Since the discovery of HIV, there have been multiple reports of small groups maintaining natural resistance against HIV despite continued high-risk behavior. These groups are referred as HIV-1–exposed seronegative (HESN) subjects and include the following: discordant couples,1–3 sex workers,4–6 perinatally exposed infants,7,8 and people who inject drugs.9–12 Host-mediated immune mechanisms of HIV-1 resistance have been proposed to explain the occurrence of HESN subjects; however, a unified protective mechanism in HESN subjects remains unlikely, as both immune quiescence13–16 and immune activation1,2,8,9,17–19 have been identified depending on the route of exposure.
HIV-exposed seronegative people who inject drugs (HESN-PWID) and share needles have been identified to possess activated myeloid dendritic cells,12 activated NK cells, and higher NK cell cytotoxic activity.10–12 Recently, we identified that low-risk nonsharing PWID subjects exhibit low levels of innate immune activation similar to control donors,12 suggesting that high-risk needle-sharing activity, not the opioid drugs themselves, likely drives the activation state associated with HESN-PWID subjects. Here, we investigated the mechanism of immune activation in PWID subjects using proteomic analysis of isolated NK cells from HESN-PWID subjects and control donors. We identified that NK cells from high-risk needle-sharing HESN-PWID subjects possessed significantly increased expression of interferon-related proteins along with several proteins members of the S100 family, including S100A4, S100A6, S100-P, and S100A14.
S100 proteins are involved in many cellular processes including regulation of proliferation, differentiation, apoptosis, Ca2+ homeostasis, inflammation, and migration. Secreted S100s, such as S100A14, may function as danger-associated molecular patters and mediate innate and adaptive immune responses by signaling mainly through Receptor for Advanced Glycosylation End (RAGE) Products and TLR4.20 The S100 family of proteins has been routinely described in cancer, but very little is known about their potential impact on NK cells or HIV. S100P was initially purified from placenta, and it has been described to be involved in cell proliferation, survival, and differentiation,21,22 while also serving as a prognostic biomarker in cancer.23 S100A4 and S100A6 were shown to be overexpressed specifically in CD57highCD56dim NK cells, a more mature subpopulation of NK cells, and recruited into the NK immune synapse.24,25 S100A14 has been described in cancer,26–33 and according to its sequence and structure, it differs from other members of the S100 family, as it cannot bind Ca2+.34 The primary receptor for S100A14 in a cancer cell line has been identified as RAGE.33 Human NK cells express detectable RAGE on their surface, while TLR4 is generally expressed in NK cells at very low levels, but it can be enhanced by infection.35,36
In this study, we describe S100A14 to be upregulated in NK cells by proteomic analysis and in the plasma of HESN-PWID compared with controls donors. We further investigated the role of S100A14 as a potential mechanism for the sustained activation of the NK cell in HESN-PWID. In control donors, in vitro treatment of peripheral blood mononuclear cells (PBMCs) with extracellular S100A14 demonstrated that activation of NK cells is not direct, but it occurs in a monocyte-dependent manner and that monocyte activation is through TLR4 signaling.
HIV-exposed, seronegative people who inject drugs (HESN-PWID) were enrolled from the city of Philadelphia through community-based street outreach in specific neighborhoods previously identified as “risk pockets.”37 Risk pockets are defined as locations within neighborhoods with a high HIV-1 prevalence where injectable drugs are sold, used, and at times exchanged for sex.12 We used the “Prognostic Model for Sero-conversion Among Injection Drug Users”38 to identify high-risk HESN-PWID subjects for our study based on their frequency of injection and needle-sharing behavior. Briefly, subjects from known risk pockets were identified as HESN-PWID if they remained HIV-1 IgG seronegative despite a history of greater than 2 years of daily injection and frequent (weekly or greater) needle sharing with partners of unknown HIV status. Demographic characteristics and risk factors of the HESN-PWID subjects are summarized in Table 1. All HESN-PWID subjects screened for the study were referred to drug treatment programs, counseled to use the local needle-exchange program services to reduce their risk of exposure to blood-borne pathogens, and referred to additional health services as needed. Healthy control donors were recruited from The Wistar Institute Phlebotomy Program. Six ART-suppressed and 2 viremic HIV-1–infected subjects were recruited from the Presbyterian Hospital of Philadelphia; the age mean for these patients is 47.25 years, and their CD4 counts range from 112 to 994 cells/mm3. All protocols were approved by institutional review boards of the National Institutes of Health and The Wistar Institute, and all participants provided informed consent before the blood draw. S100A14 plasma levels were measured by ELISA (Abexxa, Cambridge, United Kingdom) following the manufacturer instructions.
