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1.
Carbohydr Polym ; 339: 122256, 2024 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-38823922

RESUMO

Recently, the intestinal lymphatic transport based on Peyer's patches (PPs) is emerging as a promising absorption pathway for natural polysaccharides. Herein, the aim of this study is to investigate the PP-based oral absorption of a pectic polysaccharide from Smilax china L. (SCLP), as well as its uptake and transport mechanisms in related immune cells. Taking advantages of the traceability of fluorescently labeled SCLP, we confirmed that SCLP could be absorbed into PPs and captured by their mononuclear phagocytes (dendritic cells and macrophages) following oral administration. Subsequently, the systematic in vitro study suggested that the endocytic mechanisms of SCLP by model mononuclear phagocytes (BMDCs and RAW264.7 cells) mainly involved caveolae-mediated endocytosis, macropinocytosis and phagocytosis. More importantly, SCLP directly binds and interacts with toll-like receptor 2 (TLR2) and galectin 3 (Gal-3) receptor, and was taken up by mononuclear phagocytes in receptor-mediated manner. After internalization, SCLP was intracellularly transported primarily through endolysosomal pathway and ultimately localized in lysosomes. In summary, this work reveals novel information and perspectives about the in vivo fate of SCLP, which will contribute to further research and utilization of SCLP and other pectic polysaccharides.


Assuntos
Nódulos Linfáticos Agregados , Smilax , Animais , Camundongos , Células RAW 264.7 , Nódulos Linfáticos Agregados/metabolismo , Smilax/química , Endocitose , Pectinas/química , Pectinas/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Fagocitose/efeitos dos fármacos , Fagócitos/metabolismo , Fagócitos/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo , Camundongos Endogâmicos BALB C , Masculino , Células Dendríticas/metabolismo , Células Dendríticas/efeitos dos fármacos , Administração Oral
2.
Proc Natl Acad Sci U S A ; 121(25): e2312499121, 2024 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-38857395

RESUMO

Ex vivo expansion of human CD34+ hematopoietic stem and progenitor cells remains a challenge due to rapid differentiation after detachment from the bone marrow niche. In this study, we assessed the capacity of an inducible fusion protein to enable sustained ex vivo proliferation of hematopoietic precursors and their capacity to differentiate into functional phagocytes. We fused the coding sequences of an FK506-Binding Protein 12 (FKBP12)-derived destabilization domain (DD) to the myeloid/lymphoid lineage leukemia/eleven nineteen leukemia (MLL-ENL) fusion gene to generate the fusion protein DD-MLL-ENL and retrovirally expressed the protein switch in human CD34+ progenitors. Using Shield1, a chemical inhibitor of DD fusion protein degradation, we established large-scale and long-term expansion of late monocytic precursors. Upon Shield1 removal, the cells lost self-renewal capacity and spontaneously differentiated, even after 2.5 y of continuous ex vivo expansion. In the absence of Shield1, stimulation with IFN-γ, LPS, and GM-CSF triggered terminal differentiation. Gene expression analysis of the obtained phagocytes revealed marked similarity with naïve monocytes. In functional assays, the novel phagocytes migrated toward CCL2, attached to VCAM-1 under shear stress, produced reactive oxygen species, and engulfed bacterial particles, cellular particles, and apoptotic cells. Finally, we demonstrated Fcγ receptor recognition and phagocytosis of opsonized lymphoma cells in an antibody-dependent manner. Overall, we have established an engineered protein that, as a single factor, is useful for large-scale ex vivo production of human phagocytes. Such adjustable proteins have the potential to be applied as molecular tools to produce functional immune cells for experimental cell-based approaches.


Assuntos
Diferenciação Celular , Fagócitos , Humanos , Fagócitos/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteína de Leucina Linfoide-Mieloide/metabolismo , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia/genética , Leucemia/patologia , Leucemia/metabolismo , Engenharia de Proteínas/métodos , Fagocitose
3.
Sci Adv ; 10(23): eadl6083, 2024 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-38838151

RESUMO

Hepatocellular carcinoma (HCC) acquires an immunosuppressive microenvironment, leading to unbeneficial therapeutic outcomes. Hyaluronan-mediated motility receptor (HMMR) plays a crucial role in tumor progression. Here, we found that aberrant expression of HMMR could be a predictive biomarker for the immune suppressive microenvironment of HCC, but the mechanism remains unclear. We established an HMMR-/- liver cancer mouse model to elucidate the HMMR-mediated mechanism of the dysregulated "don't eat me" signal. HMMR knockout inhibited liver cancer growth and induced phagocytosis. HMMRhigh liver cancer cells escaped from phagocytosis via sustaining CD47 signaling. Patients with HMMRhighCD47high expression showed a worse prognosis than those with HMMRlowCD47low expression. HMMR formed a complex with FAK/SRC in the cytoplasm to activate NF-κB signaling, which could be independent of membrane interaction with CD44. Notably, targeting HMMR could enhance anti-PD-1 treatment efficiency by recruiting CD8+ T cells. Overall, our data revealed a regulatory mechanism of the "don't eat me" signal and knockdown of HMMR for enhancing anti-PD-1 treatment.


