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Keywords:

  • CD64;
  • Colitis;
  • Dendritic cells;
  • Intestine;
  • MΦs

Abstract

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

Dendritic cells (DCs) and monocyte-derived macrophages (MΦs) are key components of intestinal immunity. However, the lack of surface markers differentiating MΦs from DCs has hampered understanding of their respective functions. Here, we demonstrate that, using CD64 expression, MΦs can be distinguished from DCs in the intestine of both mice and humans. On that basis, we revisit the phenotype of intestinal DCs in the absence of contaminating MΦs and we delineate a developmental pathway in the healthy intestine that leads from newly extravasated Ly-6Chi monocytes to intestinal MΦs. We determine how inflammation impacts this pathway and show that T cell-mediated colitis is associated with massive recruitment of monocytes to the intestine and the mesenteric lymph node (MLN). There, these monocytes differentiate into inflammatory MΦs endowed with phagocytic activity and the ability to produce inducible nitric oxide synthase. In the MLNs, inflammatory MΦs are located in the T-cell zone and trigger the induction of proinflammatory T cells. Finally, T cell-mediated colitis develops irrespective of intestinal DC migration, an unexpected finding supporting an important role for MLN-resident inflammatory MΦs in the etiology of T cell-mediated colitis.


Introduction

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

The intestinal lamina propria (LP) contains cells that express high levels of CX3CR1, the receptor for the fractalkine chemokine [1, 2]. Based on their monocytic origin and on their inability to migrate to the mesenteric lymph nodes (MLNs) such CX3CR1hi cells have been defined as macrophages (MΦs) [1-3]. CX3CR1hi MΦs contribute to the intestinal LP homeostasis through the production of anti-inflammatory cytokines and the clearance of commensal bacteria that breach the epithelial barrier [4]. In contrast, during intestinal inflammation, microenvironmental signals promote the differentiation of extravasated monocytes into proinflammatory MΦs with the ability to produce interleukin (IL)-12, IL-23, tumor necrosis (TNF)-α and inducible nitric oxide synthase (iNOS) [5-7]. However, little is known about the developmental trajectories that lead extravasated monocytes to either anti- or proinflammatory intestinal MΦs. This is primarily due to the fact that a surface marker permitting unequivocal identification of MΦs within the intestine and their distinction from dendritic cells (DCs) is lacking.

The interstitial DCs (Int-DCs) present throughout the LP derive from blood precursors known as pre-DCs [2]. Under steady-state conditions, the Int-DCs found in the intestinal LP induce oral tolerance by carrying antigens originating from food or from harmless bacteria to the MLNs [8, 9]. The CD103+ Int-DCs found in the steady-state LP have the selective ability to express aldehyde dehydrogenase (ALDH) and thereby produce retinoic acid (RA). As a result, upon migration to MLNs they trigger the differentiation of naive CD4+ T cells specific for food and microbiota antigens into induced Foxp3+ regulatory T (iTreg) cells [10-13]. In contrast, the Int-DCs that develop in inflamed LP upon exposure to pathogens lose their capacity to generate iTreg cells and, upon migration to the MLNs, trigger the differentiation of naive, antigen-responsive CD4+ T cells into T helper type 1 (Th1) cells that are specific for the invading pathogen and produce mainly IFN-γ [9, 14].

We have previously reported that the monocyte-derived cells that are present in skeletal muscle express high levels of CD64, the high-affinity IgG receptor FcγR [15]. By using the expression of CD64, we also succeeded in distinguishing MΦs from DCs in the LP of the large and small intestine and the MLNs of both mice and humans. We thus reinvestigated at a high resolution MF and DC development in the intestinal LP under healthy conditions and studied how it was affected by inflammation. In this process, we found that during T cell-mediated colitis a similar developmental pathway gives rise to inflammatory MΦs in both the intestinal LP and in the MLNs. Moreover, we made the highly unanticipated observation that T cell-mediated colitis unfolds in absence of a normal influx of LP-derived migratory DCs (Mig-DCs) and showed that the numerically dominant inflammatory MΦs that develop within the MLNs also contribute to the induction of proinflammatory CD4+ T cells during T cell-mediated colitis.

Results

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

CD64 expression distinguishes MΦs from DCs

We sought to identify a surface marker that would allow the distinction of MΦs from DCs in the intestine and the MLNs. Using mice expressing an enhanced green fluorescent protein (EGFP) under the control of the gene coding for the CX3CR1 chemokine receptor (CX3CR1-EGFP mice; [16]), we found that all the CX3CR1hi MΦs of the LP of the small and large intestine expressed CD64 (Fig. 1A). Moreover, by combining CD64 and CD11c expression, we observed that the MHC class II (MHCII)-positive cells present in the LP of the small and large intestine could be readily separated into a CD11chiCD64 and a CD11c− to intCD64+ subset (Fig. 1B and C).

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Figure 1. CD64 expression distinguishes monocyte-derived MΦs from DCs in the intestinal LP and the MLNs. Cells were prepared from (A) the LP of the small and large intestine of CX3CR1-EGFP mice and (B–E) the LP of the small and large intestine, the MLNs and the blood of B6 (CD45.1) WT + B6 (CD45.2) Ccr2−/− [RIGHTWARDS ARROW] B6 (CD45.1-CD45.2) chimeras. (A–D) After excluding NK cells, B cells, T cells, eosinophils, and neutrophils, the specified cells were analyzed by flow cytometry. (A) MHCII+ cells of the specified organs were analyzed for CX3CR1(EGFP) and CD64 expression. Percentages of CX3CR1hiCD64+ cells are indicated. (B–D) MHCII+ cells from the LP of the small and large intestine and from the MLNs were analyzed for CD11c and CD64 expression. The CD11chiCD64 and CD11c− to intCD64+ subsets were then analyzed for the percentages of CD45.1+ (WT) and CD45.2+ (Ccr2−/−) donor cells. (E) CD11b+ blood cells were divided into Ly-6G+ neutrophils and Ly-6ChiLy-6G monocytes and analyzed for the percentages of CD45.1+ (WT) and CD45.2+ (Ccr2−/−) donor cells. The percentages of cells found in each of the specified gates are indicated. (A) Data are representative of three independent experiments and (B–E) data are representative of at least 12 chimeric mice corresponding to three independent experiments.

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Ly-6Chi monocytes express CCR2, a chemokine receptor that promotes their egress from the bone marrow into the blood, and as a result CCR2-deficient (Ccr2−/−) mice show markedly reduced numbers of circulating Ly-6Chi monocytes and tissue MΦs [17]. In contrast, DCs are not affected by CCR2 deficiency [18]. Therefore, we used this differential CCR2 requirement to determine whether the presence of CD11chiCD64 and CD11c− to intCD64+ cells in the intestinal LP depended on CCR2 expression. Accordingly, mice coexpressing CD45.1 and CD45.2 were lethally irradiated and reconstituted with a 1 to 1 mixture of BM cells isolated from CD45.1+ wild-type (WT) mice and from CD45.2+ Ccr2−/− mice. These B6 (CD45.1) WT + B6 (CD45.2) Ccr2−/− [RIGHTWARDS ARROW] B6 (CD45.1-CD45.2) competitive chimeras were analyzed 8 weeks after BM transfer. As expected, Ly-6Chi blood monocytes were primarily composed of CD45.1+ Ccr2+ donor cells whereas neutrophils were comprised of almost equal percentages of CD45.1 and CD45.2 donor cells (Fig. 1E). CCR2-deficient and WT donor cells contributed equally to the CD11chiCD64 intestinal LP cells (Fig. 1B and C). The capacity to develop in a CCR2-independent manner combined with a specific absence in mice lacking the ligand for the Fms-like tyrosine kinase receptor 3 (Flt3L; Supporting Information Fig. 1A), strongly suggest that the CD11chiCD64 intestinal LP cells correspond to Int-DCs. In contrast, the CD11c− to intCD64+ intestinal LP cells showed percentages of CD45.1 and CD45.2 donor cells identical to those of Ly-6Chi blood monocytes (Fig. 1B and C). Consistent with the view that CD11c− to intCD64+ intestinal LP cells constitute monocyte-derived MΦs, they developed independently of Flt3L (Supporting Information Fig. 1A). A similar conclusion applied to the few CD11c− to intCD64+ cells found among the MHCII+ cells present in the MLNs of mice kept under specific pathogen-free conditions (Fig. 1D). Therefore, by combining CD64 and CD11c expression, it is possible to unequivocally distinguish CD11c− to intCD64+ MΦs from CD11chiCD64 DCs among the MHCII+ cells of the intestinal LP and of the MLNs without having to rely on the use of CX3CR1-EGFP reporter mice.

