IL-10+ NK and TGF-β+ NK cells play negative regulatory roles in HIV infection

Background Natural killer (NK) cells play cytotoxic roles by targeting tumor cells or virus infected cells, they also play regulatory roles by secreting cytokines and chemokines. Transforming growth factor (TGF)-β and interleukin (IL)-10 are important immunosuppressive cytokines potentially related to the immune dysregulation that occurs in the infection of human immunodeficiency virus (HIV). NK cells are an important source of TGF-β and a main early producer of IL-10 in response to viral infection. Here, we evaluated the percentages of IL-10+ and TGF-β+ NK cells in HIV-infected patients relative to healthy controls (HCs). Methods Study participants (n = 63) included 31 antiretroviral treatment (ART)-naïve HIV-infected patients, 17 ART-treated HIV-infected patients, and 15 HIV-negative HCs. Expression of IL-10 or TGF-β in NK cells was examined by flow cytometry, and the influences of recombinant IL-10 (rIL-10) or recombinant TGF-β (rTGF-β) on NK cell function were investigated in vitro. Results Compared with HCs, ART-naïve HIV-infected patients had increased percentages of IL-10+ (2.0% vs. 0.4%, p = 0.015) and TGF-β+ (4.5% vs. 2.1%, p = 0.022) NK cells, and ART-treated patients also had a higher percentage of IL-10+ NK cells (2.5% vs. 0.4%, p = 0.002). The percentages of IL-10+ and TGF-β+ NK cells were positively correlated (r = 0.388; p = 0.010). The results of in vitro experiments demonstrated that rIL-10 and rTGF-β inhibited NK cell CD107a expression (p = 0.037 and p = 0.024, respectively), IFN-γ secretion (p = 0.006, p = 0.016, respectively), and granzyme B release after stimulation (p = 0.014, p = 0.040, respectively). Conclusions Our data suggest that the percentages of IL-10+ or TGF-β+ NK cells are increased in HIV-infected patients, and that rIL-10 and/or rTGF-β can inhibit NK cell functions in vitro, providing a potential therapeutic target for strategies aimed at combating HIV infection.


