Inorganic Nanoparticles and the Immune System: Detection, Selective Activation and Tolerance

The immune system is the responsible for body integrity and prevention of external invasion. On one side, nanoparticles are no triggers that the immune system is prepared to detect, on the other side it is known that foreign bodies, not only bacteria, viruses and parasites, but also inorganic matter, can cause various pathologies such as silicosis, asbestosis or inflammatory reactions. Therefore, nanoparticles entering the body, after interaction with proteins, will be either recognized as self-agents or detected by the immune system, encompassing immunostimulation or immunosuppression responses. The nature of these interactions seems to be dictated not specially by the composition of the material but by modifications of NP coating (composition, surface charge and structure). Herein, we explore the use of gold nanoparticles as substrates to carry multifunctional ligands to manipulate the immune system in a controlled manner, from undetection to immunostimulation. Murine bone marrow macrophages can be activated with artificial nanometric objects consisting of a gold nanoparticle functionalized with peptides. In the presence of some conjugates, macrophage proliferation was stopped and pro-inflammatory cytokines were induced. The biochemical type of response depended on the type of conjugated peptide and was correlated with the degree of ordering in the peptide coating. These findings help to illustrate the basic requirements involved in medical NP conjugate design to either activate the immune system or hide from it, in order to reach their targets before being removed by phagocytes. Additionally, it opens up the possibility to modulate the immune response in order to suppress unwanted responses resulting from autoimmunity, or allergy or to stimulate protective responses against pathogens.

Antimycobacterial activity of Indigofera suffruticosa with activation potential of the innate immune system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

On the transcriptional role of NLRC5 and the function of NLRC5-driven MHC class I expression in the immune system

Major histocompatibility complex (MHC) molecules are of crucial importance for the immune system to recognize and defend the body against external attacks. Foreign antigens are presented by specialized cells, called antigen presenting cells, to T lymphocytes in the context of MHC molecules, thereby inducing T cell activation. In addition, MHC molecules are essential for Natural Killer (NK) cell biology, playing a role in NK cell education and activation. Recently, the NOD-like receptor (NLR) family member NLRC5 (NLR caspase recruitment domain containing protein 5) was found to act as transcriptional regulator of MHC class I, in particular in T and NK cells. Its role in MHC class I expression is however minor in dendritic cells (DCs). This raised the question of whether inflammatory conditions, which augment the levels of NLRC5 in DCs, could increase its contribution to MHC class I expression. Our work shows that MHC class I transcript and intracellular levels depend on NLRC5, while its role in MHC class I surface expression is instead negligible. We describe however a general salvage mechanism that enables cells with low intracellular MHC class I levels to nevertheless maintain relatively high MHC class I on the cell surface. In addition, we lack a thorough understanding of NLRC5 target gene specificity and mechanism of action. Our work delineates the unique consensus sequence in MHC class I promoters required for NLRC5 recruitment and pinpoints conserved features conferring its specificity. Furthermore, through genome-wide analyses, we confirm that NLRC5 regulates classical MHC class I genes and identify novel target genes all encoding non-classical MHC class I molecules exerting an array of functions in immunity and tolerance. We finally asked why a dedicated factor co-regulates MHC class I expression specifically in T and NK lymphocytes. We show that deregulated NLRC5 expression affects the education of NK cells and alters the crosstalk between T and NK cells, leading to NK cell-mediated killing of T lymphocytes. Altogether this thesis work brings insights into molecular and physiological aspects of NLRC5 function, which might help understand certain aspects of immune responses and disorders. -- Les molécules du complexe majeur d'histocompatibilité (CMH) sont essentielles au système immunitaire pour l'initiation de la réponse immunitaire. En effet, l'activation des lymphocytes T nécessite la reconnaissance d'un antigène étranger présenté par les cellules présentatrices d'antigènes sur une molécule du CMH. Les molécules du CMH ont également un rôle fondamental pour la fonction des cellules Natural Killer (NK) puisqu'elles sont nécessaires à leur processus d'éducation et d'activation. Récemment, NLRC5 (NLR caspase recruitment domain containing protein 5), un membre de la famille des récepteurs de type NOD (NLRs), a été décrit comme un facteur de transactivation de l'expression des gènes du CMH de classe I. A l'état basai, cette fonction transcriptionnelle est essentielle dans les lymphocytes T et NK, alors que ce rôle reste mineur pour l'expression des molécules du CMH de classe I dans les cellules dendritiques (DCs). Dans des conditions inflammatoires, l'expression de NLRC5 augmente dans les DCs. Notre travail démontre que, dans ces conditions, les transcrits et les niveaux intracellulaires des molécules du CMH de classe I augmentent aussi d'une façon dépendante de NLRC5. A contrario, le rôle de NLRC5 sur les niveaux de molécules de surface reste minoritaire. Cette observation nous a conduits à l'identification d'un mécanisme général de compensation qui permet aux cellules de maintenir des niveaux relativement élevés de molécules de CMH de class I à leur surface malgré de faibles niveaux intracellulaires. De plus, il semblait nécessaire de s'orienter vers une approche plus globale afin de déterminer l'étendue de la fonction transcriptionnelle de NLRC5. Par une approche du génome entier, nous avons pu décrire une séquence consensus conservée présente dans les promoteurs des gènes du CMH de classe I, sur laquelle NLRC5 est spécifiquement recruté. Nous avons pu également identifier de nouveaux gènes cibles codant pour des molécules de CMH de classe I non classiques impliqués dans l'immunité et la tolérance. Finalement, nous nous sommes demandé quel est l'intérêt d'avoir un facteur transcriptionnel, en l'occurrence NLRC5, qui orchestre l'expression du CMH de classe I dans les lymphocytes T et NK. Nous montrons que la dérégulation de l'expression de NLRC5 affecte l'éducation des cellules NK et conduit à la mort cellulaire des lymphocytes T médiée par les cellules NK. Dans l'ensemble ce travail de thèse contribue à la caractérisation du rôle de NLRC5, tant au niveau moléculaire que physiologique, ce qui présente un intérêt dans le cadre de la compréhension de certains aspects physiopathologique de la réponse immunitaire.

