Osteopontin in the host response to Leishmania amazonensis.

BACKGROUND: Leishmania (L.) spp are intracellular eukaryotic parasites responsible for cutaneous or visceral leishmaniasis, replicating predominantly in macrophages (MF). In C57BL/6 mice virulence with L. amazonensis has been associated with inhibition of Th1 immune responses and an uncontrolled lesion development, whereas DBA/2 mice control any lesion. Parasitic clearance by the MFs requires the activation of proper immune responses. One of the immune related genes expressed in immune cells including MF, codes for osteopontin (OPN). OPN is a secreted glycoprotein, acting as an immune regulator. Its implication in promoting Th1 immunity in response to infectious microorganisms and its known protective effect against viral and bacterial infections via activation of the immune response, render OPN a molecule of interest in the study of the host response to L. amazonensis. RESULTS: We examined the host response to L. amazonensis of opn mutant and wild type C57BL/6 mice. Bone marrow derived MFs were infected with the parasites in vitro, and opn mutant and wild type mice were inoculated in vivo by intradermal injection in the ears. The DBA/2 strain known to control L. amazonensis infection was also used for comparison. Our data indicate that the parasites increased opn gene expression and OPN protein while parasitic proliferation was contained in the presence of OPN. In the presence of parasites the expression of inflammation-related transcripts was inhibited. Interleukin-1-beta (IL-1ß), and transcripts of the NLR-family (NLRC4, NLRP3) were down regulated after L. amazonensis infection. In the absence of OPN, the inhibition by the parasites of IL-1ß transcripts was less efficient and a pyroptosis-like cell phenotype was detected in vitro, suggesting a central role of OPN in the host-response to L. amazonensis. Similarly, in vivo, in the absence of OPN, while the clinical inflammatory phenotype is more severe, an increase of these transcripts was observed. CONCLUSIONS: L. amazonensis infection induces opn gene expression and protein, which in turn participates in shaping the host response to the parasites, seemingly by decreasing the activation of inflammation. OPN, further evaluated as a target for Leishmaniasis control represents an additional interest in improving vaccination strategies against the parasites.

Bone marrow monocytes sustain NK cell-poiesis during non-alcoholic steatohepatitis.

Natural killer (NK) cells are the predominant lymphocyte population in the liver. At the onset of non-alcoholic steatohepatitis (NASH), an accumulation of activated NK cells is observed in the liver in parallel with inflammatory monocyte recruitment and an increased systemic inflammation. Using in vivo and in vitro experiments, we unveil a specific stimulation of NK cell-poiesis during NASH by medullary monocytes that trans-present interleukin-15 (IL-15) and secrete osteopontin, a biomarker for patients with NASH. This cellular dialogue leads to increased survival and maturation of NK precursors that are recruited to the liver, where they dampen the inflammatory monocyte infiltration. The increase in the production of both osteopontin and the IL-15/IL-15Rα complex by bone marrow monocytes is induced by endotoxemia. We propose a tripartite gut-liver-bone marrow axis regulating the immune population dynamics and effector functions during liver inflammation.

Investigation of type 1 diabetes in NOD mice knockout for the osteopontin gene.

