r1918 virus-induced weight loss, time to death, and pathology are dependent upon mouse genotype.
WT (n = 19), IL1RKO (n = 12), and TNFRKO (n = 13) mice were infected i.n. with 106 PFU of r1918 virus, and weight loss and mortality were monitored for 8 and 10 dpi, respectively. WT and IL1RKO mice exhibited significant and sustained weight loss as early as 1 dpi and weighed significantly less than the TNFRKO mice at all time points (P < 0.05) (Fig. ). In contrast, weight loss was not consistently observed in all TNFRKO mice until 6 dpi, at which point the TNFRKO mice achieved statistically significant weight loss (P < 0.05). Median time to death was longer for TNFRKO mice (8 dpi) than for WT (7 dpi) or IL1RKO mice (6 dpi) (Fig. ). The differences in time to death between the WT and IL1RKO mice did not reach statistical significance (P = 0.06); however, the TNFRKO mice statistically survived longer than the WT (P = 0.0005) and IL1RKO mice (P < 0.0001).
FIG. 1. Characterization of mortality, weight loss, and lung viral titers in r1918-infected WT, TNFRKO, and IL1RKO mice at 1, 3, and 5 dpi. (A) Average weight of surviving animals at each time point (expressed as mean ± standard deviation SD; n = (more ...)
Viral titer in the lungs of infected animals was measured at 1, 3, and 5 dpi (n = 6 for TNFRKO and IL1RKO mice; n = 9 for WT mice). There were no significant differences in viral titers in the lungs at 1 and 3 dpi between the three mouse strains (Fig. ). However, IL1RKO mice had higher levels of virus in the lung than WT and TNFRKO mice at 5 dpi (P < 0.05). Although it is difficult to concretely determine if this is biologically significant, the occurrence of earlier mortality within the IL1RKO mice suggests that this difference in viral load may be biologically relevant. Viral titers were not measured in the blood. However, virus was not detected in the brain or spleen of any of the mice at any of the time points examined, suggesting that the virus did not become systemic in these animals at this inoculation dose (data not shown). Virus replication in the lungs of infected mice was confirmed by real-time, quantitative RT-PCR (qRT-PCR) with probes specific to the influenza virus matrix gene (data not shown).
Histopathology was examined by hematoxylin and eosin staining of lung tissue sections from infected animals, and viral antigen was detected by immunohistochemistry (Fig. ). We found that at 1 dpi, WT mice either lacked airway lesions or had mild epithelial cell degeneration, but they had mild diffuse histiocytic alveolitis with some perivascular cuffs of neutrophils and lymphocytes. No viral antigen staining was detected in lung and airway samples at this time point (Fig. ). At 3 and 5 dpi, moderate necrotizing bronchiolitis and bronchitis with associated neutrophilic to histiocytic luminal and peribronchiolar inflammation were present, accompanied by mild to moderate diffuse histiocytic to neutrophilic alveolitis. Viral antigen was commonly seen in respiratory epithelium of the upper and lower airways and, slightly less commonly, within peribronchiolar alveolar macrophages (Fig. ). Viral antigen staining declined slightly at 5 dpi in the airway epithelium.
FIG. 2. Photomicrographs of hematoxylin-and-eosin-stained and immunohistochemically stained lung tissue sections from mice intranasally inoculated with r1918 virus. (A) Mild epithelial cell degeneration and associated mild histiocytic alveolitis in WT mouse at (more ...)
TNFRKO mice at 1 dpi had mild to moderate necrosis in bronchi and bronchioles, with minimal to no associated inflammation and mild to moderate peribronchiolar histiocytic to neutrophilic alveolitis. Viral antigen was infrequent in respiratory epithelial cells and rare in alveolar macrophages (Fig. ). At 3 and 5 dpi, in comparison to the lungs from WT mice, the necrosis in TNFRKO mice was progressively less prominent, and inflammation was minimal or lacking (Fig. ). On day 3, viral antigen staining in TNFRKO mice was less severe than in WT mice (Fig. insert), and on day 5, minimal viral antigen was detected, less than in any of the other mouse groups.
