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Background: Restoring impaired peripheral immune tolerance is the primary challenge in treating autoimmune diseases. Our previous research demonstrated the effectiveness of small spleen peptides (SSPs), a fraction of low molecular weight proteins, in inhibiting the progression of psoriatic arthritis, even in the presence of high levels of the proinflammatory cytokine TNFα in the bloodstream. When specifically targeting dendritic cells (DCs), SSPs transform them into tolerogenic cells, which efficiently induce the development of regulatory Foxp3 + Treg cells. In this study, we provide further insights into the mechanism of action of SSPs., Results: We found that SSPs stimulate the activation of the mTOR signaling pathway in dendritic cells, albeit in a different manner than the classical immunogenic stimulus LPS. While LPS-induced activation is rapid, strong, and sustained, the activity induced by SSPs is delayed, less intense, yet still significant. These distinct patterns of activation, as measured by phosphorylation of key components of the pathway are also observed in response to other immunogenic and tolerogenic stimuli such as GM-CSF + IL-4 or IL-10 and TGFβ. The disparity in mTOR activation between immunogenic and tolerogenic stimuli is quantitative rather than qualitative. In both cases, mTOR activation primarily occurs through the PI3K/Akt signaling axis and involves ERK and GSK3β kinases, with minimal involvement of AMPK or NF-kB pathways. Furthermore, in the case of SSPs, mTOR activation seems to involve adenosine receptors. Additionally, we observed that DCs treated with SSPs exhibit an energy metabolism with high plasticity, which is typical of tolerogenic cells rather than immunogenic cells., Conclusion: Hence, the decision whether dendritic cells enter an inflammatory or tolerogenic state seems to rely on varying activation thresholds and kinetics of the mTOR signaling pathway., (© 2024. The Author(s).)

Restoring peripheral immune tolerance is crucial for addressing autoimmune diseases. An ancient mechanism in maintaining the balance between inflammation and tolerance is the ratio of extracellular ATP (exATP) and adenosine. Our previous research demonstrated the effectiveness of small spleen peptides (SSPs) in inhibiting psoriatic arthritis progression, even in the presence of the pro-inflammatory cytokine TNFα, by transforming dendritic cells (DCs) into tolerogenic cells and fostering regulatory Foxp3 + Treg cells. Here, we identified thymosins as the primary constituents of SSPs, but recombinant thymosin peptides were less efficient in inhibiting arthritis than SSPs. Since Tβ4 is an ecto-ATPase-binding protein, we hypothesized that SSPs regulate exATP profiles. Real-time investigation of exATP levels in DCs revealed that tolerogenic stimulation led to robust de novo exATP synthesis followed by significant degradation, while immunogenic stimulation resulted in a less pronounced increase in exATP and less effective degradation. These contrasting exATP profiles were crucial in determining whether DCs entered an inflammatory or tolerogenic state, highlighting the significance of SSPs as natural regulators of peripheral immunological tolerance, with potential therapeutic benefits for autoimmune diseases. Finally, we demonstrated that the tolerogenic phenotype of SSPs is mainly influenced by adenosine receptors, and in vivo administration of SSPs inhibits psoriatic skin inflammation.

Sphingosine 1-phosphate (S1P), a lysosphingolipid associated with high-density lipoprotein (HDL), contributes to the anti-atherogenic potential attributed to this lipoprotein. This study examined whether a reduction of S1P plasma levels affects atherosclerosis in a murine model of disease. LDL-R(-/-)mice on Western diet were given ABC294640, an inhibitor of sphingosine kinase (SphK) for 16 weeks. ABC294640 decreased plasma S1P by approximately 30%. However, ABC294640 failed to affect atherosclerotic lesion formation. Plasma triglycerides were reduced whereas total and HDL-cholesterol remained unchanged in course of ABC294640 treatment. ABC294640 increased plasma interleukin (IL)-12p70 and RANTES concentration as well as IL-12p70, RANTES and interferon (IFN)-γ production by peritoneal cells and this was paralleled by enhanced activity of peritoneal and spleen dendritic cells as evidenced by up-regulation of CD86 and MHC-II on CD11c(+) cells. As a consequence, increased T-cell activation was noted in ABC294640-treated mice as indicated by enhanced CD4(+) splenocyte proliferation, IFN-γ and IL-2 production, and CD69 expression. Concomitantly, however, ABC294640 treatment redistributed CD4(+) and CD8(+) cells from blood to lymphatic organs and reduced T-cell number within atherosclerotic lesions. In addition, plasma sVCAM-1, sICAM-1, and MCP-1 levels as well as in vivo leukocyte adhesion and CCL19-induced T-cell penetration into peritoneum were lower in ABC294640-treated animals. In vitro experiments demonstrated reduced VCAM-1 and ICAM-1 expression and lymphocyte adhesion to endothelial cells exposed to ABC294640. In conclusion, treatment with SphK inhibitor leads to both pro- and anti-atherogenic effects in LDL-R(-/-) mice. As a consequence, SphK inhibition fails to affect atherosclerosis despite significant S1P reduction in plasma.

