Dependence of intestinal granuloma formation on unique myeloid DC-like cells

Granulomas represent a localized inflammatory reaction that is characteristically observed in many inflammatory conditions. However, the mechanisms of granuloma formation have not been fully defined. Herein we demonstrate, by using experimental models of intestinal inflammation, that a unique CD11c⁺ DC-like cell subset that exhibits phenotypic and functional features of immature myeloid DCs and is characterized by the expression of a macrophage marker (F4/80) produces large amounts of IL-23 and directly induces the development of granulomas under a Th1-predominant intestinal inflammatory condition. Importantly, both IL-4 and IgG contribute to the suppression of F4/80⁺ DC-like cell–mediated granuloma formation by regulating the function and differentiation of this cell subset. In addition, enteric flora is required for the F4/80⁺ DC-like cell–mediated granuloma formation. Collectively, our data provide what we believe are novel insights into the involvement of F4/80⁺ DC-like cells in intestinal granuloma formation and demonstrate the role of host (IL-4 and IgG) and environmental (enteric flora) factors that regulate this function.

Granulomatous inflammation is a chronic inflammatory reaction, usually in response to foreign material (e.g., microorganisms, parasites), which is characterized by nodular collections of epithelioid cells (which are generally considered to represent activated macrophages) surrounded by lymphocytes (1–3). However, lesions that are classified as granulomas have diverse appearances. Although most granulomas are believed to be T cell mediated, several types of immune reactions are involved in the development or suppression of granuloma formation (1–3). Interestingly, recent studies have proposed that DCs contribute to the development of granulomas in the liver and lung (4–6).

DCs represent a phenotypically heterogeneous population that is classified into several subsets, notably myeloid DCs (CD11c⁺CD11b⁺CD4⁺CD8α^– and CD11c⁺CD11b⁺CD4^–CD8α^–), lymphoid DCs (CD11c⁺CD8α⁺CD11b^–), and plasmacytoid DCs (CD11c⁺CD11b^–B220⁺Gr-1⁺). DCs are crucial components of the immune system owing to their essential role in the induction and regulation of immune responses (7–9). Even in a given subset, DCs possess considerable functional plasticity, depending on their maturation stage, environmental conditions, and location (7–9). In the intestine, several DC subsets are found depending on anatomical structures; both lymphoid and myeloid DCs are present in lymphoid follicles, such as Peyer patches (10), whereas CD11c⁺CD11b⁺ DCs with immature phenotype represent a major DC subset in the lamina propria (LP) of small and large intestines (11–13). Intestinal DCs play a critical role in initiating local and systemic immune responses by capturing antigens (e.g., from bacteria or apoptotic epithelial cells (14–16), by producing effector and regulatory cytokines (10, 17), and by activating B cells (18). Furthermore, specific products from enteric bacteria and intestinal epithelial cells have been shown to regulate the function of intestinal DCs (13, 17, 19). In addition to maintaining the state of health, there are functionally distinct DC subsets that play pathogenic versus regulatory roles in the development of intestinal inflammation (11, 20–22). DCs also play an important role in the development of inflammatory bowel disease, a term that encompasses a group of chronic intestinal inflammatory conditions that are initiated and perpetuated by exaggerated immune responses to luminal bacterial antigens (23, 24). Of the 2 major forms of inflammatory bowel disease, only Crohn disease is characterized by granulomatous inflammation; ulcerative colitis is not (2, 23). However, the role of DCs in the development of intestinal granulomas has not yet been defined.

In this study, we demonstrate in experimental models of intestinal inflammation that a unique immature myeloid DC–like (IMD–like) subset characterized by the coexpression of F4/80 and by the production of IL-23 directly contributes to the development of intestinal granulomas and, importantly, that host (IL-4 and IgG) and environmental (enteric flora) factors determine the ability of these cells to form granulomas.

Absence of B cells and IL-4 induces development of intestinal granulomas. Activation of the Th1 pathway is required for the development of disease in most murine models of intestinal inflammation, some of which develop granulomas with low frequencies (25, 26). In contrast, TCRαKO mice spontaneously develop chronic intestinal inflammation that shares some features with human ulcerative colitis; this experimental colitis is mediated by Th2 cytokines (27–29). Herein we demonstrate an unexpected observation, namely, that lack of both IL-4 and B cells in TCRαKO mice results in development of a distinctive type of intestinal inflammation characterized by the presence of granulomas. Granulomatous inflammation was observed in the mucosa (Figure 1A) as well as in the submucosa (Figure 1B) of the colon and ileocecal junction area of 39.8% (64/161 mice) of IL-4– and B cell–deficient TCRα triple-KO (αμIL4TKO) mice at 24 weeks of age but not in 1102 TCRαKO, 548 B cell–deficient TCRα double-KO (αμDKO), 144 IL-4–deficient TCRα double-KO (αIL4DKO), or 113 WT mice (Figure 1, C and D, and data not shown). Granulomas were more frequently observed in mice with marked inflammation as compared with mice with mild inflammation, but there was no statistically significant difference between these groups. The inflammation associated with αμIL4TKO mice was located primarily in the proximal colon, especially the ileocecal area (Figure 1E), whereas the inflammation associated with αμDKO mice involved the entire colon and was especially marked in the distal regions (Figure 1F). These observations are consistent with the findings that expression of proinflammatory cytokine (IL-1β) was detectable in the ileocecal junction area of αμIL4TKO but not αμDKO mice (Figure 1G). At 24 weeks of age, there was no significant difference in the frequency of inflammation between αμDKO and αμIL4TKO mice (Figure 1H). The frequency of disease was much higher in αμIL4TKO mice as compared with TCRαKO and αIL4DKO mice. These results suggest that both B cells and IL-4 are required for the suppression of intestinal inflammation–associated granuloma formation. Indeed, B cells suppress Schistosoma mansoni– and Leishmania donovani–mediated granulomatous inflammation (3, 30).

