VEGF-C–dependent stimulation of lymphatic function ameliorates experimental inflammatory bowel disease

Crohn’s disease (CD) and ulcerative colitis (UC) are chronic inflammatory bowel diseases (IBDs) of unknown etiology that are associated with an aberrant mucosal immune response. Neoangiogenesis and vascular injury are observed in IBD along with increased lymphangiogenesis. While the pathogenic role of angiogenesis in IBD is well characterized, it is not clear how or if increased lymphangiogenesis promotes disease. Here, we determined that enhancing lymphangiogenesis and lymphatic function reduces experimental IBD. Specifically, we demonstrated that adenoviral induction of prolymphangiogenic factor VEGF-C provides marked protection against the development of acute and chronic colitis in 2 different animal models. VEGF-C–dependent protection was observed in combination with increased inflammatory cell mobilization and bacterial antigen clearance from the inflamed colon to the draining lymph nodes. Moreover, we found that the VEGF-C/VEGFR3 pathway regulates macrophage (MΦ) plasticity and activation both in cultured MΦs and in vivo, imparting a hybrid M1-M2 phenotype. The protective function of VEGF-C was meditated by the so-called resolving MΦs during chronic experimental colitis in a STAT6-dependent manner. Together, these findings shed light on the contribution of lymphatics to the pathogenesis of gut inflammation and suggest that correction of defective lymphatic function with VEGF-C has potential as a therapeutic strategy for IBD.

The inflammatory bowel diseases (IBDs), consisting of ulcerative colitis (UC) and Crohn’s disease (CD), are characterized by chronic inflammation of the gastrointestinal tract in genetically susceptible individuals exposed to environmental risk factors. The etiology of IBD has been extensively investigated in the past few decades, but its pathogenesis is not fully understood (1–3). While the study of IBD has been largely focused on the roles played by the classical components of the immune system, other reports have called attention to the significant contribution of nonimmune cells, such as epithelial, endothelial, mesenchymal, and vascular cells (4–6). In particular, there is a growing body of evidence showing that IBD is associated with the development of new blood (angiogenesis) and lymph (lymphangiogenesis) vessels from the existing vascular network (7–10). Although the pathogenic role of angiogenesis in IBD is well understood (11), whether lymphangiogenesis plays a protective or pathogenic role is still unknown. Recent studies demonstrated that lymphatic vessels (LVs) are functionally important for the resolution of intestinal inflammation. For example, mice deficient for angiopoietin-2 show intestinal lymphatic dysplasia and exacerbated colitis in the dextran sulfate sodium (DSS) colitis model (12, 13), while selective ablation of the LVs leads to distortion of the intestinal villi architecture and severe bowel inflammation (14). Although the precise pathophysiology of IBD is unknown, LV obstruction and dysfunction are long-recognized features observed in humans with this disease (15, 16). The existence of an abnormal lymphatic system is further supported by a recent article describing defective drainage capacity of LVs in human CD bowel strictures (17). Moreover, among patients with CD, relatively lower LV density was associated with a higher risk of endoscopic disease recurrence after surgery, suggesting that patients could benefit from improved lymphatic flow (18). In an animal model of intestinal ileitis, impaired contractile function of mesenteric collecting lymphatics was observed both in vivo and in vitro, again showing a strong correlation with the degree of mucosal inflammation (19). Nevertheless, together with LV dysfunction, macrophages (MΦs) may also have a fundamental contribution to IBD pathogenesis. They have been found to play opposing roles in experimental models of intestinal inflammation, such as the DSS and IL-10–KO models of colitis (20, 21). In fact, while classically activated M1 MΦs contribute to the pathogenesis of these 2 models of experimental colitis primarily by secreting proinflammatory cytokines, such as IL-6, TNF-α and IFN-γ, and causing tissue damage (22), alternatively activated M2 MΦs contribute to the resolution of colitis primarily by expressing low levels of proinflammatory cytokines, but high levels of arginase 1 (Arg-1), FIZZ1, YM-1, and IL-10 (20, 23, 24). Thus, the factors that modulate MΦ polarization could affect the severity of human and experimental colitis. These observations suggest that resolution of chronic inflammation may require restoration of proper lymphatic function and proper MΦ activation because of the essential importance of maintaining normal flow balance and removing inflammatory cells, mediators, and bacterial antigens away from inflamed sites (25).

Lymphangiogenesis is mediated by binding of the lymphatic vascular endothelial selective growth factors VEGF-C and VEGF-D to VEGFR3 (26, 27). VEGFR3, also known as Flt4, was the first lymphatic-specific growth factor receptor identified (28), and its neutralization by specific antibodies reduces bacterial infection associated–airway inflammation (29) and surgical blockade–induced lymphangiogenesis (30, 31). Moreover, antilymphatic treatment with anti-VEGFR3 antibodies in an animal model of IBD aggravates inflammation and submucosal edema, increases leukocyte infiltration, and causes the LVs to become tortuous (13, 32). VEGF-C is also chemotactic for MΦs during pathological conditions, and its receptor VEGFR3 is expressed by a substantial fraction of peripheral blood monocytes and activated tissue MΦs (33, 34). Overall, these studies have established a direct role for VEGF-C/VEGFR3 signaling in inflammation-induced lymphangiogenesis and immune response and suggest that therapies aimed at promoting lymphatic function, e.g., with prolymphangiogenic factors, such as VEGF-C, may provide a novel strategy for the treatment of inflammatory conditions, including IBD. In the current study, we examined the effect of stimulating lymphatic function and adaptive immune response via VEGF-C/VEGFR3 signaling on the severity of intestinal inflammation, on lymphatic drainage, as well as on bacterial antigen clearance and MΦ activation during inflammatory conditions. Furthermore, we evaluated the mechanism through which this pathway acts in experimental disease progression.

