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Lymphology 47 (2014) 3-27

LYMPHATIC DYSREGULATION IN INTESTINAL : NEW INSIGHTS INTO INFLAMMATORY BOWEL DISEASE PATHOMECHANISMS

F. Becker, P. Yi, M. Al-Kofahi, V.C. Ganta, J. Morris, J.S. Alexander

Departments of Molecular and Cellular Physiology (FB,PY,MA- K,VCG,JSA) and Internal Medicine, Gastroenterology (JM), Louisiana State University Health Sciences Center - Shreveport, Louisiana, USA, and Department of General and Visceral Surgery (FB), University Hospital Muenster, Germany

ABSTRACT Keywords: inflammatory bowel disease, lymphatic remodeling, lymphatic pumping, Alterations in the intestinal lymphatic VEGFR-3, VIP, histamine, intestinal network are well-established features of , NF-κB, TLR, endothelin human and experimental inflammatory bowel disease (IBD). Such lymphangiogenic The two most prominent forms of expansion might enhance classic intestinal inflammatory bowel disease (IBD), ulcerative lymphatic transport, eliminating excess colitis (UC) and Crohn’s disease (CD), accumulations of fluid, inflammatory cells remain serious and globally increasing and mediators, and could therefore be medical conditions, now affecting ~1.4 interpreted as an ‘adaptive’ response to acute million Americans (1). Currently, the precise and chronic inflammatory processes. However, etiology of IBD has not yet been elucidated whether these new lymphatic vessels are whereas it is commonly accepted that in functional, unregulated or immature (and genetically susceptible individuals, altered what factors may promote ‘maturation’ of and inappropriate intestinal interactions these vessels) is currently an area under between unspecified environmental factors intense investigation. It is still controversial and host immunity (e.g., NOD2 /ATG16L1 whether impaired lymphatic function in IBD polymorphisms) contribute to the fundamen- is a direct consequence of the intestinal tal pathophysiology of IBD (2). Rather than inflammation, or a preceding - additive factors, mechanisms contributing to like event. Current research has uncovered IBD are understood to represent multiple novel regulatory factors as well as new roles pivotal factors, which interact closely and for familiar signaling pathways, which appear modify each other synergically. Therefore, to be linked to inflammation-induced understanding complexities in IBD patho- lymphatic alterations. The current review genesis requires identification and integration summarizes mechanisms amplifying lymphatic of host related processes, effects of individual dysregulation and remodeling in intestinal microbiomes and environmental influences, inflammation at the , cell and molecular which when combined, lead to improper and levels and discusses the influence of lymphan- intense host immune responses (3,4). giogenesis and intestinal lymphatic transport However, despite different individual function as they relate to IBD pathophysiology. ranges of clinical manifestations and courses

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 4 in IBD, the primary and cardinal charac- function (efferent arm) contributing to both teristic symptom of these two idiopathic initiation and perpetuation. Therefore, inflammatory disorders still remains robust besides angiogenesis, disturbances intestinal inflammation, whose attendant in intestinal function and pathological origins have now been widely remodeling are important vascular contribu- investigated (3,5). The , tions to the pathophysiology of IBD (11). more specifically the gut mucosa, is the In 2008, van Kruiningen and Colombel principle ‘frontier barrier’ to bacterial flora reintroduced the as a and foreign antigens. Consequently, the ‘forgotten’ factor in IBD pathophysiology initial regional mucosal immune response when they, among others, reminded the IBD likely originates at the mucosal border community that alterations in lymphatic and represents the primary initiator for vessels have been observed since the first subsequent disruptions in the intestinal description of regional ileitis by Crohn et al immune homeostasis and accompanying in 1932 (12,13). Over ensuing decades chronic bowel inflammation in the surgeons, pathologists and researchers have pathophysiology of IBD (6,7). repeatedly recognized increased lymphatic Within the mucosal , vessel density as a characteristic feature in several key factors contributing to IBD CD, and more recently UC, with evidence of pathology have thus far been identified. lymphatic expansion in both the inflamed Contemporary research has been primarily and non-affected areas of the gut. focused on components of the innate and Importantly, the key functional impact of acquired arms of the immune system, lymphatics in maintaining gut immune whereas non-immune cells have received less homeostasis has at least been underrated in but increasing attention (8). It is now widely the pathogenesis of IBD (11,13-15). New established that the formation of new blood experimental methods and recent findings vessels (hemangiogenesis) in IBD contributes now have re-positioned intestinal lymphatics to increased intestinal inflammation, since as an important component of the mucosal both angiogenesis and inflammatory immune response, whereas lymphatic failure mediators (e.g., VEGF-A, TNF-α) help to is closely and causally linked to intestinal mobilize leukocytes and increase premature inflammation. By now, disturbances in the fibrotic remodeling (9,10). Where intestinal lymphatic transport system have different components of the intestinal been equally documented as important microvasculature (, , factors in the initiation and development of ) have been extensively studied for IBD (11,16,17). their response towards mediators and Acute inflammatory conditions have mechanisms of leukocyte recruitment and been traditionally recognized to increase extravasation, the removal of infiltrated lymphatic flow to balance the associated immune cells and transport of antigen- profound tissue edema through an active and presenting cells to appropriate downstream unidirectional transport at least under acute compartments through the lymphatic system conditions (18,19). However, accumulating has been comparatively neglected (11). reports reveal disturbances in intestinal For example, accumulating evidence now lymphatic transport function (and even indicates that intestinal edema (a major retrograde flow) in IBD, which may clinical sign in IBD) is not only caused by be caused by obstruction or pumping failure. inflammatory mediators which increase Recently, Colombel et al 2011 studied vascular solute permeability (afferent arm) regional obstruction of lymphatics in CD, but also reflects negative influences of these reporting increased inflammatory lymphoid mediators on intestinal lymphatic transport follicles, and lymphocytic

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 5 perilymphangitis in all CD samples, cytokines may even disturb junction archi- demonstrating the strong correlation between tecture to increase permeability in ‘mature’ inflammation and lymphatic transport failure lymphatic vessels (11). Whether impaired caused by obstruction (20). This concept is lymphatic function in IBD is a direct supported by observations in experimental consequence of the intestinal inflammation models of lymphatic obstruction: deliberately or a lymphangitis like preceding event is still impaired intestinal lymphatic transport up for prospective debate. This review capacity alone was found to be able to summarizes different factors amplifying recapitulate the anatomic and clinical lymphatic dysregulation in intestinal fundamental features of CD (e.g., leukocyte inflammation and discusses the influence of extravasation, edema, and even the develop- lymphangiogenesis and intestinal lymphatic ment of enteroenteric and enterocutaneous transport function on IBD pathophysiology. fistulae) (21). It is therefore possible that lymphatic obstruction and subsequent Intestinal Lymphatic Transport Function remodeling may further impair lymphatic transport capacity, leading to sustained Intestinal lymphatic vessels are the lymphangiogenesis (11). Interestingly, and in central conduit for efferent removal of agreement with this, Zampfell et al have interstitial fluid, perivascularly infiltrated demonstrated that lymphatic stasis is a highly immune cells, lipids and cholesterol from potent inducer of lymphatic expansion (22). digestive visceral organs (25-28). Lymph Additionally, von der Weid et al found that collection begins at blind-ended initial heterogeneous factors like cytokines or lymphatics which are structurally adapted to reactive oxygen/nitrogen species can suppress be ‘pulled open’ by increased tissue turgor lymphatic pumping (16,23,24). Therefore, it allowing lymphatic entry of interstitial fluid, seems as though lymphostasis promotes the particularly during acute edema (11). In accumulation of several diverse heterogeneous contrast to the structurally unique, discon- lymphatic pump-suppressing factors in IBD, tinuous “button”-like junctions in initial some of which may provoke lymphangio- vessels, which contain adherens and tight genesis. Still, the gap between cause and junction proteins (VE-cadherin, occludin, consequence in IBD pathophysiology and claudin-5, zonula occludens-1, junctional whether increased lymphangiogenesis should adhesion molecule-A, and endothelial cell- be considered as pathological or protective selective adhesion molecule) (29), adherens is still debatable. and tight junctions in collecting lymphatics An important challenge for future are relatively continuous with zipper-like lymphatic endeavors will be to characterize junctions. Intestinal lymphatic transport is the nature of these observed alterations in at least partially an active process, accom- lymphatic vessel function and formation in plished by the ‘lymphangions’ (literally IBD. The lymphangiogenic expansion in lymph-‘’), which are valve-containing IBD might contribute to classic intestinal contractile subunits of lymphatic vessels lymphatic transport, eliminating accumula- invested with lymphatic medial smooth tions of fluid, inflammatory cells as well as muscle cells in close approach with mediators and could therefore be interpreted terminals which can influence lymphangion as adaptive to the inflammatory process. contractility and periodicity (30). Accordingly, Whether these new lymphatic vessels are the ability of intestinal lymphatic vessels to functional, unregulated or immature (and adequately remove interstitial fluid and cells what factors are needed to ‘mature’ these from the gut interstitium depends on both vessels) is currently an area under concen- adequate patent lymphatic conduits and trated investigation, and inflammatory active pumping of these lymphangions (24).

