Although half of all children become infected with RSV during the first year of life, only a small percentage go on to develop severe disease requiring hospitalization or long-term respiratory sequelae of infection (recurrent cough or wheeze). Several developmental, environmental, and genetic differences can contribute to the susceptibility of these children to RSV-induced illness.
Our previous studies with mice demonstrated that RSV titers peaked on day 4 following RSV infection and that there were quantitative differences in the titers obtained from AKR/J and C57BL/6J mouse strains (56
). RSV titers are the result of complicated interactions between the virus and host, including attachment, entry, replication, release from the respiratory epithelium, and the early host inflammatory responses to this infection. Although titer is not definitively related to disease, RSV titer has been associated with disease severity in animals or humans. RSV infection does induce clinical illness in mice (e.g., predictable and reproducible ruffling and weight loss and inflammatory responses that are more difficult to quantify). In our initial report, we described a difference in weight loss between AKR/J and C57BL/6J mice, with AKR/J mice demonstrating higher titers and greater weight loss (56
). One of the most convincing demonstrations of the association between RSV titers and clinical indicators of disease in mice was described by Ramilo and associates (41
). Their study demonstrated that reduced RSV replication (lower titer) was associated with significant modulation of inflammatory and clinical markers of acute disease.
Clinical reports also provide evidence for the association between viral titer and disease in humans. Studies measuring passive antibody against RSV and disease in children demonstrate decreased symptoms associate with decreased titers (24
). Children with immune deficiency (primary or secondary) do not limit RSV replication and have increased and prolonged viral shedding and increased clinical illness and mortality from the infection (22
). Premature infants treated with palivizumab have decreased clinical illness (presumably due to limitation of virus spread in the lungs) compared to control infants (43
). Although it is important to recognize that these are associations and not proven causal relationships between RSV titer and disease severity, differences in titer levels can be a useful biologic marker and provide insights into the underlying disease pathogenesis.
In this study, we used RSV titer levels at 96 h postinfection as a quantitative trait for RSV susceptibility and identified a genetic locus (QTL) containing one or more genes that control the differential susceptibility of AKR/J and C57BL/6J mice. This significant QTL, named Rsvs1 (LOD = 7.8 for backcross studies, LOD = 5.2 for F2 crosses, and LOD = 12.5 for the combined backcross and F2 data), maps to the proximal end of mouse chromosome 6. One additional putative QTL on chromosome 2 was suggestive of a linkage, but only when the total backcross and F2 populations were combined in the analysis. Further analysis suggested joint interactions between genes on chromosomes 2 and 6 (backcrosses) and between genes on chromosomes 6 and 10 (F2 crosses). Microarray analysis identified genes differentially expressed in a time-dependent and strain-dependent fashion following RSV infection. Integrating the data from the microarray and QTL studies identified 18 candidate genes for RSV susceptibility within the defined Rsvs1 interval on mouse chromosome 6.
RSV assembly requires interaction with cellular actin and microtubules (15
). Differences in the expression or function of genes involved in actin-cytoskeleton interactions in the C57BL/6J and AKR/J mice could result in the differences in viral titers. RSV filament and syncytium (cell-cell fusion) formation following infection is dependent on RhoA signaling activity (44
). For example, RSV F associates with RhoA and caveolin-1 in plasma membrane microdomains (lipid rafts) (39
). Eight genes within the chromosome 6 interval encompassing Rsvs1
have involvement in cytoskeleton elements and actin-G-protein interactions.
The QTL and microarray data suggest that early biochemical and cellular events following RSV infection determine the level of RSV titer and the ongoing cellular and immune responses to the acute infection. Moreover, strain-specific differences in these early events were consistent with the differential RSV susceptibility demonstrated by C57BL/6J and AKR/J mice. These differences could be determined, in part, by the genes that map within Rsvs1. The QTL region on mouse chromosome 6 is large (30 Mbp) and contains over 200 genes. Further studies are needed to fine map Rsvs1. Nonetheless, to begin to assess how genes in this region might respond during infection, transcriptional changes were used to identify genes that are responsive to infection and to delineate the role of mechanistic pathways and processes related to RSV pathogenesis.
