TLR8 regulation of LILRA3 in monocytes is abrogated in human immunodeficiency virus infection and correlates to CD4 counts and virus loads
© Low et al. 2016
Received: 15 September 2015
Accepted: 29 February 2016
Published: 12 March 2016
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.
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.
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.
Extensive work has been done to functionally characterize members of the leukocyte immunoglobulin like receptor (LILR) family [1–11]. In comparison, the function of leukocyte immunoglobulin like receptor A3 (LILRA3; ILT6; CD85e) has been less well characterized. LILRA3 is a 70 kDa highly glycosylated protein secreted by monocytes. The differentiation of monocytes into osteoclasts and dendritic cells has been shown to upregulate LILRA3 [12, 13]. It is the only member of the LILR family that exists constitutively as a soluble protein due to a lack of a transmembrane domain and cytoplasmic tail. LILRA3 binds to HLA-G and classical HLAs with a much lower affinity than LILRB1 and LILRB2 , although it seems to bind to free heavy chain forms of HLA-C with higher affinity .
Around 30 % of healthy Caucasians carry the LILRA3 deletion of 6,7 Kb which includes nearly the complete coding sequence [8, 16]. The homozygous deletion is found in 3 % of the healthy Caucasian population and confers susceptibility to some autoimmune diseases [17–19], HIV-infection (in revision), and B cell non-hodgkin lymphoma .
Other members of the LILR family were demonstrated to play important roles in HIV infection . Myeloid dendritic cells in elite controllers, who spontaneously maintain low viremia, have a selective upregulation of LILRB1 and LILRB3, whereas patients with a progressive infection showed a downregulation of LILRA2 and upregulation of LILRB2 .
As we have shown in a previous report, LILRA3 is an immunostimulatory molecule that specifically induces the proliferation of CD8 T-cells and NK-cells in the presence of allogeneic stimulation, as well as the production of pro-inflammatory cytokines . Based on these properties, we suspect that LILRA3 could be involved in immune responses against viruses. However, nothing is known about the processes upstream of LILRA3 and their regulation.
Pattern recognition receptors (PRRs) are widely expressed on a variety of immune sensing cells. They recognize pathogen associated molecular patterns to initiate the innate immune response and direct adaptive immunity [23, 24]. The best characterized PRRs are the toll-like receptors (TLR). Among the 10 members which sense a variety of infection types, those associated with virus infections are intracellular TLR3 (dsRNA), -7, -8 (both ssRNA) and -9 (unmethylated CpG DNA).
We used TLR-agonists as surrogates for whether virus infections could induce expression of LILRA3. We wanted to determine which TLR agonist could regulate LILRA3 expression and showed that ssRNA40, a TLR8 agonist, is a most prominent regulator of LILRA3. Since HIV is an ssRNA virus which has been documented to induce aberrant TLR responses in hosts [25, 26], we analyzed the TLR8 regulation of LILRA3 expression in the context of HIV infection.
TLR-8 induces LILRA3 expression in CD14+ monocytes
We determined monocytes to be the major producer of LILRA3 under ssRNA40 stimulation, as LILRA3 mRNA was detected only in CD14+ monocytes after 22-h ssRNA40 stimulation in PBMCs. In all three donors tested, almost all of the LILRA3 expression occurred in CD14+ monocytes, with two of the three donors expressing higher LILRA3 under ssRNA40 stimulation compared to LPS (Fig. 1d).
LILRA3 is upregulated after other proinflammatory cytokines
TLR8 regulation of LILRA3 is abrogated in HIV infection
HIV infection selectively abrogates ssRNA40, but not LPS, activation of LILRA3
Both ssRNA40 and LPS-induced LILRA3 expression correlates with viral load and CD4 counts
Since we believe that LILRA3 can be helpful in a virus infection due to its ability to induce proliferation in cytotoxic cell subsets , we hypothesize that expression of LILRA3 could indicate better disease status of the patients, i.e. lower viral load, higher CD4 and CD8 T-cell and NK-cell counts. We performed a one-tailed Spearman correlation analysis of both ssRNA40 and LPS-induced LILRA3 expression with the abovementioned clinical parameters and observed that higher induction of LILRA3 was significantly correlated with lower viral load of HIV-untreated patients. Virus loads were not analysed for treated patients because they were almost uniformly below 20 virus copies/mL. LILRA3-induction correlated positively with CD4 T-cell count in untreated patients (Fig. 4c), but not for treated patients (Additional file 2: Figure S2).
