Regulatory immune cytokines in RSV infection

Al Turaiki, Wael (2014) Regulatory immune cytokines in RSV infection. PhD thesis, University of Liverpool.

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Abstract

Antibody production in the lungs is an essential defence mechanism against respiratory pathogens. However, little is known about the local activation of B cells in the lung.The production of BAFF and APRIL by airway epithelial cells could contribute to local recruitment, activation, class switch recombination and antibody production by B cells in the lung. In vitro, BEAS-2B cells were used to characterize BAFF and APRIL production simulated either by RSV infection or addition of cytokines. RSV and IFN-β significantly induced expression of BAFF mRNA and protein but not APRIL. BAFF mRNA reached significantly high levels at 12h and declined at 48h after either RSV infection or IFN-β stimulation. Western blot analysis of resting epithelial cells showed that membrane BAFF was expressed by resting cells. On RSV infection or IFN-β stimulation, expression of membrane BAFF increased at 12 and 24hours and disappeared at 48h, which suggests soluble BAFF was cleaved from the membrane and released into the culture supernatant by 48h, where it was measured by ELISA. When BEAS-2B cells were infected with RSV after pre-incubation with anti-IFN β, expression of BAFF was blocked, which indicates that airway epithelial cells can produce BAFF in an interferon dependent manner. BEAS-2B cells did not express CXCL12, CXL13, CCL19 or CCL21, which indicates there are other potential sources that express these chemokines during RSV infection rather than the airway epithelium. A murine model of RSV infection was used to examine expression of BAFF, APRIL and of the chemokines CXCL12, CXCL13, CCL19 and CCL21. Cytokine mRNA and RSV N gene expression were measured by Taqman PCR in lung tissue from control mice at day 0 and mice challenged with RSV (A2 strain) or control UV-treated RSV at days 1, 2, 4, 7, 8, 10, 14 and 21 days after RSV infection by ELISA. RSV N RNA was significantly detected at day 1, 2 and 4 after RSV infection compared to UV RSV control. BAFF mRNA expression was increased significantly after RSV infection on day 1 ,7 and 8 in comparison to UV treated RSV control at the same time points. Equally, BAFF protein was also elevated significantly after RSV infection at days 1, 2, 4, 7 and 8 in comparison to UV- RSV control at the same time points. CXCL13 mRNA expression was increased significantly after RSV infection on day 1 and 7 in comparison to UV-RSV control at the same time points. Moreover, CXCL13 protein was increased significantly after RSV infection at day 1, 2 and 7 in comparison to UV RSV control at the same time points. CXCL12, CCL19 and CCL21 mRNA and protein levels were not increased significantly after RSV infection, which may indicate they are not active during RSV infection. Examination of mouse lung sections showed strong positive staining of B cells (CD20) following RSV infection at day 1, 2, 4, 7 and 8 and FACS analysis B cells numbers were increased significantly at day 6 and 8 following RSV infection relative to UV-RSV control. RSV infection results in up-regulated BAFF and CXCL13 expression, consistent with a role for CXCL13 in recruiting B cells and BAFF in promoting airway B cell survival or differentiation. Collectively, these results suggest that the airway epithelial could help recruit and support B cell growth and development and Ab production in the lung.

Item Type:	Thesis (PhD)
Subjects:	R Medicine > R Medicine (General) R Medicine > RA Public aspects of medicine
Depositing User:	Symplectic Admin
Date Deposited:	18 Aug 2015 08:25
Last Modified:	01 Aug 2018 01:30
URI:	http://livrepository.liverpool.ac.uk/id/eprint/2008039

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