Cross-reactive, natural IgG recognizing L. major promote parasite internalization by dendritic cells and promote protective immunity

In cutaneous leishmaniasis, infection of dendritic cells (DC) is essential for generation of T cell-dependent protective immunity. DC acquires Leishmania major through Fc receptor (FcR)-mediated uptake of complexes comprising antibodies bound to parasites. We now assessed the development of the initial B cell and DC response to the parasite itself and if natural IgG play a role. L. major parasites display large numbers of phospholipids on their surface. Parasites were opsonized with normal mouse serum (NMS), or serum containing anti-phospholipid IgG (PL). We found that L. major bound to PL which significantly enhanced parasite phagocytosis by DC as compared to NMS. Similar results were obtained with cross-reactive human PL antibodies using myeloid primary human DC. In addition, mice infected with PL-opsonized parasites showed significantly improved disease outcome compared to mice infected with NMS-opsonized parasites. Finally, IgMi mice, which produce membrane-bound IgM only and no secreted antibodies, displayed increased susceptibility to infection as compared to wild types. Interestingly, once NMS was administered to IgMi mice, their phenotype was normalized to that of wild types. Upon incubation with IgG-opsonized parasite (IgG derived from infected mice or using PL antibodies), also the IgMi mice were able to show superior immunity. Our findings suggest that “natural” cross-reactive antibodies (e.g., anti-PL Ab) in NMS bind to pathogens to facilitate phagocytosis, which leads to induction of protective immunity via preferential DC infection. Prior L. major-specific B cell-priming does not seem to be absolutely required to facilitate clearance of this important human pathogen in vivo

Mature oligodendrocytes actively increase in vivo cytoskeletal plasticity following CNS damage

Background: Oligodendrocytes are myelinating cells of the central nervous system which support functionally, structurally, and metabolically neurons. Mature oligodendrocytes are generally believed to be mere targets of destruction in the context of neuroinflammation and tissue damage, but their real degree of in vivo plasticity has become a matter of debate. We thus investigated the in vivo dynamic, actin-related response of these cells under different kinds of demyelinating stress. Methods: We used a novel mouse model (oLucR) expressing luciferase in myelin oligodendrocyte glycoproteinpositive oligodendrocytes under the control of a beta-actin promoter. Activity of this promoter served as surrogate for dynamics of the cytoskeleton gene transcription through recording of in vivo bioluminescence following diphtheria toxin-induced oligodendrocyte death and autoimmune demyelination. Cytoskeletal gene expression was quantified from mature oligodendrocytes directly isolated from transgenic animals through cell sorting. Results: Experimental demyelinating setups augmented oligodendrocyte-specific in vivo bioluminescence. These changes in luciferase signal were confirmed by further ex vivo analysis of the central nervous system tissue from oLucR mice. Increase in bioluminescence upon autoimmune inflammation was parallel to an oligodendrocytespecific increased transcription of beta-tubulin. Conclusions: Mature oligodendrocytes acutely increase their cytoskeletal plasticity in vivo during demyelination. They are therefore not passive players under demyelinating conditions but can rather react dynamically to external insults

Tolerance without clonal expansion: Self-antigen-expressing B cells program self-reactive T cells for future deletion

B cells have been shown in various animal models to induce immunological tolerance leading to reduced immune responses and protection from autoimmunity. We show that interaction of B cells with naive T cells results in T cell triggering accompanied by the expression of negative costimulatory molecules such as PD-1, CTLA-4, B and T lymphocyte attenuator, and CD5. Following interaction with B cells, T cells were not induced to proliferate, in a process that was dependent on their expression of PD-1 and CTLA-4, but not CD5. In contrast, the T cells became sensitive to Ag-induced cell death. Our results demonstrate that B cells participate in the homeostasis of the immune system by ablation of conventional self-reactive T cells

Neuroinflammation by cytotoxic T-lymphocytes impairs retrograde axonal transport in an oligodendrocyte mutant mouse

