Apoptosis is induced in Chlamydia trachomatis infected HEp-2 cells by the addition of a combination innate immune activation compounds and the inhibitor wedelolactone

Problem: Innate immune activation of human cells, for some intracellular pathogens, is advantageous for vacuole morphology and pathogenic viability. It is unknown whether innate immune activation is advantageous to Chlamydia trachomatis viability. ----- ----- Method of study: Innate immune activation of HEp-2 cells during Chlamydia infection was conducted using lipopolysaccharide (LPS), polyI:C, and wedelolactone (innate immune inhibitor) to investigate the impact of these conditions on viability of Chlamydia. ----- ----- Results: The addition of LPS and polyI:C to stimulate activation of the two distinct innate immune pathways (nuclear factor kappa beta and interferon regulatory factor) had no impact on the viability of Chlamydia. However, when compounds targeting either pathway were added in combination with the specific innate immune inhibitor (wedelolactone) a major impact on Chlamydia viability was observed. This impact was found to be due to the induction of apoptosis of the HEp-2 cells under these conditions. ----- ----- Conclusion: This is the first time that induction of apoptosis has been reported in C. trachomatis-infected cells when treated with a combination of innate immune activators and wedelolactone.

Investigations into the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy and maternal immune activation

The amygdala is a limbic structure that is involved in many of our emotions and processing of these emotions such as fear, anger and pleasure. Conditions such as anxiety, autism, and also epilepsy, have been linked to abnormal functioning of the amygdala, owing to improper neurodevelopment or damage. This thesis investigated the cellular and molecular changes in the amygdala in models of temporal lobe epilepsy (TLE) and maternal immune activation (MIA). The kainic acid (KA) model of temporal lobe epilepsy (TLE) was used to induce Ammon’s-horn sclerosis (AHS) and to investigate behavioural and cytoarchitectural changes that occur in the amygdala related to Neuropeptide Y1 receptor expression. Results showed that KA-injected animals showed increased anxiety-like behaviours and displayed histopathological hallmarks of AHS including CA1 ablation, granule cell dispersion, volume reduction and astrogliosis. Amygdalar volume and neuronal loss was observed in the ipsilateral nuclei which was accompanied by astrogliosis. In addition, a decrease in Y1 receptor expressing cells in the ipsilateral CA1 and CA3 sectors of the hippocampus, ipsi- and contralateral granule cell layer of the dentate gyrus and ipsilateral central nucleus of the amygdala was found, consistent with a reduction in Y1 receptor protein levels. The results suggest that plastic changes in hippocampal and/or amygdalar Y1 receptor expression may negatively impact anxiety levels. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain and tight regulation and appropriate control of GABA is vital for neurochemical homeostasis. GABA transporter-1 (GAT-1) is abundantly expressed by neurones and astrocytes and plays a key role in GABA reuptake and regulation. Imbalance in GABA homeostasis has been implicated in epilepsy with GAT-1 being an attractive pharmacological target. Electron microscopy was used to examine the distribution, expression and morphology of GAT-1 expressing structures in the amygdala of the TLE model. Results suggest that GAT-1 was preferentially expressed on putative axon terminals over astrocytic processes in this TLE model. Myelin integrity was examined and results suggested that in the TLE model myelinated fibres were damaged in comparison to controls. Synaptic morphology was studied and results suggested that asymmetric (excitatory) synapses occurred more frequently than symmetric (inhibitory) synapses in the TLE model in comparison to controls. This study illustrated that the amygdala undergoes ultrastructural alterations in this TLE model. Maternal immune activation (MIA) is a risk factor for neurodevelopmental disorders such as autism, schizophrenia and also epilepsy. MIA was induced at a critical window of amygdalar development at E12 using bacterial mimetic lipopolysaccharide (LPS). Results showed that MIA activates cytokine, toll-like receptor and chemokine expression in the fetal brain that is prolonged in the postnatal amygdala. Inflammation elicited by MIA may prime the fetal brain for alterations seen in the glial environment and this in turn have deleterious effects on neuronal populations as seen in the amygdala at P14. These findings may suggest that MIA induced during amygdalar development may predispose offspring to amygdalar related disorders such as heightened anxiety, fear impairment and also neurodevelopmental disorders.

