RESUMO
Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.
Assuntos
Vesículas Extracelulares , Mycobacterium tuberculosis , Tuberculose , Humanos , Células Th1 , Neutrófilos , Monócitos , Células DendríticasRESUMO
The Gram-negative bacteria Brucella abortus is a major cause of brucellosis in animals and humans. The host innate immune response to B. abortus is mainly associated with phagocytic cells such as dendritic cells, neutrophils, and macrophages. However, as mast cells naturally reside in the main bacterial entry sites they may be involved in bacterial recognition. At present, little is known about the role of mast cells during B. abortus infection. The role of the innate immune receptors TLR2 and TLR4 in activation of mast cells by B. abortus (strain RB51) infection was analyzed in this study. The results showed that B. abortus did not induce mast cell degranulation, but did induce the synthesis of the cytokines IL-1ß, IL-6, TNF-α, CCL3, CCL4, and CCL5. Furthermore, B. abortus stimulated key cell signaling molecules involved in mast cell activation such as p38 and NF-κB. Blockade of the receptors TLR2 and TLR4 decreased TNF-α and IL-6 release by mast cells in response to B. abortus. Taken together, our results demonstrate that mast cells are activated by B. abortus and may play a role in inducing an inflammatory response during the initial phase of the infection.
Assuntos
Brucella abortus , Brucelose , Humanos , Animais , Receptor 2 Toll-Like , Receptor 4 Toll-Like , Mastócitos , Fator de Necrose Tumoral alfa , Interleucina-6RESUMO
The clinical presentation of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ranges between mild respiratory symptoms and a severe disease that shares many of the features of sepsis. Sepsis is a deregulated response to infection that causes life-threatening organ failure. During sepsis, the intestinal epithelial cells are affected, causing an increase in intestinal permeability and allowing microbial translocation from the intestine to the circulation, which exacerbates the inflammatory response. Here we studied patients with moderate, severe and critical COVID-19 by measuring a panel of molecules representative of the innate and adaptive immune responses to SARS-CoV-2, which also reflect the presence of systemic inflammation and the state of the intestinal barrier. We found that non-surviving COVID-19 patients had higher levels of low-affinity anti-RBD IgA antibodies than surviving patients, which may be a response to increased microbial translocation. We identified sFas and granulysin, in addition to IL-6 and IL-10, as possible early biomarkers with high sensitivity (>73 %) and specificity (>51 %) to discriminate between surviving and non-surviving COVID-19 patients. Finally, we found that the microbial metabolite d-lactate and the tight junction regulator zonulin were increased in the serum of patients with severe COVID-19 and in COVID-19 patients with secondary infections, suggesting that increased intestinal permeability may be a source of secondary infections in these patients. COVID-19 patients with secondary infections had higher disease severity and mortality than patients without these infections, indicating that intestinal permeability markers could provide complementary information to the serum cytokines for the early identification of COVID-19 patients with a high risk of a fatal outcome.
Assuntos
COVID-19 , Coinfecção , Sepse , Humanos , COVID-19/diagnóstico , SARS-CoV-2 , Interleucina-6 , Interleucina-10 , Permeabilidade , Biomarcadores , IntestinosRESUMO
Mast cells (MC) play a central role in the early containment of bacterial infections, such as that caused by Listeria monocytogenes (L.m). The mechanisms of MC activation induced by L.m infection are well known, so it is possible to evaluate whether they are susceptible to targeting and modulation by different drugs. Recent evidence indicates that valproic acid (VPA) inhibits the immune response which favors L.m pathogenesis in vivo. Herein, we examined the immunomodulatory effect of VPA on L.m-mediated MC activation. To this end, bone marrow-derived mast cells (BMMC) were pre-incubated with VPA and then stimulated with L.m. We found that VPA reduced MC degranulation and cytokine release induced by L.m. MC activation during L.m infection relies on Toll-Like Receptor 2 (TLR2) engagement, however VPA treatment did not affect MC TLR2 cell surface expression. Moreover, VPA was able to decrease MC activation by the classic TLR2 ligands, peptidoglycan and lipopeptide Pam3CSK4. VPA also reduced cytokine production in response to Listeriolysin O (LLO), which activates MC by a TLR2-independent mechanism. In addition, VPA decreased the activation of critical events on MC signaling cascades, such as the increase on intracellular Ca2+ and phosphorylation of p38, ERK1/2 and -p65 subunit of NF-κB. Altogether, our data demonstrate that VPA affects key cell signaling events that regulate MC activation following L.m infection. These results indicate that VPA can modulate the functional activity of different immune cells that participate in the control of L.m infection.
