RESUMO
BACKGROUND: Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. OBJECTIVE: We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma. METHODS: We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-like (ptges1-/-) mice with aspirin. RESULTS: RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. CONCLUSIONS: RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.
Assuntos
Aspergilose/metabolismo , Aspergillus fumigatus/imunologia , Asma Induzida por Aspirina/metabolismo , Pulmão/patologia , Músculo Liso/metabolismo , Proteínas RGS/metabolismo , Mucosa Respiratória/metabolismo , Animais , Espasmo Brônquico , Células Cultivadas , Dinoprostona/biossíntese , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Músculo Liso/patologia , Prostaglandina-E Sintases/genética , Proteínas RGS/genética , Transdução de SinaisRESUMO
Regulatory T cells (Tregs) play a cardinal role in the immune system by suppressing detrimental autoimmune responses, but their role in acute, chronic infectious diseases and tumor microenvironment remains unclear. We recently demonstrated that IFN-α/ß receptor (IFNAR) signaling promotes Treg function in autoimmunity. Here we dissected the functional role of IFNAR-signaling in Tregs using Treg-specific IFNAR deficient (IFNARfl/flxFoxp3YFP-Cre) mice in acute LCMV Armstrong, chronic Clone-13 viral infection, and in tumor models. In both viral infection and tumor models, IFNARfl/flxFoxp3YFP-Cre mice Tregs expressed enhanced Treg associated activation antigens. LCMV-specific CD8+ T cells and tumor infiltrating lymphocytes from IFNARfl/flxFoxp3YFP-Cre mice produced less antiviral and antitumor IFN-γ and TNF-α. In chronic viral model, the numbers of antiviral effector and memory CD8+ T cells were decreased in IFNARfl/flxFoxp3YFP-Cre mice and the effector CD4+ and CD8+ T cells exhibited a phenotype compatible with enhanced exhaustion. IFNARfl/flxFoxp3YFP-Cre mice cleared Armstrong infection normally, but had higher viral titers in sera, kidneys and lungs during chronic infection, and higher tumor burden than the WT controls. The enhanced activated phenotype was evident through transcriptome analysis of IFNARfl/flxFoxp3YFP-Cre mice Tregs during infection demonstrated differential expression of a unique gene signature characterized by elevated levels of genes involved in suppression and decreased levels of genes mediating apoptosis. Thus, IFN signaling in Tregs is beneficial to host resulting in a more effective antiviral response and augmented antitumor immunity.
Assuntos
Infecções por Arenaviridae/imunologia , Neoplasias do Colo/imunologia , Interferon Tipo I/farmacologia , Coriomeningite Linfocítica/imunologia , Melanoma Experimental/imunologia , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Antivirais/farmacologia , Infecções por Arenaviridae/tratamento farmacológico , Infecções por Arenaviridae/metabolismo , Infecções por Arenaviridae/virologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Cultivadas , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Neoplasias do Colo/virologia , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Interferon gama/metabolismo , Coriomeningite Linfocítica/tratamento farmacológico , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/metabolismo , Melanoma Experimental/virologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta/fisiologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/virologia , Microambiente Tumoral/efeitos dos fármacosRESUMO
Eos (lkzf4) is a member of the Ikaros family of transcription factors and is preferentially expressed in T-regulatory (Treg) cells. However, the role of Eos in Treg function is controversial. One study using siRNA knock down of Eos demonstrated that it was critical for Treg suppressor function. In contrast, Treg from mice with a global deficiency of Eos had normal Treg function in vitro and in vivo. To further dissect the function of Eos in Tregs, we generated mice with a conditional knock out of Eos in Treg cells (lkzf4fl/fl X Foxp3YFP-cre, Eos cKO). Deletion of Eos in Treg resulted in activation of CD4+Foxp3- and CD8+ T cells at the age of 3 months, cellular infiltration in non-lymphoid tissues, hyperglobulinemia, and anti-nuclear antibodies. While Tregs from Eos cKO mice displayed normal suppressive function in vitro, Eos cKO mice developed severe Experimental Autoimmune Encephalomyletis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG) and Eos cKO Treg were unable to suppress Inflammatory Bowel Disease (IBD). Eos cKO mice had decreased growth of the transplantable murine adenocarcinoma MC38 tumor accompanied by enhanced IFN-γ/TNF-α production by CD8+ T cells in tumor draining lymph nodes. Mice with a global deficiency of Eos or a deficiency of Eos only in T cells developed autoimmunity at a much older age (12 months or 7-8 months, respectively). Taken together, Eos appears to play an essential role in multiple aspects of Treg suppressor function, but also plays an as yet unknown role in the function of CD4+Foxp3- and CD8+ T cells and potentially in non-T cells.
