Cytokine production and signaling pathways in respiratory virus infection.

It has been confirmed that respiratory virus infections can induce abberant cytokine production in the host. These cytokines may be associated with both elimination of the virus and complications in the host, such as virus-induced asthma. Representative host defense mechanisms against pathogens, including bacteria and viruses, are mediated by the innate immune system. Cells of the innate immune system express essential molecules, namely pattern recognition receptors (PRRs), such as Toll-like receptors, nucleotide-binding oligomerization domain-like receptors, and retinoic acid-inducible gene-I-like receptors. These PRRs can recognize components of pathogens such as bacterial lipopolysaccharide, viral antigens, and their genomes (DNA and RNA). Furthermore, PRRs activate various signaling pathways resulting in cytokine production against pathogen infection. However, the exact mechanisms remain unknown. In this review, we mainly focus on the representative mechanisms of cytokine production through PRRs and signaling pathways due to virus infections, including respiratory virus infections. In addition, we describe the relationships between respiratory infections and virus-induced asthma.


INTRODUCTION
Coordination between innate and adaptive immunity against pathogens is indispensable in higher organisms including humans (Medzhitov, 2007). In particular, innate immunity plays a critical role during primary infection with various bacteria and viruses (Barbalat et al., 2011;Jarchum and Pamer, 2011;Kumar et al., 2011). The specific recognition of microorganisms may represent the basis of innate immunity (Barbalat et al., 2011;Jarchum and Pamer, 2011;Kumar et al., 2011). Specific recognition systems have gradually been clarified and the common platforms are Toll-like receptors (TLRs), the NLR family (nucleotide-binding oligomerization domain-like receptors), and the RLR family [RIG (retinoic acid-inducible gene)-I-like receptors] (Kumar et al., 2011;Yu and Levine, 2011). These molecules are called pattern recognition receptors (PRRs). PRRs can recognize lipopolysaccharides (LPS), viral antigens, and bacterial/viral genomes, leading to the activation of intrinsic signaling pathways (e.g., myeloid differentiation factor 88; MyD88) and the production of various cytokines (Barbalat et al., 2011;Jarchum and Pamer, 2011;Kumar et al., 2011;Ting Tan et al., 2013). The production of such cytokines may activate leukocytes and eliminate the infective agents (Chehadeh and Alkhabbaz, 2013;Ting Tan et al., 2013).
At present, over 50 cytokines have been discovered. They form networks and play pivotal roles in infectious and allergic diseases (Barnes, 2008;Desai and Brightling, 2012;Holgate, 2012). These cytokines are mainly produced by blood cells, lymphoid tissues, and epithelial cells. For example, interferons (IFNs), which are anti-viral cytokines produced by lymphocytes and epithelial cells, are dramatically induced by various viral infections such as influenza (Qin et al., 2011b;Högner et al., 2013;Lopušná et al., 2013). This induction may contribute to the elimination of viruses in vivo. Indeed, we use recombinant IFNs to treat chronic viral infections such as hepatitis C (Nagao et al., 2012;Slim and Afridi, 2012). On the other hand, aberrant induction of other cytokines such as interleukin (IL)-4 may induce various allergic diseases, such as virus-induced asthma (Baraldo et al., 2012;Krishnamoorthy et al., 2012). In addition, aberrant induction and an imbalance of various proinflammatory cytokines, for example, IL-1β, IL-6, and tumor necrosis factor (TNF), may induce severe systemic inflammatory response syndrome (Watanabe et al., 2003;Xu et al., 2012). Thus, various cytokines may be associated with the pathophysiology of inflammation and remodeling of the airways post-infection.
Acute respiratory illnesses (ARI) are the most common diseases in humans. Accumulating evidence suggests that around 80% of the causative agents of ARI may be respiratory viruses (Heymann et al., 2004;Fujitsuka et al., 2011). The prognosis is good in most patients with viral ARI; however, viruses causing ARI may be responsible for more severe diseases like bronchitis, bronchiolitis, and pneumonia (Domachowske and Rosenberg, 1999;Sigurs, 2002;Kusel et al., 2007). Furthermore, representative respiratory viruses such as respiratory syncytial virus (RSV) may induce bronchiolitis or pneumonia with wheezing in infants (Stein et al., 1999;Sigurs et al., 2000).
To better understand host defense mechanisms against viruses, it is important to clarify these molecular mechanisms. In this review, we focus on cytokine production and signaling pathways during viral infection. We also discuss the relationships between cytokine profiles and virus-induced asthma under the main theme "virus-induced asthma."

