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Keywords:

  • Dendritic cells;
  • IL-6;
  • Mucosal immunity;
  • Intestinal immunity;
  • Ulcerative colitis

Abstract

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

Dendritic cells (DCs) control the type and location of immune responses. Ulcerative colitis (UC) is considered a Th2 disease mediated by IL-13 where up to one third of patients can develop extraintestinal manifestations. Colonic biopsies from inflamed and noninflamed areas of UC patients were cultured in vitro and their supernatants were used to condition human blood enriched DCs from healthy controls. Levels of IL-13 in the culture supernatants were below the detection limit in most cases and the cytokine profile suggested a mixed profile rather than a Th2 cytokine profile. IL-6 was the predominant cytokine found in inflamed areas from UC patients and its concentration correlated with the Mayo endoscopic score for severity of disease. DCs conditioned with noninflamed culture supernatants acquired a regulatory phenotype with decreased stimulatory capacity. However, DCs conditioned with inflamed culture supernatants acquired a proinflammatory phenotype with increased expression of the skin-homing chemokine CCR8. These DCs did not have decreased T-cell stimulatory capacity and primed T cells with the skin-homing CLA molecule in an IL-6-dependent mechanism. Our results highlight the role of IL-6 in UC and question the concept of UC as a Th2 disease and the relevance of IL-13 in its etiology.


Introduction

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

The gastrointestinal tract is in contact with a wide variety of commensal microbiota and diverse pathogens, and therefore requires a balance to be maintained between immunity and immune tolerance; the lack of immune responses against food antigens and/or the commensal microbiota is essential to maintain the homeostasis of the gastrointestinal tract [[1]].

Ulcerative colitis (UC) is a form of inflammatory bowel disease (IBD), traditionally related to a Th2 cytokine profile mediated by IL-13 [[2, 3]], where immune homeostasis of the gastrointestinal tract is compromised. Up to 1/3rd of UC patients can develop extraintestinal manifestations with the skin being one of such tissues [[4, 5]].

Dendritic cells (DCs) are the most potent antigen presenting cells and determine the nature and type of immune responses [[6, 7]]. Intestinal DCs control immune tolerance in the gastrointestinal tract [[8-10]]. DCs also maintain immune responses localized to specific tissues, since they imprint specific tissue-homing profiles on stimulated T cells [[11]]. Retinoic acid (RA), the active form of vitamin A following dehydrogenization by the RALDH2 enzyme controls some of the mechanisms of immune homeostasis of the gut [[12-14]]. RA-producing DCs mediate the IgA switching of B cells [[15]], the generation of T cells with a regulatory phenotype [[10]], and the imprinting of gut-homing markers on B and T cells [[16, 17]], thereby keeping tolerogenic immune responses compartmentalized to the gastrointestinal tract. A tolerogenic role has also been recently described for fractalkine (CX3CR1) since knockout mice failed to develop oral tolerance [[18]]. Invariant Vα24 restricted T cells (iNKT cells) also play a role in oral tolerance although their exact role remains unclear [[19-21]].

In healthy individuals, gut microenvironment controls the phenotype and function of human DCs. Thus, tolerogenic “gut-like” DCs can be generated when they are exposed to such microenvironments [[22-26]]. In UC patients, immune homeostasis in the gastrointestinal tract is compromised. Compared with biopsies from healthy controls, biopsies from inflamed and noninflamed areas of the colon from UC patients exhibit increased production of proinflammatory cytokines, with higher levels within inflamed areas. However, the expression of mediators of tissue damage was restricted to inflamed areas [[27]].

We hypothesized that local factors controlling intestinal homeostasis in UC patients are either lost or masked by ongoing inflammation, driving DCs towards a proinflammatory phenotype, and orchestrating the dysregulated immune response in the gut. We characterized the local expression of soluble cytokines and the gene expression profile of molecules involved in intestinal homeostasis in the gut. Secondly, we studied the effect of conditioning human blood enriched DCs with the intestinal microenvironment from both inflamed and noninflamed areas of the gut, from UC patients. Our results confirmed that noninflamed areas of the gut from UC patients rendered DCs less stimulatory. However, DCs conditioned with inflamed areas from the same patients acquired a proinflammatory skin-homing profile and imprinted a skin-homing phenotype on T cells in an IL-6 dependent mechanism.