CD8α+-type and CD11b+-type intestinal DCs

Conventional DCs can be categorized into CD8α+- and CD11b+-type DCs [19, 20]. The CD11chiCD64 Int-DCs of the LP and their migratory counterparts present in the MLNs showed the same dichotomy and comprised CD8α+- and CD11b+-type DCs that can be unambiguously identified by their CD24+CD11b and CD11b+CD64 phenotype, respectively (Supporting Information Fig. 1B). As expected, CD11b+-type DCs expressed CD172α (SIRPα) whereas CD8α+-type DCs were CD172α (Supporting Information Fig. 1B). In contrast to CD64+ MΦs that lacked CD103 (data not shown), a substantial fraction of CD24+CD11b and CD11b+CD64 DCs found in the LP and the MLNs expressed CD103 (Supporting Information Fig. 1C). Notably, CD24+CD11b DCs were CX3CR1, whereas CD11b+CD64 DCs were CX3CR1int (Supporting Information Fig. 1C), emphasizing that in the intestine CX3CR1 expression is not restricted to monocyte-derived cells (Fig. 1A). Likewise, F4/80 was expressed at high and intermediate levels on CD11c− to intCD64+ MΦs and CD11b+CD64 DCs, respectively (Supporting Information Fig. 1C). Therefore, by identifying MΦs on the basis of their CD11c− to intCD64+ phenotype and subsequently subdividing CD11chiCD64 DCs into CD24+CD11b and CD11b+CD64 cells, it is possible to overcome several of the limitations previously encountered in the identification of MΦs and DC subsets in the intestinal LP and the MLNs.

CD64 marks MΦs in humans

To evaluate whether CD64 could be also used to identify intestinal MΦs in humans, we generated humanized mice and determined whether the HLA-DR+ cells found in their intestinal LP also contained CD64+ MΦs in addition to DCs. CD34+ human stem cells from cord-blood were transfected with GFP and then injected into NOD-scid-IL-2Rγ−/− (NSG) mice. Eleven weeks after reconstitution, the GFP+HLA-DR+ cells found in the intestinal LP could be readily divided into CD64− to low and CD64hi cells (Supporting Information Fig. 2A). The CD64− to low cells were composed of BDCA3+ and BDCA1+ cells, which represent the human equivalent of mouse CD8α+- and CD11b+-type DCs, respectively [19]. In contrast, the CD64hi cells were low for both BDCA3 and BDCA1 and likely corresponded to LP MΦs (data not shown).

To support our conclusion that human intestinal MΦs expressed CD64, we analyzed colon biopsies obtained from patients suffering from ulcerative colitis. The HLA-DR+ LP cells found in healthy sections of the human colon contained BDCA3+ and BDCA1+ DCs as well as CD64hi cells that based on expression of CD14 likely corresponded to monocyte-derived cell (Supporting Information Fig. 2B). Interestingly, analysis of diseased tissue samples from the same patients showed a dramatic increase in the proportion of CD14+CD64hi MΦs among HLA-DR+ LP cells as compared with healthy tissue samples. Therefore, HLA-DR+ MΦs were CD64hi in both steady state and inflamed human colon whereas colonic DCs were CD64− to low.

Connecting recently extravasated Ly-6Chi monocytes to intestinal MΦs

The CD11b+ cells found in the LP of the small intestine comprised CX3CR1int and CX3CR1hi cells (Fig. 2A). In contrast to the CX3CR1hi cells that are only constituted of MΦs (Fig. 2B), the CX3CR1int cells were heterogeneous and contained CD11b+-type Int-DCs that can be readily gated out on the basis of their CD11chiCD64 phenotype (Fig. 2C). Analysis of the remaining, Int-DC-deprived, CX3CR1int cells on a Ly-6C-MHCII plot led to the identification of three populations (Fig. 2C). Population 1 (P1) resembled Ly-6Chi blood monocytes in terms of FSC-SSC profile (data not shown) and of its Ly-6Chi, CD64low, MHCII, CD11c− to low, CX3CR1int, and CD11b+ phenotype (Fig. 2C and D). P1 cells in the LP can also be identified on the basis of their unique CD11b+Ly-6Chi phenotype (Fig. 2E). Population 2 (P2) was composed of Ly-6Cint to hiMHCII+ cells whereas population 3 (P3) had a Ly-6ClowMHCII+ phenotype identical to that of the CX3CR1high MΦs that are denoted as P4 cells (Fig. 2B and C). Note that the P3 and P4 cells can only be distinguished on the basis of their distinct levels of expression of the CX3CR1(EGFP) reporter (Fig. 2). Therefore, in WT mice, P3 and P4 cells cannot be distinguished and are considered below as a single Ly-6ClowMHCII+CD64+ P3/P4 population. The P2 cells “bridged” the diagonally opposite positions occupied by the P1 and the P3/P4 cells, a feature expected for developmental intermediates linking recently extravasated Ly-6ChiMHCII monocytes to CX3CR1int and CX3CR1high MΦs.

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Figure 2. CD64, Ly-6C, CD11b, CD11c, MHCII, and CX3CR1 expression in blood monocytes and in CX3CR1int and CX3CR1hi cells of the LP of the small intestine. Cells were prepared from the specified organs of CX3CR1-EGFP mice. (A) After excluding NK cells, B cells, T cells, eosinophils, and neutrophils, the remaining cells were analyzed by flow cytometry for CD11b and CX3CR1 expression. (B) Analysis of CD11b+CX3CR1hi cells for CD11c, CD64, Ly-6C, MHCII, and CX3CR1 expression. (C) Analysis of CD11b+CX3CR1int cells for CD11c, CD64, Ly-6C, MHCII, and CX3CR1 expression. In the CX3CR1int ΔDCs plots, CD11chiCD64 Int-DCs were excluded from CD11b+CX3CR1int cells prior to analysis. The P1 (Ly-6ChiMHCIICD64lowCX3CR1int), P2 (Ly-6Cint to hiMHCII+CD64lowCX3CR1int), P3 (Ly-6ClowMHCII+CD64+CX3CR1int) and P4 (Ly-6ClowMHCII+CD64+CX3CR1hi) stages are highlighted. (D) Analysis of CD11b+Ly-6Chi blood monocytes for CD64, Ly-6C, CD11c, MHCII, and CX3CR1 expression. (E) Analysis of CD11b+Ly-6Chi monocytes of the small intestine for CD64, Ly-6C, CD11c, MHCII, and CX3CR1 expression. The percentages of cells found in each of the specified gates are indicated. Data are representative of at least three independent experiments.

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When CD11b+ LP cells from WT mice were analyzed on a Ly-6C-CD64 plot they comprised Ly-6CCD64 cells that were CCR2 independent and corresponded to CD11b+ Int-DCs (Fig. 3A). The remaining cells (red gate; Fig. 3A) strictly depended on CCR2 expression for their development and were thus of monocytic origin (Fig. 3A). They adopted a similar waterfall-shaped distribution on Ly-6C-CD64 and Ly-6C-MHCII plots (Fig. 3A), a feature consistent with the fact that CD64 and MHCII expression followed the same trend along the putative P1 [RIGHTWARDS ARROW] P2 [RIGHTWARDS ARROW] P3 [RIGHTWARDS ARROW] P4 developmental series. For the sake of brevity, the gate including the P1, P2, and P3/P4 cells is thus referred here as the “Mo-waterfall gate” and the waterfall-shaped distribution as the “Mo-waterfall” (Fig. 3B) A similar Mo-waterfall was also observed in the LP of the large intestine (Fig. 3C).

