Flow-dependent regulation of rat mesenteric lymphatic vessel contractile response requires activation of endothelial TRPV4 channels.

OBJECTIVES: The objective of our study is to evaluate the involvement of the transient receptor potential vanilloid 4 (TRPV4) in the alteration of lymphatic pumping in response to flow and determine the signaling pathways involved. METHODS: We used immunofluorescence imaging and western blotting to assess TRPV4 expression in rat mesenteric lymphatic vessels. We examined inhibition of TRPV4 with HC067047, nitric oxide synthase (NOS) with L-NNA and cyclooxygenases (COXs) with indomethacin on the contractile response of pressurized lymphatic vessels to flow changes induced by a stepwise increase in pressure gradients, and the functionality of endothelial TRPV4 channels by measuring the intracellular Ca2+ response of primary lymphatic endothelial cell cultures to the selective agonist GSK1016790A. RESULTS: TRPV4 protein was expressed in both the endothelial and the smooth muscle layer of rat mesenteric lymphatics with high endothelial expression around the valve sites. When maintained under constant transmural pressure, most lymphatic vessels displayed a decrease in contraction frequency under conditions of flow and this effect was ablated through inhibition of NOS, COX or TRPV4. CONCLUSIONS: Our findings demonstrate a critical role for TRPV4 in the decrease in contraction frequency induced in lymphatic vessels by increases in flow rate via the production and action of nitric oxide and dilatory prostanoids.

Not just fibrotic: endothelial-derived TGFß maintains contractile function and lymphatic muscle phenotype during homeostasis.

Cell-cell communication within the lymphatic vasculature during homeostasis is incompletely detailed. Although many discoveries highlight the pathological roles of transforming growth factor-beta (TGFß) in chronic vascular inflammation and associated fibrosis, only a small amount is known surrounding the role of TGFß-signaling in homeostatic lymphatic function. Here, we discovered that pharmacological blockade of TGFß receptor 1 (TGFßR1) negatively impacts rat mesenteric lymphatic vessel pumping, significantly reducing vessel contractility and surrounding lymphatic muscle coverage. We have identified mesenteric lymphatic endothelial cells themselves as a source of endogenous vascular TGFß and that TGFß production is significantly increased in these cells via activation of a number of functional pattern recognition receptors they express. We show that a continuous supply of TGFß is essential to maintain the contractile phenotype of neighboring lymphatic muscle cells and support this conclusion through in vitro analysis of primary isolated lymphatic muscle cells that undergo synthetic differentiation during 2-D cell culture, a phenomenon that could be effectively rescued by supplementation with recombinant TGFß. Finally, we demonstrate that lymphatic endothelial production of TGFß is regulated, in part, by nitric oxide in a manner we propose is essential to counteract the pathological over-production of TGFß. Taken together, these data highlight the essential role of homeostatic TGFß signaling in the maintenance of lymphatic vascular function and highlight possible deleterious consequences of its inhibition.NEW & NOTEWORTHY The growth factor TGFß is commonly associated with its pathological overproduction during tissue fibrosis rather than its homeostatic functions. We expose the lymphatic endothelium as a source of endogenous TGFß, the impact of its production on the maintenance of surrounding lymphatic muscle cell phenotype, and internally regulated mechanisms of its production. Overall, these results highlight the intricate balance of TGFß-signaling as an essential component of maintaining lymphatic contractile function.

Increased contractile activity and dilation of popliteal lymphatic vessels in the TNF-α-overexpressing TNFΔARE/+ arthritic mouse.

