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RSV immunizes bystander cells to IAV infection using interferons β and λ

We observed the interference between two prevalent respiratory viruses, respiratory syncytial virus (RSV) and influenza A virus (IAV, H1N1), and characterized its molecular underpinnings in alveolar epithelial cells (A549). We found that RSV induces higher interferon (IFN) β production than IAV and that IFNβ priming confers higher protection against infection with IAV than with RSV. Consequently, we focused on the sequential infection scheme: RSV-then-IAV. Using the A549 WT, IFNAR1 KO, IFNLR1 KO, and IFNAR1–IFNLR1 double KO cell lines we found that both IFNβ and IFNλ are necessary for maximum protection against subsequent infection. Immunostaining revealed that preinfection with RSV partitions the cell population into a subpopulation susceptible to subsequent infection with IAV and an IAV-proof subpopulation. Strikingly, the susceptible cells turned out to be those already compromised and efficiently expressing RSV, whereas the bystander, interferon-primed cells are resistant to IAV infection. Thus, the virus–virus exclusion at the cell population level is not realized through a direct competition for a shared ecological niche (single cell) but rather achieved with the involvement of specific cytokines induced within the host innate immune response.


Introduction
Human respiratory tract infections are predominantly caused by viruses. Out of a plethora of respiratory viruses, recently expanded by the novel beta coronavirus SARS-CoV-2 [1], the three most common recurrent infectants, originating from different viral families, are rhinovirus (human RV, HRV), influenza A virus (IAV), and the respiratory syncytial virus (RSV, human orthopneumovirus) [2], with the latest being the most common pathogen in severe disease in young children and the elderly [3][4][5]. In temperate areas of the northern hemisphere, the incidence of respiratory infections surges in the winter season and subsides during summer. Detailed examination of temporal patterns of disease outbreaks reveals not only seasonal coincidence but also apparent avoidance of some types of cocirculating respiratory viruses [6][7][8][9]. It is unclear whether the epidemiological observations are population-level consequences of an interplay of multiple viral cooperation and competition mechanisms that operate at a single-host level [10]. Intriguingly, the epidemiological avoidance patterns are visible also for taxonomically different groups of viruses, suggesting their potential reliance on mechanisms that are not related to specific antibody-mediated cross-protection. Although viral interference can be readily observed in vitro [11], its underlying mechanisms are necessarily inextricably intertwined with the response of the host organism and await elucidation from a systemic perspective.
Non-antigenic competitive interactions among respiratory viruses have been suggested based on clinical data analysis [12,13] and were recapitulated within animal models [14,15]. Two very recent studies [16,17] demonstrated that an innate immunity mechanism, specifically antiviral cytokine signaling, is implicated in the interference between RV and IAV: blocking the type-I interferon (IFN) response restores IAV replication following RV infection. Whereas type-I IFNs (IFNα and IFNβ) were originally discovered specifically because of their activity against the influenza virus [18], their roles in RSV infection and disease have been controversial [19][20][21][22][23][24][25]. A recent animal model study [14] suggested that the RSV-IAV viral interference is antigen-independent, however direct involvement of IFNs has not been demonstrated.
Type I IFNs, together with the more recently identified type III IFNs (IFNλ1-λ4) [26,27], are produced and secreted by infected cells to trigger transcription of an array of IFNstimulated genes (ISGs) in both the infected and the bystander cells [28][29][30]. Elevated ISGs restrict viral replication until virus-specific host defense mechanisms develop [31]. IFNα and IFNβ additionally attract specific immune system cells to the site of inflammation [22,32].
In this study, we sought to determine molecular underpinnings of the interactions between RSV and IAV. We used a human alveolar epithelial cell line (A549) and characterized direct inter-viral relations and interferon-mediated interference in vitro at the single-cell level. We found that RSV induces higher IFNβ production than IAV and that IFNβ is a more potent inducer of STAT1/2 signaling than IFNλ1. Moreover, IFNβ-stimulated cells are more resistant to infection with IAV than to infection with RSV. In light of these findings, we focused on the sequential coinfection scheme, in which cells are preinfected with RSV and then infected with IAV. We developed A549-derived cell lines with knockout (KO) of   type I IFN receptor (IFNAR1 KO), type III IFN receptor subunit (IFNLR1 KO), and double KO   (IFNAR1-IFNLR1 dKO). They enabled us to find that IFNβ is the main inducer of the antiviral state in RSV-infected A549 cells and both interferons, IFNβ and IFNλ, are simultaneously necessary for building maximum protection against subsequent infection with IAV. Immunostaining revealed that preinfection with RSV partitions the cell population into a subpopulation susceptible to subsequent infection with IAV and an IAV-proof subpopulation.
Strikingly, the susceptible cells turned out to be those already compromised and efficiently expressing the priming virus. Overall, we conclude that in the sequential RSV-then-IAV infection scheme, the virus-virus exclusion at the cell-population level is not realized through a direct competition for a shared ecological niche (single cell) but rather achieved with the involvement of specific cytokines induced within the host innate immune response.