Background
Natural killer (NK) cells serve as the first line of immune defense in host protection against viruses and tumors [1]. In humans, NK cells account for 2%-18% of the lymphocytes in peripheral blood and express various inhibitory and activating receptors, including C-type lectin-like, natural cytotoxicity, and killer cell immunoglobulin-like receptors [2,3]. NK cell functions include killing target cells, cytokine production, and antibody-dependent cellular cytotoxicity (ADCC) [2]. Moreover, NK cells are critical effectors mediating cytotoxicity, and regulators modulating the activation and development of other immune response components [1]. NK cells are identified via their lack of CD3 and expression of CD56 cell surface markers, and they can be further divided into CD56 dim and CD56 bright subsets [3]. Generally, CD56 dim NK cells release perforin or granzymes, which play a key role in killing target cells, whereas CD56 bright NK cells secrete interleukin (IL)-10, interferon (IFN)-γ, transforming growth factor (TGF)-β and other cytokines, to exert immunomodulatory effects [4][5][6].
IL-10 and TGF-β are important immunoregulatory cytokines in vivo [7,8], which suppress adaptive and innate immunity [9]. IL-10 is produced by multiple cell types, including T cells, NK cells, monocytes, and B cells; NK cells are a major early source of this cytokine in response to viral infection [10][11][12][13]. IL-10 is involved in the impairment of T cell function during persistent viral infections, and blockage of the IL-10 pathway alone is sufficient to restore T cell activities and increase viral control [14]. TGF-β is also secreted by various cell types, particularly NK cells, which are the only lymphocyte population that constitutively produces this cytokine [15]. TGF-β plays important roles in immunomodulation, inflammation, and tissue repair [16], and can inhibit T cell proliferation and cytotoxicity [17]. IL-10 is reported to cause harmful effects during human immunodeficiency virus (HIV) infection by reducing IL-2 and IL-12 production, thereby inhibiting antigen-presentation and cellular immune responses [18][19][20]. HIV-infected CD4 + T cells can produce IL-10, leading to persistent viral infection [11]. High levels of TGF-β in the plasma were reported in HIV-infected patients compared with healthy controls (HCs) [21]; however, the cell types producing TGF-β in this context remain to be determined.
IL-10 + NK cells play significant modulatory roles in various viral, bacterial, and parasitic infections [12,[22][23][24]. TGF-β + NK cells have been reported to serve as an important co-stimulatory signal to induce suppressive T cells [15]. In HIV infection, multiple cells can produce IL-10 and TGF-β. The majority of research has focused only on T cells, rather than NK cells, which are a major source of these cytokines and play important roles during acute HIV infection. The percentage of IL-10 + or TGF-β + NK cells in HIV-infected patients and the regulatory effect of IL-10 and TGF-β have yet to be elucidated.
In the present study, we determined the percentages of IL-10 + and TGF-β + NK cells in HIV-infected patients and healthy controls (HCs). We also explored the immunomodulatory effects of recombinant IL-10 (rIL-10) and recombinant TGF-β (rTGF-β) on NK cell functions, including the expression of lysosomal-associated membrane glycoprotein-1 (LAMP1; also known as CD107a), and IFN-γ secretion. The results indicated that IL-10 + and TGF-β + NK cells may be risk factors for HIV disease progression, and are potential therapeutic targets in combating HIV infection.
Detection of IL-10 + or TGF-β + NK cells by flow cytometry Peripheral blood mononuclear cells (PBMCs) were isolated from HIV-infected patients and HCs by Ficoll™ density gradient centrifugation. PBMCs were cultured for 6 h with recombinant IL-12 (rIL-12, 10 ng/ml) and recombinant IL-15 (rIL-15, 50 ng/ml) in a 5% CO 2 incubator at 37°C. Total NK cells and NK cell subsets were defined by their expression of CD3 and CD56, detected using the anti-CD3 PerCP and anti-CD56 PE-Cy7 antibodies (BD Biosciences, San Jose, CA, USA), respectively. Cells were stained with intracellular anti-TGF-β-PE and anti-IL-10-APC (Biolegend, San Diego, CA, USA) and fixed in 1% paraformaldehyde before detection using a flow cytometer (LSR II; Becton Dickinson, Franklin Lakes, NJ, USA) and analysis with FACSDiva™ software (Becton Dickinson); the gating strategy is shown in Fig. 1.

Detection of CD4 + T cell count
CD4 + T cell counts were determined using a flow cytometer (FACS Calibur; Becton Dickinson). High, medium, and low Trucount™ Control Beads (BD Biosciences, San Jose, CA, USA) were used to confirm the accuracy and quality of CD4 + T cell counts. A single-platform lyse-nowash procedure was conducted with TriTEST™ anti-CD3-PerCP/CD4-FITC/CD8-PE reagents and Trucount tubes (BD Biosciences, San Jose, CA, USA) according to the manufacturer's instructions.

Determination of HIV viral load
Plasma HIV RNA levels were determined with by reverse transcription-polymerase chain reaction (RT-PCR) using the COBAS®AmpliPrep®/COBAS TaqMan™ HIV test v2.0 (Roche Diagnostic Systems, Basel, Switzerland). The detection range of this method is 20-10,000,000 copies/ml. The number of HIV RNA copies was calculated according to the manufacturer's reference standard.

Statistical analyses
The non-parametric Mann-Whitney U-test was used for evaluation of differences between two groups, and the Kruskall-Wallis test employed for comparisons of multiple groups. The Friedman's test was used for multiple pairwise comparisons. Correlation analysis was performed using the Spearman's rank correlation test. All analyses were undertaken using SPSS v19.0 (IBM, Armonk, NY, USA). p < 0.05 was considered significant.