AIMS: TNF-α acute treatment has been found to disrupt lymphatic drainage in the setting of arthritis through the NF-kB-iNOS- signaling pathway. We examined whether popliteal lymphatic vessels (pLVs) contractile activity was altered in 12- and 24- week-old females of an arthritic mouse model overexpressing TNF-α (TNFΔARE/+). MAIN METHODS: pLVs were prepared for intravital imaging to measure lymph flow speed, and ex vivo functional responses to a stepwise increase in transmural pressure in the absence or presence of the non-selective NOS inhibitor (L-NNA) or the selective iNOS inhibitor (1400W) were compared between TNFΔARE/+ and WT mice. Total eNOS (t-eNOS) and eNOS phosphorylated at ser1177 (p-eNOS) were evaluated by western blotting. KEY FINDINGS: In vivo imaging revealed a significantly increase in lymph flow speed in TNFΔARE/+ mice in comparison to WT at both ages. Pressure myography showed an increase in contraction frequency, diameters and fractional pump flow at both ages, whereas amplitude and ejection fraction were significantly decreased in older TNFΔARE/+ mice. Additionally, contraction frequency was increased in the presence of 1400W, and systolic diameter was abolished with L-NNA in TNFΔARE/+ mice compared to WT. Significant increases in p-eNOS expression and neutrophil recruitment (MPO activity) were observed in TNFΔARE/+ mice compared to WT. SIGNIFICANCE: Our data reveal functional changes in pLVs, especially in advanced stage of arthritis. These alterations may be related to eNOS and iNOS response, which can affect drainage of the inflammatory content from the joints.

The dietary fibre rhamnogalacturonan improves intestinal epithelial barrier function in a microbiota-independent manner.

BACKGROUND AND PURPOSE: Dietary fibre comprises a complex group of polysaccharides that are indigestible but are fermented by gut microbiota, promoting beneficial effects to the intestinal mucosa indirectly through the production of short chain fatty acids. We found that a polysaccharide, rhamnogalacturonan (RGal), from the plant Acmella oleracea, has direct effects on intestinal epithelial barrier function. Our objective was to determine the mechanism whereby RGal enhances epithelial barrier function. EXPERIMENTAL APPROACH: Monolayers of colonic epithelial cell lines (Caco-2, T84) and of human primary cells from organoids were mounted in Ussing chambers to assess barrier function. The cellular mechanism of RGal effects on barrier function was determined using inhibitors of TLR-4 and PKC isoforms. KEY RESULTS: Apically applied RGal (1000 µg ml-1 ) significantly enhanced barrier function as shown by increased transepithelial electrical resistance (TER) and reduced fluorescein isothiocyanate (FITC)-dextran flux in Caco-2, T84 and human primary cell monolayers, and accelerated tight junction reassembly in Caco-2 cells in a calcium switch assay. RGal also reversed the barrier-damaging effects of inflammatory cytokines on FITC-dextran flux and preserved the tight junction distribution of occludin. RGal activated TLR4 in TLR4-expressing HEK reporter cells, an effect that was inhibited by the TLR4 inhibitor, C34. The effect of RGal was also dependent on PKC, specifically the isoforms PKCδ and PKCζ. CONCLUSION AND IMPLICATIONS: RGal enhances intestinal epithelial barrier function through activation of TLR4 and PKC signalling pathways. Elucidation of RGal mechanisms of action could lead to new, dietary approaches to enhance mucosal healing in inflammatory bowel diseases.

Off-Target Effect of Lovastatin Disrupts Dietary Lipid Uptake and Dissemination through Pro-Drug Inhibition of the Mesenteric Lymphatic Smooth Muscle Cell Contractile Apparatus.

Previously published, off-target effects of statins on skeletal smooth muscle function have linked structural characteristics within this drug class to myopathic effects. However, the effect of these drugs on lymphatic vascular smooth muscle cell function, and by proxy dietary cholesterol uptake, by the intestinal lymphatic network has not been investigated. Several of the most widely prescribed statins (Atorvastatin, Pravastatin, Lovastatin, and Simvastatin) were tested for their in-situ effects on smooth muscle contractility in rat mesenteric collecting lymphatic vessels. Lovastatin and Simvastatin had a concentration-dependent effect of initially increasing vessel contraction frequency before flatlining the vessel, a phenomenon which was found to be a lactone-ring dependent phenomenon and could be ameliorated through use of Lovastatin- or Simvastatin-hydroxyacid (HA). Simvastatin treatment further resulted in mitochondrial depolymerization within primary-isolated rat lymphatic smooth muscle cells (LMCs) while Lovastatin was found to be acting in a mitochondrial-independent manner, increasing the function of RhoKinase. Lovastatin's effect on RhoKinase was investigated through pharmacological testing and in vitro analysis of increased MLC and MYPT1 phosphorylation within primary isolated LMCs. Finally, acute in vivo treatment of rats with Lovastatin, but not Lovastatin-HA, resulted in a significantly decreased dietary lipid absorption in vivo through induced disfunction of mesenteric lymph uptake and trafficking.

