The innate immune system depends for its effectiveness on the function of specialized pattern recognition receptors which enable it to target pathogens for destruction on the basis of conserved molecular patterns such as flagellin or lipopolysaccharide (LPS). Specifically, LPS is recognized by the Toll-like receptor 4 (TLR4), activating a signaling pathway which triggers the production of both pro- and anti-inflammatory mediators. Very low doses of LPS, however, preferentially induce pro-inflammatory cytokines, which can lead to persistent low-grade inflammation, a contributing factor in a host of chronic diseases. The mild pro-inflammatory skewing induced by super-low-dose LPS also potentiates the inflammatory response to later challenge with a higher dose of LPS in a phenomenon known as the "Shwartzman reaction" or "endotoxin priming". We investigated the mechanisms involved in pro-inflammatory skewing by super-low-dose LPS in THP-1 cells and found it to be governed by a regulatory circuit of competitive inhibition between glycogen synthase kinase 3 (GSK3) and Akt, which promote the activity of the transcription factors FoxO1 and CREB, respectively. Super-low-dose LPS mildly activated FoxO1 and pro-inflammatory gene transcription without inducing anti-inflammatory genes or activating CREB, and this pro-inflammatory skewing could be abolished by inhibition of GSK3 or direct activation of CREB. We then examined the dynamics of the LPS response at various different dosages in murine bone-marrow-derived macrophages (BMDM). The pro-inflammatory cytokine IL-12 was most strongly induced by intermediate LPS dosages, with very low or high doses inducing less robust IL-12 production. Knockout of the inhibitory TLR4 pathway molecules Lyn or IRAK-M resulted in sustained induction of IL-12 by high doses of LPS. By activating CREB, we were able to reduce inflammation in WT BMDM, and saw that this corresponded with increased phosphorylation of CREB. Overall, we are confident that this subnetwork is an important switch regulating the resolution of inflammation in response to TLR4 stimulation. Furthermore, we propose that endotoxin priming is an example of the generalized capacity of all signaling networks to recall prior states, and that an appreciation for the history and context of exposure to stimuli is critical for the understanding of signaling behavior. / Ph. D.

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Master of Science / Department of Biology / Sherry D. Fleming / Helicobacter is a common intestinal pathogen of most laboratory mice from both commercial and academic sources worldwide. Not previously thought to have an effect, recent evidence indicates Helicobacter infection alters cytokine, chemokine, and gene expression in the stomach, intestine, and colon. Though the in vivo cell types responsible for these changes are currently unknown, in vitro results suggest macrophages are the likely source. In addition to detection and elimination of pathogens, intestinal macrophages play a role in maintaining homeostasis. By altering gene expression and cytokine production in the microenvironment, we hypothesized that Helicobacter infection altered the phenotype and inflammatory response of submucosal intestinal macrophages. To test this hypothesis, we examined macrophages within whole mounts of intestinal muscle as well as isolated macrophages from Helicobacter-infected or uninfected mouse intestine. Macrophages from the intestinal muscle of Helicobacter-infected mice showed increased expression of F4/80 and CD11b, altered gene expression, and increased phagocytosis when compared to macrophages from uninfected mice. Infection also altered the macrophage response to stimuli. Macrophages from infected mice produced significantly lower concentrations of cytokines, chemokines, and PGE[subscript]2 in response to stimulation with either IFN and LPS or IL-4 and IC. These data support our hypothesis demonstrating that the intestinal muscle macrophage phenotype, function, and response to stimulation are altered by Helicobacter infection both in vivo and in vitro.

