Graduation Year

2016

Document Type

Thesis

Degree

Ph.D.

Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Shyam Mohapatra, Ph.D.

Committee Member

Neeraj Vij, Ph.D.

Committee Member

Burt Anderson, Ph.D.

Committee Member

Srinivas Bharadwaj, Ph.D.

Committee Member

Kenneth Ugen, Ph.D.

Keywords

Respiratory infections, Stromal cells, Immune regulation, Viral response, IDO1, IFN- β

Abstract

Respiratory syncytial virus (RSV) is the most common cause of acute lower respiratory tract infection in young children worldwide, accounting for an estimated 33.8 million cases of respiratory disease, over 3 million of which require hospitalization, and between 66,000 and 199,000 deaths in this susceptible population. Additionally, severe RSV infection early in life is associated with an increased risk of wheeze and other airway disorders later in life. Despite this, there is currently no vaccine or economically reasonable prophylactic regimen to prevent infection. While disease is typically more severe in infancy RSV can infect throughout the lifespan repeatedly as the body does not develop protective immunity during primary or subsequent infection. The mechanisms behind this incomplete immunity are unclear.

RSV has been reported to infect numerous extra-epithelial cell types. Interestingly, viral infection in human mesenchymal stem cells (MSCs) has been reported, but the consequences are poorly understood. MSCs are an immune regulatory cell population present in nearly every organ including the nasal mucosa and the lung. They play a role in regulating immune responses and mediating tissue repair. In the following studies we sought to determine whether RSV infection of MSCs enhances their immune regulatory functions and contributes to RSV-associated lung disease.

RSV was shown to replicate in human MSCs by fluorescence microscopy, plaque assay, and expression of RSV transcripts. RSV-infected MSCs showed differentially altered expression of cytokines and chemokines such as IL-1β, IL-6, IL-8 and SDF-1 compared to normal human bronchial epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (~100-fold) and indoleamine-2,3-dioxygenase (IDO) (~70-fold) compared with mock-infected MSCs. IDO was identified in cytosolic protein of infected cells by Western blots and enzymatic activity was detected by tryptophan catabolism assay. Treatment of PBMCs with culture supernatants from RSV-infected MSCs reduced their proliferation in a dose dependent manner. This effect on PBMC activation was reversed by treatment of MSCs with the IDO inhibitors 1-methyltryptophan and vitamin K3 during RSV infection.

We also demonstrated the pathway leading to IDO expression in RSV infected MSCs. Neutralizing IFN-β prevented IDO expression and activity indicating its role as a viral response factor perhaps “high jacked” by the virus in immune escape. Treatment of MSCs with an endosomal TLR, but not a RIG-I, inhibitor prevented IFN-β and IDO expression. Additionally, TLR3/dsRNA complex inhibitor was able to block IFN-β stimulation, while a TLR7/8/9 inhibitory ODN did not, suggesting that endosomal TLR3 detection of RSV dsRNA was leading to IFN-β and IDO expression. Together, these findings indicate that RSV infection of MSCs triggers their immune regulatory function via TLR3 recognition of dsRNA, upregulating IFN-β expression and IDO activity, ultimately affecting immune cell proliferation. This finding may account for the lack of protective RSV immunity and consequent repeated infections throughout one's lifetime.

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