CpG methylation patterns in the IFNgamma promoter in naive T cells: variations during Th1 and Th2 differentiation and between atopics and non-atopics.

Interferon-gamma (IFNgamma) gene expression is tightly regulated in early life, and exaggerated negative control of IFNgamma production in CD4(+) T cells has been associated with risk for subsequent development of atopy. Recent studies have demonstrated hypermethylation of CpG sites in the IFNgamma promoter in neonates, a mechanism which in mice leads to strong suppression of IFNgamma gene transcription. In the present study, the methylation status of six CpG sites in the proximal promoter of the human IFNgamma gene was determined by bisulphite sequencing. Cell populations studied were Th1 or Th2 polarized cell lines derived from neonatal and adult CD4(+)/CD45RA(+) T cells, CD4(+) and CD8(+) naive T cells from cord blood of children followed to outcome age 2 for assessment of atopy status, and CD4(+) and CD8(+) naive T cells from 6 yr old and adult atopics and controls. We demonstrate that in vitro differentiation of CD4(+) T cells down the Th1 pathway (but not the Th2 pathway) is accompanied by progressive demethylation of CpG sites in the IFNgamma promoter, which is most marked in neonatal cells. Atopy development by age 2 was not associated with variations in methylation patterns in cord blood T cells. However, IFNgamma promoter methylation was reduced in CD8(+) T cells from atopic children in the age range in which hyperproduction of IFNgamma as recently been identified as a common feature of the atopic phenotype. The findings demonstrate the potency of IFNgamma promoter methylation as a mechanism for control of human IFNgamma gene expression, particularly during early life. Differential regulation of IFNgamma promoter methylation in T cells may be an important contributory factor in atopy development in childhood, and this possibility warrants further detailed investigation.

Functional maturation of CD4+CD25+CTLA4+CD45RA+ T regulatory cells in human neonatal T cell responses to environmental antigens/allergens.

A number of laboratories have reported cord blood T cell responses to ubiquitous environmental Ags, including allergens, by proliferation and cytokine secretion. Moreover, the magnitude of these responses has been linked with risk for subsequent expression of allergy. These findings have been widely interpreted as evidence for transplacental priming and the development of fetal T memory cells against Ags present in the maternal environment. However, we present findings below that suggest that neonatal T cell responses to allergens (and other Ags) differ markedly from those occurring in later life. Notably, in contrast to allergen-responsive adult CD4(+) T cell cultures, responding neonatal T cell cultures display high levels of apoptosis. Comparable responses were observed against a range of microbial Ags and against a parasite Ag absent from the local environment, but not against autoantigen. A notable finding was the appearance in these cultures of CD4(+)CD25(+)CTLA4(+) T cells that de novo develop MLR-suppressive activity. These cells moreover expressed CD45RA and CD38, hallmarks of recent thymic emigrants. CFSE-labeling studies indicate that the CD4(+)CD25(+) cells observed at the end of the culture period were present in the day 0 starting populations, but they were not suppressive in MLR responses. Collectively, these findings suggest that a significant component of the reactivity of human neonatal CD4(+) T cells toward nominal Ag (allergen) represents a default response by recent thymic emigrants, providing an initial burst of short-lived cellular immunity in the absence of conventional T cell memory, which is limited in intensity and duration via the parallel activation of regulatory T cells.

Flavohemoglobin Hmp, but not its individual domains, confers protection from respiratory inhibition by nitric oxide in Escherichia coli.

