RESUMO

BACKGROUND: Although important information concerning COVID-19 vaccination is available, the effects of the CoronaVac and ChadOx-1 vaccines on immunity and the redox balance in the upper airway mucosa of the aged population are not fully understood. Therefore, the aim of this study was to investigate the impacts of two doses of the CoronaVac or ChadOx-1 vaccine on immune/inflammatory responses and oxidative stress in the airway mucosa of older adults. METHODS: Seventy-six older adults of both sexes, with a mean age of 75.1 ± 6.4 years, were separated according to vaccination status into the CoronaVac (n = 52) and ChadOx-1 (n = 24) groups. Saliva samples were collected before (pre) and 30 days after (post) the administration of the second dose of the CoronaVac or ChadOx-1 vaccine to assess the levels of antibodies (sIgA and IgG), antimicrobial peptides, cytokines, and oxidant/antioxidant agents. RESULTS: The immunogenicity in the ChadOx-1 group was 37.5% for sIgA and 25% for IgG, while that in the CoronaVac group was 18.9% for sIgA and 13.2% for IgG. Intergroup analysis revealed that (1) lower levels of IFN-α, IFN-γ, and IL-10 and a greater IFN-γ/IL-10 ratio, in addition to a greater IL-6/IL-10 ratio, were found in both the pre- and postvaccination periods, and (2) lower levels of total sIgA, IL-12p70, IL-17A, TNF-α, and the IL-12p70/IL-10 ratio, in addition to higher levels of specific sIgA for SARS-CoV-2 antigens and lysozyme, were observed only in the postvaccination period in the ChadOx-1 group than in the CoronaVac group. Intragroup analysis revealed (1) a significant increase in the salivary levels of total peroxides in the postvaccination period compared to those in the prevaccination period in both volunteer groups; (2) a decrease in the levels of lysozyme and the ratio between total antioxidant capacity (TAC) and total peroxides in the postvaccination period in the CoronaVac group compared with those in the prevaccination period; and (3) decreases in the TNF-α, IL-6, and IL-12p70 levels, and the IL-12p70/IL-10 ratio in the ChadoX-1 group, as well as a higher lactoferrin concentration in the postvaccination period than in the prevaccination period. Several positive and negative correlations between the parameters assessed here were found. CONCLUSIONS: In general, the ChadOx-1 group exhibited improvements in both immune/inflammatory responses and redox balance and greater immunogenicity than did the CoronaVac group.

Assuntos

Vacinas contra COVID-19 , COVID-19 , Estresse Oxidativo , Saliva , Humanos , Feminino , Masculino , Idoso , Estresse Oxidativo/fisiologia , Estresse Oxidativo/efeitos dos fármacos , Saliva/metabolismo , Saliva/imunologia , Vacinas contra COVID-19/imunologia , COVID-19/prevenção & controle , COVID-19/imunologia , Idoso de 80 Anos ou mais , Citocinas/metabolismo , SARS-CoV-2/imunologia , Imunoglobulina G , Inflamação/metabolismo , Vacinas de Produtos Inativados

RESUMO

BACKGROUND: Encephalitozoon cuniculi is an opportunistic intracellular pathogen that establishes a balanced relationship with immunocompetent individuals depending on the activity of their CD8+ T cells lymphocytes. However, lower resistance to experimental infection with E. cuniculi was found in B-1 deficient mice (Xid), besides increased the number of CD8 T lymphocytes. Here, we evaluated the profile of CD8+ T lymphocytes from Balb/c wild-type (WT) or Balb/c Xid mice (with B-1 cell deficiency) on the microbicidal activity of macrophages challenged with E. cuniculi. METHODS: Naïve CD8 T lymphocytes from WT or Xid mice uninfected and primed CD8 T lymphocytes from WT or Xid mice infected with E cuniculi were co-cultured with macrophages previously challenged with E. cuniculi. We evaluated macrophages viability and microbicidal activity, and CD8 T lymphocytes viability and presence of activating molecules (CD62L, CD69, and CD107a). RESULTS: Macrophages co-cultured with naïve CD8 T lymphocytes from WT demonstrated high microbicidal activity. Naïve CD8 T lymphocytes obtained from WT mice had a higher expression of CD69 and LAMP-1-activating molecules compared to Xid CD8+ T lymphocytes. Primed CD8 T lymphocytes from Xid mice proliferated more than those from WT mice, however, when the expression of the activating molecule CD69 associated with the expression of CD62L was kept low. In conclusion, naïve CD8+ T lymphocytes from Xid mice, deficient in B-1 cells, they had reduced expression of activation molecules and cytotoxic activity.

