Rhinovirus reduces the severity of subsequent respiratory viral infections, which is associated with dampened inflammatory responses

Coinfection by unrelated viruses in the respiratory tract is common and can result in changes in disease severity compared to infection by individual virus strains. We have previously shown that inoculation of mice with rhinovirus (RV) two days prior to inoculation with a lethal dose of influenza A virus (PR8), provides complete protection against mortality. In this study, we extend that finding to a second lethal respiratory virus, pneumonia virus of mice (PVM) and characterize the differences in inflammatory responses and host gene expression in single virus infected vs. coinfected mice. RV prevented mortality and weight loss associated with PVM infection, suggesting that RV-mediated protection is more effective against PVM than PR8. Major changes in host gene expression upon PVM infection were delayed compared to PR8, which likely provides a larger time frame for RV-induced gene expression to alter the course of disease. Overall, RV induced earlier recruitment of inflammatory cells, while these populations were reduced at later times in coinfected mice. Findings common to both coinfection conditions included upregulated expression of mucin-associated genes in RV/PR8 and RV/PVM compared to mock/PR8 and mock/PVM infected mice and dampening of inflammation-related genes late during coinfection. These findings, combined with differences in virus replication levels and disease severity, suggest that the suppression of inflammation in RV/PVM coinfected mice may be due to early suppression of viral replication, while in RV/PR8 coinfected mice may be due to a direct suppression of inflammation. Thus, a mild upper respiratory viral infection can reduce the severity of a subsequent severe viral infection in the lungs through virus-dependent mechanisms. Author Summary Respiratory viruses from diverse families co-circulate in human populations and are frequently detected within the same host. Though clinical studies suggest that coinfection by more than one unrelated respiratory virus may alter disease severity, animal models in which we can control the doses, timing, and strains of coinfecting viruses are critical to understand how coinfection affects disease severity. In this study, we compared gene expression and immune cell recruitment between two pairs of coinfecting viruses (RV/PR8 and RV/PVM) that both result in reduced severity compared to infection by PR8 or PVM alone in mice. Reduced disease severity was associated with suppression of inflammatory responses in the lungs. However, differences in disease kinetics and host and viral gene expression suggest that protection by coinfection with RV may be due to distinct molecular mechanisms.


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The detection of more than one virus in respiratory samples is quite common, especially 52 among pediatric patients (1-4). There are differences in the outcomes of coinfection -whether it 53 results in increased, decreased, or no effect on disease severity -that likely reflect different virus 54 parings, patient populations, and study criteria. For example, coinfection with influenza B virus 124 Infection by PR8 and PVM induce different gene expression signatures in mouse lungs over 125 time 126 127 To determine potential mechanisms of protection mediated by RV against PR8 and PVM, 128 we undertook a comprehensive transcriptome analysis of mouse lungs (Fig 2). Mice were 129 coinfected with RV two days before PR8 or PVM and total lung RNA was analyzed on days 0, 2, 130 4, and 6 after PR8 or PVM inoculation. Single virus-infected mice were mock-inoculated two days 131 before PR8 or PVM and total lung RNA was isolated at the same time points. Weight loss was 132 monitored daily to test for consistency with our previous morbidity and mortality analyses. RV-133 mediated protection against PR8 was not evident by 6 days post-infection (S1 Fig) though both 134 mock/PR8 and RV/PR8 groups experienced weight loss at a rate similar to our previous study (9).
135 In contrast, complete protection against weight loss was evident in RV/PVM coinfected mice (S1 136 Fig).

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Many reads mapped to PR8 and PVM genomes from the mice infected with these viruses 187 (Fig 4). Coinfection by RV did not prevent PR8-specific gene expression, but reads mapped to 188 PR8 were significantly lower in coinfected mice at all time points (Fig 4). Similarly, we previously 189 showed that infectious PR8 titers in the lungs were equivalent in mock/PR8 and RV/PR8 infected 190 mice on days 2 and 4 after PR8 inoculation (9). However, coinfection with RV led to earlier

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The proportions of neutrophils and interstitial macrophages followed the same trends as 295 total CD11b+ cells in PVM-infected mice, with lower proportions of these cells on days 4 and 6 296 in coinfected mice (Fig 7F, 7G). In contrast, interstitial macrophages were increased in RV/PVM, 297 compared to mock/PVM-infected mice, early in infection. This indicates that coinfection by RV 298 stimulates early recruitment of CD11b+ cells, specifically interstitial macrophages, while limiting 299 recruitment of inflammatory cells later in infection. PR8-infected mice had similar trends, however 300 the differences between mock/PR8 and RV/PR8 groups were less dramatic (Fig 7B, 7C).
301 Neutrophil numbers were suppressed in RV/PR8 coinfected mice compared to mock/PR8 infected 302 mice through-out the time course (Fig 7b). The interstitial macrophage proportions in mock/PR8-303 and RV/PR8-infected mice increased over time similarly to the total CD11b+ populations (Fig   304 7C). The lower proportions of neutrophils and interstitial macrophages at later time points in RV-305 coinfected mice corresponded with mRNA levels for chemokines. This was predominantly the 306 case for neutrophil chemokines Cxcl1 and Cxcl2 (Fig 7I, 7K) and macrophage chemokines Ccl2 307 and Ccl7 (Fig 7J, 7L). These chemokines were generally lower in RV/PR8 coinfected mice on day 308 6 and RV/PVM coinfected mice on days 4 and 6 compared to mock/PR8 and mock/PVM infected 309 mice, respectively.

