RESUMO
GroEL is a chaperonin that helps other proteins fold correctly. However, alternative activities, such as acting as an insect toxin, have also been discovered. This work evaluates the chaperonin and insecticidal activity of different GroEL proteins from entomopathogenic nematodes on G. mellonella. The ability to synergize with the ExoA toxin of Pseudomonas aeruginosa was also investigated. The GroELXn protein showed the highest insecticidal activity among the different GroELs. In addition, it was able to significantly activate the phenoloxidase system of the target insects. This could tell us about the mechanism by which it exerts its toxicity on insects. GroEL proteins can enhance the toxic activity of the ExoA toxin, which could be related to its chaperonin activity. However, there is a significant difference in the synergistic effect that is more related to its alternative activity as an insecticidal toxin.
Assuntos
Inseticidas , Mariposas , Nematoides , Animais , Inseticidas/toxicidade , Inseticidas/metabolismo , Chaperonina 60/metabolismo , Chaperonina 60/farmacologia , Insetos/metabolismo , Bactérias/metabolismo , Larva/metabolismoRESUMO
Cytokines are secretion proteins that mediate and regulate immunity and inflammation. They are crucial in the progress of acute inflammatory diseases and autoimmunity. In fact, the inhibition of proinflammatory cytokines has been widely tested in the treatment of rheumatoid arthritis (RA). Some of these inhibitors have been used in the treatment of COVID-19 patients to improve survival rates. However, controlling the extent of inflammation with cytokine inhibitors is still a challenge because these molecules are redundant and pleiotropic. Here we review a novel therapeutic approach based on the use of the HSP60-derived Altered Peptide Ligand (APL) designed for RA and repositioned for the treatment of COVID-19 patients with hyperinflammation. HSP60 is a molecular chaperone found in all cells. It is involved in a wide diversity of cellular events including protein folding and trafficking. HSP60 concentration increases during cellular stress, for example inflammation. This protein has a dual role in immunity. Some HSP60-derived soluble epitopes induce inflammation, while others are immunoregulatory. Our HSP60-derived APL decreases the concentration of cytokines and induces the increase of FOXP3+ regulatory T cells (Treg) in various experimental systems. Furthermore, it decreases several cytokines and soluble mediators that are raised in RA, as well as decreases the excessive inflammatory response induced by SARS-CoV-2. This approach can be extended to other inflammatory diseases.
Assuntos
Artrite Reumatoide , Chaperonina 60 , Humanos , COVID-19 , Citocinas/metabolismo , Inflamação/tratamento farmacológico , Peptídeos/farmacologia , Peptídeos/uso terapêutico , SARS-CoV-2/metabolismo , Chaperonina 60/farmacologia , Chaperonina 60/uso terapêuticoRESUMO
AIMS: Allergic asthma is a chronic inflammatory lung disease characterized by a Th2-type immune response pattern. The development of nonspecific immunotherapy is one of the primary goals for the control of this disease. METHODS AND RESULTS: In this study, we evaluated the therapeutic effects of Lactococcus lactis-producing mycobacterial heat shock protein 65 (LLHsp65) in an ovalbumin (OVA)-induced allergic asthma model. OVA-challenged BALB/c mice were orally administrated with LLHsp65 for 10 consecutive days. The results demonstrate that LLhsp65 attenuates critical features of allergic inflammation, like airway hyperresponsiveness and mucus production. Likewise, the treatment decreases the pulmonary eosinophilia and the serum level of OVA-specific IgE. In addition to deviating immune responses towards Th1-cytokine profile, increase regulatory T cells, and cytokine levels, such as IL-6 and IL-10. CONCLUSIONS: Our results reveal that the mucosal immunotherapy of LLHsp65 significantly reduces the overall burden of airway allergic inflammation, suggesting a promising therapeutic strategy for allergic asthma treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This research reveals new perspectives on nonspecific immunotherapy based on the delivery of recombinant proteins by lactic acid bacteria to treat of allergic disorders.