Nr4A1 modulates inflammation-associated intestinal fibrosis and dampens fibrogenic signaling in myofibroblasts.

Intestinal fibrosis is a common complication of the inflammatory bowel diseases (IBDs), contributing to tissue stiffening and luminal narrowing. Human nuclear receptor 4A 1 (NR4A1) was previously reported to regulate mesenchymal cell function and dampen fibrogenic signaling. NR4A1 gene variants are associated with IBD risk, and it has been shown to regulate intestinal inflammation. Here, we tested the hypothesis that NR4A1 acts as a negative regulator of intestinal fibrosis through regulating myofibroblast function. Using the SAMP1/YitFc mouse, we tested whether two pharmacological agents known to enhance NR4A1 signaling, cytosporone B (Csn-B) or 6-mercaptopurine (6-MP), could reduce fibrosis. We also used the dextran sulfate sodium (DSS) model of colitis and assessed the magnitude of colonic fibrosis in mouse nuclear receptor 4A 1 (Nr4a1-/-) and their wild-type littermates (Nr4a1+/+). Lastly, intestinal myofibroblasts isolated from Nr4a1-/- and Nr4a1+/+ mice or primary human intestinal myofibroblasts were stimulated with transforming growth factor-ß1 (TGF-ß1), in the presence or absence of Csn-B or 6-MP, and proliferation and ECM gene expression assessed. Csn-B or 6-MP treatment significantly reduced ileal thickness, collagen, and overall ECM content in SAMP1/YitFc mice. This was associated with a reduction in proliferative markers within the mesenchymal compartment. Nr4a1-/- mice exposed to DSS exhibited increased colonic thickening and ECM content. Nr4a1-/- myofibroblasts displayed enhanced TGF-ß1-induced proliferation. Furthermore, Csn-B or 6-MP treatment was antiproliferative in Nr4a1+/+ but not Nr4a1-/- cells. Lastly, activating NR4A1 in human myofibroblasts reduced TGF-ß1-induced collagen deposition and fibrosis-related gene expression. Our data suggest that NR4A1 can attenuate fibrotic processes in intestinal myofibroblasts and could provide a valuable clinical target to treat inflammation-associated intestinal fibrosis.NEW & NOTEWORTHY Fibrosis and increased muscle thickening contribute to stricture formation and intestinal obstruction, a complication that occurs in 30%-50% of patients with CD within 10 yr of disease onset. More than 50% of those who undergo surgery to remove the obstructed bowel will experience stricture recurrence. To date, there are no drug-based approaches approved to treat intestinal strictures. In the current submission, we identify NR4A1 as a novel target to treat inflammation-associated intestinal fibrosis.

Ultra-purification of Lipopolysaccharides reveals species-specific signalling bias of TLR4: importance in macrophage function.

TLR4 location, and bacterial species-derived lipopolysaccharides, play a significant role in the downstream activation of transcription factors, accessory molecules, and products. Here, this is demonstrated through the use of classically-activated and alternatively-activated macrophages. We show that, when polarized, human macrophages differentially express and localize TLR4, resulting in biased recognition and subsequent signalling of LPS derived from Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica. Analysis of activation demonstrated that in classically activated macrophages, P. aeruginosa signals from the plasma membrane via TLR4 to p65 dependent on TAK1 and TBK1 signalling. E. coli signals dependent or independent of the endosome, utilizing both TAK1- and TBK1-signalling to induce P65 and IRF3 inducible genes and cytokines. S. enterica however, only induces P65 and IRF3 phosphorylation through signalling via the endosome. This finding outlines clear signalling mechanisms by which innate immune cells, such as macrophages, can distinguish between bacterial species and initiate specialized responses through TLR4.

Lipopolysaccharides modulate intestinal epithelial permeability and inflammation in a species-specific manner.

