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Part one of this chapter established and optimised genotyping techniques for each of the knock-out colonies of mice, which are to be used as tools for investigating the role of TRP channels in the in vivo murine models of airway inflammation. Robust and reliable genotyping protocols were developed for the wild-type and disrupted version of each of the relevant TRP channel genes.

The species Mus musculus was selected for use, as this was the first laboratory animal to have its complete genome published, thus making it possible to develop targeting vectors to disrupt specific genes in mouse blastocysts, and therefore breed knock-out mice. Whilst the genomes of other animals have now been published, the mouse is still the main animal used for genetic knock-out studies. Mice have a similar number of genes to humans, and 80% of human genes directly correspond to an equivalent mouse gene, making them a good in vivo model of the function of most human genes. It should be noted, however, that there are some disadvantages to using developmentally ‘knock-out’ animals for in vivo studies. In particular it is possible that certain genes perform different functions in the developing embryos than they do in the adult animal. Thus knocking-out a particular gene may have un-foreseen developmental effects which could change the physiology of the adult mouse in ways that do not necessarily pertain to the functions of the protein in the mature animal.

The risk of this is minimised by observing the knock-out animal’s behaviour and development compared to the wild-type.

Genotyping techniques were established in this chapter as a quick and efficient way to breed colonies of knock-out mice where enough age-matched animals were produced alongside wild-types for the in vivo experiments in this thesis. Genotyping will also be used to check the integrity of knock-out mice used in each of the studies in the following chapters. Phenotypic analysis was also performed on mice from the TRPA1-/-, TRPV1-/- and TRPV4-/- colonies by other members of the Respiratory Pharmacology group, using isolated vagus nerve preparations, which is a tissue expressing high levels of both TRPA1, TRPV1 and TRPV4 protein. These studies confirmed that TRPV1-/- mice did not respond to specific

99 agonist capsaicin, TRPA1-/- mice did not respond to specific agonist acrolein (Grace et al.

2012), and that TRPV4-/- mice did not respond to specific agonist GSK1016790A.

Colonies of TRPC6, M2, M8, V1 and V4 negative mice were obtained already backcrossed 9 or 10 times onto a pure C57BL/6 genetic background, resulting in genetically modified mice with a more than 99.9% wild-type genetic background. This allows direct comparison to be made between the genetically deficient mice and the C57BL/6 wild-type colony in in vivo models, with a high degree of certainty that any phenotypic differences are caused only by the disrupted gene, and not by other genetic differences which may occur between strains.

TRPA1-/- mice, however, were purchased with a mixed genetic background of B6;129. As the LPS and smoke in vivo models of airway inflammation were previously characterised in C57BL/6 mice, it was decided to backcross these mice onto that genetic background, rather than re-characterise the models with wild-types possessing the TRPA1-/- genetic background. Backcrossing is a time-consuming process, with every cycle increasing the genetic background by half of the previous cycle, i.e. 1 backcross = 50%, 2 = 75%, 3 = 87.5%, 4 = 93.75% and so on. When the background of the mice was 93.75%, it was decided to breed littermate controls for LPS and cigarette smoke in vivo studies. A higher number of back-crosses would have been desirable however this was not possible within the time-constraints of a PhD project. Heterozygous TRPA1-/- were therefore bred together, to yield homozygous knock-out and homozygous wild-type offspring for profiling in vivo against models of airway inflammation. These wild-type mice provide the perfect controls for their knock-out littermates, and their genetic background is close enough to C57BL/6 that they should behave similarly to the wild-type mice previously used to characterise the models, when exposed to the inflammatory challenges.

3.4.2 Measurement of TRP channel expression in murine models and human tissue RT-PCR is an excellent tool for accurately measuring the relative amount of a specific gene transcript, and TaqMan is one of the most accurate methods of quantitative RT-PCR. The main RT-PCR competitor to TaqMan is SYBR-Green, and it has been shown that TaqMan is more accurate of a broader range of input cDNA concentrations (Matsenko et al. 2008).

100 The TaqMan method was first reported in 1991 by researchers at Cetus Corporation, and utilises primers and a probe designed with high specificity for individual genes. The forward and reverse primers anneal to the 5’ and 3’ ends of the relevant gene respectively, facilitating the amplification of the gene transcript during successive PCR cycles. The oligonucleotide probe, which has a fluorophore covalently attached to its 5’-end and a quencher dye at its 3’ end, anneals within the amplified region of cDNA, i.e. between the two primers. When the probe is intact the quencher is sufficiently close to the fluorophore to quench the fluorescence emitted by fluorescence resonance energy transfer (FRET) after excitation by the TaqMan machine’s light source. However once annealed to the cDNA sequence, the probe will be degraded by the exonuclease action of the Taq-polymerase enzyme during the next PCR cycle. Thus the fluorophore is freed from its quencher and begins to emit fluorescence when stimulated, which is detected by the TaqMan machine.

The amount of fluorescence is directly proportional to the amount of genetic template created, and therefore increases with each successive PCR cycle. A critical threshold for fluorescence can therefore be set during the exponential phase of cDNA amplification, and the cycle number at which the fluorescence crosses this threshold is inversely proportional to the amount of cDNA template, for a given gene, that was present in the original sample.

TRPC6 is the predominantly expressed canonical TRP channel on human neutrophils and may be involved in cytokine induced neutrophil migration (Damann et al. 2009; Heiner et al.