Assuntos

Linfócitos T CD8-Positivos , Encephalitozoon cuniculi , Macrófagos , Animais , Linfócitos T CD8-Positivos/imunologia , Camundongos , Macrófagos/imunologia , Encephalitozoon cuniculi/imunologia , Camundongos Endogâmicos BALB C , Ativação Linfocitária/imunologia , Encefalitozoonose/imunologia , Linfócitos B/imunologia , Técnicas de Cocultura

RESUMO

INTRODUCTION: Aspirin is one of the most commonly used drugs worldwide. It is known to present antipyretic, anti-inflammatory and anti-thrombotic actions, making it extremely useful in a wide range of clinical contexts. Interestingly, homeopathically prepared Aspirin 15cH has been found to have a pro-thrombotic effect in rats, raising the hypothesis that Aspirin 15cH could also modulate the activity of inflammatory cells in different pathological processes. OBJECTIVE: Our objective was to assess what effect Aspirin 15cH has on RAW 264.7 macrophages in vitro. METHODS: The effects of Aspirin 15cH on biochemical and morphological activities of lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages were evaluated. These effects were compared with unchallenged macrophages (negative control), untreated LPS-stimulated macrophages, macrophages treated with succussed water (vehicle control), or aspirin 200 µg/mL (pharmacological inhibitor of LPS activity). Cell morphology (adhered cell area and cytoskeleton arrangements), cell viability, toll-like receptor-4 (TLR-4) expression, and the production of nitric oxide, cytokines and intracellular reactive oxygen species were assessed. RESULTS: Aspirin 15cH reduced the number of cells expressing TLR-4 on the surface (p = 0.03) and induced a "columnar" morphology of macrophage pseudopods, indicating changes in cytoskeleton arrangement. When cells were treated with both Aspirin 15cH and LPS, cell morphology became heterogeneous, suggesting that sub-populations of cells had differing sensitivities to LPS or Aspirin 15cH. Exposure of the cells to LPS alone, succussed water or aspirin 200 µg/mL produced effects consistent with the literature. CONCLUSION: Aspirin 15cH, aspirin 200 µg/mL, LPS and succussed water appear to act as independent stimuli able to induce different patterns of macrophage response. Aspirin 15cH induced changes suggestive of M2 polarization of the macrophages (i.e., toward a wound healing or tissue repair, rather than inflammatory, phenotype). These preliminary findings need to be confirmed in further specific studies.

Assuntos

Homeopatia , Lipopolissacarídeos , Ratos , Animais , Lipopolissacarídeos/farmacologia , Lipopolissacarídeos/metabolismo , Aspirina/farmacologia , Receptor 4 Toll-Like/metabolismo , Macrófagos , Citocinas , Água

RESUMO

Encephalitozoon cuniculi is a unicellular, spore-forming, obligate intracellular eukaryote belonging to the phylum Microsporidia. It is known to infect mainly immunocompromised and immunocompetent mammals, including humans. The parasite-host relationship has been evaluated using both in vitro cell culturing and animal models. For example, Balb/c and C57BL/6 mouse strains have been used interchangeably, although the latter has been considered more susceptible due to the higher fungal load observed after infection. In the present study, we identified the characteristics of the immune response of C57BL/6 mice treated or not with the immunosuppressant cyclophosphamide (Cy) and challenged with E. cuniculi by intraperitoneal route. After 14 days of infection, serum was collected to analyze Th1, Th2, and Th17 cytokine levels. In addition, peritoneal washes were performed, and the spleen sample was collected for immune cell phenotyping, whereas liver, spleen, kidney, lung, intestine, and central nervous system (CNS) samples were collected for histopathological analysis. Although infected mice displayed a reduced absolute number of macrophages, they showed an M1 profile, an elevated number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (interferon [IFN]-γ, tumor necrosis factor [TNF]-α, and interleukin [IL]-2) and Th17. Furthermore, Cy-Infected mice showed a reduced absolute number of macrophages with an M1 profile but a reduced number of CD4+T, CD8+T, B-1, and B-2 lymphocytes, with a predominance of Th1 inflammatory cytokines (IFN-γ, TNF-α, and IL-2) and Th2 (IL-4). This group displayed a higher fungal burden as well and developed more severe encephalitozoonosis, which was associated with a reduced number of T and B lymphocytes and a mixed profile of Th1 and Th2 cytokines.

