Development of a new cellular immunological detection method for tuberculosis based on HupB protein induced IL-6 release test

Abstract

Objective: As a virulence factor, HupB plays important roles in the survival of MTB after infection and modulates the host immune response. In the current study, we aim to explore a new cellular immunological detection method for tuberculosis infection detection based on HupB protein.

Methods: HupB was used to stimulate PBMCs extracted from pulmonary tuberculosis (PTB) patients, and secreted cytokines was examined. Then, we constructed a single center and a multi-center clinical trials to collect PBMCs from PTB patients, nPTB patients, or healthy volunteers to verify our findings.

Results: Cytokine's screening illustrated that IL-6 was the only cytokine released after HupB stimulation. Single-center and multi-center clinical trials showed that HupB stimulation significantly increased the level of IL-6 in the supernatant of PBMCs from PTB patients. Then we compared the specificity and sensitivity of HupB induced IL-6 release assay with ESAT-6 and CFP10 induced interferon γ release assay (IGRA), and found in smear positive PTB patients, the specificity and sensitivity of HupB induced IL-6 release assay was better than IGRA, and in smear negative PTB patients, the sensitivity was better. Combination of both assays provided an improved specificity and sensitivity for tuberculosis diagnosis.

Conclusion: This study explored an immunological detection method for tuberculosis infection cells based on HupB protein-induced IL-6 release test, which can be used to enhance the diagnosis diagnostic accuracy of TB.

Keywords: HupB; IGRA; IL-6; PBMCs; tuberculosis.

FIGURE 1

Secretion of PBMCs cytokines in samples before and after HupB induction. CON: Cytokines release in PBMCs isolated from healthy people and PTB patients. After 24 h culture in vitro, the secretion levels of IL-4, IL-12p70, TGF-β1, TNF-α, IFN-γ, IL-6, and IL-17A in PBMCs from PTB patients; HupB: HupB induced cytokines release in PBMCs isolated from healthy people and PTB patients. A total of 24 h after 10 μg/ml H37Rv HupB stimulation, the secretion levels of IL-4, IL-12p70, TGF-β1, TNF-α, IFN-γ, IL-6, and IL-17A in PBMC from PTB patients. LPS: LPS induced cytokines release in PBMCs isolated from healthy people and PTB patients. A total of 24 h after 10 μg/ml H37Rv HupB stimulation, the secretion levels of IL-4, IL-12p70, TGF-β1, TNF-α, IFN-γ, IL-6, and IL-17A in PBMC from PTB patients. PTB, tuberculosis group; HC, healthy group; LPS, positive control; ***p < 0.001; “ns” represents P-value > 0.05.

FIGURE 2

Different antigens stimulated secretion of IL-6 in PMBCs isolated from tuberculosis patients. A total of 64 blood samples from PTB patients were collected in Wuhan Jinyintan Hospital PBMCs were separated and then co-cultured with LPS, BJ-LAM, BCG-LAM, HtdY, ESAT-6, and CFP10, HupB or untreated control to induce IL-6 secretion. Control: Uninduced group.

FIGURE 3

Secretion of IL-6 in PBMCs samples before and after HupB induction. (A) HupB induced IL-6 release in PBMCs isolated from healthy control (HC), non-tuberculous pulmonary disease patients (nPTB) and pulmonary tuberculosis patients (PTB), single center clinical validation; (B) HupB induced IL-6 release in PBMCs isolated from healthy control (HC), non-tuberculous pulmonary disease patients (nPTB) and pulmonary tuberculosis patients (PTB), multi-center clinical validation. PTB, tuberculosis group; nPTB, group; HC, healthy group; Control, uninduced control group; ****p < 0.0001.

FIGURE 4

Comparison of HupB induced IL-6 release assay and IGRA for smear positive/negative PTB diagnosis. Diagnosis results of 25 of smear negative PTB patients with HupB (A) and E6C10 (C); Diagnosis results of 28 of smear positive PTB patients with HupB (B) and E6C10 (D). SP PTB, smear positive PTB group; SN PTB, smear negative PTB group; HC, healthy group; ****p < 0.0001; ***p < 0.001; **p < 0.01.