Drake LaFace

IL-23 induces spondyloarthropathy by acting on ROR-γt + CD3 + CD4 − CD8 − entheseal resident T cells

The spondyloarthropathies are a group of rheumatic diseases that are associated with inflammation at anatomically distal sites, particularly the tendon-bone attachments (entheses) and the aortic root. Serum concentrations of interleukin-23 (IL-23) are elevated and polymorphisms in the IL-23 receptor are associated with ankyosing spondylitis, however, it remains unclear whether IL-23 acts locally at the enthesis or distally on circulating cell populations. We show here that IL-23 is essential in enthesitis and acts on previously unidentified IL-23 receptor (IL-23R)+, RAR-related orphan receptor γt (ROR-γt)+CD3+CD4−CD8−, stem cell antigen 1 (Sca1)+ entheseal resident T cells. These cells allow entheses to respond to IL-23 in vitro—in the absence of further cellular recruitment—and to elaborate inflammatory mediators including IL-6, IL-17, IL-22 and chemokine (C-X-C motif) ligand 1 (CXCL1). Notably, the in vivo expression of IL-23 is sufficient to phenocopy the human disease, with the specific and characteristic development of enthesitis and entheseal new bone formation in the initial complete absence of synovitis. As in the human condition, inflammation also develops in vivo at the aortic root and valve, which are structurally similar to entheses. The presence of these entheseal resident cells and their production of IL-22, which activates signal transducer and activator of transcription 3 (STAT3)-dependent osteoblast-mediated bone remodeling, explains why dysregulation of IL-23 results in inflammation at this precise anatomical site.

MyD88-dependent expansion of an immature GR-1+CD11b+ population induces T cell suppression and Th2 polarization in sepsis

Polymicrobial sepsis alters the adaptive immune response and induces T cell suppression and Th2 immune polarization. We identify a GR-1+CD11b+ population whose numbers dramatically increase and remain elevated in the spleen, lymph nodes, and bone marrow during polymicrobial sepsis. Phenotypically, these cells are heterogeneous, immature, predominantly myeloid progenitors that express interleukin 10 and several other cytokines and chemokines. Splenic GR-1+ cells effectively suppress antigen-specific CD8+ T cell interferon (IFN) γ production but only modestly suppress antigen-specific and nonspecific CD4+ T cell proliferation. GR-1+ cell depletion in vivo prevents both the sepsis-induced augmentation of Th2 cell–dependent and depression of Th1 cell–dependent antibody production. Signaling through MyD88, but not Toll-like receptor 4, TIR domain–containing adaptor-inducing IFN-β, or the IFN-α/β receptor, is required for complete GR-1+CD11b+ expansion. GR-1+CD11b+ cells contribute to sepsis-induced T cell suppression and preferential Th2 polarization.

A paradoxical role for myeloid-derived suppressor cells in sepsis and trauma.

Myeloid-derived suppressor cells (MDSCs) are a heterogenous population of immature myeloid cells whose numbers dramatically increase in chronic and acute inflammatory diseases, including cancer, autoimmune disease, trauma, burns and sepsis. Studied originally in cancer, these cells are potently immunosuppressive, particularly in their ability to suppress antigen-specific CD8+ and CD4+ T-cell activation through multiple mechanisms, including depletion of extracellular arginine, nitrosylation of regulatory proteins, and secretion of interleukin 10, prostaglandins and other immunosuppressive mediators. However, additional properties of these cells, including increased reactive oxygen species and inflammatory cytokine production, as well as their universal expansion in nearly all inflammatory conditions, suggest that MDSCs may be more of a normal component of the inflammatory response (“emergency myelopoiesis”) than simply a pathological response to a growing tumor. Recent evocative data even suggest that the expansion of MDSCs in acute inflammatory processes, such as burns and sepsis, plays a beneficial role in the host by increasing immune surveillance and innate immune responses. Although clinical efforts are currently underway to suppress MDSC numbers and function in cancer to improve antineoplastic responses, such approaches may not be desirable or beneficial in other clinical conditions in which immune surveillance and antimicrobial activities are required.

