Influenza A viruses primarily target and infect airway and alveolar epithelial cells, which contain the SA glycans as receptors, thus causing alveolar epithelial injury and eventually failure of gas exchange (30, 31). Hence, human IAV infection may lead to acute respiratory distress syndrome (ARDS) and even death (32, 33). Various subtypes of IAVs have different abilities to attach human upper respiratory tract (URT). For example, H1N1 adsorbs abundant ciliated epithelial cells and goblet cells, whereas H5N1 hardly attaches to these cells in human URT (30). In contrast, H5N1 infects alveolar macrophages as well as alveolar epithelial cells (34, 35). Additionally, human and avian IAVs could target and infect various cells in the lower respiratory tract (LRT). It has been observed that H1N1 and H3N2 attach more abundantly to human trachea and bronchi and adsorb more cell types than H5N1 (36). Of note, low pathogenic (LP) avian IAVs generally do not cause a severe pneumonia because they bind human submucosal gland cells and their mucus which can restrain and remove these viruses before approaching LRT (37). However, high pathogenic (HP) H5N1 is able to infect type II pneumocytes as these cells possess an active metabolism, therefore providing a possibility to develop severe pneumonia (35). Moreover, it has been shown that H5N1 reduces proliferation of infected endothelial cells and causes excessive production of cytokines, leading to the lung damage (38, 39).

Hemagglutinin protein on the viral envelope can recognize SA receptors on the surface of the host cells, which is the most crucial step in the process of IAV invasion into an organism (40, 41). Influenza viruses have two common cellular receptors: SA α-2,3 galactose (SAα-2,3-Gal) and SA α-2,6 galactose (SAα-2,6-Gal) (42). It has been found that human influenza strains preferentially attach to the SAα-2,6-Gal receptor (43). The 2,6 SA receptors are present on respiratory epithelial cells of the human URT, while 2,3 SA receptors are found on epithelial cells of the birds, pigs, and in the LRT of humans (44). Thus, variation of cell surface receptors contributes as a major barrier to cross-species and zoonotic transmissions of influenza virus. Hydrolysis of HA0 precursor gives rise to a dimer HA1–HA2 linked by disulfide bonds. HA1 binds to cellular receptors and HA2 facilitates the fusion of the virus to cellular membrane (45). It is also found that HA of influenza virus binds to C-type lectin as an alternative of SA (46). Virus attachment to host cell induces endocytosis using the clatherin and clavoline-dependent mechanism. Following the endocytosis, release of viral particle is pH-dependent physiological event that occurs at late lysosome (47). Low pH of endosome opens the M2 proton channel for proton flux into the virus and triggers the uncoating and subsequent release of viral RNPs (48). The free viral RNA is imported to the nucleus by interacting with the cellular importin-α/β for replication (49, 50). Although it is recognized that both virus preference for SA receptor and host restriction factors are critical for IAV binding and entry into host cells, further studies are needed to unravel the complicated mechanisms underlying the interaction of IAVs with human airway cells.

The innate immune response is the first line of defense against viral infection which is rapid in response, but nonspecific. During the IAV infection, viral conserved components called pathogen associated molecular patterns (PAMPs) are recognized by host pathogen recognition receptors (PRRs), such as retinoic acid-inducible gene-I protein (RIG-I) and toll-like receptor (TLR), leading to activation of innate immune signaling that finally induces the production of various cytokines and antiviral molecules (51, 52). These PAMPs have certain characteristic of viral RNA that are not shared by cellular RNAs, such as regions of double-stranded RNA (dsRNA) or the presence of a 5’-triphosphate group (53, 54).

Pathogen recognition receptors have the ability to distinguish self from non-self molecules within the infected cells. RIG-I is the main receptor to recognize the intracellular ssRNA and transcriptional intermediates of IAVs in the infected host cells (Figure 1). Non-self RNA and transcriptional products of IAVs in the cytoplasm are also sensed by melanoma differentiation-associated gene 5 (55). Following the recognition of PAMPs, RIG-I is activated and its caspase activation and recruitment domains (CARDs) are exposed. Then the CARD is modulated by dephosphorylation or ubiquitination by E3 ligases, such as TRIM-containing protein 25 (TRIM25) (56). Thus, CARD-dependent association of RIG-I and MAVS trigger the downstream transduction signaling at the outer mitochondrial membrane (57). Subsequently, the transcription factors, including interferon regulatory factor 3 (IRF3) and IRF7, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) are activated, causing the expression of a variety of IFNs and cytokines (Figure 1) (58).