Assuntos
Asma/tratamento farmacológico , Proteínas de Bactérias/farmacologia , Chaperonina 60/farmacologia , Inflamação/tratamento farmacológico , Lactococcus lactis/imunologia , Administração Oral , Animais , Asma/imunologia , Líquido da Lavagem Broncoalveolar/citologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Hipersensibilidade/tratamento farmacológico , Imunoglobulina E/sangue , Imunoglobulina G/sangue , Imunoterapia , Lactococcus lactis/metabolismo , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina , Linfócitos T Reguladores/imunologiaRESUMO
Intestinal fibrosis associated with Crohn's disease (CD), which a common and serious complication of inflammatory bowel diseases. In this context, heat shock proteins (HSPs) might serve as an alternative treatment because these antigens play important roles in the regulation of effector T cells. We thus evaluated the anti-inflammatory and antifibrotic capacities of an invasive and Hsp65-producing strain-Lactococcus lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65)-in chronic intestinal inflammation to assess its potential as an alternative therapeutic strategy against fibrotic CD. Experimental colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in BALB/c mice, and the mice were treated orally with L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) via intragastric gavage. The oral administration of this strain significantly attenuated the severity of inflammation and intestinal fibrosis in mice (p < 0.05). These results are mainly justified by reductions in the levels of the pro-fibrotic cytokines IL-13 and TGF-ß and increases in the concentration of the regulatory cytokine IL-10. The L. lactis NCDO2118 FnBPA+ (pXYCYT:Hsp65) strain contributed to reductions in the severity of inflammatory damage in chronic experimental CD, and these findings confirm the effectiveness of this new antifibrotic strategy based on the delivery of therapeutic proteins to inside cells of the host intestinal mucosa.
Assuntos
Proteínas de Bactérias/farmacologia , Chaperonina 60/farmacologia , Colite/tratamento farmacológico , Lactococcus lactis/genética , Animais , Proteínas de Bactérias/administração & dosagem , Chaperonina 60/administração & dosagem , Colite/induzido quimicamente , Colite/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Fibrose/tratamento farmacológico , Fibrose/patologia , Imunoglobulina A/metabolismo , Camundongos Endogâmicos BALB C , Microrganismos Geneticamente Modificados , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.
Assuntos
Proteínas de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Disenteria Bacilar/terapia , Serina Proteases/imunologia , Shigella flexneri/imunologia , Sistemas de Secreção Tipo V/imunologia , Adjuvantes Imunológicos/farmacologia , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/uso terapêutico , Chaperonina 60/imunologia , Chaperonina 60/farmacologia , Biologia Computacional , Simulação por Computador , Disenteria Bacilar/microbiologia , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunogenicidade da Vacina , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Domínios Proteicos/imunologia , Receptor 2 Toll-Like/agonistas , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/uso terapêuticoRESUMO
Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/patologia , Receptores Toll-Like/metabolismo , Animais , Chaperonina 60/farmacologia , Expressão Gênica , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , NF-kappa B/metabolismo , RNA Interferente Pequeno , Ratos , Ratos Wistar , Transdução de Sinais/fisiologiaRESUMO
Inflammation plays an important role in the development of cardiovascular diseases (CVDs), suggesting that the immune system is a target of therapeutic interventions used for treating CVDs. This study evaluated mechanisms underlying inflammatory response and cardiomyocyte hypertrophy associated with bacterial lipopolysaccharide (LPS)- or heat shock protein 60 (HSP60)-induced Toll-like receptor (TLR) stimulation and the effect of a small interfering RNA (siRNA) against Ca2+/calmodulin-dependent kinase II delta B (CaMKIIδB) on these outcomes. Our results showed that treatment with HSP60 or LPS (TLR agonists) induced cardiomyocyte hypertrophy and complement system C3 and factor B gene expression. In vitro silencing of CaMKIIδB prevented complement gene transcription and cardiomyocyte hypertrophy associated with TLR 2/4 activation but did not prevent the increase in interleukin-6 and tumor necrosis factor-alfa gene expression in primary cultured cardiomyocytes. Moreover, CaMKIIδB silencing attenuated nuclear factor-kappa B expression. These findings supported the hypothesis that CaMKIIδB acts as a link between inflammation and cardiac hypertrophy. Furthermore, the present study is the first to show that extracellular HSP60 activated complement gene expression through CaMKIIδB. Our results indicated that a stress stimulus induced by LPS or HSP60 treatment promoted cardiomyocyte hypertrophy and initiated an inflammatory response through the complement system. However, CaMKIIδB silencing prevented the cardiomyocyte hypertrophy independent of inflammatory response induced by LPS or HSP60 treatment.
