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ALTERNATIVELY ACTIVATED MACROPHAGES IN <em>PSEUDOMONAS AERUGINOSA</em> PNEUMONIA: MODULATION OF THE NF-ΚB SIGNALING PATHWAY AND THE IMMUNOMODULATORY ROLE OF ARGINASE-1Haydar, Dalia 01 January 2018 (has links)
Background: Azithromycin polarizes macrophages into an alternative phenotype and promotes a regulated immunity. Arginase is an important effector of these macrophages believed to play an essential role in decreasing injury and promoting repair.
Hypothesis: Decreases in inflammation in response to Pseudomonas aeruginosa (PA) pneumonia achieved by polarizing macrophages to an alternative phenotype is dependent upon the production of arginase.
Methods: Requirement of arginase was examined by pharmacological inhibition using S-(2-boronoethyl)- l-cysteine (BEC) or l-norvaline and by infecting arginase-1 conditional knock-out mice (Arg1flox/flox;Lyz2-cre (Arg1Δm)) with PA intratracheally. Arg1ΔM and control Arg1flox/flox mice were then dosed with azithromycin daily via oral gavage beginning four days prior to infection. Analysis of weight loss in addition to characterization of inflammatory cells and cytokine production via flow cytometry was performed. Macrophages were then stimulated with LPS and polarized with IL4/13, IFNγ, or azithromycin plus IFNγ. Western blot for signaling mediators, p65 translocation assay, and immunofluorescence were performed.
Results: Myeloid arginase-1 deletion resulted in greater morbidity along with more severe inflammatory response compared to the Arg1flox/flox mice. Arg1Δm mice had greater numbers of neutrophils, macrophages, and lymphocytes in their airways and lymph nodes compared to the Arg1flox/flox mice. Conversely, global arginase inhibition resulted in greater weight loss along with greater neutrophil and macrophage infiltration compared to Arg1Δm mice. BEC and l-norvaline treated mice had higher numbers of lymphocytes in their lymph nodes with variable effects on airway lymphocyte counts. Azithromycin treatment comparably reduced the acute inflammatory responses in both Arg1Δm and Arg1flox/flox mice. To evaluate this mechanism, we show in vitro that azithromycin decreases NF-κB activation by preventing p65 nuclear translocation and by decreasing STAT1 activation in a concentration-dependent manner. These effects were reversed with IKKβ inhibition.
Conclusions: Myeloid arginase is essential for control of inflammatory responses in PA pneumonia with potentially different effects of other cellular sources demonstrated with global arginase inhibition. Azithromycin reduces excessive inflammation even in the absence of arginase, potentially through a cross-inhibitory mechanism involving STAT1 and NF-κB pathways through IKKβ.
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Immune profiling of keloid diseaseBagabir, Rania January 2013 (has links)
Keloid disease (KD) is a benign fibroproliferative dermal disease of unknown aetiopathogenesis that occurs in genetically susceptible individuals. KD shows high heterogeneity within the lesion, harbouring different immune cell profiles, which are poorly characterised in KD at different lesional sites. Although, it has long been appreciated that chronic inflammation and dermal fibrosis is associated with other fibrotic diseases (e.g. scleroderma), this link has not, yet, been established in KD through direct evidence. Additionally, the limited availability of a simple KD animal model has hindered our understanding of the underlying pathogenesis of KD. Therefore, the main objectives were a) to identify and profile different immune cells at defined KD lesional and histological sites, b) to further characterize the potential contribution of viral particles in KD by investigating the gene and protein expression profile of toll like receptors that recognise viral particles in KD, and c) to develop an optimized long-term serum-free organ culture (OC) model for KD research as a tool for probing novel hypotheses in KD pathobiology deduced from a) and b) and to also validate the reliability and instructiveness of this novel ex vivo KD model with conventional (e.g. dexamethasone) and potential future anti-KD compounds [(-)-epigallocatechin-3-gallate (EGCG) and plasminogen activator inhibitor-1 (PAI-1) knock-down by siRNA]. To achieve above objectives, different cellular and molecular techniques were applied. Immune profiling of KD (chapter 2) at defined lesional and histological sites generated the first comprehensive analysis of KD-associated inflammatory cell infiltrates. This work demonstrated for the first time the presence of specific type of chronic inflammation in KD that resembles the formation of tertiary lymphoid tissues (TLTs) (in 14.7%, out of 68 KD cases). Although, these TLTs are not strictly linked to defined lesional sites within the KD, they are similar in structure to mucosa-associated lymphoid tissue (MALT). Therefore, we named this phenomenon as keloid-associated lymphoid tissue (KALT). Immunophenotyping of KD lesional sites also showed a predominance of T-cells, B-cells, M2 macrophages and OX40L+ degranulated mast cells in intralesional and perilesional sites of KD compared to normal skin and normal scar tissue. In the epidermis, Langerhans cells showed no changes, whereas the intra-epidermal T-cells were significantly increased in both the intralesional and perilesional sites of KD with an increased CD4:CD8 ratio. Intra-epidermal B-cells were only rarely found in KD. Interestingly, there was no significant statistical difference between intralesional and perilesional sites of KD immunophenotyping. These abnormal immune profiles suggest the persistence of non-resolving inflammation presence towards unknown stimuli, which require further investigation. The chronic inflammation could be followed by a reparative phase in a repetitive manner leading to KD formation. Evaluation of toll-like receptor (TLR) gene and protein expression in KD showed a significant increase in the expression of intra-epidermal TLR-6, -7 and dermal TLR-8. Since these TLRs are typically up regulated during anti-viral responses, these results further support the hypothesis that certain viruses or yet unidentified ligand may play a role in KD pathogenesis (chapter 3). A successful long-term, serum-free keloid OC model was established using a 4 mm sized punch biopsy embedded in collage matrix as air liquid interface in supplemented William’s E medium for up to 6 weeks (Chapter 4).
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