Lactococcus lactis subsp. cremoris YRC3780 modifies function of mesenteric lymph node dendritic cells to modulate the balance of T cell differentiation inducing regulatory T cells.

Introduction: The intestinal immune system plays a pivotal role in the induction of immune responses against food. In the case of T cell response, dendritic cells (DCs) are especially important. However, the regulation of immune responses to food by intestinal DCs has been poorly described. In this study, we analyzed the effect of Lactococcus lactis subsp. cremoris YRC3780, a lactic acid bacterial strain isolated from kefir, a traditional fermented milk product, on the immune responses induced by antigen presentation by intestinal DCs to T cells as well as the mechanism of action of these immunomodulatory effects. It has been shown that L. cremoris YRC3780 ameliorates the symptoms of pollinosis in both animal and human studies. Methods: CD11c+ cells from mesenteric lymph nodes (MLNs) of BALB/c mice were cultured as MLN DCs with L. cremoris YRC3780 and expression of genes inducing regulatory T cells (Tregs) was examined by qPCR. In addition, MLN DCs were cocultured with CD4+ T cells from DO11.10 transgenic mice expressing an ovalbumin (OVA)-specific TCR and the OVA antigen peptide and L. cremoris YRC3780. Induction of Tregs was examined by flow cytometry, gene expression was analyzed by DNA microarray and qPCR, and the production of cytokines was measured by ELISA. MLN DCs from TLR2-deficient mice and components of L. cremoris YRC3780 were used to examine the recognition of YRC3780 by MLN DCs. Results: L. cremoris YRC3780 enhanced the expression of genes involved in Treg induction in MLN DCs and induced Foxp3+CD4+T cells in an MLN DC and CD4+ T-cell co-culture system. The effect on MLN DCs was likely mediated by receptors other than TLR2. Together with microarray analyses of CD4+ T cell gene expression and cytokine ELISA, it was demonstrated that L. cremoris YRC3780 promoted the induction of Th1 and Tregs, and regulated the balance of Th1/Th2 and Treg/Th17 cells involving multiple genes via the antigen-presentation of MLN DCs. Discussion: Our findings provide insights into the modulation of intestinal immune responses mediated by DCs and the antiallergic effects of lactic acid bacteria.

Ileitis-associated tertiary lymphoid organs arise at lymphatic valves and impede mesenteric lymph flow in response to tumor necrosis factor.

Lymphangitis and the formation of tertiary lymphoid organs (TLOs) in the mesentery are features of Crohn's disease. Here, we examined the genesis of these TLOs and their impact on disease progression. Whole-mount and intravital imaging of the ileum and ileum-draining collecting lymphatic vessels (CLVs) draining to mesenteric lymph nodes from TNFΔARE mice, a model of ileitis, revealed TLO formation at valves of CLVs. TLOs obstructed cellular and molecular outflow from the gut and were sites of lymph leakage and backflow. Tumor necrosis factor (TNF) neutralization begun at early stages of TLO formation restored lymph transport. However, robustly developed, chronic TLOs resisted regression and restoration of flow after TNF neutralization. TNF stimulation of cultured lymphatic endothelial cells reprogrammed responses to oscillatory shear stress, preventing the induction of valve-associated genes. Disrupted transport of immune cells, driven by loss of valve integrity and TLO formation, may contribute to the pathology of Crohn's disease.

The microbiota is dispensable for the early stages of peripheral regulatory T cell induction within mesenteric lymph nodes.

Intestinal Foxp3+ regulatory T cell (Treg) subsets are crucial players in tolerance to microbiota-derived and food-borne antigens, and compelling evidence suggests that the intestinal microbiota modulates their generation, functional specialization, and maintenance. Selected bacterial species and microbiota-derived metabolites, such as short-chain fatty acids (SCFAs), have been reported to promote Treg homeostasis in the intestinal lamina propria. Furthermore, gut-draining mesenteric lymph nodes (mLNs) are particularly efficient sites for the generation of peripherally induced Tregs (pTregs). Despite this knowledge, the direct role of the microbiota and their metabolites in the early stages of pTreg induction within mLNs is not fully elucidated. Here, using an adoptive transfer-based pTreg induction system, we demonstrate that neither transfer of a dysbiotic microbiota nor dietary SCFA supplementation modulated the pTreg induction capacity of mLNs. Even mice housed under germ-free (GF) conditions displayed equivalent pTreg induction within mLNs. Further molecular characterization of these de novo induced pTregs from mLNs by dissection of their transcriptomes and accessible chromatin regions revealed that the microbiota indeed has a limited impact and does not contribute to the initialization of the Treg-specific epigenetic landscape. Overall, our data suggest that the microbiota is dispensable for the early stages of pTreg induction within mLNs.

