Patterns of inflammation and immune activation by coreceptor use in people living with HIV-1

Abstract

Introduction: Human immunodeficiency virus type 1 (HIV-1) utilizes either the CCR5 (R5) or CXCR4 (X4) coreceptor for host cell entry. Coreceptor switching from R5 to X4 and elevated immune activation have been associated with disease progression. X4-tropic HIV-1 is predominantly observed in the late stage of infection, when the immune environment characterized by chronic activation is optimal for their replication. The aim of this study was to determine viral tropism in late HIV presenters and who have not previously received treatment in Mexico City and its relationship with markers of chronic immune activation.

Methods: A cross-sectional study was conducted on 122 people living with HIV (PLWH) recruited from two public health services. Viral tropism was determined using next-generation sequencing (NGS) and the geno2pheno algorithm. Immune activation was assessed through flow cytometry (CD38+, HLA-DR+), and soluble markers (sCD14, sCD163, IL-6) were quantified using enzyme-linked immunosorbent assays (ELISA). Differences in immune activation patterns between R5 and X4 group were explored using Mann-Whitney Wilcoxon test and t-test, and a principal component analysis (PCA). Logistic regression was used to evaluate associations between immune activation profiles and the presence of X4-tropic viruses.

Results: Ninety-eight individuals had high-quality V3 loop sequences, 81.6% harbored only R5 variants (R5 group), while 18.4% had mixed R5/X4 populations (X4 group). Most PLWH had CD4+ T cell counts below 200 cells/µL, showing no significant difference between groups. Elevated levels of IL-6 were significantly associated with the R5 group (p = 0.01), while the X4 group showed increased expression of CD38+ and HLA-DR+CD38+ markers, although not statistically significant. Furthermore, IL-6 emerges as a negative predictor for the presence of X4 viruses (OR = 0.06, p = 0.006).

Conclusion: R5-tropic viruses are associated with elevated inflammatory responses in early stages, as indicated by higher IL-6 levels, while X4-tropic viruses may contribute to CD4+ T cell depletion through immune activation. Consequently, elevated levels of IL-6 emerge as a negative predictor for the presence of X4 viruses. The relationship between viral tropism and chronic immune activation in HIV-1 infection reflects a complex interplay which appears to be bidirectional.

Keywords: CCR5; CD38; CXCR4; HIV-1 tropism; HLA-DR; IL-6; chronic immune activation; next generation sequencing.

Figure 1

Distribution of R5 and X4 viral variants among the viral population for each individual. All samples were obtained from late HIV presenters. (A) The 20th most frequent viral variants from each individual were plotted, as the remaining variants had a frequency of less than 1% of the viral population (see Supplementary Material ). In order to infer the use of CXCR4 or CCR5 coreceptor in each of these sequence populations, the geno2pheno tool was employed, with a FPR threshold of <3.75%. It was observed that the majority of the viral variants present in each sample clustered below 2% of the viral population. The minority variants, which clustered between 2% and 20%, represented the second most abundant viral variants. (B) A closer examination of 19 samples, of which 10 were stratified as X4 (red) and 9 as R5 (blue). This figure confirmed that the majority of viral variants had a frequency below 2% of viral population.

Figure 2

Principal Component Analysis (PCA). Dimension reduction analysis of the transform clinical and immune set of variables into a smaller one that still contains most of the information in the original set. PCAs colored by tropism assessment: individuals with only R5 viral variants (red), individuals with presence of X4 viral variants (blue) and loading values represented by green arrows. (A) PCA plot using data from PC1 (principal component) and PC2. It is observed that the loading value of IL-6 was inversely correlated with CD4 and CD8; and positively correlated with pVL. (B) PCA plot of three dimensions (PC1, PC2 and PC3). The group clustered along the left side of the PC1 are the individuals who had increased levels of IL-6 as the mayor variable that contributes to PC1 (Orange circle).

Figure 3

Comparisons of cellular markers of immune activation between R5 group (red) and X4 group (blue). The percentages of activated CD4+ T cells and CD8 T cells are plotted among 98 in PLWH. (A) %CD4+ HLA-DR+. (B) %CD4+ CD38+. (C) %CD4+ HLA-DR+ CD38+. (D) %CD8+ HLA-DR+. (E) %CD8+ CD38+. (F) %CD8+ HLA-DR+ CD38+. CD, cluster of differentiation; HLA-DR, Human Leukocyte Antigen – DR. Comparisons were made using Mann-Whitney U test or t-test as appropriate and using significant p-values <0.05.

Figure 4

Comparisons of cellular markers of immune activation between R5 group (red) and X4 group (blue). The percentages of activated CD4+ T cells and CD8 T cells are plotted among 98 in PLWH. (A) %CD4+ CCR5+ HLA-DR+. (B) %CD4+ CCR5+ CD38+. (C) %CD4+ CCR5+ HLA-DR+ CD38+. (D) %CD4+ CXCR4+ HLA-DR+. (E) %CD4+ CXCR4+ CD38+. (F) %CD4+ CXCR4+ HLA-DR+ CD38+. (G) %CD4+ CCR5+ CXCR4+ HLA-DR+. (H) %CD4+ CCR5+ CXCR4+ CD38+. (I) %CD4+ CCR5+ CXCR4+ HLA-DR+ CD38+. CD, cluster of differentiation; HLA-DR, Human Leukocyte Antigen – DR; CCR5, C-C chemokine receptor 5; CXCR4, C-X-C chemokine receptor 4. Comparisons were made using Mann-Whitney U test or t-test as appropriate and using significant p-values <0.05.