Assuntos
Listeria monocytogenes , Listeriose , Citocinas/metabolismo , Humanos , Lipopeptídeos/metabolismo , Listeriose/tratamento farmacológico , Listeriose/metabolismo , Mastócitos/metabolismo , NF-kappa B/metabolismo , Peptidoglicano/metabolismo , Receptor 2 Toll-Like/metabolismo , Ácido Valproico/metabolismo , Ácido Valproico/farmacologiaRESUMO
Most individuals infected with Mycobacterium tuberculosis (Mtb) have latent tuberculosis (TB), which can be diagnosed with tests (such as the QuantiFERON-TB Gold test [QFT]) that detect the production of IFN-γ by memory T cells in response to the Mtb-specific antigens 6 kDa early secretory antigenic target EsxA (Rv3875) (ESAT-6), 10 kDa culture filtrate antigen EsxB (Rv3874) (CFP-10), and Mtb antigen of 7.7 kDa (Rv2654c) (TB7.7). However, the immunological mechanisms that determine if an individual will develop latent or active TB remain incompletely understood. Here we compared the response of innate and adaptive peripheral blood lymphocytes from healthy individuals without Mtb infection (QFT negative) and from individuals with latent (QFT positive) or active TB infection, to determine the characteristics of these cells that correlate with each condition. In active TB patients, the levels of IFN-γ that were produced in response to Mtb-specific antigens had high positive correlations with IL-1ß, TNF-α, MCP-1, IL-6, IL-12p70, and IL-23, while the proinflammatory cytokines had high positive correlations between themselves and with IL-12p70 and IL-23. These correlations were not observed in QFT-negative or QFT-positive healthy volunteers. Activation with Mtb-soluble extract (a mixture of Mtb antigens and pathogen-associated molecular patterns [PAMPs]) increased the percentage of IFN-γ-/IL-17-producing NK cells and of IL-17-producing innate lymphoid cell 3 (ILC3) in the peripheral blood of active TB patients, but not of QFT-negative or QFT-positive healthy volunteers. Thus, active TB patients have both adaptive and innate lymphocyte subsets that produce characteristic cytokine profiles in response to Mtb-specific antigens or PAMPs. These profiles are not observed in uninfected individuals or in individuals with latent TB, suggesting that they are a response to active TB infection.
Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Antígenos de Bactérias , Citocinas , Humanos , Imunidade Inata , Interleucina-17 , Interleucina-23 , Interleucina-6 , Linfócitos , Moléculas com Motivos Associados a Patógenos , Fator de Necrose Tumoral alfaRESUMO
Listeria monocytogenes (L.m) is efficiently controlled by several cells of the innate immunity, including the Mast Cell (MC). MC is activated by L.m inducing its degranulation, cytokine production and microbicidal mechanisms. TLR2 is required for the optimal control of L.m infection by different cells of the immune system. However, little is known about the MC receptors involved in recognizing this bacterium and whether these interactions mediate MC activation. In this study, we analyzed whether TLR2 is involved in mediating different MC activation responses during L.m infection. We found that despite MC were infected with L.m, they were able to clear the bacterial load. In addition, MC degranulated and produced ROS, TNF-α, IL-1ß, IL-6, IL-13 and MCP-1 in response to bacterial infection. Interestingly, L.m induced the activation of signaling proteins: ERK, p38 and NF-κB. When TLR2 was blocked, L.m endocytosis, bactericidal activity, ROS production and mast cell degranulation were not affected. Interestingly, only IL-6 and IL-13 production were affected when TLR2 was inhibited in response to L.m infection. Furthermore, p38 activation depended on TLR2, but not ERK or NF-κB activation. These results indicate that TLR2 mediates only some MC activation pathways during L.m infection, mainly those related to IL-6 and IL-13 production.