Inflammatory myofibroblastic tumour causing intestinal obstruction in a patient with neurofibromatosis type 1.

Inflammatory myofibroblastic tumours (IMTs) are rare tumours with unpredictable biological behaviour ranging from benign to locally invasive and rarely, distant metastasis. While neurofibromatosis type 1 (NF1) may manifest with gastrointestinal soft tissue tumours, this is the first report in the literature that describes an IMT occurring in a NF1 patient who presented with intestinal obstruction. Our patient presented with intestinal obstruction secondary to an obstructing terminal ileum mesenteric tumour. En bloc bowel resection was performed, with histology revealing an IMT and an adjacent neurofibroma. The resection margins were clear and the patient was free of recurrence at six months.

Infection With Clostridioides difficile Attenuated Collagen-Induced Arthritis in Mice and Involved Mesenteric Treg and Th2 Polarization.

Objectives: Rheumatoid arthritis is an autoimmune disease with multifactorial etiopathogenesis. Among the environmental factors, mucosal infections and the inducing pathobionts are gaining increasing attention. We here set out to explore the gut-joint-axis and the impact of Clostridioides difficile infection on subsequent arthritis. Methods: We combined C. difficile infection in DBA/1J × B10.Q F1 mice with collagen induced arthritis (CIA). Mice were infected via oral gavage and infection was monitored by weight loss, colonic histology, and antibodies against bacteria. Scoring of arthritis was performed macroscopically. Intestinal microbiomes were analyzed and immune responses were monitored via quantification of transcription factor-specific mRNA isolated from the inguinal and mesenteric lymph nodes. Results: Infection with C. difficile VPI 10463 resulted in significant weight loss and severe colitis yet accelerated the reversal towards the original microbiome after antibiotic treatment. Spontaneous clearance of VPI 10463 infection reduced the incidence of subsequent CIA and led to mesenteric Treg and Th2 polarization. However, this attenuating effect was abrogated if VPI 10463 was eradicated via vancomycin followed by fecal microbiota transplantation. Moreover, VPI 10463 infection following the onset of CIA lacked therapeutic potential. Conclusion: Our results demonstrate that infection with C. difficile VPI10463 induced an inflammation of the gut that protected from subsequent arthritis development in mice. Both, microbial changes to the gut and immune cell mobilization and/or polarization may have contributed to arthritis protection. The prospect of potential therapeutic benefits resulting from C. difficile infections or some byproduct thereof call for further experiments that help elucidate exact mechanisms.

Gut Ischemia Reperfusion Injury Induces Lung Inflammation via Mesenteric Lymph-Mediated Neutrophil Activation.

Gut ischemia/reperfusion (I/R) injury is a common clinical problem associated with significant mortality and morbidities that result from systemic inflammation and remote organ dysfunction, typically acute lung injury. The mechanisms underlying the dissemination of gut-derived harmful mediators into the circulation are poorly understood. The objective of our study was to determine the role of mesenteric lymphatic circulation in the systemic and pulmonary inflammatory response to gut I/R. Using a murine intestinal I/R model, we evaluated whether and how blocking mesenteric lymph flow affects the inflammatory response in local tissues (gut) and remote organs (lungs). We further explored the mechanisms of post-I/R lymph-induced systemic inflammation by examining neutrophil activity and interaction with endothelial cells in vitro. Mice subjected to intestinal I/R displayed a significant inflammatory response in local tissues, evidenced by neutrophil infiltration into mucosal areas, as well as lung inflammation, evidenced by increased myeloperoxidase levels, neutrophil infiltration, and elevated microvascular permeability in the lungs. Mesenteric lymph duct ligation (MLDL) had no effect on gut injury per se, but effectively attenuated lung injury following gut I/R. Cell experiments showed that lymph fluid from post-I/R animals, but not pre-I/R, increased neutrophil surface CD11b expression and their ability to migrate across vascular endothelial monolayers. Moreover, post-I/R lymph upregulated neutrophil expression of pro-inflammatory cytokines and chemokines, which was mediated by a mechanism involving nuclear factor (NF)-κB signaling. Consistently, gut I/R activated NF-κB in lung neutrophils, which was alleviated by MLDL. In conclusion, all these data indicate that mesenteric lymph circulation contributes to neutrophil activation and lung inflammation following gut I/R injury partly through activating NF-κB.