Many immune activation genes are upregulated by LILRA3
LILRA3 effects on the MHC and costimulatory molecules
Since LILRA3 binds to an undetermined ligand expressed on the surface of monocytes and B-lymphocytes [20, 27], we tested if there is a direct effect of LILRA3 binding in the regulation of the MHC and costimulatory molecules expression levels on these cells. On monocytes, incubation with LILRA3 selectively upregulated HLA class I and CD80 surface expression. Notably, CD86 was conversely downregulated, whereas there was no significant change in HLA class II expression (Fig. 5b). On B-cells, there was a selective upregulation of HLA class II and CD86. However, no significant difference in the expression levels of HLA class I and CD80 were detected.
TLRs and LILRs are two families of receptors which are widely and distinctly expressed on immune cells. Other members of the LILR family of receptors have been implicated in the control of TLR activity to several different bacterial infections (reviewed in ). The LILRs have also been shown play a role in viral infections like HIV [29–31]. We showed that ssRNA40, a TLR8-agonist, is the most prominent regulator of LILRA3 expression among the TLRs, and that this was mainly mediated by the monocyte population. ssRNA40 is a uridine-rich single stranded RNA sequence derived from the U5 region of HIV-1. Although ssRNA40 is an activating ligand for TLR7 in mice, in humans it has been shown to stimulate TLR8 responses in TLR8, but not TLR7, transfected HEK 293 cells . Therefore, in our system, the ability of TLR7 and TLR9 to stimulate LILRA3 might be overlooked as the cell populations that express these receptors are underrepresented among the PBMCs. In human macrophages and dendritic cells, TLR3-ligand poly(I:C) has been shown to induce IFN-regulated genes, but not proinflammatory genes like TNF and IL6 , which might explain the missing IL6 response to poly(I:C) in our system.
Although LILRA3 expression peaks after 24 h, neutralization of IL-1R, TNF, IL-6R and interferon-γ did not lead to an appreciable change in LILRA3 (Fig. 2b), meaning that it is regulated independently of these cytokines. This could imply that LILRA3 expression is regulated directly by TLR8 signalling pathways or by other indirect pathways/cytokines, for example type I interferons, which is as yet unknown. This should be of interest for further investigation.
In HIV-untreated patients, we observed a significant abrogation of the monocytic TLR8-induced LILRA3 in comparison to the robust response of healthy controls, as well as a reconstitution of this response in treated patients (Fig. 3). It is crucial to note that in this study, we do not measure the constitutive LILRA3 expression in HIV patients, but rather the strength of the LILRA3 response to TLR4 and TLR8 stimuli. This is more reflective of the immune status of the patients, rather than the constitutive expression due to viral loads. Nevertheless, the strength of the LILRA3 induction seems to be correlated with the viral load in untreated patients (Fig. 4c), suggesting a link between the strength of LILRA3 induction and the controlling of the infection. This would be consistent with previous studies showing that the strength of the TLR3, -7 and -8 responses are more robust among individuals who remain seronegative even after repeated sexual exposure to HIV , and that a mutant TLR8 allele which is more “functional” than the wild-type allele confers protection against HIV disease progression . Considering that conventional dendritic cells also express TLR8 and are demonstrated play a role in control of HIV [36–38], it would be interesting to analyse their LILRA3 production in future experiments.
In order to assess whether the abrogation of LILRA3 expression in HIV infection is TLR8-specific or a general dampening of the monocytic response, we looked at the LPS induction of LILRA3 expression via TLR4 in a second cohort. ssRNA40-induced LILRA3 correlated well with LPS-induced LILRA3 expression across the three groups, suggesting that they act on similar signalling pathways to induce LILRA3 (Fig. 5b). However, while TLR8 stimulation gave a more robust LILRA3 response than TLR4 stimulation in healthy controls, this trend is paradoxically skewed towards LPS in HIV patients, who might actually benefit more from the TLR8-ssRNA response rather than the TLR4-LPS response. It is tempting initially to attribute this to immune dysregulation in chronically HIV-1 infected patients. However, the correlation between viral load and CD4 counts to LILRA3 expression that was present under TLR8 stimulation was even more strongly correlated under TLR4 stimulation (Fig. 4c), suggesting that the strength of the LPS response is also a factor in containing HIV-1. It has been shown that sensitization of monocytes with HIV-1 or TLR8-ligands led to higher production of proinflammatory cytokines by subsequent stimulation by LPS . One explanation could be that because the TLR8 response was exhausted in these chronically HIV-1 infected patients, another TLR(s) was used as a compensatory mechanism. When we analyzed TNF production after ssRNA40 stimulation, we did not observe any significant decrease in TNF expression in HIV untreated patients, suggesting other factors at play other than TLR-8 exhaustion.