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Figure 3. A developmental series links extravasated Ly-6Chi monocytes to intestinal MΦs. Cells were prepared from the LP of the small intestine of the specified mice. After excluding NK cells, B cells, T cells, eosinophils, and neutrophils, the remaining CD11b+ cells were analyzed by flow cytometry. (A) CD11b+ cells from B6 (CD45.1) WT + B6 (CD45.2) Ccr2−/− [RIGHTWARDS ARROW] B6 (CD45.1-CD45.2) chimeras were analyzed for Ly-6C and CD64 expression. They comprise Ly-6CCD64 Int-DCs and cells that adopt a waterfall-shaped flow cytometric distribution. The gate including those last cells is shown in red and denoted as the “Mo-waterfall gate.” Ly-6CCD64 Int-DCs and cells belonging to the Mo-waterfall gate were analyzed for the percentages of CD45.1+ (WT) and CD45.2+ (Ccr2−/−) donor cells. Expression of Ly-6C, CD11c, MHCII, CD64, and CX3CR1 is also shown for cells belonging to the Mo-waterfall gate. Red arrows highlight the putative developmental path followed during Ly-6Chi monocyte differentiation in the LP. (B, C) The same Mo-waterfall is also observed in the LP of the small (B) and large (C) intestine of CX3CR1-EGFP mice using Ly-6C-MHCII plot. The levels of CX3CR1, CD172α, and CCR7 are shown for the P1, P2, and P3/P4 populations. The percentages of cells found in each of the specified gates are indicated and are representative of three independent experiments. (D, E) Sorted Ly6-Chi monocytes from B6 mice were CFSE labeled and transferred into healthy Ccr2−/− mice (monocyte transfer) or kept in culture (monocytes in culture). Twelve hours and 72 h after transfer, expression of Ly-6C, MHCII, CD11c, and CD64 were analyzed on donor-derived CFSE+ cells isolated from the LP (D) or from the cultures (E). The percentages of cells found in each of the specified gates are indicated. Data shown are representative of two experiments with three recipient mice at each time point.

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To validate the hypothesis that a precursor-product relationship exists between the P1, P2, and P3/P4 stages that composed the Mo-waterfall (specified by red arrows in Figure 3A, bottom panels), Ly-6Chi monocytes were labeled with CFSE and adoptively transferred into healthy Ccr2−/− mice. We used Ccr2−/− hosts because they have a selective reduction in circulating Ly-6Chi monocytes that should facilitate the engraftment of the transferred CFSE+ monocytes. Analysis of the CFSE+ donor cells found in the LP of the small intestine 12 h after transfer showed some increase in MHCII and CD64 expression (Fig. 3D). By 72 h after transfer, most donor cells expressed both MHCII and CD64 and had downregulated Ly-6C expression. A small fraction of the Ly-6ClowMHCII+ CD64+ donor cells also expressed CD11c. In contrast, when the CFSE-labeled Ly-6Chi monocytes were kept in culture, they did not change their original Ly-6ChiCD64lowMHCII phenotype upon time (Fig. 3E). These data demonstrate that upon entry into the intestinal LP, Ly-6Chi monocytes sequentially give rise to the P1, P2, and P3/P4 populations and that this progression was accompanied by the upregulation of CX3CR1, MHCII, CD64, and CD11c molecules and by the downregulation of Ly-6C (Fig. 3A and B). Moreover, the cells traversing this developmental series maintained CD172α expression and, importantly for the rest of this study, did not express CCR7 (Supporting information Fig. 3B and C). Therefore, by combining CD64 expression with Ly-6C and MHCII expression it is possible to track the progressive differentiation of newly extravasated Ly-6Chi monocytes into MΦs within healthy intestinal LP (Supporting Information Fig. 3).

T cell-mediated colitis induces a massive differentiation of Ly-6Chi monocytes into MΦs

To analyze the effect of inflammation on the dynamics of MF and DC differentiation in the intestinal LP and the MLNs, we used a model of T cell-mediated colitis that relied on Cd3eΔ5/Δ5 mice that lack T cells but not B cells [21]. Transfer of CD4+Foxp3 T cells into Cd3eΔ5/Δ5 mice allows their extensive proliferation and differentiation into effector CD4+ T cells that produce mainly IFN-γ and that home to the large intestine where they trigger a severe colonic inflammation, typified by weight loss, diarrhea, and rectal prolapse (Supporting Information Fig. 4; [22]). Using this model, we first analyzed the evolution of MF and DC populations in the LP during the development of T cell-mediated colitis. Under steady-state conditions, WT and Cd3eΔ5/Δ5 mice contained similar large numbers of CD64+ MΦs in the LP of the large intestine (Fig. 4A and data not shown). Upon transfer of CD4+ Foxp3 T cells into Cd3eΔ5/Δ5 mice, the numbers of CD64+ MΦs increased in a rapid and massive manner in the LP of the large intestine (Fig. 4A and B and Supporting Information Fig. 5A). The numbers of CD8α+- and CD11b+-type Int-DCs also increased although to a lesser extent (Fig. 4A and B). Therefore, as observed in patients suffering from ulcerative colitis (Supporting Information Fig. 2B), the colitis observed in Cd3eΔ5/Δ5 mice is also associated with a large increase in the proportion of CD64+ MΦs among MHCII+ LP cells.

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Figure 4. Blood Ly-6Chi monocytes are continuously recruited into the large intestine of colitic Cd3eΔ5/Δ5 mice where they differentiate into MΦs. (A) Pie charts show the relative percentage of CD64+ MΦs, CD24+CD11b, and CD11b+CD64 Int-DCs in the LP of the large intestine of WT B6 mice and of Cd3eΔ5/Δ5 mice that had received CD4+Foxp3 T cells (IBD). Cd3eΔ5/Δ5 mice were analyzed 4 weeks after transfer of CD4+Foxp3 T cells. (B) Kinetics of accumulation of the specified cells in Cd3eΔ5/Δ5 mice that had received CD4+Foxp3 T cells. (A, B) Data are representative of at least three independent experiments with a minimum of three mice per group in each experiment. Error bars correspond to the SEM. (C, D) Cells were prepared from the LP of the large intestine of Cd3eΔ5/Δ5 mice that had received CD4+Foxp3 T cells 3 weeks before. Four days prior to analysis, blood monocytes were labeled in vivo through intravenous injection of Alexa-647 beads. The Mo-waterfall (Fig. 3A) was analyzed for the presence of cells containing beads. Data shown are representative of at least three independent experiments. (E) Sorted Ly6-Chi monocytes were CFSE labeled and transferred into colitic Cd3eΔ5/Δ5 × Ccr2−/− mice. Twelve hours and 72 h after transfer, expression of Ly-6C, MHCII, CD11c, and CD64 were analyzed on donor-derived CFSE+ cells isolated from the LP of the large intestine. The percentages of cells found in each of the specified gates are indicated. Data shown are representative of two experiments with two recipient mice at each time point.

