Effect of Aspergillus Fumigatus Infection on Vitamin D Receptor Expression - Book Report/Review Example

? Table of Contents Introduction 4 Type of Research 5 Central Question for Research 5 Research Activity on the 6 Aims and Objectives of Research 7 Description of Methodology 7 Results and Conclusions 9 References 18 Article Review and Discussion of Results: THE EFFECT OF ASPERGILLUS FUMIGATUS INFECTION ON VITAMIN D RECEPTOR EXPRESSION IN CYSTIC FIBROSIS (Coughlan, Chotirmall, Renwick, et al. 2012) Introduction Pathogenesis in cystic fibrosis, which is a systemic heritable disorder, results from reduction in chloride secretion and increased absorption of sodium. As a result, dehydrated mucus is retained in the airways. Subsequently, chronic infection through pathogenic colonization occurs in the airways, which may result in respiratory failure. Aspergillus fumigatus is one of the most commonly observed fungi that colonize the respiratory tract in people suffering from cystic fibrosis. Allergic bronchopulmonary aspergillosis (ABPA) is an exaggerated immune response to A. fumigatus and causes the deterioration of pulmonary function. Prior to the study under review, number of gaps existed in our knowledge of cystic fibrosis and colonization by A. fumigatus. These include: The impact of A. fumigatus on inflammation and airway disease in patients with cystic fibrosis was unclear. The role of A. fugmigatus in the absence of allergic bronchopulmonary aspergillosis (ABPA) was unclear. While work on azole based agents such as itraconazole in cystic fibrosis accompanied by ABPA were underway, the anti-inflammatory effects of vitamin D and other vitamin D receptor (VDR) agonists in case of A. fumigatus colonization in cystic fibrosis in absence of ABPA are unknown. This study therefore aimed at filling in these gaps in our knowledge about cystic fibrosis and A. fumigatus colonization. The current paper aims to review this study on the effect of A. fumigatus on VDR expression in patients with cystic fibrosis, conducted by Coughlan et al. in 2012. The study is reviewed in terms of its objectives, methodology, type and significance of research and the significance of its results in the wider context of research. Type of Research The study does not formulate a hypothesis. Therefore, it is not hypothesis driven. Instead, it is more of an observational study. Since the aim of the study is to understand the interaction of A. fumigatus infection with vitamin D and VDR agonists in cystic fibrosis patients lacking ABPA, it observes the interaction of azoles such as itraconazole, in addition to A. fumigatus and its toxins both in vitro and in vivo. The VDR gene and its protein expression in culture filtrates and tracheal epithelial and bronchial epithelial cells of patients with cystic fibrosis have been observed. The findings and results have been discussed in terms of the observations made in this regard. The researchers did not formulate a hypothesis to test, and have based their results on experimental observations made both in vitro and in vivo. Central Question for Research As already stated, there is no central hypothesis in this research. However, the entire research has been based on one central question: what is the “effect of A. fumigatus on VDR expression in the CF airway” and what is the “treatment effectiveness of itraconazole in A. fumigatus colonization without ABPA” (1 p1000)? Earlier studies in mice have shown that in even in the absence of ABPA, the fungus A. fumigatus is harmful as it leads to severe Th2-biased pulmonary inflammation. Moreover, it is also seen that deficiency of vitamin D may be a risk factor for cystic fibrosis accompanied with ABPA. Experimentally, in vitro treatment using vitamin D has been shown to reduce the expression of IL-5 and IL-13, which is favorable. Other studies have also shown that IL-5 and IL-13 production is elevated and pulmonary inflammation is high when VDR is deleted. Therefore, there is a link between vitamin D and IL-5 and IL-13 levels in cystic fibrosis and A. fumigatus infection. This forms the central basis for research in this study. The effect of A. fumigatus on VDR expression in an airway affected with cystic fibrosis has thus been clarified by this study. In addition, the study evaluated the effectiveness of itraconazole treatment in colonization with A. fumigatus in absence of ABPA. Research Activity on the Subject The authors suggest that not many studies have been conducted on this subject. Studies