Role of Nitric Oxide-Signalling in Hypoxia-Induced Immune Escape in Cancer

A key step in malignant progression is the acquired ability of tumour cells to escape immune-mediated lysis. A potential mechanism by which tumour cells avoid immune destruction involves the shedding of MHC Class I Chain-Related Protein A (MICA), a Natural Killer (NK) cell-activating ligand, from the tumour cell membrane. Hypoxia has been shown to cause increased MICA shedding; however, this hypoxia-induced effect can be attenuated by pharmacological activation of the cyclic guanosine monophosphate (cGMP)-dependent nitric oxide (NO)-signalling pathway in cancer cells. The primary objective of the present study was to determine whether treatment of tumour-bearing nude mice with the NO-mimetic glyceryl trinitrate (GTN) attenuates in vivo tumour growth and if so, whether this effect is dependent on the presence of an intact NK cell compartment. Results indicated that continuous transdermal administration of GTN (1.8 µg/h) can significantly attenuate the growth of transplanted human DU-145 prostate tumours but that this effect of GTN is lost in mice whose NK-cells have been depleted. Tumours and serum from the mice in this study were analysed to determine whether GTN treatment had any effect on the expression levels of proteins integral to the proposed MICA shedding mechanism; however, the results of these studies were inconclusive. As phosphodiesterase (PDE) inhibition represents a potential method to enhance NO-signalling, experiments were performed to determine whether treatment with the PDE5/6 inhibitor zaprinast could also attenuate hypoxia-induced MICA shedding and decrease in vivo growth of DU-145 tumours. Results demonstrated that treatment with zaprinast (10 mg/kg) significantly attenuates MICA shedding in DU-145 cancer cells and significantly decreases in vivo tumour growth. Taken together, the results of these experiments indicate that GTN attenuates tumour growth by sensitising tumour cells to innate immunity, likely by increasing membrane-associated tumour cell MICA levels through the reactivation of NO-signalling, and that zaprinast decreases tumour growth likely through a similar mechanism. These findings are important because they indicate that agents capable of reactivating NO-signalling, such as NO-mimetics and PDE inhibitors, can potentially be used as immunosensitisers in the treatment and/or prevention of cancer.

Maternal immune activation in late gestation enhances locomotor response to acute but not chronic amphetamine treatment in male mice offspring: Role of the D1 receptor

Exposure to elevated levels of maternal cytokines can lead to functional abnormalities of the dopaminergic system in the adult offspring, including enhanced amphetamine (AMPH)-induced locomotion. Therefore, it seems reasonable to consider that offspring of challenged mothers would behave differently in models of addictive behavior, such as behavioral sensitization. Thus, we sought to evaluate the effects of prenatal exposure to lipopolysaccharide (LPS) on the locomotor response to acute and chronic AMPH treatment in male mice offspring. For this purpose, LPS (Escherichia coli 0127:B8; 120 mu g/kg) was administered intraperitoneally to pregnant Swiss mice on gestational day 17. At adulthood, male offspring were studied under one of the following conditions: (1) locomotor response to acute AMPH treatment (2.5 or 5.0 mg/kg) in an open field test; (2) behavioral sensitization paradigm, which consists of a daily injection of AMPH (1.0 mg/kg) for 10 days and observation of locomotion in the open field on days 1, 5, 10 (development phase), 15 and 17 (expression phase). The LPS stimulated offspring showed enhancement of the locomotor-stimulant effect after an acute AMPH challenge in comparison to baseline and saline pre-treated mice. They also showed development of behavioral sensitization earlier than the saline pre-treated group, although no changes between saline and LPS pre-treated groups were observed on development or expression of locomotor behavioral sensitization to AMPH. Furthermore, there was up-regulation of D1 receptor protein level within striatum in the LPS-stimulated offspring which was strongly correlated with increased grooming behavior. Taken together, our results indicate that motor and dopaminergic alterations caused by maternal immune activation are restricted to the acute AMPH challenge, mostly due to up-regulation of the D1 receptor within the mesolimbic and nigrostriatal pathways, but no locomotor differences were observed for behavioral sensitization to AMPH. (C) 2012 Elsevier B.V. All rights reserved.