Escherichia coli possesses a two-domain flavohemoglobin, Hmp, implicated in nitric oxide (NO) detoxification. To determine the contribution of each domain of Hmp toward NO detoxification, we genetically engineered the Hmp protein and separately expressed the heme (HD) and the flavin (FD) domains in a defined hmp mutant. Expression of each domain was confirmed by Western blot analysis. CO-difference spectra showed that the HD of Hmp can bind CO, but the CO adduct showed a slightly blue-shifted peak. Overexpression of the HD resulted in an improvement of growth to a similar extent to that observed with the Vitreoscilla hemeonly globin Vgb, whereas the FD alone did not improve growth. Viability of the hmp mutant in the presence of lethal concentrations of sodium nitroprusside was increased (to 30% survival after 2 h in 5 mM sodium nitroprusside) by overexpressing Vgb or the HD. However, maximal protection was provided only by holo-Hmp (75% survival under the same conditions). Cellular respiration of the hmp mutant was instantaneously inhibited in the presence of 13.5 microM NO but remained insensitive to NO inhibition when these cells overexpressed Hmp. When HD or FD was expressed separately, no significant protection was observed. By contrast, overexpression of Vgb provided partial protection from NO respiratory inhibition. Our results suggest that, despite the homology between the HD from Hmp and Vgb (45% identity), their roles seem to be quite distinct.

Association of IL12B promoter polymorphism with severity of atopic and non-atopic asthma in children.

BACKGROUND: Severe asthma is a frequent cause of hospital admission, especially among children. The main environmental triggers of airway inflammation in asthma are viruses and aeroallergens. These agents elicit reciprocal immune responses, characterised by production of T helper 1 and T helper 2 cytokines, respectively. There is no genetic explanation for how hyper-responsiveness to these disparate environmental stimuli develops among individuals with asthma. Our aim was to assess relation between an IL12B promoter polymorphism and asthma. METHODS: We did a cohort study in which we initially genotyped 411 6-year olds for the IL12B promoter polymorphism. We then assessed the relation between this polymorphism and asthma severity. A further 85 asthmatic children in an additional sample of 433 children from the same cohort were then assessed to confirm these findings. We also examined in-vitro interleukin-12 responses in a subgroup of individuals. FINDINGS: Heterozygosity for the IL12B promoter polymorphism was observed in 76% (16) of atopic and non-atopic individuals with severe asthma in the initial sample. By comparison, heterozygotes comprised only 31% (17) of the moderate asthma group, and 48% (20) of individuals with mild asthma were heterozygous, as were unaffected controls. These findings were confirmed in the second sample (overall p<0.0001). Our data suggest that IL12B promoter heterozygosity contributes to asthma severity rather than susceptibility per se. The severity-predisposing genotype was associated with reduced interleukin 12 p40 gene transcription and decreased interleukin 12 p70 secretion. INTERPRETATION: Interleukin 12 plays a key part in antagonism of T helper 2 differentiation, and in induction of antiviral host defense. Genetically determined attenuation of interleukin-12 response capacity would, therefore, provide a plausible common immunological pathway to disease severity for the two major forms of asthma.

Differential patterns of methylation of the IFN-gamma promoter at CpG and non-CpG sites underlie differences in IFN-gamma gene expression between human neonatal and adult CD45RO- T cells.

IFN-gamma is a potent pleiotropic Th1 cytokine, the production of which is tightly regulated during fetal development. Negative control of fetal/neonatal IFN-gamma production is generally attributed to the Th1-antagonistic effect of mediators produced by the placenta, but evidence exists of additional and more direct transcriptional regulation. We report that neonatal (cord blood) CD3(+)/CD45RO(-) T cells, in particular the CD4(+)/CD45RO(-) subset, are hypermethylated at CpG and non-CpG (CpA and CpT) sites within and adjacent to the IFN-gamma promoter. In contrast, CpG methylation patterns in cord blood IFN-gamma-producing CD8(+)/CD45RO(-) T cells and CD56(+)/CD16(+)/CD3(-) NK cells did not differ significantly from those in their adult counterparts. Consistent with this finding, IFN-gamma production by stimulated naive cord blood CD4(+) T cells is reduced 5- to 10-fold relative to adult CD4(+) T cells, whereas production levels in neonatal and adult CD8(+) T cells are of a similar order. Evidence of significant CpA and CpT methylation was not discovered in promoter sequence from other cytokines (IL-4, TNF-alpha, or IFN-gammaR alpha-chain). We additionally demonstrate that overexpression of DNA methyltransferase 3a in embryonic kidney carcinoma cells is accompanied by CpA methylation of the IFN-gamma promoter.