Despite the frequency of ocular symptoms following human infection with H7 viruses, the properties which confer the ability of this influenza virus subtype to use the eye as a portal of entry are not known. As previous studies have shown that viruses of multiple subtypes are capable of binding to and replicating in ocular tissue, we chose to examine the early induction of innate immune responses elicited by influenza viruses of multiple subtypes in human ocular cells to determine whether H7 subtype-specific host responses contribute to the ocular tropism of this virus subtype. Unlike viruses of the H5N1 or H1N1 subtype, we identified an HPAI H7N7 virus which elicited elevated levels of IL-1β and upregulated genes associated with NF-κB signal transduction following infection of ocular but not respiratory cells, representing the first in vitro evidence of an H7 subtype-specific and tissue-specific response in ocular tissue.
The development of conjunctival symptoms following influenza virus exposure in humans has been documented following both laboratory exposures (e.g., by liquid containing influenza virus being splashed on a laboratorian's face or infected animals sneezing in the face and eye of a laboratorian) and occupational exposures (e.g., by direct ocular exposure to infected poultry or eye abrasions from fomites during culling operations) (5
). Both corneal and conjunctival epithelial cells express glycoconjugates containing terminal sialic acids on their surfaces, and it is credible that virus comes into contact with both tissue surfaces during an ocular exposure (8
). Furthermore, both corneal and conjunctival epithelial cells are exposed to the lacrimal drainage system in the human eye, which governs drainage from tear ducts to nasal passages (40
). As such, both cell types are likely candidates to be exposed to virus and/or support virus replication during the course of an ocular infection. While cell types located in the interior of the eye would be expected to be less accessible to influenza virus during initial environmental exposure than surface epithelial cells, their ability to support high-titer influenza virus replication as shown for retinal pigment epithelial cells (36
) and trabecular meshwork cells () underscores the diversity of cell types in ocular tissue which are potential sources of infection.
While similar kinetics of virus infection and replication were observed for both ocular epithelial cell types, HCEpiC cultures secreted 10-fold-higher levels of select cytokines and chemokines following virus infection than HConEC cultures (), indicating that virus infection of these cell types can elicit differential host responses. This finding is of particular importance when assessing the permissiveness of ocular cells to 2009 pandemic viruses. Documented instances of ocular symptoms following exposure to seasonal or 2009 H1N1 viruses are rare, suggesting that these viruses are poorly suited to use the eye as a portal of entry (19
). In support of this, we demonstrated previously that seasonal and 2009 H1N1 viruses do not readily infect mice when inoculated by the ocular route and do not replicate to high titer in human corneal cells (7
). However, a recent publication demonstrated that a 2009 H1N1 pandemic virus, but not a seasonal H1N1 virus, was capable of high-titer replication in human conjunctival tissue (13
). Interestingly, our study found that the 2009 H1N1 virus Mex/4482 replicated to titers up to 100-fold higher than those of the seasonal virus Brisbane by 72 h p.i. in HConEC cultures but not in other ocular cell types examined (). Additionally, infection with Mex/4482 virus was capable of producing elevated levels of TNF-α and IP-10 in human conjunctival but not corneal cell cultures (). The finding of differences in the magnitudes of certain host responses following influenza virus infection in general and the potential preference of particular viruses for select ocular cells underscore the necessity of studying numerous ocular cell types when evaluating the ocular tropism of influenza viruses. Further study is needed to ascertain what properties of 2009 H1N1 viruses facilitate this apparent enhanced replication and proinflammatory cytokine and chemokine production in conjunctival but not corneal cells.