Patients presenting with Inflammatory bowel disease have been shown to exhibit an altered microbiome in both Crohn's disease and Ulcerative colitis. This shift in the microbial content led us to question whether several of these microbes are important in inflammatory processes present in these diseases and more specifically whether lipopolysaccharides from the gram-negative cell wall differentially stimulates resident cells. We, therefore, investigated the possible contribution of five major species of gram-negative bacteria found to be altered in presence during disease progression and evaluate their pathogenicity through LPS. We demonstrated that LPS from these different species had individual capacities to induce NF-κB and pro-inflammatory IL-8 production from HEK-TLR4 cells in a TLR4 dependent manner. Additional work using human intestinal colonic epithelial cell monolayers (Caco-2) demonstrated that the cells responded to the serotype specific LPS in a distinct manner, inducing many inflammatory mediators such as TNF-α and IL-10 in significantly altered proportions. Furthermore, the permeability of Caco-2 monolayers, as a test for their ability to alter intestinal permeability, was also differentially altered by the serotype specific LPS modulating trans-epithelial electrical resistance, small molecule movement, and tight junction integrity. Our results suggest that specific species of bacteria may be potentiating the pathogenesis of IBD and chronic inflammatory diseases through their serotype specific LPS responses.

Lymphatic Vessel Pumping.

The lymphatic system extends its network of vessels throughout most of the body. Lymphatic vessels carry a fluid rich in proteins, immune cells, and long-chain fatty acids known as lymph. It results from an excess of interstitial tissue fluid collected from the periphery and transported centrally against hydrostatic pressure and protein concentration gradients. Thus, this one-way transport system is a key component in the maintenance of normal interstitial tissue fluid volume, protein concentration and fat metabolism, as well as the mounting of adequate immune responses as lymph passes through lymph nodes. In most cases, lymph is actively propelled via rhythmical phasic contractions through a succession of valve-bordered chambers constituting the lymphatic vessels. This contraction/relaxation cycle, or lymphatic pumping, is initiated in the smooth muscle cells present in the vessel wall by a pacemaker mechanism generating voltage-gated Ca2+ channel-induced action potentials. The action potentials provide the depolarization and Ca2+ influx essential for the engagement of the contractile machinery leading to the phasic constrictions of the lymphatic chambers and forward movement of lymph. The spontaneous lymphatic constrictions can be observed in isolated vessels in the absence of any external stimulation, while they are critically regulated by physical means, such as lymph-induced transmural pressure and flow rate, as well as diffusible molecules released from the lymphatic endothelium, perivascular nerve varicosities, blood and surrounding tissues/cells. In this chapter, we describe the latest findings which are improving our understanding of the mechanisms underlying spontaneous lymphatic pumping and discuss current theories about their physiological initiation.

Mesenteric Lymphatic Alterations Observed During DSS Induced Intestinal Inflammation Are Driven in a TLR4-PAMP/DAMP Discriminative Manner.

Background: Inflammatory bowel disease (IBD) is characterized by both acute and chronic phase inflammation of the gastro-intestinal (GI) tract that affect a large and growing number of people worldwide with little to no effective treatments. This is in part due to the lack of understanding of the disease pathogenesis and also the currently poorly described involvement of other systems such as the lymphatics. During DSS induced colitis, mice also develop a severe inflammation of terminal ileum with many features similar to IBD. As well as inflammation within the ileum we have previously demonstrated lymphatic remodeling within the mesentery and mesenteric lymph nodes of DSS-treated mice. The lymphatic remodeling includes lymphangiogenesis, lymphatic vessel dilation and leakiness, as well as cellular infiltration into the surrounding tissue and peripheral draining lymph nodes. Methods: Intestinal inflammation was induced in C57BL/6 mice by administration of 2.5% DSS in drinking water for 7 days. Mice were treated with TLR4 blocker C34 or Polymyxin-B (PMXB) daily from days 3 to 7 of DSS treatment via I.P. injection, and their therapeutic effects on disease activity and lymphatic function were examined. TLR activity and subsequent effect on lymphangiogenesis, lymphadenopathy, and mesenteric lymph node cellular composition were assessed. Results: DSS Mice treated with TLR4 inhibitor, C34, had a significantly improved disease phenotype characterized by reduced ileal and colonic insult. The change correlated with significant reduction in colonic and mesenteric inflammation, resolved mesenteric lymphangiectasia, and CD103+ DC migration similar to that of healthy control. PMXB treatment however did not resolve inflammation within the colon or associated mesenteric lymphatic dysfunction but did however prevent lymphadenopathy within the MLN through alteration of CCL21 gradients and CD103+ DC migration. Conclusions: TLR4 appears to mediate several changes within the mesenteric lymphatics, more specifically it is shown to have different outcomes whether stimulation occurs through pathogen derived factors such as LPS or tissue derived DAMPs, a novel phenomenon.

