Repurposing tetracyclines for acute respiratory distress syndrome (ARDS) and severe COVID-19: a critical discussion of recent publications.

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.

A population of naive-like CD4+ T cells stably polarized to the TH 1 lineage.

T-bet is the lineage-specifying transcription factor for CD4+ 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 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 polarized to the TH 1 lineage, predisposed to produce IFN-γ upon cell activation, and resist repolarization to other lineages in vitro and in vivo. These results demonstrate that lineage-specifying factors can polarize T cells in the absence of canonical markers of T cell activation and that this has an impact on the subsequent T-helper response.

Anti-inflammatory activity of hydroalcoholic extracts of Lavandula dentata L. and Lavandula stoechas L.

ETHNOPHARMACOLOGICAL RELEVANCE: Plants from genus Lavandula have been used as anti-inflammatory drugs in Mediterranean traditional medicine. Nowadays, there is a growing interest for complementary medicine, including herbal remedies, to treat inflammatory bowel disease (IBD). AIM OF THE STUDY: To test the anti-inflammatory properties of Lavandula dentata and Lavandula stoechas extracts in two inflammatory experimental models: TNBS model of rat colitis and the carrageenan-induced paw edema in mice, in order to mimic the intestinal conditions and the extra-intestinal manifestations of human IBD, respectively. MATERIAL AND METHODS: The extracts were characterized through the qualitative HPLC analysis. Then, they were assayed in vitro and in vivo. In vitro studies were performed in BMDMs and CMT-93 epithelial cells with different concentrations of the extracts (ranging from 0.1 to 100µg/ml). The extracts were tested in vivo in the TNBS model of rat colitis (10 and 25mg/kg) and in the carrageenan-induced paw edema in mice (10, 25 and 100mg/kg). RESULTS: L. dentata and L. stoechas extracts displayed immunomodulatory properties in vitro down-regulating different mediators of inflammation like cytokines and nitric oxide. They also showed anti-inflammatory effects in the TNBS model of colitis as evidenced by reduced myeloperoxidase activity and increased total glutathione content, indicating a decrease of neutrophil infiltration and an improvement of the oxidative state. Besides, both extracts modulated the expression of pro-inflammatory cytokines and chemokines, and ameliorated the altered epithelial barrier function. They also displayed anti-inflammatory effects in the carrageenan-induced paw edema in mice, since a significant reduction of the paw thickness was observed. This was associated with a down-regulation of the expression of different inducible enzymes like MMP-9, iNOS and COX-2 and pro-inflammatory cytokines, all involved in the maintenance of the inflammatory condition. CONCLUSION: L. dentata and L. stoechas extracts showed intestinal anti-inflammatory effect, confirming their potential use as herbal remedies in gastrointestinal disorders. In addition, their anti-inflammatory effect was also observed in other locations, thus suggesting a possible use for the treatment of the extra-intestinal symptoms of IBD.

Interleukin 6 Increases Production of Cytokines by Colonic Innate Lymphoid Cells in Mice and Patients With Chronic Intestinal Inflammation.

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.

A new therapeutic association to manage relapsing experimental colitis: Doxycycline plus Saccharomyces boulardii.

Immunomodulatory antibiotics have been proposed for the treatment of multifactorial conditions such as inflammatory bowel disease. Probiotics are able to attenuate intestinal inflammation, being considered as safe when chronically administered. The aim of the study was to evaluate the anti-inflammatory effects of doxycycline, a tetracycline with immunomodulatory properties, alone and in association with the probiotic Saccharomyces boulardii CNCMI-745. Doxycycline was assayed both in vitro (Caco-2 epithelial cells and RAW 264.7 macrophages) and in vivo, in the trinitrobenzenesulfonic acid (TNBS) model of rat colitis and the dextran sodium sulfate (DSS) model of mouse colitis. In addition, the anti-inflammatory effect of the association of doxycycline and the probiotic was evaluated in vitro and in vivo in a DSS model of reactivated colitis in mice. Doxycycline displayed immunomodulatory activity in vitro, reducing IL-8 production by intestinal epithelial cells and nitric oxide by macrophages. Doxycycline administration to TNBS-colitic rats (5, 10 and 25 mg/kg) ameliorated the intestinal inflammatory process, being its efficacy comparable to that previously showed by minocycline. Doxycycline treatment was also effective in reducing acute intestinal inflammation in the DSS model of mouse colitis. The association of doxycycline and S. boulardii helped managing colitis in a reactivated model of colitis, by reducing intestinal inflammation and accelerating the recovery and attenuating the relapse. This was evidenced by a reduced disease activity index, colonic tissue damage and expression of inflammatory mediators. This study confirms the intestinal anti-inflammatory activity of doxycycline and supports the potential use of its therapeutic association with S. boulardii for the treatment of inflammatory bowel diseases, in which doxycycline is used to induce remission and long term probiotic administration helps to prevent the relapses.

