Taenia solium excretory secretory proteins (ESPs) suppresses TLR4/AKT mediated ROS formation in human macrophages via hsa-miR-125

Abstract

Background: Helminth infections are a global health menace affecting 24% of the world population. They continue to increase global disease burden as their unclear pathology imposes serious challenges to patient management. Neurocysticercosis is classified as neglected tropical disease and is caused by larvae of helminthic cestode Taenia solium. The larvae infect humans and localize in central nervous system and cause NCC; a leading etiological agent of acquired epilepsy in the developing world. The parasite has an intricate antigenic make-up and causes active immune suppression in the residing host. It communicates with the host via its secretome which is complex mixture of proteins also called excretory secretory products (ESPs). Understanding the ESPs interaction with host can identify therapeutic intervention hot spots. In our research, we studied the effect of T. solium ESPs on human macrophages and investigated the post-translation switch involved in its immunopathogenesis.

Methodology: T. solium cysts were cultured in vitro to get ESPs and used for treating human macrophages. These macrophages were studied for cellular signaling and miR expression and quantification at transcript and protein level.

Conclusion: We found that T. solium cyst ESPs treatment to human macrophages leads to activation of Th2 immune response. A complex cytokine expression by macrophages was also observed with both Th1 and Th2 cytokines in milieu. But, at the same time ESPs modulated the macrophage function by altering the host miR expression as seen with altered ROS activity, apoptosis and phagocytosis. This leads to activated yet compromised functional macrophages, which provides a niche to support parasite survival. Thus T. solium secretome induces Th2 phenomenon in macrophages which may promote parasite's survival and delay their recognition by host immune system.

Fig 1. Taenia solium derived ESPs activate both Th1 and Th2 cytokines in macrophages.

(A) Relative gene expression of cytokines from macrophages treated with ESPs, normalized to β-Actin as housekeeping gene. (B) Absolute quantification of secreted cytokines in the milieu of T. solium ESPs treated macrophages measured using cytometric bead assay.

Fig 2. T. solium ESPs polarize macrophages to Galectin 3-subset of macrophages phenotype.

Relative expression of macrophages markers normalized to housekeeping gene β-Actin.

Fig 3. Relative expression of TLRs in ESPs treated macrophages estimated.

PC- positive control, TLR agonists used for each of the TLRs from Human TLR agonist kit -InVivoGen Cat Code # tlrl-kit1hw (TLR1/TLR2 Agonist: Pam3CSK4, TLR2 Agonist: HKLM, TLR2/TLR6 Agonist: FSL-1,TLR3 Agonist: Poly(I:C)-HMW, TLR3 Agonist: Poly(I:C)-LMW, TLR4 Agonist: LPS-EK standard (LPS E.coli K12), TLR5 Agonist: FLA-ST standard (Flagellin S. typhimurium), TLR7 Agonist: Imiquimod, TLR8 Agonist: ssRNA40/LyoVec, TLR9 Agonist: ODN2006).

Fig 4. Western blot analysis of TLR4 expression in macrophages treated with Taenia solium ESPs antigens.

The densitometry analysis of western blot bands was normalized to housekeeping protein β-Actin. The results shown are representative of three independent biological replicates.

Fig 5. Evaluation of ROS released by macrophages upon ESPs stimulation.

A) Human macrophages cells treated with ESPs+TBHP showed significantly less ROS when compared to cells treated with TBHP alone (positive control) in a flow cytometer based ROS assay (left) representative histogram plots of Cell ROX orange dye on flow cytometer, (right) qunatification of Cell ROX orange dye across the test groups B) The measurment of total ROS generation by Cytochrome C assay also confirmed reduced ROS in ESPs treated macropahges. Absorbance at 550 nm was measured 5 min after addition of cytochrome C for 10 mins at 30 sec interval (left). Total ROS respresented as bar graph from area under the curve (right).

Fig 6

Taenia solium derived ESPs treatment suppresses Akt activation in macrophages when stimulated with A) Western blot quantification for pAKT/AKT in treated cells stimulated with fMLP B) Western blot quantification for pAKT/AKT in treated cells kept with E.coli for phagocytosis. C) Taenia Solium ESPs treated macrophages had reduced bacterial (E. coli) killing capacity, AKT activator SC79 rescued the lost phenotype in ESP treated cells. D) Macrophages treated with ESPs had less Zymosan bioparticles uptake, suggesting a decrease in phagocytotic capability as seen in flouresence microscopy (left) and phagocytic index (right). The results shown are representative of three independent biological experiments, in five randomly selected high power view per slide.

Fig 7. T. solium ESP alters the epigenetic regulation in macrophages.

A) miR microarray of ESPs treated macrophages showed 57 differentially expressed miR, B) Validation of microarray identified miRs by qPCR. C). The representative graph showing miR network illustrating the interactions between miRs and their target genes (http://userver.bio.uniroma1.it/apps/mienturnet/) D) Western blot quantification of pAKT/AKT of macrophages transfected with hsa-miR-125 showed restoration of TLR4 expression in ESP treated macrophage.