Efectivo y equivalentes al efectivo

The previous sections summarize our current understanding of cystic fibrosis pathology and the available therapeutic options. Patients suffer from genetic abnormalities to the CFTR gene which predispose them for chronic and repetitive infections. Infections with PA are the most common in patients 18 years and older and they are characterized by a chronic, dysregulated, inflammatory response with aberrant T cell immunity and a predominant recruitment of neutrophils and pro-inflammatory macrophages [312]. This continual immune response lowers patients’ life expectancy by 30 years as a result of lung injury and damage, deteriorated lung function, and decreased quality of life [313].

Azithromycin, a macrolide antibiotic, exerts anti-inflammatory and

immunomodulatory effects in cystic fibrosis patients. We have previously shown that azithromycin alters macrophage polarization and shifts the macrophages into an alternatively activated phenotype, both in vitro and in a mouse model of PA pneumonia [166, 310, 311]. Murine experiments with an early polarization of macrophages into an alternative phenotype show decreased neutrophil influx and pulmonary injury in mice infected with PA. Conversely, abolishing alternative macrophage polarization was associated with a profound acute immune response along with exaggerated neutrophil influx and an altered T cell

phenotype. The absence of alternative macrophages was also associated with increased morbidity and mortality in our model of murine PA pneumonia [166, 310, 311].

These findings from in vitro, murine, and human experiments were the basis for the central hypothesis that the alternatively activated macrophages decrease pulmonary inflammation in PA infection, an effect that is dependent upon production of TGFβ and arginase-1. The objective is to determine whether alternative macrophages induced with azithromycin are essential to regulate the exaggerated inflammatory response against PA pneumonia. Additionally, our objective is to determine which specific macrophage effectors are essential for immunomodulation and whether this involves control of other immune cells like neutrophils and T cells. The second objective of our research is to define the specific mechanisms by which azithromycin modulates these macrophages. The long-term goal is to utilize these mechanisms to optimize the current therapeutic options and to identify new therapeutic targets for patients with non-resolving pulmonary inflammation.

Work presented in this dissertation is based on our previous findings and aims to identify the specific effectors of alternative macrophages that are crucial for regulating inflammation and modulating T cell disposition. Closely examining the different effectors of alternative macrophages revealed an important role of arginase-1. Arginase-1 expression and activity are increased in alternative macrophages polarized with azithromycin. Preliminary experiments using arginase-1 conditional knock-out mice verified that arginase deficiency is associated with greater morbidity in terms of more significant weight loss. Additionally, arginase-1 has unique immunomodulatory properties (discussed earlier) and include controlling NO-mediated injury, suppression of T cell function and proliferation, as well as promoting a Treg phenotype [151, 152, 154, 157, 312-316]. In cancer, myeloid suppressor cells producing arginase inhibit T cell responses against tumor cells [317, 318]. In pregnancy, arginase is essential to

prevent maternal immune reactions against the fetus [319]. Arginase also plays an important role in sepsis, trauma, surgery, certain infections, and some

inflammatory and autoimmune diseases [151, 152, 154, 157, 314-316]. However, arginase-1 mediated modulation of the inflammatory response against PA

pneumonia has not been investigated.

The work presented here also investigates the specific pathways involved with azithromycin macrophage polarization. It is unknown how azithromycin

modulates the expression of different pro- and anti- inflammatory macrophage effectors. The expression of these inflammatory cytokines and mediators is controlled via different transcription factors induced in response to distinct stimuli. Th1 cytokines can induce M1 macrophage activation by stimulation of the STAT- 1 and NF-κB transcription factors. Alternatively, Th2 cytokines activate alternative M2 macrophages through STAT-6 activation. While some reports suggest that azithromycin can inhibit NF-κB activation and polarize macrophages into an M2 phenotype, a link between these effects has not been established. Theodore Cory, a previous graduate student in the Feola lab, conducted a preliminary study of azithromycin using an in-vitro model of J774 murine macrophages. He showed that azithromycin affects non-canonical NF-κB activation. He showed that azithromycin increases the level of inactive NF-κB p105 subunit while the active p50 subunit was down-regulated. The work in this dissertation ties the preliminary data that Dr. Cory generated with specific regard to the effects of azithromycin on the canonical NF-κB subunit, p65. Additionally, the research in this dissertation evaluates cross-talk between the canonical NF-κB pathway and the STAT-1 pathway.