IL-1ß reduces cardiac lymphatic muscle contraction via COX-2 and PGE2 induction: Potential role in myocarditis.

The role of lymphatic vessels in myocarditis is largely unknown, while it has been shown to play a key role in other inflammatory diseases. We aimed to investigate the role of lymphatic vessels in myocarditis using in vivo model induced with Theiler's murine encephalomyelitis virus (TMEV) and in vitro model with rat cardiac lymphatic muscle cells (RCLMC). In the TMEV model, we found that upregulation of a set of inflammatory mediator genes, including interleukin (IL)-1ß, tumor necrosis factor (TNF)-αand COX-2 were associated with disease activity. Thus, using in vitro collagen gel contraction assays, we decided to clarify the role(s) of these mediators by testing contractility of RCLMC in response to IL-1ß and TNF-α individually and in combination, in the presence or absence of: IL-1 receptor antagonist (Anakinra); cyclooxygenase (COX) inhibitors inhibitors (TFAP, diclofenac and DuP-697). IL-1ß impaired RCLMC contractility dose-dependently, while co-incubation with both IL-1ß and TNF-α exhibited synergistic effects in decreasing RCLMC contractility with increased COX-2 expression. Anakinra maintained RCLMC contractility; Anakinra blocked the mobilization of COX-2 induced by IL-1ß with or without TNF-α. COX-2 inhibition blocked the IL-1ß-mediated decrease in RCLMC contractility. Mechanistically, we found that IL-1ß increased prostaglandin (PG) E2 release dose-dependently, while Anakinra blocked IL-1ß mediated PGE2 release. Using prostaglandin E receptor 4 (EP4) receptor antagonist, we demonstrated that EP4 receptor blockade maintained RCLMC contractility following IL-1ß exposure. Our results indicate that IL-1ß reduces RCLMC contractility via COX-2/PGE2 signaling with synergistic cooperation by TNF-α. These pathways may help provoke inflammatory mediator accumulation within the heart, driving progression from acute myocarditis into dilated cardiomyopathy.

Perinodal Adipose Tissue Participates in Immune Protection through a Lymphatic Vessel-Independent Route.

Lymphatic vessels remove and transport excess interstitial fluid to lymph nodes (LNs) for fluid balance and immune protection. LNs are typically surrounded by perinodal adipose tissue (PAT). However, PAT is a blood vessel-rich but lymphatic-rare tissue; therefore, how excess fluid in PAT is removed remains unclear. Using C57BL/6 mice, fluorescent dye tracing and transmission electron microscopy results suggest that fluid in PAT can travel to the LN via collagen I+ channels (PAT-LN conduits), merge into a collagen-rich space between the PAT and LN capsule (PAT-LN sinus), and may enter the LN via the LN capsule-associated conduits. This newly identified route of fluid flow allows fluid to enter the draining LN even when the afferent lymphatic vessels are blocked, indicating that fluid trafficking in PAT-LN conduits is not dependent on functional lymphatic vessels. Similar to lymphatic vessels, PAT-LN conduits can deliver Ags to the LN for immune protection. Additionally, Staphylococcus aureus from intradermal or i.v. infection may use PAT-LN conduits to infect PAT and stimulate PAT immune protection. Our studies revealed a new route of material exchange between PAT and the LN. Ag accumulation and bacterial infection in PAT demonstrate that PAT not only provides energy and regulatory factors, but can also directly participate in immune protection, indicating a new immune function of PAT for host immunity.

Acute small intestinal inflammation results in persistent lymphatic alterations.