INFECTION AND INNATE IMMUNITY
Host defense mechanisms against microbial infections constitute the main purpose of innate immunity (an archaic term meaning natural resistance; Jarchum and Pamer, 2011;Kumar et al., 2011). The main platforms of the molecular groups against the pathogens include TLRs, the NLR family (nucleotide-binding oligomerization domain-like receptors), and the RLR family (RIG-I-like receptors). These molecules/receptors can recognize various components including LPS derived from bacteria, viruses, and fungi, viral antigens, and the pathogen genomes (Barbalat et al., 2011;Jarchum and Pamer, 2011;Kumar et al., 2011;Yu and Levine, 2011). Subsequent events activate innate immunity involved in cytokine production in the host (Barbalat et al., 2011;Kumar et al., 2011;Yu and Levine, 2011;Ting Tan et al., 2013). The innate immune system initiates a different mechanism against each pathogen (Chehadeh and Alkhabbaz, 2013;Kemp et al., 2013). Thus, these pathogen-associated receptors are called "PRRs" (Kawai and Akira, 2007;Pang and Iwasaki, 2012). Schematic illustrations of these families are shown in Figure 1.

NUCLEOTIDE-BINDING OLIGOMERIZATION DOMAIN FAMILY AND CYTOKINE PRODUCTION
In macrophages and epithelial cells, NLRs play a pivotal role in the recognition of bacteria and viruses as PRR molecules (Figure 1; Wells et al., 2011). At present, about 20 types of NLRs have been confirmed in humans (Schroder and Tschopp, 2010). The representative pathogen PRR-related NLRs are NLRP1, NLRP3 (cryopyrin), and NLRPC4 (Schroder and Tschopp, 2010). These molecules have both signal transduction domains in the N-terminal and leucine-rich repeats in the C-terminal (Schroder and Tschopp, 2010). Thus, NLRs show the properties of both PRR molecules and signaling molecules (Schroder and Tschopp, 2010).

RETINOIC ACID-INDUCIBLE GENE-I LIKE RECEPTORS FAMILY
Retinoic acid-inducible gene-I and MDA5 (melanoma differentiation-associated protein 5) are localized in the cytosol and may be able to bind to some ssRNA viruses such as RSV, influenza virus, dengue fever viruses (DFV), and hepatitis C virus, leading to the production of type I IFN (IFN-α/β) in fibroblasts (Breiman et al., 2005;Loo et al., 2008;Jamaluddin et al., 2009;Bustos-Arriaga et al., 2011). In particular, it is known that RIG-I binds to ssRNA (5triphosphate RNA) derived from influenza virus and induces type I IFN (Loo et al., 2008). Furthermore, both RIG-I and MDA5 can bind to DFV type 2 genome and induce the production of type I IFN (Qin et al., 2011a). However, the roles of these molecules in innate immunity are not known at present.

INFLAMMASOME, RLR-ASSOCIATED SIGNALING PATHWAYS, AND CYTOKINE PRODUCTION
Inflammasome as a PRR is a concept of the inflammatory reaction-associated protein complex (Schroder and Tschopp, 2010). It is suggested that both RIG-I and MDA5 can bind to an adaptor molecule, IPS-1(interferon-1β promoter stimulator 1), and activate NF-κB, resulting in the production of type I IFN (Schroder and Tschopp, 2010;Bauernfeind and Hornung, 2013). Inflammasome is composed of some protein complexes such as Apaf-1(apoptotic protease-activating factor 1), ASC (apoptosis-associated speck-like protein containing caspase recruitment domain), NOD (nucleotide-binding domain), and NALP (NACHT, LRR and PYD domain-containing protein; Schroder and Tschopp, 2010;Bauernfeind and Hornung, 2013). The complex recognizes various components of pathogens and uric acid as"danger signals" (Schroder and Tschopp, 2010;Bauernfeind and Hornung, 2013). After recognition of the signals, these signals activate ASC, leading to the conversion of procaspase-1 to caspase-1 (Schroder and Tschopp, 2010;Bauernfeind and Hornung, 2013). The protease caspase-1 activates proinflammatory cytokine precursors such as pro-IL-1β and pro-IL-18, leading to conversion to active forms of IL-1β and IL-18 (Schroder and Tschopp, 2010;Bauernfeind and Hornung, 2013). Interestingly, very recent studies suggest that various inflammatory diseases such as atherosclerosis and rheumatoid arthritis are associated with inflammasome, although the precise mechanisms are not known.

