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant K99/R00 HL096897 (NHLBI; to N. M. H.). The authors declare that they have no conflict of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Open AccessPublished:October 14, 2016DOI:https://doi.org/10.1074/jbc.M116.756791
      Lung M2 macrophages are regulators of airway inflammation, associated with poor lung function in allergic asthma. Previously, we demonstrated that IL-4-induced M2 gene expression correlated with tyrosine phosphorylation of the insulin receptor substrate-2 (IRS-2) in macrophages. We hypothesized that negative regulation of IRS-2 activity after IL-4 stimulation is dependent upon serine phosphorylation of IRS-2. Herein, we describe an inverse relationship between tyrosine phosphorylation (Tyr(P)) and serine phosphorylation (Ser(P)) of IRS-2 after IL-4 stimulation. Inhibiting serine phosphatase activity increased Ser(P)-IRS-2 and decreased Tyr(P)-IRS-2 leading to reduced M2 gene expression (CD200R, CCL22, MMP12, and TGM2). We found that inhibition of p70S6K, downstream of TORC1, resulted in diminished Ser(P)-IRS-2 and prolonged Tyr(P)-IRS-2 as well. Inhibition of p70S6K increased expression of CD200R and CCL22 indicating that p70S6K negatively regulates some, but not all, human M2 genes. Knocking down GRB10, another negative regulatory protein downstream of TORC1, enhanced both Tyr(P)-IRS-2 and increased expression of all four M2 genes. Furthermore, GRB10 associated with IRS-2, NEDD4.2 (an E3-ubiquitin ligase), IL-4Rα, and γC after IL-4 stimulation. Both IL-4Rα and γC were ubiquitinated after 30 min of IL-4 treatment, suggesting that GRB10 may regulate degradation of the IL-4 receptor-signaling complex through interactions with NEDD4.2. Taken together, these data highlight two novel regulatory proteins that could be therapeutically manipulated to limit IL-4-induced IRS-2 signaling and polarization of M2 macrophages in allergic inflammation.

