osteopontin plays a pivotal role in in increasing severity of respiratory syncytial virus infection

The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling has a pivotal role in the spread of RSV infection

Respiratory Syncytial Virus-Infected Mesenchymal Stem Cells Regulate Immunity via Interferon Beta and Indoleamine-2,3-Dioxygenase.

Respiratory syncytial virus (RSV) has been reported to infect human mesenchymal stem cells (MSCs) but the consequences are poorly understood. MSCs are present in nearly every organ including the nasal mucosa and the lung and play a role in regulating immune responses and mediating tissue repair. 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, IL6, IL-8 and SDF-1 compared to epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (~100-fold) and indoleamine-2,3-dioxygenase (IDO) (~70-fold) than in 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, a result we confirmed by CRISPR/Cas9-mediated knockout of IDO in MSCs. Neutralizing IFN-β prevented IDO expression and activity. Treatment of MSCs with an endosomal TLR inhibitor, as well as a specific inhibitor of the TLR3/dsRNA complex, prevented IFN-β and IDO expression. Together, these results suggest that RSV infection of MSCs alters their immune regulatory function by upregulating IFN-β and IDO, affecting immune cell proliferation, which may account for the lack of protective RSV immunity and for chronicity of RSV-associated lung diseases such as asthma and COPD

Osteopontin plays a pivotal role in increasing severity of respiratory syncytial virus infection

<div><p>The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling may play a pivotal role in the spread of RSV infection.</p></div

OPN is a marker of high RSV loads in HEp-2 cells.

<p>HEp-2 cells were mock-infected or infected with 1 MOI of RSV-L19F or rgRSV-A2. RNA, supernatants and protein were isolated 24 and 48 hpi. (A) Plaque titers were obtained from supernatants of HEp-2 cells infected with RSV-L19F or rgRSV-A2. (B—D) Expression of RSV-N, IL-1β and IFN-β were determined by qPCR. Results are presented as fold-change in expression of RSV-N, IL-1β or IFN-β mRNA normalized to the control (HPRT). (E) Western blot analysis of OPN expression. 25μg of protein lysate was loaded in each lane. OPN is seen at 55 kDa and β-actin (loading control) at 42 kDa. Experiments were performed in triplicate. * p < 0.05, ** p < 0.01, **** p < 0.0001.</p

RSV replication is diminished in mice lacking OPN (OPN KO).

<p>C57BL/6 and OPN KO mice were mock-infected or infected with 3 x 10⁶ RSV-L19F. Lungs were collected 1, 3 and 5 dpi for protein and RNA extraction. (A) RSV-N mRNA expression levels were determined by qPCR and results represented as fold-change in expression of RSV-N mRNA normalized to the control (HPRT). (B and C) Levels of OPN and IL-1β in lung homogenates were determined by ELISA. BD: Below detection limits. Lung homogenates were obtained from individual mice and not pooled (n>4 per group). ** p < 0.01, **** p < 0.0001.</p

Neutralization of CD44 receptor reduces the number of RSV positive cells.

<p>Hep-2 cells were pre-treated with 10 μg of broad spectrum rat-anti human CD44 antibody (clone A020) or normal rat IgG (control). After the pre-treatment, cells were infected with RSV-KL19F (0.5 MOI) and infectious media was replaced with fresh growth media. After 24 hours, the percentage of RSV positive cells was determined by flow cytometry. Result of a representative experiment is shown. Experiments were performed in triplicate.</p

IL-1β and OPN mRNA expression in RSV infected cells.

<p>HEp-2 and HEK-293 cells were mock infected or infected with 1 MOI of RSV-L19F. RNA was isolated at 24, 48 and 72 hpi. (A-C) Expression levels of RSV-N, IL-1β and OPN were determined by qPCR. Results are presented as fold-change in expression of RSV-N, IL-1β or OPN mRNA normalized to the control (HPRT). qPCR data are represented as means ±SEM. Experiments were performed in triplicate. ** p < 0.01, *** p < 0.001**** p < 0.0001.</p

RSV infection induces OPN expression in a dose dependent manner.

<p>HEp-2 cells were mock-infected or infected with RSV-L19F (1 MOI). (A) Images of Hep-2 cells stained with polyclonal antibody against RSV and OPN at 48 hpi. Representative images of RSV positive (red) cells, OPN positive (green) cells, and DAPI (blue) nuclear staining at 200X magnification. (B) RSV titer by plaque assay of supernatants for increasing doses (0.1, 1 and 10 MOI) of UV-inactivated and native RSV-L19F at 48 hpi. (C) OPN protein (55 kDa) expression in HEp-2 cells at 48 hpi after mock, RSV and UV-inactivated RSV treatment in the indicated doses analyzed by western blots. β-actin (loading control) at 42 kDa was probed as the loading control.</p

CD44 expression mediates RSV infection.

<p>HEp-2 or HEK-293 cells were infected with 0.5 MOI of RSV-KL19F (red). After 24 hours, the cells were stained with FITC mouse anti-human CD44 antibody (green) and gated for RSV expression (red). (Top panel) Flow cytometry analysis of HEp-2 cells infected with RSV-KL19F. (Bottom panel) Flow cytometry analysis of HEK-293 cells infected with RSV-KL19F. Result of a representative experiment is shown. Experiments were performed in triplicate.</p

OPN expression is a marker of high RSV loads <i>in vivo</i>.

<p>(A) C57BL/6 mice were mock-infected or infected with 1 or 3 x 10⁶ RSV-L19F. (B) C57BL/6 mice were mock-infected or infected with 3 x 10⁶ RSV-L19F or rgRSV-A2. (A-B) Lungs were collected 3 dpi for protein isolation and levels of OPN in lung homogenates were determined by ELISA. Lung homogenates were obtained from individual mice and not pooled (n>4 per group). * p < 0.05, ** p < 0.01, **** p < 0.0001.</p