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Vitamin D may have immunomodulatory effects in the intestine. Our objective was to determine if exposure to vitamin D mitigates intestinal inflammation in IL-10 KO mice. Mice were randomized to a diet containing 25 IU (low) or 5000 IU (high) of vitamin D/kg of diet in utero and offspring were maintained on the same diet or switched to the other diet at weaning. Fecal samples were collected at 3 months of age. Vitamin D did not affect intestinal inflammation in male and female mice and did not affect KC cytokine concentration or regulate colonic gene expression in male mice. Vitamin D modulated the gut microbiota in a sex-specific manner and depending on timing of exposure. Females in the HH group had significantly higher fecal counts of C. coccoides than the other vitamin D interventions. Therefore, vitamin D may favourably modulate microbiota composition without attenuating inflammation.

Vitamin D may have immunomodulatory effects in the intestine. Our objective was to determine if exposure to vitamin D mitigates intestinal inflammation in IL-10 KO mice. Mice were randomized to a diet containing 25 IU (low) or 5000 IU (high) of vitamin D/kg of diet in utero and offspring were maintained on the same diet or switched to the other diet at weaning. Fecal samples were collected at 3 months of age. Vitamin D did not affect intestinal inflammation in male and female mice and did not affect KC cytokine concentration or regulate colonic gene expression in male mice. Vitamin D modulated the gut microbiota in a sex-specific manner and depending on timing of exposure. Females in the HH group had significantly higher fecal counts of C. coccoides than the other vitamin D interventions. Therefore, vitamin D may favourably modulate microbiota composition without attenuating inflammation.

Agrin and neuregulin are neurotrophic molecules well known for their roles at synapses in the peripheral and central nervous systems. The expression of these two molecules is not restricted to these sites however, as they are broadly expressed across multiple organ systems. What roles do agrin and neuregulin play within these alternate systems; what is the function of these molecules outside the nervous system? Here I investigate potential roles for agrin and neuregulin in inflammation. Inflammation is an immediate response by innate immune cells, primarily macrophages, to infection and is characterized by the synthesis of pro-inflammatory mediators. The innate immune system possesses multiple redundant mechanisms to locally control inflammation. The neuro-immune axis is one means of control. Often called the cholinergic anti-inflammatory pathway, it acts to regulate local inflammation via nerve-secreted acetylcholine signaling through the homopentameric α7 nicotinic acetylcholine receptors (α7nAChR) present on macrophages. Both agrin and neuregulin have been independently described to share an intricate relationship with acetylcholine receptors (AChR) in the nervous system. Agrin is best known for its role in AChR aggregation at the neuromuscular junction while neuregulin has related roles in AChR transcription, cell survival, communication and differentiation. Based on the common characteristics of synapses in the nervous and immune systems we were curious to see if agrin and neuregulin played analogous roles on macrophages. Here we show that agrin and its receptor dystroglycan are expressed on RAW264.7 macrophages. In addition, agrin treated macrophages demonstrate increased endogenous agrin and α7nAChR expression. By blocking α-dystroglycan (α-DG), a receptor for agrin, with an anti-α-DG antibody we further saw a reduction in agrin expression. We also show that agrin is able to aggregate surface α7nAChRs and transmembrane agrin co-localizes with α7nAChRs therein. Agrin appears to induce approximately a 15-fold increase in anti-inflammatory cytokine IL-10 in macrophages but does not increase pro-inflammatory cytokine TNF-α or IL-6 synthesis. Agrin-treated macrophages challenged with LPS, a potent activator of inflammation, exhibit a 57% decrease in IL-6. Macrophages treated with agrin also exhibit a 4-fold increase in STAT3, a regulator of anti-inflammatory action. The potential anti-inflammatory effects of agrin in the periphery parallel previous work describing the effects of neuregulin in the brain. Previous work completed by our lab suggests a role for neuregulin in augmenting the expression of α7nAChRs on microglia, the macrophages of the brain, but not in peripheral macrophages. Here we show that treatment of LPS challenged microglia with neuregulin produces an 88% decrease in IL-6 and a 33% decrease in TNF-α. These results indicate both agrin and neuregulin are able to induce an increase in α7nAChRs and augment the synthesis of pro- and anti-inflammatory cytokines in their respective systems. These results also further the support the evidence of neuro-immune crosstalk in the immune system. Taken together these results present two novel players in inflammatory regulation by macrophages in the periphery and CNS.

Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types. My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli. In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules. Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.

Although the etiology surrounding many autoimmune diseases remains unknown, the underlying characteristic of many of these diseases is a disruption in the balance of pro- and anti- inflammatory cytokines It is well established that the dysregulation of the IL-12 family of cytokines, an increase in IL-12/IL-23 and a decrease in IL-27 production has been implicated in these conditions. We used ELISA, RT-PCR, Immunofluorescence and Western immunoblotting in conjunction with pharmalogical inhibitors and siRNA to demonstrate the role of SHP-1/Src in the regulation of IL-12, IL-23, IL-27 and IL-10 in LPS-stimulated human THP-1 cells, monocytes and MDMs. My results show for the first time that Src kinase activity relies on SHP-1 activity, and together this complex functions in TLR4-mediated MyD88 and TRIF pathways. Furthermore Src exhibits a dual role as a positive regulator for anti-inflammatory IL-10/IL-27 and as a negative regulator of pro-inflammatory IL-12/IL-23 downstream of TLR4. Moreover, the involvement of PI3K and JNK MAPK, dependent on SHP-1/Src complex, in the regulation of IL-12 family and IL-10 downstream of TLR4 was shown.

Formation of lipid-laden macrophage foam cells and inflammation are the central components in the initiation and progression of atherosclerosis. ABCA1 is well established as an anti-atherogenic factor that facilitates cellular cholesterol and phospholipid efflux, promotes reverse cholesterol transport, and suppresses pro-inflammatory cytokine secretion. Through these functions, ABCA1 is capable of reducing the lipid burden in atherosclerotic plaque. PKA signaling is an integral factor in promoting many anti-atherogenic functions of ABCA1; however, mechanistic aspects of PKA signaling associated with ABCA1 remain poorly defined. Thus, the first part of this study investigates the involvement of spatially regulated PKA signaling in ABCA1 activities through the use of st-Ht31, a PKA de-anchoring peptide. It appears that de-anchoring PKA robustly increases ABCA1-mediated microparticle release, one of the cholesterol efflux pathways of ABCA1, and reverses macrophage foam cell formation. These results highlight the significance of subcellular compartmentalization of PKA signaling in ABCA1 functions and present PKA de-anchoring as a potential therapeutic strategy for atherosclerotic lesion regression. The second part of this study provides evidence that ABCA1 activates PKA and promotes the secretion of anti-inflammatory IL-10, a cytokine crucial for inflammation resolution. Furthermore, we provide evidence that this elevated PKA activity is the underlying mechanism in which macrophage ABCA1 promotes M2-like inflammatory response. Our results also suggest that ABCA1 activates PKA by regulating cholesterol, which poises macrophages towards an anti-inflammatory or M2-activated phenotype. Collectively, we demonstrate that PKA signaling plays a crucial multifactorial role in anti-atherogenic functions of ABCA1.

The O’Brien lab has demonstrated that Heat shock protein 27 (HSP27)shows attenuated expression in human coronary arteries as the degree of atherosclerosis progresses. Moreover, over-expression of HSP27 reduces atherogenesis in mice. The precise mechanism(s) for HSP27-mediated "atheroprotection" are incompletely understood. Nuclear Factor-kappaB (NF-κB) is a key signaling modulator in atherogenesis. Hence, this project sought to determine if recombinant HSP27 (rHSP27) alters NF-κB signaling to affect atheroprotection. Treatment of THP1 macrophages with rHSP27 resulted in degradation of IκBα, coincided with nuclear translocation of the p65 subunit and produced transcriptional evidence of activation of NF-κB signaling. When the transcriptional profile of THP1 macrophages treated with rHSP27 was analyzed using NF-κB-pathway-specific qRT-PCR arrays, among the regulated genes, IL-10 and GM-CSF mRNA levels were markedly increased, as were parallel translational effects observed. These data provide new mechanistic insights into the atheroprotective effects of HSP27.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder that occurs as a consequence of a genetic mutation that results in an overly aggressive immune response to normal bacteria. Metabolomics is a new born cousin to genomics and proteomics and involves a high throughput identification, characterization and quantification of small molecule metabolites generated by the organism. This study will show that metabolomics can be an effective tool in studying the differences between wild type and IL 10 KO mice as they age in axenic and conventional environments, and the onset of disease in a conventional environment. I show specific changes upon colonizing axenic mice with fecal bacteria that are similar to changes occurring over 16 weeks of conventional growth. Several bacterial metabolites have been identified that may play a role in the pathogenesis or provide clues to the interactions of the gut microbiota with the intestinal immune system. / Experimental Medicine

