JunD/AP-1-Mediated Gene Expression Promotes Lymphocyte Growth Dependent on Interleukin-7 Signal Transduction

Interleukin-7 (IL-7) is an essential cytokine for lymphocyte growth that has the potential for promoting immune reconstitution. This feature makes IL-7 an ideal candidate for therapeutic development. As with other cytokines, signaling through the IL-7 receptor induces the JAK/STAT pathway. However, the broad scope of IL-7 regulatory targets likely necessitates the use of other signaling components whose identities remain poorly defined. To this end, we used an IL-7 dependent T-cell line to examine how expression of the glycolytic enzyme, Hexokinase II (HXKII) was regulated by IL-7 in a STAT5-independent manner. Our studies revealed that IL-7 promoted the activity of JNK (Jun N-terminal Kinase), and that JNK, in turn, drove the expression of JunD, a component of the Activating Protein 1 (AP-1) transcription factors. Gel shifts showed that the AP-1 complex induced by IL-7 contained JunD but not c-Fos or c-Jun. Inhibition of JNK/JunD blocked glucose uptake and HXKII gene expression, indicating that this pathway was responsible for promoting HXKII expression. Because others had shown that JunD was a negative regulator of cell growth, we performed a bioinformatics analysis to uncover possible JunD-regulated gene targets. Our search revealed that JunD could control the expression of proteins involved in signal transduction, cell survival and metabolism. One of these growth promoters was the oncogene, Pim-1. Pim-1 is an IL-7-induced protein that was inhibited when the activities of JNK or JunD were blocked, showing that in IL-7 dependent T-cells JunD can promote positive signals transduced through Pim-1. This was confirmed when the IL-7-induced proliferation of CD8 T-cells was impaired upon JunD inhibition. These results show that engagement of the IL-7 receptor drives a signal that is more complex than the JAK/STAT pathway, activating JNK and JunD to induce rapid growth stimulation through the expression of metabolic and signaling factors like HXKII and Pim-1

Modulation of Macrophage Function by Lactobacillus-Conditioned Medium

Probiotics are used as microbial food supplements for health and well-being. They are thought to have immunomodulatory effects although their exact physiological mechanism of action is not clear. This study investigated the influence of probiotic Lactobacillus rhamnosus GG conditioned media (LGG-CM) on macrophage phagocytosis of non-pathogenic Escherichia coli HfrC. The gentamicin protection assay was used to study the bacterial killing phases of phagocytosis. Macrophages co-incubated with E. coli for an hour allowed them to ingest bacteria and then the rate of E. coli killing was monitored for up to 300 min to determine the killing or digestion of the bacteria by recovering them from the macrophage lysate. We found that the LGG-CM significantly increased the bacterial killing by approximately 6-fold when compared with that of controls. By contrast, this killing process was found to be associated with enhanced free radical production via the activation of NADPH oxidase, stimulated by the LGG conditioned medium. We also found that the conditioned medium had small effect on nitric oxide (NO) generation, albeit to a lesser extent. This work suggests that LGG-CM may play an important role in suppressing the total microbial load within the macrophages and hence, the extent to which pro-inflammatory molecules such as free radicals and NO are generated. The modulation of inflammation-promoting signals by LGG-CM may be beneficial as it modulates bacterial killing, and thereby prevents any collateral damage to host

Lactobacillus rhamnosus GG conditioned media modulates acute reactive oxygen species and nitric oxide in J774 murine macrophages