IFNβ and IFNλ mediate STAT1/2 activation upon RSV and IAV infection
We first characterized induction of the antiviral state by interferon signaling. We found that RSV is a more potent inducer of IFNβ than IAV at comparable multiplicities of infection (MOIs) during first two days post-infection (cf. Fig 1A, Fig 1B).
We then analyzed interferon-mediated activation of STAT1/2 during RSV or IAV infection and consecutive induction of ISGs that are the primary correlates of the antiviral state: RIG-I (viral RNA sensor [41,42]), PKR (indirect translation inhibitor [43]), and OAS1 (direct activator of RNase L, the RNA eradicator [44]). We juxtaposed results for A549 WT cells with those for the A549-derived cell lines in which we knocked out IFNβ receptor IFNAR1, heterodimeric IFNλ receptor subunit IFNLR1, and both these genes ( Fig 1C, Fig   1D). We observed that infection with RSV or IAV leads to robust activation however only IFNβ appears to be capable of attenuating the propagation of RSV.

Prestimulation with IFNβ inhibits propagation of RSV and IAV
To estimate the impact of priming with IFNβ and IFNλ1 on the infectiousness and propagation of RSV and IAV, we quantified viral RNA molecules per cell and analyzed the abundance of viral proteins. We performed infection experiments without or with a preceding, day-long stimulation with IFNβ (1000 U/ml) or with IFNλ1 (50 ng/ml). In Fig 2, we juxtaposed results for these two stimulation protocols. We found that 24 hours post-infection at MOI 0.1, prestimulation with IFNβ limits RSV progeny by nearly one order of magnitude, whereas IAV progeny is reduced by at least two orders of magnitude (cf. Fig 2A, Fig 2B). At MOI 1, when nearly all cells are infected, prestimulation with IFNβ limits RSV proliferation about five-fold, while the relative reduction of IAV proliferation is as significant as in the case of the lower MOI (cf. Fig 2A, Fig 2B). Prestimulation with IFNβ triggers an antiviral state manifested by accumulation of ISGs: RIG-I, PKR, and OAS1 and STAT1/2. These proteins maintain their elevated levels 48 hours post-infection ( Fig 2C, Fig 2D). Consequently, viral proteins (RSV fusion glycoprotein, IAV nucleoprotein) are less abundant in IFNβ-primed cells than in the naïve cells.

Impact of RSV or IAV infection on STAT1 activation
To analyze at the single-cell level how infected cells induce the antiviral state in bystander cells, we performed infections at MOI 0.1, fixed the cells 24 hours post-infection, and immunostained them for active IRF3 (as an evidence of detection of viral RNA by cell), p-STAT1 (used as an indication of the antiviral state), and viral proteins (RSV fusion glycoprotein or IAV nucleoprotein). In two-channel overlays shown in Fig 3A, Fig 3B, it can be noticed that p-STAT1 is present in most bystander cells (at various levels), whereas the infected cells exhibit p-STAT1 only sporadically. This effect indicates the capability of the viruses to suppress the response to IFN stimulation [45][46][47][48][49]. We quantified this effect by looking directly at the presence of viral proteins as the definite manifestation of productive infection, and at nuclear p-STAT1 as an indication of antiviral alertness. Based on the expression of RSV fusion glycoprotein or IAV nucleoprotein, we classified cells as either virus-positive or virus-negative and collected histograms of nuclear p-STAT1 intensity in each cell within the two groups. In Fig 3C, Fig 3D we