Assuntos
Interleucina-13/imunologia , Interleucina-6/imunologia , Listeria monocytogenes/imunologia , Mastócitos/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Degranulação Celular/imunologia , Degranulação Celular/fisiologia , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Ativação Enzimática/imunologia , Interações Hospedeiro-Patógeno/imunologia , Interleucina-13/metabolismo , Interleucina-6/metabolismo , Listeria monocytogenes/fisiologia , Mastócitos/microbiologia , Mastócitos/fisiologia , Camundongos Endogâmicos C57BL , NF-kappa B/imunologia , NF-kappa B/metabolismo , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Receptor 2 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The immune response plays a critical role in the pathophysiology of SARS-CoV-2 infection ranging from protection to tissue damage and all occur in the development of acute respiratory distress syndrome (ARDS). ARDS patients display elevated levels of inflammatory cytokines and innate immune cells, and T and B cell lymphocytes have been implicated in this dysregulated immune response. Mast cells are abundant resident cells of the respiratory tract and are able to release different inflammatory mediators rapidly following stimulation. Recently, mast cells have been associated with tissue damage during viral infections, but their role in SARS-CoV-2 infection remains unclear. In this study, we examined the profile of mast cell activation markers in the serum of COVID-19 patients. We noticed that SARS-CoV-2-infected patients showed increased carboxypeptidase A3 (CPA3) and decreased serotonin levels in their serum when compared with symptomatic SARS-CoV-2-negative patients. CPA3 levels correlated with C-reactive protein, the number of circulating neutrophils, and quick SOFA. CPA3 in serum was a good biomarker for identifying severe COVID-19 patients, whereas serotonin was a good predictor of SARS-CoV-2 infection. In summary, our results show that serum CPA3 and serotonin levels are relevant biomarkers during SARS-CoV-2 infection. This suggests that mast cells and basophils are relevant players in the inflammatory response in COVID-19 and may represent targets for therapeutic intervention.
Assuntos
COVID-19/diagnóstico , Carboxipeptidases A/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/diagnóstico , Mastócitos/imunologia , SARS-CoV-2/isolamento & purificação , Serotonina/metabolismo , Biomarcadores/análise , COVID-19/complicações , COVID-19/metabolismo , COVID-19/virologia , Humanos , Inflamação/etiologia , Inflamação/metabolismo , Inflamação/patologia , Mastócitos/patologia , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Zika virus (ZIKV) infection during pregnancy usually shows only mild symptoms and is frequently subclinical. However, it can be vertically transmitted to the fetus, causing microcephaly and other congenital defects. During pregnancy, the immune environment modifications can alter the response to viruses in general and ZIKV in particular. OBJECTIVE: To describe the role of pregnancy in the systemic pro- and anti-inflammatory response during symptomatic ZIKV infection. MATERIALS AND METHODS: A multiplex assay was used to measure 25 cytokines, chemokines, and receptors in 110 serum samples from pregnant and nonpregnant women with and without ZIKV infection with and without symptoms. Samples were collected through an epidemiological surveillance system. RESULTS: Samples from pregnant women with ZIKV infection showed a higher viral load but had similar profiles of inflammatory markers as compared with nonpregnant infected women, except for CXCL10 that was higher in infected pregnant women. Notably, the presence of ZIKV in pregnancy favored a regulatory profile by significantly increasing anti-inflammatory cytokines such as interleukin (IL)-10, receptors IL-1RA, and IL-2R, but only those pro-inflammatory cytokines such as IL-6, interferon (IFN)-α, IFN-γ and IL-17 that are essential for the antiviral response. Interestingly, there were no differences between symptomatic and weakly symptomatic ZIKV-infected groups. CONCLUSION: Our results revealed a systemic anti-inflammatory cytokine and chemokine profile that could participate in the control of the virus. The anti-inflammatory response in pregnant women infected with ZIKA was characterized by high CXCL10, a cytokine that has been correlated with congenital malformations.
