EDCs are commonly thought to bind or interfere with estrogen, androgen, progesterone, thyroid, and retinoid receptors. Oxybenzone is considered to be an endocrine-disrupting chemical and approximately 97% of people in the United States were found to have BP3 in their urine. This thesis will address how BP3 affects the innate immune system, in particular myeloid cells. My Master’s thesis aims to address two main overarching questions. Does BP3 alter macrophage polarization, cytokine/chemokine secretion, the viability in vitro? Does exposure to BP3 in vivo during pregnancy/lactation affect the RNA expression of cytokines and immunosuppressant factors associated with the myeloid population? It is unknown how BP3 impacts immune subpopulations in a neoplastic setting. Additionally, it is important to consider how these effects may contribute to malignant behaviors. My thesis evaluates the effects of BP3 on the Raw 264.7 cell lines as well as tumor tissues from mice exposed to BP3 during pregnancy and lactation. We hypothesized that BP3 exposures induce changes in myeloid cell interactions in the immune system through ER-mediated mechanisms. We anticipated that BP3 would increase the growth and migration of 4T1 cells through indirect signals imparted by myeloid cell populations. We also hypothesized that there will be a decrease in T cell proliferation following BP3 exposure and an alteration in gene expression consistent with a shift from Th1 to Th2. Finally, we expected that BP3 exposure would increase the number of myeloid cells in mouse tumors. Our research shows that oxybenzone appears to enhance the pro-inflammatory state of RAW264.7 cells and may result in the release of unidentified factors that can impact 4T1 cell anchorage-independent cell growth in these pro-inflammatory conditions. BP3 may also impact the metabolic activity of recovering RAW264.7 cells following LPS-induced activity. Additionally, BP3 may impact the release of factors from macrophages that control T cell activation-induced proliferation. By using the p53-/- mouse tumors we found that exposure to 3mg/kg/day BP3 during pregnancy and lactation did alter IDO1 RNA expression but this was not associated with markers of immunosuppressive cell types.

No description available.

Innate immune memory has become an increasingly popular area of research in the last decade. However, much of the work done on innate immune memory using inflammatory agents such as BCG, C. albicans, and β-glucan has been pursued through systemic administration, which has been shown to induce training in circulating monocytes. In addition, little is known about whether microbial ligands can induce training. Here, we show that local mucosal exposure to an acute dose of LPS induces long-lasting phenotypic changes in airway macrophage populations. LPS-exposed macrophages display increased glycolytic metabolism and differential cytokine expression upon restimulation, whereas circulating monocytes are not affected. Finally, we show that LPS exposure provides long-lasting protection against Streptococcus pneumoniae in the lung, likely due to the higher acquisition of CD11b, which is indicative of macrophage activation and phagocytosis. As much of the work on innate immune memory has been done through systemic administration of training agents, this project aims to fill existing knowledge gaps in the induction of innate immune memory upon local mucosal exposure to inflammatory agents. / Thesis / Master of Science in Medical Sciences (MSMS) / The innate immune system is one of the first defenders in our bodies that fight against a variety of pathogens. In the last decade, the innate immune system was found to be capable of having memory, meaning it reacts faster or at a heightened magnitude in response to a wide range of subsequent pathogens after it is trained by an agent. This project explores the effect a bacteria wall component, LPS, has on the lung environment and examines if it will induce memory in the lung. Our findings show that intranasal exposure to LPS changes the cellular landscape in the lung. LPS-exposed airway innate immune cells become more activated and provide subsequent protection against bacterial infections. This work has implications for using LPS as a vaccine adjuvant in order to provide protection against a variety of pathogens in addition to specific protection brought by the vaccine.

