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Basic and Translational Science

CD11c+CD123Low Dendritic Cell Subset and the Triad TNF-α/IL-17A/IFN-γ Integrate Mucosal and Peripheral Cellular Responses in HIV Patients With High-Grade Anal Intraepithelial Neoplasia

A Systems Biology Approach

Guimarães, Adriana G. D. P. MD, PhD*,†,‡; da Costa, Allysson G. MS†,‡,§,‖; Martins-Filho, Olindo A. PhD; Pimentel, João P. D. MS§; Zauli, Danielle A. G. PhD; Peruhype-Magalhães, Vanessa PhD; Teixeira-Carvalho, Andréa PhD; Béla, Samantha R. PhD; Xavier, Marcelo A. P. MD, PhD#; Coelho-dos-Reis, Jordana G. PhD; Abranches, Josilene S. MS§; Guimarães, José J. P. MD, MS*; Malheiro, Adriana PhD§,‖; Ferreira, Luiz C. L. MD, PhD†,‡

Author Information
JAIDS Journal of Acquired Immune Deficiency Syndromes: February 1, 2015 - Volume 68 - Issue 2 - p 112-122
doi: 10.1097/QAI.0000000000000412

Abstract

INTRODUCTION

The incidence of anal cancer has increased over the past 25 years by around 50% in the general population with current annual incidence rates of between 0.8 and 1.8 cases per 100,000.1–7 Among HIV-infected adults, the incidence of anal cancer is about 30-fold higher than the general population.1 HIV coinfection with the oncogenic human papilloma virus (HPV) infection is a strong risk factor for anal squamous cell carcinoma,4 as well as its severe precursor lesion, the high-grade squamous intraepithelial lesions (HSIL). Among those, HPV-associated anogenital tumors occur at increasing rates in persons with HIV/AIDS.2,4

Cell-mediated immune responses seem to play an important role in controlling HPV/HIV-associated neoplasia, but lack of data in the anal mucosal milieu due to difficulties in sampling and handling mucosal tissue obscures the search for conclusive findings. Some reports have associated increased levels of Langerhans cells (CD1a+) and intraepithelial T lymphocytes in the anal mucosa of HPV-infected patients.8 However, CD1a+ cells and DC-SIGN+Dendritic cells+ (CD209+-DC+) are reduced in HIV/HPV-coinfected patients.9–11 In situ reduction of CD3+ lymphocytes and CD4+/CD8+ ratio may also be a risk factor for susceptibility to infection and malignancy in HIV-infected patients at risk of HPV infection.10,12,13

Dissemination and progressive growth of HPV-induced lesions may be related to escape from local cytokine-mediated surveillance.14,15 Type 1 cytokines such as interleukin (IL) 2 and IFN-γ stimulate and enhance cellular and humoral immunity. Patients with HPV-associated neoplasms presenting a type 1 cytokine profile have displayed a better clinical outcome compared with others exhibiting a type-2 cytokine profile.14,15 Other cytokines such as IL-6 and TNF-α are increased in HPV-infected patients, whereas higher IL-10 and IL-12 levels were found to predict HIV/HPV coinfection.15,16 Patients with HPV-related cervical SIL present increase in HPV-inespecific IFN-g+CD8+ T cells in the context of HIV/HPV-coinfection when compared with HPV-infected patients.14 Regarding the high-grade lesions, HIV/HPV-coinfected HSIL patients were associated with assembling of a suboptimal type 1 immune response, characterized by decreased IL-2, IFN-γ, and TNF-α levels.14,17 These findings suggest an important role of cytokines in the development/morbidity of intraepithelial lesions, and studying the interplay between the proinflammatory/regulatory cytokines secreted from immune cells residing at the anal mucosa site and from peripheric cells would allow for the rational selection of putative biomarkers of disease progression to anal intraepithelial neoplasia (AIN). Thus, in this study, a new systems biology approach was used to reveal potential immunological biomarkers in AIN that would improve the diagnosis of HPV/HIV-associated disease morbidity.

MATERIALS AND METHODS

Ethical Aspects

The study had approval by the Ethics Committee from the TMFAM (Protocol# 3319/2008). All patients signed an informed written consent form in accordance to the guidelines established by the 466/2012 resolution of the Brazilian National Health Council.

