Low molecular weight dextran sulfate as complement inhibitor and cytoprotectant in solid organ and islet transplantation

Complement is an essential part of the innate immune system and plays a crucial role in organ and islet transplantation. Its activation, triggered for example by ischemia/reperfusion (I/R), significantly influences graft survival, and blocking of complement by inhibitors has been shown to attenuate I/R injury. Another player of innate immunity are the dendritic cells (DC), which form an important link between innate and adaptive immunity. DC are relevant in the induction of an immune response as well as in the maintenance of tolerance. Modulation or inhibition of both components, complement and DC, may be crucial to improve the clinical outcome of solid organ as well as islet transplantation. Low molecular weight dextran sulfate (DXS), a well-known complement inhibitor, has been shown to prevent complement-mediated damage of the donor graft endothelium and is thus acting as an endothelial protectant. In this review we will discuss the evidence for this cytoprotective effect of DXS and also highlight recent data which show that DXS inhibits the maturation of human DC. Taken together the available data suggest that DXS may be a useful reagent to prevent the activation of innate immunity, both in solid organ and islet transplantation.

Transport and uptake of immunogenic lipids

An increasing number of lipid mediators have been identified as key modulators of immunity. Among these is a family of glycolipids capable of cellular uptake, loading onto the MHC-like molecule CD1d and stimulation of NKT cells. NKT cells are particularly interesting because they bridge innate and adaptive immunity by coordinating the early events of dendritic cell maturation, recruitment of NK cells, CD4 and CD8 T cells, and B cells at the site of microbial injury. As such, their therapeutic manipulation could be of the greatest interest in vaccine design or active immunotherapy. However, the use of NKT cells as cellular adjuvant of immunity in the clinic will require a better knowledge of the pharmacology of lipid agonists in order to optimize their action and avoid potential unseen off-target effects. We have been studying extracellular transport and cellular uptake of NKT agonists for the past few years. This field is confronted to a very limited prior knowledge and a small set of usable tools. New technology must be put in place and adapted to answering basic immunology questions related to NKT cells. The intimate link between the pharmacology of glycolipids and lipid metabolism makes us believe that great variations of bioactivity could be seen in the general population when NKT agonists are used therapeutically.

Defining the role of IL-15 trans-presentation by distinct cell-types during the development and homeostasis of Natural Killer and invariant Natural Killer T cells

The immuno-regulatory functions displayed by NK and iNKT cells have highlighted their importance as key lymphocytes involved in innate and adaptive immunity. Therefore, understanding the dynamics influencing the generation of NK and iNKT cells is extremely important. IL-15 has been shown to provide a critical signal throughout the development and homeostasis of NK and iNKT cells; however, the cellular source of IL-15 has remained unclear. In this investigation, I provide evidence that the cell-type providing IL-15 to NK and iNKT cells via trans-presentation is determined by the tissue site and the maturation status of NK and iNKT cells. For NK cells, I revealed the non-hematopoietic compartment provides IL-15 to NK cells in the early stages of development while hematopoietic cells were crucial for the generation and maintenance of mature NK cells. Regarding iNKT cells in the thymus, IL-15 trans-presentation by non-hematopoietic cells was crucial for the survival of mature iNKT cells. In the liver, both hematopoietic and non-hematopoietic compartments provided IL-15 to both immature and mature iNKT cells. This IL-15 signal helped mediate the survival and proliferation of both NK and iNKT cells as well as induce the functional maturation of mature iNKT cells via enhanced T-bet expression. In conclusion, my work illustrates an important notion that the immunological niche of NK and iNKT cells is tightly regulated and that this regulation is meticulously influenced by the tissue microenvironment.

