Gene Expression Profiles from Disease Discordant Twins Suggest Shared Antiviral Pathways and Viral Exposures among Multiple Systemic Autoimmune Diseases

<div><p>Viral agents are of interest as possible autoimmune triggers due to prior reported associations and widely studied molecular mechanisms of antiviral immune responses in autoimmunity. Here we examined new viral candidates for the initiation and/or promotion of systemic autoimmune diseases (SAID), as well as possible related signaling pathways shared in the pathogenesis of those disorders. RNA isolated from peripheral blood samples from 33 twins discordant for SAID and 33 matched, unrelated healthy controls was analyzed using a custom viral-human gene microarray. Paired comparisons were made among three study groups—probands with SAID, their unaffected twins, and matched, unrelated healthy controls—using statistical and molecular pathway analyses. Probands and unaffected twins differed significantly in the expression of 537 human genes, and 107 of those were associated with viral infections. These 537 differentially expressed human genes participate in overlapping networks of several canonical, biologic pathways relating to antiviral responses and inflammation. Moreover, certain viral genes were expressed at higher levels in probands compared to either unaffected twins or unrelated, healthy controls. Interestingly, viral gene expression levels in unaffected twins appeared intermediate between those of probands and the matched, unrelated healthy controls. Of the viruses with overexpressed viral genes, herpes simplex virus-2 (HSV-2) was the only human viral pathogen identified using four distinct oligonucleotide probes corresponding to three HSV-2 genes associated with different stages of viral infection. Although the effects from immunosuppressive therapy on viral gene expression remain unclear, this exploratory study suggests a new approach to evaluate shared viral agents and antiviral immune responses that may be involved in the development of SAID.</p></div

Four distinct viral oligo probes from three HSV-2 genes that were differentially expressed between probands and unaffected twins, as well as between probands and matched, unrelated healthy controls^*.

<p>*Paired comparisons were made between probands (P) and unaffected twins (U) or matched, unrelated healthy controls (C). Statistically significant viral probes (fold change, FC >1.5, <i>q</i> <0.05) were selected.</p><p>Four distinct viral oligo probes from three HSV-2 genes that were differentially expressed between probands and unaffected twins, as well as between probands and matched, unrelated healthy controls^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142486#t003fn001" target="_blank">*</a>}.</p

Ingenuity pathway analysis of differentially expressed human genes between disease discordant twins.

<p>The analysis compared 789 oligo probes corresponding to 537 human genes significantly differentially expressed (fold change >1.5, <i>q</i> <0.05) between twins discordant for SAID. The upper horizontal axis (blue bars) describe the association of the data set with a given pathway (-log (<i>p</i> value)). The cutoff threshold value (defined as <i>p</i> = 0.05) is shown by the vertical red line. The ratio of the number of genes from the data set that map to a given pathway divided by the total number of molecules that comprise the pathway is shown on the horizontal axis (green triangles). Previously reported viral-related signaling pathways are in bold print.</p

Dot plots of three HSV-2 gene expression levels in SAID affected probands, unaffected twins, and matched, unrelated healthy controls.

<p>The individual normalized signal data for <i>UL19</i> (A, probe 1 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142486#pone.0142486.t003" target="_blank">Table 3</a>), <i>UL36</i> (B) and <i>RS1</i> (C) were plotted according to the three study groups: probands, unaffected twins, and matched, unrelated healthy controls. Statistically significant <i>p</i> values between any two groups are shown (*) and were calculated using one-way ANOVA with corrections for multiple comparisons. The proposed “normal range” values were derived from the mean plus three standard deviations (SD, dash line) of healthy control expression values.</p

Comparisons of differential gene expression values determined by relative quantitative-polymerase chain reaction and microarray analyses^*.

<p>* Fold change (FC) values indicating relative expression levels of two HSV-2 genes (<i>UL36</i> and <i>RS1</i>) between two study groups: probands (P) and matched, unrelated healthy controls (C). qPCR, quantitative polymerase chain reaction; qRT-PCR, quantitative reverse-transcription PCR; Positive, the affected probands with probe values above the proposed normal range (mean plus three standard deviation of control group); Negative, the affected probands with probe values within the proposed normal range.</p><p>Comparisons of differential gene expression values determined by relative quantitative-polymerase chain reaction and microarray analyses^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142486#t004fn001" target="_blank">*</a>}.</p

IFN-regulated genes that were significantly differentially expressed between SAID probands and their unaffected twins^*

<p>*Listed are the 41 IFN-regulated genes of 107 viral infection-related genes identified by the INTERFEROME program that were statistically significant (<i>q</i> < 0.05) and had a fold change >1.5 between disease discordant twins. Fold change values indicate increase (positive) or decreased (negative) levels of gene expression in probands related to unaffected twins.</p><p>IFN-regulated genes that were significantly differentially expressed between SAID probands and their unaffected twins^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142486#t002fn001" target="_blank">*</a>}</p

Flow chart of the experimental design and data analyses.

