**1.1 Molecular pathway of NF-κB and its activation**

In mammals, the NF-κB highly conserved protein family is composed of five members, p50 (precursor protein: p105), p52 (precursor protein: p100) [20, 21], p65 (RelA), c-Rel, and

<sup>\*</sup> Corresponding Author

Lung and Systemic Inflammation in COPD 5

Fig. 1. Schematic representation of NF-κB activation in inflammatory disease. A variety of upstream extracellular signals activate the IKK complexes, which are comprised of 3 subunits: IKKα, IKKβ, and IKKγ. Phosphorylation of IκB by the IKK complex in the Nterminal regulatory domain at two critical serine residues results in the ubiquitination and subsequent degradation of IκB by proteasome. Free NF-κB dimmers translocate into the nucleus, where they bind to specific promoters and affect gene transcription of such molecules as proinflammatory cytokines, cell adhesion molecules, COX-2, and iNOS. Some drugs and agents are able to suppress NF-κB activation via different pathways. Aspirin and sodium salicylate block IkB phosphorylation and degradation. Sulindac, Parthenolide, Aspirin inhibit activation of the NF-kB pathway by suppressing IKK activity and MRS2481

Asthma is a chronic inflammatory disease [65, 66] of the airway accompanied by reversible bronchial hyperreactivity. Increased numbers of Th2 lymphocytes [67] and eosinophils in the airway can cause chronic inflammatory response, leading to asthma [68, 69]. In addition to the existence of inflammatory cells in the airway, these patients expose changing levels in structure of airway, termed remodeling [69, 70]. As cited above, NF-κB is one of the most important transcription factors involved in the expression of wide groups of inflammatory proteins, including cytokines, adhesion molecules, and enzymes, which themselves are implicated in the pathogenesis of asthma [71]. Translocation of NF-κB and its binding activity increases in airway specimens from asthmatics, in airway epithelial cells obtained from bronchial mucosal biopsies, and in alveolar macrophages extracted from sputum.

inhibit TNF-α.

**1.2 Asthma** 

RelB [22]; these are encoded by NFKB1, NFKB2, RELA, REL, and RELB, respectively [23], which share the so-called N-terminal Rel homology domain (RHD), responsible for DNA binding and homo- and heterodimerization [24, 25]. Various combinations of dimeric complexes bind to κB sites within the DNA, where they directly regulate transcription of target genes [26]. The major form of NF-κB in cells is a p50/RelA heterodimer [27]. The diverse Rel/NF-κB proteins exhibit different abilities to shape dimers [4], dissimilar preferences for different κB sites [28, 29], and distinct abilities to interact with inhibitory subunits known as IκBs. Because different Rel/NF-κB complexes can be induced in different types of cells and via different signals, they can cooperate in diverse ways with other regulatory proteins and transcription factors to control the expression of particular gene sets [30].

In their unstimulated state, NF-κB dimers can be found in the cytoplasm of a large variety of cells as an inactive complex controlled by their interaction with the κB family of inhibitor proteins (IκB) [31, 32]. They block NF-κB nuclear localization sequences and thus cause its cytoplasmic retention [33, 34]. Numerous IκBs have been identified; there are three typical IκB proteins, IκBα [35], IκBβ [36] and IκBε [37], and two atypical IκB proteins, Bcl-3 [38] and IκBζ, which act in a different way [39]. The precursor proteins p100 (NFKB2) and p105 (NFKB1) also act as inhibitory molecules [40].

Most mediators that activate NF-κB are involved in the phosphorylation-induced degradation of IκB. Phosphorylation of IκB by the multisubunit IκB kinase (IKK) complex in N-terminal regulatory domain at two critical serine residues (S32 and S36) [41] results in the ubiquitination and subsequent degradation of IκB by the 26S proteasome [42-44]. Free NFκB dimmers translocate into the nucleus, where they bind to specific promoters and affect gene transcription [45, 46].

A variety of upstream extracellular signals, including tumor necrosis factor alpha (TNF-α) [47-50], lipopolysaccharide [51], virus infection (human T-cell leukemia virus, HIV1) [52- 54], ionizing radiation [55], interleukins such as IL-1β [48], epidermal growth factor (EGF) [3], mitogens [56], bacteria [52], reactive oxygen species (ROS) [48], environmental hazards such as cigarette smoke [57], and physical and chemical stresses [58], activate the IKK complexes, which are comprised of three subunits: IKKα, IKKβ, and IKKγ/ NEMO. IKKα and IKKβ are catalytic subunits, and IKKγ functions as a regulatory subunit [59-61].

Numerous genes associated with the inflammatory process include proinflammatory cytokines (such as TNF-α), cell adhesion molecules (such as intercellular adhesion molecule 1) [62, 63], or assumed NF-κB binding sites in their promoters that can amplify the inflammatory response and enhance the time of chronic inflammation. NF-κB also induces the expression of enzymes whose proteins have a connection to the pathogenesis of the inflammatory procedure, such as inducible cyclooxygenase (COX-2) [18], which generates prostanoids, and the inducible type of nitric oxide synthase (iNOS), which manufactures nitric oxide (NO) [64, 65]. These facts emphasize the significance of NF-κB as a regulator of inflammatory gene activation and indicate it as a predominant choice for targeted inactivation. In fact, diverse techniques intended to improve or suppress the inflammatory process related to determined pathologies have already been directed at obstructing the biological actions of NF-κB (Figure 1).

