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. 2016 Mar 12;13:15.
doi: 10.1186/s12977-016-0248-y.

TLR8 Regulation of LILRA3 in Monocytes Is Abrogated in Human Immunodeficiency Virus Infection and Correlates to CD4 Counts and Virus Loads

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Free PMC article

TLR8 Regulation of LILRA3 in Monocytes Is Abrogated in Human Immunodeficiency Virus Infection and Correlates to CD4 Counts and Virus Loads

Hui Zhi Low et al. Retrovirology. .
Free PMC article

Abstract

Background: LILRA3 is an immunostimulatory molecule which can conditionally induce the proliferation of cytotoxic cells. LILRA3 has a deletion genotype which is associated with multiple immune disorders. In this study, we wanted to analyze the regulation of LILRA3 and its significance in the context of HIV infection.

Results: We analyzed a panel of TLR agonists and found that ssRNA40, a TLR8 agonist, is a potent inducer of LILRA3 in healthy individuals. However, this regulation is much diminished in HIV. Comparison of TLR8 to TLR4 induction of LILRA3 indicated that LPS induces less LILRA3 than ssRNA40 among healthy controls, but not HIV patients. Levels of LILRA3 induction correlated to virus load and CD4 counts in untreated patients. Recombinant LILRA3 can induce a host of proinflammatory genes which include IL-6 and IL-1α, as well as alter the expression of MHC and costimulatory molecules in monocytes and B-cells.

Conclusion: Our experiments point towards a beneficial role for LILRA3 in virus infections, especially in ssRNA viruses, like HIV, that engage TLR8. However, the potentially beneficial role of LILRA3 is abrogated during a HIV infection. We believe that more work has to be done to study the role of LILRA3 in infectious diseases and that there is a potential for exploring the use of LILRA3 in the treatment of virus infections.

Keywords: HIV; LILRA3; Monocytes; TLR4; TLR8.