Maternal immune activation increases the corticosterone response to acute stress without affecting the hypothalamic monoamine content and sleep patterns in male mice offspring

Background/Aims: Early life experiences are homeostatic determinants for adult organisms. We evaluated the impact of prenatal immune activation during late gestation on the neuroimmune-endocrine function of adult offspring and its interaction with acute stress. Methods: Pregnant Swiss mice received saline or lipopolysaccharide (LPS) on gestational day 17. Adult male offspring were assigned to the control or restraint stress condition. We analyzed plasmatic corticosterone and catecholamine levels, the monoamine content in the hypothalamus, striatum and frontal cortex, and the sleep-wake cycle before and after acute restraint stress. Results and Conclusion: Offspring from LPS-treated dams had increased baseline norepinephrine levels and potentiated corticosterone secretion after the acute stressor, and no effect was observed on hypothalamic monoamine content or sleep behavior. The offspring of immune-activated dams exhibited impairments in stress-induced serotonergic and dopaminergic alterations in the striatum and frontal cortex. The data demonstrate a distinction between the plasmatic levels of corticosterone in response to acute stress and the hypothalamic monoamine content and sleep patterns. We provide new evidence regarding the influence of immune activation during late gestation on the neuroendocrine homeostasis of offspring.

Immune Activation Reduces Sperm Quality in the Great Tit

Mounting an immune response against pathogens incurs costs to organisms by its effects on important life-history traits, such as reproductive investment and survival. As shown recently, immune activation produces large amounts of reactive species and is suggested to induce oxidative stress. Sperm are highly susceptible to oxidative stress, which can negatively impact sperm function and ultimately male fertilizing efficiency. Here we address the question as to whether mounting an immune response affects sperm quality through the damaging effects of oxidative stress. It has been demonstrated recently in birds that carotenoid-based ornaments can be reliable signals of a male's ability to protect sperm from oxidative damage. In a full-factorial design, we immune-challenged great tit males while simultaneously increasing their vitamin E availability, and assessed the effect on sperm quality and oxidative damage. We conducted this experiment in a natural population and tested the males' response to the experimental treatment in relation to their carotenoid-based breast coloration, a condition-dependent trait. Immune activation induced a steeper decline in sperm swimming velocity, thus highlighting the potential costs of an induced immune response on sperm competitive ability and fertilizing efficiency. We found sperm oxidative damage to be negatively correlated with sperm swimming velocity. However, blood resistance to a free-radical attack (a measure of somatic antioxidant capacity) as well as plasma and sperm levels of oxidative damage (lipid peroxidation) remained unaffected, thus suggesting that the observed effect did not arise through oxidative stress. Towards the end of their breeding cycle, swimming velocity of sperm of more intensely colored males was higher, which has important implications for the evolution of mate choice and multiple mating in females because females may accrue both direct and indirect benefits by mating with males having better quality sperm.

Platelet-activating factor is crucial in psoralen and ultraviolet A-induced immune suppression, inflammation, and apoptosis.

Psoralen plus UVA (PUVA) is used as a very effective treatment modality for various diseases, including psoriasis and cutaneous T-cell lymphoma. PUVA-induced immune suppression and/or apoptosis are thought to be responsible for the therapeutic action. However, the molecular mechanisms by which PUVA acts are not well understood. We have previously identified platelet-activating factor (PAF), a potent phospholipid mediator, as a crucial substance triggering ultraviolet B radiation-induced immune suppression. In this study, we used PAF receptor knockout mice, a selective PAF receptor antagonist, a COX-2 inhibitor (presumably blocking downstream effects of PAF), and PAF-like molecules to test the role of PAF receptor binding in PUVA treatment. We found that activation of the PAF pathway is crucial for PUVA-induced immune suppression (as measured by suppression of delayed type hypersensitivity to Candida albicans) and that it plays a role in skin inflammation and apoptosis. Downstream of PAF, interleukin-10 was involved in PUVA-induced immune suppression but not inflammation. Better understanding of PUVA's mechanisms may offer the opportunity to dissect the therapeutic from the detrimental (ie, carcinogenic) effects and/or to develop new drugs (eg, using the PAF pathway) that act like PUVA but have fewer side effects.

