**10. Novel therapeutics in neutrophilic lung diseases**

work performed investigating the role of MMP-9 in the pathogenesis of asthma. In a report by Cundall et al the authors state that MMP-9 concentrations in BAL fluid correlate with eo‐ sinophils but not neutrophil or monocyte/macrophage counts. [78] They hypothesize that PMNs and macrophages release MMP-9 which breaks down the basement membrane, mak‐ ing it easier for the eosinophils to migrate into the airways. In another study, MMP-9 levels in BAL fluid were correlated significantly with decreases in FEV1 seen in asthma patients.

HNE, another potent neutrophil derived protease, has also been correlated with symptoms of asthma. [79] Patients with allergic rhinitis has significantly elevated levels of HNE in their nasal lavage compared to control patients in which no rhinitis was observed. To add to the myriad of evidence that neutrophils are at the very least, associated with asthma, a study by Norzila et al demonstrated that myeloperoxidase (MPO), a neutrophil mediator of the oxi‐ dative burst, is elevated in induced sputum collected from certain asthma patients com‐ pared to control patients. [80] Because MPO, HNE, and MMP-9 are all contained in intracellular granules of the neutrophil it is evidence that neutrophils present in/around the

Diagnosis of asthma is made through evaluation of symptoms and pulmonary function test‐ ing (PFT) via spirometry. An increase in FEV of ≥15% in conjunction with reported wheez‐ ing, chest tightness, and coughing is diagnostic for asthma. A difficulty arises when patients present with normal spirometry results. To address this, home PFT devices are available to record lung function data over a period of time to encapsulate more data points. Additional‐ ly, controlled exacerbation of asthma attacks with methacholine in the clinician's office is a reliable method of eliciting the necessary response to confirm a diagnosis of asthma. [81]

Similar to the other lung diseases discussed in this chapter, treatment of asthma is relegated to management of symptoms. Monitoring of frequency and severity of attacks is vital to ad‐ ministering correct dosages of medication. Patients are encouraged to keep records of at‐ tacks with information regarding date/time, location, duration, and triggers. The standard treatment of asthma is glucocorticoid (GC) inhaler with a long-acting β-agonist. [82] The GC treatment is directed at reducing the constant inflammatory state, whereas the β-agonist is a bronchodilator intended to ameliorate airway obstruction. So physicians will also prescribe the use of IgE inhibitors or neutralizing antibodies such as omaluzimab to combat the high levels of the pro-inflammatory molecule. [83] In neutrophil associated and steroid-resistant asthma, clinicians have fewer options with which to treat this potentially deadly condition. A patient's response to a two week trial of traditional asthma therapy will indicate whether or not they are a candidate for alternative asthma therapy. Because certain forms of asthma are refractory to GC therapy, the focus of treatment in such patients shifts to a more aggres‐ sive immunosuppressive approach. Treatment with cyclosporine, tacrolimus, and metho‐ trexate have been associated with some benefit, although the risk of side effects is significantly higher in these classes of medicines. Finally, IV immunoglobulin therapy is uti‐

lung in asthma patients are activated and degranulate.

