Smita Kulkarni

Sex differences in the Toll-like receptor–mediated response of plasmacytoid dendritic cells to HIV-1

Manifestations of viral infections can differ between women and men, and marked sex differences have been described in the course of HIV-1 disease. HIV-1–infected women tend to have lower viral loads early in HIV-1 infection but progress faster to AIDS for a given viral load than men. Here we show substantial sex differences in the response of plasmacytoid dendritic cells (pDCs) to HIV-1. pDCs derived from women produce markedly more interferon-α (IFN-α) in response to HIV-1–encoded Toll-like receptor 7 (TLR7) ligands than pDCs derived from men, resulting in stronger secondary activation of CD8+ T cells. In line with these in vitro studies, treatment-naive women chronically infected with HIV-1 had considerably higher levels of CD8+ T cell activation than men after adjusting for viral load. These data show that sex differences in TLR-mediated activation of pDCs may account for higher immune activation in women compared to men at a given HIV-1 viral load and provide a mechanism by which the same level of viral replication might result in faster HIV-1 disease progression in women compared to men. Modulation of the TLR7 pathway in pDCs may therefore represent a new approach to reduce HIV-1–associated pathology.

Differential microRNA regulation of HLA-C expression and its association with HIV control

The HLA-C locus is distinct relative to the other classical HLA class I loci in that it has relatively limited polymorphism, lower expression on the cell surface, and more extensive ligand–receptor interactions with killer-cell immunoglobulin-like receptors. A single nucleotide polymorphism (SNP) 35u2009kb upstream of HLA-C (rs9264942; termed −35) associates with control of HIV, and with levels of HLA-C messenger RNA transcripts and cell-surface expression, but the mechanism underlying its varied expression is unknown. We proposed that the −35 SNP is not the causal variant for differential HLA-C expression, but rather is marking another polymorphism that directly affects levels of HLA-C. Here we show that variation within the 3′ untranslated region (UTR) of HLA-C regulates binding of the microRNA hsa-miR-148 to its target site, resulting in relatively low surface expression of alleles that bind this microRNA and high expression of HLA-C alleles that escape post-transcriptional regulation. The 3′u2009UTR variant associates strongly with control of HIV, potentially adding to the effects of genetic variation encoding the peptide-binding region of the HLA class I loci. Variation in HLA-C expression adds another layer of diversity to this highly polymorphic locus that must be considered when deciphering the function of these molecules in health and disease.

Genetic interplay between HLA-C and MIR148A in HIV control and Crohn disease

Significance In the human population different HLA-C allotypes are present that have different expression levels at the cell surface. Individuals with higher expressed HLA-C allotypes demonstrate better HIV control but increased risk of Crohn disease. A microRNA, miR-148a, regulates expression of some HLA-C allotypes. We report here that this microRNA also varies in expression level between people. MIR148A variation showed significant and opposing effects on HIV viral control vs. risk of Crohn disease, specifically in subjects with HLA-C alleles that are regulated by miR-148a. These results are independent of confounding effects by other HLA loci because only HLA-C is regulated by miR-148a. Our data represent an example of gene interactions that affect immune response and thereby the risk of human disease. Variation in the 3′ untranslated region (3′UTR) of the HLA-C locus determines binding of the microRNA Hsa-miR-148a, resulting in lower cell surface expression of alleles that bind miR-148a relative to those alleles that escape its binding. The HLA-C 3′UTR variant was shown to associate with HIV control, but like the vast majority of disease associations in a region dense with causal candidates, a direct effect of HLA-C expression level on HIV control was not proven. We demonstrate that a MIR148A insertion/deletion polymorphism associates with its own expression levels, affecting the extent to which HLA-C is down-regulated, the level of HIV control, and the risk of Crohn disease only among those carrying an intact miR-148a binding site in the HLA-C 3′UTR. These data illustrate a direct effect of HLA-C expression level on HIV control that cannot be attributed to other HLA loci in linkage disequilibrium with HLA-C and highlight the rich complexity of genetic interactions in human disease.

KIR Genotyping by Multiplex PCR-SSP

Diversity across KIR haplotypes stems from differences in numbers of inhibitory and activating receptors, as well as allelic polymorphism of individual genes. The KIR locus has undergone large expansions and contractions over time and is believed to be coevolving with genes encoding its HLA class I ligands located within the MHC locus. KIR and HLA compound genotypes have been associated with susceptibility to or protection from infectious, autoimmune, reproductive, and malignant disorders. We describe here a simple and reliable multiplex PCR-SSP (sequence-specific priming) method for relatively rapid and inexpensive genotyping of 15 KIR genes using standard agarose gel electrophoresis.

