Karl M. Stuhlmeier

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.

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.

Liver X receptors interfere with cytokine-induced proliferation and cell survival in normal and leukemic lymphocytes

Liver X receptors (LXRs) are nuclear receptors regulating lipid and cholesterol metabolism. Recent data indicate an additional role of LXR in immunity by controlling dendritic cell and T‐cell function and in breast and prostate cancer cells. Here, we show that LXR activation interferes with IL‐2 and IL‐7‐induced proliferation and cell cycle progression of human T‐cell blasts mainly through inhibited phosphorylation of the retinoblastoma protein and decreased expression of the cell cycle protein cyclin B. Comparable results were obtained with IL‐2‐dependent chronic lymphoblastic leukemia (CLL) T cells. Furthermore, we show for B‐CLL cells that LXR are functionally active and inhibit expression of survival genes bcl‐2 and MMP‐9, and significantly reduce cell viability, suggesting an interference of LXR with cytokine‐dependent CLL cell survival. In conclusion, our data reveal LXR as a potent modulator of cytokine‐dependent proliferation and survival of normal and malignant T and B lymphocytes. This novel LXR action could find clinical application in immunosuppressive and antileukemic therapies.

Bacterial metabolite interference with maturation of human monocyte-derived dendritic cells.

Dendritic cells (DC), the most potent APC, are central to antimicrobial immunity. Because of evolutionary pressure, it is reasonable that pathogens have evolved strategies to also subvert this host‐defense mechanism. In the present study, we describe a novel way of bacterial interference with DC maturation. The bacterial metaboliten‐butyrate, which occurs physiologically in high concentrations in the gastrointestinal tract and has well‐known anti‐inflammatory effects, is able to prevent LPS‐induced maturation of DC resulting in a reduced capability to stimulate T cells. In particular, n‐butyrate prevents homotypic DC clustering, inhibits IL‐12 while sparing IL‐10 production, and at the molecular level, blocks NF‐κB translocation. These results demonstrate efficient targeting of DC function by a bacterial metabolite, which might explain the particular type of immune responsiveness in the presence of this bacterial agent as exemplified in the gastrointestinal tract.

Apis Mellifera Venom and Melittin Block neither NF-κB-p50-DNA Interactions nor the Activation of NF-κB, Instead They Activate the Transcription of Proinflammatory Genes and the Release of Reactive Oxygen Intermediates

Many alternative treatment approaches, originating from Asia, are becoming increasingly popular in the Western hemisphere. Recently, an article published in a renowned journal reported that venom of apis mellifera (bee venom (BV)) and melittin mediate immune-modulating effects by blocking the activation of the transcription factor NF-κB. Such a modus operandi would corroborate the many claims of beneficial effects of BV treatment and give immediate credit to this form of therapy. Fibroblast-like synoviocytes from rheumatoid arthritis patients and dermal fibroblast cells and white blood cells from healthy volunteers were used to study the effects of BV and melittin on the activation of NF-κB and a series of genes that are markers of inflammation. EMSAs demonstrate that neither BV nor melittin blocked IL-1β-induced NF-κB activation; neither did they affect phosphorylation or degradation of IκB. Contrary to published data, even high concentrations of BV and melittin were without any effect on NF-κB-p50-DNA interactions. More importantly, in fibroblast-like synoviocytes, but also in dermal fibroblasts as well as in mononuclear cells exposed to BV or melittin, mRNA levels of several proinflammatory genes are significantly increased, and Western blot data show elevated cyclooxygenase-2 protein levels. Furthermore, exposure to BV higher than 10 μg/ml resulted in disintegration of all cell types tested. In addition, large quantities of oxygen radicals are produced in a dose-dependent manner in leukocytes exposed to BV. Taken together, data presented in this work do not corroborate an earlier report regarding the effectiveness of BV as an inhibitor of the transcription factor NF-κB.

Epigenetic regulation of dendritic cell differentiation and function by oxidized phospholipids.

Dendritic cells (DCs) are the key cell type in the regulation of an adaptive immune response. Under inflammatory conditions monocytes can give rise to immunostimulatory DCs, depending on microenvironmental stimuli. Here we show that oxidized phospholipids (Ox-Pls), which are generated during inflammatory reactions, dysregulate the differentiation of DCs. DCs generated in the presence of Ox-Pls up-regulated the typical DC marker DC-SIGN but did not express CD1a, CD1b, and CD1c. These DCs generated in the presence of Ox-Pls had a substantially diminished T cell-stimulating capacity after stimulation with Toll-like receptor ligands. Toll-like receptor ligand-induced production of interleukin-12 also was strongly diminished, whereas induction of CD83 was not altered. In addition, we found that Ox-Pls strongly inhibit inflammatory stimuli-induced phosphorylation of histone H3, a key step of interleukin-12 production, yet leaving activation of nuclear factor-kappaB unaltered. Taken together, Ox-Pls present during differentiation yielded DCs with a reduced capacity to become immunostimulatory mature DCs. Furthermore, the presence of Ox-Pls blocked histone modifications required for full activation of DCs. Therefore, inflammation-derived Ox-Pls control DC functions in part by epigenetic mechanisms.