Analysis of key molecules of the innate immune system in mammary epithelial cells isolated from marker-assisted and conventionally selected cattle

Mastitis is the most prevalent infectious disease in dairy herds. Breeding programs considering mastitis susceptibility were adopted as approaches to improve udder health status. In recent decades, conventional selection criteria based on phenotypic characteristics such as somatic cell score in milk have been widely used to select animals. Recently, approaches to incorporate molecular information have become feasible because of the detection of quantitative trait loci (QTL) affecting mastitis resistance. The aims of the study were to explore molecular mechanisms underlying mastitis resistance and the genetic mechanisms underlying a QTL on Bos taurus chromosome 18 found to influence udder health. Primary cell cultures of mammary epithelial cells from heifers that were selected for high or low susceptibility to mastitis were established. Selection based on estimated pedigree breeding value or on the basis of marker-assisted selection using QTL information was implemented. The mRNA expression of 10 key molecules of the innate immune system was measured using quantitative real-time PCR after 1, 6, and 24 h of challenge with heat-inactivated mastitis pathogens (Escherichia coli and Staphylococcus aureus) and expression levels in the high and low susceptibility groups were compared according to selection criteria. In the marker-assisted selection groups, mRNA expression in cells isolated from less-susceptible animals was significantly elevated for toll-like receptor 2, tumor necrosis factor-alpha, IL-1beta, IL-6, IL-8, RANTES (regulated upon activation, normal t-cell expressed and secreted), complement factor C3, and lactoferrin. In the estimated pedigree breeding value groups, mRNA expression was significantly elevated only for V-rel reticuloendotheliosis viral oncogene homolog A, IL-1 beta, and RANTES. These observations provide first insights into genetically determined divergent reactions to pathogens in the bovine mammary gland and indicate that the application of QTL information could be a successful tool for the selection of animals resistant to mastitis.

