Galectin-3 orchestrates the histology of mesentery and protects liver during lupus-like syndrome induced by pristane.

Galectin-3 (Gal-3) controls intercellular and cell-extracellular matrix interactions during immunological responses. In chronic inflammation, Gal-3 is associated with fibrotic events, regulates B cell differentiation and delays lupus progression. Gal-3 deficient mice (Lgals3-/-) have intense germinal center formation and atypical plasma cell generation correlated to high levels IgG, IgE, and IgA. Here, we used pristane (2,6,10,14-tetramethylpentadecane) to induce lupus-like syndrome in Lgals3-/- and Lgals3+/+ BALB/c mice. Mesentery and peritoneal cells were monitored because promptly react to pristane injected in the peritoneal cavity. For the first time, mesenteric tissues have been associated to the pathogenesis of experimental lupus-like syndrome. In Lgals3+/+ pristane-induced mice, mesentery was hallmarked by intense fibrogranulomatous reaction restricted to submesothelial regions and organized niches containing macrophages and B lymphocytes and plasma cells. In contrast, Lgals3-/- pristane-treated mice had diffuse mesenteric fibrosis affecting submesothelium and peripheral tissues, atypical M1/M2 macrophage polarization and significant DLL1+ cells expansion, suggesting possible involvement of Notch/Delta pathways in the disease. Early inflammatory reaction to pristane was characterized by significant disturbances on monocyte recruitment, macrophage differentiation and dendritic cell (DC) responses in the peritoneal cavity of pristane-induced Lgals3-/- mice. A correlative analysis showed that mesenteric damages in the absence of Gal-3 were directly associated with severe portal inflammation and hepatitis. In conclusion, it has suggested that Gal-3 orchestrates histological organization in the mesentery and prevents lupoid hepatitis in experimental lupus-like syndrome by controlling macrophage polarization, Notch signaling pathways and DC differentiation in mesenteric structures.

ER stress activation in the intestinal mucosa but not in mesenteric adipose tissue is associated with inflammation in Crohn's disease patients.

Chronic/abnormal activation of endoplasmic reticulum (ER) stress is linked to the exacerbation of the inflammatory process and has been recently linked to Crohn's disease (CD) pathophysiology. We investigated the intestinal mucosa and the mesenteric adipose tissue (MAT) collected from CD patients with active disease (CD group) and from non-IBD patients (CTR group) to study ER stress activation and to address tissue-specific modulation in CD. The intestinal mucosa of CD patients showed an upregulation in the expression of ER stress related genes, including ATF3, DNAJC3, STC2, DDIT3, CALR, HSPA5 and HSP90B1. Results showed that EIF2AK3 gene was upregulated, along with increased protein expression of p-eIF2α and p-eIF2α/eIF2α ratio. Additionally, ERN1 gene expression was upregulated, along with an increased spliced/activated form sXBP1 protein. Despite the upregulation of ATF6 gene expression in the intestinal mucosa of CD patients, no differences were found in ATF6 protein expression. Lastly, the analysis of MAT revealed unchanged levels of ER stress markers along with no differences in the activation of UPR. However, chaperone gene expression was modulated in the MAT of CD patients. To conclude, our results address tissue-specific differences in UPR activation in CD and point the ER stress as an important pro-inflammatory mechanism in CD, specifically in the intestinal mucosa.

A selective window after the food-intake period favors tolerance induction in mesenteric lymph nodes.

Biological rhythms are periodic oscillations that occur in the physiology of the organism and the cells. The rhythms of the immune system are strictly regulated and the circadian alteration seems to have serious consequences. Even so, it is not clear how the immune cells of the intestinal mucosa synchronize with the external environment. Besides, little is known about the way in which biological rhythms affect the critical functions of intestinal immunity, such as oral tolerance. We studied fluctuations in the relevant parameters of intestinal immunity at four different times throughout the day. By using multivariate statistical tools, we found that these oscillations represent at least three different time frames with different conditions for tolerance induction that are altered in Per2ko mice lacking one of the clock genes. Our results allowed us to characterize a window in the final stage of the dark phase that promotes the induction of specific regulatory populations and favors its location in the lamina propria. We show here that, at the end of the intake, the entry of luminal antigens, soluble factors, and leukocyte populations converge in the mesenteric lymph nodes (MLN) and display the greatest potential of the tolerogenic machinery.