IL1RKO mice had mild to moderate necrosis in bronchi and bronchioles at 1 dpi with associated heterophilic to histiocytic inflammation and mild peribronchiolar histiocytic alveolitis. Viral antigen was commonly seen in degenerating or necrotic respiratory epithelium of the pulmonary bronchioles and associated luminal debris and frequently within alveolar epithelium and alveolar macrophages (Fig. ). At 3 and 5 dpi, the necrosis and inflammation in IL1RKO mice were slightly less severe than at 1 dpi but much less severe than in the corresponding WT mice. The IL1RKO mice had similar alveolar and airway lesions at 1 and 3 dpi as TNFRKO mice except that the histiocytic alveolitis was only peribronchiolar and was less severe in the IL1RKO mice. At 3 dpi, viral antigen was present in necrotic cellular debris within bronchiolar lumina and especially in alveoli and alveolar macrophages. Virus-positive respiratory epithelial cells were less frequently observed than at 1 dpi (Fig. ). At 5 dpi, the IL1RKO mice retained mild to moderate lesions in alveoli and airways while such lesions were reduced in TNFRKO mice.
In summary, at 1 dpi, pathology and viral antigen detection were greatest in IL1RKO and TNFRKO mice and least in WT animals. However, at 3 and 5 dpi, pathology continued to increase in WT mice but became less severe in IL1RKO mice. TNFRKO mice exhibited an intermediate level of pathology at 3 dpi and the least amount of pathology and viral antigen at the later time points. Overall, these histopathological findings are consistent with the reduced amount of weight loss and the longer median time to death observed for TNFRKO mice. Although it is not clear why the IL1RKO mice exhibited less pathology at 3 and 5 dpi and died sooner than the WT mice, this observation has been previously reported (27
). It is possible that, although the damage lessened over time, the IL1RKO mice were not able to recover from the overwhelming, early tissue damage.
IL1RKO and TNFRKO mice exhibit dramatic differences in global gene expression in response to infection.
To better understand the role of IL-1R1 and TNFR signaling in the host response to r1918 virus infection, we examined global gene expression within the lungs of infected animals at 1, 3, and 5 dpi. We observed that IL1RKO mice had the greatest number of genes differentially expressed in response to infection and that a large number of these genes were differentially expressed at each time point (Table ). In contrast, fewer genes were differentially expressed in WT and TNFRKO mice across these time points, and the number, identity, and relative expression of differentially expressed genes were more variable. This suggests that IL-1R1 signaling may be required for effective modulation of the host transcriptional response over time.
Number of genes differentially expressed in the lungs of mice infected with r1918 virus
In all three strains of mice at all three time points, a significant number of differentially expressed genes were related to cell death (32 to 38% of genes with annotation) and cellular growth and proliferation (28 to 42% of genes with annotation) (see Table S1 in the supplemental material). When we examined the genes that increased with infection separately from those that decreased, we observed an increase in inflammatory response, cell death, cell growth and proliferation, and hematological system development and function. Further, there was a decrease in the transcription of genes related to lipid metabolism and small-molecule biochemistry in all three strains of mice at the time points examined. These common biological responses to the r1918 virus may be in part due to the increase in immune cell recruitment and tissue remodeling occurring in the lung. However, although the three mouse strains had altered expression levels of genes within these categories, the expression patterns of genes also showed unique differences in the precise genes regulated, the extent of differential expression, or the kinetics of the response (see Table S2).
To address whether differences in the response to the r1918 virus in the three strains might be due to patterns of cytokine gene expression, we next examined differential cytokine expression across 1, 3, and 5 dpi (see Fig. S1 in the supplemental material). In contrast to what might be expected under the general hypothesis of a cytokine storm, we observed that overall patterns of expression for these genes were similar between the WT and TNFRKO mice. In contrast, IL1RKO mice exhibited an increase in the expression of many chemokines (TNF-α, gamma interferon [IFN-γ], and IFN-β), which occurred earlier than in the WT or TNFRKO mice. This suggests that absence of IL-1R1 signaling has a more widespread impact on cytokine expression than the absence of TNFR signaling. However, the similarities in expression between the WT and TNFRKO mice also indicated that expression of other genes may be influencing differences in the phenotypes observed in these mice.
