key: cord-0326907-5iq5jlqc
authors: Lozhkov, Alexey A; Yolshin, Nikita D; Baranovskaya, Irina L; Plotnikova, Marina A; Sergeeva, Mariia V.; Gyulikhandanova, Natalia E; Klotchenko, Sergey A; Vasin, Andrey V
title: Kinetics Of Interferon-λ And Receptor Expression In Response To In Vitro Respiratory Viral Infection
date: 2021-12-30
journal: bioRxiv
DOI: 10.1101/2021.12.30.474514
sha: 49f33362598f28d484f6088db2beaa6f8b93c7d1
doc_id: 326907
cord_uid: 5iq5jlqc

The major protective immune response against viruses is production of type I and III interferons (IFNs). IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs) that block viral replication and further viral spread. The ability of respiratory viruses to suppress induction of IFN-mediated antiviral defenses in infected epithelial cells may be a factor contributing to the particular pathogenicity of several strains. In this report, we analyzed expression of IFNs and some ISGs in an alveolar epithelial cell subtype (A549) in response to infection with: influenza A viruses (A/California/07/09pdm (H1N1), A/Texas/50/12 (H3N2)); influenza B virus (B/Phuket/3073/13); adenovirus type 5 and 6; or respiratory syncytial virus (strain A2). IFNL and ISGs expression significantly increased in response to infection with all RNA viruses 24 hpi. Nevertheless, only IBV led to early increase in IFNL and ISGs mRNA level. IBV and H1N1 infection led to elevated proinflammatory cytokine production. We speculate that augmented IFN-α, IFN-β, IL-6 levels negatively correlate to SOCS1 expression. Importantly, we showed a decrease in IFNLR1 mRNA in case of IBV infection that implies the existence of negative ISGs expression regulation at IFNλR level. It could be either a specific feature of IBV or a consequence of early IFNL expression.

In response to viral infection, components of the innate immune response are activated (Cole and Ho, 2017) . The most important components of the innate immune response are type I and III interferons (IFNs) (Cole and Ho, 2017) . IFNs induce activation of defense mechanisms and prepare cells for possible viral invasion. While the antiviral properties of type I IFNs have been widely studied (Randall and Goodbourn, 2008) , much less is known about the features of type III IFNs (IFN-λ). In humans, four IFN-λ subtypes have been found: IFN-λ 1 (IL-29); IFN-λ 2 (IL-28A); IFN-λ 3 (IL-28B); and IFN-λ 4 . IFN-λ are encoded by IFNL1-4 genes. Among IFN-λ 1-3 , there is high conservation of amino acid sequence (Miknis et al., 2010) . The actions of IFN-λ on the cell are carried out by binding to the heterodimeric receptor (IFNλR). IFN-λ functions significantly overlap with those of type I IFNs and induce the expression of analogous interferon-stimulated genes (ISGs) (Crotta et al., 2013) . Expression of both type I and type III IFNs is induced by the activation of the two most important cytosolic sensors, retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5). RIG-I and MDA5 appear to differentially stimulate IFNs in response to different virus-derived structures, with RIG-I generally responding most potently to 5′ di and tri-phosphate double-stranded RNAs (dsRNA); MDA5 preferentially associates with long dsRNA (Brisse and Ly, 2019) .

Despite obvious similarities in mechanisms of induction and downstream signaling, there are obviously some differences in the functioning of type I and type III IFNs. Presumably, type I IFNs have the potential to induce inflammation in addition to antiviral function, while type III IFNs promote the production of antiviral ISGs without the function of inducing inflammation (Sun Y et al., 2018) .

The selectivity of type III IFNs is due to peculiarities of receptor subunit expression. IFN-λ actions are carried out through the heterodimeric IFNλR receptor, consisting of the IFNλR1 and IL10R2 subunits. The IL10R2 subunit is also part of the receptor complexes for IL-10, IL-22, and IL-26; it is expressed in cells of various tissues (Miknis et al., 2010) . Expression of the IFNλR1 subunit demonstrates a more limited cellular distribution and is present in epithelial cells (Sommereyns et al., 2008) , keratinocytes (Zahn et al., 2011) differentiated dendritic cells (Yin et al., 2012; Zhang et al., 2013) and hepatocytes (Dickensheets et al., 2013) .

Consequently, the mucous membranes of the respiratory and gastrointestinal tracts are tissues that are mainly targeted by IFN-λ (Sommereyns et al., 2008) . This tissue specificity correlates with IFN-λ antiviral activity, which manifests itself mainly in relation to viruses with high tropism for epithelial tissues (Hermant and Michiels, 2014; Lozhkov et al., 2020) . This class of viruses includes respiratory viruses such as influenza A and B virus (IAV, IBV), respiratory syncytial virus (RSV), and some types of adenovirus (AdV).

