Thomas Weichhart

The TSC-mTOR Signaling Pathway Regulates the Innate Inflammatory Response

The innate inflammatory immune response must be tightly controlled to avoid damage to the host. Here, we showed that the tuberous sclerosis complex-mammalian target of rapamycin (TSC-mTOR) pathway regulated inflammatory responses after bacterial stimulation in monocytes, macrophages, and primary dendritic cells. Inhibition of mTOR by rapamycin promoted production of proinflammatory cytokines via the transcription factor NF-kappaB but blocked the release of interleukin-10 via the transcription factor STAT3. Conversely, deletion of TSC2, the key negative regulator of mTOR, diminished NF-kappaB but enhanced STAT3 activity and reversed this proinflammatory cytokine shift. Rapamycin-hyperactivated monocytes displayed a strong T helper 1 (Th1) cell- and Th17 cell-polarizing potency. Inhibition of mTOR in vivo regulated the inflammatory response and protected genetically susceptible mice against lethal Listeria monocytogenes infection. These data identify the TSC2-mTOR pathway as a key regulator of innate immune homeostasis with broad clinical implications for infectious and autoimmune diseases, vaccination, cancer, and transplantation.

The multiple facets of mTOR in immunity.

The mammalian target of rapamycin (mTOR) is an evolutionarily conserved serine-threonine kinase that is known to sense the environmental and cellular nutrition status to control cell growth. In immunity, mTOR is essential for both the proper activation and subsequent proliferation of effector T cells, yet also restrains the development of regulatory T cells. However, in monocytes/macrophages and peripheral myeloid dendritic cells, mTOR restricts proinflammatory and promotes anti-inflammatory responses, whereas, in plasmacytoid dendritic cells, mTOR fosters type I interferon production. These results place mTOR in a novel immunoregulatory context that highlights the potential of mTOR inhibitors as both immunosuppressant and anti-cancer agents.

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.

Regulation of innate immune cell function by mTOR

The innate immune system is central for the maintenance of tissue homeostasis and quickly responds to local or systemic perturbations by pathogenic or sterile insults. This rapid response must be metabolically supported to allow cell migration and proliferation and to enable efficient production of cytokines and lipid mediators. This Review focuses on the role of mammalian target of rapamycin (mTOR) in controlling and shaping the effector responses of innate immune cells. mTOR reconfigures cellular metabolism and regulates translation, cytokine responses, antigen presentation, macrophage polarization and cell migration. The mTOR network emerges as an integrative rheostat that couples cellular activation to the environmental and intracellular nutritional status to dictate and optimize the inflammatory response. A detailed understanding of how mTOR metabolically coordinates effector responses by myeloid cells will provide important insights into immunity in health and disease.

A Versatile Role of Mammalian Target of Rapamycin in Human Dendritic Cell Function and Differentiation

The mammalian target of rapamycin (mTOR) regulates cell growth and survival and exists as rapamycin-sensitive mTOR complex (mTORC) 1 and as rapamycin-insensitive mTORC2. Although mTOR is a well-known regulator of diverse immune cells, its detailed role in human dendritic cell (DC) function and differentiation is only incompletely understood. In this study, we demonstrate divergent roles of mTOR during activation and differentiation of myeloid DCs (mDCs) and monocyte-derived DCs (moDCs). Inhibition of mTORC1 in mDCs activated with TLR-dependent or -independent stimuli increased proinflammatory cytokines and NF-κB, whereas IL-10 and STAT3 were blocked. Rapamycin regulated the costimulatory/surface molecules CD86, programmed death ligand-1, and CD25 on mDCs and significantly increased the T cell allostimulatory potential of mDCs. In contrast, rapamycin suppressed immunostimulatory molecules and the allostimulatory potential of LPS-stimulated moDCs by an inability to augment NF-κB signaling. In differentiating moDCs, the PI3K/Akt-dependent mTOR pathway was constitutively activated by GM-CSF to induce DC differentiation in an mTORC1-dependent manner. Inhibition of mTORC1 or mTORC1/2 during moDC differentiation decreased moDC survival and markedly hampered its immunostimulatory phenotype. Analyzing the fate of DCs in vivo, we found that kidney transplant patients treated with rapamycin displayed an increased immunostimulatory potential of mDCs compared with patients treated with calcineurin inhibitors. Furthermore, rapamycin did not interfere with mDC differentiation in these patients. Collectively, mTOR exerts divergent immunoregulatory functions during DC activation and differentiation depending on the DC type that lead to opposing T cell responses, which might be of clinical importance in transplantation, cancer, and also for novel vaccination strategies.

