Endogenous Specialized Proresolving Mediator Profiles in a Novel Experimental Model of Lymphatic Obstruction and Intestinal Inflammation in African Green Monkeys
2019; Elsevier BV; Volume: 189; Issue: 10 Linguagem: Inglês
10.1016/j.ajpath.2019.05.013
ISSN1525-2191
AutoresFelix Becker, Emily Romero, Jason Goetzmann, Dana L. Hasselschwert, Beth K. Dray, John A. Vanchiere, Jane Fontenot, J. Winny Yun, Paul C. Norris, Luke A. White, Melany Musso, Charles N. Serhan, J. Steven Alexander, Felicity N. E. Gavins,
Tópico(s)Therapeutic Uses of Natural Elements
ResumoChanges in the intestinal lymphatic vascular system, such as lymphatic obstruction, are characteristic features of inflammatory bowel diseases. The lymphatic vasculature forms a conduit to enable resolution of inflammation; this process is driven by specialized endogenous proresolving mediators (SPMs). To evaluate contributions of lymphatic obstruction to intestinal inflammation and to study profiles of SPMs, we generated a novel animal model of lymphatic obstruction using African green monkeys. Follow-up studies were performed at 7, 21, and 61 days. Inflammation was determined by histology. Luminex assays were performed to evaluate chemokine and cytokine levels. In addition, lipid mediator metabololipidomic profiling was performed to identify SPMs. After 7 days, lymphatic obstruction resulted in a localized inflammatory state, paralleled by an increase in inflammatory chemokines and cytokines, which were found to be up-regulated after 7 days but returned to baseline after 21 and 61 days. At the same time, a distinct pattern of SPMs was profiled, with an increase for D-series resolvins, protectins, maresins, and lipoxins at 61 days. These results indicate that intestinal lymphatic obstruction can lead to an acute inflammatory state, accompanied by an increase in proinflammatory mediators, followed by a phase of resolution, paralleled by an increase and decrease of respective SPMs. Changes in the intestinal lymphatic vascular system, such as lymphatic obstruction, are characteristic features of inflammatory bowel diseases. The lymphatic vasculature forms a conduit to enable resolution of inflammation; this process is driven by specialized endogenous proresolving mediators (SPMs). To evaluate contributions of lymphatic obstruction to intestinal inflammation and to study profiles of SPMs, we generated a novel animal model of lymphatic obstruction using African green monkeys. Follow-up studies were performed at 7, 21, and 61 days. Inflammation was determined by histology. Luminex assays were performed to evaluate chemokine and cytokine levels. In addition, lipid mediator metabololipidomic profiling was performed to identify SPMs. After 7 days, lymphatic obstruction resulted in a localized inflammatory state, paralleled by an increase in inflammatory chemokines and cytokines, which were found to be up-regulated after 7 days but returned to baseline after 21 and 61 days. At the same time, a distinct pattern of SPMs was profiled, with an increase for D-series resolvins, protectins, maresins, and lipoxins at 61 days. These results indicate that intestinal lymphatic obstruction can lead to an acute inflammatory state, accompanied by an increase in proinflammatory mediators, followed by a phase of resolution, paralleled by an increase and decrease of respective SPMs. Inflammatory bowel diseases (IBDs), including its two main entities [Crohn disease (CD) and ulcerative colitis (UC)], represent chronic relapsing immune-mediated inflammatory disorders of the gastrointestinal tract. Despite a worldwide increase in prevalence and incidence, the exact etiology is still unknown. The current concept of IBD etiopathogenesis is that in genetically susceptible individuals, environmental factors and a dysregulated intestinal microbiome initiate an inappropriate inflammatory response by the host's mucosal immunity, leading to chronic intestinal inflammation.1Ananthakrishnan A.N. Epidemiology and risk factors for IBD.Nat Rev Gastroenterol Hepatol. 2015; 12: 205-217Crossref PubMed Scopus (883) Google Scholar This multifactorial hypothesis is based on the complex interplay of various immune and nonimmune cells within the mucosal immune system. Among these nonimmune components, intestinal lymphatic vessels have gained recent attention among the IBD research community, especially for CD.2Van Kruiningen H.J. Colombel J.F. The forgotten role of lymphangitis in Crohn's disease.Gut. 2008; 57: 1-4Crossref PubMed Scopus (103) Google Scholar The intestinal lymphatic vasculature is a unidirectional drainage system for interstitial fluids, lipids, small soluble antigens, and perivascularly infiltrated immune cells, organized into a network of capillaries and collecting vessels.3Bernier-Latmani J. Petrova T.V. Intestinal lymphatic vasculature: structure, mechanisms and functions.Nat Rev Gastroenterol Hepatol. 