Immunoregulatory Effects of Porcine Plasma Protein Concentrates on Rat Intestinal Epithelial Cells and Splenocytes

This study was funded by APC Europe and the Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds [SAF2017-88457-R, AGL2017-85270- R, BFU2014-57736-P, AGL2014-58883-R], and by Junta de Andalucía [CTS235, CTS164]. CHC, CJA and BO were supported by the University of Granada (Contrato Puente Program-Plan Propio) and the Ministry of Education [Spain], respectively. CIBERehd is funded by Instituto de Salud Carlos III.Blood contains proteins which have interest as products that may regulate immune function. For this reason some protein-based products are currently used as nutritional supplements for animals, for instance two porcine concentrates, spray dried serum (SDS), and an immunoglobulin concentrate (IC). These products have shown to protect against colonic inflammation in rodents. In the present study we characterize the ability of these products to modulate immune function in isolated cells, namely intestinal epithelial cells (IEC18 cells) and rat spleen cells. Our data indicate that both porcine protein concentrates indeed alter immune cell function, based on the secretion of the modulators known as cytokines. In intestinal epithelial IEC18 cells they promoted the secretion of GRO alpha and MCP-1 cytokines. In spleen cells they mainly inhibited the production of TNF, a key proinflammatory cytokine. In addition, the IC product augmented the release of IL-10, an anti-inflammatory cytokine. Taken together, our data indicate that the immunomodulatory effects observed in vivo are consistent with the direct actions of the protein concentrates on epithelial cells, T lymphocytes, and monocytes. Serum protein concentrates have been shown to exert in vivo anti-inflammatory effects. Specific effects on different cell types and their mechanism of action remain unraveled. We aimed to characterize the immunomodulatory effect of two porcine plasma protein concentrates, spray dried serum (SDS) and an immunoglobulin concentrate (IC), currently used as animal nutritional supplements with established in vivo immunomodulatory properties. Cytokine production by the intestinal epithelial cell line IEC18 and by primary cultures of rat splenocytes was studied. The molecular pathways involved were explored with specific inhibitors and gene knockdown. Our results indicate that both products induced GRO alpha and MCP-1 production in IEC18 cells by a MyD88/NF-kappa B-dependent mechanism. Inhibition of TNF production was observed in rat primary splenocyte cultures. The immunoglobulin concentrate induced IL-10 expression in primary splenocytes and lymphocytes. The effect on TNF was independent of IL-10 production or the stimulation of NF-kB, MAPKs, AKT, or RAGE. In conclusion, SDS and IC directly regulate intestinal and systemic immune response in murine intestinal epithelial cells and in T lymphocytes and monocytes.APC EuropeMinistry of Economy and CompetitivityEuropean Commission SAF2017-88457-R AGL2017-85270R BFU2014-57736-P AGL2014-58883-RJunta de Andalucia CTS235 CTS164University of Granada (Contrato Puente Program-Plan Propio)Ministry of Education [Spain]Instituto de Salud Carlos IIIEuropean Commissio

Epithelial deletion of the glucocorticoid receptor protects the mouse intestine against experimental inflammation

Intestinal epithelium glucocorticoid receptor knockout mice (NR3C1 IEC) were treated with dextran sulfate sodium (DSS, 2.5%) to induce colitis. Inflammatory status was assessed by morphological and biochemical methods and corticoid production was measured in colonic explants. Key Results. After 7 days of DSS NR3C1 mice exhibited lower weight loss and tissue damage, reduced colonic expression of S100A9, attenuated phosphorylation of STAT3 and a better overall state compared with WT. Ki67 immunoreactivity was also shifted, indicating an effect on epithelial proliferation. A subgroup of mice were treated with budesonide and showed completely prevented budesonide induced weight loss. Epithelial deletion of the glucocorticoid receptor also protected mice in a protracted colitis protocol. Conversely knockout mice presented a worse status compared to the control group at 1 day post DSS, as shown by blood in feces and increased inflammatory parameters. In a separate experiment colonic corticosterone production was shown to be significantly increased in knockout mice at 7 days of colitis but not at earlier stages. Conclusions and Implications. The intestinal epithelial glucocorticoid receptor has deleterious effects in experimental colitis induced by dextran sodium sulfate, probably related to inhibition of epithelial proliferative responses leading to impaired wound healing and reduced endogenous corticosterone production.Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds [BFU2014-57736-P, AGL2014-58883-R, SAF2017-88457-R, AGL2017-85270-R]Junta de Andalucía [CTS235, CTS164]Ministry of EducationCIBERehd is funded by Instituto de Salud Carlos II

A Mucosal and Cutaneous Chemokine Ligand for the Lymphocyte Chemoattractant Receptor GPR15

