THE ROLE OF MATRIX METALLOPROTEINASES (MMP) AND THEIR INHIBITOR IN INFLUENZA A VIRUS-INDUCED HOST LUNG INJURY NG HUEY HIAN (B.Sc.(Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2011 PUBLICATIONS Narasaraju T, Sim MK, Ng HH, Phoon MC, Shanker N, Lal SK and Chow VT (2009) ―Adaptation of human influenza H3N2 virus in a mouse pneumonitis model: insights into viral virulence, tissue tropism and host pathogenesis.‖ Microbes and Infection 11(1): 2-11 Narasaraju T, Ng HH, Phoon MC and Chow VT (2010) ―MCP-1 antibody treatment enhances damage and impedes repair of the alveolar epithelium in influenza pneumonitis.‖ American Journal of Respiratory Cell and Molecular Biology 42(6): 732-743 Narasaraju T, Yang E, Perumalsamy R, Ng HH, Poh WP, Liew AA, Phoon MC, Rooijen NV, Chow VT (2011) ―Excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of Influenza pneumonitis.‖ The American Journal of Pathology 179(1): 199-210 POSTERS PRESENTED AT INTERNATIONAL CONFERENCES Adaptation of Human Influenza H3N2 Virus in a Mouse Pneumonitis Model: Insights into Viral Virulence, Tissue Tropism and Host Pathogenesis Presented at the X International Symposium on Respiratory Viral Infections by The Macrae Group, Sentosa, Singapore, 28th Feb – 2nd March 2008 The Role of Matrix Metalloproteases in the Pathogenesis of Influenza Pneumonitis Presented at the 2010 Annual Scientific Meeting and Exhibition of the Australian Society of Microbiology, Sydney, Australia, 4th – 8th July 2010 i ACKNOWLEDGEMENTS I would like to express my heartfelt gratitude to: A/Prof Vincent Chow, who has been a most encouraging supervisor and for having faith in me and allowing me the opportunity to undertake this project His encouragement and supervision has allowed me to develop valuable critical thinking and skills of scientific reasoning which has benefited me greatly A/Prof Sim Meng Kwoon, for co-supervising me on this project, and for his invaluable guidance, constant support and understanding throughout the whole project, providing a platform for me to learn Dr Teluguakula Narasaraju, for being such an inspiring and important mentor for this project He thought me almost all the techniques I learnt for my honors and masters year and I am very grateful to be able to turn to him for guidance and advice whenever I am unsure Dr Seet Ju Ee, for taking time off her busy schedule to the scoring for the histopathology slides and for agreeing to my requests which could be quite confusing and tough at times Mrs Phoon, for being a motherly figure throughout my years in Microbiology and for her administrative and technical assistance Kelly, for being such a great help as the Laboratory Officer, constantly procuring reagents and helping in the day-to-day administrative matters for me Yong Chiat, Wu Yan, Audrey-Ann, Meilan, Jung Pu, Edwin, Youjin, Kai Sen, Wai Chii, Wee Peng, Fiona, Fabian, Hui Ann, Cynthia, Ivan - My past and present labmates, for their unfailing help and support We are like a big family and all the laughter and fun we shared will stay with me throughout my life Thank you all for being there when I needed advice or just needed a friend to talk to It has been quite a ride and I‘m glad we were all in this journey together Dad, Mum, Sis, Bro and my family – Thanks for understanding my crankiness and absence from several family events because of lab It is comforting to know that after a long day‘s work, I have a blissful home I can return to everyday Thank you for all the support ii TABLE OF CONTENTS Content Pages Publications i Posters presented at international conferences i Acknowledgements ii Table of Contents iii-vii Summary viii-ix List of tables x List of figures xi-xiii List of abbreviations xiv-xv Chapter 1: Introduction Chapter 2: Literature review 1-4 5-45 2.1 Background of