Toll-like receptors are critical PRRs that sense the pathogen outside of cell membrane and internally at endosomes and lysosomes (59). TLRs expressed on cell membrane are TLR1, 2, 4, 5, and 6 that recognize PAMPs derived from bacteria, fungi, and protozoa, while TLR3, 7, 8, and 9 are expressed on the surface of endosomes and lysosomes and exclusively recognize nucleic acid PAMPs derived from various viruses, including IAVs (60–62). TLR3, TLR7, and TLR8 are involved in sensing the IAV components in cytoplasmic endosomes during the virus replication. It is known that TLR3 recognizes dsRNA in endosomes (63). Interestingly, it was shown that TLR3 may recognize recently unidentified RNA structures that are present in phagocytosed cells infected with IAVs (64). In plasmacytoid dendritic cells (pDCs), TLR7 recognizes the ssRNA of the influenza virions that are taken up into the endosomes (65). Then downstream signaling of TLR7 is activated via the adaptor protein myeloid differentiation factor 88 in pDCs, which results in the activation of either NF-κB or IRF7 to induce the expression of pro-inflammatory cytokines and type I IFNs, respectively (65). In macrophages and DCs, TLR3 interacts with TIR-domain-containing adapter-inducing interferon-β. Such interaction results in activation of the serine–threonine kinases IκKε (IKKε) and TBK1 that phosphorylate IRF3 to regulate the expression of IFN-β (66). In human monocytes and macrophages, TLR8 is stimulated by its ligand ssRNA, leading to the production of IL-12. However, the relationship between TLR8 and IAV infection has not been defined (67).

Moreover, some NOD-like receptors, such as NOD-like receptor family pyrin domain containing 3 (NLRP3, also known as cryopyrin) and NLR apoptosis inhibitory protein 5, have been observed to be activated upon cellular infection with IAV (68). NLRP3 is expressed by number of cells, including DCs, macrophages, neutrophils, monocytes, and human pulmonary epithelial cells (69, 70). Three signals are required for activation of inflamosome to trigger cytokine production. First, NLRP3 is activated through pathogen detection, which induces the expression of the genes encoding pro-IL-1β, pro-IL-18, and pro-caspase-1 (71). Second, the IAV-encoded M2 ion channel is required to trigger NLRP3 inflammasome activation and cleavage of pro-IL-1β and pro-IL-18 (72). Recently, it was revealed that accumulation of IAV PB1-F2 in the lysosome of macrophages acted as the third signal leading to activation of the NLRP3 inflammasome (73).

Activation of specific transcription factors including, NF-κB, IRF3, and IRF7 during IAV infection results in translocation of these factors into the nucleus where they initiate the transcription of genes encoding IFNs and pro-inflammatory cytokines (TNF, IL6, IL1β, etc.). It is well known that type I IFNs, such as IFN-α and IFN-β, and type III IFNs also known as interferon lambdas (IFN-λ1, IFN-λ2, IFN-λ3, IFN-λ4) play important roles in antiviral response in both virus-infected and uninfected cells (74). Infection with IAV induces robust expression of type I and type III IFN genes (75). Following the expression, IFN-α and IFN-β interact with IFN-α/β receptors (IFNAR), while IFN-λs interact with IFNL receptors (IFNLR) in an autocrine or paracrine manner, which activate Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway. Phosphorylated STAT1 and STAT2 bind with IRF9 to form a complex ISG factor 3 (ISGF3). ISGF3 translocates into nucleus and binds with IFN-stimulated response element, which triggers the transcription of numerous IFN-stimulated genes (ISGs) (Figure 1) (76). Previous studies suggest that type I and type III IFNs provide similar defense against IAV infection in wild-type mice (77). It was further shown that IAV infection induced expression of same type of ISGs in epithelial cells of wild type, IFNAR- or IFNLR-deficient mice (78). Only when both IFN-α/β and IFN-λ receptors in mice were knocked out, the animals failed to restrict non-pathogenic influenza virus (77). In spite of similar role of IFNα/β and IFN-λs to a certain extent, some notable differences exist. For example, it has been observed that mice infected with influenza virus showed higher pulmonary inflammation and mortality after treatment with IFNα, while IFN-λ remained protective (79, 80).