IL-1R1 and TNFR have distinct roles in complement and coagulation response.
To gain insight into why TNFRKO mice were able to live longer than WT and IL1RKO mice and maintain their body weight, we next performed pairwise comparisons of gene expression at 1, 3, and 5 dpi to determine the gene expression patterns that specifically distinguished TNFRKO mice from the other mouse strains. From this analysis, we identified 383 genes at 1 dpi, 1,056 genes at 3 dpi, and 905 genes at 5 dpi that distinguished the TNFRKO mice from the other two stains. Functional analysis of these genes suggests distinct regulation of several biological pathways within the TNFRKO animals which may contribute to decreased pathology and increased time to death (Table ). For example, TNFRKO mice exhibited an expression profile for genes associated with acute-phase signaling that distinguished them from the other two mouse strains at 1 and 3 dpi. Activation of acute-phase signaling results in a range of biological activities in response to viral infection, including many inflammatory processes. Acute-phase signaling is particularly relevant to the present study because signaling through the pathway is initiated by TNFR, IL-1R1, and IL-6 expression (reviewed in reference 10
). When we compared the genes that were differentially expressed in TNFRKO mice with genes differentially expressed in the other two strains, we identified genes downstream of all three cytokine receptors (IL-1R1, TNFR, or IL-6R) that had unique expression patterns in the TNFRKO mice (Fig. ; see also Fig. S2 in the supplemental material). The alteration of gene expression downstream of IL-1R1 and IL-6R in TNFRKO mice suggests that the loss of TNFR signaling during infection has a substantial impact on acute-phase signaling, including an impact on signaling mediated through these receptors.
Top canonical pathways distinguishing TNFRKO mice from IL1RKO and WT mice at each day postinfection
FIG. 3. Differential host transcriptional responses within the acute-phase response pathway in r1918-infected WT, TNFRKO, and IL1RKO mice at 1, 3, and 5 dpi. Diagram shows the acute-phase response signaling pathway including cascades initiated through the TNF-α (more ...)
We next expanded our view of acute-phase signaling beyond what was specifically different in the TNFRKO mice. As shown in blue in Fig. , the majority of genes within the pathway were differentially expressed between strains on at least one time point. This included an upregulation of negative acute-phase response genes in WT mice at 1 and 3 dpi that was not observed in the knockout animals (Fig. ). This finding suggests that both IL-1 and TNF-α signaling are required for the increased expression of these genes following r1918 infection. It is notable that the relative decrease in negative acute-phase molecule gene expression in the IL1RKO mice coincided with a decrease in the expression of TCF3 and TCF4, transcription factors that regulate the expression of genes that code for negative acute-phase molecules including apolipoproteins (11
). This may be one potential mechanism by which the animals in this group suppress the expression of negative acute-phase response genes.
FIG. 4. Differential activation of acute-phase response signaling pathways in r1918-infected WT, TNFRKO, and IL1RKO mice. One-dimensional clustering of differentially expressed genes within the categories of negative acute-phase molecules, complement, and coagulation (more ...)
