A new coding mutation in the Tnf-α leader sequence in tuberculosis-sensitive I/St mice causes higher secretion levels of soluble TNF-α

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Abstract
I/St and A/Sn mice are polar extremes in terms of several parameters defining sensitivity to Mycobacterium tuberculosis. TNF-α, mainly produced by activated macrophages, can mediate both physiological and pathophysiological processes. Adequate TNF-α levels are essential for a forceful protective response to M. tuberculosis. We have functionally characterized a nonsynonymous substitution, Arg8His, in the highly conserved cytoplasmic domain of the pro-TNF-α leader peptide from extremely M. tuberculosis-sensitive I/St mice. This was compared to the common pro-TNF-α variant found in A/Sn mice. Using cDNA constructs, both variants were constitutively expressed in HEK293A cells. A significantly higher secretion level of Arg8His TNF-α was shown using flow cytometry and ELISA analysis (P=0.0063), while intracellular levels were similar for both protein variants. An even TNF-α distribution throughout the cells was seen using confocal microscopy. This suggests that the Arg8His substitution affects pro-TNF-α processing. The I/St mouse may serve as a model to further explore the function of the well-conserved cytoplasmic region of TNF-α. However, other identified substitutions in the I/St promoter, introns and 3′UTR of Tnf-α, as well as the cellular environment in vivo may affect the balance between soluble and intracellular Arg8His TNF-α before and during M. tuberculosis infection.