Assuntos
Autoimunidade/imunologia , Proteínas de Ligação a DNA/imunologia , Proteínas do Tecido Nervoso/imunologia , Linfócitos T Reguladores/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Encefalomielite Autoimune Experimental/imunologia , Feminino , Fatores de Transcrição Forkhead/imunologia , Doenças Inflamatórias Intestinais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glicoproteína Mielina-Oligodendrócito/imunologiaRESUMO
Rheumatoid arthritis (RA) is associated with the presence of certain HLA class II genes. However, why some individuals carrying RA non-associated alleles develop arthritis is still unexplained. The trans-heterodimer between two RA non-associated HLA genes can render susceptibility to develop arthritis in humanized mice, DQA1*0103/DQB1*0604, suggesting a role for DQ α chains in pathogenesis. In this study we determined the role of DQA1 in arthritis by using mice expressing DQA1*0103 and lacking endogenous class II molecules. Proximity ligation assay showed that DQA1*0103 is expressed on the cell surface as a dimer with CD74. Upon immunization with type II collagen, DQA1*0103 mice generated an antigen-specific cellular and humoral response and developed severe arthritis. Structural modelling suggests that DQA1*0103/CD74 form a pocket with similarity to the antigen binding pocket. DQA1*0103 mice present type II collagen-derived peptides that are not presented by an arthritis-resistant DQA1*0103/DQB1*0601 allele, suggesting that the DQA1*0103/CD74 dimer may result in presentation of unique antigens and susceptibility to develop arthritis. The present data provide a possible explanation by which the DQA1 molecule contributes to susceptibility to develop arthritis.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Artrite Experimental/metabolismo , Cadeias alfa de HLA-DQ/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Baço/metabolismo , Animais , Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/química , Antígenos de Diferenciação de Linfócitos B/imunologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Sítios de Ligação , Membrana Celular/imunologia , Membrana Celular/metabolismo , Proliferação de Células , Células Cultivadas , Colágeno Tipo II , Feminino , Cadeias alfa de HLA-DQ/química , Cadeias alfa de HLA-DQ/genética , Cadeias alfa de HLA-DQ/imunologia , Antígenos de Histocompatibilidade Classe II/química , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Ativação Linfocitária , Masculino , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Multimerização Proteica , Baço/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de TempoRESUMO
CD2 and CD58 are two important costimulatory molecules involved in generating the signal II required for normal immune signaling. However, this interaction can be targeted to be of benefit in cases of abnormal immune signaling seen in autoimmune diseases. Our objective in this study was to design a peptidomimetic (compound 7) based on a ß-strand structure of the adhesion domain of CD2 protein to inhibit CD2-CD58 protein-protein interaction and its effect on immunomodulation in the collagen-induced arthritis (CIA) model. The ability of compound 7 to bind to CD58 protein was assessed using flow cytometry. The effect of compound 7 on modulating the immune response was evaluated in an autoimmune disease using CIA in mice. The stability of compound 7 was evaluated in mouse serum using mass spectrometry. Antibody (Ab) binding inhibition studies suggested that compound 7 binds to CD58 protein. Compound 7 was successful in modulating immune responses when administered in the CIA mouse model along with reducing anti-collagen Ab levels and decreasing the level of interferon gamma (IFN-γ) relative to control treatments. Compound 7 was found to be nonimmunogenic and stable in mouse serum up to 48 h. Results suggest that compound 7 can serve as a lead compound for immunomodulation, and could be a therapeutic agent for the autoimmune disease rheumatoid arthritis (RA).