Influenza virus 2.2 Influenza Pathogenesis 2.3 Occurrence and geographical distribution 6-7 2.4 Clinical Pathology of Influenza Virus 7-8 2.5 Influenza virus and host defences 2.6 Neutrophils 11-16 2.7 Neutrophils and Influenza virus-induced lung injury 17-20 2.8 Neutrophilic enzymes 20-24 2.9 Matrix Metalloproteinases 25-34 2.10 Gelatinases 34-39 2.11 Existing therapies for Influenza 39-43 2.12 Doxycycline 44-45 8-10 iii Chapter 3: Materials and Methods 46-68 3.1 Use of BALB/c Mice and Animal Husbandry 3.2 Intranasal infection of mice 46-47 3.3 Doxycycline treatment 47-48 3.4 Broncho-Alveolar Lavage Fluid (BALF) Collection from mice 3.5 BALF cell counts 3.5.1 Total BALF cell count using trypan blue exclusion 48-49 3.5.2 Differential BALF cell count using giemsa staining 49 3.6 Lowry Protein estimation assay of BALF and lung homogenate 50 3.7 Gelatinase Zymography 50-51 3.8 Western Blot Analysis 51-52 3.9 Extraction and preparation of lungs for histopathology 52-53 3.10 Immunohistochemistry 54-55 3.11 Homogenization of Lungs 53-54 3.12 Myeloperoxidase (MPO) Enzyme Activity Assay 54-55 3.13 Streaking of blood agar plate with lung homogenate 55-56 3.14 Total RNA Purification from animal tissues and mammalian cells 3.15 46 48 56 Quantitation and determination of purity and integrity of total RNA 56-57 3.16 Reverse Transcription 57-58 3.17 Classical PCR for viral gene detection 3.18 SYBR Green Real time analysis of genes 3.19 Cell Culture 3.20 Virus infection of LA-4 cells 58 58-60 61 iv 3.20.1 Seeding of cells in 24-well place or 6-well plate 3.20.2 Virus infection of LA-4 cells 3.20.3 Harvesting of cells and supernatant for subsequent experiments 3.21 Plaque assay for virus titre 3.21.1 Seeding of cells in 24-well plate 3.21.2 Infection of MDCK cells with virus 3.21.3 Preparation and addition of Avicel Overlay 64 3.21.4 Fixation and staining to visualise plaques 64 3.22 Statistical analysis 65 3.23 Summary of methodology 3.23.1 Summary of methodology (In Vitro) 66 3.23.2 Summary of methodology (In Vivo) 67-68 3.23.2a Mice Infection Experiment 67 3.23.2b Doxycycline Treatment Experiment 68 Chapter 4: Modulation of gelatinases by Influenza A virus 61-62 62 62-63 63 63-64 69-105 4.1 Results 4.1.1 Microarray analysis of MMP gene expression 69 4.1.2 Average weight of mice 70 4.1.3 Virus titres of mice lung homogenates determined by plaque assay 72 Immunohistochemical detection for influenza virus antigen in lung tissues 72 4.1.4 4.1.5 Histopathology of lung tissues of mice 4.1.6 Myeloperoxidase (MPO) assay in mice lung homogenates 69-93 75-76 79 v 4.1.7 Real-Time PCR analysis of gelatinases gene expression in lung Tissues 79 4.1.8 Western Blot analysis of gelatinases protein levels in BALF 82 4.1.9 Gelatinase zymography analysis of gelatinases protein activity in BALF 82 4.1.10 Cytopathic effect (CPE) of LA-4 cells 85 4.1.11 Classical PCR to detect virus presence in LA-4 cells 85 4.1.12 Virus titres of supernatant from LA-4 cells determined by plaque assay 86 Real-Time PCR analysis of gelatinases gene expression in LA-4 cells 89 4.1.14 Western Blot analysis of gelatinases protein levels in LA-4 cells 91 4.1.15 Gelatinase zymography of gelatinases protein activity in LA-4 cells 91 4.1.13 4.2 Discussion 4.2.1 Expression of MMPs in influenza pneumonitis 94-95 4.2.2 Mouse-adapted Influenza A/Aichi/2/68 P10 (H3N2) virus infection in mice 95-96 Mouse-adapted Influenza A/Aichi/2/68 P10 (H3N2) virus causes productive replication in LA4 cells 97 Evaluation of acute lung injury in mice infected with the mouse-adapted Influenza A/Aichi/2/68 P10 (H3N2) virus 97-98 Increase in MMPs expression and activity and their role in influenza virus-induced host lung injury 99-104 4.2.3 4.2.4 4.2.5 4.3 Conclusion 94-104 104-105 vi Chapter 5: Effects of doxycycline on