Gender-specific modulation of immune system complement gene expression in marine medaka Oryzias melastigma following dietary exposure of BDE-47

BDE-47 is one of the most widely found congeners of PBDEs in marine environments. The potential immunomodulatory effects of BDE-47 on fish complement system were studied using the marine medaka Oryzias melastigma as a model fish. Three-month-old O. melastigma were subjected to short-term (5 days) and long-term (21 days) exposure to two concentrations of BDE-47 (low dose at 290 +/- 172 ng/day; high dose at 580 +/- 344 ng/day) via dietary uptake of BDE-47 encapsulated in Artemia nauplii. Body burdens of BDE-47 and other metabolic products were analyzed in the exposed and control fish. Only a small amount of debrominated product, BDE-28, was detected, while other metabolic products were all under detection limit. Transcriptional expression of six major complement system genes involved in complement activation: C1r/s (classical pathway), MBL-2 (lectin pathway), CFP (alternative pathway), F2 (coagulation pathway), C3 (the central component of complement system), and C9 (cell lysis) were quantified in the liver of marine medaka. Endogenous expression of all six complement system genes was found to be higher in males than in females (p < 0.05). Upon dietary exposure of marine medaka to BDE-47, expression of all six complement genes were downregulated in males at day 5 (or longer), whereas in females, MBl-2, CFP, and F2 mRNAs expression were upregulated, but C3 and C9 remained stable with exposure time and dose. A significant negative relationship was found between BDE-47 body burden and mRNA expression of C1r/s, CFP, and C3 in male fish (r = -0.8576 to -0.9447). The above findings on changes in complement gene expression patterns indicate the complement system may be compromised in male O. melastigma upon dietary exposure to BDE-47. Distinct gender difference in expression of six major complement system genes was evident in marine medaka under resting condition and dietary BDE-47 challenge. The immunomodulatory effects of BDE-47 on transcriptional expression of these complement components in marine medaka were likely induced by the parent compound instead of biotransformed products. Our results clearly demonstrate that future direction for fish immunotoxicology and risk assessment of immunosuppressive chemicals must include parallel evaluation for both genders.

SNAREing immunity: the role of SNAREs in the immune system

The trafficking of molecules and membranes within cells is a prerequisite for all aspects of cellular immune functions, including the delivery and recycling of cell-surface proteins, secretion of immune mediators, ingestion of pathogens and activation of lymphocytes. SNARE (soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptor)-family members mediate membrane fusion during all steps of trafficking, and function in almost all aspects of innate and adaptive immune responses. Here, we provide an overview of the roles of SNAREs in immune cells, offering insight into one level at which precision and tight regulation are instilled on immune responses.

The role of the innate immune system in the clearance of apoptotic cells

Rapid clearance of dying cells is a vital feature of apoptosis throughout development, tissue homeostasis and resolution of inflammation. The phagocytic removal of apoptotic cells is mediated by both professional and amateur phagocytes, armed with a series of pattern recognition receptors that participate in host defence and apoptotic cell clearance. CD14 is one such molecule. It is involved in apoptotic cell clearance (known to be immunosuppressive and anti-inflammatory) and binding of the pathogen-associated molecular pattern, lipopolysaccharides (a pro-inflammatory event). Thus CD14 is involved in the assembly of two distinct ligand-dependent macrophage responses. This project sought to characterise the involvement of the innate immune system, particularly CD14, in the removal of apoptotic cells. The role of non-myeloid CD14 was also considered and the data suggests that the expression of CD14 by phagocytes may define their professional status as phagocytes. To assess if differential CD14 ligation causes the ligand-dependent divergence in macrophage responses, a series of CD14 point mutants were used to map the binding of apoptotic cells and lipopolysaccharides. Monoclonal antibodies, 61D3 and MEM18, known to interfere with ligand-binding and responses, were also mapped. Data suggests that residue 11 of CD14, is key for the binding of 61D3 (but not MEM18), LPS and apoptotic cells, indicating lipopolysaccharides and apoptotic cells bind to similar residues. Furthermore using an NF-kB reporter, results show lipopolysaccharides but not apoptotic cells stimulate NF-kB. Taken together these data suggests ligand-dependent CD14 responses occur via a mechanism that occurs downstream of CD14 ligation but upstream of NF-?B activation. Alternatively apoptotic cell ligation of CD14 may not result in any signalling event, possibly by exclusion of TLR-4, suggesting that engulfment receptors, (e.g. TIM-4, BAI1 and Stablin-2) are required to mediate the uptake of apoptotic cells and the associated anti-inflammatory response.

