Although markers of microbial translocation have been associated with immune activation in HIV disease, the clinical significance of these relationships is poorly defined [17
]. We measured markers of microbial translocation (LPS), humoral immune response (EndoCAb), LPS bioactivity (sCD14), and enterocyte damage (I-FABP) among participants in a study of continuous versus intermittent ART. Using a case-control study design, we found that (1) high sCD14 levels were associated with an increased risk of all-cause mortality; (2) the association of sCD14 level with mortality persisted after adjustment for baseline CD4+
T cell count and HIV RNA level and for markers of inflammation and was evident for subjects in both the DC and VS groups; (3) sCD14 levels correlated with levels of IL-6, hsCRP, SAA, and D-dimer; (4) I-FABP, a specific indicator of enterocyte death, was detectable in many SMART subjects; and (5) detectable I-FABP levels were associated with lower nadir and baseline CD4+
T cell counts. Collectively, these observations are consistent with a model in which HIV infection causes ongoing damage to the gut mucosa, leading to increased microbial translocation, increased systemic inflammation, and increased mortality.
Subjects with the highest sCD14 levels, reflecting the greatest monocyte activation, had a 6-fold higher rate of death than did those with the lowest levels of sCD14. The likelihood of type I error is increased, given comparisons of 5 variables for 3 outcomes, but the P
value of <.001 makes it unlikely that this finding was attributable to chance. sCD14 level has been associated with disease progression in chronic viral hepatitis (N.G.S., unpublished data) and mortality in other clinical situations characterized by endotoxemia, including hemodialysis [29
]. High sCD14 levels could lead to increased CD38 expression on CD4+
T cells, which is a predictor of CD4+
T cell loss and death [30
]. In this study, the causes of death included CVD, AIDS, hepatic and renal disease, malignancy, and others [19
]. Although higher levels of sCD14 were not associated with increased risk of CVD or AIDS, subjects may not have been followed up long enough to detect the impact that sCD14 could have on progression to these particular end points. Nonetheless, high levels of LPS-induced monocyte activation may point to a common mechanistic pathway that underlies the different pathological conditions that culminate in death in HIV-infected individuals.
sCD14 is unlikely to be merely a marker of an acute phase response. The LPS binding site on CD14 is well-described [32
]. Endotoxin challenge in humans upregulates CD14 expression 2.5-fold [33
], and LPS stimulation induces sCD14 secretion by monocytes [25
]. sCD14 increases macrophage and neutrophil responses to LPS 50–100-fold, compared with LPS alone [34
]. sCD14 is elevated in other diseases characterized or exacerbated by endotoxemia, such as hepatitis [35
], rheumatoid arthritis, and systemic lupus erythematosus [36
], but not in all situations with elevated acute phase reactants, including viral infections or after cardiac surgery [37
]. The weak correlation between sCD14 and IL-6 levels suggests an indirect relationship in which the factors that cause increased sCD14 levels–namely, microbial products–also cause increased levels of inflammatory markers.
Adjusting for inflammatory markers, CD4+
T cell count, or HIV RNA level did not reduce the association of sCD14 levels with death, suggesting that sCD14 independently predicts mortality. Although the increased risk of death in the VS arm was not significant (likely because it was underpowered), the lack of interaction between the DC arm and the VS arm suggests that the effect of sCD14 is independent of treatment interruption, intermittent high-level viremia, and low CD4+
T cell counts. Surprisingly, sCD14 levels were higher in subjects receiving ART with HIV RNA level ≤ 400 copies/mL than they were in untreated individuals. This could reflect an effect of treatment on sCD14 levels, but it contrasts with published data showing no change in sCD14 level after 48 weeks of ART [17
] and a decrease in sCD14 levels after>1 year of ART [39
]. Because the P
for this comparison was .04, this finding could be a chance occurrence, or it could reflect differences in disease severity between these unmatched groups of subjects. Similarly, lower EndoCAb levels in treated patients and the lack of difference in LPS levels between patients receiving ART and those not receiving ART may reflect these confounding factors.
Contrary to what we hypothesized, neither LPS nor 16S rDNA was directly associated with clinical end points. 16S rDNA degrades easily after freeze-thaw cycles, which may have affected our results [41
]. The lack of association between LPS and sCD14 levels may be related to the biology of LPS and the host response to it. LPS is cleared rapidly from the blood by numerous mechanisms, including transfer to high-density lipoprotein, formation of LBP–LPS complexes [42
], scavenging by EndoCAb [43
], and clearance by sCD14 [44
]. LBP, EndoCAb, and HDL and other plasma proteins inhibit LPS measurement [45
], and plasma turbidity and interference from triglycerides can complicate LPS measurement [46
]. Alternatively, a given level of LPS may result in differing levels of monocyte activation in different individuals. There may be a genetic component to the response to LPS, akin to the polymorphism in the peptidoglycan receptor NOD2 in Crohn disease [47
]. Indeed, polymorphisms in both NOD2 and TLR4 have been associated with bacteremia and mortality [47
]. Furthermore, a polymorphism in the promoter region of the gene encoding CD14 (-159C/T) has been associated with higher sCD14 levels in heavy alcohol drinkers [48
] and in those with hepatitis C infection [49
]. Thus, when considering the consequences of microbial translocation, measures of the host response to the microbial product (bioreactivity), rather than measures of the microbial product itself, may be more accurate and relevant.
Histological analysis of intestinal biopsy specimens from HIV-infected patients shows enterocyte damage [50
]. Because only dying enterocytes release I-FABP [24
], the elevated I-FABP levels detected in most subjects may reflect ongoing structural damage to the gastrointestinal tract. Indeed, the association of lower CD4+
T cell counts with higher I-FABP levels also supports the hypothesis that damage to the gut, immune activation, and CD4+
T cell loss are intimately linked. Because our HIV-negative volunteer group was not matched for factors that could affect enterocyte death, such as liver disease and age, further investigation with longitudinal samples in a matched, diverse patient population is needed to confirm these findings.
Taken together, our data show that high plasma levels of the monocyte-expressed LPS receptor sCD14 are associated with an increased risk of all-cause mortality in HIV-infected subjects in the SMART study. The diversity of the patients enrolled in this study, who were from virtually every continent, and many of whom had hepatitis co-infection and other co-morbidities and were receiving various ART regimens (or none at all) [19
], makes the results relevant to many patient populations. The markers of inflammation that confer an increased risk of death in this patient population correlate with the level of monocyte stimulation by LPS. In addition, we show that I-FABP, a marker of enterocyte death, is increased in these HIV-infected subjects and that detectable levels are associated with lower baseline and nadir CD4+
T cell counts. These results suggest that attenuation of the inflammation induced by microbial products, in addition to viral suppression, may be necessary to maximally reduce mortality and improve clinical outcomes.