The Influence of the Autoimmunity-Associated Ancestral HLA Haplotype AH8.1 on the Human Gut Microbiota: A Cross-Sectional Study

<div><p>Multiple immune-related genes are encoded in the HLA complex on chromosome 6p21. The 8.1 ancestral haplotype (AH8.1) include the classical HLA alleles HLA-B*08:01 and HLA-DRB1*03:01, and has been associated with a large number of autoimmune diseases, but the underlying mechanisms for this association are largely unknown. Given the recently established links between the gut microbiota and inflammatory diseases, we hypothesized that the AH8.1 influences the host gut microbial community composition. To study this further, healthy individuals were selected from the Norwegian Bone Marrow Donor Registry and categorized as either I. AH8.1 homozygote (n=34), II. AH8.1 heterozygote (n=38), III. Non AH8.1 heterozygote or IV. HLA-DRB1 homozygote but non AH8.1 (n=15). Bacterial DNA from stool samples were subjected to sequencing of the V3–V5 region of the 16S rRNA gene on the 454 Life Sciences platform and data analyzed using Mothur and QIIME. The results showed that the abundances of different taxa were highly variable within all pre-defined AH8.1 genotype groups. Using univariate non-parametric statistics, there were no differences regarding alpha or beta diversity between AH8.1 carriers (categories I and II) and non-carriers (categories III and IV), however four different taxa (<i>Prevotellaceae</i>, <i>Clostridium XVIII</i>, <i>Coprococcus</i>, <i>Enterorhabdus</i>) had nominally significant lower abundances in AH8.1 carriers than non-carriers. After including possible confounders in a multivariate linear regression, only the two latter genera remained significantly associated. In conclusion, the overall contribution of the AH8.1 haplotype to the variation in gut microbiota profile of stool in the present study was small.</p></div

Genus level taxonomic distribution in 117 Norwegian stool samples according to ancestral HLA haplotype AH8.1 status.

<p>(A) The genus abundances sorted according to the relative abundance of <i>Bacteroides</i>, of n = 72 AH8.1 carriers (left) are shown compared with the n = 45 non-AH8.1 samples (right). In (B) the genus abundances are shown according to all four pre-defined genotype groups; Left: AH8.1 homozygotes, i.e. homozygosity for both HLA-B*08 and HLA-DRB1*03; Middle left: AH8.1 heterozygotes, i.e. at least 1 copy of HLA-B*08 and HLA-DRB1*03; Middle right: Non AH8.1 heterozygotes, i.e. HLA-B*08 and HLA-DRB1*03 both not present; Right: <i>HLA-DRB1</i> homozygous but non AH8.1.</p

Phylogenetic diversity according to the ancestral HLA haplotype AH8.1 status.

<p>Phylogenetic diversity, an alpha diversity measure of bacterial richness, is shown according to carrier status of the AH8.1 haplotype (left), as well as the pre-defined HLA genotype groups (right, AH8.1 homozygotes, i.e. homozygosity for both HLA-B*08 and HLA-DRB1*03; AH8.1 heterozygotes, i.e. at least 1 copy of HLA-B*08 and HLA-DRB1*03; Non AH8.1 heterozygotes, i.e. HLA-B*08 and HLA-DRB1*03 both not present. Non AH8.1 homozygotes, i.e. HLA-DRB1 homozygous but non AH8.1). There were no significant differences between the groups.</p

Beta diversity according to the ancestral HLA haplotype AH8.1 status.

<p>A principal coordinate plot of the beta diversity measure unweighted unifrac shows all samples colored according to the HLA genotype groups. There were no significant differences between the groups. <i>Yellow</i>: AH8.1 homozygous, i.e. homozygosity for both HLA-B*08 and HLA-DRB1*03. <i>Violet</i>: AH8.1 heterozygotes, i.e. at least 1 copy of HLA-B*08 and HLA-DRB1*03. <i>Turquoise</i>: Non AH8.1 heterozygotes, i.e. HLA-B*08 and HLA-DRB1*03 both not present. <i>Grey</i>: HLA-DRB1 homozygous but non AH8.1.</p

Characteristics of the study population according to HLA genotype^a.

