Complexes of Oligoribonucleotides with d-Mannitol Modulate the Innate Immune Response to Influenza A Virus H1N1 (A/FM/1/47) In Vivo

Rapid replication of the influenza A virus and lung tissue damage caused by exaggerated pro-inflammatory host immune responses lead to numerous deaths. Therefore, novel therapeutic agents that have anti-influenza activities and attenuate excessive pro-inflammatory responses that are induced by an influenza virus infection are needed. Oligoribonucleotides-d-mannitol (ORNs-d-M) complexes possess both antiviral and anti-inflammatory activities. The current research was aimed at studying the ORNs-d-M effects on expression of innate immune genes in mice lungs during an influenza virus infection. Expression of genes was determined by RT-qPCR and Western blot assays. In the present studies, we found that the ORNs-d-M reduced the influenza-induced up-expression of Toll-like receptors (TLRs) (tlr3, tlr7, tlr8), nuclear factor NF-kB (nfkbia, nfnb1), cytokines (ifnε, ifnk, ifna2, ifnb1, ifnγ, il6, il1b, il12a, tnf), chemokines (ccl3, ccl4, сcl5, cxcl9, cxcl10, cxcl11), interferon-stimulated genes (ISGs) (oas1a, oas2, oas3, mx1), and pro-oxidation (nos2, xdh) genes. The ORNs-d-M inhibited the mRNA overexpression of tlr3, tlr7, and tlr8 induced by the influenza virus, which suggests that they impair the upregulation of NF-kB, cytokines, chemokines, ISGs, and pro-oxidation genes induced by the influenza virus by inhibiting activation of the TLR-3, TLR-7, and TLR-8 signaling pathways. By impairing activation of the TLR-3, TLR-7, and TLR-8 signaling pathways, the ORNs-d-M can modulate the innate immune response to an influenza virus infection.


Introduction
The influenza A virus causes pandemics, which makes it responsible for high mortality rates and great economic losses every year [1]. Currently, annual trivalent or quadrivalent vaccines are the main anti-influenza therapeutics. However, rapid antigenic drift and the shift in influenza viruses make it difficult to select appropriate vaccine strains [2][3][4][5]. Furthermore, the anti-influenza licensed drugs are currently limited to oseltamivir, zanamivir [6], amantadine, and rimantadine [7,8]. However, the emergence of drug-resistant influenza variants [8][9][10] and co-infection with influenza and other respiratory viruses decrease with regard to the efficiency of these drugs [11,12]. Therefore, new anti-influenza therapeutics with novel mechanisms of action are urgently required to combat the persistent threat of influenza viruses.
Infection by the influenza A virus is frequently characterized by considerable inflammation [13]. Lung tissue damage is a consequence of diseases associated with influenza A virus infections since inflammation results from the release of pro-inflammatory chemokines and the recruitment of To determine the influence of the ORNs-D-M on up-expression of some pro-oxidation genes induced by the influenza virus, the mRNA levels of nos2, arg2, and xdh in mice lungs were determined by RT-qPCR after prevention and treatment with the ORNs-D-M of the influenza virus infection. As shown in Figure 1a, the overexpression of these investigated genes was detected in mice lungs 48 h after infection with the influenza virus when compared to the control. Conversely, the ORNs-D-M injection into healthy mice as a positive control of the ORNs-D-M, the mRNA expression levels of nos2, arg2, and xdh remained unchanged when compared to the healthy ones. The ORNs-D-M injection for prevention and treatment of the influenza infection reduced the mRNA level of nos2, arg2, and xdh expression in comparison with the virus-infected mice.
After 48 h of infection with the influenza virus and both prevention and treatment with the ORNs-D-M of the influenza infection, the level of lipid peroxidation (LPO) products in mice lungs was measured by thiobarbituric acid reactive species (TBARS). The level of TBARS in influenza-infected mice lungs was found to be 43% higher than the control while both prevention and treatment with ORNs-D-M of the influenza virus infection decreased the TBARS level by 18% and 15%, respectively, when compared to the infected mice ( Figure 1b). An unchanged TBARS level was observed in with ORNs-D-М of the influenza virus infection decreased the TBARS level by 18% and 15%, respectively, when compared to the infected mice ( Figure 1b). An unchanged TBARS level was observed in mice lungs after the ORNs-D-М injection without the influenza virus infection and was compared to the control.