Inflammatory bowel disease (IBD) has a complex pathophysiology with limited treatments. Structural and functional changes in the intestinal lymphatic system have been associated with the disease, with increased risk of IBD occurrence linked to a history of acute intestinal injury. To examine the potential role of the lymphatic system in inflammation recurrence, we evaluated morphological and functional changes in mouse mucosal and mesenteric lymphatic vessels, and within the mesenteric lymph nodes during acute ileitis caused by a 7-day treatment with dextran sodium sulfate (DSS). We monitored whether the changes persisted during a 14-day recovery period and determined their potential consequences on dendritic cell (DC) trafficking between the mucosa and lymphoid tissues. DSS administration was associated with marked lymphatic abnormalities and dysfunctions exemplified by lymphangiectasia and lymphangiogenesis in the ileal mucosa and mesentery, increased mesenteric lymphatic vessel leakage, and lymphadenopathy. Lymphangiogenesis and lymphadenopathy were still evident after recovery from intestinal inflammation and correlated with higher numbers of DCs in mucosal and lymphatic tissues. Specifically, a deficit in CD103+ DCs observed during acute DSS in the lamina propria was reversed and further enhanced during recovery. We concluded that an acute intestinal insult caused alterations of the mesenteric lymphatic system, including lymphangiogenesis, which persisted after resolution of inflammation. These morphological and functional changes could compromise DC function and movement, increasing susceptibility to further gastrointestinal disease. Elucidation of the changes in mesenteric and intestinal lymphatic function should offer key insights for new therapeutic strategies in gastrointestinal disorders such as IBD. NEW & NOTEWORTHY Lymphatic integrity plays a critical role in small intestinal homeostasis. Acute intestinal insult in a mouse model of acute ileitis causes morphological and functional changes in mesenteric and intestinal lymphatic vessels. While some of the changes significantly regressed during inflammation resolution, others persisted, including lymphangiogenesis and altered dendritic cell function and movement, potentially increasing susceptibility to the recurrence of gastrointestinal inflammation.

The pro-inflammatory cytokine TNF-α inhibits lymphatic pumping via activation of the NF-κB-iNOS signaling pathway.

OBJECTIVE: Mesenteric lymphatic vessel pumping, important to propel lymph and immune cells from the intestinal interstitium to the mesenteric lymph nodes, is compromised during intestinal inflammation. The objective of this study was to test the hypothesis that the pro-inflammatory cytokine TNF-α, is a significant contributor to the inflammation-induced lymphatic contractile dysfunction, and to determine its mode of action. METHODS: Contractile parameters were obtained from isolated rat mesenteric lymphatic vessels mounted on a pressure myograph after 24-hours incubation with or without TNF-α. Various inhibitors were administered, and quantitative real-time PCR, Western blotting, and immunofluorescence confocal imaging were applied to characterize the mechanisms involved in TNF-α actions. RESULTS: Vessel contraction frequency was significantly decreased after TNF-α treatment and could be restored by selective inhibition of NF-кB, iNOS, guanylate cyclase, and ATP-sensitive K+ channels. We further demonstrated that NF-кB inhibition also suppressed the significant increase in iNOS mRNA observed in TNF-α-treated lymphatic vessels and that TNF-α treatment favored the nuclear translocation of the p65 NF-κB subunit. CONCLUSIONS: These findings suggest that TNF-α decreases mesenteric lymphatic contractility by activating the NF-κB-iNOS signaling pathway. This mechanism could contribute to the alteration of lymphatic pumping reported in intestinal inflammation.

TNFΔARE Mice Display Abnormal Lymphatics and Develop Tertiary Lymphoid Organs in the Mesentery.

Chronic inflammatory diseases are associated with a persistent and enhanced response to environmental antigens. As an adaptive response to this exaggerated immune state, affected tissue typically develops tertiary lymphoid organs. Studies of Crohn disease (CD), a chronic inflammatory disease of the intestinal tract, report tertiary lymphoid organs present within the mucosal wall, along with other lymphatic diseases, such as lymphangiogenesis and obstructed lymphatic vessels. These observations suggest that downstream mesenteric lymphatic vessels and lymph drainage into mesenteric lymph nodes may be compromised. However, information is lacking on the morphologic features and functional status of mesenteric lymphatics in CD. Using confocal imaging, PCR, flow cytometry, and functional strategies, we addressed these questions in the established TNFΔARE mouse model of CD and found that this mouse model had many lymphatic abnormalities reminiscent of human CD. These abnormalities include intestinal lymphangiectasia, mesenteric lymph node lymphadenopathy, and lymphangiogenesis in both the mesentery and mucosa. Critically, TNFΔARE mice also present mesenteric tertiary lymphoid organs and have altered lymphatic transport of dendritic cells to mesenteric lymph nodes, two features likely to actively modulate immunity. Our findings provide key insights into lymphatic remodeling in the TNFΔARE mouse model. They shed light on the involvement of these lymphatic changes in immune dysfunctions observed in CD and suggest the lymphatic system as new target for therapeutic options.

