3.2 Plasma proteome profiling
Group-wise comparison identified 53 soluble factors which differed between the groups (P < .05; Mann–Whitney U test), of which 47 factors were different between HC and VP, 45 between long-term ART and VP, and 3 between HC and ART (Fig. 2A and Supplementary file 1, http://links.lww.com/MD/C93). Among those factors, 5 factors were unique between ART and VP (CCL13, CCL4, CXCL1, CD4, and CSF-1), while 7 factors were unique between HC and VP (DCN, PlGF, HO-1, CCL23, TNFRSF21, CXCL11, and VEGFR-2), and only 1 secretory factor between HC and ART, namely angiopoietin-2 (ANG-2). The 2 chemokines CCL4 and CCL13 were significantly elevated in ART-treated patients compared to VP, and even a trend was observed compared to HC (Fig. 2B). In patients on long-term suppressive ART, all of the pro-inflammatory molecules examined (IL-7, IL-12, and soluble IL receptor IL12RB1) went back to levels of healthy controls. Among the 29 cytokines tested, 93% (27/29) of them went back to physiological levels in the long-term ART group, but not the levels of the non-cytokine molecules soluble killer cell lectin-like receptor subfamily D member 1 (KLRD1, also CD94) (P = .02) and platelet-derived growth factor subunit B (PGDFB) (P = .0485) (Fig. 2B). ANG-2, an endothelial activation marker, did also not normalize to the healthy state following long-term suppressive therapy.
To further dissect which biomarkers can differentiate the groups, cluster analysis (k-means) was performed at significance level FDR < .05. Cluster analyses identified higher levels of CCL13, SDF-1, TWEAK, CXCL5, CD40-L, CASP8, TNFRSF12A, DCN, and TNFRSF21 in ART and HC, while CXCL11, Gal-9, IL-12, CXCL13, CXCL9, CXCL10, CCL19, KLRDI, CD8A, IL12RB1, CD27, Granzyme-H, and Granzyme-A in VP (Fig. 3A). The patients with successful long-term ART clustered and networked with HC, while VP grouped separately. We further used a stringent statistical significance to find the precise network and potential markers between the VP and ART/HC groups. Cluster and network analysis identified 13 soluble factors (CD8A, CRTAM, CXCL13, EGF, CD5, CD40, CXCL9, Gal-1, IL12RB1, KLRD1, PD-1, CASP-8, and TNFRSF9) which were significantly discrete at false discovery rate (FDR) adjusted to P(q) <0.001 using ANOVA (Fig. 3B).
3.3 Functional enrichment analysis
We performed functional enrichment analysis by Kyoto Encyclopedia of Genes and Genomes (KEGG) using 53 proteins (defined by Mann–Whitney U test significant differences between groups). In total, 32 pathways are enriched with FDR <0.05 (Supplementary data file 2, http://links.lww.com/MD/C94). Among these pathways, cytokine–cytokine receptor interaction (FDR = 3.62 × 10−29) and chemokine signaling pathway (FDR = 1.26 × 10−10) included together 29 soluble factors. Apart from that, antigen processing and presentation (FDR = 0.007), and natural killer cell mediated cytotoxicity (FDR = 0.037) are enriched. The CIRCOS plot indicates the difference of soluble marker levels between groups and subsequent signaling pathways the molecules are involved in (Fig. 4A). There are several factors, which are present in more than 1 pathway. Further Ingenuity Pathway analysis (IPA) identifies that the expression of KLRD1 is regulated by proinflammatory molecules (IL15, IL21, IL2, IL4, IL12 complex, etc) and it regulates the tumor necrosis factor (TNF) and cytokine signaling pathways (Fig. 4B).
Our study investigated the levels of soluble biomarkers in patients with nearly 2 decades successful ART with the highest number of proteins studied so far. Very long-term ART normalizes the level of most cytokines and chemokines explored to the degree of healthy individuals. However, some biomarkers, like KLRD1 and ANG-2, do not go back to healthy physiological levels indicating that immunological events still take place in HIV-1-infected patients despite long-term suppressive ART.
In concordance with other studies, elevations of pro-inflammatory cytokines and chemokines were seen in VP. As observed earlier, the 2 chemokines CCL4 and CCL13 were significantly elevated in ART-treated patients compared to VP, and even a trend was observed compared to HC. CCL4 is known as a major HIV-suppressive factor for CCR5-tropic strains, which is produced by CD8+ T-cells, as it acts as a competitor for the viral binding site. Since VP patients fail to control the infection, reduced CCL4 levels are explainable, either as one of the causes or the consequences of sustained viremia. But it is surprising, that CCL4 levels in patients on long-term ART were higher than for HC, though it was not statistically significant. The CCL13 ligand for receptor CCR2/CCR3 is a chemotactic factor for monocytes and macrophages. However, its involvement during HIV infection is not evident. SDF-1 binds to CXCR4 and therefore competes with X4-tropic virus strains, but to a less extent than CCL4 does with CCR5-tropic strains. In our study SDF-1 levels in ART were comparable with those in HC, but were significantly reduced in VP. Therefore, apart from suppressing the virus, ART might also boost the host's non-cytolytic antiviral immunity.