Tribbles (Trib) protein was first described in Drosophila as a regulator of proliferation, later being implicated as a G2/M modulator. In mammalian systems, three Trib gene family members have been identified, which share a conserved motif similar to the catalytic domain of serine/threonine kinases. However, they lack several conserved residues in the ATP-binding pocket and the core motif of the catalytic domain necessary for catalytic function. Tribbles 1 (Trib1) is involved in inflammation through its ability to regulate MAPK, NF-κB and the CCAAT Enhancer Binding Protein (C/EBP). Moreover, Trib1 is associated with human disease, such as atherosclerosis and acute myeloid leukaemia. In this thesis, I investigated the functional role of Trib1 in Toll-like Receptor (TLR)-induced inflammatory responses together with pro- or anti-inflammatory cytokines. The RAW264.7 myeloid cell line was stimulated with TLR2/9 ligands in the presence or absence of IFN-γ or IL-10. I observed a high level of Trib1 expression in the presence of IFN-γ and TLR2 ligands, but weak Trib1 expression following treatment with IL-10 and TLR9 ligands. In gene knock-down experiments using small interfering RNAs (siRNA) to reduce Trib1 expression, C/EBPβ was up-regulated in both stimulated (by IFN-γ and TLR2 ligands) and resting macrophage populations. TNF-α production was increased following Trib1 knockdown after treatment with IFN-γ and/or TLR2 ligands but IL-6 secretion remained unchanged. Furthermore, ERK1/2 expression was reduced in Trib1 siRNA-treated cells and failed to induce chemokinesis in macrophages. Finally, Trib1 was demonstrated to act as a modulator of cell cycle (G2/M) transition and displays a delayed apoptotic phenotype. The work in this thesis demonstrates that mammalian Trib1 contributes to the pro-inflammatory response and functions as a regulator of the ERK1/2 and C/EBPβ pathways following TLR ligand-mediated activation. Its novel functions include acting as a modulator of G2/M arrest and suppressing macrophage migration.

When I first arrived in Liège, the scientific activities of our laboratory focused on the identification of candidate-genes whose different alleles interfere with the resistance/susceptibility of animals against infectious diseases. Two genes were being intensively studied (Mx and OAS) due to their theoretical potential in interfering with the replicative cycle of several viruses responsible for bovines viral pneumonias (Baise et al., 2004 ; Gerardin et al., 2004 ; Leroy et al., 2005 ; 2006). My work consisted then, in the identification of other genes potentially implicated in the resistance against the Paramyxoviruses. The Paramyxoviridae family includes some of the great and ubiquitous disease-causing viruses of animals, including the bovine parainfluenza type 3, the bovine respiratory syncytial virus, the Newcastle disease virus, the distemper virus, etc. Evidence was accumulating that genetic factors were involved both in the control of infectious diseases (Abel et al., 1991 ; 1995 ; Alcais et al., 1997 ; Jin et al., 1999 ; Martinson et al. 1997 ; Shaw et al., 1995) and in the regulation of infection levels and clinical presentation (Garcia et al., 1999 ; Plancoulaine et al., 2000 ; 2003). Thus, identifying genes that control the organism response to paramyxoviruses was a crucial step in elucidating how they might affect the pathophysiological processes underlying the severity of the disease induced. Other experiences done in this laboratory showed us how risky and difficult was any extrapolation of the mouse results to another species if these results were brought through infection with a heterologous virus, so we decided to implement this strategy with Sendai virus (SeV), the archetype organism of the Paramyxoviridae family, from which most of the basic biochemical, molecular and biologic properties of the whole family were derived from (Chanock et al., 2001). With this goal in mind my thesis was divided in five successive steps: In order to establish a standard model of SeV infection, the first step consisted in determining the best volume of inoculum that was needed to achieve a safe, reproducible pulmonary deposition of Sendai virus in the mice lungs. Secondly we developed a murine model of SeV infection using a series of different and sophisticated procedures that allowed a quantitative assessment of disease severity and progression. Then we compared SeV infections among 6 strains of mice that were deliberately chosen because they originated from different lineages, as deduced from known genealogical and phylogenetic data. Applying these procedures to distinct inbred strains of mice, revealed highly significant differences in susceptibility between them. More specifically 129/Sv were highly susceptible while BALB/c were particularly resistant, BALB/c exhibiting a benign and asymptomatic affection of the epithelium of the airways, with no functional impact, generating slight mononucleated cell infiltration, in which viral replication is repressed and the virus swiftly eliminated. As a result, in the fourth part of my study we discussed a series of hypotheses that should be tested in the future to improve our understanding of why BALB/c is so resistant to SeV infection. Practically speaking, our studies led to the gathering of a genomic DNA collection from the parental extreme lines in terms of susceptibility (129/Sv) and resistance (BALB/c) and their F1 and F2 offspring. Within this bank, each of the 263 DNA sample is associated with a portfolio of phenotypic values that are estimators of the resistance each mouse opposed to the SeV. We hope that, between expert hands, this bank will allow the detection of genes of which the alleles contribute, at least in part, to the spectacular resistance of BALB/c. The last part of my thesis consisted in applying our model to establish if the receptor TLR4 influenced the pathophysiology of the Paramyxoviridae in general. Because the role of this receptor had already been excluded for SeV, we tested another virus of the same family and homologous for the mice, the PVM (standing for pneumonia virus of mice). This work showed, in contradiction of what had been found in heterologous models in the past, that TLR4 is not involved in host defense against respiratory tract infection with the Paramyxoviridae.