Sample Preparation and Proteome Analysis
Purified NK cells from 6 HESN-PWID and 6 control donors were prepared for proteomic analysis by negative selection using magnetic beads (Miltenyi Biotech, Bergisch Gladbach, Germany) to greater than 97% purity. NK cells (2 × 106 cells per donor) were lysed using 50 mM Tris-Cl, 1% SDS, pH 8.0. After centrifugation, the supernatant was loaded onto a 12% sodium dodecyl sulfate (SDS)-gel and separated for 2.0 cm followed by fixing and staining with colloidal Coomassie. The region of the gel containing protein was excised into 10 equal pieces and digested with trypsin. Tryptic peptides were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) on a Q Exactive Plus mass spectrometer (ThermoFisher Scientific, Waltham, MA) coupled to a Nano-ACQUITY UPLC system (Waters, Milford, MA). Samples were injected onto a UPLC Symmetry trap column (180 μm i.d. × 2 cm packed with 5-μm C18 resin; Waters), and tryptic peptides were separated by reversed phase high-performance liquid chromatography (RP-HPLC) on a BEH C18 nanocapillary analytical column (75 μm i.d. × 25 cm, 1.7-μm particle size; Waters) using a 2-hour gradient. Eluted peptides were analyzed in data-dependent mode where the mass spectrometer obtained full MS scans from 400 to 2000 m/z at a 70,000 resolution. Full scans were followed by MS/MS scans at a 17,500 resolution on the 20 most abundant ions. Peptide match option was set as “preferred,” the exclude isotopes option, and charge-state screening option, were “enabled” to reject singly and unassigned charged ions. MS/MS spectra were searched using MaxQuant 1.539 against the human Uniref database plus common contaminants. MS/MS spectra were searched using full tryptic specificity with up to 2 missed cleavages, static carboxamidomethylation of Cys, and variable oxidation of Met and protein N-terminal acetylation. Consensus identification lists were generated with false discovery rates of 1% at peptide level and an initial 5% at the protein level. LFQ ratio was set at 1, main search at 4.5 ppm. Razor+unique and min ratio counts were both set at 1. Resulting data were processed in PERSEUS 1.5 and exported to Excel for further analysis. Data were further filtered by removing proteins identified by a single peptide and known contaminants. The final protein false discovery rate was approximately 1%. Proteins exhibiting at least a 2-fold change between groups with a P-value less than P < 0.05 (student T test) were considered significantly changed.
Isolation and Stimulation of Cells
PBMCs were isolated from whole blood by density gradient centrifugation on Ficoll (GE Healthcare, Pittsburgh, PA). Initial NK cell enrichment was performed with the Negative selection EasySep Human NK Cell Isolation Kit (StemCell; Vancouver, Canada). NK cells were then purified further to reduce the presence of contaminating myeloid cells by staining with CD56 PerCP Cy5.5 (BD Biosciences, San Jose, CA) and CD3 APC (BD). CD56+CD3− cells were sorted using a FACSAria (BD Biosciences) to greater than 99.5% purity. Monocytes were isolated using Negative selection EasySep Human Monocyte Isolation Kit (StemCell). To determine NK cell activation (from within PBMC or after NK cell isolation), cells were stimulated with either 10 μg/mL of recombinant S100A14 (ACROBiosystems; Newark, DE) or 5000 U/mL of interferon-alpha (IFN-alpha) (PBL Assay Science; Piscataway, NJ) for 18 hours. To determine monocyte activation (from within PBMC or after monocyte isolation), cells were stimulated for 5 hours with either 10 μg/mL of recombinant S100A14 (ACRO Biosystems), or 100 ng/mL of Lipopolysaccharide (LPS) in the presence of monensin (BD Golgi Stop; 0.266 µL/106 cells). To inhibit LPS, we added 15 μg/mL of polymyxin B (Calbiochem; San Diego, CA) before addition of S100A14. For inhibiting TLR4, 10 μM of TAK-242 (Calbiochem) was added 30 minutes before the addition of S100A14. Cells were stained with the appropriate antibodies for 15 minutes at room temperature in the dark, washed twice with 0.09% sodium azide in PBS, and then fixed with Cytofix Buffer (BD). All antibodies were used at manufacturer recommended concentrations including: CD3 BV510 (BD, UCHT1), CD56 PerCP Cy5.5 (BD, B159), CD69 BV421 (BD, FN50) for NK cells, and CD3 APC-H7 (BD, SK7), CD14 APC (BD, M5E2), and TNF-alpha PE (BD, Mab11) for monocytes. Samples were run in a 14-color LSR II (BD Biosciences), and a minimum of 100,000 events were recorded. Flow cytometry data were analyzed with the FlowJo Software v9.9.4 (TreeStar, Ashland, OR).
