Immune cell characteristics and cytokine responses in adult HIV-negative tuberculous meningitis: an observational cohort study

Abstract

Immunopathology contributes to high mortality in tuberculous meningitis (TBM) but little is known about the blood and cerebrospinal fluid (CSF) immune response. We prospectively characterised the immune response of 160 TBM suspects in an Indonesian cohort, including 67 HIV-negative probable or definite TBM cases. TBM patients presented with severe disease and 38% died in 6 months. Blood from TBM patients analysed by flow cytometry showed lower αβT and γδT cells, NK cells and MAIT cells compared to 26 pulmonary tuberculosis patients (2.4-4-fold, all p < 0.05) and 27 healthy controls (2.7-7.6-fold, p < 0.001), but higher neutrophils and classical monocytes (2.3-3.0-fold, p < 0.001). CSF leukocyte activation was higher than in blood (1.8-9-fold). CSF of TBM patients showed a predominance of αβT and NK cells, associated with better survival. Cytokine production after ex-vivo stimulation of whole blood showed a much broader range in TBM compared to both control groups (p < 0.001). Among TBM patients, high ex-vivo production of TNF-α, IL-6 and IL-10 correlated with fever, lymphocyte count and monocyte HLA-DR expression (all p < 0.05). TBM patients show a strong myeloid blood response, with a broad variation in immune function. This may influence the response to adjuvant treatment and should be considered in future trials of host-directed therapy.

Figure 1

Patient inclusion diagram. Of the 26 patients with pulmonary tuberculosis, one had no ex-vivo cytokine response available. Of the 27 healthy controls, one had no ex-vivo cytokine response and one had no flow cytometry results available. LP = lumbar puncture.

Figure 2

Flow cytometry results. (A) Blood flow cytometry results for 40 tuberculous meningitis (TBM) patients showing concentrations of individual cell types as depicted in the legend. (B) Median concentrations in 26 healthy controls, 26 pulmonary tuberculosis and TBM patients for myeloid (left) and lymphoid (right) cell types. (C) CSF flow cytometry results for 41 individual TBM patients. Patients with >200 leukocytes/μL are displayed in the left subplot and patients with ≤200 leukocytes/μL in the right subplot. (D) Median CSF cell composition of all TBM patients combined. Note: CD3⁻ lymphocytes without NK cell markers, most likely B cells (14%, IQR 11–43 of lymphocytes), were not included in the analysis because the flow cytometry panels lacked B-cell markers to formally confirm their phenotype.

Figure 3

Blood versus CSF leukocyte activation. Median fluorescence intensity of activation markers in blood (x-axis) versus CSF (y-axis) for myeloid (A) and lymphoid (B) cell types. These modified ‘bag plots’ show the 50% median data-points and can thereby be compared to a two-dimension box plot without whiskers.

Figure 4

Ex-vivo whole blood cytokine results. (A) IL-1β response after stimulation of whole blood with BCG, M. tuberculosis and S. pneumoniae for each of the three patient groups tuberculous meningitis (TBM), pulmonary tuberculosis (PTB) and healthy controls (HC). (B) Comparison of these patient groups on principal component (PC) 1 versus PC2 in principal component analysis of all six measured cytokines for the afore-mentioned stimuli for three patient groups. (C) Heatmap showing the combination of six cytokines and three stimuli (y-axis; B = BCG, M = M. tuberculosis and S = S. pneumoniae) for all 50 included TBM patients (x-axis) sorted on their score on PC1.