Assuntos
Antígeno CD47 , Carcinoma Hepatocelular , Receptores de Hialuronatos , Neoplasias Hepáticas , Fagócitos , Fagocitose , Animais , Humanos , Camundongos , Carcinoma Hepatocelular/patologia , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/genética , Antígeno CD47/metabolismo , Antígeno CD47/genética , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linhagem Celular Tumoral , Quinase 1 de Adesão Focal/metabolismo , Quinase 1 de Adesão Focal/genética , Receptores de Hialuronatos/metabolismo , Receptores de Hialuronatos/genética , Evasão da Resposta Imune , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/imunologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/genética , Camundongos Knockout , NF-kappa B/metabolismo , Fagócitos/metabolismo , Fagócitos/imunologia , Transdução de Sinais , Evasão Tumoral , Microambiente Tumoral/imunologia
4.
Front Immunol ; 15: 1401294, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38720899

RESUMO

Inhibitory natural killer (NK) cell receptors recognize MHC class I (MHC-I) in trans on target cells and suppress cytotoxicity. Some NK cell receptors recognize MHC-I in cis, but the role of this interaction is uncertain. Ly49Q, an atypical Ly49 receptor expressed in non-NK cells, binds MHC-I in cis and mediates chemotaxis of neutrophils and type I interferon production by plasmacytoid dendritic cells. We identified a lipid-binding motif in the juxtamembrane region of Ly49Q and found that Ly49Q organized functional membrane domains comprising sphingolipids via sulfatide binding. Ly49Q recruited actin-remodeling molecules to an immunoreceptor tyrosine-based inhibitory motif, which enabled the sphingolipid-enriched membrane domain to mediate complicated actin remodeling at the lamellipodia and phagosome membranes during phagocytosis. Thus, Ly49Q facilitates integrative regulation of proteins and lipid species to construct a cell type-specific membrane platform. Other Ly49 members possess lipid binding motifs; therefore, membrane platform organization may be a primary role of some NK cell receptors.


Assuntos
Esfingolipídeos , Animais , Humanos , Esfingolipídeos/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Fagocitose , Fagócitos/imunologia , Fagócitos/metabolismo , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Membrana Celular/metabolismo , Ligação Proteica
5.
EMBO Rep ; 25(6): 2550-2570, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38730209

RESUMO

Transmembrane protein 268 (TMEM268) is a novel, tumor growth-related protein first reported by our laboratory. It interacts with the integrin subunit ß4 (ITGB4) and plays a positive role in the regulation of the ITGB4/PLEC signaling pathway. Here, we investigated the effects and mechanism of TMEM268 in anti-infectious immune response in mice. Tmem268 knockout in mice aggravated cecal ligation and puncture-induced sepsis, as evidenced by higher bacterial burden in various tissues and organs, congestion, and apoptosis. Moreover, Tmem268 deficiency in mice inhibited phagocyte adhesion and migration, thus decreasing phagocyte infiltration at the site of infection and complement-dependent phagocytosis. Further findings indicated that TMEM268 interacts with CD11b and inhibits its degradation via the endosome-lysosome pathway. Our results reveal a positive regulatory role of TMEM268 in ß2 integrin-associated anti-infectious immune responses and signify the potential value of targeting the TMEM268-CD11b signaling axis for the maintenance of immune homeostasis and immunotherapy for sepsis and related immune disorders.


Assuntos
Antígeno CD11b , Proteínas de Membrana , Camundongos Knockout , Sepse , Transdução de Sinais , Animais , Camundongos , Antígeno CD11b/metabolismo , Antígeno CD11b/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Sepse/genética , Sepse/imunologia , Sepse/metabolismo , Fagocitose , Adesão Celular/genética , Regulação para Baixo , Movimento Celular/genética , Deleção de Genes , Fagócitos/metabolismo , Fagócitos/imunologia , Camundongos Endogâmicos C57BL , Humanos , Lisossomos/metabolismo , Endossomos/metabolismo
6.
Cells ; 13(10)2024 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-38786045

RESUMO

Macrophages and microglia are professional phagocytes that sense and migrate toward "eat-me" signals. The role of phagocytic cells is to maintain homeostasis by engulfing senescent or apoptotic cells, debris, and abnormally aggregated macromolecules. Usually, dying cells send out "find-me" signals, facilitating the recruitment of phagocytes. Healthy cells can also promote or inhibit the phagocytosis phenomenon of macrophages and microglia by tuning the balance between "eat-me" and "don't-eat-me" signals at different stages in their lifespan, while the "don't-eat-me" signals are often hijacked by tumor cells as a mechanism of immune evasion. Using a combination of bioinformatic analysis and spatial profiling, we delineate the balance of the "don't-eat-me" CD47/SIRPα and "eat-me" CALR/STC1 ligand-receptor interactions to guide therapeutic strategies that are being developed for glioblastoma sequestered in the central nervous system (CNS).