Figure 1

Development of intestinal inflammation–associated granulomas in the absence of IL-4 and B cells. (A–D) In αμIL4TKO mice (24 weeks of age), granulomas are detectable within the colonic mucosa (A) as well as in the ileocecal areas (B). In contrast, granulomas are undetectable in the age-matched WT (C) and αμDKO (D) mice. (E and F) Gross features of the large intestines of αμIL4TKO (E) and αμDKO (F) mice at 24 weeks of age are shown. The inflammation is mostly restricted to the ileocecal junction (arrow) and proximal colon in αμIL4TKO mice whereas the inflammation involves the entire colon, in particular the distal colon (arrowheads), in αμDKO mice. (G) RPA shows expression of proinflammatory cytokine (IL-1β) in the ileocecal junction of αμIL4TKO (αμIL4) but not αμDKO (αμ) mice (24 weeks of age). Data are representative of 2 individual experiments. (H) Frequency of colitis in WT (open squares, n = 168), TCRαKO (open circles, n = 222), αIL4DKO (open triangles, n = 144), αμDKO (closed circles, n = 218) and αμIL4TKO (closed triangles, n = 104) mice at various ages are shown. MIF, Macrophage migration inhibitory factor.

Presence of CD11c⁺CD11b⁺F4/80^low cells in granuloma. RNase protection assay (RPA) analysis using RNA from the ileocecal regions revealed a significant increase in F4/80 expression in αμIL4TKO mice compared with αμDKO mice (Figure 2A). Immunohistochemical analysis showed diffuse infiltration of CD4⁺TCRα^–β⁺ T cells and aggregates of CD11b⁺ cells in the areas of granulomatous inflammation (Figure 2, B and C). Interestingly, recent studies have proposed that DCs contribute to the development of granulomas in the liver and lung (4–6). Indeed, a large number of CD11c⁺ cells were also identified within granulomas of αμIL4TKO mice (Figure 2D). To characterize further the CD11c⁺ cells present in granulomas, cells were isolated from grossly recognizable granulomas and subjected to flow cytometric analysis. Within granulomas, 18.3% ± 1.5% (n = 12) of the cells had the CD11c⁺CD11b⁺ phenotype without expression of B220 or CD8α (Figure 3A). Unexpectedly, the majority of CD11c⁺CD11b⁺ cells coexpressed a specific marker for macrophages, F4/80, without expression of another macrophage marker, MOMA-1 (Figure 3B and Supplemental Figures 1 and 2; supplemental material available online with this article; doi:10.1172/JCI30150DS1). The F4/80 expression was also confirmed by quantitative polymerase chain reaction (QPCR) analysis using purified CD11c⁺ cells from granulomas (Figure 3C). To determine whether the F4/80⁺CD11c⁺ cells represented macrophages or DCs, purified F4/80⁺CD11c⁺ and F4/80^–CD11c^– cells were subjected to morphological analysis. Of note, fine dendrites, a specific feature of DCs, were observed on the F4/80⁺CD11c⁺ cells (Figure 3D). In addition, F4/80⁺CD11c⁺ cells were characterized by low expression of CD86 compared with WT BM–derived mature DCs (Figure 3E). Furthermore, unlike BM-derived mature DCs, F4/80⁺CD11c⁺ cells did not present antigens to T cells as assessed by mixed lymphocyte reactions (Figure 3F). Moreover, phagocytic activity as judged by the FITC-dextran uptake was observed in F4/80⁺CD11c⁺ cells although the activity of these cells was relatively low compared with that observed in BM-derived immature DCs (Figure 3G). In addition, the purified CD11c⁺ cells from the colonic LP of αμIL4TKO, αIL4DKO, αμDKO, TCRαKO, and WT mice exhibited the properties of immature DCs (data not shown). These findings identify a granuloma-associated unique cell subset that shares some phenotypic and functional properties with IMD and, unexpectedly, also expresses a macrophage marker, F4/80.