RESUMO

Background: Inflammaging is a phenomenon that has been associated with the development and progression of sarcopenia and frailty syndrome. According to the literature, on the one side, the increase in body fat is associated with a systemic pro-inflammatory status, which consequently favors inflammaging, and on the other side, the regular practice of physical exercise can mitigate the development of this scenario. Therefore, here, we aimed to evaluate the association between inflammaging and physical factors, both body and functional, in a group of physically active older women. Methods: Seventy older women (mean age 72.66 ± 6.17 years) participated in this observational cross-sectional and were separated into the eutrophic, overweight, and obese groups. It was assessed: by bioimpedance-body fat percentage (Fat%) and total (Fat kg), skeletal muscle mass (muscle), and free fat mass both in percentage (FFM%) and total (FFMkg); by the International Physical Activity Questionnaire (IPAQ)-the time of moderate-intensity physical activity per week; by physical tests-handgrip (HG), sit-up-stand-on-the-chair in 5 repetitions (Sit-up) and vertical squat jump test (SJ); in addition to the determination of serum cytokine concentration (IL-6, TNF-α, IL-10, and IL-8), and also body mass index (BMI) and calf circumference (Calf). Results: Higher FFM% and lower body fat (both kg and %) were found in the eutrophic group than in the other groups. The eutrophic group also performed more weekly physical activity, jumped higher, and presented not only higher serum IL-6 concentration but also an increased ratio of IL-10/IL-6, IL-10/TNF-α, IL-10/IL-8 as compared to the values found in the overweight group. The obese group presented higher body fat (kg and %) and lower FFM% than the other groups and also higher serum IL-6 concentration than the overweight group. Interestingly, several significant negative and positive correlations between body composition, physical tests, and serum cytokine concentrations were found in the eutrophic and obese groups. Conclusion: While the eutrophic older women group showed a remarkable regulation of the systemic inflammatory status with positive associations in the physical parameters assessed, the overweight and obese groups presented impairment regulations of the inflammaging, which could be related to less weekly physical activity and higher body fat.

RESUMO

Lymphocytes act as regulatory and effector cells in inflammation and infection situations. A metabolic switch towards glycolytic metabolism predominance occurs during T lymphocyte differentiation to inflammatory phenotypes (Th1 and Th17 cells). Maturation of T regulatory cells, however, may require activation of oxidative pathways. Metabolic transitions also occur in different maturation stages and activation of B lymphocytes. Under activation, B lymphocytes undergo cell growth and proliferation, associated with increased macromolecule synthesis. The B lymphocyte response to an antigen challenge requires an increased adenosine triphosphate (ATP) supply derived mainly through glycolytic metabolism. After stimulation, B lymphocytes increase glucose uptake, but they do not accumulate glycolytic intermediates, probably due to an increase in various metabolic pathway 'end product' formation. Activated B lymphocytes are associated with increased utilization of pyrimidines and purines for RNA synthesis and fatty acid oxidation. The generation of plasmablasts and plasma cells from B lymphocytes is crucial for antibody production. Antibody production and secretion require increased glucose consumption since 90% of consumed glucose is needed for antibody glycosylation. This review describes critical aspects of lymphocyte metabolism and functional interplay during activation. We discuss the primary fuels for the metabolism of lymphocytes and the particularities of T and B cell metabolism, including the differentiation of lymphocytes, stages of development of B cells, and the production of antibodies.

Assuntos

Linfócitos B , Metabolismo dos Lipídeos , Glicosilação , Transporte Biológico , Anticorpos , Glucose

RESUMO

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens, such as Encephalitozoon cuniculi, an obligate intracellular fungus that causes emerging encephalitozoonosis in humans and other animals. Malassezia has immunomodulatory effects and can modulate the production of pro- and anti-inflammatory cytokines via keratinocytes and human monocytes. In this study, we evaluated the modulatory effects of heat-killed Malassezia pachydermatis suspension on macrophages challenged with Encephalitozoon cuniculi. Macrophages were treated with heat-killed M. pachydermatis suspension before being infected with spores of E. cuniculi. The cultures were stained with calcofluor, and the spores, internalized or not, were counted to determine their phagocytic capacity and index (PC and PI, respectively). Microbicidal and phagocytic activities were evaluated by transmission electron microscopy (TEM). The untreated macrophages had higher PC and PI and number of phagocytosed spores than treated macrophages. However, TEM revealed that treated macrophages had higher microbicidal activity because there were few spores in different degrees of degeneration and amorphous materials in the phagocytic vacuoles. Macrophages treated with heat-killed M. pachydermatis suspension had lower PC and PI and incipient presence of E. cuniculi in phagosomes. Treated macrophages had a mixed pattern of cytokine release with Th1, Th2, and Th17 profiles, with emphasis on interleukin (IL)-10, IL-4, IL-17, IL-6, and interferon (IFN)-γ secretion, and particularly high production of anti-inflammatory cytokines. Our results suggest that treatment with heat-killed M. pachydermatis suspension increases the release of cytokines and decreases the phagocytic activity of macrophages challenged with E. cuniculi.