LV density and the VEGF-C/VEGFR3 pathway are altered in active IBD–involved colons. To assess whether lymphangiogenesis occurs in human IBD, we performed immunohistochemical staining of control and active CD– and UC–involved colons with antibodies recognizing podoplanin, a widely accepted marker of lymphatic vasculature (9, 10, 35). Normal colon mucosa only showed sporadic, thin LVs in the lamina propria and submucosa, whereas CD and UC tissues contained numerous, readily detectable dilated LVs (Figure 1A). Quantitative analysis of the same sections revealed that the total number of LVs per field in the lamina propria and submucosa of both CD and UC specimens was significantly increased compared with that of the control non-IBD tissue (Figure 1B), thus confirming the occurrence of lymphangiogenesis in IBD patients.

Figure 1

LV density and VEGF-C/VEGFR3 signaling are increased in the colon of IBD patients. (A and B) Immunohistochemical staining with an Ab recognizing podoplanin was performed on control (NL) and active CD– and UC–involved colons (A). Quantitative analysis of LV density in the same sections is shown in B. Data are expressed as the mean percentage of LV per area ± SEM. Scale bar: 100 μm. (C) Control (n = 15) and CD (n = 16) and UC (n = 16) patients with active inflammation were investigated for VEGF-C and VEGF-D content in mucosal extracts. VEGF-C and VEGF-D were measured by ELISA. (D) Frozen sections from the colons of control (NL, n = 15) or CD (IBD, n = 16) patients with active inflammation stained with both anti-LYVE1 and anti-VEGFR3 Abs. Original magnification, ×40. (E) VEGFR3⁺ LVs were quantified by stereological point counting of 10 regions per section, and the numbers were normalized per total section area expressed in mm². Results are presented as the mean vessel density per group ± SEM. (F and G) MFI per vessel was analyzed on VEGFR3⁺ (F) and LYVE1⁺ (G) LVs (10–15 vessels per section). MFI is expressed as relative units normalized per vascular area expressed in μm². *P < 0.01; **P < 0.001 (B, C, E, and F).

The molecular mechanisms of inflammation-induced lymphangiogenesis remain largely unknown. Reports showing inhibition of LV growth by blockade of VEGFR3 signaling demonstrate the involvement of this receptor in inflammation-driven lymphangiogenesis (29). To verify whether the VEGF-C/VEGFR3 pathway is involved in intestinal inflammation–dependent lymphangiogenesis, control and active CD– and UC–involved colons were analyzed for the expression of these 2 molecules. Figure 1C (upper panel) shows that compared with control tissues, VEGF-C expression was upregulated in the mucosal extracts of patients with IBD. The higher VEGF-C levels observed in UC patients compared with those in CD patients may be the result of a greater degree of mucosal inflammation in the UC samples. Notably, the other VEGFR3 ligand VEGF-D showed comparable amounts in all groups (Figure 1C, lower panel), and for this reason, it was subsequently excluded from the study. Frozen sections double-stained with anti-LYVE1 and anti-VEGFR3 Abs showed increased VEGFR3⁺ vessel density in inflamed tissue compared with that seen in quiescent LVs in control sections (Figure 1, D and E). Coexpression of VEGFR3 and LYVE1 was quantified using the method described by Tammela et al. (36), which measures the fluorescent intensity of target expression normalized per vascular area. This revealed a lymphatic-specific increase in VEGFR3 (Figure 1F), but not LYVE1 (Figure 1G), in CD and UC sections compared with that seen in control tissues, suggesting that inflammation also increases VEGFR3 expression in individual vessels. Collectively, these data demonstrate that mucosal lymphatic vascularization is increased in active IBD–involved tissues and suggest that chronic inflammation stimulates the VEGF-C/VEGFR3 pathway, a finding compatible with inflammation-associated lymphangiogenesis.

Role of VEGFR3 signaling in human intestinal lymphatic endothelial cell behavior in vitro. Proliferation, migration, and capillary reorganization are all processes necessary for lymphatic regeneration (37). To assess the role of VEGFR3 and its ligand VEGF-C in human intestinal lymphatic endothelial cell (HILEC) behavior, these cells were isolated from normal and IBD mucosa, as described in the Supplemental Information (supplemental material available online with this article; doi:10.1172/JCI72189DS1), and their biological functions were characterized in vitro. Medium supplemented with VEGF-C promoted the proliferation (Supplemental Figure 1A), migration (Supplemental Figure 1B), and capillary formation (Supplemental Figure 1, C and D) of both control (NL) and IBD HILECs, with a significantly greater response by IBD HILECs when compared with that by NL cells. VEGF156 mutant, a selective agonist that specifically binds VEGFR3, induced similar results, indicating that the effects of VEGF-C do not require VEGFR2 binding. After 96 hours of VEGF-C/VEGF156 treatment, blocking the activation of VEGFR3 (SAR131675) (38) significantly inhibited both basal and VEGF-C– or VEGF156-induced proliferation (Supplemental Figure 1A), migration (Supplemental Figure 1B), and tube formation (Supplemental Figure 1, C and D), with similar effects in NL and IBD HILECs, showing that VEGFR3 activation is necessary for these biological functions. We found that cell lysates from VEGF-C–stimulated NL and IBD HILECs displayed increased VEGFR3 phosphorylation (p-Tyr), which in turn was inhibited upon SAR131675 treatment (Supplemental Figure 1E). Moreover, overexpression of VEGFR3 in IBD HILECs confirmed what we observed by immunofluorescence (see Figure 1) and demonstrated that our method of isolation could represent a novel tool for in vitro study of lymphatics in IBD.

