Exacerbations of respiratory diseases such as asthma and COPD are often associated with concomitant respiratory viral infections. Since TLR3 is activated by viral dsRNA, the purpose of the current study was to better understand the functional consequences of TLR3 activation in vivo. Administration of poly(I:C), a synthetic TLR3 ligand, to the lungs of mice induced marked inflammation accompanied by impaired lung function. TLR3 KO mice were partially protected from the effects of poly(I:C) demonstrating the involvement of TLR3. These data provide further support for a role of TLR3 in respiratory diseases and suggest a potential mechanisitic pathway for viral exacerbations.
Upon activation, TLR3 recruits a Toll-IL-1 receptor (TIR) – related adaptor protein inducing interferon (TRIF), which activates both IFN-regulatory factor 3 (IRF3) and NF-kB  and . In our model, following poly(I:C) administration to the lungs, there was an up regulation of TLR3, -2, -7, and 9 gene expression and their associated signaling molecules. Previous in vitro studies have demonstrated that activation of TLR3 with poly(I:C) induces up regulation of its own expression as well as the expression of other TLRs. For example, poly(I:C) up regulates mRNA for TLR2, 3 and 4 in airway smooth muscle cells  and TLR2, 3, 6 and 10 in lung epithelial cells . In vivo, the up regulation of TLR mRNA expression may have occurred as a result of expression of TLRs on infiltrating cells or through up regulation on resident lung cells. Indeed, monocytes express all of the known TLRs . In contrast, neutrophils have been shown to express all the TLRs except TLR3 . Within the lung, all of the known TLRs have been found to be expressed by human primary bronchial epithelial  and smooth muscle cells . The up regulation of multiple members of the TLR family, as a consequence of activation of one TLR, may indicate the creation of an environment of hyper-responsiveness to pathogen insult whereby, an exacerbation event could be triggered in the event that the lung is exposed to other toll-ligands. In support of this hypothesis, it has been shown that infection of airway epithelial cells with Hemophilus influenza induced the secretion of CXCL-8, up regulated TLR3 expression and increased the responsiveness to a secondary challenge of Rhinovirus. Interestingly, inhibition of TLR3 with small interfering RNA, inhibited the Rhinovirus-induced CXCL-8 production . In addition this same group demonstrated that pretreatment with Rhinovirus resulted in delayed bacterial clearance when a secondary infection was induced using nontypeable Hemophilus influenza. Sajjan et al. showed that this may be the result of decreases in transepithelial resistance or compromised tight junctions and loss of zona occludins-1 and junctional adhesion molecule-1 . Taken together these studies suggest that activation of TLRs, such as TLR3 can result in a perturbation of the local environment, specifically dysregulation of the airway epithelium thereby supporting an environment primed for an exacerbation. We are currently focusing efforts in our laboratory toward identifying the composition of the mononuclear cell populations in this model including the activation state of various cell types including dendritic cells. In a review by Fe et. al. it is summarized that TLR3 can induce a variety of cytokines in human dendritic cells including IFNβ, and CXCL10 .
In vivo TLR3 agonism by synthetic dsRNA also resulted in a profound up regulation of the expression and secretion of multiple pro-inflammatory cytokines, chemokines, and growth factors. In vitro studies have demonstrated that activation of TLR3 by dsRNA on different cell types including natural killer cells , epithelial cells [3, 21, 22], and smooth muscle cells  results in increased expression and/or secretion of pro-inflammatory cytokines including IL-6, CXCL-8, CCL-2, CCL-5, CXCL-10, GM-CSF, TNFα and IFNγ. A likely source of cytokines following poly(I:C) administration may be the airway epithelium since activation of BEAS-2B cells in vitro induced a profile of pro-inflammatory cytokines similar to that observed following in vivo poly(I:C) challenge. TLR3 has been identified and functionally characterized in mouse tracheal muscle  and in primary human small airway epithelial cells [21, 3, 22]. Previous in vitro studies have also demonstrated the secretion of inflammatory mediators following TLR3 activation of epithelial cells[3, 3, 21]. The up regulation of pro-inflammatory cytokines and chemokines provides an inflammatory milieu supporting the infiltration of inflammatory cells into the airways and lung interstitium. Accompanying the inflammation-rich pathology was the presence of bronchial epithelial cell hypertrophy. The hypertrophic cells extended into the secondary and tertiary airways. Epithelial cell hypertrophy is normally associated with increased mucus production . However, in the current study, there was no evidence for increased mucus production by PAS staining. Given the distribution of goblet cells in normal mouse airways, which is restricted to the main bronchi and primary bronchioles, the data suggest that the hypertrophic epithelial cells are not mucus-producing goblet cells.
