The modulation of macrophage apoptosis during S. pneumoniae infection by HIV-1 orantiretroviral agents.

HIV patients are more susceptible to lung infection with a bacterium called the pneumococcus. Since
effective HIV therapy became available, many secondary infections have become less common in
HIV. However, pneumococcal infection occurs more frequently in these individuals despite effective
HIV treatment. A cell called a macrophage has an important role clearing bacteria from the lung. Its
capacity to do this is regulated by the time at which it dies following exposure to bacteria. HIV
infects macrophages and can alter how they perform this protective role. In the laboratory, I will
address whether HIV affects macrophage survival (and thus its function) during pneumococcal
infection and I will determine precisely which point in the sequence of events which leads to
macrophage death is affected by HIV. I will also determine whether HIV has an effect on a specific
signalling pathway in macrophages which can alter levels of factors that regulate susceptibility to
cell death. I will examine whether some medicines used to treat HIV itself affect the macrophage?s
survival and consequently its activity against pneumococcal infection. I will repeat certain
experiments using macrophages taken from the lungs of volunteers.
Understanding these interactions will hopefully identify possible targets for new HIV treatments

There is a high incidence of invasive pneumococcal disease in HIV-1. Despite anti-retroviral therapy
the incidence of invasive bacterial disease remains elevated and accounts for a third of non-AIDS
deaths. The alveolar macrophage is essential to the lung?s response to Streptococcus pneumoniae
(Spn) infection, with key roles in bacterial clearance and regulation of inflammation, which are
controlled by the timing of apoptosis induction.
My hypothesis is that both HIV-1 and antiretroviral therapy modulate the macrophage
apoptotic response to Spn, enhancing susceptibility to Spn in HIV-1 infected individuals.
My objectives will be to:
1. Establish which of the sequential steps, that culminate in macrophage apoptosis during Spn
infection, is altered by HIV-1 infection of macrophages.
2. Investigate the role of the PI3-k/Akt pathway in the manipulation of apoptosis susceptibility,
during Spn infection, by HIV-1.
3. Determine if HIV-1 protease or reverse transcriptase inhibitors also directly modulate
macrophage apoptosis.
I will observe macrophage apoptosis during Spn infection using HIV-1 or sham-infected cell lines
and primary cells. Apoptosis will be quantified by cell morphology and hypodiploid DNA
measurement using flow cytometry. To determine which points in the apoptotic cascade are altered,
markers of critical stages in the evolution of apoptosis in macrophages during Spn infection
(including lysosomal membrane permeabilisation, cathepsin D activation, cytochrome C release from
mitochondria, Mcl-1 expression and caspase activation) will be measured at key time points. Akt
phosphorylation will be measured by western blot in the same experimental conditions to determine
the effect of HIV-1 on the PI3-k/Akt pathway. Using PI3Kinase inhibitors and by transfecting cells
with constitutively active Akt I will establish whether enhancement or inhibition of the PI3-k/Akt
pathway affects the apoptotic response of cells to Spn. Finally I will determine if protease inhibitors
and the non-nucleoside reverse transcriptase inhibitor efavirenz alter HIV-1 related changes in
macrophage apoptosis induction during Spn infection by repeating experiments in the presence of
these drugs. To confirm the validity of findings in the in vitro macrophage models, I will repeat key
experiments using primary monocyte-derived macrophages and alveolar macrophages from HIV-1
infected individuals commencing antiretroviral therapy and HIV-negative controls.
The scientific and medical opportunities which this work will create will arise from the identification
of a new mechanism by which HIV-1 manipulates the macrophage?s response to common bacterial
pathogens. The findings will inform therapeutic strategies to minimise the impact of HIV-1 on the
susceptibility to Spn and related bacterial infections.