Description

Mammalian STE20 kinase (Mst1/2) and IL-27 Regulate Activity of Primary Macrophages in response to Mycobacterium tuberculosis
Priyanka Namdev, Jasmine Canlas and Ankita Garg
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA 30606, USA,
 
A better understanding of host responses to M. tuberculosis (Mtb) infection is essential for developing host directed therapies for controlling tuberculosis (TB). In this study, we investigated two related phagolysosomal pathways- LC3-associated phagocytosis (LAP) and autophagy in primary human macrophages, and its regulation by IL-27.   
Primary monocyte derived macrophages (MDM) were generated by culturing CD14+ monocytes from HIV (-) TB QFT (-) healthy individuals in the presence of M-CSF, and infected with Mtb Erdman bacilli in the presence or absence of recombinant IL-27 (rIL-27). In some experiments MDM were treated with TLR- 2, -4 blocking or isotype matched control antibodies, or MRT68921 (Unc-51 like autophagy activating kinase (ULK1/2) inhibitor) or rapamycin prior to infection. Expression of pMst1/2, IL-27, LC3B, p62, cathepsin B and GAPDH proteins in the cellular lysates were determined by immunoblotting. Data were analyzed using Student’s t test.
Infection of MDM with Mtb bacilli resulted in an increased phosphorylation of Mst1/2 kinase (pMst1/2) and IL-27 expression as compared to uninfected controls (p=0.03 and p=0.04, respectively). The increase in pMst1/2 was observed as early as 3-hrs post-infection. Pre-treatment with blocking TLR-2 antibody prior to infection downregulated pMst1/2 (p=0.04) and IL-27 (p= 0.03), TLR-4 blocking had no effect on pMst1/2 and IL-27 expression. With regard to phagolysosomal function, LC3B-II expression increased in MDM at 3 -hours post-infection (p.i.) with a concomitant increase in cathepsin B; however, autophagic flux measured by p62 degradation was observed at 24-hours p.i. Treatment with MRT68921 marginally inhibited LC3B-II without affecting cathepsin B expression at 3-hrs p.i, and significantly increased LC3B-II accumulation due to inhibition in autophagic flux at 24-hours p.i., suggesting regulation of LC3B-II mediated anti-mycobacterial activity at 3-hours p.i. through a mechanism other than ULK1/2. Importantly, treatment of Mtb-infected MDM with rIL-27 decreased pMst1/2 (p=0.03), LC3B-II (p-0.02) and cathepsin B (p=0.04) expression, but did not affect p62 degradation (p=0.09).
These findings demonstrate the differential regulation of LAP and autophagic activity of MDM during early and late infection with Mtb, and suggest that IL-27 inhibits the protective immunological response to Mtb. An improved understanding of macrophage responses can provide important insights into strategies to control Mtb early replication.