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T-bet is the lineage-specifying transcription factor for CD4+ T helper type 1 (TH 1) cells. T-bet has also been found in other CD4+ T cell subsets, including TH 17 cells and Treg, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell differentiation and function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells that have naïve cell surface markers and a naïve cell transcriptional profile and that this novel cell population is phenotypically and functionally distinct from previously described populations of naïve and memory CD4+ T cells. Naïve-like T-bet-experienced cells are polarised to the TH 1 lineage, predisposed to produce IFNγ upon cell activation, and resist repolarisation to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarise T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T helper response. This article is protected by copyright. All rights reserved.

Introduction: Drug repurposing can be a successful approach to deal with the scarcity of cost-effective therapies in situations such as the COVID-19 pandemic. Tetracyclines have previously shown efficacy in preclinical acute respiratory distress syndrome (ARDS) models and initial predictions and experimental reports suggest a direct antiviral activity against SARS-CoV2. Furthermore, a few clinical reports indicate their potential in COVID-19 patients. In addition to the scarcity and limitations of the scientific evidence, the effectiveness of tetracyclines in experimental ARDS has been proven extensively, counteracting the overt inflammatory reaction and fibrosis sequelae due to a synergic combination of pharmacological activities. Areas covered: This paper discusses the scientific evidence behind the application of tetracyclines for ARDS/COVID-19. Expert Opinion: The benefits of their multi-target pharmacology and their safety profile overcome the limitations, such as antibiotic activity and low commercial interest. Immunomodulatory tetracyclines and novel chemically modified non-antibiotic tetracyclines have therapeutic potential. Further drug repurposing studies in ARDS and severe COVID-19 are necessary.

Innate lymphoid cells (ILC) play a critical role in regulating immune responses at mucosal surfaces. Various subsets exist resembling T cell lineages defined by the expression of specific transcription factors. Thus, T-bet is expressed in ILC1 and Th1 cells. In order to further understand the functional roles of T-bet in ILC, we generated a fate-mapping mouse model that permanently marks cells and their progeny that are expressing, or have ever expressed T-bet. Here we have identified and characterised a novel ILC with characteristics of ILC1 and ILC2 that are “fate-mapped” for T-bet expression and arise early in neonatal life prior to establishment of a mature microbiome. These ILC1-ILC2 cells are critically dependent on T-bet and are able to express type 1 and type 2 cytokines at steady state, but not in the context of inflammation. These findings refine our understanding of ILC lineage regulation and stability and have important implications for the understanding of ILC biology at mucosal surfaces.SUMMARYInnate lymphoid cells (ILC) play a critical role in regulating immune responses at mucosal surfaces. Three distinct ILC groups have been described according to expression of subset defining transcription factors and other markers. In this study we characterize a novel ILC subset with characteristics of group 1 and group 2 ILC in vivo.

T-bet is the lineage-specifying transcription factor for CD4+ T helper type 1 (TH1) cells. T-bet has also been found in other CD4+ T cell subsets, including TH17 cells and TREG, where it modulates their functional characteristics. However, we lack information on when and where T-bet is expressed during T cell differentiation and how this impacts T cell function. To address this, we traced the ontogeny of T-bet-expressing cells using a fluorescent fate-mapping mouse line. We demonstrate that T-bet is expressed in a subset of CD4+ T cells with naïve cell surface markers and that this novel cell population is phenotypically and functionally distinct from conventional naïve CD4+ T cells. These cells are also distinct from previously described populations of memory phenotype and stem cell-like T cells. Naïve-like T-bet-experienced cells are polarised to the TH1 lineage, predisposed to produce IFNγ upon cell activation, and resist repolarisation to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can function to polarise T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T helper response.