Percentages of total NK cells and NK subsets in HIV-infected patients and HCs
The clinical characteristics of ART-naïve and ARTtreated patients, and HCs, are presented in Table 1. The sex and age of HIV-infected patients were matched with Gating strategy for identification of NK cells and their subsetsP1 represents lymphocytes identified using a side and forward scatter dot plot. P2 represents CD56 bright NK cells; gating, CD3 − CD56 bright . P3 represents CD56 dim NK cells; gating, CD3 − CD56 dim . P2 + P3 represents total NK cells. P4 represents IL-10 + NK cells. P5 represents TGF-β + NK cells those of HCs. The percentages of total NK cells and NK subsets among lymphocytes in peripheral blood samples from the three groups were measured by flow cytometry.
The percentages of total NK cells in ART-naïve and ART-treated patient samples were lower than that in HCs (p = 0.014, p = 0.024) (Fig. 2a). In addition, the percentages of CD56 dim NK cells in ART-naïve patients were lower than those in HCs (p = 0.006) (Fig. 2b) and there was a tendency towards lower percentages of CD56 bright NK cells in ART-naïve patients compared with those of HCs (p = 0.067) (Fig. 2c).

Percentages of IL-10 + or TGF-β + NK cells in HIV-infected patients
PBMCs obtained from HCs, and ART-naïve, and ARTtreated patients, were stimulated with rIL-12 and rIL-15. The percentage of IL-10 + or TGF-β + NK cells was examined by intracellular staining and multi-color flow cytometry. Representative flow cytometry plots of IL-10 or TGF-β expression in NK cells from each participant group are shown in Fig. 3a. The percentage of IL-10 + NK cells in ART-naïve patients was higher than that in HCs (p = 0.015); moreover, the percentage of IL-10 + NK cells was also higher in the ART-treated HIV patient group than that in HCs (p = 0.002) (Fig. 3b). The percentage of TGF-β + NK cells in ART-naïve patients was elevated compared with that of HCs (p = 0.022), while in ART-treated patients, the percentage of TGF-β + NK cells still exhibited a tendency to be higher than that of HCs (p = 0.060) (Fig. 3c). There was no difference in the percentage of IL-10 + CD56 dim NK cells among the three groups (Fig. 3d), whereas the percentage of TGF-β + CD56 dim NK cells in ART-naïve patients was higher than that in HCs (p = 0.024) (Fig. 3e). Moreover, there were no differences in the percentages of IL-10 + CD56 bright and TGF-β + CD56 bright NK cells among the three groups (Fig. 3f, g). In ART-naïve patients, the percentage of IL-10 + NK cells was positively correlated with that of TGF-β + NK cells (r = 0.388, p = 0.010) (Fig. 3h) and the percentage of IL-10 + CD56 dim NK cells correlated positively with that of TGF-β + CD56 dim NK cells (r = 0.438, p = 0.003) (Fig. 3i).
Comparison of the percentages of IL-10 + and TGF-β + NK cells in different groups according to CD4 + T cell count or HIV viral load CD4 + T cell counts and viral loads are important markers for HIV disease progression. We analyzed the correlation between the percentage of IL-10 + or TGF-β + NK cells and CD4 + T cell counts and viral loads and found no significant correlations between them. Furthermore,  we analyzed groups subdivided by CD4 + T cell count or viral load. The percentage of IL-10 + NK cells in the CD4 + T < 350 cells/μl group of ART-naïve patients was higher than that of the HC group (p = 0.032), and there was an increasing trend in the CD4 + T ≥ 350 cells/μl group of ART-naïve patients compared with HCs (p = 0.075) (Fig. 4a). There was no difference in the percentages of TGF-β + NK cells among groups (Fig. 4b). When grouped Fig. 3 Comparison of the percentages of IL-10 + and TGF-β + NK cells in HIV-infected patients and HCsPBMCs from HCs, ART-naïve, and ART-treated patients were stimulated using rIL-12 (10 ng/ml) and rIL-15 (50 ng/ml). IL-10 + and TGF-β + NK cells were examined by multi-color flow cytometry, with gates defined according to isotype controls. Representative flow-cytometric plots of IL-10 + and TGF-β + NK cells in HIV-infected patients and HCs (a). Comparison of the percentages of IL-10 + NK cells (b), TGF-β + NK cells (c), IL-10 + CD56 dim NK cells (d), TGF-β + CD56 dim NK cells (e), IL-10 + CD56 bright NK cells (f), and TGF-β + CD56 bright NK cells (g), among the three groups. Correlation between the percentage of IL-10 +