Effects of leflunomide on hyaluronan synthases (HAS) : NF-κB-independent suppression of IL-1-induced HAS1 transcription by leflunomide

Despite evidence that points to unfettered hyaluronic acid (HA) production as a culprit in the progression of rheumatic disorders, little is known about differences in regulation and biological functions of the three hyaluronan synthase (HAS) genes. Testing the effects of drugs with proven anti-inflammatory effects could help to clarify biological functions of these genes. In this study, we demonstrate that leflunomide suppresses HA release in fibroblast-like synoviocytes (FLS) in a dose-dependent manner. We further demonstrate that leflunomide suppresses HA synthase activity, as determined by 14C-glucuronic acid incorporation assays. Additional experiments revealed that in FLS, leflunomide specifically blocked the induction of HAS1. HAS2 and HAS3, genes that are, in contrast to HAS1, constitutively expressed in FLS, are not significantly affected. Leflunomide can function as a NF-κB inhibitor. However, EMSA experiments demonstrate that at the concentrations used, leflunomide neither interferes with IL-1β- nor with PMA-induced NF-κB translocation. Furthermore, reconstituting the pyrimidine synthase pathway did not lead to the restoration of IL-1β-induced HAS1 activation. More importantly, two tyrosine kinase inhibitors mimicked the effect of leflunomide in that both blocked IL-1β-induced HAS1 activation without affecting HAS2 or HAS3. These data point at HAS1 activation as the possible cause for unfettered HA production in rheumatoid arthritis and might explain, at least in part, the beneficial effects of leflunomide treatment. These findings also support the concept that IL-1β-induced HAS1 activation depends on the activation of tyrosine kinases, and indicate that leflunomide blocks HA release by suppressing tyrosine kinases rather than through inhibition of NF-κB translocation.

Cytokine release and dynamics of leukocyte populations after CD3/TCR monoclonal antibody treatment

Cytokine release and clinical side effects resulting from the use of OKT3 and BMA 031 monoclonal antibodies in the treatment of kidney graft recipients were evaluated and compared. The rise observed in serum levels of interferon γ, TNFα, and IL-8 was similar after administration of either monoclonal antibody. Furthermore, both OKT3 and BMA 031 resulted in rapid disappearance not only of virtually all T cells, but also of substantial percentages of all major leukocyte populations from the circulation; this effect is probably due to cytokine release activating endothelial cells and thereby causing extravasation even of leukocytes not specifically recognized by the administered antibodies. Evidence has thus been obtained that BMA 031 is as potent as OKT3 in inducing unequivocal signs of T cell activationin vivo. However, while OKT3 therapy was accompanied by adverse side effects in our study as in previous ones, we saw no such reactions in any of the patients receiving BMA 031. This contrast might be due to different mechanisms of leukocyte activation possibly inducing other mediators in the case of OKT3, which then, in combination with the cytokines, could generate treatment-associated morbidity.

The ssRNA Genome of Human Rhinovirus Induces a Type I IFN Response but Fails to Induce Maturation in Human Monocyte-Derived Dendritic Cells

Dendritic cells (DCs) use pattern recognition receptors to sense invading viruses and triggering of these receptors induces a maturation program. Human rhinoviruses (HRVs) belong to the family of Picornaviridae, which have a single-stranded, coding RNA genome. Because HRV does not replicate in DCs, we used genomic RNA from HRV in this study to analyze the impact of natural occurring viral ssRNA on DC function. We found that transfection of human monocyte-derived DCs with viral ssRNA induced type I IFN production but failed to activate the NF-κB pathway in DCs. In line with this observation, the up-regulation of typical maturation markers such as CD83 or the production of the proinflammatory cytokines IL-12p40, IL-6, and TNF-α was not detectable. Most importantly, the T cell stimulatory capacity of viral ssRNA-treated DCs was not enhanced and remained at the level of immature DCs. Taken together, our results demonstrate that viral ssRNA efficiently activates the innate defense arm of DCs, whereas it is insufficient to activate the stimulatory capacity of DCs for the adaptive defense responses.

Immunoregulation in Urinary Tract Inflammation—A Role of Tamm-Horsfall Glycoprotein Tamm-Horsfall Glycoprotein Links Innate Immune Cell Activation with Adaptive Immunity via a Toll-Like Receptor-4–Dependent Mechanism. J Clin Invest 115: 468–475, 2005

The Tamm-Horsfall protein (THP), the most abundant protein in normal urine, was discovered five decades ago ([1][1]), or possibly even much earlier ([2][2]), but the role for the most abundant protein in normal urine had remained enigmatic for a long time. It is expressed in the thick ascending limb