      Introduction

      Both genetic and environmental factors influence the onset and exacerbation of allergic asthma, which remains one of the most costly, non-communicable diseases of the human population. Allergic inflammation is characterized by airway hyper-reactivity, type 2 cytokines (IL-4, -5, and -13), IgE, and the overproduction of mucus in the lung (
      • Genuneit J.
      • Cantelmo J.L.
      • Weinmayr G.
      • Wong G.W.
      • Cooper P.J.
      • Riikjärv M.A.
      • Gotua M.
      • Kabesch M.
      • von Mutius E.
      • Forastiere F.
      • Crane J.
      • Nystad W.
      • El-Sharif N.
      • Batlles-Garrido J.
      • García-Marcos L.
      • et al.
      A multi-centre study of candidate genes for wheeze and allergy: the International Study of Asthma and Allergies in Childhood Phase 2.
      ,
      • Vercelli D.
      Advances in asthma and allergy genetics in 2007.
      • Binia A.
      • Kabesch M.
      Respiratory medicine - genetic base for allergy and asthma.
      ). Macrophages present at the site of allergic inflammation can be polarized by Th2-cytokines to become “alternatively activated” or M2 macrophages. In addition, the abundance of M2 macrophages in the lungs of asthmatics correlates with symptom severity (
      • Melgert B.N.
      • ten Hacken N.H.
      • Rutgers B.
      • Timens W.
      • Postma D.S.
      • Hylkema M.N.
      More alternative activation of macrophages in lungs of asthmatic patients.
      ,
      • Viksman M.Y.
      • Bochner B.S.
      • Peebles R.S.
      • Schleimer R.P.
      • Liu M.C.
      Expression of activation markers on alveolar macrophages in allergic asthmatics after endobronchial or whole-lung allergen challenge.
      • St-Laurent J.
      • Turmel V.
      • Boulet L.P.
      • Bissonnette E.
      Alveolar macrophage subpopulations in bronchoalveolar lavage and induced sputum of asthmatic and control subjects.
      ). As M2 macrophage polarization is dependent upon IL-4 stimulation, this cytokine and its signaling pathways represent excellent targets for asthma therapies.
      Our previous work has identified IL-4 signaling through the type I IL-4 receptor complex and activation of insulin receptor substrate 2 (IRS-2)
      The abbreviations used are: IRS-2, insulin receptor substrate 2; IP, immunoprecipitation; qPCR, quantitative PCR; ANOVA, analysis of variance.
      as important for the degree of polarization of M2 macrophages (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ). IL-4 binds first to the IL-4Rα chain with high affinity followed by recruitment of the common γ chain (IL-2Rγ or γC) to create a functional type I receptor. Assembly of this receptor complex leads to recruitment and phosphorylation of the JAK1 and -3 proteins followed by robust, yet transient, tyrosine phosphorylation of IRS-2 and STAT6. Phosphorylated STAT6 homodimerizes and translocates to the nucleus, where it activates transcription of M2 macrophage-specific genes (ArgI, Retnla, and Chi3l3 in the mouse and TGM2, MMP12, CCL22, CD200R in humans). Activation of the IRS-2 pathway by IL-4 binding the type I IL-4 receptor, but not the type II IL-4 receptor, further enhances the degree of M2 macrophage gene expression (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ). Although Shp-1 and SHIP-1 have been implicated in negative regulation of IL-4-induced STAT6 signaling (
      • Haque S.J.
      • Harbor P.
      • Tabrizi M.
      • Yi T.
      • Williams B.R.
      Protein-tyrosine phosphatase Shp-1 is a negative regulator of IL-4- and IL-13-dependent signal transduction.
      ), nothing is known of the negative regulatory processes that suppress the IL-4-induced IRS-2 signaling pathway.
      Serine phosphorylation of the IRS proteins is one mechanism by which insulin-induced IRS signaling is terminated (
      • Boura-Halfon S.
      • Zick Y.
      Serine kinases of insulin receptor substrate proteins.
      • Boura-Halfon S.