Isolated NK cells were cultured in 24-well plates; a transwell insert with a 0.4-μm filter was placed in each well, and autologous isolated monocytes were added in a 1:1 ratio in media with or without S100A14 (10 μg/mL). Expression of surface CD69 on NK cells was analyzed after 18 hours using multiparameter flow cytometry using the antibodies describe above.
All statistical analyses were performed using Prism 7 software (GraphPad Software, La Jolla, CA). No data were purposefully excluded from the analysis. Any missing data from any subject were solely due to technical/cell yield reasons. All graphs are presented using the mean and the ± SEM. The Mann–Whitney test was used to investigate differences between 2 groups with an unpaired nonparametric analysis. To determine the differences between more than 2 groups with an unpaired nonparametric analysis, the Kruskal–Wallis test was used. For statistical analysis of more than 2 groups with a paired, nonparametric analysis, the Friedman test was used. Correlations between 2 variables were performed using Spearman correlation of untransformed data with a 95% confidence interval. All tests were performed using a two-tailed analysis, and results with a P < 0.05 were considered statistically significant.
Proteome of Purified NK Cells From HESN-PWID Subjects and Control Donors
To investigate the protein profile of activated NK cells from HESN-PWID subjects, we performed label-free quantitative proteomics using Gel/LC-MS/MS on purified NK cells from 6 HESN-PWID subjects and 6 control donors. We identified a total of 5818 proteins, from which 108 proteins had a fold change greater than ±2.0 and a P value lower than 0.05 (see Table 1, Supplemental Digital Content 1, http://links.lww.com/QAI/B242). As shown in Figure 1, we observed the significantly increased expression of various interferon-related proteins (depicted in blue) and proteins involved in NK cytotoxicity (depicted in green). We also observed an unexpected strong signature of S100 family proteins (depicted in red) including S100A4, S100A6, S100-P, and S100A14 (Fig. 1). To investigate whether the increased expression of proteins from the S100 family was linked to interferon stimulation, we performed a separate proteomic analysis of NK cells from control donors exposed to 5000 U/mL of IFN-alpha for 18 hours. Purified NK cells from control donors stimulated with IFN-alpha in vitro displayed a strong proteomic signature of interferon-related proteins and proteins involved in NK cytotoxicity but did not show evidence of S100 proteins (see Table 2, Supplemental Digital Content 1, http://links.lww.com/QAI/B242). Together, these data suggest that the interferon-related response and S100 signature observed in the NK cells from HESN-PWID may represent distinct pathways linked to NK activation.
Of note, the median NK CD69 activation of the 6 HESN-PWID subjects and 6 control donors used for proteomic analysis was 7.7% and 1.8%, respectively, as determined by flow cytometry. We analyzed CD69 expression on CD56dimCD3- NK cell population using the gating strategy depicted in Figure 2A. This differential level of activation was representative of significant differences in NK activation evaluated in a larger cohort of samples between the HESN-PWID and control cohorts (Fig. 2B). Because of the limited sample size of proteomic specimens, however, we could not determine whether the intracellular expression of any of S100 proteins or IFN-induced proteins correlated directly with surface levels of NK activation.