Systemic delivery of VEGF-C significantly improved experimental colitis both clinically and histologically. Since VEGFR3 signaling is essential for lymphangiogenesis in various conditions (26, 39), we sought to determine whether the pathway was also important for lymphangiogenesis in chronic experimental colitis. For this purpose, we studied the functional role of the VEGF-C/VEGFR3 pathway in vivo by systemic inhibition of VEGFR3 (15) or by delivery of the human lymphangiogenic factor VEGF-C in DSS and IL-10–KO models of chronic colitis using a blocking Ab or adenoviral transfer, respectively. Mice undergoing 2 cycles of DSS treatment and 15-week-old IL-10–KO animals with established colitis were injected with adenoviruses encoding human VEGF-C (Ad-hVEGF-C) or with the anti-VEGFR3 Ab mF431C1. Western blot and immunoprecipitation analyses of tissue lysates obtained from the colons of each experimental group clearly showed the efficient inhibition of VEGFR3 phosphorylation and upregulation of Ad-hVEGF-C at the indicated time points (Supplemental Figure 2A and data not shown). Notably, in the DSS model, we found that VEGFR3 protein levels were increased during both the acute and chronic phases of intestinal inflammation (Supplemental Figure 2A).

Systemic delivery of Ad-hVEGF-C significantly reduced the severity of chronic intestinal inflammation in both experimental models in terms of percentage of body weight (Figure 2, A and B), endoscopic evaluation (Figure 2C and Supplemental Figure 2B), and disease activity index (DAI, a combinatorial score including stool blood, form, and body weight) (Supplemental Figure 2, C and D), whereas mF431C1 worsened the clinical course compared with that of control groups (only DSS-treated and IL-10 WT mice). Notably, DSS-treated mice showed similar effects also in the acute phase of inflammation (Figure 2, A and E, and Supplemental Figure 2, B and D). Protection against chronic colitis by VEGF-C in these 2 experimental models was paralleled by histological examination, in which we also observed reduced inflammatory cell infiltration into the intestinal mucosa and diminished submucosal edema (Figure 2, D and E).

Figure 2

Systemic delivery of human VEGF-C significantly improves colitis both clinically and histologically. Mice undergoing 2 cycles of DSS treatment and 15-week-old IL-10–KO mice with established colitis were injected with Ad-hVEGF-C (n = 8/time point) or anti-VEGFR3 Ab (mF431C1, n = 8/time point). Systemic delivery of VEGF-C significantly reduced the severity of colitis based on percentage of body weight (A and B), endoscopic evaluation (C), and histological examination (D and E), whereas mF431C1 worsened the colitis compared with that seen in control mice, which received DSS alone. UN, no DSS; WT, IL-10 WT. Black dashed lines represent the 2 DSS cycles. Red and green dashed lines indicate the first VEGF-C and mF431C1 administration, respectively. (C) Representative endoscopic images of colons from the indicated groups of mice after the second cycle of DSS (day 28). (D) Representative H&E-stained images of colons from the indicated groups of mice at the end of the experiment (day 40 for DSS-treated mice and week 20 for IL-10–KO animals). (E) Histological scores of the entire section were quantified blindly by an expert pathologist and were expressed as the mean value per group ± SEM. All other graphs represent the mean value ± SEM. *P < 0.05 versus DSS alone; **P < 0.001 versus DSS alone.

The VEGF-C/VEGFR3 pathway is required for lymphangiogenesis in experimental colitis. Lymphatics expanded dramatically during DSS-induced acute and chronic colitis. Frozen sections from the colon of control (untreated) or DSS-treated (colitic) mice were double stained with anti-LYVE1 and anti-VEGFR3 Abs, and VEGFR3⁺ LVs were quantified in 10 regions per section. The results showed an increase in VEGFR3⁺ vessel density in inflamed tissues compared with LVs in control tissues (Supplemental Figure 3, A and B). Coexpression of VEGFR3 and LYVE1 was quantified, and the fluorescent intensity of target expression was normalized per vascular area. This revealed a lymphatic-specific increase in VEGFR3 (Supplemental Figure 3C) in colitic sections compared with that observed in control tissues, suggesting that inflammation also increases VEGFR3 levels in individual vessels. In parallel, protein extracts from the colon of colitic mice showed increased VEGF-C expression levels compared with those in healthy animals (Supplemental Figure 3D), as observed in human tissues (see Figure 1). We obtained Similar results with IL-10–KO animals (data not shown).

Whole mounts of proximal, distal, and rectal colonic segments from DSS-treated and untreated mice were stained with Abs against LYVE1 and CD31 to measure the area density and dimension of LVs during acute and chronic colitis. We found that systemic delivery of VEGF-C significantly increased lymphangiogenesis at the indicated time points, both in terms of LV density (Figure 3, A and B) and size (Supplemental Figure 3E) in comparison with the DSS-only control group. In contrast, mF431C1 had an inhibitory effect on LV dimension (Supplemental Figure 3E) and the number of LVs per area (Figure 3B). Frozen sections from colons of WT and colitic IL-10–KO mice were also double stained with anti-LYVE1 and anti-CD31 Abs (Figure 3C), and LYVE1⁺ LVs were counted in 10 regions per section at the indicated time points. Our results showed that while systemic delivery of VEGF-C significantly increased LV density, mF431C1 reduced the number of LVs per area compared with that in the WT control group (Figure 3D). Notably, neither systemic inhibition of VEGFR3 nor delivery of the human lymphangiogenic factor VEGF-C affected the number of CD31⁺ blood vessels in the inflamed colon (Figure 3E and data not shown). This indicates that this pathway does not influence angiogenesis during experimental colitis. Besides, except for increased lymphangiogenesis in various organs, such as the liver and spleen, we failed to find any evidence of inflammation or organ failure (data not shown), thus allowing us to conclude that no major systemic effects are expected upon treatment with this prolymphangiogenic factor. To determine the proliferative status of LVs at different stages of intestinal inflammation, untreated and VEGF-C– or mF431C1-treated colitic mice were injected with BrdU 2 hours prior to sacrifice. We costained the control and inflamed sections for BrdU in combination with anti-LYVE1 Ab and quantified the number of BrdU⁺ cells per LV (Figure 4A). The results showed that systemic delivery of Ad-hVEGF-C enhanced LV proliferation in both the acute and chronic phases of DSS-induced colitis (Figure 4B) and in colitic IL-10–KO animals (Figure 4C) when compared with control groups (UN and WT, respectively). In contrast, mF431C1 treatment completely inhibited LV growth in both experimental models (Figure 4, B and C). LV modulation correlated with disease activity and weight loss, demonstrating that the VEGF-C/VEGFR3 pathway is not only required for proper lymphangiogenesis during intestinal inflammation, but that lymphangiogenesis per se might be important for the resolution of inflammation.