Study Population

Detailed information of the study population is described in supplemental material and methods (see Supplemental Digital Content, https://links.lww.com/QAI/A587). Supplemental table 1 (see Supplemental Digital Content, https://links.lww.com/QAI/A587) displays the details of the study population, which was categorized as follows: (1) group AIN(−)/HIV(−), (2) group AIN(−)/HIV(+), and (3) group AIN(+)/HIV(+). Group AIN(+) was subcategorized based on the histopathology analysis of anal mucosa and classified as low-grade squamous intraepithelial lesion (LSIL) or HSIL.

Immunophenotypic and Intracytoplasmic Cytokine Analysis

Immunophenotypic and intracytoplasmic cytokine staining of peripheral blood mononuclear cells (PBMC) and anal mucosa mononuclear cells was performed as described in supplemental material (see Supplemental Digital Content,https://links.lww.com/QAI/A587).

The FlowJo software version 9.4 was used for data analysis. For analysis of PBMC, the identification of total lymphocytes was performed, initially by a lymphocyte scatter gate set-up, using FSC versus SSC contour plot, as illustrated in Figure 1A. Monocyte analysis was performed by FITC anti-CD14 versus SSC contour plots gated on SSClowCD14high cells (Fig. 1B).

F1-2
FIGURE 1:
Representative flow cytometric analysis of mononuclear cell subsets. For analysis of peripheral blood mononuclear cells, the identification of total lymphocytes was performed by a lymphocyte scatter gate set-up, using FSC versus SSC pseudo-color plot (A). The selection of the monocyte subset was made after setting the gate on high CD14+ versus SSC pseudo-color plot (B). Peripheral blood dendritic cell subsets were detected using specific anti-CD11c versus anti-CD123 dual color contour plot (C). The nonoverlapping mDC and pDC were identified as CD11c+CD123 and CD11cCD123+ cells, respectively. The overlapping DC was characterized as CD11c+CD123Low DCs. For analysis of anal mucosa mononuclear cells, the identification of total lymphocytes was performed using anti-CD45 versus SSC contour plot (D). The selection of CD1a+ DC subset was done using anti-CD1a versus SSC contour plot (E). Analysis of anal mucosal Langheran DC subsets was performed using anti-CD209 contour plot (F).

Identification of the DC subsets was performed by immunophenotypic analysis using PE anti-human CD11c and FITC anti-human CD123 monoclonal antibodies. As shown in Figure 1C, the nonoverlapping myeloid DC (mDC) and plasmacytoid DC (pDC) subsets were identified as CD11c+ CD123 and CD123HighCD11c cells, respectively.19,20 The overlapping DC subset was characterized as CD11c+CD123Low cells.21,22 Subsequently, the selection of CD1a+ cells was done by anti-FITC-CD1a versus SSC contour plots. The CD4+ T cells were determined by anti-PerCP-CD3 versus anti-FITC-CD4 contour plots. All results were expressed as percentage of cells for each cell subset. After the selection of CD4+ T-cell subset and CD1a+ cells, the frequency of cytokine-producing cells was determined using quadrant statistics on PE anti-IFN-γ or APC anti-IL-10 contour plots.

Selective analysis of anal mucosa mononuclear cells was achieved by side scatter-SSC versus anti-CD45/anti-CD4 or anti-CD1a or anti-CD209. Identification of CD4+ T cells was performed initially by a lymphocyte scatter gate set-up, using SSC versus anti-PercP-CD45 (Fig. 1D). After that, intraepithelial CD4+ T cells were characterized within CD45high cells. Analysis of DC subsets was performed using SSC versus anti-FITC-CD1a (Fig. 1E) or anti-FITC-CD209 (Fig. 1F) contour plots.

Flow Cytometric Quantitative Analysis of Cytokines Secreted by Culture of PBMC and Anal Mucosal Cells

The cytokine levels of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ, and IL-17A were measured in the culture supernatant of PBMC and anal mucosal mononuclear cells from all patients by Cytometric Bead Array (BDM HU TH1, TH2, TH17 CBA Kit—code 560484; BD Biosciences, San Jose, CA). The cytokine levels were measured as per manufacturer's instructions and modified according to Peruhype-Magalhães et al.23 Data acquisition was performed by flow cytometry using an FACSCalibur flow cytometer (Becton Dickinson, LA Jolla, CA). The results were expressed as picograms per milliliter, as assessed by the standard curve.