Results

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

An increased pro-inflammatory cytokine profile in inflamed areas of UC patients

Biopsies from inflamed areas of UC patients, compared with paired healthy noninflamed areas, produced higher levels of proinflammatory cytokines (Fig. 1A). Neither gender, nor age, nor extension of disease showed a significant effect on cytokine production (data not shown). IL-13, considered the effector cytokine in UC [[2, 3]], was not differentially secreted between inflamed and noninflamed areas of gut of UC patients and in most cases (10 out of 11 from healthy and 6 out of 11 from inflamed areas) it was below detection limit so it did not correlate with severity of disease. IL-6 was the predominant cytokine found in inflamed areas of UC patients (2760 ± 813 pg/mL) (Fig. 1B) and its concentration correlated with the Mayo endoscopic score (Fig. 1C) being the only cytokine displaying such characteristic. Th2-related cytokines, like IL-4 and IL-7, were also increased in inflamed areas. However, their concentration was low (IL-4: 3.8 ± 0.7 pg/mL; IL-7: 21.3 ± 3.3 pg/mL) compared with IL-6 (2760 ± 813 pg/mL) and other Th1-related cytokines that were also increased in inflamed areas. These Th1-related cytokines included IFN-α, (245.9 ± 27.3 pg/mL), IFN-γ (328.5 ± 72.63 pg/mL), and TNF-α (153.1 ± 36.5 pg/mL), suggesting a mixed rather than a Th2 cytokine profile (Fig. 1B).

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Figure 1. Inflamed areas from UC biopsies have increased proinflammatory cytokine secretion. (A) Secretion of soluble cytokines quantified following culture of colonic biopsies for 24 h, from inflamed affected areas of UC patients and paired biopsies from healthy unaffected areas from the same patients. Each point represents a sample from one individual. (B) Cytokine profile in inflamed areas from UC patients. Data are shown as mean + SEM of n = 12 independent patients. (C) Correlation between secreted IL-6 from inflamed biopsies and Mayo endoscopic score. Paired t-test (A) and Pearson's correlation (C) were applied. P-values below 0.05 were considered statistically significant (*p < 0.05, **p < 0.01).

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IL-6 controls the dysregulated cytokine profile in UC patients

Since IL-13 is considered to be the effector cytokine in UC [[2, 3]] we performed blocking experiments in our culture system. Addition of anti-IL-13 to inflamed areas of UC patients did not decrease the secretion of the assayed cytokines. On the contrary, it increased further secretion of IL-4 and IL-17 (Fig. 2). These results might suggest that in UC patients, the role of IL-13 could be elicited in an auto and/or paracrine manner. This may explain why IL-13 was not found in culture supernatants (Fig. 1A) and why its effect was not abrogated by soluble blocking antibody (Fig. 2). Addition of soluble recombinant IL-13 to healthy intestinal areas of UC patients failed to increase secretion of IFN-γ, TNF-α, IL-4, and IL-6 as seen on inflamed areas and only restored secretion of IFN-α, IL-7, and IL-17 (Fig. 3). IL-17 was induced both in healthy areas exposed to IL-13 and in inflamed areas blocked with anti IL-13 probably reflecting different mechanisms of control in different cell types.

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Figure 2. IL-13 blockade has no relevant effect in the cytokine profile of inflamed areas from UC patients. Biopsies from inflamed areas of UC patients were cultured for 24 h with or without 2.5 μg/mL of blocking anti-IL-13 and the secretion of cytokines assayed. Each point represents a sample from one individual. *p < 0.05, **p < 0.01, paired t-test.

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image

Figure 3. IL-13 supplementation in healthy areas from UC patients does not promote a Th2 microenvironment. Biopsies from noninflamed or healthy areas of UC patients were cultured with or without 50 ng/mL of recombinant IL-13. 24 h later, cytokine secretion was assayed. Each point represents a sample from one individual. ***p < 0.001, paired t-test.

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IL-6 was the predominant cytokine found in culture supernatants of inflamed areas in UC patients (Fig. 1B) and its concentration correlated with the Mayo endoscopic score (Fig. 1C). We explored therefore its role controlling the intestinal cytokine milieu in UC patients. Anti-IL-6 supplementation to biopsy cultures from inflamed areas of UC patients effectively blocked the available IL-6 in the biopsy culture supernatants (Fig. 4). Secreted

cytokines which were upregulated in inflamed areas from UC patients (Fig. 1A) were decreased following biopsy incubation in the presence of anti-IL-6 (Fig. 4) and their levels restored (IFN-α, TNF-α, IL-7) or even decreased (IL-4, p < 0.01; and IFN-γ, p < 0.05) compared with those found in healthy areas. Similarly, we cultured noninflamed intestinal areas from UC patients in the presence of IL-6. After IL-6 stimulation, all assayed secreted cytokines (except regulatory IL-10), were induced in the biopsy cultures (Fig. 5) to the levels displayed in the paired inflamed areas (Fig. 1A) while IFN-γ and IL-4 acquired higher values than those identified in inflamed areas from such patients (p < 0.001 in both cases). Together, these findings highlight a central role of soluble IL-6 for controlling the dysregulated cytokine milieu in inflamed areas of UC patients in the absence of any external challenge.

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Figure 4. IL-6 blockade in inflamed areas from UC patients decreases the proinflammatory microenvironment. Biopsies from inflamed areas of UC patients were cultured with or without 0.5 μg/mL of blocking anti-IL-6 antibody. The secretion of cytokines was then assayed following 24 h of culture. Each point represents a sample from one individual. **p < 0.01, ***p < 0.001, paired t-test.