Assuntos
Quimiocina CXCL10/metabolismo , Citocinas/metabolismo , Complicações Infecciosas na Gravidez/metabolismo , Complicações Infecciosas na Gravidez/virologia , Carga Viral , Infecção por Zika virus/metabolismo , Infecção por Zika virus/virologia , Zika virus/fisiologia , Adulto , Biomarcadores , Feminino , Humanos , Imunomodulação , Gravidez , Complicações Infecciosas na Gravidez/imunologia , Trimestres da Gravidez , Adulto Jovem , Infecção por Zika virus/imunologiaRESUMO
Valproic acid (VPA) is a drug commonly used for epileptic seizure control. Recently, it has been shown that VPA alters the activation of several immune cells, including Natural Killer (NK) cells, which play an important role in the containment of viruses and intracellular bacteria. Although VPA can increase susceptibility to extracellular pathogens, it is unknown whether the suppressor effect of VPA could affect the course of intracellular bacterial infection. This study aimed to evaluate the role of VPA during Listeria monocytogenes (L.m) infection, and whether NK cell activation was affected. We found that VPA significantly augmented mortality in L.m infected mice. This effect was associated with increased bacterial load in the spleen, liver, and blood. Concurrently, decreased levels of IFN-γ in serum and lower splenic indexes were observed. Moreover, in vitro analysis showed that VPA treatment decreased the frequency of IFN-γ-producing NK cells within L.m infected splenocytes. Similarly, VPA inhibited the production of IFN-γ by NK cells stimulated with IL-12 and IL-18, which is a crucial system for early IFN-γ production in listeriosis. Finally, VPA decreased the phosphorylation of STAT4, p65, and p38, without affecting the expression of IL-12 and IL-18 receptors. Altogether, our results indicate that VPA increases the susceptibility to Listeria monocytogenes infection and suggest that NK cell is one of the main targets of VPA, but further work is needed to ascertain this effect.
Assuntos
Interferon gama/metabolismo , Células Matadoras Naturais/imunologia , Listeria monocytogenes/fisiologia , Listeriose/imunologia , Ácido Valproico/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Humanos , Imunomodulação , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos BALB C , Fator de Transcrição STAT4/metabolismo , Transdução de Sinais , Ácido Valproico/imunologiaRESUMO
"Transferon Oral" is a peptide-derived product with immunomodulatory properties obtained from the lysis and dialysis of human buffy coat. Its active pharmaceutical ingredient, generically known as Dialyzable Leucocyte Extract, is a mixture of peptide populations with reproducible proportions among batches. "Transferon Oral" modulates IFN-γ, TNF-α, and IL-6 and increases the survival rate in a herpes infection murine model when oropharyngeally (ORO) administered, which correlate with clinical observations where "Transferon Oral" is used as a therapeutic auxiliary in inflammatory diseases. Notwithstanding, how a peptide-derived product elicits systemic modulation of cytokines when ORO administered remains unclear. To shed light on the pharmacology of "Transferon Oral" its peptide components must be known. Ten "Transferon Oral" batches were sequenced by mass spectrometry and the intact peptides were identified. The most abundant peptides were the monomeric human Ubiquitin (Ub), a globular low-molecular mass protein, and an Ub variant which lacks the two-terminal Gly (Ub-GG). Recombinant Ub prevented murine death when ORO administered in a herpes infection murine model. Besides, the percentage of survival increased in groups treated with Transferon Oral+Ub and decreased in groups treated with Ub-depleted "Transferon Oral" respect to the group treated with "Transferon Oral" only. Our findings indicate that the biological properties of "Transferon Oral" are partially associated to the Ub content. They suggest that Ub may activate its extracellular receptor (CXCR-4) in the stomach eliciting systemic immunomodulatory effects via vagus nerve. This is the first report that identifies an active component of "Transferon Oral" with the potential for the development of oral peptide immunomodulators.