Phagocytes such as macrophages are capable of detecting and killing pathogenic bacteria by producing reactive oxygen and nitrogen species. Formation of free radicals in macrophages may be regulated by probiotics or by factors released by probiotics but yet to be identified. Thus, studies were carried out to determine whether cell-free conditioned medium obtained from cultures of Lactobacillus rhamnosus GG (LGG-CM) regulate production of reactive oxygen species (ROS) and/or nitric oxide (NO) in macrophages. J774 macrophages in culture were loaded with either H2DCFDA for monitoring ROS or with DAFFM-DA for NO detection. Free radical production was measured on a fluorescence microplate reader and changes were analysed by Cumulative sum (CuSum) calculations. Low concentration of LGG-CM (10% LGG-CM) or LPS did not cause any significant change in basal levels of ROS or NO production. In contrast, high concentration of LGG-CM (75% and 100%) significantly enhanced ROS generation but also significantly reduced NO level. These findings are novel and suggest for the first time that probiotics may release factors in culture which enhance ROS production and may additionally reduce deleterious effects associated with excessive nitrogen species by suppressing NO level. These events may account, in part, for the beneficial bactericidal and anti-inflammatory actions ascribed to probiotics and may be of clinical relevance

Cytokine-dependent and cytokine-independent roles for Mcl-1: genetic evidence for multiple mechanisms by which Mcl-1 promotes survival in primary T lymphocytes

Myeloid cell leukemia sequence-1 (Mcl-1) is a critical anti-apoptotic factor in T lymphocytes. However, in spite of the many pro-apoptotic proteins with proposed binding to Mcl-1, the specific interactions by which Mcl-1 regulates primary T-cell survival under different conditions have not been fully explored. Further, how different trophic cytokines modulate the specific role(s) of Mcl-1 is unknown. Here, we use genetic mouse models to dissect the roles of Mcl-1 in primary T lymphocytes. Using the inducible Mcl-1-floxed estrogen receptor-Cre fusion protein (Mcl-1f/fERCre) deletion system in combination with genetic modification of other B-cell lymphoma 2 (Bcl-2) family members, we show that loss of pro-apoptotic Bcl-2 homologous antagonist/killer (Bak) rescues the survival of Mcl-1-deficient T cells in the presence of IL-7. Without IL-7, the survival of Mcl-1-deficient cells cannot be rescued by loss of Bak, but is partially rescued by overexpression of Bcl-2 or loss of Bcl-2-interacting mediator of cell death (Bim). Thus, Mcl-1 and Bcl-2 have a shared role, the inhibition of Bim, in promoting T-cell survival during cytokine withdrawal. Finally, we show that other common gamma-chain (γc) cytokines differentially modulate the roles of Mcl-1. IL-15 has effects similar to those of IL-7 in memory T cells and naïve CD8+ cells, but not naïve CD4+ cells. However, IL-4 maintains Mcl-1 and Bcl-2 but also upregulates Bim and Bcl-2-associated X protein (Bax), thus altering the cell's dependence on Mcl-1

Prevalence of Allergic Disorders among Primary School-Aged Children in Madinah, Saudi Arabia: Two-Stage Cross-Sectional Survey

There are limited data on the epidemiology of allergic disorders in Saudi Arabia. Such data are needed for, amongst other things, helping to plan service provision at a time when there is considerable investment taking place in national healthcare development. We sought to estimate the prevalence of atopic eczema, allergic rhinitis and asthma in primary school children in Madinah, Saudi Arabia.We conducted a two-stage cross-sectional survey of schoolchildren in Madinah. Children were recruited from 38 randomly selected schools. Questionnaires were sent to the parents of all 6,139 6-8 year old children in these schools. These parental-completed questionnaires incorporated questions from the International Study of Asthma and Allergies in Childhood (ISAAC), which had previously been validated for use in Arab populations. We undertook descriptive analyses, using the Generalized Estimating Equation (GEE) to calculate 95% confidence intervals. The overall response rate was 85.9% (n = 5,188), 84.6% for girls and 86.2% for boys, respectively. Overall, parents reported symptoms suggestive of a history of eczema in 10.3% (95%CI 9.4, 11.4), rhinitis in 24.2% (95%CI 22.3, 26.2) and asthma in 23.6% (95%CI 21.3, 26.0) of children. Overall, 41.7% (95%CI 39.1, 44.4) of children had symptoms suggestive of at least one allergic disorder, with a substantial minority manifesting symptoms indicative of co-morbid allergic disease. Comparison of these symptom-based prevalence estimates with reports of clinician-diagnosed disease suggested that the majority of children with eczema and asthma had been diagnosed, but only a minority (17.4%) of children had been diagnosed with rhinitis. International comparisons indicated that children in Madinah have amongst the highest prevalence of allergic problems in the world.Symptoms indicative of allergic disease are very common in primary school-aged children in Madinah, Saudi Arabia, with figures comparable to the highest risk regions in the world

CRP identifies homeostatic immune oscillations in cancer patients: a potential treatment targeting tool?