Figure 2

Cellular composition in granulomas. (A) Analysis of the cellular infiltrate at the ileocecal junction of αμIL4TKO mice with granulomas and αμDKO mice was performed by RPA using total RNA (10 μg). All mice were 24 weeks of age. The bands were visualized by 12-hour exposure (top panel) and 6-hour exposure (bottom panel). (B–E) Immunohistochemical analysis of granulomas in the ileocecal junction (B–D) and colonic LP (E) of αμIL4TKO was performed using anti-CD11b (B), anti-CD4 (C), and anti-CD11c (D and E) mAbs. Findings are representative of 8 individual experiments.

Figure 3

Granuloma-associated CD11c⁺ cells exhibit a unique IMD-like phenotype with F4/80 expression. (A) Staining of CD11c versus CD11b (left panel) and B220 or CD8α expression (solid red histograms, right panels) on gated CD11b⁺CD11c⁺ cells from grossly recognizable granulomas (n = 12) are shown. (B) Expression (n = 9) of F4/80 or MOMA-1 on gated CD11b⁺CD11c⁺ cells are shown as solid green histograms. Black lines represent isotype control staining. (C) QPCR shows F4/80 expression by purified CD11c⁺ cells (n = 3) from granulomas and splenic CD4⁺ cells (n = 3). (D) Morphology of FACS-purified F4/80⁺CD11c⁺ cells from granulomas is shown. Fine dendrites are recognized on these cells (arrows). (E) Expressions of CD86 on granuloma-derived CD11c⁺CD11b⁺ cells (solid orange histograms) and BM-derived mature DCs (blue line) are shown. Black line represents isotype control staining. The averages of CD86 mean fluorescent intensity (MFI) on BM-derived immature (iDC, n = 3) and mature (mDC, n = 3) and granuloma-derived CD11c⁺CD11b⁺ cells (gDC, n = 10) are summarized. (F) T cells (T) from BALB/c mice were cultured without or with C57BL/6 mDC or gDC (n = 3) for 80 hours and pulsed with 1 μCi ³H for 16 hours. (G) iDC (n = 3), mDC (n = 3), and gDC (n = 3) were cultured with FITC-dextran at 4°C or 37°C and subjected to flow cytometric analysis. The averages of phagocytic activity are indicated as FITC-dextran MFI at 37°C minus FITC-dextran MFI at 4°C. Bottom panel shows gDC cultured with FITC-dextran at 37°C. Statistical significances are indicated by asterisks. **P < 0.001; *P < 0.05.

IL-12p40 but not IFN-γ is required for the development of granulomas. The experimental colitis models of Crohn disease that are characterized by the presence of granulomas have been shown to exhibit upregulation of IL-12p40 and IFN-γ in the colon (25, 26, 31). Indeed, RPA and ELISA analyses showed that the expression levels of IL-12p40 and IFN-γ were significantly upregulated in the colons of αμIL4TKO mice compared with αμDKO, αIL4DKO, TCRαKO, and WT mice (Figure 4, A and B). To determine whether IFN-γ is involved in the development of granulomas, TCRα quadruple-KO mice deficient in B cells, IL-4, and IFN-γ (αμIL4IFNQKO) were generated. Like αμIL4TKO mice, αμIL4IFNQKO mice spontaneously developed granulomatous colitis (Figure 4C).

Figure 4

IL-12p40 but not IFN-γ is involved in the development of intestinal granulomas. (A) RPA using RNA from ileocecal junction areas shows that IL-12p40 but not p35 is detectable in αμIL4TKO but not TCRαKO (TCRα) mice (24 weeks of age). (B) Colonic LP cells (2 × 10⁵) from WT (n = 5), TCRαKO (n = 7), αμDKO (n = 7), αIL4DKO (αIL4) (n = 7), and αμIL4TKO (n = 9) mice were cultured for 48 hours, and the culture supernatants were subjected to ELISA. There was a significant increase (**P < 0.001) of IL-12p40 and IFN-γ production by colonic LP cells from αμIL4TKO mice compared with αμDKO, αIL4DKO, TCRαKO, and WT mice. (C) Typical granulomatous inflammation characterized by nodular collections of epithelioid cells surrounded by lymphoid cells is still detectable in αμIL4IFNQKO mice. The frequency and severity of granulomas were similar in αμIL4TKO and αμIL4IFNQKO mice (data not shown). (D and E) Anti–IL-12p40 mAbs (0.5 mg) were administered weekly to αμIL4TKO mice (16 weeks of age) that had developed colitis, as indicated by the presence of diarrhea, and the mice were sacrificed at 24 weeks of age. Granulomatous inflammation, identified grossly as nodular lesions, is present in the ileocecal junction area of the αμIL4TKO mice administered control Igs (arrow) but not those administered anti–IL-12p40 mAbs (arrowhead). Granulomatous inflammation was graded as follows: 0, no granulomas present; 1, aggregation of cells without definite granuloma formation that is characterized by nodular collections of epithelioid cells surrounded by lymphocytes; 2, an occasional small granuloma present; 3, a few large granulomas present; and 4, many granulomas present. Results are summarized in E. Gran, granulomas.