It is well known that respiratory viruses induce IFNs and ISGs production. However, a vast majority of research is focused on features of one or several viral strains, meanwhile matching the data from unrelated research that were carried out in different cell lines should be approached with caution. The number of works that are devoted to direct comparison of the kinetics of IFNs expression stimulated by a wide panel of respiratory viruses is limited. In present research paper we evaluated the dynamics of IFNL expression using A549 cells infected with RNA-viruses (IAV, IBV, RSV) and DNA-virus (AdV). AdV and RNA-viruses are quite different in pathogenesis, so that we observed distinct IFNL expression.

All viral strains were obtained from the Virus and Cell Culture Collection of the Smorodintsev Research Institute of Influenza (St. Petersburg, Russia). Influenza viruses were grown in 11-day-old embryonated eggs, purified by sucrose gradient, and stored at −80°C.

The infectivity values of the viral stocks in MDCK cells were: 3.2×10 7 TCID 50 /ml for A/California/07/09 ((A)H1N1pdm09); 3.2×10 7 TCID 50 /ml for A/Texas/50/12 ((A)H3N2); and 3.2×10 5 TCID 50 /ml for B/Phuket/3073/13 (Yamagata lineage).

Adenovirus working stocks were generated by infecting A549 cells at a multiplicity of infection (MOI) of 0.001 for 72 h. Supernatant was then clarified by centrifugation, aliquoted, and stored at −80°C. The infectivity of the viral stocks in A549 cells was 3.2×10 6 TCID 50 /ml for both serotype 5 (AdV-5) and serotype 6 (AdV-6). The RSV A2 strain was grown in HEp-2 cells. The infectivity of the RSV stocks in HEp-2 cells was 3.2×10 7 TCID 50 /ml.

The A549 (CCL-185) cell line was obtained from the American Type Culture Collection (ATCC). A549 cells (human type II alveolar epithelial line) were cultured in F12K medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA). For infection, cells were seeded onto 12-well plates (Thermo Scientific Nunc, USA) at 5×10 5 cells per well. Around 100% confluent monolayers were washed with DPBS (Gibco, USA) and infected at a multiplicity of infection (MOI) of 1. After 60 min of adsorption at 37°C, viruscontaining inoculum was removed, and 1 ml of fresh medium was added. Every plate contained at least three replicates of uninfected cells. The infected cells and non-infected controls were incubated at 37°C (5% CO 2 with humidification) and harvested at 4, 8, and 24 hours after infection.

Primers and fluorescent oligonucleotide probes, containing fluorescent reporter dyes at the 5'-end and a quencher at the 3'-end (Table 1) , were commercially synthesized and HPLCpurified (Evrogen, Russia).

Total RNA was isolated from A549 cells using TRIZol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA concentrations and integrity were analyzed using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA).

Two micrograms of total RNA were treated by DNase (Promega) and then directly reverse transcribed using oligo-dT 16 primers and MMLV reverse transcriptase (Promega). Complementary DNA synthesis was carried out at 42°C for 60 min; products were stored at −20°C until use.

Real-time PCR assays were performed using the CFX96 Real-Time PCR System (Bio-Rad, USA). Evaluation of IFNL1, IFNL2-3, IL10RB, IFNLR1 

Data processing was carried out in Microsoft Excel. GraphPad Prism was used to evaluate the statistical significance of differences.

In our study, we examined cellular immune responses to infection of A549 epithelial cells with RNA viruses (IBV, IAV H1N1pdm09, IAV H3N2, RSV) and a DNA virus (AdV serotype 5 and 6) at the same MOI. The replicative cycle of influenza is about 8 hours. The production of mRNA and proteins of RSV reaches its peak by 15-20 hpi, while AdV life cycle is a bit longer. Cells were infected without trypsyn to exclude the possibility of infection with viral offspring. So, IFNs and ISGs production refers to a single replicative cycle of any virus. We choose 4 hpi, 8 hpi, and 24 hpi timepoints for comparison of IFNL expression kinetics. At first, we showed that the viral genomes are capable of effectively replicating in the A549 cell culture selected (Supplementary Materials, Figure S1 ). Hence, all of the selected viruses exhibited the ability to replicate and to form new viral particles in A549 cells.