RelB [22]; these are encoded by NFKB1, NFKB2, RELA, REL, and RELB, respectively [23], which share the so-called N-terminal Rel homology domain (RHD), responsible for DNA binding and homo- and heterodimerization [24, 25]. Various combinations of dimeric complexes bind to κB sites within the DNA, where they directly regulate transcription of target genes [26]. The major form of NF-κB in cells is a p50/RelA heterodimer [27]. The diverse Rel/NF-κB proteins exhibit different abilities to shape dimers [4], dissimilar preferences for different κB sites [28, 29], and distinct abilities to interact with inhibitory subunits known as IκBs. Because different Rel/NF-κB complexes can be induced in different types of cells and via different signals, they can cooperate in diverse ways with other regulatory proteins and transcription factors to control the expression of particular

In their unstimulated state, NF-κB dimers can be found in the cytoplasm of a large variety of cells as an inactive complex controlled by their interaction with the κB family of inhibitor proteins (IκB) [31, 32]. They block NF-κB nuclear localization sequences and thus cause its cytoplasmic retention [33, 34]. Numerous IκBs have been identified; there are three typical IκB proteins, IκBα [35], IκBβ [36] and IκBε [37], and two atypical IκB proteins, Bcl-3 [38] and IκBζ, which act in a different way [39]. The precursor proteins p100 (NFKB2) and p105

Most mediators that activate NF-κB are involved in the phosphorylation-induced degradation of IκB. Phosphorylation of IκB by the multisubunit IκB kinase (IKK) complex in N-terminal regulatory domain at two critical serine residues (S32 and S36) [41] results in the ubiquitination and subsequent degradation of IκB by the 26S proteasome [42-44]. Free NFκB dimmers translocate into the nucleus, where they bind to specific promoters and affect

A variety of upstream extracellular signals, including tumor necrosis factor alpha (TNF-α) [47-50], lipopolysaccharide [51], virus infection (human T-cell leukemia virus, HIV1) [52- 54], ionizing radiation [55], interleukins such as IL-1β [48], epidermal growth factor (EGF) [3], mitogens [56], bacteria [52], reactive oxygen species (ROS) [48], environmental hazards such as cigarette smoke [57], and physical and chemical stresses [58], activate the IKK complexes, which are comprised of three subunits: IKKα, IKKβ, and IKKγ/ NEMO. IKKα

Numerous genes associated with the inflammatory process include proinflammatory cytokines (such as TNF-α), cell adhesion molecules (such as intercellular adhesion molecule 1) [62, 63], or assumed NF-κB binding sites in their promoters that can amplify the inflammatory response and enhance the time of chronic inflammation. NF-κB also induces the expression of enzymes whose proteins have a connection to the pathogenesis of the inflammatory procedure, such as inducible cyclooxygenase (COX-2) [18], which generates prostanoids, and the inducible type of nitric oxide synthase (iNOS), which manufactures nitric oxide (NO) [64, 65]. These facts emphasize the significance of NF-κB as a regulator of inflammatory gene activation and indicate it as a predominant choice for targeted inactivation. In fact, diverse techniques intended to improve or suppress the inflammatory process related to determined pathologies have already been directed at obstructing the

and IKKβ are catalytic subunits, and IKKγ functions as a regulatory subunit [59-61].

gene sets [30].

(NFKB1) also act as inhibitory molecules [40].

gene transcription [45, 46].

biological actions of NF-κB (Figure 1).

Fig. 1. Schematic representation of NF-κB activation in inflammatory disease. A variety of upstream extracellular signals activate the IKK complexes, which are comprised of 3 subunits: IKKα, IKKβ, and IKKγ. Phosphorylation of IκB by the IKK complex in the Nterminal regulatory domain at two critical serine residues results in the ubiquitination and subsequent degradation of IκB by proteasome. Free NF-κB dimmers translocate into the nucleus, where they bind to specific promoters and affect gene transcription of such molecules as proinflammatory cytokines, cell adhesion molecules, COX-2, and iNOS. Some drugs and agents are able to suppress NF-κB activation via different pathways. Aspirin and sodium salicylate block IkB phosphorylation and degradation. Sulindac, Parthenolide, Aspirin inhibit activation of the NF-kB pathway by suppressing IKK activity and MRS2481 inhibit TNF-α.