Assuntos
Artrite Experimental/tratamento farmacológico , Antígenos CD2/imunologia , Antígenos CD58/imunologia , Adesão Celular/efeitos dos fármacos , Colágeno/efeitos dos fármacos , Peptidomiméticos/uso terapêutico , Animais , Artrite Experimental/imunologia , Artrite Experimental/patologia , Ligação Competitiva , Colágeno/imunologia , Feminino , Camundongos , Camundongos Endogâmicos DBA , Peptidomiméticos/farmacologiaRESUMO
The current approach to treating HER2-overexpressed breast cancer is the use of monoclonal antibodies or a combination of antibodies with traditional chemotherapeutic agents or kinase inhibitors. Our approach is to target clinically validated HER2 domain IV with peptidomimetics and inhibit the protein-protein interactions (PPI) of HERs. Unlike antibodies, peptidomimetics have advantages in terms of stability, modification, and molecular size. We have designed peptidomimetics (compounds 5 and 9) that bind to HER2 domain IV, inhibit protein-protein interactions, and decrease cell viability in breast cancer cells with HER2 overexpression. We have shown, using enzyme fragment complementation and proximity ligation assays, that peptidomimetics inhibit the PPI of HER2:HER3. Compounds 5 and 9 suppressed the tumor growth in a xenograft mouse model. Furthermore, we have shown that these compounds inhibit PPI of HER2:HER3 and phosphorylation of HER2 as compared to control in tissue samples derived from in vivo studies. The stability of the compounds was also investigated in mouse serum, and the compounds exhibited stability with a half-life of up to 3 h. These results suggest that the novel peptidomimetics we have developed target the extracellular domain of HER2 protein and inhibit HER2:HER3 interaction, providing a novel method to treat HER2-positive cancer.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Mama/efeitos dos fármacos , Peptidomiméticos/uso terapêutico , Mapas de Interação de Proteínas/efeitos dos fármacos , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-3/antagonistas & inibidores , Animais , Antineoplásicos/sangue , Antineoplásicos/química , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Modelos Moleculares , Peptidomiméticos/sangue , Peptidomiméticos/química , Multimerização Proteica/efeitos dos fármacos , Receptor ErbB-2/análise , Receptor ErbB-2/metabolismo , Receptor ErbB-3/análise , Receptor ErbB-3/metabolismoRESUMO
Peptides and peptidomimetics can function as immunomodulating agents by either blocking the immune response or stimulating the immune response to generate tolerance. Knowledge of B- or T-cell epitopes along with conformational constraints is important in the design of peptide-based immunomodulating agents. Work on the conformational aspects of peptides, synthesis and modified amino acid side chains have contributed to the development of a new generation of therapeutic agents for autoimmune diseases and cancer. The design of peptides/peptidomimetics for immunomodulation in autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, systemic lupus and HIV infection is reviewed. In cancer therapy, peptide epitopes are used in such a way that the body is trained to recognize and fight the cancer cells locally as well as systemically.
Assuntos
Doenças Autoimunes/imunologia , Fatores Imunológicos/imunologia , Neoplasias/imunologia , Peptídeos/imunologia , Peptidomiméticos/imunologia , Doenças Autoimunes/tratamento farmacológico , Desenho de Fármacos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Fatores Imunológicos/química , Fatores Imunológicos/uso terapêutico , Neoplasias/tratamento farmacológico , Peptídeos/química , Peptídeos/uso terapêutico , Peptidomiméticos/química , Peptidomiméticos/uso terapêutico , Conformação ProteicaRESUMO
Targeting co-stimulatory molecules to modulate the immune response has been shown to have useful therapeutic effects for autoimmune diseases. Among the co-stimulatory molecules, CD2 and CD58 are very important in the early stages of generation of an immune response. Our goal was to utilize CD2-derived peptides to modulate protein-protein interactions between CD2 and CD58, thereby modulating the immune response. Several peptides were designed based on the structure of the CD58-binding domain of CD2 protein. Among the CD2-derived peptides, peptide 6 from the F and C ß-strand region of CD2 protein exhibited inhibition of cell-cell adhesion in the nanomolar concentration range. Peptide 6 was evaluated for its ability to bind to CD58 in Caco-2 cells and to CD48 in T cells from rodents. A molecular model was proposed for binding a peptide to CD58 and CD48 using docking studies. Furthermore, in vivo studies were carried out to evaluate the therapeutic ability of the peptide to modulate the immune response in the collagen-induced arthritis (CIA) mouse model. In vivo studies indicated that peptide 6 was able to suppress the progression of CIA. Evaluation of the antigenicity of peptides in CIA and transgenic animal models indicated that this peptide is not immunogenic.
