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ABSTRACT Immune exhaustion and senescence are scarcely studied in HIV-pediatric patients. We studied the circulatory CD8 T cells activation/exhaustion and senescent phenotype of children and adolescents vertically infected with HIV or uninfected controls based on the expression of human leukocyte antigen (HLA-DR), CD38, T cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif (ITIM) domain (TIGIT), programmed death 1 (PD-1) and CD57 by flow cytometry, during approximately one year. Eleven HIV-infected (HI) and nine HIV-uninfected (HU) children/adolescents who received two doses or one dose of meningococcal C conjugate vaccine (MenC), respectively, were involved in this study. Blood samples were collected before the immunization (T0), 1-2 months after the first dose (T1), and 1-2 months after the second dose (T2), which was administered approximately one year after the first one. HI patients not receiving combined antiretroviral therapy (cART) showed a higher frequency of CD8 T cells TIGIT+, PD-1+ or CD57+, as well as a higher frequency of CD8 T cells co-expressing CD38/HLA-DR/TIGIT or CD38/HLA-DR/PD-1 when compared to HI treated or HU individuals, at all times that they were assessed. CD8 T cells co-expressing CD38/DR/TIGIT were inversely correlated with the CD4/CD8 ratio but positively associated with viral load. The co-expression of CD38/DR/TIGIT or CD38/DR/PD-1 on CD8 T cells was also inversely associated with the CD4 T cells expressing co-stimulatory molecules CD127/CD28. The results showed a higher expression of exhaustion/senescence markers on CD8 T cells of untreated HI children/adolescents and its correlations with viral load.
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ABSTRACT BACKGROUND: Enteroaggregative Escherichia coli (EAEC) is an E. coli pathotype that presents aggregative adhesion patterns on in vitro cultivated cells, mainly related to persistent diarrhea cases in children. EAEC virulence factors are important for host colonization and pathogenicity. Intestinal epithelial cells (IECs) recognize pathogen-associated molecular patterns (PAMPs) and initiate an immune response. Several studies using in vivo and in vitro models emphasize the probiotic activity and immunomodulatory capacity of Lactobacillus and Bifidobacterium species. OBJECTIVE To evaluate the modulation of cytokine production by probiotics Bifidobacterium animalis and Lactobacillus casei in human intestinal Caco-2 cells exposed to different strains of EAEC. METHODS: Caco-2 cells were incubated with EAEC strains in the presence or absence of probiotics. The production of cytokines IL-8, TNF-α, IL-1β and IL-10 was evaluated in the supernatants by a sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: Cytokine production did not change when uninfected and EAEC-infected Caco-2 cells were exposed to probiotics separately. All EAEC induced a significant increase in IL-8 production by Caco-2 cells, but the probiotics, even together, could not reduce its production. On the other hand, the synergic activity of probiotic strains significantly increased TNF-α production but decreased the basal production of IL-1ß. Also, probiotics induced a significant increase in the production of the anti-inflammatory cytokine IL-10 during EAEC infection. CONCLUSION: Our results reinforce the synergistic immunomodulatory activity of probiotics during EAEC infection.
