Inflammatory macrophages and renal tubular epthelial cell apoptosis

Macrophages (Mφ) play a key role in renal inflammation and may be cytotoxic to resident cells within tissues. I began this thesis by examining the effect of Mφ upon the level of apoptosis and proliferation in tubular epithelial cells in vitro. I used a microscopically quantifiable co-culture assay to determine whether inflammatory Mφ modulated apoptosis and proliferation of (i) Madine-Darby canine kidney (MDCK) cells and (ii) primary murine tubular epithelial cells. Bone marrow derived Mφ and target tubular cells were differentially labeled with fluorochromes and interacted for 24 hrs. Apoptosis and proliferation was quantified by fluorescent microscopy after fixation and Hoechst staining. Quiescent non-activated Mφ did not induce significant apoptosis of tubular cells whilst cytokine activated (LPS/IFN-γ) Mφ induced marked apoptosis of both MDKC and primary tubular cells. Pharmacological inhibition of nitric oxide (NO) production with L-NAME or inclusion of cytokine activated Mcj) derived from inducible nitric oxide synthase (iNOS) knockout (KO) mice reduced apoptosis in both MDKC and primary tubular cells. MDCK cell proliferation was significantly suppressed by both non-activated and activated Mφ from iNOS WT and iNOS KO mice indicating that it was an NOindependent effect. In contrast, no effect upon primary tubular cell proliferation was evidentI then went on to examine the role of NO in vivo in the murine model of unilateral ureteric obstruction (UUO) characterised by tubular cell apoptosis and interstitial fibrosis. Initially UUO was performed in iNOS KO and wild-type mice but iNOS KO mice exhibited significantly increased Mφ infiltration. Therefore, the specific iNOS inhibitor L-NIL (control D-NIL) was administered between days 5 to 7 following UUO. Mice were sacrificed at day 7 and the obstructed kidney removed for histological analysis. L-NIL treatment did not affect Mφ infiltration but did reduce both tubular and interstitial cell apoptosis. Proliferation of tubular cells and interstitial cells was unaffected. Although myofibroblast accumulation was unaffected, interstitial fibrosis was significantly increased by L-NIL treatment.I then went on to examine the role of NO in vivo in the murine model of unilateral ureteric obstruction (UUO) characterised by tubular cell apoptosis and interstitial fibrosis. Initially UUO was performed in iNOS KO and wild-type mice but iNOS KO mice exhibited significantly increased Mφ infiltration. Therefore, the specific iNOS inhibitor L-NIL (control D-NIL) was administered between days 5 to 7 following UUO. Mice were sacrificed at day 7 and the obstructed kidney removed for histological analysis. L-NIL treatment did not affect Mφ infiltration but did reduce both tubular and interstitial cell apoptosis. Proliferation of tubular cells and interstitial cells was unaffected. Although myofibroblast accumulation was unaffected, interstitial fibrosis was significantly increased by L-NIL treatment

Inhibition of cyclin-dependent kinase 9 downregulates cytokine production without detrimentally affecting human monocyte-derived macrophage viability

Cyclin-dependent kinase (CDK) inhibitor drugs (CDKi), such as R-roscovitine and AT7519, induce neutrophil apoptosis in vitro and enhance the resolution of inflammation in a number of in vivo models. This class of compounds are potential novel therapeutic agents that could promote the resolution of acute and chronic inflammatory conditions where neutrophil activation contributes to tissue damage and aberrant tissue repair. In this study we investigated CDKi effects on macrophage pro-inflammatory mediator production and viability. Treatment of human monocyte-derived macrophages (MDMs) with the CDKi AT7519 and R-roscovitine at concentrations that induce neutrophil apoptosis had no significant effect on control or LPS-activated MDM apoptosis and viability, and did not detrimentally affect MDM efferocytosis of apoptotic cells. In addition, enhanced efferocytosis, induced by the glucocorticoid dexamethasone, was also unaffected after a short time treatment with R-roscovitine. Macrophage cytokine responses to inflammatory stimuli are also of importance during inflammation and resolution. As a key target of CDKi, CDK9, is involved in protein transcription via the RNA polymerase II complex, we investigated the effect of CDKi drugs on cytokine production. Our data show that treatment with AT7519 significantly downregulated expression and release of key MDM cytokines IL-6, TNF, IL-10 and IL-1β, as well as markers of pro-inflammatory macrophage polarisation. R-Roscovitine was also able to downregulate inflammatory cytokine protein secretion from MDMs. Using siRNA transfection, we demonstrate that genetic knock-down of CDK9 replicates these findings, reducing expression and release of pro-inflammatory cytokines. Furthermore, overexpression of CDK9 in THP-1 cells can promote a pro-inflammatory phenotype in these cells, suggesting that CDK9 plays an important role in the inflammatory phenotype of macrophages. Overall, this study demonstrates that pharmacological and genetic targeting of CDK9 inhibits an inflammatory phenotype in human MDMs. As such these data indicate that CDK9 may be key to therapeutically targeting pro-inflammatory macrophage functions during chronic inflammation

