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1.
FASEB J ; 35(12): e21997, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34719814

RESUMO

The deadliest complication of infection by Plasmodium parasites, cerebral malaria, accounts for the majority of malarial fatalities. Although our understanding of the cellular and molecular mechanisms underlying the pathology remains incomplete, recent studies support the contribution of systemic and neuroinflammation as the cause of cerebral edema and blood-brain barrier (BBB) dysfunction. All Plasmodium species encode an orthologue of the innate cytokine, Macrophage Migration Inhibitory Factor (MIF), which functions in mammalian biology to regulate innate responses. Plasmodium MIF (PMIF) similarly signals through the host MIF receptor CD74, leading to an enhanced inflammatory response. We investigated the PMIF-CD74 interaction in the onset of experimental cerebral malaria (ECM) and liver stage Plasmodium development by using a combination of CD74 deficient (Cd74-/- ) hosts and PMIF deficient parasites. Cd74-/- mice were found to be protected from ECM and the protection was associated with the inability of brain microvessels to present parasite antigen to sequestered and pathogenic Plasmodium-specific CD8+ T cells. Infection of WT hosts with PMIF-deficient sporozoites or infection of Cd74-/- hosts with WT sporozoites impacted the survival of infected hepatocytes and subsequently reduced blood-stage associated inflammation, contributing to protection from ECM. We recapitulated these finding with a novel pharmacologic PMIF-selective antagonist that reduced PMIF/CD74 signaling and fully protected mice from ECM. These findings reveal a conserved mechanism for Plasmodium usurpation of host CD74 signaling and suggest a tractable approach for new pharmacologic intervention.


Assuntos
Antígenos de Diferenciação de Linfócitos B/química , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe II/química , Inflamação/prevenção & controle , Fígado/patologia , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Malária Cerebral/prevenção & controle , Plasmodium berghei/fisiologia , Animais , Antígenos de Diferenciação de Linfócitos B/fisiologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Fígado/imunologia , Fígado/parasitologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Malária Cerebral/etiologia , Malária Cerebral/metabolismo , Malária Cerebral/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
2.
FASEB J ; 33(6): 6919-6932, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30817226

RESUMO

T cells expressing invariant γδ antigen receptors (γδ T cells) bridge innate and adaptive immunity and facilitate barrier responses to pathogens. Macrophage migration inhibitory factor (MIF) is an upstream mediator of host defense that up-regulates the expression of pattern recognition receptors and sustains inflammatory responses by inhibiting activation-induced apoptosis in monocytes and macrophages. Surprisingly, Mif-/- γδ T cells, when compared with wild type, were observed to produce >10-fold higher levels of the proinflammatory cytokine IL-17 after stimulation with gram-positive exotoxins. High-IL-17 expression was associated with the characteristic features of IL-17-producing γδ T (γδ17) cells, including expression of IL-23R, IL-1R1, and the transcription factors RORγt and Sox13. In the gram-positive model of shock mediated by toxic shock syndrome toxin (TSST-1), Mif-/- mice succumbed to death more quickly with increased pulmonary neutrophil accumulation and higher production of cytokines, including IL-1ß and IL-23. Mif-/- γδ T cells also produced high levels of IL-17 in response to Mycobacterium lipomannan, and depletion of γδ T cells improved survival from acutely lethal Mycobacterium infection or TSST-1 administration. These data indicate that MIF deficiency is associated with a compensatory amplification of γδ17 cell responses, with implications for innate immunity and IL-17-mediated pathology in situations such as gram-positive toxic shock or Mycobacterium infection.-Kim, H. K., Garcia, A. B., Siu, E., Tilstam, P., Das, R., Roberts, S., Leng, L., Bucala, R. Macrophage migration inhibitory factor regulates innate γδ T-cell responses via IL-17 expression.


