The innate immune system is the first line of defense against potentially dangerous microbes, and its main role is to recognize pathogens and initiate fast defensive responses. Because the human respiratory tracts are exposed to a myriad of pathogens daily, the immune system needs to recognize and initiate host defenses against these pathogens. Akt may regulate multiple points of the airway innate immune system as well as the airway’s ability to detect pathogens.
Mucociliary clearance: The physical defense of the airways
The primary physical innate defense mechanism of the airways is mucociliary clearance (MCC) (Figure 2). The main functional components of MCC are mucus production by airway secretory cells and mucus transport by airway ciliated cells[11,24-26]. Cilia are specialized organelles lining airway epithelial cells. Mucus traps inhaled particulates and pathogens, and coordinated ciliary beating drives debris-laden mucus toward the pharynx, where it is swallowed or expectorated. The airway surface liquid (ASL) is composed of the mucus layer that rides on top of the periciliary liquid (PCL) that surrounds the cilia. The composition of the PCL (volume, viscosity, and pH) mainly depends on epithelial ion channels. Dysregulation of epithelial ion channels in CF is associated with increased mucus viscosity and PCL depletion that impairs MCC. Direct cilia motor protein defects in primary ciliary dyskinesia (PCD) also impair MCC. Both CF and PCD patients are more susceptible to airway infections[30-32], supporting the importance of effective MCC to airway defense. A reduction of ciliated cells is also observed in patients with inflammatory diseases like chronic rhinosinusitis[32,33] as well as after exposure to compounds in cigarette smoke.
Figure 2 Mucociliary clearance and innate immunity in the lung.
A: Trachea, bronchi, and conducting bronchioles comprise the conducting zone of the airways; B: The conducting airway epithelium is lined with columnar motile ciliated cells and secretory goblet cells. Goblet cells secrete mucins like MUC5AC that polymerize to form mucus, which traps inhaled pathogens and debris; The mucus layer rides on top of a less viscous PCL composed of salt, water, and antimicrobials; Together, the mucus and PCL comprise the airway surface liquid; Coordinated metachronal beating of the motile cilia within the PCL layer pushes the sticky mucus layer up to respiratory tree to the oropharynx, where it is expectorated or swallowed; This process is termed MCC, and is the physical defense of the airway against infection; Epithelial cells also secrete antimicrobial peptide and radicals (NO, H2O2) to directly kill pathogens and produce cytokines and chemokines to activate inflammation; Shown is a representative diagram of tracheal or bronchial epithelium; In lower conducting airways (non-cartilaginous bronchioles < approximately 1 mm in diameter), secretory club cells (also known as bronchiolar exocrine cells) are found instead of goblet cells; As described in the text, there are several potential mechanisms by which protein kinase B may regulate MCC and other innate immune pathways. Figure made using Biorender.com. PCL: Periciliary layer; MCC: Mucociliary clearance; MUC5AC: Mucin 5AC.
The normal mucus layer is composed of mainly water, mucins, proteins, lipids, and salts. However, the gel properties of mucus are produced by mucins, large cross-linked glycoproteins, including mucin 5AC (MUC5AC) produced by surface goblet cells and MUC5B produced by mucus cells of submucosal glands. Elevated MUC5AC levels are linked to asthma and may contribute to airway obstruction[36-38]. Akt has been suggested to be linked to MUC5AC production, though the data are conflicting. In human bronchial epithelial cells, direct inhibition of Akt upregulates MUC5AC production. Activation of the PI3K/Akt pathway may also significantly reduced influenza-induced MUC5AC overproduction via negative cross-talk with the mitogen-activated protein kinase (MAPK) pathway. In contrast, other studies showed that inhibition of Akt reduces MUC5AC levels[41,42]. The discrepancies in these studies might be due to different experimental models used. However, because Akt may play a role in regulating MCC by controlling MUC5AC levels, Akt inhibitors or activators may be a novel therapeutic strategy to manipulate MUC5AC levels to reduce mucus hypersecretion in asthma or chronic obstructive pulmonary disease (COPD).