#### **1.2 Asthma**

Asthma is a chronic inflammatory disease [65, 66] of the airway accompanied by reversible bronchial hyperreactivity. Increased numbers of Th2 lymphocytes [67] and eosinophils in the airway can cause chronic inflammatory response, leading to asthma [68, 69]. In addition to the existence of inflammatory cells in the airway, these patients expose changing levels in structure of airway, termed remodeling [69, 70]. As cited above, NF-κB is one of the most important transcription factors involved in the expression of wide groups of inflammatory proteins, including cytokines, adhesion molecules, and enzymes, which themselves are implicated in the pathogenesis of asthma [71]. Translocation of NF-κB and its binding activity increases in airway specimens from asthmatics, in airway epithelial cells obtained from bronchial mucosal biopsies, and in alveolar macrophages extracted from sputum.

Lung and Systemic Inflammation in COPD 7

(ICAM-1). It has been illustrated that epithelial upregulation of ICAM-1, which has an important role in cellinteraction, exists in asthmatics. Active bronchial asthma is matched by an amplified level of soluble ICAM-1 in serum and thereby is associated with the pathogenesis of asthma. When rhinoviruses activate NF-κB, it amplifies the gene expression ofICAM-1 in bronchial epithelial cells, because rhinovirus utilizes ICAM-1 as a cellular

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow obstruction which is irreversible. COPD is a complex of two chronic lung diseases: chronic bronchitis and emphysema both caused mainly by a familiar irritant, cigarettes [74]. The inflammatory response in smokers' lungs is not fully understood [75]. One theory is that cigarette smoke disturbs the oxidant/antioxidant balance by induction of oxidative stress, which stimulates activation of redox-sensitive transcription factors such as NF-κB. Transcription factors, including NF-κB (Table 1) and activator protein 1 (AP-1), have a key role through gene transcription of wide range of inflammatory cytokines that cause airway inflammation, including TNF-α interleukin (IL)-8, and interleukin (IL)-6 [41, 76]. As well, NF-κB has been demonstrated to be a mediator of cigarette smoke effects on gene transcription in various cell types. Its activated dimer has been revealed to be induced in

Previous studies have reported that cigarette smoke increases DNA damage in lung fibroblasts and human bronchial epithelial cells; however, this does not lead to necrosis or apoptosis. Lung fibroblasts and human bronchial epithelial cells are capable of repairing DNA damage and forming colonies after sub-culturing in normal medium. Cigarettesmoke-induced DNA damage is involved in modulating cell survival or apoptosis via numerous signaling pathways. It has been elucidated that NF-κB plays a significant role in

Transcription of genes is not only dependent upon transcription factor bindings; it is also related to the alteration of core histone proteins which adjust the availability of the genome to cofactors and nuclear factors. Octamers are composed of two copies of each histone core protein, H2A, H2B, H3, and H4, and DNA covers them. Post-translational modification of N-terminal side chains of each histone cause conformational changes via phosphorylation,

Histone acetyltransferases (HATs) acetylate lysine residues in histones, neutralize their positive charge, and lead to chromatin relaxation, increasing binding of transcription factors

The imbalance of acetylation/deacetylation and increase in acetylation might cause transcription of proinflammatory genes mediated by NF-κB and therefore initiate chronic inflammation. Consequently, the imbalance of histone acetylation/deacetylation may have a role in the inflammatory response in "susceptible" smokers who progress toCOPD [76].

When NF-κB translocates into the nucleus and acetylates histone H4, the sequence leads to DNA relaxation and transcriptional accessibility. Research has shown that smoking cessation in patients suffering from COPD causes increased histone H3 acetylation,

and RNA polymerase II, which unwinds DNA and increases gene amplification [76].

receptor [73].

**1.3 Chronic obstructive pulmonary disease** 

bronchial biopsies of smokers [77].

mediating cell survival [78].

methylation and acetylation[76].

Results show that the agents that are coordinate with deterioration of asthma generally activate NF-κB. Viral infections, allergens [72], and ozone, all of which can cause activation of NF-κB, are related to aggravation of asthma [73].

Viral infections of the upper respiratory airway might intensify asthma by activation of NFκB. In cell cultures of bronchial epithelial cells, rhinovirus causes induction of oxidative stress and NF-κB activation and increases expression of IL-8, which can in turn participate in neutrophil recruitment into the upper respiratory tract. Respiratory syncytial virus (RSV) has been involved in stimulation of NF-kB and consequent expression of IL-8 and IL-1 in human type II–like alveolar epithelial cells (A549 cells). Thus NF-κB seems to be activated during replication of RSV (Table 1)[73].

In vitro research has revealed that allergens activate NF-κB in bronchial epithelial cells of asthmatic patients. For example, exposure to aerosolized ovalbumin causes profound activation of NF-κB and transcription of inducible nitric oxide synthase in the respiratory tract of sensitized Brown Norway rats [73]. Mice lacking the NF-κB subunits p50 or c-Rel exhibit less airway inflammation in response to an antigen challenge, signifying the fundamental role of NF-κB in allergic respiratory disease [68].

Furthermore, activation of NF-κB has also been illustrated in animal models of allergic airway inflammation in airway epithelium. However, inhibition of NF-κB activation in airways did not ameliorate airway hyperresponsiveness, a key characteristic of asthma. These findings reveal that NF-κB activation in airway epithelium is essential to the airways in response to allergen activity via recruitment of inflammatory cells but also exhibits a different segregation between hyperresponsiveness and airway inflammation [68].