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The Mo-waterfall found in the inflamed LP differed from the one observed under healthy conditions in that it consisted primarily of Ly-6ChiMHCII (P1) and Ly-6Cint to hiMHCII+ (P2) cells and yielded comparably few Ly-6ClowMHCII+ (P3/P4) cells (compare Fig. 3C and Fig. 4C). Consistent with this last observation, adoptive transfer of CFSE-labeled Ly-6Chi BM monocytes into colitic Cd3eΔ5/Δ5 × Ccr2−/− mice gave rise to CD64+ LP MΦs that had a predominant Ly-6Cint to hiMHCII+ phenotype (Fig. 4E). The same conclusion can be reached after marking the Ly-6Chi blood monocytes present in Cd3eΔ5/Δ5 mice undergoing T cell-mediated colitis via intravenous injection of latex beads [23]. Four days after injection of latex beads, the bead-labeled cells showed a blunted waterfall-shaped distribution (Fig. 4D). Therefore, in mice undergoing colitis, blood monocytes are constantly recruited to the large intestine LP where they predominantly differentiate into Ly-6Cint to hiMHCII+CD64+ MΦs.

Colitis is associated with the differentiation of Ly-6Chi monocytes into MΦs in the MLNs

Unexpectedly, colitic Cd3eΔ5/Δ5 mice also displayed a rapid accumulation of CD64+ MΦs in the MLNs (Fig. 5 A and B and Supporting Information Fig. 5B). To determine whether these MΦs corresponded to LP-derived migratory MΦs, we determined if they expressed CCR7, a chemokine receptor required for the migration of intestinal DCs to the MLNs [24]. In contrast to the LP-derived Mig-DCs, the MΦs found in the MLNs of colitic Cd3eΔ5/Δ5 mice were CCR7 (Fig. 5C). It is thus likely that the MΦs that accumulate in the MLNs of colitic Cd3eΔ5/Δ5 mice do not represent the migratory counterpart of intestinal MΦs and originate from blood monocytes that seeded the MLNs and differentiated in situ. Consistent with this view, after excluding CD11b+-type DCs, the remaining CD11b+ monocytic cells present in the MLNs of colitic Cd3eΔ5/Δ5 mice showed a waterfall-shaped distribution that consisted primarily of Ly-6ChiMHCII and Ly-6Cint to hiMHCII+ cells and that resembled the blunted Mo-waterfall found in the LP of colitic mice (Fig. 5D). Moreover, the Ly-6ChiMHCII cells had an FSC-SSC profile typical of undifferentiated blood monocytes and their progression through the Mo-waterfall was associated with an increase in both cell size and granularity (Fig. 5D). Consistent with this last observation, adoptive transfer of CFSE-labeled Ly-6Chi monocytes into colitic Cd3eΔ5/Δ5 Ccr2−/− mice gave rise in the MLNs to CD64+ MΦs that were predominantly Ly-6Cint to hiMHCII+ (Fig. 5E). Importantly, in both the LP and the MLNs, the differentiation of adoptively transferred Ly-6Chi monocytes into CD64+ MΦs followed the same kinetics (compare Fig. 4E and 5E). In the case the MΦs found in the MLNs of colitic Cd3eΔ5/Δ5 mice will have represented migratory counterpart of intestinal MΦs, a protracted kinetic of reconstitution would have been expected in the MLNs as compared with that in the LP. Therefore, in Cd3eΔ5/Δ5 mice undergoing T cell-mediated colitis, Ly-6Chi blood monocytes are massively recruited to the MLNs where they differentiate into MΦs. The fact that they do not represent the migratory counterpart of intestinal MΦs is congruent with previous studies demonstrating that intestinal MΦs do not migrate to the MLNs under healthy or TLR-induced inflammatory conditions [3].

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Figure 5. Extravasated Ly-6Chi monocytes differentiate into MΦs in colitic MLNs. Cells were prepared from the MLNs of WT B6 mice and of Cd3eΔ5/Δ5 mice that had received CD4+Foxp3 T cells (IBD), and defined as CD64+ MΦs, CD24+CD11b, and CD11b+CD64 Mig-DCs (see Supporting Information Fig. 5B). (A) Pie charts show the relative percentage of the specified cells for the two groups of mice. Cd3eΔ5/Δ5 mice were analyzed 4 weeks after transfer of CD4+Foxp3 T cells. (B) Kinetics of accumulation of the specified cells. (A, B) Data shown are representative of at least three independent experiments with a minimum of three mice per group in each experiment. Error bars correspond to the SEM. (C) CCR7 expression on CD64+ MΦs, CD24+CD11b, and CD11b+CD64 Mig-DCs found in the MLNs of Cd3eΔ5/Δ5 mice 4 weeks after transfer of CD4+Foxp3 T cells. (D) The FSC-SSC profile of the cells belonging to the specified sections of the Mo-waterfall found in the MLNs of Cd3eΔ5/Δ5 mice was analyzed 4 weeks after transfer of CD4+Foxp3 T cells. (C, D) Data shown are representative of at least three independent experiments. (E) Sorted Ly6-Chi monocytes were CFSE labeled and transferred into colitic Cd3eΔ5/Δ5 × Ccr2−/− mice. Twelve hours and 72 h after transfer, expression of Ly-6C, MHCII, CD11c, and CD64 were analyzed on donor-derived CFSE+ cells isolated from the MLNs. The percentages of cells found in each of the specified gates are indicated. Data shown are representative of two experiments with two recipient mice at each time point.

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Ly-6Chi monocytes differentiate into iNOS-expressing inflammatory MΦs during T cell-mediated colitis

A fraction of the CD64+ MΦs that developed in the MLNs of colitic Cd3eΔ5/Δ5 mice that had received CD4+Foxp3 T cells became iNOS+ (Fig. 6A and B). iNOS+ MΦs also developed in the LP of the large intestine, but with slower kinetics compared with MLNs (Fig. 6A and B). The majority of iNOS+ MΦs present in the large intestine LP and the MLNs were Ly-6Cint to hiMHCII+ (Fig. 6C). In contrast, DCs remained iNOS at all time points (Fig. 6A). Therefore, during T cell-mediated colitis, a fraction of the blood monocytes that are recruited to the large intestine LP and the MLNs differentiate into iNOS-expressing inflammatory CD64+ MΦs.

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Figure 6. MLN-resident inflammatory MΦs are iNOS+, located in the T-cell zone and excel in the induction of IFN-γ-producing effector T cells in vitro. Cells were prepared from the MLNs and the large intestine LP of Cd3eΔ5/Δ5 mice that had received CD4+Foxp3 T cells. After excluding NK cells, B cells, T cells, eosinophils, and neutrophils, the remaining MHCII+ cells were analyzed for CD64, CD11c, CD24, CD11b, CCR7, and intracellular iNOS expression. The distinct cell subsets were identified as specified in Supporting Information Fig. 5. (A) Percentage of iNOS+ cells within the specified subsets at various time points after T-cell transfer. (B) iNOS expression within the MΦs found in the MLNs and the LP of the large intestine of colitic Cd3eΔ5/Δ5 mice. (C) iNOS+ cells from the MLNs and the large intestine of colitic Cd3eΔ5/Δ5 mice were analyzed for their position on the Mo-waterfall using Ly-6C-MHCII plot. (A–C) Data shown are representative of at least three independent experiments corresponding to three mice per group. The error bars correspond to the SEM. *p < 0.05, Student's t-test. (D) MLNs from colitic Cd3eΔ5/Δ5 mice were analyzed by confocal microscopy 4 weeks after transfer of CD4+Foxp3 T cells. Sections were stained with anti-CD11c, anti-CD3ε, anti-CD45R (B220), and anti-iNOS antibodies. As shown in the insets (lower right panel), iNOS+ cells were in close contact with CD3ε+ T cells. Scale bars, 20 μm. Data shown are representative of three independent experiments. (E) Cells isolated from MLNs of B6 mice (steady state) and of Cd3eΔ5/Δ5 mice that had received CD4+Foxp3 T cells 2 weeks earlier (IBD Day 15), were subjected to an assay measuring ALDH at the single-cell level and Mig-DC subsets were FACS-sorted into ALDH+ and ALDH fractions. The sorted MΦs and DCs were loaded with OVA323–339-peptide and cocultured with CFSE-labeled OT-II T cells. After 5 days of culture, OT-II T cells were restimulated with PMA and ionomycin and analyzed for intracellular IFN-γ and IL-17. The very small numbers of MΦs found in steady-state MLNs prevented their sorting. Data are shown as mean + SEM of triplicate cultures pooled from at least 3 independent experiments. *p < 0.05, Student's t-test.