Assuntos

Encephalitozoon cuniculi , Malassezia , Animais , Humanos , Encephalitozoon cuniculi/fisiologia , Temperatura Alta , Macrófagos , Citocinas

RESUMO

Lymphocytes act as regulatory and effector cells in inflammation and infection situations. A metabolic switch towards glycolytic metabolism predominance occurs during T lymphocyte differentiation to inflammatory phenotypes (Th1 and Th17 cells). Maturation of T regulatory cells, however, may require activation of oxidative pathways. Metabolic transitions also occur in different maturation stages and activation of B lymphocytes. Under activation, B lymphocytes undergo cell growth and proliferation, associated with increased macromolecule synthesis. The B lymphocyte response to an antigen challenge requires an increased adenosine triphosphate (ATP) supply derived mainly through glycolytic metabolism. After stimulation, B lymphocytes increase glucose uptake, but they do not accumulate glycolytic intermediates, probably due to an increase in various metabolic pathway ‘end product’ formation. Activated B lymphocytes are associated with increased utilization of pyrimidines and purines for RNA synthesis and fatty acid oxidation. The generation of plasmablasts and plasma cells from B lymphocytes is crucial for antibody production. Antibody production and secretion require increased glucose consumption since 90% of consumed glucose is needed for antibody glycosylation. This review describes critical aspects of lymphocyte metabolism and functional interplay during activation. We discuss the primary fuels for the metabolism of lymphocytes and the particularities of T and B cell metabolism, including the differentiation of lymphocytes, stages of development of B cells, and the production of antibodies.

RESUMO

B-1 lymphocytes are a subtype of B cells with functional and phenotypic features that differ from conventional B lymphocytes. These cells are mainly located in mice's pleural and peritoneal cavities and express unconventional B cell surface markers. B-1 cells participate in immunity by producing antibodies, cytokines, and chemokines and physically interacting with other immune cells. In addition, B-1 cells can differentiate into mononuclear phagocyte-like cells and phagocytize several pathogens. However, the activation and differentiation of B-1 cells are not entirely understood. It is known that several factors can influence B-1 cells, such as pathogens components and the immune response. This work aimed to evaluate the influence of chronic stress on B-1 cell activation and differentiation into phagocytes. The experimental sleep restriction was used as a stress model since the sleep alteration alters several immune cells' functions. Thus, mice were submitted to sleep restriction for 21 consecutive days, and the activation and differentiation of B-1 cells were analyzed. Our results demonstrated that B-1 cells initiated the differentiation process into mononuclear phagocytes after the period of sleep restriction. In addition, we detected a significant decrease in lymphoid lineage commitment factors (EBF, E2A, Blnk) (*P < 0.05) and an increase in the G-CSFR gene (related to the myeloid lineage commitment factor) (****P < 0.0001), as compared to control mice no submitted to sleep restriction. An increase in the co-stimulatory molecules CD80 and CD86 (**P < 0.01 and *P < 0.05, respectively) and a higher production of nitric oxide (NO) (*P < 0.05) and reactive oxygen species (ROS) (*P < 0.05) were also observed in B-1 cells from mice submitted to sleep restriction. Nevertheless, B-1 cells from sleep-restricted mice showed a significant reduction in the Toll-like receptors (TLR)-2, -6, and -9, and interleukine-10 (IL-10) cytokine expression (***P < 0.001) as compared to control. Sleep-restricted mice intraperitoneally infected withL. amazonensispromastigotes showed a reduction in the average internalized parasites (*P < 0.05) by B-1 cells. These findings suggest that sleep restriction interferes with B-1 lymphocyte activation and differentiation. In addition, b-1 cells assumed a more myeloid profile but with a lower phagocytic capacity in this stress condition.

Assuntos

Subpopulações de Linfócitos B , Ativação Linfocitária , Camundongos , Animais , Diferenciação Celular , Linfócitos B , Citocinas/metabolismo , Sono

RESUMO

Background: Relevant aspects regarding the SARS-CoV-2 pathogenesis and the systemic immune response to this infection have been reported. However, the mucosal immune response of the upper airways two months after SARS-CoV-2 infection in patients with mild/moderate symptoms is still not completely described. Therefore, we investigated the immune/inflammatory responses of the mucosa of the upper airways of mild/moderate symptom COVID-19 patients two months after the SARS-CoV-2 infection in comparison to a control group composed of non-COVID-19 healthy individuals. Methods: A cohort of 80 volunteers (age 37.2 ± 8.2), including non-COVID-19 healthy individuals (n=24) and COVID-19 patients (n=56) who presented mild/moderate symptoms during a COVID-19 outbreak in Brazil in November and December of 2020. Saliva samples were obtained two months after the COVID-19 diagnosis to assess the levels of SIgA by ELISA and the cytokines by multiplex analysis. Results: Salivary levels of SIgA were detected in 39 volunteers into the COVID-19 group and, unexpectedly, in 14 volunteers in the control group. Based on this observation, we distributed the volunteers of the control group into without SIgA or with SIgA sub-groups, and COVID-19 group into without SIgA or with SIgA sub-groups. Individuals with SIgA showed higher levels of IL-10, IL-17A, IFN-γ, IL-12p70, IL-13, and IFN-α than those without SIgA. In intergroup analysis, the COVID-19 groups showed higher salivary levels of IL-10, IL-13, IL-17A, and IFN-α than the control group. No statistical differences were verified in the salivary levels of IL-6 and IFN-ß. Lower IL-12p70/IL-10 and IFN-γ/IL-10 ratios were found in the control group without SIgA than the control group with SIgA and the COVID-19 group with SIgA. Conclusion: We were able to present, for the first time, that associations between distinct immunological profiles can help the mucosal immunity to maintain the salivary levels of SIgA in COVID-19 patients two months after the SARS-CoV-2 infection.