Effects of ex vivo γ-tocopherol on airway macrophage function in healthy and mild allergic asthmatics

Elevated inflammation and altered immune responses are features found in atopic asthmatic airways. Recent studies indicate γ-tocopherol (GT) supplementation can suppress airway inflammation in allergic asthma. We studied the effects of in vitro GT supplementation on receptor-mediated phagocytosis and expression of cell surface molecules associated with innate and adaptive immunity on sputum-derived macrophages. Cells from nonsmoking healthy (n = 6) and mild house dust mite-sensitive allergic asthmatics (n = 6) were treated ex vivo with GT (300 µM) or saline (control). Phagocytosis of opsonized zymosan A bioparticles (Saccharomyces cerevisiae) and expression of surface molecules associated with innate and adaptive immunity were assessed using flow cytometry. GT caused significantly decreased (p < 0.05) internalization of attached zymosan bioparticles and decreased (p < 0.05) macrophage expression of CD206, CD36 and CD86 in allergic asthmatics but not in controls. Overall, GT caused downregulation of both innate and adaptive immune response elements, and atopic status appears to be an important factor.

Thrombosis during pregnancy: Risks, prevention, and treatment for mother and fetus--harvesting the power of omic technology, biomarkers and in vitro or in vivo models to facilitate the treatment of thrombosis

Maternal thromboembolism and a spectrum of placenta-mediated complications including the pre-eclampsia syndromes, fetal growth restriction, fetal loss, and abruption manifest a shared etiopathogenesis and predisposing risk factors. Furthermore, these maternal and fetal complications are often linked to subsequent maternal health consequences that comprise the metabolic syndrome, namely, thromboembolism, chronic hypertension, and type II diabetes. Traditionally, several lines of evidence have linked vasoconstriction, excessive thrombosis and inflammation, and impaired trophoblast invasion at the uteroplacental interface as hallmark features of the placental complications. "Omic" technologies and biomarker development have been largely based upon advances in vascular biology, improved understanding of the molecular basis and biochemical pathways responsible for the clinically relevant diseases, and increasingly robust large cohort and/or registry based studies. Advances in understanding of innate and adaptive immunity appear to play an important role in several pregnancy complications. Strategies aimed at improving prediction of these pregnancy complications are often incorporating hemodynamic blood flow data using non-invasive imaging technologies of the utero-placental and maternal circulations early in pregnancy. Some evidence suggests that a multiple marker approach will yield the best performing prediction tools, which may then in turn offer the possibility of early intervention to prevent or ameliorate these pregnancy complications. Prediction of maternal cardiovascular and non-cardiovascular consequences following pregnancy represents an important area of future research, which may have significant public health consequences not only for cardiovascular disease, but also for a variety of other disorders, such as autoimmune and neurodegenerative diseases.

Simultaneous induction of NKG2D and NKG2D ligand for promoting immune surveillance by IL-12 plus doxorubicin treatment

NKG2D (natural killer group 2, member D) and its ligands interaction in tumor microenvironment directs tumor infiltrating immune cells to recognize tumor cells, stimulate cytotoxic effector immune cells, and therefore eradicate tumor cells. IL-12, a cytokine produced by antigen presenting cells, has remarkable antitumor effect by activating innate and adaptive immunity. Doxorubicin, a commonly used chemotherapeutic agent also boosts the host antitumor immune response to cause tumor cell death. Our previous publication suggests that IL-12 plus doxorubicin enhances NKG2D function-dependent inhibition of tumor progression and promotes CD8+T cells infiltrating into tumors. The purpose of this study is to determine the underlying mechanism. Our study reveals a novel function of doxorubicin, which is to augment IL-12–induced NKG2D expression in CD8+T cells but not in NK or CD4+T cells. This observation was further validated by NK and CD8+T cell-depletion studies, in which only depletion of CD8+T cells abolished the expression of NKG2D in lymphocytes. The induced NKG2D expression in CD8+T cells is tightly associated with tumor-specific localization of CD8+T cells and improved antitumor efficacy. The IL-12 plus doxorubicin treatment-induced antitumor efficacy is also due to NKG2D ligand Rae-1 induction in tumors. Rae-1 induction in tumors is a long term effect in multiple tumor models, but not in normal tissues. A novel CD8+T cell direct contact dependent mechanism accounts for Rae-1 induction in vivo and in vitro, and CD80 is the receptor through which CD8+T cells interplay with tumor cells to upregulate Rae-1 on tumor cells. In summary, increased NKG2D expression in CD8+T cells in response to IL-12 plus doxorubicin was closely associated with tumor-specific localization of CD8+T cells and greater antitumor efficacy of the combined regimen than either agent alone. NKG2D ligand Rae-1 induction is triggered by the interaction of CD80 on tumor cells with tumor infiltrating CD+8 T cells.