Airway irritants such as ozone may also exacerbate asthma symptoms and trigger inflammation through NF-κB activation. Exposure of A549 cells to ozone affects activation of NF-κB and transcription of IL-8. Another study revealed that rats exposed to ozone subsequently show time- and dose-dependent activation of NF-κB and modulate penetration of neutrophils and monocytes into lavageable airspace via expression of CXC and CC chemokines, respectively [73].

Cre/lox molecular techniques have been examined whether inhibiting NF-κB expression only in airway epithelial cells in a mouse model would diminish levels of airway remodeling. In selective airway epithelial cells frominhibitor of κB kinase β (Ikkβ) knockout mice, peribronchial fibrosis had considerably reduced levels of TGF-β in BAL, and numbers of cells had positive peribronchial TGF-β1. Airway epithelial Ikk-β ablation also leads to reduction in levels of mucus and eosinophils in the airway [69].

Reduction in expressions of NF- κB-regulated chemokines such as eotaxin-1 and Th2 cells can diminish airway inflammatory response in the airway as well. These findings support the key role of NF-κB pathway the in bronchial epithelium and its significance in the process of remodeling [69].

As cited above, expression of some cytokines and adhesion molecules as a result of NF-κB activation exacerbates inflammation in airway cells. For example, tumor necrosis factor alpha (TNF-α) is a cytokine produced by macrophages and associated with inflammation. It increases the expression of adhesion molecules for recruitment of immune cells to damaged tissue. TNF-α may also be involved in expression of intercellular adhesion molecule 1

Results show that the agents that are coordinate with deterioration of asthma generally activate NF-κB. Viral infections, allergens [72], and ozone, all of which can cause activation

Viral infections of the upper respiratory airway might intensify asthma by activation of NFκB. In cell cultures of bronchial epithelial cells, rhinovirus causes induction of oxidative stress and NF-κB activation and increases expression of IL-8, which can in turn participate in neutrophil recruitment into the upper respiratory tract. Respiratory syncytial virus (RSV) has been involved in stimulation of NF-kB and consequent expression of IL-8 and IL-1 in human type II–like alveolar epithelial cells (A549 cells). Thus NF-κB seems to be activated

In vitro research has revealed that allergens activate NF-κB in bronchial epithelial cells of asthmatic patients. For example, exposure to aerosolized ovalbumin causes profound activation of NF-κB and transcription of inducible nitric oxide synthase in the respiratory tract of sensitized Brown Norway rats [73]. Mice lacking the NF-κB subunits p50 or c-Rel exhibit less airway inflammation in response to an antigen challenge, signifying the

Furthermore, activation of NF-κB has also been illustrated in animal models of allergic airway inflammation in airway epithelium. However, inhibition of NF-κB activation in airways did not ameliorate airway hyperresponsiveness, a key characteristic of asthma. These findings reveal that NF-κB activation in airway epithelium is essential to the airways in response to allergen activity via recruitment of inflammatory cells but also exhibits a

Airway irritants such as ozone may also exacerbate asthma symptoms and trigger inflammation through NF-κB activation. Exposure of A549 cells to ozone affects activation of NF-κB and transcription of IL-8. Another study revealed that rats exposed to ozone subsequently show time- and dose-dependent activation of NF-κB and modulate penetration of neutrophils and monocytes into lavageable airspace via expression of CXC

Cre/lox molecular techniques have been examined whether inhibiting NF-κB expression only in airway epithelial cells in a mouse model would diminish levels of airway remodeling. In selective airway epithelial cells frominhibitor of κB kinase β (Ikkβ) knockout mice, peribronchial fibrosis had considerably reduced levels of TGF-β in BAL, and numbers of cells had positive peribronchial TGF-β1. Airway epithelial Ikk-β ablation also leads to

Reduction in expressions of NF- κB-regulated chemokines such as eotaxin-1 and Th2 cells can diminish airway inflammatory response in the airway as well. These findings support the key role of NF-κB pathway the in bronchial epithelium and its significance in the process

As cited above, expression of some cytokines and adhesion molecules as a result of NF-κB activation exacerbates inflammation in airway cells. For example, tumor necrosis factor alpha (TNF-α) is a cytokine produced by macrophages and associated with inflammation. It increases the expression of adhesion molecules for recruitment of immune cells to damaged tissue. TNF-α may also be involved in expression of intercellular adhesion molecule 1

different segregation between hyperresponsiveness and airway inflammation [68].

of NF-κB, are related to aggravation of asthma [73].

fundamental role of NF-κB in allergic respiratory disease [68].

reduction in levels of mucus and eosinophils in the airway [69].

during replication of RSV (Table 1)[73].

and CC chemokines, respectively [73].

of remodeling [69].