Statistical Analysis

The frequency of cells and cytokine-producing PBMC subsets and the frequency of cytokine-producing anal mucosal mononuclear cell subsets were compared by nonparametric tests based on analysis of variance, Kruskal–Wallis test followed by Dunn multiple comparison test. The Graphpad Prism software version 5.0 (Graph-Pad Software, San Diego, CA) was used for data analysis. Statistically significant differences were considered if P < 0.05 and are highlighted by connecting lines. Cytokine signature analysis was performed as described previously18 and in the supplemental material (see Supplemental Digital Content, https://links.lww.com/QAI/A587).

Systems Biology Analysis

To model the complex interactions between the biomarkers evaluated in the study, networks were assembled by, first, assessing the association within the cytokines secreted in the culture supernatant of either PBMC with/and anal mucosal mononuclear cells for each clinical group. Spearman correlation test was performed to assess the association between the levels of each cytokine (picograms per milliliter) and the percentage of cell subsets tested. The positive and negative correlations were significant when the P < 0.05. The correlation index (r) was used to categorize the correlation strength as negative (r < 0), moderate (0.36 > r < 0.67), and strong (r > 0.68) and shown in Figure 5. The Graphpad Prism software version 5.0 was used for data analysis, and the Cytoscape (version 2.8) (Cytoscape Consortium, San Diego, CA) was used for computing the subsystems composed by networks of the biomolecules.24

RESULTS

Variations in the Frequency of Circulating IFN-γ+ or IL-10+ CD4+ T Cells, IFN-γ+ or IL-10+ CD1a+ Cells, CD11c+CD123Low, and pDC Are Associated With the HIV Infection Rather Than the Presence of AIN

Aiming to identify cell biomarkers associated with AIN, the analysis of phenotypic/functional features in the peripheral blood of groups AIN(−)/HIV(−), AIN(−)HIV(+), and AIN(+)HIV(+) were performed (Fig. 2). Data analysis demonstrated that despite the presence of AIN, all HIV(+) patients presented decreased percentage of circulating lymphocytes, mainly CD4+ T cells along with decreased frequency of CD11cCD123+-pDC and increased levels of circulating CD1a+ cells (Fig. 2A). Moreover, regardless of the presence of AIN, higher frequency of IFN-γ+ or IL-10+-CD4+ T cells and CD11c+CD123LowDCs as well as lower frequency of IFN-γ+ or IL-10+ CD1a+ cells was observed in HIV(+) patients with or without AIN (Fig. 2B).

F2-2
FIGURE 2:
Phenotypic and functional features of peripheral blood and anal mucosal mononuclear cells from AIN(−)HIV(−) ([INCREMENT]), AIN(−)HIV(+) ([Black up-pointing triangle]), and AIN(+)HIV(+) ([Black up-pointing triangle]) individuals. (A) Frequency of peripheral blood mononuclear cell subsets (lymphocytes, CD4+ T cells, CD14+ monocytes, CD1a+ cells, CD11c+CD123-mDC, CD11cCD123+-pDC, and -CD11c+CD123Low DC. (B) Intracellular cytokine profile (IFN-γ and IL-10) of circulating CD4+ T cells and CD1a+ cells. (C) Frequency of anal mucosal mononuclear cell subsets (CD45High lymphocytes, CD1a+-DC, and -CD209+-DC). The results are expressed as scattering plots of individual values, and median percentage of mononuclear cells represented as a line. Significant differences (P < 0.05) are highlighted by connecting lines.

Single Quantitative Alteration in Intraepithelial Lymphocyte and DC Subsets in Anal Mucosa of HIV-Infected Patients Are Unrelated to the Presence of AIN

We have characterized the phenotypic features of anal mucosal intraepithelial mononuclear cells of the groups: AIN(−)/HIV(−), AIN(−)HIV(+), and AIN(+)HIV(+) (Fig. 2C). Data analysis revealed an increased frequency of intraepithelial CD45High lymphocytes as well as increased frequency of CD1a+-DCs and CD209+-DCs in all HIV(+) patients regardless of the presence of AIN (Fig. 2C).