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T cell-mediated colitis develops in mice with impaired migration of DCs to the MLNs

To evaluate the requirement of LP-derived Mig-DCs in the development of T cell-mediated colitis, we generated Cd3eΔ5/Δ5 × Ccr7−/− mice in which the CCR7-dependent migration of Int-DCs from the intestinal LP to the MLNs is impaired [24]. Cd3eΔ5/Δ5 × Ccr7−/− mice and Cd3eΔ5/Δ5 control mice were reconstituted with CD4+Foxp3 T cells isolated from CCR7-sufficient mice to avoid adventitious effects due to the lack of CCR7 on T cells, and analyzed 4 weeks after transfer. As expected, while both types of mice had comparable numbers of Int-DCs in the LP of the large intestine (Fig. 7A), the MLNs of Cd3eΔ5/Δ5 × Ccr7−/− mice contained numbers of CD8α+-type and CD11b+-type Mig-DCs that were reduced fourfold and sevenfold respectively, as compared with that in the Cd3eΔ5/Δ5 mice (Fig. 7A). In contrast, the numbers of MLN-resident MΦs were comparable in both types of mice (Fig. 7A). Unexpectedly, preventing a normal influx of LP-derived Mig-DCs resulted in exacerbated T cell-mediated colitis as shown by the increased percentage of IFN-γ-producing effector T cells in the large intestine and the MLNs (Fig. 7B and C). This amplified inflammatory response was also associated with a more rapid weight loss (Fig. 7D). Therefore, in the Cd3eΔ5/Δ5model, T cell-mediated colitis unfolds irrespective of a normal influx of LP-derived Mig-DCs.

image

Figure 7. Cd3eΔ5/Δ5 × Ccr7−/− mice develop severe T cell-mediated colitis. (A–C) 4 weeks after transfer of CD4+Foxp3 T cells into Cd3eΔ5/Δ5 mice and Cd3eΔ5/Δ5 × Ccr7−/− mice, cells from the MLNs and the LP of the large intestine were analyzed. (A) Cells were subdivided into the specified subsets and their numbers determined. (B, C) Cells from (B) the MLNs and (C) the large intestine LP were restimulated in vitro with PMA plus ionomycin to assess for their capacity to produce IFN-γ and IL-17. Percentages of IFN-γ+ and IL-17+ cells among CD4+ T cells from Cd3eΔ5/Δ5 mice and Cd3eΔ5/Δ5 × Ccr7−/− mice are shown. (D) Weight fluctuation of Cd3eΔ5/Δ5 mice and Cd3eΔ5/Δ5 × Ccr7−/− mice transferred with CD4+ Foxp3 T cells. Data are shown as mean +− SEM of at least three (A–C) and six (D) mice per group and are pooled from three independent. * p < 0.05, Student's t-test.

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MLN-resident inflammatory MΦs induce IFN-γ-producing effector T cells in vitro

We next analyzed whether the numerically dominant inflammatory MΦs that develop within the MLNs can participate in the induction of colitogenic T cells. First, we assessed whether they were located within the T-cell zone of the MLNs, a prerequisite for the activation of naive CD4+ T cells. Although CD64 expression would have constituted the best way of localizing MLN-resident inflammatory MΦs, our fixation protocol prevented the use of this marker. Considering that iNOS+ inflammatory MΦs expressed high levels of CD11c and that iNOS expression is restrained to these cells in the MLNs of colitic mice (Fig. 6A and B), we relied on iNOS and CD11c coexpression to locate MLN-resident inflammatory MΦs. Confocal microscopy showed that iNOS+CD11c+ inflammatory MΦs were primarily found within the T-cell zone of the MLNs of colitic Cd3eΔ5/Δ5 mice (Fig. 6D). Moreover, MLN-resident inflammatory MΦs had a high phagocytic activity and lacked a DC-morphology (Supporting Information Fig. 6).

Next, we assessed whether the inflammatory MΦs that develop in the MLNs of colitic Cd3eΔ5/Δ5 mice were capable of converting naïve CD4+ T cells into effector T cells in vitro and we compared them with the LP-derived Mig-DCs that are also found in colitic MLNs. Considering that colitic MLNs still contains some tolerogenic ALDH+ Mig-DCs (data not shown) that may have led us to underestimate the IFN-γ-producing capacity of ALDH Mig-DCs, LP-derived Mig-DCs were sorted into ALDH+ and ALDH cells. The various sorted cell types were pulsed with an ovalbumin (OVA)-derived peptide and cocultured with OVA-specific OT-II CD4+ T cells. After 5 days of culture, OT-II T cells were analyzed for the production of IFN-γ and IL-17 (Fig. 6E). Surprisingly, among the analyzed MLN cells, inflammatory MΦs, together with ALDH CD8α+-type Mig-DCs, possessed the strongest capacity to induce IFN-γ production by naive OT-II T cells. Therefore, the numerically dominant inflammatory MΦs that develop in the MLNs of colitic mice were located in the T-cell zone and excelled in the generation of Th1 effectors in vitro.

MLN-resident MΦs from colitic mice induce IFN-γ-producing effector T cells in vivo

To determine whether MLN-resident inflammatory MΦs were capable of triggering the differentiation of CD4+ T cells into IFN-γ-producing effector T cells in vivo, we developed the adoptive transfer experiment outlined in Fig. 8A. Consistent with previous data [25], when CD4+ T cells were labeled with CFSE and transferred into Cd3eΔ5/Δ5 host, two coincident and independent phenomena occurred [22]. A fraction of the transferred CD4+ T cells proliferated very slowly, accounting for the CFSEhigh cells observed at day 8 after transfer (Fig. 8B, upper panels). In contrast, a fraction of them proliferated extensively, yielding a prominent population of CFSElow cells. These fast-proliferating, CFSElow CD4+ T cells were capable of producing IFN-γ and their generation was strictly dependent on interaction with MHCII molecules expressed on the surface of the antigen-presenting cells of the host (Fig. 8B, middle panels). It is those fast-proliferating T cells that are responsible for the induction of colitis when transferred in the absence of Treg cells. Antigenic peptides derived from the enteric bacteria and presented by MHCII molecules have been shown to be responsible for this TCR-driven fast proliferation whereas the slow proliferation is independent of MHCII molecules and IL-7-driven [26-30].

image

Figure 8. MLN-resident CD64+ MΦs from colitic Cd3eΔ5/Δ5 mice trigger the differentiation of Th1 cells in vivo. (A) Outline of the adoptive transfer model. Cd3eΔ5/Δ5 mice were injected i.v. with 2 × 106 CD4+Foxp3 T cells to induce colitis. Two weeks later, when colitis was largely established, inflammatory CD64+ MΦs were sorted from the MLNs. Cd3eΔ5/Δ5 × MHCIIΔ/Δ hosts were injected i.v. with the sorted inflammatory CD64+ MΦs (2 × 106) and 1 day later with CFSE-labeled CD4+ T cells (2 × 106). In parallel experiments, CFSE-labeled CD4+ T cells (2 × 106) were injected into Cd3eΔ5/Δ5 (positive control) and Cd3eΔ5/Δ5 × MHCIIΔ/Δ (negative control) hosts. (B) Eight days after T-cell transfer, CD4+ T-cell proliferation was assessed by measuring CFSE dilution and IFN-γ and IL-17 production evaluated after a step of in vitro stimulation with PMA plus ionomycin. Percentages of IFN-γ+ and IL-17+ cells among slow (CFSEhigh) and fast (CFSElow) proliferating CD4+ T cells isolated from the specified mice are shown. Data shown are representative of three independent experiments consisting of five mice per group.