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.<p>The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is primed for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.

Toll-like receptor 9 (TLR9) activates the innate immune system in response to microbial DNA or mimicking oligodeoxynucleotides. While the discrimination of host and microbial DNA is presumed to reflect TLR9-mediated recognition of CpG motifs, little information is available to verify this hypothesis. Cell stimulation experiments demonstrate preferential activation of TLR9 by CpG-containing nucleic acids, however direct binding investigations have reached contradictory conclusions with respect to the ability of TLR9 to bind nucleic acids in a sequence-specific fashion. Here we report expression of the soluble, ectodomain of human TLR9 with characterization of its ligand-binding properties. TLR9 has a high degree of ligand specificity in being able to discriminate not only CpG dinucleotides, but also higher order six nucleotide motifs that mediate species-specific activation. However, TLR9 ligand binding is also functionally influenced by nucleic acids in a sequence-independent manner both in vitro and in cell proliferation experiments. A model is proposed in which TLR9 activation is mediated specifically by CpG-containing ligands while sensitivity is mediated specifically by the absolute concentration of nucleic acids in a sequence-independent manner.<p>The bovine hsp70A promoter was used to direct the heat-regulated synthesis of the ectodomain of human TLR9 in transfected cultured bovine cells. The protein was efficiently secreted from transfected cells in a temperature-dependent manner and the recombinant receptor produced was found to be relatively pure. A stably transfected cell line with regulated expression of the protein was obtained and repeated thermal cycling of the cultures enabled high-yield production of the receptor in an active ligand-binding form. Using this recombinant receptor to study the ligand binding properties of TLR9, a model of positive cooperativity is proposed in which the sensitivity of TLR9 ligand binding is modulated by the absolute concentration of nucleic acids in a sequence-independent fashion, while activation of TLR9 is highly dependent on DNA sequence. That is to say that TLR9 is primed for activation by interaction with non-activating sequences but activation itself occurs in a sequence-specific fashion.

Drosophila TGFbeta-Activating-Kinase 1 (dTAK1) is an essential component of both the Immune Deficiency (IMD) innate immune and TNF/Eiger apoptotic cascades. The IMD and JNK pathways bifurcate at the level of dTAK1. Hence, elucidating the regulatory mechanism of dTAK1 is pertinent to understanding the regulation of both innate immunity and apoptosis. In this study, Trabid was identified as a novel negative regulator of the Drosophila IMD pathway. Trabid interacted with dTAK1 and decreased K63-linked ubiquitination, thereby reducing immune signalling. Three tandem Npl4 Zinc Fingers (NZF) and C518 were required for Trabid activity. Lysines 142 &amp; 156 were identified as the K63 Ub acceptor sites of dTAK1, required for K63-linked ubiquitination and signalling. Also, results show Lys 156 functioned as the K48 Ub acceptor site. Further, the ZF domain of TAK1-associated Binding Protein 2 (dTAB2) was important in modulating dTAK1 K63-linked ubiquitination and thereby the immune signal. These results indicate an elaborate and multi-tiered mechanism for regulating dTAK1 activity and modulating the immune signal. Further, Ariadne-2 (Ari-2) was identified as a novel component of the Drosophila TNF/Eiger pathway which functioned at the level of dTAK1. Results indicate that Ari-2 is essential for normal development and longevity. It enhances the apoptotic signal when concomitantly over-expressed with Eiger. Further, Ari-2 interacts with dTAK1, dTAB2 and dTRAF2 which are all implicated in TNF/Eiger signalling. Thus, evidence supports the hypothesis that Ari-2 functions as an adaptor, involved in assembling a distinct signalling complex which transduces the apoptotic signal without activating immunity.