The viability of Lactobacillus fermentum CECT5716 is not essential to exert intestinal anti-inflammatory properties.

Probiotics have been used as alternative therapies in intestinal inflammatory disorders. Many studies have shown that different bacterial probiotic strains possess immuno-modulatory and anti-inflammatory properties. However, there is an increasing interest in the use of non-viable bacteria to reduce the risk of microbial translocation and infection. The aim of this study was to evaluate whether the viability of L. fermentum CECT5716 is essential to exert its intestinal anti-inflammatory effect. We compared the preventative effects of viable and non-viable probiotic in the TNBS model of rat colitis. In vitro studies were also performed in Caco-2 and RAW 264.7 cells to evaluate the probiotic effects on IL-8, IL-1ß and nitrite production, and p44/42 and p38 MAP kinase protein expressions. In vitro results revealed a decrease in the stimulated production of pro-inflammatory mediators regardless of the viability of the probiotic. Likewise, both forms of the probiotic administered to colitic rats produced a significant reduction of IL-1ß and TNF-α levels and colonic iNOS expression. In conclusion, both live and dead L. fermentum CECT5716 have been demonstrated to attenuate the inflammatory process and diminish the production of some of the inflammatory mediators. In fact, the viability of this probiotic did not affect its immuno-modulatory and anti-inflammatory properties.

Silk fibroin nanoparticles constitute a vector for controlled release of resveratrol in an experimental model of inflammatory bowel disease in rats.

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 anti-inflammatory effects of oligosaccharides derived from lactulose in the trinitrobenzenesulfonic acid model of rat colitis.

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.

The probiotic Lactobacillus coryniformis CECT5711 reduces the vascular pro-oxidant and pro-inflammatory status in obese mice.

Obesity is associated with intestine dysbiosis and is characterized by a low-grade inflammatory status, which affects vascular function. In the present study, we evaluated the effects of a probiotic with immunomodulatory properties, Lactobacillus coryniformis CECT5711, in obese mice fed on an HFD (high-fat diet). The probiotic treatment was given for 12 weeks, and it did not affect the weight evolution, although it reduced basal glycaemia and insulin resistance. L. coryniformis administration to HFD-induced obese mice induced marked changes in microbiota composition and reduced the metabolic endotoxaemia as it decreased the LPS (lipopolysaccharide) plasma level, which was associated with a significant improvement in gut barrier disruption. Furthermore, it lowered TNFα (tumour necrosis factor α) expression in liver, improving the inflammatory status, and thus the glucose metabolism. Additionally, the probiotic reversed the endothelial dysfunction observed in obese mice when endothelium- and NO (nitric oxide)-dependent vasodilatation induced by acetylcholine in aortic rings was studied. It also restored the increased vessel superoxide levels observed in obese mice, by reducing NADPH oxidase activity and increasing antioxidant enzymes. Moreover, chronic probiotic administration for 2 weeks also improved endothelial dysfunction and vascular oxidative stress induced by in vivo administration of LPS in control mice fed on a standard chow diet. The results of the present study demonstrate an endothelial-protective effect of L. coryniformis CECT5711 in obese mice by increasing NO bioavailability, suggesting the therapeutic potential of this gut microbiota manipulation to prevent vasculopathy in obesity.

Intestinal anti-inflammatory activity of the Serpylli herba extract in experimental models of rodent colitis.

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-1ß, 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.

Functional plasticity of Th17 cells: implications in gastrointestinal tract function.