At this stage, it is still unclear whether the robustness of the LILRA3 induction is a cause or an effect of improved viral control and we cannot rule out in our study that the stronger LILRA3 responses are due to a general healthier immune status of the patients with lower viral loads. However, in light of a recent study of the genetics of LILRA3 deletion in HIV patients by Ahrenstorf et al. (in revision), HIV patients have higher percentages of LILRA3 −/− donors and there is a faster progression of the infection with decreasing gene dosage. Taken together, there is evidence to suggest that LILRA3 is helpful in immune control of HIV.
LILRA3 expression level is confirmed to be dependent on its gene-dosage, with LILRA3 +/− donors expressing substantially less LILRA3 than LILRA3+/+ donors (Fig. 4b). The untreated HIV patients had such depressed levels of LILRA3 induction that both genotypes gave similar low levels of gene expression. This is in line with a previous study that revealed decreased LILRA3 expression in LILRA3 +/− rheumatoid arthritis patients . However, it is unclear whether this is the result of haploinsufficiency or the deletion allele being dominant negative.
Previously, we have shown that LILRA3 can induce a host of proinflammatory cytokines. We also show now in this paper that LILRA3 can upregulate transcription of IL-6, IL-1A and IL-1B, but not LILRA3 itself and IL-10. Previous studies have shown that IL6 have suppressive effects on TNF production in vitro and in vivo [41, 42]. As LILRA3 upregulates IL6, but not TNF, the IL6 expression might have had repressive effects on TNF production, which could explain why Lee et al.  saw a repression of LPS-induced TNF by LILRA3. Interestingly, two out of the four donors we stimulated with LILRA3 responded best at 1000 ng/mL, whereas the other 2 responded at 500 ng/mL, but not at all at 1000 ng/mL (Fig. 5a). This means that LILRA3 activation is under very tight control, with different donors having different activation thresholds. The abrogation of its stimulatory effects at too high concentrations is most likely a mechanism against overactivation.
LILRA3 can modulate the expression MHC and costimulatory molecules in monocytes and B-cells. Although monocytes are the major producers of LILRA3 (Fig. 1d), the as yet unknown receptor for LILRA3 was found on monocytes and it exerts its immune modulating properties via the monocytes [20, 27]. One possibility is that this is a mechanism for infected monocytes to alert nearby monocytes about the presence of virus particles in the vicinity.
Gene set enrichment analysis showed that LILRA3 regulates many genes involved in the pathology of RA (Additional file 3: Figure S3B). In the Han Chinese population, the genetic presence of LILRA3 is associated with increased risk of rheumatoid arthritis .
Our data point towards a role of LILRA3 in virus immunity. ssRNA40 is the most prominent inducer of LILRA3, which is produced by monocytes. Although HIV-1 patients express significantly less LILRA3 in response to ssRNA40, the LPS-induced LILRA3 remained unchanged. We also revealed that both TLR8 and TLR4 mediated LILRA3 expression can be correlated with viral loads and CD4 T-cell counts in untreated patients and that LILRA3 can upregulate proinflammatory cytokines, as well as induce changes MHC and costimulatory molecule expression monocytes and B-cells. Based on our findings, LILRA3 could potentially be exploited in the treatment of viral diseases like HIV.
PBMC and monocyte isolation and culture
PBMCs were isolated using density gradient centrifugation by layering blood onto Biocoll (Millipore) and centrifugation at 1200×g for 20 min without brake. The resulting buffy layer was then washed twice with PBS before use in subsequent experiments. Monocyte enrichment was performed using the RosetteSep™ Human Monocyte Enrichment Cocktail (Stemcell Technologies). Positive enrichment of CD14+ monocytes was performed using CD14 Magnetic Particles (Becton–Dickinson). Cells were counted using trypan blue staining on a Neubauer hemocytometer and cultured at 37 °C with 5 % CO2, in RPMI 1640 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 2 mM l-glutamine (all Biochrom), and 10 % AB serum.
RNA extraction and reverse transcription
Total RNA was extracted using Zymo Research Quick-RNA Microprep (Zymo Research), utilizing RNase-Free Dnase Set (Qiagen) for in-column genomic DNA removal. Reverse transcription was perform using High-Capacity cDNA Reverse Transcription Kit (Life Technologies).