Chemoattractants control lymphocyte recruitment from the blood, contributing to the systemic organization of the immune system. The G protein-linked receptor GPR15 mediates lymphocyte homing to the large intestines and skin. Here we show that the 9 kDa CC-motif containing cationic polypeptide AP57/colon-derived sushi containing domain-2 binding factor (CSBF), encoded by C10orf99 in the human and 2610528A11Rik in the mouse, functions as a chemokine ligand for GPR15 (GPR15L). GPR15L binds GPR15 and attracts GPR15-expressing T cells including lymphocytes in colon-draining lymph nodes and Vγ3+ thymic precursors of dermal epithelial T cells. Patterns of GPR15L expression by epithelial cells in adult mice and humans suggest a homeostatic role for the chemokine in lymphocyte localization to the large intestines, as well as a role in homing to the epidermis during wound healing or inflammation. GPR15L is also significantly expressed in squamous mucosa of the oral cavity and esophagus with still poorly defined regulation. Identification of the chemotactic activity of GPR15L adds to its reported antibacterial and tumor cell growth regulatory functions and suggests the potential of targeting GPR15L–GPR15 interactions for modulation of mucosal and cutaneous inflammation

Modulación ejercida por los glucocorticoides sobre la función de barrera intestinal

Tesis Univ. Granada. Programa Oficial de Doctorado en: Medicina Clínica y Salud Públic

Automatic Adaptation of Model Neurons and Connections to Build Hybrid Circuits with Living Networks

Hybrid circuits built by creating mono- or bi-directional interactions among living cells and model neurons and synapses are an effective way to study neuron, synaptic and neural network dynamics. However, hybrid circuit technology has been largely underused in the context of neuroscience studies mainly because of the inherent difficulty in implementing and tuning this type of interactions. In this paper, we present a set of algorithms for the automatic adaptation of model neurons and connections in the creation of hybrid circuits with living neural networks. The algorithms perform model time and amplitude scaling, real-time drift adaptation, goal-driven synaptic and model tuning/calibration and also automatic parameter mapping. These algorithms have been implemented in RTHybrid, an open-source library that works with hard real-time constraints. We provide validation examples by building hybrid circuits in a central pattern generator. The results of the validation experiments show that the proposed dynamic adaptation facilitates closed-loop communication among living and artificial model neurons and connections, and contributes to characterize system dynamics, achieve control, automate experimental protocols and extend the lifespan of the preparationsThis work was supported by MINECO/ FEDER PGC2018-095895-B-I00, DPI2015-65833-P, TIN2017-84452-R and ONRG grant N62909-14-1-N27

Germ-free and Antibiotic-treated Mice are Highly Susceptible to Epithelial Injury in DSS Colitis

Background and Aims: Intestinal microbiota is required to maintain immune homeostasis and intestinal barrier function. At the same time, intraluminal bacteria are considered to be involved in inflammatory bowel disease and are required for colitis induction in animal models, with the possible exception of dextran sulphate sodium [DSS] colitis. This study was carried out to ascertain the mechanism underlying the induction of colitis by DSS in the absence of bacteria. Methods: Conventional and germ-free [GF] Naval Medical Research Institute [NMRI] mice were used, plus conventional mice treated with an antibiotic cocktail to deplete the intestinal microbiota [‘pseudo-GF’ or PGF mice]. The differential response to DSS was assessed. Results: Conventional mice developed DSS-induced colitis normally, whereas GF mice showed only minimal inflammation [no colonic thickening, lower myeloperoxidase activity, IL-6, IL-17, TNF- α, and IFN-γ secretion by splenocytes and mesenteric cell cultures, etc.]. However, these mice suffered enhanced haemorrhage, epithelial injury and mortality as a consequence of a weakened intestinal barrier, as shown by lower occludin, claudin 4, TFF3, MUC3, and IL-22. In contrast, PGF mice had a relatively normal, albeit attenuated, inflammatory response, but were less prone to haemorrhage and epithelial injury than GF mice. This was correlated with an increased expression of IL-10 and Foxp3 and preservation barrier-related markers. Conclusions: We conclude that enteric bacteria are essential for the development of normal DSSinduced colitis. The absence of microbiota reduces DSS colonic inflammation dramatically but it also impairs barrier function, whereas subtotal microbiota depletion has intermediate effects at both levels.Ministerio de Economía y Competividad [Spain]European Union (EU) SAF2008-01432 AGL2008-04332 SAF2011-22922 SAF2011-22812 BFU2014-57736-P AGL2014-58883-RFundación Ramón Areces [Spain]Junta de Andalucía CTS164 CTS235 CTS6736Ministerio de Educación [Spain]Swedish Research CouncilInstituto de Salud Carlos IIISpanish GovernmentEuropean Union (EU) BFU2007-30688-E/BF

Tissue Non-specific Alkaline Phosphatase Expression is Needed for the Full Stimulation of T Cells and T Cell-Dependent Colitis.