The efficient replication in all ocular cell types of HPAI viruses bearing a lysine at position 627 in PB2 suggests that this residue confers a replication advantage in both respiratory and ocular cells, as similar patterns of replication with HPAI H7N7 viruses bearing a lysine at this position have been observed in both murine corneal epithelial sheets and human bronchial and lung cells (8
). The abundant expression of α2-3-linked sialic acids on the surfaces of the primary ocular cell types tested here may have further facilitated high-titer replication of viruses with an avian receptor binding preference, including subtypes such as H5N1, which are only infrequently associated with ocular disease (4
). Despite their high sequence homology, the HPAI H7N3 virus Can/504 replicated to titers >1,000-fold higher than those of the LPAI Can/444 virus in HCEpiC and HTMC cultures, and >100-fold higher in HConEC cultures, by 48 h p.i., indicating the presence of H7 virus molecular determinants of ocular replication that are independent of position 627 () (24
). Heightened levels of apoptosis and necrosis following infection of HCEpiC cultures with the LPAI viruses Can/444 and NY/107 compared with the HPAI viruses Can/504 and NL/230 highlight a distinct heterogeneity in the behavior of H7 subtype viruses in ocular cells and demonstrate that influenza viruses can elicit pronounced host responses in this tissue in the absence of high-titer virus replication. Interestingly, the attenuated proliferation and induction of nucleosomes detected following infection of ocular cell types with NY/107 virus were also observed in human respiratory cell types; this H7N2 virus possesses receptor binding and transmissibility properties distinct from those of other North American H7 viruses, and further study is warranted to better understand the atypical behavior of this strain (4
Direct comparisons between virus infection of respiratory and ocular cells allow for greater identification of features specific to one tissue type and can provide clues regarding the tropism of individual viruses for one cell type over another. Despite different epidemiologic profiles of human infection, the infectivity and replication kinetics of HPAI H5N1 and H7N7 viruses are consistent between both ocular and respiratory cells (9
). The attenuated production of proinflammatory cytokines and chemokines following HPAI H7N7 virus infection is further maintained in both tissue types. While HPAI H7 and H5 viruses are detrimental to cell viability in both ocular and respiratory cells, we have shown that subtype-specific differences affect levels of necrosis but not apoptosis in Calu-3 cells, while more pronounced subtype-specific differences in apoptosis but not necrosis predominate in HCEpiC cultures (B and C) (9
). Notably, the production of IL-1β in HCEpiC cultures following NL/219 virus infection is not observed in Calu-3 cells; this respiratory cell type does not produce substantial levels of IL-1β following influenza virus infection (data not shown). Previous studies have similarly found increased mRNA expression of IL-1β in alveolar and monocyte-derived macrophages, but not primary human bronchial epithelial cells, following lung injury or influenza virus infection (14
). IL-1β, a cytokine involved in inflammatory responses, cell growth, and tissue repair, is frequently associated with ocular disease processes (10
). Similar to our findings, a previous study found that RSV infection of corneal epithelial cells resulted in an upregulation of secreted IL-1β cytokine without a substantial change in cellular IL-1β mRNA levels (10
Previous studies have demonstrated an association between dysregulated host innate immune responses and ocular surface inflammation; in particular, many of the documented effects of IL-1β in ocular inflammation and pathology occur in an NF-κB-dependent manner (2
). The heightened expression of genes associated with NF-κB signal transduction following NL/219 virus infection (relative to other virus subtypes) in HCEpiC and the corresponding decreased expression of these genes following infection of Calu-3 cells mirror the tropism for ocular and not respiratory disease observed following H7 virus infection in humans. In addition to heightened expression of NF-κB-related genes, HCEpiC cultures infected with NL/219 virus possessed levels of cytokine mRNA transcript comparable to those in H5N1 virus-infected cells, in contrast to the delayed and attenuated levels of cytokine mRNA transcript detected following HPAI H7N7 virus infection in respiratory Calu-3 cells, with reduced expression of NF-κB-related genes (9
). However, divergent patterns of NF-κB-related gene activation following infection of HCEpiC cultures with NL/219 and Can/504 viruses indicate that other, yet-unknown host factors likely contribute toward ocular tropism. Ongoing research on potential virus- and strain-specific posttranscriptional regulation of cytokine production to ascertain the disassociation of mRNA and cytokine secretion into the supernatant (notably with regard to TNF-α) will allow for a better understanding of innate immune responses in ocular tissue following influenza virus infection. Furthermore, additional study is needed to explore the potential correlation and implications of heightened transcriptional activity of cytokine and NF-κB-related genes in ocular cells following H7 virus infection. Augmented NF-κB signal transduction in human corneal cells following H7 virus infection identifies a prospective target for immunomodulatory agents to mitigate conjunctival symptoms following H7 virus infection in humans (10
Understanding the properties which govern the ocular tropism of influenza A viruses has implications which reach beyond H7 subtype viruses. Reports of conjunctivitis concurrent with influenza-like illness have been reported for both H5 and H7 subtype viruses (15
). The presence of both α2-3- and α2-6-linked sialic acids on epithelial cells of the human eye suggests that a broad range of influenza viruses could bind to this tissue, including virus subtypes (such as H5N1) which may use the eye as an initial site of virus replication before establishment of a respiratory infection (30
). Continued study of the interplay between influenza viruses, the human eye, and the link between ocular infection and development of respiratory disease will provide much-needed information to guide prevention, treatment, and control strategies for all influenza virus subtypes.