In this study, we demonstrate for the first time that RV infection delays bacterial clearance in vivo. Delayed bacterial clearance was accompanied by attenuated expression of the neutrophil-attracting chemokines KC/CXCL1 and MIP-2/CXCL2. We also show that, despite increasing bacterial binding, RV suppresses chemokine responses to subsequent NTHi infection in cultured bronchial epithelial cells. RV infection also attenuated TLR2- and TLR5-stimulated chemokine responses in cultured bronchial epithelial cells and alveolar macrophages, suggesting that RV infection interferes with TLR-dependent innate immune defenses. Most importantly, we define a mechanism by which RV infection desensitizes TLR signaling in both bronchial epithelial cells and alveolar macrophages. We demonstrate that RV infection causes degradation of IRAK-1, a key adaptor protein in MyD88-dependent TLR signaling, thereby suppressing chemokine responses to subsequent NTHi challenge. Further, we show that IRAK-1 degradation was dependent on TLR2, but not on other TLRs which activate MyD88-dependent signaling. Based on these observations, we speculate that suppression of TLR-dependent innate immune responses increases the risk of acquiring secondary bacterial infection and promotes invasiveness of bacterial flora that is present in the nasopharynx/lower airways of CF and COPD patients.
Although there is clinical evidence indicating that RV infection precedes bacterial infection-associated disease exacerbations 
, until now there were no experimental data demonstrating an increased risk of bacterial infection following RV infection in vivo
. Using a novel mouse model of RV infection that we developed 
, we found that RV infection promotes NTHi persistence in the lungs, likely by attenuating KC and MIP-2 expression and impairing recruitment of neutrophils to the airways. Appropriate neutrophil recruitment is required for bacterial clearance, particularly under circumstances that exceed the antimicrobial capacity of airway epithelial cells and alveolar macrophages. Mice with a delayed neutrophil response fail to clear P. aeruginosa
efficiently from their lungs 
. Recently, influenza virus was also shown to suppress KC and MIP-2 responses to subsequent bacterial infection; suppression was dependent on virus-stimulated type I interferons 
. Since RV infection also stimulates type I interferon responses both in vivo
and in airway epithelial cells in vitro
, it is plausible that RV suppresses chemokine responses via this mechanism. However, this is unlikely, because replication-deficient UV-irradiated RV, which does not stimulate expression of type I interferons, also attenuated chemokine response to NTHi infection in vitro
Both bronchial epithelial cells and alveolar macrophages express TLRs. TLRs play a crucial role in the establishment of appropriate innate immune responses, including expression of chemokines, upon recognition of pathogen associated molecular patterns. Previously, RV infection was shown to suppress subsequent cytokine responses to LPS, a TLR4 ligand in human alveolar macrophages 
. Suppression was dependent on viral replication. In contrast, we found that attenuation of TLR-dependent signaling in both bronchial epithelial cells and alveolar macrophages was independent of viral replication. These data suggest that suppression is initiated by an early event in the viral life cycle, for example, binding or endocytosis. This is not surprising, because RV binding itself has been shown to activate signaling mechanisms leading to phosphatidylinositol 3-kinase activation, IL-8 expression and generation of reactive oxygen species 
In the present study, we found that RV infection not only attenuated chemokine expression due to subsequent challenge with NTHi infection, but also to TLR2 and TLR5 ligands. NTHi stimulates TLR2 signaling leading to pro-inflammatory cytokine expression 
. In addition, both TLR2 and TLR5 initiate a MyD88-dependent signaling pathway leading to chemokine and pro-inflammatory cytokine expression. We therefore hypothesized that RV attenuates MyD88-dependent TLR signaling. Accordingly, we found that RV infection significantly increased IRAK-M while dramatically reducing the levels of IRAK-1 protein in both airway epithelial cells and murine alveolar macrophages. IRAK-M is thought to be a negative regulator of MyD88-dependent TLR signaling 
. However, genetic silencing of IRAK-M did not restore NTHi-induced chemokine expression in RV-infected airway epithelial cells, suggesting that IRAK-M does not contribute to RV-induced attenuation of IL-8 expression. This is not entirely unexpected, as IRAK-M has been demonstrated to play a vital role in the suppression of a non-canonical TLR signaling pathway that relies on activation of NF-κB-inducing kinase rather than IκB kinase 
In contrast, blocking of RV-induced reduction in IRAK-1 by using a proteasomal inhibitor restored NTHi-induced IL-8 responses in RV-infected bronchial epithelial cells, indicating a role for IRAK-1. Previously, it has been shown that post-translational modification of IRAK-1, such as hyperphosphorylation and ubiquitination that occurs following MyD88-dependent TLR or IL-1 signaling, leads to IRAK-1 degradation 
. IRAK-1 degradation constitutes a mechanism for limiting exaggerated innate immune responses. In the present study, we found that, in vitro
, IRAK-1 is degraded as early as 4 h post-RV infection and the IRAK-1 protein levels remain low for at least 24 h. Pre-treatment with lactacystin prevented reductions in IRAK-1 protein abundance, suggesting that RV promotes proteasomal degradation of IRAK-1. Reduced IRAK-1 was also observed in mice infected with RV 48 h post-infection. These observations suggest that RV induces IRAK-1 degradation via the activation of MyD88-dependent IL-1/TLR signaling cascades. However, since lactacystin can also inhibit proteasomal degradation of other proteins of TLR pathway, further studies using IRAK-1 mutants which are not amenable to ubiquitation and proteasomal degradation are necessary to further confirm the specific role of IRAK-1 in RV-induced suppression of NTHi-stimulated chemokine responses.
Recently, RV infection of BEAS-2B cells was shown to elicit low levels of IL-1β release, which in turn induced MyD88-dependent potentiation of IL-8 production 
. However, in the present study, inhibition of IL-1 receptor by IL-1ra neither blocked RV-induced degradation of IRAK-1 nor reversed suppression of NTHi-stimulated IL-8, suggesting that the IRAK-1 degradation that we observed is not due to RV-induced IL-1β signaling.
RV single-stranded RNA may activate MyD88-dependent TLR signaling directly via TLR7 and TLR8. In our studies we found that airway epithelial cells do not express TLR7 and express TLR8 at very low levels. Further, knockdown of TLR8 neither prevented RV-induced degradation of IRAK-1 nor reversed suppressive effects of RV on NTHi-stimulated IL-8. Similar to our results, recently it TLR7/8 agonist, imiquimod was found to be inefficient in activating airway epithelial cells suggesting that these cells may not express TLR7 or 8 
. On the other hand, it has been demonstrated that both intact and UV-irradiated RV-6 stimulate NF-κB transactivation in TLR2-transfected HEK-293 cells 
. Since we observed that both replication-competent and replication-deficient RV were equally capable of causing IRAK-1 degradation, we posit that TLR2 plays a role in RV-induced IRAK-1 degradation. Consistent with this notion, we found that knockdown of TLR2 significantly reduced RV-induced IRAK-1. However, we could not determine the effect of TLR2 knockdown on suppression of NTHi-stimulated IL-8 in RV-infected cells, because NTHi also requires TLR2 to stimulate IL-8 
. Also, the precise mechanism by which RV activates TLR2 remains to be elucidated.
In summary, we have shown that RV suppresses NTHi-induced IL-8 expression in airway epithelial cells and alveolar macrophages by inducing TLR2-dependent degradation of IRAK-1. Our results suggest that RV may increase the risk of acquiring secondary bacterial infection by attenuating TLR-dependent innate immune responses.