Assuntos
Imunidade Inata/imunologia , Inflamação/imunologia , Interleucina-17/metabolismo , Oxirredutases Intramoleculares/fisiologia , Fatores Inibidores da Migração de Macrófagos/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Células Th17/imunologia , Tuberculose Pulmonar/imunologia , Animais , Toxinas Bacterianas/administração & dosagem , Enterotoxinas/administração & dosagem , Inflamação/metabolismo , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mycobacterium bovis/imunologia , Receptores de Interleucina/metabolismo , Choque Séptico/induzido quimicamente , Choque Séptico/imunologia , Choque Séptico/patologia , Superantígenos/administração & dosagem , Tuberculose Pulmonar/microbiologia , Tuberculose Pulmonar/patologia
3.
Proc Natl Acad Sci U S A ; 114(40): E8421-E8429, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28923927

RESUMO

Little is known about mechanisms that drive the development of progressive multiple sclerosis (MS), although inflammatory factors, such as macrophage migration inhibitory factor (MIF), its homolog D-dopachrome tautomerase (D-DT), and their common receptor CD74 may contribute to disease worsening. Our findings demonstrate elevated MIF and D-DT levels in males with progressive disease compared with relapsing-remitting males (RRMS) and female MS subjects, with increased levels of CD74 in females vs. males with high MS disease severity. Furthermore, increased MIF and D-DT levels in males with progressive disease were significantly correlated with the presence of two high-expression promoter polymorphisms located in the MIF gene, a -794CATT5-8 microsatellite repeat and a -173 G/C SNP. Conversely, mice lacking MIF or D-DT developed less-severe signs of experimental autoimmune encephalomyelitis, a murine model of MS, thus implicating both homologs as copathogenic contributors. These findings indicate that genetically controlled high MIF expression (and D-DT) promotes MS progression in males, suggesting that these two factors are sex-specific disease modifiers and raising the possibility that aggressive anti-MIF treatment of clinically isolated syndrome or RRMS males with a high-expresser genotype might slow or prevent the onset of progressive MS. Additionally, selective targeting of MIF:CD74 signaling might provide an effective, trackable therapeutic approach for MS subjects of both sexes.


Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/fisiologia , Fatores Inibidores da Migração de Macrófagos/metabolismo , Fatores Inibidores da Migração de Macrófagos/fisiologia , Esclerose Múltipla/patologia , Índice de Gravidade de Doença , Adulto , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Feminino , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Oxirredutases Intramoleculares/genética , Fatores Inibidores da Migração de Macrófagos/genética , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Esclerose Múltipla/genética , Esclerose Múltipla/metabolismo , Polimorfismo Genético
4.
Mol Microbiol ; 88(4): 702-12, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23347134

RESUMO

Resistance to antimalarials targeting the folate pathway is widespread. GTP-cyclohydrolase (gch1), the first enzyme in this pathway, exhibits extensive copy number variation (CN) in parasite isolates from areas with a history of longstanding antifolate use. Increased CN of gch1 is associated with a greater number of point mutations in enzymes targeted by the antifolates, pyrimethamine and sulphadoxine. While these observations suggest that increases in gch1 CN are an adaptation to drug pressure, changes in CN have not been experimentally demonstrated to directly alter drug susceptibility. To determine if changes in gch1 expression alone modify pyrimethamine sensitivity, we manipulated gch1 CN in several parasite lines to test the effect on drug sensitivity. We report that increases in gch1 CN alter pyrimethamine resistance in most parasites lines. However we find evidence of a detrimental effect of very high levels of gch1 overexpression in parasite lines with high endogenous levels of gch1 expression, revealing the importance of maintaining balance in the folate pathway and implicating changes in gch1 expression in preserving proper metabolic flux. This work expands our understanding of parasite adaptation to drug pressure and provides a possible mechanism for how specific mutations become fixed within parasite populations.