The ORNs-D-М Inhibit the Overexpression of Cytokines, Chemokines, and ISGs Induced by the Influenza Virus In Vivo
We also investigated the influence of the ORNs-D-М on expression of cytokines, chemokines, and ISGs at the influenza virus infection. As shown in Figures 2 and 3, the increased mRNA expression level of cytokines ifnε, ifnk, ifna2, ifnb1, and ifnγ and ISGs oas1a, oas2, oas3, and mx1 was determined 48 hours after influenza virus infection in comparison with the control. It was also detected to increase the mRNA level of the pro-inflammatory cytokines il6, il1b, il12a, and tnf and chemokines ccl3, ccl4, сcl5, cxcl9, cxcl10, and cxcl11 induced by the influenza virus infection (see Figures 4 and 5). However, unchanged mRNA expression of these investigated genes was observed in lungs of ORNs-D-М-treated mice without the influenza virus infection in comparison with the control (Figures 2-5).

The ORNs-D-M Inhibit the Overexpression of Cytokines, Chemokines, and ISGs Induced by the Influenza Virus In Vivo
We also investigated the influence of the ORNs-D-M on expression of cytokines, chemokines, and ISGs at the influenza virus infection. As shown in Figures 2 and 3, the increased mRNA expression level of cytokines ifnε, ifnk, ifna2, ifnb1, and ifnγ and ISGs oas1a, oas2, oas3, and mx1 was determined 48 h after influenza virus infection in comparison with the control. It was also detected to increase the mRNA level of the pro-inflammatory cytokines il6, il1b, il12a, and tnf and chemokines ccl3, ccl4, ccl5, cxcl9, cxcl10, and cxcl11 induced by the influenza virus infection (see Figures 4 and 5). However, unchanged mRNA expression of these investigated genes was observed in lungs of ORNs-D-M-treated mice without the influenza virus infection in comparison with the control (Figures 2-5). In addition, both prevention and treatment with the ORNs-D-M during the influenza virus infection led to a reduced mRNA level of ifnε, ifnk, ifna2, ifnb1, ifnγ, oas1a, oas2, oas3, mx1 (Figure 4), il6, il1b, il12a, tnf, ccl3, ccl4, ccl5, cxcl9, cxcl10, and cxcl11 ( Figure 5) compared with the influenza control. Conversely, the rnasel mRNA expression remained unchanged in lungs of the infected mice, the treated mice with the ORNs-D-M without the influenza virus infection, and the treated mice with the ORNs-D-M for prevention and treatment of the influenza virus infection in comparison with the control (see Figure 3). In addition, both prevention and treatment with the ORNs-D-М during the influenza virus infection led to a reduced mRNA level of ifnε, ifnk, ifna2, ifnb1, ifnγ, oas1a, oas2, oas3, mx1 (Figure 4), il6, il1b, il12a, tnf, ccl3, ccl4, сcl5, cxcl9, cxcl10, and cxcl11 ( Figure 5) compared with the influenza control. Conversely, the rnasel mRNA expression remained unchanged in lungs of the infected mice, the treated mice with the ORNs-D-М without the influenza virus infection, and the treated mice with the ORNs-D-М for prevention and treatment of the influenza virus infection in comparison with the control (see Figure 3).       Investigating mRNA of the tlr3, tlr7, tlr8, nfkbia, and nfnb1, we found the same tendency as during studding of the mRNA level of xdh, nos2, arg2, oas1a, oas2, oas3, mx1, ifnε, ifnk, ifna2, ifnb1, ifnγ, ccl3, ccl4, сcl5, cxcl9, cxcl10, cxcl11, il6, il1b, il12a, and tnf (Figures 1a and 2 -5). For example, it was shown that the mRNA of nfkb1, nfkbiα, tlr3, tlr7, and tlr8 increased in mice lungs 48 hours after infection with the influenza virus and compared to the control (see Figure 6a). However, the mRNA of nfkb1, nfkbiα, tlr3, tlr7, and tlr8 after both prevention and treatment with ORNs-D-M of the influenza virus infection decreased vs. the influenza-infected mice. Additionally, unchanged mRNA expression of these investigated genes was observed in lungs of the ORNs-D-М-treated mice without the influenza virus infection and compared to the control.
