Central IRAK-4 kinase inhibition for the treatment of pain following nerve injury in rats.

There is ample evidence for the role of the immune system in developing chronic pain following peripheral nerve injury. Especially Toll-like receptors (TLRs) and their associated signaling components and pro-inflammatory cytokines such as IL-1ß, induced after injury, are involved in nociceptive processes and believed to contribute to the manifestation of chronic neuropathic pain states. Whereas the inhibition of the kinase function of IRAK-4, a central kinase downstream of TLRs and IL-1 receptors (IL-1Rs), seems efficacious in various chronic inflammatory and autoimmune models, it's role in regulating chronic neuropathic pain remained elusive to date. Here, we examined whether pharmacological inhibition of IRAK-4 kinase activity using PF-06650833 and BMS-986147, two clinical-stage kinase inhibitors, is effective for controlling persistent pain following nerve injury. Both inhibitors potently inhibited TLR-triggered cytokine release in human peripheral blood mononuclear cell (PBMC) as well as human and rat whole blood cultures. BMS-986147 showing favorable pharmacokinetic (PK) properties, significantly inhibited R848-triggered plasma TNF levels in a rat in vivo cytokine release model after single oral dosing. However, BMS-986147 dose dependently reversed cold allodynia in a rat chronic constriction injury (CCI) model following intrathecal administration only, supporting the notion that central neuro-immune modulation is beneficial for treating chronic neuropathic pain. Although both inhibitors were efficacious in inhibiting IL-1ß- or TLR-triggered cytokine release in rat dorsal root ganglion cultures, only partial efficacy was reached in IL-1ß-stimulated human glial cultures indicating that inhibiting IRAK-4́'s kinase function might be partially dispensable for human IL-1ß driven neuroinflammation. Overall, our data demonstrate that IRAK-4 inhibitors could provide therapeutic benefit in chronic pain following nerve injury, and the central driver for efficacy in the neuropathic pain model as well as potential side effects of centrally available IRAK-4 inhibitors warrant further investigation to develop effective analgesia for patients in high unmet medical need.

Immature dendritic cells convert anergic nonregulatory T cells into Foxp3- IL-10+ regulatory T cells by engaging CD28 and CTLA-4.

Anergic T cells can survive for long time periods passively in a hyporesponsive state without obvious active functions. Thus, the immunological reason for their maintenance is unclear. Here, we induced peptide-specific anergy in T cells from mice by coculturing these cells with immature murine dendritic cells (DCs). We found that these anergic, nonsuppressive IL-10(-) Foxp3(-) CTLA-4(+) CD25(low) Egr2(+) T cells could be converted into suppressive IL-10(+) Foxp3(-) CTLA-4(+) CD25(high) Egr2(+) cells resembling type-1 Treg cells (Tr1) when stimulated a second time by immature DCs in vitro. Addition of TGF-ß during anergy induction favored Foxp3(+) Treg-cell induction, while TGF-ß had little effect when added to the second stimulation. Expression of both CD28 and CTLA-4 molecules on anergic T cells was required to allow their conversion into Tr1-like cells. Suppressor activity was enabled via CD28-mediated CD25 upregulation, acting as an IL-2 sink, together with a CTLA-4-mediated inhibition of NFATc1/α activation to shut down IL-2-mediated proliferation. Together, these data provide evidence and mechanistical insights into how persistent anergic T cells may serve as a resting memory pool for Tr1-like cells.

Similar inflammatory DC maturation signatures induced by TNF or Trypanosoma brucei antigens instruct default Th2-cell responses.

DCs represent the major cell type leading to polarized T-helper (Th) cell responses in vivo. Here, we asked whether the instruction of murine Th2 responses by DCs matured with the proinflammatory cytokine TNF is qualitatively different from maturation by different types of TLR4/MyD88-dependent variant-specific surface glycoproteins (VSGs) of Trypanosoma brucei (T. brucei). The results obtained by analyzing DC surface markers, Notch ligand mRNA, cytokines, asthma, and experimental autoimmune encephalomyelitis (EAE) models as well as performing microarrays indicate that both types of stimuli induce similar inflammatory, semi-mature DC profiles. DCs matured by TNF or VSG treatment expressed a common inflammatory signature of 24 genes correlating with their Th2-polarization capacity. However, the same 24 genes and 4498 additional genes were expressed by DCs treated with LPS that went on to induce Th1 cells. These findings support the concept of a default pathway for Th2-cell induction in DCs matured under suboptimal or inflammatory conditions, independent of the surface receptors and signaling pathways involved. Our data also indicate that quantitative differences in DC maturation might direct Th2- vs Th1-cell responses, since suboptimally matured inflammatory DCs induce default Th2-cell maturation, whereas fully mature DCs induce Th1-cell maturation.