The gastrointestinal tract is an active player of the human immune system, participating in the innate and adaptive immune responses, keeping the homeostasis of the human being in a healthy status. However, most intestinal conditions are associated with an altered immune response, which implies the activation of CD4(+) T helper (Th) cells. Based on their cytokine secretion, transcription factor expression and immunological functions, the differentiated Th cells were initially subdivided into different lineages: Th1 (that express the transcription factor T-box (T-bet), secrete interferon (IFN)-γ and protect the host against intracellular infections) and Th2 (that express GATA binding protein 3 (GATA-3), secrete interleukin (IL)-4, IL-5 and IL-13, and mediate host defense against helminths). Later, a new subset was identified, the Th17, which selectively produces IL-17A and is crucial for host defense against extracellular pathogens. More recently, a functional plasticity between the Th1 and Th17 lineages has been described, a process sometimes controversial that seems to play a key role in different inflammatory conditions, including those affecting the gastrointestinal system. This review will summarize the current knowledge regarding the regulation and functional role of Th17 cells in the gut, focusing on these newly identified features of this T cell subset, including plasticity, their relationship with regulatory T cells and their heterogeneity in the inflammatory microenvironment. A better understanding of these issues is critical to elucidate the role of Th17 cells in intestine immunity, and so for the design of novel therapeutic approaches for intestinal diseases specifically targeting Th17 cells.

Active colitis exacerbates immune response to internalized food antigens in mice.

BACKGROUND: Previous studies have indicated that colitis increases intestinal permeability to food antigens. This condition also generates an immunoreactive milieu in the gut, which may exacerbate or counteract allergy reactions. This, along with the fact that both colitis and allergy are being codiagnosed more frequently, means the scientific interest on the immune relation between these pathologies is increasing. We evaluated the immune response to an internalized food antigen that was initiated during a concomitant active intestinal inflammatory response. METHODS: An ovalbumin (OVA)-induced immune response was analyzed in healthy mice and in mice suffering from colitis induced by the administration of dinitrofluorobenzene/dinitrosulfonic acid (DNFB/DNS) at the moment of OVA challenge. The OVA-induced clinical score and allergy response both in plasma and in splenocyte cultures from these animals were compared. RESULTS: Although no differences were observed in the allergy clinical score, the concomitant active colitis led to an increase in the immune response to OVA antigen, as shown by increased spleen size and OVA-induced splenocyte proliferation, exacerbated expression of total and OVA-specific IgG1 levels, increased colonic IL-4 expression and OVA-induced IL-4 and IL-5 cytokine expression in spleen cells. CONCLUSIONS: Our results indicate that animals with active colitis undergo an exacerbated immune response to an internalized antigen. This finding could be relevant for the allergy management of patients presenting simultaneously with chronic colitis.

Intestinal anti-inflammatory activity of hydroalcoholic extracts of Phlomis purpurea L. and Phlomis lychnitis L. in the trinitrobenzenesulphonic acid model of rat colitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Different species from genus Phlomis, frequently native from the the eastern Mediterranean zone, have been used in traditional medicine as an anti-inflammatory remedy. Among other constituents, they contain polyphenols that show antioxidant properties, which are interesting for the treatment of inflammatory pathologies associated with oxidative stress in humans, such as inflammatory bowel disease (IBD). The aim of this study was to evaluate the intestinal anti-inflammatoy effect of hydroalcoholic extracts of Phlomis lychnitis and P. purpurea in the trinitrobenzenesulphonic acid (TNBS) model of rat colitis, a well characterized experimental model with some resemblance to human IBD. MATERIALS AND METHODS: Hydroalcoholic extracts of both plants were characterized by determining their polyphenolic content and then assayed in the TNBS model of rat colitis. For this purpose, female Wistar rats were assigned to seven groups (n=10): healthy control, untreated TNBS-colitis and five TNBS- colitis groups treated with Phlomis lychnitis (10 and 20mg/kg), P. purpurea (10 and 25mg/kg) and sulphasalazine (200mg/kg), as a positive control. Treatments started the same day of TNBS colitis induction, and rats were sacrificed one week later. Colonic inflammation was evaluated both histologically and biochemically. RESULTS: The histological (macroscopic and microscopic) analysis of colonic samples revealed that both extracts showed an anti-inflammatory effect, which was confirmed biochemically by a decreased colonic MPO activity, a maker of neutrophil infiltration, an increased colonic glutathione content, which counteracts the oxidative status associated with the inflammatory process, and a down-regulated iNOS expression. However, only the extract of P. purpurea reduced the expression of the proinflammatory cytokines IL-1ß and IL-17, the chemokines CINC-1 and MCP-1, as well as the adhesion molecule ICAM-1, ameliorating the altered immune response associated with the colonic inflammation. Furthermore, both P. lychnitis and P. purpurea extracts were able to significantly increase the expression of markers of epithelial integrity such as MUC-2, MUC-3 and villin, thus revealing an improvement in the altered colonic permeability that characterizes colonic inflammation. CONCLUSIONS: Both extracts showed intestinal anti-inflammatory activity in the TNBS model of rat colitis, thus confirming their traditional use in digestive inflammatory complaints. In addition to their antioxidant properties, other mechanisms can contribute to this beneficial effect, like an improvement in the intestine epithelial barrier and a downregulation of the immune response.