These ISGs target different steps of IAV life cycle. For example, viral entry into cells can be restricted by several ISGs, including Mx family, interferon-induced transmembrane protein family (IFITMs), cholesterol 25-hydroxylase (CH25H), and TRIM proteins. Mx family is comprised of MxA and MxB in human, Mx1 and Mx2 in mice. Mx proteins are produced by various cells, such as hepatocytes, DCs, endothelial cells, and immune cells (81). Mx proteins were the first ISGs identified to restrict IAV infection of mice (82). Recently, a study showed that MxA could retain incoming viral genome in the cytoplasm of human cell (83). In addition, nuclear Mx1 in mice impedes the process of early transcription of IAV activated by the polymerase in the nucleus (84). It is thought that the sensitivity of IAVs to MxA depends on their nucleocapsid proteins, and usually avian strains of IAVs are more sensitive to MxA than human strains (85, 86). However, role of MxB in humans or Mx2 in mice during IAV infection is poorly understood (87).

Interferon-induced transmembrane protein families are known as new ISGs that restrict early viral entry by altering the cellular membrane properties like cell adhesion, fluidity, and spontaneous curvatures (88, 89). It has been found that IFITM proteins restrict the replication of IAVs by interfering virus–host cell fusion following viral attachment and endocytosis (90). Another antiviral ISG, CH25H is an integral element of cellular membranes and upregulated by IFN signaling. CH25H enzymatic activity converts cholesterol to soluble 25-hydroxycholesterol (25HC), which is involved in antiviral defense against enveloped viruses, including influenza virus through blocking viral fusion. Recently, it was suggested that high concentration of 25HC causes physical changes of cellular membrane properties to prevent viral fusion (91). Moreover, previous studies showed that IFN-activated STAT1 bound to the promoter proximal region of the Ch25h gene to stimulate the production of 25HC that enhanced innate immune response against IAV (92, 93). In addition, TRIM proteins play multiple roles in antiviral immunity. TRIM25, an E3 ubiquitin ligase, is considered to regulate the re-localization of RIG-I to mitochondrion and signal transduction to MAVS for innate immune response against the viral infection (94). TRIM22 blocks IAV genome encapsidation and degrades nucleoprotein of IAV by polyubiquitination (95). TRIM32 binds with influenza PB1 RNA polymerase, reduces the polymerase activity, and thus restricts the viral replication (96).

There are increasing number of ISGs that regulate viral mRNA expression and protein translation. For example, zinc finger antiviral protein (ZAP), oligoadenylate synthase and ribonuclease L (OAS-RNase L), PKR, and ISG15 are involved in the regulation of IAV mRNA levels and protein synthesis. ZAPs inhibit the expression of IAV PB2 and PA proteins by reducing the viral mRNA expression and blocking its translation (97, 98). OAS-RNase L can destroy viral RNA in the cytosol of host cells and finally halt the protein synthesis process and viral replication. Mice with reduced expression of RNase L are more prone to influenza virus infection (99). PKR is expressed by all kind of cells and upregulated by type I and type III IFNs (100). PKR expressed in inactive form is activated by influenza virus infection. PKR is a known anti-IAV factor that binds to viral dsRNA and suppresses viral protein synthesis. Genetically deficient PKR mice are highly susceptible to influenza virus (101). ISG15 is an ubiquitin-like protein and restrict viral replication by interfering with virus release and translation of viral proteins (102).

In addition, many other ISGs are also involved in innate immunity against IAV infection. These include viperin, tetherin, and so on (103, 104). It has been shown that overexpression of viperin (also known as RSAD2) restricts the release of influenza virus by affecting the formation of lipid rafts specific microdomains that are particular budding sites of the virus (105). Tetherin is another potential host antiviral factor. In 2008, it was reported that tetherin inhibited retrovirus release (106). Tetherin appeared to limit cellular export of viral progenies by internalizing and degrading them exported to the surface of infected cells (107). It was also found that tetherin restrained the influenza virus by tethering and degrading newly budded viral particles (108). Recently, it has been known that tetherin was able to suppress the budding of several laboratory oriented and seasonal influenza strains, but unable to restrict pandemic influenza A/Hamburg/4/2009 and wild-type influenza virus particles (103, 104, 108).