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There were no clear trends in alveolar macrophage numbers in mock/PR8-and RV/PR8-311 infected mice (Fig 7D), though their proportions were significantly higher in RV/PVM-coinfected 312 mice compared to mock/PVM-infected mice on days 4 and 6 ( Fig 7H). This is likely due to Discussion 320 Previously, we found that inoculation of mice with a mild respiratory viral pathogen, RV 321 or murine coronavirus MHV-1, two days before PR8 provided significant protection against PR8-322 mediated disease (9). In this study, we expanded these results to show that RV-mediated protection 323 was not specific to PR8, but also provided significant disease protection against a respiratory virus 324 from another viral family, PVM. This is in agreement with other studies showing protection 325 afforded by viral coinfection (10-12). Despite the commonality of coinfection resulting in reduced 326 disease severity, there are differences between the virus combinations in the kinetics of disease 327 and viral replication. Coinfection by RV provided more effective protection against PVM than 328 PR8. RV/PVM coinfected mice had little to no signs of disease (Fig 1) and significantly limited 329 PVM replication (Fig 4). In contrast, coinfection by RV prevented mortality, but not morbidity, 330 associated with PR8 infection, and reduced viral gene expression but did not prevent infection by 331 PR8 (Fig 4) (9). Further, RV given concurrently with PVM was as effective as when it was given 332 two days before PVM (Fig 1). In contrast, RV was less effective at reducing the severity of PR8 333 when given concurrently and also exacerbated disease when it was given two days after PR8 (9). 334 We also observed differences in the kinetics of gene expression in response to these virus pairs.
335 Host (Fig 3) and viral (Fig 4) gene expression changes in response to PVM were delayed compared 336 to PR8, thereby giving a larger window for RV-mediated protection. Thus, RV may be inducing 337 antiviral mechanisms that are more effective against PVM, or different mechanisms may be 338 responsible for inhibiting PVM infection and mediating effective clearance of PR8. We used 339 transcriptomic and flow cytometry analyses to identify potential mechanisms that mediate 340 protection against PR8 and PVM in mice that were coinfected with RV.

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Analysis of RV-inoculated mice on day 0 (two days after RV inoculation) revealed up-342 regulation of 327 genes. These genes were highly enriched in GO categories that involved cell 343 division or chemokine signaling (S2 Table). Despite expression of several chemokine and 344 chemokine receptor genes, we did not observe a dramatic increase in immune cells in the lungs of 345 RV-infected mice on day 0 (Fig 7). Although our flow cytometry results had variability in RV-346 infected mice on day 0 between our studies, we detected a significant increase in interstitial 347 macrophages in the RV/PVM study (Fig 7G

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In contrast to early recruitment of neutrophils, RV-coinfected mice had reduced numbers 357 of neutrophils and interstitial macrophages later during infection (Fig 7). This decrease in 358 inflammatory cell recruitment could be a result of reduced viral infection (Fig 4) and  We analyzed flow cytometry (Fig 7) and viral read count (Fig 4) data resulting from our 508 experiments to identify time-varying differences between mice infected with PR8 or PVM alone 509 or coinfected with RV. To this end, we used negative binomial regression on each response 510 variable with an explanatory model that had a main effect of days post-infections, a main effect of 511 treatment (single or coinfection), and the interaction between the two main effects. Response 512 variables were the number of CD11b cells, neutrophils, interstitial macrophages, alveolar 513 macrophages, and viral read counts; all response variables were normalized versus total cell count 514 except for viral RNA read count which was normalized against total RNA read count. Due to a 515 prior visual investigation of our data, it seemed some of our response variables might be better fit 516 with a quadratic time term. Because of this we fit an alternative model that included an orthogonal 517 polynomial of degree 2 for time. We assessed whether the quadratic model was better than the 518 linear model using a likelihood ratio test and chose the quadratic model if it offered a significant 519 improvement in fit over the simpler linear model. The significance of treatment and time was 520 determined using a type-I ANOVA. To detect differences between treatments at a given time, we 521 also performed post hoc pairwise comparisons of the modeled mean at our observational time 522 points (days 0, 2, 4, and 6) using the emmeans package in R. Supplemental Table 3