Pneumonia is the second leading cause of disability-adjusted life-years lost worldwide and the eighth leading cause of death in the United States. Traumatic brain injury (TBI) patients have classically been considered immunosuppressed, but recent research reported that mild head trauma patients have reduced incidence of pneumonia compared to blunt trauma patients. Using our mild TBI model followed by bacterial pneumonia, we investigated the effect of neuronal signaling on innate immune function. To test whether any mild injury primes host immune responses to pneumonia, we generated a mild tail trauma (TT) model. mTBI mice showed protection from bacterial pneumonia while TT mice did not. Using an FDA-approved neurokinin-1 receptor (NK1R) antagonist, aprepitant, we confirmed our previous findings that substance P (SP) is a key mediator of enhanced resistance to pneumonia. Blocking NK1R showed that mTBI-induced release of SP augments pulmonary neutrophil recruitment and microbicidal activity to pulmonary bacterial pathogens. In TT mice, NK1R agonism enhanced the same neutrophil functions, further supporting the hypothesis. No differences were found between mTBI and TT neutrophils’ ability to phagocytose, generate oxidative burst, or acidify phagosomes. However, neutrophils from mTBI mice produced more neutrophil extracellular traps in response to bacterial challenge. These studies show that neurokinin signaling in our model contributes to enhanced bacterial clearance. Cholinergic anti-inflammatory pathway signaling though the α7 nicotinic acetylcholine receptor (α7 nAChR) is also a critical component of improved survival. Blockade of α7 nAChR abrogated the mTBI survival benefit. Mimicking cholinergic signaling using α7 nAChR agonist recapitulated the mTBI reduced pro-inflammatory cytokine production and improved survival. No physiologic differences emerged within 24h following pneumonia, but mTBI and α7 agonist treated mice had significantly lower TNFα in bronchoalveolar fluid, suggesting reduced injurious pulmonary inflammation. However, replacing early TNFα during pneumonia did not increase mortality. Western blot analysis showed downregulation of HMGB1 release in mTBI mice, suggesting that vagal cholinergic signaling reduces late mediators of organ damage. Our experiments show that mTBI enhances resistance to pneumonia by activating the vagus nerve signaling through neurokinin and cholinergic pathways. Translation of these findings could be innovative solutions to fighting or preventing infections.

Introdução: Colite Ulcerativa (UC) é uma Doença Inflamatória Intestinal (DII) caracterizada por uma resposta imune exacerbada, com sintomas como diarreia, perda de peso e sangue nas fezes. Apesar dos medicamentos disponíveis, a remissão da doença nem sempre consegue ser alcançada e há a necessidade de terapias alternativas. A colite induzida por DSS (Dextran Sulfate Sodium) é um modelo animal utilizado na investigação de novas terapias por sua semelhança à UC humana. DSS provoca dano à barreira epitelial do cólon, induzindo uma resposta imune exacerbada; entretanto, o exato mecanismo não está totalmente esclarecido. O Ultrassom Terapêutico (TUS) foi utilizado para tratamento de injúria renal em modelo experimental, sua ação se dá através da estimulação do nervo vago (VN) e consequente ativação da via antiinflamatória colinérgica (CAIP). Uma vez que pacientes com DII podem exibir atividade disfuncional do VN, TUS pode ser investigado como terapia alternativa. Objetivos: Investigar temporalmente o perfil clínico, proteômico, histológico e imunológico da colite aguda induzida por DSS; e determinar os efeitos de TUS na colite induzida por DSS. Métodos: No primeiro estudo, a severidade da colite foi avaliada pela administração de DSS 1-3%, observando a resposta clínica e histológica. A análise temporal de DSS 3% incluiu uma avaliação proteômica e histológica do cólon, e a resposta imune celular no baço, linfonodo mesentérico (MLN) e cólon. No segundo estudo, utilizando o modelo de DSS 2%, TUS foi aplicado no abdômen dos animais e foram observados os sintomas clínicos, dano histológico, proteômica do cólon e respostas imunes celulares no baço, MLN e cólon. Animais esplenectomizados ou knockout para a7nAChR (marcador clássico para ativação de CAIP) foram utilizados. Resultados: No primeiro estudo, observou-se que a severidade da doença foi aumentada seguindo concentrações de 1-3% DSS. A análise temporal de DSS 3% demonstrou que os macrófagos (F4/80+) se apresentam como a primeira resposta