The innate immune system and the clearance of apoptotic cells

Apoptosis, programmed cell death, is used by multicellular organisms to remove cells that are in excess, damaged or diseased. Activation of the apoptosis programme generates "eat me" signals on the surface of the apoptotic cell that mediate recognition and clearance by the innate immune system. CD14, a pattern recognition receptor expressed on macrophages, is widely known for its ability to recognise the pathogen-associated molecular pattern lipopolysaccharide (LPS) and promote inflammation. However, CD14 has also been shown to mediate binding and removal of apoptotic cells in a process that is anti-inflammatory suggesting CD14 is capable of producing two distinct, ligand-dependent macrophage responses. Whilst the molecular basis for this dichotomy has yet to be defined it is clear that CD14 defines a point of interest on the macrophage surface where we may study ligand-specific responses of macrophages. Our work seeks to define the molecular mechanisms underlying the involvement of CD14 in the non-inflammatory clearance of apoptotic cells. Here we used three different differentiation strategies to generate macrophages from the monocytic cell line THP-1. The resultant macrophage models were characterised to assess the expression and function of CD14 within each model system. Whilst each macrophage model shows increased levels of surface CD14 expression, our results demonstrate significant differences in the various models’ abilities to respond to LPS and clear apoptotic cells in a CD14-dependent manner. TLR4 levels correlated positively with LPS responsiveness but not CD14-dependent apoptotic cell clearance or anti-inflammatory responses to apoptotic cells. These observations suggest CD14-dependent apoptotic cell clearance is not dependent on TLR4. Taken together our data support the notion that the CD14 ligand-dependent responses to LPS and apoptotic cells derive from changes at the macrophage surface. The nature and composition of the CD14-co-receptor complex for LPS and apoptotic cell binding and responses is the subject of further study.

High levels of T lymphocyte activation in Leishmania-HIV-1 co-infected individuals despite low HIV viral load

Background: Concomitant infections may influence HIV progression by causing chronic activation leading to decline in T-cell function. In the Americas, visceral (AVL) and tegumentary leishmaniasis (ATL) have emerged as important opportunistic infections in HIV-AIDS patients and both of those diseases have been implicated as potentially important co-factors in disease progression. We investigated whether leishmaniasis increases lymphocyte activation in HIV-1 co-infected patients. This might contribute to impaired cellular immune function. Methods: To address this issue we analyzed CD4(+) T absolute counts and the proportion of CD8(+) T cells expressing CD38 in Leishmania/HIV co-infected patients that recovered after anti-leishmanial therapy. Results: We found that, despite clinical remission of leishmaniasis, AVL co-infected patients presented a more severe immunossupression as suggested by CD4(+) T cell counts under 200 cells/mm(3), differing from ATL/HIV-AIDS cases that tends to show higher lymphocytes levels (over 350 cells/mm(3)). Furthermore, five out of nine, AVL/HIV-AIDS presented low CD4(+) T cell counts in spite of low or undetectable viral load. Expression of CD38 on CD8(+) T lymphocytes was significantly higher in AVL or ATL/HIV-AIDS cases compared to HIV/AIDS patients without leishmaniasis or healthy subjects. Conclusions: Leishmania infection can increase the degree of immune system activation in individuals concomitantly infected with HIV. In addition, AVL/HIV-AIDS patients can present low CD4(+) T cell counts and higher proportion of activated T lymphocytes even when HIV viral load is suppressed under HAART. This fact can cause a misinterpretation of these laboratorial markers in co-infected patients.

The role of local and systemic renin angiotensin system activation in a genetic model of sympathetic hyperactivity-induced heart failure in mice