Bei der Altersabhängigen Makuladegeneration (AMD) handelt es sich um eine Erkrankung des Auges, die die Macula lutea, die Stelle des schärfsten Sehens betrifft. Sie ist verbunden mit der Degeneration von RPE-Zellen, die zur Dystrophie von Photorezeptoren und damit zum Verlust des zentralen Sehvermögens führt. Eine ähnliche Pathophysiologie ist bei der sogenannten Retinalen Pigmentepitheldystrophie (RPED) des Hundes zu beobachten. Die Transplantation von gesunden RPE-Zellen in das betroffene Gebiet stellt eine vielversprechende Therapiemöglichkeit dar. Die Transplantatabstoßung als Kom-plikation schränkt die klinische Anwendung ein. Eine beim Patienten nach Transplantation lebenslang durchgeführte systemische Immunsuppression ist mit erheblichen Nebenwirkungen verbunden. Deshalb bietet die Gentherapie unter Einbezug immunsuppressiver Zytokine wie beispielsweise des Interleukin-10 (IL-10) eine Lösung. In der vorliegenden Arbeit wurde ein selbst konstruierter IL-10-Expressionsvektor (Plasmid pCIneoIL-10) mittels Gentransfer in humane RPE-Zellen in vitro eingebracht. Untersucht wurde die Wirkung des sezernierten IL-10 auf die Proliferation von allogenen T-Lymphozyten mit und ohne allogene Makrophagen als professionelle antigenpräsentierende Zellen (APC). Neben humanen Spender RPE-Zellen (Spender-hRPE-Zellen) wurde eine immortalisierte Permanent-Zelllinie (hTERT-RPE1-Zellen) eingesetzt, deren Hauptvorteil in einer gleichbleibend hohen Wachstumsrate lag. Als transientes Transfektions-system für den Transfer von IL-10-DNA in hRPE-Zellen wurden kationische Lipide gewählt. Drei verschiedene Lipidformulierungen wurden miteinander verglichen und das optimale Transfektionsreagenz:DNA-Verhältnis, mit dem die höchste Transfektionseffizienz erreicht werden konnte, evaluiert. Eine Transfektionseffizienz von 23,3 ± 9,0 % (hTERT-RPE1-Zellen) beziehungsweise 10,3 ± 4,5 % (Spender-hRPE-Zellen) konnte erreicht werden. Die Transfektion hatte weder einen negativen Einfluss auf die Vitalität der hRPE-Zellen, noch wurde der natürliche Zelltod, die Apoptose, erhöht. Die IL-10-mRNA-Expression wurde mittels RT-PCR nachgewiesen. Lediglich bei den transfizierten hRPE-Zellen konnte IL-10-mRNA gefunden werden. Mittels ELISA konnte das IL-10-Protein gemessen werden. Die Sekretion des IL-10 in den Kulturüberstand von transfizierten hRPE-Zellen wurde dafür über einen Zeitraum von 7 Tagen untersucht. Es konnte festgestellt werden, dass die maximale IL-10-Proteinkonzentration bei beiden Zelllinien am Tag 3 mit Werten von 10,3 ± 0,8 ng/ml (hTERT-RPE1-Zellen) und 3,1 ng/ml (Spender-hRPE-Zellen) lag. Es bestand überdies eine positive Korrelation zwischen Transfektionseffizienz und synthetisiertem IL-10. Es wurde außerdem gezeigt, dass durch Stimulation mit dem immunmodulatorischen Zytokin Interferon-gamma (IFN-g) hRPE-Zellen MHC Klasse II-Moleküle vermehrt exprimierten. Damit sind sie ebenso wie die Makrophagen zur Antigenpräsentation fähig. Die Wirkung des von den transfizierten hRPE-Zellen sezernierten IL-10 auf die Proliferation von T-Lymphozyten wurde zwischen Tag 2 und Tag 6 (hTERT-RPE1-Zellen) beziehungsweise zwischen Tag 2 und Tag 4 (Spender-hRPE-Zellen) photometrisch untersucht. Die Proliferation allogener T-Lymphozyten mit beziehungsweise ohne Makrophagen konnte durch das sezernierte IL-10 supprimiert werden. Bei den hTERT-RPE1-Zellen lag ohne die Anwesenheit von professionellen APC am Tag 6 eine signifikante Reduktion der T-Lymphozytenproliferation vor, während bei Kokultivierung mit Makrophagen Signifikanzen am Tag 5 und