Antigen-specific immunotherapy with apitopes suppresses generation of FVIII inhibitor antibodies in HLA-transgenic mice.

Hemophilia A (HA) is a blood clotting disorder that is caused by various genetic deficiencies in the factor VIII (FVIII)-encoding F8 gene. Patients receiving FVIII-replacement therapy are at risk for developing neutralizing antibodies (FVIII inhibitors), rendering the FVIII-replacement therapy ineffective. Immunological tolerance toward FVIII can be achieved through immune tolerance induction protocols in some patients, but this is a lengthy and costly desensitization program. Long-term eradication of inhibitors in patients with HA could be achieved by antigen-specific immunotherapy targeting CD4+ T-cells, because formation of FVIII inhibitors is T-cell dependent. Here, we report a peptide-based antigen-specific immunotherapy that is designed to specifically reestablish immune tolerance to FVIII through the development of antigen-processing-independent epitopes (apitopes). We identified 2 FVIII immunodominant peptides in immunized HLA-DRA*0101/DRB1*1501 transgenic (HLA-DR2tg) mice that were optimized for tolerogenicity. These modified peptide analogs were initially screened for recognition using FVIII-specific T-cell hybridoma clones from FVIII-immunized HLA-DR2tg mice. The FVIII apitopes were promiscuous and bound common human HLA-DRB1* allelic variants. The combination of these 2 FVIII apitopes (ATX-F8-117), administered according to a dose-escalation protocol, promoted T-cell tolerance toward FVIII in HLA-DR2tg mice. Furthermore, treatment with ATX-F8-117 significantly reduced FVIII inhibitor formation. ATX-F8-117 regulates anti-FVIII T-cell and B-cell responses, specifically the generation of FVIII inhibitors, revealing peptide-based antigen-specific immunotherapy as a promising approach to suppress and treat inhibitor formation in susceptible patients with HA.

Neuromelanin is an immune stimulator for dendritic cells in vitro.

BACKGROUND: Parkinson's disease (PD) is characterized at the cellular level by a destruction of neuromelanin (NM)-containing dopaminergic cells and a profound reduction in striatal dopamine. It has been shown recently that anti-melanin antibodies are increased in sera of Parkinson patients, suggesting that NM may act as an autoantigen. In this study we tested whether NM is being recognized by dendritic cells (DCs), the major cell type for inducing T- and B-cell responses in vivo. This recognition of NM by DCs is a prerequisite to trigger an adaptive autoimmune response directed against NM-associated structures. RESULTS: Murine DCs were treated with NM of substantia nigra (SN) from human subjects or with synthetic dopamine melanin (DAM). DCs effectively phagocytized NM and subsequently developed a mature phenotype (CD86(high)/MHCII(high)). NM-activated DCs secreted the proinflammatory cytokines IL-6 and TNF-α. In addition, they potently triggered T cell proliferation in a mixed lymphocyte reaction, showing that DC activation was functional to induce a primary T cell response. In contrast, DAM, which lacks the protein and lipid components of NM but mimics the dopamine-melanin backbone of NM, had only very little effect on DC phenotype and function. CONCLUSIONS: NM is recognized by DCs in vitro and triggers their maturation. If operative in vivo, this would allow the DC-mediated transport and presentation of SN antigens to the adaptive immune system, leading to autoimmmunity in susceptible individuals. Our data provide a rationale for an autoimmune-based pathomechanism of PD with NM as the initial trigger.

Highly Selective Sub-Nanomolar Cathepsin S Inhibitors by Merging Fragment Binders with Nitrile Inhibitors.

Pharmacological inhibition of cathepsin S (CatS) allows for a specific modulation of the adaptive immune system and many major diseases. Here, we used NMR fragment screening and crystal structure-aided merging to synthesize novel, highly selective CatS inhibitors with picomolar enzymatic Ki values and nanomolar functional activity in human Raji cells. Noncovalent fragment hits revealed binding hotspots, while the covalent inhibitor structure-activity relationship enabled efficient potency optimization.

Inhibiting the immunoproteasome's ß5i catalytic activity affects human peripheral blood-derived immune cell viability.

