Natural killer (NK) cells are innate immune cells found throughout the body in both lymphoid and nonlymphoid compartments that contribute to the first lines of defense against invading pathogens.1,2 NK cell responses are driven by finely tuned interactions between NK cell–associated inhibitory and activating receptors and include a potent, fast-acting cytotoxic capability to directly kill infected cells but not healthy normal cells. In addition, NK cells can be activated by cytokines, including interleukin-12 (IL-12) in combination with IL-15 or IL-18, to produce proinflammatory cytokines such as tumor necrosis factor alpha and interferon gamma (IFN-γ) and thereby also impact antigen-presenting cell (APC) function and induction of adaptive immune responses.1,2
Human blood NK cell subsets are identified based on the differential expression of the surface markers CD56 and CD16 with the vast majority (>90%) of blood NK cells defined as CD56+/dimCD16+.2,3 Traditionally, this population of NK cells is considered to be the predominant cytotoxic subset,2,4 but recent studies have indicated that CD56dim NK cells are also capable of cytokine production.5–7 A smaller fraction within the blood, approximately 10% of total NK cells, expresses high levels of CD56 (CD56bright), but lack the expression of CD16 and produces cytokines in response to stimulation by cytokines.2–4,6 A third typically minor NK cell subset lacks expression of CD56, but maintains the expression of CD16. Expansion of this particular NK cell subset has been observed in a number of chronic viral infections, including HIV-1 and hepatitis C virus,8–10 and is less functional compared with the other NK cell subsets.11
The antitumor and antiviral properties of NK cells have long been known, but NK cells also play a prominent role in antibacterial immune responses through an ability to directly lyse infected cells and provide early sources of various proinflammatory cytokines.12 The importance of these innate immune cells for controlling bacterial infections in humans is uniquely demonstrated by the increased susceptibility of humans with NK cell deficiencies to multiple types of bacterial infections.13 Intrinsic and extrinsic factors contribute to the activation of NK cells in response to bacterial challenge. Early studies demonstrated an ability of human NK cells to lyse bacteria-infected Hela cells14 and Legionella pneumophilia–15 and Mycobacterium avium–infected monocytes.16 Bacteria-induced IFN-γ production by NK cells has been demonstrated in response to a number of pathogenic strains of bacteria, including Staphylococcus aureus,17,18 Helicobacter pylori,19,20 Escherichia coli,20 and Mycobacterium tuberculosis.21–23 Moreover, NK cells can also respond to nonpathogenic bacteria, including nonpathogenic E. coli and strains of Lactobacillus, by upregulating activation markers, producing IFN-γ, and increasing cytolytic activity.17,24–27 The direct activation of NK cells by bacterial products occurs through the expression of specific bacterial Toll-like receptors (TLRs), including TLR2, TLR4, and TLR5,28–34 whereas indirect activation occurs through accessory cells, such as dendritic cells or monocytes, typically in response to the cytokines produced by the APC themselves, such as IL-12, in conjunction with IL-15 or IL-18.28,30,35–38
Much of the work addressing NK cell function during HIV-1 infection has focused on the role of NK cells in antiviral immunity, and it is not known whether the ability of NK cells to respond to bacteria is compromised during chronic HIV-1 infection. This question is important as dysfunctional antibacterial NK cell responses may, in part, contribute to the increased prevalence of bacteria-associated opportunistic infections39 or the high incidence of coinfection with M. tuberculosis in immune-compromised, HIV-1–infected individuals.40 The antibacterial response of NK cells may also be impacted by the increase in HIV-associated microbial translocation41 either by inducing NK cells to produce proinflammatory cytokines in vivo and thus contribute to a state of chronic immune activation or, conversely, by leading to defective bacteria-associated NK cell responses through overstimulation or exhaustion. To address these possibilities, we investigated the cytokine responses of peripheral blood NK cells to commensal and pathogenic whole bacteria in antiretroviral therapy (ART)-treated and -untreated subjects with chronic HIV-1 infection.