Quantitative real-time PCR
Real-time PCR primers
Annealing temp. (°C)
Mean age (SD)
Mean age (SD)
TLR-agonist stimulation experiments
The Human TLR1-9 Agonist Kit (Invivogen) was used for the pilot study. 106/mL PBMCs were stimulated for 24 h with 1 μg/mL Pam3CSK4 (P3C), 108 cells/mL heat killed Listeria monocytogenes (HKLM), 1 μg/mL polyinosinic-polycytidylic acid (Poly I:C) in high molecular weight (HMW) and low molecular weight (LMW) forms, 100 ng/mL lipopolysaccharide (LPS), 100 ng/mL flagellin, 100 ng/mL synthetic diacylated lipoprotein FSL-1, 1 μg/mL iniquimod, 1 μg/mL ssRNA40/LyoVec and 5 μM CpG oligonucleotide ODN 2006. For the kinetics, PBMC were stimulated with ssRNA40/Lyovec and ssRNA41 and harvested for real-time PCR analysis at various time points up to 48 h. For the rest of the stimulation experiments, PBMCs or monocytes were stimulated overnight for 20–22 h with respective stimulants. In later samples, the supernatants from monocyte stimulation was analyzed for TNF production was analyzed using the TNF elisa kit (Biolegend). Anakinra (Sobi), Certolizumab (UCB) and Tocilizumab (Roche) were obtained as leftovers from clinical treatments and used to neutralize IL-1R, TNF and IL-6R respectively. Functional grade purified anti-human IFN gamma (eBioscience) was used to neutralize IFN-γ.
Western blot to detect LILRA3 secretion upon ssRNA40 stimulation
Purified PBMCs from LILRA3 positive and deleted persons were cultured in Optimem +0.5 % AB-serum in the presence or absence of 5 μg/mL ssRNA40, using 3 × 106 cells per well per 100 µL in a 96-well plate. After 48 h incubation, the supernatant was harvested, spun down at 13,000 rpm for 5 min in a microcentrifuge and run on a 4–12 % reducing SDS-PAGE gel (Life Technologies), and transferred onto a nitrocellulose membrane. LILRA3 antibody clone 2E9 (Abnova) was used for detection, using Peroxidase Conjugated Stabilized Goat Anti-Mouse IgG (H + L) (Pierce) as secondary antibody. After stripping the membrane with Restore Western Blot Stripping Buffer (Thermo Scientific), IL6 was detected using clone MQ2-13A5 (Biolegend) together with Peroxidase Conjugated Stabilized Goat Anti-Rat IgG (H + L) (Pierce). SuperSignal West Dura Chemiluminescent Substrate (Pierce) was used in conjunction with Versadoc MP4000 (Biorad) to visualize the bands.
DNA extraction and LILRA3 deletion genotyping
Genomic DNA was extracted from whole blood using Quick-gDNA MiniPrep kit (Zymo Research) according to manufacturer’s instructions. LILRA3 deletion genotyping was done by PCR using reverse primer 5′-GACAGCAGATTCTAAAACAGTG-3′ for both genotypes; forward primers 5′-CCCCTGGAGCTCGTGG-3′ for the presence genotype and 5′-CATCTCGATCTGCCACTGACAC-3′ for the deletion genotype. PCR for both genotypes was performed in multiplex using Amplitaq Gold DNA Polymerase (Applied Biosystems) using the following cycling conditions on the Biometra T3 thermocycler: initial denaturation at 95 °C for 20 min, followed by 43 cycles of denaturation (95 °C for 10 s), annealing and elongation (62 °C for 2 min) and a final elongation step of 62 °C for 10 min. PCR products were analysed on a 1.5 % agarose ethidium bromide gel for the presence (1150 bp) and deletion (250 bp) genotypes.