RELATIONSHIPS BETWEEN PRRs, SIGNALING PATHWAYS, AND CYTOKINE PRODUCTION IN RESPIRATORY VIRUS-INFECTED CELLS
In general, cytokine production in immunological cells such as lymphocytes may be induced through each cytokine receptor on the cells (Salek-Ardakani and Croft, 2010;Rossol et al., 2011). Certainly, this process may occur in virus-infected cells (He and Greenberg, 2002). As mentioned previously, cytokine production may trigger innate immunity through PRRs including TLRs, RLRs, and inflammasomes (NLRPs-pro-caspase-1 complex; Figure 1; Yu and Levine, 2011). These receptors and/or intracellular protein complexes induce phosphorylation of the signaling molecules. Although the precise mechanisms are not known, the phosphorylation cascades of the molecules lead to cytokine production in virus-infected cells (Yu and Levine, 2011). The representative data of virus infection-associated signaling pathways is shown in Figure 4. Briefly, a previous report showed that RSV infection in human fetal lung fibroblasts (MRC-5 cells) induces various cytokines through the activation (phosphorylation) of Akt (murine thymoma viral oncogene homolog/protein kinase B), p38MAPK (mitogen activated protein kinase), ERK1/2 (extracellular signal-regulated kinase), and IκB-α (Seki et al., 2013). Human rhinovirus (HRV) infection in human bronchial epithelium cells (BEAS-2B cells) activated p38MAPK, ERK1/2, and NF-κB (nuclear factor kappa B protein). Human parainfluenza virus (HPIV) infection in MRC-5 cells activated p38MAPK and IκB-α (Yoshizumi et al., 2010). However, it is not currently known how PRRs are associated with the production of these cytokines.
Previous in vitro studies have demonstrated that elevated IL-6, IL-8, and RANTES are found in sputum and serum in influenza virus infection (Yamaya, 2012). IL-6 and IL-8 were elevated in sputum and serum in HRV infection (Yamaya, 2012). Systemic avian influenza virus [subtype A(H5N1) virus] infection induced excessive production of proinflammatory cytokine, namely a cytokine storm (Ramos and Fernandez-Sesma, 2012). These results imply that cytokine production profiles may vary. Although the detailed information of the signaling pathways is not yet known, these differences may be associated with the pathophysiology of each respiratory virus infection (Schwarze and Mackenzie, 2013).

RELATIONSHIP BETWEEN CYTOKINE PRODUCTION DUE TO RESPIRATORY VIRUS INFECTION AND THE PATHOPHYSIOLOGY OF VIRUS-INDUCED ASTHMA
Viral infections clearly induce inflammation at infected sites. A variety of complicated pathophysiological events occur at these sites. In broad terms, these events may constitute converged cell death and regeneration (Rennard and von Wachenfeldt, 2011). The process of events has been named "remodeling" (Al-Muhsen et al., 2011). Cytokines derived from respiratory virus infections may be associated with airway remodeling (Kuo et al., 2011). It is suggested that the major production sources of cytokines are airway epithelium, fibroblasts, myofibroblasts, and leukocytes within infected regions (Westergren-Thorsson et al., 2010). These cytokines may be associated with remodeling processes following respiratory virus infections (Holtzman et al., 2002).

www.frontiersin.org FIGURE 4 | PRRs and virus infection-associated signaling pathways.
The innate immune response to the fusion protein of an important respiratory pathogen of humans, RSV, is mediated by TLR4. The RSV F protein induces translocation of TLR4 to the endosome together with TRAM. TLR3 is expressed in intracellular endosomes and responds to the presence of double-stranded RNA (dsRNA), which forms as a product of the replication of the majority of RNA viruses such as RSV. TLR3 and TLR4 activate TRIF-dependent signaling, which activates NF-κB and IRF-3, and results in the induction of proinflammatory cytokines and type I IFNs. Moreover, TLR4 is able to signal via both MyD88-dependent and -independent pathways and is able to activate a response via IRF-3, NF-κB, AP-1, ERK and IKK. These receptors recognize RSV and induce an appropriate antiviral innate immune response in the infected cells. Akt, protein kinase B; ERK1/2, extracellular signal regulating kinase1/2; PDK1, phosphoinositide-dependent kinase 1; PI3K: phosphatidylinositol-3 kinase.

CONCLUSION
Since the discovery of PRRs, remarkable progress has been made toward understanding the role of innate immunity against pathogens. However, the precise roles of PRRs, the mechanisms of intrinsic signaling pathways, and cytokine production with regard to PRRs are not fully understood. In addition, recent studies suggest that PRRs may be associated with various inflammatory diseases such as gout, rheumatoid arthritis, and atherosclerosis. It would be beneficial to clarify the functional relevancy of infectious diseases and other inflammatory diseases in the near future.