By using a Western blot assay, protein levels of the nfkb1, nfkbiα were studied in mice lungs that had been infected with the influenza virus and had been treated with the ORNs-D-М for prevention Investigating mRNA of the tlr3, tlr7, tlr8, nfkbia, and nfnb1, we found the same tendency as during studding of the mRNA level of xdh, nos2, arg2, oas1a, oas2, oas3, mx1, ifnε, ifnk, ifna2, ifnb1, ifnγ, ccl3, ccl4, ccl5, cxcl9, cxcl10, cxcl11, il6, il1b, il12a, and tnf (Figures 1a and 2, Figures 3-5). For example, it was shown that the mRNA of nfkb1, nfkbiα, tlr3, tlr7, and tlr8 increased in mice lungs 48 h after infection with the influenza virus and compared to the control (see Figure 6a). However, the mRNA of nfkb1, nfkbiα, tlr3, tlr7, and tlr8 after both prevention and treatment with ORNs-D-M of the influenza virus infection decreased vs. the influenza-infected mice. Additionally, unchanged mRNA expression of these investigated genes was observed in lungs of the ORNs-D-M-treated mice without the influenza virus infection and compared to the control.
By using a Western blot assay, protein levels of the nfkb1, nfkbiα were studied in mice lungs that had been infected with the influenza virus and had been treated with the ORNs-D-M for prevention and treatment of the influenza virus infection (see Figure 6b). The protein level of nfkb1, nfkbiα increased in the influenza-infected mice lungs when compared to the control while both ORNs-D-M prevention and treatment of the influenza infection reduced the protein level of nfkb1 and nfkbiα compared to the influenza-infected mice. Unchanged protein expression of these genes was observed in lungs of the uninfected mice that had been treated with the ORNs-D-M in comparison with the control. and treatment of the influenza virus infection (see Figure 6b). The protein level of nfkb1, nfkbiα increased in the influenza-infected mice lungs when compared to the control while both ORNs-D-М prevention and treatment of the influenza infection reduced the protein level of nfkb1 and nfkbiα compared to the influenza-infected mice. Unchanged protein expression of these genes was observed in lungs of the uninfected mice that had been treated with the ORNs-D-М in comparison with the control. Additionally, the infectious titer of the influenza virus after prevention and treatment with the ORNs-D-М was investigated using the TCID50 assay. As shown in Table 1, 48 hours after infection with the influenza virus, both prevention and treatment with the ORNs-D-М decreased the infectious titer of the influenza virus in comparison with the influenza control. Similar tendency was observed at studding of weight loss. Additionally, the infectious titer of the influenza virus after prevention and treatment with the ORNs-D-M was investigated using the TCID 50 assay. As shown in Table 1, 48 h after infection with the influenza virus, both prevention and treatment with the ORNs-D-M decreased the infectious titer of the influenza virus in comparison with the influenza control. Similar tendency was observed at studding of weight loss.