The multifunctional role of mTOR in innate immunity: implications for transplant immunity.

The mammalian target of rapamycin (mTOR) is an evolutionary conserved serine–threonine kinase that senses various environmental stimuli in most cells primarily to control cell growth. Restriction of cellular proliferation by mTOR inhibition led to the use of mTOR inhibitors as immunosuppressants in allogeneic transplantation as well as novel anticancer agents. However, distinct inflammatory side effects such as fever, pneumonitis, glomerulonephritis or anemia of chronic disease have been observed under this treatment regime. Apart from the mere cell‐cycle regulatory effect of mTOR in dividing cells, recent data revealed a master regulatory role of mTOR in the innate immune system. Hence, inhibition of mTOR promotes proinflammatory cytokines such as IL‐12 and IL‐1β, inhibits the anti‐inflammatory cytokine IL‐10 and boosts MHC antigen presentation via autophagy in monocytes/macrophages and dendritic cells. Moreover, mTOR regulates type I interferon production and the expression of chemokine receptors and costimulatory molecules. These results place mTOR in a complex immunoregulatory context by controlling innate and adaptive immune responses. In this review, we discuss the clinical consequences of mTOR‐inhibitor therapy and aim to integrate this recent data into our current view of the molecular mechanisms of clinically employed mTOR inhibitors and discuss their relevance with special emphasis to transplantation.

Serum Amyloid A in Uremic HDL Promotes Inflammation

Uremia impairs the atheroprotective properties of HDL, but the mechanisms underlying why this occurs are unknown. Here, we observed that HDL isolated from healthy individuals inhibited the production of inflammatory cytokines by peripheral monocytes stimulated with a Toll-like receptor 2 agonist. In contrast, HDL isolated from the majority of patients with ESRD did not show this anti-inflammatory property; many HDL samples even promoted the production of inflammatory cytokines. To investigate this difference, we used shotgun proteomics to identify 49 HDL-associated proteins in a uremia-specific pattern. Proteins enriched in HDL from patients with ESRD (ESRD-HDL) included surfactant protein B (SP-B), apolipoprotein C-II, serum amyloid A (SAA), and α-1-microglobulin/bikunin precursor. In addition, we detected some ESRD-enriched proteins in earlier stages of CKD. We did not detect a difference in oxidation status between HDL isolated from uremic and healthy patients. Regarding function of these uremia-specific proteins, only SAA mimicked ESRD-HDL by promoting inflammatory cytokine production. Furthermore, SAA levels in ESRD-HDL inversely correlated with its anti-inflammatory potency. In conclusion, HDL has anti-inflammatory activities that are defective in uremic patients as a result of specific changes in its molecular composition. These data suggest a potential link between the high levels of inflammation and cardiovascular mortality in uremia.

Neutralization of Osteopontin Inhibits Obesity-Induced Inflammation and Insulin Resistance