MATERIALS AND METHODS
Blood samples were obtained from 40 HIV-1–infected subjects who were receiving care at the University of Colorado Infectious Disease Group Practice Clinic and the University of Colorado Hospital (Aurora, CO). Blood samples were also obtained from 24 healthy adults, self-identifying as HIV-1 uninfected, who served as normal controls. HIV-1–infected subjects were either untreated with plasma viremia (ART-naive or had not been on ART for at least 1 year at the time of screening; “untreated”; n = 23) or were receiving ART for >2 years with suppression of plasma viral load to <48 copies HIV-1 RNA/mL at the time of screening (“treated,” n = 17).
All untreated HIV-1–infected patients were chronically infected and showed no signs of acute illness at the time of enrollment into the study. The clinical characteristics of the cohorts are detailed in Table 1. All the study subjects participated voluntarily and gave written, informed consent. This study was approved by the Colorado Multiple Institutional Review Board at the University of Colorado Anschutz Medical Campus.
Isolation of Human Peripheral Blood Mononuclear Cells
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using standard Ficoll-Hypaque (Amersham Biosciences, Piscataway, NJ) density gradient centrifugation and were cryopreserved and stored in liquid nitrogen as detailed elsewhere.42,43
Whole Bacterial Preparations
E. coli (no. 25,922; ATCC, Manassas, VA) and Salmonella typhimurium (no. 35,986, ATCC) were grown, heat-inactivated, and stored as previously described.43,44
Surface and Intracellular Flow Cytometry Staining Assays, Acquisition, and Analysis
Standard flow cytometry staining protocols for surface markers and intracellular IFN-γ are detailed elsewhere.44–46 NK cells were identified within CD3− lymphocytes (PE-Texas Red CD3, ECD; Beckman Coulter, Fullerton, CA) using V450 or PE-Cy5 CD56 and APC-H7 or AF700 CD16 (both BD Biosciences, San Jose, CA). AF700 IFN-γ (BD Biosciences) was used to evaluate the frequencies of IFN-γ+ cells after in vitro stimulation. Monocytes were evaluated using V450 CD14, and myeloid dendritic cells (mDCs) were evaluated using fluorescein isothiocyanate Lineage (CD3, CD14, CD16, CD19, CD20, and CD56), APC-Cy7 HLA-DR, PE-Cy5 CD11c (all BD Biosciences), and APC CD123 (Miltenyi Biotec, Auburn, CA) as previously described.42,43,47 All flow cytometry data were acquired on an LSRII Flow Cytometer (BD Biosciences) and analyzed using BD FACSDiva software version 6.1.2 (BD Biosciences).
NK cell subsets were identified by the expression of CD56 and CD16. In our initial studies, we noted a reduction in the fraction of CD56brightCD16− NK cells and a corresponding increase in the CD56dimCD16− NK cells in culture relative to preculture frequencies (see Figure S1a and S1b, Supplemental Digital Content, http://links.lww.com/QAI/A474). Overall CD56 expression levels on CD56+CD16− NK cells were also reduced after both culture and stimulation (see Figure S1c, Supplemental Digital Content, http://links.lww.com/QAI/A474). Thus, going forward, we used a published gating strategy that included all CD56+CD16− cells48 rather than gating only on CD56bright NK cells to avoid the exclusion of CD56bright NK cells that subsequently decreased CD56 expression during the in vitro culture period. A representative example of the gating strategy used is shown in Figure S1d (see Supplemental Digital Content, http://links.lww.com/QAI/A474).
In Vitro Stimulation of PBMCs
PBMCs were thawed and either assessed for baseline percentages of NK cells by flow cytometry or cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA) + 10% human AB serum (Gemini Bioproducts, West Sacramento, CA) + 1% penicillin–streptomycin–L-glutamine (Sigma-Aldrich, St Louis, MO; complete media) with or without heat-inactivated bacteria (6 bacteria: 1 PBMC) for 4 hours. To accommodate the early production of IFN-γ as reported by others,5 Brefeldin A (1 μg/mL; BD Biosciences) was added for the remainder of the culture (12–18 hours).
In Vitro Stimulation of Purified NK Cells and mDCs or Monocyte-Depleted Cultures
NK cells were isolated from PBMCs by negative selection using a magnetic bead kit (NK cell isolation kit; Miltenyi Biotec) as per the manufacturer's instructions. NK cells accounted for 90.8%–97.5% (n = 3) of the isolated cells.
