Immunology and Cell Biology (2011) 89, 437–446 & 2011 Australasian Society for Immunology Inc. All rights reserved 0818-9641/11 www.nature.com/icb ORIGINAL ARTICLE Bystander inhibition of dendritic cell differentiation by Mycobacterium tuberculosis-induced IL-10 Maria Elena Remoli1,3, Elena Giacomini1,3, Elisa Petruccioli1, Valerie Gafa1, Martina Severa1, Maria Cristina Gagliardi1, Elisabetta Iona1, Richard Pine2, Roberto Nisini1 and Eliana Marina Coccia1 Mycobacterium tuberculosis (Mtb) evades the immune response by impairing the functions of different antigen-presenting cells. We have recently shown that Mtb hijacks differentiation of monocytes into dendritic cells (DCs). To further characterize the mechanisms underlying this process, we investigated the consequences of inducing dendritic cell differentiation using interferon-a and granulocyte-macrophage colony-stimulating factor in the presence of supernatants (SNs) obtained from monocyte cultures treated with or without heat-inactivated Mtb. Although the SNs from control cultures do not interfere with the generation of fully differentiated DCs, monocytes stimulated with SNs from Mtb-stimulated cells (SN Mtb) remained CD14+ and poorly differentiated into CD1a+ cells. Among cytokines known to affect dendritic cell differentiation, we observed a robust production of interleukin-1b, interleukin-6, interleukin-10 and tumor necrosis factor-a upon Mtb stimulation. However, only interleukin-10 neutralization through the addition of soluble interleukin-10 receptor reversed the inhibitory activity of SN Mtb. Accordingly, the addition of recombinant interleukin-10 was able to significantly reduce CD1a expression. The interaction of Mtb with differentiating monocytes rapidly activates p38 mitogen-activated protein kinase, signal transducer and activator of transcription pathways, which are likely involved in interleukin-10 gene expression. Taken together, our results suggest that Mtb may inhibit the differentiation of bystander non-infected monocytes into DCs through the release of interleukin-10. These results shed light on new aspects of the host–pathogen interaction, which might help to identify innovative immunological strategies to limit Mtb virulence. Immunology and Cell Biology (2011) 89, 437–446; doi:10.1038/icb.2010.106; published online 31 August 2010 Keywords: dendritic cell differentiation; immunoevasion strategy; IL-10; monocyte; Mycobacterium tuberculosis Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens and tuberculosis still remains one of the major health threats to mankind.1,2 The success of Mtb as a highly adapted human pathogen also relies on the ability to evade the immune response and, in turn, to persist in an immunocompetent host. This phenomenon is based on the capacity of Mtb to switch to a dormant status of latency, as well as on the ability to survive within macrophages by arresting phagosomal maturation.3,4 Besides the well-characterized immunoevasion strategies occurring in Mtb-infected macrophages, more recently, a new mechanism of Mtb immune evasion that relies on the capacity of Mtb to interfere with monocyte differentiation into dendritic cells (DCs) has been characterized.5,6 The importance of DCs in regulating the immune response against pathogens, including Mtb, has been largely demonstrated7,8 given their capacity to act as antigen-presenting cells for naı̈ve T lymphocytes and, hence, to play a crucial role in the induction of adaptive immunity. Thus, by limiting the generation of functionally active DCs, Mtb could block the afferent limb of specific adaptive immunity. Previous findings 1Department showed that despite the presence of interferon (IFN)-a and granulocyte macrophage colony-stimulating factor (GM-CSF), Mtb-infected monocytes do not differentiate into DCs, rather they develop into macrophage-like cells, which retain CD14 without acquiring CD1a, partially express CD86 and do not upregulate CD80 and HLA-DR.6 Moreover, Mtb promotes the differentiation of subverted CD83+ DCs characterized by a selective failure in the expression of CD1 molecules and no upregulation of CD80 and HLA-DR molecules in the presence of interleukin (IL)-4 and GM-CSF.5 As a consequence T lymphocytes stimulated by macrophage-like cells or subverted DCs showed a reduced ability to proliferate and to produce IFN-g. Thus, the expansion of T-lymphocytes lacking effector function would reduce the T-cell help provided to infected alveolar macrophages for killing the intracellular pathogen. Furthermore, given their low expression of stimulatory and co-stimulatory molecules, Mtb-infected macrophage-like cells could turn into immunoprivileged host cells for pathogen replication. Cytokines represent key molecules involved in the regulation of the immune response directed against Mtb.9 Indeed, cross-talk between of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome, Italy and 2UMDNJ-New Jersey