ENCONTRO INFECÇÕES E TB GOIANIA 2012 MECANISMOS IMUNOLÓGICOS DAS INFECÇÕES RESPIRATÓRIAS E TB José Roberto Lapa e Silva Faculdade de Medicina Universidade Federal do Rio de Janeiro

DELINEAMENTO Relevância do tema Resposta imune inflamatória na porta de entrada - Papel dos neutrófilos Resposta imune na infecção latente - Papel do granuloma Resposta imune na doença ativa - Imunossupressão na TB ativa Conclusões

RELEVÂNCIA DA PESQUISA EM IMUNOLOGIA DA TB

A transmissão aerógena da tuberculose Raios solares ultra-violetas Matam bacilos Partículas levitantes Partículas infectantes CONTATO FOCO (+++) Partículas maiores que se depositam Fernando Fiuza de Mello

Em 70% das pessoas que inalam bacilos, eles são detidos na porta de entrada por barreiras e imunidade inata A depuração mucociliar é importante mecanismoss de defesa contra a TB Células e substâncias contribuem para o controle da infecção

Fagocitose pelo macrófago alveolar Infecção: uma transmissão bem sucedida Nidação do bacilo no alvéolo Crescimento livre Fagocitose pelo macrófago alveolar Fernando Fiuza de Mello

Migração de monócito-macrófagos Maior atividade inicial contra o bacilo Diseminação hematogênica intra-monócito Alvéolo Capilar Macrófago Polimorfo Monócito Linfócito Fernando Fiuza de Mello

Neutrófilos participam da fase inicial do estabelecimento da infecção por TB Ectossomas são vesículas dos neutrófilos que suprimem a ação micobactericida dos macs (Duarte TA, Tuberculosis 2012)

bacilo intra macrófago Fagocitose e multiplicação bacilar intra-macrófago Rompimento do fagossoma e do lisofagosoma Lisossoma Fagocitose Fagossoma Lisofagossoma (de fusão) Crescimento livre do bacilo intra macrófago Atividade bacilar bloqueando a fusão fagossoma+lisossoma e novas fagocitoses Macrófago Fernando Fiuza de Mello

4 Imunidade na TB: Sinalização MHC1 Sinalização MHC2 L.T-CD3/8 Macrófago 4 L.T-CD8 Ativação L.T-CD4 Th 0 L.Tm Th 1 Th 2 Diferenciação Ativação Grânulos bactericidas bacteriostáticos Ativação do macrófago produção de peróxidos limitação das lesões IL2 + g IT IL8 e outras  TFN -a -  das lesões IL4, 5,12 e outras Atividade citotóxica e ação de grânulos

Modelo esquemático do granuloma Necrose caseosa Capilar Ptose vascular Macrófago Célula de Langhans Linfócito auxiliar Polimorfo nuclear modulador Fernando Fiuza de Mello

Granulomas tuberculosos com necrose caseosa (HE) José Roberto Lapa e Silva

Células epitelióides formando paliçada em torno do granuloma (IPX, RFD9) José Roberto Lapa e Silva

Células dendríticas presentes no infiltrado em torno do granuloma (IPX, RFD1) José Roberto Lapa e Silva

DIFERENTES ESTÁGIOS DO GRANULOMA Reece & Kaufmann. Curr Opin Microbiol 2012

HYPOTHESIS Active TB is marked by a relative immunodepression in the affected host Despite the increased expression of defense molecules, some individuals evolve to uncontrolled disease Possible explanation: increased production of deactivating cytokines in the lungs, such as IL-10 or TGF-beta

STRATEGY Use of bronchoalveolar lavages to investigate lung immune response during the development of active TB Investigation of phenotypic changes in lung cells, production of cytokines, expression of cytokine genes, enzymes and receptors during the development of active disease

Co-expression of activating and deactivating cytokines in BAL lysates Bonecini-Almeida MG et al. Inf. Immun. 2004; 72:2628

Presence of biologically active TGF-b ligand in BAL supernatants Bonecini-Almeida MG et al. Inf. Immun. 2004; 72:2628

Presence of cytokines in BAL supernatants Bonecini-Almeida MG et al. Inf. Immun. 2004; 72:2628

Correlation Between CFP32 (Rv0577) and IL-10 in Sputum of Patients with Active Pulmonary Tb R2 = 0.58 Huard RC et al. Inf. Immun. 2003; 71:8871

STRATEGY Collection of induced sputum from bacteriologically confirmed TB patients at the time of diagnosis and at the following times during treatment: 15 DAYS 30 DAYS 60 DAYS 180 DAYS

Host immune markers for TB We compared cases of TB (n=30), other lung diseases/OLD (n=11), health workers/HCW (n=16) at time of diagnosis; TB cases were followed at 15, 30, 60, and 180 days of treatment; Cell pellets of induced sputum were lysed, mRNA extracted, and real-time PCR performed; Gene expression relative to GAPDH for each mediator was established. An.