Since the generation of TCRα quadruple-KO mice deficient in B cells and IL-4 as well as IL-12p40 was difficult because of the colocalization of the TCRα and IL-12p40 loci in the same chromosome, the role of IL-12p40 in the development of granulomas was investigated by in vivo neutralization of IL-12p40 activity by administration of specific mAbs (32). The administration of anti–IL-12p40 mAbs but not of control rat Igs contributed to the suppression of granulomas (Figure 4, D and E). These results suggest that IL-12p40 but not IFN-γ is required for the development of intestinal inflammation-associated granulomas, consistent with a previous report showing a reduction in the size and number of polyvinyl­pyrrolidone-induced skin granulomas in IL-12p40 KO mice (7).

IL-23 that is produced by CD11c⁺ cells is regulated by IgG and IL-4. To determine whether CD11c⁺ cells are a source of IL-12p40 production in αμIL4TKO mice, freshly isolated CD11c⁺ and CD11c^– cells were subjected to real-time PCR analysis. A significantly higher level of IL-12p40 expression was detectable in CD11c⁺ than in CD11c^– cells from these mice (Figure 5A). This finding was further confirmed by using ELISA (Figure 5B). Of note, considerably greater enhancement of IL-12p40 production was observed when purified CD11c⁺ cells from grossly recognizable granulomas were examined (Figure 5B). IL-12p40 associates with IL-12p35 to form IL-12 or with p19 to form IL-23 (33), and DCs localized to the distal small intestine have been shown to express predominantly IL-23 (34). Importantly, IL-23 has been recently found to be a crucial factor in the development of granulomas (35). Interestingly, ELISA assay showed the spontaneous production of IL-23 but not IL-12 by granuloma-derived CD11c⁺ cells (Figure 5C).

Figure 5

IL-4 and IgG suppress IL-12p40 production by IMD-like cells. (A) Expression of β-actin and IL-12p40 by CD11c⁺ and CD11c^– fractions from the ileocecal junction of αμIL4TKO mice. (B) IL-12p40 production by CD11c⁺ and CD11c^– colonic fractions and CD11c⁺ granuloma fractions from αμIL4TKO mice (n = 4–6). *P < 0.005. (C) IL-12p40, IL-12, and IL-23 expression by CD11c⁺ granuloma fractions (n = 3). **P < 0.001. (D) IL-12p40 expressions by CD11⁺ granuloma cells cultured without (no) or with IL-4, IgG, F(ab)², immunocomplex (IC), IgG with anti-FcRγII/III mAb (IgG/αFC), or combination of IL-4 and IgG (IL-4/IgG). Values are averages (n = 6–9) of percentages of IL-12p40 production with additive divided by IL-12p40 production without additive. ^#P < 0.01. (E) FcγRIIb/III and IL-4Rα expression (solid histograms) on CD11c⁺CD11b⁺ granuloma cells. (F) IL-12p40 productions by purified CD11⁺ cells (n = 3–6) from the ileocecal junction of αμIL4TKO mice with (black bars) or without (white bars) granulomas, in response to PBS (no), peptidoglycan (PEG), poly(I:C), LPS, flagellin (Fra), loxoribine (Lox), CpG, or muramyl dipeptide (MDP). (G) IL-12p40 production by CD11⁺ granuloma cells in response to PBS (no), CpG, or CpG plus IL-4 (CpG/IL-4), IgG (CpG/IgG), or F(ab)² fragment [CpG/F(ab)]. Data represent averages of percentages of IL-12p40 production with additive divided by IL-12p40 production without additive. (H) QPCR shows IL-12p40, p35, and p19 expressions by CD11c⁺ granuloma cells (n = 4) stimulated with CpG. (I) IL-12p40, IL-12, and IL-23 production by CD11⁺ granuloma cells stimulated with CpG. (J) QPCR shows expression of IL-17a by purified CD4⁺ T cells (n = 3) from ileocecal junction of mice indicated. dRn, baseline-corrected, normalized fluorescence. ^#P < 0.01.

Since the absence of B cells as well as IL-4 was necessary for the development of granulomas, the effect of IL-4 and Igs on IL-12p40 production by IMD-like cells was also examined. Interestingly, the addition of IL-4 significantly suppressed the production of IL-12p40 by CD11c⁺ cells (Figure 5D). Addition of IgG purified from TCRαKO mice with colitis also reduced the IL-12p40 production by CD11c⁺ cells (Figure 5D). In addition, immunocomplexes made of OVA and anti-OVA IgG exhibited a suppressive effect on IL-12p40 production (Figure 5D). In contrast, F(ab)² fragments failed to reduce IL-12p40 production, and addition of anti-FcRγII/III mAbs inhibited IgG-mediated suppression of IL-12p40 production (Figure 5D). Interestingly, the strongest effect on the suppression of IL-12p40 production was observed when both IL-4 and IgG were added to the cultures of granuloma-derived CD11c⁺ cells (Figure 5D). Consistent with this response to IL-4 and IgG, CD11c⁺ cells from αμIL4TKO mice expressed IL-4Rα and FcγRII/III as defined by flow cytometric analysis (Figure 5E). These in vitro findings suggest that both IL-4 and the Fc portion of IgG contribute to the downregulation of IL-12p40 production by granuloma-associated IMD-like cells.

Since dysregulated host/enteric bacteria interactions are involved in the pathogenesis of chronic colitis (24), we determined whether specific microorganism-derived products stimulate IL-12p40 production by IMD-like cells. Purified CD11c⁺ cells from the colon with or without granulomas were cultured with several bacterial products that are recognized by specific pattern recognition receptors, such as TLRs and NOD2 (CARD15) (36). Interestingly, among these products, CpG (TLR9 ligand) and, to a lesser extent, loxoribine (TLR7 ligand) specifically stimulated CD11c⁺ cells from granulomatous intestine to produce IL-12p40 (Figure 5F). In contrast, CpG only slightly enhanced the production of IL-12p40 by CD11c⁺ cells from nongranulomatous intestine (Figure 5F). The induction of IL-12p40 production by CpG was downregulated in the presence of IL-4 or intact IgG but not by IgG F(ab)² fragments (Figure 5G). Furthermore, QPCR analysis revealed that both CpG-stimulated and freshly isolated CD11c⁺ cells from αμIL4TKO mice produced significantly more p19 (IL-23) and p40 than p35 (IL-12) (Figure 5H). This result was further confirmed by ELISA (Figure 5I). Since IL-23 has been demonstrated to induce IL-17–producing CD4⁺ T cells termed Th17 (37, 38), IL-17A and IL-17F expression by purified CD4⁺ T cells from the ileocecal junction of αμIL4TKO, αμDKO, αIL4DKO, TCRαKO, and WT mice was examined. Interestingly, a significant increase in IL-17A and IL-17F expression was observed in the CD4⁺ T cells from αμIL4TKO mice as compared with other mouse groups tested (Figure 5J and data not shown). These studies suggest that bacterial CpG stimulates granuloma-associated CD11c⁺ cells to produce IL-23 and that IgG and IL-4 contribute to the regulation of this production.

IMD-like cells can induce granuloma formation. To determine whether granuloma-derived IMD-like cells have the ability to induce granulomas, purified CD11c⁺ cells (3 × 10⁵; more than 90% of these cells expressed F4/80) from granulomas of αμIL4TKO mice (24 weeks of age) were directly injected into the ileocecal junction of young αμIL4TKO mice at 9 weeks of age, a time when granulomas have not yet developed. Interestingly, the local transfer of CD11c⁺ cells but not mock PBS injection resulted in the development of granulomas in the recipient ileocecal junction (Figure 6, A, B, and G). In contrast with αμIL4TKO mice, RAG-1KO mice (which do not have T or B cells), 6-month-old αμDKO mice (which develop colitis in the presence of IL-4), B cell–deficient IL-4 DKO mice (which lack both B cells and IL-4 but are not prone to develop colitis), and WT mice did not develop granulomas after the local injection of CD11c⁺ cells from granulomas of αμIL4TKO mice (Figure 6, C–E, and I). These findings indicate that IMD-like cells are capable of directly inducing granuloma formation in intestine and that the host factors, including absence of IL-4, impaired B cell function, and susceptibility to inflammation, are important determinants in granuloma formation.

Figure 6

Granuloma-derived IMD-like cells possess an ability to directly induce granuloma formation. (A–H) Purified IMD-like cells (3 × 10⁵) from granulomatous lesions of αμIL4TKO mice (24 weeks of age) (B–E, G, and H) or PBS (A) were injected into the intestinal wall at the ileocecal junction (A–E), subcapsular areas of the liver (G), and the kidneys (H) in young αμIL4TKO mice (9 weeks of age) (A, B, G, and H), RAG-1 KO mice (C), old αμDKO mice (24 weeks of age) (D), μIL4DKO mice (E). Recipient mice were sacrificed 3 weeks after injection and the organs were subjected to histological examination. (F) Purified CD11c⁺ cells from granulomatous lesions of αμIL4TKO mice (24 weeks of age) were labeled with CSFE and directly injected into the ileocecal junction. Recipient mice were sacrificed 4 days after injection and tissues were subjected to fluorescence microscopic analysis. Accumulation of green fluorescence⁺ cells is observed in the injection site. Data are representative of 2 individual experiments. (I) Summary of results of these local cell transfer experiments. Frequencies of granuloma induction are indicated on the bottom line. Red stars indicate tissues in which granuloma formation was induced.