on A. fumigatus and effectiveness of azole agents in presence of ABPA are abundant. However, studies on cystic fibrosis accompanied by A. fumigatus infection in absence of ABPA have not been performed. This study is therefore a pioneer in the field. It is the first study to evaluate the effectiveness of treatment with itraconazole in A. fumigatus colonization in cystic fibrosis patients without ABPA. This is an area of low research activity, unlike studies on A. fumigatus colonization with ABPA. Furthermore, not many studies have been conducted thereafter on this subject. Considering the less amount of time that has passed since this research was originally published, it is yet to be seen whether further intensive research on this important area of cystic fibrosis will ensue or not. Aims and Objectives of Research While the aims and objectives of this research have been made clear in the preceding sections, this section breaks down the objectives further in order to make the research goals very clear. The aims of the study, in terms of the central research question [what is the “effect of A. fumigatus on VDR expression in the CF airway” and what is the “treatment effectiveness of itraconazole in A. fumigatus colonization without ABPA” (1 p1000)] described above are broken down as follows: Investigation of the effect of culture filtrates of A. fumigatus on VDR gene and protein expression in cystic fibrosis infected cells. Evaluation of the regulation of VDR expression by gliotoxin, a mycotoxin from A. fumigatus. Evaluation of the effect of gliotoxin on the production of Th2 cytokines after itraconazole treatment. Investigation of A. fumigatus bioburden in cystic fibrosis airways after itraconazole treatment. Study of the mosaic pattern on HRCT associated with A. fumigatus after itraconazole treatment. Description of Methodology The cystic fibrosis patients for this research study were recruited from Beaumont Hospital, Dublin, Ireland. The patients attended the hospital for two years. They were screened based on eligibility criteria formulated for the study. They had a confirmed diagnosis for cystic fibrosis and were colonized with A. fumigatus. They were free from exacerbation and had no prior diagnosis with ABPA. Patients who had allergies to azoles or related substances, had lung transplantations, had a confirmed or suspected diagnosis with ABPA, or had been administered with systemic corticosteroid therapy were excluded from the study. Ethical approval was acquired from Beaumont Hospital Insti-tutional Review Board. Collection of expectorated sputum released spontaneously through deep cough was done during each visit. Four study samples were collected from each patient in addition to bronchial brushings both prior to and after treatment with itraconazole. Presence of other airway colonizers was detected through appropriate standard microbiological procedures. Cell cultures of A. fumigatus were prepared. Statistical analysis was performed through the standard procedures. Standard deviations, means and medians were determined. Data was tested through normality tests such as Shapiro Wilk test or Kolmogorov-Smirnoff as and when appropriate. Paired and unpaired student t test was also used for normal data. The Mann-Whitney U test was used for comparisons in case of non-normal data. ANOVA was used for normal distribution to study the trends over time. Tests such as Tukey’s test were used for post hoc multiple comparisons. Trends over time for non-normal data were compared through Friedmans chi-square test. Other statistical tools employed include regression analysis and Generalized Estimating Equations. The PRISM 4.0 software package and SAS were used for statistical analysis. Western blotting was used to study VDR protein expression. Furthermore, qRT-PCR was used to study VDR gene expression in cell cultures of A. fumigatus colonized patients. The immunomodulatory Aspergillus toxin – gliotoxin – was characterized. Quantification of the toxin in samples obtained from Aspergillus ­positive patients was done through HPLC analysis of chloroform extracts. Radiological changes in cystic fibrosis airway before and after the eradication of A. fumigatus were observed using HRCT scans in patients who were colonized with the fungus but had no prior history of corticosteroid treatment or ABPA. The HRCT scans were performed before treatment with itraconazole at baseline, then ten weeks after completion of itraconazole therapy, and then during the follow up after