Anatomical Variation in Mesenteric Macrophage Phenotypes in Crohn's Disease.

INTRODUCTION: Clinical trials are currently investigating whether an extended mesenteric resection for ileocecal resections could reduce postoperative recurrence in Crohn's disease. Resection of the mesorectum, which contains proinflammatory macrophages, during proct(ocol)ectomy, is associated with reduced recurrent inflammation and improved wound healing. We aimed to characterize the macrophages in the ileocecal mesentery, which were compared with those in the mesorectum, to provide a biological rationale for the ongoing trials. METHODS: In 13 patients with Crohn's disease and 4 control patients undergoing a proctectomy, tissue specimens were sampled at 3 locations from the mesorectum: distal (rectum), middle, and proximal (sigmoid). In 38 patients with Crohn's disease and 7 control patients undergoing ileocecal resections, tissue specimens also obtained from 3 locations: adjacent to the inflamed terminal ileum, adjacent to the noninflamed ileal resection margin, and centrally along the ileocolic artery. Immune cells from these tissue specimens were analyzed by flow cytometry for expression of CD206 to determine their inflammatory status. RESULTS: In the mesorectum, a gradient from proinflammatory to regulatory macrophages from distal to proximal was observed, corresponding to the adjacent inflammation of the intestine. By contrast, the ileocecal mesentery did not contain high amounts of proinflammatory macrophages adjacent to the inflamed tissue, and a gradient toward a more proinflammatory phenotype was seen in the central mesenteric area. DISCUSSION: Although the mesentery is a continuous structure, the mesorectum and the ileocecal mesentery show different immunological characteristics. Therefore, currently, there is no basis to perform an extended ileocecal resection in patients with Crohn's disease.

Myeloperoxidase instigates proinflammatory responses in a cecal ligation and puncture rat model of sepsis.

Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.

Engagement of Posthemorrhagic Shock Mesenteric Lymph on CD4+ T Lymphocytes In Vivo and In Vitro.

BACKGROUND: Immune dysfunction is associated with posthemorrhagic shock mesenteric lymph (PHSML) return. To determine the proliferation and cytokine production capacity of CD4+ T lymphocytes, the effect of PHSML drainage on spleen CD4+ T lymphocytes in a mouse model of hemorrhagic shock was assessed. METHODS: The normal spleen CD4+ T lymphocytes were in vitro incubated with either drained normal mesenteric lymph (NML), PHSML during hypotension (PHSML-H), or PHSML from 0 h to 3 h after resuscitation (PHSML-R) to verify direct proliferation effects of PHSML. RESULTS: Hemorrhagic shock led to reduction of proliferation and mRNA expression of interleukin 2 (IL-2) and IL-2 receptor in CD4+ T lymphocytes and to decrease in IL-2 and interferon γ (IFN-γ) levels in supernatants. In contrast, the interleukin-4 levels were increased. These effects were reversed by PHSML drainage. Moreover, NML incubation promoted CD4+ T lymphocyte proliferation, whereas both PHSML-H and PHSML-R treatment had a biphasic effects on CD4+ T lymphocyte proliferation, exhibiting an enhanced effect at early stages and an inhibitory effect at later stages. Compared with NML, PHSML-H increased IL-2 expression at 12 h, but decreased expression of both IL-2 and IFN-γ at 24 h. By contrast, PHSML-R induced significant increases in IL-2 and IFN-γ levels at 24 h. Interleukin-4 expression in CD4+ T lymphocytes was reduced at 12 h, but augmented at 24 h after incubation with either PHSML-H or PHSML-R. CONCLUSIONS: The results indicate that PHSML has a direct inhibitory effect on CD4+ T lymphocyte proliferation that induces an inflammatory response, which is associated with cellular immune dysfunction.

Correlation Analysis of Morphological Changes of Structural and Functional Areas of Mesenteric Lymph Nodes with Cytokines of Lymph of Thoracic Lymphatic Duct in Experimental Breast Cancer.