Assuntos

COVID-19 , Imunoglobulina A Secretora , Adulto , Teste para COVID-19 , Humanos , Imunidade nas Mucosas , Interleucina-10 , Interleucina-13 , Interleucina-17 , Pessoa de Meia-Idade , SARS-CoV-2

RESUMO

Opportunistic fungal pneumonia is a cause of concern in immunocompromised patients due to its high morbidity and mortality rates. One such opportunistic agent affecting immunocompromised patients is the microsporidia called Encephalitozoon cuniculi. This study aimed to evaluate pneumonia caused by E. cuniculi in mice treated with the immunosuppressive agent cyclophosphamide (Cy). This study also aimed to describe the immune cells associated with the microsporidial pneumonia. C57BL/6 mice were infected intravenously with E. cuniculi spores and treated with Cy (75 mg/kg/week, intraperitoneally). Thirty days post-infection, the fungal burden (qPCR), histopathological lesions, cytokine production, and the phenotype of the immune cells in the lung parenchyma were evaluated. Histologically, interstitial pneumonia with lymphocytic infiltrate was observed in the infected animals. The infiltrate mainly consisted of CD8+ and CD4+ T lymphocytes, with reduced populations of B lymphocytes and macrophages. The production of tumor necrosis factor-alpha (TNF-α) was significant in the animals of the infected groups. Also, the fungal burden was higher in the Cy-treated animals, which was confirmed by the immunohistochemical observation of spores. These results demonstrated that E. cuniculi infection of C57BL/6 mice caused lymphocytic interstitial pneumonia (characterized by a predominant lymphocytic infiltrate), which was aggravated by Cy-induced immunosuppression. Thus, these results can be used to understand the different pathological, immunological, and therapeutic aspects of lymphocytic interstitial pneumonia.

Assuntos

Cuniculidae , Encefalitozoonose , Pneumonia , Animais , Ciclofosfamida/efeitos adversos , Humanos , Hospedeiro Imunocomprometido , Camundongos , Camundongos Endogâmicos C57BL , Pneumonia/tratamento farmacológico

RESUMO

Encephalitozoon cuniculi, an intracellular pathogen, lives in a balanced relationship with immunocompetent individuals based on the activity of T lymphocytes. We previously highlighted the greater susceptibility of B-1 cell-deficient mice (XID mice) to encephalitozoonosis. This study aimed to develop a model of disseminated and severe encephalitozoonosis in mice with combined immunodeficiency to elucidate the role of B cells. To address this objective, cyclophosphamide (Cy)-treated BALB/c and XID mice were inoculated with E. cuniculi, followed by the evaluation of the immune response and histopathological lesions. Immunosuppressed BALB/c mice manifested no clinical signs with an increase in the populations of T lymphocytes and macrophages in the spleen. Immunosuppressed and infected XID mice revealed elevated T cells, macrophages populations, and pro-inflammatory cytokines levels (IFN-γ, TNF-α, and IL-6) with the presence of abdominal effusion and lesions in multiple organs. These clinical characteristics are associated with extensive and severe encephalitozoonosis. The symptoms and lesion size were reduced, whereas B-2 and CD4+ T cells populations were increased in the spleen by transferring B-2 cells adoptive to XID mice. Moreover, B-1 cells adoptive transfer upregulated the peritoneal populations of B-2 cells and macrophages but not T lymphocytes and decreased the symptoms. Herein, we speculated the consistency in the development of severe and disseminated encephalitozoonosis in mice with genetic deficiency of Bruton's tyrosine kinase (Btk) associated with Cy immunosuppression develop with that of the models with T cell deficiency. Taken together, these data emphasized the crucial role of B cells in the protective immune response against encephalitozoonosis.

Assuntos

Encephalitozoon cuniculi , Encefalitozoonose , Doenças dos Roedores , Transferência Adotiva/veterinária , Animais , Encefalitozoonose/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Baço

