Septicaemia models using Streptococcus pneumoniae and Listeria monocytogenes: understanding the role of complement properdin

Streptococcus pneumoniae and Listeria monocytogenes, pathogens which can cause severe infectious disease in human, were used to infect properdin-deficient and wildtype mice. The aim was to deduce a role for properdin, positive regulator of the alternative pathway of complement activation, by comparing and contrasting the immune response of the two genotypes in vivo. We show that properdin-deficient and wildtype mice mounted antipneumococcal serotype-specific IgM antibodies, which were protective. Properdin-deficient mice, however, had increased survival in the model of streptococcal pneumonia and sepsis. Low activity of the classical pathway of complement and modulation of FcγR2b expression appear to be pathogenically involved. In listeriosis, however, properdin-deficient mice had reduced survival and a dendritic cell population that was impaired in maturation and activity. In vitro analyses of splenocytes and bone marrow-derived myeloid cells support the view that the opposing outcomes of properdin-deficient and wildtype mice in these two infection models is likely to be due to a skewing of macrophage activity to an M2 phenotype in the properdin-deficient mice. The phenotypes observed thus appear to reflect the extent to which M2- or M1-polarised macrophages are involved in the immune responses to S. pneumoniae and L. monocytogenes. We conclude that properdin controls the strength of immune responses by affecting humoral as well as cellular phenotypes during acute bacterial infection and ensuing inflammation

Role of Hypoxia and Toll-like Receptor Ligands in Matrix Metalloproteinase-7 Regulation in Primary Human Macrophages

Many solid tumours and other pathological sites such as infected wounds are characterised by hypoxic regions (defined by an O2 tension of <1%), which are often heavily infiltrated by macrophages. Macrophages respond to hypoxia by up-regulating a number of genes likely to promote tumour growth and spread, including MMP-7. MMP-7 has been shown to be up-regulated in various tumours and it also has important roles in protection against microbial infections including release of the pro-inflammatory cytokine TNF and activation of pro-defensins. The aim of this project was therefore to determine the mechanisms of hypoxic up-regulation of matrix metalloproteinase-7 (MMP-7) in primary human macrophages. An important aspect of this project was the analysis of the MMP-7 promoter in an attempt to identify the DNA elements required for hypoxic up-regulation, using wild-type and mutated luciferase reporter constructs transfected into primary human macrophages. A - 296 bp construct was shown to be up-regulated 3-fold in primary human macrophages exposed to 5 days of hypoxia. The luciferase expression from the constructs containing mutations in the Ets and AP-1 transcription factor binding sites was not detectable, suggesting that these sites were essential for basal MMP-7 gene expression. In this project, it was shown that MMP-7 mRNA was indeed up-regulated in severe hypoxia (0.2% O2 for 18 hrs) in primary human macrophages. However, further experiments produced the surprising finding that hypoxia alone was not able to up-regulate MMP-7 mRNA; rather, the gene was induced by co-stimulation with hypoxia and TLR ligands such as LPS. The use of Polymyxin B, which neutralises LPS, blocked MMP-7 hypoxic up-regulation. Therefore, my data indicate that the observed and previously published “hypoxic” up-regulation of MMP-7 mRNA is actually most likely to be due to the synergistic interaction of hypoxia with LPS or other TLR ligands. MMP-7 has previously been shown to be induced by TLR ligands, but my finding of synergy between these and hypoxia in up-regulation of MMP-7 mRNA and protein is novel, and challenges current opinion on MMP-7 regulation by hypoxia. Since the PI3K/Akt pathways is involved in TLR signaling and has been reported to be involved in hypoxic up-regulation of MMP-7, this pathway was investigated using two inhibitors, LY294002 and wortmannin. LY294002, and to a lesser extent wortmannin, inhibited LPS-induced MMP-7 up-regulation, linking MMP-7 LPS-regulation with the PI3K pathway. Another TLR signaling pathway, NF-κB, was investigated as a possible MMP-7 regulating pathway. NF-κB seems to be involved in MMP-7 up-regulation. Therefore, both PI3K and NF-κB pathways can be essential in MMP-7 up-regulation. These findings regarding the regulation MMP-7 expression will expand knowledge of its important role, especially in innate immunity in the context of hypoxia and infection

Monocyte-derived macrophages matured under prolonged hypoxia transcriptionally up-regulate HIF-1? mRNA