Galectin-3, a unique member of the growing family of β-galactoside binding lectins, contains a single carbohydrate recognition domain and a glycine rich N-terminal domain through which it can form oligomers and functions to cross-link both carbohydrate and non-carbohydrate ligands. Galectin-3 is widely expressed in adult tissues, particularly on and secreted by activated macrophages and monocytes. Galectin-3 has been implicated in many facets of the inflammatory response including neutrophil and macrophage activation and function. In this thesis I have examined the role of galectin-3 during fibrosis, alternative activation of macrophages and pneumonia. Galectin-3 expression is upregulated in established human fibrotic liver disease and in a mouse model of liver fibrosis induced by carbon tetrachloride. Galectin-3 expression is temporally and spatially related to the induction and resolution of experimental hepatic fibrosis in this model. In addition, disruption of the galectin-3 gene markedly attenuates liver and kidney fibrosis, induced by unilateral ureteric obstruction, with reduced collagen deposition and myofibroblast activation. Results suggest that galectin-3 may promote fibrosis by stimulating myofibroblast activation by a transforming growth factor-β (TGF-β)-independent mechanism. Recent reports suggest that alternative macrophage activation is one of the key steps toward the progression of fibrosis. Disruption of the galectin-3 gene specifically restrains interleukin-4 (IL-4)/IL-13-induced alternative macrophage activation in vitro. My results suggest that the key mechanism required for activation of an alternative macrophage phenotype is an IL-4-stimulated galectin-3 feed back loop which directly activates CD98 causing sustained phosphatidylinositol 3-kinase (PI3-K) activation. The gram-positive Streptococcus pneumoniae (S. pn) is the leading cause of community acquired pneumonia worldwide, resulting in high mortality. Galectin-3-/- mice demonstrate a clearance defect of S. pn with increased septicaemia and a greater extent of lung damage compared to wild type mice. This phenotype is markedly reduced in pneumonia induced by the gram-negative Escherichia coli (E.coli). I have shown that presence of galectin-3 reduces the severity of pneumonia induced by S. pn and this is achieved through a number of processes: 1) Galectin-3 has bactericidal properties towards S. pn in vitro. 2) Galectin-3-/- macrophages show reduced production of nitrite following incubation with both S. pn and E. coli and hence a reduction in bacterial killing. 3) Galectin-3 activates neutrophils to produce reactive oxygen species which enhances the bactericidal activity of neutrophils. 4) Activation of neutrophils by galectin-3 augments phagocytosis of bacteria. 5) Finally, initial data suggests that galectin-3-/- neutrophils apoptose more readily than wild type neutrophils in vitro and galectin-3-/- macrophages phagocytose apoptotic neutrophils less efficiently compared to wild type. In vivo this would result in an accumulation of dying cells in the lung. The damage these apoptotic cells would have on the lung tissue may enable the bacteria to enter the blood stream resulting in sepsis. In summary, in response to chronic tissue injury, persistant upregulation of galectin-3 causes myofibroblast and alternative macrophage activation, thus enhancing collagen deposition and scarring. However during an acute S. pn infection, galectin-3 plays a benefitial role to aid the clearance of bacteria through a variety of processes. Therefore, galectin-3 plays a critical role in a variety of inflammatory disorders.

Macrophages (Mφ) have previously been implicated in both acute and chronic renal allograft rejection however the mechanisms remain unclear. In this thesis I set out to explore the effect of the Mφ on the endothelium in the context of renal graft rejection. Initial studies focussed upon human renal allograft tissue from transplant nephrectomies performed because of chronic allograft nephropathy (CAN). Immunostaining was carried out on these tissues (n=29) and control kidney tissue obtained from nephrectomies performed for renal cell carcinoma (n=19). An increased interstitial Mφ infiltrate was found compared to control tissue. Immunostaining for the T cell marker CD3 and the B cell marker CD20 demonstrated that both lymphocyte populations were present in the CAN tissue with almost negligible numbers seen in control tissue. Previous work in the group had demonstrated a reduced number of CD31 positive peritubular capillaries in the tissues used in these studies. In the work undertaken in this thesis, additional analysis was performed to study lymphatic vessels. Immunostaining of control tissue with the lymphatic endothelial cell (LEC) marker podoplanin demonstrated a normal distribution of lymphatic vessels around large interlobular arteries. CAN tissue, however, exhibited an increased lymphatic density with lymphatic vessels evident within the interstitium; a finding verified with two additional LEC markers (LYVE-1 and VEGFR-3). Further investigations examined possible mediators that could be responsible for the reduced microvascular peritubular capillary network and increased lymphatic vessels present in tissues affected by CAN. Previous work had implicated nitric oxide (NO) generated by the enzyme inducible nitric oxide synthase (iNOS) in cardiac allograft rejection. Double immunolabelling for iNOS and the Mφ marker CD68 revealed evidence of Mφ expression of iNOS. No obvious reduction in vascular endothelial growth factor (VEGF)-A was evident although marked expression of VEGF-A was found in CD20 positive B cells within CAN tissue. Occasional interstitial cells expressed the lymphangiogenic growth factor VEGF-C, with double labelling studies indicating occasional CD68 +ve Mø that were positive for VEGF-C. In vitro studies were undertaken to dissect the interaction between Mø and microvascular endothelial cells (MCEC-1) using well established in vitro co-culture techniques. Co-culture of cytokine activated bone marrow derived Mø with MCEC-1 cells (a murine cardiac microvascular endothelial cell line) resulted in increasing levels of MCEC-1 apoptosis and a reduced cell number over a 24-hour time course. Non-activated Mø or cytokines alone were not cytotoxic. Co-cultures were performed in the presence of L-Nimino- ethyl lysine (L-Nil), a specific inhibitor of iNOS (control D-N6- (1-iminoethyl)-lysine (D-Nil)). L-Nil significantly inhibited MCEC-1 apoptosis and preserved cell number implicating a major role for NO in Mø-mediated MCEC-1 death. Importantly, L-Nil treatment did not affect TNFα production by cytokines suggesting that TNFα is not involved in MCEC-1 death in this in vitro experimental system. Experiments were then undertaken involving the depletion of Mø in a murine model of acute renal allograft rejection. Renal transplants were performed between donor Balb/c mice and either FVB/N CD11b-DTR mice transgenic for the diphtheria toxin receptor (DTR) under the CD11b promoter or control non-transgenic FVB/N mice. Diphtheria toxin (DT) was administered on days 3 and 5 to induce Mø depletion and mice sacrificed at day 7. Isograft controls were also performed between FVB/N mice. Murine allografts exhibited marked interstitial F4/80 positive Mø infiltration with expression of iNOS in the allografts. There was significant loss of peritubular capillaries (PTC) in allografts compared to isografts, indicating microvascular injury. DT treated CD11b-DTR mice exhibited 75% reduction in Mø infiltration and this was associated with dramatic microvascular protection. B and T cells were not evident in the isograft but significant accumulation of B and T cells was present in the allograft and not affect by Mø depletion. Interestingly, there was an increase in the number of podoplanin positive lymphatic vessels in the allograft compared to the isograft, which was significantly inhibited following Mø depletion. The final area of study focussed upon attempts to isolate lymphatic endothelial cells in vitro. Two types of vascular cells (HUVECs and HDMECs) were analysed by flow cytometry for LEC markers and immunofluorescence to phenotype the cells. Magnetic bead sorting was then undertaken to isolate discrete populations of endothelial cells expressing LEC markers. The murine studies reinforce the cytotoxic potential of Mø and supports a role for Mø in the deleterious rarefaction of microvascular interstitial vessels with resultant tissue hypoxia and ischaemia. Furthermore, these data support the involvement of Mø in the interstitial lymphangiogenesis that may occur in renal allografts. Furthermore, the study of human allograft tissue indicates that microvascular rarefaction and an increase in intrarenal lymphatic vessels occurs in human disease. Lastly, Mø expression of iNOS and VEGF-C suggests that Mø are involved in key processes that may adversely affect graft outcome.