To determine whether the locally transferred cells were retained at the injected sites, CFSE-labeled CD11c⁺ cells were utilized. Interestingly, CFSE⁺ cells with granuloma-like structure were detected at the injection sites 4 days after the cell injection (Figure 6F). To determine whether the ability of IMD-like cells to induce granuloma formation is intestine specific, granuloma-derived CD11c⁺ cells were directly injected into the liver and kidney where spontaneous granuloma development was not observed even in older αμIL4TKO mice (24 weeks of age). Surprisingly, the local transfer of CD11c⁺ cells was able to induce the development of granulomas in the liver and kidney of recipient young αμIL4TKO mice (9 weeks of age) (Figure 6, G–I).

IL-4 and IgG contribute to the suppression of IMD-like cell–mediated granuloma formation. Since intestinal inflammation–associated granulomas specifically develop in the absence of both IL-4 and B cells, studies were performed to determine whether IL-4 and/or IgG is actually required for the suppression of IMD-like cell–mediated granuloma formation. Purified F4/80⁺ CD11c⁺ cells (3 × 10⁵) from granulomas of αμIL4TKO mice (24 weeks of age) were directly injected into the ileocecal junction of young αμIL4TKO mice (9 weeks of age). IL-4 (10 μg) or PBS was then continuously supplied to some recipient mice for 3 weeks through a catheter from an osmotic pump that was implanted within the dorsa, as previously described (29). In other recipient mice, IgG (1 mg) purified from TCRαKO or WT mice was repeatedly injected i.p. once every 3 days for 3 weeks. Notably, the IMD-like cell–mediated granuloma formation was completely abolished by the delivery of IL-4 (Figure 7, A and E) but not PBS (Figure 7, B and E). In addition, the administration of IgG from not only TCRαKO but also WT mice resulted in significant suppressive effect on granuloma development; however, complete abolishment of granuloma formation was not achieved, as indicated by the presence of small granuloma-like structure and lymphoid aggregates (Figure 7, C–E). Furthermore, significant downregulation (more than 4-fold) of IL-12p40 and p19 expression in the ileocecal junction area was observed in the recipient αμIL4TKO mice who received administration of IL-4 or IgG compared with those who received PBS administration (Figure 7F). These findings suggest that the ability of IMD-like cells to form intestinal granulomas is reciprocally regulated by both IL-4 and IgG.

Figure 7

Both IL-4 and IgG are required for the regulation of IMD-like cell–mediated granuloma formation. (A–E) CD11c⁺ cells from granulomatous of old αμIL4TKO mice were injected into the intestinal wall at the ileocecal junction in young αμIL4TKO mice. IL-4 (A, n = 5) or PBS (B, n = 5), or IgG from TCRαKO mice (C, n = 5) or WT mice (D, n = 5), were administered into recipient mice. Recipient mice were sacrificed at 3 weeks after local cell transfer. Scores of granulomas are summarized in E. Statistical significance is indicated by asterisks. *P < 0.05; **P < 0.0001. (F) RNA isolated from ileocecal junction of recipient mice with administration of PBS (black line, n = 5), IL-4 (red line, n = 5), or IgG (blue line, n = 5) was subjected to QPCR analysis for the detection of β-actin, IL-12p40, and p19. Lines represent the averages of 5 mice in each group. (G) Purified CD11c⁺ cells from granulomas of old αμIL4TKO mice were injected into the intestinal wall at the ileocecal junction in young αμIL4TKO mice without (top panels) or with (bottom panels) depletion of enteric bacteria by treatment with a combination of antibiotics. No granulomas were detectable in the decontaminated recipient mice (n = 8). Gross findings (left panels) show no recognizable nodular formation in the ileocecal junction with enlargement of cecum in the mouse with depleted enteric flora (bottom left) versus presence of granuloma formation in the ileocecal junction (arrow) without enlargement in the control mouse with enteric flora (top left).

Enteric bacteria are required for IMD-like cell–mediated granuloma formation. Since IL-12p40 and p19 expression by IMD-like cells was significantly enhanced by stimulation with the bacterial product CpG (Figure 5G), we next examined the role of enteric flora in the IMD-like cell–induced granuloma formation by locally transferring granuloma-derived IMD-like cells into the ileocecal junction of αμIL4TKO mice in which enteric flora had been depleted by a combination of broad-spectrum antibiotics in their drinking water, as previously described (39). As shown in Figure 7G, the antibiotic-treated mice exhibited a dramatic enlargement of the cecum, a characteristic feature of germ-free mice (40). Of note, no granuloma was identified in the ileocecal junction of 8 antibiotic-treated αμIL4TKO mice that received local transfer of IMD-like cells (Figure 7G). In contrast, local IMD-like cell transfer induced granuloma formation in 3 out of 8 αμIL4TKO mice that did not receive antibiotic treatment (Figure 7G). These findings suggest that, in addition to altered host factors (IL-4 and IgG), enteric flora is required for IMD-like cell–induced intestinal granuloma formation.