SAP expression in invariant NKT cells is required for cognate help to support B-cell responses.

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 SAP(fl/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.

A shorter and more specific oral sensitization-based experimental model of food allergy in mice.

Cow's milk protein allergy (CMPA) is one of the most prevalent human food-borne allergies, particularly in children. Experimental animal models have become critical tools with which to perform research on new therapeutic approaches and on the molecular mechanisms involved. However, oral food allergen sensitization in mice requires several weeks and is usually associated with unspecific immune responses. To overcome these inconveniences, we have developed a new food allergy model that takes only two weeks while retaining the main characters of allergic response to food antigens. The new model is characterized by oral sensitization of weaned Balb/c mice with 5 doses of purified cow's milk protein (CMP) plus cholera toxin (CT) for only two weeks and posterior challenge with an intraperitoneal administration of the allergen at the end of the sensitization period. In parallel, we studied a conventional protocol that lasts for seven weeks, and also the non-specific effects exerted by CT in both protocols. The shorter protocol achieves a similar clinical score as the original food allergy model without macroscopically affecting gut morphology or physiology. Moreover, the shorter protocol caused an increased IL-4 production and a more selective antigen-specific IgG1 response. Finally, the extended CT administration during the sensitization period of the conventional protocol is responsible for the exacerbated immune response observed in that model. Therefore, the new model presented here allows a reduction not only in experimental time but also in the number of animals required per experiment while maintaining the features of conventional allergy models. We propose that the new protocol reported will contribute to advancing allergy research.

The immunomodulatory properties of viable Lactobacillus salivarius ssp. salivarius CECT5713 are not restricted to the large intestine.

PURPOSE: The aim of this study was to better characterise the biological effects of Lactobacillus salivarius ssp. salivarius CECT5713, a probiotic with immunomodulatory properties. METHODS: Live or dead probiotic was assayed in the TNBS model of rat colitis to determine whether viability was a requisite to exert the beneficial effects. In vitro studies were also performed in Caco-2 cells to evaluate its effects on epithelial cell recovery and IL-8 production. Finally, the probiotic was assayed in the LPS model of septic shock in mice to establish its effects when there is an altered systemic immune response. RESULTS: The viability of the probiotic was required for its anti-inflammatory activity. The probiotic inhibited IL-8 production in stimulated Caco-2 cells and facilitated the recovery of damaged intestinal epithelium. In LPS-treated mice, the probiotic inhibited the production of TNFα in plasma and lungs and increased the hepatic glutathione content. These effects were associated with an improvement in the altered production of the T-cell cytokines in splenocytes, by reducing IL-2 and IL-5 and by increasing IL-10. Finally, it reduced the increased plasma IgG production in LPS-treated mice. CONCLUSION: The anti-inflammatory effects of viable L. salivarius ssp. salivarius CECT5713 are not restricted to the gastrointestinal tract.

Suppression of TNBS-induced colitis in rats by 4-methylesculetin, a natural coumarin: comparison with prednisolone and sulphasalazine.

