Modulation of the Phagosome Proteome by Interferon-γ

Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-γ (IFN-γ), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-γ-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-γ includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-γ-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-γ-activated macrophages. Macrophages are immune cells that function in the clearance of infectious particles. This process involves the engulfment of microbes into phagosomes where these particles are lysed and degraded. In the current study, we used a large scale quantitative proteomics approach to analyze the changes in protein abundance induced on phagosomes by interferon-γ (IFN-γ), an inflammatory cytokine that activates macrophages. Our analysis identified 167 IFN-γ-modulated proteins on phagosomes of which more than 90% were up-regulated. The list of phagosomal proteins regulated by IFN-γ includes proteins expected to alter phagosome maturation, enhance microbe degradation, trigger the macrophage immune response, and promote antigen loading on major histocompatibility complex (MHC) class I molecules. A dynamic analysis of IFN-γ-sensitive proteins by Western blot indicated that newly formed phagosomes display a delayed proteolytic activity coupled to an increased recruitment of the MHC class I peptide-loading complex. These phagosomal conditions may favor antigen presentation by MHC class I molecules on IFN-γ-activated macrophages. The first line of defense against microbial infection involves the direct removal of pathogens by a variety of phagocytic cells including macrophages, polymorphonuclear neutrophils, and dendritic cells. These cells have evolved unique functions enabling them to engulf microorganisms in a specialized organelle, the phagosome, where they are killed and degraded (1Underhill D.M. Ozinsky A. Phagocytosis of microbes: complexity in action.Annu. Rev. Immunol. 2002; 20: 825-852Crossref PubMed Scopus (847) Google Scholar). The innate ability of phagosomes to perform this task relies on the coordinated assembly of a variety of molecular machines through a complex process of organelle maturation (2Gotthardt D. Blancheteau V. Bosserhoff A. Ruppert T. Delorenzi M. Soldati T. Proteomics fingerprinting of phagosome maturation and evidence for the role of a Gα during uptake.Mol. Cell. Proteomics. 2006; 12: 2228-2243Abstract Full Text Full Text PDF Scopus (86) Google Scholar). For instance, newly formed phagosomes acquire the vacuolar ATPase (V-ATPase) 1The abbreviations used are: V-ATPase, vacuolar ATPase; ER, endoplasmic reticulum; IFN-γ, interferon-γ; MHC, major histocompatibility complex; MDS, multidimensional scaling; TLR, Toll-like receptor; mAb, monoclonal antibody; pAb, polyclonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; 2-D, two-dimensional; IGTP, interferon-γ-induced GTPase; GBP, guanylate-binding protein; LRP, low density lipoprotein receptor-related protein; VAMP, vesicle-associated membrane protein. complex, a proton pump that acidifies the phagosome lumen (3Nishi T. Forgac M. The vacuolar (H+)-ATPases: nature's most versatile proton pumps.Nat. Rev. Mol. Cell Biol. 2002; 3: 94-103Crossref PubMed Scopus (1005) Google Scholar). This acidification process activates several lysosomal hydrolases delivered to phagosomes through fusion events with endosomes and lysosomes. Furthermore peptides derived from the degradation of microorganisms are loaded on both MHC class I and class II molecules for their presentation at the cell surface, a process that triggers an efficient adaptive immune response (4Jutras I. Desjardins M. Phagocytosis: at the crossroads of innate and adaptive immunity.Annu. Rev. Cell Dev. Biol. 2005; 21: 511-527Crossref PubMed Scopus (179) Google Scholar). Phagosomes are therefore pivotal platforms in linking both the innate and adaptive immune responses. IFN-γ is a crucial factor in the clearance of infection as impaired production of IFN-γ or defects in the IFN-γ signaling pathway result in increased susceptibility to various bacterial (5Boehm U. Klamp T. Groot M. Howard J.C. Cellular responses to interferon-γ.Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2505) Google Scholar) and viral infections (6Novelli F. Casanova J.L. The role of IL-12, IL-23 and IFN-γ in immunity to viruses.Cytokine Growth Factor Rev. 2004; 5: 367-377Crossref Scopus (88) Google Scholar). During the innate inflammatory response, IFN-γ is produced mainly by natural killer cells and subsets of T lymphocytes, including natural killer T cells and CD8+ T cells (7Schroder K. Hertzog P.J. Ravasi T. Hume D.A. Interferon-γ: an overview of signals, mechanisms and functions.J. Leukoc. Biol. 2004; 75: 163-189Crossref PubMed Scopus (2965) Google Scholar). Production of IFN-γ by these cells is stimulated by interleukin-12, a cytokine secreted by macrophages and dendritic cells in response to microbial stimulation of Toll-like receptors (TLRs) (8Trinchieri G. Interleukin-12 and the regulation of innate resistance and adaptive immunity.Nat. Rev. Immunol. 2003; 3: 133-146Crossref PubMed Scopus (2994) Google Scholar). On binding to its receptor, IFN-γ alters the expression of hundreds of genes in activated macrophages (5Boehm U. Klamp T. Groot M. Howard J.C. Cellular responses to interferon-γ.Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2505) Google Scholar, 9Ehrt S. Schnappinger D. Bekiranov S. Drenkow J. Shi S. Gingeras T.R. Gaasterland T. Schoolnik G. Nathan C. Reprogramming of the macrophage transcriptome in response to interferon-γ and Mycobacterium tuberculosis: signaling roles of nitric oxide synthase-2 and phagocyte oxidase.J. Exp. Med. 2001; 194: 1123-1140Crossref PubMed Scopus (401) Google Scholar, 10Yan W. Lee H. Yi E.C. Reiss D. Shannon P. Kwieciszewski B.K. Coito C. Li X.J. Keller A. Eng J. Galitski T. Goodlett D.R. Aebersold R. Katze M.G. System-based proteomic analysis of the interferon response in human liver cells.Genome Biol. 2004; 5: R54Crossref PubMed Google Scholar) by triggering complex signaling cascades, notably the JAK-STAT (Janus kinase-signal transducers and activators of transcription) signal transduction pathway (7Schroder K. Hertzog P.J. Ravasi T. Hume D.A. Interferon-γ: an overview of signals, mechanisms and functions.J. Leukoc. Biol. 2004; 75: 163-189Crossref PubMed Scopus (2965) Google Scholar). The ensuing IFN-γ-induced protein expression program enhances the microbicidal capacity of macrophages, which also respond to IFN-γ by increasing antigen presentation and by secreting inflammatory cytokines that contribute to the recruitment of immune cells to the site of infection (7Schroder K. Hertzog P.J. Ravasi T. Hume D.A. Interferon-γ: an overview of signals, mechanisms and functions.J. Leukoc. Biol. 2004; 75: 163-189Crossref PubMed Scopus (2965) Google Scholar). As the microbicidal capacity of macrophages involves many phagosome-associated functions, significant modifications in the protein content of phagosomes are thus expected to occur in response to IFN-γ. Several studies have examined the IFN-γ-induced expression program both at the transcriptional and the translational levels (5Boehm U. Klamp T. Groot M. Howard J.C. Cellular responses to interferon-γ.Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2505) Google Scholar, 10Yan W. Lee H. Yi E.C. Reiss D. Shannon P. Kwieciszewski B.K. Coito C. Li X.J. Keller A. Eng J. Galitski T. Goodlett D.R. Aebersold R. Katze M.G. System-based proteomic analysis of the interferon response in human liver cells.Genome Biol. 2004; 5: R54Crossref PubMed Google Scholar, 11Der S.D. Zhou A. Williams B.R. Silverman R.H. Identification of genes differentially regulated by interferon α, β, or γ using oligonucleotide arrays.Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 15623-15628Crossref PubMed Scopus (1544) Google Scholar); however, the IFN-γ-induced protein expression profile has never been assessed on isolated phagosomes. In the present study, we took advantage of a unique proteomics platform developed to perform large scale comparative analyses and characterize the changes occurring to phagosomes in IFN-γ-treated cells. The results obtained reveal a complex series of modifications in a wide range of phagosomal molecular machines coordinated to enhance both innate and adaptive immunity. The rat anti-Lamp1 lumenal ID4B mAb from the The against The a from The a from The and the were from The and were from The mAb from The mAb from The from The from macrophage were in with and at in For IFN-γ in the to the of phagosomes. Phagosomes were formed by the of M. G. H. A. G. of Biol. Full Text PDF PubMed Google Scholar). For or quantitative proteomics cells were to for with and in for For the dynamic analysis of phagosome and were as indicated in Phagosomes were isolated on a as I. A. J. S. G. Desjardins M. is a of Biol. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). using this have been to of major J. S. I. G. C. S. H. P. F. C. Desjardins M. A analysis of the PubMed Scopus Google Scholar). phagosomes or cell were in The of proteins and in the were by and were by to Western which using The obtained from Western were using and IFN-γ-treated were in and first with for at with of using at for The were to and and with at with were to a of to protein were to and at a of in and were using a with a and a using a of in at a of The were to the and were on line with the during loading and line during on the were from a at a of for using of and from both and using a of in at a of to and analysis and as were with using The were into by in both the and by to a and The of is on of and for a to to the of and were to a on the of to that by of and for large from PubMed Scopus Google Scholar) and a than at this or abundance changes were to The were for using a dynamic and and of the This or more or of the or of the analysis and is to the to than peptides were in the and to and using to the proteomics A and were to the of the in the the of peptides that were by IFN-γ. IFN-γ-modulated peptides were on the of their using a and of the to IFN-γ-treated in of The for peptides with these by of the expression and a used to and using developed with a significant in were for by For this the peptides were into an list and for of for using and the for protein A of protein were in that with and with of and were for both and The identified proteins were by using at of the proteins that are are by the identified identified by a with than were The for protein are The for has been identified through of several and of the of with The protein of the IFN-γ-modulated peptides by using The list to and on protein The protein abundance were using an to abundance The platform than is to and protein we have that the and is used for the is by peptides and therefore a more For for a with a of the is This is the of protein abundance the phagosomal proteins were by and identified as J. R. S. S. R. C. Desjardins M. The phagosome into phagosome functions.J. Cell Biol. 2001; PubMed Scopus Google Scholar, M. S. S. G. A. P. D. Desjardins M. Phagosomes are for antigen 2003; PubMed Scopus Google Scholar). isolated phagosomes were in and and for loading in the first by of the first the were in a for and in a for The using The were and the protein were using the were obtained from to abundance changes in protein were and with the peptides with in by using a coupled to a characterize the phagosomal proteins that are by IFN-γ, we used a quantitative proteomics platform to as on of peptides to and J. H. J. M. C. M. P. D. I. cell is with in PubMed Scopus Google Scholar). its ability to low proteins and the protein content of a complex the platform is also as in protein in the scale analysis of the of phagosomes from or IFN-γ-treated macrophages using the platform identified in at of and IFN-γ-treated phagosome of significant in their and were thus as by IFN-γ. the of complex proteomics by the and and in The in content the and IFN-γ-treated were to on the for peptides in the As in the and IFN-γ-treated is This that the peptides and the for are and unique to a the of the IFN-γ-modulated peptides the were is evidence of the unique and of the IFN-γ-modulated proteomics analysis using and IFN-γ-treated of the proteomics using a for the profile of of and the and IFN-γ-treated The of the the peptides the in the and IFN-γ-treated with the most and peptides on the and that a of the IFN-γ-modulated peptides were by IFN-γ. the of the for the of the IFN-γ-modulated peptides as a function of their the peptides to by IFN-γ in this study, were IFN-γ were A of the peptides were increased by or with a and most of the peptides were by or with a and Our thus that macrophage by IFN-γ through the of the significant of peptides a complex of protein by IFN-γ on the The of the IFN-γ-modulated peptides were to and the peptides were to the proteins in I and This process the of IFN-γ-modulated peptides to peptides to the of peptides with low and to with modifications as and These IFN-γ-modulated peptides to that were and that were by IFN-γ I and proteins more than and of proteins peptides with that were with a of are several peptides to the protein may in the of regulation including or in The is for protein in and the identified are in the obtained using of proteins in the IFN-γ-treated were and the As in the peptides to a of by IFN-γ. peptides were for the and these a of Western blot analysis the of both and on phagosomes from IFN-γ-treated macrophages and phagosome proteins identified by the large scale quantitative proteomics protein to to the factor membrane as into of MHC class I molecules. in antigen also as the assembly of MHC class I the assembly of MHC class I as protein and as cell as a phagocytic for interferon as cell as the in the of to I factor in in the assembly of MHC class I by of as an membrane protein of protein of protein of as as guanylate-binding protein protein as as in antigen protein as guanylate-binding protein protein as membrane protein during to the to the to the to the protein A membrane membrane as membrane protein in and to the induced density lipoprotein receptor-related protein. as as a in the of complex functions in class I in antigen as to as a protein that and as as a protein for to the a fusion cell as a in the in and regulation of during lysosomal and and membrane protein to the in the assembly of MHC class I to the and in an to and phagosomal to endosomes and to the and function in membrane the and to with the with the to the protein as as a protein as in complex functions in function in protein function as in membrane fusion in membrane fusion in membrane fusion and and membrane protein. to endosomes and factor in membrane fusion in in membrane fusion events in the with in membrane fusion events in the in membrane fusion events in the to to several and in various and in innate immunity. to viral in innate immunity. to viral in innate immunity. to bacterial protein in the of of protein protein in membrane fusion events endosomes and as in membrane fusion events and A A in phagosome a through the membrane that of protein protein protein protein protein protein protein in a phagosome proteins identified by the large scale quantitative proteomics factor protein protein protein protein A class I fusion membrane protein protein protein factor protein protein protein A A protein protein protein protein protein protein protein in a and the IFN-γ-modulated proteins identified with we used As in phagosomes from or IFN-γ-treated macrophages complex protein protein abundance were obtained by the of from A of were and a of at a in protein abundance IFN-γ to proteins were identified by analysis of the proteins identified by analysis also been identified using the proteomics The in the proteins identified by analysis and by quantitative proteomics analysis to the and of both proteomics For instance, the of the approach an in of on IFN-γ-treated most to differentially of the The however, to membrane a of this In more than of the IFN-γ-modulated proteins identified by the platform were membrane The of membrane proteins in phagosomes is expected to that the large scale approach efficient in membrane these results the of the IFN-γ-modulated proteins identified using phagosome proteins identified by protein the assembly of MHC class I to as an as I. in the removal of the from MHC class II protein in the assembly of MHC class I an in antigen protein as as an of genes and factor receptors to the signaling in the assembly of MHC class I protein protein protein the of the as lysosomal protein and during lysosomal in the of in antigen for MHC class I in a The IFN-γ-modulated proteins identified in proteomics analysis and were for their in as in The protein that from of the IFN-γ-modulated proteins and were by proteins identified in proteomics This the of the proteins that we identified as regulated by IFN-γ and that of these may by IFN-γ. Our proteomics analysis identified many proteins with the microbicidal function of with the of IFN-γ on macrophages (5Boehm U. Klamp T. Groot M. Howard J.C. Cellular responses to interferon-γ.Annu. Rev. Immunol. 