Small molecule inhibitors selectively targeting the immunoproteasome subunit ß5i are currently being developed for the treatment of autoimmune disorders. However, patients carrying loss-of-function mutations in the gene encoding ß5i (Psmb8) suffer from the proteasome-associated autoinflammatory syndromes (PRAAS) emphasizing the need to study pharmacological inhibition of immunoproteasome function in human cells. Here, we characterized the immunomodulatory potential of the selective ß5i inhibitor ONX 0914 and Bortezomib, a pan-proteasome inhibitor, in human peripheral blood mononuclear cells (PBMCs). Both compounds efficiently blocked pro-inflammatory cytokine secretion in human whole blood and PBMC cultures stimulated with toll-like receptor (TLR) agonists. Furthermore, the compounds inhibited T cell cytokine production induced by recall antigen CMVpp65 or by polyclonal stimulation. The viability of PBMCs, however, was rapidly decreased in the presence of ONX 0914 and Bortezomib demonstrated by decreased residual cytosolic ATP and increased Annexin V surface binding. Interestingly, HLA-DR + monocytes were rapidly depleted from the cultures in the presence of ONX 0914 as a ß5i-selective inhibitor and Bortezomib. In conclusion, the anti-inflammatory potential of ß5i-selective inhibitors is correlating with a cytotoxicity increase in human PBMC subsets ex vivo. Our results provide important insights into the anti-inflammatory mechanism of action of ß5i-inhibitors which currently hold the promise as a novel therapy for autoinflammatory diseases.

Low doses of cholera toxin and its mediator cAMP induce CTLA-2 secretion by dendritic cells to enhance regulatory T cell conversion.

Immature or semi-mature dendritic cells (DCs) represent tolerogenic maturation stages that can convert naive T cells into Foxp3+ induced regulatory T cells (iTreg). Here we found that murine bone marrow-derived DCs (BM-DCs) treated with cholera toxin (CT) matured by up-regulating MHC-II and costimulatory molecules using either high or low doses of CT (CThi, CTlo) or with cAMP, a known mediator CT signals. However, all three conditions also induced mRNA of both isoforms of the tolerogenic molecule cytotoxic T lymphocyte antigen 2 (CTLA-2α and CTLA-2ß). Only DCs matured under CThi conditions secreted IL-1ß, IL-6 and IL-23 leading to the instruction of Th17 cell polarization. In contrast, CTlo- or cAMP-DCs resembled semi-mature DCs and enhanced TGF-ß-dependent Foxp3+ iTreg conversion. iTreg conversion could be reduced using siRNA blocking of CTLA-2 and reversely, addition of recombinant CTLA-2α increased iTreg conversion in vitro. Injection of CTlo- or cAMP-DCs exerted MOG peptide-specific protective effects in experimental autoimmune encephalomyelitis (EAE) by inducing Foxp3+ Tregs and reducing Th17 responses. Together, we identified CTLA-2 production by DCs as a novel tolerogenic mediator of TGF-ß-mediated iTreg induction in vitro and in vivo. The CT-induced and cAMP-mediated up-regulation of CTLA-2 also may point to a novel immune evasion mechanism of Vibrio cholerae.

Dendritic cells generated with Flt3L and exposed to apoptotic cells lack induction of T cell anergy and Foxp3⁺ regulatory T cell conversion in vitro.

Removal of apoptotic cells, which appear during the steady state, is a pre-requisite to prevent generation of secondary necrotic cells that may lead to autoimmunity. The recognition of apoptotic material by dendritic cells (DCs) has been proposed to convert them into tolerogenic DCs equipped with specialized tolerogenic mechanisms on T cells. However, comparative studies to demonstrate functional alterations of DCs upon exposure to apoptotic cells have not been performed so far. Here we show that immature murine bone marrow-derived DCs generated with GM-CSF (GM-DCs) or Flt3L (FL-DCs) interact with live or apoptotic syngeneic thymocytes. As expected, GM-DCs phagocytose apoptotic but not live cells, FL-DCs only show trogocytosis of membrane parts. Interaction with live or apoptotic thymocytes did not lead to DC maturation. Both GM-DCs and FL-DCs present OVA as protein, peptide and membrane-associated antigens. Interestingly, only GM-DCs were able to induce T cell anergy or convert naïve T cells into FoxP3⁺ regulatory T cells (Tregs) but FL-DCs did not show either of these effects. Unexpectedly, exposure of immature GM-DCs to live or apoptotic thymocytes did not improve DC functions in both types of in vitro T cell tolerance induction assays. Together, our data suggest that these tolerogenic in vitro measures of immature BM-DCs are not further enhanced by exposure to apoptotic cells and may depend on the generating cytokine.

