SlanDCs comprise a major subpopulation of proinflammatory human blood monocytes, with characteristics different from other monocytes and close to some DC subsets, and selective expression of 6-sulfo LacNAc1 (a carbohydrate modification of P-selectin glycoprotein ligand-1).1–3 In healthy individuals, circulating slanDCs are the major subpopulation of blood DCs, representing 0.6%–2% of peripheral blood mononuclear cells (PBMCs).1,2 Frequency of slanDCs and their potential to release proinflammatory cytokines elevates during inflammatory disorders such as rheumatoid arthritis and Crohn disease.4 SlanDCs have also been identified as inflammatory dermal DCs in active psoriasis skin lesions5 and a novel effector cell type in the immunopathogenesis of lupus erythematosus.6 Recently, slanDCs were uncovered to frequently accumulate in the metastatic tumor-draining lymph nodes of cancer patients.3 Accumulation of proinflammatory slanDCs in intestinal tissues of acute GvHD patients was also demonstrated.7
Activated slanDCs are a prominent source of cytokines such as IL-1β.8,9 IL-1β is a member of the IL-1 family proinflammatory cytokine synthesized as mature bioactive form.10,11 Through binding to its receptor (IL-1R1), IL-1β plays an important role in body defense during infection.11,12 It has also been reported that IL-1β is involved in chronic inflammatory disorders such as HIV-1 infection.10,13 Different cell types such as monocytes, DCs, and macrophages release IL-1β during chronic HIV-1 infection.14–16
The frequency and activity of myeloid dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs) are disturbed during HIV-1 infection. It has been shown that both mDCs and pDCs deteriorate in frequency and functions in HIV-1–infected patients,17,18 and that slanDCs increase in numbers and tumor necrosis factor-alpha (TNF-α) response to microbial stimuli in viremic patients during chronic infection.16 However, the functional profile of slanDCs is not well investigated in HIV-1 infections. In this study we evaluated slanDCs' frequency and potential to produce IL-1β in HIV-1–infected patients. Our observation point to an accumulation of activated slanDCs in viremic HIV-1–infected individuals, which produce more IL-1β compared with slanDCs of healthy controls. However, in patients with successful antiretroviral therapy (ART), relative numbers and extracellular IL-1β secretion of slanDCs were not different from controls, indicating restoration by ART.
Blood samples were collected from 13 healthy donors and 31 HIV-1–infected patients [13 ART–treated patients (median age: 47 years, male/female ratio: 8/5, median viral load: 20 copies/mL and median CD4+ T-cell counts: 508 cells/μL) and 18 untreated patients (median age: 44.5 years, male/female ratio: 13/5, median viral load: 4395 copies/mL and median CD4+ T-cell counts: 474 cells/μL)] at Hannover Medical School. All donors were informed and gave written consent before their participation in this study. Characteristics of the patients included in this study are shown (see Table S1, Supplemental Digital Content, http://links.lww.com/QAI/A839). The study was approved by the local ethical committee of Hannover Medical School.
Absolute Count and Plasma HIV-1 RNA Analysis
To assess the absolute numbers of slanDCs, we used Trucount beads (BD Biosciences, Leimen, Germany) according to the manufacturer's protocol. CD4+ T-lymphocyte counts were performed by flow cytometry using Cyto-stat tetraCHROME (Beckman coulter, Krefeld, Germany). Plasma HIV-1 RNA levels were measured using the Cobas TaqMan HIV-1 test (Roche Diagnostics, Mannheim, Germany).
Isolation of Mononuclear Cells and Culture
PBMCs were separated using Ficoll density gradient centrifugation as described before.19,20 PBMCs were cultured in 24-well plates at a concentration of 3 × 106 cells/mL using RPMI 1640 media (Biochrom, Berlin, Germany) supplemented with 10% FCS, 1 mM L-glutamine, 50 U/mL penicillin, 0.5 mM sodium pyruvate, and 50 μg/mL streptomycin. The cells were cultured for 16 hours and 25 ng/mL lipopolysaccharide (LPS, Sigma, Seelze, Germany) was used to stimulate slanDCs.