NK cells and TGF-β + NK cells (h). Correlation between the percentage of IL-10 + CD56 dim NK cells and TGF-β + CD56 dim NK cells (i)
according to the level of viral load, the percentage of IL-10 + NK cells in ART-naïve patients with viral load > 10 4 copies/ml was higher than that in the HC group (p = 0.023) (Fig. 4c), and that in the group with viral load < 10 4 copies/ml, while the percentage of TGF-β + NK cells was higher than that in the HC group (p = 0.027) (Fig. 4d).

Effect of rIL-10 and rTGF-β on the NK cell CD107a expression and IFN-γ secretion
The expression of CD107a and IFN-γ secretion can be used to assess the functional anti-viral ability of NK cells. The inhibitory effects of rIL-10 or rTGF-β on NK cell functions were evaluated, and the percentages of IFN-γ + and CD107a + NK cells determined. Representative flow cytometry plots, demonstrating the effects of rIL-10 or rTGF-β on CD107a expression by NK cells are presented in Fig. 5a. CD107a expression in NK cells was inhibited by high concentrations of rIL-10 or rTGF-β in vitro (p = 0.037, p = 0.024), and the rate of inhibition increased gradually with increasing concentrations of these cytokines (Fig. 5b, c). Moreover, the effect of rIL-10 and rTGF-β on CD107a expression in NK cells was synergistic, demonstrating inhibitory effects much greater than those of either cytokine individually (p = 0.006) (Fig. 5d).

Effects of rIL-10 and rTGF-β on granzyme B and perforin release by NK cells
Granzyme B and perforin are important functional markers for NK cells, which indicate the killing capability of NK cells. The percentages of IL-10 + and TGF-β + NK cells were elevated in HIV infected patients; however, their influence on the release of granzyme B and perforin by NK cells is not known. Our results demonstrate that a low concentration of rIL-10 or rTGF-β did not inhibit the release of granzyme B, whereas high concentrations of either cytokine did inhibit the release of this factor (p = 0.014, p = 0.040) (Fig. 7a, b). A synergistic effect of rIL-10 and rTGF-β on inhibition of the release of granzyme B was also noted (p = 0.037) (Fig. 7c). Similarly, rIL-10 and rTGF-β inhibited perforin release by NK cells (Fig. 8a-c).