Enlarged colitogenic T cell population paradoxically supports colitis prevention through the B-lymphocyte-dependent peripheral generation of CD4(+)Foxp3(+) Treg cells.

Intestinal inflammation can be induced by the reconstitution of T/B cell-deficient mice with low numbers of CD4(+) T lymphocytes depleted of CD25(+)Foxp3(+) regulatory T cells (Treg). Using RAG-knockout mice as recipients of either splenocytes exclusively depleted of CD25(+) cells or FACS-purified CD4(+)CD25(-)Foxp3(-) T cells, we found that the augmentation of potentially colitogenic naïve T cell numbers in the inoculum was unexpectedly beneficial for the suppression of colon disease and maintenance of immune homeostasis. Protection against T cell-mediated colitis correlated with a significant increment in the frequency of peripherally-induced CD4(+)CD25(+)Foxp3(+) T (pTreg) cells, especially in the mesenteric lymph nodes, an effect that required the presence of B cells and CD4(+)CD25(-)Foxp3(+) cells in physiological proportions. Our findings support a model whereby the interplay between B lymphocytes and a diversified naïve T cell repertoire is critical for the generation of CD4(+)CD25(+)Foxp3(+) pTreg cells and colitis suppression.

A Hyperlipidic Diet Combined with Short-Term Ovariectomy Increases Adiposity and Hyperleptinemia and Decreases Cytokine Content in Mesenteric Adipose Tissue.

Four-week-old female Wistar rats were divided into two groups and fed a control diet (C) or a hyperlipidic diet (H) for 4 weeks. Rats from each group underwent ovariectomy (OVX) or sham surgery (SHAM). They received C or H for the next four weeks. The body weight gain (BW), food efficiency (FE), and carcass lipid content were higher in the OVX H than in the SHAM H. The OVX H exhibited a higher serum leptin level than other groups. IL-6, TNF-α, and IL-10 content of mesenteric (MES) adipose tissue was lower in the OVX H than in the OVX C. IL-6, TNF-α, and IL-10 content of retroperitoneal (RET) adipose tissue was lower in the SHAM H than in the SHAM C. The SHAM H showed decreased TG relative to the SHAM C. Similar results were obtained in relation to IL-6Rα, TNFR1, TLR-4, and MyD88 contents in the MES and RET white adipose tissue among the groups. A hyperlipidic diet for 8 weeks combined with short-term ovariectomy decreases the cytokine content of MES adipose tissues but increases BW, enhancing FE and elevating serum leptin levels. These suggest that the absence of estrogens promotes metabolic changes that may contribute to installation of a proinflammatory process induced by a hyperlipidic diet.

Inhibition of advanced glycation end products by aminoguanidine restores mast cell numbers and reactivity in alloxan-diabetic rats.

Mast cell number and reactivity have been shown to be down-regulated under diabetic conditions. This study was undertaken in order to investigate the role of the advanced glycation end products in the reduction of mast cell number and reactivity in diabetic rats. The effect of aminoguanidine on mast cell apoptosis was also evaluated. Diabetes was induced by intravenous injection of alloxan into fasted rats and aminoguanidine was administered after 3 days of diabetes induction, once daily for 18 consecutive days. Mast cell apoptosis and levels of Bax, a pro-apoptotic member of Bcl-2 family, were evaluated by TUNEL and western blot, respectively. Diabetes led to increased levels of fructosamine and AGEs in the plasma, an effect prevented by aminoguanidine. Treatment with aminoguanidine restored mast cell numbers in the pleural cavity and in mesenteric tissue of diabetic rats. Aminoguanidine also significantly reversed the diabetes-induced reduction in histamine release, as measured by fluorescence, following activation with substance P or antigen in vitro. Increased apoptosis and levels of Bax in mast cells from diabetic rats were inhibited by aminoguanidine. In conclusion, our findings showed that aminoguanidine restored the number and reactivity of mast cells in diabetic rats, accompanied by suppression of apoptosis, evidencing that advanced glycation end product formation has a critical role in mast cell behavior of diabetic rats.