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Besides autonomous, spontaneous myogenic family include Prostaglandin-H2 (PGH2), -F2 contractions which actively maintain (PGF2), -D2 (PGD2), -E2 (PGE2), -I2 (PGI2 unidirectional lymph outflow, the contractile ‘Prostacyclin’) and Thromboxane (TXA). behavior of lymphangion pumping is also These agents modulate numerous pathways modified by multiple extrinsic influences in both normal physiology and pathological including external tissue compression, conditions (especially inflammation) intraluminal pressure and fluid load, nervous including vascular permeability, leukocyte inputs as well as contributions by many local infiltration, T-cell activation or nociceptor and circulating autocrine and paracrine sensitization (32,33). Accordingly, it is well signal mediators (e.g., cytokines or documented that elevated prostaglandin chemokines) (16). Consequently, lymphatic levels are found in UC, CD and in experi- pumping represents an active and highly mental models of intestinal inflammation regulated process whose failure interrupts (34,35). Whereas the main source of AA lymphatic transport function, often with derivatives seems to be inflammatory cells, disastrous consequences. However, lymphatic studies also demonstrate that lymphatic vessels exhibit an astonishing ability to adapt vessels themselves are able to release prosta- to enormous changes in the interstitial glandins, regulating lymphatic contractility environment, whose major challenge to these with a more significant role in increasing mechanisms is inflammation. rhythmical constriction for PGH2, PGF2 and The intestinal inflammation occurring in TXA2 whereas PGI2 and PGE2 were found the clinical course of IBD is not only accom- to decrease lymphatic pumping (36-40). plished by a persistent imbalance between However, this regulatory mechanism seems pro- and anti-inflammatory but is also to be disturbed in inflammation. associated with a distinct disturbance in Recently, the influence of prostaglandins intestinal fluid load, which both affect on intestinal lymphatic pumping under lymphatic transport function in UC and CD. inflammatory conditions has been most Whereas it is well described that lymph flow exhaustively studied by the group of von der and intestinal morphologic lymphatic expan- Weid et al (16,40-42). Using the trinitro- sion (lymphangiogenesis) are consistently benzene sulfonic acid (TNBS) induced ileitis increased in IBD, how intestinal lymphatic model in guinea pigs, the group likewise contractile function is altered under inflam- identified EP4 and IP as associated receptors matory conditions still remains unclear (11). in the PGI2, and PGE2 mediated decrease of However, the malfunction of intestinal intestinal lymphatic vessel contraction lymphangions, either congenitally or acquired frequency and established mechanistic links through blockade, distension or lymphatic to protein kinase A activation and ATP- medial disturbances, sensitive K+ channels (41). Although now influences the onset and persistence of several coincident reports have found that lymphostasis, and may contribute to the PGI2 and PGE2 formed by endothelial etiology of IBD (16,23,24,31). cyclooxygenase significantly impair normal lymphatic pumping (promoting edema and Prostanoids lymphostasis), cyclooxygenase inhibitors are not beneficial in IBD, more often intensifying Among the various paracrine signal bleeding, protein- losing enteropathy and mediators composing the intestinal mucosal dysregulating epithelial barrier, which is immune system, prostanoids [(cyclooxygenase normally enhanced by PGE2 (23,42-48). (COX) derived metabolites of arachidonic acid (AA)] are exceptionally relevant to IBD Nitric Oxide pathogenesis. Members of the prostanoid

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Among the numerous mediators released of KATP channel blockers might be able to in response to the dysregulated intestinal restore lymphatic pump activity (as has been immune response in IBD, the ubiquitous shown in vitro) (56). intercellular messenger and potent vasodilator nitric oxide (NO), has been closely linked to Histamine impaired lymphatic transport function. NO• (a small gas gasotransmitter) is enzymatically As already stated, gut interstitial fluid produced by several nitric oxide synthases and immune cell overload not only depends (eNOS, iNOS) (49). NO• flux closely reflects on afferent influx from blood vessels, but is NOS isoform expression: inducible NOS also linked to efferent intestinal lymphatic (iNOS) produces high tissue levels of NO•, transport function. Both pathways, vascular whereas eNOS produces low, calcium permeability and active lymphatic transport, regulated bursts of NO• (50). Interestingly, are adjusted by various forms of paracrine iNOS is expressed in a species and - and systemic signaling. Among these, specific fashion by endothelial, smooth histamine is a mediator which influences both muscle and various immune cells (macro- arms of this tightly regulated system (57). phages, neutrophils) and can be induced by Histamine storage and release by mast cells inflammatory cytokines like IL-1β, TNF-α plays an important role in gastrointestinal or IFN-γ. Furthermore, it has been established secretion and immune modulation in allergic that elevated tissue levels of iNOS are present diseases (58). The response to histamine within inflamed regions in intestinal samples reflects the type and distribution of histamine from IBD patients, and that increased NO• receptors (H1R-H4R) (59). Interestingly, production contributes to tissue injury, the first description of physiological effects mucosal vasodilation or enhanced vascular of histamine in 1910 by Dale et al included and epithelial permeability at these iNOS+ intestinal smooth muscle contraction and foci (51). Conversely, iNOS may paradoxi- vasodilatation (60). In IBD, it is well estab- cally be beneficial in the setting of IBD as lished that increased mast cell recruitment intestinal microvessel basally (leading to elevated, potentially degranulating, express iNOS, which appears to suppress local histamine levels) within the mucosa, is a inflammation induced leukocyte adhesion characteristic sign of intestinal inflammation (52,53). Importantly, NO• is known to in CD and UC (61,62). Histamine levels are hyperpolarize lymphatic smooth muscle cells greatly increased in IBD patients, and and reduces lymphatic contractility, strongly correlate with disease severity (63,64). Since inhibiting spontaneous pumping (39,54,55). histamine secretion and H-receptors are Recent studies in animal models of found in macrophages, dendritic cells, T- experimental erosive e.g. TNBS-colitis, and B-, histamine may not only (known to robustly and reproducibly depress mediate vasodilatation, but also immune intestinal lymphatic function), shows highly processes in inflammatory disorders (65). upregulated iNOS expression in intestinal Recently, studies have investigated the effects lymphatic smooth muscle cells themselves. of histamine on intestinal lymphatic smooth This iNOS mediated NO•-flux accompanies muscle function. Direct application of increased smooth muscle hyperpolarization histamine decreases mesenteric lymphatic and consequently, impaired lymphatic contractility (via H1 receptors expressed on contractility (40). Mechanistically, KATP lymphatic smooth muscle), whereas channel activation has been shown to be an histamine binding to H2 receptors can induce important mechanism underlying NO•- lymphatic contractility. However, based on induced lymphatic relaxation and hyper- the observed receptor distributions on polarization. Therefore, the therapeutic usage intestinal lymphatics, the dominant effect