This study tested the hypothesis that prolonged severe hypoxia during monocyte to macrophage differentiation results in macrophages with a pattern of gene expression and phenotype distinct from those maturing in normal oxygen levels. Macrophages accumulate in hypoxic and anoxic areas within pathological sites such as tumours, wounds, and arthritic joints, and have been proposed as vehicles for gene therapy delivery to such tissues. Several non-pathological tissues are also hypoxic. We therefore argue that differentiation from monocyte to macrophage in hypoxic conditions is a common occurrence. However, the effect of long term severe hypoxia on monocyte to macrophage differentiation has not been studied. Here, using primary human peripheral blood monocytes, we show that maturation for 5 days in 0.2% oxygen results in decreased phagocytosis, and decreased CD40 and CD206 expression. Chronic hypoxia induced much higher mRNA levels of the pro-angiogenic cytokine, VEGF, in adherence-purified macrophages (27-fold), CD14-magnetic bead purified monocytes (90-fold), and PBMC (104-fold) compared to acute (24h) hypoxia (11, 17 and 9-fold, respectively). This suggests that macrophages may play an even greater role in angiogenesis than previously appreciated. Furthermore, chronic hypoxia resulted in up-regulation of HIF-1? mRNA, in all monocyte-derived macrophage types studied. Actinomycin D experiments indicate that the increases in HIF-1? mRNA were not due to increased mRNA stability. To our knowledge this is the first study demonstrating up-regulation of HIF-1? mRNA by hypoxia in macrophages. Taken together, the data support the hypothesis that hypoxia affects monocyte to macrophage maturation, resulting in a distinct gene expression pattern and phenotype

Properdin deficiency promotes increased macrophages in atherosclerotic lesions after feeding on a low fat diet.

<p>Quantitative immunohistochemistry of (<b>A</b>) % smooth muscle cells in aortic root plaques of male and female LDLR^−/−P^KO versus LDLR^−/−P^WT mice. Random areas in 15–20 plaques from n = 4–5 mice/group were analysed under high magnification using NIS elements software. Data presented are mean and IQR (<b>B</b>) Macrophage (MAC 387) index (percentage of the total cells/high power field within plaques) from the relevant groups, n = 5 mice/group were examined, mean and IQR, p<0.05, intergenotype comparisons were by two way ANOVA with a Mann Whitney U post test. (<b>C</b>) M2 (CD206) macrophage index, n = 5 mice/group examined, mean +/− SEM, p<0.05 (2 way ANOVA and Mann Whitney U test).</p

Properdin deficiency promotes macrophage mRNA synthesis typical of M2 macrophages under conditions of atherosclerosis.

<p>(<b>A</b>) Spleens from male LDLR^−/−P^KO and LDLR^−/−P^WT fed a LFD were analysed for various mRNA species including C3 (500 bp), sPLA2-V (329 bp) and MCP-1 (490 bp) by RTPCR. β-actin (540 bp) was control. (<b>B</b>) Arginase 1 mRNA expression was enhanced 50 fold in splenic macrophages from male LDLR^−/−P^KO compared to LDLR−/−P^WT fed a LFD. Representative images and analyses of matched pairs are shown.</p

Body weight, complement and lipid data in male and female LDLR^−/−P^KO and LDLR−/−P^WT fed LF and HFDs for 12 weeks.

<p>Data are means +/− SEM, n = 7–8 each group, *P<0.05.</p

Development of atherosclerotic plaques in LDLR−/− PWT and LDLR−/−PKO mice fed LFD and HFD.

<p><b>A</b> therosclerosis-prone mice (males and females) lacking properdin were fed low fat or high fat diets for 12 weeks. Atherosclerosis burden was assessed in aortae and at aortic roots. (<b>A)</b> lesion burden in aortae identified by Oil Red O staining (stains lipid) was calculated from light microscopic images using NIS elements software (Nikon), (8 per group, mean and IQR), *p<0.05, **p<0.01, intergenotype comparisons were by one way ANOVA with post test. (<b>B</b>) Plaque area within aortic roots measured from histological images (NIS elements), n = 5–8, data are mean and IQR **p<0.01, intergenotype comparisons were by one way ANOVA with post test. (<b>C</b>) Representative Oil Red O images of whole aortae from each group. (<b>D</b>) Visualisation of plaques at the aortic root in each of the groups, arrowhead indicates a large plaque in male LDLR^−/−P^KO, arrows indicate typical plaques in the other mouse groups that were studied.</p

Enhanced lipid storage in properdin-deficient macrophages isolated from pro-atherosclerotic mice.

<p>Bone marrow derived macrophages (BMDM) from male LDLR^−/−P^KO mice have the capacity to store more LDL as lipid droplets within cells than LDLR^−/−P^WT. 610–648 cells counted.</p