(ICAM-1). It has been illustrated that epithelial upregulation of ICAM-1, which has an important role in cellinteraction, exists in asthmatics. Active bronchial asthma is matched by an amplified level of soluble ICAM-1 in serum and thereby is associated with the pathogenesis of asthma. When rhinoviruses activate NF-κB, it amplifies the gene expression ofICAM-1 in bronchial epithelial cells, because rhinovirus utilizes ICAM-1 as a cellular receptor [73].

#### **1.3 Chronic obstructive pulmonary disease**

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow obstruction which is irreversible. COPD is a complex of two chronic lung diseases: chronic bronchitis and emphysema both caused mainly by a familiar irritant, cigarettes [74]. The inflammatory response in smokers' lungs is not fully understood [75]. One theory is that cigarette smoke disturbs the oxidant/antioxidant balance by induction of oxidative stress, which stimulates activation of redox-sensitive transcription factors such as NF-κB. Transcription factors, including NF-κB (Table 1) and activator protein 1 (AP-1), have a key role through gene transcription of wide range of inflammatory cytokines that cause airway inflammation, including TNF-α interleukin (IL)-8, and interleukin (IL)-6 [41, 76]. As well, NF-κB has been demonstrated to be a mediator of cigarette smoke effects on gene transcription in various cell types. Its activated dimer has been revealed to be induced in bronchial biopsies of smokers [77].

Previous studies have reported that cigarette smoke increases DNA damage in lung fibroblasts and human bronchial epithelial cells; however, this does not lead to necrosis or apoptosis. Lung fibroblasts and human bronchial epithelial cells are capable of repairing DNA damage and forming colonies after sub-culturing in normal medium. Cigarettesmoke-induced DNA damage is involved in modulating cell survival or apoptosis via numerous signaling pathways. It has been elucidated that NF-κB plays a significant role in mediating cell survival [78].

Transcription of genes is not only dependent upon transcription factor bindings; it is also related to the alteration of core histone proteins which adjust the availability of the genome to cofactors and nuclear factors. Octamers are composed of two copies of each histone core protein, H2A, H2B, H3, and H4, and DNA covers them. Post-translational modification of N-terminal side chains of each histone cause conformational changes via phosphorylation, methylation and acetylation[76].

Histone acetyltransferases (HATs) acetylate lysine residues in histones, neutralize their positive charge, and lead to chromatin relaxation, increasing binding of transcription factors and RNA polymerase II, which unwinds DNA and increases gene amplification [76].

The imbalance of acetylation/deacetylation and increase in acetylation might cause transcription of proinflammatory genes mediated by NF-κB and therefore initiate chronic inflammation. Consequently, the imbalance of histone acetylation/deacetylation may have a role in the inflammatory response in "susceptible" smokers who progress toCOPD [76].

When NF-κB translocates into the nucleus and acetylates histone H4, the sequence leads to DNA relaxation and transcriptional accessibility. Research has shown that smoking cessation in patients suffering from COPD causes increased histone H3 acetylation,

Lung and Systemic Inflammation in COPD 9

viscosity of respiratory secretions and chronic lung infection by *Pseudomonas aeruginosa* and other bacterial species, resulting in considerable morbidity in cystic fibrosis subjects

*Pseudomonas aeruginosa* supposedly causes activation of NF-κB and may play an important role in overproduction of mucin caused by the increase in MUC2 mucin transcription (Table 1) [73]. Even though there is not enough data in vivo, enhanced activation of NF-κB and amplification of IL-8 can be observed in bronchial epithelial cells that display CFTR mutations (IB3 cells) in comparison with normal bronchial epithelial cells line (C38 cells). To decrease sputum viscosity in CF patients, inhibition of NF-κB activation might be a useful procedure for decreasing airway inflammation and improve lung function [82]. These findings show that CFTR mutations are related to modification of NF-κB levels and airway inflammation [73]. Another research revealed that, in either wild-type (WT) or mutant (CFTR) isogenic bronchial epithelial cell lines infected by *Pseudomonas aeruginosa*, transcriptional changes occur in cytokine production. For example, NF-κB activates transcription of four -regulated cytokines include ICAM-1, CXCL1, IL-8 and IL-6, but protein expression in both cell lines involves only enhancement of IL-6 and IL-8 expressions. Inhibition of NF-κB prior to countering t *Pseudomonas aeruginosa* revealed different levels of

T. Joseph and colleagues demonstrated that in vitro activation of NF-κB in human airway epithelial cells isolated from CF (DeltaF508/DeltaF508) and non-CF (NCF) patients when infected by *Pseudomonas aeruginosa* elevated nuclear levels of IkBα in CF cells, although this increase was transient. They also showed increased baseline translocation of NF-κB to nuclei in primary CF epithelial cell cultures; following *Pseudomonas aeruginosa* infection, activation