The Effects of Physical and Chemical Modulating Agents on the Induction of Vaccine-Induced Immune Responses

After the development of the viral-based prostate cancer vaccine, Ad5-PSA, much research has been orientated to help enhance the induced immune response by combining the vaccine with physical and chemical modulating agents, more specifically the polymers polyethylenimine (PEI), chitosan, and chitosan coated with CD3 complex antibodies; all previously shown to stimulate an immune response as isolated gene carriers. To compare the vaccine-induced immune responses between the naked vaccine and the polymer-vaccine combinations, a mouse model using the ovalbumin- specific Ad-OVA vaccine was tested using intracellular cytokine staining (ICS), tetramer staining, and cytotoxic T-cell lymphocyte assays to measure the activation of CD8+ T-cells, interferon gamma proteins (INFƒ×), and the induced cytotoxicity to ovalbumin. The Ad-OVA vaccine combined with both chitosan and chitosan with CD3 complex antibodies, both natural polymers, were found to induce similar immune responses to the naked vaccine while the vaccine combined with the synthetic polymer, PEI, diminished the immune response.

Effects of the microenvironment on Fas/Fas ligand interactions and their implications for UV-induced immune suppression

Skin cancer is the most common malignancy in humans. Although highly treatable, non-melanoma skin cancer is commonly followed by other non-cutaneous malignancies. Ultraviolet radiation (UVR) acts as both tumor initiator and promoter, and also results in the suppression of specific immune responses. The systemic suppression of immune responses is initiated by DNA damage, which promotes IL-10 production, an important cytokine as anti-IL-10 can abrogate the suppression, and upregulates the pro-apoptotic proteins Fas and Fas ligand (FasL). FasL is a critical factor for UV-induced immune suppression, and the suppressor cell induced by UV expresses FasL. ^ We hypothesized that the microenvironment affects Fas/FasL interactions, and that these interactions are important to the phenomenon of UV induced immune suppression. To determine the effects of the interaction of FasL and IL-10, splenocytes isolated from C57Bl/6 mice were cultured in the presence or absence of IL-10 post-mitogenic activation. We determined that IL-10 protects from Fas-mediated apoptosis by lowering Fas sensitivity and lowering the levels of either Fas or FasL. This protection is stronger when IL-10 is given immediately after mitogenic activation, and does not increase any of the inhibitors of apoptosis studied. In vivo, splenocytes from UV-irradiated mice are resistant to Fas-mediated apoptosis and present very high levels of IL-10, lowered Fas sensitivity and lowered caspase cleavage despite higher expression of Fas and FasL than non-irradiated mice. ^ UV-induced immune suppression affects female mice preferentially, which led us to look at prolactin as a possible component of this suppression since this hormone has also been associated with increased skin carcinogenesis. The interaction of FasL and prolactin results in suppression of the delayed type hypersensitivity response to Candida albicans. This lack of response depends on FasL as is not seen in gld mice. Similar to UV-induced immune suppression, the suppression is caused by a Th2 deviation, and correlates with a significant increase in Fas expression. In the presence of UV, the effects of prolactin seemed to be protective, and UV actually restores the DTH response.^ Taken together, these observations suggest that the microenvironment dictates the outcome of the interaction of FasL with Fas going from promoting apoptosis to preventing apoptosis or mediating a Th2 deviation and suppression of a Th1 response. ^