Assuntos
Antígeno CD47 , Calreticulina , Glioblastoma , Fagócitos , Fagocitose , Humanos , Glioblastoma/patologia , Glioblastoma/terapia , Glioblastoma/metabolismo , Antígeno CD47/metabolismo , Fagócitos/metabolismo , Calreticulina/metabolismo , Receptores Imunológicos/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Microglia/metabolismo , Microglia/patologia , Morte Celular , Animais , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Antígenos de Diferenciação
7.
mBio ; 15(6): e0086224, 2024 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-38767353

RESUMO

Mammalian target of rapamycin (mTOR) is a key regulator of metabolism in the mammalian cell. Here, we show the essential role for mTOR signaling in the immune response to bacterial infection. Inhibition of mTOR during infection with Staphylococcus aureus revealed that mTOR signaling is required for bactericidal free radical production by phagocytes. Mechanistically, mTOR supported glucose transporter GLUT1 expression, potentially through hypoxia-inducible factor 1α, upon phagocyte activation. Cytokine and chemokine signaling, inducible nitric oxide synthase, and p65 nuclear translocation were present at similar levels during mTOR suppression, suggesting an NF-κB-independent role for mTOR signaling in the immune response during bacterial infection. We propose that mTOR signaling primarily mediates the metabolic requirements necessary for phagocyte bactericidal free radical production. This study has important implications for the metabolic requirements of innate immune cells during bacterial infection as well as the clinical use of mTOR inhibitors.IMPORTANCESirolimus, everolimus, temsirolimus, and similar are a class of pharmaceutics commonly used in the clinical treatment of cancer and the anti-rejection of transplanted organs. Each of these agents suppresses the activity of the mammalian target of rapamycin (mTOR), a master regulator of metabolism in human cells. Activation of mTOR is also involved in the immune response to bacterial infection, and treatments that inhibit mTOR are associated with increased susceptibility to bacterial infections in the skin and soft tissue. Infections caused by Staphylococcus aureus are among the most common and severe. Our study shows that this susceptibility to S. aureus infection during mTOR suppression is due to an impaired function of phagocytic immune cells responsible for controlling bacterial infections. Specifically, we observed that mTOR activity is required for phagocytes to produce antimicrobial free radicals. These results have important implications for immune responses during clinical treatments and in disease states where mTOR is suppressed.


Assuntos
Transportador de Glucose Tipo 1 , Fagócitos , Transdução de Sinais , Infecções Estafilocócicas , Staphylococcus aureus , Serina-Treonina Quinases TOR , Staphylococcus aureus/imunologia , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/microbiologia , Fagócitos/imunologia , Fagócitos/metabolismo , Fagócitos/microbiologia , Humanos , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , Animais , Radicais Livres/metabolismo , Camundongos , Camundongos Endogâmicos C57BL
8.
Proc Natl Acad Sci U S A ; 121(23): e2320388121, 2024 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-38805284

RESUMO

Essential for reactive oxygen species (EROS) protein is a recently identified molecular chaperone of NOX2 (gp91phox), the catalytic subunit of phagocyte NADPH oxidase. Deficiency in EROS is a recently identified cause for chronic granulomatous disease, a genetic disorder with recurrent bacterial and fungal infections. Here, we report a cryo-EM structure of the EROS-NOX2-p22phox heterotrimeric complex at an overall resolution of 3.56Å. EROS and p22phox are situated on the opposite sides of NOX2, and there is no direct contact between them. EROS associates with NOX2 through two antiparallel transmembrane (TM) α-helices and multiple ß-strands that form hydrogen bonds with the cytoplasmic domain of NOX2. EROS binding induces a 79° upward bend of TM2 and a 48° backward rotation of the lower part of TM6 in NOX2, resulting in an increase in the distance between the two hemes and a shift of the binding site for flavin adenine dinucleotide (FAD). These conformational changes are expected to compromise superoxide production by NOX2, suggesting that the EROS-bound NOX2 is in a protected state against activation. Phorbol myristate acetate, an activator of NOX2 in vitro, is able to induce dissociation of NOX2 from EROS with concurrent increase in FAD binding and superoxide production in a transfected COS-7 model. In differentiated neutrophil-like HL-60, the majority of NOX2 on the cell surface is dissociated with EROS. Further studies are required to delineate how EROS dissociates from NOX2 during its transport to cell surface, which may be a potential mechanism for regulation of NOX2 activation.