In a systematic search for drugs for therapeutic treatment that may be utilized for inhibition of IL-8 secretion from these cells, a series of amphiphilic pyridinium salts was examined. The most effective of these salts is a (R)-1- phenylpropionic acid ester known as MRS2481. For optimal activity, it has been demonstrated that the ester ought to be joined to the pyridinium derivative by an eight-carbon chain. MRS2481 seems to be able to suppress signaling of the NF-κB and AP-1 to the IL-8 promoter . Another therapeutic feature is that MRS2481 is an effective inhibitor of TNF-α, which leads to suppression of phosphorylation and proteosomal destruction of IκBα (Figure 1). In this way, IκBα is maintained and keeps the IL-8 promoter silent [85]. Another pharmaceutical strategy against the inflammatory phenotype of the CF lung is Parthenolide, which is sesquiterpene lactone derived from the feverfew plant. Numerous researchers have controversially proposed that this compound suppresses the NF-κB pathway by attenuation of IκBα degradation. As we show in Figure 1, parthenolide inhibits IκB kinase, ensuring the stabilization of IκBα in cytoplasm, hence causing inhibition of NF-κB translocation and reduction of following inflammatory responses, so parthenolide can be an effective treatment for the excessive inflammation in

another therapeutic medicine, Azithromycin (AZM), has been shown to modulate airway inflammation in CF subjects. AZM suppressed IL-8 expression in a CF cell line. Because the IL-8 gene is transcripted by NF-κB, it can be concluded that this is the probable pathway by which AZM activates NF-κB in the cell line. Such findings indicate the anti-inflammatory

followed by the lack of innate immune responses [73].

dependence on NF-κB for expression of the cytokines [83].

of IκBα might suppress that of NF-κB [84].

CF [86].

illustrating that the stability of the inflammation in the lungs in COPD after smoking cessation may be regulated by H3 acetylation. As cited above, this study shows that cigarette smoking affects chromatin remodeling in the lungs [76]. Smoking has been found to reduce expression of IκB protein dramatically and thus affects regulation of NF-κB. Unexpectedly, in ex-smokers with COPD, a notable depletion of IκBα has been detected. Nevertheless, the NF-κB DNA binding in these patients was similar to that in nonsmokers [76]. Other investigations confirm the enhanced activation of NF-κB in cigarette smoke. Cigarette-smoke-exposed Guinea pigs increase expression of IL-8 in response to NF-κB activation. Furthermore, studies of smokers and number of pack-years reveal a positive correlation with NF-κB activation. Smokers with COPD and currently healthy smokers both increase DNA binding activity of NF-κB [76]. NF-κB expression and its translocation in lung tissue and sputum increase in COPD patients in comparison with non-smoking controls, and this seems to be related to exacerbation [79].

Caramori and coworkers investigated p65 expression in leucocytes extracted from sputum patients with exacerbated COPD and revealed p65 transcription in macrophages but not in neutrophils [80].

Even though an enhanced proinflammatory molecule whose expression is vitally dependent on NF-κB activation has been formerly described in COPD, the role of NF-κB activation has not been determined. We hypothesize that, through COPD exacerbations, initiation factors including viral and bacterial infections could activate NF-κB, generate cytokines and chemokines, and lead to inflammatory cell penetration of the airways. Sputum immunocytochemistry methods have evidenced activation of p65 in alveolar macrophages through COPD exacerbations [80].

As a sign of oxidative stress activation, Di Stefano and colleagues demonstrated increases in activation of NF-κB in segmental and subsegmental bronchial biopsies in COPD subjects and healthy smokers accompanied by enhanced lipid peroxidation products. They reported increased localization of p65 and its immunoreactivity in bronchial epithelium but not in submucosa. Nevertheless, they could not diagnose any difference between healthy smokers and COPD smoking subjects [81]. Similarly, Yagi and coworkers investigated IκBα expression by an immunostaining method to measure NF-κB activation indirectly in airway epithelial cells. They revealed increased levels of phosphorylated IκBα in both ex-smokers with COPD and subjects without COPD. Phosphorylated IκBα underwent degradation and freed NF-κB to bind to enhancers of related genes [76].

Inflammatory molecules in COPD cause increased neutrophils and inflammatory agents in the airways and bronchial tissue of patients [79]. Mishra and colleagues reported that NF-κB can be inhibited independently from IκBα and may be inhibited via a peroxisome proliferator-activated receptor α (PPAR-α). The interaction of PPARα with the p65 and c-Jun subunits of NF-κB and AP-1, respectively, may block their activation, suppressing expression of cytokines such as IL-6 [76].

#### **1.4 Cystic fibrosis**

Cystic fibrosis (CF) is a chronic inflammatory airway disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lung disease in CF expresses a profoundly proinflammatory phenotype related to increased constitutive

illustrating that the stability of the inflammation in the lungs in COPD after smoking cessation may be regulated by H3 acetylation. As cited above, this study shows that cigarette smoking affects chromatin remodeling in the lungs [76]. Smoking has been found to reduce expression of IκB protein dramatically and thus affects regulation of NF-κB. Unexpectedly, in ex-smokers with COPD, a notable depletion of IκBα has been detected. Nevertheless, the NF-κB DNA binding in these patients was similar to that in nonsmokers [76]. Other investigations confirm the enhanced activation of NF-κB in cigarette smoke. Cigarette-smoke-exposed Guinea pigs increase expression of IL-8 in response to NF-κB activation. Furthermore, studies of smokers and number of pack-years reveal a positive correlation with NF-κB activation. Smokers with COPD and currently healthy smokers both increase DNA binding activity of NF-κB [76]. NF-κB expression and its translocation in lung tissue and sputum increase in COPD patients in comparison with non-smoking controls,