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 8 appears to be an increase in contractility (66). in an in vivo model of intestinal ischemia Interestingly, other studies showed that low reperfusion (I/R) injury increased intestinal concentrations of histamine increase lymph flow (by 80%) (76). Interestingly, lymphatic contractility by binding to smooth their group found that the increase in lymph muscle H1 receptors which are highly sensi- flow produced by 0.2pmol/g VIP (i.p.) was tive to histamine, while higher concentrations also associated with a significant decrease in of histamine decrease lymphatic contractility lymph and serum concentrations of LPS, α through H2 receptors (expressed on lymphatic TNF- as well as reduced gut histopathology, endothelial cells) via stimulation of the NO• ALT, D-lactate and creatinine levels. Impor- production (66,67). In conclusion, effects of tantly, this did not appear to reflect a lymph histamine on gut edema formation may “dilution” effect. Most importantly perhaps reflect tissue histamine receptor expression was the observation that VIP administration and histamine abundance, which modulate led to a reduction of T-cells in Peyer’s both vascular permeability as well as patches, mesenteric lymph nodes, lymphatic transport function. and (76). As previously stated, it is worth noting that under inflammatory Vasoactive Intestinal Peptide (VIP) conditions VIP can be secreted by several immune cells (characteristically elevated in The wide spectrum between physiological the intestinal inflammatory mucosa and in function and inflammation-induced distur- lymph in IBD). Therefore, it could be bances in intestinal lymphatic transport are anticipated that increased intestinal VIP illustrated by responses to vasoactive production by mucosal resident cells might intestinal peptide (VIP), a 28-amino acid have paracrine effects on lymphatic vessels to immunomodulating and anti-inflammatory modulate lymphatic transport function in a neuropeptide (68-72). VIP is usually released time and spatially regulated fashion. There by peptidergic , but also can be released appears to be a marked decrease in VIP- (infrequently) by immune cells. In the setting immunoreactive nerve fibers in the lamina of inflammation, VIP has been shown to propria and of patients with IBD, inhibit LPS induced release of TNF-α and indicating that neutrally-derived VIP levels IL-6 by macrophages (71,73) suppresses the may be depressed in these patients (79). release of IL-2, -4 and -10 in peripheral However, in addition to the aforementioned T-cells (74), and decreases GI mucosal injury vascular effects, it has also been unexpectedly (75), possibly through enhancement of barrier reported that VIP can act as a potent anti- function (76). In the context of lymphatic inflammatory agent which beneficially homeostatic regulation, VIP is mainly modulates innate and adaptive immunity, secreted by nerve fibers in the of suggesting its potential therapeutic use in lymphatics, which modulate both vessel immune and inflammatory disorders (69). relaxation and contractility in the control of Interestingly, systemic VIP treatment showed interstitial fluid balance (77,78). Von der broad prophylactic and therapeutic benefit in Weid et al studied the effect of VIP on an in vivo murine model of TNBS-induced porcine lymphatic pumping capacity in an colitis (80,81). In this model, VIP treatment in vitro model and found that topical VIP showed potent local and systemic effects e.g., administration diminished lymphatic attenuating the severity of colitis, reducing pumping by decreasing the frequency of systemic and colonic pro-inflammatory lymphatic contractions and by hyperpolarizing cytokines and increasing anti-inflammatory lymphatic muscle membrane potentials (77). cytokines and reducing inflammatory Conversely, Yang et al found that sys- induced TLR2/4 overexpression (80,82). temic (i.p.) administration of VIP (0.2pmol/g) However, it is also worth noting that

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Newmann et al were not able to perceive this possible that decreased lymph flow during protective effect of VIP in a similar model of inflammation could be beneficial by limiting experimental TNBS-colitis (83). Interestingly, transfer of toxins into the general circulation. when VIP treatment increased intestinal The apparent disconnect between the lymph flow (after experimental intestinal I/R findings of von der Weid et al and Yang et al injury), there was an observed decrease in the could indicate differing effects between direct concentration of inflammatory gut-derived VIP actions on lymphatic smooth muscle mediators, including endotoxin and TNF-α (decreased lymphatic smooth muscle (76). The apparently contradictory results of contractility) compared with indirect smooth increased lymph flow with a concomitant muscle cell-independent effects of VIP decrease in the concentration of inflamma- (endothelial, immune cells). tory mediators (76) might reflect local immune modulating and anti-inflammatory Alcohol effects of VIP. However, these optimistic results of therapeutic VIP usage in experi- In addition to the above-listed mental models of inflammation (especially in ‘endogenous’ factors, exogenous and environ- models of experimental colitis) demonstrate mental factors (e.g., Western diet) may also the need for additional interpretive research modulate extent and activity of intestinal and translational studies. So far, VIP has inflammation in IBD (85,86). However, the been positively tested in one clinical trial for role of environmental factors as modifying sarcoidosis, a related inflammatory-immune “triggers” in IBD is often complex and state disorder, with good effect (84). Further efforts dependent. For example, cigarette smoking are also needed to clarify the role of VIP in decreases the extent of disease in UC, but intestinal lymphatic transport function in the worsens symptoms in CD (87). Another setting of IBD. Endogenous VIP might environmental factor that increases abdominal therefore play an important regulatory role symptoms in IBD and that affects intestinal in controlling intestinal inflammation and lymphatic transport capacity (and also shows represent a potential treatment for IBD. contradictory results), is alcohol consumption. The effects of VIP in these models may also Cohort studies of IBD patients have shown maintain lymph flow through several possible that alcohol consumption generally worsens mechanisms unrelated to lymphatic pumping intestinal symptoms in CD and UC (65,88), (e.g., barrier function and immunomodula- apparently via enhanced immune responses. tion). Paradoxically, Yang et al showed that In particular, alcohol consumption can in I/R injury increased lymph flow is associ- increase intestinal epithelial permeability, ated with improved clinical features in the an established pathomechanism in IBD (89). intestine, although lymph has been shown to The effects of alcohol consumption on transfer inflammatory cells and mediators lymphatic transport function are more from sites of injury to the general circulation, complex in IBD. Mesenteric lymph flow in e.g., during shock (“shock lymph syndrome”), rats and thoracic duct lymph flow in human often provoking systemic inflammatory are both increased by alcohol consumption response syndrome/ multi-organ dysfunction (90,91). However, other in vitro studies show syndrome (SIR/MODS) (76). One interpre- that alcohol administration directly onto tation of the findings by Yang et al is that mesenteric lymphatic vessels inhibits a reduction in the residence time of lymph lymphatic myogenic responses. Conversely, within the inflamed gut may reduce the “digestive” alcohol consumption (possibly content (or composition) of inflammatory mediated by acetaldehyde) may indirectly mediators in the interstitium or lymph, which increase myogenic contraction and lymph favorably affects clinical outcomes. It is also flow in mesenteric lymphatic vessels, which