PBMCs were depleted of BDCA-1+ and BDCA-3+ mDC or CD14+ monocytes using positive magnetic bead selection protocols (Antibiotin MicroBeads or CD14 MicroBeads, respectively; both from Miltenyi Biotec) as previously described.43 PBMCs were depleted of mDC (defined as Lineage-HLA-DR+CD123loCD11c+)47 by 86.8%–97.0% (n = 6) and depleted of monocytes by 87.9%–98.3% (n = 6).
Monocytes were separated from total PBMCs using CD14 microbeads and accounted for 92.9%–98.4% of the isolated cells. Monocyte-depleted PBMCs were plated on the bottom wells of a 24-well Costar transwell plate (Corning Inc, Corning, NY), and then monocytes were added onto membrane inserts (0.4-μm pore size) placed into wells. To account for any effect of attached microbeads on CD14+ monocyte function, the same number of isolated monocytes was mixed back with the monocyte-depleted PBMCs and used as control PBMCs (final CD14+ percentage within control PBMC: 10.8%–22.9%, n = 5). E. coli was added either directly to the monocytes in the inserts or to the wells containing control PBMC for 4 hours before the addition of Brefeldin A. After overnight culture, the cells were collected from the bottom wells, and the frequencies of IFN-γ+ NK cells were evaluated by the intracellular flow cytometry assay.
Allogeneic Monocyte–NK Cell Cocultures
To have the same monocytes used for the stimulation of NK cells from multiple donors, monocytes were isolated from 1 uninfected donor using CD14+ microbeads (final monocyte purity: 99.3%), cryopreserved, and stored in liquid nitrogen. NK cells were isolated from uninfected (n = 4) and HIV-1–infected (n = 4) donors as described above. Monocytes were then thawed and cultured at a ratio of 1:1 with purified NK cells in complete media with or without E. coli (6:1) for 19–22 hours, and culture supernatants were collected and stored at −20°C. IFN-γ production within culture supernatants was evaluated by an enzyme-linked immunosorbent assay (eBioscience, San Diego, CA).
For nonparametric analysis, comparisons between independent groups were made using the Mann–Whitney t test and the Friedman test with a multiple Dunn comparison test for matched–paired comparisons across multiple groups. To determine the differences between groups of paired data, the Wilcoxon matched-pairs signed-rank test was performed. Correlations between variables were assessed using the Spearman test. For small sample sizes (n < 7), comparisons between independent groups were made using the unpaired t test. The paired t test was used for the analysis of matched–paired groups and the repeated measures analysis of variance with a Dunnett multiple comparison test was used for matched–paired comparisons across multiple groups. In all analyses, a P value of <0.05 was considered significant. All statistical analyses were performed using GraphPad Prism Version 6 for Windows (GraphPad Software, San Diego, CA).
NK Cells That Produce IFN-γ in Response to Commensal Bacteria Are Reduced in HIV-1–Infected Individuals
Frequencies of total NK cells (CD56±CD16±) producing IFN-γ were determined within PBMCs with and without stimulation with heat-killed E. coli. In the absence of exogenous stimulation, low frequencies of IFN-γ+ NK cells were observed in all the study groups without statistical differences observed between them (data not shown). However, the frequencies of IFN-γ+ NK cells in response to the stimulation of PBMCs with E. coli were significantly reduced when the cells were obtained from HIV-1-infected donor groups as opposed to when the PBMCs were from uninfected subjects (Fig. 1A).
For all 3 NK cell subsets, in vitro stimulation of uninfected donor PBMCs with E. coli resulted in IFN-γ production (Fig. 1B). The CD56+CD16− NK cells and CD56+CD16+ NK cells had similarly high percentages of IFN-γ+ cells in response to E. coli with most IFN-γ production coming from the CD56dim populations, regardless of CD16 expression, as has been previously reported.5–7 Fractions of CD56+CD16− and CD56+CD16+ NK cells producing IFN-γ were significantly greater than those of the CD56−CD16+ NK cell subset (Fig. 1B).