twelve months. Other routine standard tests were performed for assessment of pulmonary function and levels of immunological significant molecules such as IgE in serum. Radioallergosorbent test was performed for validating the absence of development of ABPA during the course of the study. Results and Conclusions This study has filled in necessary gaps in our knowledge of cystic fibrosis, especially that which is not accompanied by ABPA. The findings and their respective significances are summarized as follows: Firstly, this study has shown that approximately one third of the people suffering from cystic fibrosis also suffer from Aspergillus fumigatus colonization. Most importantly, this study has shown that the downregulation of VDR gene and protein expression occurs both in vitro and in vivo by A. fumigatus (figure 1). The immune and inflammatory state in cystic fibrosis is highly affected by vitamin D. It functions though the nuclear vitamin D receptors (VDRs). Deficiency of vitamin D is a risk factor for ABPA in cystic fibrosis patients who are colonized with Aspergillus fumigatus. Therefore, administration of vitamin D supplements may reduce the risk of ABPA in such individuals. Figure 1: Down-regulation of VDR gene and protein expression by culture ?ltrates of Aspergillus fumigatus Secondly, this study has also shown that VDR expression is overwhelmingly inhibited by gliotoxin (figure 2 and 3). In the absence of ABPA, A. fumigatus colonization enhances IL-5 and IL-13 production and downregulation of VDR through gliotoxin. The inflammatory state in cystic fibrosis is disrupted in the presence of fungal colonization in absence of ABPA and presence of vitamin D deficiency, wherein, vitamin D is not able to exert its positive impact. In case of patients with cystic fibrosis accompanied with ABPA, vitamin D beneficially acts by attenuating the IL-5 and IL-13 cytokines. This cannot be seen in individuals in whom ABPA is absent. Furthermore, the positive effects of vitamin D on expression of VDR are also overcome by gliotoxin. Therefore, while administration of vitamin D to prevent ABPA is highly vital, the elimination of gliotoxin is equally important. Figure 2: Effect of puri?ed gliotoxin on VDR expression. Figure 3: Characterization of the Aspergillus virulence factor responsible for down-regulation of VDR. By showing that the levels of IL-5 and IL-13 are significantly reduced after itraconazole treatment, this study has led to the important conclusion that itraconazole plays an important role in the reduction of Th2 inflammatory response in cystic fibrosis patients (figure 4). Figure 4: Reduction of Gliotoxin (Gt)-enhanced production of Th2 cytokines after itraconazole treatment. Another important finding of this study is that colonization with A. fumigatus is greatly reduced in the airways affected with cystic fibrosis upon treatment with itraconazole (figure 5). This result is sustained even after a 1-year follow up. It also reduced the levels of gliotoxin and resulted in upregulation of VDR expression. Figure 5: Reduction of Aspergillus bioburden in cystic ?brosis (CF) lung after treatment with itraconazole, resulting in increased VDR expression This study has also shown that the radiological HRCT appearance was improved upon eradication of A. fumigatus colonization from the airway of cystic fibrosis affected individuals. It also resulted in less infective exacerbations, improved pulmonary function and led to a reduced need for intravenous antibiotics. The radiological evaluation showed a novel mosaic pattern unrelated to ABPA (figure 6). Figure 6: Radiological and clinical improvement demonstrated after itraconazole therapy in Aspergillus fumigatus-colonized patients. Inflammatory benefit can be incurred by studying the mosaic pattern after itraconazole treatment. Itraconazole treatment was seen to produce positive outcomes in all clinical parameters. None of the patients in the study developed ABPA. Moreover, it was seen that there was a reduced likelihood for the occurrence of ABPA in the patients as radioallergosorbent test to Aspergillus showed reduction in the levels of IgE and attenuation of Th2 cytokine response after itraconazole therapy (figure 6). Itraconazole has thus not only been implicated in antifungal effects but also anti-inflammatory effects. While prior studies have suggested that vitamin D has a therapeutic potential for the prevention of ABPA, the present study has shown that this is only useful upon elimination of