We performed a correlation analysis of the morphometric parameters of mesenteric lymph nodes and cytokine content in the lymph of thoracic duct in rats with chemically induced breast cancer. The study showed that activity of the local immune response in the lymph nodes in breast cancer is aimed at antitumor protection. In breast cancer, the area of the paracortical zone remained at the level of the intact group, while the area of lymphoid nodules with germinative centers and the area of medullary substance increased; the number of macrophages in the thymus-dependent zone and zone responsible for humoral immunity also increased. The following positive correlations were revealed: in germinative centers and medullary substance, number of mitotic cells correlated with cytokine IL-5 content and the number of medium lymphocytes correlated with the content of chemokine MIP-1α; in the germinative centers, the number of immunoblasts correlated with the level of cytokine GRO/KC, in the paracortical zone, the number of macrophages correlated with the level of chemokine MCP-1, the number of reticular cells correlated with IL-6 and M-CSF content; in medullary substance, the number of small lymphocytes and mature cells plasma cells (their content was reduced) correlated with the level of chemokine GRO/KC, which can be caused by their migration from the lymph node.

Dendritic Cells of Mesenteric and Regional Lymph Nodes Contribute to Yersinia enterocolitica O:3-Induced Reactive Arthritis in TNFRp55-/- Mice.

Dendritic cells (DCs) participate in the pathogenesis of several diseases. We investigated DCs and the connection between mucosa and joints in a murine model of Yersinia enterocolitica O:3-induced reactive arthritis (ReA) in TNFRp55-/- mice. DCs of mesenteric lymph nodes (MLN) and joint regional lymph nodes (RLN) were analyzed in TNFRp55-/- and wild-type mice. On day 14 after Y. enterocolitica infection (arthritis onset), we found that under TNFRp55 deficiency, migratory (MHChighCD11c+) DCs increased significantly in RLN. Within these RLN, resident (MHCintCD11c+) DCs increased on days 14 and 21. Similar changes in both migratory and resident DCs were also detected on day 14 in MLN of TNFRp55-/- mice. In vitro, LPS-stimulated migratory TNFRp55-/- DCs of MLN increased IL-12/23p40 compared with wild-type mice. In addition, TNFRp55-/- bone marrow-derived DCs in a TNFRp55-/- MLN microenvironment exhibited higher expression of CCR7 after Y. enterocolitica infection. The major intestinal DC subsets (CD103+CD11b-, CD103-CD11b+, and CD103+CD11b+) were found in the RLN of Y. enterocolitica-infected TNFRp55-/- mice. Fingolimod (FTY720) treatment of Y. enterocolitica-infected mice reduced the CD11b- subset of migratory DCs in RLN of TNFRp55-/- mice and significantly suppressed the severity of ReA in these mice. This result was associated with decreased articular IL-12/23p40 and IFN-γ levels. In vitro FTY720 treatment downregulated CCR7 on Y. enterocolitica-infected bone marrow-derived DCs and purified MLN DCs, which may explain the mechanism underlying the impairment of DCs in RLN induced by FTY720. Taken together, data indicate the migration of intestinal DCs to RLN and the contribution of these cells in the immunopathogenesis of ReA, which may provide evidence for controlling this disease.

Reference intervals of mesenteric lymph node size according to lymphocyte counts in asymptomatic children.

There is no acknowledged reference interval of mesenteric lymph node size in healthy children, and the size criterion for mesenteric lymph node enlargement (MLNE) has long been controversial. This study aimed to explore the reference intervals of mesenteric lymph node size according to lymphocyte counts in asymptomatic children and to develop a more appropriate definition of MLNE. The asymptomatic children included were divided into five age strata: 2 to 3 yr; 3 to 4 yr; 4 to 5 yr; 5 to 6 yr; and 6 to 7 yr. Correlation analyses between lymphocyte counts and the long-axis diameter, short-axis diameter, and average diameter of the largest mesenteric lymph node (LMLN) were performed. A reference interval of the short-axis diameter of LMLN was established according to this correlation analysis in each age group. We also report a reference interval of lymphocyte count in each age group. This study revealed significant correlations between the short-axis diameter of LMLN and lymphocyte count in all age groups, as well as in subdivided boy groups and girl groups. The overall reference interval of the short-axis diameter of LMLN in children was 0.54 cm-1.03 cm, with mean value of 0.75 cm. This study supports the use of the short-axis diameter greater than 8-10 mm as the diagnostic criterion for primary mesenteric lymphadenitis based on the presence of a cluster of three or more mesenteric lymph nodes and in the absence of other abnormalities.

