Estresse e periodontite: avaliação da inervação simpática no periodonto e da resposta adrenérgica após infecção por Porphyromonas gingivalis
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Data
2022-01-25
Autores
Moraes, Renata Mendonça [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O estresse agrava a doença periodontal por vários mecanismos, sendo a estimulação do sistema nervoso simpático (SNS) um deles. A literatura mostra que a estimulação de receptores β-adrenérgicos (β-AR) aumenta a angiogênese em ossos longos, e a expansão microvascular agrava a periodontite. Ainda, catecolaminas aumentam a virulência de periodontopatógenos e agem na resposta imune. Assim, o objetivo deste trabalho foi avaliar: (1) a inervação simpática no periodonto e a influência da ativação do SNS na vascularização periodontal em camundongos e (2) a influência do sistema adrenérgico nos fatores de virulência de Porphyromonas gingivalis (Pg) e na resposta imunológica a este patógeno in vivo (Galleria mellonella). Na primeira parte, camundongos receberam injeção intraperitoneal de solução salina (PBS) ou isoproterenol (ISO; agonista não seletivo β adrenérgico) por 1 mês, para detecção in situ de tirosina hidroxilase, neuropeptídeo Y, transportador de norepinefrina (NET) e endomucina em mandíbulas. Expressão de mRNA de Vegf-a, Il-1β, Il-6, Adrb2 e Rankl foi quantificada 2 h após administração de ISO/PBS em mandíbula e tíbias, que serviram como controle positivo. Diferentemente das tíbias, não houve alteração na expressão dos genes analisados em mandíbula. Por outro lado, NET foi mais expresso no osso alveolar do que na tíbia, sendo detectado nos osteoblastos, osteócitos e células do ligamento. Embora o padrão de inervação e a expressão de Adrb2 sejam semelhantes entre mandíbula e tíbia, o tratamento com ISO não influenciou no número e área de vasos positivos para endomucina. Na segunda parte, investigamos a influência adrenérgica na resposta imune de G. mellonella durante infecção por Pg utilizando norepinefrina (NE; agonista α e β adrenérgico) e ISO. Pg também foi cultivada na presença de ISO (PgISO) ou NE para avaliação da ação direta dos compostos na bactéria. ISO sistêmico protegeu as larvas da infecção por Pg, aumentando o número de hemócitos e reduzindo a contagem de células de Pg na hemolinfa, exclusivamente pelo β-AR. Diferentemente, NE aumentou mortalidade, diminuiu o número de hemócitos. Apenas PgISO aumentou a morte das larvas, apesar de ambos, NE e ISO, terem aumentado a expressão de fatores de virulência na bactéria in vitro. ISO circulante, concomitante com PgISO, reduziu parcialmente a mortalidade das larvas. A influência do estresse na doença periodontal envolve diversas vias que alteram os dois pilares da periodontite (microbiota e sistema imune). No entanto, a ação na resposta do hospedeiro parece ser superior, uma vez que a estimulação β-AR em osso alveolar saudável não alterou a produção de citocinas pró-inflamatórias ou microvascularização e a modulação da resposta imune em G. mellonella por compostos adrenérgicos foi mais importante para o desfecho da infecção que sua ação direta sobre a bactéria.