2005). TRPC6 expression was shown to be significantly increased in macrophages from COPD patients compared to control subjects (Finney-Hayward et al. 2010). TRPC6 is also highly expressed in lung tissue in both humans and mice. There was however, very little difference in TRPC6 expression between smoke exposed mice and time-matched air-exposed controls at any of the measured time-points, in either the 3 or 14 day smoke exposure model. This observation also holds true for the LPS model. There does seem to be, however, a small trend towards increased TRPC6 expression in human emphysema patients compared to both non-smokers and smokers. It must be stressed however that there were no statistically significant differences in TRPC6 expression between any of the groups. This may be due to an outlier in the ‘donor’ group. An increase in TRPC6 expression in the emphysema group would conform to the observations of Finney-Hayward et al 2010 in

101 macrophages, although it should be regarded with some caution as the changes are not statistically significant when using Dunn’s multiple comparisons post-test.

TRPM2 is predominantly expressed in inflammatory cells, including macrophages, lymphocytes and neutrophils which are thought to be integral to driving the progression of COPD. The only study to look at the expression of this channel in relation to COPD and cigarette smoke, showed that neither disease nor smoking status affected TRPM2 expression (Finney-Hayward et al. 2010). This observation is paralleled by the data presented in Figure 3.5, where TRPM2 expression measured in lung parenchyma was not affected by inflammatory disease (emphysema) or smoking status. These observations, however, are in marked contrast to the data obtained in the murine models, where TRPM2 was significantly increased from the first time-point (2 hours) in the 3-day smoke, 14-day smoke and LPS exposed animals compared to their time-matched controls. The level of TRPM2 up-regulation compared to control also correlates with relative severity of BALF cellular infiltration in each of the models. However the amount of TRPM2 expression in these models cannot be attributed to the increased numbers of TRPM2 expressing cells, as the up-regulation occurs before the cellular infiltration.

TRPV1 is a heat activated thermoceptor, expressed highly in neurons, but also widely in other tissues and cell-types, including lung alveolar epithelial cells. Finney-Hayward et al.

measured TRPV1 expression in macrophages, and found no difference between COPD patients, smokers and controls. However another study looking at human primary bronchial fibroblasts found that TRPV1 was induced by three separate inflammatory stimuli: TNFα, LPS, and IL-1α. The fibroblasts which predominantly did not express TRPV1 before treatment, consistently expressed the channel at 24 and 48 hour time points after inflammatory stimulation (Sadofsky et al. 2012). This TRPV1 mRNA was shown to be functional as the specific TRPV1 agonists, capsaicin and resiniferatoxin, stimulated Ca²⁺

signalling in the fibroblasts treated with inflammatory mediators.

However in the current experiments, LPS exposure of mice in vivo did not induce increased TRPV1 expression in lung tissue samples, but in fact appeared to ablate the low levels of expression measureable in the saline exposed animals at the 2, 6, 24 and 48 hour time-points (Figure 3.4). The expression also appeared to be decreased, albeit slightly and

non-102 significantly, in the smoke exposed animals (3-day and 14-day). A number of the inflammatory mediators induced by LPS and cigarette smoke are known to activate TRPV1, both directly and indirectly. It is feasible that these mediators may induce a down-regulation of TRPV1 message, which is proportional to the severity of inflammation. However this would be in conflict with the in vitro data presented by Sadofsky et al. 2012.

In contrast to TRPV1, TRPV4 is appeared slightly (but non-significantly) increased at 2 hours after both the 3-day and 14-day smoke model. There is however a significant down-regulation of TRPV4 expression at the 6 hour time-point after LPS expression. TRPV4 was significantly increased in emphysema patients compared to donors. A search of the literature yielded no relevant published documents detailing expression changes of TRPV4 in models of lung inflammation, either in vitro or in vivo, however TNF-α was shown to cause up-regulation of TRPV4 (and TRPV1) mRNA in synoviocytes (cells found in the synovial membrane) (Denadai-Souza et al. 2012). It is also worth noting that up to seven single nucleotide polymorphisms of TRPV4 were identified in a genome-wide association study, as being associated with COPD (Zhu et al. 2009). The authors postulate that at least three of the associated polymorphisms may affect TRPV4 splicing and so result in loss of TRPV4 function, while other evidence suggests that TRPV4 may play a pro-inflammatory role, indicating a gain of function mutation may contribute to COPD. The observed increase in TRPV4 expression in emphysema patients in Figure 3.5 suggests that a gain of function mutation is more likely to have a role in the development of COPD than a loss of function.

TRPA1 and TRPM8 were below the detection threshold in all of the above murine models and controls. As previously mentioned, this is not unexpected as these two channels are predominantly expressed in the neuronal cell bodies then trafficked to the lungs. Non-neuronal expression of TRPA1 has however been described in both human and mouse lungs or associated cell types (Kunert-Keil et al. 2006; Stokes et al. 2006). TRPM8 expression is not reported in whole lung tissue, however it has been reported in human bronchial epithelial cells (Sabnis, Shadid, et al. 2008; Tsavaler et al. 2001). Using the reagents and methods described in this chapter, however, non-neuronal expression of these channels was below the detection threshold in all control, LPS-exposed and CS-exposed murine groups. This is not to say that TRPA1 and TRPM8 are not present or active in the non-neuronal murine lung

103 tissues under control and disease conditions, rather just that the available tools were not sensitive enough under these experimental conditions.

3.4.3 Summary

In summary this chapter has characterised the genotyping techniques necessary to test the role of six TRP channels in murine models of inflammation. TRPA1-/- mice were backcrossed onto a 93.75% C57BL/6 genetic background, to allow littermate controls to be used in the previously characterised murine models. TaqMan RT-PCR was used to measure the expression of TRP channels C6, M2, V1 and V4 in the murine models and human tissue samples, implicating a role for each of the channels, whilst highlighting some similarities and differences between the three murine models, as well as between the murine models and the human disease. TRPA1 and TRPM8 were not expressed at detectable levels in the lung parenchyma in either the murine models or the human examples.

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Acute Cigarette Smoke Exposure