Probiotics confer health benefits through many mechanisms including modulation of the gut immune system. Gut mucosal macrophages play a pivotal role in driving mucosal immune responses. The local environment and macrophage subset determine immune response: tolerance, associated with an M2-like, regulatory macrophage phenotype and inflammatory activation with an M1-like phenotype. The aims of this study were firstly to investigate the immunomodulatory effects of a panel of heat-killed (HK) probiotic bacteria and their secreted proteins (SP) of Bifidobacterium breve (BB), Lactobacillus rhamnosus GG (LR), L. salivarius (LS), L. plantarum (LP), L. ferrmentum (LF), and L. casei strain Shirota (LcS) on cytokine production and TLR expression in monocultures of monocytes, macrophage subsets, and intestinal epithelial cells. Normally, mucosal gut macrophages resemble the M2 subset and fail to express CD14, a co-receptor for LPS signalling. Therefore, probiotic modulation of LPS-induced NF-kB activity and cytokine expression was investigated using a THP-1 monocyte-derived reporter cell line, model of CD14 hi / lo M1 and M2 macrophages. Secondly, a transwell co-culture system was developed to investigate probiotic modulation of macrophage-influenced epithelial barrier function. Parameters investigated included cytokine, TLR and hBD-2 expression, TEER and IHC staining of the tight junction protein, ZO-1. Probiotics selectively modulated monocyte and macrophage subset cytokine expression. Probiotics (HK and SP) suppress CD14 lo , augment CD14 hi M1, and differentially regulated TNF-α production in M2s. M2 macrophage IL-6 production was suppressed by both HK and SPs, and differentially regulated in CD14 lo and CD14 hi M1s. NF-κB activation failed to parallel probiotic regulation of TNF-α and IL-6. Probiotics (HK-LF and HK-LcS) selectively modulated both endogenous and exogenous TNF-α and IL-10, as well as their induction of epithelial cell expression of TLR and hBD-2. Epithelial expression of TEER, ZO-1 and the endogenous TLR signal regulator, Tollip, were suppressed upon co-culture with pro-inflammatory M1 macrophages paralleled by a suppression of IL-10 and up-regulation of TNF-α and IL-8. In the presence of LPS, HK-LF enhanced TEER, ZO-1 and partially rescued Tollip expression, whereas HK-LcS had no effect on TEER and ZO-1 and displayed a weaker rescue effect on Tollip compared with LF. In the M2/epithelial cell co-culture, both probiotics enhanced TEER and ZO-1 in the presence of LPS, whilst displaying a differential modulation of Tollip, dependant on the format of probiotic (HK or SP). In conclusion, probiotic strains can differentially exert immune activatory or suppressive functions and immunomodulation is determined by strain, inflammatory environment, and mucosal macrophage effector phenotype. Future probiotic development must consider prophylactic use in healthy individuals or therapeutic treatment of defined pathological conditions, strain-specific effects, gut mucosal integrity, and immune phenotype of mucosal macrophages.

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