Targeted Mutation of Nuclear Bone Morphogenetic Protein 2 Impairs Secondary Immune Response in a Mouse Model

Abstract

We recently identified a nuclear variant of the BMP2 growth factor, called nBMP2. In an effort to understand the function of this variant protein, we generated a mouse line in which BMP2 is expressed and functions normally, but nBMP2 is excluded from the nucleus. This novel mutation allows the study of nBMP2 without compromising BMP2 function. To determine whether nBMP2 plays a role in immune function, we performed a series of experiments in which we compared mouse survival, organ weights, immune cells numbers, and bacterial load in wild type and nBmp2NLStm mice following primary and secondary challenges with Staphylococcus aureus. Following primary challenge with S. aureus, wild type and nBmp2NLStm mice showed no differences in survival or bacterial load and generated similar numbers and types of leukocytes, although mutant spleens were smaller than wild type. Secondary bacterial challenge with S. aureus, however, produced differences in survival, with increased mortality seen in nBmp2NLStm mice. This increased mortality corresponded to higher levels of bacteremia in nBmp2NLStm mice and to a reduced enlargement of mutant spleens in response to the secondary infection. Together, these results suggest that the recently described nuclear variant of BMP2 is necessary for efficient secondary immune responses.

The nuclear variant of bone morphogenetic protein 2 (nBMP2) is expressed in macrophages and alters calcium response

“…To determine whether nBMP2 is expressed in macrophages, BMD macrophages and splenic macrophages were isolated from naïve (uninfected) wild type and nBmp2NLS tm mutant mice and differentiated in vitro , and immunocytochemistry was performed using an anti-BMP2 antibody that binds to both BMP2 and nBMP2. Consistent with our prior observation of impaired immune response in nBmp2NLS tm mutant mice 11 , nBMP2 was detected in the nuclei of wild type BMD (Fig. 1a) and splenic (Fig.…”

“…In our prior study, immune deficiencies in nBMP2NLS tm mice were detectable only after the mice received a secondary infection with S. aureus 11 . Because our current experiments revealed no significant differences in Ca 2+ response in naïve BMD macrophages from mutant compared to wild type mice, we decided to replicate the in vivo conditions of our previous work by examining splenic macrophage harvested from mice after a secondary infection with S. aureus , and by using S. aureus supernatant as the stimulus to trigger Ca 2+ flux 11 . Although S. aureus is a gram positive bacteria that does not produce LPS, it does produce liphoteichoic acid (LTA), which is similarly able to activate macrophages 20,21 .…”

“…Previously, we demonstrated that deficiency of nBMP2 in the nucleus impairs secondary immune response as evidenced by diminished spleen enlargement, poor clearance of S. aureus from the bloodstream, and increased mortality after secondary infection 11 . We have also shown that deficiency of nBMP2 in myonuclei is correlated with slowed skeletal muscle relaxation after contraction, and deficiency of nBMP2 in the nuclei of hippocampal neurons is correlated with learning/memory deficits 2,3 .…”

“…To see if nBmp2NLS tm mutants had compromised immune response, mice were challenged by systemic infection with Staphylococcus aureus . While the mutants’ immune response to a primary infection appeared normal, their immune response to a secondary infection challenge 30 days later resulted in higher levels of bacteremia, increased mortality, and failure of spleens to enlarge normally 11 . Although we did not observed differences in the total number of macrophages in spleen, thymus, or lymph node from wild type compared to mutant mice, we did observe that after the secondary infection, spleen from nBmp2NLS tm mutant mice showed fewer hemosiderin-laden macrophages than spleen from wild type mice 11 .…”

“…While the mutants’ immune response to a primary infection appeared normal, their immune response to a secondary infection challenge 30 days later resulted in higher levels of bacteremia, increased mortality, and failure of spleens to enlarge normally 11 . Although we did not observed differences in the total number of macrophages in spleen, thymus, or lymph node from wild type compared to mutant mice, we did observe that after the secondary infection, spleen from nBmp2NLS tm mutant mice showed fewer hemosiderin-laden macrophages than spleen from wild type mice 11 . Macrophages in the spleen accumulate hemosiderin by phagocytosing damaged red blood cells and hemoglobin, which would be present in the blood stream of S. aureus -challenged mice due to the hemolysins that S. aureus expresses 12–14 .…”