Günther Staffler

Tamm-Horsfall glycoprotein links innate immune cell activation with adaptive immunity via a Toll-like receptor-4–dependent mechanism

Tamm-Horsfall glycoprotein (THP) is expressed exclusively in the kidney and constitutes the most abundant protein in mammalian urine. A critical role for THP in antibacterial host defense and inflammatory disorders of the urogenital tract has been suggested. We demonstrate that THP activates myeloid DCs via Toll-like receptor-4 (TLR4) to acquire a fully mature DC phenotype. THP triggers typical TLR signaling, culminating in activation of NF-kappaB. Bone marrow-derived macrophages from TLR4- and MyD88-deficient mice were nonresponsive to THP in contrast to those from TLR2- and TLR9-deficient mice. In vivo THP-driven TNF-alpha production was evident in WT but not in Tlr4-/- mice. Importantly, generation of THP-specific Abs consistently detectable in urinary tract inflammation was completely blunted in Tlr4-/- mice. These data show that THP is a regulatory factor of innate and adaptive immunity and therefore could have significant impact on host immunity in the urinary tract.

Selective Inhibition of T Cell Activation Via CD147 Through Novel Modulation of Lipid Rafts

The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling molecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorganization of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation-associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor α-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling.

Vascular–Endothelial Cadherin (CD144)– but Not PECAM–1 (CD31)–Based Cell–to–Cell Contacts Convey the Maintenance of a Quiescent Endothelial Monolayer

Background: In vivo, all blood vessels are lined by a single layer of flattened noncycling endothelial cells. We tested the hypothesis that the maintenance of such a quiescent endothelial monolayer depends on homotypic contacts between not yet defined growth–inhibitory molecules located at interendothelial junctions. Methods: ECV304 cells, which lack endogenous vascular endothelial cadherin (VE cadherin) or CD31 expression, were transfected with cDNA encoding for the respective proteins or with the empty vector. Results: In VE cadherin transfectants, β–catenin was targeted to junctional regions and the F–actin–based cytoskeleton formed parallel bundles reaching from one cell border to the other. In contrast, in CD31 transfectants and in empty vector cells, β–catenin was dispersed throughout the cytoplasm, and F–actin formed short, plump and criss–cross bundles. On a two–dimensional plastic matrix, both, VE cadherin and CD31 transfectants formed clusters of polygonal cells, whereas in three–dimensional gels, only VE cadherin cells were able to form tubes. Empty vector cells grew in a fibroblast–like pattern and neither formed clusters nor tubes. Most importantly, whereas CD31 and empty vector cells grew on top of each other, formed polylayers and maintained cycling even after reaching confluence, VE cadherin cells strictly maintained a single layer of flattened cells and the numbers of cycling cells dramatically dropped after reaching a continuous monolayer. Conclusion: The insertion of VE cadherin into ECV304 cells produces a cell type which mimics endothelial growth characteristics seen in vivo.

Osteopontin is a key player for local adipose tissue macrophage proliferation in obesity

Objective Recent findings point towards an important role of local macrophage proliferation also in obesity-induced adipose tissue inflammation that underlies insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine highly upregulated in adipose tissue (AT) of obese and has repeatedly been shown to be functionally involved in adipose-tissue inflammation and metabolic sequelae. In the present work, we aimed at unveiling both the role of OPN in human monocyte and macrophage proliferation as well as the impact of OPN deficiency on local macrophage proliferation in a mouse model for diet-induced obesity. Methods The impact of recombinant OPN on viability, apoptosis, and proliferation was analyzed in human peripheral blood monocytes and derived macrophages. Wild type (WT) and OPN knockout mice (SPP1KO) were compared with respect to in vivo adipose tissue macrophage and in vitro bone marrow-derived macrophage (BMDM) proliferation. Results OPN not only enhanced survival and decreased apoptosis of human monocytes but also induced proliferation similar to macrophage colony stimulating factor (M-CSF). Even in fully differentiated monocyte-derived macrophages, OPN induced a proliferative response. Moreover, proliferation of adipose tissue macrophages in obese mice was detectable in WT but virtually absent in SPP1KO. In BMDM, OPN also induced proliferation while OPN as well as M-CSF-induced proliferation was similar in WT and SPP1KO. Conclusions These data confirm that monocytes and macrophages not only are responsive to OPN and migrate to sites of inflammation but also they survive and proliferate more in the presence of OPN, a mechanism also strongly confirmed in vivo. Therefore, secreted OPN appears to be an essential player in AT inflammation, not only by driving monocyte chemotaxis and macrophage differentiation but also by facilitating local proliferation of macrophages.