Caramori and coworkers investigated p65 expression in leucocytes extracted from sputum patients with exacerbated COPD and revealed p65 transcription in macrophages but not in

Even though an enhanced proinflammatory molecule whose expression is vitally dependent on NF-κB activation has been formerly described in COPD, the role of NF-κB activation has not been determined. We hypothesize that, through COPD exacerbations, initiation factors including viral and bacterial infections could activate NF-κB, generate cytokines and chemokines, and lead to inflammatory cell penetration of the airways. Sputum immunocytochemistry methods have evidenced activation of p65 in alveolar macrophages

As a sign of oxidative stress activation, Di Stefano and colleagues demonstrated increases in activation of NF-κB in segmental and subsegmental bronchial biopsies in COPD subjects and healthy smokers accompanied by enhanced lipid peroxidation products. They reported increased localization of p65 and its immunoreactivity in bronchial epithelium but not in submucosa. Nevertheless, they could not diagnose any difference between healthy smokers and COPD smoking subjects [81]. Similarly, Yagi and coworkers investigated IκBα expression by an immunostaining method to measure NF-κB activation indirectly in airway epithelial cells. They revealed increased levels of phosphorylated IκBα in both ex-smokers with COPD and subjects without COPD. Phosphorylated IκBα underwent degradation and

Inflammatory molecules in COPD cause increased neutrophils and inflammatory agents in the airways and bronchial tissue of patients [79]. Mishra and colleagues reported that NF-κB can be inhibited independently from IκBα and may be inhibited via a peroxisome proliferator-activated receptor α (PPAR-α). The interaction of PPARα with the p65 and c-Jun subunits of NF-κB and AP-1, respectively, may block their activation, suppressing

Cystic fibrosis (CF) is a chronic inflammatory airway disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Lung disease in CF expresses a profoundly proinflammatory phenotype related to increased constitutive

and this seems to be related to exacerbation [79].

freed NF-κB to bind to enhancers of related genes [76].

expression of cytokines such as IL-6 [76].

**1.4 Cystic fibrosis** 

neutrophils [80].

through COPD exacerbations [80].

viscosity of respiratory secretions and chronic lung infection by *Pseudomonas aeruginosa* and other bacterial species, resulting in considerable morbidity in cystic fibrosis subjects followed by the lack of innate immune responses [73].

*Pseudomonas aeruginosa* supposedly causes activation of NF-κB and may play an important role in overproduction of mucin caused by the increase in MUC2 mucin transcription (Table 1) [73]. Even though there is not enough data in vivo, enhanced activation of NF-κB and amplification of IL-8 can be observed in bronchial epithelial cells that display CFTR mutations (IB3 cells) in comparison with normal bronchial epithelial cells line (C38 cells). To decrease sputum viscosity in CF patients, inhibition of NF-κB activation might be a useful procedure for decreasing airway inflammation and improve lung function [82]. These findings show that CFTR mutations are related to modification of NF-κB levels and airway inflammation [73]. Another research revealed that, in either wild-type (WT) or mutant (CFTR) isogenic bronchial epithelial cell lines infected by *Pseudomonas aeruginosa*, transcriptional changes occur in cytokine production. For example, NF-κB activates transcription of four -regulated cytokines include ICAM-1, CXCL1, IL-8 and IL-6, but protein expression in both cell lines involves only enhancement of IL-6 and IL-8 expressions. Inhibition of NF-κB prior to countering t *Pseudomonas aeruginosa* revealed different levels of dependence on NF-κB for expression of the cytokines [83].

T. Joseph and colleagues demonstrated that in vitro activation of NF-κB in human airway epithelial cells isolated from CF (DeltaF508/DeltaF508) and non-CF (NCF) patients when infected by *Pseudomonas aeruginosa* elevated nuclear levels of IkBα in CF cells, although this increase was transient. They also showed increased baseline translocation of NF-κB to nuclei in primary CF epithelial cell cultures; following *Pseudomonas aeruginosa* infection, activation of IκBα might suppress that of NF-κB [84].

