Multivariate Analysis of Chlamydia pneumoniae Lung Infection in Two Inbred Mouse Strains
Type of DegreeDissertation
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This investigation was aimed at dissecting the mechanisms of C. pneumoniae pathogenesis by multivariate analysis of challenge experiments in a mouse model of C. pneumoniae lung infection. To facilitate these analyses, a platform method for real-time, single-step, duplex reverse transcriptase quantitative PCR (RT-qPCR) for quantification of mRNA was established on the LightCycler® platform as the first part of this study. This method allows simultaneous reverse transcription and real-time PCR amplification of analyte genes and mRNA of reference genes in a single-tube reaction. Twenty-minute RT reactions at 55°C followed by 18 high-stringency step-down thermal cycles and 25 relaxed-stringency fluorescence acquisition cycles produced sensitive and accurate RT-PCR results. C. pneumoniae is an agent of community-acquired respiratory infection, and is strongly associated with atherosclerotic coronary heart disease. In the second part of this study, a balanced multivariate experimental design investigated the major factors that influence disease (measured as lung weight increase), chlamydial lung burden, and transcript levels of 25 genes. The influence of the categorical factors i) host genetic background, ii) pre-challenge immunity against C. pneumoniae, iii) time after challenge infection, iv) dietary protein content, and v) dietary antioxidant content was investigated. The combined effects of the main factors mouse strain, immune status, and time after challenge inoculation resulted in opposite outcomes for lung disease and elimination of C. pneumoniae, while dietary protein and antioxidant content had little overall influence. A/J mice prioritized minimizing disease at the cost of low pathogen elimination while C57BL/6 mice prioritized elimination of C. pneumoniae at the cost of high disease. C57BL/6 mice had generally higher transcript levels than A/J for most cellular markers except for T cell maturation markers. Most cytokines, inflammatory modulators, and cellular markers were significantly lower in naïve than in immune mice and significantly higher on day-3 than on day-10 post inoculation. The high disease outcome in C57BL/6 mice correlated with lower GATA3 and higher arginase 2 transcripts than in A/J, while low C. pneumoniae load correlated with high Tim3 and arginase 2 transcripts. A simple best-fit partial least square regression model using day-3 Tim3, GATA3, and arginase 2 transcript concentrations predicted 85% of the day-10 disease and 72% of the day-10 C. pneumoniae lung load. Thus, host-dependent levels of early Th1 and Th2 responses, and their balance, significantly correlated with late disease and pathogen load of C. pneumoniae lung infection.