An increase in complement expression is a downstream consequence of acute-phase response signaling. We found that genes associated with the complement canonical pathway generally had lower expression in TNFRKO mice than in the IL1RKO and WT animals, particularly at 3 dpi (Table ), and the highest expression in the IL1RKO mice on all 3 days (Fig. ). One exception to this trend was the marked decrease in the expression of the complement protein C5 gene in the IL1RKO mice across 1, 3, and 5 dpi. Although the expression of the C5 gene was also decreased in the WT and TNFRKO mice compared to expression in uninfected mice, this decrease in expression was not apparent until 3 and 5 dpi in the TNFRKO mice and 5 dpi in the WT mice. This suggests that IL-1R1 signaling plays a role in the relative repression of complement genes following r1918 virus infection. The differential impact of IL-1R1 and TNFR signaling on complement is interesting in light of recent of work indicating a role for complement in the activation and infiltration of influenza virus-specific T cells in the lung during infection (17
The regulation of coagulation is closely linked to complement, and we also identified a delay in (fibrinogen B [FGB] and SERPINA1) or relative absence of differential expression (F2, FGA, and SERPING1) of coagulation genes in the TNFRKO mice at 1 and 3 dpi compared to WT (Fig. ). This suggests that the initiation of the coagulation response may be one mechanism by which TNFR signaling impacts the severity of influenza virus infection. This was distinct from IL-1R1 signaling, as all of these molecules and the coagulation-associated gene FGG were notably downregulated or unchanged compared to their levels in mock infections in the IL1RKO mice.
TNFRKO mice exhibit a lower specific antiviral and innate immune response.
Expression of a highly interconnected network of interferon-related antiviral genes was significantly lower in TNFRKO mice than in WT and IL1RKO mice at 5 dpi (Fig. ). When we examined the temporal expression patterns of genes in this network, we found that their expression increased in the context of infection for each strain. To understand how the extent of upregulation of this network differed between mouse strains, we calculated the average change in expression for this network by taking the change in expression for all molecules at each day postinoculation and dividing this number by the number of molecules within the network. This indicated that the average expression of genes within the network was lower in the TNFRKO mice than in the other two strains (Fig. ). This could be due in part to the relative absence of signaling through TNF receptor (TNFRSF1a) to STAT1 in the TNFRKO mice. It is of note that the IL1RKO mice had the highest average expression level of these molecules, and this degree of upregulation was maintained over time. Combined, these data suggest that TNFRKO mice have a delayed or decreased transcriptional response within key pathways, including antiviral and innate immune signaling, complement, coagulation, and regulation of negative acute-phase response genes. The differential regulation of these host responses within the TNFRKO mice suggest several mechanisms by which TNFR signaling influences the degree of tissue damage and the survival time in mice infected with the r1918 virus.
FIG. 5. Differential gene expression of interferon-related innate responses distinguishes TNFRKO mice from WT and IL1RKO strains during r1918-infection. (A) IPA of gene expression differences that distinguished TNFRKO from WT and IL1RKO mice at each time point (more ...) IL1RKO mice exhibit enhanced inflammatory and dendritic cell (DC)-related gene expression at 1 dpi.
We next examined the transcriptional pathways specifically impacted by the loss of IL-1R1 signaling during r1918 virus infection to understand why the IL1RKO mice died sooner than the other mouse strains. This was done by evaluating the genes that were uniquely expressed in the IL1RKO mice using pairwise comparisons of the one-way ANOVA (P < 0.05) between IL1RKO mice and WT mice and between IL1RKO mice and TNFRKO mice at 1, 3, and 5 dpi.
At 1 dpi, a significant proportion of the genes related to immune cell trafficking and cell-to-cell signaling interactions distinguished IL1RKO mice from the other two mouse strains. These included genes that regulate intercellular communication between DC and NK cells, TREM1 signaling, and DC maturation (Table ). Within the TREM1 pathway, molecules that mediate pathogen recognition and subsequent inflammation (TREM1, NOD2, and TYROBP) (2
) and chemoattractant molecules (MIP-1α, MCP-1, and MCP-3) had the highest expression in IL1RKO mice among all mouse strains (Fig. ). In the IL1RKO mice, these changes were concurrent with a downregulation of TREM2, which suppresses TNF-α, thereby promoting an anti-inflammatory state (32
). Concurrent with TREM1 signaling activation was the upregulation of genes related to DC maturation and communication between DC and NK cells, including killer cell lectin-like receptors (KLRK1 and KLRD1) and molecules associated with the activation of T cells (IFN-γ, CD86, and ICAM-1) at 1 dpi.