Common dysregulated pathways in obese adipose tissue and atherosclerosis

BackgroundThe metabolic syndrome is becoming increasingly prevalent in the general population that is at simultaneous risk for both type 2 diabetes and cardiovascular disease. The critical pathogenic mechanisms underlying these diseases are obesity-driven insulin resistance and atherosclerosis, respectively. To obtain a better understanding of molecular mechanisms involved in pathogenesis of the metabolic syndrome as a basis for future treatment strategies, studies considering both inherent risks, namely metabolic and cardiovascular, are needed. Hence, the aim of this study was to identify pathways commonly dysregulated in obese adipose tissue and atherosclerotic plaques.MethodsWe carried out a gene set enrichment analysis utilizing data from two microarray experiments with obese white adipose tissue and atherosclerotic aortae as well as respective controls using a combined insulin resistance-atherosclerosis mouse model.ResultsWe identified 22 dysregulated pathways common to both tissues with p values below 0.05, and selected inflammatory response and oxidative phosphorylation pathways from the Hallmark gene set to conduct a deeper evaluation at the single gene level. This analysis provided evidence of a vast overlap in gene expression alterations in obese adipose tissue and atherosclerosis with Il7r, C3ar1, Tlr1, Rgs1 and Semad4d being the highest ranked genes for the inflammatory response pathway and Maob, Bckdha, Aldh6a1, Echs1 and Cox8a for the oxidative phosphorylation pathway.ConclusionsIn conclusion, this study provides extensive evidence for common pathogenic pathways underlying obesity-driven insulin resistance and atherogenesis which could provide a basis for the development of novel strategies to simultaneously prevent type 2 diabetes and cardiovascular disease in patients with metabolic syndrome.

Osteopontin affects macrophage polarization promoting endocytic but not inflammatory properties.

Macrophages are the main drivers of obesity‐induced adipose tissue (AT) inflammation that causes insulin resistance. Macrophages polarize toward different inflammatory (M1) or protective (M2) phenotypes. Osteopontin (OPN) is an inflammatory cytokine highly expressed in AT in obesity and known to be involved in chronic inflammatory processes. It was hypothesized that OPN polarizes macrophages into a proinflammatory phenotype.

Immunological blockade of adipocyte inflammation caused by increased matrix metalloproteinase-cleaved osteopontin in obesity.

Osteopontin (OPN) is upregulated in adipose tissue (AT) in obesity and contributes to subclinical inflammation, adipocyte dysfunction, and insulin resistance. OPN effects can be increased by cleavage by matrix metalloproteinases (MMP). This study aimed at investigating the presence of OPN cleavage products in human AT in obesity and their impact on adipocyte function and immunological blockade of these effects.

Identification of matrix metalloproteinase-12 as a candidate molecule for prevention and treatment of cardiometabolic disease.