AIN Is Associated With Prominent Secretion of Proinflammatory/Regulatory Cytokines From Anal Mucosal Mononuclear Cells as Opposed to PBMC

Aiming to further characterize the immunological profile associated with AIN during HIV infection, we have assembled, for each clinical group, the biomarker signature with the frequency of high producers of each cytokine secreted in the supernatant of anal mucosa (m) and peripheral blood (pb) mononuclear cells (Fig. 3). Initially, the number of subjects with “High” cytokine levels was compiled in gray-scale diagrams to determine the frequency of High producers in each clinical group (Fig. 3A). Our data demonstrated that whereas AIN(−)HIV(−) subjects presented more relevant cytokine production by PBMC, AIN(−)HIV(+) presented a balanced contribution of both compartments (Fig. 3A). Moreover, AIN(+)HIV(+) group presented significantly increased cytokine production in the anal mucosa compartment (Fig. 3A).

F3-2
FIGURE 3:
In vitro cytokine secretion profile (IL-6, TNF, IL-2, IFN-γ, IL-4, IL-10, and IL-17A) of anal mucosa (m) and peripheral blood (pb) mononuclear cells from AIN(−)HIV(−), AIN(−)HIV(+), and AIN(+)HIV(+) individuals. (A) Black-and-white diagrams representing low (□) and high (▬) cytokine producers. Each lane represents a cytokine, and each block represents the pattern of cytokine production of each patient. The numbers bellow each lane represent the frequency of high producers of the cytokine tested. (B) Biomarker radar graphs summarize the percentage of high cytokine producers from each studied group. When the frequency of high producers was above the 50th percentile (in a scale of 0%–100%), the result was highlighted by underline and bold format in both diagrams and radar graphs. ND, not determined.

Comparative analysis was also performed after assembling radar graphs of each clinical group, aiming to identify the most relevant biomarkers. Data analysis showed that AIN(−)HIV(−) subjects showed an overall IL-10-modulated proinflammatory profile (pbIL-6, pbTNF-α, and pbIFN-γ) in peripheral blood with discrete cytokine production in anal mucosa (Fig. 3B). However, AIN(−)HIV(+) patients displayed a balanced proinflammatory/regulatory profile in anal mucosa (mIL-6, mIL-2, and mIL-10), and peripheral blood (pbIL-6, pbTNF-α, pbIL-2, pbIFN-γ, and pb-IL-17A) (Fig. 3B). Anal mucosal mononuclear cells from AIN(+)HIV(+) patients showed significant capacity to secrete proinflammatory/regulatory cytokines, specifically mTNF-α > mIL-4 > mIL-10 > mIL-6 = mIL-17A, with minor secretion (pbIL-2) from PBMC (Fig. 3B).

Higher Levels of Circulating CD11c+CD123Low and CD1a+ DCs Along With Elevated Levels of IFN-γ-Secreting CD4+ T Cells Are Major Features Associated With HSIL in AIN(+)HIV(+) Patients

Aiming to further characterize the localized and systemic immune response in AIN(+)HIV(+)patients, we have subcategorized these patients based on the histopathology analysis of anal mucosa, classified as LSIL and HSIL (Fig. 4).

F4-2
FIGURE 4:
Histopathology, phenotypic, and functional features associated with AIN. (A) Histopathology analysis of anal mucosa from AIN(+)HIV(+) patients defining LSIL and high-grade squamous intraepithelial lesion (HSIL). LSIL: anal transition zone with acanthosis, papillomatosis, and diffuse mononuclear inflammatory infiltrate. The highlight shows the presence of koilocytes, enlarged cells with a cytoplasmic halo, surrounding the nucleus that is indicative of active HPV replication. HSIL: squamous mucosa with disorders of maturation and polarity across its thickness and abnormal basaloid cells with an increased nuclear to cytoplasmic ratio. Left panel: hematoxylin and eosin ×20; right panel: hematoxylin and eosin ×63. (B) Phenotypic and functional features of peripheral blood (pb) and anal mucosa (m) mononuclear cells from LSIL+ and HSIL+ HIV(+) patients. The data are represented in bar charts as median percentage of mononuclear cells and interquartile range. Significant differences (P < 0.05) are highlighted by connecting lines. (C) Biomarker radar graphs were plotted with the percentage of high cytokine producers (scale 0%–100%) in LSIL and HSIL subgroups. Relevant changes are considered when the frequency of high producers is above the 50th percentile and highlighted by underline and bold format.