The purpose of the work presented in this dissertation is to specifically investigate the role of arginase-1 in the immunomodulatory functions of alternative macrophages and to define the molecular anti-inflammatory

mechanism of azithromycin. In addition to evaluating my hypotheses in murine models of PA pneumonia, I also address the translatability of my observations by

evaluating the immunomodulatory properties of azithromycin in cystic fibrosis patients.

The long-term goal is to define key regulators of the exaggerated immune response that can be of therapeutic value for immunotherapy in cystic fibrosis. The objective of this project is to investigate the regulatory role of arginase-1 in the immune response to PA pneumonia and its modulation of T cell immunity. Additionally, this project examines the effects of azithromycin on the transcription factors involved with macrophage polarization, specifically NF-κB and STAT-1.

The central hypotheses to be tested in the following chapters are:

I- Decreases in inflammation in response to PA pneumonia achieved by polarizing macrophages to an alternatively-activated phenotype is dependent upon the production of arginase-1.

II- Azithromycin polarizes macrophages to an M2 phenotype via inhibition of STAT1 through cross-talk from NF-κB signaling mediators.

III- The ability of azithromycin-induced M2 macrophages to control

inflammation in response to PA pneumonia is dependent on arginase-1 expression.

I found that decreases in inflammation in response to PA pneumonia are dependent upon the production of arginase-1. Arginase-1 deletion resulted in exaggerated neutrophil influx and in skewing of the T cell responses towards a Th1 and Th17 responses.

Additionally, azithromycin polarized macrophages to an M2 phenotype via inhibition of STAT1 through cross-talk from the NF-κB signaling mediators.

Azithromycin inhibited p65 nuclear translocation which resulted in IKKβ

accumulation due to suppressed negative feedback. Thus, inhibiting the NF-κB signaling pathway cross-inhibited the STAT-1 pathway.

Finally, azithromycin regulated the immune responses in PA pneumonia via mechanisms independent of arginase. Azithromycin protected against excessive morbidity and exaggerated inflammation by controlling the influx of neutrophils and inflammatory macrophages in mice with macrophages that could not express arginase-1. Additionally, azithromycin balanced the Th17/Treg responses in mice infected with PA, an effect that was also independent of arginase production.

Figure 1.1a

Th17

Th2

Treg Th1

Defective epithelium Defective macrophages Defective neutrophils Defective adaptive immunity

- Impaired mucociliary clearance - Increased NF-κB signaling - Decreased antigen presentation - Inactivation of antimicrobial substances - Loosened junctions and increased paracellular permeability - Impaired phagocytic function - Impaired antigen presentation - Impaired clearance of dead neutrophils - Altered M1/M2 macrophage polarization - Exaggerated NF-κB activation - Exaggerated pro- inflammatory cytokine release - Impaired phagocytic function - Excessive release of granule products and superoxide anions - Increased neutrophil elastases and proteases - Protease >>> anti- protease - Destruction of

connective tissue and matrix proteins

- Unregulated cell death and release of intracellular DNA - Impaired antigen presentation and necessary costimulation - Impaired membrane depolarization and signaling transduction - Intrinsic skewing towards Th17 predominant phenotype - Th17 >>> Treg Mutated CFTR 59

Figure 1.1b

Figure 1.1. Cystic fibrosis pathology.

Cystic fibrosis patients suffer from a defective and dysregulated immune

response. (a) The main alterations of the cystic fibrosis epithelium, macrophages, neutrophils, and adaptive immune cells. (b) Figure depicts the progression of cystic fibrosis pathogenesis from a functional CFTR mutation (1) to the development of end-stage bronchiectasis (11).