The pathogenesis of RSV infection has been studied in mouse models for over 20 years. After internalization of the RSV virion, lungs of infected mice manifest an eclipse period with significantly decreased virus titers. This is followed by increasing titers that peak by 4 to 5 days postinoculation (19
). Despite initial viral clearing by day 7, visible illness peaked on day 7 or 8, as evidenced by ruffled fur, reduced activity, and weight loss (19
). Mice (7 to 8 weeks old) infected with RSV typically lose a small amount of weight initially (56
) but can lose up to 10% of body weight by day 7, with a slow recovery by 12 to 14 days postinfection (19
). Lung inflammation is observable at days 2 to 4 after infection, manifested as lymphocyte margination within the pulmonary vasculature and mononuclear cell infiltrates in the perivascular spaces (19
), and includes NK cell immigration to the lung (28
). The pathology becomes most severe between days 5 and 8, with progression of perivascular, peribronchial, and diffuse alveolar macrophage and lymphocyte infiltrates (18
). During this time, both CD4- and CD8-positive T lymphocytes play major roles in RSV clearance, but they also contribute to lung pathology and clinical illness in the mice (7
). By day 10, pneumonia resolves and progression of the perivascular and peribronchiolar infiltrates ceases, and by day 15, the alveolar spaces become relatively free of lymphocytes and macrophages, but the perivascular lymphoid aggregates remain. Serum neutralizing antibody to RSV can first be detected 7 to 10 days following RSV infection and peaks at 4 to 6 weeks (17
Previous studies have demonstrated that RSV infection results in the activation of numerous transcription factors, including NF-κB and c/EBP, which in turn coordinate the expression of a cascade of proinflammatory cytokines and adhesion proteins (8
). Thus, it was reasonable to examine the lung transcriptome during infection. Alterations in transcript levels noted in the microarray analysis are consistent with the pathological time course outlined above. A limitation of this approach is that the cellular source of the mRNA is uncertain. RSV infection can result in alterations in gene expression in resident cells and can cause the immigration of lymphocytes and other inflammatory cells into the lungs, resulting in changes in the RNA levels observed. Despite this limitation, several informative patterns of gene expression were identified by this method.
We used two approaches to contrast transcriptomes of the mouse strains. The first involved identifying the transcripts that differed between C57BL/6J and AKR/J mouse lungs at each time during infection; this yielded 1,350 unique transcripts, including 1,121 annotated genes and 229 nonannotated cDNAs. Interestingly, the transcripts encoded by Cftr
differed between the AKR/J and C57BL/6J mice. Mice deficient in Cftr
) have a decreased ability to clear RSV infection, increased inflammation, and decreased production of NO in the airway (10
). The effect of Cftr
alteration on RSV susceptibility could be the result of decreased production of NO species such as peroxynitrite, which inhibits viral replication. Therefore, Cftr
deficiency or alteration in CFTR protein function could lead to the titer differences observed following RSV infection in the AKR/J and C57BL/6J mice.
The second approach was to evaluate the within-strain lung transcriptome, comparing untreated controls to infected mice. This yielded 535 unique transcripts (including 479 annotated genes and 49 nonannotated cDNAs). To better characterize the temporal transcriptome response to RSV, these transcripts were analyzed further using self-organizing map and GO enrichment tools. The initial response involved early antiviral defense changes, including transcripts associated with T-cell differentiation and activation, innate immune responses, antigen presentation, early response to virus, inflammatory response and chemotaxis, and IFN type I biosynthesis. For example, the expression of several essential transcription factors responsible for the virus-induced transcription of the type-I IFNs (Stat1, Stat2, and Irf7) and of chemokine genes (Ccl2 [MCP-1], Ccl4 [MIP1-β], and Cxcl10 [IP10]) increased during the initial 12 to 24 h following RSV exposure. This group also contained transcripts of genes with antiviral properties, including Adar, Eif2ak2, Mx1, Mx2, Oas1a, Oasl1 Isg15, Ifit1, and Tyki. Taken together, these changes are consistent with augmented early host cellular antiviral responses and were similar in both resistant (C57BL/6J) and sensitive (AKR/J) mouse strains. This was unexpected, because it would have been predicted that the C57BL/6J mouse would be more efficient in these early responses, resulting in the lower RSV titers observed subsequently. Although the resistant and sensitive mouse strains had many similar transcript levels during the initial response, by 6 h the AKR/J mice had more decreased transcripts involved in cytoskeletal organization and epidermis development, suggestive of a greater degree of initial epithelial injury (possibly due to preceding syncytium formation) in the sensitive AKR/J strain than the resistant C57BL/6J strain.
These initial transcriptional changes were followed by a cluster of transcripts that increased more in C57BL/6J mice than in AKR/J mice at 48 h. Significant increases were noted in biologic processes contributing to T-cell differentiation and activation, suggesting a more robust immune response in the resistant strain. Representative transcripts that increased more in the C57BL/6J mouse included CD antigens (Cd3d, Cd4, Cd8a, and Cd247 [CD3h]), T-cell receptors (Tcra and Tcrb-V8.2), cytokine signal transduction molecules (Itk and Ikzf1), and T antigen receptor-activated T-cell signaling molecules (Ptprc [CD45]). Other increased transcripts occurred for transcription factors (Vav1), motility factors (Dock2), ancillary molecules (Coro1a and Arhgdib), and a larger group of histones (18 transcripts were found in this group, including Hist1h3b). The differences in these transcripts at 48 h suggested activation of resident T cells or rapid T-cell immigration, which may account for the significant reduction in RSV titer seen in the C57BL/6J mice at 96 h postinfection.