      • Zick Y.
      Phosphorylation of IRS proteins, insulin action, and insulin resistance.
      ,
      • Copps K.D.
      • White M.F.
      Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2.
      • Fritsche L.
      • Neukamm S.S.
      • Lehmann R.
      • Kremmer E.
      • Hennige A.M.
      • Hunder-Gugel A.
      • Schenk M.
      • Häring H.U.
      • Schleicher E.D.
      • Weigert C.
      Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser-675 and Ser-907.
      ). Serine phosphorylation of IRS-1 and IRS-2 prevents p85 binding and PI3K activation (
      • Hançer N.J.
      • Qiu W.
      • Cherella C.
      • Li Y.
      • Copps K.D.
      • White M.F.
      Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation.
      ), promotes IRS degradation, and promotes dissociation of IRS molecules from the insulin receptor (
      • Monami G.
      • Emiliozzi V.
      • Morrione A.
      Grb10/Nedd4-mediated multiubiquitination of the insulin-like growth factor receptor regulates receptor internalization.
      ,
      • Greene M.W.
      • Sakaue H.
      • Wang L.
      • Alessi D.R.
      • Roth R.A.
      Modulation of insulin-stimulated degradation of human insulin receptor substrate-1 by serine 312 phosphorylation.
      ). A number of different serine/threonine kinases (e.g. ERK1/2, TORC1/2, p70S6K, GSK-3α/β, JNK) have been shown to phosphorylate-specific serine residues of IRS-1 to inhibit insulin signaling (
      • Copps K.D.
      • White M.F.
      Regulation of insulin sensitivity by serine/threonine phosphorylation of insulin receptor substrate proteins IRS1 and IRS2.
      ,
      • Fritsche L.
      • Neukamm S.S.
      • Lehmann R.
      • Kremmer E.
      • Hennige A.M.
      • Hunder-Gugel A.
      • Schenk M.
      • Häring H.U.
      • Schleicher E.D.
      • Weigert C.
      Insulin-induced serine phosphorylation of IRS-2 via ERK1/2 and mTOR: studies on the function of Ser-675 and Ser-907.
      • Hançer N.J.
      • Qiu W.
      • Cherella C.
      • Li Y.
      • Copps K.D.
      • White M.F.
      Insulin and metabolic stress stimulate multisite serine/threonine phosphorylation of insulin receptor substrate 1 and inhibit tyrosine phosphorylation.
      ). Recent publications, however, highlight the importance of the TOR complexes and TOR-activated proteins in regulating M2 polarization in mouse macrophages in response to IL-4 (
      • Byles V.
      • Covarrubias A.J.
      • Ben-Sahra I.
      • Lamming D.W.
      • Sabatini D.M.
      • Manning B.D.
      • Horng T.
      The TSC-mTOR pathway regulates macrophage polarization.
      • Zhu L.
      • Yang T.
      • Li L.
      • Sun L.
      • Hou Y.
      • Hu X.
      • Zhang L.
      • Tian H.
      • Zhao Q.
      • Peng J.
      • Zhang H.
      • Wang R.
      • Yang Z.
      • Zhang L.
      • Zhao Y.
      TSC1 controls macrophage polarization to prevent inflammatory disease.
      ,
      • Festuccia W.T.
      • Pouliot P.
      • Bakan I.
      • Sabatini D.M.
      • Laplante M.
      Myeloid-specific Rictor deletion induces M1 macrophage polarization and potentiates in vivo pro-inflammatory response to lipopolysaccharide.
      ,
      • Hallowell R.W.
      • Collins S.
      • Zhang Y.
      • Chan-Li Y.
      • Powell J.
      • Horton M.R.
      Mtor signaling pathways regulate macrophage differentiation and function.
      • Mercalli A.
      • Calavita I.
      • Dugnani E.
      • Citro A.
      • Cantarelli E.
      • Nano R.
      • Melzi R.
      • Maffi P.
      • Secchi A.
      • Sordi V.
      • Piemonti L.
      Rapamycin unbalances the polarization of human macrophages to M1.
      ). Because our previous work showed that IRS-2 tyrosine phosphorylation correlated with M2 polarization, we sought to determine whether serine phosphorylation of IRS-2 and TOR-activated regulatory pathways were responsible for controlling IL-4 signaling through IRS-2 in human macrophages.