In earlier studies, levels of proinflammatory cytokines in patients on ART have not been reduced to levels of healthy individuals despite successful therapy.[18,19] In our study, we observed, that in patients on long-term suppressive ART, all of the proinflammatory molecules examined (IL-7, IL-12, and soluble IL receptor sIL12RB1) went back to normal physiological levels. Among the 29 cytokines tested, 93% (27/29) of them went back to normal physiological levels in long-term ART groups, This confirms, that very long-term successful ART does not only lead to a pronounced decline in viral load but also reconstitutes the inflammatory state almost to the physiological level of healthy individuals.
Together all analysis demonstrated, that in patients on ART, KLRD1 levels were in between those of HC and VP. This receptor is expressed mainly on NK cells and plays a significant role in mediating NK cytotoxicity. KLRD1 (CD94) forms heterodimers with NKG2 resulting in a receptor complex expressed on NK cells, and also on some CD8+ T cells. So far, 5 different members of the CD94/NKG2 family are known which are inhibitory receptors, CD94/NKG2A and CD94/NKG2B, or activating receptors, CD94/NKG2C, CD94/NKG2E, and CD94/NKG2H. It has further been shown, that some of these complexes recognize the non-classical Human Leukocyte Antigen (HLA) class I molecules, namely HLA-E, with different binding affinities.[20,21] Contradictory results have been observed in HIV-1 infection, for example increase and stabilization of the expression of HLA-E on lymphocytes in vivo, which might be a mechanism of immune evasion used by the virus. Since HLA-E can bind to CD94/NKG2A, this interaction could lead to impaired NK cell function. In concordance, it has also been reported, that expression of inhibitory CD94/NKG2A is increased in viraemic patients. In our data, an increased level of sKLRD1 was seen in VP. However, we do not know which heterodimer types are involved in that upregulation. Other groups have also observed increased expression of CD94 and NKG2A on NK cells and T-cells, which was associated with disease progression in HIV-infected patients.[22,23] That would support the hypothesis of upregulated CD94/NKG2A in VP leading to inhibition of NK cell activity and NK cell dysfunction. In contrast, there are also studies proposing a decrease in cells expressing CD94/NKG2A, along with an increase of cell expressing the activating receptor CD94/NKG2C in viremic HIV-infected patients.[24,25] Despite these contradictory results, it can be concluded that CD94/NKG2 signaling is altered in viral progressors and that long-term ART could not fully re-establish the physiological state of healthy individuals in our cohort.
Angiopoietin-2 binds to the cell surface receptor for angiopoietin-1 (ANG-1), namely TEK/TIE2; and thus, modulates ANG-1 signaling. An earlier study in females from Kenya observed, that increased ANG-2 plasma levels in chronic HIV-1 infection decrease after ART. A higher level of ANG-2 is also associated with higher mortality. However, in our study with male populations both in VP and ART, the median plasma level is lower than in HC, with statistical significance between ART and HC group. The study in Kenya also detected significant associations between the use of oral contraceptive pills and higher plasma ANG-2 levels in pregnancy. They also observed that estrogen stimulates ANG-2 mRNA expression. We, therefore, conclude that endothelial activation marker ANG-2 was lower in a male with advanced HIV infection and had no effect on ART initiation, though it does not increase to a healthy status.
The study has limitations that merit comments. First, the numbers of patients were relatively low. This is mainly because of limited numbers of HIV-infected individuals with very long-term suppressive therapy for whom adequate clinical and demographical information is available. These groups of patients were identified from nearly 10,000 patients who got treatment care in Sweden and availability of the plasma samples. Second, we only looked into 92 plasma soluble factors. However, to best of our knowledge, this is the most substantial amount of markers that have been studied to date.
In conclusion, this is the first study, which investigated the levels of soluble biomarkers in patients with nearly 2 decades successful ART with the highest number of proteins studied so far. Very long-term ART normalizes the level of most cytokines and chemokines explored to the degree of healthy individuals. However, some biomarkers do not go back to healthy physiological levels indicating that immunological events still take place in HIV-1-infected patients, despite long-term suppressive ART. Future analyses of cellular subsets other than T-lymphocyte populations, like NK cells, are likely to help us gain further insights into the long-term restoration of the immune system by ART.
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chemokines; cytokines; decade long ART; HIV-1
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