Lipopolysaccharide modulates neutrophil recruitment and macrophage polarization on lymphatic vessels and impairs lymphatic function in rat mesentery.

Impairment of the lymphatic system is apparent in multiple inflammatory pathologies connected to elevated endotoxins such as LPS. However, the direct mechanisms by which LPS influences the lymphatic contractility are not well understood. We hypothesized that a dynamic modulation of innate immune cell populations in mesentery under inflammatory conditions perturbs tissue cytokine/chemokine homeostasis and subsequently influences lymphatic function. We used rats that were intraperitoneally injected with LPS (10 mg/kg) to determine the changes in the profiles of innate immune cells in the mesentery and in the stretch-mediated contractile responses of isolated lymphatic preparations. Results demonstrated a reduction in the phasic contractile activity of mesenteric lymphatic vessels from LPS-injected rats and a severe impairment of lymphatic pump function and flow. There was a significant reduction in the number of neutrophils and an increase in monocytes/macrophages present on the lymphatic vessels and in the clear mesentery of the LPS group. This population of monocytes and macrophages established a robust M2 phenotype, with the majority showing high expression of CD163 and CD206. Several cytokines and chemoattractants for neutrophils and macrophages were significantly changed in the mesentery of LPS-injected rats. Treatment of lymphatic muscle cells (LMCs) with LPS showed significant changes in the expression of adhesion molecules, VCAM1, ICAM1, CXCR2, and galectin-9. LPS-TLR4-mediated regulation of pAKT, pERK pI-κB, and pMLC20 in LMCs promoted both contractile and inflammatory pathways. Thus, our data provide the first evidence connecting the dynamic changes in innate immune cells on or near the lymphatics and complex cytokine milieu during inflammation with lymphatic dysfunction.

Colonic Insult Impairs Lymph Flow, Increases Cellular Content of the Lymph, Alters Local Lymphatic Microenvironment, and Leads to Sustained Inflammation in the Rat Ileum.

BACKGROUND: Lymphatic dysfunction has been linked to inflammation since the 1930s. Lymphatic function in the gut and mesentery is grossly underexplored in models of inflammatory bowel disease despite the use of lymphatic occlusion in early models of inflammatory bowel disease. Activation of the innate and adaptive immune system is a hallmark of TNBS-induced inflammation and is linked to disruption of the intrinsic lymph pump. Recent identification of crosstalk between lymphatic vessel resident immune cells and regulation of lymphatic vessel contractility underscore the importance of the timing of lymphatic dysfunction during tissue inflammation in response to TNBS. METHODS: To evaluate lymphatic function in TNBS induced inflammation, lymph was collected and flow measured from mesenteric lymphatics. Cellularity and cytokine profile of the lymph was also measured. Histopathology was performed to determine severity of injury and immunofluorescent staining of the mesentery was done to evaluate changes in the population of immune cells that reside near and on gastro-intestinal collecting lymphatics. RESULTS: Lymph transport fell 24 hours after TNBS administration and began recovering at 72 hours. Significant reduction of lymph flow preceded significant increase in histopathological score and occurred simultaneously with increased myeloperoxidase activity. These changes were preceded by increased MHCII cells surrounding mesenteric lymphatics leading to an altered lymphatic environment that would favor dysfunction. CONCLUSIONS: Alterations in environmental factors that effect lymphatic function occur before the development of gross GI inflammation. Reduced lymphatic function in TNBS-mediated inflammation is likely an early factor in the development of injury and that recovery of function is associated with resolution of inflammation.

Experimental ileitis alters prostaglandin biosynthesis in mesenteric lymphatic and blood vessels.