      Discussion

      IRS-2 has been described as a key regulator of M2 macrophage polarization (
      • Heller N.M.
      • Qi X.
      • Junttila I.S.
      • Shirey K.A.
      • Vogel S.N.
      • Paul W.E.
      • Keegan A.D.
      Type I IL-4Rs selectively activate IRS-2 to induce target gene expression in macrophages.
      ,
      • Heller N.M.
      • Qi X.
      • Gesbert F.
      • Keegan A.D.
      The extracellular and transmembrane domains of the γC and interleukin (IL)-13 receptor α1 chains, not their cytoplasmic domains, dictate the nature of signaling responses to IL-4 and IL-13.
      ). Given the correlation between the presence of M2 macrophages in the asthmatic lung and asthma severity in humans (
      • Melgert B.N.
      • ten Hacken N.H.
      • Rutgers B.
      • Timens W.
      • Postma D.S.
      • Hylkema M.N.
      More alternative activation of macrophages in lungs of asthmatic patients.
      ,
      • Viksman M.Y.
      • Bochner B.S.
      • Peebles R.S.
      • Schleimer R.P.
      • Liu M.C.
      Expression of activation markers on alveolar macrophages in allergic asthmatics after endobronchial or whole-lung allergen challenge.
      • St-Laurent J.
      • Turmel V.
      • Boulet L.P.
      • Bissonnette E.
      Alveolar macrophage subpopulations in bronchoalveolar lavage and induced sputum of asthmatic and control subjects.
      ), understanding the regulation of IL-4-activated signaling pathways that lead to M2 polarization is critical for interrupting asthma progression.
      The goal of this study was to determine mechanisms that negatively regulate the activity of IRS-2 in response to IL-4. Our current results show that down-regulation of IRS-2 signaling by serine phosphorylation follows a paradigm previously established for insulin responses (
      • Sun X.J.
      • Miralpeix M.
      • Myers Jr, M.G.
      • Glasheen E.M.
      • Backer J.M.
      • Kahn C.R.
      • White M.F.
      Expression and function of IRS-1 in insulin signal transmission.
      • Paz K.
      • Hemi R.
      • LeRoith D.
      • Karasik A.
      • Elhanany E.
      • Kanety H.
      • Zick Y.
      A molecular basis for insulin resistance. Elevated serine/threonine phosphorylation of IRS-1 and IRS-2 inhibits their binding to the juxtamembrane region of the insulin receptor and impairs their ability to undergo insulin-induced tyrosine phosphorylation.
      ,
      • Greene M.W.
      • Garofalo R.S.
      Positive and negative regulatory role of insulin receptor substrate 1 and 2 (IRS-1 and IRS-2) serine/threonine phosphorylation.
      • Zick Y.
      Ser/Thr phosphorylation of IRS proteins: a molecular basis for insulin resistance.
      ). Various well defined mechanisms, including serine/threonine kinase activation, steric hindrance, and ubiquitin-mediated degradation of IRS proteins (
      • Rui L.
      • Yuan M.
      • Frantz D.
      • Shoelson S.
      • White M.F.
      SOCS-1 and SOCS-3 block insulin signaling by ubiquitin-mediated degradation of IRS1 and IRS2.
      ), are known to limit insulin-induced IRS-1 activation. Our findings similarly show an on-to-off tyrosine/serine phosphorylation switch for IRS-2 in response to IL-4 (summarized in Fig. 5). IL-4 activates tyrosine phosphorylation and M2 macrophage polarization, whereas serine phosphorylation inhibits IL-4-activated IRS-2 signaling. Using pharmacological and siRNA knockdown approaches, we found that two TORC1-activated proteins, p70S6K and GRB10, were important negative regulators of IL-4-induced IRS-2 signaling and M2 gene expression in human monocytes.
      Figure thumbnail gr5
      FIGURE 5.Regulatory pathways of IL-4 signaling and M2 macrophage differentiation. IL-4 stimulation is a potent inducer of M2 macrophage polarization. 1, IL-4 can bind two kinds of IL-4 receptor complexes on monocyte-macrophages, type I (shown here) and type II. 2, both STAT6 and IRS-2 are recruited to the IL-4 receptor α chain after JAK phosphorylation of key tyrosine residues in the cytoplasmic domain. 3, phosphorylation of tyrosine residues of IRS-2 occurs, presumably by proximity to the activated JAKs. Tyrosine phosphorylation of IRS-2 occurs most potently as a result of engagement of IL-4 with type I receptor complexes. Engagement of type II receptor complexes by either IL-4 or IL-13 does not elicit robust tyrosine phosphorylation of IRS-2. 4, PI3K is activated through docking and activation of p85α with tyrosine phosphorylated IRS-2. PIP3 activation of PDK1 leads to phosphorylation of AKT Thr308. 5a, AKT activates mTORC1 as well as possibly other pathways downstream of AKT that directly contribute to M2 gene expression. 5b. PIP3-activated mTORC2 fully activates AKT function by phosphorylation of Ser-473. 6a, mTORC1 phosphorylates p70S6K, which interacts with IRS-2 to phosphorylate serine residues and thereby limit IL-4 signaling. 6b, GRB10 is directly activated and stabilized by mTORC1. 7, GRB10 interacts with IRS-2, common γ chain (γC), IL-4Rα and NEDD4.2 to target these proteins for ubiquitination (Ub) and, most likely, proteasomal degradation. Our current study establishes that the mTORC-1 activated proteins p70S6K and GRB10 limit signaling of tyrosine-phosphorylated IRS-2 through serine phosphorylation of IRS-2 and ubiquitination, respectively. The effect of these two negative regulatory mechanisms is a reduction in M2 polarization of human macrophages.
      Based upon the results of the Ser(P)- and Tyr(P)-IRS-2 studies, we expected induction of the human M2 gene panel after amplifying Tyr(P)-IRS-2 tyrosine with p70S6K inhibition. Indeed, two of four M2 genes were enhanced with p70S6K inhibition, indicating a greater complexity to regulation of M2 gene expression than anticipated. Many M2 genes share common activating and inhibitory pathways (
      • Covarrubias A.J.
      • Aksoylar H.I.
      • Horng T.
      Control of macrophage metabolism and activation by mTOR and Akt signaling.
      ,
      • Dasgupta P.
      • Dorsey N.J.
      • Li J.
      • Qi X.
      • Smith E.P.
      • Yamaji-Kegan K.
      • Keegan A.D.
      The adaptor protein insulin receptor substrate 2 inhibits alternative macrophage activation and allergic lung inflammation.
      ) and can be AKT-dependent or -independent (
      • Covarrubias A.J.
      • Aksoylar H.I.
      • Yu J.
      • Snyder N.W.
      • Worth A.J.
      • Iyer S.S.
      • Wang J.
      • Ben-Sahra I.
      • Byles V.
      • Polynne-Stapornkul T.
      • Espinosa E.C.
      • Lamming D.
      • Manning B.D.
      • Zhang Y.
      • Blair I.A.
      • Horng T.
      Akt-mTORC1 signaling regulates Acly to integrate metabolic input to control of macrophage activation.
      ). For example, studies of macrophage polarization using macrophages derived from Tsc1 (
      • Byles V.
      • Covarrubias A.J.
      • Ben-Sahra I.
      • Lamming D.W.
      • Sabatini D.M.
      • Manning B.D.
      • Horng T.
      The TSC-mTOR pathway regulates macrophage polarization.
      ,
      • Zhu L.
      • Yang T.
      • Li L.
      • Sun L.
      • Hou Y.
      • Hu X.
      • Zhang L.
      • Tian H.
      • Zhao Q.
      • Peng J.
      • Zhang H.
      • Wang R.
      • Yang Z.
      • Zhang L.
      • Zhao Y.
      TSC1 controls macrophage polarization to prevent inflammatory disease.
      )-, Raptor (

      Robert, H., Samuel, C., Yee, C.-L., Jonathan, P., and Maureen, R. H., (May 18–23, 2012) MTOR regulates the differentiation of classically and alternatively activated macrophages. In C92. Control of Macrophage Polarization and Effector Functions, American Thoracic Society, pp. A5061–A5061, 2012 International Conference, San Francisco,.