Effect of prestimulation with IFNβ or IFNλ1 on IAV spread in A549 WT cells
Thus far, we have used cell-population techniques to show that IFNβ is a more potent inducer of the antiviral state than IFNλ1 (Fig 1) and, as a consequence, in A549 cells viral proliferation is more suppressed after prestimulation with IFNβ than with IFNλ1 (Fig 2A).
Since RSV turned out to elicit stronger IFNβ response than IAV, to characterize the potential interferon-mediated cross-protection between the two viruses we decided to investigate specifically the dual infection scheme in which the cells are first preinfected with RSV and after 24 hours infected with IAV.
To gain insight into the interferon-mediated interaction at the single-cell level, we quantified the protective effect of IFNβ or IFNλ1 in the context of IAV infection based on imaging data ( Fig 4A). In Fig 4B we show proportions of cells expressing viral nucleoprotein 24 hours after treatment with IAV at MOI = 1 in each field of view in the case of naïve (that is, devoid of any prior IFN stimulation) A549 WT cells and A549 WT cells that were prestimulated with either IFNβ or IFNλ1. We estimated that more than 40% nonprestimulated cells express nucleoprotein 24 hours after infection. This proportion drops to about 3% after prestimulation with IFNβ and about 10% after prestimulation with IFNλ1.
Relations between these proportions provide additional evidence for strong IAV spread attenuation in A549 cells conferred by IFNs. gives a tenfold reduction of IAV nucleoprotein-expressing cells, from ~40% to ~4%). The protective effect conferred by RSV-priming is significantly weaker in IFNAR1 KO cells incapable of responding to IFNβ (reduction from ~40% to ~20%) and only slightly weaker in IFNLR1 cells incapable of responding to IFNλ (reduction from ~40% to ~10%). The priming effect is virtually non-existent in cells devoid of receptors for both IFNβ and IFNλ. In Fig 5C we show dPCR results of corresponding experiments that are in qualitative agreement with results of single-cell analysis in Fig 5B. Overall, this demonstrates that preinfection with RSV protects from infection with IAV in an interferon-dependent manner in A549 cells. The protective effect may be attributed nearly entirely to the paracrine stimulation with both IFNβ and IFNλ, with a major contribution from IFNβ.

IAV infects preferentially the cells preinfected by and expressing RSV
Visual inspection of WT cells suggested that IAV nucleoprotein is preferentially expressed in the cells with apparent expression of RSV fusion glycoprotein (see Fig 6A). To quantitatively verify this hypothesis, as in Fig 3D, we classified cells as virus-positive and virus-negative.
We decided to use the measured level of IAV NP as the binary criterion because of its mostly nuclear location, which facilitates image analysis. We then collected histograms of intensity of RSV fusion glycoprotein in the cells belonging to these two groups. Clearly, in A549 WT cells, the IAV+ cells are associated with higher abundance of RSV fusion glycoprotein than IAV− cells (KS of 0.62 indicates good discernibility). This effect is much weaker in IFNAR1-IFNLR1 dKO cells, which cannot respond to neither IFNβ nor IFNλ (KS of 0.15 indicates that the two distributions are nearly overlapping). This clearly shows that the RSV-IAV exclusion is not realized through a direct competition for a shared ecological niche, a single cell, but rather achieved with the involvement of interferon signaling.