Background and Purpose: Immunomodulatory tetracyclines are well-characterised drugs with a pharmacological potential beyond their antibiotic properties. Particulaarly, minocycline and doxycycicline have shown beneficial effects in experimental colitis, although pro-inflammatory actions have also been described in macrophages. Therefore, we aimed to characterise the mechanism behind their effect in acute intestinal inflammation. Experimental Approach: A comparative pharmacological study was first used to elucidate teh most relevant actions of immunomodulatory tetracyclines: doxycycline, minocycline, tigecycline and other antibiotic or immunomodulatory drugs were assessed in bone-marrow derived macrophages and in DSS-induced mouse colitis, where different barrier markers, inflammatory mediators, microRNAs, TLRs, and the gut microbiota composition were evaluated. Then, the sequential immune events that mediate the intestinal anti-inflammatory effect of minocycline in DSS-colitis were characterised. Key Results: We have identified a novel immunomodulatory activity of tetracyclines, potentiating the innate immune response and leading to an enhanced resolution of inflammation. This is also the first report describing the intestinal anti-inflammatory effect of tigecycline. A minor therapeutic benefit seems to derive from their antibiotic properties. Conversely, immunomodulatory tetracyclines potentiate macrophage cytokine release in vitro and, while improving mucosal recovery in colitic mice, they up-regulate Ccl2, miR-142, miR-375 and Tlr4. In particular, minocycline initially enhances IL-1β, IL-6, IL-22, GM-CSF and IL-4 colonic production and monocyte recruitment to the intestine, subsequently increasing Ly6C−MHCII+ macrophages, Tregs and type-2 intestinal immune responses. Conclusion and Implications: Immunomodulatory tetracyclines potentiate protective immune pathways leading to mucosal healing and resolution, representing a promising drug reposition strategy for the treatment of intestinal inflammation.

Objective: The use of immunomodulatory antibiotics to simultaneously target different factors involved in intestinal inflammatory conditions is an interesting but understudied pharmacological strategy. A great therapeutic potential has been obtained with minocycline and doxycycline in experimental colitis. Therefore, understanding the contribution of the different activities of immunomodulatory tetracyclines is crucial for the improvement and translation of their use into clinic. Design: A comparative pharmacological study including tetracyclines and other antibiotic or immunomodulatory drugs was performed in 2,4-dinitrobenzene sulfonic acid (DNBS)-induced colitis in mice. The correlation between the therapeutic efficacy of each drug and changes in the gut microbiota composition, markers of barrier integrity, inflammatory mediators, microRNAs and TLRs was analysed to identify the main mechanisms of action. Results: Tetracyclines counteracted most of the markers found altered in DNBS-colitis, which differed from effects of corticosteroid treatment. Of note, administration of tetracyclines led to increased mucosal protection, associated with up-regulated expression of CCL2, miR-142 and miR-375. All drugs with antibiotic activity ameliorated the progression of inflammation and reduced neutrophil-related genes, such as miR-223, despite their effects were not associated with restored intestinal dysbiosis. However, reduced bacterial richness was correlated with increased expression of TLR2 and TLR9 in antibiotic-treated groups and TLR6 was also up-regulated by the immunomodulatory tetracyclines with higher efficacy (doxycycline, minocycline and tigecycline). Conclusion: The anti-inflammatory effect of tetracyclines involves specific modifications in TLR and microRNA expression leading to an improved microbial-derived signalling and mucosal protection. These results support the potential of immunomodulatory tetracyclines to prevent inflammation-associated tissue damage in acute intestinal inflammation.

Alternative herbal therapy is increasingly used for inflammatory bowel disease (IBD), but it is essential to prove its safety and efficacy. We have assayed the anti-inflammatory effects of Amanda®, a standardized water extract of Spanish Marcona almond skin very rich in polyphenols, in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis. Amanda® reduced the inflamed/ulcerated colonic area and preserved the mucosal architecture. The extract decreased the inflammatory infiltrate and the myeloperoxidase activity, as well as improved the oxidative state by increasing the glutathione content. Amanda® also modulated the expression of pro-inflammatory cytokines and inducible nitric oxide synthase. Besides, Amanda® improved the epithelial barrier function by restoring the levels of villin and mucin MUC3. Thus, the data from this work prove that Amanda® extract possesses therapeutic activity in TNBS-induced colitis in rats, targeting different pathological factors of IBD like oxidative stress, neutrophil mucosal infiltration and loss of intestinal epithelial barrier function.