celular, seguidos por células T CD25+, CD4+ e CD8+. A piora clínica da doença correspondeu ao aumento progressivo de fatores pró-inflamatórios e dano tecidual no cólon, exceto no dia 8. Foram observados menores níveis dos marcadores de células T CD25+, CD4+ e CD8+ no MLN e/ou baço, sugerindo a ocorrência de tropismo destas células para o intestino. No segundo estudo, a aplicação de TUS diminuiu a severidade da doença através da melhora de sintomas clínicos, danos teciduais e encurtamento do cólon. A proteômica do cólon demonstrou uma resposta anti-inflamatória durante a fase de injúria (D0-7), induzindo uma resolução acelerada da doença na fase de recuperação (D8-14). TUS diminuiu os níveis de células T CD8+ e normalizou os níveis de células T CD25+ no cólon. Animais esplenectomizados não demonstraram melhora clínica ou histológica, enquanto animais a7nAChR KO apresentaram piora da colite experimental. Além disso, TUS aumentou os níveis de células F4/80+a7nAChR+ no intestino de animais WT DSS 2%. Conclusão: Nossos resultados demonstram que a severidade da doença depende da concentração de DSS, relacionada com as respostas clínica, proteômica e imune no modelo animal de DSS 3%; e TUS diminuiu a severidade da colite induzida por DSS presumidamente pela da estimulação do VN e consequente ativação de CAIP através do baço. / Introduction: Ulcerative Colitis (UC) is an Inflammatory Bowel Disease (IBD) characterized by uncontrolled immune response, presenting with symptoms of diarrhea, weight loss and bloody stools. Despite available treatments, UC sustained remission is not achievable and there is still the need for alternative therapies. Dextran Sulfate Sodium (DSS)-induced colitis is a mouse model used to investigate novel therapies, since it closely mimics human UC. DSS damages the colonic epithelial barrier, leading to an exacerbated immune response. However, the exact mechanism is not totally understood. Previous studies showed the use of Therapeutic Ultrasound (TUS) to prevent kidney injury in mice through stimulation of the vagus nerve (VN) and activation of the cholinergic anti-inflammatory pathway (CAIP). Since IBD patients can present with dysfunctional VN activity, TUS could be studied as an alternative therapy. Objectives: To investigate the temporal clinical, proteomic, histological and cellular immune profiles of DSS-induced acute colitis; and to determine the effects of TUS directed toward the VN and spleen in the course of DSS-induced colitis. Methods: First, we analyzed DSS-induced colitis severity by administration of 1-3% DSS, observing the clinical course and histological damage. A time course analysis was performed at 3% DSS, including colon proteomics, colon histology and immune cell responses in the spleen, MLN (mesenteric lymph node) and colon. Next, utilizing 2% DSS in drinking water, we applied TUS over the mice abdomen and analyzed clinical symptoms, histological damage, colon proteomics and immune cell responses in the spleen, MLN and colon. Splenectomized and a7nAChR (key indicator of CAIP activation) KO animals were also used. Results: In the first study, we observed worsening of the disease when increasing DSS concentrations from 1 to 3%. Time course analysis of 3% DSS revealed macrophages to be the first responders, followed by CD25+, CD4+ and CD8+ T cells. Worsening of the disease corresponded to a progressive increase in pro-inflammatory colonic factors and histological damage, except at day 8. Lower levels of CD25+, CD4+ and CD8+ T cells in MLN and/or spleen suggest an immune cell tropism to the gut. In the second study, TUS attenuated DSS induced colitis through amelioration of clinical symptoms, histological damage and colon shortening. Proteomic colon analysis demonstrated an antiinflammatory profile during the injury phase (D0-7), whilst inducing an early resolution of the disease during the recovery phase (D8-14). TUS decreased CD8+ and normalized CD25+ T cell levels in the gut. Splenectomized animals demonstrated no improved clinical and pathological outcomes, and a7nAChR KO mice presented with worsening of the disease. Furthermore, there were increased levels of F4/80+a7nAChR+ cells in the colon of 2% DSS WT mice under TUS treatment. Conclusion: Our results demonstrate that the severity of colitis is dependent on DSS concentration, correlated with clinical, proteomic and cellular immune responses on 3% DSS; and TUS significantly improved DSS-induced acute colitis presumably through stimulation of the VN and consequent activation of CAIP through the spleen.