Conditional ablation of macrophages disrupts ovarian vasculature

Macrophages are the most abundant immune cell within the ovary. Their dynamic distribution throughout the ovarian cycle and heterogenic array of functions suggest the involvement in various ovarian processes, but their functional role has yet to be fully established. The aim was to induce conditional macrophage ablation to elucidate the putative role of macrophages in maintaining the integrity of ovarian vasculature. Using the CD11b-diphtheria toxin receptor (DTR) mouse, in which expression of human DTR is under the control of the macrophage-specific promoter sequence CD11b, ovarian macrophages were specifically ablated in adult females by injections of diphtheria toxin (DT). CD11b-DTR mice were given DT treatment or vehicle and ovaries collected at 2, 8, 16, 24 and 48 h. Histochemical stains were employed to characterise morphological changes, immunohistochemistry for F4/80 to identify macrophages and the endothelial cell marker CD31 used to quantify vascular changes. In normal ovaries, macrophages were detected in corpora lutea and in the theca layer of healthy and atretic follicles. As macrophage ablation progressed, increasing amounts of ovarian haemorrhage were observed affecting both luteal and thecal tissue associated with significant endothelial cell depletion, increased erythrocyte accumulation and increased follicular atresia by 16 h. These events were followed by necrosis and profound structural damage. Changes were limited to the ovary, as DT treatment does not disrupt the vasculature of other tissues likely reflecting the unique cyclical nature of the ovarian vasculature and heterogeneity between macrophages within different tissues. These results show that macrophages play a critical role in maintaining ovarian vascular integrity

Resident pleural macrophages are key orchestrators of neutrophil recruitment in pleural inflammation

Rationale: The role played by resident pleural macrophages in the initiation of pleural inflammation is currently unclear. Objective: To evaluate the role of resident pleural macrophages in the initiation of inflammation. Methods: We have used a conditional macrophage ablation strategy to determine the role of resident pleural macrophages in the regulation of neutrophil recruitment in a murine model of experimental pleurisy induced by the administration of carrageenan and formalin- fixed Staphylococcus aureus. Measurements and Main Results: Conditional macrophage ablation mice express the human diphtheria toxin receptor under the control of the CD11b promoter such that the administration of diphtheria toxin induces ablation of nearly 97% of resident macrophages. Ablation of resident pleural macrophages before the administration of carrageenan or S. aureus dramatically reduced neutrophil influx into the pleural cavity. In the carrageenan model, the reduction in neutrophil infiltration was associated with marked early reduction in the level of macrophage inflammatory protein 2 as well as reduced levels of various cytokines, including tumor necrosis factor α, interleukin 6, and interleukin 10. Adoptive transfer of nontransgenic macrophages partially restored neutrophil infiltration. We also stimulated macrophage-depleted and nondepleted pleural cell populations with carrageenan in vitro and determined the production of chemokines and cytokines. Chemokine and cytokine production was markedly reduced by macrophage depletion, reinforcing the role of resident pleural macrophages in the generation of mediators that initiate acute inflammation. Conclusion: These studies indicate a critical role for resident pleural macrophages in sensing perturbation to the local microenvironment and orchestrating subsequent neutrophil infiltration

11β-Hydroxysteroid Dehydrogenase type 1 is expressed in neutrophils and restrains an inflammatory response in male mice

Endogenous glucocorticoid action within cells is enhanced by prereceptor metabolism by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts intrinsically inert cortisone and 11-dehydrocorticosterone into active cortisol and corticosterone, respectively. 11β-HSD1 is highly expressed in immune cells elicited to the mouse peritoneum during thioglycollate-induced peritonitis and is down-regulated as the inflammation resolves. During inflammation, 11β-HSD1-deficient mice show enhanced recruitment of inflammatory cells and delayed acquisition of macrophage phagocytic capacity. However, the key cells in which 11β-HSD1 exerts these effects remain unknown. Here we have identified neutrophils (CD11b(+),Ly6G(+),7/4(+) cells) as the thioglycollate-recruited cells that most highly express 11β-HSD1 and show dynamic regulation of 11β-HSD1 in these cells during an inflammatory response. Flow cytometry showed high expression of 11β-HSD1 in peritoneal neutrophils early during inflammation, declining at later states. In contrast, expression in blood neutrophils continued to increase during inflammation. Ablation of monocytes/macrophages by treatment of CD11b-diphtheria-toxin receptor transgenic mice with diphtheria toxin prior to thioglycollate injection had no significant effect on 11β-HSD1 activity in peritoneal cells, consistent with neutrophils being the predominant 11β-HSD1 expressing cell type at this time. Similar to genetic deficiency in 11β-HSD1, acute inhibition of 11β-HSD1 activity during thioglycollate-induced peritonitis augmented inflammatory cell recruitment to the peritoneum. These data suggest that neutrophil 11β-HSD1 increases during inflammation to contribute to the restraining effect of glucocorticoids upon neutrophil-mediated inflammation. In human neutrophils, lipopolysaccharide activation increased 11β-HSD1 expression, suggesting the antiinflammatory effects of 11β-HSD1 in neutrophils may be conserved in humans