Airway epithelial cells are the first target of IAVs. These cells produce antiviral and chemotactic molecules that initiate immune responses by rapid recruitment of innate effector cells, such as NK cells, monocytes, and neutrophils. All cell types have their own unique mechanisms to interact with virus-infected cells to limit viral replication, and also prime adaptive immune cells for antigen-specific immunity and memory. Tumor necrosis factor alpha (TNF-α) and IL-1 induce endothelial adhesion molecules, which trigger the migration of innate immune cells, such as macrophages, blood borne DCs, and natural killer (NK) cells to the site of infection.

Alveolar macrophages are critical for limiting viral spread. Activated macrophages phagocytose IAV-infected cells and thus limit viral spread and regulate the following adaptive immune response (109). Monocytes derived from bone marrow precursors circulate in bloodstream. During IAV infection, MCP-1 (CCL-2) produced by infected epithelial cells attracts alveolar macrophages and monocytes via their CCR2 receptors (110). NK cells are important cytotoxic lymphocytes of innate immune system to eliminate IAV infection. It has been revealed that lysis of IAV-infected cells is mediated by the binding of IAV-HA with the cytotoxicity NKp44 and NKp46 receptors (111). Expression of IAV-HA on the surface of infected cells is recognition signal for NK cells, and thereby NK cells target and lyse the infected cells (112, 113).

Dendritic cells, the specialized antigen-presenting cells, bridge up the innate and adaptive immune responses during the IAV infection. Adaptive immune response begins when naïve and memory T lymphocytes recognize viral antigens presented by DCs. In the naïve steady state, DCs are orchestrated underneath the respiratory tract, including the airway epithelial tissue, lung parenchyma, and the alveolar spaces of the lungs (114, 115), where they constantly monitor for invading pathogens by their dendrites that are extended to airway lumen through the tight junctions of epithelial cells. Upon infection with IAV, the conventional DCs (cDCs) migrate from lungs to lymph nodes through interaction between CCR7 and its ligand CCL19 and CCL21 (116). In the lymph nodes, cDCs present antigens derived from IAV to T lymphocytes (117, 118). The self-infected DCs degrade the viral protein into immune peptides. Immune peptides (epitopes) in the cytosol are exported to the endoplasmatic reticulum, where they bind with major histocompatibility complex (MHC) class I molecule. Following the binding with epitopes, MHC class I is transported to the cell membrane via the Golgi complex for recognition by virus-specific CD8⁺ cytotoxic T cells (CTL). However, viral proteins degraded in endosomes/lysosomes are associated with MHC class II molecule. These complexes are presented on the cell membrane for recognition by CD4⁺ T helper (Th) cells. This process may lead to B cell proliferation and maturation to antibody producing plasma cells (119). In addition, DCs can exert cytolytic activity and contribute to the formation of bronchus-associated lymphoid tissue (BALT) during the IAV infection (120).

The nasal openings and URT are the main entry sites for IAVs and mucosal immune system also acts as the first line to limit the IAV infection apart from innate immunity. Secretory IgA (s-IgA) and IgM are the major neutralizing antibodies present on mucosa to prevent viral entry. Nasal secretions contain IgA which can neutralize HA and NA of IAVs (129, 158). During primary infection with IAVs, all three major immunoglobulin classes (IgG, IgA, and IgM) are present in mucosal secretion to limit the infection, though IgA and IgM are higher in concentration than IgG (159). It is thought that IgM response is dominant during primary infection, whereas during secondary infection IgG response is dominant for immunoglobin secretion (2, 119, 121). In the URT, mucosal response is induced in the nasopharyngeal-associated lymphoid tissues (NALT) (160–162). When antigens are pinocytosed or phagocytosed by macrophages present on the NALT, they interact with local T and B cells, resulting in development of a large number of IgA Ab-forming cell (IgA-AFC) precursors (163, 164). The primed T and B cells migrate from NALT to the lungs via general circulation, where they differentiate into specific IgA-AFC to secrete antiviral antibodies. Thus, NALT appears to be initial inductive site for secretion of s-IgA against IAV infection. In the LRT, mucosal immune responses occur in the BALT (165). BALT is the site for AFC development and production of mucosal s-IgA against IAV infection (166).