Top 10 IPA canonical pathways that distinguish IL1RKO mice from WT and TNFRKO mice following infection with r1918 influenza virus
FIG. 6. IL1RKO mice show differential gene expression of TREM signaling-related genes and DC maturation at day 1 postinfection. (A) Expression of genes related to TREM1 and DC signaling was unique in the IL1RKO animals at 1 dpi. Average expression for each group (more ...)
Many of the molecules related to DC function and TREM1 signaling form an interconnected network (Fig. ), and TNF-α is a central molecule within these three pathways. Although expression of TNF-α is increased in the other strains, the expression of the TNF-α gene was upregulated to the greatest extent in the IL1RKO mice, specifically distinguishing the IL1RKO mice from the other strains at all time points measured. This may suggest that in the absence of IL-1R1 signaling the IL1RKO mice disproportionally increase TNF-α gene expression, perhaps reflecting a level of redundancy in proinflammatory response. Increased expression of TREM1 and increased inflammation and cell-specific responses at 1 dpi correlate with the greater degree of inflammation noted in the lungs of IL1RKO mice at 1 dpi by histopathology.
IL1RKO mice exhibit increased expression of cell-cell communication and cellular movement genes.
Expression of genes related to cellular movement differentiated IL1RKO from WT and TNFRKO mice at 3 and 5 dpi (Table ). This included a significant number of genes related to tight-junction signaling and actin cytoskeleton signaling at 3 dpi (Fig. ). In addition to these pathways, there was a significant enrichment of genes related to integrin and integrin-linked kinase (ILK) that distinguished IL1RKO from WT and TNFRKO mice at 5 dpi. Although there were some downregulated genes (CCND1 and DSP) that had the lowest expression in the IL1RKO mice among all strains, in general the expression of the genes related to both integrin and ILK signaling were upregulated and at 5 dpi had the highest expression in IL1RKO mice.
FIG. 7. IL1RKO mice show differential expression of cellular movement genes at 3 and 5 dpi. Transcription of genes related to cellular movement differentiated the IL1RKO mice from WT and TNFRKO mice at 3 and 5 dpi. Two-dimensional clustering of differentially (more ...)
Highly pathogenic H5N1 avian influenza virus and r1918 pandemic influenza virus infections have a dramatic impact on immune cell infiltration into the mouse lung, causing excessive macrophage and neutrophil infiltration that likely affects immunopathological outcome (24
). Our analyses revealed that mice deficient in IL-1R1 signaling show enhanced gene expression changes related to cellular movement and cell-to-cell signaling. These transcriptional events, which influence tight-junction and actin cytoskeleton signaling, precede leukocyte extravasation and infiltration. Integrin and ILK signaling are closely related and play a key role in translating extracellular signals to intracellular changes in signaling and structure. These differences in inflammation and cell-to-cell signaling support our histopathology findings (Fig. ). The histopathology shows that at day 1 the IL1RKO mice had greater inflammation in the airways, which was accompanied by a higher infiltration of both polymorphonuclear leukocytes (PMN) and histiocytes (HC) than in WT or TNFRKO mice. Interestingly, by day 3 postinfection, the WT mice had more severe inflammation with more PMN and HC than the other two groups. This may suggest that the early increase in infiltration in these mice was a more important determinant of lethality than the later levels of infiltration.
The coordinated expression within these functionally related pathways suggests that a deficiency in IL-1R1 signaling leads to a compensatory increase in proinflammatory signaling at 1 dpi and cell-to-cell communication and movement at 3 and 5 dpi (see Fig. S3 in the supplemental material), which may be due in part to the greater increase in TNF-α gene expression in the IL1RKO mice. In combination with the histopathology data, this may suggest that the combination of early inflammatory response and increased TNF-α-associated signaling contributed to the early mortality in the IL1RKO mice. Overall, we observed that the TNFRKO mice exhibited a decrease in the expression of acute-phase genes and a decrease in the expression of early antiviral genes throughout r1918 virus infection. Combined, these transcriptional data suggest that, despite redundancy in the actions of IL-1 and TNF-α, during r1918 infection these animals show distinct responses that may in part contribute to the accelerated mortality in the IL1RKO mice and delayed mortality in the TNFRKO mice.