Assuntos
Antígenos CD/metabolismo , Antígenos CD2/metabolismo , Antígenos CD58/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/química , Anticorpos/imunologia , Antígenos CD/química , Antígenos CD/imunologia , Artrite Experimental/tratamento farmacológico , Artrite Experimental/patologia , Sítios de Ligação , Antígenos CD2/química , Antígeno CD48 , Antígenos CD58/química , Antígenos CD58/imunologia , Células CACO-2 , Adesão Celular/efeitos dos fármacos , Corantes Fluorescentes , Humanos , Terapia de Imunossupressão , Células Jurkat , Camundongos , Camundongos Endogâmicos DBA , Camundongos Transgênicos , Simulação de Acoplamento Molecular , Peptídeos Cíclicos/química , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Cell adhesion molecule CD2 and its ligand CD58 provide good examples of protein-protein interactions in cells that participate in the immune response. To modulate the cell adhesion interaction, peptides were designed from the discontinuous epitopes of the ß-strand region of CD2 protein. The two strands were linked by a peptide bond. ß-Strands in the peptides were nucleated by inserting a ß-sheet-inducing (D)-Pro-Pro sequence or a dibenzofuran (DBF) turn mimetic with key amino acid sequences from CD2 protein that binds to CD58. The solution structures of the peptides (5-10) were studied by NMR and molecular dynamics simulations. The ability of these peptides to inhibit cell adhesion interaction was studied by E-rosetting and lymphocyte epithelial assays. Peptides 6 and 7 inhibit the cell adhesion activity with an IC(50) of 7 and 11 nM, respectively, in lymphocyte epithelial adhesion assay. NMR and molecular modeling results indicated that peptides 6 and 7 exhibited ß-hairpin structure in solution.
Assuntos
Antígenos CD2/química , Antígenos CD58/química , Adesão Celular/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Sequência de Aminoácidos , Animais , Antígenos CD2/imunologia , Antígenos CD58/imunologia , Células CACO-2 , Adesão Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Humanos , Células Jurkat , Camundongos , Simulação de Dinâmica Molecular , Ressonância Magnética Nuclear Biomolecular , Peptídeos/síntese química , Peptídeos/imunologia , Conformação Proteica , Mapeamento de Interação de Proteínas , Formação de Roseta , Relação Estrutura-Atividade , Linfócitos T/efeitos dos fármacosRESUMO
Cell adhesion molecules play a central role at every step of the immune response. The function of leukocytes can be regulated by modulating adhesion interactions between cell adhesion molecules to develop therapeutic agents against autoimmune diseases. Among the different cell adhesion molecules that participate in the immunologic response, CD2 and its ligand CD58 (LFA-3) are two of the best-characterized adhesion molecules mediating the immune response. To modulate the cell adhesion interaction, peptides were designed from the discontinuous epitopes of the beta-strand region of CD2 protein. The two strands were linked by a peptide bond. beta-Strands in the peptides were nucleated by inserting a beta-sheet-inducing Pro-Gly sequence with key amino acid sequences from CD2 protein that binds to CD58. Using a fluorescence assay, peptides that exhibited potential inhibitory activity in cell adhesion were evaluated for their ability to bind to CD58 protein. A model for peptide binding to CD58 protein was proposed based on docking studies. Administration of one of the peptides, P3 in collagen-induced arthritis in the mouse model, indicated that peptide P3 was able to suppress rheumatoid arthritis in mice.