The neonatal period represents a critical time period in the development of the immune system. Adaptive human immune responses are generally viewed as immature at birth. However little is known about innate immune capacity at birth. The TLR system plays an integral role as pattern recognition receptors in the innate immune response. It is also critical in initiating and regulating the adaptive immune response. Preterm birth is associated with increased risk of developing infections in early life and has been associated with increased risk of development of other chronic disorders in later life; however the underlying mechanisms are not at all well understood. Recently, late preterm neonates (34-36 weeks gestation vs full term, 37+ weeks) have been identified as having significantly greater risks of morbidity and mortality in the perinatal period than their full term counterparts. Hence, we focus on examination of TLR responses in late preterm and full term neonates to better understand immune potential and function in these populations. We examined cord blood cytokine and chemokine responses following stimulation with a broad range of TLR agonists. Our results show for the first time that late preterm neonates have reduced capacity to produce both pro- and anti-inflammatory cytokines following stimulation with a panel of TLR agonists. This reduced responsiveness was not due to a reduction in the number of responding cells, but instead appears to be mediated by a reduction in the intrinsic levels of expression of TLRs and associated adaptor proteins. Because little is known about how the innate immune system develops throughout life, we next compared TLR responses in full term neonates to 7 children, adolescents and adults. We found that neonates had selective impairments in TLR responses, most notably in anti-inflammatory cytokine production and anti-viral immune responses compared to the other age groups. Epigenetic modifications, such as the addition or removal of acetyl groups to histone proteins by histone acetyl transferase (HAT) and histone deactylase (HDAC) respectively, are able to modify the expression of genes. Hence, environmental stimuli have been shown to influence gene expression in part by modifying the level or activity of these epigenetic regulators. Currently there are no studies which have examined how epigenetic modifications may influence neonatal innate immune responses. Hence, we sought to determine how modulation of endogenous HDAC activity would affect neonatal innate immune responses. We found that inhibition of HDAC had both inhibitory and enhancing effects on cytokine expression depending on the TLR pathway activated, indicating that the endogenous HDAC expression does not have a global inhibitory impact on all TLR-dependent responses. In summary, this body of work demonstrates that neonatal innate immune responses vary depending on gestational age, indicating that the final few weeks of gestation are crucial for maturation of responses to both bacteria and viruses. Neonates respond differently to TLR stimuli than do older individuals, further highlighting a maturation process of the innate immune system which continues throughout life. Finally, we have shown that environmental exposures may have powerful effects on immune responses in early life.

The neonatal period represents a critical time period in the development of the immune system. Adaptive human immune responses are generally viewed as immature at birth. However little is known about innate immune capacity at birth. The TLR system plays an integral role as pattern recognition receptors in the innate immune response. It is also critical in initiating and regulating the adaptive immune response. Preterm birth is associated with increased risk of developing infections in early life and has been associated with increased risk of development of other chronic disorders in later life; however the underlying mechanisms are not at all well understood. Recently, late preterm neonates (34-36 weeks gestation vs full term, 37+ weeks) have been identified as having significantly greater risks of morbidity and mortality in the perinatal period than their full term counterparts. Hence, we focus on examination of TLR responses in late preterm and full term neonates to better understand immune potential and function in these populations. We examined cord blood cytokine and chemokine responses following stimulation with a broad range of TLR agonists. Our results show for the first time that late preterm neonates have reduced capacity to produce both pro- and anti-inflammatory cytokines following stimulation with a panel of TLR agonists. This reduced responsiveness was not due to a reduction in the number of responding cells, but instead appears to be mediated by a reduction in the intrinsic levels of expression of TLRs and associated adaptor proteins. Because little is known about how the innate immune system develops throughout life, we next compared TLR responses in full term neonates to 7 children, adolescents and adults. We found that neonates had selective impairments in TLR responses, most notably in anti-inflammatory cytokine production and anti-viral immune responses compared to the other age groups. Epigenetic modifications, such as the addition or removal of acetyl groups to histone proteins by histone acetyl transferase (HAT) and histone deactylase (HDAC) respectively, are able to modify the expression of genes. Hence, environmental stimuli have been shown to influence gene expression in part by modifying the level or activity of these epigenetic regulators. Currently there are no studies which have examined how epigenetic modifications may influence neonatal innate immune responses. Hence, we sought to determine how modulation of endogenous HDAC activity would affect neonatal innate immune responses. We found that inhibition of HDAC had both inhibitory and enhancing effects on cytokine expression depending on the TLR pathway activated, indicating that the endogenous HDAC expression does not have a global inhibitory impact on all TLR-dependent responses. In summary, this body of work demonstrates that neonatal innate immune responses vary depending on gestational age, indicating that the final few weeks of gestation are crucial for maturation of responses to both bacteria and viruses. Neonates respond differently to TLR stimuli than do older individuals, further highlighting a maturation process of the innate immune system which continues throughout life. Finally, we have shown that environmental exposures may have powerful effects on immune responses in early life.