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 10 would be expected to have beneficial effects affect the unidirectional lipid clearance. on ISF conduction or intestinal immunity ‘Fat wrapping’ (creeping fat) at the surface (92). However, it remains unclear if alcohol of the large and small bowel plus mesenteric related modulation of intrinsic lymphatic fat deposition are characteristic findings in pumping significantly impacts tissue edema CD, which are accompanied by increased and inflammation or negatively regulates numbers of mesenteric adipocytes (96,97). gut barrier function in IBD. In spite of the However, this condition is accompanied by controversy resulting from differences in increased abdominal but not total body fat in vivo and in vitro studies, recent patient (BMI is usually reduced in forms of IBD) targeted dietary recommendations still (98). Since the intestinal localization of recommend alcohol abstinence for CD and accumulating fat correlates with transmural UC patients (93). inflammation, ulceration and strictures in Beyond the aforementioned results CD, a direct contribution of fat wrapping in (mostly generated under experimental IBD pathophysiology has been suggested conditions), a translational clinical study (99). The role of intestinal fat as an important reexamining the role of deliberately impaired extraluminal factor in IBD is also supported intestinal lymphatic transport function in by observations that increased gut adiposity IBD was accomplished by Tonelli et al (94). increases local and systemic levels of TNF-α, Based on the proposed role of intestinal leptin, adiponectin, macrophage colony- lymphatics in the pathogenesis in CD, those stimulating factor (M-CSF), macrophage authors measured the intestinal lymphatic migration inhibitory factor (MMIF) and C- transport capacity to distinguish between reactive protein (CRP), therefore influencing inflammation-affected and ‘healthy’ areas in intestinal inflammatory responses in IBD the small intestines of CD patients. The (96,98,100,101). Interestingly, fat deposition functional capacity of lymphatic integrity and found in CD also resembles fat accumulation transport was established by subserosally in experimental models of impaired lymph injecting Evan’s blue (a potent marker of transport and (22,102). lymphatic vessels) into the intestinal wall of Moreover, fat deposition in these settings is patients undergoing surgery (95). Preliminary often accompanied by fibrosis, another data showed that slowed lymphatic outflow common finding in IBD (103). Importantly, (requiring >2 minutes) is correlated with CD- related studies showed that disturbances in like macroscopic features like fat-wrapping, lymphatic specification genes like Prox-1 wall thickening, wall edema or ulcers while can trigger a loss of lymphatic integrity, an outflow time of ≤ 2 minutes was correlated which reciprocally induces fat leakage from with macroscopically normal findings. As of lymphatics to exacerbate lymphatic structural yet, the long-term goals of the study, i.e., defects in IBD, and may be an underlying establishing rates of lymphatic outflow as a cause of mesenteric fat (102). In addition, predictive marker of recurrence after surgery fatty acids also differentially modulate the for CD, still await further validation (94). activity of NOD2, a signaling module com- monly dysregulated in CD, which controls Intestinal lipid clearance NF-κB activity and alpha-, beta-defensin expression (104,105). Thus, it is worth noting While the previously described signs of that it is still unknown whether changes in inflammatory-induced intestinal lymphatic the intestinal lipid milieu might influence transport failure concern fluid removal human IBD activity through altered (edema) and immune cell clearance (leuko- regulation of NOD2 signals. stasis), disturbances in lymphatic vessel Lim et al demonstrated that cholesterol integrity and transport function might also also might interfere with lymphatic

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 11 functioning (106). Hypercholesterolemic mice These observations of lymphatic vessel exhibit disturbances in lymphatic vasculature expansion in IBD have been recently and lymphatics actually undergo degenera- confirmed by studies showing that intestinal tion in response to elevated cholesterol (107). inflammation in UC, CD and experimental Considering the complex gap between IBD is correlated with extensive gut lymphatic rarefaction and accumulation of lymphangiogenesis (111-114). Interestingly, intestinal fat, cholesterol accumulations Rahier et al, pointed out that lymphatic might significantly deteriorate lymphatic density increases transmurally in CD, but is functioning in IBD through several molecular limited to the inflamed mucosa in UC, and mechanisms, especially lipid rafts. Therefore, closely parallels the extent of inflammation in anticholesteremic HMG-coA reductase these conditions (15). Geleff et al concluded inhibitors (statins) have been tested in models that lymphatic proliferation in both forms of of intestinal inflammation and have been IBD is activated by inflammatory processes shown to prevent cholesterol synthesis and that affect all regions of the gut, irrespective moreover, improve experimental IBD (108). of the disease process, and also stated that Following these results, Pravastatin has lymphatic expansion can also be observed in been studied in clinical trials for IBD fibrotic end-stage IBD (111). This widespread (NCT00599625, B. Behm, MD, Univ. of induction of lymphatics (now commonly Virginia) but so far not yet been shown to visualized using lymphatic biomarkers like directly influence or stabilize lymphatic podoplanin) is seen even in ‘quiescent’ structure or function in IBD. segments of the ileum and colon in CD, Adequate intestinal lymphatic transport indicating that processes and mediators pro- may depend on maintenance of lymphatic moting lymphatic expansion may be present vessel ‘specification,’ which is also responsible throughout the intestine before clinically for lymphatic-blood vascular partitioning active disease can be recognized (15). during lymphangiogenesis. In the intestine, Lymphatic vessels are also very rapidly fasting induced adipose factor (Fiaf) is an increased in experimental colitis and may important factor, which helps to maintain represent an adaptation, at least in acute postnatal lymphatic vessel specification inflammation (14,111,114,115). We found through induction of Prox-1 (109). Fiaf is an that acute experimental colitis (induced by adipocytokine lipase inhibitor, DSS 3%) in mice was characterized by a expressed by adipocytes as well as several dramatic expansion of lymphatic vessels at other cell types including nerves, epithelial 7 days (114). The inability to remodel these cells, which acts as a stimulus for fatty acid vessels (produced by a genetic deficiency in oxidation and uncoupling in fat metabolism Angiopoetin-2 (Ang-2-/-)) was found to (110). Since Fiaf suppression by locally produce greater disease activity, despite lower accumulated fat might secondarily repress levels of intestinal leukocyte (neutrophil) lymphatic specifying programs (via Prox-1 infiltration, also revealing an important role pathways), disturbances of gut lipid of Ang-2 in gut leukocyte recruitment. These metabolism in IBD might provide an findings suggest that defects in the ability to additional mechanistic basis for lymphatic remodel lymphatic alterations, as well as dysregulation. Furthermore, a diminished their normal distribution or abundance, may capacity of lymphatics to clear intestinally increase gut susceptibility to injury. Whether absorbed fat might also further suppress Fiaf increased lymphatic density (present prior to expression and Fiaf-dependent lymphatic active disease) is a basis of disease or a vessel expansion. compensatory physiological response that suppresses IBD development remains Intestinal lymphangiogenesis controversial and complex. Although there is