Mice overexpressing proteolipid protein (PLP) develop a leukodystrophy-like disease involving cytotoxic, CD8+ T-lymphocytes. Here we show that these cytotoxic T-lymphocytes perturb retrograde axonal transport. Using fluorogold stereotactically injected into the colliculus superior, we found that PLP overexpression in oligodendrocytes led to significantly reduced retrograde axonal transport in retina ganglion cell axons. We also observed an accumulation of mitochondria in the juxtaparanodal axonal swellings, indicative for a disturbed axonal transport. PLP overexpression in the absence of T-lymphocytes rescued retrograde axonal transport defects and abolished axonal swellings. Bone marrow transfer from wildtype mice, but not from perforin- or granzyme B-deficient mutants, into lymphocyte-deficient PLP mutant mice led again to impaired axonal transport and the formation of axonal swellings, which are predominantly located at the juxtaparanodal region. This demonstrates that the adaptive immune system, including cytotoxic T-lymphocytes which release perforin and granzyme B, are necessary to perturb axonal integrity in the PLP-transgenic disease model. Based on our observations, so far not attended molecular and cellular players belonging to the immune system should be considered to understand pathogenesis in inherited myelin disorders with progressive axonal damage

Hepatocyte Bcl-3 protects from death-receptor mediated apoptosis and subsequent acute liver failure

Acute liver failure (ALF) is a rare entity but exhibits a high mortality. The mechanisms underlying ALF are not completely understood. The present study explored the role of the hepatic B cell leukemia-3 (Bcl-3), a transcriptional regulator of nuclear factor-kappa B (NF-κB), in two independent models of ALF. We employed a recently developed transgenic mouse model in a C57BL6/J background comparing wild-type (WT) and transgenic littermates with hepatocyte-specific overexpression of Bcl-3 (Bcl-3Hep) in the ALF model of d-galactosamine (d-GalN) and lipopolysaccharide (LPS). Additionally, the apoptosis-inducing CD95 (FAS/APO-1)-ligand was explored. Bcl-3Hep mice exhibited a significant protection from ALF with decreased serum transaminases, decreased activation of the apoptotic caspases 8, 9, and 3, lower rates of oxidative stress, B-cell lymphoma 2 like 1 (BCL2L1/BCL-XL) degradation and accompanying mitochondrial cytochrome c release, and ultimately a decreased mortality rate from d-GalN/LPS compared to WT mice. d-GalN/LPS treatment resulted in a marked inflammatory cytokine release and stimulated the activation of signal transducer and activator of transcription (STAT) 3, c-Jun N-terminal kinases (JNK) and extracellular signal-regulated kinase (ERK) signaling comparably in the hepatic compartment of Bcl-3Hep and WT mice. However, in contrast to the WT, Bcl-3Hep mice showed a diminished rate of IkappaB kinase-beta (IKK-β) degradation, persistent receptor interacting protein kinase (RIPK) 1 function and thus prolonged cytoprotective nuclear factor-kappa B (NF-κB) p65 signaling through increased p65 stability and enhanced transcription. Likewise, Bcl-3 overexpression in hepatocytes protected from ALF with massive hepatocyte apoptosis induced by the anti-FAS antibody Jo2. The protection was also linked to IKK-β stabilization. Overall, our study showed that Bcl-3 rendered hepatocytes more resistant to hepatotoxicity induced by d-GalN/LPS and FAS-ligand. Therefore, Bcl-3 appears to be a critical regulator of the dynamics in ALF through IKK-β

Generation of a novel T cell specific interleukin-1 receptor type 1 conditional knock out mouse reveals intrinsic defects in survival, expansion and cytokine production of CD4 T cells

Interleukin-1 (IL-1) plays a crucial role in numerous inflammatory diseases via action on its only known signaling IL-1 receptor type 1 (IL-1R1). To investigate the role of IL-1 signaling in selected cell types, we generated a new mouse strain in which exon 5 of the Il1r1 gene is flanked by loxP sites. Crossing of these mice with CD4-Cre transgenic mice resulted in IL-1R1 loss of function specifically in T cells. These mice, termed IL-1R1ΔT, displayed normal development under steady state conditions. Importantly, isolated CD4 positive T cells retained their capacity to differentiate toward Th1 or Th17 cell lineages in vitro, and strongly proliferated in cultures supplemented with either anti-CD3/CD28 or Concanavalin A, but, as predicted, were completely unresponsive to IL-1β administration. Furthermore, IL-1R1ΔT mice were protected from gut inflammation in the anti-CD3 treatment model, due to dramatically reduced frequencies and absolute numbers of IL-17A and interferon (IFN)-γ producing cells. Taken together, our data shows the necessity of intact IL-1 signaling for survival and expansion of CD4 T cells that were developed in an otherwise IL-1 sufficient environment

Regulatory T Cells and IL-10 Independently Counterregulate Cytotoxic T Lymphocyte Responses Induced by Transcutaneous Immunization

