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Keywords:

  • CD163;
  • IL-10;
  • Leprosy;
  • Macrophages

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References

Lepromatous macrophages possess a regulatory phenotype that contributes to the immunosuppression observed in leprosy. CD163, a scavenger receptor that recognizes hemoglobin–haptoglobin complexes, is expressed at higher levels in lepromatous cells, although its functional role in leprosy is not yet established. We herein demonstrate that human lepromatous lesions are microenvironments rich in IDO+CD163+. Cells isolated from these lesions were CD68+IDO+CD163+ while higher levels of sCD163 in lepromatous sera positively correlated with IL-10 levels and IDO activity. Different Myco-bacterium leprae (ML) concentrations in healthy monocytes likewise revealed a positive correlation between increased concentrations of the mycobacteria and IDO, CD209, and CD163 expression. The regulatory phenotype in ML-stimulated monocytes was accompanied by increased TNF, IL-10, and TGF-β levels whereas IL-10 blockade reduced ML-induced CD163 expression. The CD163 blockade reduced ML uptake in human monocytes. ML uptake was higher in HEK293 cells transfected with the cDNA for CD163 than in untransfected cells. Simultaneously, increased CD163 expression in lepromatous cells seemed to be dependent on ML uptake, and contributed to augmented iron storage in lepromatous macrophages. Altogether, these results suggest that ML-induced CD163 expression modulates the host cell phenotype to create a favorable environment for myco-bacterial entry and survival.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References

Leprosy is an infectious disease caused by Mycobacterium leprae (ML) in which susceptibility to the mycobacteria and its clinical manifestations are attributed to the host immune response. The clinical and immunological patterns of this unique chronic infectious disease clearly demonstrate a continuous scale of changes in histological lesions. Disease classification is defined within two poles (tuberculoid to lepromatous) with transitions between these clinical forms. While typical epithelioid macrophages predominate at the paucibacillary tuberculoid pole of the disease, inactivated foamy macrophages predominate at the lepromatous end [1].

In lepromatous leprosy (LL), the lack of systemic inflammatory signals and corresponding local ones strongly indicates that a complex anti-inflammatory network is at work. In this regard, neuroendocrine system involvement, in conjunction with the existence of multiple suppressive pathways under the control of the innate and adaptive immune response, has been reported [2-7].

We have suggested that IDO may play a role in a hitherto unknown suppressive mechanism in leprosy [6]. It has also been reported that accumulated oxidized host phospholipids in lepromatous macrophages downregulate the innate immune response [8]. Foamy macrophages seem to sustain intracellular mycobacteria in a physiological state similar to a nonreplicating vegetative one [9]. In this context, Montoya et al. [10] demonstrated that lepromatous macrophages exhibit a high expression of the cysteine-rich superfamily scavenger receptor (SRCR), which increases the phagocytic capacity of macrophages and leads to a reduction in bactericidal activity.

CD163, a receptor only expressed in monocytes and macrophages, is a member of the class B SRCR superfamily with immunomodulatory properties. Likewise, CD163 is a receptor of hemoglobin (Hb) and hemoglobin–haptoglobin (Hp, Hb–Hp) complexes. The metabolites resulting from intracellular Hb degradation exhibit potent antioxidative and anti-inflammatory effects. It has been described that the binding of Hb to CD163 induces the release of IL-10 and other anti-inflammatory mediators from macrophages in vivo [11]. It has also been demonstrated that IL-10 enhances CD163 expression by creating a feedback arm of regulation [12, 13] and that the CD163 levels in plasma inversely correlate with the expression of CD163 in blood monocytes [14]. In addition, increased CD163 shedding seems to be associated with the immunosuppressive control of inflammation [15]. The role of CD163 as a bacterial sensor has also been proposed, raising the possibility that a different extracellular domain in this receptor is responsible for triggering proinflammatory cytokines, in contrast to what has been considered its traditional endocytic role [16].

Recent reports have demonstrated ongoing interaction between CD163 and IDO in bone marrow-derived dendritic cells (BMDCs), perhaps indicating that different CD163 signals lead to IDO expression [17]. In an oligonucleotide array gene analysis in human macrophages subsequent to treatment with commercial Hb, a gene expression pattern with increased TNF and IDO expression was revealed [18].

A number of authors have recently attempted to classify the different subsets of monocyte-derived cells by exploring their functional and phenotypical characteristics [19]. Among the differential markers, macrophage polarization dictates iron handling by “inflammatory” and “alternatively active” macrophages, the latter showing larger intracellular labile iron deposits in association with high CD163 expression [20]. The presence of intracellular iron deposits has been documented in the foamy macrophages present in atherosclerotic lesions also in conjunction with high CD163 expression [21].