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We took advantage of this model to determine whether upon transfer into Cd3eΔ5/Δ5 x MHCIIΔ/Δ mice, MLN-resident inflammatory MΦs isolated from MHCII-sufficient, colitic mice were capable of rescuing the fast proliferation of cotransferred CD4+ T cells and their differentiation into IFN-γ-producing effectors. As shown in Fig. 8B, transfer of CD64+ MΦs from MLNs of colitic mice was sufficient to trigger the fast proliferation of a fraction of the cotransferred CD4+ T cells and their differentiation into IFN-γ-producing effectors. The fact that we could only transfer relatively small numbers of CD64+ MΦs per mouse likely explained that the magnitude of the CD4+ T-cell responses was smaller than that observed after injecting T cells into MHCII-sufficient Cd3eΔ5/Δ5 mice. Regardless of this limitation, our data demonstrate that inflammatory CD64+ MΦs isolated from the MLNs of colitic mice can induce the proliferation and differentiation of CD4+ T cells into IFN-γ-producing effector T cells in vivo.

Discussion

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

We have shown that in healthy and inflamed intestinal LP and MLNs, CD64 expression makes it possible to accurately identify MΦs and to delineate a developmental series — called the Mo-waterfall — that starts from extravasated Ly-6Chi monocytes and leads to intestinal MΦs. Using Ly-6C-MHCII and Ly-6C-CD64 plots, the corresponding stages of this monocyte-derived developmental series have been denoted as P1 (Ly-6ChiMHCIICD64lowCX3CR1int LP monocytes), P2 (Ly-6Cint to hiMHCII+CD64lowCX3CR1int intermediate cells), P3 (Ly-6ClowMHCII+CD64+CX3CR1int MF), and P4 (Ly-6ClowMHCII+CD64+CX3CR1hi MF) (Supporting Information Fig. 3). Interestingly, we found that in humanized mice and humans, CD64 was highly expressed by HLA-DR+ cells that coexpressed CD14; a finding strongly suggesting that high CD64 expression marks cells of monocytic origin in humans. In support of this conclusion, a recent study demonstrated that the CD14hi and CD14lo subsets of HLA-DR+ MΦs were uniformly CD64+ in both healthy and inflamed human intestine, whereas MHCII+CD14 cells that include DCs were mostly CD64 [31]. Therefore, CD64 can be used in both mice and human to distinguish MΦs from CD8α+/BDCA-3+-type and CD11b+/BDCA-1+-type DCs, and to study how MΦs originate from extravasated Ly-6Chi monocytes in healthy and inflamed conditions (Fig. 9). Although monocyte-derived MHCII+ cells found in the intestinal LP have been subdivided into CD11c MΦs and CD11c+ monocyte-derived DCs (Mo-DCs) by some authors [[2], [32], [33]], CD11c shows a continuous density distribution from negative to low among monocyte-derived MHCII+ LP cells (Fig. 2 and 3), a finding emphasizing the difficulty in using CD11b to define discrete stages amongst monocyte-derived MHCII+ LP cells.

image

Figure 9. A model of the independent developmental processes that generate MΦs and DCs in the intestine and the MLNs under healthy and inflammatory conditions. (A) Two independent processes continuously occur in the healthy (steady state) intestine. First, CD8α+- and CD11b+-type Int-DCs develop in the LP from blood-derived pre-DC precursors and constitutively migrate to the MLNs in a CCR7-dependent manner. The resulting Mig-DCs contain ALDH+ DCs that are primarily endowed with tolerogenic properties. Second, a developmental series — called the Mo-waterfall — unfolds in the LP. It starts with extravasated Ly-6Chi MHCII monocytes (P1) and leads to LP-resident anti-inflammatory MΦs termed P3 (Ly-6ClowMHCII+CD64+CX3CR1int) and P4 (Ly-6ClowMHCII+CD64+CX3CR1hi) via a P2 intermediate stage. (B) Under inflammatory conditions (e.g. T cell-mediated colitis), three independent processes occur in the intestinal LP and in the MLNs. First, CD8α+- and CD11b+-type Int-DCs develop in the intestinal LP and subsequently migrate to the MLNs. Among those Mig-DCs, ALDH CD8α+-, and CD11b+-type DCs are capable of inducing proinflammatory CD4+ T cells. Second, massive numbers of Ly-6Chi monocytes (inf P1) seed the inflamed LP and predominantly develop into inflammatory Ly-6Cint to hiMHCII+CD64hi MΦs (inf P2) that do not migrate to the MLNs during T cell-mediated colitis. Third, Ly-6Chi blood monocytes are massively recruited to the MLNs where they locally differentiate into inflammatory Ly-6Cint to hi MHCII+CD64+ MΦs (inf P2). These MLN-resident inflammatory MΦs are located in the T-cell zone and capable of generating Th1 cells in vitro and in vivo. As a result, during T cell-mediated colitis, MLN-resident inflammatory MΦs likely synergize with ALDH Mig-DCs to induce the differentiation of naïve T cells into proinflammatory T cells.

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During T cell-mediated colitis, extravasated Ly-6Chi monocytes differentiate into CD64+MHCII+ inflammatory MΦs that are CCR7 and, consistent with results from Pabst et al. [3], cannot migrate to the MLNs. This characteristic prevents their participation in the activation of naïve T cells in the MLNs. However, giving their high MHCII expression, inflammatory MΦs can potentially present antigens to tissue-resident memory T cells and to the iTreg cells that reach the LP [32, 34]. Along that line, due to its high affinity for IgG, the expression of CD64 may possibly arm LP MΦs with IgG specific for previously encountered pathogens and allow them to efficiently initiate recall responses upon subsequent pathogen reexposure [15]. Although the inflammatory conditions achieved during T cell-mediated colitis do not induce LP MΦs to migrate to MLNs, it remains possible that some bacterial adjuvants confer MLN-homing property to some LP MΦs as shown for muscle Mo-DCs [33].

Unexpectedly, T cell-mediated colitis was also found associated with a massive recruitment of blood Ly-6Chi monocytes in the MLNs, where they differentiated into inflammatory MΦs via a blunted Mo-waterfall similar to the one observed in the LP of inflamed intestine. These MLN-resident inflammatory MΦs did not display a dendritic morphology and were phagocytic, a hallmark of MΦs. In addition, they expressed high levels of MHCII molecules and were located in the T-cell zone, which are two prerequisites for proper interactions with naive CD4+ T cells. Consistent with these attributes, MLN-resident inflammatory MΦs were capable of generating IFN-γ-producing T cells in vitro and in vivo. Therefore, during T cell-mediated colitis, MLN-resident inflammatory MΦs likely synergize with LP-derived ALDH Mig-DCs to induce the differentiation of naïve T cells into proinflammatory T cells.

Upon transfer of CD4+Foxp3 T cells, we showed that Cd3eΔ5/Δ5 × Ccr7−/− recipient mice developed an exacerbated colitis as compared with Cd3eΔ5/Δ5 recipient mice. This result is consistent with our hypothesis that MLN-resident inflammatory MΦs actively participate in the induction of T cell-mediated colitis irrespective of the presence of Mig-DCs and with the view that the RA-producing ALDH+ LP-derived Mig-DCs that are still present in colitic mice contribute to dampen colitic disease (Fig. 6E). It is important to stress that our results do not question a role for ALDH LP-derived Mig-DCs in the generation of proinflammatory T cells during colitis. However, they differ from previous studies in that they reveal that the inflammatory MΦs that expand massively in the MLNs of colitic mice and outnumber Mig-DCs are also capable of inducing proinflammatory CD4+ T cells. It remains to be determined how the inflammatory MΦs that permanently reside in the MLNs gather enteric antigens in the absence of LP-derived Mig-DCs. Akin to the situation recently described for mice depleted of innate lymphoid cells [35], the lack of T cells in Cd3eΔ5/Δ5 × Ccr7−/− mice may favor the dissemination of commensal bacteria to the MLNs and their capture by the resident MΦs.