Analysis of Surface Markers and Intracellular Cytokine
Peripheral blood slanDCs were analyzed for surface expression of CD40, CD80, CD86, HLA-DR, and mTNF-α. To evaluate the intracellular IL-1β expression in monocytes, mDCs, pDCs, and slanDCs, PBMCs were cultured with 2 μg/mL Brefeldin A in the presence or absence of LPS. Slan (M-DC8)+ cells were gated from CD45+CD3−CD19− cells after exclusion of dead cells and doublets. The detailed gating strategy for mDCs, monocytes, pDCs, and slanDCs was as shown (see Fig. S1, Supplemental Digital Content, http://links.lww.com/QAI/A839). Expressions of surface receptors and intracellular cytokines were analyzed by flow cytometry using FACSCanto II and the following monoclonal antibodies were used: anti-CD3-BD Horizon V500, anti-CD16-PE, anti-CD45-PerCP, (all from BD Biosciences), anti-CD3-APC, anti-CD11c-APC-Cy7, anti-CD19-APC-Cy7/BV510, anti-CD40-APC-Cy7, anti-CD56-BV510/PE-Cy7, anti-CD80-BV421, anti-CD86-PE-Cy7, anti-CD95-FITC, anti-CD123-PE-Cy7, anti-CD178-PE, anti-HLA-DR-BV421 (all from Biolegend), anti-CD14-FITC, anti-slan (M-DC8)-APC/PE (both from Milteny), anti-IL-1β-PerCp, and anti-mTNF-α-PE (both from R&D Systems). As negative controls, fluorochrome-conjugated isotype-matched antibodies from the respective companies were included.
To measure IL-1β in culture supernatants, cytometric bead array flex set (BD Biosciences) was used according to the company's instruction and FCAP array software V.3 was used for data analysis.
Unpaired t-tests (to compare 2 groups) or 1-way analysis of variance followed by Bonferroni posttests (to compare three or more groups) were applied to evaluate the statistical significance of the observed differences. The Spearman test was used to perform correlation analysis. All statistical tests were performed using Graphpad prism V5 software (GraphPad inc).
High Frequency of slanDCs With Enhanced Activation in HIV-1 Infection
In this study, we first assessed the relative and absolute numbers of slanDCs in PBMCs of healthy donors, as well as HIV-1 patients, which included treated and untreated subjects. We observed a higher percentage of M-DC8+ slanDCs in the CD45+ PBMCs of untreated HIV-1–infected individuals compared with healthy donors (Fig. 1A). The increased frequency of slanDCs in untreated HIV-1–infected individuals again restored to the level of healthy controls after 1 year of ART treatment (Fig. 1B, Table S1, Supplemental Digital Content, http://links.lww.com/QAI/A839). The absolute counts of slanDCs were also higher in untreated subjects compared with healthy donors (Fig. 1C), which was also restored to the level of healthy controls after 1 year of ART (Fig. 1C). Moreover, slanDCs in HIV-1–infected patients presented more activity through upregulated surface expression of CD40, CD80, CD86, HLA-DR, and mTNF-α compared with healthy controls (Fig. 1D). The increased frequency and absolute numbers of slanDCs with higher immune activation in HIV-1–infected individuals led us to perform further analyses of slanDCs with respect to cytokine secretion.
Enhanced IL-1β Secretion by slanDCs in HIV-1 Infection
IL-1β is released by different cell types such as DCs, monocytes, and macrophages,14,15 which serve as immunoregulatory cells through secretion of several proinflammatory mediators in response to HIV-1 infection.13,16 To assess the role of different DC subsets in IL-1β production, freshly isolated PMBCs from untreated, treated, and healthy donors were stimulated with LPS for 16 hours. Compared with healthy donors, significantly more slanDCs in both treated and untreated HIV-1–infected patients produced IL-1β in response to LPS (Figs. 2A, B). Similarly, we have also measured significantly higher levels of IL-10, IL-12, and TNF-α expression in slanDCs from HIV-1–infected individuals compared with slanDCs from healthy controls (see Fig. S2, Supplemental Digital Content, http://links.lww.com/QAI/A839). A correlation analysis revealed that the proportion of IL-1β producing slanDCs were higher in patients with higher viral load (Fig. 2C). The absolute CD4+ T-cell counts show inverse correlation with IL-1β producing slanDCs, although it was not to the level of significance (data not shown). In addition, the proportion of IL-1β+ slanDCs were significantly higher in patients with activated slanDCs that expressed CD40 or mTNF-α (Figs. 2D, E). The MFI of slan (M-DC8) of slanDCs were directly correlated to the IL-1β expression of slanDCs in HIV-1–infected individuals (Fig. 2F). In healthy individuals, mDCs were as competent as slanDCs to produce IL-1β (Fig. 2G). In contrast, compared with mDCs, slanDCs from the same donors produced significantly higher levels of intracellular lL-1β in untreated HIV-1–infected individuals (Fig. 2H). After ART, mDCs significantly restored their ability to produce intracellular IL-1β, however, significantly less when compared with slanDCs (Fig. 2I). The percentage of intracellular IL-1β+ classical CD14+ monocytes after LPS stimulation was high and remained similar between HIV-1–infected individuals and healthy controls, whereas the proportion of IL-1β expressing CD16+ monocytes was higher in HIV-1–infected individuals compared with healthy controls (see Fig. S3, Supplemental Digital Content, http://links.lww.com/QAI/A839). Furthermore, significantly higher IL-1β levels were measured after 16 hours of LPS stimulation in PBMCs culture supernatants of untreated but not in treated HIV-1–infected individuals compared with healthy controls (Fig. 2J).