A. fumigatus using itraconazole treatment to facilitate the expression of VDR. Therefore, in cystic fibrosis patients, mere supplementation with vitamin D is not enough to prevent ABPA. It can only be achieved if vitamin D is supplemented along with antifungal treatment to eliminate colonization of A. fumigatus. Several studies on the subject have investigated it further. While Kreindler et al., in a study conducted in 2010, had shown that Th2 responses to A. fumigatus were attenuated by vitamin D3 in cystic fibrosis patients with ABPA, another study in 2013 by Nguyen et al. has shown that vitamin D could help prevent ABPA by modulating the OX40L protein that is associated with increased immune cell response (Th2 response, IL-5 and IL-13 cytokine secretion) (2, 3). This study is a further advancement of a previous study conducted by Nguyen et al. in 2012 that investigated the role of OX40L protein Th2 immune responses to A. fumigatus in humans and mice and its regulation by vitamin D (4). Another clinical trial is currently underway. The trial titled “Open-label Vitamin D Trial for Patients with Cystic Fibrosis and Allergic Bronchopulmonary Aspergillosis” is an interventional nonrandomized safety/efficacy study whose purpose is to investigate if administration of vitamin D in patients with cystic fibrosis and ABPA resulted in a reduction of the allergic response and improves their respiratory symptoms (5). As is evident from the preceding discussion, vitamin D plays an important role in cystic fibrosis and ABPA, especially in terms of anti-inflammatory action. In case of patients with cystic fibrosis in absence of ABPA, vitamin D supplementation has to be accompanied with elimination of A. fumigatus colonization through azoles. The reviewed study has several shortcomings such as small study population, open-label masking and uncontrolled nature of the trial. However, it is the first study that investigated the effectiveness of treatment with itraconazole in A. fumigatus colonization in cystic fibrosis patients without ABPA. It is thus a very significant study that sets stage for further studies on the subject. Summary of findings: This study has illustrated that VDR downregulation occurs upon colonization with A. fumigatus in airways of individuals suffering from cystic fibrosis. Gliotoxin has been identified as a fungal agent that further mediates this effect. In addition, it was also found that colonization by A. fumigatus is associated with high levels of gliotoxin, mosaic pattern in HRCT and increased Th2 inflammatory response by higher production of IL-5 and IL-13 cytokines. Therapy using itraconazole resulted in higher VDR expression, attenuated gliotoxin levels and decreased levels of IL-5 and IL-13 cytokines, all of which are significantly involved in causing the allergic process that causes ABPA. Elimination of the fungus from the respiratory tract of patients with cystic fibrosis was found to result in diminished mosaic pattern in HRCT, improved respiratory symptoms, and better pulmonary function. References 1. Coughlan CA, Chotirmall SH, Renwick J, Hassan T, Low TB, Bergsson G, et al. The Effect of Aspergillus fumigatus Infection on Vitamin D Receptor Expression in Cystic Fibrosis. Am J Respir Crit Care Med [Internet]. 2012 Nov [cited 2013 Apr 8]; 186(10):999-1007. Available from Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/22904183. 2. Kreindler JL, Steele C, Nguyen N, Chan YR, Pilewski JM, Alcorn JF, et al. Vitamin D3 attenuates Th2 responses to Aspergillus fumigatus mounted by CD4+ T cells from cystic fibrosis patients with allergic bronchopulmonary aspergillosis. J Clin Invest [Internet]. 2010 Sep [cited 2013 Apr 8]; 120(9):3242-54. Available from Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/20714107. 3. Nguyen NL, Chen K, McAleer J, Kolls JK. Vitamin D regulation of OX40L in immune responses to Aspergillus fumigatus. Infect Immun [Internet]. 2013 Feb [cited 2013 Apr 8]; [Epub ahead of print]. Available from Pubmed: http://www.ncbi.nlm.nih.gov/pubmed/23439304. 4. 3. Nguyen NL, Chen K, Alcorn J, Kolls JK. Vitamin D regulation of Th2 Immune responses to Aspergillus fumigatus in mice and humans: role of OX40L. J Immunol [Internet]. 2012 [cited 2013 Apr 8]; 188. Available from: http://www.jimmunol.org/cgi/content/meeting_abstract/188/1_MeetingAbstracts/164.19. 5. Clinical Trials.gov [Internet]. US: A National Institutes of Health; 2013 [cited 2013 Apr 8]. Available from: http://clinicaltrials.gov/show/NCT01222273. Read More