Potential function of granulysin, other related effector molecules and lymphocyte subsets in patients with TB and HIV/TB coinfection

Abstract

Background: Host effector mechanism against Mycobacterium tuberculosis (Mtb) infection is dependent on innate immune response by macrophages and neutrophils and the alterations in balanced adaptive immunity. Coordinated release of cytolytic effector molecules from NK cells and effector T cells and the subsequent granule-associated killing of infected cells have been documented; however, their role in clinical tuberculosis (TB) is still controversy.

Objective: To investigate whether circulating granulysin and other effector molecules are associated with the number of NK cells, iNKT cells, Vγ9(+)Vδ2(+) T cells, CD4(+) T cells and CD8(+) T cells, and such association influences the clinical outcome of the disease in patients with pulmonary TB and HIV/TB coinfection.

Methods: Circulating granulysin, perforin, granzyme-B and IFN-γ levels were determined by ELISA. The isoforms of granulysin were analyzed by Western blot analysis. The effector cells were analyzed by flow cytometry.

Results: Circulating granulysin and perforin levels in TB patients were lower than healthy controls, whereas the granulysin levels in HIV/TB coinfection were much higher than in any other groups, TB and HIV with or without receiving HAART, which corresponded to the number of CD8(+) T cells which kept high, but not with NK cells and other possible cellular sources of granulysin. In addition, the 17kDa, 15kDa and 9kDa isoforms of granulysin were recognized in plasma of HIV/TB coinfection. Increased granulysin and decreased IFN-γ levels in HIV/TB coinfection and TB after completion of anti-TB therapy were observed.

Conclusion: The results suggested that the alteration of circulating granulysin has potential function in host immune response against TB and HIV/TB coinfection. This is the first demonstration so far of granulysin in HIV/TB coinfection.

Keywords: Granulysin; HIV; HIV/TB Coinfection; Lymphocytes Subsets.; TB.

Fig 1

Circulating granulysin (ng/ml) (A), perforin (pg/ml) (B), granzyme-B (pg/ml) (C) and IFN-γ (pg/ml) (D) levels before anti-TB therapy in Thai patients with HIV/TB coinfection and TB in comparison with healthy controls (HC), HIV+HAART- and HIV+HAART+. Each dot represented one individual. A horizontal bar indicated the median of each group. *, p <0.05; **, p <0.01.

Fig 2

Isoforms of granulysin expression in plasma from Thai patients by Western blot analysis. Lane 1-3, HIV/TB coinfection plasma, 3 bands of ∼17kDa, 15kDa and 9kDa isoforms; Lanes: 4-6, TB plasma, I band of ∼17kDa isoform (Lane 4) and 2 bands of ∼15kDa and 9kDa isoforms (Lane 5-6); Lane 7, 2 bands of HC ∼15kDa and 9kDa isoforms.

Fig 3

The number of NK cells (A), iNKT cells (B), Vγ9⁺Vδ2⁺ T cells (C), CD4⁺ T cells (D) and CD8⁺ T cells (E) per microliter (µl) in Thai patients with HIV/TB coinfection and TB in comparison with healthy controls (HC), HIV+HAART- and HIV+HAART+ determined by flow cytometric analysis. Each dot represented one individual. A horizontal bar indicated the median of each group. *, p <0.05; **, p <0.01.

Fig 4

Circulating granulysin (ng/ml) and IFN-γ (pg/ml) in Thai patients with HIV/TB coinfection (A and C) and patients with TB (B and D) before and after completion of anti-TB therapy. Each dot represented one individual. *, p <0.05.

Fig 5

Circulating granulysin (ng/ml) in Japanese patients with HIV/TB coinfection before anti-TB therapy, HIV+HAART- and healthy controls (HC). Each dot represented one individual. A horizontal bar indicated the median of each group. *, p <0.05.