The beta2 integrins are important for transendothelial migration of leukocytes as well as for T-cell activation during antigen presentation. Despite abundant expression of beta2 integrins on antigen-presenting cells (APCs), their functional relevance for antigen presentation is completely unclear. We show here that dendritic cells (DCs) from CD18-deficient mice, which lack all functional beta2 integrins, have no defect in antigen presentation. Moreover, DCs from normal mice express inactive beta2 integrins that do not become activated on contact with T cells, at least in vitro. Pharmacologic activation of beta2 integrins on DCs results in a significant reduction of their T cell-activating capacity. This effect is mediated by Mac-1 (CD11b/CD18) on DCs because it could be reversed via blocking antibodies against CD18 and CD11b. Furthermore, the antigen-presenting capacity of macrophages, which express constitutively active beta2 integrins, is significantly enhanced on Mac-1 blockade. We therefore conclude that active CD11b/CD18 (Mac-1) on APCs directly inhibits T-cell activation.

The adaptive immune response is initiated when naive T cells interact with dendritic cells (DC). However, the physicodynamics as well as the molecules that constitute the contact plane (immunological synapse) between DC and T cells are not well understood. We show here that for the formation of stable conjugates, T cells need to be preactivated by DC in a CD80/86- and antigen dose-dependent manner. When activated, T cells induce cytoskeletal reorganization within DC via CD40-CD40L signaling. Polarization of the actin and fascin cytoskeleton in DC is associated with sustained DC-T cell contacts, strong T cell proliferation and a Th1 response. Organized contact planes with clearly separated patches containing TCR or CD11a are also formed. Thus, DC-T cell interactions take place in a sequential, interdependent fashion: first, DC "license" naive T cells to engage DC in an antigen dose- and CD80/86-dependent fashion. Then, these preactivated T cells induce cytoskeletal reorientation in DC, resulting in sustained DC-T cell contacts and subsequent T cell activation. These results demonstrate that T cells control the mode of interaction based on information gathered from DC.

Immature dendritic cells (DC) reside in tissues where they initiate immune responses by taking up foreign antigens. Since DC have a limited tissue half-life, the DC pool in tissues has to be replenished constantly. This implies that precursor/immature DC must be able to cross non-activated endothelium using as yet unknown mechanisms. Here we show that immature, but not mature bone marrow-derived murine DC migrate across resting endothelial monolayers in vitro. We find that endothelial intercellular adhesion molecule-2 (ICAM-2) is a major player in transendothelial migration (TEM) of immature DC, accounting for at least 41% of TEM. Surprisingly, the ICAM-2-mediated TEM was independent of beta2-integrins, the known ICAM-2 ligands, since neither blocking of beta2-integrins with antibodies nor the use of CD18-deficient DC affected the ICAM-2-specific TEM. In humans, the C-type lectin DC-specific ICAM-3-grabbing nonintegrin (DC-SIGN) was shown to interact with ICAM-2, suggesting a similar role in mice. However, we find that none of the murine DC-SIGN homologues mDC-SIGN, murine DC-SIGN-related molecule-1 (mSIGN-R1) and mSIGN-R3 is expressed on the surface of bone marrow-derived mouse DC. Taken together, this study shows that ICAM-2 strongly supports transmigration of immature DC across resting endothelium by interacting with ligands that are distinct from beta2-integrins and DC-SIGN homologues.