<p>Flow chart of the experimental design and data analyses.</p

Matriptase Deletion Initiates a Sjögren’s Syndrome-Like Disease in Mice

<div><p>Objective</p><p>The objective of this study was to determine the effect of epithelial barrier disruption, caused by deficiency of the membrane-anchored serine protease, matriptase, on salivary gland function and the induction of autoimmunity in an animal model.</p><p>Methods</p><p>Embryonic and acute ablation of matriptase expression in the salivary glands of mice was induced, leading to decreased epithelial barrier function. Mice were characterized for secretory epithelial function and the induction of autoimmunity including salivary and lacrimal gland dysfunction, lymphocytic infiltration, serum anti-Ro/SSA, anti-La/SSB and antinuclear antibodies. Salivary glands immune activation/regulation, barrier function as well as tight junction proteins expression also were determined. Expression of matriptase in minor salivary gland biopsies was compared among pSS patients and healthy volunteers.</p><p>Results</p><p>Embryonic ablation of matriptase expression in mice resulted in the loss of secretory epithelial cell function and the induction of autoimmunity similar to that observed in primary Sjögren’s syndrome. Phenotypic changes included exocrine gland dysfunction, lymphocytic infiltrates, production of Sjögren’s syndrome-specific autoantibodies, and overall activation of the immune system. Acute ablation of matriptase expression resulted in significant salivary gland dysfunction in the absence of overt immune activation. Analysis of the salivary glands indicates a loss of electrical potential across the epithelial layer as well as altered distribution of a tight junction protein. Moreover, a significant decrease in matriptase gene expression was detected in the minor salivary glands of pSS patients compared with healthy volunteers.</p><p>Conclusions</p><p>Our findings demonstrate that local impairment of epithelial barrier function can lead to loss of exocrine gland dysfunction in the absence of inflammation while systemic deletion can induce a primary Sjögren’s syndrome like phenotype with autoimmunity and loss of gland function.</p></div

T cell activation and regulation changes in draining lymph nodes and spleens from <i>St14</i>^– mice.

<p>Salivary gland draining lymph nodes (DLN) and spleens were collected from <i>St14⁺</i> and <i>St14^–</i> animals. Samples from 2–3 mice were pooled per group and used to detect CD4, CD8 and CD62L expression by flow cytometry. (A, B) Both CD4+ and CD8+ cells were significantly decreased in both the DLN and spleens from <i>St14^–</i> mice compared with <i>St14⁺</i> mice. CD62L expression was also decreased on both CD4+ and CD8+ cells in <i>St14^–</i> mice compared with <i>St14⁺</i> mice. (C, D, E) The percentage and total number of CD4+CD25+Foxp3+ natural T regulatory cells (nTreg) were altered in the DLN and spleen cells. Data shown are the mean ± SEM for each animal group (<i>N</i> = 4 for both groups). Statistical significance was determined using an unpaired Student’s <i>t</i>-test. *<i>P</i><0.05, **<i>P</i>≤0.001, ***<i>P</i><0.0001.</p

Decreased epithelial electrical potential as well as changes in protein distribution in mice with acute salivary gland-specific ablation of matriptase.

<p>Electrical potential (EP) of salivary glands was measured 22 weeks post-AAV2 delivery to glands in live AAV2-Cre (<i>N = </i>5) and AAV2-LacZ (<i>N = </i>3) mice. Membrane potential of pierced ducts of <i>St14^LoxP/LoxP</i> mice (<i>N = </i>2) was used as a control for impaired ductal epithelium. (A) Local depletion of matriptase in SMG from AAV2-Cre animals resulted in a significant EP decrease compared with SMG from AAV2-LacZ control mice. The EP of AAV2-Cre mice was similar to that of pierced glands. (B) Correlation between saliva production and EP of salivary glands from AAV2-Cre mice (<i>N = </i>3) and AAV2-LacZ mice (<i>N = </i>3). Data shown are the mean ± SEM for each group. Statistical significance was determined using unpaired Student’s <i>t</i> test and Pearson’s rank correlation test, respectively. NS, <i>P = </i>0.1978, **<i>P = </i>0.0044, ***<i>P</i><0.001. (C) Changes in distribution of proteins associated with tight junctions or salivary gland function induced by the loss of matriptase were visualized by immunofluorescent staining of paraffin-embedded SMG tissue samples from <i>St14^–</i> and <i>St14⁺</i> control mice 28 to 40 weeks of age (<i>N = </i>2 both groups). Representative confocal images are shown. Apical staining of ductal cells for claudin 3 is shown by arrowheads in both <i>St14⁺</i> and <i>St14⁻</i> animals. An increase in the signal for claudin 3 was detected in the cytoplasm and basal membrane of ductal cells in <i>St14^–</i> mice compared with <i>St14⁺</i> mice, as indicated with arrow; original magnification 40X.</p