112 Oncogenesis, Inflammatory and Parasitic Tropical Diseases of the Lung

**9.5. Diagnosis of asthma**

**9.6. Traditional asthma therapeutics**

With better understanding of neutrophilic lung disease has come more advanced and target‐ ed therapeutics. Towards that end, recent work by the Blalock and Gaggar groups at the University of Alabama at Birmingham (UAB) has expanded the role of PMNs in multiple chronic inflammatory lung diseases, including COPD, CF, and BOS. They described a novel concept of neutrophils proteases producing a neutrophil chemokine from extra-cellular col‐ lagen that acted in a feed-forward mechanism of disease. Seminal papers by Weathington et al and Gaggar et al detail the step-wise manner in which IL-8 draws PMNs into the intersti‐ tium, upon activation they release MMP-8 and MMP-9 which perform an initial digestion of collagen from macromolecule size. Subsequently, neutrophils release prolyl endopeptidase (PE), a serine protease previously only known to be a processor of neuropeptides. PE per‐ forms the final digestion of collagen to the tri-peptide proline-glycine-proline (PGP) from the PPGP amino acid motif that is repeated over 40 times throughout a single collagen mole‐ cule. [84],[85] PGP binds to the same receptors as IL-8, CXCR1 and CXCR2 acting a neutro‐ phil chemoattractant and activator. [86] The authors showed that not only are the proteases responsible for PGP production present and elevated in BAL fluid collected from COPD and CF patients, both stable and in exacerbations, but PGP is also measurably elevated by mass spectrometry in the BAL fluid of such patients and correlates with PMN burden in disease. [39],[87],[88] These data indicate that not only is PGP a potential biomarker for chronic in‐ flammatory neutrophilic lung disease, but the system of proteases responsible for PGP's production, and the receptors upon which it acts are potential targets for the development of novel precise therapeutics. Furthermore, work by Hardison et al, and Braber et al have dem‐ onstrated that cigarette smoke and its constituents are capable of acetylating PGP into the more potent and stable n-terminal acetylated form, AcPGP. [89],[90] AcPGP has proven to be resistant to degradation by leukotriene A4 hydrolase (LTA4H), a hydrolase/amino-pepti‐ dase also produced a number of cells, including neutrophils. In a 2010 Science paper, Snel‐ grove et al described a novel function for the dual purpose enzyme in resolving acute neutrophilic inflammation in a mouse model of influenza. [91] It would be extremely useful to have pharmaco-interventions able to modulate the PGP system of neutrophil inflamma‐ tion, either at the genesis (MMP, PE) or terminus (CXCR, LTA4H).

Although any therapeutics derived from such work may be years away from fruition, there are other recent advancements that are already making an impact on patient morbidity and mortality. Kalydeco, a drug produced by Vertex Pharmaceuticals is the first drug developed that addresses the underlying genetic cause of CF. First released on the market in January of 2012, it is effective in patients that carry the G115D amino acid change. [92] This is a class III mutation in which the protein traffics the cell surface but the channel does not function properly. Kalydeco interacts with the channel and increases the open probability of the channel. Another Vertex product, currently titled VX-809, is designed to act in patients with class II mutations (i.e., ΔF508). VX-809 acts in the endoplasmic reticulum, allowing improp‐ erly folded CF protein to pool and undergo corrected folding which results in trafficking to the cell membrane. [93] Both Vertex products are the result of so-called high throughput small molecule screening in which hundreds of thousands of small molecules are screened in a recombinant cell-based assay for an effect on cell function. The discovery of drugs that address the underlying genetics cause is an exciting advancement in any genetic disease, but made even more so by the fact that CF is one of the more common, and fatal diseases caused by a genetic malformation. Whether any of these drugs change the number or activation state of PMNs in the airway is currently unknown

Many of the therapies previously mentioned are also in use in the treatment of PMN-related or glucocorticoid resistant asthma. The PDE4 inhibitors, along with ultra-long acting β2 ago‐ nists have begun to be used in combating the airway dysfunction associated with asthma. [98] There is work being done to abrogate the ability of inflammatory cells such as neutro‐ phils to bind adhesion molecules such as the integrin VLA-4. [99] Furthermore, kinase inhib‐ itors being investigated that target p38 MAPK and PI3K would also effect neutrophil

New Frontiers in the Diagnosis and Treatment of Chronic Neutrophilic Lung Diseases

http://dx.doi.org/10.5772/53834

115

Chronic neutrophilic airway inflammation is a clinically similar, but foundationally hetero‐ geneous cohort of disease. Although neutrophils are necessary and effective components of the innate immune system in resolving infection, when dysregulated, they can be potent me‐ diators of devastating inflammation. Current therapeutics in a variety of neutrophilic lung diseases fail to address the underlying causes of the conditions and yield questionable bene‐ fit to patients. Fortunately, advances in identifications of biomarkers such as PGP and others afford the opportunity to develop targeted therapeutics aimed at resolving and preventing

and Matthew T. Hardison3\*

2 Division of Pulmonary, Allergy, and Critical Care Medicine, Department of Medicine, Uni‐

3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX,

[1] Chen, G., Zhuchenko, O. & Kuspa, A. Immune-like phagocyte activity in the social