Changes in IFN expression were virus specific. Significant increases in IFNL (1, 2/3) as well as IFNB mRNA levels were observed upon infection with all RNA viruses ( Figure  1a ,b). Increases in IFNL expression correlated with accumulation of RNA from these viruses. The kinetics of IFNL expression, in response to IBV infection, was fundamentally different compared to the other RNA viruses. Already at 4 hpi, an increase in IFNL level, by more than 10,000-fold, was observed compared with the control.

IFN-λ protein level in cell culture supernatants was assessed. It was found that a significant increase in IFN-λ is observed only with IBV or RSV infection (Figure 2a) . The levels of IFN-α and IFN-β increased in response to IBV and (A)H1N1pdm09 (Figure 2b,c) . Despite the fact that IFNs are the primary, universal link in the activation of the innate immune response, we have shown that stimulation of the type I and type III IFN systems is not only virus specific, but also strain specific.

We also evaluated changes in the expression of several ISGs in response to viral infection. In both virus-infected and uninfected cells, IFNs induce the expression of myxovirus resistance protein (MxA), which makes MxA an excellent marker for detecting activation of an IFN-dependent response (Haller et al., 2015) . We evaluated MxA expression kinetics at 4, 8, and 24 hpi (Figure 3a ). In general, MxA expression by 24 hpi increased significantly upon infection with all viruses. However, the MxA mRNA level in AdV infected cells was significantly lower than those with RNA virus infections. At 4 hpi with IBV, a significant increase in MxA expression (about two orders of magnitude) was noted. By 8 hpi with IBV, MxA expression had reached its maximum and was also significantly increased compared to all other groups. By 24 hpi with RSV, there was a significant increase in MxA expression.

The expression pattern for IFIT1 looked similar ( Figure 3b ). However, with AdV infection, no significant change in IFIT1 expression was observed. Therefore, it can be concluded that the increase in IFNL expression was largely synchronized with increased MxA and IFIT1 mRNA levels.

We also measured the expression of SOCS1 (Figure 3c ), which is an inducible negative regulator of IFN lambda. By 4 hpi, we did not find any significant changes in expression. By 24 hpi, however, SOCS1 expression was significantly increased in cells when infected with (A)H3N2, IBV, or RSV.

The levels of specific cytokines (IL-6, IL-8, IL-10) in cell culture supernatants were analyzed ( Figure 4 ). IL-6 levels were significantly increased with IBV and (A)H1N1pdm09. The IL-6 levels for RSV and (A)H3N2 also exceeded control values. Differences in IL-10 levels were found for IBV and (A)H1N1pdm09. With the (A)H3N2, RSV, and AdV5 viruses, infection led to an increase in the level of chemokine IL-8. Thus, secreted levels of IL-6 and IL-10 generally correlated with type I IFN production. A significantly increased IL-8 level was a specific feature of (A)H3N2 infection in A549 cells.

It should be noted that MDA5 and RIG-1 expression is induced by autocrine or paracrine action of IFNs, so that an increase in mRNA level of these genes can be considered as positive feedback loop that could further augments IFNs production. Already by 4 hpi, IBV infection led to an increase in the expression of both the RIG-1 and MDA5 cytosolic sensors (Fig. 5) . By 24 hpi, RIG-1 expression was also significantly increased in response to infection with IBV, A/H3N2, or RSV. Notably, an increase in MDA5 expression was observed for all RNA viruses.

Possible regulation of IFN-λ-dependent signaling activation by variation in IFNλR subunit expression was evaluated. The expression levels of IFNLR1 and IL10R2 were assessed. A significant decrease in the expression of IFNLR1 subunit (more than 5-fold compared to the control) was noted one day after IBV infection ( Figure 4) . Presumably, the decrease in IFNLR1 level was associated with a rapid increase in IFNL expression. There were no significant changes in the expression levels of IL10R2, a nonspecific IFNλR subunit.

In our previous work (Plotnikova et al., 2021) we showed that IFN-λ 1 exhibits antiviral activity against various RNA viruses (IAV, SARS-CoV-2, CHIKV). IFNs are a major component of innate defense against viruses. The production of endogenous IFN-λ by epithelial cells is a natural defense mechanism that limits the growth and spread of RNA viruses. In this work, we assessed the dynamics of IFNL and several ISGs expression in response to infection of A549 cells with respiratory viruses (H1pdm09, H3, IBV, RSV, AdV types 5 and 6). In present study it was shown that stimulated by IBV early induction of IFNL and ISGs expression is associated with a decrease in mRNA level of IFNLR1, the specific subunit of IFNλR.