influenza-induced inflammation and host lung injury 106-137 5.1 Results 5.1.1 Average weight of mice 106 5.1.2 Western Blot analysis of gelatinases protein levels in BALF 108 5.1.3 Gelatinase zymography analysis of gelatinases protein activity in BALF 108 5.1.4 Total inflammatory cell count in BALF 111 5.1.5 Differential inflammatory cell count in BALF 5.1.6 Myeloperoxidase (MPO) assay in mice lung homogenates 112 5.1.7 Virus titres of mice lung homogenates determined by plaque assay 115 5.1.8 BALF protein concentrations 115 5.1.9 Histopathology of lung tissues of mice 5.1.10 Western Blot of T1-α and Thrombomodulin protein levels in BALF 122 5.1.11 Blood agar streaking of mice lung homogenates 124 5.2 Discussion 5.2.1 Influenza as a Public Health Concern 5.2.2 Use of doxycycline (MMP inhibitor) in alleviating pulmonary conditions 127-128 5.2.3 Doxycycline treatment of mice infected with mouse-adapted Influenza A/Aichi/2/68 P10 (H3N2) virus 128-134 Conclusion 135-137 5.3 106-125 111-112 117-118 126-134 126 Chapter 6: Reference 138-157 Chapter 7: Appendices 158-161 vii SUMMARY Influenza pneumonitis has always been a considerable concern as it is associated with substantial morbidity and mortality and could lead to post-infection sequelae such as acute lung injury (ALI) or in more severe cases, acute respiratory distress syndrome (ARDS) Matrix metalloproteinases (MMPs), especially the gelatinases, contribute to the initial stage of ALI or ARDS pathogenesis due to their eminent ability to degrade major components of the basement membrane such as gelatin and collagen IV, thus resulting in damage of the epithelium and endothelium and consequentially, alveolarcapillary barrier disruption In this present study, we observed an increase in gelatinases MMP-2 and MMP-9 upon mouse-adapted influenza A/Aichi/2/68 (H3N2) P10 virus infection in a murine pneumonitis in vivo model and the acute inflammatory response elicited by virus infection results in massive infiltration of macrophages and neutrophils, which are sources of gelatinases In addition, in vitro infection of murine LA-4 alveolar epithelial cells demonstrates another source of gelatinases during influenza virus infection The host reponse to increase expression of gelatinases was accompanied by augmented epithelial and endothelial damage, as determined by respective elevated T1-α and thrombomodulin protein markers in the BALF and protein leakage into the airspaces We show here that oral administration of a low dose of doxycycline, a MMP inhibitor which inhibits gelatinases MMP-2 and MMP9, not only reduces inflammation following influenza virus infection in mice, but also leads to significant assauge of host lung injury by minimising the destruction of pulmonary endothelium and epithelium, thus lessening leakage of proteinaceous viii material into the airways Influenza-induced host lung injury is effectively improved by lower doses of doxycycline but when a higher dose of the drug is administered, inflammation was reduced to such a substantial level that renders the viral clearance inefficient, resulting in high virus load which has direct cytopathic effects on the host cells and eventually, further pulmonary damage It is 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17.95 17.87 16.55 16.45 18.84 18.99 19.77 19.61 15.74 15.72 23.48 23.43 MMP 24.13 24.44 23.47 23.65 22.31 22.21 24.97 25.03 24.93 25.25 21.94 21.67 29.16 29.2 Sample Relative MMP2-β-actin Average reference expression 4.92 5.305 5.52 5.78 5.76 5.809 0.504 0.705149 5.76 6.13 6.04 5.16 5.64 6.2 5.95 5.68 5.77 Fold Change -1.41814 Table 7.1 Ct values obtained from Real-time PCR of MMP-2 gene for control uninfected and influenza virus-infected BALB/c mice on day post-infection timepoint (in vivo experiment) Data above was analyzed by