OBJECTIVE Obesity is associated with a state of chronic low-grade inflammation mediated by immune cells that are primarily located to adipose tissue and liver. The chronic inflammatory response appears to underlie obesity-induced metabolic deterioration including insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine, the expression of which is strongly upregulated in adipose tissue and liver upon obesity. Here, we studied OPN effects in obesity-induced inflammation and insulin resistance by targeting OPN action in vivo. RESEARCH DESIGN AND METHODS C57BL/6J mice were fed a high-fat diet to induce obesity and were then intravenously treated with an OPN-neutralizing or control antibody. Insulin sensitivity and inflammatory alterations in adipose tissue and liver were assessed. RESULTS Interference with OPN action by a neutralizing antibody for 5 days significantly improved insulin sensitivity in diet-induced obese mice. Anti-OPN treatment attenuated liver and adipose tissue macrophage infiltration and inflammatory gene expression by increasing macrophage apoptosis and significantly reducing c-Jun NH2-terminal kinase activation. Moreover, we report OPN as a novel negative regulator for the activation of hepatic signal transducer and activator of transcription 3 (STAT3), which is essential for glucose homeostasis and insulin sensitivity. Consequently, OPN neutralization decreased expression of hepatic gluconeogenic markers, which are targets of STAT3-mediated downregulation. CONCLUSIONS These findings demonstrate that antibody-mediated neutralization of OPN action significantly reduces insulin resistance in obesity. OPN neutralization partially decreases obesity-associated inflammation in adipose tissue and liver and reverses signal transduction related to insulin resistance and glucose homeostasis. Hence, targeting OPN could provide a novel approach for the treatment of obesity-related metabolic disorders.

Prevention of high-fat diet-induced adipose tissue remodeling in obese diabetic mice by n-3 polyunsaturated fatty acids

Objective:Obesity is associated with reduced insulin sensitivity and extensive reorganization of adipose tissue. As polyunsaturated fatty acids (PUFA) appear to inhibit diabetes development, we investigated PUFA effects on markers of matrix remodeling in white adipose tissue.Methods and procedure:Male obese diabetic (db/db) mice were treated with either a low-fat standard diet (LF), or high-fat diets rich in saturated and monounsaturated fatty acids (HF/S), n-6 PUFA (HF/6) or the latter including marine n-3 PUFA (HF/3). White adipose tissue was analyzed for gene expression, fatty acid composition and by immunofluorescence.Results:HF/S treatment increased adipose tissue expression of a number of genes involved in matrix degradation including matrix metalloproteinase (MMP)-12, -14 and cathepsin K, L and S compared with LF. MMP-12 gene was expressed in macrophages and adipocytes, and MMP-12 protein colocalized with both cell types. In addition, mean adipocyte area increased by 1.6-fold in HF/S-treated mice. Genes essential for collagen production, such as procollagen I, III, VI, tenascin C and biglycan were upregulated in HF/S-treated animals as well. N-3 PUFA supplementation resulted in enrichment of these fatty acids in adipose tissue. Moreover, n-3 PUFA inhibited the HF/S-induced upregulation of genes involved in matrix degradation and production I restored mean adipocyte area and prevented MMP-12 expression in macrophages and adipocytes.Conclusion:N-3 PUFA prevent high-fat diet-induced matrix remodeling and adipocyte enlargement in adipose tissue of obese diabetic mice. Such changes could contribute to diabetes prevention by n-3 PUFA in obese patients.

Mammalian target of rapamycin: a signaling kinase for every aspect of cellular life.

The mammalian (or mechanistic) target of rapamycin (mTOR) is an evolutionarily conserved serine-threonine kinase that is known to sense the environmental and cellular nutrition and energy status. Diverse mitogens, growth factors, and nutrients stimulate the activation of the two mTOR complexes mTORC1 and mTORC2 to regulate diverse functions, such as cell growth, proliferation, development, memory, longevity, angiogenesis, autophagy, and innate as well as adaptive immune responses. Dysregulation of the mTOR pathway is frequently observed in various cancers and in genetic disorders, such as tuberous sclerosis complex or cystic kidney disease. In this review, I will give an overview of the current understanding of mTOR signaling and its role in diverse tissues and cells. Genetic deletion of specific mTOR pathway proteins in distinct tissues and cells broadened our understanding of the cell-specific roles of mTORC1 and mTORC2. Inhibition of mTOR is an established therapeutic principle in transplantation medicine and in cancers, such as renal cell carcinoma. Pharmacological targeting of both mTOR complexes by novel drugs potentially expand the clinical applicability and efficacy of mTOR inhibition in various disease settings.