The Role of Cytokine Immune Responses in Pulmonary Tuberculosis in Patients Co-Infected With Human Immunodeficiency Virus

Abstract

The gold standard for pulmonary tuberculosis (PTB) diagnosis is either isolation of Mycobacterium tuberculosis (Mtb) by culture or detection of Mtb-specific nucleic acids by molecular methods. However, apart from laboratory infrastructure, culture take longer time to get results, while molecular methods are expensive and require a lot of human expertise. Despite its limitation of lower specificity and sensitivity, Acid Fast Bacilli (AFB) microscopy smear remains the most widely used and cost effective laboratory diagnostic technique for PTB diagnosis in low-and-middle income (LMIC) countries. The use of Interferon Gamma Release Assays (IGRAs) have shown promise in diagnosis of TB in immune-competent as compared to immunocompromised individuals. However, IGRA is limited in distinguishing between active and latent TB. Therefore, measuring of Mtb antigen-specific immune responses and cytokines levels produced by T cell after exposure to the pathogen would be useful. As such, the overall aim of this study was to evaluate pulmonary tuberculosis smear status with cellular immune profile and cytokine response in adult patients with pulmonary tuberculosis (PTB) co-infected with HIV in AMPATH, Moi Teaching and Referral Hospital (MTRH). The specific objectives were to: compare Th1 (IFN-γ, TNF-α, IL-2, IL-8 and IL-12p70) and Th2 (IL-4, IL-6 and IL-10) cytokine responses with culture status in patients with PTB co-infected with HIV; determine diagnostic accuracy of Th1 (IFN-γ, TNF-α, IL-2, IL-8 and IL-12p70) and Th2 (IL-4, IL-6 and IL-10) cytokine responses, and to assess the cofounding effect of lymphocytes in the production of these cytokines, in AFB microscopy smear negative patients with PTB co-infected with HIV and relate T cells (CD3/CD4/CD8), B cells (CD 19), NK cells (CD16/CD56) and Th1 (IFN-γ, TNF-α, IL-2, IL-8 and IL-12p70) /Th2 (IL-4, IL-6 and IL-10) cytokines with sputum smear status in patients with PTB co-infected with HIV in AMPATH, MTRH. Study participants were adults with HIV attending TB clinic within AMPATH. In a longitudinal study, a total of 86 study participants were recruited: 46 culture-negative and 40 culture-positive. Blood and sputum samples were collected from all the study participants. The blood samples were analyzed using a four color FACSCalibur flow cytometer to determine immunophenotypic characteristics, and the same samples cultured and supernatant (plasma) harvested to evaluate cytokine profiles using Enzyme-Linked Immunosorbent Assay (ELISA). Sputum samples were analyzed to determine AFB smear status using direct microscopy and Lowenstein Jensen medium. Statistical analyses were performed using R software. The overall mean age of the participants was 39 years (SD=12), with 48.8% being male. Although, lymphocytes counts were higher in culture-positive relative to culture-negative, the CD8, CD19, and CD16/CD56 were comparable in the two groups. The CD4 counts differed between the two groups (P=0.012). The Th1 cytokines showed a better discrimination between culture-positive and culture-negative PTB individuals; IFN-γ (P=0.001), TNF-α (P=0.001), IL-2 (P=0.001) and IL-12(p70) (P=0.016). The Th2 cytokines (IL-4, IL-6 and IL-10) were comparable between the culture-positive and culture-negative groups. When, PTB culture-positive (AFB microscopy smear negative) and PTB culture-negative (AFB microscopy smear negative) cytokines median levels were compared; IFN-γ (P<0.001), TNF-α (P=0.004), IL-2 (P=0.004) and IL-4 (P=0.009) were elevated. Lastly, when Th1 cytokines (IFN-γ, TNF-α and IL-2), Th2 cytokines (IL-6 and IL-10) and T cells were included in the logistic regression fit for PTB outcome, the predictive power of discriminating between those who were AFB smear negative in the diagnosis of PTB was better with cross-validated area under the curve (AUC) being 0.87 (95% CI: 0.78, 0.96). A direct correlation was reported between, IFN-γ and IL-2 cytokines (r=0.48, P<0.001, power = 99.1%); IL-2 and TNF-α (r=0.27, P=0.22, power=62.6%); IL-4 and IL-10 (r=0.29, P=0.015, power= 69.1%) cytokines; and IL-6 and TNF-α cytokines (r=0.68, P<0.001, power=97.3%), a concomitant increase in one cytokine resulted in an increase of the other when analyzed by Pearson correlation. On the contrary, an inverse correlation was observed between median levels of; TNF-α and IL-8 cytokines in (r=-0.41, P=0.022, power=93.9%); and IL-6 and IL-8 cytokines (r=-0.44, P<0.001, power>99.9) demonstrating an antagonistic relationship between the two sets cytokines. This study provided evidence for the ability of combination of Th1 cytokines in discriminating against culture-positive and culture-negative PTB, Th1 and Th2 cytokines to determine PTB status in AFB microscopy smear negative patients co-infected with HIV.