Medical School, Public Health Research Institute, Newark, NJ, USA 3These two authors contributed equally to this work. Correspondence: Dr EM Coccia, Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, Rome 00161, Italy. E-mail: eliana.coccia@iss.it Received 13 February 2010; revised 3 July 2010; accepted 4 July 2010; published online 31 August 2010 Mtb bystander inhibition of DC differentiation ME Remoli et al 438 immune cells is mainly based on the release of immune mediators, such as IFN-g and tumor necrosis factor (TNF)-a, which activate macrophages and promote bacterial killing.10 However, a broader panel of regulatory and inflammatory cytokines are released from Mtb-infected macrophages and DCs, from innate cells (mainly natural killer cells) and specific T cells. Their influence on the inflammatory environment and the impact on the killing capacity of macrophages have been well elucidated.9 On the contrary, the influence of the inflammatory milieu on the capacity of recruited monocytes to differentiate into DCs has been poorly investigated so far despite the importance of DCs in controlling the immune response against Mtb. To this aim, we sought to investigate whether, besides the direct interaction with monocytes,6 Mtb would affect DC differentiation through the secretion of a variety of cytokines that would act in a paracrine manner on the surrounding uninfected monocytes. Within the cytokine storm released by Mtb-infected cells,11,12 type I IFN has a key role given the importance of these cytokines in promoting monocyte differentiation into DCs13,14 and in enhancing a protective Th1 immune response against Mtb by acting on DC immunological functions.15 In addition to that, our previous studies showing the release of type I IFN from Mtb-infected DCs11and the capacity of Mtb to inhibit type I IFN responses in infected monocytes and macrophages16,17 suggested that Mtb has established a complex interplay with the type I IFN system. Based on this evidence, we investigated the impact of the inflammatory milieu induced locally by Mtb on DC differentiation promoted by IFN-a and GM-CSF. Having found that supernatants (SNs) obtained from monocyte cultures stimulated for 5 days with IFN-a and GM-CSF in the presence of heat-inactivated Mtb (SN Mtb) inhibited DC differentiation, a set of experiments were conducted to identify the cytokine(s) responsible for this phenomenon. Interestingly, we found that only IL-10 was able to inhibit significantly CD1a expression. In addition, the molecular mechanisms underlying IL-10 gene expression in Mtb-challenged monocytes were investigated to further characterize the immunoevasion strategies of Mtb through IL-10. RESULTS Inhibition of DC differentiation by SNs obtained from IFN-a-differentiating monocytes upon Mtb stimulation To investigate whether cytokines released from cells interacting with Mtb could affect DC differentiation, monocyte cultures were stimulated for 5 days with IFN-a (1000 U ml1) and GM-CSF (50 ng ml1) in the absence or presence of heat-killed Mtb (Mtb:cell ratio 5:1). Supernatants from control cultures (SN Ctr) or Mtb-treated cultures (SN Mtb) were harvested and filtered to remove extracellular bacteria (Figure 1). As both live and heat-killed Mtb are able to inhibit DC differentiation,6 we chose to treat monocyte cultures with heatkilled Mtb to avoid the possible cytopathic effects induced by live bacteria that could alter the composition of SN. Preliminary experiments were performed to find out the optimal dilution of SN required to investigate their effect on DC differentiation (data not shown). Having found that 50% of heterologous SN exhibited the best effect, the viability of DC cultures was evaluated to exclude any possible artifact due to the addition of SN. A comparative analysis between monocytes induced to differentiate into DCs with IFN-a and GM-CSF alone or in the presence of 50% of heterologous SN was performed. Both non-stimulated cultures (Ctr) and cultures treated either with SN Ctr or with SN Mtb displayed a similar viability as evaluated by propidium iodide staining (data not shown), indicating that the replacement of 50% cell medium with heterologous SN did not affect the viability of these cultures. Similarly, the addition of heat-inactivated Mtb to monocyte-differentiating cultures did not affect the percentage of live cells in the cultures (data not shown). The effect of SN Ctr and SN Mtb addition to cultures of monocytes induced to differentiate into DCs with IFN-a and GM-CSF was then investigated (Figure 2). No effect on monocyte differentiation into DCs was observed when the heterologous SN Ctr was tested, as the monocytes fully differentiated into CD14 and CD1a+ cells as occurred in control cultures (Figure 2a, left panel). Conversely, a strong reduction of CD1a+ cell frequency was observed after 5 days of culture in the presence of 50% SN Mtb or when monocytes were