Immature DCs differentiate into IMD-like cells under immune-compromised conditions. To determine whether the ability of IMD-like cells developing in αμIL4TKO mice to form granulomas is specific for these mice, BM-derived DCs from WT mice were expanded in vitro and separated into CD86^–CD11c⁺CD11b⁺ (immature) and CD86⁺CD11c⁺CD11b⁺ (mature) myeloid DC subsets, then injected into the ileocecal junction of young αμIL4TKO mice. The local transfer of immature (Figure 8, A and E) but not mature (Figure 8, B and E) myeloid DCs led to the development of granulomas in the recipient αμIL4TKO mice. In contrast, the local transfer of BM-derived immature DCs from WT mice failed to induce granuloma formation in the recipient αIL4DKO (Figure 8, C and E) and αμDKO (Figure 8, D and E) mice. These findings suggest that IMD-derived cells are capable of inducing granuloma formation when provided with a granuloma-favoring microenvironment (absence of IL-4 and impaired B cell function).

Figure 8

Potential ability of immature DCs to differentiate IMD-like cells capable of inducing granuloma formation. (A–D) BM-derived DCs from WT mice were expanded in in vitro culture with GM-CSF in the presence or absence of LPS. After culture, immature (CD11c⁺CD11b⁺CD86^–, purity of more than 98%) (A, C, and D) and mature (CD11c⁺CD11b⁺CD86^high, purity of more than 98%) (B) myeloid DCs were purified by FACS sorting. We directly injected 3 × 10⁵ cells into the ileocecal junction of young αμIL4TKO (A and B), αIL4DKO (C), and αμDKO (D) mice (9 weeks of age). Recipient mice were sacrificed 3 weeks after injection. (E) Results of these local cell transfer experiments are summarized. Frequencies of granuloma induction are indicated on the bottom line. Red star indicates the tissues in which granuloma formation was induced. (F) BM-derived DCs from GFP transgenic mice were expanded in culture with GM-CSF in vitro. After culture, CD11c⁺ cells were purified through the MACS system. We injected 3 × 10⁵ cells directly into the ileocecal junction of αμIL4TKO mice, and recipient mice were sacrificed 3 weeks after injection. Cells were isolated from the ileocecal junction and subjected to flow cytometric analysis. Top panel shows F4/80 expression on gated GFP⁺CD11c⁺ cells before cell injection (blue line) and from the recipient ileocecal junction (red line). F4/80 versus CD11c expression on gated GFP⁺ cells among the cells isolated from the recipient ileocecal junction is shown (bottom panel). Data are representative of 2 individual experiments.

To track donor-derived DCs in the recipient mice, IMDs from the BM of GFP transgenic mice were locally transferred into ileocecal junctions of recipient αμIL4TKO, αμDKO, and αIL4DKO mice. Notably, F4/80 expression was induced on the transferred immature DCs in the recipient αμIL4TKO mice, as indicated by the expression of F4/80 on the transferred GFP⁺CD11c⁺ cells isolated from the ileocecal junction of the recipient mice as compared with the lack of F4/80 expression by GFP⁺CD11⁺ cells before the transfer (Figure 8F). In contrast, GFP⁺ cells were not detectable in the cells isolated from the ileocecal junctions of recipient αμDKO and αIL4DKO mice following local transfer of immature DCs. These findings suggest that BM-derived immature DCs differentiate into IMD-like cells in the absence of both IL-4 and B cells. In addition, the presence of both IL-4 and B cell deficiencies enhances the retention of immature DCs within the intestine, presumably to induce their differentiation into IMD-like cells.

A unique DC-like subset that coexpresses CD11c and F4/80 with an immature phenotype and exhibits morphological similarity with foamy macrophages has recently been identified in the lung after infection of Mycobacterium tuberculosis, which characteristically causes granulomatous inflammation (6, 41). We demonstrate herein that an increase in the similar cell subset is also associated with the development of intestinal granulomas in TCRαKO mice in the absence of both IL-4 and B cells. This CD11c⁺F4/80⁺ cell subset present in intestinal granulomas shares several features with IMDs. These features include CD11b expression, low CD86 expression, reduced antigen-presenting capability, and enhanced phagocytic activity. Of note, this unique subset is also characterized by the presence of fine dendrites (Figure 3D). After the capture of pathogens, inflammatory monocytes rapidly differentiate into DCs or macrophages; monocytes that remain in the inflammatory site generally differentiate into macrophages (42, 43), and intradermal injection of Salmonella blocks the differentiation of monocytes into functional DCs and favors their differentiation into macrophages (44). Although in granulomatous colitis, classical macrophage populations without DC markers exist (data not shown), the majority of CD11c⁺ cells express the macrophage marker F4/80 (Figure 3 and Supplemental Figure 1). Therefore, it is possible that granuloma-favoring conditions (with impaired IL-4 and IgG production) may disturb the appropriate differentiation pathway of inflammatory monocytes into DCs/macrophages. Indeed, Igs have been shown to induce maturation of DCs (45, 46). Furthermore, IL-4 has been widely used to generate BM-derived DCs in vitro, and recent reports suggest that IL-4–induced signaling is required for the maturation rather than expansion of myeloid DCs (47, 48). Recent studies have also demonstrated that the majority of DCs present in the intestinal LP, where these cells are constitutively exposed to enteric microorganisms, exhibit a CD11c^lowCD11b⁺CD86^low phenotype (11–13). Interestingly, the immature phenotype of intestinal LP DCs is preserved even after development of inflammation or after exposure to LPS (11–13), suggesting that the immature phenotype of CD11c⁺ cells represents an intestinal LP–specific DC subset that, after migration into the draining lymph nodes, is induced to complete differentiation/maturation (14, 42). We herein show that the locally transferred BM-derived IMDs are retained within the recipient intestine and differentiate into IMD-like cells in the absence of both IL-4 and B cells. Therefore, it is also possible that B cell and IL-4 deficiency alters programmed intestinal DC differentiation pathways and induces the development of unique IMD-like cells capable of forming granulomas.

