Gregory Hanley

Chronic restraint stress promotes immune suppression through toll-like receptor 4-mediated phosphoinositide 3-kinase signaling.

Stress, either psychological or physical, can have a dramatic impact on the immune system. Toll-like receptors (TLRs) play a pivotal role in the induction of innate and adaptive immune response. We have reported that stress modulates the immune response in a TLR4-dependent manner. However, the mechanisms underlying TLR4-mediated signaling in stress modulation of immune system have not been identified. Here, we demonstrate an essential role for the TLR4-mediated phosphoinositide 3-kinase (PI3K)/Akt signaling. PI3K inhibition by inhibitors wortmannin or LY294002 abrogated protection of stress-induced immune suppression in TLR4-deficient mice compared with TLR4-deficient mice that did not receive the inhibitors. The mechanisms by which PI3K are increased in the TLR4-deficient lymphocytes may involve increased phosphorylation of Akt as well as increased phosphorylation of glycogen synthase kinase-3 beta (GSK-3 beta). The stress-mediated suppression of T help 1 (Th1) cytokine and increased production of Th2 cytokine was greatly reduced in TLR4 deficient mice compared with the wild type mice. Moreover, inhibition of PI3K diminished protection of the above Th1 and Th2 changes caused by stress in TLR4-deficient mice compared with non-stressed mice and the wild type mice. Our data demonstrated that TLR4 negatively regulates PI3K activity in wild type mice, leading to the observed the stress-induced immune response. The higher levels of PI3K prevent TLR4 deficient mice from the stress-induced immune response. Therefore, stress modulates the immune system through TLR4-mediated PI3K/Akt signaling.

Essential role of toll-like receptor 2 in morphine-induced microglia activation in mice

Opioids are powerful pain relievers, but also potent inducers of dependence and tolerance. Chronic morphine administration (via subcutaneous pellet) induces morphine dependence in the nucleus accumbens, an important dependence region in the brain, yet the cellular mechanisms are mostly unknown. Toll-like receptor 2 (TLR2) plays an essential function in controlling innate and inflammatory responses. Using a knockout mouse lacking TLR2, we assessed the contribution of TLR2 to microglia activation and development of morphine dependence. We report here that mice deficient in TLR2 inhibit morphine-induced the levels of microglia activation and proinflammatory cytokines. Moreover, in TLR2 knockout mice the main symptoms of morphine withdrawal were significantly attenuated. Our data reveal that TLR2 plays a critical role in morphine-induced microglia activation and dependence.

Morphine promotes apoptosis via TLR2, and this is negatively regulated by β-arrestin 2

We have previously reported that morphine induces apoptosis. However, the underlying molecular mechanisms remain to be elucidated. Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, modulates cell survival and cell death in various systems. Evidence indicates that beta-arrestin 2 acts as a negative regulator of innate immune activation by TLRs. Here, we investigated the roles of TLR2, the downstreaming mediator MyD88, and beta-arrestin 2 in morphine-induced apoptosis. We showed that overexpression of TLR2 in HEK293 cells caused a significant increase in apoptosis after morphine treatment. Inhibition of MyD88 by transfecting dominant negative MyD88 or overexpression of beta-arrestin 2 by transfecting beta-arrestin 2 full length plasmid in TLR2 overexpressing HEK293 cells attenuated morphine-induced apoptosis. Our study thus demonstrates that TLR2 signaling mediates the morphine-induced apoptosis, and beta-arrestin 2 is a negative regulator in morphine-induced, TLR2-mediated apoptosis.

Toll-like receptor 4 mediates chronic restraint stress-induced immune suppression

Stress, either physical or psychological, can have a dramatic impact on the immune system. Little progress, however, has been made in understanding stress-induced immune suppression. We report here that mice subjected to chronic 12-hour daily physical restraint for two days significantly increased the expression of Toll-like receptor 4 (TLR4). Interestingly, TLR4-deficient mice are resistant to stress-induced lymphocyte reduction. In addition, restraint stress caused dramatic decrease in T help 1 (Th1) cytokine IFN-gamma and IL-2 levels but increase in Th2 cytokine IL-4 in wild type mice. Moreover, the restraint stress significantly inhibits changes of Th1 and Th2 cytokines in TLR4-deficient mice compared with the wild type mice. Therefore, stress modulates the immune system through a TLR4-dependent mechanism.

