Interrelationship between natural resistance gene (NRAMP-1) function and nitric oxide expression:Molecular mechnisms and pathophysiological background

Resistance to intracellular pathogens is in part genetically determined. One of such host resistance gene, named NRAMP-1 (for natural resistance associated macrophages), is a transmembrane protein expressed in the late phagolysosom of neutrophils and macrophages of mammals. Although, it is known that NRAMP-1 is a putative transporter of divalent metals including iron and of protons across the phagolysosomal membrane, the underlying mechanism by which NRAMP-1 confers resistance is still elusive. In mice a mutation within the NRAMP-1 gene leads to loss of function and reduced production of nitric oxide (NO). This is of relevance for host defense since the formation of NO is a pivotal effector pathway for immune defense against intracellular microbes such as Mycobacteria or Leishmania spp. The aim of our project is thus to characterize the molecular mechanisms by which NRAMP-1 functionality regulates NO formation. We will study this by using murine macrophage cells which are stable transfected either with a NRAMP-1 expressing plasmid (NRAMP1r) or a plasmid over-expressing an antisense (non-functional) NRAMP-1s. Based on preliminary data also of our group showing differences in inducible NO synthase (iNOS) activity, the protein being responsible for high output formation of NO following cytokine stimulation of macrophages, between NRAMP1r and NRAMP1s cells, we will then investigate the divergent expression of iNOS from the translational and posttranscriptional levels down to the transcriptional level trying to identify target transcription factors whose binding affinity is altered by NRAMP-1 function leading to altered iNOS gene expression. Moreover, being aware of the fact that NRAMP-1 is a putative transporter of iron and protons we then want to study the pathophysiological background by which NRAMP-1 influences the molecular expression of iNOS. Possibilities which will we investigated include a NRAMP-1 mediated regulation (i) of cellular iron homeostasis with subsequent effects on iNOS expression since iron is a strong inhibitor of iNOS transcription and (ii) of intracellular pH and/or formation of reactive oxygen species both of which are potent regulators of transcription factor binding affinities and iNOS expression.Our results may help to understand by which mechanisms NRAMP-1 strengthens host response of macrophages and will contribute to disentangle the complex interrelationship between NRAMP-1 function, changes in host defense mechanism (NO and oxygen radical formation) and iron homeostasis thus providing information for favorable therapeutic approaches to modulate this network during infections with intracellular pathogens.