Dendritic Cell Subsets in Intestinal Immunity and Inflammation.

The mammalian intestine is a complex environment that is constantly exposed to Ags derived from food, microbiota, and metabolites. Intestinal dendritic cells (DC) have the responsibility of establishing oral tolerance against these Ags while initiating immune responses against mucosal pathogens. We now know that DC are a heterogeneous population of innate immune cells composed of classical and monocyte-derived DC, Langerhans cells, and plasmacytoid DC. In the intestine, DC are found in organized lymphoid tissues, such as the mesenteric lymph nodes and Peyer's patches, as well as in the lamina propria. In this Brief Review, we review recent work that describes a division of labor between and collaboration among gut DC subsets in the context of intestinal homeostasis and inflammation. Understanding relationships between DC subtypes and their biological functions will rationalize oral vaccine design and will provide insights into treatments that quiet pathological intestinal inflammation.

B cell hyperactivation in an Ackr4-deficient mouse strain is not caused by lack of ACKR4 expression.

The majority of genetically modified C57BL/6 mice contain congenic passenger DNA around the targeted gene locus as they were generated from 129-derived embryonic stem cells (ESCs) with subsequent backcrossing to the C57BL/6 genetic background. When studying the role of atypical chemokine receptor 4 (ACKR4) in the immune system, we realized that the two available Ackr4-deficient mouse strains (Ackr4-/- and Ackr4GFP/GFP ) show profoundly different phenotypes: Compared to wild-type and Ackr4GFP/GFP mice, Ackr4-/- mice show a strong accumulation of plasma blasts in mesenteric lymph node and spleen as well as increased B cell proliferation after in vitro activation. This phenotype was maintained after further backcrossing to C57BL/6 mice and was even present in heterozygous Ackr4+/- animals, suggesting that a gene variant on the targeted chromosome might cause this phenotype. Exome sequencing revealed that a region of approximately 20 Mbp around the Ackr4 locus on chromosome 9 still originates from the 129 background based on high variant density observed. In activated Ackr4-/- and Ackr4GFP/GFP B cells, transcripts of genes around the Ackr4 locus were equally deregulated compared to C57BL/6 B cells, whereas increased expression of IL-6 was selectively observed in B cells of Ackr4-/- mice. Because the gene encoding for IL-6 is placed on chromosome 5 these findings suggest that passenger DNA around the Ackr4 locus has an indirect effect on B cell activation and IL-6 production. Results of the present study should not only lead to the reinterpretation of data from earlier studies using Ackr4-/- mice but should remind the scientific community about the limitations of mouse models using mice created by gene-targeting of nonsyngeneic ESCs.

SIV-specific CD8+ T cells are clonotypically distinct across lymphoid and mucosal tissues.

CD8+ T cell responses are necessary for immune control of simian immunodeficiency virus (SIV). However, the key parameters that dictate antiviral potency remain elusive, conceivably because most studies to date have been restricted to analyses of circulating CD8+ T cells. We conducted a detailed clonotypic, functional, and phenotypic survey of SIV-specific CD8+ T cells across multiple anatomical sites in chronically infected rhesus macaques with high (>10,000 copies/mL plasma) or low burdens of viral RNA (<10,000 copies/mL plasma). No significant differences in response magnitude were identified across anatomical compartments. Rhesus macaques with low viral loads (VLs) harbored higher frequencies of polyfunctional CXCR5+ SIV-specific CD8+ T cells in various lymphoid tissues and higher proportions of unique Gag-specific CD8+ T cell clonotypes in the mesenteric lymph nodes relative to rhesus macaques with high VLs. In addition, public Gag-specific CD8+ T cell clonotypes were more commonly shared across distinct anatomical sites than the corresponding private clonotypes, which tended to form tissue-specific repertoires, especially in the peripheral blood and the gastrointestinal tract. Collectively, these data suggest that functionality and tissue localization are important determinants of CD8+ T cell-mediated efficacy against SIV.

Early-life programming of mesenteric lymph node stromal cell identity by the lymphotoxin pathway regulates adult mucosal immunity.