β-Arrestin 2 regulates toll-like receptor 4-mediated apoptotic signalling through glycogen synthase kinase-3β

Toll‐like receptor 4 (TLR4), a key member of the TLR family, has been well characterized by its function in the induction of inflammatory products of innate immunity. However, the involvement of TLR4 in a variety of apoptotic events by an unknown mechanism has been the focus of great interest. Our investigation found that TLR4 promoted apoptotic signalling by affecting the glycogen synthase kinase‐3β (GSK‐3β) pathway in a serum‐deprivation‐induced apoptotic paradigm. Serum deprivation induces GSK‐3β activation in a pathway that leads to subsequent cell apoptosis. Intriguingly, this apoptotic cascade is amplified in presence of TLR4 but greatly attenuated by β‐arrestin 2, another critical molecule implicated in TLR4‐mediated immune responses. Our data suggest that the association of β‐arrestin 2 with GSK‐3β contributes to the stabilization of phospho‐GSK‐3β, an inactive form of GSK‐3β. It becomes a critical determinant for the attenuation of TLR4‐initiated apoptosis by β‐arrestin 2. Taken together, we demonstrate that the TLR4 possesses the capability of accelerating GSK‐3β activation thereby deteriorating serum‐deprivation‐induced apoptosis; β‐arrestin 2 represents an inhibitory effect on the TLR4‐mediated apoptotic cascade, through controlling the homeostasis of activation and inactivation of GSK‐3β.

Toll-like receptor 2 is required for opioids-induced neuronal apoptosis

Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, is widely expressed in various systems, including the immune and nervous systems and plays a critical role in controlling innate and adaptive immune responses. We previously reported that opioids inhibit cell growth and trigger apoptosis. However, the underlying mechanism by which TLR2 mediates apoptosis in response to opioids is not yet known. Here we show that chronic morphine treatment in primary neurons dramatically increased the expression of TLR2 at both the messenger RNA and protein levels. In addition, TLR2 deficiency significantly inhibited chronic morphine-induced apoptosis in primary neurons. Activation of caspase-3 after morphine treatment is impaired in TLR2 deficient primary neurons. Moreover, morphine treatment failed to induce an increased level of phosphorylated glycogen synthase kinase 3 beta (GSK3beta) in TLR2 deficient primary neurons, suggesting an involvement of GSK3beta in morphine-mediated TLR2 signaling. These results thus demonstrate that opioids prime neurons to undergo apoptosis by inducing TLR2 expression. Our data suggest that inhibition of TLR2 is capable of preventing opioids-induced damage to neurons.

Toll-like receptor 9 is required for opioid-induced microglia apoptosis.

Opioids have been widely applied in clinics as one of the most potent pain relievers for centuries, but their abuse has deleterious physiological effects beyond addiction. However, the underlying mechanism by which microglia in response to opioids remains largely unknown. Here we show that morphine induces the expression of Toll-like receptor 9 (TLR9), a key mediator of innate immunity and inflammation. Interestingly, TLR9 deficiency significantly inhibited morphine-induced apoptosis in microglia. Similar results were obtained when endogenous TLR9 expression was suppressed by the TLR9 inhibitor CpGODN. Inhibition of p38 MAPK by its specific inhibitor SB203580 attenuated morphine-induced microglia apoptosis in wild type microglia. Morphine caused a dramatic decrease in Bcl-2 level but increase in Bax level in wild type microglia, but not in TLR9 deficient microglia. In addition, morphine treatment failed to induce an increased levels of phosphorylated p38 MAPK and MAP kinase kinase 3/6 (MKK3/6), the upstream MAPK kinase of p38 MAPK, in either TLR9 deficient or µ-opioid receptor (µOR) deficient primary microglia, suggesting an involvement of MAPK and µOR in morphine-mediated TLR9 signaling. Moreover, morphine-induced TLR9 expression and microglia apoptosis appears to require μOR. Collectively, these results reveal that opioids prime microglia to undergo apoptosis through TLR9 and µOR as well. Taken together, our data suggest that inhibition of TLR9 and/or blockage of µOR is capable of preventing opioid-induced brain damage.

