GSK3β: A plausible molecular target in the cytokinemodulating effect of exogenous insulin in a murine model of malarial 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.

Potential of repurposing chloroquine as an adjunct therapy for melioidosis based on a murine model of Burkholderia 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.

The anti-malarial chloroquine modulated cytokine levels and increased animal survivability via Akt-mediated inhibition of GSK3β in Burkholderia pseudomalleiinfected mice

@#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.

Anti-malarial and anti-inflammatory effects of Gleichenia truncata mediated through inhibition of GSK3β

Gleichenia truncata is a highland fern from the Gleicheniaceae family known for its traditional use among indigenous communities in Asia to treat fever. The scientific basis of its effect has yet to be documented. A yeast-based kinase assay conducted in our laboratory revealed that crude methanolic extract (CME) of G. truncata exhibited glycogen synthase kinase-3 (GSK3)-inhibitory activity. GSK3β is now recognized to have a pivotal role in the regulation of inflammatory response during bacterial infections. We have also previously shown that lithium chloride (LiCl), a GSK3 inhibitor suppressed development of Plasmodium berghei in a murine model of malarial infection. The present study is aimed at evaluating G. truncata for its anti-malarial and anti-inflammatory effects using in vivo malarial and melioidosis infection models respectively. In a four-day suppressive test, intraperitoneal injections of up to 250 mg/kg body weight (bw) G. truncata CME into P.berghei-infected mice suppressed parasitaemia development by >60%. Intraperitoneal administration of 150 mg/kg bw G. truncata CME into Burkholderia pseudomallei-infected mice improved survivability by 44%. G. truncata CME lowered levels of pro-inflammatory cytokines (TNF-α, IFN-γ) in serum and organs of B. pseudomallei-infected mice. In both infections, increased phosphorylations (Ser9) of GSK3β were detected in organ samples of animals administered with G. truncata CME compared to controls. Taken together, results from this study strongly suggest that the anti-malarial and anti-inflammatory effects elicited by G. truncata in part were mediated through inhibition of GSK3β. The findings provide scientific basis for the ethnomedicinal use of this fern to treat inflammation-associated symptoms.

Regulatory role of GSK3β in the activation of NF-κB and modulation of cytokine levels in Burkholderia pseudomallei-infected PBMC isolated from Streptozotocin-induced diabetic animals

Increased susceptibility of diabetics to melioidosis, a disease caused by the Burkholderia pseudomallei bacterium is believed to be attributed to dysfunction of the innate immune system. However, the underlying mechanism of the innate susceptibility is not well-understood. Glycogen synthase kinase-3β (GSK3β) plays an important role in the innate inflammatory response caused by bacterial pathogens. The present study was conducted to investigate the effects of GSK3β inhibition by LiCl on levels of pro- and anti-inflammatory cytokines; and the activity of transcription factor NF-κB in B. pseudomallei-infected peripheral blood mononuclear cells (PBMC) derived from diabetic-induced and normal Sprague Dawley rats. In addition, the effects of LiCl on intracellular bacterial counts were also investigated. Infection of PBMC from diabetic and normal rats with B. pseudomallei resulted in elevated levels of cytokines (TNF-α, IL-12 and IL-10) and phosphorylation of NF-κB in both cell types. Intracellular bacterial counts decreased with time in both cell types during infection. However bacterial clearance was less prominent in diabetic PBMC. Burkholderia pseudomallei infection also caused inactivation (Ser9 phosphorylation) of GSK3β in normal PBMC, an effect absent in infected diabetic PBMC. Inhibition of GSK3β by LiCl lowered the levels of pro-inflammatory cytokines (TNF-α and IL-12) in both normal and diabetic PBMC. Similarly, phosphorylated NF- κB (pNF-κB) levels in both cell types were decreased with LiCl treatment. Also, LiCl was able to significantly decrease the intracellular bacterial count in normal as well as diabetic PBMC. Interestingly, the levels of anti-inflammatory cytokine IL-10 in both normal and diabetic PBMC were further elevated with GSK3β inhibition. More importantly, GSK3β in infected diabetic PBMC was inactivated as in their non-diabetic counterparts upon LiCl treatment. Taken together, our results suggest that inhibition of dysregulated GSK3β in diabetic PBMC resulted in the inactivation of NF-κB and modulation of inflammatory cytokine levels. This is evidence that dysregulation of GSK3β is a contributing factor in the molecular basis of innate dysfunction and susceptibility of diabetic host to melioidosis infection.