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 12 clearly an increase in the abundance of modulators of lymphatic vascularity (124). mucosal lymphatic vessels in human IBD sVEGFR-2 levels regulate transplant success and experimental erosive intestinal injury, and represent both mediators and potential it is unclear whether, and when, lymphatic therapeutic modalities in IBD and other expansion is adaptive (or not). For example, chronic inflammatory diseases. ovarian cancer induces a remarkable increase Lymphangiogenesis and LEC in lymphangiogenesis (in response to CD11b+ tube formation critically depend on signaling macrophage recruitment), which is, however, mediated by VEGFR-3 activity, ERK and largely dysfunctional in terms of fluid small GTPase activity (125,126). A soluble drainage (116). VEGFR-2 variant of ‘sVR2’ was found to not affect hemangiogenesis in tumors but Vascular Endothelial Growth Factor suppressed lymphangiogenesis, an effect Receptor-3 thought to represent VEGF-R2 binding of VEGF-C (124). In fibroblasts obtained from Postnatal lymphangiogenesis is mainly strictured segments of bowel from Crohn’s mediated through stimulation of Vascular disease, VEGF-A production was higher in Endothelial Growth Factor Receptor-3 the strictured segments than in the adjacent (VEGFR-3) which is expressed principally non-strictured segments, or normal controls; on lymphatic endothelial cells (LEC) and how VEGF’s types and levels are altered in activated by growth factors VEGF-D and IBD to affect both blood and lymphatic VEGF-C (117,118). Stimulation of VEGFR-3 vessels is an important area of investigation signaling increases LEC proliferation, (127). migration, and survival, whereas blocking As the central regulator of lymphangio- the VEGFR-3 pathway inhibits both genesis, VEGFR-3 activity depends on its inflammation-induced and tumor mediated level of expression, availability of ligand lymphangiogenesis (117,119-121). (VEGF-D and C) and downstream signals. Several additional signals can epigenet- VEGFR-3 is not only a growth factor, but ically control LEC phenotype through serves as a potent signal modulator affecting VEGFR-3. Capillary tube formation by LEC barrier function and lymphatic pumping. depends on mTOR (mammalian target of As in blood vessels where VEGF-A decreases rapamycin), and the mTOR inhibitor blood vessel barrier, VEGFR-3 activation of rapamycin diminishes LEC tube formation lymphatic endothelial cells decreases their in vitro, an effect which was blocked in cells barrier function, potentially through changes expressing rapamycin sensitive mTOR. in monolayer or junctional organization Mechanistically, rapamycin decreased (128). Most interestingly, while VEGFR-3 VEGFR-3 synthesis and lability in LEC stimulation increases permeability, laminar (122). This effect was recapitulated in LEC fluid shear in lymphatics improves lymphatic by intentional suppression of VEGFR-3, endothelial barrier, a phenomenon that and demonstrates a role of VEGFR-3 in involves Rac-1-actin microfilament dynamics lymphatic ‘capillary’ tube formation. This (129). Therefore chronic lymphostasis in IBD finding has important implications for would be anticipated to increase lymphatic lymphatic suppression in Rapamycin permeability and interfere with containment (‘sirolimus’) therapy for cancer. It is unclear (and hence) transport of lymph. if or how this lymphatic suppression modulates rapamycin treatment of refractory Nuclear Transcription Factor Kappa-B (NF-κB) CD (123). Similarly, soluble VEGF receptors like sVEGFR-2 also appear to represent a The nuclear transcription factor kappa-B class of important emerging endogenous (NF-κB) has been comprehensively described

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 13 as a key regulator in the physiological perpet- organs including the intestine, where uation of innate and acquired immunity. lymphatic endothelial cells (LEC) persistently Multiple intracellular signaling cascades rely express nuclear NF-κB (136). NF-κB expres- on NF-κB to orchestrate the transcriptional sion in LEC is necessary for maintenance of control of a variety of pro- and anti- lymphatic endothelial programming, survival inflammatory mediators (e.g., chemokines, and lymphangiogenesis through the essential cytokines, adhesion molecules) shaping induction of Prox1 and VEGFR-3, particu- inflammatory responses and homeostatic larly during episodes of active inflammation processes like growth control and apoptosis (135,137). Lymphatic endothelial NF-κB (130,131). At the molecular level, the main (p50) interacts with the transcription factor mediators of an appropriate inflammatory Prox1 to activate VEGFR-3 transcription, response through rapid gene expression supporting VEGFR-3 dependent phenotype reprogramming are members of the NF-κB and lymphatic survival signals. Conversely, family (present in virtually every cell type), silencing of NF-κB reduces Prox1 and consisting of five mammalian proteins VEGFR-3 expression in cultured LEC (137). NF-κB1 (p105/p50), NF-κB2 (p100/p52), Because NF-κB is normally persistently RelA (p65), RelB and c-Rel, which all share expressed, nuclearly localized, and active in a highly conserved 300-amino acid Rel lymphatic endothelial cells to maintain their homology region (RHR) responsible for normal physiological functions, disturbances homo- and hetero-dimerization and sequence- in NF-κB function or signaling (which coin- specific DNA binding (132). In unstimulated cides in the setting of intestinal inflammation cells, NF-κB is sequestered in an inactive in IBD) might lead to alterations in lymphatic form in the cytoplasm bound to specific character and formation, which could exag- inhibitory proteins of the inhibitor (I)-B gerate forms of IBD (136,138). Therefore, family. Following stimulation through TLRs, while inflammation promotes lymphangio- proinflammatory cytokines or antigen recep- genesis, suggesting that increased lymphatics tors I-κB are phosphorylated, ubiquitinated are adaptive, inflammatory factors (IL-1β, and finally proteolytically degraded, leading TNF-α, IFN-γ) also present during active to de-repression of NF-κB, which rapidly IBD might limit the full functionality of translocates to the nucleus and activates the inflammation induced lymphatic networks, transcription of several target genes (130,133). (at least as long as they are present) (114). It has also been thoroughly established that Thus, whereas an increase in lymphatic intestinal inflammation occurring in IBD is vessel density (LVD) has been concordantly accompanied by an NF-κB driven overexpres- described as a constant feature of CD and sion of pro-inflammatory adhesion molecules UC, the exact molecular triggers have and mediators, leading to disturbances in remained unidentified (13,15). The NF-κB mucosal immunity (134). Beside classic pathway presents an auspicious mechanistic targets (chemokines and cytokines), it has connection between a variety of inflammatory been recently described that inflammation stimuli and inflammation-induced lymphatic induced NF-B directly upregulates two major hyperplasia as well as lymphangiogenesis transcripts involved in lymphangiogenesis, through a Prox-1 regulated increase of vascular endothelial growth factor receptor VEGFR-3 expression, amplifying cellular (VEGFR)-3 and its key transcription factor responses to local growth factors like VEGF- Prospero-related homeobox-1 (Prox1), C/D (135,137,139). These interactions also resulting in a sustained inflammatory- underscore the independence of lymphangio- induced lymphatic formation (135). genesis in the pathophysiology of IBD Unlike most cells, NF-κB is constitutively because paradoxically, proliferation of blood active in the lymphatic endothelium in most vessel endothelial cells in angiogenesis may

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 14 in some cases be suppressed by activation of tion of lymphatic NF-κB, it is interesting to NF-κB (140-142). speculate that lymphatic disturbances in CD However and intriguingly, the previously (and perhaps also UC) might increase IBD mentioned main lymphangiogenic growth activity through lymphatic suppression as factors (VEGF-C/D) actually appear to be described by Flister et al and in some depressed in IBD, while VEGFR-3 appears experimental IBD models (11,137,143). to be increased. Rahier et al suggest that As shown above, the role of NF-κB in VEGF-C and VEGF-D expression was absent IBD related intestinal inflammation is by or greatly decreased in inflamed as well as in now clearly established, although there are non-inflamed intestinal tissues specimens of gaps in our knowledge still regarding IBD patients whereas the abundance of inflammatory induced lymphangiogenesis VEGFR-3 in Crohn’s disease and ulcerative through Prox-1 and VEGFR-3 and their colitis is less clear (15,17). The upregulation activating growth factors (VEGF-C, VEGF- of VEGFR-3 expression by inflammatory D), which remains a new and promising area. cytokines through NF-κB and Prox-1 should Besides the sometimes conflicting findings for result in an increased receptor availability, lymphangiogenesis in IBD, it is also still but a lower net availability of local VEGF- unclear what effects standard IBD therapies C/D might potentially contribute to “aberrant” (corticosteroids, sulfasalazine or metho- IBD associated intestinal lymphatic charac- trexate, etc.) have on NF-κB mediated teristics (14,17,135,137). This might lead to lymphatic vessel function and formation a shift in IBD toward deficient VEGFR-3 (since some are known to at least partially signaling. Although there is clearly an inhibit NF-κB activity) (134,144,145). In increase in lymphatic vessel density in IBD conclusion, these findings suggest that (14,15,111), the mechanisms which account identification of mechanisms which support for lymphangiogenesis in IBD may therefore and induce expression of lymphatics in the differ somewhat from those active in normal inflamed intestine may reveal novel physiology (and suggest eminent roles for the therapeutic approaches for treating IBD. NF-κB pathway). This finding suggests that the sources of lymphangiogenic growth Transforming Growth Factor (TGF)-β1 factors in IBD may be suppressed or blocked (whereas the corresponding receptor is The alterations in lymphatic integrity, sensitized). It is furthermore still uncertain remodeling, and especially function, found whether and when lymphatic vessel expan- in CD and UC, find their basis to some sion in IBD is accompanied with impaired or extent in unbalanced equilibrium in pro/anti- improved lymphatic function. These findings lymphangiogenic factors (22,146). In addition may indicate that an IBD associated to members of the vascular endothelial depression in lymphatic activation (possibly growth factor family, which support lymphatic through NF-κB pathway suppression) could proliferation and capillary formation, recent lead to either insufficient lymphatic density, studies identify potent anti-lymphangiogenic loss of lymphatic specification, inappropriate mediators in the process of lymphangio- patterning or remodeling, any of which genesis, for instance, interferon-γ (IFN-γ), would be anticipated to intensify IBD disease transforming growth factor-β and endostatin activity (15,24). (147,148). Among these, the cytokine TGF-β1 Recent reports also connect the NF-κB appears to play an important role in blocking pathway with another aspect of IBD patho- lymphatic vessel regeneration and reorgani- physiology. Whereas genetic mutations zation, and is linked closely to several events associated with the susceptibility for IBD in the pathophysiology of chronic intestinal might interfere with the normal tonic activa- disorders such as IBD (146,149-151).