Figure 3

Systemic delivery of Ad-hVEGF-C increases LV density in colitic mice. Whole mounts of proximal, distal, and rectal colonic segments from DSS-treated mice with acute and chronic colitis were stained with Abs against LYVE1 (green) and CD31 (red) to measure LV area density. (A) Representative images of whole-mount staining of distal colons from the indicated groups at day 10. Scale bar: 75 μm. (B) LV density was quantified at the indicated time points and reported as the mean values ± SEM for each group (n = 8 mice/group with 5 fields/colonic segment). Scale bar: 40 μm. *P < 0.05; **P < 0.001 versus DSS alone. (C) Representative images of frozen sections from colons of treated control (WT, n = 8) and IL-10–KO (colitic, n = 8) mice stained for LYVE1 (green), CD31 (red), and DAPI (blue). Scale bar: 75 μm. LYVE1⁺ (D) and CD31⁺ (E) vessels were counted in 10 regions per section, and the numbers were normalized per total section area expressed in mm². Results are presented as the mean vessel density per group ± SEM. Black dashed lines represent the 2 DSS cycles. Red and green dashed lines indicate the first VEGF-C and mF431C1 administration, respectively. *P < 0.05; **P < 0.001 versus DSS alone.

Figure 4

LV proliferation is increased in VEGF-C–treated colitic mice and blocked upon mF431C1 administration. Untreated (n = 8/time point) and VEGF-C– (n = 8/time point) or mF431C1-treated (n = 8) colitic mice were injected with BrdU 2 hours prior to sacrifice at the indicated time points. (A) Representative images of paraffin-embedded inflamed sections from DSS-treated mice were costained for BrdU (brown) in combination with anti-LYVE1 Ab (red), and the number of BrdU⁺ cells per LV was quantified in each group (B and C). Counts were performed on consecutive and randomly selected fields (n = 10 fields/section for each group), and data are reported as the mean ± SEM. Black dashed lines represent the 2 DSS cycles. Red and green dashed lines indicate the first VEGF-C and mF431C1 administration, respectively. Scale bar: 28 μm. *P < 0.05; **P < 0.001 versus DSS alone.

Exacerbation of experimental colitis by VEGFR3 blockade is associated with decreased afferent lymph flow and inflammatory cell migration to draining nodes. Studies reporting abnormalities, such as submucosal edema, extensive dilation of lacteals (40, 41), lymphangitis, immune cell trafficking, and lymphangiogenesis (9, 42) in IBD patients suggest impaired lymph drainage and lymphatic pumping, implicating the lymphatic system as a player in various inflammatory disorders, including IBD. For this reason, we next investigated whether inhibition of lymphangiogenesis in mF431C1-treated mice is related to reduced lymphatic drainage and decreased inflammatory cell mobilization from the inflamed tissue to the draining lymph nodes (DLNs). To this end, we injected 10 μg of Evans blue dye into the colonic mucosa of Ad-hVEGF-C–treated and mF431C1-treated mice undergoing 2 cycles of DSS treatment, using DSS only–treated mice as a control group. Evans blue dye, which is specifically taken up by the lymphatic vasculature (43), was then extracted 16 hours after its injection. The extraction of Evans blue dye from distal colons of comparable weight revealed that although lymphatic drainage was decreased in the chronic phase of colitis when compared with healthy (untreated) mice, it was enhanced by systemic delivery of VEGF-C (Figure 5A). By contrast, mF431C1-treated mice had significantly more dye remaining in the inflamed tissue than did mice treated with DSS alone (Figure 5A), suggestive of reduced lymphatic clearance function. We obtained similar results with Ad-hVEGF-C– and mF431C1-treated IL-10–KO mice, using WT animals as a control group (Supplemental Figure 3F). We further determined the rate of inflammatory cell mobilization from healthy and inflamed colons to the DLNs by intramucosal injection of GFP⁺ inflammatory cells (Figure 5B). Quantification of GFP⁺ cells in the DLNs by immunofluorescence (Figure 5C, upper panels) or FACS analysis (Figure 5C, lower panels) showed that systemic delivery of VEGF-C considerably increased inflammatory cell migration, whereas systemic inhibition of the receptor VEGFR3 reduced the mobilization rate of inflammatory cells from the inflamed colon (Figure 5, C and D). Overall, these data suggest that during experimental chronic colitis, the VEGF-C/VEGFR3 pathway plays a fundamental role in increasing lymph flow and inflammatory cell mobilization from the inflamed site to the DLNs, both of which are important events for inflammatory resolution.