Plasma S100A14 is Higher in HESN-PWID Subjects and Correlates With Intracellular NK Cell S100A14 Expression
Among the unique S100 protein signature in the NK cells from HESN-PWID subjects, we set out to investigate the S100A14 protein because of its uniformly high fold change among HESN-PWID subjects, and it is reported potential to be secreted.31,33 We measured plasma levels of S100A14 in 27 HESN-PWID subjects (Table 1), 15 control donors, and 8 HIV-1–infected subjects (2 viremic and 6 ART-suppressed). We detected significantly higher levels of S100A14 protein in the plasma of HESN-PWID subjects when compared with either control donors or HIV-1–infected subjects (Fig. 2C). Although we did not observe a correlation between surface CD69 protein expression in NK cells and S100A14 plasma levels (data not shown), we did observe that S100A14 levels in plasma by ELISA had a strong correlation (r = 0.951, P < 0.0001, n = 12) with the expression of S100A14 in NK cells as detected by proteomic analysis (Fig. 2D). These data suggest that S100A14 plasma levels are associated with the upregulation of S100A14 in NK cells; yet, our data do not address whether other cells or immune processes may also contribute to increased S100A14 plasma levels in HESN-PWID.
Recombinant S100A14 can Activate NK Cells in a Monocyte-Dependent Fashion
To investigate whether extracellular S100A14 has a role in mediating innate immune activation observed in HESN-PWID subjects, we stimulated PBMCs from control donors with increasing doses of recombinant S100A14 for 18 hours and assessed NK activation by flow cytometry. After stimulation of PBMC with S100A14, we observed a dose-dependent increase in CD69 activation among CD56dimCD3- gated NK cells (data not shown). Treatment of PBMC with 10-μg/mL S100A14 induced significantly higher (P = 0.011, n = 10) CD69 activation on CD56dimCD3- gated NK cells compared with untreated PBMC (Fig. 3A). As a positive control, we stimulated PBMCs with 5000-U/mL recombinant IFN-alpha. We observed that S100A14 and IFN-alpha both can induce comparable levels of CD69 upregulation (Fig. 3A). Because we observed that S100A14 did not represent an interferon-inducible protein by proteomic analysis, we suspected that S100A14 induced activation might synergize with IFN-alpha stimulation. Indeed, when we combined S100A14 and IFN-alpha together, we observed an additive effect in the activation of NK cells that was greater than either stimulation alone (Fig. 3A).
We next tested whether recombinant S100A14 could directly activate NK cells or whether it required the actions of other cells in the PBMC. We performed 2 rounds of NK isolation to achieve NK purity greater than 99.5% and stimulated NK cells directly with 10-μg/mL S100A14. Unlike NK cells in a PBMC mixture, S100A14 did not directly activate isolated NK cells (Fig. 3B). By contrast, IFN-alpha stimulation induced isolated NK cells to significantly upregulate CD69 (P = 0.0008, n = 6) as expected (Fig. 3B). These data led us to test whether other cell types in the PBMC respond to S100A14 and trigger NK cell activation indirectly. Because of the well-described crosstalk between myeloid cells and NK cells, we tested whether S100A14 could activate monocytes. We stimulated PBMCs from control donors with 10-μg/mL S100A14 for 5 hours and then measured production of TNF-alpha in CD14+HLA-DR+ gated monocytes by intracellular staining (Fig. 3C). As shown in Figure 3D, the percentage of monocytes producing TNF-alpha was significantly increased after S100A14 exposure (P = 0.018, n = 8). The level of TNF-alpha produced by monocytes in response to S100A14 was comparable with levels induced by LPS stimulation, which is a potent trigger for monocyte activation. Unlike purified NK cells, S100A14 was able to induce production of TNF-alpha on isolated monocytes (P = 0.0099, n = 7) (Fig. 3E). As activated monocytes can provide potent triggers for NK activation, we next measured whether S100A14-activated monocytes could trigger NK activation. We co-cultured autologous purified monocytes and purified NK cells in a 1:1 ratio and observed that the presence of monocytes and S100A14 could induce NK activation (Fig. 3F). By contrast, the addition of monocytes to NK cells in the absence of S100A14 alone failed to trigger NK activation (Fig. 3F). To investigate whether monocyte-mediated activation was dependent on soluble factors (eg, IL-12) or on cell-to-cell contact, we used a transwell system for our NK–monocyte co-cultures. We observed that separation of NK cells and monocytes decreased NK cell activation in the presence of S100A14 (Fig. 3F), indicating that S100A14-mediated activation of NK cells is augmented by direct contact with monocytes.
