Chronic low-grade inflammation is associated the Metabolic Syndrome (MetS) and may contribute to its development. A diet high in saturated fat (SFA) has been associated with increased inflammation and development of the MetS. SFAs have been shown to elicit pro-inflammatory signaling through proteins of innate immunity, TLR4 and Nods 1 and 2. We determined whether common polymorphisms in the genes of these proteins could modify the association between fat intake and biomarkers of the MetS. Fat intake was measured using a food frequency questionnaire and genotyping was completed using real-time PCR. The TLR4 Asp299Gly (rs4986790) polymorphism was associated with decreased insulin sensitivity while an intronic polymorphism (rs5030728) modified the association between dietary SFA and HDL-cholesterol. The Nod1 Glu266Lys polymorphism modified the association between dietary SFA and HOMA-IR. These results suggest a role for innate immunity in mediating some of the effects of dietary SFAs on factors associated with the MetS.

Chronic low-grade inflammation is associated the Metabolic Syndrome (MetS) and may contribute to its development. A diet high in saturated fat (SFA) has been associated with increased inflammation and development of the MetS. SFAs have been shown to elicit pro-inflammatory signaling through proteins of innate immunity, TLR4 and Nods 1 and 2. We determined whether common polymorphisms in the genes of these proteins could modify the association between fat intake and biomarkers of the MetS. Fat intake was measured using a food frequency questionnaire and genotyping was completed using real-time PCR. The TLR4 Asp299Gly (rs4986790) polymorphism was associated with decreased insulin sensitivity while an intronic polymorphism (rs5030728) modified the association between dietary SFA and HDL-cholesterol. The Nod1 Glu266Lys polymorphism modified the association between dietary SFA and HOMA-IR. These results suggest a role for innate immunity in mediating some of the effects of dietary SFAs on factors associated with the MetS.

Lung dysfunction due to exposure to air in high intensity livestock barn operations is a common problem for workers in these facilities. Exposure to this air has been linked to disorders such as chronic bronchitis, occupational asthma, organic dust toxic syndrome, and chronic cough and phlegm. These symptoms have been linked to higher levels of endotoxins in air in chicken and swine barns. However, there are many other toxic molecules such as bacterial DNA and gases capable of inducing respiratory inflammation. Bacterial molecules are recognized through highly conserved pattern recognition molecules called Toll-like receptors (TLR). While lipopolysaccharides are recognized by TLR4, bacterial unmethylated DNA binds to and signals through TLR9. As a prelude to understanding the biology of TLR9 in lung inflammation, it is important to precisely clarify their in situ expression in the lung. I determined expression of TLR9 in intact lungs from cattle, pigs, dogs, horses, mice, and humans. Two samples from normal lungs of cattle, pigs, dogs, three from horses, and two from inflamed calf lungs were tested. Five normal mouse and three normal human lungs were similarly tested as well as 5 human lungs with diagnosis of asthma. The expression was determined with multiple methods such as Western blots, immunohistology, immunogold electron microscopy and in situ hybridization. Lungs from all the species showed TLR9 expression in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells. Immuno-electron microscopy detected TLR9 on the plasma membrane, cytoplasm and the nucleus of various cells including macrophages. In situ hybridization demonstrated TLR9 mRNA in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells of mouse and human. Asthmatic human lungs showed many more inflammatory cells expressing TLR9 compared to healthy lungs. In cattle and horses, pulmonary intravascular macrophages showed robust expression of TLR9. Depletion of pulmonary intravascular macrophages in horses resulted in significant reduction in total TLR9 mRNA in the lungs. Having determined that TLR9 expression is similarly expressed on many lung cell types in mice and humans, I determined the role of TLR9 in barn air induced lung inflammation by exposing TLR9-/- and wild-type mice (6 per group) to single or multiple days (5 and 20) in a chicken barn. Each exposure was of 8 hours/day duration. The TLR9-/- mice exposed 5 and 20 times showed significant reductions in TNF-alpha and IFN-gamma expression in lung lavages as well as cellular changes consistent with reduced lung inflammation such as reductions in the number of lung neutrophils. This suggests that barn dust DNA, acting through TLR9, contributes to lung inflammation seen in response to exposure to chicken barn air. These fundamental data advance our knowledge on the cell-specific expression of TLR9 across a range of species including the humans and demonstrate that TLR9-/- partially regulates lung inflammation induced following exposure to chicken barn air.

