Jordi Xaus

Macrophages require different nucleoside transport systems for proliferation and activation

To evalúate the mechanisms involved in macrophage proliferation and activation, we studied the regulation of the nucleoside transport systems. In murine bone marrow‐derived macrophages, the nucleo‐sides required for DNA and RNA synthesis are recruited from the extracellular medium. M‐CSF induced macrophage proliferation and DNA and RNA synthesis, whereas interferon γ (IFN‐γ) led to activation, blocked proliferation, and induced only RNA synthesis. Macrophages express at least the concentrative systems N1 and N2 (CNT2 and CNT1 genes, respectively) and the equilibrative systems es and ei (ENT1 and ENT2 genes, respectively). Incubation with M‐CSF only up‐regulated the equilibrative system es. Inhibition of this transport system blocked M‐CSF‐dependent proliferation. Treatment with IFN‐γ only induced the concentrative N1 and N2 systems. IFN‐γ also down‐regulated the increased expression of the es equilibrative system induced by M‐CSF. Thus, macrophage proliferation and activation require selective regulation of nucleoside transporters and may respond to specific requirements for DNA and RNA synthesis. This report also shows that the nucleo‐side transporters are critical for macrophage proliferation and activation.

Butyrate in vitro immune-modulatory effects might be mediated through a proliferation-related induction of apoptosis

Survival and proliferation signals are two processes closely interrelated and finely controlled in most cell types, whose deregulation may lead to carcinogenesis. In the last decade, different studies have suggested that both cellular functions are also intimately associated with other cellular activities such as differentiation and cellular activation, especially in immune cells. The aim of this study was to evaluate the effects of the short-chain fatty acid (SCFA) butyrate on the proliferation and activation state of different cell types involved in inflammatory bowel disease. We focused on intestinal epithelial cells, macrophages and T-lymphocytes, using both primary non-transformed cultures and established cell lines. The results showed that low concentrations of butyrate inhibited the proliferation of all the immune cell types tested in this work, whereas it only induced apoptosis in activated T-lymphocytes, non-differentiated epithelial cells and macrophage cell lines, but not in differentiated epithelial cells or primary macrophages. Butyrate apoptosis induction was mediated by caspase-3/7 activation. This SCFA was only able to modify cell activation, measured as expression of inflammatory cytokines, in those cell types in which apoptosis was induced. In conclusion, our results suggest a cell type-specificity of the immune-modulatory effects of butyrate based on the proliferation/activation characteristic physiology of these processes in different cells types.

Decorin Reverses the Repressive Effect of Autocrine-Produced TGF-β on Mouse Macrophage Activation

Several cytokines or growth factors induce macrophages to proliferate, become activated, differentiate, or die through apoptosis. Like the major macrophage activator IFN-γ, the extracellular matrix protein decorin inhibits proliferation and protects macrophages from the induction of apoptosis. Decorin enhances the IFN-γ-induced expression of the IAα and IAβ MHC class II genes. Moreover, it increases the IFN-γ- or LPS-induced expression of inducible NO synthase, TNF-α, IL-1β, and IL-6 genes and the secretion of these cytokines. Using a number of extracellular matrix proteins, we found a negative correlation between adhesion and proliferation. However, the effects of decorin on macrophage activation do not seem to be mediated through its effect on adhesion or proliferation. Instead, this proteoglycan abolishes the binding of TGF-β to macrophages, as shown by Scatchard analysis of 125I-labeled TGF-β, which, in the absence of decorin, showed a Kd of 0.11 ± 0.03 nM and ∼5000 receptors/cell. This was confirmed when we treated macrophages with Abs to block the endogenously produced TGF-β, which enhanced macrophage activation in a way similar to decorin. The increase in activation mediated by decorin demonstrates that macrophages are under negative regulation that can be reversed by proteins of the extracellular matrix.

Lactobacillus fermentum CECT 5716 prevents and reverts intestinal damage on TNBS-induced colitis in mice.