RESUMO
Mast cell activation through the high-affinity IgE receptor (FcεRI) plays a central role in allergic reactions. FcεRI-mediated activation triggers multiple signaling pathways leading to degranulation and synthesis of different inflammatory mediators. IgE-mediated mast cell activation can be modulated by different molecules, including several drugs. Herein, we investigated the immunomodulatory activity of the histone deacetylase inhibitor valproic acid (VPA) on IgE-mediated mast cell activation. To this end, bone marrow-derived mast cells (BMMC) were sensitized with IgE and treated with VPA followed by FcεRI cross-linking. The results indicated that VPA reduced mast cell IgE-dependent degranulation and cytokine release. VPA also induced a significant reduction in the cell surface expression of FcεRI and CD117, but not other mast cell surface molecules. Interestingly, VPA treatment inhibited the phosphorylation of PLCγ2, a key signaling molecule involved in IgE-mediated degranulation and cytokine secretion. However, VPA did not affect the phosphorylation of other key components of the FcεRI signaling pathway, such as Syk, Akt, ERK1/2, or p38. Altogether, our data demonstrate that VPA affects PLCγ2 phosphorylation, which in turn decreases IgE-mediated mast cell activation. These results suggest that VPA might be a key modulator of allergic reactions and might be a promising therapeutic candidate.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Fosfolipase C gama/antagonistas & inibidores , Receptores de IgE/efeitos dos fármacos , Ácido Valproico/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Interleucina-13/metabolismo , Interleucina-6/metabolismo , Mastócitos/citologia , Camundongos , Fosfolipase C gama/fisiologia , Receptores de IgE/biossíntese , Receptores de IgE/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
hScrib and hDlg belong to the PDZ family of proteins. Since the identification of these highly phylogenetically conserved scaffolds, an increasing amount of experiments has elucidated the roles of hScrib and hDlg in a variety of cell functions. Remarkably, their participation during the establishment of polarity in epithelial cells is well documented. Although the role of both proteins in the immune system is scantly known, it has become a growing field of investigation. Here, we summarize the interactions and functions of hScrib and hDlg1, which participate in diverse functions involving cell polarization in immune cells, and discuss their relevance in the immune cell biology. The fundamental role of hScrib and hDlg1 during the establishment of the immunological synapse, hence T cell activation, and the recently described role of hScrib in reactive oxygen species production in macrophages and of hDlg1 in cytokine production by dendritic cells highlight the importance of both proteins in immune cell biology. The expression of these proteins in other leukocytes can be anticipated and needs to be confirmed. Due to their multiple interaction domains, there is a wide range of possible interactions of hScrib and hDlg1 that remains to be explored in the immune system.
Assuntos
Polaridade Celular/imunologia , Células Dendríticas/imunologia , Proteína 1 Homóloga a Discs-Large/metabolismo , Macrófagos/imunologia , Proteínas de Membrana/metabolismo , Linfócitos T/imunologia , Proteínas Supressoras de Tumor/metabolismo , Citocinas/metabolismo , Células Dendríticas/metabolismo , Humanos , Imunidade Celular , Sinapses Imunológicas/imunologia , Sinapses Imunológicas/metabolismo , Ativação Linfocitária/imunologia , Macrófagos/metabolismo , Espécies Reativas de Oxigênio , Linfócitos T/metabolismoRESUMO
We recently reported, for the first time, the expression and regulation of the PDZ polarity proteins Scrib and Dlg1 in human APCs, and also described the viral targeting of these proteins by NS1 of influenza A virus in human dendritic cells (DCs). Scrib plays an important role in reactive oxygen species (ROS) production in MÏs and uropod formation and migration in T cells, while Dlg1 is important for T cell downstream activation after Ag recognition. Nevertheless, the functions of these proteins in human DCs remain unknown. Here, we knocked-down the expression of both Scrib and Dlg1 in human DCs and then evaluated the expression of co-stimulatory molecules and cytokine production during maturation. We demonstrated that Scrib is necessary for adequate CD86 expression, while Dlg1 is important for CD83 up-regulation and IL-6 production upon maturation, suggesting that Scrib and Dlg1 participate in separate pathways in DCs. Additionally, both proteins are required for adequate IL-12 production after maturation. Furthermore, we showed that the inefficient maturation of DCs induced by Scrib or Dlg1 depletion leads to impaired T cell activation. Our results revealed the previously unknown contribution of Scrib and Dlg1 in human DCs pivotal functions, which may be able to impact innate and adaptive immune response.
