Immunopathological aspects of human cytomegalovirus in chronic disease

Background Human cytomegalovirus (HCMV) is a ubiquitous, ancient virus that has co- evolved with its host through the ages. As such, it has developed a host of ways to evade eradication by our immune system. This has resulted in a sort of equilibrium in the immunocompetent host -where the infection dwells asymptomatically for the lifetime of the host, but once the equilibrium is disturbed by immune activation or heavy immunosuppression, HCMV can be detrimental to the health of the infected individual. While these are well-known facts, less is known about the characterisation of HCMV infection in individuals with minor immunodeficiencies, or those with a low-grade immune activation such as chronic inflammation. Whether HCMV infection is activated in these cases, and what its role may be in driving pathological processes in autoimmune diseases, cardiovascular diseases, the bile duct injury seen in neonatal cholestasis, especially biliary atresia, and cancer, have been subject to my studies. The thesis consists of six studies covering the above-mentioned topics. Methods In Study I, we determined presence of HCMV proteins immediate early antigen (IEA) and pp65, and DNA in peripheral mononuclear leukocytes from synovial fluid and peripheral blood, respectively, from 34 patients with rheumatoid arthritis (RA) using immunofluorescence (IF) and real-time PCR. In Study II, we determined presence of HCMV proteins in 28 salivary gland tissue samples and HCMV antibodies in 28 serum samples, respectively, from patients with Sjögren's disease (SD) using immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA), compared to patients with sicca symptoms without SD. In Study III, we investigated correlations between HCMV IgG serostatus and titres with a panel of metabolic and pro-inflammatory biomarkers as well as ApoB100 autoantibodies, in 324 patients with prior myocardial infarction. In Study IV, we determined the presence of HCMV proteins and DNA using IHC, in situ hybridisation (ISH) and real-time PCR, in 24 patients with biliary atresia. In Study V, we studied the presence of HCMV-induced CD13 autoantibodies, known to inhibit the differentiation from monocytes to macrophages, in 33 serum samples from mothers of children with neonatal cholestasis, using fluorescence activated cell sorting (FACS) after incubating the serum samples with CD13-expressing mouse cells. Serum samples from mothers of healthy full-term infants and blood donors were used as controls. We also tested if maternal sera could inhibit monocyte-macrophage differentiation, and whether that was dependent on CD13 autoantibodies. In Study VI, we studied the impact of HCMV infection on ARG2 expression in three different malignant glioblastoma (GBM) cell lines by real-time PCR of cDNA synthesised from RNA isolated from the infected cells. We investigated the role of HCMV in ARG2 expression by measuring the effect of short interfering RNA (siRNA) on HCMV IE72 and IE86, and determined the effect of silencing ARG2 in GBM cell line U-251 MG. We also assayed the expression and activity of MMP-2 and MMP-9 upon infection with HCMV with qPCR and ELISA, and investigated how this was impacted by the silencing of ARG2. We transfected one of the cell lines to stably overexpress ARG2 and measured the effects of ARG2 overexpression on proliferation, migration, invasion and vasculogenesis, using MTT cell proliferation assay, in vitro wound healing assay, migration and invasion assay and tube-forming assay. Finally, we determined the presence of ARG2 in GBM tissue samples by IHC, compared to samples from normal aging brains. ARG2 expression in GBM was also investigated with in silico analysis of gene expression data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets, linking the expression to survival data. Results and Discussion In Study I, HCMV tegument protein pp65 was prevalent in 65% PMNL samples from synovial fluid, compared to 18% peripheral blood, in RA patients. HCMV-IE and -pp150 DNA was detected in similar proportions of RA patients and patients with other types of arthritis. HCMV-IgG seropositivity was 75%-76% in RA patients, arthritis control group, and healthy controls. The results add to evidence that active HCMV infection is present at the site of inflammation. In Study II, we found that HCMV antigens IEA, pp65 and LA were highly abundant in tissue specimens from SD patients, who also had higher HCMV IgM prevalence compared to controls. The level of positive staining for each antigen was differentiated in four tiers. The SD group scored higher levels of positive staining compared to the sicca control group for all tested antigens: HCMV-IE (p=0.016), -LA (p=0.01), -pp65 (p=0.032). There was no correlation between presence of HCMV antigens in the salivary glands and autoantibody occurrence or clinical symptoms. In the SD group, HCMV-IgG seropositivity was 75%, while HCMV-IgM positivity was 32.1%, which was higher than the control groups. HCMV-IgG titres were significantly higher in SD patients, compared to healthy controls (p