Figure 5

Systemic delivery of VEGF-C markedly increases lymphatic drainage and mobilization of inflammatory cells from the inflamed colon to DLNs. (A) 10 μg of Evans blue dye was injected into the colonic mucosa of healthy (UN, n = 4/time point), Ad-hVEGF-C– (n = 5/time point), and mF431C1-treated mice (n = 5/time point) undergoing 2 cycles of DSS treatment. Mice treated with DSS alone (n = 5/time point) were used as a control group. Evans blue was then extracted 16 hours after the dye injection from distal colons of comparable weight. Graph shows the total dye remaining in the colon expressed in μg. Data represent the mean per group ± SEM. Black dashed lines represent the 2 DSS cycles. (B–D) GFP⁺ peritoneal inflammatory cells (~10⁶ cells) were injected into the rectal mucosa of the inflamed and noninflamed colons of healthy (UN, n = 5) and DSS-treated mice (n = 5 per experimental group, at day 5 after the second DSS cycle) and IL-10 WT (n = 4) and IL-10–KO mice (n = 4 per group at day 21 after the first Ad-hVEGF-C administration). Twelve hours later, the DLNs were sampled and sectioned for histologic and FACS analysis. (C) Upper panels: Representative frozen sections of DLNs from the indicated experimental groups coimmunostained for LYVE1 (red) and DAPI (blue) and merged. Lower panels: Representative FACS from the indicated groups. The number in each FACS histogram represents the percentage of GFP⁺ cells. Scale bar: 50 μm. (D) GFP⁺ inflammatory cells in the entire frozen section were quantified and are presented as AU. Error bars represent the mean per group ± SEM. *P < 0.05; **P < 0.001.

Systemic delivery of VEGF-C accelerates antigen clearance from the inflamed colon to the DLNs through macrophage mobilization. One of the etiological theories on the pathogenesis of IBD is the impaired clearance of foreign material, leading to a sustained activation of innate immune cells and a compensatory induction of the adaptive immune response (44, 45). Indeed, delayed or ineffective clearance of bacteria that penetrate the intestinal mucosal barrier could prolong innate immune responses and enhance the presentation of bacterial antigens to facilitate the development of adaptive immune responses. Thus, while IBD may be due to an aberrant immune response against intestinal bacteria, slow or ineffective eradication of local bacteria may also be a key associated event contributing to disease pathogenesis (46). To ascertain whether the VEGF-C/VEGFR3 pathway is involved in antigen clearance through local LVs and DLNs during intestinal inflammation, we administered a single intramucosal injection of carboxylated crimson fluorescent LPS–coated beads into colitic mice untreated or treated with Ad-hVEGF-C and mF431C1 and monitored the clearance of the LPS-coated beads into the DLNs 1 and 3 days after injection. Two different populations of MΦs and DCs were characterized based on the expression of CD11b, CD11c, F4/80, and CD103, and, to avoid overestimation due to cells that had taken up more beads, we selected only cells that had taken up 1 bead over all bead⁺ cells (Figure 6A). While systemic delivery of VEGF-C accelerated the clearance of single-bead⁺ cells from the inflamed colon (Figure 6, B and C, left panels) to the DLNs (Figure 6, B and C, right panels) both 1 and 3 days after injection, systemic inhibition of VEGFR3 blocked this effect. As a control, we also determined whether the uptake of coated beads differed between the various cell populations. Coated beads were taken up efficiently by both CD45⁺ intestinal MΦs and DCs 12 hours after the injection (Supplemental Figure 4A).

Figure 6

Systemic delivery of Ad-hVEGF-C accelerates bacterial antigen clearance from the inflamed colon to DLNs. (A–C) A single intramucosal injection of carboxylated crimson fluorescent LPS-coated beads was administered on day 5 after the second DSS cycle (chronic inflammation) and on day 21 after the first Ad-hVEGF-C/mF431C1 administration to IL-10–KO mice. Colitic mice treated with Ad-GFP and injected with LPS-coated beads were used as controls. (A) FACS plots showing DCs (R1 and R4) and MΦs (R3 and R5) within the bead⁺ cell population based on the expression of CD11b (R3), CD11c (R1), F4/80 (R5, gated on R2), and CD103 (R4, gated on R2). (B and C) Clearance of single-bead⁺ cells from the inflamed colon (left panels) to the DLNs (right panels) was monitored, and quantification is reported as the mean per group ± SEM. n = 5 mice per group. *P < 0.05 versus LPS-coated beads plus Ad-GFP.

We then analyzed the phenotype of cleared LPS-coated bead⁺ cells in the DLNs 3 days after injection and found that systemic delivery of Ad-hVEGF-C accelerated the migration of both CD11c^– and CD11c⁺ MΦs, but had no effect on CD11b– and CD11b⁺ DCs (Supplemental Figure 4, B and C). In contrast, blocking of VEGFR3 activation led to the inhibition of both CD11c^– and CD11c⁺ MΦ migration toward the DLNs without influencing DC clearance (Supplemental Figure 4, B and C), thus demonstrating the specific role of the VEGF-C/VEGFR3 signaling pathway in bacterial antigen clearance by MΦs.

Depleting MΦs eliminates protection in VEGF-C–treated mice during chronic experimental colitis. To determine whether the protection seen in VEGF-C–treated mice during disease progression was at least in part the result of VEGF-C–induced MΦ activation, clinical parameters, lymph flow, and edema formation were monitored after treatment with clodronate-containing liposomes (CDLs), which have previously been shown to deplete MΦs in mouse colons (23, 24, 47). As shown in Supplemental Figure 5A, CDLs were effective at depleting different MΦ subpopulations when compared with PBS-injected mice. Interestingly, while in healthy mice, none of the MΦ subpopulations expressed VEGFR3, during inflammation, both CD11c^– and CD11c⁺ MΦs upregulated receptor levels on the cell membrane (Supplemental Figure 5, B and C), whereas DCs did not, thus explaining the specific effect of VEGF-C on the biological functions of these cells. While depletion of MΦs by CDLs in VEGF-C–treated mice did not affect DSS-induced LV size (Figure 7A), number (Figure 7B, left panel), or lymph flow (Figure 7B, right panel), it did partially restore the phenotype of untreated colitic animals in terms of DSS-induced weight loss (Figure 7C), histological score (Figure 7D and Supplemental Figure 6A left panel), and DAI (Supplemental Figure 6A, right panel). This indicates that the denser and larger VEGF-C–stimulated LV network in the gut is not sufficient per se to resolve inflammation but is accompanied by the role of MΦs. We achieved comparable results with IL-10–KO animalas (data not shown).