Genes modulating innate immunity in mammals are generally considered the first line of defense with respect to invading pathogens and therefore it has become important to characterize naturally occurring genetic variation, and subsequently determine whether this variation is likely to be benign, beneficial, or detrimental to the host. Relevant to this study, the mammalian Toll-like receptor proteins (TLR), encoded by members of the TLR gene family, have the capacity to recognize a wide variety of pathogen ligands, and mutations within these genes have been shown to influence disease susceptibility or resistance within mammalian species. Two studies which sought to determine the frequency and distribution of naturally occurring genetic variation within the bovine and equine TLR genes revealed a large number of discrete point mutations, which were subsequently used to reconstruct haplotypes for each investigated gene across a large number of samples. Detailed analyses of haplotypes provided evidence for extensive haplotype sharing among specialized breeds, subspecies, and even divergent species. Classical and new tests of selection provided evidence for significant deviations from a strictly neutral model of molecular evolution for both cattle as well as equids, with some of the same TLR genes deviating from a strictly neutral model among divergent species. As a first step toward determining whether naturally occurring bovine TLR variation is likely to be benign, beneficial, or detrimental, we tested validated variation from bovine TLR genes capable of recognizing components of Mycobacteria for associations with Mycobacterium avium subspecies paratuberculosis (MAP) infection in dairy cattle, and found several SNPs that were nominally associated with disease status, thereby providing evidence for small-effect loci potentially influencing risk for differential susceptibility to Johne's disease.

Lung dysfunction due to exposure to air in high intensity livestock barn operations is a common problem for workers in these facilities. Exposure to this air has been linked to disorders such as chronic bronchitis, occupational asthma, organic dust toxic syndrome, and chronic cough and phlegm. These symptoms have been linked to higher levels of endotoxins in air in chicken and swine barns. However, there are many other toxic molecules such as bacterial DNA and gases capable of inducing respiratory inflammation. Bacterial molecules are recognized through highly conserved pattern recognition molecules called Toll-like receptors (TLR). While lipopolysaccharides are recognized by TLR4, bacterial unmethylated DNA binds to and signals through TLR9. As a prelude to understanding the biology of TLR9 in lung inflammation, it is important to precisely clarify their in situ expression in the lung. I determined expression of TLR9 in intact lungs from cattle, pigs, dogs, horses, mice, and humans. Two samples from normal lungs of cattle, pigs, dogs, three from horses, and two from inflamed calf lungs were tested. Five normal mouse and three normal human lungs were similarly tested as well as 5 human lungs with diagnosis of asthma. The expression was determined with multiple methods such as Western blots, immunohistology, immunogold electron microscopy and in situ hybridization. Lungs from all the species showed TLR9 expression in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells. Immuno-electron microscopy detected TLR9 on the plasma membrane, cytoplasm and the nucleus of various cells including macrophages. In situ hybridization demonstrated TLR9 mRNA in the bronchial epithelium, vascular endothelium, alveolar septa, alveolar macrophages, and type-II alveolar epithelial cells of mouse and human. Asthmatic human lungs showed many more inflammatory cells expressing TLR9 compared to healthy lungs. In cattle and horses, pulmonary intravascular macrophages showed robust expression of TLR9. Depletion of pulmonary intravascular macrophages in horses resulted in significant reduction in total TLR9 mRNA in the lungs. Having determined that TLR9 expression is similarly expressed on many lung cell types in mice and humans, I determined the role of TLR9 in barn air induced lung inflammation by exposing TLR9-/- and wild-type mice (6 per group) to single or multiple days (5 and 20) in a chicken barn. Each exposure was of 8 hours/day duration. The TLR9-/- mice exposed 5 and 20 times showed significant reductions in TNF-alpha and IFN-gamma expression in lung lavages as well as cellular changes consistent with reduced lung inflammation such as reductions in the number of lung neutrophils. This suggests that barn dust DNA, acting through TLR9, contributes to lung inflammation seen in response to exposure to chicken barn air. These fundamental data advance our knowledge on the cell-specific expression of TLR9 across a range of species including the humans and demonstrate that TLR9-/- partially regulates lung inflammation induced following exposure to chicken barn air.