Minocycline is a second-generation, semi-synthetic tetracycline that has been in therapeutic use for over 30 years because of its antibiotic properties against both gram-positive and gram-negative bacteria. It is mainly used in the treatment of acne vulgaris and some sexually transmitted diseases. Recently, it has been reported that tetracyclines can exert a variety of biological actions that are independent of their anti-microbial activity, including anti-inflammatory and anti-apoptotic activities, and inhibition of proteolysis, angiogenesis and tumour metastasis. These findings specifically concern to minocycline as it has recently been found to have multiple non-antibiotic biological effects that are beneficial in experimental models of various diseases with an inflammatory basis, including dermatitis, periodontitis, atherosclerosis and autoimmune disorders such as rheumatoid arthritis and inflammatory bowel disease. Of note, minocycline has also emerged as the most effective tetracycline derivative at providing neuroprotection. This effect has been confirmed in experimental models of ischaemia, traumatic brain injury and neuropathic pain, and of several neurodegenerative conditions including Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Alzheimer's disease, multiple sclerosis and spinal cord injury. Moreover, other pre-clinical studies have shown its ability to inhibit malignant cell growth and activation and replication of human immunodeficiency virus, and to prevent bone resorption. Considering the above-mentioned findings, this review will cover the most important topics in the pharmacology of minocycline to date, supporting its evaluation as a new therapeutic approach for many of the diseases described herein.

BACKGROUND AND PURPOSE Dersalazine sodium (DS) is a new chemical entity formed by combining, through an azo bond, a potent platelet activating factor (PAF) antagonist (UR-12715) with 5-aminosalicylic acid (5-ASA). DS has been demonstrated to have anti-inflammatory effects on trinitrobenzene sulphonic acid (TNBS)-induced colitis in rats and recently in UC patients in phase II PoC. There is Increasing evidence that Th17 cells have an important role in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to further characterize the anti-inflammatory effects of DS. EXPERIMENTAL APPROACH Effect of DS (10 or 30 mg·kg−1 b.i.d.) on TNBS-induced colitis in rats was studied after 2 and 7 days with special focus on inflammatory mediators. Additionally, its anti-inflammatory properties were analysed in two different models of dextran sodium sulphate (DSS)-induced colitis, BALB/c and C57BL/6 mice, the latter being dependent on IL-17. KEY RESULTS DS, when administered for 7 days, showed intestinal anti-inflammatory effects in TNBS-induced colitis; these effects were observed both macroscopically and through the profile of inflammatory mediators (TNF, IL-1β, IL-6 and IL-17). Although the 2 day treatment with DS did not induce intestinal anti-inflammatory effects, it was sufficient to reduce the enhanced IL-17 expression. DS showed beneficial effects on DSS-induced colitis in C57BL/6 mice and reduced colonic pro-inflammatory cytokines IL-1β, IL-6 and IL-17. In contrast, it did not exert intestinal anti-inflammatory effects on DSS-induced colitis in BALB/c mice. CONCLUSIONS AND IMPLICATIONS DS exerts intestinal anti-inflammatory activity in different rodent models of colitis through down-regulation of IL-17 expression.

Purpose We aimed to evaluate the intestinal anti-inflammatory properties of silk fibroin nanoparticles, around 100 nm in size, when loaded with the stilbene compound resveratrol, in an experimental model of rat colitis. Methods Nanoparticles were loaded with resveratrol by adsorption. The biological effects of the resveratrol-loaded nanoparticles were tested both in vitro, in a cell culture of RAW 264.7 cells (mouse macrophages), and in vivo, in the trinitrobenzenesulfonic acid model of rat colitis, when administered intracolonically. Results The resveratrol liberation in 1× phosphate-buffered saline (PBS; pH 7.4) was characterized by fast liberation, reaching the solubility limit in 3 hours, which was maintained over a period of 80 hours. The in vitro assays revealed immunomodulatory properties exerted by these resveratrol-loaded nanoparticles since they promoted macrophage activity in basal conditions and inhibited this activity when stimulated with lipopolysaccharide. The in vivo experiments showed that after evaluation of the macroscopic symptoms, inflammatory markers, and intestinal barrier function, the fibroin nanoparticles loaded with resveratrol had a better effect than the single treatments, being similar to that produced by the glucocorticoid dexamethasone. Conclusion Silk fibroin nanoparticles constitute an attractive strategy for the controlled release of resveratrol, showing immunomodulatory properties and intestinal anti-inflammatory effects.