treated with heatkilled Mtb (Figure 2a, right panel). Accordingly, the majority of Mtb-stimulated cells maintained the CD14 expression whereas the addition of SN Mtb only partially reduced the expression of CD14 on differentiating monocytes. The effect exerted by the SN derived from Mtb-stimulated cultures was specific for the differentiation into DCs because no modulation of costimulatory and MHC molecule expression (CD80, CD86, HLA-DR and HLA-ABC) was observed on monocytes stimulated with SN Ctr and SN Mtb (Supplementary Figure 1). Furthermore, a higher percentage of double-negative CD1a and CD14 cells was observed in SN Mtb-stimulated cultures compared with Mtb-treated cells (37.6 vs 18.6%, respectively), whose phenotype resulted similar to CD1a+ cells as shown by fluorescence-activated Figure 1 Schematic diagram of the experimental design. The cartoon represents the experimental plan used to investigate the effect of cytokines released from Mtb-stimulated cultures on the differentiation of monocytes into DCs induced by IFN-a and GM-CSF. Immunology and Cell Biology Mtb bystander inhibition of DC differentiation ME Remoli et al 439 Figure 2 Inhibition of monocyte differentiation into DCs in response to treatment with SN Ctr and SN Mtb. Monocytes were cultured for 5 days with GM-CSF and IFN-a, in the presence or not of heat-inactivated Mtb (Mtb:cell ratio 5:1) or 50% of SN Ctr or SN Mtb. The expression of CD1a and CD14 was then analyzed by flow cytometry. (a) Dot plots of CD1a and CD14-stained cells from a representative experiment are shown. (b) The mean values ± s.e. of the percentage of CD1a+ and CD14+ cells obtained from different donors are shown (n¼10). The SNs used to treat GM-CSF and IFN-a differentiating monocytes were derived from seven different donors. CD1a expression: *P¼0.0003 Mtb vs Ctr and **P¼0.002 SN Mtb vs SN Ctr. CD14 expression: *P¼0.011 Ctr vs Mtb and **P¼0.007 SN Ctr vs SN Mtb. (c) Kinetics of CD1a expression. The percentage of CD1a+ cells was analyzed after 1, 2, 3, 4 or 5 days of culture with GM-CSF and IFN-a in the presence or not of heat-inactivated Mtb or 50% of SN Ctr or SN Mtb. Each value is the mean ± s.e. of the percentage of CD1a+ cells obtained from three experiments (n¼3) performed with different donors treated with SNs collected from two different cultures. cell sorter analysis of CD86, CD80, HLA-DR and CD38 (data not shown). Given these results, it is likely that the SN Mtb blocks the final differentiation of CD14+ monocytes into CD1a+ DC, leading to the development of an intermediate population of CD1a and CD14 cells. The same effect was observed using monocytes isolated from 10 different donors (Figure 2b). Indeed, the percentage of CD1a+ cells was significantly reduced from 78 to 27% in SN Mtb- or Mtbstimulated cells, while roughly 30% of cells remained CD14. A kinetic study was also performed to follow the effect of Mtb and the cytokines released from Mtb-stimulated cultures on the time course of monocyte differentiation into DCs (Figure 2c). In both cases CD1a expression did not arise after 1 day of culture and the cultures remained CD1a compared with those stimulated with SN Ctr, in which the percentage of CD1a+ cells reached nearly 90%. Real-time reverse transcriptase (RT)-PCR performed on RNA obtained from three different DC cultures showed that, in line with the surface expression of CD1a and CD14, SN Mtb also reduced CD1a mRNA content and had little effect on the level of CD14 mRNA as compared with the expression in monocytes (Figure 3). A similar profile of CD1a and CD14 mRNA expression was observed in cells interacting with heat-killed Mtb, suggesting that the expression of mRNA encoding cell surface antigens associated with DC differentiation represents an important target of mycobacterial immune evasion strategies. Cytokine production from differentiating monocytes upon Mtb stimulation It has been previously demonstrated by several groups that pro- and anti-inflammatory cytokines, such as IL-1b, IL-6, IL-10 and TNF-a, can inhibit the differentiation of monocytes into DC.18–22 To investigate whether Mtb may affect DC differentiation through the release of these factors, SNs from Ctr and Mtb-stimulated cultures were Immunology and Cell Biology Mtb bystander inhibition of DC differentiation ME Remoli et al 440 Figure 3 Expression of CD1a and CD14 mRNA in IFN-a and GM-CSF differentiating monocytes stimulated with heat-inactivated Mtb, SN Ctr and SN Mtb. Total RNA was extracted after 5 days of treatment with heat-inactivated Mtb or 50% SN Ctr or SN Mtb. Freshly isolated monocytes were used as internal control. CD1a and CD14 mRNA expression was analyzed by real-time RT-PCR in three different donors (a–c). All quantification data are presented as a ratio to the GAPDH level and represent the mean±s.e. of triplicate values. The standard errors (95% confidence limits) were calculated using the Student’s t test. Figure 