Figures

Fig. 1
Fig. 1
LILRA3 induction by TLRs. a Induction of LILRA3 and IL6 expression by a panel of TLR agonists. LILRA3 and IL6 expression was measured, using qPCR, as fold change to the unstimulated control from PBMCs stimulated for 24 h with Pam3CSK4 (P3C), heat killed Listeria monocytogenes (HKLM), polyinosinic-polycytidylic acid (Poly I:C) in high molecular weight (HMW) and low molecular weight (LMW) forms, lipopolysaccharide (LPS), flagellin, synthetic diacylated lipoprotein FSL-1, Imiquimod (R837), ssRNA40/LyoVec and CpG oligonucleotide ODN 2006. b Induction of LILRA3 by ssRNA40 on the transcriptional level. LILRA3 expression was measured as fold change to the unstimulated control from PBMCs stimulated for 24 h with uridine-rich HIV-derived ssRNA40, using ssRNA41 as control (n = 8). Wilcoxon matched-pairs signed rank test was used to compare the median between ssRNA41 and ssRNA40 stimulated LILRA3 expression. Scatter dot plot overlayed on bar graph displayed as median with interquartile range. c Induction of LILRA3 in PBMCS by ssRNA40 on the protein level. 30 × 106/mL PBMCs from LILRA3 +/+ and LILRA3 / donors were stimulated with 5 μg/mL ssRNA40. 40 µL from each supernatant was loaded and Ponceau S staining was used as loading control. The membrane was blotted for LILRA3 and IL6, using recombinant LILRA3 as protein control. d Expression of ssRNA40-induced LILRA3 by monocytes. PBMCs stimulated overnight with ssRNA40 and LPS were purified for CD14+ monocytes using magnetic-activated cell sorting. The resulting CD14+ positive and depleted fractions were analysed via qPCR for LILRA3 expression, normalized to RPLP0 and relatively quantified to calibrator cDNA (*p < 0.05; **p < 0.01)
Fig. 2
Fig. 2
Features of ssRNA40-induced LILRA3 expression. a Kinetics of LILRA3 expression was compared to other ssRNA40 inducible cytokines. PBMCs from two donors were stimulated with ssRNA40 or ssRNA41 and the RNA was harvested at various time points. qPCR for LILRA3, TNF, IFNb, IFNg, and IL1b expression, normalized to RPLP0 and relatively quantified to calibrator cDNA (pooled cDNA from 10. b Effect of cytokine inhibition on LILRA3 expression. PBMCs from two donors were stimulated overnight with ssRNA40 together with neutralizing agents anakinra, certolizumab, tocilizumab and IFN-γ neutralizing antibodies and analysed for LILRA3 and IL6 expression by qPCR. Repeated measures ANOVA was used to calculate difference of ssRNA40+ neutralization to ssRNA40 alone and results displayed as mean ± SD
Fig. 3
Fig. 3
Analysis of ssRNA40-induced LILRA3 expression in HIV. a Monocytes of healthy donors, HIV-untreated and HIV-treated patients were enriched using the Rosettesep system and stimulated overnight with ssRNA40 and analysed using qPCR for LILRA3. Wilcoxon matched-pairs signed rank test was used to compare intra-group unstimulated and ssRNA40-stimulated LILRA3 expression. Kruskal–Wallis test was used to compare inter-group ssRNA40-stimulated LILRA3 expression. Results displayed as median with interquartile range. b The values for the ssRNA40-induced LILRA3 (without unstimulated controls) were segregated according to genotype (LILRA3 +/+ and LILRA3 +/). Mann–Whitney test was used to compare intra-group LILRA3 expression. Kruskal–Wallis test was used to compare inter-group LILRA3 expression among LILRA3 +/+ donors. Results displayed as median with interquartile range (*p < 0.05; **p < 0.01; ***p < 0.001)
Fig. 4
Fig. 4
Relationship between ssRNA40 and LPS-induced LILRA3. a Monocytes of healthy donors, HIV-untreated and HIV-treated patients were enriched using the Rosettesep system and stimulated overnight with ssRNA40 and LPS. LILRA3 expression was analyzed using qPCR and TNF expression was analyzed using ELISA. Kruskal–Wallis test was used to compare inter-group ssRNA40-stimulated LILRA3 and TNF expression. Bar graph depicts median with interquartile range. b Correlation analysis of ssRNA40 versus LPS induced LILRA3 expression. Two-tailed spearman analysis was used to calculate correlation. Non-linear regression straight line robust fit was used to calculate the trend lines. c Relationship between ssRNA40/LPS induced LILRA3 expression and disease parameters in HIV-untreated patients. One-tailed Spearman analysis was used to calculate correlation between LILRA3 expression and virus loads, CD4 and CD8 T-cell and NK-cell counts in HIV-untreated patients. Non-linear regression straight line robust fit was used to calculate the trend lines
Fig. 5
Fig. 5
LILRA3 stimulation of cytokines and antigen presenting cells. a qPCR of LILRA3 induced gene expression of LILRA3, IL-6, IL-1A, IL-1B and IL-10. Due to the different optimal concentrations of LILRA3 for various donors, the gene expression of all four donors were shown, with the corresponding mean ± SD. b Effect of LILRA3 on the antigen presentation mechanism of monocytes and B-cells. PBMCs were stimulated with varying concentrations of LILRA3 for 48 h and analysed by flow cytometry for expression of CD80, CD86, HLA-DR and HLA-ABC on monocytes (CD14+CD33+) and B-cells (CD3+CD19+). On monocytes, upregulation of HLA-ABC and CD80 was observed, whereas CD86 was downregulated. On B-cells, there was a slight but significant upregulation of HLA-DR and CD86. 1-way ANOVA with repeated measures Dunett post test was used to compare to 0 ng/mL LILRA3 control. Results expressed as mean ± SD from six donors. (*p < 0.05; **p < 0.01; ***p < 0.001)

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