Sains Malaysiana 42(6)(2013): 725–735

Inhibition of GSK3 Attenuates the Intracellular Multiplication of Burkholderia pseudomallei and Modulates the Inflammatory Response in Infected Macrophages and A549

Epithelial Lung Cells

(Perencatan GSK3 Mengurangkan Penggandaan B. pseudomallei Intrasel dan Memodulasi Respons

Inflamasi dalam Makrofaj dan Sel Peparu Epitelium A549 Terinfeksi)

Pramila Maniam, Aishah Farliani Shirat, Hasidah Mohd Sidek, Ghazally Ismail & Noor Embi*

School of Biosciences and Biotechnology, Faculty of Science and Technology

Universiti Kebangsaan Malaysia, 43600 UKM  Bangi, Selangor D.E. Malaysia

Received: 13 June 2012/Accepted: 18 September 2012

ABSTRACT

Burkholderia pseudomallei, the causative agent of melioidosis, is an intracellular pathogen capable of invading and multiplying in both phagocytic and non-phagocytic cells. Infection results in an inflammatory response involving production of both pro- and anti-inflammatory cytokines. The cellular mechanism regulating this response, believed to play an important role in the pathogenesis of meliodoisis, is not fully understood. In recent years, glycogen synthase kinase-3 (GSK3) has been shown to assume a pivotal role in regulating production of these cytokines. Bacterial infection of host cells activates Toll-like receptors (TLRs) and results in the phosphorylation of GSK3β through activation of the phosphoinositide 3-kinase (PI3K) pathway. In this study, we investigated the effects of GSK3 inhibition in regulating B. pseudomallei-induced inflammatory response in macrophages and A549 epithelial lung cells. Our results showed that infection of cells with B. pseudomallei resulted in the increase of anti-inflammatory cytokine, IL-10 and pro-inflammatory cytokine, TNF-α. Pre-treatment of infected cells with GSK3 inhibitors caused further increase in the level of IL-10 but a significant decrease in TNF-α. These changes corresponded with the detection of phosphorylated GSK3β in infected cells treated with LiCl; suggesting that modulation of inflammatory response in B. pseudomallei infection involves phosphorylation of GSK3β (Ser 9). This could explain our observations from the invasion assays that pre-treatment of B. pseudomallei-infected cells with GSK3 inhibitors resulted in decreased intracellular replication of bacteria within macrophages and A549 epithelial lung cells. In summary, our results demonstrate a regulatory function of GSK3 in the modulation of cytokine levels during B. pseudomallei infection.

Keywords: Burkholderia pseudomallei; glycogen synthase kinase-3; inflammation; macrophage; melioidosis

ABSTRAK

Burkholderia pseudomallei, patogen penyebab melioidosis merupakan bakteria intrasel yang mampu menginfeksi dan mengganda dalam sel fagosit serta sel bukan fagosit. Infeksi mengakibatkan respons inflamasi yang melibatkan penghasilan sitokin pro- dan anti-inflamasi. Mekanisme pengawalan respons tersebut yang dipercayai memainkan peranan penting dalam patogenesis melioidosis masih belum difahami sepenuhnya. Glikogen sintase kinase-3 (GSK3) kini diketahui mempunyai peranan utama dalam pengawalan penghasilan sitokin pro- dan anti-inflamasi. Infeksi sel hos oleh bakteria mengaktifkan reseptor Toll-like (TLR) dan mencetuskan pemfosfatan GSK3β melalui pengaktifan tapak jalan fosfoinositid-3-kinase (PI3K). Kami mengkaji kesan perencatan GSK3 dalam pengawalan respons inflamasi yang diaruh oleh B. pseudomallei dalam makrofaj dan sel epitelium peparu A549. Hasil yang kami peroleh menunjukkan peningkatan penghasilan sitokin anti-inflamasi, IL-10 dan sitokin pro-inflamasi, TNF-α dalam sel diinfeksi B. pseudomallei. Pra-perlakuan sel terinfeksi dengan perencat GSK3 menyebabkan aras sitokin IL-10 meningkat dengan lebih tinggi tetapi penghasilan TNF-α berkurangan secara signifikan. Perubahan aras sitokin IL-10 dan TNF-α berpadanan dengan pengesanan GSK3β terfosfat dalam sel diinfeksi yang diberi perlakuan LiCl. Ini mencadangkan bahawa modulasi respons inflamasi semasa infeksi B. pseudomallei melibatkan pemfosfatan GSK3β (Ser 9). Ini juga menjelaskan cerapan daripada asai penaklukan yang menunjukkan pra-perlakuan perencat GSK3 menyebabkan pengurangan penggandaan bakteria intrasel dalam makrofaj dan sel epitelium peparu A549 yang diinfeksi B. pseudomallei. Secara keseluruhan hasil kami menunjukkan GSK3 terlibat dalam modulasi aras sitokin semasa sel diinfeksi B. pseudomallei.

Kata kunci: Burkholderia pseudomallei; glikogen sintase kinase-3; inflamasi; makrofaj; melioidosis

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*Corresponding author; email: noormb@ukm.my