4 Production of IL-10, IL-6, IL-1b and TNF-a from IFN-a and GM-CSF differentiating monocytes upon Mtb stimulation. The concentration of cytokines present in SN Ctr and SN Mtb was determined by cytometric bead array. The values represent the means±s.e. of the cytokine concentrations detected in the SN of cultures collected from nine independent experiments. IL-10 production: *P¼0.011; IL-6 production: *P¼0.002; IL-1b production: *P¼0.0015; TNF-a production: *P¼0.035. harvested at day 5 and cytometric bead array analysis was performed. A robust production of IL-1b, IL-6, IL-10 and TNF-a was induced, although to different extents, by Mtb stimulation (Figure 4), supporting our working hypothesis on the inhibitory role of Mtb-stimulated cytokines in DC differentiation. Immunology and Cell Biology Definition of the inhibitory role of IL-10 in DC differentiation To identify the cytokines that could be responsible for the inhibition of DC differentiation, recombinant IL-1b, IL-6, IL-10 and TNF-a cytokines were added to the monocytes cultured in differentiation medium and the expression of CD1a and CD14 was evaluated after Mtb bystander inhibition of DC differentiation ME Remoli et al 441 Figure 5 Effects of recombinant cytokines on DC differentiation. Monocytes were stimulated to differentiate with IFN-a and GM-CSF in the presence of IL-6 (20 ng ml1), IL-10 (20 ng ml1), TNF-a (50 ng ml1) and IL-1b (10 ng ml1). CD1a and CD14 expression was evaluated by flow cytometry after 5 days. (a) Dot plots of CD1a- and CD14-stained cells from a representative donor are shown. (b) The mean value ± s.e. of the percentage of CD1a+ and CD14+ cells obtained with eight different donors are shown (n¼8). CD1a expression: *P¼0.0006 Ctr vs IL-10. CD14 expression: *P¼0.011 Ctr vs IL-10. 5 days of culture (Figures 5a and b). We found that only IL-10 reproduced the effect of SN Mtb, limiting the induction of CD1a (to about 50%) and preserving the level of CD14 (at about 40%). Dose–response experiments showed that the inhibitory effect of IL-10 on CD1a expression was not observed when 0.1 and 1 ng ml1 doses were used, while it started with 2 ng ml1 and was stronger at the dose of 10 ng ml1 as evaluated by immunofluorescence at day 5 (data not shown). This apparent discrepancy between the dose of recombinant IL-10 required to inhibit monocyte differentiation and the amount of IL-10 found in the SN Mtb could be ascribed to the higher bioactivity of natural cytokines compared with the recombinant counterpart produced in a prokaryotic expression system. Moreover, the capacity of live and heat-inactivated Mtb to inhibit the differentiation of monocytes into DCs correlates well with the observation that IL-10 production from differentiating monocytes was independent of the vitality of Mtb6 (and data not shown). To further define the role of IL-10 in DC differentiation, both SN Ctr and SN Mtb were incubated with soluble IL-10 receptor (5 mg ml1) to neutralize IL-10 activity (Figure 6). The addition of soluble IL-10 receptor significantly reversed the inhibitory activity of Immunology and Cell Biology Mtb bystander inhibition of DC differentiation ME Remoli et al 442 Figure 6 Recovery of CD1a expression upon the neutralization of IL-10. SN Ctr and SN Mtb were incubated for 1 h with sIL10r (5 ng ml1) to neutralize the released IL-10 and, then, added to monocytes cultured with GM-CSF and IFN-a. The percentage of cells expressing CD1a and CD14 was evaluated by flow cytometry after 5 days of culture. The mean values±s.e. of the percentage of CD1a+ and CD14+ cells obtained from four different donors, each stimulated with SN collected from four different donors, are shown (n¼4). CD1a expression: *P¼0.005 SN Mtb vs SN Mtb+sIL-10r. CD14 expression: *P¼0.003 SN Mtb vs SN Mtb+sIL-10r. SN Mtb on CD1a expression, although it partially affected the expression of CD14 as evaluated by fluorescence-activated cell sorter. Thus, IL-10 is necessary and largely sufficient to mediate the effect of Mtb and SN Mtb on differentiation of monocytes to DCs evidenced by monitoring CD1a and CD14 cell surface expression. Contribution of the p38 mitogen-activated protein kinase (MAPK) pathway and STAT-3 activation to IL-10 gene expression in differentiating monocytes upon Mtb stimulation Having found that IL-10 is an important player in CD1a inhibition observed in differentiating monocytes, we focused our studies on the regulation of IL-10 gene expression in IFN-a and GM-CSF cultured monocytes upon Mtb stimulation. To study whether the release of IL-10 correlated with an increased expression of the IL-10 gene, RNA was extracted from differentiating monocytes at different times following Mtb stimulation and the IL-10 mRNA expression was then analyzed by real-time RT-PCR (Figure 7a). An increased mRNA level was observed as early as 4 h post-Mtb treatment and was maximally detected at 16 h. Immunology and Cell Biology Based