Excretory/secretory-products of Echinococcus multilocularis larvae induce apoptosis and tolerogenic properties in dendritic cells in vitro.

BACKGROUND: Alveolar echinococcosis, caused by Echinococcus multilocularis larvae, is a chronic disease associated with considerable modulation of the host immune response. Dendritic cells (DC) are key effectors in shaping the immune response and among the first cells encountered by the parasite during an infection. Although it is assumed that E.multilocularis, by excretory/secretory (E/S)-products, specifically affects DC to deviate immune responses, little information is available on the molecular nature of respective E/S-products and their mode of action. METHODOLOGY/PRINCIPAL FINDINGS: We established cultivation systems for exposing DC to live material from early (oncosphere), chronic (metacestode) and late (protoscolex) infectious stages. When co-incubated with Echinococcus primary cells, representing the invading oncosphere, or metacestode vesicles, a significant proportion of DC underwent apoptosis and the surviving DC failed to mature. In contrast, DC exposed to protoscoleces upregulated maturation markers and did not undergo apoptosis. After pre-incubation with primary cells and metacestode vesicles, DC showed a strongly impaired ability to be activated by the TLR ligand LPS, which was not observed in DC pre-treated with protoscolex E/S-products. While none of the larvae induced the secretion of pro-inflammatory IL-12p70, the production of immunosuppressive IL-10 was elevated in response to primary cell E/S-products. Finally, upon incubation with DC and naïve T-cells, E/S-products from metacestode vesicles led to a significant expansion of Foxp3+ T cells in vitro. CONCLUSIONS: This is the first report on the induction of apoptosis in DC by cestode E/S-products. Our data indicate that the early infective stage of E. multilocularis is a strong inducer of tolerance in DC, which is most probably important for generating an immunosuppressive environment at an infection phase in which the parasite is highly vulnerable to host attacks. The induction of CD4+CD25+Foxp3+ T cells through metacestode E/S-products suggests that these cells fulfill an important role for parasite persistence during chronic echinococcosis.

Role of dendritic cell maturity/costimulation for generation, homeostasis, and suppressive activity of regulatory T cells.

Tolerogenicity of dendritic cells (DCs) has initially been attributed exclusively to immature/resting stages, while mature/activated DCs were considered strictly immunogenic. Later, all different subsets among the myeloid/conventional DCs and plasmacytoid DCs have been shown to bear tolerogenic potential, so that tolerogenicity could not be attributed to a specific subset. Immunosuppressive treatments of immature DC subsets could prevent re-programming into mature DCs or upregulated inhibitory surface markers or cytokines. Furthermore, the different T cell tolerance mechanisms anergy, deletion, immune deviation, and suppression require different quantities and qualities of costimulation by DCs. Since expansion of regulatory T cells (Tregs) has been shown to be promoted best by fully mature DCs the role of CD80/B7-1 and CD86/B7-2 as major costimulatory molecules for Treg biology is under debate. In this review, we discuss the role of these and other costimulatory molecules on myeloid DCs and their ligands CD28 and CD152/CTLA-4 on Tregs for peripheral conversion from naive CD4⁺ T cells into the major subsets of Foxp3⁺ Tregs and Foxp3⁻ IL-10⁺ regulatory type-1 T cells (Tr1) or Tr1-like cells and their role for peripheral maintenance in the steady state and after activation.

Functional T-cell responses generated by dendritic cells expressing the early HIV-1 proteins Tat, Rev and Nef.

The limitations of highly active anti-retroviral therapy (HAART) have necessitated the development of alternative therapeutic strategies. One of the approaches that has gained prominence in recent years is therapeutic vaccination. We decided to assess the capacity of mature dendritic cells, derived from blood monocytes of HIV-1 infected patients, to generate functional T-cell responses. For this purpose, we constructed a chimeric mRNA encoding the proteins Tat, Rev and Nef. The TaReNef encoding information was linked to the HLA class II-targeting sequence of DC-LAMP. Broadly directed HIV-specific CD4(+) and CD8(+) cytotoxic T cells exhibiting a poly-functional cytokine secretion pattern were generated by co-culturing with autologous chimeric mRNA electroporated dendritic cells. Thus, administration of ex vivo generated dendritic cells expressing the early proteins Tat, Rev and Nef might offer a promising approach for therapeutic vaccination in HIV-1 infection.