1997; 15: 749-795Crossref PubMed Scopus (2505) Google Scholar, K. Hertzog P.J. Ravasi T. Hume D.A. Interferon-γ: an overview of signals, mechanisms and functions.J. Leukoc. Biol. 2004; 75: 163-189Crossref PubMed Scopus (2965) Google Scholar). The proteins in and more than lysosomal of the complex, the proton pump that acidifies the phagosome and of the protein complex that phagosomes. IFN-γ several phagosomal proteins in fusion with including and as and function in phagosome maturation S. and function at during Immunol. 2002; PubMed Scopus Google Scholar, A. J. phagosome maturation in Cell Sci. 2001; Google to in the and of T. H. P. M. A role for the lysosomal membrane protein in the and of and lysosomal Cell Sci. 2002; PubMed Scopus Google and functions in the fusion and endosomes W. C. R. S. R. The fusion of endosomes and Biol. Cell. PubMed Scopus Google Scholar). has been to occur on isolated phagosomes and to fusion with endosomes R. M. A. M. P. M. P. A. H. G. and a role for in fusion Biol. Cell. 2004; 15: PubMed Scopus Google a process that may a protein that assembly on U. V. 2004; 5: PubMed Scopus Google Scholar). The of on phagosomes from IFN-γ-treated cells may also promote phagosome maturation as of and as results in increased I. S. is to and their Cell Sci. 2006; PubMed Scopus Google Scholar). these that IFN-γ alters the of phagosomes endosomes and and therefore phagosome Phagosomes from IFN-γ-treated cells were to have increased levels of several IFN-γ-induced notably of the and These roles in innate immunity against bacterial and infections of by 2003; PubMed Scopus Google Scholar, S. I. J. G. G. G. Howard J.C. of by the 2005; PubMed Scopus Google Scholar). The mechanisms for the of against infections are however, are to function in phagosome maturation of by 2003; PubMed Scopus Google Scholar, V. to 2006; PubMed Scopus Google Scholar). The are and to endoplasmic or in IFN-γ-treated however, during to phagosomes S. K. R. R. Howard J.C. the of resistance and on to membrane induced by Immunol. 2004; PubMed Scopus Google Scholar). is IFN-γ-induced that we identified for the first on phagosomes. to the of large and display The function of is they activity The guanylate-binding proteins of the with unique 2005; PubMed Scopus Google Scholar). IFN-γ several phagosomal proteins that are in the of The IFN-γ-modulated proteins on phagosomes several receptors and signaling as a protein to receptors to the I. A. M. R. S. P. G. J. C. F. The with a protein Immunol. 2005; PubMed Scopus Google Scholar). and the receptor, were to by IFN-γ on phagosomes. The in a in the of cells A. D. and binding of cell and and of Exp. Med. 2001; 194: PubMed Scopus Google may that activated macrophages their of cells in inflammatory The of the is with the role of IFN-γ in the of as an that the of A. Mycobacterium from both and of interferon-γ and Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The IFN-γ-modulated receptors on phagosomes also Toll-like receptors and in the immune response to These to bacterial or viral present in in to as that to microbial at the cell In line with the of a protein to function as an for J. Li protein is a for receptors and is in the innate function of Full Text Full Text PDF PubMed Scopus Google by IFN-γ on phagosomes. a in the production of and molecular Rev. PubMed Scopus Google also on phagosomes IFN-γ These results that phagosomal proteins a role in several of the macrophage immune response to IFN-γ. The of antigen presentation is a of IFN-γ on macrophages and phagosomes from IFN-γ-treated cells increased levels of several proteins in MHC class I antigen presentation (7Schroder K. Hertzog P.J. Ravasi T. Hume D.A. Interferon-γ: an overview of signals, mechanisms and functions.J. Leukoc. Biol. 2004; 75: 163-189Crossref PubMed Scopus (2965) Google Scholar). As in phagosomes from IFN-γ-treated cells increased levels of a MHC class I molecules of an and of and of the MHC class I peptide-loading complex and P. Cellular mechanisms of Immunol. 