In conclusion, we have defined a novel gating strategy that is based on the differential expression of CD64 and that allows the unequivocal identification of MΦs in the intestine of mice and humans in both healthy and inflamed conditions (Fig. 9). On that basis, we demonstrated that T cell-mediated colitis is associated with a massive recruitment of monocytes to the intestinal LP where they differentiated into LP-resident inflammatory MΦs. Coincidentally, T cell-mediated colitis also induced the massive recruitment of monocytes to the MLNs and their differentiation into MLN-resident inflammatory MΦs capable of converting naïve T cells into effector Th1 cells. In the case functionally similar inflammatory MΦs develop in the MLNs of patients suffering from inflammatory bowel disease, they might constitute potential targets for the management of inflammatory digestive diseases.

Materials and methods

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

Mice

CX3CR1-EGFP [16], Foxp3-EGFP [25], Cd3eΔ5/Δ5 [21], OT-II [36], Ccr2−/− [37], CCR7−/− [38], and Cd3eΔ5/Δ5 × MHCIIΔ/Δ mice [25] were maintained on a C57BL/6 background and under specific pathogen-free conditions. All experiments were done in accordance with French and European guidelines for animal care.

Cell preparation

DCs were isolated from the MLNs and the LP of the small and large intestine as described in [11].

Flow cytometry

Cells were stained and analyzed using a FACS LSRII system (BD Biosciences). Allophycocyanin-Cy7-conjugated anti-NK1.1 (PK136), anti-CD3 (17A2), anti-Ly-6G (1A8), anti-CD19 (6D5), and PE-conjugated anti-CD64 (X54–5/7.1) were all from Biolegend, allophycocyanin-conjugated anti-CCR3 (83103) was from R&D, PE-Cy7-conjugated anti-CD11c (N418), Alexa-700-conjugated anti-MHC Class II (I-A/I-E) (M5/114.15.2), PE-Cy5.5-conjugated anti-CD45.2 (104), allophycocyanin-conjugated anti-CD45.1 (A20), PE-Cy5-conjugated anti-CD24 (M1/69), and PE-Cy5-conjugated anti-CD5 (53–7.3) were all from eBioscience, Pacific-Blue-conjugated anti-CD11b (M1/70), PE- or biotin-conjugated anti-CD103 (M290), allophycocyanin-conjugated anti-CD172α (P84), FITC-conjugated anti-Ly6C (AL21), Pacific-Blue-conjugated anti-CD4 (RM4–5), biotin-conjugated anti-CCR7 (4B12) were all from BD Pharmingen. Biotin-conjugated antibodies were detected using streptavidin conjugated with Quantum-Dot605 (Invitrogen). Intracellular iNOS staining was performed using unlabeled anti-rabbit iNOS (M19, Santa Cruz) and allophycocyanin-conjugated anti-rabbit IgG (Invitrogen). ALDH+ cells were identified as described [11], and intracellular cytokines staining performed as described [33]. Prior to DC and MF analysis, B cells, T cells, NK cells, eosinophils and neutrophils were systematically gated out using a “dump-channel” corresponding to cells positive for CD19, CD3, NK1.1, CCR3 or Ly-6G cells. Analysis was performed using FlowJo software (Tree Star, Inc.).

Assessment of phagocytosis

FACS-sorted DC and MF subsets were seeded on Alcian blue-treated coverslips and incubated with 5% FCS supplemented RPMI containing yellow green fluorescent 0.5 μm Fluoresbrite microspheres (Polysciences, Inc.) for 1 h at 37°C before washing, fixation, staining for MHCII and iNOS and analysis by confocal microscopy.

Generation of BM chimeras

Seven to –8-week-old B6 CD45.1 × CD45.2 mice were lethally irradiated with two doses of 550 rads, 5 h apart, and then injected i.v. with 2 × 106 BM cells. BM cells were obtained from femurs and tibias of WT B6 CD45.1 or of Ccr2−/− CD45.2 mice.

T cell-mediated colitis

T cells were purified by magnetic separation from spleens and LNs of Foxp3-EGFP mice using a CD4 negative isolation kit (Dynal). Cells were then stained with PE-conjugated anti-CD4 (RM4–5) antibody and CD4+Foxp3 T cells (2 × 106) were sorted and injected intravenously into Cd3eΔ5/Δ5 mice.

In vivo labeling of Ly-6Chi monocytes

Blood monocytes were labeled with latex beads as described [23].

Adoptive transfer of Ly-6ChiMHCII monocytes

Bone marrow and spleens from RAG−/− mice were harvested. Cells were stained for CD11b, Ly6C, Ly6G, MHCII, CCR3, and CD11c. Ly-6Chi monocytes were sorted as CD11b+Ly-6GCCR3CD11cMHCIILy-6Chi cells with a purity of more than 95% and labeled with CFSE (Molecular Probes). A 2 × 106 CFSE-labeled Ly-6Chi monocytes were transferred intravenously into healthy Ccr2−/− mice or Cd3eΔ5/Δ5 × Ccr2−/− colitic mice. In parallel 2 × 104 CFSE-labeled Ly-6Chi monocytes were maintained in vitro in complete medium in the presence of 1 × 106 WT BM cells.

Adoptive transfer of inflammatory CD64+ MΦs

Cd3eΔ5/Δ5 mice were injected i.v. with 2 × 106 CD4+Foxp3 T cells to induce colitis. Two weeks later, when colitis was largely installed, inflammatory CD64+ MΦs were isolated from the MLNs. Cd3eΔ5/Δ5 × MHCIIΔ/Δ hosts were first injected i.v. with 2 × 106 inflammatory CD64+ MΦs and then, 1 day later, with 2 × 106 CFSE-labeled CD4+ T cells. In parallel experiments, 2 × 106 CFSE-labeled CD4+ T cells were injected into Cd3eΔ5/Δ5 and Cd3eΔ5/Δ5 × MHCIIΔ/Δ. Eight days after T-cell transfer, CD4+ T-cell proliferation and cytokine production were evaluated.

Preparation of CFSE-labeled T cells

OT-II T cells were purified by magnetic separation from pooled spleen and LN using a CD4 negative isolation kit (Dynal). For CFSE labeling, purified cells were resuspended in PBS and labeled with 2.5 μM 5- and 6-carboxy-fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes) for 3 min at room temperature.

Culture of CFSE-labeled OT-II T cells

3 × 103 DCs or MΦs were cocultured with 2 × 104 CFSE-labeled OT-II T cells in 200 μL in the presence of ovalbumin (323–339) peptide (0.06 μg/mL). After 5 days of culture, cells were restimulated with PMA and ionomycin for 5 h before intracellular cytokine staining. Proliferation was measured by loss of CFSE staining.

Immunofluorescence staining

MLNs were fixed with 3.2% paraformaldehyde for 1 h, washed in PBS, infused overnight in 35% sucrose, and frozen in Tissue-Tek OCT compound (Electron Microscopy Sciences, Hatfield, PA). After permeabilization for 5 min in PBS containing 0.5% saponin, 2% bovine serum albumin, 1% fetal calf serum, and blockade of Fc binding with 1% goat serum for 30 min, 12 μm cryostat tissue sections were labeled overnight at 4°C with rabbit anti-iNOS (Santa Cruz Biotechnology), hamster anti-CD11c (N418, Biolegend) and rat anti-CD45R (RA3–6B2, Biolegend) antibodies or control antibodies followed by incubation for 1 h at room temperature with secondary antibodies and SYTOX Blue for nuclei staining. Slides were mounted in Prolong Gold (Invitrogen) and observed with a Zeiss LSM 510 or a Zeiss LSM 780 confocal microscope (Carl Zeiss, Jena, Germany).