In summary, the present study describes a predominant subset of macrophages in lepromatous lesions exhibiting high expressions of CD163 and IDO connected to foamy aspects and iron deposits. Furthermore, ML was able to increase CD163 expression in human monocytes, making it likely that this scavenger receptor is involved in mycobacterium uptake and survival. These data support the idea that IDO and CD163 are the main mediators in the regulation of ML infection in lepromatous macrophages. Our study also demonstrates that these systems cooperate in consort with other cell systems in a double-edge, exchangeable manner to generate an anti-inflammatory microenvironment favoring mycobacterium persistence and survival.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References

Lepromatous macrophages express higher protein and CD163 mRNA levels parallel with IDO protein

To investigate the possibility of characterizing an in vivo subset of macrophages in LL lesions, we stained six LL skin biopsies with anti-CD163 and anti-IDO antibodies and compared them with six BT (Borderline Tuberculoid) skin biopsies. In BT skin lesions, lower numbers of CD163+ and IDO+ cells (0 to 20% of cells) were distributed within inflammatory infiltrates compared with the LL skin lesions in which higher numbers of cells were CD163+ and IDO+ (Fig. 1A; 50% and >50% of cells; p = 0.02 and p = 0.01). Double immunofluorescence showed that 40% of IDO+ cells also expressed CD163 (Fig. 1B). To validate increased CD163 protein expression, we obtained protein extracts from four LL and four BT skin lesions and submitted these samples to a SDS page under denaturation conditions. As demonstrated in Figure 1C, there was a significant difference in the CD163 protein levels in LL lesion extracts when compared with BT extracts. As previously demonstrated by De Souza Sales et al. [6] IDO expression was higher in LL lesion extracts in comparison to BT ones when evaluated by both mono- and polyclonal antibodies (Supporting Information Fig. 1).

image

Figure 1. Expression of CD163 and IDO in leprosy skin lesions. (A) CD163 (top) and IDO (bottom) expression was evaluated in borderline tuberculoid (BT) and lepromatous (LL) skin lesions by immunohistochemical staining. Arrows indicate marker-positive cells. Scale bar = 100 μm. Data shown are representative of six sections from BT and six sections from LL evaluated and are representative of two experiments performed. (B) CD163 and IDO coexpression in LL skin lesions was determined by double immunofluorescence. CD163 (green, Alexa Fluor 488®), IDO (red, Alexa Fluor 568®), and DAPI (nuclei). Images were visualized and obtained by a laser confocal microscope (Zeiss). Scale bars = 100 μm. Data shown are representative of four samples. (C) The expression of CD163 in skin biopsies was evaluated by immunoblotting. A densitometry analysis of each blot was performed and is shown as a CD163/tubulin ratio. Results are expressed as mean ± SE (n = 4). (D) Biopsies were obtained from LL (n = 5) and BT (n = 5) lesions. Real-time PCR was performed to measure the mRNA expression of CD163, IDO, and IL-10. Results are shown as the means ± SE of five independent experiments. An ANOVA test was used for statistical analysis, *p < 0.05.

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CD163 mRNA levels were significantly higher in LL as compared with BT lesions (0.54 ± 0.24 in LL versus 0.08 ± 0.025 in BT, p < 0.05). With respect to IDO mRNA, no significant difference between the two groups was observed ([6]; Fig. 1D). To verify if CD163 mRNA expression correlated with IL-10 expression, IL-10 mRNA levels were evaluated in the same skin lesions. As demonstrated in Fig. 1D, IL-10 mRNA was significantly higher in LL lesions (0.50 ± 0.12 in LL versus 0.07 ± 0.02 in BT, p < 0.05) in addition to correlating with CD163 and IDO.

Isolated cells from LL skin lesions were evaluated by flow cytometry to identify their phenotype and placed in culture. Flow cytometry revealed that after 24 h of culture, 41.74 ± 0.17% of the isolated cells were CD163+ (n = 6). Analysis of other cell markers revealed that these same cells also expressed CD209 (56.22 ± 0.66%, n = 4), HLA-DR (81.42 ± 0.94%, n = 5), and IDO (40.01 ± 2.50%, n = 3) (Fig. 2A). As observed by confocal microscopy, almost all cells were CD68+ (data not shown), confirming a macrophage phenotype. In addition, most of the cells were CD163+ while some coexpressed with IDO after 6 days of culture (Fig. 2B).

image

Figure 2. Phenotype of isolated LL skin lesion cells. (A) Isolated cells derived from LL skin lesions were marked with the antibodies: CD163-allophycocyanin (n = 6), HLA-DR-PE (n = 5), CD209-FITC (n = 4), and IDO-PE (n = 3), and analyzed by flow cytometry. A representative overlay is demonstrated. The black line histogram represents the negative control and the red line histogram represents the percentage of positive cells for each marker. (B) Double immunofluorescence for CD163 (red, Alexa Fluor 568®) and IDO (green, Alexa Fluor 488®) expression (arrows, see insert) in isolated cells from LL skin lesions after 6 days of culture. The results shown are representative of a single experiment with three replicates/cells. Images were visualized and obtained by Zeiss Colibri fluorescence microscope. Scale bars = 100 μm.