Assuntos
Microscopia Crioeletrônica , NADPH Oxidase 2 , NADPH Oxidases , Fagócitos , Humanos , NADPH Oxidase 2/metabolismo , NADPH Oxidase 2/genética , NADPH Oxidase 2/química , Fagócitos/metabolismo , NADPH Oxidases/metabolismo , NADPH Oxidases/genética , NADPH Oxidases/química , Ligação Proteica , Sítios de Ligação , Doença Granulomatosa Crônica/metabolismo , Doença Granulomatosa Crônica/genética , Modelos Moleculares , Espécies Reativas de Oxigênio/metabolismo
9.
Front Immunol ; 15: 1372904, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38742116

RESUMO

Introduction: The California purple sea urchin, Strongylocentrotus purpuratus, relies solely on an innate immune system to combat the many pathogens in the marine environment. One aspect of their molecular defenses is the SpTransformer (SpTrf) gene family that is upregulated in response to immune challenge. The gene sequences are highly variable both within and among animals and likely encode thousands of SpTrf isoforms within the sea urchin population. The native SpTrf proteins bind foreign targets and augment phagocytosis of a marine Vibrio. A recombinant (r)SpTrf-E1-Ec protein produced by E. coli also binds Vibrio but does not augment phagocytosis. Methods: To address the question of whether other rSpTrf isoforms function as opsonins and augment phagocytosis, six rSpTrf proteins were expressed in insect cells. Results: The rSpTrf proteins are larger than expected, are glycosylated, and one dimerized irreversibly. Each rSpTrf protein cross-linked to inert magnetic beads (rSpTrf::beads) results in different levels of surface binding and phagocytosis by phagocytes. Initial analysis shows that significantly more rSpTrf::beads associate with cells compared to control BSA::beads. Binding specificity was verified by pre-incubating the rSpTrf::beads with antibodies, which reduces the association with phagocytes. The different rSpTrf::beads show significant differences for cell surface binding and phagocytosis by phagocytes. Furthermore, there are differences among the three distinct types of phagocytes that show specific vs. constitutive binding and phagocytosis. Conclusion: These findings illustrate the complexity and effectiveness of the sea urchin innate immune system driven by the natSpTrf proteins and the phagocyte cell populations that act to neutralize a wide range of foreign pathogens.


Assuntos
Fagócitos , Fagocitose , Proteínas Recombinantes , Animais , Fagocitose/imunologia , Fagócitos/imunologia , Fagócitos/metabolismo , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Ligação Proteica , Strongylocentrotus purpuratus/imunologia , Strongylocentrotus purpuratus/genética , Imunidade Inata , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Ouriços-do-Mar/imunologia , Vibrio/imunologia , Proteínas Opsonizantes/metabolismo , Proteínas Opsonizantes/imunologia
10.
Front Immunol ; 15: 1379798, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38756777

RESUMO

Introduction: Cryptosporidiosis is a poorly controlled zoonosis caused by an intestinal parasite, Cryptosporidium parvum, with a high prevalence in livestock (cattle, sheep, and goats). Young animals are particularly susceptible to this infection due to the immaturity of their intestinal immune system. In a neonatal mouse model, we previously demonstrated the importance of the innate immunity and particularly of type 1 conventional dendritic cells (cDC1) among mononuclear phagocytes (MPs) in controlling the acute phase of C. parvum infection. These immune populations are well described in mice and humans, but their fine characterization in the intestine of young ruminants remained to be further explored. Methods: Immune cells of the small intestinal Peyer's patches and of the distal jejunum were isolated from naive lambs and calves at different ages. This was followed by their fine characterization by flow cytometry and transcriptomic analyses (q-RT-PCR and single cell RNAseq (lamb cells)). Newborn animals were infected with C. parvum, clinical signs and parasite burden were quantified, and isolated MP cells were characterized by flow cytometry in comparison with age matched control animals. Results: Here, we identified one population of macrophages and three subsets of cDC (cDC1, cDC2, and a minor cDC subset with migratory properties) in the intestine of lamb and calf by phenotypic and targeted gene expression analyses. Unsupervised single-cell transcriptomic analysis confirmed the identification of these four intestinal MP subpopulations in lamb, while highlighting a deeper diversity of cell subsets among monocytic and dendritic cells. We demonstrated a weak proportion of cDC1 in the intestine of highly susceptible newborn lambs together with an increase of these cells within the first days of life and in response to the infection. Discussion: Considering cDC1 importance for efficient parasite control in the mouse model, one may speculate that the cDC1/cDC2 ratio plays also a key role for the efficient control of C. parvum in young ruminants. In this study, we established the first fine characterization of intestinal MP subsets in young lambs and calves providing new insights for comparative immunology of the intestinal MP system across species and for future investigations on host-Cryptosporidium interactions in target species.