NK subset IFN-γ responses to E. coli were next compared between untreated and ART-treated HIV-1–infected donors and uninfected control subjects (Fig. 1C). In PBMCs from untreated donors, fewer NK cells in all 3 subsets produced IFN-γ after stimulation with E. coli than did NK cells from control donors. Among treated donors, the percentage of IFN-γ+ CD56+CD16− NK cells did not statistically differ from uninfected controls. In contrast, the percentages of IFN-γ+ NK cells within the CD56+CD16+ and CD56−CD16+ NK cell subsets in treated donors were statistically lower than in controls and were not significantly different from that in the untreated donors, suggesting that these responses had not normalized despite viral suppression on ART.
Previous studies have shown that the frequencies of blood NK cell subsets are altered during HIV-1 infection.11,49–51 To determine whether HIV-1–associated decreases in E. coli–induced IFN-γ+ NK cell responses were simply a reflection of altered blood NK cell frequencies, we measured NK cell subset frequencies directly ex vivo (before stimulation). Percentages of CD56+CD16− NK cells were similar across all cohorts (see Figure S2, Supplemental Digital Content, http://links.lww.com/QAI/A474). CD56+CD16+ NK cells were reduced, and CD56−CD56+ NK cells were increased in untreated subjects compared with that in uninfected controls, whereas frequencies of those NK cell subsets in treated subjects did not differ statistically from those of controls. Despite the significant increase in the frequency of CD56−CD16+ NK cells in the untreated subjects (see Figure S2, Supplemental Digital Content, http://links.lww.com/QAI/A474), there was no concurrent increase in the fraction of CD56−CD16+ NK cells producing IFN-γ (Fig. 1C) suggesting that the expansion of this subset during untreated HIV-1 infection was dominated by CD56−CD16+ NK cells that failed to produce IFN-γ in response to E. coli. Moreover, despite the normalization of CD56+CD16+ and CD56−CD16+ NK cell numbers in the treated subjects, the frequency of bacteria-reactive IFN-γ+ NK cells among these subsets was reduced (Fig. 1C). Taken together, these observations suggest that altered baseline frequencies of NK cells do not fully account for the reduced frequencies of bacteria-reactive IFN-γ+ NK cells in HIV-1 infection.
NK Cell IFN-γ Responses to Pathogenic Bacteria Are Also Reduced Within HIV-1–Infected Individuals
NK cell responses within PBMCs to pathogenic S. typhimurium were evaluated using cells from uninfected and HIV-1–infected donors. In uninfected subjects, S. typhimurium induced statistically higher frequencies of IFN-γ+ NK cells than did stimulation with E. coli (Fig. 2A). Reduced percentages of IFN-γ–producing NK cells were detected in all 3 NK cell subsets from untreated subjects after stimulation with S. typhimurium relative to NK cell subsets within S. typhimurium stimulated PBMCs from uninfected donors (Fig. 2B). Among treated donors, the percentage of IFN-γ+ CD56+CD16+ and CD56−CD16+ NK cells in response to S. typhimurium was also lower than for the same NK cell subset from uninfected subjects (Fig. 2B).
NK Cell IFN-γ Production in Response to Bacteria Requires Monocytes in a Contact-Dependent Manner
Because NK cells produced IFN-γ after stimulation with bacteria, we wanted to determine whether the bacteria acted directly on the NK cells or through the bacteria action on accessory cells. Stimulation of purified normal donor NK cells with E. coli failed to induce significant IFN-γ production relative to NK cells stimulated within PBMCs (Fig. 3A). This finding indicates that the ability of bacteria to stimulate NK cell production of IFN-γ was not due to the direct interaction of the bacteria with NK cells. Because the bacteria did not stimulate the NK cells directly, we wanted to determine if mDCs or monocytes were required for the bacteria-induced NK cell production of IFN-γ, PBMCs were depleted of either mDCs or monocytes before adding the bacteria. The percentage of NK cells producing IFN-γ within the accessory cell-depleted PBMCs and total PBMCs was compared (Fig. 3B). The removal of monocytes from PBMCs resulted in a significant decrease in the frequency of IFN-γ–producing NK cells after stimulation with E. coli, whereas minimal differences were observed in mDC-depleted PBMC cultures (Fig. 3B). After monocyte depletion, the percentage of IFN-γ+ cells within the CD56+CD16−, CD56+CD16+, and CD56−CD16+ NK cell subsets was decreased, on average, by 41.1% ± 18.4%, 86.2% ± 6.2%, and 79.2% ± 12.5%, respectively.