[2] Ribeiro, C. & Brehelin, M. Insect haemocytes: what type of cell is that? *J Insect Physiol*

the progressive destruction that is a hallmark of chronic neutrophilic lung disease.

1 University of Alabama at Birmingham Medical School, Birmingham, AL, USA

recruitment and activation in asthma. [100]

, Amit Gaggar<sup>2</sup>

\*Address all correspondence to: mthardis@bcmedu

amoeba. *Science* 317, 678-681 (2007).

52, 417-429 (2006).

versity of Alabama at Birmingham, Birmingham, AL, USA

**11. Conclusion**

**Author details**

T. Andrew Guess1

USA

**References**

Patients with COPD, an even larger cohort than those with CF may also soon benefit from new therapies targeted at resolving the underlying cause rather than merely treating symp‐ toms. There is currently only a single phosphodiesterase 4 (PDE4) inhibitor, Daliresp that is approved for treatment of COPD in the United States. However, there are clinical trials cur‐ rently underway researching the effects of multiple other PDE4 inhibitors. [94] PD4 is a cAMP specific phosphodiesterase present, primarily, in inflammatory cells and also in epi‐ thelial cells. Treatment with Daliresp has been shown to reduce the release of pro-inflamma‐ tory cytokines by neutrophils and resident monocyte/macrophages. Unfortunately, there are several side effects associated with Daliresp and thus the need for better, more targeted PDE4 inhibitors is apparent. Additionally, there have been recent advancements made in traditional COPD therapies. The development of ultra-long acting β2 agonists has proved beneficial in a number of lung diseases, including COPD, A1AT, and asthma. [95] Research is also underway into the identification of biomarkers for smokers who will develop COPD, allowing treatment or prevention to possibly begin earlier. Investigators at Weill Cornell College of Medicine are using a metabolomics approach in a cohort of smokers to establish a thorough catalogue of abnormal cell changes in airway epithelium after cigarette smoking. (weill.cornell.edu) Utilizing serum, epithelial lining fluid, and airway epithelial samples, Dr. Crystal's group aims to identify the early changes in airway epithelium that indicate if a pa‐ tient will develop COPD later.

McNab et al recently published work detailing their investigation of "compound cg," a small molecule that assists in reducing aggregates of abnormal A1AT protein. [96] GC was effective in an *in vitro* model of A1AT deficiency and showed significant reduction in At1AT aggregates by both immunohistochemistry and Western blot analysis. Gene therapy is an‐ other approach, also applicable to CF that is being investigated as a potential source of cur‐ ing the disease in A1AT deficiency. A group at UMass has pioneered a dual gene therapy approach that addressed both the lung malfunction and liver disease so often associated with aggregation of mutant protein. In utilizing an adeno-associated virus (AAV) to intro‐ duce corrected protein product in the lung, and microRNAs (miRNA) in the liver to reduce production of dysfunctional protein, the investigators have presented the possibility of cura‐ tive therapy for patients with A1AT deficiency. [97]

Many of the therapies previously mentioned are also in use in the treatment of PMN-related or glucocorticoid resistant asthma. The PDE4 inhibitors, along with ultra-long acting β2 ago‐ nists have begun to be used in combating the airway dysfunction associated with asthma. [98] There is work being done to abrogate the ability of inflammatory cells such as neutro‐ phils to bind adhesion molecules such as the integrin VLA-4. [99] Furthermore, kinase inhib‐ itors being investigated that target p38 MAPK and PI3K would also effect neutrophil recruitment and activation in asthma. [100]
