Maaike J. Smelt

Immunological Properties of Inulin-Type Fructans

Beneficial effects of inulin-type fructans are discussed in view of studies that applied the oligosaccharides in colon cancer, chronic inflammatory diseases, vaccination efficacy, and prevention of infection and allergy. In the present paper, we discuss their immunomodulating effects. It is suggested that immunomodulation is elicited through indirect and direct mechanisms. Indirect mechanisms encompass stimulation of growth and activity of lactic acid bacteria, but can also be caused by fermentation products of these bacteria, i.e., short chain fatty acids. Evidence for direct effects on the immune system generally remains to be confirmed. It is suggested that inulin-type fructans can be detected by gut dendritic cells (DCs), through receptor ligation of pathogen recognition receptors (PRRs) such as Toll-like receptors, nucleotide oligomerization domain containing proteins (NODs), C-type lectin receptors, and galectins, eventually inducing pro- and anti-inflammatory cytokines. DCs may also exert antigen presenting capacity toward effector cells, such as B cells, T cells, and natural killer cells locally, or in the spleen. Inulin-type fructans may also ligate PRRs expressed on gut epithelium, which could influence its barrier function. Inulin-type fructans are potent immunomodulating food components that hold many promises for prevention of disease. However, more studies into the mechanisms, dose-effect relations, and structure-function studies are required.

L. plantarum, L. salivarius, and L. lactis Attenuate Th2 Responses and Increase Treg Frequencies in Healthy Mice in a Strain Dependent Manner

Many studies on probiotics are aimed at restoring immune homeostasis in patients to prevent disease recurrence or reduce immune-mediated pathology. Of equal interest is the use of probiotics in sub-clinical situations, which are characterized by reduced immune function or low-grade inflammation, with an increased risk of infection or disease as a consequence. Most mechanistic studies focus on the use of probiotics in experimental disease models, which may not be informative for these sub-clinical conditions. To gain better understanding of the effects in the healthy situation, we investigated the immunomodulatory effects of two Lactobacillus probiotic strains, i.e. L. plantarum WCFS1 and L. salivarius UCC118, and a non-probiotic lactococcus strain, i.e. L. lactis MG1363, in healthy mice. We studied the effect of these bacteria on the systemic adaptive immune system after 5 days of administration. Only L. plantarum induced an increase in regulatory CD103+ DC and regulatory T cell frequencies in the spleen. However, all three bacterial strains, including L. lactis, reduced specific splenic T helper cell cytokine responses after ex vivo restimulation. The effect on IFN-γ, IL5, IL10, and IL17 production by CD4+ and CD8+ T cells was dependent on the strain administered. A shared observation was that all three bacterial strains reduced T helper 2 cell frequencies. We demonstrate that systemic immunomodulation is not only observed after treatment with probiotic organisms, but also after treatment with non-probiotic bacteria. Our data demonstrate that in healthy mice, lactobacilli can balance T cell immunity in favor of a more regulatory status, via both regulatory T cell dependent and independent mechanisms in a strain dependent manner.

Susceptibility of human pancreatic β cells for cytomegalovirus infection and the effects on cellular immunogenicity.

Objectives Human cytomegalovirus (HCMV) infection has been suggested to be a causal factor in the development of type 1 diabetes, posttransplantation diabetes, and the failure of islet allografts. This effect of CMV has been interpreted as an indirect effect on the immune system rather than direct infection–induced cell death. In the present study, we investigated (i) the susceptibility of &bgr; cells to HCMV infection, (ii) regulation of immune cell–activating ligands, (iii) release of proinflammatory cytokines, and (iv) the effects on peripheral blood mononuclear cell (PBMC) activation. Methods CM insulinoma cells and primary &bgr; cells were HCMV-infected in vitro using a laboratory and a clinical HCMV strain. The susceptibility to infection was measured by the expression of viral genes and proteins. Furthermore, expression levels of Major Histocompatibility Complex I, Intracellular Adhesion Molecule-1, and Lymphocyte Function Associated Antigen-3 and the release of proinflammatory cytokines were determined. In addition, PBMC activation to HCMV-infected &bgr; cells was determined. Results &bgr; Cells were susceptible to HCMV infection. Moreover, the infection increased the cellular immunogenicity, as demonstrated by an increased MHC I and ICAM-1 expression and an increased proinflammatory cytokine release. Human cytomegalovirus–infected CM cells potently activated PBMCs. The infection-induced effects were dependent on both viral “sensing” and viral replication. Conclusions In vivo &bgr;-cell HCMV infection and infection-enhanced cellular immunogenicity may have important consequences for native or transplanted &bgr;-cell survival.