Multiparameter flow cytometric analysis of C -reactive protein binding to human-derived cell lines and peripheral blood monocytes

C-reactive protein (CRP), a normally occurring human plasma protein may become elevated as much as 1,000 fold during disease states involving acute inflammation or tissue damage. Through its binding to phosphorylcholine in the presence of calcium, CRP has been shown to potentiate the activation of complement, stimulate phagocytosis and opsonize certain microorganisms. Utilizing a flow cytometric functional ligand binding assay I have demonstrated that a monocyte population in human peripheral blood and specific human-derived myelomonocytic cell lines reproducibly bind an evolutionarily conserved conformational pentraxin epitope on human CRP through a mechanism that does not involve its ligand, phosphorylcholine. ^ A variety of cell lines at different stages of differentiation were examined. The monocytic cell line, THP-1, bound the most CRP followed by U937 and KG-1a cells. The HL-60 cell line was induced towards either the granulocyte or monocyte pathway with DMSO or PMA, respectively. Untreated HL-60 cells or DMSO-treated cells did not bind CRP while cells treated with PMA showed increased binding of CRP, similar to U-937 cells. T cell and B-cell derived lines were negative. ^ Inhibition studies with Limulin and human SAP demonstrated that the binding site is a conserved pentraxin epitope. The calcium requirement necessary for binding to occur indicated that the cells recognize a conformational form of CRP. Phosphorylcholine did not inhibit the reaction therefore the possibility that CRP had bound to damaged membranes with exposed PC sites was discounted. ^ A study of 81 normal donors using flow cytometry demonstrated that a majority of peripheral blood monocytes (67.9 ± 1.3, mean ± sem) bound CRP. The percentage of binding was normally distributed and not affected by gender, age or ethnicity. Whole blood obtained from donors representing a variety of disease states showed a significant reduction in the level of CRP bound by monocytes in those donors classified with infection, inflammation or cancer. This reduction in monocyte populations binding CRP did not correlate with the concentration of plasma CRP. ^ The ability of monocytes to specifically bind CRP combined with the binding reactivity of the protein itself to a variety of phosphorylcholine containing substances may represent an important bridge between innate and adaptive immunity. ^