2017; 14: 510-526Crossref PubMed Scopus (111) Google Scholar, 4Alitalo K. The lymphatic vasculature in disease.Nat Med. 2011; 17: 1371-1380Crossref PubMed Scopus (665) Google Scholar The intestine contains lymphatic capillaries, whereas in the mesentery, only collecting vessels can be found, which run parallel with mesenteric arteries and veins.3Bernier-Latmani J. Petrova T.V. Intestinal lymphatic vasculature: structure, mechanisms and functions.Nat Rev Gastroenterol Hepatol. 2017; 14: 510-526Crossref PubMed Scopus (111) Google Scholar Studies in humans have revealed structural and functional alterations in the intestinal lymphatic vasculature in CD.5Becker F. Yi P. Al-Kofahi M. Ganta V.C. Morris J. Alexander J.S. Lymphatic dysregulation in intestinal inflammation: new insights into inflammatory bowel disease pathomechanisms.Lymphology. 2014; 47: 3-27PubMed Google Scholar Indeed, among the long recognized fundamental histopathologic hallmarks of CD are intralymphatic lymphocyte stasis, lymphangiectasia, and inflammatory granulomas resembling a chronic lymphocytic and granulomatous lymphangitis.2Van Kruiningen H.J. Colombel J.F. The forgotten role of lymphangitis in Crohn's disease.Gut. 2008; 57: 1-4Crossref PubMed Scopus (103) Google Scholar Using modern imaging techniques, a growing body of evidence points toward lymphatic expansion and altered lymphatic drainage in the small and large intestine of CD patients.6Geleff S. Schoppmann S.F. Oberhuber G. Increase in podoplanin-expressing intestinal lymphatic vessels in inflammatory bowel disease.Virchows Arch. 2003; 442: 231-237Crossref PubMed Scopus (70) Google Scholar, 7Rahier J.F. De Beauce S. Dubuquoy L. Erdual E. Colombel J.F. Jouret-Mourin A. Geboes K. Desreumaux P. Increased lymphatic vessel density and lymphangiogenesis in inflammatory bowel disease.Aliment Pharmacol Ther. 2011; 34: 533-543Crossref PubMed Scopus (74) Google Scholar, 8Pedica F. Ligorio C. Tonelli P. Bartolini S. Baccarini P. 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Lymphatic dysregulation in intestinal inflammation: new insights into inflammatory bowel disease pathomechanisms.Lymphology. 2014; 47: 3-27PubMed Google Scholar Blocking intestinal inflammation–associated lymphangiogenesis has been shown to result in reduced vessel density and decreased intestinal lymph flow, which subsequently causes an attenuated antigen clearance, all of which exacerbated experimental colitis.11Jurisic G. Sundberg J.P. Detmar M. Blockade of VEGF receptor-3 aggravates inflammatory bowel disease and lymphatic vessel enlargement.Inflamm Bowel Dis. 2013; 19: 1983-1989PubMed Google Scholar, 12D'Alessio S. Correale C. Tacconi C. Gandelli A. Pietrogrande G. Vetrano S. Genua M. Arena V. Spinelli A. Peyrin-Biroulet L. Fiocchi C. Danese S. VEGF-C-dependent stimulation of lymphatic function ameliorates experimental inflammatory bowel disease.J Clin Invest. 2014; 124: 3863-3878Crossref PubMed Scopus (156) Google Scholar, 13Sato H. Higashiyama M. Hozumi H. Sato S. Furuhashi H. 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Downregulation of FoxC2 increased susceptibility to experimental colitis: influence of lymphatic drainage function?.Inflamm Bowel Dis. 2015; 21: 1282-1296PubMed Google Scholar In agreement with this, promotion of intestinal inflammation–associated lymphangiogenesis via adenoviral induction of vascular endothelial growth factor-C improved lymph flow, immune cell drainage, and bacterial antigen clearance, which resulted in diminished intestinal inflammation.12D'Alessio S. Correale C. Tacconi C. Gandelli A. Pietrogrande G. Vetrano S. Genua M. Arena V. Spinelli A. Peyrin-Biroulet L. Fiocchi C. Danese S. VEGF-C-dependent stimulation of lymphatic function ameliorates experimental inflammatory bowel disease.J Clin Invest. 2014; 124: 3863-3878Crossref PubMed Scopus (156) Google Scholar This growing body of evidence from human and experimental data supports the role of lymphatics as an integral part of the intestinal immune-surveillance system by enabling efficient drainage to downstream lymph nodes of fluid, infiltrated immune cells, and antigen-presenting cells regulating immunity and subsequently promoting the resolution of inflammation. An acute inflammatory response is the host's physiological and protective answer to invading pathogens, which is self-limited and ends with resolution of inflammation and ultimately tissue homeostasis.16Serhan C.N. Pro-resolving lipid mediators are leads for resolution physiology.Nature. 2014; 510: 92-101Crossref PubMed Scopus (1832) Google Scholar However, if these well-controlled resolution pathways fail, the acute inflammatory response becomes self-sustaining and chronic. Although the intestinal lymphatic vasculature forms the conduit to end anti-inflammatory programs and enable resolution of inflammation, this dynamic process is driven by specialized endogenous proresolving mediators (SPMs). These include the lipoxins17Colgan S.P. Serhan C.N. Parkos C.A. Delp-Archer C. Madara J.L. Lipoxin A4 modulates transmigration of human neutrophils across intestinal epithelial monolayers.J Clin Invest. 