To determine whether contact between monocytes and NK cells was required, a transwell system was used to separate bacteria-stimulated monocytes from NK cells in monocyte-depleted PBMCs. Fewer IFN-γ+ NK cells were detected in response to E. coli stimulation when the stimulated monocytes were separated than when they were in contact with NK cells during stimulation (Fig. 3C). Separation of bacteria-stimulated monocytes resulted in an 82.2% ± 2.9% decrease in the percent of IFN-γ+ cells within the CD56+CD16− NK cell subset and a decrease of 92.8% ± 2.5% and 92.0% ± 3.1% within the CD56+CD16+ and CD56−CD16+ NK cell subsets, respectively. These results show that contact between monocytes and NK cells is required for IFN-γ production by NK cells in response to bacteria.
IFN-γ Production by NK Cells From HIV-1–Infected Donors Is Not Restored by Exposure to Uninfected Donor Monocytes
To determine whether the defect in NK cell IFN-γ production observed in HIV-1–infected individuals was because of dysfunction of monocytes or NK cells, monocytes were isolated from an allogeneic, uninfected donor and cultured with purified NK cells from either uninfected or untreated, HIV-1–infected donors in the presence of E. coli, and IFN-γ levels were measured in culture supernatant. E. coli stimulation of purified monocytes did not induce IFN-γ production (data not shown). Normal donor monocytes stimulated normal donor NK cells to produce IFN-γ (Fig. 3D). However, in the presence of normal donor monocytes, levels of IFN-γ that were detected in culture supernatants of NK cells from HIV-1–infected donors were reduced relative to culture supernatants from uninfected donor NK cells (Fig. 3D).
Numeric and functional NK cell defects have been observed during both acute and chronic HIV-1 infection and may contribute to HIV-1 pathogenesis.11,49–52 Specifically, HIV-1–associated changes in NK cell phenotype, including altered expression of activating and inhibitory receptors, have been associated with impaired cytotoxicity against NK-sensitive cell lines, reduced cytokine production in response to known NK cell activation-inducing cytokines and defective antibody-dependent cellular cytotoxicity responses.9,11,53–59 Impaired ability of NK cells to kill HIV-infected cells is likely mediated through HIV-induced selective alteration of major histocompatibility complex class I expression in conjunction with modulating ligands important in triggering NK cell cytotoxic responses.60–62 Long-term ART (>2 years) typically results in the restoration of NK cell phenotype and function.11,54,63,64 However, some studies have demonstrated a persistent impairment in IFN-γ production in treated subjects despite the normalization of phenotype and cytotoxic function.65,66 In addition, a recent study demonstrated that NK cells remained in an activated state, defined by the coexpression of HLA-DR and CD38, despite subjects having received ART for a median duration of 11.5 years.67 Moreover, NK cell inhibitory or activation receptors generally do not return to normal levels when viremia is suppressed by ART, although in some cases normalization occurs after prolonged viral suppression.59,68
In this study, we used an in vitro assay to evaluate the production of IFN-γ by NK cells in response to the bacterial stimulation of PBMCs. In agreement with previous studies where isolated CD56dim NK cells produced IFN-γ in response to receptor-mediated and cytokine-mediated activation,5–7 the majority of bacteria-reactive IFN-γ–producing NK cells were found within CD56dim NK cells, irrespective of CD16 expression. We further demonstrate that chronic, untreated HIV-1 infection results in the impairment of NK cells to produce IFN-γ in response to both commensal and pathogenic bacteria. Furthermore, limited functional improvement was observed in NK cells from subjects on long-term ART, despite evidence of effective viral suppression and improved CD4 counts. Although we observed relative changes in the frequency of blood NK cell subsets within HIV-1-infected subjects before stimulation, these changes could only partly account for the reduced numbers of IFN-γ+ NK cells. Induction of IFN-γ was dependent on contact with monocytes, yet HIV-associated NK cell function was not restored by exposure to normal monocytes, suggesting that at least a component of the dysfunction is an intrinsic NK cell defect. We believe that our study is the first to show that antibacterial NK cell responses are impacted by chronic HIV-1 infection with limited restoration in function after ART.