Central Nervous System Expression of IL-10 Inhibits Autoimmune Encephalomyelitis

Multiple sclerosis, an inflammatory, demyelinating disease of the CNS currently lacks an effective therapy. We show here that CNS inflammation and clinical disease in experimental autoimmune encephalomyelitis, an experimental model of multiple sclerosis, could be prevented completely by a replication-defective adenovirus vector expressing the anti-inflammatory cytokine IL-10 (replication-deficient adenovirus expressing human IL-10), but only upon inoculation into the CNS where local infection and high IL-10 levels were achieved. High circulating levels of IL-10 produced by i.v. infection with replication-deficient adenovirus expressing human IL-10 was ineffective, although the immunological pathways for disease are initiated in the periphery in this disease model. In addition to this protective activity, intracranial injection of replication-deficient adenovirus expressing human IL-10 to mice with active disease blocked progression and accelerated disease remission. In a relapsing-remitting disease model, IL-10 gene transfer during remission prevented subsequent relapses. These data help explain the varying outcomes previously reported for systemic delivery of IL-10 in experimental autoimmune encephalomyelitis and show that, for optimum therapeutic activity, IL-10 must either access the CNS from the peripheral circulation or be delivered directly to it by strategies including the gene transfer described here.

Interleukin-17A upregulates receptor activator of NF-κB on osteoclast precursors

IntroductionThe interaction between the immune and skeletal systems is evidenced by the bone loss observed in autoimmune diseases such as rheumatoid arthritis. In this paper we describe a new mechanism by which the immune cytokine IL-17A directly affects osteoclastogenesis.MethodsHuman CD14+ cells were isolated from healthy donors, cultured on dentine slices and coverslips and stimulated with IL-17A and/or receptor activator of NF-κB ligand (RANKL). Osteoclast differentiation was evaluated by gene expression, flow cytometry, tartrate-resistant acid phosphatase staining, fluorescence and electron microscopy. Physiologic bone remodelling was studied in wild-type (Wt) and IL-17A-/- mice using micro-computer tomography and serum RANKL/osteoprotegerin concentration. Functional osteoclastogenesis assays were performed using bone marrow macrophages isolated from IL-17A-/- and Wt mice.ResultsIL-17A upregulates the receptor activator for NF-κB receptor on human osteoclast precursors in vitro, leading to increased sensitivity to RANKL signalling, osteoclast differentiation and bone loss. IL-17A-/- mice have physiological bone homeostasis indistinguishable from Wt mice, and bone marrow macrophages isolated from these mice develop fully functional normal osteoclasts.ConclusionsCollectively our data demonstrate anti-IL-17A treatment as a selective therapeutic target for bone loss associated with autoimmune diseases.

Targeted adenovirus-induced expression of IL-10 decreases thymic apoptosis and improves survival in murine sepsis

Sepsis remains a significant clinical conundrum, and recent clinical trials with anticytokine therapies have produced disappointing results. Animal studies have suggested that increased lymphocyte apoptosis may contribute to sepsis-induced mortality. We report here that inhibition of thymocyte apoptosis by targeted adenovirus-induced thymic expression of human IL-10 reduced blood bacteremia and prevented mortality in sepsis. In contrast, systemic administration of an adenovirus expressing IL-10 was without any protective effect. Improvements in survival were associated with increases in Bcl-2 expression and reductions in caspase-3 activity and thymocyte apoptosis. These studies demonstrate that thymic apoptosis plays a critical role in the pathogenesis of sepsis and identifies a gene therapy approach for its therapeutic intervention.

IL-10 Directly Activates and Expands Tumor-Resident CD8+ T Cells without De Novo Infiltration from Secondary Lymphoid Organs

The presence of activated intratumoral T cells correlates clinically with better prognosis in patients with cancer. Although tumor vaccines can increase the number of tumor-specific CD8(+) T cells in systemic circulation, they frequently fail to increase the number of active and tumor reactive T cells within the tumor. Here we show that treatment with the pleiotropic cytokine interleukin-10 (IL-10) induces specific activation of tumor-resident CD8(+) T cells as well as their intratumoral expansion in several mouse tumor models. We found that inhibition of T-cell trafficking from lymphoid organs did not impair IL-10-induced tumor rejection or the activation of tumor-resident CD8(+) T cells. Tumor-resident CD8(+) T cells expressed elevated levels of the IL-10 receptor and were directly activated by IL-10, resulting in prominent phosphorylation of STAT3 and STAT1. Although CD4(+) T cells, regulatory T cells, NK cells, and dendritic cells have been reported as prominent targets of IL-10 in the tumor microenvironment, we found that expression of the IL-10R was required only on CD8(+) T cells to facilitate IL-10-induced tumor rejection as well as in situ expansion and proliferation of tumor-resident CD8 T cells. Together, our findings indicate that IL-10 activates CD8(+) T-cell-mediated tumor control and suggest that IL-10 may represent a potential tumor immunotherapy in human patients with cancer.