Intestinal microbiota modulation is becoming an interesting approach to manage inflammatory bowel disease and can be achieved by the administration of prebiotics. Previous studies showed the intestinal anti-inflammatory effects of the prebiotic lactulose. The aim of the present study was to test the preventative effects of oligosaccharides derived from lactulose with prebiotic properties (OsLu) in the trinitrobenzenesulfonic acid model of rat colitis and compare them with those of lactulose. Both treatments modified bacterial profile in intestinal contents, increasing the bifidobacteria and lactobacilli counts and up-regulating the production of short-chain fatty acids, although OsLu generated a larger amount. OsLu also inhibited to a greater extent different pro-inflammatory markers such as interleukins (IL) 1, 6, 12, and 23 and chemokines (MCP-1 and CINC-1). However, both prebiotics equally restored colonic epithelial integrity, evaluated both with a histological score (OsLu, 9.8 ± 2.2; and lactulose, 12.1 ± 2.1, vs colitic control, 27.3 ± 3.3) and by measuring several key proteins of the mucosal barrier (MUC-2, MUC-3, and TTF-3). OsLu effect was also associated with an inhibition of iNOS expression and a reduction of Th17 cell activity in the inflamed tissue that facilitated the intestinal mucosa barrier recovery. In conclusion, OsLu showed a better anti-inflammatory profile than lactulose in this model of experimental colitis. © 2014 American Chemical Society., This work was supported by the Spanish Ministry of Economy and Competitivity (SAF2011-29648), by Consolider-Ingenio 2010: FUNC-C-FOOD CSD 2007-00063, and by Junta de Andalucia (AGR-6826 and CTS 164) with funds from the European Union. F.A. is a predoctoral fellow of the Junta de Andalucia; J.G.-M. is a predoctoral fellow of the Spanish Ministry of Education and Science; N.G.-M. is a postdoctoral fellow of the Ramon Areces Foundation; M.E.R.-C. is a postdoctoral fellow of CIBEREHD. CIBEREHD is funded by the Instituto de Salud Carlos III.

Introduction: Nowadays, there is an increasing interest for alternative options in the treatment of inflammatory bowel diseases (IBDs) that combine efficacy and an adequate safety profile. Methods: The intestinal anti-inflammatory effects of Serpylli herba, the officinal drug in the European Pharmacopeia composed by the aerial parts of wild thyme (Thymus serpyllum), were evaluated in the trinitrobenzenesulfonic acid (TNBS)-induced rat colitis and dextran sodium sulfate (DSS)-induced mouse colitis, which are well characterized experimental models with some resemblance to human IBD. Results: S. herba extract exerted an intestinal anti-inflammatory effect in both experimental models of colitis, as evidenced both histologically, since it facilitated the tissue recovery of the damaged colon, and biochemically as showed by the improvement of the different inflammatory markers evaluated, including myeloperoxidase activity, glutathione content, and leukotriene B4 levels as well as the expression of the inducible proteins iNOS and COX-2. This beneficial effect was associated with the reduction in the expression of different cytokines, like TNFα, IL-1β, IFNγ, IL-6 and IL-17, the chemokine MCP-1, and the adhesion molecule ICAM-1, thus ameliorating the altered immune response associated with the colonic inflammation. Conclusion: S. herba extract displays an anti-inflammatory effect on different models of rodent colitis that could be attributed to its immunomodulatory properties.