In summary, we identified higher absolute numbers and frequency of peripheral blood slanDCs in HIV-1–infected individuals that lead to enhanced secretion of IL-1β. In contrast, mDCs and pDCs did not contribute to IL-1β production in untreated HIV-1–infected patients.
During chronic HIV-1 infection, circulating plasmacytoid dendritic cell (pDC) and myeloid dendritic cell (mDC) numbers are reduced, and this has been well studied.21,22 SlanDCs, which represent a major subpopulation of proinflammatory blood monocytes, presented higher numbers and produced more TNF-α in response to LPS in patients with viremic, chronic HIV-1 infection.16 However, these cells have not been studied yet in terms of their potential to produce IL-1β during HIV-1 infection. Hence, we determined the relative numbers and absolute counts of slanDCs and their potential to produce IL-1β in peripheral blood of HIV-1–infected individuals. We could show that HIV-1–infected individuals display higher frequencies and absolute numbers of slanDCs compared with healthy controls, which again normalized after successful ART, indicating restoration by treatment. The observed increase of slanDCs number in HIV-1–infected viremic individuals may be due to a higher migration and recirculation rate from one organ to another, specific proliferation and differentiation, or resistance to apoptosis during HIV-1 infection. Immune activation is the hallmark of HIV-1 infection, therefore we could also detect activated slanDCs in HIV-1–infected individuals. Our finding corroborates the previously published report by Dutertre et al,16 where higher absolute numbers and frequency of nonclassical monocyte expressing CD16 have been reported.
IL-1β is known to evolve in chronic inflammation during HIV-1 infection, which subsequently leads to pyroptosis of CD4+ T cells.13 We determined a higher intracellular content of IL-1β in LPS stimulated slanDCs of HIV-1–infected individuals, revealing direct correlation with viral load, MFI of M-DC8 marker, and activation of slanDCs (Figs. 2C–F). To show the robustness of the correlation, we have included treated and untreated patients for our correlation analysis. The higher percentage of intracellular IL-1β+ slanDCs in treated patients might be due to the previous activation by microbial products translocating from gut-associated lymphoid tissue into the circulation and ongoing residual viral replication. In addition, absolute CD4+ T-cell counts were not showing significant correlation with the IL-1β+ slanDCs in our cohorts, this might be due to low CD4+ T-cell counts despite the suppressed viral load in some of the treated patients (see Table S1, Supplemental Digital Content, http://links.lww.com/QAI/A839). Patients whose slanDCs were producing higher amounts of intracellular IL-1β also produced higher intracellular TNF-α in response to LPS, corroborating the Dutertre et al study.16 After ART, the frequency of IL-1β producing slanDCs remained similar compared with untreated patients suggesting that immune restoration may require more time. Other DC subsets (mDCs and pDCs) were not active producers of IL-1β in untreated HIV-1 patients. In contrast, mDCs of healthy donors were as competent as slanDCs in producing IL-1β, whereas mDCs from ART-treated patients expressed lower levels of IL-1β compared with their counter slanDCs, indicating a partial immune restoration.
The increase of IL-1β in the PBMC culture supernatants of untreated HIV-1 patients may be due to increased numbers of slanDCs contributing to the higher amount of IL-1β. This might be the reason why a lower IL-1β concentration was detected in the culture supernatant from ART-treated patients who displayed less slanDC counts compared with untreated patients while exerting similar frequencies of IL-1β expressing slanDCs. In fact, in addition to slanDCs, our experiments suggested that monocytes might also contribute to the higher IL-1β concentration in PBMC culture supernatant of untreated HIV-1 patients. Elevated proinflamatory cytokines such as IL-1β and TNF-α were demonstrated in the serum of HIV-1 patients and blood monocytes were reported as the contributing cells.15,16,23 It has been reported that IL-1β plays a role in body defense by activating cells of both adaptive and innate immunity during chronic inflammatory disorders such as HIV-1 infection.10,12,13
In conclusion, we identified slanDCs as one of the main cellular IL-1β sources apart from monocytes in chronic HIV-1 infection especially during LPS stimulation, an in vitro reflection of the translocation of gut luminal Gram-negative bacterial products.24 Therefore, this adds another piece of knowledge for further understanding the mechanisms behind the pathogenesis of HIV-1 infection in vivo.
The authors would like to appreciate the cell sorting core facility, Hannover Medical School for their support. The Institute of Transfusion Medicine, Hannover Medical School has provided blood samples from healthy donors. The authors also thank the HIV outpatient clinic at Hannover Medical School for providing the samples from HIV-1 patients.
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