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Figure 5. IL-6 supplementation in healthy areas from UC patients promotes a proinflammatory microenvironment. Colonic biopsies from non-inflamed or healthy areas of UC patients were cultured with or without 50 ng/mL of recombinant IL-6 and cytokine secretion was measured after 24 h. Each point represents a sample from one individual. *p < 0.05, **p < 0.01, ***p < 0.001, paired t-test.

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Inflamed areas of the gut from UC patients have decreased expression of CX3CR1 and RALDH2

We then studied expression of several mRNA molecules related to mechanisms of intestinal tolerance, such as bacterial load (16s), Muc2 (main protein from the mucus layer), FoxP3 (representative of T cells with a regulatory phenotype), Vα24 (invariant chain of Vα24-restricted invariant NKT cells), CX3CR1 (fractalkine) and RALDH2 (necessary to metabolize retinoic acid from dietary vitamin A) (Fig. 6). Neither gender, nor age, nor extension of the disease had any effect on the mRNA expression profile of any of the assayed molecules (data not shown). None of the assayed molecules correlated with the Mayo endoscopic score for severity of disease or with concentration of secreted cytokines (data not shown). Inflamed intestinal areas from UC patients had decreased mRNA expression of both RALDH2 and CX3CR1. Having characterized both the local cytokine milieu (Fig. 1) and gene expression profile (Fig. 6) of both inflamed and noninflamed areas from UC patients, we next studied the effect of intestinal microenvironment in conditioning phenotype and function of human blood enriched DCs.

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Figure 6. mRNA profile of healthy and inflamed colonic biopsies from UC patients. Colonic biopsies from inflamed affected or healthy unaffected areas of UC patients were cultured for 24 h and mRNA expression of 16s, MUC2, FOXP3, Vα24, CX3CR1 and RALDH2 quantified. mRNA expression was normalized to the housekeeping gene GADPH and expressed in arbitrary units (U). Each point represents a sample from one individual. *p < 0.05, paired t-test.

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DCs conditioned with inflamed areas of the gut from UC patients become skin-homing not regulatory DCs

Following validation of the methodology [[26]], DCs from each healthy control were conditioned in the presence of basal medium and supernatant (SN) from paired healthy and inflamed colonic areas from a single UC patient. DCs conditioned with healthy areas acquired a regulatory phenotype, while those conditioned with inflamed areas from the same patients did not. Thus, although exposure to both inflamed and healthy areas increased CD40 expression on DCs, together with an increased potential to migrate to the lymph nodes (CCR7 upregulation) (Fig. 7A and B), HLA-DR expression was specifically decreased in DCs exposed to noninflamed areas (Fig. 7A and B). Expression of other activation/maturation markers (CD83, CD86) was unaffected, as were TLR2 and TLR4 (data not shown). Since CX3CR1 mRNA expression was specifically decreased in inflamed areas, we studied its expression on DCs following conditioning with either inflamed or healthy intestinal microenvironments. CX3CR1 expression on DCs was increased on DCs exposed to both microenvironments with no statistically significant differences (Fig. 7A and B).

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Figure 7. Inflamed areas from UC biopsies activate DCs. (A) Blood DCs were conditioned with colonic biopsy culture supernatant (SN) from healthy or inflamed areas of UC patients for 24 h. As a control DCs were cultured with media alone (basal DCs). The HLA-DR and CD40 intensity ratio (IR) and percentage of CCR7, CX3CR1, and CCR8 were then quantified by flow cytometry. Shaded area represents positive events after subtraction from isotype histograms. Each histogram is representative of several independent experiments (HLA-DR, n = 12; CD40, n = 10; CX3CR1, n = 12; CCR7, n = 11; CCR8, n = 7). (B) Summary of experiments shown in (A) displayed as mean + SEM of replicates. One-way ANOVA repeated measures and secondary paired t-test following Bonferroni correction were applied. *p < 0.05, **p < 0.01 paired t-test.

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We have previously identified that when cultured in vitro in culture medium, DCs lose expression of homing markers [[26]]. However, if DCs are cultured in the presence of a tissue microenvironment, DCs acquire characteristics of local tissue DCs, including expression of specific tissue-associated homing markers. Thus, a gut-homing profile can be induced on DCs when exposed to gut SNs from healthy colonic biopsies [[26]]. However DCs exposed to gut SNs from UC patients (either inflamed or healthy areas) did not acquire a gut-homing profile. Gut-homing β7 and gut-retaining CD103 (mean expression below 10% in all cases) were not induced in any case. Similarly, expression of skin-homing markers CLA and CCR4 (mean expression below 10% in all cases) did not change (data not shown). However, the skin-homing chemokine receptor CCR8 was specifically induced on DCs following conditioning with inflamed areas from UC patients (Fig. 7A and B), providing DCs with potential to migrate to cutaneous sites.

