Dendritic cells (DCs) have been implicated in the pathogenesis of psoriasis but the roles for specific DC subsets are not well defined. Here we show that DCs are required for psoriasis-like changes ...in mouse skin induced by the local injection of IL-23. However, Flt3L-dependent DCs and resident Langerhans cells are dispensable for the inflammation. In epidermis and dermis, the critical DCs are TNF-producing and IL-1β-producing monocyte-derived DCs, including a population of inflammatory Langerhans cells. Depleting Ly6C
blood monocytes reduces DC accumulation and the skin changes induced either by injecting IL-23 or by application of the TLR7 agonist imiquimod. Moreover, we find that IL-23-induced inflammation requires expression of CCR6 by DCs or their precursors, and that CCR6 mediates monocyte trafficking into inflamed skin. Collectively, our results imply that monocyte-derived cells are critical contributors to psoriasis through production of inflammatory cytokines that augment the activation of skin T cells.
Psoriasis is a common immune-mediated chronic inflammatory skin disorder, but the mechanisms of pathogenesis are still poorly understood. IL-23 is expressed in psoriatic skin, and IL-23 injection ...produces IL-22-dependent psoriasiform changes in mouse skin. Th17 cells produce IL-22 and display CCR6, the CCL20 receptor; CCR6+ T cells and CCL20 are abundant in psoriatic skin. We investigated a possible role for CCR6 in recruiting Th17 cells and producing psoriasiform pathology by injecting IL-23 into the skin of WT and Ccr6-/- mice. Unlike for WT mice, IL-23-injected ears of Ccr6-/- mice showed neither substantial epidermal/dermal changes nor increased Il22 mRNA expression. However, injection of IL-22 yielded equivalent psoriasiform changes in WT and Ccr6-/- mice. Surprisingly, IL-23-injected ears of WT and Ccr6-/- mice contained similar numbers of Th cells able to make IL-17A and/or IL-22. Furthermore, in ears of Rag1-/- mice, IL-23 initially induced skin changes and levels of Il22 mRNA that were indistinguishable from WT mice, revealing at least one non-T cell source for IL-22. We conclude that CCR6 is essential in a model of IL-23-induced, IL-22-mediated dermatitis, which develops in sequential T cell-independent and T cell-dependent phases. These findings reveal an expanded role for CCR6 in IL-23-related responses and identify CCR6 as a potential therapeutic target in psoriasis.
Some pathways of T cell differentiation are associated with characteristic patterns of chemokine receptor expression. A new lineage of effector/memory CD4+ T cells has been identified whose signature ...products are IL-17 cytokines and whose differentiation requires the nuclear receptor, RORgammat. These Th17 cells are critical effectors in mouse models of autoimmune disease. We have analyzed the association between chemokine receptor expression and IL-17 production for human T cells. Activating cord blood (naive) CD4+ T cells under conditions driving Th17 differentiation led to preferential induction of CCR6, CCR9, and CXCR6. Despite these data, we found no strong correlation between the production of IL-17 and expression of CCR9 or CXCR6. By contrast, our analyses revealed that virtually all IL-17-producing CD4+ T cells, either made in our in vitro cultures or found in peripheral blood, expressed CCR6, a receptor found on approximately 50% of CD4+ memory PBL. Compared with CD4+CD45RO+CCR6- cells, CD4+CD45RO+CCR6+ cells contained at least 100-fold more IL-17A mRNA and secreted 100-fold more IL-17 protein. The CCR6+ cells showed a similar enrichment in mRNA for RORgammat. CCR6 was likewise expressed on all IL-17-producing CD8+ PBL. CCR6 has been associated with the trafficking of T, B, and dendritic cells to epithelial sites, but has not been linked to a specific T cell phenotype. Our data reveal a fundamental feature of IL-17-producing human T cells and a novel role for CCR6, suggesting both new directions for investigating IL-17-related immune responses and possible targets for preventing inflammatory injury.
The interaction of Borrelia burgdorferi, the causative agent of Lyme borreliosis, with phagocytic cells induces the activation of NF-κB and the expression of proinflammatory cytokines including tumor ...necrosis factor alpha (TNF-α). B. burgdorferi-induced TNF-α production is also dependent on the activation of p38 mitogen-activated protein (MAP) kinase. The specific contribution of these signaling pathways to the response of phagocytic cells to the spirochete and the molecular mechanisms underlying this response remain unresolved. We now show that p38 MAP kinase activity regulates the transcriptional activation of NF-κB in response to spirochetal lysate stimulation of phagocytic cells. The regulation occurs at the nuclear level and is independent of the translocation of the transcription factor to the nucleus or its capacity to bind to specific DNA target sequences. In RAW264.7 cells, p38α MAP kinase regulates the phosphorylation of NF-κB RelA. p38 MAP kinase phosphorylates the nuclear kinase mitogen- and stress-activated protein kinase 1 (MSK1). MSK1 in turn phosphorylates the transcriptionally active subunit of NF-κB, RelA. The repression of MSK1 expression with small interfering RNA results in reduced RelA phosphorylation and a significant decrease in the production of TNF-α in response to B. burgdorferi lysates. Overall, these results clarify the contribution of the signaling pathways that are activated in response to the interaction of spirochetes with phagocytic cells to TNF-α production. Our results situate p38 MAP kinase activity as a central regulator of the phagocytic proinflammatory response through MSK1-mediated transcriptional activation of the transcription factor NF-κB.
