This is the first report of differences in cytokine expression mediated at the transcriptional level in HIV-infected individuals treated with an ABC-containing regimen. Previous studies chose to assess circulating inflammatory mediators based on their reported associations with CVDs and systemic inflammation. Our approach allowed us to simultaneously profile the expression of a large number of cytokines in two treatment groups, and these results suggest a heightened transcriptional response for inflammatory mediators in those treated with ABC.
One of the four cytokines that showed a significant change in expression, CD40LG, has also been shown to be elevated in the plasma of an ABC-treated animal model [17
]. CD40 ligand is expressed primarily on activated CD4+
T cells, platelets and vascular smooth muscle cells and, under inflammatory conditions, is also induced on monocytes and NK cells [18
]. Ligation with CD40 leads to the production of reactive oxygen species and adhesion molecules that promote a local inflammatory reaction. Both of these molecules are increased in atherosclerotic plaques and their coupling triggers atherogenic signalling events and the upregulation of VCAM-1, ICAM-1, various interleukins, including IL-8 and members of the TNF superfamily such as LTA. LTA itself mediates a large number of inflammatory responses and could be associated with CVD via the concomitant induction of adhesion molecules and cytokines from endothelial and vascular smooth muscle cells [19
This study found significantly lower levels of CCL5 transcription in women on an ABC-containing regimen. Low baseline CCL5 (RANTES) levels were an independent predictor of cardiac mortality in male patients followed up prospectively for 24-months for the occurrence of cardiac mortality and MI [20
]. This followed an earlier study that associated increased serum levels of IL-8 and reduced levels of CCL5 with the risk of CHD, after adjustment for known risk factors [21
]. Although there are conflicting reports correlating elevated levels of CCL5 with adverse cardiac outcomes, it should be noted that measurement of circulating CCL5 can be error prone as it can be readily released by activated platelets during sample collection [22
]. Nevertheless, when examining CCL5 transcription, single nucleotide polymorphisms that are known to downregulate CCL5 expression have been linked to cardiac events in a number of studies [24
]. CCL5 can bind to both chemokine receptors CCR1 and CCR5, and the loss of either of these receptors is associated with the positive and negative development of atherosclerosis, respectively, with the absence of CCR5 demonstrating a protective effect by reducing atherosclerotic plaque formation [26
]. However, a well-defined consequence of reduced CCL5 expression is the coupled upregulation of CCR5 [20
]. This feedback mechanism may lead to adverse cardiovascular outcomes, as a protective effect for CVD, CAD and MI has been associated with lower levels of CCR5.
IL-6 is a classical marker of systemic inflammation, yet our preliminary data showed that approximately 50% of samples in both arms had levels of IL-6 transcription that were below the limit of detection. This cytokine was subsequently excluded from the expanded analysis, as it would have resulted in data from only 15 samples from each arm. Furthermore, excluding those that had undetectable IL-6 would have introduced unwanted bias into the data analysis.
There are several limitations to the work outlined here. This sub-group analysis was matched by CD4 count and viral load in an attempt to ensure the groups are alike but there may be unknown biases introduced that would be mitigated if we were able to use all randomized samples. All of the women in this study were breastfeeding at the six-month interval and previous data demonstrate that breastfeeding raises inflammatory cytokines, although it should increase cytokines equally in both [27
]. However, it may underestimate the change between cytokine expression between the 6- and 12-month period. The period of virologic suppression was relatively short, and it is unknown if both groups would have viral suppression in all reservoirs equally, and this may contribute to differences between the groups. While the lopinavir/ritonavir group may have side effects such as insulin resistance and lipid abnormalities contributing to increased inflammation, they would more likely bias towards the null and fail to demonstrate differences in inflammation rather than the results observed here.
Our data demonstrate the presence of an intracellular inflammatory response in those treated with ABC-based antiretrovirals. All of the differentially expressed genes identified in this study are under the transcriptional control of NF-κB, a well-established mediator of inflammation, and these data suggest its possible involvement in mediating inflammation during ABC-containing ART [28
]. This correlates with our recently presented findings that Toll-like receptors may recognize ABC and stimulate an inflammatory response in vitro
]. However, the interplay between multiple signalling pathways makes it difficult to isolate the transcriptional changes that are possibly attributable to ABC. It is possible that these differences in cytokine expression are a reflection of a greater reduction in systemic inflammation in those treated with a PI-based regiment, but we consider this to be unlikely as women in both arms attained full virologic suppression after nine months of ART. Our study was not designed to evaluate clinical cardiovascular events, and it is important to note that few cardiovascular events (and no MIs) occurred in either study arm in this group of young women with higher CD4 cell counts receiving PMTCT. In conclusion, our data suggest that ABC may directly stimulate the expression of proinflammatory cytokines or it may produce a secondary immunomodulatory effect of upregulated mediators such as CD40L. There is a need to expand the scope of research on ABC beyond the traditional biomarkers of systemic inflammation to understand cellular messaging and, ultimately, the mechanism of inflammation. While the pathways involved in mediating inflammation will need to be the subject of future investigation, it appears that ABC-containing ART regimens have the capacity to upregulate intracellular inflammatory processes.