The molecular origin and consequences of escape from miRNA regulation by HLA-C alleles.

Differential expression of human leukocyte antigen C (HLA-C) allotypes is mediated by the binding of a microRNA, miR-148a, to the 3 untranslated region of some, but not all, HLA-C alleles. The binding results in lower levels of HLA-C expression, which is associated with higher levels of HIV-1 viral load among infected individuals. The alternative set of HLA-C alleles has several substitutions in the miR-148a binding site that prevent binding and HLA-C downregulation; these high-expression alleles associate with control of HIV-1 viral load. We show that the common ancestor of all extant HLA-C alleles was suppressed by miR-148a. Substitutions that prevent miR-148a binding arose by a sequence exchange event between an HLA-C allele and an HLA-B (MIM 142830) allele of a B(∗)07-like lineage. The event occurred 3-5 million years ago, resulting in an HLA-C variant that escape from miR-148a downregulation. We present evidence suggesting that selection played a role in the successful spread of the HLA-C escape alleles, giving rise to 7 of the 14 extant HLA-C lineages. Notably, critical peptide and KIR binding residues of the escape variants have selectively converged to resemble the sequence of their inhibited counterparts, such that the inhibited and escape groupings differ primarily by their levels of expression.

Lack of Duffy antigen receptor for chemokines: no influence on HIV disease progression in an African treatment-naive population.

Dear Editors: He et al recently reported an association between a single-nucleotide polymorphism (SNP, rs2814778, -46T→C) in the erythroid specific promoter region of the Duffy antigen receptor for chemokine (DARC) and HIV-1 acquisition and disease progression in African Americans (He et al., 2008). The authors described a higher frequency of the null homozygous genotype (-46C/C) in HIV-infected (70%) versus non-infected individuals (60%). Based on these findings they argued that the null allele confers susceptibility to infection with HIV-1 and suggested that up to 11% of the HIV-1 burden in sub-Saharan Africa could be attributed to homozygosity for the null allele. Oddly, however, these authors reported that the null genotype associated with better outcomes amongst those who became infected, including longer survival and more particularly decelerated loss of CD4+ T-lymphocytes. Another report showed an association between the -46C/C genotype and significantly reduced total white blood cells (WBC) in African Americans explaining ∼20% of population variation in WBC (Nalls et al., 2008). The -46C/C genotype is nearly fixed in those sub-Saharan African populations where Plasmodium vivax is endemic, apparently because it confers resistance against P. vivax malaria, but -46C is virtually absent in individuals of European descent (Tournamille et al., 1995; Hamblin and Di Rienzo, 2000). In those regions of sub-Saharan Africa that are not endemic for P. vivax, the -46C allele is not fixed, however, and the wildtype (-46T) allele is frequently observed (Rosenberg et al., 2007). Considering the potential importance of DARC on HIV-1 disease progression in Africans, we studied the effect of the DARC -46 genotype on outcomes to HIV infection in a cohort of untreated black South Africans. This region of Africa is not endemic for P. vivax and lies outside of the area of 96% mean fixation for -46C; thus, the null allele of DARC is not fixed in our study population and the allele frequency of -46T (the wildtype allele) is about 20%. We assessed the effects of the null (C/C), heterozygous (T/C), and wildtype (T/T) genotypes on rates of CD4 decline and mean HIV-1 viral loads in a group of 381 untreated black South Africans. Since the -46C/C genotype has previously been associated significantly with reduced total WBC (Nalls et al., 2008), we also determined WBC in our cohort in order to verify that our cohort is sufficiently powered to detect an effect of the -46 genotype, where -46C/C is expected to associate with low WBC as previously demonstrated.

Epigenetic Regulation of Differential HLA-A Allelic Expression Levels

MHC class I expression levels influence the strength of immune responses and represent another variable in determining outcome to disease beyond peptide binding alone. Identification of the HLA loci that vary in allelic expression levels and delineating the mechanism responsible for expression variation may provide the opportunity to modify their expression therapeutically. We have examined the expression levels of allelic lineages at the HLA-A locus in a sample of 216 European Americans using a real-time polymerase chain reaction assay, which amplifies all HLA-A lineages specifically with equal efficiency, and observed a gradient of expression that associates with HLA-A allelic lineage (R = 0.6, P = 5 × 10(-25)). DNA methylation of the HLA-A gene appears to contribute to the variation in HLA-A mRNA expression levels, as a significant inverse correlation was observed between HLA-A mRNA expression levels in untreated cells and the degree to which expression is increased after treatment of the cells with a DNA methyltransferase inhibitor (R = 0.6, P = 2.8 × 10(-6)). Further, deep-sequencing and immunoprecipitation assays revealed allelic lineage-specific methylation patterns within the HLA-A promoter region where increased DNA methylation levels correlated significantly with reduced HLA-A expression levels (R = 0.89, P = 3.7 × 10(-9)). These data demonstrate HLA-A allelic lineage-specific variation in expression levels, and DNA methylation as a likely factor in contributing to this variation.