real-time PCR Gene expression of MMP-2 was normalized to gene expression levels of housekeeping gene β-actin Graph summarizing the fold change of gene expression levels of the MMP-2 gene is shown in figure 4.8 (A) 158 Sample Con R3 Con R3 Con R4 Con R4 Con R5 Con R5 INF RB1 INF RB1 INF RB2 INF RB2 INF RB3 INF RB3 INF RB4 INF RB4 INF RB5 INF RB5 β-actin 16.82 16.72 19.73 19.8 17.99 18.12 22.73 22.66 14.76 14.77 18.19 18.16 27.78 27.8 27.46 27.14 MMP 22.03 22.42 25.65 25.55 23.23 23.23 27.76 28.03 20.9 20.94 23.94 24.14 35.36 34.31 36 34.43 MMP2-β-actin 5.21 5.7 5.92 5.75 5.24 5.11 5.03 5.37 6.14 6.17 5.75 5.98 7.58 6.51 8.54 7.29 Average 5.488333 6.436 Sample Relative reference expression Fold Change 0.947667 -1.92875 0.51847 Table 7.2 Ct values obtained from Real-time PCR of MMP-2 gene for control uninfected and influenza virus-infected BALB/c mice on day post-infection timepoint (in vivo experiment) Data above was analyzed by real-time PCR Gene expression of MMP-2 was normalized to gene expression levels of housekeeping gene β-actin Graph summarizing the fold change of gene expression levels of the MMP-2 gene is shown in figure 4.8 (A) 159 Sample Con R1 Con R1 Con R2 Con R2 INF B1 INF B1 INF B2 INF B2 INF B3 INF B3 INF B4 INF B4 INF B5 INF B5 β-actin 19.21 19.44 17.95 17.87 16.55 16.45 18.84 18.99 19.77 19.61 15.74 15.72 23.48 23.43 MMP 25.13 25.02 24.92 24.89 23.61 23.64 25.74 25.68 25.49 25.54 23.62 23.67 25.72 25.87 MMP9-β-actin 5.92 5.58 6.97 7.02 7.06 7.19 6.9 6.69 5.72 5.93 7.88 7.95 2.24 2.44 Average 6.3725 Sample reference Relative expression Fold Change -0.3725 1.294594249 1.295 Table 7.3 Ct values obtained from Real-time PCR of MMP-9 gene for control uninfected and influenza virus-infected BALB/c mice on day post-infection timepoint (in vivo experiment) Data above was analyzed by real-time PCR Gene expression of MMP-9 was normalized to gene expression levels of housekeeping gene β-actin Graph summarizing the fold change of gene expression levels of the MMP-9 gene is shown in figure 4.8 (B) 160 Sample Con R3 Con R3 Con R4 Con R4 Con R5 Con R5 INF RB1 INF RB1 INF RB3 INF RB3 INF RB4 INF RB4 INF RB5 INF RB5 β-actin 16.82 16.72 19.73 19.8 17.99 18.12 22.73 22.66 18.19 18.16 27.78 27.8 27.46 27.14 MMP 24.03 24.02 25.97 26.1 25.25 25.28 28.01 28 24.87 25 28.47 28.71 26.94 27.19 MMP-β-actin 7.21 7.3 6.24 6.3 7.26 7.16 5.28 5.34 6.68 6.84 0.69 0.91 -0.52 0.05 Average 6.911667 3.15875 Sample reference Relative expression Fold Change -3.75292 13.48157 13.48157 Table 7.4 Ct values obtained from Real-time PCR of MMP-9 gene for control uninfected and influenza virus-infected BALB/c mice on day post-infection timepoint (in vivo experiment) Data above was analyzed by real-time PCR Gene expression of MMP-9 was normalized to gene expression levels of housekeeping gene β-actin Graph summarizing the fold change of gene expression levels of the MMP-9 gene is shown in figure 4.8 (B) 161 Con 0h Con 0h Con 12h Con 12h Con 24h Con 24h Con 48h Con 48h Inf 0h Inf 0h Inf 12h Inf 12h Inf 24h Inf 24h Inf 48h Inf 48h MMP Mean 178.97 236.07 163.06 233.16 173.82 231.84 178.59 226.58 156.84 229.91 186.35 239.89 186.05 240.25 186.51 239.4 MMP Pixels 2444 4058 2665 4782 2625 3988 2289 4869 2300 4407 3414 5710 3435 5694 3804 6801 Absolute Intensity 437402.68 957972.06 434554.9 1114971.12 456277.5 924577.92 408792.51 1103218.02 360732 1013213.37 636198.9 1369771.9 639081.75 1367983.5 709484.04 1628159.4 Average Intensity % Relative to Control 697687.37 100 774763.01 100 690427.71 100 756005.265 100 686972.685 98.46425699 1002985.4 129.4570581 1003532.625 145.3494132 1168821.72 154.6049709 Table 7.5 Absolute intensities of bands obtained from densitometric analyses of MMP-2 Western blot bands for control uninfected and influenza virus-infected LA-4 cells (in vitro