Sympathetic hyperactivity (SH) and renin angiotensin system (RAS) activation are commonly associated with heart failure (HF), even though the relative contribution of these factors to the cardiac derangement is less understood. The role of SH on RAS components and its consequences for the HF were investigated in mice lacking alpha(2A) and alpha(2C) adrenoceptor knockout (alpha(2A)/alpha(2C) ARKO) that present SH with evidence of HF by 7 mo of age. Cardiac and systemic RAS components and plasma norepinephrine (PN) levels were evaluated in male adult mice at 3 and 7 mo of age. In addition, cardiac morphometric analysis, collagen content, exercise tolerance, and hemodynamic assessments were made. At 3 mo, alpha(2A)/alpha(2C)ARKO mice showed no signs of HF, while displaying elevated PN, activation of local and systemic RAS components, and increased cardiomyocyte width (16%) compared with wild-type mice (WT). In contrast, at 7 mo, alpha(2A)/alpha(2C)ARKO mice presented clear signs of HF accompanied only by cardiac activation of angiotensinogen and ANG II levels and increased collagen content (twofold). Consistent with this local activation of RAS, 8 wk of ANG II AT(1) receptor blocker treatment restored cardiac structure and function comparable to the WT. Collectively, these data provide direct evidence that cardiac RAS activation plays a major role underlying the structural and functional abnormalities associated with a genetic SH-induced HF in mice.

The Kinin System in Patients with Systemic Lupus Erythematosus Exhibiting Mucocutaneous Lesions: A Clinical Study

In the present study, we evaluated the kinin system components in the plasma of patients with systemic lupus erythematosus exhibiting mucocutaneous lesions. Fifteen women with active cutaneous lupus (P) and 15 normal healthy women (C) were studied. Low molecular (LKg) and high molecular (HKg) weight kininogen were determined by ELISA (expressed mu g Bk/ml). The activities of tissue kallikrein (TKal), plasma kallikrein (PKal) and kininase II were assayed by their action on selective substrates. Statistical analysis was performed using the Mann-Whitney test. The patients presented increased plasma levels of LKg (P = 2.98, C = 0.79) and HKg (P = 1.78, C = 0.5) associated with the increased activity of PKal (P = 2.50, C = 1.63 U/ml), TKal (P = 1.87, C = 1.30 mu M pNa/ml) and kininase II (P = 1.50, C = 0.51 mu M Hys-Leu/ml), when compared to the values observed in the control group (P < 0.0001 for each comparison). Thus, the increased concentration of all parameters of the kinin system in these patients indicate an overactivity of the kinin system in the acute phase of lupus, corroborating with the participation of these mediators in lupus pathogenesis.

A Dynamical Modeling to Study the Adaptive Immune System and the Influence of Antibodies in the Immune Memory

Immunological systems have been an abundant inspiration to contemporary computer scientists. Problem solving strategies, stemming from known immune system phenomena, have been successfully applied to chall enging problems of modem computing. Simulation systems and mathematical modeling are also beginning use to answer more complex immunological questions as immune memory process and duration of vaccines, where the regulation mechanisms are not still known sufficiently (Lundegaard, Lund, Kesmir, Brunak, Nielsen, 2007). In this article we studied in machina a approach to simulate the process of antigenic mutation and its implications for the process of memory. Our results have suggested that the durability of the immune memory is affected by the process of antigenic mutation.and by populations of soluble antibodies in the blood. The results also strongly suggest that the decrease of the production of antibodies favors the global maintenance of immune memory.

Behavior: A Relevant Tool for Brain-immune System Interaction Studies

Neuroimmunomodulation describes the field focused on understanding the mechanisms by which the central nervous system interacts with the immune system, potentially leading to changes in animal behavior. Nonetheless, not many articles dealing with neuroimmunomodulation employ behavior as an analytical endpoint. Even fewer papers deal with social status as a possible modifier of neuroimmune phenomena. In the described sets of experiments, we tackle both, using a paradigm of social dominance and subordination. We first review data on the effects of different ranks within a stable hierarchical relationship. Submissive mice in this condition display more anxiety-like behaviors, have decreased innate immunity, and show a decreased resistance to implantation and development of melanoma metastases in their lungs. This suggests that even in a stable, social, hierarchical rank, submissive animals may be subjected to higher levels of stress, with putative biological relevance to host susceptibility to disease. Second, we review data on how dominant and submissive mice respond differentially to lipopolysaccharide (LPS), employing a motivational perspective to sickness behavior. Dominant animals display decreased number and frequency in several aspects of behavior, particularly agonistic social interaction, that is, directed toward the submissive cage mate. This was not observed in submissive mice that maintained the required behavior expected by its dominant mate. Expression of sickness behavior relies on motivational reorganization of priorities, which are different along different social ranks, leading to diverse outcomes. We suggest that in vitro assessment of neuroimmune phenomena can only be understood based on the behavioral context in which they occur.