Discussion
To replicate, an invading pathogen has to evade or compromise the innate immunity. When challenged by the infectant, the innate immune system acts to impede pathogen replication before a more specific adaptive response is evoked. In the context of sequential viral coinfections, the priming virus both breaks through the innate protection in infected cells and, indirectly, via interferon secreted by these cells, triggers the protective antiviral state in bystander cells. These two antagonistic processes determine the fate of both the primary and the secondary infection at the cell-population level.
To obtain insight into this interplay at the single-cell level, we studied how upon a priming infection with RSV, individual cells become either cross-protected or, conversely, more vulnerable to the second virus, IAV. The choice of these two pathogens has been motivated by the fact that RSV and influenza viruses show relatively high coincidence [50].
The particular order of infections that we focused on results from our observations that (1) RSV induces higher IFNβ production than IAV, (2) IFNβ-stimulated cells are more resistant to infection with IAV than to infection with RSV, and (3) IAV is sensitive to IFNλ and this sensitivity does not rely solely on STAT1/2 signaling. We thus inferred that the potential cross-protection may be more pronounced in the RSV-then-IAV protocol, which is consistent with a comparison of RSV-then-IAV and IAV-then-RSV coinfections in a mouse model, showing that the IAV-then-RSV coinfection is associated with higher IAV loads and mouse mortality [51] (see also [52]).
We found that IFNβ is the main inducer of the STAT1/2-associated antiviral state in RSV-infected A549 cells and both interferons, IFNβ and IFNλ, are simultaneously necessary for building maximum protection against a subsequent infection with IAV. A synergistic effect of type I and type III interferon, consistent with our findings, has been previously reported in mice by Mordstein et al. [53]. In our coinfection experiments, we infected the epithelial alveolar cells with RSV at MOI of 0.1 to readily observe their stratification into the compromised infected cells and the reinforced bystander cells. Immunostaining revealed that preinfection with RSV partitions the cell population into a subpopulation susceptible to a subsequent infection with IAV and an IAV-proof subpopulation. This dynamic functional differentiation of cells may be amplified in vivo by differential propensity of specific cell types to permit or restrict viral replication, produce interferons, and build up the antiviral state [54].
We found that the susceptible cells turned out to be predominantly those already The natural question is to what extent the described cross-protection mechanism reduces the incidence of coinfections or severity of the disease featured by a secondary infection in vivo. Answering the question is especially important, but appears complex, in the context of secondary SARS-CoV-2 infections. An early study by Ziegler et al. [55] showed that the receptor for SARS-CoV-2, ACE2, is a protein coded by ISG, but later Onabajo et al. [56] identified the transcriptionally independent truncated isoform of ACE2, deltaACE2, not ACE2, as an ISG. Since deltaACE2 may not bind SARS-CoV-2 spike protein, the latter study suggests that interferon signaling is not hijacked by this virus to enhance its proliferation. Several studies suggested beneficial roles of type I and type III interferon at the early stage of infection [57]. It is thus possible that interferons secreted in response to a primary infection exhibit a protective role against a secondary infection with SARS-CoV-2 (see also [58]).
At the clinical level, one may expect that the occurrence of coinfections may result from defective work of the immune system (which aggravates the course of the disease and also imaginably cannot mediate antagonistic viral interactions). Although this effect cannot be excluded and coinfection data should be treated with caution, impact of coinfections on disease severity is unclear. Most respiratory viral coinfections, detected usually in a small proportion of patients, appear not to increase severity of clinical outcomes [59][60][61][62], with two noteworthy exceptions of a coinfection by IAV and IBV [50] and coinfections with RSV in paediatric patients [63]. It should be noted that interactions of two respiratory viruses within the host can hardly be translated to their coincidence or avoidance in human populations. This is because at the population level there are two additional and antagonistic effects: on one hand, viral coincidence of contagious diseases can be seasonal (the effective contact rate is modulated by weather and behavioral patterns); on the other hand, infected and ill individuals change their behavior to avoid contact. A significant behavioral reduction of the contact rate may result from an outbreak of an epidemic of a threatening virus, decreasing the risk of propagation of other viruses, as observed during the COVID-19 pandemic in 2020 [64,65].
In conclusion, our study demonstrates that the infection with RSV cross-protects alveolar epithelial cells against infection with IAV. Whereas RSV-infected cells are more vulnerable to infection with IAV, priming with RSV indirectly protects bystander non-infected cells from IAV. The cross-protection mechanism relies on induction of and paracrine stimulation with both IFNβ and IFNλ.

Cell lines and cultures
The A549 and MDCK cell lines were purchased from ATTC. A549 cells were cultured in

Data analysis Image analysis
Confocal images obtained from immunostaining were analyzed using our in-house software (https://pmbm.ippt.pan.pl/software/shuttletracker). Nuclear outlines were detected based on DAPI staining. Outlines of nuclei that were partially out of frame or mitotic were excluded from analysis; outlines of overlapping nuclei were split based on geometric convexity defects when possible. Cells were classified as either RSV-negative or RSV-positive based on the combination of the sum of intensities of pixels inside nuclear outlines (weight 5) and the perinuclear region (weight 1, decreasing sigmoidally with the distance from the nuclear contour). Cells were classified as either IAV-negative or IAV-positive based on the sum of intensities of pixels inside nuclear outlines.

Credible interval
Credible interval (CrI) of the Kolmogorov-Smirnov statistics (Fig 3C, Fig 3D, Fig 6B) has been estimated by resampling data points 10 5 times. In each resampling, data points were drawn at random with probabilities proportional to the number of data points in each category (virus-positive, virus-negative) and the number of resampled data points was equal to the total number of data points in both categories.