OBJECTIVE: Type 1 diabetes onset is preceded by a pre-inflammatory stage leading to insulitis and followed by targeted destruction of the insulin-producing beta cells of the pancreas. Osteopontin (OPN) is a secreted phosphoprotein with cytokine properties, implicated in many physiological and pathological processes, including infection and autoimmunity. We have previously identified up-regulated osteopontin transcripts in the pancreatic lymph nodes of the NOD (Non-Obese Diabetic) mouse at the pre-diabetic stages. Investigating the underlined disease initiating mechanisms may well contribute to the development of novel preventive therapies. Our aim was to construct opn null mice in a NOD autoimmune-prone genetic background and address the pathogenic or protective role of the osteopontin molecule in the early stages of type 1 diabetes. METHODS: We generated opn null mutant mice in a NOD genetic background by serial backcrossing to the existing C57BL/6 opn knockout strain. The presence of opn wild type or null alleles in the congenic lines was evaluated by PCR amplification. We used NOD opn-null mice to assess the phenotypic evolution of type 1 diabetes. The presence of OPN in the serum was evaluated by ELISA and by immunostaining on the mouse tissues. The primary gene structure of the NOD opn encoding gene and protein sequences were compared to the known alleles of other mouse strains. Evaluation of Single Nucleotide Polymorphisms (SNPs) variation between opn alleles of the opn gene is reported. RESULTS: In the absence of OPN, type 1 diabetes is accelerated, suggesting a protective role of this cytokine on the insulin-producing cells of the pancreatic islets. Conversely, in the presence of the opn gene, an increase of the OPN protein in the serum of young NOD mice indicates that this molecule might be involved in the immune regulatory events taking place at early stages, prior to disease onset. Our data support that OPN acts as a positive regulator of the early islet autoimmune damage, possibly by a shift of the steady-state of T1D pathogenesis. We report that the OPN protein structure of the NOD/ShiLtJ strain corresponds to the a-type allele of the osteopontin gene. Comparative analysis of the single nucleotide polymorphisms between the a-type and b-type alleles indicates that the majority of variations are within the non-coding regions of the gene. CONCLUSIONS: The construction of opn null mice in an autoimmune genetic background (NOD.B6.Cg-spp1-/-) provides important tools for the study of the implication of the OPN in type 1 diabetes, offering the possibility to address the significance of this molecule as an early marker of the disease and as a therapeutic agent in preclinical studies.

RNA Extraction from Ears and Draining Lymph Nodes of Mice Infected with Leishmania amazonensis.

Parasites of the genus Leishmania infect the mammalian hosts, including mice and humans and cause cutaneous or visceral leishmaniasis depending upon the parasite species transmitted by the vector sandfly. Leishmania amazonensis is one of the Leishmania species responsible for the cutaneous form of the disease. We have inoculated with these parasites the ear dermis of mice. RNA preparations were performed from fragmented tissues using a buffer containing guanidin isothiocynate (RLT buffer, RNeasy Mini Kit, Qiagen, SAS, France) and ß-mercaptoethanol. Both reagents facilitate the isolation of intact RNA from tissues and the use of the RNeasy Kits present with several advantages that facilitate the isolation of pure non-degraded total RNA: i) This method allows to avoid the presence of phenol in the RNA extraction buffer, commonly used in alternative protocols; ii) Moreover Diethylpyrocarbonate (DEPC) treatment of glassware, to avoid RNAses contamination of the samples, is not required with this protocol; iii) Finally, it is a fast procedure and the isolated total RNA may be concentrated in a small volume thus facilitating its use for downstream experimental procedures.

Autoimmunity and autoinflammation: A systems view on signaling pathway dysregulation profiles.

INTRODUCTION: Autoinflammatory and autoimmune disorders are characterized by aberrant changes in innate and adaptive immunity that may lead from an initial inflammatory state to an organ specific damage. These disorders possess heterogeneity in terms of affected organs and clinical phenotypes. However, despite the differences in etiology and phenotypic variations, they share genetic associations, treatment responses and clinical manifestations. The mechanisms involved in their initiation and development remain poorly understood, however the existence of some clear similarities between autoimmune and autoinflammatory disorders indicates variable degrees of interaction between immune-related mechanisms. METHODS: Our study aims at contributing to a holistic, pathway-centered view on the inflammatory condition of autoimmune and autoinflammatory diseases. We have evaluated similarities and specificities of pathway activity changes in twelve autoimmune and autoinflammatory disorders by performing meta-analysis of publicly available gene expression datasets generated from peripheral blood mononuclear cells, using a bioinformatics pipeline that integrates Self Organizing Maps and Pathway Signal Flow algorithms along with KEGG pathway topologies. RESULTS AND CONCLUSIONS: The results reveal that clinically divergent disease groups share common pathway perturbation profiles. We identified pathways, similarly perturbed in all the studied diseases, such as PI3K-Akt, Toll-like receptor, and NF-kappa B signaling, that serve as integrators of signals guiding immune cell polarization, migration, growth, survival and differentiation. Further, two clusters of diseases were identified based on specifically dysregulated pathways: one gathering mostly autoimmune and the other mainly autoinflammatory diseases. Cluster separation was driven not only by apparent involvement of pathways implicated in adaptive immunity in one case, and inflammation in the other, but also by processes not explicitly related to immune response, but rather representing various events related to the formation of specific pathophysiological environment. Thus, our data suggest that while all of the studied diseases are affected by activation of common inflammatory processes, disease-specific variations in their relative balance are also identified.