Assuntos
Apresentação de Antígeno , Células Dendríticas/imunologia , Proteína 1 Homóloga a Discs-Large/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Imunidade Adaptativa , Antígenos CD/biossíntese , Antígenos CD/genética , Antígeno B7-2/biossíntese , Antígeno B7-2/genética , Células Cultivadas , Citocinas/biossíntese , Citocinas/genética , Proteína 1 Homóloga a Discs-Large/antagonistas & inibidores , Proteína 1 Homóloga a Discs-Large/genética , Técnicas de Silenciamento de Genes , Humanos , Imunidade Inata , Imunoglobulinas/biossíntese , Imunoglobulinas/genética , Interleucina-12/metabolismo , Interleucina-6/biossíntese , Interleucina-6/genética , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Densidade Pós-Sináptica/fisiologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidores , Proteínas Supressoras de Tumor/genética , Regulação para Cima , Antígeno CD83RESUMO
Gaining knowledge of the neoplastic side of the three main cells-B cells, Follicular Helper T (Tfh) cells, and follicular dendritic cells (FDCs) -involved in the germinal center (GC) reaction can shed light toward further understanding the microuniverse that is the GC, opening the possibility of better treatments. This paper gives a review of the more complex underlying mechanisms involved in the malignant transformations that take place in the GC. Whilst our understanding of the biology of the GC-related B cell lymphomas has increased-this is not reviewed in detail here-the dark side involving neoplasms of Tfh cells and FDCs are poorly studied, in great part, due to their low incidence. The aggressive behavior of Tfh lymphomas and the metastatic potential of FDCs sarcomas make them clinically relevant, merit further attention and are the main focus of this review. Tfh cells and FDCs malignancies can often be misdiagnosed. The better understanding of these entities linked to their molecular and genetic characterization will lead to prediction of high-risk patients, better diagnosis, prognosis, and treatments based on molecular profiles.
RESUMO
Persistent infection with high-risk human papillomavirus (HR-HPV) is the main factor in the development of cervical cancer (CC). The presence of immunosuppressive factors plays an important role in the development of this type of cancer. To determine whether CD39 and CD73, which participate in the production of immunosuppressive adenosine (Ado), are involved in the progression of CC, we compared the concentrations and hydrolytic activity of these ectonucleotidases in platelet-free plasma (PFP) samples between patients with low-grade squamous intraepithelial lesions (LSILs) (n = 18), high-grade squamous intraepithelial lesions (HSILs) (n = 12), and CC (n = 19) and normal donors (NDs) (n = 15). The concentrations of CD39 and CD73 in PFP increased with disease progression (r = 0.5929, p < 0.001). The PFP of patients with HSILs or CC showed the highest concentrations of CD39 (2.3 and 2.2 times that of the NDs, respectively) and CD73 (1.7 and 2.68 times that of the NDs, respectively), which were associated with a high capacity to generate Ado from the hydrolysis of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The addition of POM-1 and APCP, specific inhibitors of CD39 and CD73, respectively, inhibited the ADPase and AMPase activity of PFP by more than 90%. A high level of the 90 kD isoform of CD73 was detected in the PFP of patients with HSILs or CC. Digestion with endoglycosidase H and N-glycanase generated CD73 with weights of approximately 90 kD, 85 kD, 80 kD, and 70 kD. In addition, the levels of transforming grow factor-ß (TGF-ß) in the PFPs of patients with LSIL, HSIL and CC positively correlated with those of CD39 (r = 0.4432, p < 0.001) and CD73 (r = 0.5786, p < 0.001). These results suggest that persistent infection by HR-HPV and the concomitant production of TGF-ß promote the expression of CD39 and CD73 to favor CC progression through Ado generation.
Assuntos
5'-Nucleotidase/metabolismo , Antígenos CD/metabolismo , Apirase/metabolismo , Neoplasias do Colo do Útero/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Adulto , Ensaio de Imunoadsorção Enzimática , Feminino , HumanosRESUMO
BACKGROUND: Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells. METHODS: The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection. RESULTS: Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60 days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice. CONCLUSION: J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.