The Impact of Lactobacillus plantarum WCFS1 Teichoic Acid D-Alanylation on the Generation of Effector and Regulatory T-cells in Healthy Mice

To date it remains unclear how probiotics affect the immune system. Bacterial envelope components may play an essential role, as these are the first to establish bacterial-host cell interactions. Teichoic acids (TAs), and especially lipoteichoic acids, are the most pro-inflammatory components of the gram-positive bacterial envelope. This effect is dependent on D-alanyl substitution of the TA backbone and interactions with TLR2 on host cells. Although the pro-inflammatory properties of TAs have been established in vitro, it remains unclear how TAs affect immunomodulation in vivo. In this study, we investigated the role of TA D-alanylation on L. plantarum–induced intestinal and systemic immunomodulation in vivo. For this, we compared the effect of L. plantarum WCFS1 and its TA D-Alanylation negative derivative (dltX-D) on the distribution of dendritic cell and T cell populations and responses in healthy mice. We demonstrated that the majority of the L. plantarum-induced in vivo immunomodulatory effects were dependent on D-alanylation (D-Ala), as some L. plantarum WCFS1-induced immune changes were not observed in the dltX-D-treated group and some were only observed after treatment with dltX-D. Strikingly, not only pro-inflammatory immune responses were abolished in the absence of D-Ala substitution, but also anti-inflammatory responses, such as the L. plantarum-induced generation of regulatory T cells in the spleen. With this study we provide insight in host-microbe interactions, by demonstrating the involvement of D-alanylation of TAs on the bacterial membrane in intestinal and systemic immunomodulation in healthy mice.

Pancreatic Beta-Cell Purification by Altering FAD and NAD(P)H Metabolism

Isolation of primary beta cells from other cells within in the pancreatic islets is of importance for many fields of islet research. However, up to now, no satisfactory method has been developed that gained high numbers of viable beta cells, without considerable alpha-cell contamination. In this study, we investigated whether rat beta cells can be isolated from nonbeta endocrine cells by manipulating the flavin adenine dinucleotide (FAD) and nicotinamide-adenine dinucleotide phosphate (NAD(P)H) autofluorescence. Beta cells were isolated from dispersed islets by flow cytometry, based on their high FAD and NAD(P)H fluorescence. To improve beta cell yield and purity, the cellular FAD and NAD(P)H contents were altered by preincubation in culture media containing varying amounts of D-glucose and amino acids. Manipulation of the cellular FAD and NAD(P)H fluorescence improves beta cell yield and purity after sorting. This method is also a fast and reliable method to measure beta cell functional viability. A conceivable application is assessing beta cell viability before transplantation.

Structural surface changes and inflammatory responses against alginate‐based microcapsules after exposure to human peritoneal fluid

Microencapsulation of cells is a promising approach to prevent rejection in the absence of immunosuppression. Clinical application, however, is hampered by insufficient insight in factors influencing biocompatibility of the capsules in humans. In the present study we exposed alginate-based capsules prepared of different types of alginate to human peritoneal fluid. Subsequently we studied the physicochemical changes of the capsules surface by applying micro-Fourier Transform Infrared Spectroscopy. We did test alginate-beads and alginate-poly-L-lysine capsules prepared of different types of alginate. In all tested capsule formulations we found adsorption of components from human peritoneal fluid and clear physicochemical changes of the surface. These changes were alginate-dependent. The adsorption had no significant effects on the permselective properties of the capsule but we found a strong increase of TNFα production by human peripheral blood mononuclear cells when exposed to alginate-beads treated with human peritoneal fluid. This elevated responsiveness was not observed with alginate-PLL capsules. The results show that alginate-based capsule surfaces always undergo physicochemical changes of the surface when exposed to human peritoneal fluid. This adsorption may lead to enhancement of the inflammatory responses against the microcapsules. Our result implicate that biocompatibility measurements should not only been done with freshly prepared capsules but also with capsules that have been exposed to fluid from the implantation site in order to predict the in vivo responses.

Probiotics Can Generate FoxP3 T-Cell Responses in the Small Intestine and Simultaneously Inducing CD4 and CD8 T Cell Activation in the Large Intestine

Most studies on probiotics aim to restore intestinal homeostasis to reduce immune-pathology in disease. Of equal importance are studies on how probiotics might prevent or delay disease in healthy individuals. However, knowledge on mechanisms of probiotic actions in healthy individuals is scarce. To gain more insight in how different bacterial strains may modulate the healthy intestinal immune system, we investigated the effect of the food derived bacterial strains L. plantarum WCFS1, L. salivarius UCC118, and L. lactis MG1363, on the intestinal regulatory immune phenotype in healthy mice. All three bacterial strains induced an upregulation of activity and numbers of CD11c+ MHCII+ DCs in the immune-sampling Peyer’s Patches. Only L. salivarius UCC118 skewed towards an immune regulatory phenotype in the small intestinal lamina propria (SILP). The effects were different in the large intestine lamina propria. L. salivarius UCC118 induced activation in both CD4 and CD8 positive T-cells while L. plantarum WCFS1 induced a more regulatory phenotype. Moreover, L. plantarum WCFS1 decreased the Th1/Th2 ratio in the SILP. Also L. lactis MG1363 had immunomodulatory effects. L. lactis MG1363 decreased the expression of the GATA-3 and T-bet in the SILP. As our data show that contradictory effects may occur in different parts of the gut, it is recommended to study effects of probiotic in different sites in the intestine. Our strain-specific results suggest that unspecified application of probiotics may not be very effective. Our data also indicate that selection of specific probiotic strain activities on the basis of responses in healthy mice may be a promising strategy to specifically stimulate or suppress immunity in specific parts of the intestine.