Along with the demonstration that poly(I:C), acting as a TLR3 ligand, results in an inflammatory response in vivo, the study presents a novel finding that stimulation of TLR3 results in a measurable impairment of lung function both without provocation and characterized by increased AHR to methacholine. Similar changes in baseline lung function have also been described in mice exposed to Respiratory Syncytial virus (RSV) . Recent studies have demonstrated that pre-exposure of mouse tracheas to poly(I:C) in vitro increases the expression of bradykinin B1 and B2 receptors on the smooth muscle and confers AHR to bradykinin . Notably, inhibition of the bradykinin B1 receptor confers protection from acetylcholine-induced AHR following allergen sensitization and challenge . In contrast, AHR to histamine following parainfluenza-3 infection in guinea pigs was inhibited by a bradykinin B2 receptor antagonist . Taken together these data suggest a role for bradykinin in TLR3-induced airway dysfunction. In the current study some, but not all, functional responses were protected in TLR3 KO mice following multiple administrations of poly(I:C). Specifically, they were protected from baseline lung function changes in response to poly(I:C), however protection from AHR in response to provocation with methacholine did not result in significant protection. Further, the pro-inflammatory mediators produced following poly(I:C) administration were not modulated in TLR3 KO mice. Unpublished data from our laboratory has shown that TLR3 KO mice were significantly protected from a single administration of poly(I:C) with respect to pro-inflammatory mediators in the bronchoalveolar lavage (data not shown), indicating that mediators released in response to acute activation with poly(I:C) may be more TLR3 dependent. This data suggests that another receptor for poly(I:C) may be available. Indeed, since a percentage of TLR3 KO mice succumb to poly(I:C)-induced shock, it suggests that poly(I:C) may still signal in the absence of TLR3 . Indeed, dsRNA can also signal through dsRNA-dependent protein kinase (PKR) , RIGI  and MDA-5 . The potential redundancy in the dsRNA downstream pathways may be an explanation for the incomplete protection observed in TLR3 KO mice.
Understanding the different signaling pathways involved in recognition of dsRNA by the host has been a major area of focus by many researchers. Le Goffic et al. demonstrated that sensing of influenza A virus by TLR3 and RIG-I regulates a pro-inflammatory response. In contrast, RIG-I but not MDA-5 also mediates type I IFN-dependent antiviral signaling response. Use of non-poly(I:C) TLR3 ligands is necessary to further define the impact of TLR3-specific signaling on pulmonary pathophysiology. Interestingly, TLR3 KO mice demonstrate protection from influenza A virus-induced lung function impairment accompanied by reduced inflammation and improved survival .
These data taken along with the inflammatory consequences of TLR3 activation suggest that sustained TLR3 activation may also contribute to severe exacerbations of chronic pulmonary diseases. In summary, the data presented in this study suggest that sustained TLR3 activation may play an important role in respiratory disease pathogenesis. A better understanding of the effects of TLR3 activation will provide additional insight into the mechanisms underlying virus-induced exacerbations associated with respiratory diseases. Additionally, these studies provide an opportunity to identify suitable targets for therapeutic intervention for respiratory disease exacerbations.