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High levels of CD163 were detected in the blood of lepromatous patients

Increased levels of CD163 in the sera of LL patients were observed in comparison with what was ascertained in the sera of healthy controls (HC) (6017.0 ± 593.9 in LL versus 1435.0 ± 129.6 in HC, p < 0.001) and BT (6017.0 ± 593.9 in LL versus 2150.0 ± 112.1 in BT, p < 0.001) (Fig. 3A). Interestingly, the higher levels of sCD163 correlated with our recent report of higher IDO activity in LL patient sera [6].

image

Figure 3. CD163 expression in serum samples from leprosy patients. (A) sCD163 levels were verified in HC (n = 10), LL (n = 10), and BT (n = 10) sera by ELISA. (B) IL-10 levels were verified in HC (n = 15), LL (n = 22), and BT (n = 23) sera by ELISA. Data are shown as mean + SE of two replicates of the indicated number of samples. An ANOVA test was performed, ***p < 0.001 and **p < 0.01.

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IL-10 levels in sera were also examined (Fig. 3B). The data confirmed previous reports showing higher levels of IL-10 in LL sera in comparison with BT and HC sera (36.08 ± 11.80 in LL versus 3.88 ± 1.27 in HC, p < 0.01; 36.08 ± 11.80 in LL versus 9.48 ± 4.93 in BT, p < 0.01).

ML induces CD163 expression in human monocytes through an IL-10-dependent pathway

We evaluated the ability of pathogenic mycobacteria such as ML and M. bovis BCG to induce CD163 and compared them to another pathogenic species Eschericia coli. ML (5: 1)-induced high CD163 expression in human monocytic culture (ML = 5.07 ± 2.32 versus the nonstimulated (n.s.) = 0.69 ± 0.38, p < 0.05), in contrast to BCG and E. coli, which did not (data not shown).

Both dead and live ML were able to induce increased expressions of CD163, IDO, and CD209 in human monocytes (Fig. 4A and B), which were accompanied by an uptick in TNF (46.91 ± 10.44 in nonstimulated versus 206.8 ± 21.78 in ML-stimulated, p < 0.01), TGF-β (71.3 ± 12.9 in nonstimulated versus 1093 ± 386.5 in ML-stimulated, p < 0.01), and IL-10 (154.4 ± 71.34 in nonstimulated versus 571.5 ± 199.5 in ML-stimulated, p < 0.05) in ML (MOI 10:1)-stimulated cultures (Fig. 4B). As explained in our previous report, IDO expression observed by increased ML MOI was met by an increase in IDO activity and a decrease in nitrate levels in cell supernatants [6].

image

Figure 4. CD163 induction by ML is partially dependent on IL-10. Adherent monocytes from HCs were stimulated or not with live or dead ML at the indicated MOIs for 24 h. The percentages of positive cells in (A) dead ML-stimulated cells or (B) live ML-stimulated cells are shown as mean ± SE (n = 5) by flow cytometry. (C) Cytokine profiles were measured by ELISA in dead ML-stimulated cultures (MOI 10). Results are shown as mean ± SE of five independent experiments performed in duplicate. (D) Monocytes from HCs stimulated with ML (MOI 5) were incubated in the presence of neutralizing IL-10 (anti-IL-10) and its isotype (IgG1) for 24 h. The percent CD163+ cells was determined by flow cytometry and expressed as mean ± SE (n = 3) and are representative of three experiments performed in triplicate. (E) Monocytes were cultured with ML (5: 1) in the presence or absence of neutralizing IL-10 (1 μg/mL) or isotype control. Alternatively, rIL-10 (10 ng/mL) was added to the cultures, in the presence or absence of ML; and IDO activity (Kyn/Trp) was evaluated in supernatants after 24 h of culture. Data are shown as mean ± SE (n = 10) and are pooled of five experiments performed. An ANOVA test was performed and used for statistical analysis, ***p < 0.001 and *p < 0.05.