RESUMO

Phagocytic responses are critical for effective host defense against opportunistic fungal pathogens, such as Encephalitozoon cuniculi, an obligate intracellular fungus that causes emerging ncephalitozoonosis in humans and other animals. Malassezia has immunomodulatory effects and can modulate the production of pro- and anti-inflammatory cytokines via keratinocytes and human monocytes. In this study, we evaluated the modulatory effects of heat-killed Malassezia pachydermatis suspension on macrophages challenged with Encephalitozoon cuniculi. Macrophages were treated with heat-killed M. pachydermatis suspension before being infected with spores of E. cuniculi. The cultures were stained with calcofluor, and the spores, internal- ized or not, were counted to determine their phagocytic capacity and index (PC and PI, respectively). Micro-bicidal and phagocytic activities were evaluated by transmission electron microscopy (TEM). The untreated macrophages had higher PC and PI and number of phagocytosed spores than treated macrophages. However, TEM revealed that treated macrophages had higher microbicidal activity because there were few spores in different degrees of degeneration and amorphous materials in the phagocytic vacuoles. Macrophages treated with heat-killed M. pachydermatis suspension had lower PC and PI and incipient presence of E. cuniculi in phagosomes. Treated macrophages had a mixed pattern of cytokine release with Th1, Th2, and Th17 profiles, with emphasis on interleukin (IL)-10, IL-4, IL-17, IL-6, and interferon (IFN)-g secretion, and particularly high production of anti-inflammatory cytokines. Our results suggest that treatment with heat-killed M. pachyder-matis suspension increases the release of cytokines and decreases the phagocytic activity of macrophages challenged with E. cuniculi.

RESUMO

B-1 lymphocytes are a subtype of B cells with functional and phenotypic features that differ from conventional B lymphocytes. These cells are mainly located in mice’s pleural and peritoneal cavities and express unconventional B cell surface markers. B-1 cells participate in immunity by producing antibodies, cytokines, and chemokines and physically interacting with other immune cells. In addition, B-1 cells can differentiate into mononuclear phagocyte-like cells and phagocytize several pathogens. However, the activation and differentiation of B-1 cells are not entirely understood. It is known that several factors can influence B-1 cells, such as pathogens components and the immune response. This work aimed to evaluate the influence of chronic stress on B-1 cell activation and differentiation into phagocytes. The experimental sleep restriction was used as a stress model since the sleep alteration alters several immune cells' functions. Thus, mice were submitted to sleep restriction for 21 consecutive days, and the activation and differentiation of B-1 cells were analyzed. Our results demonstrated that B-1 cells initiated the differentiation process into mononuclear phagocytes after the period of sleep restriction. In addition, we detected a significant decrease in lymphoid lineage commitment factors (EBF, E2A, Blnk) (*P < 0.05) and an increase in the G-CSFR gene (related to the myeloid lineage commitment factor) (****P < 0.0001), as compared to control mice no submitted to sleep restriction. An increase in the co-stimulatory molecules CD80 and CD86 (**P < 0.01 and *P < 0.05, respectively) and a higher production of nitric oxide (NO) (*P < 0.05) and reactive oxygen species (ROS) (*P < 0.05) were also observed in B-1 cells from mice submitted to sleep restriction. Nevertheless, B-1 cells from sleep-restricted mice showed a significant reduction in the Toll-like receptors (TLR)-2, −6, and −9, and interleukine-10 (IL-10) cytokine expression (***P < 0.001) as compared to control. Sleep-restricted mice intraperitoneally infected with L. amazonensis promastigotes showed a reduction in the average internalized parasites (*P < 0.05) by B-1 cells. These findings suggest that sleep restriction interferes with B-1 lymphocyte activation and differentiation. In addition, b-1 cells assumed a more myeloid profile but with a lower phagocytic capacity in this stress condition.

RESUMO

Opportunistic fungal pneumonia is a cause of concern in immunocompromised patients due to its high morbidity and mortality rates. One such opportunistic agent affecting immunocompromised patients is the microsporidia called Encephalitozoon cuniculi. This study aimed to evaluate pneumonia caused by E. cuniculi in mice treated with the immunosuppressive agent cyclophosphamide (Cy). This study also aimed to describe the immune cells associated with the microsporidial pneumonia. C57BL/6 mice were infected intravenously with E. cuniculi spores and treated with Cy (75 mg/kg/week, intraperitoneally). Thirty days post-infection, the fungal burden (qPCR), histopathological lesions, cytokine production, and the phenotype of the immune cells in the lung parenchyma were evaluated. Histologically, interstitial pneumonia with lymphocytic infiltrate was observed in the infected animals. The infiltrate mainly consisted of CD8+ and CD4+ T lymphocytes, with reduced populations of B lymphocytes and macrophages. The production of tumor necrosis factor-alpha (TNF-α) was significant in the animals of the infected groups. Also, the fungal burden was higher in the Cy-treated animals, which was confirmed by the immunohistochemical observation of spores. These results demonstrated that E. cuniculi infection of C57BL/6 mice caused lymphocytic interstitial pneumonia (characterized by a predominant lymphocytic infiltrate), which was aggravated by Cy-induced immunosuppression. Thus, these results can be used to understand the different pathological, immunological, and therapeutic aspects of lymphocytic interstitial pneumonia.