Discussion
The influenza virus induces lung tissue damage by causing overproduction of free radicals including reactive nitrogen intermediates (RNIs) (NO, NO 2 , HNO 2 ) and reactive oxygen species (ROSs) (O 2 − , OH, H 2 O 2 − ) [32]. During acute influenza virus infection, an increased level of free radicals can directly contribute to cell death of infected lung tissue and exacerbate pathology caused by the influenza virus replication [33]. The important pro-oxidation genes, which are responsible for generating free radicals, are xdh and nos2 [34]. The pathogenic role of RNIs and ROSs during influenza virus infection realizes by increasing the enzyme activity of NOS, XO, and mRNA expression of nos2 and xdh in influenza-infected lungs [34][35][36]. Arginase activation in the airway epithelial cells causes a reduction in nos2 expression, which reduces NO generation [37]. In the presented study, we found the overexpression of xdh, nos2, and arg2 genes induced by the influenza virus FM147 infection (Figure 1a). In our previous studies, we found that the ORNs-D-M have antiviral activity against RNA and DNA viruses with a wide spectrum of antiviral action [24]. The total yeast RNA possesses an anti-inflammatory action and stabilize nitric oxide synthase (NOS) activity in vitro and in vivo [28][29][30][31]. In this study, we found that the ORNs-D-M injection for prevention and treatment reduced the nos2, arg2, and xdh up-expression induced by the influenza virus infection (Figure 1a).
Oxidative stress induced by overproduction of the free radical increases the LPO level during influenza virus H1N1 infection [38]. Next, we estimated an ability of the ORNs-D-M to affect the level of LPO products in mice lungs during the influenza virus infection and detected that the ORNs-D-M injection for prevention and treatment can decrease the level of LPO in influenza-infected mice (Figure 1b), which indicates that these ORNs-D-M likely decrease the protein level of nos2, arg2, and xdh during the influenza virus infection. These results suggest that the ORNs-D-M can impair the up-regulation of nos2, xdh, and arg2 genes and increase LPO products induced by the influenza virus, which suppresses NOS activity and stabilizes the membrane [28,39].
The protein complex NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) regulates transcription of a large number of genes associated with inflammatory cytokines and downstream ISGs [61][62][63]. Influenza virus infection of airway epithelium cells is dependent on an active NF-kB signaling pathway [64,65]. Cells with low NF-kB activity were virtually resistant to influenza virus infection while activation of the NF-kB signaling pathway by influenza virus infection is not sufficient for allowing infection in these cells [64]. During the influenza virus infection, NF-kB-dependent gene expression is mediated by overexpression of the viral proteins, overproduction ROSs, and activation of the IkB kinase [64]. In the presented study, we found upregulation of the NF-kB1 and the NFKBia-induced influenza virus FM147 (Figure 6a,b). We also estimated an ability of the ORNs-D-M to effect upregulation of the NF-kB1. NFKBia induced the influenza virus and found that the ORNs-D-M, which had been injected into mice for prevention and treatment of the influenza virus infection, have an inhibition effect on the upregulation of the NF-kB1. NFKBia induced the influenza virus. Our results also suggest that, by inhibiting overexpression of the NF-kB1 and NFKBia, the ORNs-D-M can impair influenza-induced overexpression of the cytokines, chemokines, ISGs, and pro-oxidation genes and inhibit influenza virus replication dependent on an active NF-kB signaling pathway [64,65]. The innate immune system is the first stage of protection of an organism against invading pathogens and is associated with a highly conserved host-cell signaling mechanism [66]. Different pattern-recognition receptors are expressed to recognize pathogen-associated molecular patterns, which initiate the signaling cascades inducing cytokine production [66]. The innate immune system recognizes the influenza virus by pattern-recognition receptors such as TLR3 (double-stranded RNA), TLR7 (single-stranded RNA), TLR8 (single-stranded RNA), retinoic acid-inducible gene I (RIG-I) (5 -triphosphate RNA), and the NOD-like receptor family member (various stimuli) [67][68][69]. Activation of TLRs triggers a cascade of signals leading to the activation of NF-kB and the activation of NF-kB dependent production of pro-inflammatory cytokines including IL-6 and TNF-α and the production of type I IFNs [70,71]. In this study, we also found that the influenza virus FM147 induced upregulation of the tlr3, tlr7, and tlr8. Furthermore, in our study, we found that the ORNs-D-M injection for prevention and treatment inhibit the up-expression of tlr3, tlr7, and tlr8 induced by the influenza virus infection (Figure 6a,b). These results suggest that the ORNs-D-M can effectively antagonize TLR-3, TLR-7, and TLR-8, inhibit NF-kB activity, and suppress the secretion of the cytokines, chemokines, and pro-oxidants during the influenza virus infection [16,72].
In addition, we evaluated a decreasing replication of the influenza virus by the ORNs-D-M in this in vivo experiment. Both the ORNs-D-M injection for prevention and treatment reduced the infectious titer of influenza virus by 1.4 and 2.2 lgTCID 50 during the influenza infection (Table 1) [26]. Data analysis of the influenza virus replication and gene expression of innate immune responses shows that a single injections of the ORNs-D-M for the influenza prevention and treatment inhibit partial replication of the influenza virus and normalize the up-expression genes of innate immune responses induced by the influenza infection (for example infβ1, nfkbiα). In our previous and current studies, we found out that the ORNs-D-M injected for treatment decrease the influenza virus replication better than the ORNs-D-M injected for prevention [26]. Conversely, the ORNs-D-M injection for prevention inhibits the up-expression of genes (nos2, arg2, ifnk, ifnγ, oas3, il6, il1b, il12a, ccl3, ccl4, cxcl9, cxcl11, nfkbiα, tlr3, tlr7, and tlr8) induced by the influenza virus better than the ORNs-D-M injection for treatment. The obtained results suggest that, besides inhibiting activity of influenza viral proteins (NA, HA) [26,27], modulating the innate immune response to influenza virus infection by the ORNs-D-M can be another mechanism responsible for their anti-influenza activity.
Complexes of nurture ORNs with D-mannitol are total yeast RNA with a dominant fraction of 3-8 nucleotides modified with D-mannitol. The nurture ORNs bind with low affinity and non-specificity to some target molecules [73]. We believe that in the complexes, there are sequences that bind to the viral protein and change their conformation and activity as well as sequences that bind to the Toll-like receptors. We suggested that different sequences with different actions provide the complexes with a wide range of biological activities. In future research, ORNs-D-M sequences and their binding regions to the Toll-like receptors should be identified and characterized.