RESUMO CONTEXTO: Escherichia coli enteroagregativa (EAEC) é um patotipo de E. coli que apresenta o padrão de aderência agregativa em células cultivadas in vitro, sendo comumente relacionada a casos de diarreia persistente em crianças. Fatores de virulência presentes em EAEC são importantes para a colonização do hospedeiro e patogenicidade. As células epiteliais intestinais (IECs) reconhecem padrões moleculares associados a patógenos (PAMPs) e iniciam uma resposta imune. Vários estudos usando modelos in vivo e in vitro enfatizam a atividade probiótica e a capacidade imunomoduladora de espécies de Lactobacillus e Bifidobacterium. OBJETIVO: Este estudo avaliou a modulação da produção de citocinas pelos probióticos Bifidobacterium animalis and Lactobacillus casei em células intestinais humanas Caco-2 expostas a diferentes cepas de EAEC. MÉTODOS: As células Caco-2 foram incubadas com as cepas de EAEC na presença ou ausência dos probióticos. A produção das citocinas IL-8, TNF-α, IL-1β e IL-10 foi avaliada nos sobrenadantes por ELISA sanduíche. RESULTADOS: Não houve alteração na produção de citocinas quando as células não infectadas e as células infectadas com EAEC foram expostas aos probióticos separadamente. Todas as cepas de EAEC induziram aumento significativo na produção de IL-8 pelas células Caco-2, mas os probióticos, ainda que em conjunto, não foram capazes de reduzir a produção desta citocina. Por outro lado, as cepas de probióticos aumentaram significativamente a produção de TNF-α mas diminuíram a produção basal de IL-1ß. Além disso, os probióticos induziram um aumento significativo na produção da citocina anti-inflamatória IL-10 durante a infecção por EAEC. CONCLUSÃO: Nossos resultados reforçam a atividade imunomodulatória sinérgica dos probióticos durante a infecção de EAEC.
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ABSTRACT BACKGROUND: The competence of enteroaggregative Escherichia coli (EAEC) to adhere to the intestinal epithelium of the host is a key role to the colonization and disease development. The virulence genes are crucial for EAEC pathogenicity during adherence, internalization and persistence in the host. The overwhelming majority of antigen encounters in a host occurs on the intestine surface, which is considered a part of innate mucosal immunity. Intestinal epithelial cells (IECs) can be activated by microorganisms and induce an immune response. OBJECTIVE: The present study investigated the interaction of invasive EAEC strains with T84 intestinal epithelial cell line in respect to bacterial invasiveness, persistence and cytokines production. METHODS: We evaluated intracellular persistence of invasive EAEC strains (H92/3, I49/3 and the prototype 042) and production of cytokines by sandwich ELISA in T84 cells upon 24 hours of infection. RESULTS: The survival rates of the prototype 042 was 0.5x103 CFU/mL while survival of I49/3 and H92/3 reached 3.2x103 CFU/mL and 1.4x103 CFU/mL, respectively. Infection with all EAEC strains tested induced significant amounts of IL-8, IL-6 and TNF-α compared to uninfected T84 cells. CONCLUSION: These data showed that infection by invasive EAEC induce a proinflammatory immune response in intestinal epithelial T84 cells.
RESUMO CONTEXTO: A competência de Escherichia coli enteroagregativa (EAEC) para aderir ao epitélio intestinal do hospedeiro é um papel fundamental para a colonização e o desenvolvimento da doença. Os genes de virulência são cruciais para a patogenicidade de EAEC durante a aderência, a internalização e a persistência no hospedeiro. A grande maioria dos encontros de antígenos em um hospedeiro ocorre na superfície do intestino, que é considerada parte da imunidade inata da mucosa. As células epiteliais intestinais (IECs) podem ser ativadas por micro-organismos e induzir uma resposta imune. OBJETIVO: O presente estudo investigou a interação de cepas invasoras de EAEC com a linhagem celular epitelial intestinal T84 em relação a invasão bacteriana, a persistência e a produção de citocinas. MÉTODOS: Avaliamos a persistência intracelular de cepas invasoras de EAEC (H92/3, I49/3 e o protótipo 042) e a produção de citocinas por ELISA "sanduíche" em células T84 após 24 horas de infecção. RESULTADOS: As taxas de sobrevivência da cepa protótipo 042 foi de 0,5x103 UFC/mL, enquanto a sobrevivência de I49/3 e H92/3 atingiu 3,2x103 UFC/mL e 1,4x103 UFC/mL, respectivamente. A infecção com todas as cepas EAEC testadas induziu quantidades significativas de IL-8, IL-6 e TNF-α em comparação com células T84 não infectadas. CONCLUSÃO: Estes dados mostraram que a infecção por EAEC invasoras induzem uma resposta imune pró-inflamatória em células epiteliais intestinais T84.