William Walker

Sex-Associated Hormones and Immunity to Protozoan Parasites

SUMMARY Numerous epidemiological and clinical studies have noted differences in the incidence and severity of parasitic diseases between males and females. Although in some instances this may be due to gender-associated differences in behavior, there is overwhelming evidence that sex-associated hormones can also modulate immune responses and consequently directly influence the outcome of parasitic infection. Animal models of disease can often recreate the gender-dependent differences observed in humans, and the role of sex-associated hormones can be confirmed by experimentally altering their levels. Under normal circumstances, levels of sex hormones not only differ between males and females but vary according to age. Furthermore, not only are females of reproductive age subject to the regular hormonal cycles which control ovulation, they are also exposed to dramatically altered levels during pregnancy. It is thus not surprising that the severity of many diseases, including those caused by parasites, has been shown to be affected by one or more of these circumstances. In addition, infection with many pathogens has been shown to have an adverse influence on pregnancy. In this article we review the impact of sex-associated hormones on the immune system and the development and maintenance of immunity to the intracellular protozoan parasites Toxoplasma gondii, Plasmodium spp., and Leishmania spp.

The CD40/CD40 Ligand Interaction Is Required for Resistance to Toxoplasmic Encephalitis

ABSTRACT Since the CD40/CD40 ligand (CD40L) interaction is involved in the regulation of macrophage production of interleukin 12 (IL-12) and T-cell production of gamma interferon (IFN-γ), effector cell functions associated with resistance to Toxoplasma gondii, the role of CD40L in immunity to this parasite was assessed. Infection of C57BL/6 mice with T. gondii results in an upregulation of CD40 expression on accessory cell populations at local sites of infection as well as in lymphoid tissues. Splenocytes from C57BL/6 mice infected with T. gondii for 5 days produced high levels of IL-12 and IFN-γ when stimulated with toxoplasma lysate antigen, and blocking CD40L did not significantly alter the production of IFN-γ or IL-12 by these cells. Similar results were observed with splenocytes and mononuclear cells isolated from the brains of chronically infected mice. Interestingly, although CD40L−/− mice infected withT. gondii produced less IL-12 than wild-type mice, they produced comparable levels of IFN-γ but succumbed to toxoplasmic encephalitis 4 to 5 weeks after infection. The inability of CD40L−/− mice to control parasite replication in the brain correlated with the ability of soluble CD40L, in combination with IFN-γ, to activate macrophages in vitro to control replication ofT. gondii. Together, these results identify an important role for the CD40/CD40L interaction in resistance to T. gondii. However, this interaction may be more important in the control of parasite replication in the brain rather than the generation of protective T-cell responses during toxoplasmosis.

Prostaglandin E2 is a potent regulator of interleukin-12- and interleukin-18-induced natural killer cell interferon-γ synthesis

Synthesis of interferon (IFN)‐γ by natural killer (NK) cells is an important pro‐inflammatory event with interleukin (IL)‐12 and IL‐18 playing major inductive roles. However, other temporal events are likely to regulate such processes and as prostaglandin E2 (PGE2) is ubiquitous during inflammation this study tested the hypothesis that PGE2 was capable of directly modulating cytokine‐induced NK cell IFN‐γ synthesis in the absence of other immune cells. Using homogenous NK cell lines to establish direct effects, PGE2 (0·1–1u2003µm) was found to suppress NK cell IFN‐γ synthesis and antagonized the potent synergistic IFN‐γ‐inducing effects of IL‐12 and IL‐18. The actions of PGE2 were mimicked by synthetic PGE2 analogues including misoprostol and butaprost. The selective EP2 receptor agonist butaprost, but not the EP1/EP3 agonist sulprostone, suppressed IFN‐γ synthesis and exclusively competed with PGE2 for receptor binding on NK cells. Further analysis showed that PGE2 did not modulate IL‐12 receptor mRNA expression and the effects of PGE2 could be mimicked by the phosphodiesterase inhibitor 3‐iosobutyl‐1‐methylxanthine. The absence of demonstrable receptor modulation coupled with the observed suppression of IFN‐γ synthesis by both EP2 receptor‐selective agonists and IBMX suggest that PGE2 acts directly on NK cells via EP2 receptors with its downstream effects on cAMP metabolism. This conclusion is further supported by findings that PGE2 and its analogues consistently elevated levels of cAMP in NK cells. The ability of PGE2 to antagonize the potent inductive signal provided by the combination of IL‐12 and IL‐18 supports the concept that PGE2 may play an important role in limiting innate inflammatory processes in vivo through direct suppression of NK cell IFN‐γ synthesis.

