Persistent infection due to a small colony variant of Burkholderia pseudomallei leads to PD 1 upregulation on circulating immune cells and mononuclear infiltration in viscera of experimental BALB/c mice
Melioidosis is a neglected tropical disease endemic across South East Asia and Northern Australia. The etiological agent, Burkholderia pseudomallei (B.pseudomallei), is a Gram-negative, rod-shaped, motile bacterium residing in the soil and muddy water across endemic regions of the tropical world. The bacterium is known to cause persistent infections by remaining latent within host cells for prolonged duration. Reactivation of the recrudescent disease often occurs in elders whose immunity wanes. Moreover, recurrence rates in melioidosis patients can be up to ~13% despite appropriate antibiotic therapy, suggestive of bacterial persistence and inefficacy of antibiotic regimens. The mechanisms behind bacterial persistence in the host remain unclear, and hence understanding host immunity during persistent B. pseudomallei infections may help designing potential immunotherapy. A persistent infection was generated using a small-colony variant (SCV) and a wild-type (WT) B. pseudomallei in BALB/c mice via intranasal administration. Infected mice that survived for >60 days were sacrificed. Lungs, livers, spleens, and peripheral blood mononuclear cells were harvested for experimental investigations. Histopathological changes of organs were observed in the infected mice, suggestive of successful establishment of persistent infections. Moreover, natural killer (NK) cell frequency was increased in SCV- and WT-infected mice. We observed programmed death-1 (PD-1) upregulation on B cells of SCV- and WT-infected mice. Interestingly, PD-1 upregulation was only observed on NK cells and monocytes of SCV-infected mice. In contrast, cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) downregulation was seen on NK cells of WT-infected mice, and on monocytes of SCV- and WT-infected mice. The SCV and the WT of B. pseudomallei distinctly upregulated PD-1 expression on B cells, NK cells, and monocytes to dampen host immunity, which likely facilitates bacterial persistence. PD-1/PD-L1 pathway appears to play an important role in the persistence of B. pseudomallei in the host. B. pseudomallei is a bacterium that causes melioidosis, a disease endemic in Southeastern Asia and Northern Australia. It is estimated that melioidosis leads to 89,000 deaths worldwide each year. Nevertheless, melioidosis continues to remain a neglected tropical disease that is not even on the list of neglected tropical diseases of the World Health Organization. Furthermore, the disease has a high mortality and recurrence rate, which can be up to 40% and 13%, respectively. It has also been well documented that B. pseudomallei causes latent/persistent infections for a prolonged period without showing apparent symptoms in the infected individual. The mechanisms that are responsible for bacterial persistence in the host remain unclear. Our results demonstrated that B. pseudomallei were able to upregulate PD-1 expression on B cells, NK cells, and/or monocytes during persistent diseases, which likely diminish optimal host immunity. The weakened host immunity in turns facilitates persistence of the bacterium. Interestingly, the SCV had a higher PD-1 expression on distinct immune cells compared to the WT, which might explain its frequent association with persistent infections. Immunotherapies by targeting PD-1/PD-L1 pathway could serve as a better treatment than the conventional antibiotic regimens, which cause a high rate of recurrence in melioidosis patients.
[…] PBMCs (1x106 cells in each tube) were stained with Alexa Fluor 488 hamster anti-mouse CD3e (BD Biosciences, clone 145-2C11), Pe-Cy7 rat anti-mouse CD4 (BD Biosciences, clone GK1.5), APC-H7 rat anti-mouse CD8a (BD Biosciences, clone 53–6.7), APC hamster anti-mouse PD-1 (BD Biosciences, clone J43), and PE hamster anti-mouse CTLA (BD Biosciences, clone UC10-4F10-11), Fixable Viability Stain 510 (BD Biosciences, cloneR35-95). Corresponding isotype control for each antibody was prepared for appropriate setting of gates during multicolor flow cytometry analysis. All antibodies were pre-titrated for optimal working concentration. Data were acquired on an 8-color FACSCanto II immunocytometry system (BD Biosciences) with BD FACSDiva software (BD Bioscience). Data were exported from BD FACSDiva and analyzed using Flowjo software version 10 (Tree Star, Oregon, USA). […]