CSF-1–dependant donor-derived macrophages mediate chronic graft-versus-host disease

Chronic GVHD (cGVHD) is the major cause of late, nonrelapse death following stem cell transplantation and characteristically develops in organs such as skin and lung. Here, we used multiple murine models of cGVHD to investigate the contribution of macrophage populations in the development of cGVHD. Using an established IL-17–dependent sclerodermatous cGVHD model, we confirmed that macrophages infiltrating the skin are derived from donor bone marrow (F4/80+CSF-1R+CD206+iNOS–). Cutaneous cGVHD developed in a CSF-1/CSF-1R–dependent manner, as treatment of recipients after transplantation with CSF-1 exacerbated macrophage infiltration and cutaneous pathology. Additionally, recipients of grafts from Csf1r–/– mice had substantially less macrophage infiltration and cutaneous pathology as compared with those receiving wild-type grafts. Neither CCL2/CCR2 nor GM-CSF/GM-CSFR signaling pathways were required for macrophage infiltration or development of cGVHD. In a different cGVHD model, in which bronchiolitis obliterans is a prominent manifestation, F4/80+ macrophage infiltration was similarly noted in the lungs of recipients after transplantation, and lung cGVHD was also IL-17 and CSF-1/CSF-1R dependent. Importantly, depletion of macrophages using an anti–CSF-1R mAb markedly reduced cutaneous and pulmonary cGVHD. Taken together, these data indicate that donor macrophages mediate the development of cGVHD and suggest that targeting CSF-1 signaling after transplantation may prevent and treat cGVHD

Deletion of Wntless in myeloid cells exacerbates liver fibrosis and the ductular reaction in chronic liver injury

Background: Macrophages play critical roles in liver regeneration, fibrosis development and resolution. They are among the first responders to liver injury and are implicated in orchestrating the fibrogenic response via multiple mechanisms. Macrophages are also intimately associated with the activated hepatic progenitor cell (HPC) niche or ductular reaction that develops in parallel with fibrosis. Among the many macrophage-derived mediators implicated in liver disease progression, a key role for macrophage-derived Wnt proteins in driving pro-regenerative HPC activation towards a hepatocellular fate has been suggested. Wnt proteins, in general, however, have been associated with both pro-and anti-fibrogenic activities in the liver and other organs. We investigated the role of macrophage-derived Wnt proteins in fibrogenesis and HPC activation in murine models of chronic liver disease by conditionally deleting Wntless expression, which encodes a chaperone essential for Wnt protein secretion, in LysM-Cre-expressing myeloid cells (LysM-Wls mice)

The role of c-jun N-terminal kinase (JNK) in human T cell function.

T cells are involved in cellular pathways which enable the immune system to protect us against infection and cancer. However, the same mechanisms also allow T cells to generate chronic inflammatory conditions, including autoimmunity and allergy. Thus a concerted effort has been made to try to understand how the immune system functions in order to inhibit responses which may have harmful effects on tissues and organs. There is a continued search for new immunosuppressants which can only be accomplished through a better understanding of the pathways that regulate T cell function. This includes the intracellular signalling pathways which modulate T cell proliferation and cytokine production. While the Mitogen-Activated Protein Kinases (MAPK), extracellular signal-regulated protein kinases (ERK) and p38 have received attention, the role of the stress-activated protein kinases or c-jun N-terminal kinases (JNK) remains controversial. To overcome some of the limitations in studying the role of JNK, a new approach was taken in this thesis. The investigations used recently described peptides (TAT-JIP[subscript]153-163 and TAT-JIP[subscript]153-172) derived from the scaffold protein, JIP-1, which have previously been demonstrated to act as JNK pathway inhibitors. The research characterised the specificity of these inhibitors to enable the appropriate interpretation of data. Using these inhibitors, we were able to show that JNK regulated human T cell proliferation and cytokine production in T cell responses induced independently of TCR ligation (PHAPMA) or via the TCR (anti-CD3-anti-CD28 antibodies, Mixed Lymphocyte Reaction (MLR), Tetanus Toxoid and Der p 2). The data demonstrated that JNK primarily regulated the Th1 cytokine patterns (IFNγ, IL2 and LT) with minimal effect on Th2 cytokine production (IL4, IL10) in response to all stimulatory models. However, while the JNK signalling pathway promoted T cell proliferation and cytokine production in response to PHA-PMA, the pathway depressed these responses following stimulation with anti-CD3-anti-CD28 antibodies and Tetanus Toxoid. Thus activation of JNK with microbial pathogens such as Pseudomonas aeruginosa (PA), which non-specifically activate T cells, may promote lymphocyte proliferation and the release of Th1 cytokines, such as IFNγ. In contrast, JNK activation resulting from engagement of the T cell receptor (TCR) (i.e. Tetanus Toxoid), down-regulates Th1 cytokine production. Therefore, it is likely that the JNK signalling pathway may dampen the development of chronic inflammatory conditions resulting from infection with intracellular parasites and autoimmune diseases. In contrast to Tetanus Toxoid, responses to the recombinant house dust mite allergen, Dermatophagoides pteronyssinus (Der p 2) were promoted by JNK, leading to an increase in Th1 cytokine production. Thus the results suggest that the use of JNK inhibitors could exacerbate both inflammatory conditions (autoimmunity and allergy) and this may also apply to p38 but not the ERK signalling pathway.Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 200