Noninflamed areas from UC patients promote a regulatory cytokine phenotype on DC

Since DCs have been conditioned with cultured SNs that are loaded with cytokines (Fig. 1A), assessing cytokine secretion by DCs is not a feasible approach. Therefore, we studied their natural ongoing intracellular cytokine production, in the absence of any external challenge [[8, 26]], following conditioning with intestinal microenvironments.

None of the assayed cytokines in the culture SNs correlated with the ongoing cytokine production of DCs following SN conditioning (data not shown). When exposed to a noninflamed microenvironment, DCs decreased ongoing production of proinflammatory cytokines IL-12 and IL-6, and increased regulatory cytokine IL-10, when compared with DCs exposed to paired inflamed areas (Fig. 8A and B). Ongoing production of TGF-β was not changed in any case. Therefore, while DCs conditioned with healthy SNs acquired a regulatory cytokine profile, those DCs conditioned with inflamed SNs from the same donors were driven toward a Th1/Th17 cytokine profile.

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Figure 8. DCs conditioned with inflamed areas from UC patients fail to acquire a regulatory cytokine profile. (A) Blood DCs were conditioned with colonic biopsy culture supernatant (SN) as before, and ongoing production of IL-10, IL-12 (p40/p70), IL-6 and TGF-β in DCs was measured by flow cytometry in comparison with unconditioned (basal) DCs. The shaded area represents the percentage of positive cells following subtraction from paired DCs incubated in the absence of monensin. Histograms are representative of several independent experiments (IL-10, n = 11; IL-12(p40/p70), n = 12; IL-6, n = 12; TGF-β, n = 12). (B) Summary of experiments shown in (A) displaying mean + SEM. One-way ANOVA repeated measures and secondary paired t-test following Bonferroni correction were applied. *p < 0.05 paired t-test.

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Inflammation conditions DCs to increase their stimulatory capacity and prime Tcells with a skin-homing profile

The stimulatory capacity of DCs was specifically modified following SN conditioning (Fig. 9A and B). Healthy-SN conditioned DCs were less stimulatory than basal (p < 0.01 at 3%) and inflamed-conditioned DCs (p < 0.05 at 2% and p < 0.001 at 3%) with no differences among the latter. β7 integrin is induced in all stimulated human T cells irrespective of the source of human DCs, while CLA expression on stimulated T cells is dependent on the tissue source of human DCs [[26]]. DCs conditioned with healthy intestinal areas from UC patients decreased their stimulatory capacity and promoted the generation of gut-homing T cells (Fig. 9C). Thus, stimulated (CFSElow) T cells maintained the gut-homing β7 integrin and decreased expression of the skin-homing CLA, compared with unconditioned (basal) DCs as previously reported when using cultured SNs from healthy controls [[26]]. However, when DCs had been conditioned with inflamed areas from the same patients, DCs did not decrease their stimulatory capacity (Fig. 9B). Also, their acquired skin-homing capacity (Fig. 7B) was reflected in an increased capacity to prime skin-homing CLA on stimulated T cells, compared with DCs conditioned with healthy areas from the same patients (Fig. 9D).

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Figure 9. DCs conditioned with inflamed areas from UC patients increase their stimulatory capacity and prime T cells with skin-homing capacity. (A) T cells were labeled with CFSE and cultured for 5 days alone (resting) or in the presence of 3% allogeneic unconditioned DCs (basal) or DCs which had been previously conditioned with culture supernatants (SNs) from healthy and inflamed areas of UC patients. T-cell proliferation was quantified by flow cytometric measurement of CFSE dilution. (B) Summary of nine independent experiments displaying mean + SEM of T-cell proliferation in the three culture conditions. (C) Flow cytometry plots demonstrating expression of β7 (gut-homing) and CLA (skin-homing) on resting T cells (unconditioned) and T cells stimulated (CFSElow) by 3% basal, healthy-SNs, or inflamed-SN DCs. (D) Summary of eight independent experiments showing expression of CLA (top) or β7 (bottom) on T-stimulated cells conditioned as before. Healthy or inflamed areas from the same patients were used to condition DCs from the same healthy donor in all cases. Each point represents data from one patient. Two-way ANOVA repeated measures (B) and paired t-test (D) were applied. *p < 0.05, **p < 0.01.

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IL-6 plays a central role in the dysregulated immune response in UC patients

IL-6 was the predominant cytokine found in inflamed areas from UC patients (Fig. 1B) and its concentration correlated with the Mayo endoscopic score for severity of disease (Fig. 1C). Ongoing production of IL-6 in DCs correlated with their stimulatory capacity (Fig. 10A); this was the only studied cytokine that displayed such characteristics. Therefore, we studied the effect on DC phenotype and function of blocking IL-6 in inflamed supernatants from UC patients. This restored the cytokine profile to normal (Fig. 4) and DCs conditioned with this microenvironment were less stimulatory than their unblocked counterparts (p < 0.001 at 3% DCs) (Fig. 10B). Generation of stimulated skin-homing T cells was also inhibited (Fig. 10D).