Variation in dinucleotide (GT) repeat sequence in the first exon of the STAT6 gene is associated with atopic asthma and differentially regulates the promoter activity in vitro

Upregulation of the IL-4/IL-13 mediated Th2 response is a characteristic of allergic diseases such as asthma, a common and often debilitating disease.1 STAT6 is a critical signalling molecule in the Th2 signalling pathway, and mice lacking STAT6 are protected from allergic pulmonary manifestations.2 The importance of STAT6 in asthma is also evident from studies showing that STAT6 gene expression is markedly upregulated in airway epithelial cells in asthma.3 STAT6 is important in the expression of VCAM-1 in endothelial cells and of chemokines, such as eotaxin, in epithelial cells following stimulation with IL-4 and IL-13.4 As a consequence, STAT6 has been considered a strong candidate for predisposition to atopic asthma. Indeed, the human STAT6 gene is mapped to chromosome 12q13.3−q14.1, a region linked to total serum IgE concentration and atopy in several populations.5 A number of common polymorphisms have been identified, including a GT repeat in exon 1 and three common SNPs (G4219A, A4491G, and A4671G; GenBank AF067575) in the 3′ untranslated region of the human STAT6 gene.6–9 Although all four of these polymorphisms have been shown to be associated with allergic phenotypes in various populations, their functional relevance remains unclear.nnDinucleotide repeats are the most frequent of the simple repeats distributed throughout the human genome, and many of these exhibit length polymorphisms. Investigations into the effect of dinucleotide repeats on gene expression have shown enhanced10 or decreased transcriptional activity11,12 in the context of different genes and cell types. These regulatory effects have been proposed to be due, in part, to the fact that dinucleotide repeats have potential to form alternative DNA structures, such as Z-, H- and cruciform DNA.13 The Z-DNA sequences in human genes tend to be located near transcription sites, which makes it possible that they play …

An essential role for IL-13 in maintaining a non-healing response following leishmania mexicana infection

A comparison of the growth of Leishmania mexicana in IL‐4–/–, IL‐4Rα–/– and wild‐type BALB/c mice demonstrated a disease exacerbative role for IL‐13 as well as IL‐4. Thus, while both IL‐4–/– and IL‐4Rα–/– mice were more resistant than wild‐type controls to infection with L. mexicana, IL‐4Rα–/– mice, which are unresponsive to IL‐13 as well as IL‐4, were significantly more resistant to parasite growth than their IL‐4–/– counterparts. Cytokine and antibody analysis revealed a Th1‐biased specific response in both infected IL‐4–/– and IL‐4Rα–/– mice compared with wild‐type animals. Reconstituting SCID mice with IL‐4–/–, IL‐4Rα–/– or wild‐type splenocytes prior to infection demonstrated that the early onset of lesion growth was dependent on the presence of lymphocytes responding to IL‐4 and/or IL‐13, as lesions failed to develop in only the SCID IL‐4Rα–/– reconstituted mice. An independent role for IL‐13 in L. mexicana infection was demonstrated by comparing disease progression in IL‐13–/–, IL‐4–/–/IL‐13–/– and wild‐type B6/129 mice. In contrast to IL‐4–/–/IL‐13–/– mice, which were resistant, IL‐13–/– mice developed lesions similar in size to wild‐type animalsup to weeku20048 post infection. However, in contrast to wild‐type mice in which disease continued to progress, lesions eventually healed in IL‐13–/– mice, in association with the development of a Th1 response. Collectively our results suggest that IL‐4 plays a critical role in early lesion development, and that IL‐13 plays a crucial part in maintaining a chronic non‐healing infection.

Chemokine receptor 7 (CCR7) gene expression is regulated by NF-κB and activator protein 1 (AP1) in metastatic squamous cell carcinoma of head and neck (SCCHN).