Interferon-alpha as a mediator of polyinosinic:polycytidylic acid-induced type 1 diabetes.

A number of studies and clinical case reports have implicated interferon (IFN)-alpha as a potential mediator of type 1 diabetes pathogenesis. Administration of polyinosinic:polycytidylic acid (poly I:C), a mimic of viral double-stranded RNA, induces diabetes in C57BL/6 mice expressing the B7.1 costimulatory molecule in islets. We investigated the potential role of IFN-alpha in this disease model. The quantitative correlation between IFN-alpha levels and time to diabetes, diabetes prevention with anti-IFN-alpha antibody, and ability of IFN-alpha itself to induce diabetes are consistent with the hypothesis that poly I:C in this model acts by induction of IFN-alpha in a genetically susceptible host. Numerous recent studies highlight the importance of the innate immune system and toll receptors in determining adaptive immune responses, and we speculate that for type 1 diabetes, viral and other environmental factors may act through induction of IFNs.

Functional genomics in early autoimmunity.

The molecular mechanisms initiating the autoimmune process in type 1 diabetes mellitus (T1DM) remain unknown, and studies aiming to address this question have been compromised by the difficulty of predicting the disease at an early age both in humans and in animal models. An additional hindrance in selecting individuals at an early age has been the complex genetic inheritance of autoimmune diabetes, implicating not only several genes but also environmental factors. We have previously demonstrated the predictive value of insulin autoantibodies (IAAs) at an early age, between three to five weeks in the NOD mouse. Animals positive for early appearance of IAAs (E-IAAs) develop autoimmune diabetes earlier. We showed a correlation between the presence of IAAs in the mothers during pregnancy, E-IAAs in the litters, and the early appearance of T1DM. NOD mice, E-IAA-positive, within litters from IAA-positive mothers during pregnancy, develop diabetes earlier and at a much greater rate than animals that are IAA-negative and from IAA-negative mothers. The molecular mechanisms responsible for this early autoimmune subphenotype were addressed by a global approach to differential gene expression analysis in the pancreatic lymph nodes (PaLNs). Although the data analysis is currently in progress, gene expression signatures were observed that are characteristic for PaLNs with regard to the presence or absence of IAAs. Overall, these data are consistent with the hypothesis of an early environmental influence from the autoimmune maternal environment on the genetic predisposition of the offspring, characterized by specific gene signatures leading to autoimmune disease.

Genetic architecture of early pre-inflammatory stage transcription signatures of autoimmune diabetes in the pancreatic lymph nodes of the NOD mouse reveals significant gene enrichment on chromosomes 6 and 7.

Autoimmune diseases are characterized by the stimulation of an excessive immune response to self-tissues by inner and/or outer organism factors. Common characteristics in their etiology include a complex genetic predisposition and environmental triggers as well as the implication of the major histocompatibility (MHC) locus on human chromosome 6p21. A restraint number of non-MHC susceptibility genes, part of the genetic component of type 1 diabetes have been identified in human and in animal models, while the complete spectrum of genes involved remains unknown. We elaborate herein patterns of chromosomal organization of 162 genes differentially expressed in the pancreatic lymph nodes of Non-Obese Diabetic mice, carefully selected by early sub-phenotypic evaluation (presence or absence of insulin autoantibodies). Chromosomal assignment of these genes revealed a non-random distribution on five chromosomes (47%). Significant gene enrichment was observed in particular for two chromosomes, 6 and 7. While a subset of these genes coding for secreted proteins showed significant enrichment on both chromosomes, the overall pool of genes was significantly enriched on chromosome 7. The significance of this unexpected gene distribution on the mouse genome is discussed in the light of novel findings indicating that genes affecting common diseases map to recombination "hotspot" regions of mammalian genomes. The genetic architecture of transcripts differentially expressed in specific stages of autoimmune diabetes offers novel venues towards our understanding of patterns of inheritance potentially affecting the pathological disease mechanisms.