Assuntos
Animais , Ratos , Miócitos Cardíacos/patologia , Receptores Toll-Like/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Transdução de Sinais/fisiologia , Expressão Gênica , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Ratos Wistar , Chaperonina 60/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , RNA Interferente Pequeno , Inflamação/metabolismoRESUMO
BACKGROUND: Allergic asthma is a chronic pulmonary disease characterized by a Th2 inflammatory response. The modulation of a Th2 immune response based on immune deviation to a Th1 pattern or induction and migration of regulatory T cells to the lungs constitutes one of the major therapeutic approaches that is being investigated for the treatment of allergic asthma. The potentials of Mycobacterium leprae 65-kD heat-shock protein or Toll-like receptor 9 ligand (CpG oligodeoxynucleotides) as immune modulators for the treatment of airway allergic disease have been studied individually. OBJECTIVE: Mycobacterial protein combined with CpG was used as immunotherapy for airway allergy. METHODS: Using an ovalbumin-induced asthma model, mice were sensitized and challenged, and then treated with mycobacterial heat-shock protein (Hsp65) combined with CpG. RESULTS: The treatment of mice with established allergy led to the attenuation of eosinophilia, Th2 cytokines and airway hyperresponsiveness. Hsp65 plus CpG treatment also induced an increase in OVA-specific IFN-γ levels and in the frequency of lung inflammatory monocytes. Moreover, we show that the reduction of eosinophilia and the recruitment of inflammatory monocytes to the lungs required early triggering of TLR9, IFN-γ and CCR2 by immunotherapy components. CONCLUSION: In addition to immune deviation to a Th1 response in the modulation of Th2 allergic inflammation, our findings also attribute an important role to the innate response mediated by TLR9, associated with the recruitment of CCR2-dependent monocytes. CLINICAL RELEVANCE: Our findings show that the Hsp65/CpG treatment is a promising strategy for consideration in translational studies.
Assuntos
Asma/tratamento farmacológico , Proteínas de Bactérias/farmacologia , Chaperonina 60/farmacologia , Interferon gama/imunologia , Mycobacterium leprae , Oligodesoxirribonucleotídeos/farmacologia , Receptores CCR2/imunologia , Transdução de Sinais/efeitos dos fármacos , Receptor Toll-Like 9/imunologia , Animais , Asma/genética , Asma/imunologia , Imunoterapia , Interferon gama/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptores CCR2/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/patologia , Receptor Toll-Like 9/genéticaRESUMO
Heat shock proteins (Hsp) are families of highly conserved molecules and immunodominant antigens in some infections and in autoimmune diseases. Some reports suggest that different regions of the Hsp60 molecule induce distinct immune responses. However, there are no reports comparing physiological T-cell reactivity to Hsp60 in mice. In this study, we have analyzed T-cell proliferation and cytokine production induced by Hsp60, under physiological conditions, in three mouse strains bearing distinct major histocompatibility complex (MHC) backgrounds. Proliferative response predominantly was found in C57BL/6 mice, mostly induced by N-terminal and intermediate Hsp60 peptides (P < 0.0001). Interferon-gamma (IFNgamma) production was broadly induced by different regions of Hsp60 in all three mouse strains, although response was focused in different peptide groups in each strain. We did not observe an exclusive Th1 or Th2 cytokine profile induced by any particular region of Hsp60. However, we identified a strain hierarchy in IL-10 production induced by Hsp60 peptides from different regions, mostly detected in C3H/HePas, and in BALB/c, but not in C57BL/6 mice. In contrast, IL-4 production only was induced by the intermediate and C-terminal region peptides in both C3H/HePas and BALB/c mice. Our data give original information on physiological cellular reactivity to Hsp60. We also have identified peptides with the capacity to induce the production of anti-inflammatory cytokines, bringing perspectives for their use in immunotherapy of chronic inflammatory diseases and allograft rejection.
Assuntos
Chaperonina 60/farmacologia , Sequência de Aminoácidos , Animais , Antígenos , Proliferação de Células/efeitos dos fármacos , Chaperonina 60/química , Humanos , Interferon gama/biossíntese , Interleucina-10/biossíntese , Interleucina-4/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/farmacologia , Baço/citologia , Baço/efeitos dos fármacosRESUMO
Heat shock proteins (Hsp) are moving from the category of basically intracellular chaperone molecules to important proteins in both innate and acquired immune responses, with great potential for clinical application as immunomodulators. Both proinflammatory and regulatory Hsp-reactive T cells have been described in animal models of autoimmune diseases. To investigate the role of autoreactivity to Hsp60 and Hsp70 in human transplantation, we analyzed, sequentially, peripheral blood mononuclear cell proliferation and cytokine production before and at different time points after renal transplantation, as well as the modulation of proliferation to Hsp in the presence of exogenous cytokines. Proliferation to Hsp60 and Hsp70 in the pretransplantation (pre-Tx) period was significantly associated with rejection episodes in the first months post-Tx. In contrast, IL-4 production was significantly associated with absence of rejection. Addition of exogenous IL-4 distinctly modulated the proliferative response to Hsp60; inhibiting proliferation in 83% of patients in the early post-Tx period (0-6 months), in which rejection episodes occurred, and inducing proliferation in 62.5% of patients in the later period (>12-24 months), when no rejection was observed. Characterization of autoreactive anti-Hsp60 regulatory T cells may permit new approaches to control the proinflammatory response to the graft, as well as aggressive autoimmunity.