The aim of the present study was to compare the effects of the 4-methylesculetin with those produced by prednisolone and sulphasalazine and to elucidate the mechanisms involved in its action. Colitis was induced in rat by instillation of trinitrobenzenesulphonic acid (TNBS). The colon damage was evaluated using macroscopic, microscopic and biochemical analysis. In addition, in vitro studies were performed to evaluate cytokine production in cell cultures using the murine macrophage cell line RAW264.7, mouse splenocytes and the human colonic epithelial cell line Caco-2. 4-Methylesculetin produced a reduction of the macroscopic damage score and the recovery of the intestinal cytoarchitecture. These effects were associated with a prevention of the GSH depletion and an inhibition in AP activity. After colitis relapse, 4-methylesculetin improved the colonic inflammatory status as evidenced by histological findings, with a reduction in apoptosis, as well as biochemically by inhibition of colonic myeloperoxidase, alkaline phosphatase and metalloproteinase 9 activities. Paired with this inhibitive activity, there was a decrease in malondialdehyde content and in IL-1ß levels. In vitro assays revealed that 4-methylesculetin promoted an inhibition in IL-1ß, IL-8, IL-2 and IFN-γ production in cell cultures. In conclusion, 4-methylesculetin showed similar efficacy to that obtained with either prednisolone or sulphasalazine, both in the acute phase of colitis as well as following a curative protocol. The intestinal anti-inflammatory activity by 4-methylesculetin is likely related to its ability in reduce colonic oxidative stress and inhibit pro-inflammatory cytokine production.

The association of minocycline and the probiotic Escherichia coli Nissle 1917 results in an additive beneficial effect in a DSS model of reactivated colitis in mice.

Antibiotics have been empirically used for human inflammatory bowel disease, being limited to short periods. Probiotics are able to attenuate intestinal inflammation due to its immunomodulatory properties, being considered as safe when chronically administered. The aim was to test the association of minocycline, a tetracycline with immunomodulatory properties, and the probiotic Escherichia coli Nissle 1917 (EcN) in a mouse model of reactivated colitis. For this purpose, female C57BL/6J mice were assigned to different groups: non-colitic and dextran sodium sulfate (DSS)-control groups (without treatment), minocycline (50 mg/kg/day; p.o.), EcN (5×10(8) CFU/day; p.o.), and minocycline plus EcN treated groups. Colitis was induced by adding DSS in the drinking water (3%) for 5 days; 2 weeks later, colitis was reactivated by subsequent exposure to DSS. The inflammatory status was evaluated daily by a disease activity index (DAI); colonic damage was assessed histologically and biochemically by evaluating mRNA relative expression of different mediators by qPCR. Finally, a microbiological analysis of the colonic contents was performed. Minocycline and EcN exerted intestinal anti-inflammatory effect and attenuated the reactivation of the colitis, as shown by the reduced DAI values, being these effects greater when combining both treatments. This was evidenced histologically and biochemically, by reduced expression of TNFα, IL-1ß, IL-2, MIP-2, MCP-1, ICAM-1, iNOS and MMP-9, together with increased MUC-3 and ZO-1 expression. Finally, the altered microbiota composition of colitic mice was partially restored after the different treatments. In conclusion, EcN supplementation to minocycline treatment improves the recovery of the intestinal damage and prevents the reactivation of experimental colitis.

DNFB-DNS hapten-induced colitis in mice should not be considered a model of inflammatory bowel disease.

BACKGROUND: The dinitrofluorobenzene/dinitrosulfonic acid (DNFB/DNS) model was originally described as an experimental model of intestinal inflammation resembling human ulcerative colitis (UC). Due to the absence of acceptable UC experimental models for pharmacological preclinical assays, here we examine the immune response induced in this model. METHODS: Balb/c mice were sensitized by skin application of DNFB on day 1, followed by an intrarectal challenge with DNS on day 5. We further expanded this model by administering a second DNS challenge on day 15. The features of colonic inflammation and immune response were evaluated. RESULTS: The changes observed in colonic tissue corresponded, in comparison to the trinitrobenzene sulfonic acid (TNBS) colitis model, to a mild mucosal effect in the colon, which spontaneously resolved in less than 5 days. Furthermore, the second hapten challenge did not exacerbate the inflammatory response. In contrast to other studies, we did not observe any clear involvement of tumor necrosis factor alpha (TNF-α) or other Th1 cytokines during the initial inflammatory response; however, we found that a more Th2-humoral response appeared to mediate the first contact with the hapten. An increased humoral response was detected during the second challenge, although an increased Th1/Th17-cytokine expression profile was also simultaneously observed. CONCLUSIONS: On the basis of these results, although the DNFB/DNS model can display some features found in human UC, it should be considered as a model for the study of the intestinal hypersensitivity seen, for example, during food allergy or irritable bowel syndrome but not intestinal inflammation per se.