In a systematic search for drugs for therapeutic treatment that may be utilized for inhibition of IL-8 secretion from these cells, a series of amphiphilic pyridinium salts was examined. The most effective of these salts is a (R)-1- phenylpropionic acid ester known as MRS2481. For optimal activity, it has been demonstrated that the ester ought to be joined to the pyridinium derivative by an eight-carbon chain. MRS2481 seems to be able to suppress signaling of the NF-κB and AP-1 to the IL-8 promoter . Another therapeutic feature is that MRS2481 is an effective inhibitor of TNF-α, which leads to suppression of phosphorylation and proteosomal destruction of IκBα (Figure 1). In this way, IκBα is maintained and keeps the IL-8 promoter silent [85]. Another pharmaceutical strategy against the inflammatory phenotype of the CF lung is Parthenolide, which is sesquiterpene lactone derived from the feverfew plant. Numerous researchers have controversially proposed that this compound suppresses the NF-κB pathway by attenuation of IκBα degradation. As we show in Figure 1, parthenolide inhibits IκB kinase, ensuring the stabilization of IκBα in cytoplasm, hence causing inhibition of NF-κB translocation and reduction of following inflammatory responses, so parthenolide can be an effective treatment for the excessive inflammation in CF [86].

another therapeutic medicine, Azithromycin (AZM), has been shown to modulate airway inflammation in CF subjects. AZM suppressed IL-8 expression in a CF cell line. Because the IL-8 gene is transcripted by NF-κB, it can be concluded that this is the probable pathway by which AZM activates NF-κB in the cell line. Such findings indicate the anti-inflammatory

Lung and Systemic Inflammation in COPD 11

Sulphur mustard (SM) is a chemical weapon used during the Iraq war against Iran of the late 1980s [93, 94]. It can produce damage in skin, eyes, and, , most importantly, in lung. 2- Chloroethyl ethyl sulphide (CEES) is a sulphur vesicating agent and an analogue of SM. Both of these agents are alkylating agents that affect cellular thiols and are highly toxic. CEES appears to decrease iNOS expression by associating with the LPS-induced stimulation of transcription factor NF-κB. CEES also alkylates the NF-κB consensus sequence, thus suppressing the binding of the NF-kB to the iNOS promoter. Even though the activation of NF-κB due to SM or CEES countering has been elucidated in different cell lines, the exact mechanism of this pathway is still poorly understood, and the question of whether activated

Diesel exhaust (DE) is a major pollutant;exposure increases a prominent inflammatory response in the airways, with induction of cytokines such as IL-8, IL-13 and activation of redox sensitive nuclear factors (NF-κB, AP-1) in the bronchial epithelium, including upregulation in the transcription of ICAM-1 and vascular endothelial adhesion molecules (VCAM-1). It has been established that DE activates the p38 and JNK MAPK pathways and

Several strategies have been proposed to block the activation of NF-κB. An extensive diversity of molecules (both natural and synthetic) has been highlighted as having an effect on activation of NF-κB and being able suppress it. These compounds suppress NF-κB

Table 1. The implication of NF-κB in inflamatory lung disease.

NF-κB induces an inflammatory pathway remains to be elucidated [95].

**2.1 Sulphur mustard Inhalation** 

**2.2 Diesel exhaust** 

causes the activation of NF-κB and AP-1 [96].

**3. Strategies to block NF-κB activation** 

task of this macrolide. Suppression of NF-κB activity reveals other proinflammatory molecules regulated by this factor as an AZM effect relevant to the treatment of CF [87].

#### **1.5 Acute respiratory distress syndrome**

Acute respiratory distress syndrome (ARDS) is known for enormous infiltration of neutrophils into the lungs accompanied by leak of serum proteins, especially albumin, into the alveolar space, blood loss in the intra-alveolar space, and interstitial edema, all important and frequent signs in exacerbation of ARDS. In spite of the occurrence of ARDS in all over the world, the precise pathophysiology mechanisms remain to be detailed [88].

Varying expression levels of proinflammatory cytokines are associated with the progression of ARDS. overexperssion of proinflammatory cytokines such as TNF-α, IL-6 and IL-8 in the lung has been demontrated in bronchoalveolar lavage (BAL) of ARDS patients and is correlated with poor outcome [88].

Patients with proved ARDS revealed increased activation of NF-κB in alveolar macrophages, in comparison with control subjects without acute lung injury [73]. Because there were no notable increases in the levels of transcription factors, including CREB, AP-I , or SP- I activation, in alveolar macrophages from patients with ARDS, NF-κB is suggested to be a significant upstream regulator for cytokine gene expression in ARDS patients, because of its existence on the enhancer of proinflammatory cytokines (Table 1). The level of subunits p50, p65, and c- Rel decreased in cytoplasm of alveolar macrophages in ARDS subjects, proving the existence of an ongoing stimulus for NF-κB activation. Increased levels of oxygen radicals, proinflarnmatory cytokines, and endotoxin in ARDS might be associated with NF-κB activation. TNF-a and IL-8 are increased in BAL of ARDS subjects [88].

NF-κB activation can alsobe caused by oxygen radicals. Our in vivo data from a hemorrhage-induced murine model of ARDS indicates an outstanding role for xanthine oxidase, a kind of oxygen radical, in stimulation of NF-κB in lung cells [88]. Cytoplasmic and nuclear levels of IκBa are not notibly dissimilar in alveolar macrophages from ARDS subjects and controls, so these findings are rather unexpected, because signals that cause activation of NF-κB would be expected to generate phosphorylation. Alveolar macrophages have a significant protective role in mediating NF-κB activation in the lung and in initiation of neutrophilic inflammation [73, 88].