Assuntos
Adaptação Biológica , Antimaláricos/farmacologia , Resistência a Medicamentos , Antagonistas do Ácido Fólico/farmacologia , Dosagem de Genes , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Genes de Protozoários , Pirimetamina/farmacologia
5.
Bone ; 184: 117086, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38552893

RESUMO

PURPOSE: Mitofusin 2 (Mfn2) is one of two mitofusins involved in regulating mitochondrial size, shape and function, including mitophagy, an important cellular mechanism to limit oxidative stress. Reduced expression of Mfn2 has been associated with impaired osteoblast differentiation and function and a reduction in the number of viable osteocytes in bone. We hypothesized that the genetic absence of Mfn2 in these cells would increase their susceptibility to aging-associated metabolic stress, leading to a progressive impairment in skeletal homeostasis over time. METHODS: Mfn2 was selectively deleted in vivo at three different stages of osteoblast lineage commitment by crossing mice in which the Mfn2 gene was floxed with transgenic mice expressing Cre under the control of the promoter for Osterix (OSX), collagen1a1, or DMP1 (Dentin Matrix Acidic Phosphoprotein 1). RESULTS: Mice in which Mfn2 was deleted using DMP1-cre demonstrated a progressive and dramatic decline in bone mineral density (BMD) beginning at 10 weeks of age (n = 5 for each sex and each genotype from age 10 to 20 weeks). By 15 weeks, there was evidence for a functional decline in muscle performance as assessed using a rotarod apparatus (n = 3; 2 males/ 1 female for each genotype), accompanied by a decline in lean body mass. A marked reduction in trabecular bone mass was evident on bone histomorphometry, and biomechanical testing at 25 weeks (k/o: 2 male/1 female, control 2 male/2 female) revealed severely impaired femur strength. Extensive regional myofiber atrophy and degeneration was observed on skeletal muscle histology. Electron microscopy showed progressive disruption of cellular architecture, with disorganized sarcomeres and a bloated mitochondrial reticulum. There was also evidence of neurodegeneration within the ventral horn and roots of the lumbar spinal cord, which was accompanied by myelin loss and myofiber atrophy. Deletion of Mfn2 using OSX-cre or Col1a1-cre did not result in a musculoskeletal phenotype. Where possible, male and female animals were analyzed separately, but small numbers of animals in each group limited statistical power. For other outcomes, where sex was not considered, small sample sizes might still limit the strength of the observation. CONCLUSION: Despite known functional overlap of Mfn1 and Mfn2 in some tissues, and their co-expression in bone, muscle and spinal cord, deletion of Mfn2 using the 8 kB DMP1 promoter uncovered an important non-redundant role for Mfn2 in maintaining the neuromuscular/bone axis.


Assuntos
Densidade Óssea , GTP Fosfo-Hidrolases , Animais , Feminino , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Masculino , Camundongos , Densidade Óssea/genética , Densidade Óssea/fisiologia , Camundongos Transgênicos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Osso e Ossos/patologia , Osso e Ossos/metabolismo , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Osteoblastos/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética
6.
Endocrinology ; 162(5)2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33640975

RESUMO

Sphingosine-1-phosphate (S1P) is an anabolic clastokine. Sphingosine kinase (SPHK) is the rate-limiting enzyme in S1P production and has 2 isoforms. To evaluate the roles of SPHK1 and SPHK2 in bone, we examined the skeletal phenotype of mice with selective deletion of SPHK1 in osteoclasts (SPHK1-Oc-/-) and mice in which the SPHK2 gene was deleted in all tissues (SPHK2-/-). SPHK1-Oc-/- had normal bone mass. By contrast, SPHK2-/- female mice had a 14% lower spinal bone mineral density (BMD; P < 0.01) and males a 22% lower BMD at the same site (P < 0.001). SPHK2-/- and control mice were subsequently treated either with daily parathyroid hormone [PTH](1-34) or vehicle for 29 days. The response to PTH was significantly attenuated in the SPHK2-/-mice. The mean femoral bone volume to total volume fraction (BV/TV) increased by 24.8% in the PTH-treated female control animals vs 10.6% in the vehicle-treated female controls (P < 0.01). In contrast, in the SPHK2-/- female mice the difference in femoral trabecular BV/TV at the end of treatment was not significant (20.5 vs13.3%, PTH vs vehicle, P = NS). The anabolic response to PTH was significantly attenuated in the spine of male SPHK2-/- mice (29.7% vs 23.1%, PTH vs vehicle, in controls, P < 0.05; 26.9% vs 19.5% PTH vs vehicle in SPHK2-/- mice, P = NS). The spine responded normally in the SPHK2-/- female mice. Interestingly, suppression of sclerostin was blunted in the SPHK2-/- mice when those animals were treated with an anabolic PTH regimen. We conclude that SPHK2 has an important role in mediating both normal bone remodeling and the anabolic response to PTH.