Host immune markers for TB The following set of genes were evaluated: - Genes that impair Th-1 immunity: IL-10, TGFβ-RI and II, IRAK-M, SOCS 1 and 3, IL1Rn, IDO - Genes that promote Th-1 immunity: IFN-γ,IL-12-p35 and p40, IL-23-p19, TNF-α, NEMO - Genes that affect both Th-1 and Th-2 responses: CD80, CD86, TLR2 An.

Host Mediators as Surrogate Markers for TB Diagnosis and Response to Anti-TB Treatment Almeida et al. J Immunol 2009

PROTEIN EXPRESSION IN SPUTUM SUPS IN TB AND CONTROL SUBJECTS Almeida et al. J Immunol 2009

Sumário dos achados Casos de TB expressaram > níveis de mediadores que se contrapõem à resposta tipo Th1 Altos níveis de mediadores intra (SOCS e IRAK-M) e extracelulares (IL-10, , IDO, etc.) que suprimem a resposta imune Estes mediadores são uma resposta direta ao Mtb, pois diminuem muito aos 30 D de tratamento. Almeida et al. J Immunol 2009

PROPOSED SCHEMATIC VIEW OF IMMUNE REGULATION IN TB Ho & Lapa e Silva. Discov Med 2010

Hypothesis High levels of IL-10 in the lungs of M.tb. infected subjects could facilitate the development of active disease and recurrence of TB. Aim To investigate the levels of IL-10 and IFN-gamma in the lungs at TB diagnosis and during anti-TB chemotherapy and to correlate these levels to relapse rates over a long follow-up period In the present work, we pursued the hypothesis that high levels of IL-10 etc. To answer this question we designed a longitudinal study in which TB patients were evaluated at the time of diagnosis, during and after TB treatment and sputum samples collected at these time points and the patients were followed for over five years. Lago PM et al. Int J Tuberc Lung Dis 2012

Results IL-10 and IFN-gamma were both present in the lungs of TB patients at all time points but only IL-10 showed two patterns at the end of the treatment An Lago PM et al. Int J Tuberc Lung Dis 2012

TWO PATTERNS OF IL-10 IN TB PATIENTS IN SERIAL INDUCED SPUTUM Pattern 1: IL-10 levels decreased until Day 60 of treatment and increased at Day 180 Pattern 2: IL-10 levels decreased until Day 60 of treatment and decreased even further at Day 180 Pattern 1 revealed a large increase in the levels of IL-10 at diagnosis and a decrease until T60 with a new increase at the end of treatment (T180). Pattern 2 showed a decrease from the IL-10 levels until T60 with a lower level at the end of treatment (T180). Red bars represent the median of IL-10 values for each time point. Limit of detection 5 pg/ml. Lago PM et al. Int J Tuberc Lung Dis 2012

TB recurrence and the two IL-10 patterns 20 patients had a follow up of 69.8 + 1.3 months: six had other TB episodes and 14 didn’t; Out of the six cases with TB recurrence, - FIve had Pattern 1 - One had Pattern 2 [OR: 30,0 (CI 95% = 2,19 - 411,3), p= 0,0072 (Fisher’s exact test)]; 20 patients had a follow up of 69.8 + 1.3 months: six had other TB episodes and 14 didn’t; Out of the six cases with TB recurrence, five had Pattern 1 versus one with Pattern 2 with a p value of less than 0,01; The six TB cases with recurrence had a total of 15 episodes of TB, including the one that had serial sputum collection. All episodes were treated with standard Rifampin, Isoniazid and Pyrazinamide for six months. At the last visit, all were well and the chest X-R had limited fibrosis compatible with TB sequelae. Lago PM et al. Int J Tuberc Lung Dis 2012

CONCLUSÕES A TB latente (LTBI) é um dos maiores desafios da saúde pública mundial (reservatório de 2 bi de pessoas, do qual sairão os casos ativos) Os mecanismos de transição da LTBI para TB ativa são largamente desconhecidos A imunossupressão própria da TB ativa resulta em maior morbidade e mortalidade O estudo da imunopatogenia da TB ajudará a encontrar respostas para estas questões