Prostaglandins are important mediators responsible for many changes that occur during the inflammatory response. Specifically, in inflammatory bowel disease (IBD), prostaglandins are key players in maintenance of blood flow and mucosal defense. In blood vessels, prostaglandins modulate and inhibit transmigration. In lymphatic vessels, on the other hand, prostaglandin E2 (PGE2) and prostacyclin (PGI2) have been shown to potently inhibit lymphatic contractility. Inhibition of lymphatic contractility could impair proper tissue fluid drainage during inflammation, consequently leading to the submucosal oedema observed in IBD. Alterations in production of PGE2 and PGI2 during inflammation could have severe implications on lymphatic and vascular functions within the small intestine. Using the 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced ileitis guinea pig and rat models, we assessed by quantitative PCR changes in mRNA transcript of enzymes and receptors involved in the production and actions of prostaglandins in mesenteric lymphatic and blood vessels as well as in the affected ileum. Furthermore, we also assessed lymphatic tissue levels of PGE2 and PGI2 during inflammation. We observed significant changes in lymphatic mRNA expression of COX-1, COX-2, MPGES-1, PGIS, EP4 and IP and increases in PGE2 and PGI2 in tissues of TNBS-treated animals. Changes in mRNA in blood vessels from TNBS-treated animals included differences in COX-1, COX-2, MPGES-1, PGIS, EP1, EP2 and IP expression. Prostaglandin metabolites are differentially regulated in both lymphatic and blood vessels during intestinal inflammation.

Distinct roles of L- and T-type voltage-dependent Ca2+ channels in regulation of lymphatic vessel contractile activity.

Lymph drainage maintains tissue fluid homeostasis and facilitates immune response. It is promoted by phasic contractions of collecting lymphatic vessels through which lymph is propelled back into the blood circulation. This rhythmic contractile activity (i.e. lymphatic pumping) increases in rate with increase in luminal pressure and relies on activation of nifedipine-sensitive voltage-dependent Ca(2+) channels (VDCCs). Despite their importance, these channels have not been characterized in lymphatic vessels. We used pressure- and wire-myography as well as intracellular microelectrode electrophysiology to characterize the pharmacological and electrophysiological properties of L-type and T-type VDCCs in rat mesenteric lymphatic vessels and evaluated their particular role in the regulation of lymphatic pumping by stretch. We complemented our study with PCR and confocal immunofluorescence imaging to investigate the expression and localization of these channels in lymphatic vessels. Our data suggest a delineating role of VDCCs in stretch-induced lymphatic vessel contractions, as the stretch-induced increase in force of lymphatic vessel contractions was significantly attenuated in the presence of L-type VDCC blockers nifedipine and diltiazem, while the stretch-induced increase in contraction frequency was significantly decreased by the T-type VDCC blockers mibefradil and nickel. The latter effect was correlated with a hyperpolarization. We propose that activation of T-type VDCCs depolarizes membrane potential, regulating the frequency of lymphatic contractions via opening of L-type VDCCs, which drive the strength of contractions.

Electrophysiological properties of rat mesenteric lymphatic vessels and their regulation by stretch.

BACKGROUND: In mammals, lymph is propelled centrally primarily via the phasic contractions of collecting lymphatic vessels, known as lymphatic pumping. Electrophysiological studies conducted in guinea pig and sheep mesenteric lymphatic vessels indicate that contractions are initiated in the lymphatic muscle by nifedipine-sensitive action potentials (APs). Lymphatic pumping is highly sensitive to luminal fluid loading and the mechanical properties of this stretch-induced pumping have been consistently studied, in particular in rat mesenteric lymphatic vessels. However, membrane potential (Vm) and the electrophysiological events underlying stretch-induced lymphatic pumping have not been investigated in the rat. The aim of this study was thus to examine the properties of rat mesenteric lymphatic muscle Vm under resting conditions and to assess changes in Vm caused by distension. METHODS AND RESULTS: Lymphatic muscle Vm was measured with sharp intracellular microelectrodes either in unstretched conditions or under isometric tension provided by a wire-myograph. In unstretched vessels, Vm was -48 ± 2 mV (n=30). APs (amplitude ∼25 mV) were observed at a frequency of ∼8/min and were abolished by nifedipine. Under isometric tension, Vm was less polarized (-36 ± 1 mV, n=23), even at minimum tension. Increase in tension led to increase in contraction strength and contraction/AP frequency, while Vm was slightly hyperpolarized and AP amplitude not markedly altered. CONCLUSIONS: In our experimental conditions, rat lymphatic muscle has electrophysiological characteristics similar to that in other species. It responds to an increase in isometric tension with an increase in AP frequency, but resting Vm is not significantly affected.