1993; 92: 75-82Crossref PubMed Scopus (213) Google Scholar, 18Maddox J.F. Colgan S.P. Clish C.B. Petasis N.A. Fokin V.V. Serhan C.N. 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Resolution of inflammation: a new therapeutic frontier.Nat Rev Drug Discov. 2016; 15: 551-567Crossref PubMed Scopus (490) Google Scholar Resolution of inflammation is particularly important in the gut, where the mucosal surface and its immune system are in constant contact with pathogens and in an ongoing state of basal inflammation. To maintain a balance between physiological inflammatory responses and overwhelming destructive chronic inflammatory states, a meticulously orchestrated intestinal resolution program is mandatory. It has now been demonstrated that IBD patients are characterized by an imbalance between proinflammatory agents and proresolving molecules within the mucosal immune system. Masoodi et al26Masoodi M. Pearl D.S. Eiden M. Shute J.K. Brown J.F. Calder P.C. Trebble T.M. 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In addition, issues still persist with animal models of intestinal inflammation as these current models most closely resemble clinical and histopathologic features of acute colitis, rather than CD. In the 1970s, Kalima et al33Kalima T.V. Experimental lymphatic obstruction in the ileum.Ann Chir Gynaecol Fenn. 1970; 59: 187-201PubMed Google Scholar described an animal model of intestinal inflammation based on regional lymphatic obstruction, which closely resembled anatomic and clinical features of CD in humans. Although lymphatic obstruction alone does not reproduce the entire spectrum of CD pathology, it reproduces striking inflammatory similarities and many clinical features, including submucosal edema, thickening of the bowel wall, aggregation of lymphocytes, formation of lymphocytic and granulomatous changes, and the development of enteroenteric and enterocutaneous fistulae.33Kalima T.V. Experimental lymphatic obstruction in the ileum.Ann Chir Gynaecol Fenn. 1970; 59: 187-201PubMed Google Scholar, 34Kalima T.V. Collan Y. Intestinal villus in experimental lymphatic obstruction: correlation of light and electron microscopic findings with clinical diseases.Scand J Gastroenterol. 1970; 5: 497-510PubMed Google Scholar, 35Kalima T.V. Saloniemi H. Rahko T. Experimental regional enteritis in pigs.Scand J Gastroenterol. 1976; 11: 353-362Crossref PubMed Google Scholar To date, no available rodent animal model has ever shown such clinical similarities to CD and, in addition, there is no animal model currently available to study intestinal inflammation, which is based on lymphatic obstruction. Herein, we recapitulated and further characterized the inflammatory model of regional lymphatic obstruction in nonhuman primates (NHPs), with a special focus on intestinal lymph flow, inflammatory markers, and mediators of resolution. A clinically relevant variation of the model of Kalima et al33Kalima T.V. Experimental lymphatic obstruction in the ileum.Ann Chir Gynaecol Fenn. 1970; 59: 187-201PubMed Google Scholar was recapitulated in NHPs with persistent blockade of regional lymph flow over a time course of 61 days. A severe inflammatory flare was observed, characterized by local and systemic up-regulation of inflammatory markers within the first 7 days, with a return to physiological homeostasis after 21 and 61 days, respectively. Using modern multipanel Luminex assays (Bio-Rad, Hercules, CA) and solid-phase extraction, coupled with LM-SPM liquid chromatography–tandem mass spectrometry (LC-MS/MS)–based metabololipidomics, small-bowel and colon samples were analyzed to identify unique signature profiles and relationship(s) between proinflammatory and anti-inflammatory cytokines, chemokines, and LMs and SPMs to assess their potential pathologic implications in the intestine of animals subjected to lymphatic blockade. All animal protocols were approved by the University of Louisiana at Lafayette Animal Care and Use Committee and were handled in accordance with the American Association for Accreditation of Laboratory Animal Care guidelines. Fifteen male African green monkeys (AGMs; Chlorocebus aethiops sabaeus; 2 to 4 years of age; weighing between 3.19 and 5.36 kg) were used and maintained at the New Iberia Research Center, University of Louisiana at Lafayette. Animals were socially housed during this study, except for a 1-week interval of separation postoperatively. Animals were fed daily with regular primate diet (5L2P*; Purina Lab, St. Louis, MO) in amounts appropriate for the size of the animals, which was supplemented with fruit, novel foodstuffs, and/or seed foraging at least five times as part of the New Iberia Research Center Plan for Environmental