Figure 7

MΦ depletion using CDL reduces protection of VEGF-C–overexpressing mice during DSS-induced colitis. Mice undergoing 2 cycles of DSS treatment were injected with Ad-hVEGF-C and intrarectally administered CDL or PBS, as described in Methods. (A) Representative images of whole-mount staining of distal colons from the indicated groups after the second cycle of DSS (day 28). Scale bar: 75 μm. (B, left panel) LYVE1⁺ vessels were counted in 10 regions per section, and the numbers were normalized per total section area expressed in mm². Results are presented as the mean vessel density per group ± SEM. (B, right panel) Evans blue was extracted 16 hours after the dye injection from distal colons of comparable weight. Graph shows the total dye remaining in the colon expressed in μg. Data represent the mean per group ± SEM (n = 4–6/group). (C and D) CDL administration significantly reduced the protective effect of VEGF-C on the basis of the percentage of body weight (C) and histological examination (D) compared with control mice, which received only DSS plus VEGF-C. Black dashed lines represent the 2 DSS cycles. (D) Representative H&E-stained images of colons from the indicated groups of mice at the end of the experiment (day 40). Original magnification, ×20. Results in C are presented as the mean value per group at the indicated time points ± SEM (n = 4–6/group). *P < 0.05 versus DSS plus VEGF-C alone.

The VEGF-C/VEGFR3 pathway regulates in vivo and in vitro MΦ plasticity and activation. Assessing the putative phenotype of VEGF-C–induced MΦs in colitic mice, we compared our in vivo–derived colonic MΦs from VEGF-C–treated animals with those from untreated mice, both in the inflammatory phase of colitis. The release of IL-6, TNF-α, and IFN-γ was significantly lower in the culture of colonic MΦs from VEGF-C–induced animals compared with that in the untreated colitic controls, in which cytokine levels were expectedly elevated (Supplemental Figure 6B). In contrast, MΦs isolated from the colons of mF431C1-treated mice revealed an opposite effect (Supplemental Figure 6B). This is consistent with the protective role of the VEGF-C/VEGFR3 pathway in experimental colitis induced by DSS. Interestingly, MΦs from VEGF-C–treated mice were alternatively activated, producing increased levels of the M2 markers Ym1, Fizz1, Arg1, and IL-10 compared with those detected in untreated colitic animals (Figure 8A), but these MΦs also expressed high levels of typical M1 markers, such as Cox2, MHCII, iNOS, and CD80 (Figure 8B), thus reflecting a hybrid phenotype. When we examined the expression levels of intracellular cAMP, typically increased in this hybrid so-called “resolution-phase” subpopulation (48), we found that they were significantly elevated in MΦs isolated from VEGF-C–treated colitic mice compared with the levels detected in their untreated counterparts (Supplemental Figure 6C), confirming the presence of a “resolving” MΦ subpopulation. As shown in Figure 8, A and B, and Supplemental Figure 6C, MΦs isolated from the colons of mF431C1-treated mice revealed an opposite effect. We obtained similar results with IL-10–KO animals (data not shown).

Figure 8

The VEGF-C/VEGFR3 pathway regulates in vivo and in vitro MΦ plasticity and activation. (A and B) Mice undergoing 2 cycles of DSS treatment were injected with Ad-hVEGF-C or anti-VEGFR3 Ab. Colonic expression of M2 (A) and M1 (B) MΦ-associated genes was examined by real-time PCR analysis for Ym1, Arg1, Fizz1, Cox2, and iNOS; by ELISA for IL-10; and by FACS for MHCII and CD80 after 24 hours in vitro. Data represent the means ± SEM per group (n = 4–6/group). (A and B) **P < 0.01 versus DSS alone. (C and D) BM-derived MΦs were cultured and stimulated in vitro with LPS (M1), IL-4 (M2), VEGF-C, and VEGF-C plus mF431C1 for 24 hours. Expression of the indicated genes was then examined by real-time PCR, ELISA, or FACS. Data represent the means ± SEM of 3 independent experiments. **P < 0.01 versus untreated (UN); ^§P < 0.001 versus VEGF-C alone.

To assess the putative mechanism by which VEGF-C mediates MΦ plasticity and polarized activation, we subjected BM-derived MΦs from healthy untreated animals to in vitro M1 (by LPS) and M2 (by IL-4) polarization and VEGF-C stimulation. Our results showed that VEGF-C alone strongly increased both M1 and M2 gene expression, almost comparable to that seen in control cells stimulated with LPS or IL-4 alone (Figure 8C and Supplemental Figure 6D); these outcomes were abolished upon treatment with the anti-VEGFR3 Ab (Figure 8C and Supplemental Figure 6D). Overall, these data confirm the induction of a unique in vitro mixed M1-M2 subpopulation, as observed in vivo, and demonstrate a direct effect of the VEGF-C/VEGFR3 pathway not only on MΦ-mediated bacterial antigen clearance (Figure 6) but also on MΦ plasticity and polarization (Figure 8).