Pimecrolimus (SDZ ASM981) is a non-steroid member of calcineurin inhibitors recently developed for the treatment of inflammatory skin diseases. In this study, we compared the effect of pimecrolimus and corticosteroids on the differentiation, maturation and function of murine bone marrow-derived dendritic cells (BM-DC). We added pimecrolimus at concentrations of 5-500 ng/ml or 0.5 ng/ml mometasone furoate at different timepoints to the BM-DC culture and checked (i) the number of matured cells, (ii) the expression of activation markers, (iii) the release of cytokines and (iv) the stimulatory capacity of the resulting BM-DC in vivo. Even at the highest concentration, pimecrolimus treatment resulted in only modest effects. In the pimecrolimus-treated culture, we observed a decrease in the numbers of matured cells but no significant effects on the expression of activation markers. The release of some inflammatory cytokines was reduced, but the stimulatory capacity in vivo was not affected. In contrast, mometasone furoate has pronounced effects on BM-DC at a concentration ten to 1000 times lower than those used with pimecrolimus. Furthermore, topical treatment of mice with clobetasole cream 0.05% resulted in almost complete depletion of splenic DC and a severe hyposplenia, while high-dose oral pimecrolimus treatment did not show any effects on the spleen or on splenic DC. These results support that pimecrolimus, unlike corticosteroids, has little effects on dendritic cells. To the best of our knowledge, this is the first study of this type with use of BM-DC.

Recently, we established a doxycycline‐inducible human tumor necrosis factor alpha (TNFα)‐transgenic mouse line, ihTNFtg. Non‐induced young and elderly mice showed low but constitutive expression of hTNFα due to promoter leakiness. The persistently present hTNFα stimulated endogenous pro‐inflammatory mouse mS100A8/A9 alarmins. Secreted mS100A8/A9 in turn induced the expression and release of mouse mTNFα. The continuous upregulation of pro‐inflammatory mTNFα and mS100A8/A9 proteins, due to their mutual expression dependency, gradually led to increased levels in colon tissue and blood. This finally exceeded the threshold levels tolerated by the healthy organism, leading to the onset of intestinal inflammation. Here, recombinant hTNFα functioned as an initial trigger for the development of chronic inflammation. Crossing ihTNFtg mice with S100A9KO mice lacking active S100A8/A9 alarmins or with Rag1KO mice lacking T and B lymphocytes completely abrogated the development of colonic inflammation, despite the still leaky hTNFα promoter. Furthermore, both the intensity of the immune response and the strength of immunosuppressive Treg induction was found to depend on the major histocompatibility complex (MHC) genetic composition. In summary, the onset of intestinal inflammation in elderly mice depends on at least four factors that have to be present simultaneously: TNFα upregulation, S100A8/A9 protein expression, functional T lymphocytes and genetic composition, with the MHC haplotype being of central importance. Only joint action of these factors leads to chronic intestinal inflammation, while absence of any of these determinants abrogates the development of the autoimmune disorder. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) vaccines have the potential to improve antiretroviral drug treatment by inducing cytotoxic killing of HIV-infected cells. Prophylactic vaccines utilize new antigens to initiate immunity; however, in HIV-infected individuals the load of viral antigen is not the limiting factor for the restoration of immune responses. Here we describe a novel immunization strategy with DermaVir that improves viral antigen presentation using dendritic cells (DC). DermaVir contains a distinctive plasmid DNA expressing all HIV proteins except integrase to induce immune responses with broad specificity. The DNA is formulated to a mannosilated particle to target antigen-presenting cells and to protect the DNA from intracellular degradation. After topical application, DermaVir-transduced cells migrate from the skin to the draining lymph node and interdigitate as DermaVir-expressing, antigen-presenting DC. We compared the immunogenicity of topical andex vivoDC-based DermaVir vaccinations in naïve rhesus macaques. Both vaccinations induced simian immunodeficiency virus-specific CD4 helper and CD8 memory T cells detected by anin vivoskin test and anin vitrointracellular cytokine-based assay. Topical DermaVir vaccination represents an improvement upon existingex vivoDC-based immunization technologies and may provide a new therapeutic option for HIV-infected patients. [ABSTRACT FROM AUTHOR]