Assuntos
Criptosporidiose , Cryptosporidium parvum , Homeostase , Animais , Criptosporidiose/imunologia , Criptosporidiose/parasitologia , Cryptosporidium parvum/imunologia , Ovinos , Bovinos , Homeostase/imunologia , Células Dendríticas/imunologia , Células Dendríticas/parasitologia , Fagócitos/imunologia , Fagócitos/parasitologia , Animais Recém-Nascidos , Doenças dos Ovinos/parasitologia , Doenças dos Ovinos/imunologia , Nódulos Linfáticos Agregados/imunologia , Nódulos Linfáticos Agregados/parasitologia , Macrófagos/imunologia , Macrófagos/parasitologia , Intestinos/parasitologia , Intestinos/imunologia , Ruminantes/parasitologia , Ruminantes/imunologia
11.
Methods Mol Biol ; 2775: 195-209, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38758319

RESUMO

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an encapsulated fungal pathogen found ubiquitously in the environment that causes pneumonia and life-threatening infections of the central nervous system. Following inhalation of yeasts or desiccated basidiospores into the lung alveoli, resident pulmonary phagocytic cells aid in the identification and eradication of Cryptococcus yeast through their arsenal of pattern recognition receptors (PRRs). PRRs recognize conserved pathogen-associated molecular patterns (PAMPs), such as branched mannans, ß-glucans, and chitins that are the major components of the fungal cell wall. However, the key receptors/ligand interactions required for cryptococcal recognition and eventual fungal clearance have yet to be elucidated. Here we present an imaging flow cytometer (IFC) method that offers a novel quantitative cellular imaging and population statistics tool to accurately measure phagocytosis of fungal cells. It has the capacity to measure two distinct steps of phagocytosis: association/attachment and internalization in a high-throughput and quantitative manner that is difficult to achieve with other technologies. Results from these IFC studies allow for the potential to identify PRRs required for recognition, uptake, and subsequent activation of cytokine production, as well as other effector cell responses required for fungal clearance.


Assuntos
Cryptococcus neoformans , Citometria de Fluxo , Fagocitose , Citometria de Fluxo/métodos , Cryptococcus neoformans/metabolismo , Animais , Camundongos , Fagócitos/metabolismo , Fagócitos/microbiologia , Criptococose/microbiologia , Criptococose/metabolismo , Criptococose/imunologia , Cryptococcus/metabolismo , Humanos , Citometria por Imagem/métodos , Receptores de Reconhecimento de Padrão/metabolismo
12.
mBio ; 15(5): e0342923, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38624208

RESUMO

The Hippo kinases MST1 and MST2 initiate a highly conserved signaling cascade called the Hippo pathway that limits organ size and tumor formation in animals. Intriguingly, pathogens hijack this host pathway during infection, but the role of MST1/2 in innate immune cells against pathogens is unclear. In this report, we generated Mst1/2 knockout macrophages to investigate the regulatory activities of the Hippo kinases in immunity. Transcriptomic analyses identified differentially expressed genes (DEGs) regulated by MST1/2 that are enriched in biological pathways, such as systemic lupus erythematosus, tuberculosis, and apoptosis. Surprisingly, pharmacological inhibition of the downstream components LATS1/2 in the canonical Hippo pathway did not affect the expression of a set of immune DEGs, suggesting that MST1/2 control these genes via alternative inflammatory Hippo signaling. Moreover, MST1/2 may affect immune communication by influencing the release of cytokines, including TNFα, CXCL10, and IL-1ra. Comparative analyses of the single- and double-knockout macrophages revealed that MST1 and MST2 differentially regulate TNFα release and expression of the immune transcription factor MAF, indicating that the two homologous Hippo kinases individually play a unique role in innate immunity. Notably, both MST1 and MST2 can promote apoptotic cell death in macrophages upon stimulation. Lastly, we demonstrate that the Hippo kinases are critical factors in mammalian macrophages and single-cell amoebae to restrict infection by Legionella pneumophila, Escherichia coli, and Pseudomonas aeruginosa. Together, these results uncover non-canonical inflammatory Hippo signaling in macrophages and the evolutionarily conserved role of the Hippo kinases in the anti-microbial defense of eukaryotic hosts. IMPORTANCE: Identifying host factors involved in susceptibility to infection is fundamental for understanding host-pathogen interactions. Clinically, individuals with mutations in the MST1 gene which encodes one of the Hippo kinases experience recurrent infection. However, the impact of the Hippo kinases on innate immunity remains largely undetermined. This study uses mammalian macrophages and free-living amoebae with single- and double-knockout in the Hippo kinase genes and reveals that the Hippo kinases are the evolutionarily conserved determinants of host defense against microbes. In macrophages, the Hippo kinases MST1 and MST2 control immune activities at multiple levels, including gene expression, immune cell communication, and programmed cell death. Importantly, these activities controlled by MST1 and MST2 in macrophages are independent of the canonical Hippo cascade that is known to limit tissue growth and tumor formation. Together, these findings unveil a unique inflammatory Hippo signaling pathway that plays an essential role in innate immunity.