It is known that the induction of IFNs, proinflammatory cytokines, and chemokines is associated with strain pathogenicity (Cole and Ho, 2017). When studying the IFN status of A549 cells, we showed that infection with RNA viruses led to a significant increase in mRNA, meanwhile AdV infection elicited only a weak increase in IFNL and IFNB mRNA ( Figure 1 ). With IBV, type III IFNs were extremely elevated, with a peak at 4 hpi. It has been observed, in monocyte-derived dendritic cells, that IBV induces early expression of IFNL1 and IFNB mRNA (as early as 2 hpi). IAV causes noticeable activation of these genes much later (only starting from 8-12 hpi) (Strengell et al., 2012; Sun Y et al., 2018). Differences in IFN expression kinetics, obtained here for IAV and IBV, on the whole agree with results described in the literature. In turns, AdV can evade the early IFN-dependent immune response.

In our work, we used AdVs (types 5 and 6) which belong to serotype C and exhibit high tropism for respiratory epithelial cells (Chahal et al., 2012) . Despite the absence of significant changes in IFNL1 expression, by 24 hours for both AdV5 and AdV6, an increase in IFNL2/3 mRNA (4 to 6-fold) was observed ( Figure 1 ). In the modern literature, it has been shown that AdV has a suppression system for the IFN-induced antiviral response (Chahal et al., 2012) .

We performed a qPCR analysis to determine the expression levels (4 and 24 hpi) of antiviral ISGs: MxA and IFIT1. Already by 4 hpi, both MxA and IFIT1 displayed markedly elevated mRNA levels with IBV infection. The level of IFIT1 mRNA significantly increased only when cells were infected with RNA viruses (Figure 3 ). In general, these ISGs profile was synchronized with IFNL expression. This way, we observed distinct IFNL and ISGs expression in case of the different respiratory viruses. Next, we aimed to define the regulatory factors that could influence on IFNL and ISGs expression profile.

It has been shown that IAV infection induces IFNL expression mainly through RIG-Idependent pathway (Wei et al., 2014) . Induction of IFN expression occurs already in response to IBV penetration into the cell; and RIG-I cytosolic RNA sensors play a key role in virus recognition (Mäkelä et al., 2015) . Presuming that both viral genome replication and the production of IFNs can lead to a change in the expression of cytosolic sensors by positive feedback mechanisms (RIG-I and MDA5 are also ISGs), we evaluated the expression of both RLRs at an early stage (4 hpi) and a late stage (24 hpi) of infection. Infection with RNA viruses resulted in an increase in MDA5 and RIG-1 expression by 24 hpi, with the exception of IAV H1N1 ( Figure 5) .

Type I and III IFNs can up-regulate SOCS proteins, which negatively regulate IFN signaling by inhibiting the JAK-STAT signaling pathway (Schneider et al., 2014) . Here, we found that SOCS1 expression was elevated in IAV H3N2, IBV, or RSV-infected cells 24 hpi. In general, these observations are consistent with the changes in RLR expression ( Figure 5 ). Importantly, SOCS1 mRNA level was not increased in case of IBV 4 hpi, whereas expression of other ISGs (MxA, IFIT1, RIG-I, MDA5) and IFNL was clearly elevated at this timepoint. It has been shown that the physiological role of SOCS1 proteins is to prevent tissue damage caused by the potent pro-inflammatory effects of type I IFNs (Blumer et al., 2017) . Along these lines, attenuation of SOCS1 expression can serve as a marker indicating an increased potential of IAV H1N1 and IBV to cause hypercytokinemia.

According to Sun's assumptions (Sun Y et al., 2018) , type I IFNs have the potential to induce inflammation in addition to antiviral function, while lambda IFNs promote the production of antiviral ISGs without the excessive inflammation. In our study, IBV infection was associated with a cytopathic effect and led to increases in the proinflammatory factors IFN-α, IFN-β, IFN-λ, IL-6, and IL-10. It should be noted that although IL-10 itself cannot be attributed to mediators that promote inflammation, it can be a marker of uncontrolled immunopathology (Guo and Thomas, 2017) . With influenza (A)H1N1pdm09, the production kinetics of these cytokines were generally the same, with the exception of IFN-λ. This may be evidence of a cytopathic effect of (A)H1N1pdm09 and IBV that is related to decrease in SOCS1 expression.