The search for a suitable biomarker which indicates immune system responses in cancer patients has been long and arduous, but a widely known biomarker has emerged as a potential candidate for this purpose. C-Reactive Protein (CRP) is an acute-phase plasma protein that can be used as a marker for activation of the immune system. The short plasma half-life and relatively robust and reliable response to inflammation, make CRP an ideal candidate marker for inflammation. The high- sensitivity test for CRP, termed Low-Reactive Protein (LRP, L-CRP or hs-CRP), measures very low levels of CRP more accurately, and is even more reliable than standard CRP for this purpose. Usually, static sampling of CRP has been used for clinical studies and these can predict disease presence or recurrence, notably for a number of cancers. We have used frequent serial L-CRP measurements across three clinical laboratories in two countries and for different advanced cancers, and have demonstrated similar, repeatable observations of a cyclical variation in CRP levels in these patients. We hypothesise that these L-CRP oscillations are part of a homeostatic immune response to advanced malignancy and have some preliminary data linking the timing of therapy to treatment success. This article reviews CRP, shows some of our data and advances the reasoning for the hypothesis that explains the CRP cycles in terms of homeostatic immune regulatory cycles. This knowledge might also open the way for improved timing of treatment(s) for improved clinical efficacy

Feed-Forward Microprocessing and Splicing Activities at a MicroRNA–Containing Intron

The majority of mammalian microRNA (miRNA) genes reside within introns of protein-encoding and non-coding genes, yet the mechanisms coordinating primary transcript processing into both mature miRNA and spliced mRNA are poorly understood. Analysis of melanoma invasion suppressor miR-211 expressed from intron 6 of melastatin revealed that microprocessing of miR-211 promotes splicing of the exon 6–exon 7 junction of melastatin by a mechanism requiring the RNase III activity of Drosha. Additionally, mutations in the 5′ splice site (5′SS), but not in the 3′SS, branch point, or polypyrimidine tract of intron 6 reduced miR-211 biogenesis and Drosha recruitment to intron 6, indicating that 5′SS recognition by the spliceosome promotes microprocessing of miR-211. Globally, knockdown of U1 splicing factors reduced intronic miRNA expression. Our data demonstrate novel mutually-cooperative microprocessing and splicing activities at an intronic miRNA locus and suggest that the initiation of spliceosome assembly may promote microprocessing of intronic miRNAs

Overexpression of Batf induces an apoptotic defect and an associated lymphoproliferative disorder in mice

Activator protein-1 (AP-1) is a dimeric transcription factor composed of the Jun, Fos and Atf families of proteins. Batf is expressed in the immune system and participates in AP-1 dimers that modulate gene expression in response to a variety of stimuli. Transgenic (Tg) mice overexpressing human BATF in T cells were generated using the human CD2 promoter (CD2-HA (hemagglutinin antigen) - BATF). By 1 year of age, over 90% of the mice developed a lymphoproliferative disorder (LPD). The enlarged lymph nodes characteristic of this LPD contain a polyclonal accumulation of T cells with a CD4+ bias, yet efforts to propagate these tumor cells in vitro demonstrate that they do not proliferate as well as wild-type CD4+ T cells. Instead, the accumulation of these cells is likely due to an apoptotic defect as CD2-HA-BATF Tg T cells challenged by trophic factor withdrawal in vitro resist apoptosis and display a pro-survival pattern of Bcl-2 family protein expression. As elevated levels of Batf expression are a feature of lymphoid tumors in both humans and mice, these observations support the use of CD2-HA-BATF mice as a model for investigating the molecular details of apoptotic dysregulation in LPD