Histological data showed that LSIL displayed anal transition zone with acanthosis, papillomatosis, and diffuse mononuclear inflammatory infiltrate (Fig. 4A). The presence of koilocytes, enlarged cells with a cytoplasmic halo surrounding the nucleus, was indicative of active HPV replication (Fig. 4A, left panel highlight). HSIL showed squamous mucosa with disorders of maturation and polarity across its thickness and abnormal basaloid cells with an increased nuclear to cytoplasmic ratio (Fig. 4A, right panel).

Considering the relevance of the histopathological findings of AIN to the disease outcome, we have further analyzed the phenotypic and functional profile of anal mucosa mononuclear cells from patients with either LSIL or HSIL. Although HSIL+ patients showed lower levels of circulating and intraepithelial lymphocytes, these subjects presented similar levels of CD4+ T cells, but significantly higher levels of IFN-γ+CD4+ T cells in peripheral blood as compared with LSIL (Fig. 4B). However, HSIL+ patients showed higher levels of circulating CD11c+CD123Low and CD1a+ DCs, but lower frequency of IFN-γ or IL-10-secreting CD1a+ DCs in the peripheral blood as compared with LSIL (Fig. 4B).

Radar graphs displayed that mTNF-α and mIL-4 are AIN-related elements, whereas mIL-6, mIFN-γ, mIL-10, and mIL-17A are selective HSIL-related features. Also, enhanced frequency of pbIL-2 and pbIL-10 was observed in HSIL+ patients (Fig. 4C).

HIV(+) Patients Presented a Complex Biomarkers Network Rich in Negative Connections, Regardless of the Presence of AIN

HIV(+) patients presented some preserved interactions among biomarkers as illustrated for peripheral blood and anal mucosa by the triad TNF-α/IL-10/IL-6 (Fig. 5). However, regardless the presence of AIN, HIV(+) patients presented more complex and imbricated biomarker network rich in negative connections, mainly between peripheral blood cells and anal mucosal cells, as well as soluble cytokines, when compared with the reference HIV(−) controls (Figs. 5A, B). Noteworthy, AIN was associated with a relevant loss of connections between anal mucosal CD1a+ DCs with several biomarkers from the peripheral blood, such as IFN-γ+CD4+ cells and pbIL-2, as well as anal mucosa such as, mTNF-α and the CD1a+/CD209+/CD45+ triad (Fig. 5B). Mucosal IL-17A (mIL-17A) also lost negative and positive connections with pDCs (negative), IFN-γ+CD4+ cells (positive), and CD209+ DCs (negative). Interestingly, mIL-17A gained positive correlation with mIL-6 in HIV(+)AIN(+), which is absent in HIV(+)AIN(−) and kept the strong mIL-10/mTNF-α/mIL-6 triad seen in both groups (Fig. 5B). This strong mIL-10/mTNF-α/mIL-6 triad is seen in LSIL; however it is absent in HSIL indicating the regulatory axis of mucosal IL-10 and IL-6 is important to regulate the effects of mucosal TNF-α.

F5-2
FIGURE 5:
Systemic analysis of phenotypic and functional features of peripheral blood and anal mucosa of HIV(+) patients and controls. Biomarker networks were assembled in 4 subnets (each) using the correlations among the selected biomarkers within each clinical group [AIN(−)HIV(−)—reference biomarker network in blue shades (A); AIN(−)HIV(+) in orange shades and AIN(+)HIV(+) in dark mustard shades—HIV-related biomarker network (B); LSIL and HSIL—AIN-related biomarker network in red shades (C)]. Subnets of blood (left) and anal mucosa (right) were sided with each other with cellular (top) and cytokine (bottom) biomarkers from each of these compartments. Significant correlations were compiled as described in Material and Methods. The Spearman correlation between each pair of biomarkers is illustrated if P < 0.05 and is represented by lines. The strength of the correlation was given by “r” and is illustrated as negative (r < 0; - -), moderate (0.36 > r < 0.67; □), and strong (r > 0.68; ▬).