Modulation of IFN signaling can be accomplished by alteration of receptor subunit expression (Stanifer et al., 2019) . For instance, published work has established that in nasopharyngeal swabs of children with a severe course of rhinovirus, IFNLR1 expression was increased compared to samples of children infected with RSV (Pierangeli et al., 2018) . Evaluation of IFNλR subunit expression showed that only with IBV was there a slight decrease in IFNLR1 mRNA level, while non-specific subunit IL10R2 mRNA level did not change ( Figure 5 ). At the moment, there is not much information available regarding molecular mechanisms in negative regulation of IFNLR1 expression (Stanifer et al., 2019) . In any case, decreased IFNLR1 expression appears to be a natural compensatory mechanism realized in response to excessive activation of IFNλR-mediated signaling.

In present study we compared the expression profile of IFNL and several ISGs in response to infection A549 with a panel of widely disturbed respiratory viruses. A549 cells are standard cell line that a huge amount of virological experiments are carried out. Although the study could be considered as summarizing and generalization of previously known data, our work highlighted unique features of type III IFN production that should be taken into account by studies examining viral pathogenicity. We speculate that production of proinflammatory cytokines negatively correlate to SOCS1 expression. Importantly, we showed a decrease in IFNLR1 mRNA in case of IBV infection that implies the existence of negative ISGs expression regulation at IFNλR level. It could be either a specific feature of IBV or a consequence of early IFNL expression. Gene expression was analyzed via Δ Δ Ct method (relative to GAPDH). Statistical significance (p-value) was determined by ordinary one-way ANOVA, followed by a pairwise Dunnett's multiple comparisons test: **** -Adjusted P Value < 0.0001; *** -< 0.001; ** -< 0.01; * -< 0.05 compared to Cntr. Cntr -intact cells that were cultured in the same conditions and were not infected (instead, sterile medium F12K was added). At least three biological replicates were used for each experimental data point. Data are represented as mean ± SD. IFN-α (b) , and IFN-β (c) were measured by ELISA of cell supernatants 24 hours post infection Statistical significance (p-value) was determined by ordinary one-way ANOVA, followed by a pairwise Dunnett's multiple comparisons test: **** -Adjusted P Value < 0.0001 compared to Cntr. Cntr -intact cells that were cultured in the same conditions and were not infected (instead, sterile medium F12K was added). At least three biological replicates were used for each experimental data point. Data are represented as mean ± SD. Δ Ct method (relative to GAPDH). Statistical significance (p-value) was determined by ordinary one-way ANOVA, followed by a pairwise Dunnett's multiple comparisons test: **** -Adjusted P Value < 0.0001; *** -< 0.001; ** -< 0.01 compared to Cntr. Cntr -intact cells that were cultured in the same conditions and were not infected (instead, sterile medium F12K was added). At least three biological replicates were used for each experimental data point. Data are represented as mean ± SD. , and IL-10 levels were measured by ELISA in cell supernatants 24 hours post infection. Statistical significance (p-value) was determined by ordinary one-way ANOVA, followed by a pairwise Dunnett's multiple comparisons test: **** -Adjusted P Value < 0.0001; *** -< 0.001; ** -< 0.01 compared to Cntr. Cntr -intact cells that were cultured in the same conditions and were not infected (instead, sterile medium F12K was added). At least three biological replicates were used for each experimental data point. Data are represented as mean ± SD. Δ Ct method (relative to GAPDH). The expression of IFNL receptor subunits IFNLR1 (a) and IL10RB (b), as well as the cytosolic RLRs Rig-1 (c) and MDA5 (d), were assessed by RT-PCR. Statistical significance (p-value) was determined by ordinary one-way ANOVA, followed by a pairwise Dunnett's multiple comparisons test: **** -Adjusted P Value < 0.0001; *** -< 0.001; ** -< 0.01 compared to Cntr. Cntr -intact cells that were cultured in the same conditions and were not infected (instead, sterile medium F12K was added). At least three biological replicates were used for each experimental data point. Data are represented as mean ± SD. Figure S1 : The specified respiratory viruses are capable of replication in A549 cells. Comparison of relative genomic replication rates. The y-axis is the decimal logarithm of viral gene expression. Gene expression was calculated using the Δ Δ Ct method (relative to GAPDH). When calculating expression, the viral genome expression level at 2 hpi was used for normalization of viral replication at later time points. For example, IAV expression at 4 hpi or later was normalized to IAV expression at 2 hpi, etc. The x-axis shows the time after infection. Cntr -intact cells that were cultured in the same conditions and were not infected (instead, sterile medium F12K was added). At least three biological replicates were used for each experimental data point. 

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The authors would like to acknowledge the kind help of Edward S. Ramsay for his assistance (translation, editing) in preparation of this paper.

The authors declare that there are no conflicts of interests regarding the publication of this paper.

This work was supported by a Russian State Assignment for Fundamental Research (0784-2020-0023).