Assuntos
Via de Sinalização Hippo , Imunidade Inata , Macrófagos , Proteínas Serina-Treonina Quinases , Serina-Treonina Quinase 3 , Transdução de Sinais , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Camundongos , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/metabolismo , Fagócitos/imunologia , Fagócitos/microbiologia , Fagócitos/metabolismo , Camundongos Knockout , Infecções Bacterianas/imunologia , Infecções Bacterianas/microbiologia , Infecções Bacterianas/genética , Perfilação da Expressão Gênica , Camundongos Endogâmicos C57BL , Pseudomonas aeruginosa/imunologia
13.
Sci Signal ; 17(834): eadq0353, 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38687824

RESUMO

Displacement of the glycocalyx by membrane blebbing enables macrophages to recognize apoptotic cells.


Assuntos
Apoptose , Glicocálix , Macrófagos , Humanos , Glicocálix/metabolismo , Animais , Macrófagos/metabolismo , Macrófagos/citologia , Fagócitos/metabolismo , Fagócitos/citologia , Fagocitose , Camundongos
14.
Cell Rep Med ; 5(5): 101528, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38677283

RESUMO

Stimulator of interferon genes (STING)-dependent signaling is requisite for effective anti-microbial and anti-tumor activity. STING signaling is commonly defective in cancer cells, which enables tumor cells to evade the immunosurveillance system. We evaluate here whether intrinsic STING signaling in such tumor cells could be reconstituted by creating recombinant herpes simplex viruses (rHSVs) that express components of the STING signaling pathway. We observe that rHSVs expressing STING and/or cGAS replicate inefficiently yet retain in vivo anti-tumor activity, independent of oncolytic activity requisite on the trans-activation of extrinsic STING signaling in phagocytes by engulfed microbial dsDNA species. Accordingly, the in vivo effects of virotherapy could be simulated by nanoparticles incorporating non-coding dsDNA species, which comparably elicit the trans-activation of phagocytes and augment the efficacy of established cancer treatments including checkpoint inhibition and radiation therapy. Our results help elucidate mechanisms of virotherapeutic anti-tumor activity as well as provide alternate strategies to treat cancer.


Assuntos
DNA , Fagócitos , Animais , Fagócitos/imunologia , Fagócitos/metabolismo , Humanos , Camundongos , DNA/metabolismo , DNA/imunologia , DNA/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Transdução de Sinais , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Linhagem Celular Tumoral , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Neoplasias/genética , Simplexvirus/genética , Simplexvirus/imunologia , Camundongos Endogâmicos C57BL , Terapia Viral Oncolítica/métodos
15.
J Neuroinflammation ; 21(1): 92, 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38610019

RESUMO

Glial cells are key players in the initiation of innate immunity in neurodegeneration. Upon damage, they switch their basal activation state and acquire new functions in a context and time-dependent manner. Since modulation of neuroinflammation is becoming an interesting approach for the treatment of neurodegenerative diseases, it is crucial to understand the specific contribution of these cells to the inflammatory reaction and to select experimental models that recapitulate what occurs in the human disease. Previously, we have characterized a region-specific activation pattern of CD11b+ cells and astrocytes in the α-synuclein overexpression mouse model of Parkinson´s disease (PD). In this study we hypothesized that the time and the intensity of dopaminergic neuronal death would promote different glial activation states. Dopaminergic degeneration was induced with two administration regimens of the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), subacute (sMPTP) and chronic (cMPTP). Our results show that in the sMPTP mouse model, the pro-inflammatory phenotype of striatal CD11b+ cells was counteracted by an anti-inflammatory astrocytic profile. In the midbrain the roles were inverted, CD11b+ cells exhibited an anti-inflammatory profile and astrocytes were pro-inflammatory. The overall response generated resulted in decreased CD4 T cell infiltration in both regions. Chronic MPTP exposure resulted in a mild and prolonged neuronal degeneration that generated a pro-inflammatory response and increased CD4 T cell infiltration in both regions. At the onset of the neurodegenerative process, microglia and astrocytes cooperated in the removal of dopaminergic terminals. With time, only microglia maintained the phagocytic activity. In the ventral midbrain, astrocytes were the main phagocytic mediators at early stages of degeneration while microglia were the major phagocytic cells in the chronic state. In this scenario, we questioned which activation pattern recapitulates better the features of glial activation in PD. Glial activation in the cMPTP mouse model reflects many pathways of their corresponding counterparts in the human brain with advanced PD. Altogether, our results point toward a context-dependent cooperativity of microglia/myeloid cells and astrocytes in response to neuronal damage and the relevance of selecting the right experimental models for the study of neuroinflammation.