Redundant mechanisms support immunoglobulin A (IgA) responses to intestinal antigens. These include multiple priming sites [mesenteric lymph nodes (MLNs), Peyer's patches, and isolated lymphoid follicles] and various cytokines that promote class switch to IgA, even in the absence of T cells. Despite these backup mechanisms, vaccination against enteric pathogens such as rotavirus has limited success in some populations. Genetic and environmental signals experienced during early life are known to influence mucosal immunity, yet the mechanisms for how these exposures operate remain unclear. Here, we used rotavirus infection to follow antigen-specific IgA responses through time and in different gut compartments. Using genetic and pharmacological approaches, we tested the role of the lymphotoxin (LT) pathway-known to support IgA responses-at different developmental stages. We found that LT-ß receptor (LTßR) signaling in early life programs intestinal IgA responses in adulthood by affecting antibody class switch recombination to IgA and subsequent generation of IgA antibody-secreting cells within an intact MLN. In addition, early-life LTßR signaling dictates the phenotype and function of MLN stromal cells to support IgA responses in the adult. Collectively, our studies uncover new mechanistic insights into how early-life LTßR signaling affects mucosal immune responses during adulthood.

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.

Estimating Residence Times of Lymphocytes in Ovine Lymph Nodes.

The ability of lymphocytes to recirculate between blood and secondary lymphoid tissues such as lymph nodes (LNs) and spleen is well established. Sheep have been used as an experimental system to study lymphocyte recirculation for decades and multiple studies document accumulation and loss of intravenously (i.v.) transferred lymphocytes in efferent lymph of various ovine LNs. Yet, surprisingly little work has been done to accurately quantify the dynamics of lymphocyte exit from the LNs and to estimate the average residence times of lymphocytes in ovine LNs. In this work we developed a series of mathematical models based on fundamental principles of lymphocyte recirculation in the body under non-inflammatory (resting) conditions. Our analysis suggested that in sheep, recirculating lymphocytes spend on average 3 h in the spleen and 20 h in skin or gut-draining LNs with a distribution of residence times in LNs following a skewed gamma (lognormal-like) distribution. Our mathematical models also suggested an explanation for a puzzling observation of the long-term persistence of i.v. transferred lymphocytes in the efferent lymph of the prescapular LN (pLN); the model predicted that this is a natural consequence of long-term persistence of the transferred lymphocytes in circulation. We also found that lymphocytes isolated from the skin-draining pLN have a 2-fold increased entry rate into the pLN as opposed to the mesenteric (gut-draining) LN (mLN). Likewise, lymphocytes from mLN had a 3-fold increased entry rate into the mLN as opposed to entry rate into pLN. In contrast, these cannulation data could not be explained by preferential retention of cells in LNs of their origin. Taken together, our work illustrates the power of mathematical modeling in describing the kinetics of lymphocyte migration in sheep and provides quantitative estimates of lymphocyte residence times in ovine LNs.

Differential Pathogenic Th17 Profile in Mesenteric Lymph Nodes of Crohn's Disease and Ulcerative Colitis Patients.

The drug targets IL23 and IL12 regulate pathogenicity and plasticity of intestinal Th17 cells in Crohn's disease (CD) and ulcerative colitis (UC), the two most common inflammatory bowel diseases (IBD). However, studies examining Th17 dysregulation in mesenteric lymph nodes (mLNs) of these patients are rare. We showed that in mLNs, CD could be distinguished from UC by increased frequencies of CCR6+CXCR3-RORγ+Tbet-CD4+ (Th17) memory T cells enriched in CD62Llow effector memory T cells (TEM), and their differentially expressed molecular profile. Th17 TEM cells (expressing IL17A, IL17F, RORC, and STAT3) displayed a higher pathogenic/cytotoxic (IL23R, IL18RAP, and GZMB, CD160, PRF1) gene signature in CD relative to UC, while non-pathogenic/regulatory genes (IL9, FOXP3, CTLA4) were more elevated in UC. In both CD and UC, IL12 but not IL23, augmented IFNγ expression in Th17 TEM and switched their molecular profile toward an ex-Th17 (Th1*)-biased transcriptomic signature (increased IFNG, and decreased TCF7, IL17A), suggesting that Th17 plasticity occurs in mLNs before their recruitment to inflamed colon. We propose that differences observed between Th17 cell frequencies and their molecular profile in CD and UC might have implications in understanding disease pathogenesis, and thus, therapeutic management of patients with IBD.