Background: The regulation and significance of aberrant CCR7 expression in metastatic SCCHN is poorly understood. Results: High CCR7 expression correlates with decreased patient survival. CCR7 is regulated by NF-κB and AP1 signaling pathways. Conclusion: CCR7 is a poor prognosis marker. NF-κB and AP1 signaling pathways co-regulate CCR7 expression. Significance: Understanding the regulation of CCR7 is crucial for developing therapeutic targets for treating metastatic SCCHN. The chemokine receptor CCR7 is a seven-transmembrane domain G-protein-coupled receptor that facilitates leukocyte migration to regional lymph nodes. Aberrant CCR7 expression in a number of human malignancies has been linked to pro-survival, -invasive, and -metastatic pathways. We demonstrate here that up-regulation of CCR7 in squamous cell carcinoma of the head and neck (SCCHN) patient tumors correlates with lower survival because of metastatic disease. Because of this important oncogenic phenotype, we investigated the mechanisms that regulate CCR7 expression in these tumors. Interestingly, the inflammatory transcription factor NF-κB has been associated with a more aggressive SCCHN phenotype. Immunohistochemical staining of a SCCHN tumor cohort (n = 47) strongly linked NF-κB staining and CCR7 expression in SCCHN. Thus, we investigated whether NF-κB contributes to metastatic disease by promoting CCR7 expression in SCCHN tumor cells. We characterized four novel, potential NF-κB binding sites in the 1000-bp promoter region upstream of the CCR7 gene, using luciferase, ChIP, and EMSA. However, NF-κB inhibition only resulted in partial reduction in CCR7 expression, prompting consideration of other co-regulators of CCR7. Indeed, cooperation between NF-κB and AP1 transcription factors, which are often co-activated, is crucial to the regulation of CCR7 mRNA expression in metastatic SCCHN cells. Thus, our findings support an important biological role for inflammatory NF-κB and AP1 in the regulation of CCR7 expression in metastatic SCCHN. As such, CCR7, NF-κB, and AP1 could be potentially useful therapeutic targets in controlling the progression and metastasis of SCCHN tumors.

RNA interference of STAT6 rapidly attenuates ongoing interleukin‐13‐mediated events in lung epithelial cells

Signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a central role in asthma pathogenesis, with its activation driving the development of airway hyper‐reactivity and local inflammation. Therefore, inhibition of local STAT6 expression provides a rationale for therapeutic intervention in bronchial asthma. Given the absence of specific inhibitory drugs, we tested the ability of small interfering RNAs (siRNAs) to target STAT6 gene expression through the molecular process of RNA interference (RNAi). At pico‐molar concentrations, STAT6‐specific siRNAs potently inhibited STAT6 mRNA expression in lung epithelial cells (50% inhibitory concentration rangeu2003=u2003134–861u2003pm) without inducing cellular interferon responses. Detectable STAT6 protein expression was rapidly abolished within 48u2003hr of treatment (t1/2 rangeu2003=u2003<u200312–37u2003hr) and this was unaffected by pretreatment with STAT6‐activating cytokines. Furthermore, STAT6 suppression by RNAi produced downstream functional inhibitory effects in that interleukin (IL)‐13‐ or IL‐4‐driven eotaxin chemokine family [chemokine (C‐C motif) ligand 11 (CCL11), CCL24 and CCL26] mRNA expression was markedly inhibited. Induction of detectable CCL26 protein synthesis was completely ablated by pretreating cells with STAT6‐specific siRNA. The therapeutic potential of this approach is further demonstrated by novel findings that cells pre‐exposed to IL‐13 or IL‐4 and subsequently treated with STAT6‐targeting siRNA exhibited a rapid and significant attenuation of ongoing CCL26 protein expression, suggesting that chronic asthma‐associated lung inflammation will be responsive to this approach.

Development of pre-clinical models for evaluating the therapeutic potential of candidate siRNA targeting STAT6.