Regarding the degree of lesion, LSIL+ patients presented a broader set of negative connections between PBMC and anal mucosal cytokines. However, although HSIL+ patients presented more concise biomarker subnets, these were composed by stronger and positive interactions between peripheral blood and anal mucosa environments, exemplified by the triad of mIL-17A/mTNF-α/IFN-γ+CD4+cells. In HSIL+ patients, IFN-γ+CD4+cells and CD11c+CD123Low DC form a strong direct positive interaction, which was weak in LSIL+ patients. The pbMON/mIL-10 dyad stands alone without other connections in HSIL+ patients. The strong mIL-10/mTNF-α/mIL-6 triad seen in AIN(−) and LSIL+ patients is not formed in HSIL+ patients indicating that this regulatory subnet is not operating in the higher-grade lesion (Fig. 5C). Also, HSIL+ patients presented a complete loss of connections within the mucosal CD1a+/CD209+/CD45+ triad, which is observed in AIN(−) and partially seen in LSIL+ patients (Fig. 5C).

DISCUSSION

In the context of local and systemic immune deregulation, a systems biology approach allowed for a unique analysis in a wider perspective. The frequency of cell subsets taken alone may not vary during the process of deregulation of the immune system, but their function and interaction with the microenvironment may shift from 1 stage to another. The analysis of networks composed of the biomarkers tested, indicated that these might gain or loose either positive or negative connections with each other in different clinical settings of the AIN and may indicate the presence of HSIL, which may progress to anal cancer.

Our data regarding the anal mucosa mononuclear cells from AIN(+)HIV(+) patients with HPV infection showed a prominent capacity in producing proinflammatory/regulatory cytokines, mainly mTNF-α > IL-4 > IL-10 > IL-6 = IL-17A. Mucosal TNF-α/IFN-γ/IL-17A were selective HSIL-related biomarkers. Regardless the presence of AIN, HIV(+) patients presented a complex biomarker network rich in negative connections. However, HSIL+ patients display stronger positive links between peripheral blood and anal mucosa environments, exemplified by the triad mIL-17A/mTNF-α/pbIFN-γ+CD4+. Several studies have shown that the presence of high-grade cervical intraepithelial neoplasia in HIV/HPV-coinfected patients had high statistical significance and correlation with cytokine deregulation.17,25 Indeed, the lack of immune surveillance in tumors has been associated with increased tissue inflammatory cytokines (IL-12, IL-1, TNF-α, and IL-17),25–28 which was associated with poorer outcomes in some epithelial cancers, when accompanied by the presence of regulatory T cells in tumor specimens.13,29,30

Another consequence of HIV infection that predisposes to HPV-related carcinomas is the impairment of local immunity by reducing the function of cytotoxic and regulatory T cells, CD1a+, and DC-SIGN+ DCs in anal tissue.13,14,31 Particularly, CD1a+ and DC-SIGN+ DCs have a role in protecting anal tissue from malignancy.13 The interplay of these cells with leukocytes in the anal mucosa exemplified by the CD1a+/CD209+/CD45+ triad in AIN(−) and LSIL+ patients may be important to prevent the development of high-grade lesions.

Yaghoobi et al13 have observed a higher risk of recurrence of anal cancer and HSIL in patients with low CD4+ T cells; nonetheless, they suggested that the levels of circulating CD4+ T cells taken alone do not contribute for predicting HPV-associated neoplasia. Our data demonstrated that decreased frequency of circulating CD4+ T cells, pDC, and IFN-γ+ or IL-10+-producing CD1a+ DCs as well as increased percentage of CD1a+ cells, IFN-γ+ or IL-10+ CD4+ T cells, and CD11c+CD123Low found are associated to HIV infection rather than the presence of AIN.

Regarding the HIV infection, in agreement with the present results, several reports demonstrate alteration in the percentages of DC subsets in HIV-infected patients.31–36 Myeloid DCs (CD11c+CD123Low) exhibited increase during HIV infection and in HIV-infected patients with AIN, whereas pDCs had decreased levels in the peripheral blood of HIV-infected patients, which is in agreement with previous findings.32–37

During HIV infection, plasmacytoid DCs (CD123High) strongly suppress HIV replication in autologous CD4+ T cells through a mechanism involving IFN-α as well as other antiviral factors.38 On exposure to LPS, TNF-α, and IL-1β, HIV-1 gp120 was demonstrated to sensitize cultured DC and blood DC for apoptosis. Binding of HIV-1 gp120 to DC-SIGN was shown to promote ASK-1-dependent activation–induced apoptosis of human DCs.36,37 This could be a mechanism by which HIV escapes from its surveillance in vivo. An alternative hypothesis to explain the lower percentage of pDCs in the peripheral blood of HIV-infected patients could be that pDCs with a partially activated phenotype accumulate in lymphoid tissue during asymptomatic chronic HIV-1 infection.35 pDCs display outstanding capacity on producing IFN type I and have therefore robust antiviral activity, which could drive these cells to the mucosal and lymphoid tissues on inflammatory signals.35,36,39 The decreased percentage of pDCs in the peripheral blood of HIV-infected patients with AIN also could be related to their migration to the local inflammatory site to attempt resolving the mucosal infection.