      )-, and Rictor (
      • Byles V.
      • Covarrubias A.J.
      • Ben-Sahra I.
      • Lamming D.W.
      • Sabatini D.M.
      • Manning B.D.
      • Horng T.
      The TSC-mTOR pathway regulates macrophage polarization.
      ,

      Robert, H., Samuel, C., Yee, C.-L., Jonathan, P., and Maureen, R. H., (May 18–23, 2012) MTOR regulates the differentiation of classically and alternatively activated macrophages. In C92. Control of Macrophage Polarization and Effector Functions, American Thoracic Society, pp. A5061–A5061, 2012 International Conference, San Francisco,.

      )-deficient mice have concluded that TORC1 down-regulates IL-4 signaling/M2 polarization (
      • Byles V.
      • Covarrubias A.J.
      • Ben-Sahra I.
      • Lamming D.W.
      • Sabatini D.M.
      • Manning B.D.
      • Horng T.
      The TSC-mTOR pathway regulates macrophage polarization.
      ,
      • Zhu L.
      • Yang T.
      • Li L.
      • Sun L.
      • Hou Y.
      • Hu X.
      • Zhang L.
      • Tian H.
      • Zhao Q.
      • Peng J.
      • Zhang H.
      • Wang R.
      • Yang Z.
      • Zhang L.
      • Zhao Y.
      TSC1 controls macrophage polarization to prevent inflammatory disease.
      ) and TORC2 promotes M2 polarization (

      Robert, H., Samuel, C., Yee, C.-L., Jonathan, P., and Maureen, R. H., (May 18–23, 2012) MTOR regulates the differentiation of classically and alternatively activated macrophages. In C92. Control of Macrophage Polarization and Effector Functions, American Thoracic Society, pp. A5061–A5061, 2012 International Conference, San Francisco,.

      ). However, TORC1 serves as a signaling “node” that can activate multiple downstream signaling pathways (e.g. p70S6K, GRB10, 4E-BP, and others) that may also influence macrophage polarization (
      • Laplante M.
      • Sabatini D.M.
      mTOR signaling at a glance.
      ,
      • Weichhart T.
      Mammalian target of rapamycin: a signaling kinase for every aspect of cellular life.
      ). It is clear from our study that control of IL-4 signaling and M2 gene expression downstream of TORC1 is not all mediated via p70S6K. To that end, we demonstrated that another TORC1-activated protein, GRB10, could regulate IL-4-induced IRS-2 signaling to promote human M2 gene expression. From our co-immunoprecipitation experiments we suggested a second mechanism by which GRB10 bound to receptor subunits that make up the type I IL-4 receptor complex (IL-4Rα, γC) mediates interaction with NEDD4.2. In this way IL-4 signaling could be suppressed by GRB10 by reducing cell surface availability of the type I IL-4 receptor. Future work will address the precise role of GRB10 in regulating this pathway.
      In summary, our work shows for the first time that serine phosphorylation of IRS-2 is an important mechanism for down-regulating activation of IRS-2 and M2 gene expression in response to IL-4. We show that tyrosine phosphorylation of IRS-2 and M2 gene expression is controlled by p70S6K and by GRB10, two proteins activated by TORC1 after IL-4 stimulation. Specifically, p70S6K regulated serine phosphorylation of IRS-2 in response to IL-4 and expression of some, but not all, human M2 macrophage genes. GRB10 also negatively regulated the IL-4-activated IRS-2 pathway and M2 gene expression in human monocytes, likely by regulating cell surface expression of the type I IL-4 receptor. To our knowledge we are the first to show GRB10 binding to the components of the IL-4 receptor complex and its effect on IL-4-induced signaling responses and human M2 macrophage gene expression. Our work may provide new therapeutic avenues where p70S6K or GRB10 activity could be enhanced to prevent M2 macrophage polarization in diseases like asthma.

      Author Contributions

      K. J. W. conceptualized the experiments presented in FIGURE 2., FIGURE 3., FIGURE 4. and provided data acquisition in FIGURE 1., FIGURE 2., FIGURE 3., FIGURE 4., statistical analysis throughout, and manuscript preparation. X. F. provided data acquisition and statistical analysis for FIGURE 2., FIGURE 3.. N. M. G. conceptualized the experiments and provided data acquisition in FIGURE 1., FIGURE 2.. J. J. T. provided data acquisition of Fig. 2. N. M. G. provided funding support, conceptualization of experiments throughout, figure design, and manuscript preparation.

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