<p>^aFour different groups were included to represent homozygosity and heterozygosity for the AH8.1 haplotype (as defined by the presence of HLA-B*0801 and DRB1*0301), absence of this haplotype as well as homozygosity for other DRB1 haplotypes. There were no significant differences (all p>0.23) for any of the parameters when applying Kruskal-Wallis test for continuous and 2x4 chi square test for categorical parameters.</p><p>^bTransport time as defined by time from sampling to the freezer.</p><p>Characteristics of the study population according to HLA genotype<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133804#t002fn001" target="_blank">^a</a>.</p

Pre-defined HLA genotype groups.

<p>AH8.1: Ancestral haplotype 8.1</p><p>Pre-defined HLA genotype groups.</p

Bacterial taxa associated with being a carrier of the ancestral HLA haplotype AH8.1.

<p>^aMann-Whitney U test, using non-transformed relative abundances</p><p>^bLinear regression using square-root arcsine transformed relative abundance as dependent variable and including AH8.1 carrier status, age, gender, BMI, the use of oral drugs, smoking and time in the model.</p><p>Bacterial taxa associated with being a carrier of the ancestral HLA haplotype AH8.1.</p

Enterotype groups according to ancestral HLA haplotype AH8.1 status.

<p>The figure shows the individual samples as colored symbols according to their HLA genotype groups (see below) distributed according to similarity of their distribution of bacterial genera in a two-dimensional plot according to the methods by Arumugam et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133804#pone.0133804.ref034" target="_blank">34</a>]. The samples were classified into three fractions, enterotypes, dominated by either <i>Prevotella</i> (blue), <i>Bacteroides</i> (green) or a mix of bacteria (red), where the colored square indicate the centre of the distribution of the enterotype, the straight lines connect the included samples and the colored ellipses cover the individuals near the center of gravity for each cluster (1.5σ). Bacterial taxa overrepresented in the corresponding enterotypes are listed. As evident from the symbol color of the individual dots, the different HLA genotype groups (see below) were not preferentially distributed to one particular enterotype fraction. <i>Yellow diamonds</i>: AH8.1 homozygotes, i.e. homozygosity for both HLA-B*08 and HLA-DRB1*03. <i>Violet squares</i>: AH8.1 heterozygous, i.e. at least 1 copy of HLA-B*08 and HLA-DRB1*03. <i>Turquoise circles</i>: Non AH8.1 heterozygotes, i.e. HLA-B*08 and HLA-DRB1*03 both not present. <i>Grey triangles</i>: HLA-DRB1 homozygotes but non AH8.1.</p

Conditional logistic regression analyses within the major histocompatibility complex (MHC) in primary sclerosing cholangitis (PSC).

<p>The three various conditional strategies employed and their conditional steps are indicated by Ia-Id, IIa-IIc and IIIa-IIIc. For all panels the conditional <i>P</i>-values (y-axis) are plotted against the position on chromosome 6 shown in million base pairs (Mb, x-axis). HLA alleles, <i>DRB1-DQB1</i> haplotypes, SNPs, the MICA5.1 allele and KIR ligands are distinctly designated (see legend in panel Ia). The significance threshold (<i>P</i>≤2.66×10⁻⁶) is indicated by the black, horizontal dotted line.</p

The extension of nine significantly primary sclerosing cholangitis (PSC) associated major histocompatibility complex (MHC) haplotypes, observed in the unconditional and conditional regression analyses combined.

<p>The extension of the haplotypes is determined by three categories of linkage disequilibrium (<i>r</i>²>0.2 (green), <i>r</i>²>0.5 (purple) and <i>r</i>²>0.8 (black), respectively) between a single haplotype marker (listed in the figure box) and any single nucleotide polymorphism (SNP) in the dataset. Haplotype 1; AH8.1 (A*01-C*07-B*08-DRB3*01:01-DRB1*03:01-DQA1*05:01-DQB1*02:01). Haplotype 2; HLA-DRB1*13:01-DQB1*06:03. Haplotype 3; HLA-DRB1*04-DQB1*03:01. Haplotype 4; HLA-DRB1*04-DQB1*03:02. Haplotype 5; HLA-DRB1*15:01-DQB1*06:02. Haplotype 6; HLA-B*07. Haplotype 7; HLA-DRB3*03:01-DRB1*13:02-DQB1*06:04. Haplotype 8, HLA-DRB1*07:01-DQB1*03:03. Haplotype 9; an independent LD-block centromeric of <i>NOTCH4</i> in class III, tagged by rs116212904. DRB1*15:01 is part of the DR2 serotype group.</p