Molecular mechanism of jet fuel induced immune suppression

Dermal exposure to jet fuel suppresses the immune response. Immune regulatory cytokines, and biological modifiers, including platelet activating factor, prostaglandin E2, and interleukin-10 have all been implicated in the pathway leading to immunosuppression. It is estimated that approximately 260 different hydrocarbons are found in JP-8 (jet propulsion-8) jet fuel, and the identity of the immunotoxic compound is not known. The recent availability of synthetic jet fuel (S-8), which is devoid of aromatic hydrocarbons, made it feasible to design experiments to test the hypothesis that the aromatic hydrocarbons are responsible for jet fuel induced immune suppression. Applying S-8 to the skin of mice does not up-regulate the expression of epidermal cyclooxygenase-2 nor does it induce immune suppression. Adding back a cocktail of 7 of the most prevalent aromatic hydrocarbons found in jet fuel to S-8 up-regulated cyclooxygenase-2 expression and induced immune suppression. Cyclooxygenase-2 induction can be initiated by reactive oxygen species (ROS). JP-8 treated keratinocytes increased ROS production, S-8 did not. Antioxidant pre-treatment blocked jet fuel induced immune suppression and cyclooxygenase-2 up-regulation. Accumulation of reactive oxygen species induces oxidant stress and affects activity of ROS sensitive transcription factors. JP-8 induced activation of NFκB while S-8 did not. Pre-treatment with antioxidants blocked activation of NFκB and parthenolide, an NFκB inhibitor, blocked jet fuel induced immune suppression and cyclooxygenase-2 expression in skin of treated mice. p65 siRNA transfected keratinocytes demonstrated NFκB is critically involved in jet fuel induced COX-2 expression. These findings clearly implicate the aromatic hydrocarbons found in jet fuel as the agents responsible for inducing immune suppression, in part by the production of reaction oxygen species, NFκB dependent up-regulation of cyclooxygenase-2, and the production of immune regulatory factors and cytokines. ^

Dual role of interleukin-12 on ultraviolet -induced immune suppression

Skin cancer is the most prevalent form of neoplasia, with over one million newcases diagnosed this year. UV radiation is a ubiquitous environmental agent that induces skin cancer. In addition to its carcinogenic effect, UV radiation also suppresses cell-mediated immune responses. This immune suppression is not only observed at the site of irradiation, but UV radiation also induces systemic immune suppression. Since UV radiation has a limited ability to penetrate the skin, the question of the mechanism of this systemic immune suppression arises. A number of studies have suggested that UV radiation induce systemic effects through the production of immunoregulatory cytokines, such as IL-4 and IL-10. These cytokines affect the immune response by altering systemic antigen presentation, specifically by suppressing the activation of Th1 cells while allowing the activation of Th2 cells. Because IL-12 is an important regulator of Th1 cell activation, we tested the hypothesis that administration of IL-12 could overcome UV-induced immune suppression. ^ The studies presented here are divided into dime specific aims. In the first specific aim, the ability of IL-12 to overcome UV-induced immune suppression was examined. IL-12 could overcome UV-induced immune suppression as well as prevent the generation of and neutralize the activity of preformed suppressor cells induced by UV radiation. In the second specific aim, the mechanism by which IL-12 overcomes UV-induced immune suppression was examined. IL-12 overcame UV-induced immune suppression by blocking the production of immunoregulatory cytokines such as IL-4, IL-10 and TNF-α. In the third specific aim, the effect of UV radiation on antigen presentation was investigated. UV radiation was found to decrease the production of biologically active IL-12. In addition, UV also increased the production of IL-12p40 homodimer, an antagonist of IL-12p70 heterodimer. This result suggests that IL-12 may have a dual role in the immune suppression induced by, UV radiation. On one hand the biologically active IL-12p70 heterodimer blocks UV-induced immune suppression. In contrast, IL-12p40 homodimer may mediate the suppressive effect of UV radiation. This paradox indicates that IL-12 may have a greater regulatory role in the immune response than was previously suspected. ^

