Charlotte P. Peters

Functional Differences between Human NKp44− and NKp44+ RORC+ Innate Lymphoid Cells

Human RORC+ lymphoid tissue inducer cells are part of a rapidly expanding family of innate lymphoid cells (ILC) that participate in innate and adaptive immune responses as well as in lymphoid tissue (re) modeling. The assessment of a potential role for innate lymphocyte-derived cytokines in human homeostasis and disease is hampered by a poor characterization of RORC+ innate cell subsets and a lack of knowledge on the distribution of these cells in adults. Here we show that functionally distinct subsets of human RORC+ innate lymphoid cells are enriched for secretion of IL-17a or IL-22. Both subsets have an activated phenotype and can be distinguished based on the presence or absence of the natural cytotoxicity receptor NKp44. NKp44+ IL-22 producing cells are present in tonsils while NKp44− IL-17a producing cells are present in fetal developing lymph nodes. Development of human intestinal NKp44+ ILC is a programmed event that is independent of bacterial colonization and these cells colonize the fetal intestine during the first trimester. In the adult intestine, NKp44+ ILC are the main ILC subset producing IL-22. NKp44− ILC remain present throughout adulthood in peripheral non-inflamed lymph nodes as resting, non-cytokine producing cells. However, upon stimulation lymph node ILC can swiftly initiate cytokine transcription suggesting that secondary human lymphoid organs may function as a reservoir for innate lymphoid cells capable of participating in inflammatory responses.

Transcriptional control of innate lymphoid cells.

Cells that belong to the family of innate lymphoid cells (ILCs) not only form a first line of defense against invading microbes, but also play essential roles in tissue remodeling and immune pathology. Rorγt+ ILCs, producing the cytokines IL‐22 and IL‐17, include lymphoid tissue inducer (LTi) cells which are critical for the formation of lymphoid structures. Recently another ILC subset has been identified, which is dependent on RORα for its development and is dedicated to the production of the Th2 cytokines IL‐5 and IL‐13. These ILCs have been termed type 2 ILCs. All ILC subets are considered to belong to the same family that also includes natural killer cells because they all rely on the common γ‐chain (γc) of the IL‐2 receptor for their development and function, share a lymphoid morphology and depend on the transcriptional repressor Id2 for their development. Other transcription factors, including Notch, and the aryl hydrocarbon receptor (AhR) in RORγt+ ILCs and GATA3 in type 2 ILCs, also play roles in the development, survival, and function of these ILC subpopulations. Here we review the current knowledge with regard to the transcription factors involved in the development and functions of ILCs.

Effects of infliximab retreatment after consecutive discontinuation of infliximab and adalimumab in refractory Crohn's disease.

Background:Switches between anti–tumor necrosis factor agents in the treatment of Crohns disease (CD) occur in case of treatment failure, intolerance, or patient preference. No data are currently available on the usefulness of a second infliximab treatment after earlier discontinuation and previous switch to an alternative anti–tumor necrosis factor agent. In this study, we evaluated the clinical benefit of infliximab retreatment in patients with CD after sequential use of both infliximab and adalimumab. Methods:Twenty-nine patients with CD who had received earlier treatments with sequential infliximab and adalimumab and were then restarted on infliximab were retrieved from a multicenter registry designed for the follow-up of adalimumab treatment for CD. Short-term and sustained effects of infliximab retreatment were evaluated retrospectively by reviewing clinical records. Follow-up was 18 months for all patients. Results:In 13/29 (45%) patients, infliximab was reintroduced at intensified dosing schedule (>5 mg/kg or <8 wk) for 23/29 (79%) of patients similar to the schedule who were on at time of previous discontinuation. During the second infliximab treatment course, dosing was further intensified in 11 out of 29 (38%) patients. After 18 months 18/29 (62%), patients were still on continued therapy of their second infliximab treatment. Infliximab was discontinued (after a median of 7 mo) in 11 out of 29 patients for loss of response (n = 7 [24%]), intolerance (n = 3 [10%]), or non-compliance (n = 1 [3%]). Use of induction schedule or concomitant immunomodulators were not significantly associated with treatment benefit. Conclusions:The majority of patients with CD benefit from a second treatment with infliximab after previous treatment with infliximab and adalimumab, which offer a meaningful therapeutic option in often highly refractory patients.

