Pedro Salvador

Candida albicans stimulates in vivo differentiation of haematopoietic stem and progenitor cells towards macrophages by a TLR2-dependent signalling

Toll‐like receptors (TLRs) are expressed by haematopoietic stem and progenitor cells (HSPCs), and may play a role in haematopoiesis in response to pathogens during infection. We have previously demonstrated that (i) inactivated yeasts of Candida albicans induce in vitro differentiation of HSPCs towards the myeloid lineage, and (ii) soluble TLR agonists induce in vivo their differentiation towards macrophages. In this work, using an in vivo model of HSPCs transplantation, we report for the first time that HSPCs sense C. albicans in vivo and subsequently are directed to produce macrophages by a TLR2‐dependent signalling. Purified lineage‐negative cells (Lin−) from bone marrow of C57BL/6 mice (CD45.2 alloantigen) were transplanted into B6Ly5.1 mice (CD45.1 alloantigen), which were then injected with viable or inactivated C. albicans yeasts. Transplanted cells were detected in the spleen and in the bone marrow of recipient mice, and they differentiate preferentially to macrophages, both in response to infection or in response to inactivated yeasts. The generation of macrophages was dependent on TLR2 but independent of TLR4, as transplanted Lin− cells from TLR2−/− mice did not give rise to macrophages, whereas Lin− cells from TLR4−/− mice generated macrophages similarly to control cells. Interestingly, the absence of TLR2, or in a minor extent TLR4, gives Lin− cells an advantage in transplantation assays, as increases the percentage of transplanted recovered cells. Our results indicatethat TLR‐mediated recognition of C. albicans by HSPCs may help replace and/or increase cells that constitute the first line of defence against the fungus, and suggest that TLR‐mediated signalling may lead to reprogramming early progenitors to rapidly replenishing the innate immune system and generate the most necessary mature cells to deal with the pathogen.

Autophagy stimulation prevents intestinal mucosal inflammation and ameliorates murine colitis

Defective autophagy contributes to the pathogenesis of inflammatory disorders such as inflammatory bowel disease and there are interactions between autophagy and inflammation. Here we have analysed the effects of autophagy stimulators on murine colitis.

Stimulation of autophagy prevents intestinal mucosal inflammation and ameliorates murine colitis

Defective autophagy contributes to the pathogenesis of inflammatory disorders such as inflammatory bowel disease and there are interactions between autophagy and inflammation. Here we have analysed the effects of autophagy stimulators on murine colitis.

CD16+ Macrophages Mediate Fibrosis in Inflammatory Bowel Disease

Background and Aims Fibrosis is a common complication of Crohns disease [CD], and is related to dysregulated tissular repair following inflammation, in which macrophages play a central role. We have previously observed that STAT6-/- mice present delayed mucosal recovery after 2,4,6-trinitrobenzenesulfonic acid [TNBS]-induced colitis due to a deficiency in reparatory interleukin-4 [IL4]/STAT6-dependent M2 macrophages, which can be reverted by the exogenous transfer of this cell type. In the present study, we analyse the role of STAT6-dependent macrophages in intestinal fibrosis. Methods Colitis was induced by weekly intra-rectal administration of TNBS [6 weeks] to STAT6-/- mice and wild-type [WT] animals. Colonic surgical resections were obtained from CD patients and from colon cancer patients. Results Chronic colitis provoked a fibrogenic response in STAT6-/- mice, but not in WT animals. An accumulation of M2 macrophages, defined as CD206+ cells, was observed in WT mice, but not in STAT6-/- animals. Instead, the latter group showed an increase in CD16+ macrophages that correlated with the expression of fibrogenic markers. CD16+ macrophages were also increased in the damaged mucosa of Crohns disease patients with stenotic or penetrating complications. Finally, administration of IL4-treated WT macrophages to STAT6-/- mice reduced TNBS-induced fibrosis. Conclusions Our study demonstrates that STAT6 deficiency dysregulates the macrophage response to inflammatory outbursts by increasing the presence of a population of CD16+ macrophages that seems to contribute to intestinal fibrosis.

A Single Nucleotide Polymorphism in the Vitamin D Receptor Gene Is Associated With Decreased Levels of the Protein and a Penetrating Pattern in Crohn’s Disease

Background Vitamin D signaling modulates inflammation through the vitamin D receptor (VDR). The synonymous single nucleotide polymorphism (SNP) rs731236, located in the VDR gene, has been associated with a higher risk of Crohns disease (CD). We analyzed differences in VDR expression levels among CD patients who were homozygous for allelic variants in this SNP and their relevance for disease course. Methods DNA was extracted from blood samples of CD patients, and SNP genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. Fresh blood from patients was used to isolate peripheral blood mononuclear cells (PBMCs) or to determine the expression of adhesion molecules by flow cytometry. We analyzed the gene expression of VDR and several cytokines in PBMCs using real-time polymerase chain reaction and the protein levels of VDR, NFκB, and IκBα by immunoblot. In addition, we collected complete clinical data for a group of 103 patients, including age at diagnosis, disease location, and disease behavior to compare patient characteristics with respect to genotype. Results We found that CD patients who were homozygous for the risk allele presented lower levels of VDR protein in PBMCs, and that this was associated with an upregulation of IL1β mRNA and activation of lymphocytic adhesion molecules. These patients had a higher risk of developing a B3-penetrating phenotype and of needing to undergo surgery. Conclusion Our data highlight the relevance of vitamin D/VDR signaling in modulating the subjacent inflammation that leads to CD-related complications.

Succinate receptor mediates intestinal inflammation and fibrosis

Succinate, an intermediate of the tricarboxylic acid cycle, is accumulated in inflamed areas and its signaling through succinate receptor (SUCNR1) regulates immune function. We analyze SUCNR1 expression in the intestine of Crohns disease patients and its role in murine intestinal inflammation and fibrosis. We show that both serum and intestinal succinate levels and SUCNR1 expression in intestinal surgical resections were higher in CD patients than in controls. SUCNR1 co-localized with CD86, CD206, and α-SMA+ cells in human intestine and we found a positive and significant correlation between SUCNR1 and α-SMA expression. In human isolated fibroblasts from CD patients SUCNR1 expression was higher than in those from controls and treatment with succinate increased SUCNR1 expression, fibrotic markers and inflammatory cytokines through SUCNR1. This receptor modulated the expression of pro-inflammatory cytokines in resting murine macrophages, macrophage polarization and fibroblast activation and Sucnr1−/− mice were protected against both acute TNBS-colitis and intestinal fibrosis induced by the heterotopic transplant of colonic tissue. We demonstrate increased succinate levels in serum and SUCNR1 expression in intestinal tissue of CD patients and show a role for SUCNR1 in murine intestinal inflammation and fibrosis.