experiment) Graph depicting percentage of MMP-2 band desitometries in infected cells expressed relative to control cells at 0h, 12h, 24h and 48h timepoints are shown in figure 4.15 162 Ctrl 0h Ctrl 0h Ctrl 12h Ctrl 12h Ctrl 24h Ctrl 24h Ctrl 48h Ctrl 48h Inf 0h Inf 0h Inf 12h Inf 12h Inf 24h Inf 24h Inf 48h Inf 48h MMP Mean 11.98 11.85 12.98 11.85 99.2 116.15 199.3 188.76 11.98 11.85 12.98 12.45 54.38 130.75 159.98 152.81 MMP Pixels 101 99 99 69 522 551 313 427 101 99 89 69 647 496 856 975 Absolute Intensity 1209.98 1173.15 1285.02 817.65 51782.4 63998.65 62380.9 80600.52 1209.98 1173.15 1155.22 859.05 35183.86 64852 136942.88 148989.75 Average Intensity % Relative to Control 1191.565 100 1051.335 100 57890.525 100 71490.71 100 1191.565 100 1007.135 95.7958215 50017.93 86.40089203 142966.315 199.9788714 Table 7.6 Absolute intensities of bands obtained from densitometric analyses of MMP-9 Western blot bands for control uninfected and influenza virus-infected LA-4 cells (in vitro experiment) Graph depicting percentage of MMP-9 band desitometries in infected cells expressed relative to control cells at 0h, 12h, 24h and 48h timepoints are shown in figure 4.15 163 Ctrl 0h Ctrl 0h Ctrl 12h Ctrl 12h Ctrl 24h Ctrl 24h Ctrl 48h Ctrl 48h Inf 0h Inf 0h Inf 12h Inf 12h Inf 24h Inf 24h Inf 48h Inf 48h MMP Mean 7.31 7.71 23.58 19.82 11.78 21.82 7.93 20.12 7.31 7.71 15.51 19.65 13.83 19.81 11.51 24.29 MMP Pixels 265 1010 595 2461 284 2123 176 1342 265 1010 501 2549 993 3099 230 2042 Absolute Intensity 1937.15 7787.1 14030.1 48777.02 3345.52 46323.86 1395.68 27001.04 1937.15 7787.1 7770.51 50087.85 13733.19 61391.19 2647.3 49600.18 Average Intensity % Relative to Control 4862.125 100 31403.56 100 24834.69 100 14198.36 100 4862.125 100 28929.18 92.12070224 37562.19 151.2488781 26123.74 183.9912497 Table 7.7 Absolute intensities of bands obtained from densitometric analyses of MMP-2 gelatinase zymography bands for control uninfected and influenza virus-infected LA-4 cells (in vitro experiment) Graph depicting percentage of MMP-2 band desitometries in infected cells expressed relative to control cells at 0h, 12h, 24h and 48h timepoints are shown in figure 4.16 164 Ctrl 0h Ctrl 0h Ctrl 12h Ctrl 12h Ctrl 24h Ctrl 24h Ctrl 48h Ctrl 48h Inf 0h Inf 0h Inf 12h Inf 12h Inf 24h Inf 24h Inf 48h Inf 48h MMP Mean 14.76 8.81 124.42 25.54 81.41 9.02 42.06 10.38 39.08 7.57 173.11 24.2 125.15 19.96 127.95 17.43 MMP Pixels 963 305 1142 680 1037 505 911 285 703 307 1543 760 1194 852 1361 468 Absolute Intensity 14213.88 2687.05 142087.64 17367.2 84422.17 4555.1 38316.66 2958.3 27473.24 2323.99 267108.73 18392 149429.1 17005.92 174139.95 8157.24 Average Intensity % Relative to Control 8450.465 100 79727.42 100 44488.635 100 20637.48 100 14898.615 176.3052684 142750.365 179.0480176 83217.51 187.0534126 91148.595 441.6653341 Table 7.8 Absolute intensities of bands obtained from densitometric analyses of MMP-9 gelatinase zymography bands for control uninfected and influenza virus-infected LA-4 cells (in vitro experiment) Graph depicting percentage of MMP-9 band desitometries in infected cells expressed relative to control cells at 0h, 12h, 24h and 48h timepoints are shown in figure 4.16 165 ... disruption of the capillary-alveolar barrier function results in the leakage of inflammatory exudates, edema fluid and plasma proteins into the lung interstitium and alveolar spaces and the collapse of. .. et al, 2010) 2.5 Influenza virus and host defences During an acute influenza virus infection, both arms of immunity: Innate and adaptive, are important in protecting the host against the pathogen... pathogen The former has a primary goal of limiting virus growth and activating the onset of the adaptive arm, in which viral clearance takes place (Tate et al, 2008) In the early innate phase of host