ATG16L1 and NOD2 polymorphisms enhance phagocytosis in monocytes of Crohn's disease patients

AIM To investigate if the presence of relevant genetic polymorphisms has effect on the effectual clearance of bacteria by monocytes and granulocytes in patients with Crohns disease (CD). METHODS In this study, we assessed the differential responses in phagocytosis by measuring the phagocytic activity and the percentage of active phagocytic monocytes and granulocytes in inflammatory bowel disease patients as well as healthy controls. As both autophagy related like 1 (ATG16L1) and immunity-related guanosine triphosphatase gene are autophagy genes associated with CD and more recently nucleotide-binding ligomerization domain-containing protein 2 (NOD2) has been identified as a potent inducer of autophagy we genotyped the patients for these variants and correlated this to the phagocytic reaction. The genotyping was done with restriction fragment length polymorphisms analysis and the phagocytosis was determined with the pHrodo™ Escherichia coli Bioparticles Phagocytosis kit for flowcytometry. RESULTS In this study, we demonstrate that analysis of the monocyte and granulocyte populations of patients with CD and ulcerative colitis showed a comparable phagocytic activity (ratio of mean fluorescence intensity) between the patient groups and the healthy controls. CD patients show a significantly higher phagocytic capacity (ratio mean percentage of phagocytic cells) compared to healthy controls (51.91% ± 2.85% vs 37.67% ± 7.06%, P = 0.05). The extend of disease was not of influence. However, variants of ATG16L1 (WT: 2.03 ± 0.19 vs homozygoot variant: 4.38 ± 0.37, P < 0.009) as well as NOD2 (C-ins) (heterozygous variant: 42.08 ± 2.94 vs homozygous variant: 75.58 ± 4.34 (P = 0.05) are associated with the phagocytic activity in patients with CD. CONCLUSION Monocytes of CD patients show enhanced phagocytosis associated with the presence of ATG16L1 and NOD2 variants. This could be part of the pathophysiological mechanism resulting in the disease.

Anti-Inflammatory Effects of Urocanic Acid Derivatives in Models Ex Vivo and In Vivo of Inflammatory Bowel Disease

Urocanic acid (UCA) derivatives were tested for their anti-inflammatory activity in inflammatory bowel disease (IBD) in two models: ex vivo and an experimental mouse model. Ex vivo: inflamed colonic tissue was incubated in culture medium with or without the UCA derivatives. Biopsies, incubated with UCA derivatives, produced lower levels of proinflammatory cytokines IL-6 and IL-8 as compared to control biopsies. The same compounds also showed increased levels of IL-10, providing an additional indication for anti-inflammatory properties. In vivo: a combination of two imidazoles and a combination of two of their ethyl esters were administered to mice while colitis was induced by oral administration of dextran sodium sulfate (DSS). Some parameters did not show conclusive effects, but the imidazoles and their ethyl esters reduced the area of inflammation and the number of infiltrating neutrophils. Fibrosis and the sum of all histological aspects were reduced by the imidazoles, whereas the ethyl esters reduced the colon weight to length ratio. These results suggest that the UCA derivatives have anti-inflammatory effect on IBD. In addition, fine tuning of the ex vivo model may provide an elegant way to predict anti-inflammatory effects of potential drugs in humans, which may decrease the need for animal experiments.

Mo1824 miR-511-3p, Embedded in the Macrophage Mannose Receptor Gene, Contributes to Experimental Colitis

Approximately 40% of inflammatory bowel diseases (IBD) are associated with thromboembolic events causing significant rate of morbidity and mortality. These extra intestinal events of IBD are initiated by the qualitative and quantitative abnormalities in both coagulation and fibrinolytic factors producing states of hypercoagulability. Nevertheless, the interrelationship between IBD pathogenesis and hypercoagulability condition is not well understood (Figure 1). Here we examine the role of a coagulable state triggered by impaired fibrinolysis in the development of chronic colonic inflammation. Plasminogen (Plg) is the primary fibrinolytic enzyme, becomes activated to serine protease plasmin to dissolve the fibrin clot and to counterbalance hypercoagulability. The rapidity of IBD is caused by combining the effects of genetic and environmental factors. Our data show that when fed with a high fat diet (HFD, 45% milk fat), mouse knockout of Plg (Plg-/mice) developed continuous lesions in the colon with multiple inflammatory polyps (as of 8 weeks, n=15) bearing ulcers. This pathophysiology was not observed in HFD fed wild type (Plg+/+) mice and standard chow diet fed Plg+/+ and Plg-/mice. The increased inflammatory cell infiltration in the colon of HFD fed Plg-/mice was corroborated with increased levels of pro-inflammatory cytokines (IL-6, IL-17 & TNF alpha). In addition, HFD-fed Plg-/mice expressed reduced levels of colonic epithelial tight junctions (Claudin1, Occludin and ZO1), a finding compatible with an impaired epithelial barrier function, compared to Plg+/+ mice. Moreover, HFD fed Plg-/mice showed increased levels of plasma endotoxin, an indicator of impaired intestinal barrier function, compared with HFD-fed Plg+/+ mice. All these pathologies of HFD fed Plg-/mice were associated with excessive fibrin deposition in both vascular and extravascular space in the colon compared with HFD fed Plg+/+ mice providing a link between fibrin deposition and inflammation. Altogether, our present data imply that fibrinolytic pathway plays a crucial role in colon homeostasis and defects in this pathway can cause impaired colon barrier function and sustained colonic inflammation. In depth exploration of this pathway will provide a clue towards the potential role of Plg in colon homeostasis. This study will also challenge current paradigms on the relationship between coagulation abnormalities and IBD, which presume that these abnormalities are a consequence rather than a cause of the disease.