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We attempted to clarify whether ML interference in IL-10 production positively regulates CD163. It was verified that the blockade of IL-10 reduced ML-induced CD163 expression (7.60 ± 1.93 in ML versus 1.53 ± 0.60 in ML + neutralizing IL-10, p < 0.05) (Fig. 4D), suggesting that ML-induced IL-10 is capable of upregulating CD163 expression in human monocytes. It was also shown that in ML-stimulated cultures, the IL-10 blockade reduced IDO activity, evaluated via the Kyn/Trp ratio (Fig. 4E). In the presence or not of ML, recombinant IL-10 (rIL-10) was able to increase IDO activity in comparison with unstimulated cultures. These data infer that ML is able to activate a positive feedback loop enrolling both IL-10 and CD163. Since IDO activity in human monocytes is known to increase as a result of ML exposure [6], it can be speculated that, in LL, the regulatory adaptive immune response is induced by innate IL-10, CD163, and IDO-mediated pathways.

The increased uptake of ML by monocytes is partially inhibited by anti-CD163

The effect of the phagocytosis pathway blockade on CD163 expression was investigated by testing whether inert beads were able to induce CD163 expression but, in this scenario, no effect was observed (data not shown). To verify whether live (MOI 5: 1) or dead (MOI 5: 1) ML colocalizes with CD163 in human monocytes, flow cytometry analysis was performed to ascertain the percentage of double-positive CD163 — ML cells. Although no statistical difference could be found, live mycobacteria colocalized more closely with CD163 (32.71 ± 9.04%) than dead ML (17.75 ± 1.47%) (Fig. 5A).

image

Figure 5. CD163 may function as a coreceptor for ML entry into monocytes. (A) Adherent monocytes were stimulated or not with live or dead ML. The percentage of ML+ (labeled with PKH26) CD163+ cells was evaluated by flow cytometry (n = 3). (B) Adherent monocytes were pretreated with 1 μM of cytochalasin B (n = 3) or not for 30 min prior to ML infection. After 24 h, the percentage of CD163+ cells was evaluated by flow cytometry. (C) Monocytes were pretreated for 30 min with neutralizing CD163 antibody RM3/1 (20 μg/mL) or isotype (IgG1) and infected with PKH67-labeled live ML for 2, 16, and 24 h. The percentage of cells bearing fluorescent bacteria (ML-labeling green) was measured by flow cytometry. (A–C) Data are shown as mean ± SE of three replicates and are representative of three experiments performed. (D) Immunofluorescent analysis of monocytic cell cultures labeled with PKH67 (green) treated as described in (C). The images illustrate cells internalized or not with PKH26-labeled ML (ML-labeling red) in these different treatments. Nuclei were stained with DAPI. Scale bar=20 μm. Data shown are representative of six experiments performed. (E) CD163-transfected HEK293 cells and control HEK293 cells were stained with anti-CD163 or an isotype control and analyzed by flow cytometry. Data are shown as mean ± SE of the percentage of CD163 positive cells. (F) Flow cytometric analysis of ML uptake expressed as MFI after 16 h of infection in both transfected and untransfected cells and shown as mean ± SE of three replicates and representative of five experiments performed. All experiments were performed using a MOI of 5. A Kruskal–Wallis test/Dunn's Multiple Comparison Test was performed and used for statistical analysis; *p < 0.05, **p < 0.01, and ***p < 0.001.

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Via flow cytometry, it was verified whether the addition of cytochalasin B (cyt B) could modify the expression of CD163 on the monocytic surface. Figure 5B shows that Cyt B decreased ML-induced CD163 expression, inferring that bacterial phagocytosis is an important mechanism involved in CD163 induction. Accordingly, it was then evaluated if a CD163 blockade could in any way affect mycobacterium uptake.

As detected by flow cytometric analysis, CD163-neutralizing antibody decreased ML internalization by monocytes in both early (2 h) and later (16 and 24 h) incubation times as compared to isotype pretreated (Fig. 5C and D) and nontreated (Fig. 5D) monocytes. Time course experiments showed that ML phagocytosis occurs in a similar manner (about 50% of infections) in nonpretreated and isotype-pretreated cells at the times analyzed. However, the bacterial association process in anti-CD163-preteated cells was more expressive in the shortest time slot (from 100% in ML + isotype versus 20.49 ± 3.250% in ML + neutralizing CD163 at 2 h, p < 0.0001) when compared with the later times (from 100% in ML + isotypee versus 62.27 ± 5.159% in ML + neutralizing CD163 at 16 h, p < 0.0001; and 45.31 ± 1.25% in ML + isotype versus 67.72 ± 1.13% in ML + neutralizing CD163 at 24 h, p < 0.01).