One of the manifestations of X-linked lymphoproliferative disease (XLP) is progressive agammaglobulinemia, caused by the absence of a functional signaling lymphocyte activation molecule (SLAM)–associated protein (SAP) in T, invariant natural killer T (NKT) cells and NK cells. Here we report that α-galactosylceramide (αGalCer) activated NKT cells positively regulate antibody responses to haptenated protein antigens at multiple checkpoints, including germinal center formation and affinity maturation. Whereas NKT cell–dependent B cell responses were absent in SAP−/−.B6 mice that completely lack NKT cells, the small number of SAP-deficient NKT cells in SAP−/−.BALB/c mice adjuvated antibody production, but not the germinal center reaction. To test the hypothesis that SAP-deficient NKT cells can facilitate humoral immunity, SAP was deleted after development in SAPfl/fl.tgCreERT2.B6 mice. We find that NKT cell intrinsic expression of SAP is dispensable for noncognate helper functions, but is critical for providing cognate help to antigen-specific B cells. These results demonstrate that SLAM-family receptor-regulated cell-cell interactions are not limited to T-B cell conjugates. We conclude that in the absence of SAP, several routes of NKT cell–mediated antibody production are still accessible. The latter suggests that residual NKT cells in XLP patients might contribute to variations in dysgammaglobulinemia.

9 páginas, 8 figuras, 3 tablas., In the present study we describe the preparation and chemical characterization of a caramel with a high (70%) content of difructose dianhydrides (DFAs) and glycosylated derivatives (DFAs). This product was obtained by thermal activation (90 °C) of highly concentrated (90% w/v) aqueous d-fructose solutions using the sulfonic acid ion-exchange resin Lewatit S2328 as caramelization catalyst. DFAs represent a unique family of cyclic fructans with prebiotic properties already present in low proportions (, We thank the Spanish Ministerio de Ciencia e Innovación (contract numbers CTQ2007-61180/PPQ, CTQ2006-15515-C02-01/BQU, and SAF2008-02616) and the Junta de Andalucía for financial support (CTS 164). E.S.-P. is a “Project of Excellence” Fellow (Junta de Andalucía; contract number P06-AGR-02150). CIBERehd is funded by the Instituto de Salud Carlos III.

Antonio Abel Lozano-Pérez,1 Alba Rodriguez-Nogales,2 Víctor Ortiz-Cullera,1 Francesca Algieri,2 José Garrido-Mesa,2 Pedro Zorrilla,2 M Elena Rodriguez-Cabezas,2 Natividad Garrido-Mesa,2 M Pilar Utrilla,2 Laura De Matteis,3 Jesús Martínez delaFuente,3 José Luis Cenis,1 Julio Gálvez2 1Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario, Murcia, Spain; 2Centro de Investigaciones Biomédicas en Red – Enfermedades Hepáticas y Digestivas, Department of Pharmacology, ibs Granada, Center for Biomedical Research, University of Granada, Granada, Spain; 3Instituto de Nanociencia de Aragón, Universidad de Zaragoza, Zaragoza, Spain Purpose: We aimed to evaluate the intestinal anti-inflammatory properties of silk fibroin nanoparticles, around 100nm in size, when loaded with the stilbene compound resveratrol, in an experimental model of rat colitis. Methods: Nanoparticles were loaded with resveratrol by adsorption. The biological effects of the resveratrol-loaded nanoparticles were tested both in vitro, in a cell culture of RAW 264.7cells (mouse macrophages), and in vivo, in the trinitrobenzenesulfonic acid model of rat colitis, when administered intracolonically.Results: The resveratrol liberation in 1× phosphate-buffered saline (PBS; pH 7.4) was characterized by fast liberation, reaching the solubility limit in 3hours, which was maintained over a period of 80hours. The in vitro assays revealed immunomodulatory properties exerted by these resveratrol-loaded nanoparticles since they promoted macrophage activity in basal conditions and inhibited this activity when stimulated with lipopolysaccharide. The in vivo experiments showed that after evaluation of the macroscopic symptoms, inflammatory markers, and intestinal barrier function, the fibroin nanoparticles loaded with resveratrol had a better effect than the single treatments, being similar to that produced by the glucocorticoid dexamethasone. Conclusion: Silk fibroin nanoparticles constitute an attractive strategy for the controlled release of resveratrol, showing immunomodulatory properties and intestinal anti-inflammatory effects. Keywords: immunomodulatory, cytokines, TNBS rat colitis, RAW 264.7 macrophage cells, antioxidant