Discussion
Regulation of the immune system has attracted considerable attention over the last decade, and the cytokines, IL-10 and TGF-β, have important roles in controlling immune processes. The suppression of anti-cancer immunity by TGF-β and IL-10 has been reported in several studies [25][26][27][28]. In breast cancer, circulating levels of IL-10 + and TGF-β + NK cells are increased, which inhibits the production of the NK cell effectors, IFN-γ and CD107a, and promotes the migration and invasion of breast-cancer cells [9]. Brockman et al. reported that IL-10 mRNA levels were increased in several PBMC subpopulations in HIVinfected patients compared with HCs, particularly T cells, B cells, monocytes, and NK cells, and that the inhibition of the IL-10 pathway enhanced the proliferative capacity of T cells [29]. Based on these findings regarding the levels of IL-10 mRNA in NK cells, we further explored the intracellular secretion of IL-10 and TGF-β proteins in NK cells (total and subsets) and investigated the relationship between IL-10 + /TGF-β + NK cells and CD4 + T cell counts/viral loads. Our data elucidate the effects of IL-10 and TGF-β on NK cell functions, including IFN-γ secretion, CD107a expression, and granzyme B or perforin release.
We found that ART-naïve patients had elevated percentages of IL-10 + NK cells relative to HCs; however, patients who had received ART, also had higher percentages of IL-10 + NK cells than those of HCs. Comparisons of TGF-β + NK cell percentages among study groups   Fig. 7 Effects of rIL-10 and rTGF-β on granzyme-B release by NK cellsPBMCs were co-cultured with rIL-10 and/or rTGF-β for 5 h, and then stimulated with PMA and ionomycin for 6 h. The inhibition of granzyme-B release by NK cells using different concentrations of rIL-10 (a) or rTGF-β (b) was determined. The effect of treatment with both rIL-10 and rTGF-β on granzyme-B release by NK cells was synergistic (c) generated similar results to those for IL-10 + NK cells. These data indicate that ART is insufficient to induce complete recovery of immune functions, and that specific immune therapies are required to overcome the negative regulation. Our previous study showed that HIV infection can result in impaired NK cell functions, including diminished levels of perforin and IFN-γ production [30,31], which may be related to negative regulation by cytokines. In the present study, the results of our in vitro experiments demonstrate that the expression of CD107a and secretion of IFN-γ by NK cells can be inhibited by rIL-10 or rTGF-β. High concentrations of rIL-10 or rTGF-β could also inhibit the release of granzyme B and perforin; therefore, we suspect that the increase in the percentage of IL-10 + and TGF-β + NK cells may be related to impaired NK cell cytotoxic function during HIV infection.
The mechanisms underlying the elevated percentage of IL-10 + and TGF-β + NK cells during HIV infection remain unclear; however, it was reported that the HIV Env and Tat peptides are responsible for IL-10 production by CD4 + T cells [32][33][34], and the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway was reported as involved in this process [35,36]. Further, in vitro experiments have indicated that HIV Tat can induce TGF-β production by NK cells [21], and that TGF-β may suppress NK cell functions by repressing the mTOR pathway [37], or inhibit IFN-γ secretion and the ADCC response of NK cells via SMAD3-mediated signaling [38].

Conclusions
The results of our study demonstrate that the percentages of IL-10 + and TGF-β + NK cells are elevated in ART-naïve HIV-infected patients, and that ART-treated HIV patients also have a high percentage of IL-10 + NK cells. We also show that rIL-10 and/or rTGF-β can inhibit the functions of NK cell effectors. Our research provides a potential therapeutic target for combating HIV infection, along with information useful for research into the pathogenesis and immunotherapy of HIV infection.

Acknowledgments
The authors express their gratitude for the generosity of the patients who participated in this study.

Funding
Funding for this study was provided by the Mega Projects of National Science Research for the 12th Five-Year Plan (2014ZX10001002001004), and the Platform Project for Close Combination of Basic and Clinical Medicine (YIDA FAZI [2013] no. 5). The funding sources had no role in the study design, data collection, data analyses, preparation of the manuscript, or decision to publish.

Availability of data and materials
All data generated or analyzed during this study are included in this manuscript.
Authors' contributions YJ and HS designed the experiments, interpreted the data, and wrote the manuscript. MY, XS, XC, MM, XY, SQ, and YJ carried out the NK cell experiments. ZZ and YF carried out CD4 + T cell counts. XH participated in measurement of HIV viral load. JX and JL carried out the epidemiological study and helped to recruit study participants. All authors have read and approved the manuscript.  Effects of rIL-10 and rTGF-β on perforin release by NK cellsPBMCs were co-cultured with rIL-10 and/or rTGF-β for 5 h, and then stimulated with PMA and ionomycin for 6 h. The inhibition of perforin release by NK cells by different concentrations of rIL-10 (a) or rTGF-β (b) was determined. The effects of treatment with both rIL-10 and rTGF-β on perforin release by NK cells were synergistic (c)