Additional assays were performed to confirm that the neutralization of CD163 affects ML internalization and not bacterial association alone. These results showed that neutralization with anti-CD163 blocked both bacterial adhesion and phagocytosis, indicating that the internalization process was more severely affected by this treatment than was bacterial binding (∼80% of inhibition of ML association and ∼88% of inhibition of ML internalization at 2 h; ∼40% of inhibition of ML association and ∼62% of inhibition of ML internalization at 16 h). In addition, HEK293 CD163 transfected cells were tested for their capacity to internalize mycobacteria. First, in order to determine the increase in CD163 expression in these transfected cells under the experimental conditions at hand, the percentage of CD163 using CD163 mAb EDHu-1 and isotype control was checked. The dramatic increase in CD163 expression in HEK293 CD163-transfected cells in contrast to the untransfected cells (Fig. 5E) was reflected in a significantly higher ML uptake/internalization increase (Fig. 5F). No major difference in the percentage of infected cells was found in comparison with the transfected and untransfected HEK293 cells either 2 or 16 h postinfection. However, ML association (not shown) and uptake (Fig. 5F) were more efficient in CD163-transfected cells than untransfected cells after 16 h of culture (9807 ± 235 ML MIF in untransfected cells versus 22811 ± 1724, p < 0.001). As a whole, these data strongly suggest that CD163 functions as an alternative receptor for ML entry into host cells.

Foamy macrophages loaded with bacilli show iron deposits

To verify if CD163 is involved in iron uptake by LL cells, AFB-negative BT skin lesions (n = 6) and LL skin samples (n = 9) showing bacteriological index > 5 (Wade staining, Fig. 6A) were submitted to Perls’ Prussian blue reaction. Positive iron deposits were detected intracellularly in foamy, bacilli-loaded macrophages (Fig. 6B). In BT samples, epithelioid macrophages occupying the core of the typical tuberculoid granuloma stained completely negative (Fig. 6C). Small foci of iron deposits in vaguely differentiated macrophages were seen in BT lesions.

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Figure 6. Foamy macrophages loaded with bacilli show iron deposits. (A) Alcohol-acid bacilli (Purple) were stained in LL skin lesions with the Wade method (B) Iron deposits (Blue) were stained in LL and BT skin lesions (C, and insert) with Perls’ Prussian blue reaction. The images are representative of nine LL and six BT samples. Scale bars = 20 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References

In this study, past descriptions that foamy macrophages predominate in LL lesions among a plethora of other macrophages were all but confirmed. Immunohistochemical analysis of polar LL lesions demonstrated that the majority of these cells were positive for CD68, CD163, and IDO. Interestingly, after 6 days of culture, CD68+CD163+IDO+ markers were identified in cells isolated from LL lesions, suggesting that a part of these cell populations maintains the same phenotype while simultaneously discarding their intracellular bacilli and foamy appearance.

In vitro studies have demonstrated that ML provides both positive and negative regulatory signals even when TCRs are the trigger stimuli [22]. Although live ML seems to be more efficient at inducing ML phagocytosis, heat-killed ML is more effective at inducing T-cell activation [23]. Moreover, we herein describe that CD163 scavenger receptor type 2 is induced by both live and dead ML. The increased CD163 expression triggered by ML positively correlated with IDO and CD209 expression.

The role of CD163 as a bacterial receptor was first described by Fabriek et al. [16], who considered that bacterial and cellular recognition constitutes unifying and perhaps even primordial functions of the scavenger domain as well. Both the CD163 blockade and the cythocalasin B treatment were found to inhibit ML uptake by human monocytes, leading to the conjecture that CD163 contributes to ML entry into host cells and that CD163 activity is regulated by the phagocytic machinery. This hypothesis is reinforced by the neutralization of this receptor implicated in the binding and uptake of the bacteria. In addition, ML uptake was more effective in CD163-transfected HEK293 cells, thus reinforcing its role as a mycobacterial receptor.

Previous reports have demonstrated that the shedding of CD163 increases proinflammatory cytokines [24]. Our observation showed that ML was not able to induce a significant elevation in CD163 shedding in monocytic cultures but that, after 24 h of culture, ML augmented both proinflammatory (TNF) and anti-inflammatory (IL-10 and TGF-β) cytokines in HC monocytes.

CD163 has been identified as a soluble protein in cell culture supernatants and in human plasma [25]. Soluble CD163 is released from monocytic cells in response to TLR signaling as an acute innate immune response to extracellular pathogen infections [26]. Previous studies have shown that CD163 plasma levels inversely correlate with the expression of CD163 in blood monocytes, which, under some pathophysiological conditions, are a major source of sCD163 [14]. In the same vein, higher levels of sCD163 were detected in LL patient sera, suggesting that the source of sCD163 may not be blood monocytes alone, but resident tissue macrophages as well. Besides, the increase in sCD163 in LL sera correlated positively with IL-10, TNF levels, and IDO activity.