Esculetin is a coumarin derivative with high antioxidant activity. In a rat experimental model of inflammatory bowel disease induced by trinitrobenzenesulfonic acid, esculetin at the dose of 5mg/Kg displayed intestinal anti-inflammatory activity; however, its mechanism of action needs to be elucidated. Our objective was to evaluate the effects of esculetin on the intestinal inflammatory process and to clarify the mechanism of action of this compound. We also compared its effects with prednisolone and sulphasalazine. Our results demonstrate that treatment with esculetin prevented an increase in malondialdehyde content, counteracted the depletion of glutathione content, reduced epithelial cell apoptosis, reduced the secretion of pro-inflammatory cytokines, such as IL-1β, IL-2 and IFN-γ, in vitro , and reduced the colonic levels of TNF-α and IL-1β in vivo . Additionally, esculetin treatment inhibited MPO and AP activities. These results demonstrated that esculetin produced a more effective intestinal anti-inflammatory effect than sulphasalazine because it was used at a 10-fold lower dose, and it produced effects similar to those created by prednisolone. We suggest that esculetin exerts its activity by inhibiting pro-inflammatory cytokine secretion and increasing the defences against reactive oxygen species. This leads to less migration and/or activation of inflammatory cells, resulting in the improvement of lesions and functions in the intestinal epithelium. This study confirms the intestinal anti-inflammatory activity of esculetin and demonstrates that this compound has both antioxidative and immunomodulatory properties. Therefore, esculetin may be an interesting new anti-inflammatory drug for the treatment of inflammatory bowel disease.

Background & Aims Innate lymphoid cells (ILCs) are a heterogeneous group of mucosal inflammatory cells that participate in chronic intestinal inflammation. We investigated the role of interleukin 6 (IL6) in inducing activation of ILCs in mice and in human beings with chronic intestinal inflammation. Methods ILCs were isolated from colons of Tbx21-/- × Rag2-/- mice (TRUC), which develop colitis; patients with inflammatory bowel disease (IBD); and patients without colon inflammation (controls). ILCs were characterized by flow cytometry; cytokine production was measured by enzyme-linked immunosorbent assay and cytokine bead arrays. Mice were given intraperitoneal injections of depleting (CD4, CD90), neutralizing (IL6), or control antibodies. Isolated colon tissues were analyzed by histology, explant organ culture, and cell culture. Bacterial DNA was extracted from mouse fecal samples to assess the intestinal microbiota. Results IL17A- and IL22-producing, natural cytotoxicity receptor-negative, ILC3 were the major subset of ILCs detected in colons of TRUC mice. Combinations of IL23 and IL1α induced production of cytokines by these cells, which increased further after administration of IL6. Antibodies against IL6 reduced colitis in TRUC mice without significantly affecting the structure of their intestinal microbiota. Addition of IL6 increased production of IL17A, IL22, and interferon-γ by human intestinal CD3-negative, IL7-receptor-positive cells, in a dose-dependent manner. Conclusions IL6 contributes to activation of colonic natural cytotoxicity receptor-negative, CD4-negative, ILC3s in mice with chronic intestinal inflammation (TRUC mice) by increasing IL23- and IL1α-induced production of IL17A and IL22. This pathway might be targeted to treat patients with IBD because IL6, which is highly produced in colonic tissue by some IBD patients, also increased the production of IL17A, IL22, and interferon-γ by cultured human colon CD3-negative, IL7-receptor-positive cells.