An important finding of our study is that cell-to-cell contact is required between NK cells and monocytes to induce NK cell-associated IFN-γ production in response to bacterial stimulation. Crosstalk between DC and NK cells is well described,69 but an understanding of the interactions between NK cells and monocytes/macrophages is only beginning to emerge.70 Interactions between NK cell 2B4 and CD48 expressed by low dose lipopolysaccharide (LPS)-stimulated human macrophages were shown to be necessary to induce NK cell proliferation and IFN-γ production.71 Increased NK cell IFN-γ secretion has also been observed after NKp80 activation through the myeloid-specific activation-induced C-type lectin expressed by LPS-treated monocytes.72 Furthermore, LPS stimulation of human monocytes induced the upregulation of MHC class I chain-related molecule A (MICA), the ligand for NKG2D, resulting in IFN-γ production by NK cells37 suggesting that NKG2D may also be an important activating receptor permitting the induction of bacteria-induced IFN-γ by NK cells. Although some studies observed only minimal differences in NKG2D expression by NK cells in HIV-1–infected individuals,11 a recent study found increased levels of serum MICA in subjects with chronic HIV-1 infection and associated this with reduced NKG2D expression on NK cells and aberrant NKG2D-mediated recognition of target cells.52 This latter study raises the possibility that increased levels of serum MICA, potentially secreted by bacteria-stimulated monocytes, and altered NKG2D expression may also result in reduced bacteria-associated NK cell activation and IFN-γ production.
Decreased antibacterial responses by NK cells in HIV-infected individuals may also result from NK cell exhaustion because of overstimulation after exposure to opportunistic viral and bacterial pathogens and exposure to translocated bacteria and bacterial products as has been shown to occur during HIV-1 infection.41 Other studies have shown increased PD-1 expression on blood NK cells from both viremic and aviremic HIV-1–infected donors.73 Given that increased PD-1 expression on T cells during chronic HIV-1 infection has been implicated in T-cell exhaustion,74,75 an elevated expression of PD-1 on NK cells may indicate a similar functional phenotype and contribute to reduced bacteria responsiveness. To expand on our current pilot study, investigations are now underway to further address these potential mechanisms behind the HIV-1-associated defective bacterial NK cell responses.
Understanding the impact of in vivo HIV-1 infection on the antibacterial responses has clinical implications. It was recognized early in the HIV-1 epidemic that those infected with HIV-1 had a higher prevalence of bacterial infections.39,76 Although the rates of bacterial infections have declined with the advent of ART, they remain elevated in areas with a high incidence of HIV-1 infection,39,77,78 and treatments of bacterial infections are now potentially complicated by the emergence of multidrug resistant bacteria.79,80 Moreover, the role of NK cells in antibacterial immunity may take on more importance in bacterial diseases that are predominantly controlled through T cell–mediated immunity, responses likely compromised in HIV-1–infected individuals. Indeed, NK cells from individuals with HIV-1 and pulmonary tuberculosis failed to produce IFN-γ when stimulated in vitro with live M. tuberculosis.81 Thus, understanding the mechanisms underlying HIV-1–associated NK cell dysfunction could aid in the development of therapies designed to enhance or restore innate immune responses.
The authors would like to thank the physicians, staff, and patients in the Infectious Diseases Group Practice Clinic at the University of Colorado Hospital and the uninfected donors for their participation in this study. The authors would also like to thank Zachary Dong, Kirsten Miller, Zahra Kahn, Christina Briegleb, Spenser Hansen, and Lydia Hostetler for assistance with recruiting study subjects. The authors thank Jennifer Manuzak, Lisa Rogers, and Caleb Kelly for technical assistance.
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