RESUMO

Encephalitozoon cuniculi, an intracellular pathogen, lives in a balanced relationship with immunocompetent individuals based on the activity of T lymphocytes. We previously highlighted the greater susceptibility of B-1 cell-deficient mice (XID mice) to encephalitozoonosis. This study aimed to develop a model of disseminated and severe encephalitozoonosis in mice with combined immunodeficiency to elucidate the role of B cells. To address this objective, cyclophosphamide (Cy)-treated BALB/c and XID mice were inoculated with E. cuniculi, followed by the evaluation of the immune response and histopathological lesions. Immunosuppressed BALB/c mice manifested no clinical signs with an increase in the populations of T lymphocytes and macrophages in the spleen. Immunosuppressed and infected XID mice revealed elevated T cells, macrophages populations, and pro-inflammatory cytokines levels (IFN-γ, TNF-α, and IL-6) with the presence of abdominal effusion and lesions in multiple organs. These clinical characteristics are associated with extensive and severe encephalitozoonosis. The symptoms and lesion size were reduced, whereas B-2 and CD4+ T cells populations were increased in the spleen by transferring B-2 cells adoptive to XID mice. Moreover, B-1 cells adoptive transfer upregulated the peritoneal populations of B-2 cells and macrophages but not T lymphocytes and decreased the symptoms. Herein, we speculated the consistency in the development of severe and disseminated encephalitozoonosis in mice with genetic deficiency of Bruton’s tyrosine kinase (Btk) associated with Cy immunosuppression develop with that of the models with T cell deficiency. Taken together, these data emphasized the crucial role of B cells in the protective immune response against encephalitozoonosis.

RESUMO

Microsporidia are recognized as opportunistic pathogens in individuals with immunodeficiencies, especially related to T cells. Although the activity of CD8+ T lymphocytes is essential to eliminate these pathogens, earlier studies have shown significant participation of macrophages at the beginning of the infection. Macrophages and other innate immunity cells play a critical role in activating the acquired immunity. After programmed cell death, the cell fragments or apoptotic bodies are cleared by phagocytic cells, a phenomenon known as efferocytosis. This process has been recognized as a way of evading immunity by intracellular pathogens. The present study evaluated the impact of efferocytosis of apoptotic cells either infected or not on macrophages and subsequently challenged with Encephalitozoon cuniculi microsporidia. Macrophages were obtained from the bone marrow monocytes from C57BL mice, pre-incubated with apoptotic Jurkat cells (ACs), and were further challenged with E. cuniculi spores. The same procedures were performed using the previously infected Jurkat cells (IACs) and challenged with E. cuniculi spores before macrophage pre-incubation. The average number of spores internalized by macrophages in phagocytosis was counted. Macrophage expression of CD40, CD206, CD80, CD86, and MHCII, as well as the cytokines released in the culture supernatants, was measured by flow cytometry. The ultrastructural study was performed to analyze the multiplication types of pathogens. Macrophages pre-incubated with ACs and challenged with E. cuniculi showed a higher percentage of phagocytosis and an average number of internalized spores. Moreover, the presence of stages of multiplication of the pathogen inside the macrophages, particularly after efferocytosis of infected apoptotic bodies, was observed. In addition, pre-incubation with ACs or IACs and/or challenge with the pathogen decreased the viability of macrophages, reflected as high percentages of apoptosis. The marked expression of CD206 and the release of large amounts of IL-10 and IL-6 indicated the polarization of macrophages to an M2 profile, compatible with efferocytosis and favorable for pathogen development. We concluded that the pathogen favored efferocytosis and polarized the macrophages to an M2 profile, allowing the survival and multiplication of E. cuniculi inside the macrophages and explaining the possibility of macrophages acting as Trojan horses in microsporidiosis.

Assuntos

Apoptose/genética , Encephalitozoon cuniculi/imunologia , Evasão da Resposta Imune , Macrófagos/microbiologia , Esporos Fúngicos/imunologia , Animais , Medula Óssea/imunologia , Medula Óssea/microbiologia , Diferenciação Celular , Técnicas de Cocultura , Encephalitozoon cuniculi/genética , Encephalitozoon cuniculi/crescimento & desenvolvimento , Feminino , Expressão Gênica , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-6/genética , Interleucina-6/imunologia , Células Jurkat , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Receptor de Manose , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose , Cultura Primária de Células , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Esporos Fúngicos/genética , Esporos Fúngicos/crescimento & desenvolvimento