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Figure 10. IL-6 mediates the increased stimulatory and skin-homing imprinting capacity of DCs in inflamed areas of UC patients. (A) Plot showing the correlation between IL-6 expression in DCs (as determined in Fig. 8) and the percentage of T-cell proliferation (measured by CFSE dilution). Each point represents a sample from one individual. (B, C), T cells were cultured with DCs conditioned with supernatants of (B) cultures of inflamed colonic biopsy and (C) cultures of healthy biopsy, and T-cell proliferation evaluated. Data are shown as mean of 10 (B) and 4 (C) independent experiments, respectively. Error bars represent the SEM. (D) Summary of eight and three independent experiments, respectively displaying CLA expression on stimulated T cells following culture with stimulated DCs. The percentage of divided T cells and their phenotype were determined as in Figure 9. Each point represents independent experiments and individuals. Pearson's correlation test (A), two-way ANOVA repeated measures (B and C) and paired t-test (D) were applied. P-value below 0.05 was considered statistically significant (*p < 0.05, ***p < 0.001).

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To confirm that IL-6 controls immune inflammation in UC patients, we challenged noninflamed areas from such patients with IL-6. Following IL-6 supplementation, a pro-inflammatory cytokine microenvironment was induced (Fig. 5) and protective MUC2 mRNA expression was decreased while Vα24 mRNA expression was increased (data not shown). When blood DCs were exposed to such an IL-6 rich microenvironment, they decreased their ongoing production of IL-10 (data not shown) and failed to decrease their stimulatory capacity compared to both basal and healthy-SN DCs (p < 0.05 at 3% DCs) (Fig. 10C). They also acquired the capacity to prime stimulated T cells with an increased skin-homing capacity (Fig. 10D).

Discussion

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

We have identified that in humans, inflamed areas of gut from UC patients have increased production of soluble pro-inflammatory cytokines, IL-6 being the predominant cytokine. These inflamed areas of gut have decreased mRNA expression of RALDH2 and CX3CR1. Such an intestinal microenvironment conditions the phenotype and function of blood enriched DCs. Noninflamed areas from UC patients biased DCs toward a regulatory phenotype with decreased stimulatory capacity. Paired DCs conditioned with inflamed areas from the same patients increased expression of skin-homing CCR8, did not decrease their stimulatory capacity, and primed T cells with the skin-homing CLA molecule in an IL-6 dependent mechanism. Our results question whether UC is a Th2-based disease and, if mirrored in vivo, they provide an explanation for generation of skin extraintestinal manifestations in UC patients.

DCs are sentinels and sensors of the immune system and their phenotype and function are dependent on the tissue microenvironment [[28]]. If DCs are cultured in a conditioned medium which had a previous culture of a human tissue and/or a human cell line, DCs express characteristics of resident DCs in such a tissue. Thus, human gut-like DCs can be generated from blood precursors following such a protocol [[22-26]]. Rather than studying the phenotype and function of human tissue DCs when the amount of tissue is scarce, and therefore functional experiments are not feasible, such methodology can be employed to study the effect

of tissue microenvironment on functions of more readily available blood DCs. Following conditioning with a tissue microenvironment from healthy or inflamed colonic areas from UC patients, DCs were matured in both cases as assessed by CD40 and CCR7 upregulation (Fig. 7). However, like DCs conditioned with a colonic microenvironment from healthy individuals, DCs conditioned with noninflamed areas from UC patients acquired a regulatory or less stimulatory phenotype and T cells that they stimulated acquired a gut-homing profile, characterized by a decreased CLA expression [[26]]. On the contrary, DCs conditioned with inflamed areas from UC patients matured toward a nonregulatory phenotype in an IL-6 dependent manner (Fig. 10). Similar observations regarding the capacity of intestinal IL-6 to promote immune responses in Crohn's disease patients have been recently described [[29]]. Future studies should identify specific mechanisms and signaling pathways through which IL-6-mediated effects are established on DCs.

Given the dual capacity of DCs to control both immunogenic and tolerogenic immune responses, recent mouse studies have suggested that two different tolerogenic (CD103+) and proinflammatory (CX3CR1+) nonoverlapping DCs subpopulations coexist in the gut [[30-32]]. These findings suggest that DCs responsible for regulatory and inflammatory responses might be of distinct origin, phenotype and function in resting and inflammatory conditions. If some circulating contaminating monocytes have remained in our blood enriched DCs and/or if the same functional dichotomy applies in human circulating DCs, it might be possible that different DC subtypes are involved in mediating “regulatory” and “inflammatory” responses in our culture system when exposed to different colonic microenvironments. However such studies obtained from mouse ileum remain to be validated in the human context and preliminary experiments suggest that such separation of populations may not be entirely true in human colon (data not shown). It seems more feasible that DC phenotype and function are conditioned by the surrounding microenvironment to perform a tolerogenic or nontolerogenic function depending on the required needs. In agreement with our findings, intestinal “tolerogenic” antigen presenting cells are reverted toward a proinflammatory phenotype when exposed to an inflamed microenvironment in mice models of colitis [[33, 34]]. Therefore, the UC inflamed microenvironment may modulate in vivo the phenotype of newly arrived DCs toward a proinflammatory phenotype.