Our local cell-transfer study indicates that IMD-like cells differentiated under intestinal granuloma–inducing microenvironments can induce granuloma development not only in the intestine but also in nonintestinal sites such as liver and kidneys when transferred under Th1-polarized conditions. Therefore, IMD-like cells may also be involved in the development of other Th1-mediated granulomas, such as granulomas associated with bacterial (M. tuberculosis) infection. Indeed, infection by M. tuberculosis induces the development of a unique cell subset sharing both properties of DCs and macrophages in the lung (6, 41). The IMD-like cells spontaneously produce large amounts of IL-12p40 that is involved in the development of granulomas in the intestine (Figure 4D) and the skin (7). Since the IMD-like cells mostly express IL-12p40 and p19 but not p35, IL-23 rather than IL-12 may be the crucial cytokine necessary for the development of granulomas (35). Most recently, involvement of the IL-23R–signaling cascade in the pathogenesis of Crohn disease was identified by a genome-wide association study (49). Indeed, a pathogenic role of IL-23 has recently been shown in innate and acquired immune-mediated experimental colitis models (50–53). In contrast, the roles of IL-23 may differ depending on the type of intestinal inflammation, as suggested by a regulatory role of IL-23 in acute colitis models that are induced by dextran sulfate sodium (DSS) or trinitrobenzene sulfonic acid (TNBS) (54).

Importantly, our study also indicates that several factors, including cytokine balance, B cell function, and enteric bacteria, regulate the ability of IMD-like cells to form granulomas. Host/microbial interactions are considered crucial for the development of chronic intestinal inflammation (24). The production of IL-12p40 by DCs is enhanced in B cell–deficient mice (46, 55) and suppressed by IL-4 (56, 57). Engagement of inhibitory Fc receptor FcgRIIB by IgG or as immune complexes is required for the B cell–mediated suppression of S. mansoni–induced granulomas through the activation of an immunoreceptor tyrosine-based inhibition motif (ITIM) (3). In addition, as FcγRIIB accounts for over 75% of total FcR expression on DCs (58), IgG generally contributes to the suppression of immune activation by DCs (45, 46, 58). Our studies indicate that IL-4 and the Fc portion of IgG can regulate IL-23 production of IMD-like cells, which express both FcγRII and IL-4Rα. Indeed, the administration of either IL-4 or IgG led to suppression of granuloma formation induced by local transfer of granuloma-derived IMD-like cells (Figure 7). In addition, in vitro experiments (Figure 5D) showed that both uncoupled IgG and immunocomplexes suppress IL-12p40 production by IMD-like cells.

In summary, we have identified an intestinal granuloma–associated unique CD11c⁺ subset that not only shares phenotypic and functional features with IMDs but also coexpresses a marker associated with macrophages. Importantly, the ability of these unique IMD-like cells to induce granuloma formation is modulated by IL-4, IgG, and enteric flora.

View Supplemental data

We would like to thank Robert T. McCluskey, Cathy Nagler, and Hiromichi Ishikawa for their helpful comments and advice and H.J. Winn for kindly providing anti–IL-12p40 mAbs (clone C17-8 provided by C. Trinchieri). This study was supported by NIH grant DK47677 (to A.K. Bhan); NIH grant DK064351 and the Eli and Edythe L. Broad Medical Research Program (to A. Mizoguchi); NIH grants DK44319, DK51362, and DK53056 (to R.S. Blumberg); and DK64289 (to E. Mizoguchi); and the Center for the Study of Inflammatory Bowel Disease (Massachusetts General Hospital).

Address correspondence to: Atsushi Mizoguchi, Simches 8234, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA. Phone: (617) 726-8492; Fax: (617) 643-3566; E-mail: amizoguchi@partners.org.

Nonstandard abbreviations used: αμDKO, B cell–deficient TCRα double-KO (mice); αIL4DKO, IL-4–deficient TCRα double-KO (mice); αμIL4IFNQKO, TCRα quadruple-KO (mice) deficient in B cells; αμIL4TKO, TCRα triple-KO (mice); IMD, immature myeloid DC; LP, lamina propria; QPCR, quantitative PCR; RPA, RNase protection assay.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.117:605–615 (2007). doi:10.1172/JCI30150

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