Analysis of gene expression demonstrated that CD163 mRNA was higher in LL skin biopsies in contrast to BT ones. IL-10 mRNA obtained from isolated LL macrophages also increased in these cells. Sulahian and colleagues [12, 27] have demonstrated that IL-10 directly elevates CD163 mRNA. Since previous work has described the role of IL-10 in LL pathogenesis [10], we suggest that this cytokine is responsible for the maintenance of the heightened levels of CD163 in LL cells. It has also been shown that the IL-10 induction of scavenger and opsoninic receptors may facilitate antigen loading and initiate antigen presentation and adaptive immune responses to the infectious agent [28].

The link between IDO and CD163 expression in LL cells is not yet clearly understood. It has been previously shown that IFN-γ, which induces IDO, raises the activity of glycogen synthase kinase-3 in correlation with the inhibition of the AP-1- mediated DNA binding, an important transcription factor involved in IL-10 gene induction [29]. Furthermore, it has been seen that IFN-γ also suppresses CD163 expression [12, 30]. Based on these findings, we hypothesize that IDO induction in LL cells occurs via an IFN-γ-independent pathway, is mediated by IL-10, and is part of a dual mechanism involving a microbicidal axis. However, that TGF-β or TNF may play an important role in the induction of IDO in ML-stimulated monocytes cannot be excluded. For example, it has recently been reported that IDO was involved in TGF-β-stimulated cells in the intracellular signaling events responsible for the self-amplification and maintenance of a stable regulatory phenotype, which is independent of enzymatic activity, in plasmocytoid DCs [31]. Reports have also shown that Hb-triggering CD163 can heighten gene and IDO expression in macrophages and DCs [17, 18].

Iron homeostasis is essential to the sustenance of survival and growth of host mycobacteria [32]. Both ML and M. tuberculosis produce bacterioferritins [33, 34], which could be involved in controlling iron homeostasis in these pathogens. Because CD163 is related to Hb clearance, it can be speculated that, in parasitized cells, high CD163 expression may function as a pathway for the supply of iron, which perhaps reflect some of the dissimilarities among the survival mechanisms used by the various mycobacteria. An example is the fact that whereas human Hb is not used as an iron source by M. tuberculosis, it may be used for this purpose by M. haemophilum and ML [35]. In the present work, we verified larger iron storage in LL skin biopsies than in tuberculoid ones. Of note, high amounts of iron were only found in LL macrophages and none was detected in epithelioid macrophages whereas small foci of iron deposits in vaguely differentiated macrophages were seen in BT lesions. With reference to a previous description of the accumulation of lipid droplets in LL lesions [36], we could infer that ML associates with lipid vesicles as a mechanism for transferring iron from the host to ML-rich phagosomes.

As a whole, our results seem to clearly suggest that, on the one hand, CD163 may contribute to polarize LL macrophages to an anti-inflammatory phenotype by increasing the expression and levels of the immunoregulatory molecules IL-10 and IDO, although the other primary determinants of polarity in leprosy immune responses need to be better understood. In addition CD163 also contributes to ML uptake and increased amounts of iron, thus favoring bacterial survival and persistence.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References

Patients and clinical specimens

The acquisition of all specimens was approved by the Human Ethics Committee of the Oswaldo Cruz Foundation in Brazil. Leprosy patients (LL, n = 11 and BT, n = 10) were classified according to Ridley and Jopling criteria [37].

Buffy coats were obtained from healthy donors (HC) at the Hemotherapy Service of the Clementino Fraga Filho University Hospital, associated with the Federal University of Rio de Janeiro, RJ, Brazil, in accordance with the guidelines set down in the Declaration of Helsinki.

Immunohistochemical and immunofluorescent staining

The leprosy skin cryostat sections (LL, n = 6 and BT, n = 6) were processed to detect IDO+ and CD163+ cells by immunoperoxidase labeling. Sections were then incubated with polyclonal anti-IDO (Santa Cruz Biotechnology, Santa Cruz, CA, USA (H-110), 1: 50) and anti-CD163 (Santa Cruz Biotechnology (sc-20066), 1: 25). Immunohistochemical staining was performed, as previously demonstrated by De Souza Sales et al. [6].

For immunofluorescence, cryostat sections (LL, n = 4) were incubated with primary antibody polyclonal anti-IDO (1: 100) and monoclonal anti-CD163 (1: 50), followed by goat anti-rabbit labeled with Alexa Fluor 488® (1: 1000, Molecular Probes, Eugene, OR, USA) or by goat anti-mouse labeled with Alexa Fluor 568® (1: 1000, Molecular Probes). Afterwards, slides were mounted with Vectashield (Vector Laboratories). Images were obtained via confocal laser microscopy (LSM 510 META scanning; Zeiss, Göttingen, Germany).