IL-23 Is Critical for Induction of Arthritis, Osteoclast Formation, and Maintenance of Bone Mass

The role of IL-23 in the development of arthritis and bone metabolism was studied using systemic IL-23 exposure in adult mice via hydrodynamic delivery of IL-23 minicircle DNA in vivo and in mice genetically deficient in IL-23. Systemic IL-23 exposure induced chronic arthritis, severe bone loss, and myelopoiesis in the bone marrow and spleen, which resulted in increased osteoclast differentiation and systemic bone loss. The effect of IL-23 was partly dependent on CD4+ T cells, IL-17A, and TNF, but could not be reproduced by overexpression of IL-17A in vivo. A key role in the IL-23–induced arthritis was made by the expansion and activity of myeloid cells. Bone marrow macrophages derived from IL-23p19−/− mice showed a slower maturation into osteoclasts with reduced tartrate-resistant acid phosphatase-positive cells and dentine resorption capacity in in vitro osteoclastogenesis assays. This correlated with fewer multinucleated osteoclast-like cells and more trabecular bone volume and number in 26-wk-old male IL-23p19−/− mice compared with control animals. Collectively, our data suggest that systemic IL-23 exposure induces the expansion of a myeloid lineage osteoclast precursor, and targeting IL-23 pathway may combat inflammation-driven bone destruction as observed in rheumatoid arthritis and other autoimmune arthritides.

Neutrophil Mobilization from the Bone Marrow during Polymicrobial Sepsis Is Dependent on CXCL12 Signaling

Neutrophils are essential for successful host eradication of bacterial pathogens and for survival to polymicrobial sepsis. During inflammation, the bone marrow provides a large reserve of neutrophils that are released into the peripheral circulation where they traverse to sites of infection. Although neutrophils are essential for survival, few studies have investigated the mechanisms responsible for neutrophil mobilization from the bone marrow during polymicrobial sepsis. Using a cecal ligation and puncture model of polymicrobial sepsis, we demonstrated that neutrophil mobilization from the bone marrow is not dependent on TLR4, MyD88, TRIF, IFNARα/β, or CXCR2 pathway signaling during sepsis. In contrast, we observed that bone marrow CXCL12 mRNA abundance and specific CXCL12 levels are sharply reduced, whereas splenic CXCR4 mRNA and cell surface expression are increased during sepsis. Blocking CXCL12 activity significantly reduced blood neutrophilia by inhibiting bone marrow release of granulocytes during sepsis. However, CXCL12 inhibition had no impact on the expansion of bone marrow neutrophil precursors and hematopoietic progenitors. Bone marrow neutrophil retention by CXCL12 blockade prevented blood neutrophilia, inhibited peritoneal neutrophil accumulation, allowed significant peritoneal bacterial invasion, and increased polymicrobial sepsis mortality. We concluded that changes in the pattern of CXCL12 signaling during sepsis are essential for neutrophil bone marrow mobilization and host survival but have little impact on bone marrow granulopoiesis.

Increased Survival in Sepsis by In Vivo Adenovirus-Induced Expression of IL-10 in Dendritic Cells

The dendritic cell (DC) is the most potent APC of the immune system, capable of stimulating naive T cells to proliferate and differentiate into effector T cells. Recombinant adenovirus (Adv) readily transduces DCs in vitro allowing directed delivery of transgenes that modify DC function and immune responses. In this study we demonstrate that footpad injection of a recombinant Adv readily targets transduction of myeloid and lymphoid DCs in the draining popliteal lymph node, but not in other lymphoid organs. Popliteal DCs transduced with an empty recombinant Adv undergo maturation, as determined by high MHC class II and CD86 expression. However, transduction with vectors expressing human IL-10 limit DC maturation and associated T cell activation in the draining lymph node. The extent of IL-10 expression is dose dependent; transduction with low particle numbers (105) yields only local expression, while transduction with higher particle numbers (107 and 1010) leads additionally to IL-10 appearance in the circulation. Furthermore, local DC expression of human IL-10 following in vivo transduction with low particle numbers (105) significantly improves survival following cecal ligation and puncture, suggesting that compartmental modulation of DC function profoundly alters the sepsis-induced immune response.