Generation of recombinant LILRA3
Mammalian LILRA3 was produced under a Lentivirus expression system. Briefly, LILRA3 cDNA was amplified by PCR using Phusion High Fidelity DNA Polymerase (Thermo Scientific) from total cDNA of PBMC from a LILRA3+/− donor using primers (Fwd: 5′-CGGACTAGTATGACCCCCATCCTCACG-3′; Rev: 5′- CGACTAGTTCA ATGGTGATGGTGATGGTG CTCACCAGCCTTGGAGTC) incorporating SpeI digestion site at both ends and His-tag sequence at the 3′ end. The amplified LILRA3-His cDNA was cloned into the XbaI site in the multiple cloning site of pRRL-CMV-MCS-IRES-GFP , and the sequence verified by sequencing (MWG Operon). The newly generated pRRL-CMV-LILRA3-His-IRES-GFP was used to transfect HEK-293T cells using the LENTI-Smart INT kit (Invivogen) according to manufacturer protocol. Virus particles produced were concentrated using PEG-it Virus Precipitation Solution (System Biosciences) and used to transduce fresh 293T cells to generate stable LILRA3-His expressing cell line. A stably transduced cell line was also produced using of pRRL-CMV-MCS-IRES-GFP for the mock purification control. Mammalian LILRA3-His or its equivalent mock purification was produced by culturing the transduced cells using CELLine AD 1000 bioreactor flasks (INTEGRA Biosciences) and purifying the recombinant protein using HisPur Cobalt Resin from supernatant adjusted to pH 8.2–8.5. Endotoxin level was tested using LAL Chromogenic Endotoxin Quantitation Kit (Pierce) and determined to be virtually endotoxin free at 5 × 10−6 EU/µg protein. LILRA3 purified from Baculovirus expression system was produced as described previously .
LILRA3 stimulation experiments
For microarray analysis, 106/mL PBMCs were cultured for 24 h in the absence or presence of 1 µg/mL baculovirus LILRA3 in a 25 cm2 suspension culture flask. The cells were pelleted and their total RNA isolated using the Qiagen RNeasy Minikit according to the manufacturer’s instructions. Microarray and data processing are detailed below and deposited in Gene Expression Omnibus (accession GSE61356). To confirm the microarray data, PBMCs were stimulated overnight with 100, 500 and 1000 ng/mL of mammalian LILRA3 and analyzed for IL-6, IL-1A, IL-1B, IL-10 and LILRA3 expression. For changes in B-cell and monocyte expression levels of MHC and costimulatory monecules, PBMCs incubated for 48 h with 0–1000 ng/mL baculovirus LILRA3 were stained with CD14-APC-CY7 (Biolegend), CD19-V450, CD3-V500, HLA-ABC-FITC or CD80-FITC, CD86-PE (all BD Bioscience) or HLA-DR-PE (Beckman Coulter), and analyzed via flow cytometry.
Microarray experiment and data processing
Cy3-labeled cRNA was synthesized using the one color Quick Amp Labeling kit (Agilent Technologies) according to the manufacturer’s instructions. cRNA fragmentation and hybridization onto the Whole Human Genome Oligo Microarray V2 was carried out as recommended by the Agilent One-Color Microarray-Based Gene Expression Analysis Protocol V5.7. Data were analysed using the R software and several packages from BioConductor  and other sources. Preprocessing steps comprised background correction (“normexp”), quantile normalisation, probe summarisation, and log2 transformation. For the background correction, fitted intensities were calculated by the convolution of normal and exponential distributions to observed foreground and background intensities . For a robust analysis, median values were calculated from replicate samples for each gene. Differentially expressed genes (DEG) were obtained by selecting genes with a minimal fold change of two and significant changes to untreated controls keeping false-discovery rate adjusted p values of 10 % according to the rank products method in order to apply robust statistics for small replicate sizes [45, 46]. In order to further functionally characterise the selected genes, GOstats for identifying gene ontology terms was applied . A functional analysis of the selected gene list was done with SPIA, the signaling pathway impact analysis . GSEA served for understanding the genome-wide expression of all genes  via cellHTS2 . The data sets are available under GEO accession number GSE61356.
All statistical analyses were performed using Graphpad Prism 5.
LILRA3 leukocyte immunoglobulin like receptor A3
TW, HZL participated in the conception of the project and experiments. HZL performed and analysed the bulk of the experiments. NH contributed to LILRA3 stimulation experiments on B-cells and monocytes. HZL and DOV contributed to drafting of the manuscript. RS provided the lentiviral vectors. KS, CP and EW contributed to the analysis of microarray data. All authors read and approved the final manuscript.
This work was supported by the Deutsche Forschungs Gemeinschaft grant to Klinische Forschergruppe 250, TP3, WI 1031/6-1 to Torsten Witte. Microarray raw data used or referred to in this publication were generated with much help from Oliver Dittrich-Breiholz and by the Research Core Unit Transcriptomics of Hannover Medical School.
The authors declare that they have no competing interests.
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