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TGF-β1, the canonical member of the fibrosis and interrupt lymphatic regeneration transforming growth factor superfamily, to exacerbate lymphatic dysfunction (151). is secreted by various immune cells (e.g., Given the precise and balanced role of T-cells) and transduces its intercellular TGF-β in regulation of lymphatic function activity through transmembrane receptors and formation modulated by pro/anti- TGF-βR-I/II, activating multiple downstream lymphangiogenic stimuli, it seems reasonable signals (e.g., Smad protein phosphorylation), to suggest an essential participation of ultimately regulating transcription control TGF-β signaling, leading to disturbed ligand of multiple target genes (152). and receptor distributions, in the established Besides different signaling pathways in dysfunction of intestinal lymphatic anti-inflammatory immunity and growth vasculature in IBD. control, TGF-β1 is known as a potent inducer of fibrogenesis in multiple organs (153,154). Toll Like Receptors (TLRs) The induction of fibrosis through TGF-β1 via epithelial-mesenchymal transition (EMTs) The gut is continuously exposed to an has been well-described in CD, where fibrosis enormous load of commensal and pathogenic and disturbances in anti-inflammatory stimuli which help to regulate immunity. pathways represents intestinal key features in Within the immunity related mechanisms, inflammatory pathophysiology (150,155-157). toll like receptors (TLRs) are essential pattern Accordingly several groups report increased recognition receptors (PRRs) which respond levels of TGF-β isoforms in active IBD, to pathogen and danger associated molecular with clear elevation of TGF-β1, TGF-β3 and patterns (PAMPs and DAMPs), transducing TGF-BR-I, whereas phosphorylation of intestinal immune and inflammatory downstream proteins was considerably responses at several cellular and molecular reduced (149,158). However (and paradoxi- levels and affecting various cell types, cally), it seems likely that deficiency in including lymphatic endothelial cells (LEC). TGF-β and Smad signaling contributes to So far, several TLRs (TLR-1 to 13) have development and maintenance of UC and been discovered, which show differential CD (159). expression and ligand binding specificities. Currently, an accumulating experimental TLR-1 binds the acylated NH2 termini in database introduced a possible gap between bacterial (as well as the TLR-1/2 TGF-β1, IBD and lymphatic reorganization. mimetic PAM3CSK4). TLR-2 can dimerize Avraham et al (2010) showed that TGF-β1 with TLR-1 or TLR-6 to recognize bacterial intensifies tissue fibrosis by suppressing both vs. mycoplasma lipoproteins, respectively lymphangiogenesis and lymphatic function (162,163). TLR-3 recognizes viral double- and that local blockade of TGF-β1 reduced stranded RNA (164) while TLR-4 binds tissue fibrosis, decreased chronic inflamma- lipopolysaccharide (LPS) (165). TLR-5 binds tion including Th2 cell migration, and flagellin (166); TLR-7 and TLR-8 react with importantly improved lymphatic function in single-stranded RNA (and the synthetic an experimental model of wound repair (160). immunomodulator ligand drug ‘Imiquimod’) Similarly, Yan et al (2011) showed that (167,168). Lastly, TLR-9 reacts with adipose derived stem cells stimulate unmethylated CpG motifs present in bacterial lymphangiogenesis by inhibiting TGF-β and viral DNA. (161). Zampell et al found that TLR-4 and -9 A comprehensive study by Pegu et al knockout mice showed elevated deposition evaluated TLR expression in several lymphatic of type I collagen (a marker of fibrosis) and endothelial cell (LEC) models from primary importantly, higher levels of TGF-β1 (1°) , ‘stretched’ skin and 1° expression, which appear to both promote cultures, evaluating LEC inflammatory

Permission granted for single print for individual use. Reproduction not permitted without permission of Journal LYMPHOLOGY 16 responses after exposure to TLR agonists in lung LEC, with TLR-1/2, -3, -4 agonists vitro. All LECs expressed mRNAs for TLRs potently increasing CCL20 mRNA; TLR-2/6 1-6 and 9; TLR-4 mRNA was most abundant stimulation increased CCL20 in 1° skin (followed by TLRs 1-3 and 6, expressed at LECs. CCL20 protein was expressed by skin equivalent levels). Only low levels of TLR-5 and lung LEC following TLR-1/2, -3, -4, and -9 were found; (mRNAs for TLRs-7, 8, TLR, 1° skin LECs also increased CCL20 and 10 were not observed) (169). This expres- after TLR-6/2 activation. RANTES and IL-8 sion pattern is similar to that reported by were increased in all LECs after TLR-1/2, -3, Garrafa et al who found mRNAs for TLRs -4 stimulation. TLR-6/2 stimulation also 1-4, 6, and 9, but not TLR-5 (170); thymic increased RANTES and IL-8 expression (in LECs were seen to express mRNAs for TLRs 1° skin LECs). TLR-3 stimulation increased 1-6 and 9. TLR mRNA and protein expression IP-10 in all LECs and TLR-1/2, TLR-4 and were not however uniform. TLR-6/2 stimulation inducing IP-10 in 1° At the protein level, Pegu et al also found skin LEC alone. TLR-8 also increased IP-10 that TLR-4, -5 and -6 were expressed at in stretched LEC alone. the LEC surface in skin and lung LEC, with mRNAs for IL-1β, TNF-α, and IL-6 lower expression on ‘stretched’ LECs, sug- were increased in skin LEC in response to gesting potential links between loss of growth stimulation of TLRs-1/2, 3, 4; TLR-5 control regulation and LEC phenotypic activation also increased cytokines in 1° skin maturity. TLR-5 and 6 were highly expressed LECs. Garrafa et al found that at similar levels and were even more (LN) LEC also mobilize ICAM-1 and abundant than TLR-4 (at the protein level). VCAM-1 after TLR-3 and -4, and after Interestingly, TLR-3 and -9 were TLR-3, respectively. Thymic LEC only expressed on LEC outer cell membranes as expressed ICAM-1 after TLR-3, -4 activation. well as intracellularly, (except TLR-1 and 2, Interestingly, Pegu et al described only which were only intracellular) (169). moderate increases in VEGF-C (mRNA) in Intracellular vs. extracellularly presented response to TLR-1/2,-3, -4 and -5 in 1° skin TLRs likely regulate responses towards LEC and in stretched skin LEC (following lymphatically filtered antigens, or assist LEC TLR-1/2, -3 and -8); strikingly, no changes in responses to internalized antigens. VEGF-D were observed (169). TLR-1/2 -3, -4 With respect to these signals, selective and 6/2, also increased VCAM-1 and ICAM-1 LEC TLR ligand stimulation also activated mRNAs in both skin and lung LECs; lung downstream signaling programs related to LEC increased VCAM-1 mRNA in response inflammation including chemokines, to all TLRs. TLR-3 and -4 stimulation cytokines, growth factors and adhesion increased VCAM-1 and ICAM-1 expression molecules. Pegu et al found strongest LEC on cell membranes of both skin and lung TLR responses following activation of TLR LEC. TLR agonists had little effect on D6 1/2, -3, -4, -5 and -8. mRNAs for the CXCR3 scavenger receptor expression except for lung ligands CXCL9, IP-10 and CXCL11 were LEC (D6 was modestly induced by LPS and among those most highly and consistently PAM3CSK4). Garrafa et al reported that in induced in all LEC. TLR-3 was seen to be the LN LEC, TLRs-1, 3, and 4 induced IL-8, a most active inducer, followed by TLR-8 and potent chemoattractant for lymphangiogenic then -4. In 1° skin LEC, TLR-2/6 stimulation immune cells. In thymic LEC, IL-8 was also also increased mRNAs for these ligands (an stimulated by TLRs 1-6 and 9. RANTES effect not seen in lung or stretched LECs). was induced in LN EC by TLRs 1, 3, and 4 ; In general, TLR ligands increased mRNA in thymic LEC, RANTES was induced by expression of the chemoattrac- TLRs-2 and 3. In LN LEC TNF-α was tant and CCR6 ligand CCL20 in skin and released by TLRs-1-4 and by TLRs 1-6 and 9