Resolving MΦs mediate the protective role of the VEGF-C/VEGFR3 pathway during chronic experimental colitis in a STAT6-dependent manner. At the transcriptional levels, IL-4–induced differentiation of alternatively activated MΦs and the expression of genes such as MHC class II are mediated by the transcription factor STAT6 (49, 50). For this reason, we asked whether VEGF-C induced the expression and/or activation of STAT6 in the resolution-phase subpopulation of MΦs (rMΦs). Upon VEGF-C treatment, the amount of total STAT6 remained comparable to that in the untreated cells, whereas increased levels of phosphorylated STAT6 (p-STAT6) were detected, in both in vivo– (Figure 9A, left panel) and in vitro BM–derived differentiated MΦs (Figure 9A, right panel). This effect was inhibited by mF431C1 (Figure 9A), showing that the VEGF-C/VEGFR3 pathway directly modulates STAT6 activation during experimental colitis and in cultured BM–derived MΦs. In addition, inhibition of STAT6 in VEGF-C–treated cells using a specific siRNA abolished the effect of this growth factor on the expression of M2- (Figure 9B and Supplemental Figure 7A) and M1- (Figure 9C and Supplemental Figure 7B) associated genes and on MΦ migration through matrigel-coated filters (Supplemental Figure 7C), whereas the scramble control did not, demonstrating that the VEGF-C/VEGFR3 pathway directly controls MΦ plasticity and migration in a STAT6-dependent manner. Finally, since we demonstrated that the protection seen in VEGF-C–treated mice during disease progression was at least in part the result of resolving MΦ activation, we investigated the mechanism through which this pathway confers protection during DSS-induced colonic inflammation. For this purpose, we injected in vitro VEGF-C–induced rMΦs isolated from the BM of GFP mice into the rectal mucosa of colitic mice every 96 hours and monitored the clinical parameters at different time points for the entire experiment (Figure 9D). Notably, 48 hours after the injection, we still found GFP⁺ MΦs in the colons of colitic mice, while we no longer detected these cells after 96 hours (data not shown), thus allowing us to determine the injection intervals. These animals showed ameliorated clinical parameters in terms of body weight (Figure 9E), DAI (Figure 9F), and colon length (Supplemental Figure 7D) when compared with those of the untreated animals. This protective role was abolished by STAT6-silenced VEGF-C–induced MΦs (Figure 9, E and F, and Supplemental Figure 7D), demonstrating, for the first time to our knowledge, one of the mechanisms of action through which VEGF-C/VEGFR3 signaling abates the severity of experimental colitis.

Figure 9

Resolving MΦs mediate the protective role of the VEGF-C/VEGFR3 pathway during chronic experimental colitis in a STAT6-dependent manner. (A) Mice undergoing 2 cycles of DSS treatment were injected with Ad-hVEGF-C or anti-VEGFR3 Ab. Colonic expression of p-STAT6 and total STAT6 was evaluated by Western blotting in MΦs after 4 hours in culture (in vivo) and in BM-derived differentiated MΦs (in vitro). Actin was used as a loading control. (B and C) BM-derived MΦs were cultured, transfected with a STAT6-specific siRNA or a STAT6 scramble, and stimulated in vitro with VEGF-C for 24 hours. Expression of the indicated genes was then examined by ELISA (B) or real-time PCR (C). Data represent the means ± SEM of 3 independent experiments. **P < 0.01. (D–F) GFP plus BM-derived MΦs (BMDMs) were cultured, transfected with a STAT6-specific siRNA or a STAT6 scramble, and stimulated in vitro with VEGF-C or mF431C1 (D). Cells were then injected into the rectal mucosa of DSS-treated mice (n = 4/group), and clinical parameters such as body weight (E) and DAI (F) were evaluated. Black dashed lines represent the 2 DSS cycles. Results are presented as the mean value per group at the indicated time points ± SEM. *P < 0.05 for DSS plus VEGF-C MΦs versus DSS alone; ^§P < 0.01 for DSS plus VEGF-C plus STAT6 siRNA MΦs versus DSS plus VEGF-C MΦs.

In this study, we report that the specific promotion of LV function limits experimental chronic intestinal inflammation in mice that is mediated by a unique MΦ polarization and activation and modification of the tissue cytokine milieu. These novel findings are important because, although the precise pathophysiology of IBD is unknown, LV obstruction and dysfunction are long-recognized features observed in humans with this disease (15, 16), and normalizing gut lymphatics with prolymphangiogenic factors such as VEGF-C may improve disease activity. Consistent with altered lymphatic contractile function reported in an animal model of acute intestinal inflammation (19), intestinal lymphatics are known to be grossly abnormal in the gut of CD patients (10). Moreover, mucosal and submucosal edema is a typical feature of IBD tissues despite early abundant lymphatic vasculature. Nevertheless, only limited information is available on the clearance capacity of LVs in IBD (17), and some of the lymphatics might be immature or nonfunctional, as appears to be the case in tumor-associated lymphangiogenesis (51, 52). Recent work by Tammela et al. (53) demonstrated that by stimulating the VEGFR3 tyrosine kinase by treatment with the lymphangiogenic factor VEGF-C, it is possible to regenerate functional collecting LVs in mice following lymph node (LN) dissection. As VEGFR3 activity plays important roles in both the maintenance and survival of lymphatic endothelial cells (LECs) and vascular integrity, decreased VEGFR3 expression or signaling might alter many lymphatic functions, leading to increased gut injury. Indeed, antilymphatic treatment with anti-VEGFR3 antibodies aggravated inflammation and submucosal edema, increasing leukocyte infiltration in an animal model of IBD (13). In the current study, we demonstrate activation of the VEGF-C/VEGFR3 pathway in the actively inflamed mucosa of patients with IBD. Except for VEGF-D, another VEGFR3 ligand whose levels remain stable in actively inflamed intestinal tissues, expression of both VEGF-C and VEGFR3 was significantly increased in biopsy specimens from inflamed bowel segments when compared with those in controls, a finding compatible with inflammation-associated lymphangiogenesis. Similarly, we found that the expression of VEGF-C and its receptor, both expressed in colon tissue under physiologic conditions, was markedly enhanced during experimental colitis in 2 different animal models: the chronic DSS and the IL-10–KO models, (54, 55). Thus, these models offer a good platform for manipulating the VEGF-C/VEGFR3 pathway and verify its effect on the course of experimental intestinal inflammation. Indeed, we demonstrated that systemic inhibition of VEGFR3 blocked lymphangiogenesis, reducing both area density and LV dimension and growth, while significantly increasing inflammatory edema formation and inhibiting disease resolution, as also observed by Jurisic et al. (13). In contrast, lymphatic drainage was enhanced by systemic delivery of VEGF-C, which in turn significantly induced LV density and proliferation, improving intestinal inflammation. The drainage-promoting function of VEGF-C in our models is in agreement with recent findings showing that the inhibition of VEGF-C by soluble VEGFR3 (sVEGFR3) significantly decreased lymph flow in a model of bacterial skin inflammation (56) and that genetic overexpression of sVEGFR3 in the skin of mice resulted in a lymphedema-like phenotype (57). Conversely, the application of VEGF-C or tumor-derived VEGF-C enhances lymphatic function and flow, respectively (53, 58–61).