2004; 5: PubMed Scopus Google Scholar, Lee S. K. S. K. regulation by MHC class I during antigen 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). Several studies have that the of these proteins to phagosomes a pathway that to the loading and presentation of on MHC class I a process to as antigen M. S. S. G. A. P. D. Desjardins M. Phagosomes are for antigen 2003; PubMed Scopus Google Scholar, P. M. J. P. S. fusion an MHC class I in dendritic 2003; PubMed Scopus Google Scholar, C. R. P. phagosomes in dendritic cells a for presentation of Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). IFN-γ-activated macrophages are thus expected to antigen The large scale proteomics analysis has to functions on phagosomes by IFN-γ, as microbe protein degradation, and antigen the analysis on a formed by a by a in the phagosome maturation however, with various as they into a process that their dynamic (2Gotthardt D. Blancheteau V. Bosserhoff A. Ruppert T. Delorenzi M. Soldati T. Proteomics fingerprinting of phagosome maturation and evidence for the role of a Gα during uptake.Mol. Cell. Proteomics. 2006; 12: 2228-2243Abstract Full Text Full Text PDF Scopus (86) Google Scholar). a more of the changes occurring on IFN-γ-treated a of on phagosomes from or IFN-γ-treated macrophages by Western As in proteins were during and proteins to IFN-γ at during phagosome on phagosomes from cells to levels at in phagosome maturation The in an that functions in membrane that IFN-γ may phagosome fusion with to infection is on the function of the in phagocytic cells Williams D.A. Li J. of an in phagocyte PubMed Scopus Google Scholar). In macrophages, the also on phagosomes by IFN-γ The complex that in the phagosome the acidification of phagosomes in various phagocytic cells M. R. A. R. The of phagocytic cells is with a in vacuolar PubMed Scopus Google Scholar, A. C. S. P. P. G. S. phagosomal to antigen during by dendritic 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). The recruitment of this complex to phagosomes may thus the acidification by the proton In with this and A. the capacity of the PubMed Scopus Google Scholar) have that IFN-γ the acidification of phagosomes. The function of the in phagosomal has also been to phagosomal conditions that antigen degradation and favor antigen A. C. S. P. P. G. S. phagosomal to antigen during by dendritic 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). In with this proteomics analysis the of several of MHC class I peptide-loading complex In the dynamic of these and were increased by IFN-γ the phagosome maturation process In an protein in antigen a dynamic profile with in abundance and IFN-γ-treated phagosomes These results thus that IFN-γ the of the MHC class I peptide-loading complex on phagosomes. MHC class I are by the proteolytic activity of IFN-γ is to subsets of that including P. of MHC class antigen Rev. Immunol. 1998; PubMed Scopus Google Scholar). As in IFN-γ increased the recruitment of the on phagosomes as M. S. S. G. A. P. D. Desjardins M. Phagosomes are for antigen 2003; PubMed Scopus Google Scholar). IFN-γ macrophages a delayed of and of the lysosomal with the that IFN-γ the capacity of phagosomes A. the capacity of the PubMed Scopus Google Scholar). The in phagosome maturation the efficient recruitment of a that fusion that may phagosome fusion events in IFN-γ-treated cells. an protein in is factor as the recruitment of has been to phagosome maturation I. J. J. V. is for of Mycobacterium phagosome maturation J. 2006; PubMed Scopus Google Scholar). the dynamic analysis of this of phagosomal proteins that IFN-γ enhances the conditions that favor the production of and their loading on MHC class molecules as in The current the of a quantitative large scale proteomics approach to of the response to In has to functions that are by IFN-γ on phagosomes and to in these IFN-γ proteins in phagosome maturation, microbe degradation, innate immune response, and antigen these we on the dynamic of a of IFN-γ-modulated proteins during phagosome maturation to the from the proteomics this approach to a the production of MHC class I molecules in phagosomes of IFN-γ-activated macrophages. for and for the with

Récupéré en direct depuis OpenAlex et désinversé. Les résumés ne sont pas conservés dans cette base de données : les index inversés représentent 8,6 Go des 9,3 Go de texte de la base, et le serveur dispose de 13 Go libres.