<i>Rhodococcus equi</i> (<i>R. equi</i>) is an intracellular, gram-positive coccobacillus that causes pneumonia in foals aged 2 to 4 months. Neonatal foals are susceptible to <i>R. equi</i> infection probably due to inefficient Toll-like receptor (TLR)-2 signaling and inability to produce interferon gamma. One of the reasons for inefficient receptor signaling and recognition of <i>R. equi</i> by the foals immune system may be the inefficient sequestration of TLRs in lipid rafts, which act as signaling platforms. However, there are no protocols to isolate lipid rafts from equine cells and, therefore, no data on the association of TLRs with the lipid rafts in the lung cells of normal and infected foals. Because of the clinical importance of the disease, there is considerable interest in developing effective prophylactic methods, which in turn requires a better understanding of fundamental immunology of the foals. In this study, I have examined the effect of <i>R. equi</i> vaccination on the lung inflammation induced following challenge with <i>R. equi</i>. I also developed a protocol to isolate lipid rafts from broncho-alveolar lavage (BAL) cells and investigated the association of lipid rafts with TLRs.<p> In the first study, 15 mixed breed draft-type foals up to 7 weeks of age were studied. The foals were divided into control (n=7) and a vaccinated (n=8). The control foals were given 10 mL phosphate buffered saline intramuscularly while the vaccinated group was vaccinated on day 0 of the study followed by a booster on day 14. All the foals were challenged with <i>R. equi</i> (5x106 cells/mL into the dorso-caudal region of the right lung lobe). BAL was performed on day 14, 28 and 35 and all the foals were euthanized on day 49 of the study.<p> The study design did not leave any non-infected foal at the end of the experiment. Therefore, lung samples were obtained from two untreated control (non-vaccinated non-infected) foals from the Department of Veterinary Pathology, University of Saskatchewan were used. The data showed similar levels of lung inflammation in both the control and vaccinated foal groups based on BAL cytology, gross pathology and histopathology. Gross and histopathological studies indicated that both control and vaccinated foals developed granulomatous lesions. Immunohistology showed increased expression of TLR4, TLR2 and TNF alpha in alveolar septa and in some cases in the vascular endothelium and airway epithelium in the lungs of both groups compared to the untreated control foals. Western blots showed increased expression of TLR2 but not TLR4 in the lung extracts from both the vaccinated and the control foals. Vaccinated foals showed higher concentrations of TNF alpha(p=0.0219) in their BAL on day 28 but lower concentrations of IL-10 (p=0.0172) in their lung extracts collected on day 49 compared to the controls. There were no differences in IFN gamma and protein concentrations between the two groups.<p> To understand the role of lipid rafts in TLR4 and TLR2 signaling, I developed an efficient and simpler protocol to isolate lipid rafts from BAL cells of foals and confirmed their identity by localizing Flotillin-1 and GM-1 (fractions 6-9), which are lipid raft markers, and transferrin receptor (fractions 1-4) which is present in non-lipid raft fractions. Lung macrophages from naïve foals lacked sequestration of Flotillin-1 and GM-1 in the higher fractions compared to the vaccinated foals. Further, the data showed that while TLR4 and TLR2 were localized in most of the fractions (1-9) in control foal BAL collected on day 14 and 28, the TLR4 and TLR2 association was restricted to fractions 6-9 in the lipid rafts isolated from BAL cells of vaccinated foals. These data suggest that BAL cells of neonatal foals may not have effective signaling machinery because of lack of association of TLR2 and TLR4 with lipid rafts.<p> Taken together, the data show similar levels of lung inflammation in the control and vaccinated foals upon infection with <i>R. equi</i>. The vaccination, however, appeared to have some effect on the immunohistologic expression of TLR2, TLR4 and TNFalpha in the lung tissues, and increased association of TLR2 and TLR4 with the lipid raft fractions. Based on the higher expression of TNF alpha and lower expression of IL-10, the vaccinated foals may be more competent to mount an immune response against <i>R. equi</i>.

In multicellular organisms, communication between cells relies on transmitting information across membrane barriers. Different cell types interrogate particular aspects of their surrounding environment through protein receptors that span membranes and upon ligand binding, trigger enzymatic signaling cascades that culminate in the activation of one or more transcription factors. Information transmission is bidirectional, as individual cells must be able to sense unique aspects of their surroundings, relay their specialized knowledge with others, and receive the collective knowledge of surrounding cells and tissues. This two-way communication is particularly important in the innate immune system, where potentially infectious organisms must be readily detected and identified, and their presence communicated to other cells in the vicinity. Because of the rapid generation time of microorganisms, delays between any of these steps - detection, information processing or information transmission - can make the difference between successful control of infection and pathogen outgrowth. For this reason, the receptors that identify potential pathogens must be able to detect pathogens wherever they are found, be exquisitely sensitive, and initiate a robust response. At the same time, the inflammatory response to infection is itself damaging. This requires that the same receptors are tightly controlled, both by modulating their sensitivity and by rapidly turning off responses through negative feedback pathways. Here, I show that the toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP) plays a critical role in controlling the sensitivity of toll-like receptor (TLR) signaling. First, TIRAP controls the assembly of the myddosome, a protein complex that activates signal transduction, from both the plasma membrane and within endosomes of macrophages. Though TIRAP's role at the cell surface was previously described, its endosomal function was previously unknown. Second, TIRAP is an important target for negative regulation. After stimulation with the TLR4 ligand lipopolysaccharide (LPS), macrophages induce a state known as endotoxin tolerance, in which they are refractory for additional LPS stimulation. Many mechanisms for endotoxin tolerance have been proposed, but here I show that TIRAP is degraded in endotoxin tolerance, and that the mechanism of TIRAP degradation also has implications for viral/bacterial superinfection.

