Capítulo 3. Personalidad
3.2 El Modelo PEN de Personalidad de Eysenck
I have shown that Mtb infection of the lungs of transgenic mice caused human-like lung pathology; therefore my next step was to confirm the presence of the causative agents within lung lesions. I stained lung sections of the mice with Ziehl–Neelsen (the acid-fast) stain which is mainly used to identify Mtb (figure 19). Under light microscopy, Mtb bacilli appear as bright red (arrows) in areas of macrophage infiltration in WT, MMP-1 and MMP-9 mice infected with TB.
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Figure 19: Mtb bacilli in the infected lungs of the mice.
Lung sections of WT, MMP1 and -9 mice were stained with Ziehl-Neelsen (acid fast) stain that stain Mtb bacilli in bright red colour. Acid fast bacilli (arrows) are present in areas of foamy macrophage infiltration in lungs of infected mice. Original magnification x100, scale bar 20μm. Representative images from 3 independent experiments with a total of 50 mice are shown.
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Discussion
I have examined the relationship between lung ECM and caseous necrosis in lung sections of patients with pulmonary TB and observed the co-localisation of matrix destruction and caseous necrosis. This relationship was further investigated to identify the sequence of these two events using humanised mouse model. Transgenic mice expressing human MMP-1 and -9 in activated macrophages have shown an increased level of the corresponding MMP after 22 weeks infection with virulent strain of Mtb. Post-mortem processing was designed to collect the maximum number of readouts from each mouse as described in materials and methods.
MGCs were observed in lung sections of all the infected mice suggesting that this a feature of virulence strain of Mtb rather than the genetic background of the experimental mice.
However, caseous necrosis was observed in TB granulomas of humanised-MMP-1 mice only.
It is important to highlight the fact that the only difference between MMP-1 and WT mice is the expression of the human collagenase (MMP-1) in their activated macrophages. To test this hypothesis, that it was collagenase activity, as opposed to an immunological imbalance, ECM staining was performed and cytokines were measured in the lung homogenates of the Mtb infected mice. I found that there was no significant difference between the mice strains in cytokine profile. In addition, mycobacterial lung burden was comparable in WT, MMP-1 and MMP-9 mice which further confirm that histopathological changes observed were not due to difference in immunological mediators or Mtb growth. Staining ECM components revealed the breakdown of ECM in areas of caseous necrosis. Viable cells can be observed in Mtb infected lungs where ECM is intact. I found that PIIINP, an ECM degradation product, was elevated in the lung homogenates and BAL fluid of all the infected mice compared to the
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uninfected mice, suggesting that other proteases in addition to MMP-1 are contributing to lung tissue destruction and turnover of type III collagen.
Although there are various animal models of TB, finding a suitable animal model representing human pathology in TB is challenging. The non-human primates may best reproduce human phenomena when infected with TB but they are expensive and involve logistic difficulties (Lin et al., 2006). Rabbits develop a more human-like granuloma structure with central caseous necrosis and cavity formation (Dannenberg and Sugimoto, 1976).
Although rabbits could be handled easier than the non-human primates, there are less immunological tools in the research field to assess in generating various readouts when used in TB experiments.
On the other hand, the mouse is the most frequently used model in TB with advantages of similar immune response to human TB, such as key roles for IFN-γ, TNF-α and CD4+ T cells in regulating the immune response. The availability of a wide range of chemicals and reagents as well as the ability to modulate specific genes and alter genetic background add more advantages to the mouse model and make it widely used in TB research. However, TB-infected mice develop diffuse cellular infiltration and fibrotic lung lesions with poorly structured granulomas that do not form caseation or cavitation (Helke et al., 2006). Well-structured granulomas and caseous necrosis can be seen in guinea pigs but they form a fulminant response and die rapidly. One model where widespread tissue necrosis in the mouse has been reported (Kranmick), but these mice are immunocompromised and have a very high mycobacterial load, whereas in human disease mycobacteria are scanty within the granuloma (Pan et al., 2005).
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MGCs are formed by fusion of PBMCs during inflammatory reactions and involved in granuloma formation in TB, foreign body and other granulomatous lesions (Adams, 1976).
MGCs are thought to play important roles in host defence mechanisms (Kaufmann, 1993) and their absence in associated with inadequate immune response to TB (Jagadha et al., 1985).
However, the mechanism of MGCs formation is still not understood (Chambers, 1978).
There are different Mtb strains being used in TB research, the most commonly used is the H37Rv. Our group has previously shown that infecting MMP-humanised mouse with H37Rv stain did not cause caseous necrosis in the infected lungs. A likely explanation is the attenuation of the microbe as the strain has been subjected to repeated subculturing process over many decades which may have rendered the strain with weak virulence. Therefore, the appearance of MGCs in our current studies would suggest that they result from fully virulent Mtb, not laboratory-adapted strains, and the development of caseous necrosis requires both a virulent strain and expression of the collagenase MMP-1. Consistent with this, it has been demonstrated that different Mtb strains can cause diverse pathology on rabbits (Manabe et al., 2003). Lung lesions in rabbits infected with H37Rv were healed earlier than those infected with the Erdman strain.
The principal elements of the immune response to Mtb in humans (IFN-γ, TNF-α and CD4+
T cells) were first identified in mice (Cooper, 2009). In the mouse model I used, there was no difference in these immunological mediators between the WT and the humanised transgenic mice. Therefore, it would seem that the histological difference observed were not due to differences in the immunological profile between mice strains. The only difference was the expression of human metalloproteinase in the activated macrophages of the respective mouse group.
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Lung ECM plays important roles in different physiological conditions such as morphogenesis and wound healing in addition to being critical physical scaffold for the surrounding cellular structure. Excessive ECM breakdown is associated with pathological conditions like Rheumatoid Arthritis and sarcoidosis (Parks et al., 2004). The fact that caseous necrosis observed in areas of macrophage infiltration in the infected lung of MMP-1 mice only indicate that ECM destruction may be the cause of cell death, as opposed to the result of it as suggested by the current paradigm of TB pathogenesis.
The ECM has been described as a cell survival factor in other pathological conditions, such as cancer (Meredith et al., 1993). It has been thought that the formation of caseous necrosis leads to tissue destruction and subsequent cavity formation and transmission. However, this paradigm neglects the fact that ECM must be degraded before forming the cavity and that MMPs are the only enzymes capable of degrading all components of ECM at neutral pH.
Therefore, I would propose a different paradigm of TB pathogenesis, starting with granuloma formation which induces MMP-1 upregulation that in turn cause ECM destruction, leading to cell death, as cells lose survival signal from ECM, and ultimately to cavity formation.
A central role for matrix destruction in regulating cellular behaviour has widespread implications for TB research. Cell-matrix interactions may affect phagolysosomal fusion (Mills and Frausto, 1997), pro-inflammatory cytokine secretion (Coyne and Dervan, 1997), autophagy (Kuratani et al., 1998) and immune cell activation (Sorokin, 2010), all critical processes in the immune response to TB. Tissue damage is emerging as a central determinant of the outcome of the host-pathogen interaction in other lung infections, such as bacterial-viral co-infection (Salamanca et al., 2002), supporting the hypothesis that preserving matrix integrity is fundamental to an effective response to infection.
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To conclude, the humanised mouse model has revealed that interfering with ECM integrity by MMP-1-induced lung tissue destruction causes caseous necrosis which is not seen in any other immune competent mouse model. This suggests that ECM destruction is the first step in TB immunopathogenesis, as opposed to a secondary result of cell death as currently postulated. To study this finding further, I examined the effect of ECM on cells infected with Mtb in cell culture systems.
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