Enhancement and Behavioral Management. Tap water was provided ad libitum via automatic watering devices. Preoperatively, animals were fasted overnight with free access to water. Postoperatively, animals had free access to food and water ad libitum. Before the experiments, animals were screened for exclusion behavior (abnormal food/water consumption or stool consistency) to avoid an underlying gut-related wasting disease or infectious process. This included a physical examination by a veterinarian (E.R or D.L.H.), a complete blood cell count, a serum chemistry screen, and fecal testing for bacterial and parasitic pathogens. All animals were tested for SIV as well as simian T-cell leukemia virus type 1 and were found to be negative before inclusion in the experiments. Animals were randomized to either a treatment or a sham group. To evaluate the course of intestinal injury after lymphatic obstruction, 15 animals were randomly assigned into three different experimental groups and one sham group. These groups were based on the observational period after the procedure: 7 (acute), 21 (midterm), or 61 (chronic) days. These time points were selected on the basis of earlier studies by Kalima et al,33Kalima T.V. Experimental lymphatic obstruction in the ileum.Ann Chir Gynaecol Fenn. 1970; 59: 187-201PubMed Google Scholar, 35Kalima T.V. Saloniemi H. Rahko T. Experimental regional enteritis in pigs.Scand J Gastroenterol. 1976; 11: 353-362Crossref PubMed Google Scholar who reported lymphatic obstruction as a specific initiator of intestinal disease affecting multiple clinical parameters, which intensified over time. Each experimental group consisted of four animals. In addition, three animals were used as a control (sham), with one animal being sacrificed at 7, 21, and 61 days. Animals were sedated by i.m. injection of 10 mg/kg ketamine and 0.03 mg/kg buprenorphine, and a venous catheter was placed in a peripheral vein. Next, 5 mg/kg propofol was given intravenously, followed by endotracheal intubation. Anesthesia was maintained on isoflurane and propofol (5 mg/kg). Animals were placed in a supine position. The abdomen was shaved, and all the following surgical procedures were performed under sterile conditions. Briefly, a median laparotomy (approximately 3 inches) was performed, followed by an abdominal exploration to avoid congenital intra-abdominal findings. Next, the ileocecal region was located, and the last 20 cm of ileum and the first 10 cm of the cecum/ascending colon were measured and mobilized. The next step was to identify the lymphatic drainage route of the ileocecal region. Thus, multiple ileal and cecal subserosal injections of approximately 300 μL isosulfan blue (1%, Lymphazurin; Covidien, New Haven, CT) were administered to specifically stain the lymphatic vessels and the respective draining lymph nodes. This procedure rapidly ( 2 minutes) the downstream proximal lymph nodes (Figure 1A). Next, 1% isosulfan blue was injected in all stained lymph nodes to identify bigger downstream central lymph nodes at the mesenteric radix (Figure 1B). The so identified drainage route of the ileocecal region concordantly consisted regularly of one major central mesenteric lymph node, which represents the proximal downstream target for two to three ileal and three to five smaller colonic lymph nodes. Next, one to two central lymphatic vessels, which drained only from the major previously identified central mesenteric lymph node at the mesenteric radix into the cisterna chyli, were isolated. The proximal mesentery was then carefully opened, and the central lymphatic vessels at the base of the mesentery were ligated with nonreabsorbable sutures (6-0), avoiding manipulation of ileal/ileocolic arteries and veins (Figure 1C). To complete the lymphatic obstruction, 0.01 to 0.02 mL of 4% formalin (mixed with 1% isosulfan blue) was injected into the previously identified lymph nodes, again avoiding damage to lymph node capsules or manipulation of the ileal arteries and veins. Finally, the lymphatic obstruction was verified by reinjecting 1% isosulfan blue into the ileal and cecal subserosa. At this stage, a stasis of the blue dye in the mesenteric lymphatics and a complete reuptake in the proximal lymph nodes, without any leakage or outflow in the cisterna chyli, occurred. Afterward, the abdomen was closed in two layers using a continuous suture (muscle layer) and an interrupted suture technique (skin). Sham operations were performed under the exact same conditions, but only included opening of the abdominal wall, exploration, subserosal injection of 1% isosulfan blue, and closure of the abdominal wall. Postoperatively, no abdominal drain was placed and no postoperative antibiotics or nonsteroidal anti-inflammatory drugs were given. The postoperative analgesic regimen included acetaminophen (6 mg/kg, oral suspens
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