We observed that LPS and I-FABP circulating levels fluctuated significantly over the course of 24 hours. Previous work in mouse models has shown a postprandial increase in LPS levels [28]. We observed a clinically relevant decrease of LPS after breakfast and an increase after dinner which might be explained by natural variations in circadian rhythm [29, 30]. These results suggest a need to sample LPS from fasting PLWH in order to decrease variation throughout the day. Similarly, I-FABP was subject to daily variations with the lowest level at 16:00 and highest at 4:00–8:00. However, plasma levels of BDG and REG3α showed no significant variation and were not affected by food intake, time of sampling, or day/night shifts. These findings further validate the use of BDG and REG3α as markers of microbial translocation and gut damage, respectively in ART-treated PLWH.
Translocation of bacterial and fungal products are driven by epithelial gut damage and depletion of intestinal CD4-T cells and contribute to immune activation in HIV [21, 31]. Clinical studies commonly use circulating I-FABP to evaluate gut damage as a measure of enterocyte cell lysis. However, in the absence of enterocyte lysis, I-FABP poorly correlates with microbial translocation [24]. Our results show that circulating I-FABP levels varied greatly throughout the course of a day, which limits its value as a marker of gut damage, since it is dependent on the time of sampling and fasting status. In contrast to I-FABP, REG3α appeared stable over the course of 24 hours. Therefore, our results and previous work favor REG3α as a reliable gut damage marker independent of sampling time and food intake in PLWH [24].
LPS is a bacterial translocation marker, responsible for chronic immune activation in HIV-infected patients [32]. However, increasing evidence indicates that diet and food intake affect the plasma level of LPS in mouse and human models. Cani et al.[28] first reported in 2007 that plasma levels of LPS increased after feeding mice with a high-fat diet. Furthermore, López-Moreno et al.[33] reported that the consumption of diet rich in saturated fat increased plasma levels of LPS which in turn, increase the postprandial inflammatory response in subjects with metabolic syndrome. Our results also indicated that food intake was associated with an increase in plasma level of LPS in ART-treated PLWH up to four hours after lunch and supper. Although the underlying mechanism is unclear, it may be related to changes in microbiota composition, increases in the proportion of LPS producing Gram-negative bacteria in the presence of nutrients [34]. LPS detoxification by the intestinal alkaline phosphatase [35], or fat intake promoting gut translocation of LPS [28, 33]. Therefore, monitoring LPS levels in PLWH should take into account feeding state and time of specimen acquisition.
Unlike LPS, we showed that the fungal translocation marker BDG is stable throughout the day and independent of food intake. BDG can be found in food such as mushroom and seaweed [36, 37]. Interestingly, Hashimoto et al. reported that serum BDG value was elevated due to intake of seaweed in a hematopoietic stem cell transplant recipient [38]. However, the elevation of BDG may have been linked to gut damage with increased intestinal permeability during acute graft-versus-host disease (GVHD). Nevertheless, our results showed that BDG is a reliable marker for fungal translocation in ART-treated PLWH. The food provided in our study did not comprise mushroom, seaweed or other material rich of BDG. Thus, further studies need be conducted in order to study the effects of BDG rich food on its plasma level.
We acknowledge that our study presents some limitations as we did not study the underlying mechanism of daily variation of I-FABP or LPS levels. Daily changes in the external environment may also influence those markers and studies have identified the molecular underpinnings of oscillations in circadian clock gene expression occurring over the 24-hour day [30]. Our study population only included a small sample size of male participants over the age of 50, therefore younger participants and inclusion of female participants will be needed to infer study findings on a larger population.
To our knowledge, we are the first to report the daily variation of different microbial translocation with gut damage markers in ART-treated PLWH. We showed that conversely to I-FABP and LPS, plasma levels of REG3α and BDG can be considered as reliable markers of gut damage and fungal translocation respectively, and are not influenced by food intake, time of sampling, or day/night shifts. Such findings may have immediate clinical implications for making appropriate diagnosis and prognosis assessment in care and clinical trials involving persons living with HIV.