Regarding the DC myeloid compartment, it has been shown that HIV-1 infection induces the production of endogenous lipids required for effective viral production. Previous studies have demonstrated that CD1c and CD1d molecules are downregulated by HIV-1 in a Vpu-dependent and Nef-independent manner.40 Both immature and mature myeloid DCs optimally present lipid antigens making CD11c+CD123lowDCs and CD1a+DCs possible lipid presenting cells in the anal mucosa. However, the percentages of IFN-γ+ and IL-10+CD1a+DCs are decreased in HSIL+ patients, which may suggest an immature profile of CD1a+DCs. In this regard, peripheral tolerance mediated by immature DCs could take place by inducing the proliferation of regulatory T cells or inducing T-cell anergy.41,42 Therefore, a putative hypothesis to explain the altered circulating DC profile is the compensatory mechanism by which the host tries to recover lipid presentation systemically to halt tumor growth. However, expansion of immature DCs during this process may induce peripheral tolerance and regulate IFN-γ+CD4+ T-cell function. The increased frequency of these cells may be due to accumulation rather than proliferation considering that these cells are not able to clear infection or control the tumor growth. In this context, the increase in mucosal IL-17A/TNF-α/IFN-γ could be an outcome of the Th1/Th17 cell migration to mucosa, contributing to the inflammatory process without controlling tumor growth. More studies need to be performed to understand the interplay between the CD1a+ and CD11c+CD123lowDCs with the remaining CD4+ T cells as well as Th1 and Th17 balance in mucosal tissue in vivo.

Moreover, the increased frequency of intraepithelial CD45+ lymphocytes, CD1a+ DCs, and CD209+-DCs were also not associated with the presence of AIN. Higher levels of circulating CD11c+CD123Low cells and CD1a+ DCs along with elevated levels of IFN-γ+CD4+ T cells are associated with HSIL in AIN(+)HIV(+)patients. CD1a+ cells were found in the stratified epithelium of patients with AIN arranged in an interconnected network at the suprabasal layer of the anal mucosa and exhibited the characteristics of dendritic cells.11

However, analysis of biomarker networks shows that CD1a+ DCs loose either positive or negative connections with other subsets of cells when we compare LSIL patients with HSIL AIN(+)HIV(+)patients. These results are in agreement with previous study that demonstrates that depletion of CD1a+ cells in the anal mucosa is likely to contribute to the risk of HPV-related anal cancer in HIV-infected patients.13 In previous study of our group, we demonstrated that an elevation in the percentage of CD1a+ cells and CD209+-DCs reflects a distinct role of these cells in relation to protection and vulnerability of the patients to infections and tumors.9 In the biomarker network analysis, the strong dyad CD11c+CD123Low DCs and IFN-γ+CD4+ formed in HSIL+ patients may represent an ineffective attempt to control virus-induced lesion. Our findings suggest that a quantitative and/or qualitative disturbance of DC subsets might potentially interfere with the immune surveillance of HIV/HPV-associated neoplastic lesions.

The analysis of the interactions among biomarkers revealed essential connections among subsets of DCs and cytokines. Considering the novel strategies for controlling HIV infection through dendritic cells and the importance of these interactions on them, it is important to take these findings into account when designing and testing the efficacy of these therapeutic/immunoprophylactic approaches in the mucosal context. It is important to consider, however, that future studies with proper validation of the immunological subsets as well as with their implementation for monitoring mucosal tissue in the clinical setting are still needed to support their predictive value, specificity, and applicability as putative biomarkers of progression of AIN in HIV patients.

ACKNOWLEDGMENTS

The authors thank Fundação de Amparo à Pesquisa do Amazonas (FAPEAM), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), FIOCRUZ, and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) for financial support. The authors also thank PDTIS-FIOCRUZ for the use of its facilities. A.T.-C., O.A.M.F., and A.M. thank CNPq for fellowships (PQ).

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Keywords:

immunological biomarkers; HIV; HIV/HPV coinfection; AIN

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