Rat pancreatic beta cells and cytomegalovirus infection.

Objectives: Cytomegalovirus (CMV) infection has been suggested to accelerate &bgr;-cell destruction and thereby to contribute to new-onset diabetes and failure of islet allografts in both humans and rodents. Surprisingly, direct CMV infection of &bgr; cells has received only minor attention. Therefore, we investigated the susceptibility of rat &bgr; cells for rat CMV (RCMV) infection and the direct effects on the regulation of immune cell-activating ligands. Methods: Primary rat &bgr; cells, the rat &bgr;-cell line Rin-m5F, and fibroblasts were RCMV-infected in vitro. The viral gene and protein expression levels were determined as a measure for RCMV susceptibility. Gene expression levels of intracellular adhesion molecule 1, lymphocyte function associated antigen 3, rat major histocompatibility complex region A, rat major histocompatibility complex region E, toll like receptor 2, and clustered domain 14 were determined as a measure for cellular immunogenicity. Results: We demonstrate that &bgr; cells are susceptible for RCMV infection but allow only low levels of viral gene expression. In contrast, infected fibroblasts demonstrated productive viral infection and formation of viral progeny. After RCMV infection, &bgr;-cell immunogenicity was markedly increased, as demonstrated by the increased cellular expression of immune cell-activating ligands. Conclusions: Direct &bgr;-cell infection by RCMV and subsequent low-grade viral gene expression may lead to increased immunogenicity of native or transplanted &bgr; cells in vivo. An infection-induced enhanced &bgr;-cell recognizability may have important consequences for &bgr;-cell survival and the development of diabetes or rejection of islet grafts.

Effects of acute cytomegalovirus infection on rat islet allograft survival.

Transplantation of pancreatic islets is a promising therapy for the treatment of type 1 diabetes mellitus. However, long-term islet graft survival rates are still unsatisfactory low. In this study we investigated the role of cytomegalovirus (CMV) in islet allograft failure. STZ-diabetic rats received an allogenic islet graft in combination with either an acute CMV infection or control infection. A third group received ganciclovir treatment in addition to the CMV infection. Graft function was assessed by measuring basal blood glucose levels. After sacrifice, the islet grafts were retrieved for analysis of infection and leukocyte infiltration. CMV-infected recipients demonstrated accelerated islet graft failure compared to noninfected controls. CMV infection of the graft was only observed prior to complete graft failure. Quantification of the leukocyte infiltration demonstrated increased CD8+ T-cell and NK cell infiltration in the CMV-infected grafts compared to the controls. This suggests that CMV infection accelerates immune-mediated graft destruction. Antiviral ganciclovir treatment did not prevent accelerated graft failure, despite effectively decreasing the grade of infection. Our data confirm the recently published CITR data, which state that CMV is an independent risk factor for failure of islet grafts. Also, our data demonstrate that new approaches for preventing virus-induced islet allograft failure may be required.

The role of alloresponsive Ly49⁺ NK cells in rat islet allograft failure in the presence and absence of cytomegalovirus

There are still many factors to discover to explain the low success rates of islet allografts. In this study, we demonstrate that specific subpopulations of alloreactive NK cells may be involved in the failure of islet allografts. By performing allotransplantation in rats (n = 13), we observed peripheral expansion and infiltration of alloreactive Ly49i2+ NK cells in the grafts. An effective strategy in rats to enhance the expansion of Ly49i2+ NK cells is performing a rat cytomegalovirus infection (n = 6). Cytomegalovirus infection was associated with an early expansion of the Ly49i2+ NK cells and accelerated islet graft failure. The Ly49i2+ NK cells are both alloreactive and involved in virus clearance. The expansion of this subpopulation could not be blocked by cyclosporin A immunosuppression. Also alloreactive KLRH1+ NK cells infiltrated the grafts, but nonalloreactive NKR-P1B+ cells were not observed in the islet allografts. Perforin staining of the infiltrating NK cells demonstrated the cytotoxic capacity of these cells. Our data suggest a role for this NK subpopulation in rat islet allograft destruction.