Absence of the T-bet gene coding for the Th1-related transcription factor does not affect diabetes-associated phenotypes in Balb/c mice.

The T-box expressed in T cells gene (T-bet) is a member of the T-box family of transcription factors. T-bet-deficient mice show normal lymphoid development, but exhibit profound defects in their Th1-mediated immune responses. As the balance between Th1- and Th2-mediated immune responses plays a role in autoimmune-prone diseases, we have investigated the diabetes-related insulin autoantibody (IAA) and cellular immune responses (insulitis), in the absence of Th1 lineage commitment, in T-bet KO Balb/c mice, after immunization with the B9-23 insulin peptide. We have therefore investigated whether absence of the T-bet gene influences diabetes-related phenotypes in Balb/c T-bet KO mice.

Establishing insulin 1 and insulin 2 knockout congenic strains on NOD genetic background.

As insulin is a major autoantigen in autoimmune diabetes and because the insulin gene region locus in humans has been linked to diabetes risk, we have bred insulin gene knockouts onto the NOD mouse. Mice differ from humans in terms that they express two nonallelic genes of insulin. Insulin 2 is the murine homologue of the human insulin gene and is located on mouse chromosome 7. Insulin 1 is thought to have evolved by a gene duplication event, lacks the second intron of the insulin 2 gene, and is located on mouse chromosome 19. The differential thymic expression of the insulin gene may be important for central tolerance induction. Here, we present the initial establishment of congenic knockouts and characterization of the congenic intervals corresponding to insulin 1 and insulin 2 knockout genes on mouse chromosome 19 and 7, respectively.

Investigation of secreted protein transcripts as early biomarkers for type 1 diabetes in the mouse model.

Type 1 diabetes (T1D) represents a serious health burden in the world, complicated by the fact that disease onset can be preceded by a long time period without evident clinical signs. It would be then of critical importance to detect the disease in its early stages. In this direction, we seek here to identify early preinflammatory markers for autoimmune diabetes, mining our previously reported transcriptome data relevant to distinct early sub-phenotypes in the NOD mouse, associated with early insulin autoantibodies (E-IAA). More specifically we focus on secreted or transmembrane protein transcripts, identifying in this category 71 differentially expressed transcripts which are regulated at the early preinflammatory stages of T1D in the pancreatic lymph nodes (PLN). Following the expression patterns of these 71 transcripts, correspondence analysis (a multivariate analysis method) reveals a clear-cut segregation of the individual samples according to the early subphenotype used. Thus the 71 transcripts coding for secreted proteins constitute a candidate-set of predictive biomarkers for the development of autoimmune damage of the ß cells of the pancreas. The majority of these genes have human orthologs and accordingly they represent potential candidate biomarkers for the human disease. In addition, for predictive purposes, the analysis reveals the possibility to reduce significantly the size of the candidate-set in practice, with various genes displaying identical expression profiles.

Early over expression of messenger RNA for multiple genes, including insulin, in the Pancreatic Lymph Nodes of NOD mice is associated with Islet Autoimmunity.