As reported by others (62, 63), we did not detect any changes of the blood vasculature in VEGF-C–treated mice. Therefore, the improvement of experimental colitis mediated by systemic delivery of Ad-hVEGF-C is likely not caused by effects on blood vessels. Interestingly, the enhanced lymphatic drainage by VEGF-C was observed in combination with increased inflammatory cell mobilization and bacterial antigen clearance, all LV functions that we found to be inhibited by VEGFR3 blockade. VEGF-C–dependent antigen clearance was a MΦ-specific (both CD11c^– and CD11⁺) effect, supported by the fact that these cells express VEGFR3 on the cell membrane during inflammation, whereas DCs do not. Such results suggest a potential role for VEGFR3 signaling in immunity by mediating antigen-presenting cell (APC) trafficking through MΦ recruitment. The most intriguing and novel finding in this study is that the protection we observed in VEGF-C–treated mice during disease progression was not only a consequence of increased lymphangiogenesis and enhanced lymphatic flow and function, including mobilization of inflammatory cells and bacterial antigen clearance, but was also a result of a previously unknown direct VEGF-C–induced MΦ activation through STAT6 signaling. The MΦ has long been considered part of the destructive force behind IBD as a result of its involvement in regulating inflammation through the production of inflammatory cytokines and chemokines (64–66). When activated, MΦs can be functionally divided into the classically activated, or M1, type and the alternatively activated, or M2, type in response to the different stimuli of the local microenvironment (67). Our data show that administration of VEGF-C activates a hybrid MΦ phenotype, with a direct effect on cell migration and expression of both M1 and M2 markers, with opposite effects observed upon blocking of VEGFR3. This specific subpopulation has already been observed in a peritoneal model of inflammation, in which the so-called rMΦs expressed a unique mixed M1-M2 phenotype, with a role in mediating post-resolution innate lymphocyte repopulation and restoring tissue homeostasis (48, 68). We show that adoptive transfer of VEGF-C–differentiated resolving MΦs in colitic mice inhibited the clinical parameters of DSS-induced chronic colitis, in agreement with reports demonstrating that in vitro–differentiated monocytes have antiinflammatory properties or enhance the resolution of inflammation (23, 69). In addition, we discovered that the polarization and resolving actions of MΦs are mediated by the VEGF-C/VEGFR3 pathway through STAT6 activation. This is intriguing, because some reports suggest that there may be a dysregulation of STAT6 signaling in the ungoverned immune response associated with colitis (70–72), and this transcription factor plays a regulatory role in the pathogenesis of IBD (73),

Although not assessed in our study, it would be of interest to investigate the in vivo beneficial effects of VEGF-C–activated MΦs in terms of T cell immunosuppression and the potential role of DLN lymphangiogenesis in the control of intestinal inflammation (74, 75). The increased drainage capacity and resolution of chronic intestinal inflammation might, at least in part, be mediated by enhanced VEGF-C–induced DLN lymphangiogenesis in addition to the increased lymphangiogenesis we observed in the inflamed colon itself. Nevertheless, we cannot fully exclude the possibility that other VEGF-C activities, including other direct effects on the inflamed lymphatic vasculature, are involved in the antiinflammatory properties of VEGF-C.

In conclusion, our study provides the first proof of concept to our knowledge that it may be possible to treat chronic gastrointestinal inflammatory disorders by stimulating LV functions to promote drainage and bacterial antigen clearance, together with adaptive immunity, effects achievable through modulation of the VEGF-C/VEGFR3 pathway. In fact, increased VEGF-C expression levels found in mucosal extracts of patients with IBD could be insufficient to promote proper lymphatic function and MΦ-polarized activation and migration. VEGF-C gene therapy can induce growth of new LVs in the skin of Chy mice, suggesting that some forms of lymphedema might be treated by similar approaches in humans (59). In addition, use of VEGF-C plasmid in rabbit ear and mouse tail lymphedema models results in amelioration of lymphatic function and alleviation of the signs of lymphedema (61). Thus, these and our own observations support the potential use of lymphangiogenic growth factors as a novel therapeutic approach for the treatment of IBD and other chronic inflammatory diseases.

View Supplemental data

This study was supported by grants from the Broad Medical Research Program; the Italian Ministry of Health; the Fondazione Cariplo; the Italian Association for Cancer Research; the Innovative Medicines Initiative (IMI) Joint Undertaking BTCure (115142); the Fondazione Italiana per la Ricerca Sulle Malattie Apparato Digerente (FIRMAD); and the European Crohnδs and Colitis Organisation (ECCO). We thank Ron Haskell (ViraQuest Inc.) for his expert technical assistance with adenovirus preparation. Bronislaw Pytowski (ImClone Systems Inc.) provided the VEGFR3 blocking Ab mF431C1.

Address correspondence to: Silvio Danese, IBD Center, Department of Gastroenterology, Humanitas Clinical and Research Center, Via Manzoni 56, 20089 Rozzano, Milan, Italy. Phone: 390282244771; E-mail: sdanese@hotmail.com.

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

Reference information: J Clin Invest. 2014;124(9):3863–3878. doi:10.1172/JCI72189.

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