A new coding mutation in the Tnf-alpha leader sequence in tuberculosis-sensitive I/St mice causes higher secretion...

I/St and A/Sn mice are polar extremes in terms of several parameters defining sensitivity to Mycobacterium tuberculosis. TNF-alpha, mainly produced by activated macrophages, can mediate both physiological and pathophysiological processes. Adequate TNF-alpha levels are essential for a forceful protective response to M. tuberculosis. We have functionally characterized a nonsynonymous substitution, Arg8 His, in the highly conserved cytoplasmic domain of the pro-TNF-alpha leader peptide from extremely M. tuberculosis-sensitive I/St mice. This was compared to the common pro-TNF-alpha variant found in A/Sn mice. Using cDNA constructs, both variants were constitutively expressed in HEK293A cells. A significantly higher secretion level of Arg8 His TNF-alpha was shown using flow cytometry and ELISA analysis (P=0.0063), while intracellular levels were similar for both protein variants. An even TNF-alpha distribution throughout the cells was seen using confocal microscopy. This suggests that the Arg8 His substitution affects pro-TNF-alpha processing. The I/St mouse may serve as a model to further explore the function of the well-conserved cytoplasmic region of TNF-alpha. However, other identified substitutions in the I/St promoter, introns and 3'UTR of Tnf-alpha, as well as the cellular environment in vivo may affect the balance between soluble and intracellular Arg8 His TNF-alpha before and during M. tuberculosis infection.