The Role of HBD-2 and HBD-3 in Human T Cell Development

Human β-defensins (hBDs) are a family of cationic peptides able to directly kill a wide range of microorganisms including bacteria, fungi and viruses. In addition to their antimicrobial activities, defensins also contribute to the modulation of both the host innate and adaptive immunity. In this project, we demonstrate that the αCD3/28 co-stimulation of human CD4+ T cells in the presence of 10μg/ml hBD-2 or hBD-3 together causes an up-regulation in numbers of CD4+CD69+CD25+ and CD4+CD69-CD25+ T cell subsets, indicating that the treatment of hBD-2 and 3 enhances CD4+ T cell activation. Consistent with this finding, proliferation assay using CFSE suggests that hBD-2 and hBD-3 treatment in vitro induces the proliferation of CD4+ T cells following by 96hrs culture. Analysis of expression of the regulatory T cells (Tregs) specific marker, FoxP3, reveals a shift in the CD4+CD127-CD25+ Treg subset at 18hrs. However, at the later time point, we found that the percentage of FoxP3+cells decreased in the CD4+CD127-CD25+ Treg population, whereas the presence of the FoxP3+CTLA-4+ Treg subset increased. These data indicate that Treg suppressive function may be potentially defective following the co-incubation of purified T cells with either hBD-2 or hBD-3 for 42hrs in vitro due to the apparent loss of FoxP3 expression. We further characterise the role of hBD-2 and hBD-3 in driving human CD4+ T cells polarisation. Our in vitro data suggests that treatment with hBD-2 and hBD-3 can not only induces effector T cell (Teff) differentiation into RORγt+T-bet+ (Th17/Th1) cells, but can also trigger the differentiation of Treg expressing RORγt and T-bet rather than the master controller of Treg function, FoxP3. This apparent plasticity of T cell phenotype allows them to convert from Treg to Th1/17-like effector T cell phenotype following 18hrs in culture. By 42hrs in culture, treatment with hBD-2 and hBD-3 induced both Teff cell and Treg cell differentiation towards the Th17-like phenotype. Compared with the treatment with hBD-2, treatment with hBD-3 induced a more pronounced effect to increase levels of RORγt in CD4+ T cells. This elevated expression may, in turn, be responsible for the induction of higher IL-17A secretion. Consistent with this idea, it was found that treatment with hBD-3 but not hBD-2 was capable of inducing the higher level of secretion of IL-17A. Additionally, treatment with hBD-3 induced an increased expression of IL-6, which is capable of driving the differentiation of naïve T cells towards IL-17-producing Th17 cells. Functionally, using the Treg suppression assay, the data suggested that hBD-2 may dampen down Treg cell ability to induce suppression of Teff cell activity. Interestingly, co-culture with hBD-2 would also appear to increase Teff cell resistance to Treg immunoregulation in vitro. Further investigation using microarray gene analysis revealed chemokine C-C motif ligand 1 (CCL1) as potential genes responding to hBD-2 treatment. The blockade of CCL1 has been reported to inhibit Treg suppressive function. Thus, this study explored the function of these antimicrobial candidates in regulating CD4+ T cell plasticity which could result in hBD-2 and hBD-3 being able to regulate its own production, but also may regulate Treg and Teff cell development and function, thus strengthening the link between innate and adaptive immunity

Modulation of immune cells and Th1/Th2 cytokines in insulin-treated type 2 diabetes mellitus.

Background: The role of the immune system in insulin resistance associated with type 2 diabetes has been suggested. Objectives: We assessed the profile of Th1/Th2 cytokines along with the frequencies of immune cells in insulin-treated type 2 diabetic patients (T2DP). Methods: 45 T2D patients and 43 age-matched healthy subjects were selected. Serum concentrations of T-helper type 1 (Th1) and Th2 cytokines and the frequencies of innate and adaptive immunity cells were assessed. Results: T2DP were hyperglycemic and showed high level of insulin, normal levels of triglycerides and total-cholesterol and without any change in HDL-cholesterol.Compared to healthy subjects, T2DP exhibited significant decreased frequencies of neutrophils, without any change in monocytes, eosinophils and natural killer cells. The percentages of total lymphocytes (CD3+) and CD8+-T-cells decreased whereas those of regulatory T-cells increased without any change in CD4+ T-cells in T2DP. Interestingly, the frequencies of effector CD4+-T and B-cells increased in T2DP. Serum concentrations of IL-2, IFN-γ and IL-4 decreased while IL-10 significantly enhanced in T2DP, suggesting a differentiation of CD4+T helper cells towards IL-10-producing- Teff-cells in these patients. Conclusion: Insulin-treated type 2 diabetes is associated with anti-inflammatory profile consistent with differentiation of CD4+-Th-cells towards IL-10-producing-Teff-cells, concomitant with increased frequencies of Treg and B-cells, and this may probably offer prevention against certain infections or autoimmune/inflammatory diseases.