BACKGROUND: Autoimmune diabetes (T1D) onset is preceded by a long inflammatory process directed against the insulin-secreting beta cells of the pancreas. Deciphering the early autoimmune mechanisms represents a challenge due to the absence of clinical signs at early disease stages. The aim of this study was to identify genes implicated in the early steps of the autoimmune process, prior to inflammation, in T1D. We have previously established that insulin autoantibodies (E-IAA) predict early diabetes onset delineating an early phenotypic check point (window 1) in disease pathogenesis. We used this sub-phenotype and applied differential gene expression analysis in the pancreatic lymph nodes (PLN) of 5 weeks old Non Obese Diabetic (NOD) mice differing solely upon the presence or absence of E-IAA. Analysis of gene expression profiles has the potential to provide a global understanding of the disease and to generate novel hypothesis concerning the initiation of the autoimmune process. METHODS: Animals have been screened weekly for the presence of E-IAA between 3 and 5 weeks of age. E-IAA positive or negative NOD mice at least twice were selected and RNAs isolated from the PLN were used for microarray analysis. Comparison of transcriptional profiles between positive and negative animals and functional annotations of the resulting differentially expressed genes, using software together with manual literature data mining, have been performed. RESULTS: The expression of 165 genes was modulated between E-IAA positive and negative PLN. In particular, genes coding for insulin and for proteins known to be implicated in tissue remodelling and Th1 immunity have been found to be highly differentially expressed. Forty one genes showed over 5 fold differences between the two sets of samples and 30 code for extracellular proteins. This class of proteins represents potential diagnostic markers and drug targets for T1D. CONCLUSION: Our data strongly suggest that the immune related mechanisms taking place at this early age in the PLN, correlate with homeostatic changes influencing tissue integrity of the adjacent pancreatic tissue. Functional analysis of the identified genes suggested that similar mechanisms might be operating during pre-inflammatory processes deployed in tissues i) hosting parasitic microorganisms and ii) experiencing unrestricted invasion by tumour cells.

The autoimmune contrivance: genetics in the mouse model.

Autoimmunity and inheritance of complex characters behold an explosive interest in biology over the last 15 years. Research in the genetics of autoimmunity has been impelled by the isolation of genetic markers allowing tracing of heredity. The annotation and sequencing of the human and mouse genomes provide with the potential for further advancements, through the development of new technologies. This review aims to summarize advances made in the autoimmunity field, centered in type 1 diabetes in the NOD mouse model. It also aims to demonstrate that animal models, albeit some phenotypic and genetic dissimilarities with the human diseases, still remain the best way to move towards an understanding of the molecular mechanisms involved in autoimmunity. Assessing the current state of research in this field together with the increasing potential of novel biotechnology advancements, new insights to disease pathogenesis and discovery of molecular targets for intervention strategies are anticipated in the coming years.

Lower levels of surface B-cell-receptor expression in chronic lymphocytic leukemia are associated with glycosylation and folding defects of the mu and CD79a chains.

Low levels of B-cell-receptor (BCR) expression are the hallmark of tumoral B lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL). These cells also respond inadequately to stimulation through the BCR. This receptor consists of a surface immunoglobulin associated with a CD79a/CD79b heterodimer. We previously showed that the intracellular synthesis of BCR components, from transcription onward, is normal. Here, we investigated the glycosylation status and cellular localization of mu, CD79a, and CD79b chains in 10 CLL patients differing in surface immunoglobulin M (IgM) expression. We reported a severe impairment of the glycosylation and folding of mu and CD79a. These defects were associated with the retention of both chains in the endoplasmic reticulum and lower levels of surface IgM expression. In contrast, no clear impairment of glycosylation and folding was observed for CD79b. No sequence defects were identified for BCR components and for the chaperone proteins involved in BCR folding processes. These data show, for the first time, that lower levels of BCR surface expression observed in CLL are accounted for by an impaired glycosylation and folding of the mu and CD79a chains.

Genetics of type 1A (immune mediated) diabetes.

Type 1A (immune mediated) diabetes is genetically heterogeneous with important examples for man and animal models with major mutations (autosomal recessive and X-linked recessive) identified as well as oligogenic/polygenic inheritance. For the most common forms of type 1A diabetes alleles of DQ and DR within the major histocompatibility complex are important determinants of disease and allow identification of high risk individuals at birth. Further understanding of both common and rare genetic determinants of type 1A diabetes will contribute to understanding the pathogenesis of diabetes and of autoimmunity.