Immune surveillance receptors in the airway
Beyond the airway’s physical defenses, Akt is also involved in immune surveillance in the airway. The airway utilizes a gamut of receptors such as toll-like receptors (TLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), and T2R bitter receptors to detect invading pathogens[10,43-45]. TLRs are pattern recognition receptors (PRRs) initially discovered based on homology to Drosophila toll receptors. TLRs recognize pathogen-associated microbial patterns (PAMPs) and activate signaling pathways that can lead to increased transcription of cytokines as well as production of antimicrobial peptides and iNOS. Dysfunction of TLR signaling has been linked to COPD, acute lung injury, CF, and CRS[10,48-50].
In humans, 11 TLRs have been identified and are involved in the innate sensing of microbial products. These TLRs are found in dedicated immune cells such as macrophages and dendritic cells. TLRs are also found in fibroblasts, epithelial cells in the lung, intestine, and many other cell types[10,11,52,53]. Primary and immortalized airway cells express TLRs 2 through 10 at varying expression levels[50,54-57]. Lung epithelial cell TLRs respond to a variety of factors such as Pseudomonas aeruginosa flagellin (via TLR5), gram-negative bacterial lipopolysaccharide (LPS; via TLR4), unmethylated CpG from prokaryotic DNA (via TLR9), bacterial peptidoglycan (via TLR2), gram-positive bacterial lipoteichoic acid (via TLR2), viral double-stranded RNA (via TLR3), and fungal zymosan/beta-glucan (via TLR2)[43,50,57].
The broad principles of TLR signaling are already described by several excellent reviews[58,59]. Briefly, binding of PAMPs to TLRs activate their intracellular Toll/IL-1 receptor (TIR) domains and recruits one or more TIR domain-containing adaptor proteins, including myeloid differentiation primary response protein 88 (MyD88), TIR-domain-containing adaptor protein (TIRAP), TIR-domain-containing adaptor protein inducing interferon-β (TRIF), and TRIF-related adaptor molecule (TRAM). Signaling then proceeds through a serious of adapter proteins. Association of MyD88 recruits IL-1R-associated kinase (IRAK) through interactions of N-terminal death domains in both proteins. Phosphorylation of IRAK activates tumor necrosis factor receptor-associated factor-6 (TRAF6) which in turn activates transcription factors such as NF-kB and JNK to promote the production of cytokines or initiate apoptosis signaling pathways, respectively. Some TLRs, like TLR3, can also activate MyD88-independent signaling pathways leading to NF-kB activation.
In epithelial and immune cells, experimental studies have identified both positive and negative cross-talk between TLR activation and the PI3K/Akt pathway[64-66]. It is not yet fully understood how Akt is linked to TLR signaling, and these links maybe cell type-dependent or even TLR-isoform-dependent. PI3K, upstream of Akt, is often activated by TLRs in many cells, with Akt phosphorylation peaking at approximately 20 min and decreasing by approximately 1 h after stimulation. Activation of Akt via TLR stimulation may increase NF-kB signaling and cytokine expression in macrophages, while other studies showed that the PI3K/Akt pathway suppresses TLR-induced cytokine secretion in monocytes via inhibition of NF-kB[69-71]. One study suggested that binding of vasoactive intestinal peptide (VIP) to GPCRs reduced TLR4 expression via Akt in macrophages and regulatory T cells[52,72,73]. Another group demonstrated the activation of PI3K/Akt after stimulation of TLR4 is crucial for B cell survival. The role of Akt in airway TLR signaling is relatively unexplored, but data suggest that pharmacological manipulation of PI3K or Akt signaling may be a mechanism by which NF-kB activity could be controlled during bacterial or viral infection and the resulting activation of TLRs in the airway.
Cross-talk between TLRs and Akt maybe particularly important during cellular hyperoxia in the lung. Oxygen therapy is commonly used to reduce tissue hypoxia in patients with pulmonary disease. However, hyperoxia can induce lung damage that may be tied to a reduction of Akt signaling. Expression of a constitutively active form of Akt protected mouse lungs from hyperoxic injury. In a rat model of bronchopulmonary dysplasia (BPD), exposure of neonatal lungs to high (95%) oxygen reduced the expression of Akt, while overexpression of Akt was protective against lung damage. TLR4-deficient mice showed increased lung injury, higher mortality, and reduced levels of phospho (p)-Akt after hyperoxia. Expression of anti-apoptotic BCL-2 and activation of p-Akt significantly attenuated hyperoxia-induced lung injury in these TLR4-deficient mice. Thus, activating the Akt pathway with receptor ligands or direct activators like SC-79 may be useful for treatment of lung injury during hyperoxia.