Secretory IgA is the primary isotype detected at the mucosal surface (167), which contributes to mucosal protection through its distinct ability to remove an agent before it traverses the mucosal barrier and infects the cell (168). By covering the viral surface, s-IgA prevents the influenza virions from adhering to the susceptible cells, and thus inhibits their invading host cells and neutralizes the viruses without complement participation. Investigations have been demonstrated that s-IgA plays vital roles both in protection against homologous IAV infection and in cross-protection against URT infection by the viral variants (169). Generally, parenteral administration of IAV vaccine leads to the generation of serum IgG, but not s-IgA, while s-IgA and IgG are both induced by intranasal administration (168, 170). Further, polymeric s-IgA is involved in defending against influenza in humans. Moreover, the quaternary structure of the polymeric s-IgA seems to play a key role in protecting human URT from influenza, and have more neutralizing capacity against IAVs than dimeric s-IgA (171).

Hemagglutinin of IAVs has been shown to facilitate IFNAR ubiquitination and degradation, reducing the levels of IFNAR, and thus suppressing the expression of IFN-stimulated antiviral proteins (172). It has been described that two discrete antigenic sites, H9-A and H9-B, may provide a novel mechanism for H9N2 virus to counteract humoral immunity (173). In addition, a study has shown that the escape of H5N1 from vaccine-mediated immunity is caused by the addition of N-glycosylation sites on the globular head of HA (174). In contrast, antibody response against NA of IAV cannot inhibit viral infection, but restrain its diffusion, thus lowering the severity of influenza. IAVs employ NA protein to block the recognition of HA by natural cytotoxicity receptors, NKp46, and NKp44 receptors and evade the NKp46-mediated elimination, leading to minimized clearance of infected cells by NK cells (175).

Nonstructural protein-1 of IAVs is the most important IFNs antagonist protein, acting on multiple targets and suppressing the host IFN response. Viral RNA invading the host cell causes RIG-I ubiquitination by a RING-finger E3 ubiquitin ligase named as TRIM25, which is essential for RIG-I signaling pathway to trigger host antiviral innate immunity (94, 176). However, NS1 protein can inhibit the TRIM25-mediated RIG-I ubiquitination, thereby blocking RIG-I activation (177). Moreover, NS1 has an inhibitory effect on protein kinase RNA-activated (also known as protein kinase R, PKR), but the effect relies on the induced expression of vault RNAs (a kind of small non-coding RNA with approximately 100 bases). They are initially described as fornix RNP complex components (178). Through NS1 protein, influenza virus induces the expression of vault RNA that inhibits the activation of PKR and the production of IFNs and ultimately promotes the replication of the virus. In a recent reverse genetic investigation, it was found that after interfering with NS1, the phosphorylation level of PKR dramatically increased, which was attenuated by forced expression of vault RNAs (179). These data indicate that IAV has evolved a critical mechanism by which NS1-mediated PKR inhibition is mediated by upregulation of the host factor vault RNAs that inactivates PKR and blocks the production of downstream effector molecules of IFNs.

In addition, studies have shown that through the interaction with IκB kinases (IKK) α and β, two important kinases in NF-κB pathway, NS1 protein can block the phosphorylation of these kinases and eventually destroy the NF-κB complex predominating in nucleus as well as the expression of downstream genes (180, 181). Also, through the JAK-STAT pathway, NS1 protein can block IFN-mediated downstream signaling pathway and weaken the antiviral effect mediated by the downstream effector molecules induced by IFNs. Specifically, NS1 acts mainly by lowering the phosphorylation levels of STAT1, STAT2, and STAT3, preventing STAT2 from entering into the nucleus to bind to the DNA sequence of ISGs promoter region, leading to reduced expression of ISGs (182). Importantly, NS1 is not only involved in host innate immunity, but also affects adaptive immunity via modulating the maturation and the capacity of DCs to induce T cell responses (183). Evidence also indicates that influenza virus NS1 can bind to cellular double-stranded DNA (dsDNA), counteract the recruitment of RNA polymerase II (Pol II) to DNA, and finally block the transcription of IFNs and ISGs (184).

Studies have found that PB1-F2 protein has a mitochondrial positioning signal, via interacting with MAVS, to counteract RLR-mediated activation of IFN signaling pathway (185). Investigation on the interaction between the virus and host by systematic biology analysis has revealed that PB2 protein, a member of the viral polymerase complex, also plays roles in IFN antagonism (186). Furthermore, PB2 interacts with the MAVS to evade from the host IFN antiviral response, which is similar to the action mode of PB1-F2 protein (187). Recently, viral M2 protein has been found to interfere with the host autophagy (188, 189). These studies have suggested that viral M2 may inhibit the activation of TLR pathway and the generation of IFNs via blocking the host autophagy.