Semiquantification of dermal CD163+ and IDO+ cells

A semiquantitative analysis of dermal positive cells for CD163 and IDO in skin lesions of BT (n = 6) and LL (n = 6) patients was performed and classified as: (−) no positive cells, (+) presence of few positive cells (up to 5% of cells), (++) positive cells present in focuses on the inflammatory infiltrate, comprising 20% of cells, (+++) several positive cells, comprising 50%, and (++++) numerous positive cells, representing most of the cellular infiltrate (more than 50% of cells). The analysis of results was performed twice with no disagreement on the issue.

Immunoblotting

CD163 expression was quantified by Western blot analysis. As previously described, protein extracts were obtained [6] from 30 slices (10 μm) of frozen patient skin biopsies (BT, n = 4 and LL, n = 4) after which 30 μg of the extracts were loaded in 12% SDS-PAGE and blotted onto nitrocellulose membranes (Bio-Rad) with a semi-dry transfer cell (Bio-Rad). CD163 expression was evaluated after incubation with monoclonal mouse anti-human CD163 clone EDHu-1 (AbD Serotec, EUA) (1: 100) and monoclonal mouse anti-human Tubulin (Sigma-Aldrich, St. Louis, Missouri, USA) (1: 10000). Results were visualized through an enhanced chemiluminescence detection system (ECL; Amersham Biosciences, Piscataway, NJ, USA).

RNA preparation and real-time PCR

Total RNA was extracted from frozen skin fragments (LL, n = 5 and BT, n = 5), which were repaired using the Trizol reagent (Invitrogen Corporation, Carlsbad, CA, USA). The cDNA synthesis, using the Taqman PCR, was performed as described above [6]. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an endogenous control and IDO, IL-10, and CD163 mRNA were quantified via the 2−ΔCt.

Cellular immunofluorescence

Immunofluorescence was performed to verify the expression of CD68+, CD163+, and IDO+ cells. The skin macrophage cells were fixed in paraformaldehyde 4% and then incubated with the primary antibodies for 2 h at room temperature. After washing, the secondary antibody (anti-IgG1 for CD163 and CD68 and anti-IgG for IDO) was incubated and the nucleus was marked with DAPI. The images were obtained from Microscope Axio Observer Z1 (Carl Zeiss, Göttingen, Germany) via Axiovision 4.7 software.

Cell isolation

Cell isolation from skin biopsies was performed as previously described by Moura et al. [38].

Cell culture and stimulation

Peripheral blood mononuclear cells (PBMCs) were isolated under endotoxin-free conditions from heparinized venous blood by Ficoll-Hypaque (Pharmacia Fine Chemicals, Piscataway, NJ, USA) density centrifugation. PBMC were then cultured in tissue culture plates at 37°C/5% CO2. Monocyte purification was done for 2 h adherence in 24-well plates (Costar, Cambridge, MA, USA) at 2 × 106 cells per well. Live and dead ML at an MOI (2.5; 5 and 10: 1) isolated from LL leprosy patients, E. coli (5: 1), M. bovis BCG (5: 1), and Cytochalasin-B (20 μM, Sigma Chemical Co.) were used, when necessary, for stimulation. For evaluation of cytokine secretion, supernatants from ML-stimulated monocytes were harvested after 1 day of culture and stored at −20 °C until future use.

Live/dead bacteria

For live or dead bacteria detection, the LIVE/DEAD® BacLight™ Bacterial Viability Kits were used according to the manufacturer's instructions (Invitrogen Corporation).

Effect of the IL-10 blockade on monocytic culture

To block endogenous IL-10, the neutralizing anti-IL-10 rat anti-human or isotype control—IgG1 at a final concentration of 1 μg/mL (BD PharMingen, San Diego, CA, USA) was added to the monocytic culture. The neutralizing antibody was added to the culture 30 min before ML stimulation. After 24 h, the percentage of CD163+ was evaluated by flow cytometry (AccuriTM, Ann Arbor, MI, USA) and IDO activity was evaluated in the supernatants.

Estimation of IDO activity in supernatants from monocytic cell cultures by HPLC

To detect IDO activity, supernatants from ML-stimulated monocytic cultures were collected and frozen in −20°C until HPLC analysis. When necessary, IDO activity was evaluated in rIL-10 (10 ng/mL)- or anti-IL-10 (1 μg/mL)-stimulated cell supernatants. Tryptophan (Trp) and Kynurenine (Kyn) concentrations were measured by HPLC, as previously described [6].