The imidazoquinoline derivate imiquimod induces inflammatory responses and protection against transplanted tumors when applied to the skin in combination with a cognate peptide epitope (transcutaneous immunization, TCI). Here we investigated the role of regulatory T cells (T(reg)) and the suppressive cytokine IL-10 in restricting TCI-induced cytotoxic T lymphocyte (CTL) responses.TCI was performed with an ointment containing the TLR7 agonist imiquimod and a CTL epitope was applied to the depilated back skin of C57BL/6 mice. Using specific antibodies and FoxP3-diphteria toxin receptor transgenic (DEREG) mice, we interrogated inhibiting factors after TCI: by depleting FoxP3(+) regulatory T cells we found that specific CTL-responses were greatly enhanced. Beyond this, in IL-10 deficient (IL-10(-/-)) mice or after blocking of IL-10 signalling with an IL-10 receptor specific antibody, the TCI induced CTL response is greatly enhanced indicating an important role for this cytokine in TCI. However, by transfer of T(reg) in IL-10(-/-) mice and the use of B cell deficient JHT(-/-) mice, we can exclude T(reg) and B cells as source of IL-10 in the setting of TCI.We identify T(reg) and IL-10 as two important and independently acting suppressors of CTL-responses induced by transcutaneous immunization. Advanced vaccination strategies inhibiting T(reg) function and IL-10 release may lead the development of effective vaccination protocols aiming at the induction of T cell responses suitable for the prophylaxis or treatment of persistent infections or tumors

Increased responsiveness of peripheral blood mononuclear cells to in vitro TLR 2, 4 and 7 ligand stimulation in chronic pain patients

Glial activation via Toll-like receptor (TLR) signaling has been shown in animals to play an important role in the initiation and establishment of chronic pain. However, our ability to assess this central immune reactivity in clinical pain populations is currently lacking. Peripheral blood mononuclear cells (PBMCs) are an accessible source of TLR expressing cells that may mirror similarities in TLR responsiveness of the central nervous system. The aim of this study was to characterize the IL-1β response to various TLR agonists in isolated PBMCs from chronic pain sufferers (on and not on opioids) and pain-free controls. Venous blood was collected from 11 chronic pain sufferers on opioids (≥ 20 mg of morphine / day), 8 chronic pain sufferers not on opioids and 11 pain-free controls. PBMCs were isolated and stimulated in vitro with a TLR2 (Pam3CSK4), TLR4 (LPS) or TLR7 (imiquimod) agonist. IL-1β released into the supernatant was measured with ELISA. Significantly increased IL-1β expression was found in PBMCs from chronic pain sufferers (on and not on opioids) compared with pain-free controls for TLR2 (F (6, 277) = 15, P<0.0001), TLR4 (F (8, 263) = 3, P = 0.002) and TLR7 (F (2,201) = 5, P = 0.005) agonists. These data demonstrate that PBMCs from chronic pain sufferers were more responsive to TLR agonists compared with controls, suggesting peripheral cells may have the potential to become a source of biomarkers for chronic pain.Yuen H. Kwok, Mark R. Hutchinson, Melanie G. Gentgall, Paul E. Rola

Emergence of the erythroid lineage from multipotent hematopoiesis [preprint]

Red cell formation begins with the hematopoietic stem cell, but the manner by which it gives rise to erythroid progenitors, and their subsequent developmental path, remain unclear. Here we combined single-cell transcriptomics of murine hematopoietic tissues with fate potential assays to infer a continuous yet hierarchical structure for the hematopoietic network. We define the erythroid differentiation trajectory as it emerges from multipotency and diverges from 6 other blood lineages. With the aid of a new flow-cytometric sorting strategy, we validated predicted cell fate potentials at the single cell level, revealing a coupling between erythroid and basophil/mast cell fates. We uncovered novel growth factor receptor regulators of the erythroid trajectory, including the proinflammatory IL- 17RA, found to be a strong erythroid stimulator; and identified a global hematopoietic response to stress erythropoiesis. We further identified transcriptional and high-purity FACS gates for the complete isolation of all classically-defined erythroid burst-forming (BFU-e) and colony-forming progenitors (CFU-e), finding that they express a dedicated transcriptional program, distinct from that of terminally-differentiating erythroblasts. Intriguingly, profound remodeling of the cell cycle is intimately entwined with CFU-e developmental progression and with a sharp transcriptional switch that extinguishes the CFU-e stage and activates terminal differentiation. Underlying these results, our work showcases the utility of theoretic approaches linking transcriptomic data to predictive fate models, providing key insights into lineage development in vivo