Although healthy intestinal areas from UC patients have decreased expression of proinflammatory mediators compared with expression in paired inflamed areas (Fig. 1A), such mediators are still higher than those from biopsies of healthy controls [[27]]. That increase may explain why DCs conditioned with noninflamed areas from UC patients failed to increase expression of gut-homing markers on DCs; the latter occurs when DCs are exposed to intestinal culture supernatants from healthy controls [[26]]. We also showed that inflamed areas from UC patients had decreased mRNA expression of both CX3CR1 and RALDH2 (Fig. 6) compared with expression in noninflamed areas. We do not know in which cell type they are expressed, but both molecules are essential in mechanisms of intestinal homeostasis and oral tolerance [[12-14, 18]] despite recent descriptions of a proinflammatory role for retinoic acid at low concentrations in the presence of IL-15 [[35]]. It is not clear whether such decreased expression is a consequence of the proinflammatory cytokine microenvironment (Fig. 1A) or whether the proinflammatory phenotype is a consequence of decreased CX3CR1 and/or RALDH2 (among other possible factors). Also, the role of local iNKT cannot be discounted. This cell population can display both regulatory and proinflammatory properties [[36]] and has been proposed as the original source of proinflammatory cytokines in UC [[3]]. Following IL-6 challenge of healthy tissue from UC patients, we have identified increased Vα24 expression that suggests an iNKT cell expansion.

The causes of extraintestinal manifestations in IBD are poorly understood. Such extraintestinal manifestations may be a consequence of dysregulation in lymphocyte-homing pathways [[37-39]]. In agreement, our findings demonstrate the effect of tissue microenvironment in controlling DC phenotype and function, and provide an explanation for development of extraintestinal manifestations in UC patients due to a dysregulated homing response. Thus, following conditioning with inflamed areas from UC patients DCs acquired a nonregulatory skin-homing phenotype together with an increased capacity to generate skin-homing T cells through an IL-6 dependent mechanism. If the same applies in vivo, our results provide an explanation for generation of extraintestinal manifestations in the skin in UC patients

UC has been traditionally considered as a Th2 disease, with IL-13 as the effector cytokine [[2, 3]]. IL13 mediates damage of intestinal epithelial cells in murine models of colitis [[40]], but there is no proof that IL-13 is pathological relevant in UC in humans since mouse models do not always reflect mechanisms and/or cytokine profiles found in humans [[41]]. Others have described a role for IL-13 in human UC pathogenesis characterized by mRNA expression in human colonic biopsies [[42]] or by IL-13 secretion of total lamina propria mononuclear cells following in vitro stimulation [[2]]. In contrast, our assessment of the local cytokine milieu in human samples was performed in the absence of any external stimulus allowing measurement of spontaneous cytokine production and does not support IL-13-mediated effects. Blocking IL-13 in the cultures did not have a major effect on the cytokines in inflamed areas from UC patients (Fig. 2) while addition of recombinant IL-13 to healthy intestinal areas (Fig. 3) did not have a comparable effect in production of proinflammatory cytokines to that achieved with IL-6 (Fig. 5). IL-6 was the predominant cytokine found in inflamed areas from UC patients and the most predominant cytokines were related to a Th1 profile (Fig. 1B). Functional potency of secreted cytokines is highly dependent on affinity and density of specific cytokine receptors and therefore a given cytokine might be “ignored” with lower specific receptor densities [[43]]. However, IL-6 is also correlated with the Mayo endoscopic score for severity of disease (Fig. 1C) while its blockage in inflamed areas reverted the inflammation, and its supplementation to healthy areas mimicked the inflammation. Together, our data questions the relevance of IL-13 and highlights the role of IL-6 in UC [[27, 44, 45]] suggesting that UC may be characterized by a mixed profile rather than a Th2 cytokine profile [[46, 47]]. Although current therapies for UC patients involve systemic administration of anti-TNF-α, anti-IL-6 (alone or in combination with TNF-α) is revealed as potentially more efficient as some authors have recently reported [[48-50]].

In summary, our data questions the concept of UC as a Th2 disease and the relevance of IL-13 in its etiology, highlighting the role of IL-6 as a central cytokine controlling the local immune response. Our results confirmed that the tissue microenvironment conditions the phenotype and function of DCs. DCs conditioned with inflamed areas from UC patients, unlike the noninflamed areas, did not decrease their stimulatory capacity and increased their skin-homing phenotype and skin-homing imprinting capacity on T cells in an IL-6 dependent mechanism. If mirrored in vivo, this increased potential for skin homing provides an explanation for the generation of extraintestinal manifestations in the skin in UC patients and provides us with new targets for immunomodulation.