Tag 6 erkennbar waren. Die immunsuppressive Wirkung von IL-10 konnte mittels Anti-IL-10-Antikörper neutralisiert werden. Damit wurde bewiesen, dass die proliferations-supprimierende Wirkung auf IL-10 zurückzuführen war. Diese Ergebnisse könnten demnach neue Möglichkeiten zur Verhinderung einer Abstoßungsreaktion nach RPE-Zelltransplantation bei Patienten mit AMD eröffnen / Age-related macular degeneration (AMD) is a disease of eyes affecting the macula lutea, the area of the retina with the highest density of retinal pigment epithelial cells (RPE cells). The disease is characterized by degeneration of RPE cells resulting in dystrophy of photoreceptors and finally loss of central vision. Transplantation of healthy RPE cells is a promising possibility for therapy but rejection of the allotransplant limits clinical application. One way to avoid this complications is a systemic immunosuppression of the recipient but this is combined with many side effects. In this thesis a self-constructed IL-10 expression vector (plasmid pCIneoIL-10) has been transferred into human RPE cells in vitro by gene transfer. In addition to human donor RPE cells a permanent RPE cell line (hTERT-RPE1 cells) was employed. Kationic lipids were used as transient transfection system for transfer of pCIneoIL-10 into hRPE cells. Three different lipid formulations and various ratios of transfection reagent:DNA were evaluated for highest transfection efficacy. With the optimized protocols a transfection efficacy of 23,3 ± 9,0 % (hTERT-RPE1 cells) and 10,3 ± 4,5 % (donor hRPE cells) was achieved. A negative influence on the viability of the hRPE cells after transfection was not observed. The IL-10 mRNA expression was analysed by reverse transcription-polymerase chain reaction (RT-PCR). Only in transfected hRPE cells the IL-10 mRNA-amplicon with 383 bp in size was found. Secretion of IL-10 protein in the cell culture supernatants of transfected hRPE cells was investigated using an enzyme-linked immunosorbent assay (ELISA) daily for 7 days. The IL-10 protein concentrations peaked at day 3 with 10,3 ± 0,8 ng/ml (hTERT-RPE1 cells) and 3,1 ng/ml (donor hRPE cells). The amount of secreted IL-10 positively correlated with transfection efficacy. After stimulation with the immunmodulatory cytokine interferon-gamma (IFN-g) the expression of MHC class II molecules on hRPE cells is increasing. Therefore they are able to present antigens similar to macrophages. Hence, the effects of recombinantly expressed IL-10 on the proliferation of allogeneic T lymphocytes were investigated both with and without allogeneic macrophages as professional antigen presenting cells (APC). Proliferation of T lymphocytes has been investigated colorimetrically between day 2 and day 6 (hTERT-RPE1 cells) and day 2 and day 4 (donor hRPE cells) respectively. The proliferation of allogeneic T lymphocytes with and without macrophages could be suppressed by the secreted IL-10. Signifikant reduction of proliferation was observed at day 6 in absence of professional APC (14,1 ± 1,1 % to 100% of untransfected control) and between day 5 (44,1 ± 4,9 %) and day 6 (37,4 ± 6,3%) in the presence of macrophages. It was possible to neutralize the immunosuppressive effect of IL-10 with anti-IL-10 antibodies. Proving that the suppressive effect of T lymphocyte proliferation was caused by IL-10. Thus, the specific IL-10 gene transfer into hRPE cells prior to transplantation may prevent rejection process and could prove a reliable method to help prevent loss of central vision due to AMD.