Antigenic dietary protein guides maturation of the host immune system promoting resistance to Leishmania major infection in C57BL/6 mice.

The immature immune system requires constant stimulation by foreign antigens during the early stages of life to develop properly and to create efficient immune responses against later infections. We have previously shown that intake of antigenic dietary protein is critical for inducing maturation of the immune system as well as for the development of T helper type 1 (Th1) immunity. In this study, we show that administration of an amino acid (aa)-based diet during the development of the immune system subsequently resulted in inefficient control of Leishmania major infection in adult C57BL/6 mice. Compared with mice fed a control protein-containing diet, adult aa-fed mice showed a decreased interferon (IFN)-gamma response to parasite antigens and insufficient production of nitric oxide (NO), which is crucial to parasite death. However, no deviation towards Th2-specific immunity to L. major was observed. Phenotypic analysis of antigen-presenting cells (APCs) from aa-fed mice revealed deficient levels of the costimulatory molecules CD40 and CD80, and low levels of interleukin (IL)-12 produced by peritoneal macrophages, revealing an early stage of maturation of these cells. APCs isolated from aa-fed mice were unable to stimulate a Th1 response in vitro. Both phenotypic features of T cells from aa-fed mice and their ability to produce a Th1 response in the presence of mature APCs were unaffected when compared with T cells from control mice. The results presented here support the notion that regulation of Th1 immunity to infection includes environmental factors such as dietary proteins, which provide a natural source of stimulation that contributes to the process of maturation of APCs.

Depot-specific alterations to insulin signaling in mesenteric adipose tissue during intestinal inflammatory response.

Current knowledge suggests that adipose tissue is an active organ, participating in intestinal and mesenteric disease. Additionally, adipose tissue surrounds the lymph nodes and has special properties, acting as a paracrine regulator of adjacent lymphoid tissues. These adipose tissue depots can express and secrete numerous cytokines, known as adipocytokines, which then modify the action of insulin in adipose tissue itself. Using a well-accepted model of intestinal inflammation, we studied insulin signaling in mesenteric adipose tissue (MAT) and in perinodal mesenteric adipose tissue (PAT). Our results showed that the action of insulin is modified during the intestinal inflammatory response in these adipose tissue depots. MAT became resistant to insulin signaling, as evaluated by the IRS/AKT pathway, in the inflammation. This resistant status was associated with high JNK activity and the presence of infiltrating macrophages. Conversely, the adipose tissue that involves the mesenteric lymph nodes acquired greater sensitivity to insulin signaling via IRS/AKT, probably via up-regulation of IRS during experimental colitis. We demonstrated experimentally the existence of site-specific adaptive alterations in two mesenteric adipose tissue depots to the intestinal inflammatory response, probably resulting in alterations in free fatty acids and other secretory products supplied by the adjacent tissues that could act as inflammatory modulator substances.

Imprinting of CCR9 on CD4 T cells requires IL-4 signaling on mesenteric lymph node dendritic cells.

It has recently been shown that IL-4 can educate dendritic cells (DC) to differentially affect T cell effector activity. In this study, we show that IL-4 can also act upon DC to instruct naive T cells to express the gut-associated homing receptor CCR9. Thus, effector T cells generated after coculture with mesenteric lymph node (MLN)-DC show a higher expression of CCR9 when activated in the presence of IL-4. In contrast, IL-4 had no effect on CCR9 expression when naive T cells were polyclonally activated in the absence of MLN-DC, suggesting that the effect of IL-4 on CCR9 expression passed through DC. Indeed, T cells activated by MLN-DC from IL-4Ralpha(-/-) mice showed a much lower CCR9 expression and a greatly reduced migration to the small intestine than T cells activated by wild-type MLN-DC even in the presence of IL-4. Consistent with the finding that the vitamin A metabolite retinoic acid (RA) induces gut-homing molecules on T cells, we further demonstrate that IL-4 up-regulated retinaldehyde dehydrogenase 2 mRNA on MLN-DC, a critical enzyme involved in the synthesis of RA. Moreover, LE135, a RA receptor antagonist, blocked the increased expression of CCR9 driven by IL-4-treated MLN-DC. Thus, besides the direct effect of RA on T cell gut tropism, our results show that the induction of a gut-homing phenotype on CD4(+) T cells is also influenced by the effect of IL-4 on gut-associated DC.

