Melioidosis is a common cause of fatal community-acquired septicaemia and pneumonia in endemic regions even with appropriate antibiotic treatments. The involvement of inflammatory cytokines in the manifestation of melioidosis is well-documented. Antibacterial and anti-inflammatory therapies may prove more efficacious against melioidosis rather than just anti-bacterial therapy alone. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway has a central role in regulating the host inflammatory response; and glycogen synthase kinase-3β (GSK3β), a downstream effector molecule within this axis, plays a pivotal role in regulating the production of pro- and anti-inflammatory cytokines. The anti-malarial drug, chloroquine is a novel activator of Akt, and can elicit inhibition of GSK3β via PI3K/Akt signalling. LiCl, a GSK3 inhibitor is reported to increase survivability and modulate cytokine production in B. pseudomallei-infected mice. Here we determined the effects of chloroquine administration on animal survivability, cytokine levels and phosphorylation states of GSK3β (Ser9), Akt (Ser473) and NF-κB p65 (Ser536) in a murine model of acute melioidosis infection. Administration of 50 mg/kg b w chloroquine improved survivability (mean 67.0 ± 6.3%) of mice infected with 3 X LD50 B. pseudomallei compared to controls. Bacterial loads in spleen, liver, lung and blood of infected mice administered with chloroquine were significantly lower than controls. Western blot analysis revealed that the intensities of pAkt (Ser473) and pGSK3β (Ser9) in liver samples of mice administered with chloroquine were significantly (P<0.05) higher (2.3- and 4.4-fold respectively) compared to controls. On the other hand, chloroquine treatment signicantly decreased (P<0.05) phosphorylation of NF-κB p65 (Ser536) by 0.7-fold compared to control. Chloroquine administration also resulted in significantly reduced levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β and IL-18) but increased levels of antiinflammatory cytokines (IL-4 and IL-10) in sera and liver of B. pseudomallei-infected mice. Findings from this study demonstrate that the increased survivability of B. pseudomalleiinfected mice after chloroquine administration is at least in part due to its cytokine-modulating effects elicited via Akt-mediated inhibition of GSK3β that resulted in inhibition of NF-κB activation. This study represents laboratory evidence of the use of chloroquine for cytokine modulation and a plausible effective adjunctive therapeutic for B. pseudomallei infection.

Burkholderia pseudomallei is the etiologic agent of melioidosis, a major cause of community-acquired pneumonia and sepsis in the endemic areas. The overall mortality of patients with severe melioidosis remains high due to severe sepsis attributed to overwhelming inflammatory cytokine response in spite of recommended antibiotic therapy. It is crucial that therapeutic approaches beyond just effective antibiotic treatment such as adjunct therapy be considered to mitigate the dysregulated inflammatory signaling and augment host defenses. In an acute B. pseudomallei infection model, we have previously demonstrated that treatment with anti-malarial drug, chloroquine, modulated inflammatory cytokine levels and increased animal survivability via Akt-mediated inhibition of glycogen synthase kinase-3β (GSK3β). GSK3β is a downstream effector molecule within the phosphatidylinositol 3-kinase (PI3K)/ Akt axis which plays a pivotal role in regulating the production of pro- and anti-inflammatory cytokines. Here we evaluate the effect of chloroquine treatment in combination with a subtherapeutic dose of the antibiotic doxycycline on animal survivability, cytokine levels and phosphorylation states of GSK3β (Ser9) in a murine model of acute melioidosis infection to investigate whether chloroquine could be used as an adjunct therapy along with doxycycline for the treatment of melioidosis. Our findings revealed that 50 mg/kg b.w. chloroquine treatment together with a dose of 20 mg/kg b.w. doxycycline improved survivability (100%) of mice infected with 3 X LD50 of B. pseudomallei and significantly (P<0.05) lowered the bacterial loads in spleen, liver and blood compared to controls. B. pseudomallei-infected mice subjected to adjunct treatment with chloroquine and doxycycline significantly (P<0.05) reduced serum levels of pro-inflammatory cytokines (TNF-α, IFN-γ and IL-6) but increased levels of antiinflammatory cytokines (IL-4 and IL-10). Western blot analysis demonstrated that the intensities of pGSK3β (Ser9) in liver samples from mice treated with chloroquine and doxycycline combination were significantly (P<0.05) higher suggesting that the adjunct treatment resulted in significant inhibition of GSK3β. Taken together the bacteriostatic action of doxycycline coupled with the cytokine-modulating effect of chloroquine gave full protection to B. pseudomallei-infected mice and involved inhibition of GSK3β. Findings from the present study using B. pseudomallei-infected BALB/c mice suggest that chloroquine is a plausible candidate for repurposing as adjunct therapy to treat acute B. pseudomallei infection.

Malaria is a life-threatening disease caused by the Plasmodium sp. parasite. Infection results in heightened pro-inflammatory response which contributes to the pathophysiology of the disease. To mitigate the overwhelming cytokine response, host-directed therapy is a plausible approach. Glycogen synthase kinase-3β (GSK3β), a serine/threonine kinase plays a pivotal role in the regulation of inflammatory response during pathogenic infections. The present study was conducted to investigate the chemo-suppressive and cytokine-modulating effects of insulin administration in malaria-infected mice and the involvement of GSK3β. Intraperitoneal administrations of 0.3 and 0.5 U/kg body weight insulin each for four consecutive days into Plasmodium berghei NK65 (PbN)-infected mice resulted in chemo-suppression exceeding 60% and improved median survival time of infected mice (20.5 days and 19 days respectively compared to 15.5 days for non-treated control). Western analysis revealed that pGSK3β (Ser9) intensity in brain samples from insulin-treated (0.3 and 0.5 U/kg body weight) infected mice each were 0.6 and 2.2 times respectively than that in control. In liver samples, pGSK3β (Ser9) intensity from insulin-treated infected mice were significantly higher (4.8 and 16.1 fold for 0.3 and 0.5 U/kg bw respectively) than that in control. Insulin administration decreased both brain and liver pNF-κB p65 (Ser536) intensities (to 0.8 and 0.6 times for 0.3 U/kg bw insulin; and to 0.2 and 0.1 times for 0.5 U/kg bw insulin respectively compared to control). Insulin treatment (0.5 U/kg bw) also significantly decreased the serum levels of pro-inflammatory cytokines (TNF-α (3.3 times) and IFN-γ (4.9 times)) whilst significantly increasing the levels of anti-inflammatory cytokines (IL-4 (4.9 fold) and IL-10 (2.1 fold)) in PbN-infected mice. Results from this study demonstrated that the cytokinemodulating effects of insulin at least in part involve inhibition of GSK3β and consequent inhibition of the activation of NF-κB p65 suggesting insulin as a potential adjunctive therapeutic for malaria.