on this finding suggesting a transcriptional control of IL-10 expression by Mtb, we sought to investigate STAT-3 and p38 MAPK pathways, the involvement of which in the transcription of human IL-10 gene was previously demonstrated.23–25 The contribution of p38 MAPK was evaluated in IFN-a and GM-CSF differentiating monocytes stimulated with Mtb. A kinetic experiment was performed to characterize the consequences of IFN-a- and GM-CSF-induced differentiation for the previously characterized activation of p38 by Mtb in human monocytes.26 p38 MAPK was rapidly phosphorylated 1 h after Mtb stimulation and declined after 2 h (Figure 7b), confirming our previous data.26 To determine the role of MAPK in the Mtb-driven induction of IL-10, we performed a dose–response experiment with p38 inhibitor SB203580 and Erk inhibitor PD98059, and the production of IL-10 was measured by enzyme-linked immunosorbent assay upon Mtb stimulation (Supplementary Figure 2). A clear reduction of IL-10 expression started when 3 mM SB203580 was used to treat the cultures, and a stronger effect was found with higher doses. Conversely, IL-10 release was not reduced by Erk inhibitor PD98059. However, a specific effect on CD1a expression was observed when high doses of SB203580 (5 and 10 mM) were used to treat the differentiating cultures (Supplementary Figure 3). Based on these results, the SB203580 dose of 3 mM was chosen to inhibit p38 MAPK in our experimental setting. We found that p38 was likely involved in Mtb-induced IL-10 expression because a significant reduction of IL-10 production was observed in cultures treated with p38 inhibitor SB203580, whereas the Erk inhibitor PD98059 even reinforced Mtb-stimulated IL-10 production (Figure 7c). Specificity in the effect of these inhibitors on IL-10 production is shown by the lack of effect on production of IL-6 elicited by Mtb (Figure 7c). The analysis of STAT-3 activation showed a weak tyrosine phosphorylation detected as early as 4 h of stimulation, which increased at 8 h and remained sustained until 24 h following Mtb stimulation as evaluated by western blot analysis performed with antibodies raised against the phosphorylated isoform of STAT-3 (data not shown). The kinetics of STAT-3 activation paralleled the data obtained with electro-mobility shift assay performed with nuclear cell extracts prepared at different times (4, 16 and 24 h) after Mtb treatment and an oligo containing the STAT sequence present within the IL-10 promoter. A STAT-3 DNA-binding complex with the STAT binding site from the IL-10 promoter was faintly detected 4 h after Mtb stimulation and reached a maximal level at 16–24 h (Figure 7d). The identity of the complex was confirmed by supershift experiments using an antibody raised against STAT-3. Collectively, these results suggest that STAT-3 might cooperate with p38 MAPK in IL-10 gene regulation in response to Mtb challenge. DISCUSSION Our study provides evidence for a novel mechanism of Mtb immunoevasion involving the inhibitory effect of IL-10 on the differentiation of monocytes into DC. The exploitation of IL-10 as a common mechanism of immunosuppression by a heterogeneous group of intracellular pathogens that can infect macrophages, including Mtb, has been largely demonstrated.27 In particular, the capacity of IL-10 to modulate the anti-mycobacterial immune response has been established in in vitro and animal models.27 The central molecular effect of IL-10 is to dampen the expression of inflammatory cytokines, chemokines and cell surface molecules crucial for the induction of inflammation.28 Indeed, IL-10 is known to inhibit the production of IL-12 and the antigen-presenting cell functions of monocytes and macrophages, hence suppressing the development of Th1-mediated immunity.29–31 More recently, a report addressed the question of how Mtb bystander inhibition of DC differentiation ME Remoli et al 443 Figure 7 Regulation of IL-10 gene transcription in IFN-a and GM-CSF differentiating monocytes upon Mtb stimulation. (a) The kinetics of IL-10 mRNA expression was investigated in IFN-a and GM-CSF differentiating monocytes stimulated with heat-inactivated Mtb. At the indicated time points, RNA was extracted and the expression of IL-10 was measured by real-time RT-PCR. All quantification data are presented as a ratio to the GAPDH level. The results shown are representative of one out of three experiments performed with RNA extracted from three different donors that yielded similar results. (b) Activation of p38 MAPK was studied at the indicated time points following Mtb challenge by western blotting using antibodies directed against the phosphorylated (p p38) and total p38 (p38). The results shown are from one out of three experiments that yielded similar results. (c) p38 inhibitor SB203580 (Mtb+SB) or the ERK inhibitor PD98059 (Mtb+PD) were added 30 min before Mtb stimulation. The SNs were collected after 5 days and