Patients with inflammatory or infectious conditions such as periodontitis, peri-implantitis, osteomyelitis, rheumatoid arthritis, septic arthritis and loosened joint prosthesis display varying severity of destruction in the adjacent bone tissue. Bone loss in inflammatory diseases is considered a consequence of cytokine induced RANKL and subsequent enhanced osteoclast formation. Hence, osteotropic cytokines and their receptors have been suggested to be important for the pathogenesis of inflammation-induced osteolysis. It is, here, suggested that bacterial components, so called “pathogen associated molecular patterns=PAMPs”, may also be involved. Varieties of cells express receptors for PAMPs, including Toll-like receptors (TLRs) which are the first line of defence in the innate immune system. LPS (lipopolysaccharide), fimbria and lipoproteins from pathogenic bacteria such as P. gingivalis, S. aureus are ligands for TLR2 and flagellin from pathogenic flagellated bacteria like S. typhimurium is a ligand for TLR5.   Since the susceptibility to, or the severity of inflammation-associated bone diseases are likely related to differences in the tissue response, and the mechanisms by which PAMPs interact with bone cells are not fully understood, we aimed to elucidate the importance of different TLRs for inflammation induced bone loss by conducting in vitro and in vivo investigations. Activation of TLR2 and TLR5 in organ cultured mouse parietal bones increased bone resorption in a time- and concentration-dependent manner by a process inhibited by OPG and bisphosphonate, showing the crucial role of RANKL-induced osteoclast formation. In addition, the number of osteoclasts, expression of osteoclastic genes and osteoclastogenic transcription factors were increased. In the bones and in osteoblasts isolated from the bones, TLR2 agonists increased the expression of RANKL without affecting OPG, while TLR5 activation resulted in enhanced RANKL and decreased OPG. Activation of both TLR2 and TLR5 stimulated the expression in both bones and osteoblasts of prostaglandins and pro-inflammatory cytokines, known to stimulate RANKL. By blocking the cytokines and prostaglandin, we showed that TLR2 and TLR5 induced bone resorption and RANKL expression are independent of these molecules. Activation of TLR2, but not TLR5, in mouse bone marrow macrophage cultures inhibited RANKL-induced osteoclast formation, an effect not observed in committed pre-osteoclasts. Local administration in vivo of TLR2 and TLR5 agonists on the top of mouse skull bones enhanced local and systemic osteoclast formation and bone resorption. Using knockout mice, we showed that the effects by LPS from P. gingivalis (used as TLR2 agonist) and flagellins (used as TLR5 agonists) are explicit for TLR2 and TLR5 ex vivo and in vivo, respectively. These data show that stimulation of TLR2 and TLR5 results in bone resorption in vitro and in vivo mediated by increased RANKL in osteoblasts and thus may be one mechanism for developing inflammatory bone loss. Interestingly, histological analyses of skull bones of mice treated locally with TLR2 and TLR5 agonists revealed that the bones not only reacted with locally increased osteoclastogenesis (osteoclast formation), but also with locally increased new bone formation. This was observed on both periosteal and endosteal sides of the bones, as well as in the bone marrow compartment. The formation of new bone was seen close to osteoclasts in some parts, but also in other areas, distant from these cells. The response was associated with active, cuboidal osteoblasts, extensive cell proliferation and increased expression of genes coding for bone matrix proteins and osteoblastic transcription factors. In conclusion, activation of TLR2 and TLR5 in osteoblasts results in bone loss associated with enhanced osteoclast formation and bone resorption, as well as with increased osteoblast differentiation and new bone formation, indicating that inflammation causes bone modeling. The data provide an explanation why LPS from P. gingivalis and flagellin from flagella-expressing bacteria can stimulate bone loss. Since TLR2 and TLR5 can be activated not only by bacterial components, but also by endogenous ligands produced in inflammatory processes, the data also contribute to the understanding of inflammation induced bone loss in autoimmune diseases. Hopefully, these findings will contribute to the development of treatment strategies for inflammation induced bone loss.