Two alkaline phosphatase isoforms, intestinal [IAP] and tissue non-specific alkaline phosphatase [TNAP], are coexpressed in mouse colon, with the latter predominating in colitis. We aimed to examine the role of TNAP in T lymphocytes, using heterozygous TNAP+/- mice [as TNAP-/- mice are non-viable]. In vitro primary cultures and in vivo T cell models using TNAP+/- mice were used. Stimulated splenocytes [lipopolysaccharide and concanavalin A] and T lymphocytes [concanavalin A and a-CD3/a-CD28] showed a decreased cytokine production and expression when compared with wild-type [WT] cells. Decreased T cell activation was reproduced by the TNAP inhibitors levamisole, theophylline, and phenylalanine in WT cells. Intraperitoneal administration of anti-CD3 in vivo resulted in reduced plasma cytokine levels, and decreased activation of splenocytes and T cells ex vivo in TNAP+/- mice. We further tested the hypothesis that TNAP expressed in T lymphocytes is involved in T cell activation and inflammation, using the lymphocyte transfer model of colitis. Rag1-/- mice were transferred with T naïve cells [CD4+ CD62L+] from TNAP+/- or WT mice and developed colitis, which was attenuated in the group receiving TNAP+/- cells. Compared with WT, T cells from TNAP+/- mice showed a decreased capacity for proliferation, with no change in differentiation. Our results offer clear evidence that TNAP modulates T lymphocyte function and specifically T cell-dependent colitis. This was associated with distinct changes in the type of TNAP expressed, probably because of changes in glycosylation

Epithelial deletion of the glucocorticoid receptor ( Nr3c1

Intestinal epithelium glucocorticoid receptor knockout mice (NR3C1 IEC) were treated with dextran sulfate sodium (DSS, 2.5%) to induce colitis. Inflammatory status was assessed by morphological and biochemical methods and corticoid production was measured in colonic explants. Key Results. After 7 days of DSS NR3C1 mice exhibited lower weight loss and tissue damage, reduced colonic expression of S100A9, attenuated phosphorylation of STAT3 and a better overall state compared with WT. Ki67 immunoreactivity was also shifted, indicating an effect on epithelial proliferation. A subgroup of mice were treated with budesonide and showed completely prevented budesonide induced weight loss. Epithelial deletion of the glucocorticoid receptor also protected mice in a protracted colitis protocol. Conversely knockout mice presented a worse status compared to the control group at 1 day post DSS, as shown by blood in feces and increased inflammatory parameters. In a separate experiment colonic corticosterone production was shown to be significantly increased in knockout mice at 7 days of colitis but not at earlier stages. Conclusions and Implications. The intestinal epithelial glucocorticoid receptor has deleterious effects in experimental colitis induced by dextran sodium sulfate, probably related to inhibition of epithelial proliferative responses leading to impaired wound healing and reduced endogenous corticosterone production.Ministry of Economy and Competitivity, partly with Fondo Europeo de Desarrollo Regional FEDER funds [BFU2014-57736-P, AGL2014-58883-R, SAF2017-88457-R, AGL2017-85270-R]Junta de Andalucía [CTS235, CTS164]Ministry of EducationCIBERehd is funded by Instituto de Salud Carlos II

Calprotectin protects against experimental colonic inflammation in mice.

Calprotectin is a heterodimer composed of two myeloid-related proteins, S100A8 and S100A9, that is abundant in neutrophils and monocytes/macrophages. Faecal levels of calprotectin are used routinely to monitor inflammatory bowel disease activity. We aimed to assess the role of calprotectin in intestinal inflammation, using the dextran sulfate sodium model of colitis in mice. Calprotectin was administered (50 or 100 μg·day-1 ) by the intrarectal or by i.p. injection (50 μg·day-1 only). The condition of the mice was characterized by morphological and biochemical methods. Intrarectal calprotectin protected significantly against colitis, as shown by lower levels of macroscopic and microscopic damage, colonic myeloperoxidase activity and decreased expression of TNFα and toll-like receptor 4. IL-17 production by spleen and mesenteric lymph node cells was reduced. Calprotectin had no effect on body weight loss or colonic thickening. There were no effects of calprotectin after i.p. injection. Calprotectin had virtually no effects in control, non-colitic mice. Calprotectin had almost no effect on the colonic microbiota but enhanced barrier function. Treatment of rat IEC18 intestinal epithelial cells in vitro with calprotectin induced output of the chemokines CXL1 and CCL2, involving the receptor for advanced glycation end products- and NFκB. Calprotectin exerted protective effects in experimental colitis when given by the intrarectal route, by actions that appear to involve effects on the epithelium