the production of IL-10 and IL-6 was analyzed by enzyme-linked immunosorbent assay. The values represent the means ± s.e. of the cytokine concentration detected in the SN of cultures collected in four independent experiments. *P¼0.018 Ctr vs Mtb: **P¼0.03 Mtb vs Mtb+SB. (d) Cells were treated with heat-inactivated Mtb for the indicated time points. Nuclear extracts were prepared and analyzed by EMSA using a specific radiolabeled oligonucleotide corresponding to the STAT-binding site present within the IL-10 promoter. The supershift assay was performed using an anti-STAT-3 or unrelated (Unr) antibodies, where indicated. The supershifted complex is indicated with the SS arrow. This is a representative EMSA experiment, which was repeated two additional times with cell extracts from different monocyte cultures stimulated with heat-inactivated Mtb. an increased production of IL-10 by murine macrophages allows Mtb replication.32 in spite of an active Th1 response. An elegant demonstration has been provided indicating the importance of this cytokine in the control of the chronic phase and reactivation of infection. Indeed, macrophages conditioned in vivo by high levels of IL-10 exhibited a reduced killing capacity despite a vigorous specific Th1 response. These data correlate well with the causal link from both elevated levels of IL-10 in the sera of tuberculosis patients and the polymorphism of IL-10 to tuberculosis susceptibility.33–38 Although macrophages are the main producers of IL-10 and, at the same time, the targets of its immunosuppressive effects, DCs also represent key participants in the IL-10-mediated strategies induced to evade surveillance against tumors and pathogens.39 Indeed, IL-10, which is commonly found in the microenvironment of tumors, is a potent inhibitor of DC maturation and differentiation.40,41 In line with these observations, we found that, in addition to a direct effect on the infected cells,5,6 Mtb might also inhibit monocyte differentiation into DCs by inducing a bystander effect on the surrounding Immunology and Cell Biology Mtb bystander inhibition of DC differentiation ME Remoli et al 444 non-infected monocytes through the release of IL-10. Indeed, IL-10-conditioned monocytes undergo a partial differentiation leading to the development of a heterogenous population of cells expressing CD14 and displaying variable levels of CD1a. The impact of IL-10 on DC functions was previously demonstrated by different groups, which found that the exogenous addition of IL-10 to immature DCs downregulated the expression of CD1a and their capacity to stimulate an alloreactive response.20,42 These results are consistent with our observation that CD1a induction is inhibited by the IL-10 induced with Mtb or SN Mtb stimulation of monocyte cultures during IFN-a and GM-CSF treatment. A different conclusion on the capacity of IL-10 to modulate CD1 expression was drawn by Stenger et al.,43 who demonstrated that the inhibition of CD1a expression on antigen-presenting cells by Mtb was independent of IL-10. This apparent discrepancy between these data and the results published by our group (this paper and Mariotti et al.6) could depend on the experimental model used to investigate the effect of IL-10. Indeed, although Stenger analyzed the effect of Mtb infection on differentiated antigen-presenting cells obtained from the adherent fraction of peripheral blood mononuclear cells cultivated for 5 days with GM-CSF and IL-4, in our experimental setting we studied a purified CD14+/CD1a population induced to differentiate by GM-CSF and IFN-a and stimulated at day 0 with Mtb or SN Mtb. In addition, the stronger production of IL-10 observed in cultures stimulated with IFN-a and GM-CSF in the presence of heat-inactivated Mtb compared with those induced with IL-4 and GM-CSF (roughly 20–30-fold higher; data not shown) could also explain the differential outcome of IL-10 neutralization shown in this paper and in the report by Stenger et al.,43 in which the production of IL-10 was not determined at all. Moreover, another possible reason for the differences in the observations of Stenger et al.43 and of this paper could come from the stimulation of monocyte cultures with live Mycobacteria at MOI 10 (10 Mtb for 1 cell), compared with our use of five heat-inactivated Mtb to stimulate one monocyte. Thus, the MOI and the timing of Mtb stimulation, the procedure used to isolate monocytes as well as the composition of the cytokine cocktail used to promote monocyte differentiation could account for the contrasting conclusions on the role of IL-10 in the Mtb-driven impairment of DC generation. Collectively, our results shed light on a new aspect of Mtb immunoevasion mechanism suggesting that at the site of a local immune response, such as a tuberculous granuloma, the release of IL-10 from infected monocytes/macrophages and its diffusion in the lung microenvironment might