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(thymic LEC). IL-6, (another potent immune might support the use of antibiotics in IBD cell attractant) was also induced by TLRs-3, 4 therapy (172). We found that TLR-4 expres- in LN LEC, and by TLRs 1-6 and 9 in thymic sion in the gut was increased by fat feeding LECs. Monocyte chemo-attractant protein-1 and increased NF-κB p65 phosphorylation, (MCP-1) was also released by LN LEC after while simultaneously reducing SIRT-1 TLR 1-4 and 6 activation, but only TLR-3 expression, effects which were eliminated in induced MCP-1 in thymic LEC. Lastly, TLR- antibiotic treated or TLR-4 -/- mice (86). 3 stimulated IP-10 in both LN and thymic These results with TLR-4 parallel our other LEC, with a much more prominent effect on findings in Patel et al (using gut sterilization), thymic LECs. Garrafa also found that NF-κB which also showed a suppression of inflam- p65 was activated by TLRs 1-4, 6 and 9 (in mation and lymphangiogenesis (171). While LN LEC) and by TLRs 1-6 and 9 in thymic not yet fully explored, the inverse relationship LECs. Because NF-κB is persistently active between SIRT-1 and NF-κB in inflammatory in LECs to maintain their phenotypic specifi- lymphangiogenesis is intriguing. cation, TLRs may provide important links between pattern recognition, lymphatic Chemokine Scavenging Receptor (D6) structure, maturation and function. These studies show some heterogeneity Recently, a basis for lymphatic obstruc- among lymphatic endothelial cells from tion in IBD involving the D6 chemokine differing anatomic sources, which still share scavenging receptor has been described (173). a common ability to respond to PRRs. In D6 is a CC chemokine ‘clearing’ receptor different tissues, TLRs may modulate LEC expressed by lymphatic endothelial cells as activation, immune cell binding and guidance well as stromal and B-cells (174). D6 binds to, molecule expression. Although these molecular and eliminates inflammatory CC chemokines, responses appear to be well-developed in (especially CCL2) while sparing CCL19 and lymphatic endothelial cells, inappropriate or CCL21. D6 was upregulated by the GATA1 excessive activation of LEC adhesive induc- transcription factor, (in hemopoietic cells) , tion could promote lymphoid retention of suppressed by LPS (via TLR-4) and immune cells and increase the potential for upregulated by TGF-β1 (175). Importantly, obstruction. We have found that experi- Lee et al demonstrated that D6 knockout mental elimination of gut flora using triple- mice exhibit dysfunctional lymphatics with antibiotic treatment reduced gut injury in poor interstitial fluid clearance, entrapment experimental (DSS) colitis (171). Despite an of inflammatory cells and lymphatic apparent reduction in gut epithelial barrier congestion (173). marrow transfer in this model, the suppression of gut flora studies in these mice showed that D6- significantly prevented the development of knockout mice were more vulnerable to histopathology (crypt damage, epithelial experimental colitis strongly supporting links injury), weight loss, stool bleeding, etc. between lymphangitis and D6-deficiency as a Importantly we found that elevations in blood potential exacerbating event in IBD (176). vessel density were apparently prevented by However, currently it is unclear if D6 is also gut ‘sterilization,’ while lymphatic density dysfunctional in IBD, but experimental was increased over control, albeit lower than studies support accumulation of chemokines in ‘non-sterile’ DSS treated mice. This as an important exacerbating event in colitis. suggests that factors released by gut flora which activate PRRs help to induce lymphan- Endothelin giogenic programming. The relatively high lymphatic to blood vessel ratio seen in gut Besides NO• and prostanoids, primary ‘sterilized’ mice associated with protection endothelial-derived factors e.g., endothelins

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(ET-1,-2,-3) and their apposite receptors ET-1 increases lymphatic density by (ETA, ETB1, ETB2, ETC) also might influence enhancing expression of VEGF-C and -A. lymphatic functioning in IBD. Reeder and In 2010, they showed that ET-1 binding to Ferguson and later, Marchetti et al found ETB receptors increased the activation of that lymphatic endothelial cells (like blood MMP-2 and -9 proforms, a step which may vascular endothelium) release the potent be necessary for the development of fully vasoconstrictor ET-1 (177,178). Interestingly, functional lymphatic vessels (181,184,185). ETB receptor activation on lymphatic Doboszynska et al indirectly showed the endothelial cells mediates the release of two relationship of ET to lymph flow based on potent vasodilators, NO• and PGI2 (possibly immunostaining of different lymphatic a contractile feedback mechanism), while regions (184,185). Because the homeobox ETA receptors on lymphatic vascular smooth transcription factor Nkx 2-3 has been shown muscle can mediate contraction (179). to be dysregulated in IBD (which downregu- Although ETA receptors appear not to be lates ET expression in both UC and in CD), expressed by lymphatic endothelial cells, endothelin effects on lymphatics might be a Zhao and Van Helden report that low levels novel and important mechanism in IBD of ET-1 (≤ 10nM) augmented lymphatic pathogenesis (186). pumping while higher levels (≥ 100 nM) triggered vasospasm, which would be Lymphatic Restoration expected to impair lymph flow (180). In lymphatic vessels, blocking ETA receptors Several lines of evidence support that the (using BQ-610), or ETB receptors (BQ-788) expansion and normalization of lymphatic prevented ET-1 induced contraction structure and function will improve the intes- responses with ETA blockade more effective tinal inflammation seen in IBD. Tabibiazar than ETB (180). Because ET-1 mediated et al suggest lymphatic specific growth lymphatic contraction was found to be factors might attenuate edema, while blood increased by endothelial injury, but not by vascular VEGFs intensify edema (187). NO synthase inhibition, endothelial derived Similarly, suppression of lymphatic vessels hyperpolarizing factors were also proposed in mice expressing sVEGFR-3 Ig intensifies to modulate these responses. Therefore, edema, particularly in neonates. In support coordinated interactions between ETA and of this finding, Huggenberger et al have ETB receptors (on lymphatic smooth muscle shown that induction of lymphatics improve and endothelium) could have important edema in the skin (188,189). Interestingly effects on lymphatic integrity and function in anti-TNF-α treatment with Infliximab in IBD. With regard to lymphatic remodeling, arthritis (both murine and human) has been ET-1 has also been reported to promote associated with an increase in lymphangio- lymphatic vascular invasiveness and control genesis, suggesting that TNF-α may interfere lymph flow (via ETB receptors), a process with lymphangiogenesis and inflammation which appears to involve matrix metallopro- through decreased LEC proliferation, barrier teinases (MMP)-2 and -9 (181). Endothelin-1 function and capillary network development can further promote endothelial proliferation, (114,190). Whereas the immunomodulating migration and invasion through ETB receptors effects of Infliximab in human IBD and (182,183). Based on the ability of ET antago- experimental colitis are well established and nists to block Kaposi cells (which widely used, the effect on intestinal lymphatic share lymphatic endothelial programming), vessels for now remains unclear. lymphatic endothelial proliferative responses to ETA may be at least partly autocrine in CONCLUSIONS nature. Spinella et al also suggested that