Assuntos
Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Tuberculose/terapia , Animais , Carga Bacteriana , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Vesículas Extracelulares/química , Vesículas Extracelulares/transplante , Masculino , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/patogenicidade , Tuberculose/microbiologiaRESUMO
Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (Δψ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.
Assuntos
Mitofagia/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Triterpenos/farmacologia , Células A549 , Humanos , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ácido UrsólicoRESUMO
Transferon® is an immunomodulator made of a complex mixture of peptides from human dialyzable leucocyte extracts (hDLEs). Development of surrogate antibodies directed to hDLE is an indispensable tool for studies during process control and preclinical trials. These antibodies are fundamental for different analytical approaches, such as identity test and drug quantitation, as well as to characterize its pharmacokinetic and mechanisms of action. A previous murine study showed the inability of the peptides of Transferon® to induce antibody production by themselves; therefore, in this work, two approaches were tested to increase its immunogenicity: chemical conjugation of the peptides of Transferon® to carrier proteins and the use of a rabbit model. Bioconjugates were generated with Keyhole Limpet Hemocyanin (KLH) or Bovine Serum Albumin (BSA) through maleimide-activated carrier proteins. BALB/c mice and New Zealand rabbits were immunized with Transferon® conjugated to KLH or nonconjugated Transferon®. Animals that were immunized with conjugated Transferon® showed significant production of antibodies as evinced by the recognition of Transferon®-BSA conjugate in ELISA assays. Moreover, rabbits showed higher antibody titers when compared with mice. Neither mouse nor rabbits developed antibodies when immunized with nonconjugated Transferon®. Interestingly, rabbit antibodies were able to partially block IL-2 production in Jurkat cells after costimulation with Transferon®. In conclusion, it is feasible to elicit specific and functional antibodies anti-hDLE with different potential uses during the life cycle of the product.
Assuntos
Isoanticorpos/imunologia , Fator de Transferência/efeitos adversos , Adjuvantes Imunológicos , Animais , Formação de Anticorpos , Especificidade de Anticorpos/imunologia , Antígenos/administração & dosagem , Antígenos/imunologia , Ensaio de Imunoadsorção Enzimática , Humanos , Imunização , Imunoglobulina G/imunologia , Imunoglobulina G/isolamento & purificação , Isoanticorpos/isolamento & purificação , Masculino , Camundongos , Peptídeos/administração & dosagem , Peptídeos/imunologia , Coelhos , Fator de Transferência/imunologia , Fator de Transferência/uso terapêuticoRESUMO
The development of cervical cancer (CeCa) is associated with high-risk human papilloma virus (HR-HPV) infections, mainly HPV-16, which is present in more than 50% of cases. The presence of immunosuppressive factors in the early stages of the disease is also strongly linked to CeCa progression. In this context, it is unknown whether ectonucleotidases CD39 and CD73, which are involved in the production of adenosine (Ado) that suppresses the specific antitumor immune response, are present in precursor lesions of CeCa. In this pilot study, we analyzed the presence of CD39 and CD73 and their capacity to generate Ado in 25 cervical samples from patients with grade 1 cervical intraepithelial neoplasms (CIN-1) and 25 samples from normal donors (NDs) free of HPV infection. Cells obtained from cervical samples of CIN-1 patients positive for HPV-16 showed higher CD39 and CD73 contents compared to samples obtained from CIN-1 patients negative for HPV-16 and NDs. Interestingly, solubilized cervical mucus from these patients also showed higher contents of soluble CD39 and CD73, which were associated with a greater capacity to produce Ado from the hydrolysis of adenosine triphosphate (ATP) and adenosine monophosphate (AMP). In addition, serum samples of these patients showed higher levels of TGF-ß than those of CIN-1 patients negative for HPV-16 and ND. These results suggest that persistent infection with HR-HPV, mostly HPV-16, in CIN-1 patients may promote the expression of CD39 and CD73 through the production of TGF-ß in precursor lesions to generate an immunosuppressive microenvironment and allow its progression to CeCa.