counteract the differentiation of recruited monocytes promoted by type I IFN released by infected DCs.6 These data confirmed our previous observations showing distinct roles for DCs and macrophages in driving the immune response against Mtb on the basis of the differential profile of cytokines and chemokines released following Mtb infection.12,44 Indeed, although macrophages control the granulomatous inflammatory response through the release of pro- and anti-inflammatory cytokines, DCs are primarily involved in inducing anti-mycobacterial T-cell immune responses.6 Based on these findings, understanding of how Mtb initiates the production of IL-10 in differentiating monocytes would be instrumental in revealing important targets for therapeutic intervention in tuberculosis. To this aim, the regulation of IL-10 gene expression was investigated by having as background the knowledge accumulated over the past years showing the capacity of mycobacterial antigen or mycobacteria to activate different intracellular pathways leading to IL-10 gene expression.45–52 Although our results confirmed Immunology and Cell Biology the involvement of p38 MAPK, we also observed activation of STAT-3 associated with the expression of the IL-10 gene. Indeed, upon Mtb challenge a transcription complex composed of STAT-3 binding to a functional STAT site characterized within the IL-10 promoter25 was observed in differentiating monocytes. Thus, in line with previous data describing the capacity of hyperactivated STAT-3 to promote IL-10 production,23,53 we can envisage that Mtb-activated STAT-3 likely cooperates with p38 MAPK in inducing the expression of this inhibitory cytokine. In addition to their capacity to modulate IL-10 gene transcription, the combined effect of p38 and STAT-3 pathways could directly affect the differentiation of infected monocytes, which occurs in cancer, wherein the activation of these two molecules leads to an abnormal development of myeloid cells.18,40,53–56 In line with this evidence, the capacity of mycobacteria to exploit the p38 MAPK pathway to dampen different anti-microbial mechanisms has been demonstrated by several groups.57,58 In particular, we recently showed that CR3 engagement on monocytes by mycobacteria leads to p38 and ATF-2 phosphorylation and that antibody-dependent CR3 blockade or treatment with a specific p38 inhibitor caused a notable increase in CD1 molecule expression in DCs derived from mycobacteria-infected cells.26 In conclusion, it is tempting to speculate that the Mtb-induced activation of p38 MAPK and STAT-3 may disarm DCs by inhibiting their differentiation and, in turn, subvert a protective immune surveillance. Indeed, Mtb may control the differentiation of the infected monocytes into DC both through the activation of p38 and STAT-3-mediated signaling and the release of IL-10, which acts indirectly on differentiation of uninfected monocytes. Pharmacological interventions and innovative immunological approaches aimed at counteracting the immunoevasion strategies of Mtb should carefully evaluate p38 and STAT-3 pathways to replenish DCs with full antigen presentation capacity. METHODS Antibodies and other reagents Monoclonal antibodies specific for CD1a (FITC), CD14 (PE), CD80 (PE), CD86 (FITC), HLA-ABC (FITC), HLA-DR (PE), IgG1 and IgG2a (BD Pharmingen, San Diego, CA, USA) were used. Recombinant human soluble IL-10 receptor was purchased from R&D Systems (Abingdom, UK) and used at 5 mg ml1 for 1 h preincubation at 37 1C with the SNs. Recombinant TNF-a (50 ng ml1), IL-1b (10 ng ml1), IL-6 (20 ng ml1) and IL-10 (20 ng ml1) (PeproTech EC Ltd, London, UK) were used to treat monocytes. Preliminary experiments were conducted to define the optimal dose of each cytokine (data not shown). The p38 MAPK inhibitor SB203580 and the ERK inhibitor PD98059, at a concentration of 3 mM, were added 30 min before Mtb stimulation. The inhibitors were not replenished during the 5-day incubation period. Propidium iodide (Sigma Aldrich, St Louis, MO, USA) was used to test cell viability by fluorescence. Bacterial strain, media and growth conditions All the experiments were performed with Mtb H37Rv (ATCC27294; American Type Culture Collection, Manassas, VA, USA). Bacteria were prepared as previously described.12 Mtb was heat killed at 80 1C for 30 min and used at an Mtb:cell ratio of 5:1 (MOI 5). All preparations were analyzed for LPS contamination by the Limulus lysate assay (BioWhittaker Europe, Verviers, Belgium) and H37Rv contained o1 EU ml1 LPS. DC preparation and stimulation DCs were prepared as previously described.6 Briefly, peripheral blood mononuclear cells were isolated from freshly collected buffy coats obtained from healthy voluntary blood donors (Blood Bank of University ‘La Sapienza’, Rome, Italy) by density gradient centrifugation using Lympholyte-H (Cedarlane, Hornby, Ontario, Canada). Monocytes were purified by positive sorting using