Infection & Immunity

The Leishmania Laboratory - Genetic Basis of Host Susceptibility

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The genetic basis of host susceptibility to disease

The clinical manifestations of human leishmaniasis display a spectrum of disease severity. Although it is generally accepted that pathogenicity and virulence must be understood in terms of the interaction between the individual genomes of the micro-organism and its host, dissecting out the relative contributions of host and pathogen has been difficult, especially in outbred host populations such as humans. In the case of cutaneous leishmaniasis, this problem has been addressed with a reasonable degree of success by using inbred strains of mice in which the various disease phenotypes resemble the spectrum of clinical manifestations in humans.

The disease phenotype observed in human cutaneous leishmaniasis can be mimicked in the laboratory by infection of different inbred strains of mice with L. major. Thus, the mouse model of the highly susceptible BALB/c at one end of the spectrum, and the resistant C57BL/6J at the other, has been widely used to study both the genetics and biology of the host response to infection. In this system, the marked polarisation in the immune response observed upon challenge with L. major parasites has been implicated in determining the severity of disease. BALB/c mice produce Th2 type cytokines, in particular IL-4, which has been shown to be associated with disease progression and susceptibility. In contrast, recovery from infection in resistant C57BL/6J mice depends upon the induction of a Th1 type response resulting in activation of macrophages and killing of the intracellular organisms.

Working with colleagues Simon Foote and our joint PhD student Lynden Roberts, from the Genetics and Bioinformatics Group, we have been able to define three loci which appear to determine the severity of disease manifestations in this animal model for disease. Recently, a PhD student, Colleen Elso has produced congenic mice carrying the resistance alleles from the C57BL/6 mouse on the genetic background of the susceptible BALB/c mouse and vice versa, the susceptibility alleles from the BALB/c on the resistant genetic background.

Using these mice, we have demonstrated the involvement of these alleles in disease severity. Most surprisingly, we found that at the stage of infection when the difference in disease severity between the congenic and parental mice is most pronounced, the cytokine profile correlates with the parental genetic background, not with the disease phenotype. Thus, congenic mice containing the susceptibility allele on the resistant genetic background display severe disease, but show the resistant Th1 cytokine profile. Similarly, mice containing the resistance allele on the susceptible genetic background display an attenuated disease phenotype, but a Th2 cytokine profile. This indicates that the T helper cell phenotype has little effect on the final clinical outcome and suggests that the loci that we have identified act by some other mechanism.

TH1/TH2 CYTOKINES IN THE INFECTED CONGENIC MICE
At 14 weeks after infection, when the polarisation of disease was at its peak, we examined the production of mRNA for the Th1/Th2 polarising cytokines IFN-gamma and IL-4 in the draining lymph nodes of the congenic and parental mice. RNA from 36 BALB/c, B/c.B6-(lmr1, lmr2), C57BL/6, B6.B/c-(lmr1, lmr2) mice was analysed by real time fluorescence PCR using the LightCycler. A ratio between the log-transformed starting template values of IFN-gamma and IL-4 was calculated for each mouse. BALB/c mice congenic for C57BL/6 lmr1 and lmr2 show the same IFN-gamma:IL-4 ratio as the parental BALB/c mice despite being more susceptible and vice versa, C57BL/6 congenic for the BALB/c lmr1, lmr2 show similar ratios to their parental strain.

Together with Colleen Elso, a post doctoral fellow Beena Kumar is undertaking a detailed study of the mechanisms leading to the pathology observed in the congenic mice. She is studying the cell populations arriving into the lesion and the draining lymph node during the course of infection, as well as the immune responses induced by infection.

The major challenge for the future is to isolate the genes responsible for disease susceptibility in the congenic mice. This will pave the way for the identification of human susceptibility genes.

Next: The role of cytokines and their regulation in determining the disease phenotype...

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