Apoptosis differentially regulates mesenteric and subcutaneous lymph node immune responses to Trypanosoma cruzi.

Infection with Trypanosoma cruzi causes expansion of subcutaneous (SLN) and atrophy of mesenteric (MLN) lymph nodes. Here we show that excision of MLN increased parasitemia in T. cruzi-infected mice. We then studied how apoptosis of MLN cells affects immune responses to infection. T cell apoptosis increased in the MLN compared to SLN in T. cruzi-infected mice. Absolute numbers of naïve T cells decreased, and activated T cells failed to accumulate in MLN during infection. In addition, activated T cells from MLN produced less IL-2, IFN-gamma, IL-4, and IL-10 than T cells from SLN. Treatment with IL-4 or with caspase-9 inhibitor increased the recovery of viable T cells in vitro. Treatment with caspase-9 inhibitor also increased the production of cytokines by MLN T cells from infected mice. Moreover, injection of a pan caspase inhibitor prevented MLN atrophy during T. cruzi infection. Caspase-9, but not caspase-8, inhibitor also reduced MLN atrophy and increased the recovery of naïve and activated T cells from MLN. These findings indicate that caspase-mediated apoptosis and defective cytokine production are implicated in MLN atrophy and affect immune responses to T. cruzi infection.

Kinetics of mobilization and differentiation of lymphohematopoietic cells during experimental murine schistosomiasis in galectin-3 -/- mice.

Galectin-3 (gal-3), a beta-galactoside-binding animal lectin, plays a role in cell-cell and cell-extracellular matrix interactions. Extracellular gal-3 modulates cell migration and adhesion in several physiological and pathological processes. Gal-3 is highly expressed in activated macrophages. Schistosoma mansoni eggs display a large amount of gal-3 ligands on their surface and elicit a well-characterized, macrophage-dependent, granulomatous, inflammatory reaction. Here, we have investigated the acute and chronic phases of S. mansoni infection in wild-type and gal-3(-/-) mice. In the absence of gal-3, chronic-phase granulomas were smaller in diameter, displaying thinner collagen fibers with a loose orientation. Schistosoma-infected gal-3(-/-) mice had remarkable changes in the monocyte/macrophage, eosinophil, and B lymphocyte subpopulations as compared with the infected wild-type mice. We observed a reduction of macrophage number, an increase in eosinophil absolute number, and a decrease in B lymphocyte subpopulation (B220(+/high) cells) in the periphery during the evolution of the disease in gal-3(-/-) mice. B lymphopenia was followed by an increase of plasma cell number in bone marrow, spleen, and mesenteric lymph nodes of the infected gal-3(-/-) mice. The plasma IgG and IgE levels also increased in these mice. Gal-3 plays a role in the organization, collagen distribution, and mobilization of inflammatory cells to chronic-phase granulomas, niches for extramedullary myelopoiesis, besides interfering with monocyte-to-macrophage and B cell-to-plasma cell differentiation.

Annexin 1 localisation in tissue eosinophils as detected by electron microscopy.

BACKGROUND: Human and rodent leukocytes express high levels of the glucocorticoid-inducible protein annexin 1 (ANXA1) (previously referred to as lipocortin 1). Neutrophils and monocytes have abundant ANXA1 levels. AIM: We have investigated, for the first time, ANXA1 ultrastructural expression in rat eosinophils and compared it with that of extravasated neutrophils. The effect of inflammation (carrageenin peritonitis) was also monitored. METHODS: Electron microscopy was used to define the sub-cellular localisation of ANXA1 in rat eosinophils and neutrophils extravasated in the mesenteric tissue. A pair of antibodies raised against the ANXA1 N-terminus (i.e. able to recognise intact ANXA1, termed LCPS1) or the whole protein (termed LCS3) was used to perform the ultrastructural analysis. RESULTS: The majority of ANXA1 was localised in the eosinophil cytosol (approximately 60%) and nucleus (30-40%), whereas a small percentage was found on the plasma membrane (< 10%). Within the cytosol, the protein was equally distributed in the matrix and in the granules, including those containing the typical crystalloid. The two anti-ANXA1 antibodies gave similar results, with the exception that LCPS1 gave a lower degree of immunoreactivity in the plasma membrane. Inflammation (i.e. carrageenin injection) produced a modest increase in eosinophil-associated ANXA1 reactivity (significant only in the cytoplasm compartment). Extravasated neutrophils, used for comparative purposes, displayed a much higher degree of immunoreactivity for the protein. CONCLUSION: We describe for the first time ANXA1 distribution in rat eosinophil by ultrastructural analysis, and report a different protein mobilisation from extravasated neutrophils, at least in this acute model of peritonitis.