Mtb bystander inhibition of DC differentiation ME Remoli et al 445 anti-CD14-conjugated magnetic microbeads (Miltenyi, Bergisch Gladbech, Germany). The recovered cells were 499% CD14+ as determined by flow cytometry with the anti-CD14 antibody. DCs were generated by culturing monocytes in six-well tissue culture plates (Costar Corporation, Cambridge, MA, USA) with 50 ng ml1 GM-CSF (Schering-Plough, Levallois Perret, France) and 1000 U ml1 IFN-a (Roche, Nutley, NJ, USA) for 5 days at 0.5106 cells ml1 in RPMI 1640 (BioWhittaker Europe) supplemented with 2 mM Lglutamine and 15% fetal bovine serum (BioWhittaker Europe) and antibiotics. To prepare SN Ctr and SN Mtb, freshly isolated monocytes were cultured in the presence of IFN-a (1000 U ml1) and GM-CSF (50 ng ml1) for 5 days with or without heat-killed Mtb (Mtb:cell ratio 5:1). The SN Ctr and SN Mtb were harvested, filtered to remove the extracellular bacteria and then used to treat new heterologous monocyte cultures obtained from different donors. Cell viability was assessed by propidium iodide staining (Sigma Aldrich). Cells were washed with PBS and then incubated with propidium iodide (final concentration, 50 mg ml1) for 10 min at 4 1C. The percentage of live cells (propidium iodide negative cells) was evaluated by flow-cytometric analysis. Fluorescence-activated cell sorter analysis Approximately 1–2105 cells were aliquoted into tubes and washed once in PBS containing 2% fetal bovine serum. The cells were incubated with purified mAbs at 4 1C for 30 min. The cells were then washed and fixed with 2% paraformaldehyde before analysis on a fluorescence-activated cell sorter scan using CellQuest software (Becton Dickinson, Mountain View, CA, USA). A total of 5000 cells were analyzed per sample. The percentage of positive cells for a specific antigen was obtained by subtracting the percentage obtained with the matched isotype control antibody. Cytokine determinations SNs from cultures were harvested 5 days after treatment and stored at 80 1C. IL-6, TNF-a, IL-10 and IL-1b were measured with the human inflammation cytometric bead array (BD Pharmingen). IL-10 and IL-6 levels were also determined by Instant enzyme-linked immunosorbent assay Kit (Bender MedSystems, Burlingame, CA, USA). The assays were conducted according to the manufacturer’s instructions. Western blot analysis Western blots were performed as previously described.26 Monocytes (2106) were stimulated as indicated and washed twice with cold phosphate-buffered saline. The pellet was resuspended in 200 ml of 2 SDS sample buffer (20 mM dithiothreitol, 6% SDS, 0.25 M Tris, pH 6.8, 10% glycerol, 10 mM NaF and bromophenyl blue) and boiled for 5 min. Proteins were separated by SDS/PAGE and blotted onto nitrocellulose membranes (Hybond C-Extra; GE Healthcare, Uppsala, Sweden). Blots were incubated with phospho- and total-p38 (R&D System), reacted with anti-rabbit HRP-coupled secondary antibody (GE Healthcare) and developed using an ECL system. Electro-mobility shift assay Nuclear cell extracts (15 mg) were prepared and used in electro-mobility shift assay as previously described.11 Synthetic double-stranded oligonucleotide containing the STAT-binding sequence of the IL-10 promoter was end-labeled with [g32P]-ATP using T4 polynucleotide kinase. The sequence binding STAT-3 within the IL-10 promoter was described by Unterberger et al.:25 LS4 forward 5¢-ATCCTGTGCCGGGAAACC-3¢; LS4 reverse 5¢-GGTTTCCCGGCACAGG AT-3¢. For supershift analysis, 1 mg of control antibody or anti-STAT-3 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to the reaction. RNA isolation and real-time RT-PCR quantification RNA was extracted with RNeasy Mini kit (Qiagen Inc., Valencia, CA, USA) according to the manufacturer’s instructions. Reverse transcriptions were primed with oligo (dT) and performed using the Murine Leukemia Virus Reverse Transcriptase (Invitrogen Life Technologies, Carlsbad, CA, USA). Quantitative RT-PCR assays were done in triplicate using the Platinum Taq DNA Polymerase (Invitrogen Life Technologies) and the SYBR Green I (Biowhittaker Molecular Applications, Rockland, ME, USA) on a LightCycler (Roche Diagnostics). A calibration curve of a purified positive control RT-PCR product, to which arbitrary values were assigned, was used to calculate the value of a target gene. The quantification standard curves were obtained using dilutions (4-log range) of the purified positive control RT-PCR product in 10 mg ml1 sonicated salmon sperm DNA. All quantification data of CD1a, CD14 and IL-10 transcripts are shown as a ratio to the GAPDH level present in the same sample and represent the mean + s.e. of triplicate values. The standard errors (95% confidence limits) were calculated using the Student’s t-test. The sequences of the primer pairs used for the quantification of GAPDH59 and for IL-1060 have been previously described. 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