Materials and Methods
The

Mouse In Vivo Experiment
The BALB/c mice (14-16 g), 6 to 8 weeks of age, were distributed into five groups as follows:

TCID 50 Assay
The lungs (100 mg) were homogenized by liquid nitrogen and were soluted into 0.5 mL in sterile NaCl, 0.9%. Then samples were centrifuged at 4000 g for 20 min at 4 • C and supernatants were removed. The influenza virus infectious titers were determined in the supernatants using the TCID 50 assay by the method of Reed-Muench [74]. The infectious titer of the influenza virus was evaluated by the infection of MDCK cells [27].

Lipid Peroxidation Assay
Mouse lungs were homogenized by liquid nitrogen and were soluted into 3 mL of 50 mM phosphate buffered saline (PBS) (Sigma Aldrich, St. Louis, MO, USA), pH = 7.4. After 100 µL of the homogenized sample was added into 2.5 mL of 0.025 M Tris-HCl, pH = 7.4 (with 0.175 M KCl), 1 mL of 17% of trichloroacetic acid solution was centrifuged at 4000 g for 10 min at 4 • C. The protein was measured by the method from Lowry et al. [76]. Endogenous LPO products reacting with 2-thiobarbituric acid (TBA-reactive substances, TBARS) were measured using the SPECORD 210 Plus (Analytik Jena AG, Jena, Germany) at a 532 nm wavelength, which was described by Asakawa & Matsushita [77]. The data were presented as mean ± SD.

Western Blot Analysis
The lungs (20 mg) were lysed in PBS (Sigma Aldrich, St. Louis, MO, USA) with a protease inhibitor cocktail (Sigma Aldrich, Louis, MO, USA). The concentrations of total protein in the lysates were determined by the Bradford protein assay using a Bradford Reagent (Sigma Aldrich, St. Louis, MO, USA), according to the 96 well plate assay protocol suggested by the manufacturer. A total of 30 µg of protein samples was resolved on 10% SDS-PAGE. After the electrophoretic transfer of proteins onto the nitrocellulose membrane (Amersham BioSciences, Buckinghamshire, UK), the membranes were incubated overnight at 4 • C with primary antibodies using the following dilutions: NF-κB 1 (1:500; Rabbit monoclonal; cat. no. 13586; Cell Signaling Technology, Leiden, Netherlands), IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) (1:500; mouse monoclonal; cat. no. sc-1643; Santa Cruz Biotechnology, Dallas, Texas, USA), β-actin (1:20,000; rabbit polyclonal cat. no. A2103; Sigma Aldrich, St. Louis, MO, USA). Afterward, Incubation was performed with horseradish peroxidase (HRP)-conjugated by secondary anti-mouse (1:3000; cat. no. 7076; Cell Signaling Technology, Leiden, Netherlands) and anti-rabbit antibodies (1:3000; cat. no. 7074; Cell Signaling Technology, Leiden, Netherlands) at room temperature for 1 h. The blots were developed and detected with an enhanced chemiluminescence detection kit (West Pico PLUS Chemiluminescent Substrate; Thermo Scientific, Waltham, MA, USA) by following the manufacturer's instructions. Chemiluminescent signals were captured digitally using a ChemiDoc XRS + system (BIO-RAD, Hercules, CA, USA). Relative levels of the protein were quantified by a densitometric analysis [78]. The volume (intensity) of each band was quantified using Image Lab Software (BIO-RAD,