Background: Probiotics attenuate gut inflammation when administered before experimental colitis, but data on their effect after colitis induction are scarce. We aimed to evaluate the effects of Lactobacillus fermentum CECT 5716 on gut injury when administered either before or after trinitrobencene sulfonic acid (TNBS) colitis in Balb/c mice. Methods: In a preventive study, probiotic or vehicle was administered for 2 weeks before colitis. Then mice were allocated to: probiotic + TNBS, probiotic + sham, vehicle + TNBS, or vehicle + sham, and sacrificed 72 hours later. In a therapeutic study, mice were allocated into the same groups as before. Probiotic or vehicle were administered for 3 weeks. Mice were sacrificed at weeks 1, 2, and 3 after TNBS. Histological score, myeloperoxidase activity, and eicosanoid and cytokine production in colonic explant cultures were measured. Immunohistochemistry for nitrotyrosine and MyD88 was also performed. Results: In the preventive study, colitis was milder with probiotic than with vehicle (P = 0.041). This was associated with increased PGE2, IL‐2, and IL‐4 production, as well as attenuated nitrotyrosine staining in the former. In the therapeutic study, histological score at week 1 post‐TNBS was higher in probiotic than in vehicle fed mice (P = 0.018). However, at weeks 2 and 3 the histological score was significantly lower—with decreased IL‐6 production and increased MyD88 staining—in mice receiving the probiotic. Conclusions: Pretreatment with L. fermentum CECT 5716 attenuates TNBS colitis, an effect that seems to be due to its antioxidant abilities. When administered after TNBS, this probiotic is also effective in accelerating colitis recovery, and this is associated with an enhanced Toll‐like receptor function. (Inflamm Bowel Dis 2009)

Treatment with Anti-interferon-γ Monoclonal Antibodies Modifies Experimental Autoimmune Encephalomyelitis in Interferon-γ Receptor Knockout Mice

The role of interferon-γ (IFN-γ) in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE) is still controversial. We have studied the function of IFN-γ and its receptor in the EAE model using two different IFN-γ receptor knockout (IFN-γ R−/−) mouse types: C57Bl/6×129Sv, with a disruption of the IFN-γ receptor cytoplasmic domain, and 129Sv, homozygous for a disrupted IFN-γ receptor gene. Mice were immunized with peptide 40-55 from rat myelin oligodendrocyte glycoprotein. A subgroup of mice was treated with anti-IFN-γ monoclonal antibodies (mAb) on day 8 postimmunization. Clinical scoring and both histological and immunohistochemical studies were undertaken for all groups. We hereby show that treatment with anti-IFN-γ mAb worsened the disease course of 129Sv wild-type mice. However, it decreased the mean daily score in IFN-γ R−/− 129Sv and the incidence of the disease down to 50% in C57Bl/6×129Sv IFN-γ R−/− mice. Moreover, after anti-IFN-γ mAb treatment, oxidative stress levels, metallothionein I and II antioxidant protein expression, and apoptoticneuronal death were increased in wild-type mice while decreased in IFN-γ R−/− mice. These results suggest a putative alternative mechanism of action of this cytokine that works independent of its receptor.

High expression of p21Waf1 in sarcoid granulomas: a putative role for long‐lasting inflammation

In sarcoid granulomas, apoptotic events are reduced, which explains their characteristic long‐lasting inflammation. We have described that interferon‐γ (IFN‐γ) inhibits apoptosis in macrophages through the expression of p21Waf1. Here, we explore the molecular mechanisms involved in the inhibition of apoptosis in sarcoid granulomas. We analyzed skin biopsies from 19 sarcoidosis patients and 16 controls. Total RNA was subjected to semiquantitative reverse transcriptase‐polymerase chain reaction analysis. There was no difference found in the expression of proapoptotic (Bax and Bcl‐Xs) or antiapoptotic (Bcl‐2 and Bcl‐XL) genes nor in the expression of the tumor suppressor gene p53. Furthermore, the expression of IFN‐γ and the cdk inhibitors p21Waf1 and p27Kip1 were analyzed. IFN‐γ was detected in 37% of the sarcoidosis patients, and controls were negative (P<0.02). In addition, a higher proportion of patients expressing p21Waf1 (58%) versus controls (12%) was found (P<0.005). There was a significant correlation between the expression of IFN‐γ and p21Waf1 (r=0.69) and between p21Waf1 and fibronectin (r=0.65). Finally, using immunohistochemistry, high p21Waf1 reactivity was observed inside the granuloma. We conclude that the high levels of p21Waf1 in sarcoidosis may explain the absence of apoptosis in the granuloma and the persistence of inflammation.

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

PurposeThe aim of this study was to better characterise the biological effects of Lactobacillus salivarius ssp. salivarius CECT5713, a probiotic with immunomodulatory properties.MethodsLive 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.ResultsThe 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.ConclusionThe anti-inflammatory effects of viable L. salivarius ssp. salivarius CECT5713 are not restricted to the gastrointestinal tract.

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

Cows 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 cows 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.

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‐&agr;) 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. (Inflamm Bowel Dis 2011;)

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.