Obesity is strongly associated with metabolic syndrome, a combination of risk factors that predisposes to development of the cardiometabolic diseases: atherosclerotic cardiovascular disease and type 2 diabetes mellitus. Prevention of metabolic syndrome requires novel interventions to address this health challenge. The objective of this study was to identify candidate molecules for the prevention and treatment of insulin resistance and atherosclerosis, conditions that underlie type 2 diabetes mellitus and cardiovascular disease, respectively. We used an unbiased bioinformatics approach to identify molecules that are upregulated in both conditions by combining murine and human data from a microarray experiment and meta-analyses. We obtained a pool of 8 genes that were upregulated in all the databases analyzed. This included well-known and novel molecules involved in the pathophysiology of type 2 diabetes mellitus and cardiovascular disease. Notably, matrix metalloproteinase 12 (MMP12) was highly ranked in all analyses and was therefore chosen for further investigation. Analyses of visceral and subcutaneous white adipose tissue from obese compared with lean mice and humans convincingly confirmed the upregulation of MMP12 in obesity at the mRNA, protein and activity levels. In conclusion, by using this unbiased approach, an interesting pool of candidate molecules was identified, all of which have potential as targets in the treatment and prevention of cardiometabolic diseases.

Inhibition of Cellular Adhesion by Immunological Targeting of Osteopontin Neoepitopes Generated through Matrix Metalloproteinase and Thrombin Cleavage.

Osteopontin (OPN), a secreted protein involved in inflammatory processes and cancer, induces cell adhesion, migration, and activation of inflammatory pathways in various cell types. Cells bind OPN via integrins at a canonical RGD region in the full length form as well as to a contiguous cryptic site that some have shown is unmasked upon thrombin or matrix metalloproteinase cleavage. Thus, the adhesive capacity of osteopontin is enhanced by proteolytic cleavage that may occur in inflammatory conditions such as obesity, atherosclerosis, rheumatoid arthritis, tumor growth and metastasis. Our aim was to inhibit cellular adhesion to recombinant truncated proteins that correspond to the N-terminal cleavage products of thrombin- or matrix metalloproteinase-cleaved OPN in vitro. We specifically targeted the cryptic integrin binding site with monoclonal antibodies and antisera induced by peptide immunization of mice. HEK 293 cells adhered markedly stronger to truncated OPN proteins than to full length OPN. Without affecting cell binding to the full length form, the raised monoclonal antibodies specifically impeded cellular adhesion to the OPN fragments. Moreover, we show that the peptides used for immunization were able to induce antisera, which impeded adhesion either to all OPN forms, including the full-length form, or selectively to the corresponding truncated recombinant proteins. In conclusion, we developed immunological tools to selectively target functional properties of protease-cleaved OPN forms, which could find applications in treatment and prevention of various inflammatory diseases and cancers.

Peptide-based vaccination against OPN integrin binding sites does not improve cardio-metabolic disease in mice

Obesity causes insulin resistance via a chronic low-grade inflammation. This inflammation is characterized by elevated pro-inflammatory markers and macrophage accumulation in the adipose tissue (AT). AT inflammation is a key factor causing insulin resistance and thus type 2 diabetes, both linked to atherosclerotic cardiovascular disease. Osteopontin (OPN), a well-known inflammatory cytokine, is involved in obesity-linked complications including AT inflammation, insulin resistance, atherosclerosis and CVD. During inflammation, OPN is proteolytically cleaved by matrix metalloproteinases or thrombin leading to increased OPN activity. Therefore, OPN provides a new interesting target for immunological prevention and treatment of obesity-associated diseases. The aim of our study was to evaluate peptide-based vaccines against integrin binding sites of OPN and to examine whether these active immunotherapies are functional in reducing metabolic tissue inflammation, insulin resistance, and atherosclerosis in a cardio-metabolic (Ldlr-/- mice) and a diet-induced obesity model (WT mice). However, atherosclerosis, insulin resistance and AT inflammation were not diminished after treatment with OPN-derived peptides in murine models. Lack of efficacy was based on a failure to induce antibodies capable to bind epitopes in the context of functional OPN protein. In conclusion, our data point to unexpected challenges in the immunotherapeutic targeting of adhesive motives, such as RGD containing sequences, on endogenous proteins.