Preliminary Study On The Mechanism Of Cytomegalovirus Latent Infection - Reactivation In The Course Of Ulcerative Colitis

Background and aim:Human cytomegalovirus (HCMV) is one of ubiquitous beta-herpes virus that infects immunocompetent individuals leading to more invasive disease with higher mortality and morbidity. Therefore, it has been concerned by experts in the fields of microbiology and infection.In the past few years, more and more gastroenterologists have paid attention to this interdisciplinary field, since CMV infection is associated with flare-up and steroid resistance in ulcerative colitis (UC) patients. It is known the incidence of UC is increasing in China. Glucocorticoid (GC) is commonly used in moderate and severe UC patients, which provides opportunitic conditions for CMV infection or reactivation. A recent prospective study showed that CMV infection was associated with steroid-refractory and disease exacerbations in ulcerative colitis (UC). But also, steroid resistance can be reversed after antiviral therapy. However, there are some problems not solved yet, including the influence of CMV in UC disease course and the choice timing of treatment. In this study, the aim is putting forward the clinical clues by basic studies. This study is divided into 3 parts:1. Establishing a refined cell model of CMV latency and reactivation.2. Exploring the mechanisms of steroid-resistant and its impact on UC during CMV latency and activation in cell model. We also study the impact of UV-inactivated CMV on glucocorticoid resistance and cytokines secretion.3. Finding out the alteration of microRNAs (miRNAs) and its signaling pathways in cell model and UC patients before and after anti-viral therapy.Part I The establishment of CMV latency and reactivation model in vitroMethods:CMV was propagated in the human embryonic fibroblast cell line MRC-5. Differentiation of THP-1 cells derived macrophages was performed by stimulation with phorbol 12-myristate 13-acetate (PMA) for 3 days. To establish viral latency in vitro, THP-1 cells were infected in suspension and infected cells were cultured for different time. To model reactivation, we compared latent CMV in THP-1 with differentiation and CMV infection THP-1 cells derived macrophages. Model was detected by observing cell morphology, qRT-PCR quantifying viral DNA in single cell and RT-PCR detecting viral immediate early and latency related genes.Results:THP-1 monocyte cell line grew on suspension after CMV infection. Three days later, CMV viral load was stable. Virus latency associated gene UL82 expressed after infection while immediate early 72 (IE72) gene was not detected. All of these demonstrated CMV was in latency. THP-1 monocyte cell line harboring latency CMV became neither adherent after differentiation without increasing viral load nor the expression of IE72. However, viral load increased and IE72 expressed after CMV infected THP-1 derived macrophages. This suggested a reactivation model. The expression of IE72 was remarkable 3 days after infection.Part II The mechanisms of CMV infection in steroid-refractory UC and its effect on inflammationMethods:(1) Subgroup:On the basis of latency and reactivation cell model in vitro, THP-1 cells was divided into 4 groups:control group; differentiation of THP-1 with PMA (PMA group); cytomegalovirus latency infection group (CMV group); and CMV reactivation model (PMA+CMV group). (2) The mRNA of GRβand GRα and the ratio of GRβ/α was detected by real-time PCR. The protein expression of GR and phosphorylation GRa was detected by Western blotting. Pro-inflammatory and anti-inflammatory cytokines in each group were detected by Multi-Factor ELISA. (3) After UV-inactivated CMV infection, the mRNA and protein of GR were also detected. The concentration of cytokines was detected by Multi-Factor ELISA.Results:The expression of GR was slightly elevated in latency while increased significantly in reactivation. The ratio of GRβ/α was normal in latency model and increased in lytic infection. The increasing of GRβ/α in reactivation may explain steroid refractory. IL-10 elevated (control 0.72±0.12 pg/ml vs latency 0.87±0.21 pg/ml, P=0.036) and IL-5 decreased( MFI:control 48.7±3.2 vs latency 43.6±2.6, P=0.02) slightly in latency model. During lytic infection, Pro-inflammatory cytokines IL-6 (control 1.22±0.02 pg/ml vs lytic 4877.13±31.51 pg/ml,p＜0.001) and TNF-α(control 1.35±0.15 pg/ml vs lytic 1479.88±29.8 pg/ml, P＜0.001) increased remarkably and anti-inflammatory cytokine IL-5 decrease (MFI:control 48.7±3.2 vs lytic 35.6±3.7, p=0.016) may exacerbate UC. IL-10 increased both in latency and reactivation. None of GR and cytokines changed in mock-infection group.Part III Comprehensive analysis of microRNA expression in cell model and UC patients before and after anti-CMV therapyMethods:(1) Cell model subgroup:CMV and UV-inactivated latency and mimic reactivation model in vitro CMV infection. (2) Patients:blood cells from three UC patients with cytomegalovirus infection before and after anti-virus therapy. Total RNA was isolated using the Trizol reagent and its integrity and purity was detected suitable for miRNA array analysis. The dynamic miRNA expression profiling was established and the dysregulated miRNAs were analysed by bioinformatics methods and enrichment analysis.Results:The dynamic miRNA expression profiling was established. There were 536 differentially expressing genes in latency and reactivation model in vitro.509 differentially expressing miRNAs existed in mocked-latency and mocked-reactivation models. Besides,96 genes changed in reactivation and mock-reactivation model. These differentially expressed genes mainly related to inflammation, cell adhesion and differentiation as well as nuclear protein related genes by bioinformatics analysis.Prior to and post antiviral therapy, hsa-miR-199a-5p, hsa-miR-4419b and hsa-miR-642a-3p wereup-regulated. These miRNA can regulate PMA-induced proteins, DNA-binding protein, G protein coupled receptor and transforming growth factor-related protein.Hsa-miR-4419b increased in both reactivate cell model and UC patients with CMV infection. Researches on hsa-miR-4419b function may help reveal the role of CMV infection in UC patients and mechanisms of glucocorticorid resistance. Different cell signaling pathways in cell model and UC patients with CMV infection, can provide direction in further study.Conclusions:We established CMV latency and lytic model successfully. Change in number and function of GR may account for glucocorticoid resistance. The imbalance of pro-and anti-inflammatory cytokines may cause steroid resistance and worsened mocosal inflammation. Hsa-miR-4419b increased in both cell model and UC patients. Research on hsa-miR-4419b function may help reveal the role of CMV infection in UC and mechanisms of glucocorticorid resistance.