Material and methods

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

Colonic samples and biopsy culture

Colonic biopsies were obtained from 12 active UC patients following informed consent after ethical approval from the Hospital Clínico Universitario de Valladolid (Spain) and the Outer West London Research Ethics Committee (United Kingdom). Total extension of the lesion and the Mayo endoscopic score for severity of the disease were determined for each patient (Table 1). Paired samples were collected from macroscopically inflamed and macroscopically healthy noninflamed areas of the gut in ice-chilled PBS and biopsy culture started within an hour in 1.5 mL of complete medium (Dutch modified RPMI 1640 (Sigma-Aldrich, Dorset, UK) containing 100 u/mL penicillin/streptomycin, 2 mM L-glutamine, 50 μg/mL gentamicin (Sigma-Aldrich) and 10% fetal calf serum (TCS cellworks, Buckingham, UK)) for 24 h in 12-well culture dishes (1 biopsy/well) (37°C, 5% CO2). In some cases, extra biopsies from inflamed areas were cultured in the presence of anti IL-6 (0.5 μg/mL, R&D, Minneapolis, USA) or anti IL-13 blocking antibody (2.5 μg/mL, R&D) while in others, biopsies from healthy areas were cultured in the presence of recombinant IL-6 (50 ng/mL, R&D) or IL-13 (50 ng/mL, R&D). After 24 h, media from biopsy culture were centrifuged and cell-free SNs collected, while tissue was embedded in RNAlater (Ambion Inc., Austin, Texas, USA) and snap-frozen. Total RNA was isolated from each biopsy using the TRIZOL® reagent according to the protocol provided by the manufacturer. Reverse transcription was carried out by using the SuperScript® First-Strand Synthesis System for reverse Transcriptase (RT)-PCR Kit (Invitrogen, Life Technologies, Carlsbad, CA, USA) using random hexamers as primers.

Table 1. Clinical data of UC patients enrolled in this studya
PatientGenderAgeMayo endoscopic scoreExtension
  1. a

    Data includes gender, age, Mayo endoscopic score (from 0 to 3) and extension of the disease (1: only rectal; 2: lesion affects up to the splenic flexure; 3: lesion passes the splenic flexure).

1Male5423
2Male2923
3Male3432
4Female6433
5Female5111
6Male7012
7Female5731
8Female6511
9Female8222
10Female5012
11Female6621
12Male6612

Blood samples and biopsy conditioning

Human peripheral blood was collected from healthy volunteers with no known autoimmune or inflammatory diseases, allergies, or malignancies, following informed consent. Peripheral blood mononuclear cells (PBMC) were obtained by centrifugation over Ficoll-Paque Plus (Amersham Biosciences, Chalfont St. Giles, UK). Human blood enriched DCs were obtained following NycoPrepTM centrifugation of overnight cultured PBMCs [[51, 52]]. DCs from each donor were incubated for 24 h (0.5 million cells/mL) in the presence of basal medium and SNs from paired healthy and inflamed colonic areas from a single UC patient [[26]].

Quantitative polymerase chain reaction

mRNA levels of 16s, Muc2, FoxP3, Vα24, CX3CR1, RALDH2, and the housekeeping gene GADPH, were measured by real-time PCR by using a LightCycler® instrument (Roche Applied Science, Mannheim, Germany). Reactions were performed using the FastStart SYBR Green MasterMix (Roche) with thermo-labile Uracil DNA Glycosylase (UDG) (Roche) to prevent carry-over contamination. Primer sets and PCR conditions are described in Table 2. mRNA levels are expressed as the ratio molecule/GADPH in arbitrary units (U).

Table 2. Quantitative PCR primers
MoleculePrimers sequenceTaaPrimers source
  1. a

    Annealing temperature (Ta).

GADPHFw 5′-GAAGGTGAAGGTCGGAGTC-3′60[[54]]
 Rv 5′-GAAGATGGTGATGGGATTTC-3′  
16sFw 5′-TTAAACTCAAAGGAATTGACGG-3′68[[55]]
 Rv 5′-CTCACGRCACGAGCTGACGAC-3′  
FoxP3Fw 5′-CAGCACATTCCCAGAGTTCCTC-3′68[[56]]
 Rv 5′-CGCTGTGAACCAGTGGTAGATC-3′  
Vα24Fw 5′-CTGGAGGGAAAGAACTGC-3′55[[57]]
 Rv 5′-TGTCAGGGAAACAGGACC-3′  
MUC2-60Hs00159374_Applied
CX3CR1-605532957001_Roche
RALDH2-605532957001_Roche

Cytokines in culture supernatants

Cell-free culture supernatants were analyzed by using a multiplex assay (Biorad, Hercules, CA, USA) on a Luminex TM platform (Austin, TX, USA), following the manufacturer's instructions, for the concentration of interferon (IFN)-α [detection limit (D.L.) 125 pg/mL], IFN-γ [D.L. 4.04 pg/mL], tumor necrosis factor α (TNF-α) [D.L. 3.69 pg/mL], interleukin (IL)-4 [D.L. 0.43 pg/mL], IL-6 [D.L. 25.7 pg/mL], IL-7 [D.L. 3.18 pg/mL], IL-10 [D.L. 1.39 pg/mL], IL-13 (D.L. 3.74 pg/mL), and IL-17 [D.L. 12.63 pg/mL]. Those values below the level of detection were reported as being equal to that.