Similarly, at 192 h, a cluster of transcripts representative of antigen presentation and MHC class I antigen processing were increased more in the resistant strain. Increased transcripts, including those for cytokines (Ccl5 [RANTES], Ccl8 [MCP-2], Cxcl9, Cxcr6, and Retnla) and complement components (C1qg), are consistent with the development of determinants of immunogenicity and tolerance in the lungs of C57BL/6J mice exceeding that in AKR/J mice. The resistant strain also had greater increases in transcripts reflecting improved epithelial cell function and lung remodeling, including transcripts for epithelial products (Clca3 [gob5], Reg3g, Muc5b, and Tff2) and macrophage chitinases (Chi3l4). These increases are consistent with the development of determinants of tolerance and epithelial cell repair in C57BL/6J mouse lungs exceeding that in AKR/J mouse lungs.
Having characterized the transcriptional difference, we then integrated the results obtained by the QTL and microarray approaches. While this could aid in uncovering transcripts altered during infection, a limitation is that variants of genes in this region may not lead to differences in expression or message stabilization, or other events reflected by measurement of steady-state mRNA levels. Nonetheless, changes in transcript levels suggest involvement in RSV pathogenesis and strengthen possible associations.
The role of CFTR in RSV infection has not been investigated fully. As noted above, we previously reported that Cftr−/−
mice have a decreased ability to clear RSV infection, increased inflammation, and decreased production of NO in the airway (10
). Several previous clinical studies have demonstrated that acute exacerbations in patients with cystic fibrosis (CF) can result from nonbacterial agents. Studies of infants have demonstrated that RSV infection is at least temporally associated with the initial identification (and perhaps the initial colonization) by Pseudomonas aeruginosa
in the respiratory tract for a significant portion of these patients (1
). Thus, acute viral infections, particularly those caused by RSV, are likely to contribute to a significant portion of acute pulmonary exacerbations and may be a key event in bacterial colonization in the CF lung.
In addition, CFTR dysfunction predisposes the CF airway to increased inflammation by altering intracellular signaling pathways, including NF-κB, which results in the activation of inflammatory genes (61
). Venkatakrishnan et al. demonstrated increased nuclear translocation of NF-κB in uninfected CF cells and a corresponding decrease in cytosolic IκB (which inhibits NF-κB nuclear translocation and subsequent gene activation) (60
). The signal transducer and activation of transcription-1 (STAT-1) signaling pathway is also defective in CFTR-deficient cells, due to the increased expression of the protein inhibitor of activated STAT1 (PIAS1) and defective activation of the GTPase RhoA (31
). Functional CFTR was demonstrated to be necessary for normal NF-κB regulation; blocking CFTR function in normal respiratory epithelial cells or restoration of CFTR function in CF epithelial cells restores normal NF-κB regulation. RSV infection causes NF-κB activation in respiratory epithelial cell cultures (14
). RSV infection in patients with CF therefore likely results in activation of NF-κB that cannot be inhibited adequately in the absence or malfunction of CFTR. These events could result in enhanced airway obstruction (pulmonary exacerbations), as seen in patients with CF. Another proposed mechanism whereby CFTR deficiency could lead to susceptibility to infection is that lymphocytes, which also express CFTR, have a diminished capacity to secrete antibodies and cytokines in response to antigens in vitro
). Our mouse microarray data noted key differences in T-lymphocyte function and antigen presentation and are certainly consistent with this viewpoint. Although immunosuppressive therapy may contribute to the chronic illness, the reduced ability to regulate lymphocyte secretion could reduce the response to antigen presentation and may even explain why lung-transplanted patients with CF remain chronically ill.
In summary, having previously reported that gene-targeted mice lacking Cftr were susceptible to RSV infection, we have now performed a QTL analysis using AKR/J and C57BL/6J backcross and F2 progeny. A major QTL (Rsvs1) was identified on proximal mouse chromosome 6. Integrating these QTL results with those of a microarray analysis that compared the polar-responding strains during RSV infection identified several candidate genes mapping to the Rsvs1 interval, including Cftr. These findings add to our understanding of individual RSV susceptibility and implicate a modifying role for CFTR in RSV infection, which may help to explain why viral infection is a significant cause of respiratory morbidity in patients with CF.