Restraint stress induces lymphocyte reduction through p53 and PI3K/NF-κB pathways

Restraint stress, either physical or psychological, can modulate immune function. However, the mechanisms associated with stress-induced lymphocyte reduction remains to be elucidated. We have previously shown that chronic stress induces Fas-mediated lymphocyte reduction. Here, we investigated the mechanisms by which restraint stress modulates lymphocyte reduction. Our data have shown that inhibition of p53 by the p53 inhibitor PFT-alpha attenuates stress-induced reduction in lymphocyte numbers. These results were verified using p53 knockout mice, suggesting a pivotal role of p53 in this process. In addition our data have indicated that PI3K/nuclear factor kappa B (NF-kappaB) signaling pathway plays an important role in the stress-induced lymphocyte reduction. Our study thus demonstrates that restraint stress promotes lymphocyte reduction through p53 and PI3K/NF-kappaB pathways.

Chronic stress promotes lymphocyte reduction through TLR2 mediated PI3K signaling in a β-arrestin 2 dependent manner

Physical and psychological stress can alter the immune system in both humans and animals. Stress is a known risk factor for numerous human diseases, such as infectious and autoimmune diseases, and cancer. Toll-like receptors (TLRs) play a pivotal role in the induction of innate and adaptive immune response. Our previous studies have shown that TLR4 deficiency prevents stress-induced splenocyte reduction. However, the role of TLR2 in stress-mediated lymphocyte reduction is unknown. In this study, we investigated the effects of TLR2 ligands on stress-induced lymphocyte reduction. We also defined whether the phosphoinositide 3-kinases (PI3Ks)/Akt pathway contributes to TLR2-mediated lymphocyte numbers altered by stress. Our data have shown that stimulation of TLR2 by TLR2 ligands peptidoglycan (PGN) or Pam3CSK4 (Pam3) attenuates stress-induced reduction in lymphocyte numbers. However, TLR2 ligand-induced protection from stress-induced lymphocyte reduction is lost in TLR2 deficiency in mice. Furthermore, stimulation of TLR2 by PGN induces protection from stress-induced reduction in the number of splenocytes through PI3K. Moreover, PGN dramatically increases the level of phosphorylation of Akt through a PI3K-dependent manner. Moreover, we found that stimulation of TLR2 by PGN induced protection from stress-induced reduction in splenocyte numbers is abolished in β-arrestin 2 deficient mice. In addition, PGN-induced immune protection in stress-induced changes of cytokine levels appears to require β-arrestin 2, a multifunctional adaptor and signal transducer. Collectively, our study thus demonstrates that stimulation of TLR2-mediated PI3K signaling attenuates splenocyte reduction induced by stress, and that β-arrestin 2 modulates TLR2-mediated immune response following stress.

Beta-arrestin 2 modulates resveratrol-induced apoptosis and regulation of Akt/GSK3β pathways

BACKGROUND Resveratrol is emerging as a novel anticancer agent. However, the mechanism(s) by which resveratrol exerts its effects on endometrial cancer (EC) are unknown. We previously reported that beta-arrestin 2 plays a critical role in cell apoptosis. The role of ß-arrestin 2 in resveratrol modulation of endometrial cancer cell apoptosis remains to be established. SCOPE OF REVIEW EC cells HEC1B and Ishikawa were transfected with either ß-arrestin 2 RNA interfering (RNAi) plasmid or beta-arrestin 2 full-length plasmid and control vector. The cells were then exposed to differing concentrations of resveratrol. Apoptotic cells were detected by TUNEL assay. Expression of total and phosphorylated Akt (p-Akt), total and phosphorylated glycogen synthase kinase 3 beta (p-GSK3ß), and caspase-3 were determined by Western blot analysis. Our data demonstrate that inhibition of ß-arrestin 2 increases the number of apoptotic cells and caspase-3 activation. Additionally ß-arrestin 2 exerted an additive effect on resveratrol-reduced levels of p-Akt and p-GSK3ß. Overexpression of ß-arrestin 2 decreased the percentage of apoptosis and caspase-3 activation and attenuated resveratrol-reduced levels of p-Akt and p-GSK3ß. Taken together, our studies demonstrate for the first time that ß-arrestin 2 mediated signaling plays a critical role in resveratrol-induced apoptosis in EC cells. MAJOR CONCLUSIONS Resveratrol primes EC cells to undergo apoptosis by modulating beta-arrestin 2 mediated Akt/GSK3ß signaling pathways. GENERAL SIGNIFICANCE These inspiring findings would provide a new molecular basis for further understanding of cell apoptotic mechanisms mediated by ß-arrestin 2 and may provide insights into a potential clinical relevance in EC.