Characterization of DCs and MΦs from human colon biopsies

Colon biopsy specimens were obtained from patients with a history of ulcerative colitis during colonoscopy after informed consent and with the approval of the Ethics Committee of Erasmus University Medical Centre, Rotterdam. Endoscopic signs of inflammation were used to define the biopsies as inflamed. LP mononuclear cells were isolated from the biopsies as previously described [39].

Humanized mice

Humanized mice experiments were developed and analyzed as described [40].

Statistical analysis

Comparative experiments were tested for statistical significance using the unpaired Student's t-test in GraphPad Prism software (version 4.0; GraphPad).

Acknowledgments

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

We thank M. Malissen, H. Luche, B. Lucas, G. Randolph, and L. Leserman for discussions. We thank M. Barad, P. Grenot, and A. Zouine for assistance with cell sorting and M. Fallet and M. Barad for assistance with confocal microscopy. This work was supported by CNRS, INSERM, European Communities Framework Program 7 (MASTERSWITCH Integrating Project; HEALTH-F2–2008-223404 and NANOASIT Euronanomed Project), AFM, FRM, ANR (Skin DCs) and by doctoral and postdoctoral fellowships from Ministère de la Recherche (S.T.), and a Marie Curie Fellowship from the European Communities (M.G., project number 237109). Y.R., C.R.E.S., and D.B. have funding from the Cancer Research UK. Y.R. is supported by Leukaemia and Lymphoma Research. L.F.P. is supported by a CNRS/INSERM ATIP/AVENIR program. A.M.M. and C.C.B. were supported by the Medical Research Council and Wellcome Trust (UK).

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  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgments
  8. Conflict of interests
  9. References
  10. Supporting Information
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Abbreviations
EGFP

enhanced green fluorescent protein

Int-DC

interstitial DC

iTreg cell

induced Foxp3+ regulatory T cell

LP

lamina propria

Mig-DC

migratory DC

MHCII

MHC class II

Supporting Information

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

Disclaimer: Supplementary materials have been peer-reviewed but not copyedited.

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eji2440-sup-0001-FigureS1.pdf7529K

Figure 1. Characterization of intestinal CD8⟨+-type and CD11b+-type DCs. Cells were prepared from the LP of the small and large intestine and from the MLN of Flt3L-deficient (A), B6 mice (A and B) or of CX3CR1-EGFP mice (C). After excluding NK cells, B cells, T cells, eosinophils and neutrophils, the remaining MHCII+ cells were divided into CD11chiCD64– Int-DCs and CD11c– to intCD64+ MΦs (see Fig. 1). (A) CD11chiCD64– DCs Int-DCs are absent in the LP of Flt3L-deficient mice. (B) CD11chiCD64–-gated Int-DCs present in the intestinal LP were analyzed for CD24, CD11b, CD172⟨ and CD103 expression and categorized into CD24+CD11b–CD103+or– CD8⟨+-type DCs and CD11b+CD64–CD103+or– CD11b+-type DCs. The MLN contained a migratory counterpart for each of the Int-DC subsets. (C) Expression of CX3CR1 and F4/80 on CD24+CD11b– CD8⟨+-type DCs, CD11b+CD64– CD11b+-type DCs and CD11c– to intCD64+ MΦs from the specified tissues. In the MLN, Mig-DCs were identified as MHCIIhiCD11cinter-to-hiCCR7+ cells. The percentages of cells found in each of the specified gates are indicated. Data shown are representative of 3 independent experiments.

Figure 2. CD64 marks monocyte-derived MΦs in humans. (A) Human CD34+ cord-blood progenitor cells were infected with lentivirus coding for the GFP fluorescent protein and then injected into NSG mice. 8 weeks later, cells were prepared from the LP of the small and large intestine of humanized mice and GFP+ cells were analyzed by flow cytometry. After excluding B cells, T cells and NK cells, HLA-DR+ cells were analyzed for CD11c, BDCA1, BDCA3 and CD64 expression. (B) Cells were prepared from non-inflamed and inflamed sections of colon biopsy specimens obtained from patients with a history of IBD. After excluding B cells, NK cells and T cells, HLA-DR+ cells were analyzed for CD11c, CD14, BDCA1 and BDCA3 expression. Histograms correspond to CD64 expression on BDCA1+CD11c+, BDCA3+CD11c+, and CD14+CD11c+ cells. Note that human LP MΦs expressed higher levels of CD11c than those found on mouse LP MΦs. The percentages of cells found in each of the specified gates are indicated. Data in (A) are representative of 2 groups of 3 humanized mice. Due to the small amount of GFP+ cells recovered per mouse, the small and the large intestines were pooled for the analysis. Data in (B) are representative of biopsies from 4 individual patients.

Figure 3. A model of the developmental pathway that unfolds in the healthy intestinal LP and leads from blood Ly-6Chi monocytes to MΦs. The expression of CD11c, MHCII, CD64, CX3CR1, Ly-6C, CD172⟨ and CD11b is schematized. Four stages can be distinguished: P1 (tissue monocytes: Ly-6ChiMHCII–CD64lowCX3CR1int), P2 (Intermediates: Ly-6ChiMHCII+CD64lowCX3CR1int), P3 (CX3CR1int MF: Ly-6ClowMHCII+CD64+CX3CR1int) and P4 (CX3CR1hi MF: Ly-6ClowMHCII+CD64+CX3CR1hi).

Figure 4. T cell mediated colitis in Cd3e⊗5/⊗5 mice. (A) Four weeks after transfer of CD4+Foxp3– T cells into Cd3e⊗5/⊗5 hosts, CD4+ T cells were isolated from the MLN and the LP of the large intestine and restimulated in vitro with PMA plus ionomycin. IFN-©, and IL-17 expression was assessed by intracellular staining 6 hours later. The percentages of IFN-©+ and IL-17+ cells among CD4+ T cells are shown. (B) Evolution of the weight of Cd3e⊗5/⊗5 mice transferred with or without CD4+ Foxp3– T cells. In (A) and (B), data are representative of three independent experiments involving at least 3 (A) and 6 (B) mice per group or time-point. The error bars correspond to the SEM. *, p < 0.05.

Figure 5. Gating strategy used for the identification of CD64+ MΦs and of CD24+CD11b– and CD11b+CD64– DCs in the large intestine LP and the MLN of healthy and colitic mice. Cells were prepared from the LP of the large intestine (A) and the MLN (B) of steady-state B6 mice (B6 steady-state) and of Cd3e⊗5/⊗5 mice that had received CD4+Foxp3– T cells (IBD). After excluding NK cells, B cells, T cells, eosinophils and neutrophils, the remaining MHCII+ cells of the large intestine LP were analyzed for CD64, CD11c, CD24, CD11b and CCR7 expression and divided in the following subsets: MΦs (MHCII+CD11c– to intCD64+CD11b+), CD8⟨+-type (MHCII+CD11chiCD64–CD24+CD11b–) and CD11b+-type (MHCII+CD11chiCD64–CD11b+) Int-DCs. In the MLN, MΦs were identified as MHCII+CCR7–CD11c– to intCD64+CD11b+ cells and the MHCIIhiCCR7+CD64– Mig-DCs were subdivided into CD24+CD11b– CD8⟨+-type and CD11b+CD64– CD11b+-type Mig-DCs. Data are representative of at least 3 independent experiments.

Figure 6. CD64+ MΦs excel in phagocytosis and do not display a dendritic morphology. Cd3e⊗5/⊗5 mice were sacrificed 3 weeks after transfer of CD4+Foxp3– T cells. Light density cells were isolated from the MLN and FACS-sorted into MHCIIhiCD64–CD24+CD11b– CD8⟨+-type Mig-DCs, MHCIIhiCD64–CD11b+ CD11b+-type Mig-DCs and MHCIIhiCD64+CD11b+ CD64+ MΦs. The distinct subsets were then subjected to a microsphere phagocytosis assay and uptake of fluorescent microspheres was analyzed by confocal microscopy. Scale bars, 20 μm. Data are representative of 3 independent experiments.

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