Glycogen synthase kinase-3β inhibition improved survivability of mice infected with Burkholderia pseudomallei

The disease melioidosis, caused by the soil bacteria Burkholderia pseudomallei, often manifests as acute septicemia with high fatality. Glycogen synthase kinase-3β (GSK3β) plays a key role during the inflammatory response induced by bacteria. We used a murine model of acute melioidosis to investigate the effects of LiCl, a GSK3 inhibitor on experimental animal survivability as well as TNF-α, IL-1β, IFN-γ, IL-10 and IL-1Ra cytokine levels in blood, lung, liver and spleen of B. pseudomallei-infected mice. Our results showed that administration of 100 μg/g LiCl improved survivability of mice infected with 5 X LD50 of B. pseudomallei. Bacterial counts in spleen, liver and lungs of infected mice administered with LiCl were lower than non-treated controls. Our data also revealed that GSK3β is phosphorylated in the spleen, liver and lung of animals infected with B. pseudomallei. However in infected animals administered with LiCl, higher levels of pGSK3 were detected in the organs. Levels of proinflammatory cytokines (TNF-α, IL-1β and IFN-γ) and anti-inflammatory cytokines (IL-10 and IL-1Ra) in sera and organs tested were elevated significantly following B. pseudomallei infection. With GSK3β inhibition, pro-inflammatory cytokines (TNF-α, IFN-γ, IL-1β) were significantly decreased in all the samples tested whilst the levels of anti-inflammatory cytokines, IL-10 (spleen and lung) and IL-1Ra (spleen, liver and sera) were further elevated. This study represents the first report implicating GSK3β in the modulation of cytokine production during B. pseudomallei infection thus reiterating the important role of GSK3β in the inflammatory response caused by bacterial pathogens.

Suppression of Plasmodium berghei parasitemia by LiCl in an animal infection model

Malaria, caused by the Plasmodium parasite is still a health problem worldwide due to resistance of the pathogen to current anti-malarials. The search for new anti-malarial agents has become more crucial with the emergence of chloroquine-resistant Plasmodium falciparum strains. Protein kinases such as mitogen-activated protein kinase (MAPK), MAPK kinase, cyclin-dependent kinase (CDK) and glycogen synthase kinase- 3(GSK-3) of parasitic protozoa are potential drug targets. GSK-3 is an enzyme that plays a vital role in multiple cellular processes, and has been linked to pathogenesis of several diseases such as type II diabetes and Alzheimer's disease. In the present study, the antiplasmodial property of LiCl, a known GSK-3 inhibitor, was evaluated in vivo for its antimalarial effect against mice infected with Plasmodium berghei. Infected ICR mice were intraperitoneally administered with LiCl for four consecutive days before (prophylactic test) and after (suppressive test) inoculation of P. berghei-parasitised erythrocytes. Results from the suppressive test (post-infection LiCl treatment) showed inhibition of erythrocytic parasitemia development by 62.06%, 85.67% and 85.18% as compared to nontreated controls for the 100 mg/kg, 300 mg/kg and 600 mg/kg dosages respectively. Both 300 mg/kg and 600 mg/kg LiCl showed similar significant (P<0.05) suppressive values to that obtained with chloroquine-treated mice (86% suppression). The prophylactic test indicated a significantly (P<0.05) high protective effect on mice pre-treated with LiCl with suppression levels relatively comparable to chloroquine (84.07% and 86.26% suppression for the 300 mg/kg and 600 mg/kg LiCl dosages respectively versus 92.86% suppression by chloroquine). In both the suppressive and prophylactic tests, LiCl-treated animals survived longer than their non-treated counterparts. Mortality of the non-treated mice was 100% within 6 to 7 days of parasite inoculation whereas mice administered with LiCl survived beyond 9 days. Healthy non-infected mice administered with 600 mg/ kg LiCl for four consecutive days also showed decreased mortality compared to animals receiving lower doses of LiCl; three of the seven mice intraperitoneally injected with the former dose of LiCl did not survive more than 24 h after administration of LiCl whereas animals given the lower LiCl doses survived beyond four days of LiCl administration. To date, no direct evidence of anti-malarial activity in vivo or in vitro has been reported for LiCl. Evidence of anti-plasmodial activity of lithium in a mouse infection model is presented in this study.