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Fig. 1. Intestinal lymphatics normally transport interstitial fluid, immune cells, their mediators, antigens, digested lipids and cholesterol back to the central circulation and thereby prevent their accumulation in the interstitium. Lymphatic flow starts in initial lymphatics through specialized button-like junctions, and its unidirectional flow is maintained through semilunar valves and an active pumping capacity in collecting lymphatics. In IBD, disturbances in the intestinal epithelial barrier (in conjunction with gene polymorphisms and altered microbiota ) provoke activation of inflammation through danger and pathogen associated molecular patterns, triggering immune cell infiltration which produces persistent extensive tissue injury. Tissue infiltration by immune cells can be initiated and intensified by chemokines and cytokines, which are cleared by lymphatic outflow as well as scavenger receptors present on lymphatic endothelium. Transmural inflammation activates the release of lymphangiogenic growth factors VEGF-C and VEGF-D, which induce new lymphatic growth. Transforming growth factor-beta released by immune and interstitial cells can oppose lymphangiogenic signaling, which is mediated by Prox-1, VEGFR-3 and NF-κB downstream signaling. Lymphangion contractility actively supports centripetal lymph flow and is positively modulated by prostanoids (PGH2, PGF-2α, TXA2), histamine (H2 receptors) and negatively modulated by NO, histamine (H1 receptors), prostacyclin (PGI2) whereas Vasopressin and ethanol have controversial effects on lymphatic pumping (contractility/chronicity). Adipocyte derived factors (MCSF, adiponectin, MMIF) also influence lymphatic integrity. Downmodulation of Prox-1 in response to inflammatory mediators may lead to lymph leakage or reflux. The cumulative influences of these factors and conditions may lead to development of a chronic inflam- matory state which impairs the outflow of interstitial fluid and immune cells thereby perpetuating gut inflammation.

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As in the blood vasculature, many tory bowel disease: “Brothers in arms.” Gut inflammatory signaling mechanisms mediate 60 (2011), 998-1008. the disorganization, integrity and hypofunc- 9. Chidlow, JH, Jr, D Shukla, MB Grisham, et al: Pathogenic angiogenesis in IBD and tion of intestinal lymphatics in experimental experimental colitis: New ideas and thera- and human forms of IBD (Summary Fig. 1). peutic avenues. Am. J. Physiol. Gastrointest. These changes may contribute to disturbances Physiol. 293 (2007), G5-G18. of normal gut homeostasis and restitution, 10. Langston, W, JR Chidlow Jr, BA Booth, et al: Regulation of endothelial glutathione by which initiate and maintain disease activity ICAM-1 governs VEGF-A-mediated eNOS in IBD. Studies of lymphatic mechanisms activity and angiogenesis. Free Radic. Biol. identified in CD and UC not only reveal Med. 42 (2007),720-729. mechanistic bases but may also provide novel 11. Alexander, JS, GV Chaitanya, MB Grisham, and important targets for the therapeutic et al: Emerging roles of lymphatics in inflammatory bowel disease. Ann. NY Acad. intervention in IBD and maintenance of Sci 1207 (Suppl 1) (2010), E75-85. its remission. 12. Crohn, BB, L Ginzburg, GD Oppenheimer: Regional ileitis: A pathologic and clinical ACKNOWLEDGMENTS entity. J Am Med Assoc 99 (1932), 1323-1329. 13. Van Kruiningen, HJ, J-F Colombel: The forgotten role of lymphangitis in Crohn’s This work was supported by DOD Grant disease. Gut 57 (2008),1-4. PR100451 “Lymphatic Therapy in IBD.” 14. Kaiserling, E, S Kröber, S Geleff: Lymphatic The authors wish to acknowledge Ms. vessels in the colonic mucosa in ulcerative Courtney Parker for editorial assistance with colitis. Lymphology 36 (2003), 52-61. the manuscript. Dr. Becker is supported by a 15. Rahier, J-F, S De Beauce, L Dubuquoy, et al: Increased lymphatic vessel density and fellowship grant from Abbvie Corporation. lymphangiogenesis in inflammatory bowel disease. Aliment Pharmacol. Ther. 34 (2011), REFERENCES 533-543. 16. Von Der Weid, P-Y, S Rehal: Lymphatic pump function in the inflamed gut. Ann. NY 1. Molodecky, NA, IS Soon, DM Rabi, et al: Acad. Sci. 1207 (Suppl 1) (2010), E69-74. Increasing incidence and prevalence of the 17. Linares, PM, JP Gisbert: Role of growth inflammatory bowel diseases with time, based factors in the development of lymphangio- on systematic review. Gastroenterology. 142 genesis driven by inflammatory bowel disease: (2012), 46-54.e42; quiz e30. A review. Inflamm. Bowel Dis. 17 (2011), 2. Xavier, RJ, DK Podolsky: Unravelling the 1814-1821. pathogenesis of inflammatory bowel disease. 18. Nesis, L, EE Sterns: Lymph flow from the Nature 448 (2007), 427-434. colon under varying conditions. Ann. Surg. 3. Baumgart, DC, WJ Sandborn: Crohn’s 177 (1973), 422-427. disease. Lancet 380 (2012),1590-1605. 19. Sheppard, MS, EE Sterns: The role of 4. Scharl, M, G Rogler: Inflammatory bowel vascular and lymph capillaries in the disease pathogenesis: What is new? Curr. clearance of interstitially injected albumin in Opin. Gastroenterol. 28 (2012), 301-309. acute and chronic inflammation of the colon. 5. Ordás, I, L Eckmann, M Talamini, et al: Surg. Gynecol. Obstet. 139 (1974), 707-711. Ulcerative colitis. Lancet 380 (2012), 1606-1619. 20. Colombel, J-F, BG Feagan, WJ Sandborn, 6. Baumgart, DC, SR Carding: Inflammatory et al: Therapeutic drug monitoring of bowel disease: Cause and immunobiology. biologics for inflammatory bowel disease. Lancet 369 (2007),1627-1640. Inflamm. Bowel Dis. 18 (2012), 349-358. 7. Pastorelli L, C De Salvo, JR Mercado, et al: 21. Kalima, TV: Experimental lymphatic Central role of the gut epithelial barrier in the obstruction in the ileum. Ann. Chir. Gynaecol. pathogenesis of chronic intestinal inflamma- Fenn. 59 (1970), 187-201. tion: Lessons learned from animal models 22. Zampell, JC, S Aschen, ES Weitman, et al: and human genetics. Front. Immunol. 4 Regulation of adipogenesis by lymphatic fluid (2013), 280. stasis: Part I. Adipogenesis, fibrosis, and 8. Danese, S: Role of the vascular and lymphatic inflammation. Plast Reconstr. Surg. 129 endothelium in the pathogenesis of inflamma- (2012), 825-834.

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