Other PRRs exist beyond TLRs. NLRs are PRRs that activate signaling pathways leading to activation of the inflammasome. Unlike the transmembrane TLRs, NLRs are cytosolic. NLRs can respond to microbial pathogens and stimulate the production of cytokines. Depending on the domains that are expressed, NLRs can be categorized as NOD receptors, NLRP, NLRC, or NLRB, and have been extensively reviewed[78,79]. NOD1 and NOD2 are expressed in lung epithelial cells, endothelial cells, alveolar macrophages, and airway smooth muscle cells. Binding of NOD1 and NOD2 to secreted bacterial moieties results in activation of NF-kB, and polymorphisms of these receptors may increase susceptibility to respiratory infections. NLRP3 may play a major role in recruiting neutrophils and dendritic cells during Mycoplasma pneumoniae lung infection in mice. Because NLRs are relatively novel compared with TLRs, the knowledge of NLRs/Akt/PI3K/NF-kB in the lung immunity field is still rapidly developing.
Two decades ago, the GPCRs for bitter taste (known as taste family 2 receptors or T2Rs) were discovered in taste bud type II cells on the tongue. There are 25 T2R isoforms in humans[82,83] that detect bitter compounds in food. However, in recent years, the discovery of the T2Rs in extraoral tissues has suggested other roles for these receptors beyond taste, including immune surveillance. A variety of bitter receptors are expressed in the motile cilia in human airway epithelial cells and macrophages which are stimulated by bitter molecules such as denatonium benzoate, thujone from the wormwood plant, sodium thiocyanate, phenylthiocarbamide (PTC), and bitter plant flavonoids. These T2Rs also recognize gram-negative bacterial products such as acyl-homoserine lactone (AHL) and quinolone quorum-sensing molecules, suggesting they may play a role in sensing developing biofilms.
Stimulation of bitter receptors in sinonasal epithelial cell cilia activates Ca2+-dependent nitric oxide (NO) production which is bactericidal. Additionally, NO can act as a second messenger to stimulate soluble guanylyl cyclase (sGC) and protein kinase G (PKG) to phosphorylate downstream effector proteins within the cilia and increase the ciliary beat frequency and thereby MCC. One T2R isoform expressed in respiratory cilia is T2R38. Common polymorphisms in the TAS2R38 gene that render the T2R38 receptor nonfunctional are associated with increased susceptibility to upper respiratory infection[12,88], susceptibility to chronic rhinosinusitis[89-94], and surgical outcomes after functional endoscopic sinus surgery.
T2Rs also play other roles in the airway. A different subset of T2R isoforms in non-ciliated solitary chemosensory cells (SCCs), sometimes called tuft cells[44,96], leads to the propagation of Ca2+ to neighboring ciliated cells via gap junctions, triggering the neighboring cells to release anti-microbial peptides such as beta-defensin 1 and 2[96,97], which can permeabilize fungi and both gram-positive and negative bacteria. Moreover, in mouse asthma models, bitter receptor agonists are effective in reducing airway smooth muscle contraction by modulating Ca2+ signaling[98-100].
Such studies of primary cells in vitro and patients in vivo suggest that T2Rs may contribute to the recognition of bacterial products similarly to TLR signaling[45,86]. Since T2Rs activate endothelial nitric oxide synthase (eNOS) to acutely produce NO in ciliated cells, targeting this pathway through Akt, which phosphorylates and activates eNOS[101,102] independently of Ca2+, as described below, is possibly a way to activate these innate immune responses in patients with polymorphisms that render specific T2Rs like T2R38 nonfunctional. Akt also has many other downstream targets, including Nrf-2 that play a role in the above innate immune processes. Several of these targets are reviewed below.