Immunomodulatory and antibacterial effects of cystatin 9 against Francisella tularensis

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.

Figure 1

rCST9 modulates MDM responses Ft S4. (A) MDM (5 × 10⁵ in 200 μL media/sample) were treated concurrently with 50 pg of rCST9 then immediately infected with S4 (MOI of 40:1). At 24 h after infection and/or treatment, bacterial killing was significantly higher in rCST9-treated MDM, as compared with S4-infected MDM alone (P < 0.05). (B) rCST9 significantly decreased leukocyte migration in the presence of VEGF compared with VEGF alone (P < 0.05), as shown in a modified Boyden chamber assay. (C) Cells were stained to visualize and quantify migrated cells in the modified Boyden chamber assay. Data are presented as mean ± SEM. Asterisks signify significant differences of P < 0.05.

Figure 2

rCST9 confined S4 to MDM phagosome and decreased intracellular replication. rCST9 (50 pg) treated MDM (10⁵) were infected with S4 (MOI 40:1). S4-infected MDM alone served as controls. (A) All tested conditions show that the majority of S4 (red) colocalized with LAMP-1 (green) at 2 h. At 20 h, S4 showed minimal LAMP-1 colocalization and increased cytosolic replication in infected MDM alone. However, the majority of S4 remained colocalized with LAMP-1 with minimal cytosolic replication in MDM treated with rCST9. (B) TEM analysis showed S4 in phagosomes at 2 h after infection with or without rCST9 treatment. (C) However, S4 remained in the phagosomes of MDM at 24 h after infection and/or rCST9 treatment. The images are representative of at least three replicate studies.

Figure 3

rCST9 induced autophagy. (A) MDM showed evidence of autophagy (that is, double membrane AV) in MDM infected with S4 (MOI 40:1) at 20 h after rCST9 (50 pg) treatment and S4 infection as per TEM analysis. (B) Immunofluorescent microscopy showed LC3-II protein (small green spots) in human alveolar macrophages at 20 h after rCST9 (50 pg) treatment compared with untreated macrophages. Images shown in A and B are representative of experiments repeated three times using duplicate conditions. (C) Western blot analysis revealed marked conversion of LC3-I to LC3-II in rCST9 (50 pg) treated MDM compared with MDM alone. (D) rCST9 (50 pg) induced the dephosphorylation of P70S6 kinase at 30 and 60 min after treatment. PI3-KC3 phosphorylation of was detected at 10, 30 and 60 min as well as 4, 5 and 6 h in rCST9-treated MDM. Experiments were performed three times.

Figure 4

rCST9 directly affected protein changes in S4 and MDM. (A) S4 (1 × 10⁴ CFU/sample) was incubated with various dilutions rCST9 for 4 h, then plated to determine bacteria viability via replication. 50 pg of rCST9 significantly decreased S4 viability compared with all other tested dilutions (P < 0.05). (B) A portion of the S4 incubated with 50 pg of rCST9 was given to MDM (MOI 40:1) where it was killed significantly better (P < 0.05) than infected MDM alone but not as effectively as rCST9 and S4 given concurrently to MDM. (C) The schematic showing that MS analysis identified numerous proteins involved in bacterial metabolic pathway that were either downregulated or eliminated by CST9. (D) TEM analysis of untreated S4 revealed an intact, uninterrupted cell wall. However, the membranes of S4 incubated with 50 pg of rCST9 for 4 h had the periplasmic space disrupted, and in some areas, the bacterial membrane was discontinuous. Where applicable data are presented as mean ± SEM and asterisk signifies significant differences of *P < 0.05 or **P < 0.01 as indicated. Scale bar, 0.5 μm.

Figure 5

rCST9 afforded protection against pulmonary tularemia. (A) Balb/c mice (n = 12 mice/group) were given 50 pg of rCST9 i.n. concurrently with LVS (500 CFU/mouse; 100 × LD50s). rCST9-treated/infected mice had significantly higher survival compared with infected mice alone (P < 0.01). Data are presented as mean ± SEM. (B) Parallel groups of mice (n = 6/group) were euthanized 48 h after treatment and/or infection to collect lungs, liver and spleen to determine LVS burden. Bacterial loads in all three organs of rCST9-treated/infected mice were significantly lower compared with infected mice alone (P < 0.01). (C) Ft-infected mice treated with rCST9 showed moderate cellularity and maintained the architecture of the alveolar space similar to uninfected/untreated (normal) controls. LVS-infected mice alone showed a loss of alveolar architecture and hypercellularity. Magnification, 10×.