Developing siRNA therapeutics poses technical challenges including appropriate molecular design and testing in suitable pre-clinical models. We previously detailed sequence-selection and modification strategies for siRNA candidates targeting STAT6. Here, we describe methodology that evaluates the suitability of candidate siRNA for respiratory administration. Chemically-modified siRNA exhibited similar inhibitory activity (IC50) against STAT6 in vitro compared to unmodified siRNA and apical exposure testing with Caco-2 cell monolayers showed modification was not associated with cellular toxicity. Use of a modified RNA extraction protocol improved the sensitivity of a PCR-based bio-analytical assay (lower limit of siRNA strand quantification u200a=u200a 0.01 pg/µl) which was used to demonstrate that lung distribution profiles for both siRNAs were similar following intra-tracheal administration. However, after 6 hours, modified siRNA was detected in lung tissue at concentrations >1000-fold higher than unmodified siRNA. Evaluation in a rat model of allergic inflammation confirmed the persistence of modified siRNA in vivo, which was detectable in broncho-alveolar lavage (BAL) fluid, BAL cells and lung tissue samples, 72 hours after dosing. Based upon the concept of respiratory allergy as a single airway disease, we considered nasal delivery as a route for respiratory targeting, evaluating an intra-nasal exposure model that involved simple dosing followed by fine dissection of the nasal cavity. Notably, endogenous STAT6 expression was invariant throughout the nasal cavities and modified siRNA persisted for at least 3 days after administration. Coupled with our previous findings showing upregulated expression of inflammatory markers in nasal samples from asthmatics, these findings support the potential of intranasal siRNA delivery. In summary, we demonstrate the successful chemical modification of STAT6 targeting siRNA, which enhanced bio-availability without cellular toxicity or reduced efficacy. We have established a robust, sensitive method for determining siRNA bio-distribution in vivo, and developed a nasal model to aid evaluation. Further work is warranted.

Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA‐based therapeutics for asthma

The development of siRNA‐based asthma therapeutics is currently hampered by a paucity of relevant biomarkers and the need to ascertain tissue‐specific gene targeting in the context of active disease. Epithelial STAT6 expression is fundamental to asthma pathogenesis in which inflammatory changes are found throughout the respiratory tract. Therefore, to improve preclinical evaluation, we tested the efficacy of STAT6‐targeting siRNA within nasal epithelial cells (NECs) obtained from asthmatic and non‐asthmatic donors. STAT6 expression was invariant in both donor groups and amenable to suppression by siRNA treatment. In addition, STAT6 mRNA was also suppressible by apically delivered siRNA treatment in comparative differentiated nasal epithelial cell‐line monolayer cultures. Analysis of donor NECs showed consistent elevation in CCL26 (eotaxin‐3) mRNA within the asthmatic group suggesting potential as a relevant biomarker. Furthermore, targeting of STAT6 with siRNA attenuated IL‐13‐driven CCL26 expression in these cells, pointing to the utility of this approach in preclinical testing. Finally, siRNA‐mediated suppression of STAT6 was independent of donor disease phenotype or epithelial cell differentiation status, signifying therapeutic potential.

Time-Restricted Feeding Alters the Innate Immune Response to Bacterial Endotoxin

An important entraining signal for the endogenous circadian clock, independent of light, is food intake. The circadian and immune systems are linked; forced desynchrony of the circadian clock via nighttime light exposure or genetic ablation of core clock components impairs immune function. The timing of food intake affects various aspects of the circadian clock, but its effects on immune function are unknown. We tested the hypothesis that temporal desynchrony of food intake alters innate immune responses. Adult male Swiss Webster mice were provided with food during the night, the day, or ad libitum for 4 wk, followed by administration of LPS prior to the onset of either the active phase (zeitgeber time [ZT]12: Experiment 1) or the inactive phase (ZT0: Experiment 2). Three hours after LPS administration, blood was collected, and serum was tested for bacteria-killing capacity against Escherichia coli, as a functional assay of immune function. Additionally, cytokine expression was examined in the serum (protein), spleen, and hypothalamus (mRNA). Day-fed mice suppressed bacteria-killing capacity and serum cytokine responses to LPS during the active phase (ZT12). Night-fed mice increased bactericidal capacity, as well as serum and hypothalamic mRNA responses of certain proinflammatory cytokines during the active phase. Only day-fed mice enhanced serum cytokine responses when LPS challenge occurred during the inactive phase (ZT0); this did not result in enhanced bactericidal capacity. These data suggest that mistimed feeding has functional relevance for immune function and provide further evidence for the integration of the circadian, metabolic, and immune systems.