Impairment in connective tissue mast cells degranulation in spontaneously hypertensive rats: stimulus dependent resistance.

1. Microvascular permeability in the mesentery and consequent leakage of protein into the peritoneum of spontaneously hypertensive rats (SHR) and normotensive rats (NTR) was measured in vivo by the extravasation of Evans blue dye. 2. In sensitized NTR, challenge with antigen produced extensive increases in dye extravasation in the mesentery and in peritoneal lavage fluid within 10 min. 3. In sensitized SHR there was no increase in the permeability of the mesentery and a very weak increase in dye extravasation in the peritoneal cavity following challenge. 4. The glucocorticoid antagonist RU38486 did not change the permeability response induced by antigen in sensitized NTR and SHR. 5. However, compound 48/80 was equally effective in either NTR or SHR in causing increased vasopermeability. 6. Mesenteric mast cells in the NTR were degranulated after immunological challenge, whereas those in the SHR were resistant, as measured histologically. 7. Similarly, challenge ex vivo of mesentery from sensitized NTR induced contraction of guinea-pig ileum in co-incubation experiments, whereas SHR mesentery was unresponsive. 8. Plasma levels of antigen-specific IgE and IgG2a in sensitized NTR and SHR were identical. 9. Immune serum from SHR was unable to induce a passive cutaneous anaphylaxis (PCA) reaction in the skin of NTR and SHR did not develop a PCA reaction upon passive sensitization with NTR immune serum. 10. We conclude that the mast cells of SHR are resistant to degranulation following immunological challenge, although the relevant antibodies are present.

Schistosoma mansoni: peritoneal plasmacytogenesis and polypoid transformation of mesenteric milky spots in infected mice.

We have studied inflammatory reactions in the mesenteric tissue of mice infected with Schistosoma mansoni. Perivascular tissue contained diffuse infiltrates of macrophages, eosinophilic granulocytes and lymphocytes. Angiogenesis in the perivascular adipose tissue was associated with superficial plasmacytogenic foci. Polypoid structures were occasionally formed adjacent to inflammatory foci in the adipose tissue, organized around loops of capillaries, with terminal formation of a glomerular capillary network embedded in connective tissue, covered by plasmacytes. We conclude that these structures are specialized milky spots dedicated to active plasmacytogenesis and antibody secretion into the peritoneal cavity of schistosome-infected mice.

Detection of TdT-positive cells in the mesenteric lymph nodes of immunodeficient rats: study of their antigenic phenotype.

Terminal deoxynucleotidyl transferase (TdT) containing cells were found in the mesenteric lymph nodes of protein deprived and casein re-fed rats. Double immunofluorescence was used to characterize these TdT+ cells according to their surface antigenic phenotype. TdT+ cells expressing T-cell antigen markers recognized by monoclonal antibodies: W3/13 and OX-19 indicated thymic origin. It was found that these cells represented half the existing TdT+ population in the mesenteric lymph nodes. The rest of them presented the Ia antigen which is coded for by the class II major histocompatibility complex and is recognized by the OX-6 mAb. TdT+ cells presenting the OX-6+ phenotype were ascribed to a bone marrow derived subset. These results indicate that, in some instances, i.e., immunodeficiency due to protein deprivation, TdT+ cells may appear in the mesenteric lymph nodes. Their origin may be attributed either to trafficking of immature cells from the thymus or to cells that leave the bone marrow as a consequence of the damage provoked by protein deprivation.