Early and quantal (by litter) expression of insulin autoantibodies in the nonobese diabetic mice predict early diabetes onset.

Aiming to study the early stages of type 1 diabetes phenotype, before insulitis appears, we measured insulin autoantibodies (IAA) between 3 and 5 wk of age in the NOD mouse (early-IAA (E-IAA)). We report that IAA are found as early as at 3 wk of age, at weaning, and their expression is a quantal phenotype. Maternal autoantibody status influences this early phenotype, because animals of litters issued from IAA-positive ante partum mothers develop E-IAA with a significantly higher incidence than animals issued from IAA-negative mothers. These E-IAA represent synthesized rather than transplacental autoantibodies, as evidenced by higher levels in many offspring compared with maternal IAA, and negative as well as positive offspring in the same litters and it correlates with early diabetes onset, defining the first autoimmune window in diabetes pathogenesis. Therefore, autoimmune processes leading to type 1 diabetes initiate early in life, are influenced by maternal autoantibody status, and can be revealed by the presence of IAA. Our data suggest that the mechanisms responsible for the breakdown of self-tolerance are subjected not only to genetic predisposition, but also to the physiological status of the mother. Pathological progression to autoimmunity is marked by the presence of immunological windows relating early steps with final disease onset.

Genetic differentiation of poly I:C from B:9-23 peptide induced experimental autoimmune diabetes.

Type 1 diabetes is an immune-mediated disease, in which T cells of the adaptive immune system mediate beta cell destruction. Recently the innate immune system has been linked to etiopathogenesis of several autoimmune diseases including type 1 diabetes, as innate effector cells (e.g. dendritic cells, monocytes/macrophages and NK cells) can prime and promote or regulate (auto)immune responses. We have previously developed an experimental autoimmune diabetes (EAD) model with insulin peptide B:9-23 immunization in transgenic H-2(d)mice expressing the costimulatory molecule B7.1 in their islets (under the Rat Insulin Promotor, RIP). We compared the induction of diabetes with polyinosinic-polycytidylic acid (Poly I:C), a mimic of double stranded viral RNA versus insulin B:9-23 peptide in mice following backcrossing of the B7.1 transgene on to BALB/c mice from original B7.1 C57Bl/6 mice. We find that diabetes induction by Poly I:C is C57Bl/6 associated, whereas B:9-23 peptide induced diabetes and induction of insulin autoantibodies (IAA) are dependent on BALB/c genes. This B:9-23 peptide induced diabetes is consistent with MHC class II H-2(d)being necessary for the response to this peptide. Of note Poly I:C induction of diabetes was lost while B:9-23 induction was retained with backcrossing to BALB/c mice. Interaction of genes and environment (antigenic epitope and viral mimic) can be important in the pathogenesis of immune mediated diabetes and activation of the innate immune system (e.g. Poly I:C) may be one key determinant.

Lower levels of surface B-cell-receptor expression in chronic lymphocytic leukemia are associated with glycosylation and folding defects of the U and CD79a chains

Low levels of B-cell-receptor (BCR) expression are the hallmark of tumoral B lymphocytes in B-cell chronic lymphocytic leukemia (B-CLL). These cells also respond inadequately to stimulation through the BCR. This receptor consists of a surface immunoglobulin associated with a CD79a/CD79b heterodimer. We previously showed that the intracellular synthesis of BCR components, from transcription onward, is normal. Here, we investigated the glycosylation status and cellular localization of u, CD79a, and CD79b chains in 10 CLL patients differing in surface immunoglobulin m (IgM) expression. We reported a severe impairment of the glycosylation and folding of u and CD79a. These defects were associated with the retention of both chains in the endoplasmic reticulum and lower levels of surface IgM expression. In contrast, no clear impairment of glycosylation and folding was observed for CD79b. No sequence defects were identified for BCR components and for the chaperone proteins involved in BCR folding processes. These data show, for the first time, that lower levels of BCR surface expression observed in CLL are accounted for by an impaired glycosylation and folding of the u and CD79a chains