Chronic immune activation and inflammation as the cause of malignancy

Several chronic infections known to be associated with malignancy have established oncogenic properties. However the existence of chronic inflammatory conditions that do not have an established infective cause and are associated with the development of tumours strongly suggests that the inflammatory process itself provides the prerequisite environment for the development of malignancy. This environment includes upregulation of mediators of the inflammatory response such as cyclo-oxygenase (COX)-2 leading to the production of inflammatory cytokines and prostaglandins which themselves may suppress cell mediated immune responses and promote angiogenesis. These factors may also impact on cell growth and survival signalling pathways resulting in induction of cell proliferation and inhibition of apoptosis. Furthermore, chronic inflammation may lead to the production of reactive oxygen species and metabolites such as malondialdehyde within the affected cells that may in turn induce DNA damage and mutations and, as a result, be carcinogenic. Here it is proposed that the conditions provided by a chronic inflammatory environment are so essential for the progression of the neoplastic process that therapeutic intervention aimed at inhibiting inflammation, reducing angiogenesis and stimulating cell mediated immune responses may have a major role in reducing the incidence of common cancers. © 2001 Cancer Research Campaign http://www.bjcancer.com.

Studies on immune activation in rheumatoid arthritis and reactive arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovitis, progressive joint destruction, and disability. Reactive arthritis (ReA) is a sterile joint inflammation following a distant mucosal infection. The clinical course of these diseases is variable and cannot be predicted with reasonable accuracy by clinical and laboratory markers. The predictive value of circulating soluble interleukin-2 receptor (sIL-2R), a marker of lymphocyte activation, measured by Immulite® automated immunoassay analyzer, was evaluated in two cohorts of RA patients. In 175 patients with active early RA randomized to treatment with either on disease-modifying antirheumatic drug (DMARD) or a combination of 3 DMARDs and prednisolone, low baseline sIL-2R level predicted remission after 6 months in patients treated with a single DMARD. In 24 patients with active RA refractory to DMARDs, low baseline sIL-2R level predicted rapid clinical response to treatment with infliximab, an anti-tumour necrosis factor antibody. Furthermore, in a cohort of 26 patients with acute ReA, high baseline sIL-2R level predicted remission after 6 months. Levels of circulating soluble E-selectin (sE-selectin), a marker of endothelial activation, were measured annually by enzyme-linked immunosorbent assay (ELISA) in a cohort of 85 patients with early RA. During a five-year follow-up, sE-selectin levels were associated with activity and outcome of RA. The levels of neutrophil and monocyte CD11b/CD18 expression measured by flow cytometry, and circulating levels of sE-selectin measured by ELISA, and procalcitonin by immunoluminometric assay, were compared in 28 patients with acute ReA and 16 patients with early RA. The levels of the markers were comparable in ReA, RA, and healthy control subjects. In conlusion, sIL-2R may provide a new predictive marker in early RA treated with a single DMARD and refractory RA treated with infliximab. In addition, sIL-2R level predicts remission in acute ReA.

ATM- and ATR-mediated phosphorylation of XRCC3 regulates DNA double-strand break-induced checkpoint activation and repair

The RAD51 paralogs XRCC3 and RAD51C have been implicated in homologous recombination (HR) and DNA damage responses. However, the molecular mechanism(s) by which these paralogs regulate HR and DNA damage signaling remains obscure. Here, we show that an SQ motif serine 225 in XRCC3 is phosphorylated by ATR kinase in an ATM signaling pathway. We find that RAD51C but not XRCC2 is essential for XRCC3 phosphorylation, and this modification follows end resection and is specific to S and G(2) phases. XRCC3 phosphorylation is required for chromatin loading of RAD51 and HR-mediated repair of double-strand breaks (DSBs). Notably, in response to DSBs, XRCC3 participates in the intra-S-phase checkpoint following its phosphorylation and in the G(2)/M checkpoint independently of its phosphorylation. Strikingly, we find that XRCC3 distinctly regulates recovery of stalled and collapsed replication forks such that phosphorylation is required for the HR-mediated recovery of collapsed replication forks but is dispensable for the restart of stalled replication forks. Together, these findings suggest that XRCC3 is a new player in the ATM/ATR-induced DNA damage responses to control checkpoint and HR-mediated repair.