The immunomodulatory effects of novel β-oxa, β-thia, and γ-thia polyunsaturated fatty acids on human T lymphocyte proliferation, cytokine production, and activation of protein kinase C and MAPKs

We have recently demonstrated that a novel n-3 long chain polyunsaturated fatty acid (PUFA) (β-oxa 21:3n-3) was a more potent and more selective anti-inflammatory agent than n-3 PUFA. To gain further insights into this technology, we synthesized other n

Hepatocyte-specific deletion of peroxisomal protein PEX13 results in disrupted iron homeostasis

Peroxisomes are organelles, abundant in the liver, involved in a variety of cellular functions, including fatty acid metabolism, plasmalogen synthesis and metabolism of reactive oxygen species. Several inherited disorders are associated with peroxisomal dysfunction; increasingly many are associated with hepatic pathologies. The liver plays a principal role in regulation of iron metabolism. In this study we examined the possibility of a relationship between iron homeostasis and peroxisomal integrity. We examined the effect of deleting Pex13 in mouse liver on systemic iron homeostasis. We also used siRNA-mediated knock-down of PEX13 in a human hepatoma cell line (HepG2/C3A) to elucidate the mechanisms of PEX13-mediated regulation of hepcidin. We demonstrate that transgenic mice lacking hepatocyte Pex13 have defects in systemic iron homeostasis. The ablation of Pex13 expression in hepatocytes leads to a significant reduction in hepatic hepcidin levels. Our results also demonstrate that a deficiency of PEX13 gene expression in HepG2/C3A cells leads to decreased hepcidin expression, which is mediated through an increase in the signalling protein SMAD7, and endoplasmic reticulum (ER) stress. This study identifies a novel role for a protein involved in maintaining peroxisomal integrity and function in iron homeostasis. Loss of Pex13, a protein important for peroxisomal function, in hepatocytes leads to a significant increase in ER stress, which if unresolved, can affect liver function. The results from this study have implications for the management of patients with peroxisomal disorders and the liver-related complications they may develop.</p

Ascites bacterial burden and immune cell profile are associated with poor clinical outcomes in the absence of overt infection

Bacterial infections, most commonly spontaneous bacterial peritonitis in patients with ascites, occur in one third of admitted patients with cirrhosis, and account for a 4-fold increase in mortality. Bacteria are isolated from less than 40% of ascites infections by culture, necessitating empirical antibiotic treatment, but culture-independent studies suggest bacteria are commonly present, even in the absence of overt infection. Widespread detection of low levels of bacteria in ascites, in the absence of peritonitis, suggests immune impairment may contribute to higher susceptibility to infection in cirrhotic patients. However, little is known about the role of ascites leukocyte composition and function in this context. We determined ascites bacterial composition by quantitative PCR and 16S rRNA gene sequencing in 25 patients with culture-negative, non- neutrocytic ascites, and compared microbiological data with ascites and peripheral blood leukocyte composition and phenotype. Bacterial DNA was detected in ascitic fluid from 23 of 25 patients, with significant positive correlations between bacterial DNA levels and poor 6-month clinical outcomes (death, readmission). Ascites leukocyte composition was variable, but dominated by macrophages or T lymphocytes, with lower numbers of B lymphocytes and natural killer cells. Consistent with the hypothesis that impaired innate immunity contributes to susceptibility to infection, high bacterial DNA burden was associated with reduced major histocompatibility complex class II expression on ascites (but not peripheral blood) monocytes/macrophages. These data indicate an association between the presence of ascites bacterial DNA and early death and readmission in patients with decompensated cirrhosis. They further suggest that impairment of innate immunity contributes to increased bacterial translocation, risk of peritonitis, or both

Portal, but not lobular, macrophages express matrix metalloproteinase-9: association with the ductular reaction and fibrosis in chronic hepatitis C

Background Liver macrophages are a heterogeneous cell population that produces factors involved in fibrogenesis and matrix turnover, including matrix metalloproteinase (MMP) -9. During liver injury, their close proximity to hepatic progenitor cells and the ductular reaction may enable them to regulate liver repair and fibrosis. Aims To enumerate and characterise liver macrophages in patients with chronic hepatitis C, to determine whether a distinct population of macrophages is associated with the ductular reaction and portal fibrosis. Methods Immunostaining for macrophage markers (CD68, CD163, CCR2), the ductular reaction (keratin-7) and MMP-9 was performed in liver biopsy sections from patients with chronic hepatitis C virus (HCV) (n=85). Results Portal tracts were more densely populated with macrophages (10.5 +/- 0.36 macrophages/HPF) than lobules (7.2 +/- 0.16 macrophages/HPF,