RESUMO

BACKGROUND: The homeopathic medicines Silicea terra (Sil) and Zincum metallicum (Zinc) modulate macrophage activity and were assessed in an experimental study in-vitro for their effects on macrophage-BCG (Bacillus Calmette-Guérin) interaction. METHODS: RAW 264.7 macrophages were infected with BCG, treated with different potencies of Sil and Zinc (6cH, 30cH and 200cH) or vehicle, and assessed 24 and 48 h later for bacilli internalization, hydrogen peroxide (H2O2) and cytokine production, and lysosomal activity. RESULTS: Treatment with vehicle was associated with non-specific inhibition of H2O2 production to the levels exhibited by uninfected macrophages. Sil 200cH induced significant reduction of H2O2 production (p < 0.001) compared with the vehicle and all other treatments, as well as higher lysosomal activity (p ≤ 0.001) and increased IL-10 production (p ≤ 0.05). Such effects were considered specific for this remedy and potency. The number of internalized bacilli was inversely proportional to Zinc potencies, with statistically significant interaction between dilution and treatment (p = 0.003). Such linear-like behavior was not observed for Sil dilutions: peak internalization occurred with the 30cH dilution, accompanied by cellular degeneration, and IL-6 and IL-10 increased (p ≤ 0.05) only in the cells treated with Sil 6cH. CONCLUSION: Sil and Zinc presented different patterns of potency-dependent effect on macrophage activity. Bacterial digestion and a balanced IL-6/IL-10 production were related to Sil 6cH, though reduced oxidative stress with increased lysosomal activity was related to Sil 200cH. Degenerative effects were exclusively related to Sil 30cH, and potency-dependent phagocytosis was related only to Zinc.

Assuntos

Bacillus/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Materia Medica/farmacologia , Zinco/farmacologia , Brasil , Humanos , Mycobacterium bovis/efeitos dos fármacos

RESUMO

Microsporidia are recognized as opportunistic pathogens in individuals with immunodeficiencies, especially related to T cells. Although the activity of CD8+ T lymphocytes is essential to eliminate these pathogens, earlier studies have shown significant participation of macrophages at the beginning of the infection. Macrophages and other innate immunity cells play a critical role in activating the acquired immunity. After programmed cell death, the cell fragments or apoptotic bodies are cleared by phagocytic cells, a phenomenon known as efferocytosis. This process has been recognized as a way of evading immunity by intracellular pathogens. The present study evaluated the impact of efferocytosis of apoptotic cells either infected or not on macrophages and subsequently challenged with Encephalitozoon cuniculi microsporidia. Macrophages were obtained from the bone marrow monocytes from C57BL mice, pre-incubated with apoptotic Jurkat cells (ACs), and were further challenged with E. cuniculi spores. The same procedures were performed using the previously infected Jurkat cells (IACs) and challenged with E. cuniculi spores before macrophage pre-incubation. The average number of spores internalized by macrophages in phagocytosis was counted. Macrophage expression of CD40, CD206, CD80, CD86, and MHCII, as well as the cytokines released in the culture supernatants, was measured by flow cytometry. The ultrastructural study was performed to analyze the multiplication types of pathogens. Macrophages pre-incubated with ACs and challenged with E. cuniculi showed a higher percentage of phagocytosis and an average number of internalized spores. Moreover, the presence of stages of multiplication of the pathogen inside the macrophages, particularly after efferocytosis of infected apoptotic bodies, was observed. In addition, pre-incubation with ACs or IACs and/or challenge with the pathogen decreased the viability of macrophages, reflected as high percentages of apoptosis. The marked expression of CD206 and the release of large amounts of IL-10 and IL-6 indicated the polarization of macrophages to an M2 profile, compatible with efferocytosis and favorable for pathogen development. We concluded that the pathogen favored efferocytosis and polarized the macrophages to an M2 profile, allowing the survival and multiplication of E. cuniculi inside the macrophages and explaining the possibility of macrophages acting as Trojan horses in microsporidiosis.

RESUMO

Microsporidia, including Encephalitozoon intestinalis, are emerging pathogens which cause opportunistic infections in immunocompromised patients, such as those with AIDS, cancer, the elderly and people on immunosuppressive drugs. Intestinal mucosa (IM) is crucial for developing an efficient adaptive immune response against pathogenic micro-organisms, thereby preventing their colonization and subsequent infection. As immunosuppressive drugs affect the intestinal immune response is little known. In the present study, we investigated the immune response to E. intestinalis infection in the IM and gut-associated lymphoid tissue (GALT) in cyclophosphamide (Cy) immunosuppressed mice, to mimic an immunocompromised condition. Histopathology revealed lymphoplasmacytic enteritis at 7 and 14 days-post-infection (dpi) in all infected groups, however, inflammation diminished at 21 and 28 dpi. Cy treatment also led to a higher number of E. intestinalis spores and lesions, which reduced at 28 dpi. In addition, flow cytometry analysis demonstrated CD4+ and CD8+ T cells to be predominant immune cells, with up-regulation in both Th1 and Th2 cytokines at 7 and 14 dpi, as demonstrated by histopathology. In conclusion, Cy treatment reduced GALT (Peyer's plaques and mesenteric lymph nodes) and peritoneum populations but increased the T-cell population in the intestinal mucosa and the production of pro-and anti-inflammatory cytokines, which were able to eliminate this opportunistic fungus and reduced the E. intestinalis infection.