Assuntos
Anabolizantes/metabolismo , Fêmur/metabolismo , Hormônio Paratireóideo/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Coluna Vertebral/metabolismo , Animais , Densidade Óssea , Feminino , Fêmur/química , Masculino , Camundongos , Camundongos Knockout , Osteoclastos/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Coluna Vertebral/química
7.
Methods Mol Biol ; 2080: 67-84, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31745872

RESUMO

Macrophage migration inhibitory factor (MIF) is an upstream proinflammatory cytokine encoded by a functionally polymorphic locus. The promoter region of the human MIF gene contains two polymorphisms. A variable nucleotide tandem repeat at position -794 comprises five to eight CATT repeats (referred to henceforth by numbers from 5 to 8, rs5844572). Gene reporter assays show a proportional increase in transcription with CATT repeat number; the 5-repeat allele leads to low expression, and the 6-, 7-, and 8-repeat alleles lead to correspondingly higher expression of MIF. A second MIF promoter polymorphism comprises a G-to-C single nucleotide polymorphism (SNP) at position -173 (rs755622), which is in strong linkage disequilibrium with -794 7-CATT and is associated with arthritis clinical severity and higher serum and synovial fluid MIF levels. This allele also has been reported to confer improved survival in patients with outpatient pneumonia. In this chapter, we will introduce the methods of genotyping CATT5-8 repeats and the MIF -173 G/C from human samples.


Assuntos
Alelos , Fatores Inibidores da Migração de Macrófagos/genética , Repetições de Microssatélites , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Eletroforese Capilar , Predisposição Genética para Doença , Genótipo , Técnicas de Genotipagem , Reação em Cadeia da Polimerase em Tempo Real
8.
Nat Commun ; 9(1): 2714, 2018 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-30006528

RESUMO

Plasmodium species produce an ortholog of the cytokine macrophage migration inhibitory factor, PMIF, which modulates the host inflammatory response to malaria. Using a novel RNA replicon-based vaccine, we show the impact of PMIF immunoneutralization on the host response and observed improved control of liver and blood-stage Plasmodium infection, and complete protection from re-infection. Vaccination against PMIF delayed blood-stage patency after sporozoite infection, reduced the expression of the Th1-associated inflammatory markers TNF-α, IL-12, and IFN-γ during blood-stage infection, augmented Tfh cell and germinal center responses, increased anti-Plasmodium antibody titers, and enhanced the differentiation of antigen-experienced memory CD4 T cells and liver-resident CD8 T cells. Protection from re-infection was recapitulated by the adoptive transfer of CD8 or CD4 T cells from PMIF RNA immunized hosts. Parasite MIF inhibition may be a useful approach to promote immunity to Plasmodium and potentially other parasite genera that produce MIF orthologous proteins.


Assuntos
Imunidade Adaptativa/efeitos dos fármacos , Anticorpos Antiprotozoários/biossíntese , Fatores Inibidores da Migração de Macrófagos/antagonistas & inibidores , Vacinas Antimaláricas/administração & dosagem , Malária/prevenção & controle , Proteínas de Protozoários/antagonistas & inibidores , Vacinas de DNA/administração & dosagem , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/parasitologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/parasitologia , Feminino , Expressão Gênica , Centro Germinativo/efeitos dos fármacos , Centro Germinativo/imunologia , Centro Germinativo/parasitologia , Memória Imunológica/efeitos dos fármacos , Interferon gama/genética , Interferon gama/imunologia , Interleucina-12/genética , Interleucina-12/imunologia , Fatores Inibidores da Migração de Macrófagos/genética , Fatores Inibidores da Migração de Macrófagos/imunologia , Malária/imunologia , Malária/parasitologia , Vacinas Antimaláricas/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Plasmodium berghei/efeitos dos fármacos , Plasmodium berghei/genética , Plasmodium berghei/imunologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , RNA de Protozoário/genética , RNA de Protozoário/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas de DNA/biossíntese
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