Immunomodulatory and Antibacterial Effects of Cystatin 9 against Francisella tularensis

Tonyia Eaves-Pyles,Bernard Arulanandam,Vsevolod L Popov,Aaron L Miller,Csaba Szabo,Jignesh Patel,Ciro Coletta,Yingzi Cong,Lynn Soong,Emma Arigi,Igor C Almeida,Monisha Dhiman,Richard B Pyles,Eric D Carlsen,Anthony Cao,Nisha Garg

doi:10.2119/molmed.2013.00081

Abstract

Cystatin 9 (CST9) is a member of the type 2 cysteine protease inhibitor family, which has been shown to have immunomodulatory effects that restrain inflammation, but its functions against bacterial infections are unknown. Here, we report that purified human recombinant (r)CST9 protects against the deadly bacterium Francisella tularensis (Ft) in vitro and in vivo. Macrophages infected with the Ft human pathogen Schu 4 (S4), then given 50 pg of rCST9 exhibited significantly decreased intracellular bacterial replication and increased killing via preventing the escape of S4 from the phagosome. Further, rCST9 induced autophagy in macrophages via the regulation of the mammalian target of rapamycin (mTOR) signaling pathways. rCST9 promoted the upregulation of macrophage proteins involved in antiinflammation and antiapoptosis, while restraining proinflammatory-associated proteins. Interestingly, the viability and virulence of S4 also was decreased directly by rCST9. In a mouse model of Ft inhalation, rCST9 significantly decreased organ bacterial burden and improved survival, which was not accompanied by excessive cytokine secretion or subsequent immune cell migration. The current report is the first to show the immunomodulatory and antimicrobial functions of rCST9 against Ft. We hypothesize that the attenuation of inflammation by rCST9 may be exploited for therapeutic purposes during infection.

Highlights

Cystatin 9 (CST9) is a small, ~18-kDa human protein and a member of the type 2 cystatin superfamily that is compromised of 14 members [1]
We show that a single dose of human recombinant (r)CST9 given to Schu 4 (S4)-infected human monocyte-derived macrophages (MDM) prevents intracellular S4 from escaping the phagosome, and induces macrophage autophagy while regulating the phosphorylation events of a key kinases in the mammalian target of rapamycin (mTOR) pathway that determine the induction of autophagy
At 24 h after treatment and/or infection, rCST9 significantly increased intramacrophage killing of S4 compared with S4-infected MDM alone (Figure 1A)

Summary

Introduction

Cystatin 9 (CST9) is a small, ~18-kDa human protein and a member of the type 2 cystatin superfamily that is compromised of 14 members [1]. Low molecular weight endogenous cysteine protease inhibitors found in most body compartments and fluids. They are described to function intra- and/or extracellularly to inhibit their target cysteine enzymes (for example, cathepsins) to maintain a crucial protease-inhibitor balance, regulating damaging proteolytic activities [2,3]. Cystatin C is typically secreted and acts as a strong inhibitor of papainlike proteases, while cystatin F primarily regulates intracellular cathepsin C [4,5]. Sustaining the equilibrium between cysteine proteases and cystatins serves to regulate immunomodulatory functions not related to their inhibition of proteases [6]. This, in turn, results in excessive cytokine production, unrestrained tissue breakdown (for example, degradation of the extracellular matrix [ECM]) and promotes excessive immune cell extravasation to inflammation sites exacerbating organ damage [1,3,7,8,9]

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