Assuntos
Neuroglia , Doenças Neuroinflamatórias , Humanos , Animais , Camundongos , Fagócitos , Astrócitos , Modelos Animais de Doenças , Dopamina , Anti-Inflamatórios
16.
J Autoimmun ; 145: 103197, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38447248

RESUMO

BACKGROUND AND OBJECTIVE: Understanding the regulation of efferocytosis by myeloid phagocytes is important in identifying novel targets in systemic lupus erythematosus (SLE). Cadherin-11 (CDH11), a cell adhesion molecule, is implicated in inflammatory arthritis and fibrosis and recently been shown to regulate macrophage phagocytosis. The extent and mechanism of this regulation is unknown. Our objective was to examine the extent to which CDH11 regulates myeloid phagocytes and contributes to autoimmunity and tissue inflammation. METHODS: We analyzed efferocytosis in macrophages and dendritic cells (DCs) from WT and Cdh11-/- mice and investigated the mechanisms in vitro. We investigated the role of CDH11 in disease development in vivo using the pristane induced lupus model. To translate the clinical relevance of CDH11 in human disease, we measured serum CDH11 levels in two independent pediatric SLE (pSLE) cohorts and healthy controls. RESULTS: Using bone marrow derived macrophages (BMDMs) and DCs (BMDCs), we found impaired efferocytosis in phagocytes from Cdh11-/- mice, mediated by downregulated efferocytosis receptor expression and RhoGTPase activation. Specifically, loss of CDH11 downregulated Mertk expression and Rac1 activation in BMDMs, and integrin αVß3 expression and Cdc42 activation in BMDCs, highlighting distinct pathways. In vivo, Cdh11-/- mice displayed defective efferocytosis and increased accumulation of apoptotic debris in pristane-induced lupus. Further, Cdh11-/- mice had enhanced systemic inflammation and autoimmune inflammation with increased anti-dsDNA autoantibodies, splenomegaly, type I interferons, and inflammatory cytokines. Paradoxically, at the tissue level, Cdh11-/- mice were protected against glomerulonephritis, indicating a dual role in murine lupus. Finally, SLE patients had increased serum CDH11 compared to controls. CONCLUSION: This study highlights a novel role of CDH11 in regulating myeloid cells and efferocytosis and its potential as a contributor to development in autoimmunity murine lupus. Despite the increase in autoimmunity, Cdh11-/- mice developed decreased tissue inflammation and damage.


Assuntos
Caderinas , Células Dendríticas , Modelos Animais de Doenças , Lúpus Eritematoso Sistêmico , Macrófagos , Fagocitose , Animais , Criança , Feminino , Humanos , Camundongos , Autoimunidade , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Caderinas/metabolismo , Caderinas/genética , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Inflamação/imunologia , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , Lúpus Eritematoso Sistêmico/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Knockout , Células Mieloides/imunologia , Células Mieloides/metabolismo , Fagócitos/imunologia , Fagócitos/metabolismo , Fagocitose/imunologia , Terpenos
17.
J Leukoc Biol ; 115(6): 1165-1176, 2024 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-38466819

RESUMO

The effective delivery of synthetic RNA into mononuclear phagocytes is a prerequisite for experimental research and therapeutic development. However, traditional methods are highly ineffective and toxic for these cells. Here, we aimed to optimize a transfection protocol for primary bone marrow-derived phagocytes, specifically dendritic cells and macrophages, using lipid nanoparticles generated by microfluidics. Our results show that a lipid mixture similar to that used in Moderna's COVID-19 messenger RNA vaccine outperforms the others tested. Improved messenger RNA transfection can be achieved by replacing uridine with methylpseudouridine but not methoxyuridine, which interferes with transfection. The addition of diphenyleneiodonium or apocynin can enhance transfection in a cell type-dependent manner without adverse effects, while apolipoprotein E provides no added value. These optimized transfection conditions can also be used for microRNA agonists and antagonists. In sum, this study offers a straightforward, highly efficient, reproducible, and nontoxic protocol to deliver RNA into different primary mononuclear phagocytes in culture.


Assuntos
Nanopartículas , Transfecção , Nanopartículas/química , Transfecção/métodos , Animais , Células Dendríticas , Fagócitos/metabolismo , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Lipídeos/química , Camundongos , Humanos , RNA Mensageiro/genética , MicroRNAs/genética , Células Cultivadas , Camundongos Endogâmicos C57BL , Lipossomos
18.
mBio ; 15(5): e0063224, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38534159