Evaluation of ML uptake

Monocytes were pretreated with RM3/1 CD163 antibody or its isotype control—Mouse IgG1 (20 μg/mL, Santa Cruz Biotechnology®) for 30 min on ice. Prior to bacterial interaction assays, ML was stained with PKH26 Red Fluorescence cell linker Kit (Sigma) according to the manufacturer's instructions. Adherent monocytes were infected with PKH 26-labeled ML (MOI 5: 1) and after 2, 16, and 24 h postinfection, the percentage of eukaryotic cells with bacterial association was measured using an AccuriTM flow cytometry. The index of bacterial association is expressed as percentage of cells taking up PKH26-ML.

To determine bacterial internalization, ML was labeled with PKH67 Green Fluorescence cell linker Kit (Sigma) prior to infection and the fluorescent signal of extracellular bacteria after incubation time was quenched with trypan blue, as previously described [39]. The percentage of ML phagocytosis was measured by PKH-67 and measured at the FL1 channel via flow cytometry. Alternatively, ML association and internalization were evaluated at 2 and 16 h using the human embryonic kidney cell line 293 (HEK293) cells transfected with CD163 mRNA (splice variant AC1) as previously described [40].

In parallel, microscopy images were obtained from cells pretreated with the PKH 67 Green Fluorescence cell linker Kit (Sigma) (green) to visualize the eukaryotic cell membrane, prior saturation with the antibodies, and PKH 26-labeled ML (red) infection, as described below regarding the cytometry assay. Cells were also labeled with the DAPI nuclear stain. Preparations were examined using Microscope Axio Observer Z1 (Carl Zeiss) via Axiovision 4.7 software.

Cytokine detection by immunoassay or ELISA

The sera from HC (n = 10) and LL (n = 10), and BT (n = 10) patients were used to measure sCD163 and IL-10 levels by ELISA, as recommended by the manufacturer (R&D Systems, Minneapolis, MN, USA). Alternatively, cell supernatants of ML (MOI 10 : 1)-stimulated monocytes were collected after 24 h of culture and tested for the presence of TNF, TGF-β, and IL-10, as described by the manufacturer (eBioscience, Inc., San Diego, CA, USA).

Isolated macrophages/monocytic phenotypes and IDO-expressing cells in leprosy

The isolated macrophages were obtained from LL skin lesions, and monocytes were collected with a cell scraper, both after 24 h. The cells were labeled with CD163 APC, IDO PE, CD209 FITC, or HLA-DR PE. For IDO intracellular staining after fixation and permeabilization (Fixation/Permeabilization Buffer; eBioscience), cells were stained with rabbit anti-IDO polyclonal antibody (Santa Cruz Biotechnology) followed by PE-conjugated goat anti-rabbit secondary antibody (Santa Cruz Biotechnology). Normal rabbit IgG was used as the corresponding isotype antibody control. Flow cytometry analyses were performed using a Cyan flow cytometer (Dako Cytomation, Glostrup, Denmark). Gates were set for collection and analysis of 10,000 live events. To determine the percentage of positive cells, isotype controls of the different antibodies were used. The events were analyzed via Summit Software (Dako Cytomation).

Perls’ prussian blue staining

After the skin fragments were deparaffinized and hydrated, the sections were immersed in a potassium ferrocyanide solution, washed, and subsequently immersed in Safranin- acetic acid solution. After counterstaining, the sections were washed in 1% acetic acid, followed by dehydration, clarification, and mounting with Entellan® (Merck KGaA, Darmstadt, Germany). Images were obtained via a Nikon Eclipse microscope with Infinity software.

Statistical analysis

The results were expressed as mean ± SE. Significant differences between groups were determined by an ANOVA test in which a p-value ≤ 0.05 was considered significant. Analyses were performed using Windows GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA, USA). Semiquantitative evaluation of CD163+ and IDO+ cells was performed with Fisher's exact test using SPSS version 16.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References

We would especially like to thank Helen Ferreira for her excellent technical assistance together with Drs. Flavio Alves Lara, Elizabeth Pereira Sampaio, Ariane Leite de Oliveira, and Daniel Serra for their insightful discussion of the manuscript in addition to Judy Grevan for editing the text. We also extend our heartfelt thanks to Drs. Soren Kragh Moestrup and Anders Etzerodt for kindly donating the CD163 transfected cells used in this study.

This work was supported by CNPq and FAPERJ.

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References

The authors declare no financial or commercial conflict of interest.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interest
  9. References
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Abbreviations
BT

borderline tuberculoid

Hb

hemoglobin

HC

healthy control

Hp

haptoglobin

LL

lepromatous leprosy

ML

Mycobacterium lepra