Innate immunity plays a key role in the acute defense against pathogens and has emerged as an important modulator of chronic cardiometabolic diseases including T2DM and atherosclerosis. Here, as part of the GENE study, we utilized experimental endotoxemia in healthy individuals to probe demographic influences on the extent and nature of the induced acute inflammatory response. Compared to EA, we found a pattern of lower inflammatory biomarker responses in AA participants. Although temperature and hemodynamic responses did not differ by race, these parameters are subject to homeostatic regulation that may blunt differential race influences of inflammation on these key regulators of host survival. In contrast, the liver production of acute-phase proteins, CRP and SAA, which are not subject to the same homeostatic regulation, demonstrated substantial difference by race, tracking the pattern observed for the acute cytokine response. The functional role of these cytokines in immune responses, as well as the established utility of CRP and SAA as biomarkers of cardiometabolic disease risk [9, 10], suggest clinically relevant consequences of these observed race-differences in response to endotoxemia.
Our data provide several novel insights beyond epidemiological observations. First, in the GENE study, the LPS-evoked inflammatory cytokine response was higher in EA compared with AA. In support of our finding, one small study of Duffy antigen effect on endotoxin response found that Duffy-negative males of African descent had lower inflammatory response (plasma monocyte chemoattractant protein-1) than Duffy-positive males of European descent , although separate effects of race and Duffy antigen were not addressed. Second, we identified an interesting paradox in inflammatory biomarker response compared to resting biomarker levels. In epidemiological studies, plasma levels of cytokines and inflammatory markers that predict cardiometabolic disease [10, 29] differ by race with higher resting levels of IL-6 and CRP in AA compared to EA [7, 30, 31]. Although we observed similar trends for higher pre-LPS levels of several biomarkers in AA in the GENE study, there was an opposite response to endotoxemia, with lower peak levels in AA compared to EA. For example, in AA relative to EA, the LPS-induced response of plasma IL-1RA and CRP were lower, whereas baseline levels tended to be higher. Third, regardless of race, the LPS-induced cytokine responses had greater correlations with each other and with the subsequent increases in acute-phase proteins than the correlations observed for the pre-LPS cytokines with baseline biomarkers or with LPS-induced responses. This suggests that the evoked inflammatory response may reflect more accurately the biochemical and clinical consequences of acute activation of innate immunity than resting levels of inflammatory markers. Thus, measuring resting levels of inflammatory biomarkers in epidemiological studies may provide limited insight into the stress responses that are most relevant to disease related activation of innate immunity.
We interpret these findings cautiously because we do not yet know whether inflammatory responsiveness to endotoxemia has clinical relevance in disease risk prediction. Further, it is plausible that “baseline” levels of inflammatory biomarkers may reflect an ongoing low-grade inflammation that is relevant to the development of cardiometabolic diseases while the acute responses to LPS during experimental endotoxemia may be more relevant to acute inflammatory disorders like sepsis, SIRS, or other infectious diseases. However, genetic variation in TLR4 that modulates inflammatory response and sepsis has been associated with reduced risk of atherosclerosis and CVD  supporting the relevance of modeling acute TLR4 signaling responses to risk of chronic cardiometabolic disease.
The mechanisms of race-differences in inflammatory responses are uncertain but likely complex. One possibility is that the pattern observed in AA (higher pre-LPS but reduced induced cytokines) reflects chronic conditioning or priming of innate immunity in AA individuals resulting in an attenuated responsiveness during inflammation . Lower peak levels of the inflammatory-modulating IL-1RA in AA suggest that there may be a blunted resolution response as well as attenuated pro-inflammatory signaling. A teleological basis for the race differences may lie in distinct evolutionary pathogenic pressures in African populations compared with those that migrated to northern geographic regions [33, 34] leading to race and geographic differences in the inflammatory response to innate immune antigens including those modulated by diet and lifestyle factors [28, 35]. These concepts, however, require further testing in experimental, genomic and clinical studies.
We observed modest gender differences in the response to LPS with a greater CRP and SAA response in males compared to females. However, not all responses were significant or consistent e.g., peak IL-6 showed a non-significant trend toward higher response in females. Previous small studies also have shown mixed results; CRP and TNFα were increased in response to LPS in females compared with males in a European study (N=30)  but no differences in cytokines were seen in a US study (N=24) despite differences in temperature response . The GENE study recruited a much larger sample (N=294) and included race-stratified analyses. Overall, our findings suggest small, although nominally statistically significant, differences by gender in some inflammatory responses. The gender influence was less than those observed for race and is of uncertain clinical significance given modest differences and inconsistent findings across biomarkers.
Although our data did reveal consistent racial differences in inflammatory responses, the observed race-differences were modest (< 2-fold for most biomarkers) relative to the inter-individual differences in response in both EA and AA (e.g., 100-fold differences in some plasma biomarker responses between the 95th and 5th percentile responders). Such large inter-individual differences suggest that a relatively small portion of the LPS responses is attributable to race per se. Application of unbiased genetic approaches in the GENE study sample may provide novel insights into the genetic basis of innate immune responses in human and also might help identify genomic influences specific to EA and AA populations.
Our study has several unique strengths but also limitations. To our knowledge, this is the largest human experimental endotoxemia protocol published to date and the only one specifically designed to probe differences between AA and EA in the inflammatory and metabolic responses to activation of innate immunity. A distinct advantage of the model is that it controls the temporal and directional activation of innate immunity and downstream responses and thus eliminates concerns regarding reverse causation and confounding when studying patients with established chronic disease. Experimental endotoxemia is a model of activation of innate immunity that partly approximates the acute pathophysiology of sepsis syndromes. Several lines of evidence also suggest that this model may be informative in cardiometabolic disease [11, 14] although we acknowledge that it is unknown whether inflammatory responsiveness to endotoxemia is relevant to clinical disease prediction. Observational data show that sepsis and chronic infection [38, 39] induce insulin resistance, glucose intolerance and lipid derangement resembling that observed in obesity, T2DM and atherosclerosis. We and others have shown that experimental endotoxemia induces insulin resistance [13, 14], adipose tissue inflammation  and atherogenic lipoprotein changes . TLR4 is directly implicated in diet-induced obesity and atherosclerosis  through studies in mouse models, while dietary modulation of the gastrointestinal biome has been shown to influence blood levels of LPS . Atherosclerotic plaque contains microbes, likely oral and gut-derived , while blood microbial load may be predictive of development of diabetes  providing additional support for a link between the immune response to bacteria and cardiometabolic disease.