Many common cancers have a predisposition for bone metastasis. Tumor occupation of bone is both destructive and a source of debilitating pain in cancer patients. As a result, cancer-induced bone pain (CIBP) is the single most common form of clinical cancer pain. Opioids remain the golden standard for the management of CIBP; however, >30% of cancer patients do not experience adequate pain relief with opioids. Furthermore, clinical reports have suggested that opioids can exacerbate bone loss and increase the likelihood of skeletal-related events. To date, there is no known direct mechanism for opioid-induced bone loss (OIBL). We hypothesized that opioid off-target activation of toll-like receptor 4 (TLR4), an innate immune receptor that is expressed in bone, mediates an increase bone loss and associated CIBP. In the 66.1-BALB/cfC3H murine model of breast cancer bone metastasis, TLR4 expression is upregulated in tumor-burdened bone. Chronic morphine treatment exacerbated spontaneous and evoked pain behaviors in a manner paralleled by bone loss: we identified an increase in spontaneous fracture and osteolysis markers including serum collagen-type I (CTX) and intramedullary receptor activator of nuclear κ-B ligand (RANKL). Administration of (+)naloxone, a non-opioid TLR4 antagonist, attenuated both exacerbation of CIBP and morphine-induced osteolytic changes in vivo. Morphine did not alter tumor burden in vivo or tumor cell growth in vitro. Importantly, morphine produced the in vitro differentiation and activation of osteoclasts in a dose-dependent manner that was reversible with (+)naloxone, suggesting that morphine may contribute directly to osteolytic activation. To improve opioid management of CIBP, we then posited and evaluated three novel adjunct therapeutic targets: cannabinoid receptor-2, adenosine 3 receptor and sphingosine-1-phosphate receptor 1. These pharmacological targets were identified as having a multiplicity of anti-cancer, osteoprotective and/or neuroprotective effects in addition to analgesic efficacy in chronic pain. Targets were tested in the 66.1-BALB/cfC3H model of CIBP and demonstrated to have stand-alone efficacy as antinociceptive agents. Taken together, this work provides a cautionary evaluation of opioid therapy in cancer-induced bone pain and seeks to mitigate opioid side effects through the identification of innovative adjunct therapies that can ultimately improve quality of life in patients suffering from cancer pain.

Immunosenescence results in reduced immune response to infections with Streptococcus pneumoniae as well as to pneumococcal polysaccharide vaccines. The antibody response to the capsular polysaccharide (CPS) provides protection against S. pneumoniae infection. CPS immunoresponse is T cell independent and needs the macrophage-derived cytokines such as IL-12, IL-6 and IL-1β to elicit an antibody response. We showed a cytokine dysregulation, i.e. a decrease in IL-12, IL-6 and TNF-α but an increase in IL-10, in the aged (18-24 months old comparable to >65 years in human) compared to young adult mouse (8-12 weeks less than 65 years old) splenic macrophages (SM) or bone marrow derived macrophages (BMDM) activated via TLR4, TLR2 or TLR9 as well as heat killed Streptococcus pneumoniae (HKSP). There is also an age-associated defect in splenic B cells in the production of IgG3 upon stimulation with these ligands. A microarray analysis in SM followed by validation by both qt-RTPCR and western blots indicated that this age-associated defect in aged SM, BMDM and B cells was due to a heightened activity of the PI3K-Akt signaling pathway. We hypothesized that the senescence of immune responses in macrophages and B cells is due to an increase in activity of PI3K/Akt and decrease in the activity of GSK-3, the downstream kinase. Inhibition of the PI3-kinase with either LY294002 or Wortmannin restored the TLR2, 4, 9 and HKSP induced cytokine phenotype of the aged to that of the young adult in both the SM and BMDM and an enhanced IgG3 production in aged mice. We also showed that inhibition of glycogen synthase kinase-3 (GSK-3) the downstream target of the PI3K-Akt signaling pathway with SB216763 in SM, BMDM and B cells resulted in an enhancement in production of IL-10, IL-6 and IL-1β by macrophages and in B cell activation. Treatment of B cells with SB216763 in the presence of ligands for TLR-1/2, 4 or 9 as well as HKSP under in vitro conditions led to enhanced production of IgG3 and IgA, plasma cell formation and a slight increase in the proliferation of the B-cells with no adverse effects on the viability of the cells. Therefore, targeting the PI3K-AKT-GKS-3 signaling pathway could rescue the intrinsic signaling defect in the aged macrophages, increase IL-12 and IL-6, and enhance anti-CPS antibody responses.