Suppressive action of melatonin on the TH-2 immune response in rats infected with Trypanosoma cruzi

Control of the acute phase of Trypanosoma cruzi infection is critically dependent on cytokine-mediated macrophage activation to intracellular killing, natural killer (NK) cells, CD4(+) T cells, CD8(+) T cells and B cells. Cell-mediated immunity in T. cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Here we studied the role of cytokines in the regulation of innate and adaptive immunity during the acute phase of T. cruzi infection in Wistar rats. Melatonin is an effective regulator of the immune system. Macrophages and T lymphocytes, which have melatonin receptors, are target cells for the immunomodulatory function of melatonin. In this paper melatonin was orally given via two protocols: prior to and concomitant with infection. Both treatments were highly effective against T. cruzi with enhanced action for the concomitant treatment. The data suggest an up-regulation of the TH-1 immune response as all analyzed parameters, interleukin (IL)-4, IL-10, transforming growth factor-beta 1 and splenocyte proliferation, displayed reduced levels as compared with the untreated counterparts. However, the direct effects of melatonin on immune cells have not been fully investigated during T. cruzi infection. We conclude that in light of the current results, melatonin exerted important therapeutic benefits through its immune regulatory effects.

Disturbances in microbiota - cause or effect?

IBD are a group of complex polygenetic diseases also involving environmental factors. Evidence for a role for bacteria in IBD include an increased abundance of mucosa-associated bacteria in IBD (which occurs even where there is no intestinal inflammation), and the positive impact of antibiotics on the progress of both Crohn's disease (CD) and ulcerative colitis (UC) of the pouch - pouchitis. Bacteria are necessary for most animal models of IBD. The increased abundance of mucosal bacteria in IBD is not non-specific because while some mucosal bacteria are more abundant this is not the case for all mucosal bacteria including the very abundant Bacteroides vulgatus. On the other hand, antibiotic treatments are not curative, and the humoral immune Ig response to bacterial antigens which is more evident in CD, appears to be polyclonal. While this argues against a role for specific bacteria causing a classical infection, certain mucosal bacteria may damage the mucosal barrier. This would promote invasion by other commensal mucosal bacteria triggering an immune response. Altered adaptive, and to a lesser extent, innate immunity have been extensively studied, and genetic defects in the CARD15 (or NOD2) gene that encodes a bacterial sensing protein modulating innate and adaptive immunity are strongly associated with ileal CD. However, the penetrance of the homozygous CARD15 frameshift mutation, which is the most strongly CD-associated genotype, is very low with only 4% of humans with this developing CD. Furthermore, mice with the same defects in CARD15 do not develop spontaneous ileitis or colitis. Therefore, there have to be other aetiological factor(s). Altered permeability is a consistent finding in subclinical CD. There are other data to suggest that altered mucin is an early event in UC. We propose that the pathogenesis of IBD is multifactorial involving specific mucosal bacteria, defective barrier function and altered mucosal immunity in an aetiology triangle.

Regulation of autoimmune neuroinflammation by stress-responsive genes

Dissertation presented to obtain the Ph.D degree in Biology

The effects of nitric oxide on the immune system during Trypanosoma cruzi infection

Trypanosoma cruzi infection triggers substantial production of nitric oxide (NO), which has been shown to have protective and toxic effects on the host's immune system. Sensing of trypomastigotes by phagocytes activates the inducible NO-synthase (NOS2) pathway, which produces NO and is largely responsible for macrophage-mediated killing of T. cruzi. NO is also responsible for modulating virtually all steps of innate and adaptive immunity. However, NO can also cause oxidative stress, which is especially damaging to the host due to increased tissue damage. The cytokines IFN-³ and TNF-±, as well as chemokines, are strong inducers of NOS2 and are produced in large amounts during T. cruzi acute infection. Conversely, TGF-² and IL-10 negatively regulate NO production. Here we discuss the recent evidence describing the mechanisms by which NO is able to exert its antimicrobial and immune regulatory effects, the mechanisms involved in the oxidative stress response during infection and the implications of NO for the development of therapeutic strategies against T. cruzi.

