Kiran Hina Tajuddin Juliet Spencer Abstract Methods Results Conclusions Human cytomegalovirus HCMV manipulates the immune system of its host by encoding viral proteins that imitate host cytokine and chemokine pathways The HCMV gene UL111A encodes a homolog of human interleukin10 hIL10 ID: 998595
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1. Effect of cmvIL-10 on Ubiquitin mediated C-X-C receptor 4 signaling.Kiran Hina Tajuddin, Juliet SpencerAbstractMethodsResultsConclusionsHuman cytomegalovirus (HCMV) manipulates the immune system of its host by encoding viral proteins that imitate host cytokine and chemokine pathways. The HCMV gene UL111A encodes a homolog of human interleukin-10 (hIL-10), known as cmvIL-10. cmvIL-10 has many functions, including upregulating signaling from human CXC- chemokine receptor-4 (CXCR4) signaling in response to its known ligand CXCL12. cmvIL-10 promotes increased intracellular calcium flux and cell migration in response to CXCL12/CXCR4 that could assist in viral dissemination. Recent studies have shown that extracellular ubiquitin (UB) is also a natural ligand for CXCR4. This study will focus on whether cmvIL-10 also impacts UB mediated CXCR4 signaling and will examine the effects of cmvIl-10 on UB induced cell proliferation, migration, and cell signaling pathways. This research will lead to identify the possible role of cmvIL-10 in manipulating host cell signaling pathways during virus infectionIt was observed that there is no significant difference in the migratory potential of both cell lines. This data may indicate that despite physiological differences, the NuFF(fibroblast) and ARPE-19 (epithelial) have almost identical migration rates with complete media (DMEM +10%FBS) The decreasing rate of cell growth in serum-free media indicates that added nutrients (FBS) are essential for normal cell growth. Migration Assay (wound heal assay)Department of BiologyIntroductionResultsFuture DirectionsReferencesAcknowledgementsFig 3: The effect of cmvIL-10 on UB/CXCR4 mediated cell proliferation: To determine the effects of cmvIL-10 on UB mediated CXCR4 and its potential impacts on cell proliferation, an equal number of HEK 293 cells (1 x 10^4) will be plated in triplicates on four 96-well plates labeled as 0 (control), 24 hours, 48 hours and 72 hours. The cells will be treated with UB (10μg/ml) with +/- cmvIL-10 and in the presence and absence of fetal bovine serum (FBS).. The cells will then be incubated with CellTiter-Glo to measure total ATP content, and luminescent signals will be recorded at different time points. The statistical analysis of data will be performed by applying analysis of variance (ANOVA) and the two-sample t-test. Proliferation AssayFig 2: The effect of cmvIL-10 on UB/CXCR4 mediated cell migration: The wound-healing assay will be performed to study the effect of cmvIL-10 on UB/CXCR4 mediated cell migration by using HEK-293 cells. An equal amount of (7.5 x 10^4) cells will be incubated on the four-chamber slide with +/- UB in presence and absence of cmvIL-10. The assay will be initiated by scratching the cell monolayer by sterile plastic 200 μl pipette tip or using the Incucyte wound maker. The scratch will be monitored at different time points to observe collective two-dimensional cell migration (sheet migration)..Fig 1: Depiction of cmvIL-10 mediated CXCL12/CXCR4 signaling in monocyte.The viral cytokine cmvIL-10 (dark purple) binds as a dimer to IL-10R1/2 and phosphorylates STAT3 pathway. STA3 homodimerizes and translocate to the nucleus and stimulates transcription of immune suppressors. CmvIL-10 enhances CXCL12/CXCR4 (orange) mediated calcium mobilization and chemotaxis. Infected cells secreted cmvIL-10, which can increase CXCL12/CXCR4 signaling in infected cells (autocrine signaling) but can also act in a paracrine manner on other bystander cells In addition to CXCL12, CXCR4 also interacts with Ubiquitin (UB) (yellow) and UB can serve as an alternative ligand to CXCR4.Fig 4: To determine the migratory potential of NuFF and ARPE-19 cells: The wound closure was observed for NuFF and ARPE-19 cell lines at 0, 8 and 24 hours. The comparative analysis of wound closure in both cell lines indicates that the wound closure rate for both cell lines is roughly similar (%wound area recovered in 24 hour; NuFF 77, ARPE-19 66; NuFF P = 0.03, ARPE-19 P = 0.07 ; n=3). At 48 hours, the wound area was fully recovered in both cell linesFig 5: To determine the cell proliferation rate in presence and absence of fetal bovine serum (FBS): The viability of the HEK 293 cells and rate of proliferation was measured by incubating cells with CellTiter-Glo. The luminescence (RLU) signals were observed for cells incubated in complete and serum starved media. The higher RLU signals were observed in cells incubated with complete media (+10%FBS). Whereas the cells with 24hrs and 48 hrs serum starvation (SS) showed 12 and 22 precent respectively lower proliferation rates.P < 0.05 values by paired t-test.P <0.05considered significant, ***p<0.001 Migration assay (wound heal assay)0hr8hrs24hrsNuFFARPE-19The wound closure was observed for NuFF and ARPE-19 cells at 0 (control), 8 and 24 hours. The images were analyze using imagej analysis. The specific goal of this project is to determine the effects of cmvIL-10 on UB/CXCR4 mediated cell proliferation and chemotaxis. These results will serve as preliminary data to establish controls for the future experiments. The study will lead to a better understanding of the mechanisms by which HCMV manipulates host chemokine signaling pathways to affect cell proliferation and movement that benefit virus persistence. Tu CC, Arnolds KL, O'Connor CM, Spencer JV. 2018. Human Cytomegalovirus UL111A and US27 Gene Products Enhance the CXCL12/CXCR4 Signaling Axis via Distinct Mechanisms. J Virol 92Busillo JM, Benovic JL. 2007. Regulation of CXCR4 signaling. Biochim Biophys Acta 1768:952-63.Saini V, Marchese A, Majetschak M. 2010. CXC chemokine receptor 4 is a cell surface receptor for extracellular ubiquitin. J Biol Chem 285:15566-76.Scofield SLC, Daniels CR, Dalal S, Millard JA, Singh M, Singh K. Extracellular ubiquitin modulates cardiac fibroblast phenotype and function via its interaction with CXCR4. Life Sci. 2018 Oct 15;211:8-16. Steagall RJ, Daniels CR, Dalal S, Joyner WL, Singh M, Singh K. 2014. Extracellular ubiquitin increases expression of angiogenic molecules and stimulates angiogenesis in cardiac microvascular endothelial cells. Microcirculation 21:324-32.***Studies have shown conflicting results about the effect of UB/CXCR4 signaling on cellular functions. For instance, a study has shown that UB/CXCR4 signaling significantly inhibits the migratory potential of fibroblast towards wounds (Steagall RJ, 2014). However, another study has also shown that UB/CXCR4 interaction will enhance the migration into the wound (Jackson 2018), which contradicts previous findings. Therefore, we hypothesize that cmvIL-10 will enhance either inhibitory or stimulatory effects of UB/CXCR4 mediated cell migration.