Stress aggravates periodontitis, and one possible mechanism is the activation of sympathetic nervous system (SNS). The literature shows that stimulation of β-adrenergic receptors (β-AR) induces angiogenesis in long bones, and microvasculature amplification was linked to periodontitis severity. Moreover, catecholamines increase the virulence of some periodontopathogenic bacteria in vitro and influences the innate immunity. Thus, the aim of this study was (1) evaluate the presence and influence of the SNS in the stimulation of periodontal vasculature, and (2) the influence of the adrenergic system on Porphyromonas gingivalis (Pg) virulence and on the immunological response to this pathogen in vivo (Galleria mellonella larvae). For the first part, mice received isoproterenol (ISO, a non-selective β-AR agonist) or saline (PBS) for 1 month, for in situ analysis of tyrosine hydroxylase, neuropeptide Y, norepinephrine transporter (NET) and endomucin in the mandibles. Vegfa, Il-1β, Il-6, Adrb2 and Rankl mRNA expression was assessed 2 hours after PBS/ISO treatment for mandibles and tibia, that served as positive control. We observed that, differently from the tibia, the expression of these genes did not alter on the mandible. However, NET expression was detected in osteoblasts, osteocytes, and periodontal ligament fibroblasts, and were higher expressed when compared to the tibias from the same animals. Although the pattern of sympathetic innervation and Adrb2 expression were similar between tissues, ISO treatment did not increase the area or number of endomucin+ vessels. For the second part, we addressed the adrenergic signaling influence on G. mellonella immune system during Pg infection using norepinephrine (NE, α- and β-AR agonist), ISO and octopamine (insect’s endogenous hormone). Pg was also cultivated in the presence of ISO (PgISO) or NE to investigate the direct action of the ligands on bacterial virulence. Systemic administration of ISO protected the larvae from Pg infection by increasing hemocyte density accompanied by reduction of Pg load in hemolymph, in a β-AR manner. In contrast, NE increased mortality, with decreased hemocyte count and no influence on the other parameters. Only PgISO increased larvae death, despite of ISO and NE increased virulence in vitro. The concomitant injection of systemic ISO partially reversed the toxicity of the PgISO. The influence of stress on periodontitis involves different pathways, that alter the two pillars of disease’s pathogenesis (microbiota and immune system). However, the influence on the host’s inflammatory response seems to overcome the other players, since β-AR activation on healthy alveolar bone didn’t alter cytokines production or microvasculature. Besides, the modulation of innate immunity by adrenergic signaling in G. mellonella was more important for the disease’s outcome than it’s direct action on the bacteria.
Stress aggravates periodontitis, and one possible mechanism is the activation of sympathetic nervous system (SNS). The literature shows that stimulation of β-adrenergic receptors (β-AR) induces angiogenesis in long bones, and microvasculature amplification was linked to periodontitis severity. Moreover, catecholamines increase the virulence of some periodontopathogenic bacteria in vitro and influences the innate immunity. Thus, the aim of this study was (1) evaluate the presence and influence of the SNS in the stimulation of periodontal vasculature, and (2) the influence of the adrenergic system on Porphyromonas gingivalis (Pg) virulence and on the immunological response to this pathogen in vivo (Galleria mellonella larvae). For the first part, mice received isoproterenol (ISO, a non-selective β-AR agonist) or saline (PBS) for 1 month, for in situ analysis of tyrosine hydroxylase, neuropeptide Y, norepinephrine transporter (NET) and endomucin in the mandibles. Vegfa, Il-1β, Il-6, Adrb2 and Rankl mRNA expression was assessed 2 hours after PBS/ISO treatment for mandibles and tibia, that served as positive control. We observed that, differently from the tibia, the expression of these genes did not alter on the mandible. However, NET expression was detected in osteoblasts, osteocytes, and periodontal ligament fibroblasts, and were higher expressed when compared to the tibias from the same animals. Although the pattern of sympathetic innervation and Adrb2 expression were similar between tissues, ISO treatment did not increase the area or number of endomucin+ vessels. For the second part, we addressed the adrenergic signaling influence on G. mellonella immune system during Pg infection using norepinephrine (NE, α- and β-AR agonist), ISO and octopamine (insect’s endogenous hormone). Pg was also cultivated in the presence of ISO (PgISO) or NE to investigate the direct action of the ligands on bacterial virulence. Systemic administration of ISO protected the larvae from Pg infection by increasing hemocyte density accompanied by reduction of Pg load in hemolymph, in a β-AR manner. In contrast, NE increased mortality, with decreased hemocyte count and no influence on the other parameters. Only PgISO increased larvae death, despite of ISO and NE increased virulence in vitro. The concomitant injection of systemic ISO partially reversed the toxicity of the PgISO. The influence of stress on periodontitis involves different pathways, that alter the two pillars of disease’s pathogenesis (microbiota and immune system). However, the influence on the host’s inflammatory response seems to overcome the other players, since β-AR activation on healthy alveolar bone didn’t alter cytokines production or microvasculature. Besides, the modulation of innate immunity by adrenergic signaling in G. mellonella was more important for the disease’s outcome than it’s direct action on the bacteria.
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Palavras-chave
periodontite, sistema nervoso simpático, estresse, Galleria mellonella, periodontitis, Sympathetic nervous system, stress