Antibody labeling

Table 3 shows the specificity, clone, and fluorochrome of the monoclonal antibodies used. Cells were labeled in PBS containing 1 mM EDTA and 0.02% sodium azide (FACS buffer). Labeling was performed on ice and in the dark for 20 min. Cells were washed twice in FACS buffer, fixed with 1% paraformaldehyde in 0.85% saline, and stored at 4°C prior to acquisition on the flow cytometer (within 48 h). Appropriate isotype-matched control antibodies were purchased from the same manufacturers. For intracellular staining, cells were fixed with Leucoperm A following surface staining, and permeabilized with Leucoperm B before adding antibodies for intracellular labeling. The intracellular cytokine production by nonstimulated DCs was measured using superenhanced Dmax (SED) normalized subtraction (see below) to subtract the normal cumulative histogram for cytokine staining with no added monensin from a similar histogram of staining with cytokine and added monensin for the last 4 h of cell culture (Sigma, UK) [[8, 26]]. After incubation cells were washed in FACS buffer, fixed and acquired.

Table 3. Antibodies and flow cytometry
Antibody specificityCloneConjugateManufacturer
HLA-DRL243 (G46-6)PECy5BD Biosciences
CD40LOB7/6FITCAbD Serotec
CD83HB15ePEBD Biosciences
CD86BU63PEAbD Serotec
CX3CR1528728PER&D
TLR2TLR2.3FITCAbD Serotec
TLR4HTA125FITCAbD Serotec
β7FIB504PEBD Biosciences
β7FIB504PECy5BD Biosciences
CLAHECA-452FITCBD Biosciences
CLAHECA-452BiotinBD Biosciences
Streptavidin-APCBD Biosciences
CD103Ber-ACT8FITCBD Biosciences
CCR4205410APCR&D
CCR10314315APCR&D
CCR7150503PER&D
IL-6AS12FITCBD Biosciences
IL-10JES3-19F1APCBD Biosciences
IL-12(p40/p70)C11.5PEBD Biosciences
TGF-β35409PER&D
CD3UCHT1PeCy5BD Biosciences

Flow cytometry and data analysis

Cells were acquired on a FACSCalibur cytometer (BD Biosciences) and analyzed using WinList 5.0 software (Verity, ME, USA). For single parameter analysis, WinList was used to subtract the normal cumulative histogram for isotype control staining from a similar histogram of staining with the test antibody using the superenhanced Dmax (SED) normalized subtraction [[53]]. For multiple parameter analysis, positive and negative gates were set up, determined by reference to staining with isotype-matched control antibodies.

Proliferation assays

T cells were obtained from freshly isolated PBMCs and suspended in MiniMACs buffer (PBS containing 0.5% BSA and 2 mM EDTA). PBMCs were depleted of CD14, CD19, and HLA-DR positive cells with immunomagnetic beads (Miltenyi Biotech, Bisley, UK) following manufacturer's instructions. Remaining T cells were labeled with CFSE (Invitrogen Ltd, UK) following manufacturer's instructions. CFSE-labeled T cells (400,000) were incubated for 5 days in round-bottomed 96-well microtiter plates (Becton Dickinson) with or without different concentrations of allogeneic DCs (1%, 2%, or 3% of T cells), previously conditioned in a different microenvironment. Cells were recovered and percentage and phenotype of stimulated T cells (CFSElow) were quantified by flow cytometry within total CD3+ T cells in the lymphogate.

Statistical analyses

Two-tailed paired t-test, two-tailed Pearson's correlation, and one- or two-way paired ANOVA were applied as stated in the figure legends. In the case of multiple comparisons, subsequent ad-hoc Bonferroni correction was applied. The level of significance was fixed at p < 0.05.

Acknowledgements

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

This work was supported by Marie Curie Intra European Fellowship (FP7-people-IEF-2008-235993), St Mark's Hospital Foundation, the Brigid Balfour Fund, and Junta de Castilla y León (GRS175/B/07).

Conflict of interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Material and methods
  7. Acknowledgements
  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. Material and methods
  7. Acknowledgements
  8. Conflict of interest
  9. References
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Abbreviations
IBD

inflammatory bowel disease

iNKT

invariant Vα24 restricted T-cells

RA

retinoic acid

SN

culture supernatant

UC

ulcerative colitis