Redirecting NK cells mediated tumor cell lysis by a new recombinant bifunctional protein.

Natural killer (NK) cells are at the crossroad between innate and adaptive immunity and play a major role in cancer immunosurveillance. NK cell stimulation depends on a balance between inhibitory and activating receptors, such as the stimulatory lectin-like receptor NKG2D. To redirect NK cells against tumor cells, we designed bifunctional proteins able to specifically bind tumor cells and to induce their lysis by NK cells, after NKG2D engagement. To this aim, we used the 'knob into hole' heterodimerization strategy, in which 'knob' and 'hole' variants were generated by directed mutagenesis within the CH3 domain of human IgG1 Fc fragments fused to an anti-CEA or anti-HER2 scFv or to the H60 murine ligand of NKG2D, respectively. We demonstrated the capacity of the bifunctional proteins produced to specifically coat tumor cells surface with H60 ligand. Most importantly, we demonstrated that these bifunctional proteins were able to induce an NKG2D-dependent and antibody-specific tumor cell lysis by murine NK cells. Overall, the results show the possibility to redirect NK cytotoxicity to tumor cells by a new format of recombinant bispecific antibody, opening the way of potential NK cell-based cancer immunotherapies by specific activation of the NKG2D receptor at the tumor site.

iNKT/CD1d-antitumor immunotherapy significantly increases the efficacy of therapeutic CpG/peptide-based cancer vaccine.

BACKGROUND: Therapeutic cancer vaccines aim to boost the natural immunity against transformed cancer cells, and a series of adjuvants and co-stimulatory molecules have been proposed to enhance the immune response against weak self-antigens expressed on cancer cells. For instance, a peptide/CpG-based cancer vaccine has been evaluated in several clinical trials and was shown in pre-clinical studies to favor the expansion of effector T versus Tregs cells, resulting in a potent antitumor activity, as compared to other TLR ligands. Alternatively, the adjuvant activity of CD1d-restricted invariant NKT cells (iNKT) on the innate and adaptive immunity is well demonstrated, and several CD1d glycolipid ligands are under pre-clinical and clinical evaluation. Importantly, additive or even synergistic effects have been shown upon combined CD1d/NKT agonists and TLR ligands. The aim of the present study is to combine the activation and tumor targeting of activated iNKT, NK and T cells. METHODS: Activation and tumor targeting of iNKT cells via recombinant α-galactosylceramide (αGC)-loaded CD1d-anti-HER2 fusion protein (CD1d-antitumor) is combined or not with OVA peptide/CpG vaccine. Circulating and intratumoral NK and H-2Kb/OVA-specific CD8 responses are monitored, as well as the state of activation of dendritic cells (DC) with regard to activation markers and IL-12 secretion. The resulting antitumor therapy is tested against established tumor grafts of B16 melanoma cells expressing human HER2 and ovalbumin. RESULTS: The combined CD1d/iNKT antitumor therapy and CpG/peptide-based immunization leads to optimized expansion of NK and OVA-specific CD8 T cells (CTLs), likely resulting from the maturation of highly pro-inflammatory DCs as seen by a synergistic increase in serum IL-12. The enhanced innate and adaptive immune responses result in higher tumor inhibition that correlates with increased numbers of OVA-specific CTLs at the tumor site. Antibody-mediated depletion experiments further demonstrate that in this context, CTLs rather than NK cells are essential for the enhanced tumor inhibition. CONCLUSIONS: Altogether, our study in mice demonstrates that αGC/CD1d-antitumor fusion protein greatly increases the efficacy of a therapeutic CpG-based cancer vaccine, first as an adjuvant during T cell priming and second, as a therapeutic agent to redirect immune responses to the tumor site.