S100A14 Drives TNF-Alpha Production in Monocytes Through the TLR4 Receptor
S100A14 has been reported to interact with both RAGE, and TLR4;33,40 therefore, we next set out to investigate which potential receptor for S100A14 was required for monocyte-dependent NK activation. When PBMCs were pretreated with the TLR4 inhibitor, TAK-242, before the addition of S100A14, monocyte production of TNF-alpha was inhibited (Fig. 4A). To rule out the possibility that the effects observed were due to LPS contamination independently of S100A14, we tested the effects of S100A14 in the presence of the LPS inhibitor polymyxin B (PMB) and confirmed the activity of S100A14 on increasing TNF-alpha production in monocytes (Fig. 4B). Together, these data indicate that S100A14 can activate monocytes through TLR4 which in turn can impact NK activation.
Here, we identify for the first time a unique proteome profile in NK cells from HESN-PWID subjects compared with control donors. This included an IFN-induced protein signature and the presence of a number of proteins from the S100 family, including S100A14. The S100A14 protein was found to be increased in the plasma of HESN-PWID subjects compared with controls, and plasma levels of S100A14 were positively correlated with the expression of S100A14 in NK cells by proteomic analysis. Furthermore, in our subset of HIV-infected subjects (n = 8), S100A14 levels in plasma seem to be similar to those in normal donors, supporting the interpretation that increases in plasma S100A14 may be an enriched response in HESN-PWID. More studies are needed to address the importance of S100A14 during HIV infection and pathogenesis.
In vitro, recombinant S100A14 protein was shown to activate NK cells from control donors in a monocyte-dependent manner by signaling through TLR4. Together, these findings suggest that the S100A14 may be part of an NK/monocyte activation cycle promoting the activation phenotype seen in a subset of HESN-PWID subjects. However, the exact mechanism of the crosstalk between NK cells and monocytes in the context of S100A14 stimulation is still unknown. Because the crosstalk involves cell-to-cell contact, TLR4 (LPS)-mediated expression of MICA,41 a ligand for engagement of the NKG2D receptor, in monocytes is a candidate mechanism for NK activation. NKG2D is an activator receptor mainly expressed in NK cells and CD8+ T cells, and it is used to recognize infected cells, tumor cells, or stressed cells, which leads to cell-mediated cytotoxicity.42,43 NK cell co-culture with LPS-stimulated monocytes results in a downregulation of NKG2D and increase IFN-gamma production in activated NK cells.41
Although our data show that S100A14 can induce NK activation through TLR4-mediated activation of monocytes, added antiviral mechanisms mediated by TLR4 binding are likely. Previous studies have shown that pretreatment with LPS, a TLR4 ligand, can suppress HIV-1 replication in macrophages and monocytes by several distinct mechanisms, including blocking reverse transcription,44 downregulating the CD4 and CCR5 receptors,45–47 and inducing the production of proinflammatory cytokines and chemokines.44,45,47,48 Here, we describe the presence of a host protein that interacts with TLR4 that could potentially contribute to similar antiviral outcomes.
Altogether, we found evidence of increased expression of the S100A14 protein in both activated NK cells and the plasma from HESN-PWID subjects. Future research will need to investigate the effect of S100A14 NK/monocyte crosstalk on HIV-1 replication and potential NK-mediated clearance of infected target cells through S100A14 stimulation in HESN-PWID subjects. Elucidating the biological effect of S100A14 together with the triggers of S100A14 expression could help to develop future therapies for prevention of HIV infection.
The authors gratefully acknowledge the assistance of Kaye Speicher and Huan Wang in The Wistar Institute Proteomics Core Facility as well as Jeffrey Faust in The Wistar Institute Flow Cytometry Facility.
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