RESUMO

Bordetella species that cause respiratory infections in mammals include B. pertussis, which causes human whooping cough, and B. bronchiseptica, which infects nearly all mammals. Both bacterial species produce filamentous hemagglutinin (FhaB) and adenylate cyclase toxin (ACT), prominent surface-associated and secreted virulence factors that contribute to persistence in the lower respiratory tract by inhibiting clearance by phagocytic cells. FhaB and ACT proteins interact with themselves, each other, and host cells. Using immunoblot analyses, we showed that ACT binds to FhaB on the bacterial surface before it can be detected in culture supernatants. We determined that SphB1, a surface protease identified based on its requirement for FhaB cleavage, is also required for ACT cleavage, and we determined that the presence of ACT blocks SphB1-dependent and -independent cleavage of FhaB, but the presence of FhaB does not affect SphB1-dependent cleavage of ACT. The primary SphB1-dependent cleavage site on ACT is proximal to ACT's active site, in a region that is critical for ACT activity. We also determined that FhaB-bound ACT on the bacterial surface can intoxicate host cells producing CR3, the receptor for ACT. In addition to increasing our understanding of FhaB, ACT, and FhaB-ACT interactions on the Bordetella surface, our data are consistent with a model in which FhaB functions as a novel toxin delivery system by binding to ACT and allowing its release upon binding of ACT to its receptor, CR3, on phagocytic cells.IMPORTANCEBacteria need to control the variety, abundance, and conformation of proteins on their surface to survive. Members of the Gram-negative bacterial genus Bordetella include B. pertussis, which causes whooping cough in humans, and B. bronchiseptica, which causes respiratory infections in a broad range of mammals. These species produce two prominent virulence factors, the two-partner secretion (TPS) effector FhaB and adenylate cyclase toxin (ACT), that interact with themselves, each other, and host cells. Here, we determined that ACT binds FhaB on the bacterial surface before being detected in culture supernatants and that ACT bound to FhaB can be delivered to eukaryotic cells. Our data are consistent with a model in which FhaB delivers ACT specifically to phagocytic cells. This is the first report of a TPS system facilitating the delivery of a separate polypeptide toxin to target cells and expands our understanding of how TPS systems contribute to bacterial pathogenesis.


Assuntos
Toxina Adenilato Ciclase , Fagócitos , Fatores de Virulência de Bordetella , Toxina Adenilato Ciclase/metabolismo , Toxina Adenilato Ciclase/genética , Fagócitos/metabolismo , Fagócitos/microbiologia , Fatores de Virulência de Bordetella/metabolismo , Fatores de Virulência de Bordetella/genética , Humanos , Bordetella pertussis/metabolismo , Bordetella pertussis/genética , Adesinas Bacterianas/metabolismo , Adesinas Bacterianas/genética , Bordetella bronchiseptica/metabolismo , Bordetella bronchiseptica/genética , Ligação Proteica , Animais
19.
Cells ; 13(6)2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38534379

RESUMO

Disulfiram (DSF), an anti-alcoholism medicine, exerts treatment effects in patients suffering from persistent Borreliosis and also exhibits anti-cancer effects through its copper chelating derivatives and induction of oxidative stress in mitochondria. Since chronic/persistent borreliosis is characterized by increased amounts of pro-inflammatory macrophages, this study investigated opsonin-independent phagocytosis, migration, and surface marker expression of in vivo activated and in vitro cultured human monocyte-derived phagocytes (macrophages and dendritic cells) with and without DSF treatment. Phagocytosis of non-opsonized Dynabeads® M-450 and migration of macrophages and dendritic cells were monitored using live cell analyzer Juli™ Br for 24 h, imaging every 3.5 min. To simultaneously monitor phagocyte function, results were analyzed by a newly developed software based on the differential phase contrast images of cells before and after ingestion of Dynabeads. DSF decreased the phagocytic capacities exhibited by in vitro enriched and long-lived phagocytes. Although no chemotactic gradient was applied to the test system, vigorous spontaneous migration was observed. We therefore set up an algorithm to monitor and quantify both phagocytosis and migration simultaneously. DSF not only reduced phagocytosis in a majority of these long-lived phagocytes but also impaired their migration. Despite these selective effects by DSF, we found that DSF reduced the expression densities of surface antigens CD45 and CD14 in all of our long-lived phagocytes. In cells with a high metabolic activity and high mitochondrial contents, DSF led to cell death corresponding to mitochondrial oxidative stress, whereas metabolically inactive phagocytes survived our DSF treatment protocol. In conclusion, DSF affects the viability of metabolically active phagocytes by inducing mitochondrial stress and secondly attenuates phagocytosis and migration in some long-lived phagocytes.


Assuntos
Dissulfiram , Proteínas Opsonizantes , Humanos , Dissulfiram/farmacologia , Fagocitose , Fagócitos , Macrófagos
20.
Immunol Rev ; 322(1): 71-80, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38429865

RESUMO

Since their description by Metchnikoff in 1905, phagocytes have been increasingly recognized to be the entities that traffic to sites of infection and inflammation, engulf and kill infecting organisms, and clear out apoptotic debris all the while making antigens available and accessible to the lymphoid organs for future use. Therefore, phagocytes provide the gateway and the first check in host protection and immune response. Disorders in killing and chemotaxis lead not only to infection susceptibility, but also to autoimmunity. We aim to describe chronic granulomatous disease and the leukocyte adhesion deficiencies as well as myeloperoxidase deficiency and G6PD deficiency as paradigms of critical pathways.


Assuntos
Doença Granulomatosa Crônica , Neutrófilos , Humanos , Doença Granulomatosa Crônica/metabolismo , Fagocitose , Fagócitos/fisiologia , Inflamação/metabolismo
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