Polymorphisms in interferon regulatory factor 7 reduce interferon-α responses of plasmacytoid dendritic cells to HIV-1.

Recognition of HIV-1 ssRNA by Toll-like receptor 7 induces the production of the pro-inflammatory cytokines that may contribute to the systemic immune activation associated with HIV-1 disease progression. Here, we describe a novel association between polymorphisms in interferon regulatory factor 7 (IRF7), a master regulator of interferon-α (IFN-α), and the ability of plasmacytoid dendritic cells to produce IFN-α in response to HIV-1. IRF7 polymorphisms may, therefore, affect the ability of individuals to respond to HIV-1 and modulate HIV-1 disease progression.

Variation in the Untranslated Genome and Susceptibility to Infections

The clinical outcomes of infections are highly variable among individuals and are determined by complex host-pathogen interactions. Genome-wide association studies (GWAS) are powerful tools to unravel common genetic variations that are associated with disease risk and clinical outcomes. However, GWAS has only rarely revealed information on the exact genetic elements and their effects underlying an association because the majority of the hits are within non-coding regions. Some of the variants or the linked polymorphisms are now being discovered to have functional significance, such as regulatory elements in the promoter and enhancer regions or the microRNA binding sites in the 3′untranslated region of the protein-coding genes, which influence transcription, RNA stability, and translation of the protein-coding genes. However, only 3% of the entire transcriptome is protein-coding, signifying that non-coding RNAs represent most of the transcripts. Thus, a large portion of previously identified intergenic GWAS single nucleotide polymorphisms (SNPs) is in the non-coding RNAs. The non-coding RNAs form a large-scale regulatory network across the transcriptome, greatly expanding the complexity of gene regulation. Accumulating evidence also suggests that the “non-coding” genome regions actively regulate the highly dynamic three dimensional (3D) chromatin structures, which are critical for genome function. Epigenetic modulation like DNA methylation and histone modifications further affect chromatin accessibility and gene expression adding another layer of complexity to the functional interpretation of genetic variation associated with disease outcomes. We provide an overview of the current information on the influence of variation in these “untranslated” regions of the human genome on infectious diseases. The focus of this review is infectious disease-associated polymorphisms and gene regulatory mechanisms of pathophysiological relevance.

Functional Invariant Natural Killer T Cells Secreting Cytokines Are Associated With Non-Progressive Human Immunodeficiency Virus-1 Infection but Not With Suppressive Anti-Retroviral Treatment

Background CD1d restricted invariant natural killer T (iNKT) cells are important in the activation and regulation of immune responses. Limited information is available regarding the functional role of iNKT cells in the human immunodeficiency virus (HIV) disease progression. Methodology α-GalCer stimulated iNKT cells were characterized for their functionality in terms of cytokine production (IFN-γ, TNF-α, IL-2, IL-4, and IL-21) and CD107a expression in HIV-1 infected [23 long-term non progressors (LTNPs), 28 progressors, 18 patients before and after suppressive anti-retroviral treatment (ART)] along with 25 HIV-1 negative subjects using multicolor flow cytometry. Results The functional profile of α-GalCer stimulated iNKT cells was similar in LTNPs and healthy controls. The number of LTNPs showing functional response in terms of secretion of cytokines (IFN-γ/IL2/TNF-α) and CD107a expression was significantly higher than seen in the progressors. The cytokine secretion by the stimulated iNKT cells was predominantly Th1 type. The frequencies of iNKT cells showing secretion of IFN-γ or IL2 or TNF-α or expression of CD107a were higher in LTNPs (pu2009<u20090.05 for all) and also significantly associated with lower plasma viral load (p value ranged from 0.04 to 0.003) and higher CD4 count (p value ranged from 0.02 to <0.0001). The functional profile of the iNKT cells before and after ART did not differ significantly indicating absence of restoration of iNKT cells functionality after suppressive ART. The IL-4 and IL-21 secreting iNKT cells were rare in all study populations. Conclusion The presence of functional iNKT cells secreting number of cytokines in non-progressive HIV infection could be one of the multiple factors required to achieve HIV control and hence have relevance in understanding the immunity in HIV infection. The failure of restoration of the iNKT functionality after ART should be potential area of future research.