Chimeric Antigen Receptor (CAR) T cells are recognized as efficacious therapies with demonstrated ability to produce durable responses in blood cancer patients. Regulatory approvals and acceptance of ...these unique therapies by patients and reimbursement agencies have led to a significant increase in the number of next generation CAR T clinical trials. Flow cytometry is a powerful tool for comprehensive profiling of individual CAR T cells at multiple stages of clinical development, from product characterization during manufacturing to longitudinal evaluation of the infused product in patients. There are unique challenges with regard to the development and validation of flow cytometric methods for CAR T cells; moreover, the assay requirements for manufacturing and clinical monitoring differ. Based on the collective experience of the authors, this recommendation paper aims to review these challenges and present approaches to address them. The discussion focuses on describing key considerations for the design, optimization, validation and implementation of flow cytometric methods during the clinical development of CAR T cell therapies.
Importance of the field: Psoriasis is a common, chronic autoimmune disease of the skin. Despite a number of effective treatments, new therapies are needed with enhanced efficacy, safety and ...convenience. Chemokine receptors are GPCRs that control leukocyte trafficking, and like other GPCRs, are good potential drug targets. The chemokine receptor CCR6 is expressed on the TH17 subset of CD4+ T cells, which produces IL-17A/F, IL-22, TNF-α and other cytokines, and which has been implicated in the pathogenesis of psoriasis. CCR6 and its ligand, CCL20/MIP-3α, are highly expressed in psoriatic skin and CCR6 is necessary for the pathology induced in a mouse model of psoriasis-like inflammation.
Areas covered in this review: This review summarizes the evidence for the importance of the IL-23/TH17 axis, and in particular CCR6 and CCL20 in psoriasis, dating from 2000 to the present, and discusses the possibility of inhibiting CCR6 as a treatment for the disease.
What the reader will gain: The review informs the reader of the current thinking on the mechanisms of inflammation in psoriasis and the possible roles for CCR6 (and CCL20) in disease pathogenesis.
Take home message: We conclude that CCR6 should be investigated as a potential therapeutic target in psoriasis.
Borrelia burgdorferi, the causative agent of Lyme disease, shows a great ability to adapt to different environments, including the arthropod vector, and the mammalian host. The success of these ...microorganisms to survive in nature and complete their enzootic cycle depends on the regulation of genes that are essential to their survival in the different environments. This review describes the current knowledge of gene expression by
B. burgdorferi in the tick and the mammalian host. The functions of the differentially regulated gene products as well as the factors that influence their expression are discussed. A thorough understanding of the changes in gene expression and the function of the differentially expressed antigens during the life cycle of the spirochete will allow a better control of this prevalent infection and the design of new, second generation vaccines to prevent infection with the spirochete.
The interaction of macrophages with infectious agents leads to the activation of several signaling cascades, including mitogen-activated protein (MAP) kinases, such as p38. We now demonstrate that ...p38 MAP kinase-mediated responses are critical components to the immune response to Borrelia burgdorferi. The pharmacological and genetic inhibition of p38 MAP kinase activity during infection with the spirochete results in increased carditis. In transgenic mice that express a dominant negative form of p38 MAP kinase specifically in macrophages, production of the invariant natural killer T (iNKT) cell—attracting chemokine MCP-1 and of the antigen-presenting molecule CD1d are significantly reduced. The expression of the transgene therefore results in the deficient infiltration of iNKT cells, their decreased activation, and a diminished production of interferon γ (IFN-γ), leading to increased bacterial burdens and inflammation. These results show that p38 MAP kinase provides critical checkpoints for the protective immune response to the spirochete during infection of the heart.
Lymphocyte activation leads to changes in chemokine receptor expression. There are limited data, however, on how lymphocyte activators can alter chemokine signaling by affecting downstream pathways. ...We hypothesized that B cell-activating agents might alter chemokine responses by affecting downstream signal transducers, and that such effects might differ depending on the activator. We found that activating mouse B cells using either anti-IgM or lipopolysaccharide (LPS) increased the surface expression of CCR6 and CCR7 with large increases in chemotaxis to their cognate ligands. By contrast, while anti-IgM also led to enhanced calcium responses, LPS-treated cells showed only small changes in calcium signaling as compared with cells that were freshly isolated. Of particular interest, we found that LPS caused a reduction in the level of B-cell phospholipase C (PLC)-β2 mRNA and protein. Data obtained using PLC-β2(-/-) mice showed that the β2 isoform mediates close to one-half the chemokine-induced calcium signal in resting and anti-IgM-activated B cells, and we found that calcium signals in the LPS-treated cells were boosted by increasing the level of PLC-β2 using transfection, consistent with a functional effect of downregulating PLC-β2. Together, our results show activator-specific effects on responses through B-cell chemokine receptors that are mediated by quantitative changes in a downstream signal-transducing protein, revealing an activity for LPS as a downregulator of PLC-β2, and a novel mechanism for controlling chemokine-induced signals in lymphocytes.
Although regulation of CXCR3 and CCR4 is related to Th1 and Th2 differentiation, respectively, many CXCR3⁺ and CCR4⁺ cells do not express IFN‐γ and/or IL‐4, suggesting that the chemokine receptor ...genes might be inducible by mechanisms that are lineage‐independent. We investigated the regulation of CXCR3 versus IFNG, and CCR4 versus IL4 in human CD4⁺ T cells by analyzing modifications of histone H3. In naïve cord‐blood cells, under nonpolarizing conditions not inducing IL4, CCR4 was induced to high levels without many of the activation‐associated changes in promoter histone H3 found for both IL4 and CCR4 in Th2 cells. Importantly, CCR4 expression was stable in Th2 cells, but fell in nonpolarized cells after the cells were rested; this decline could be reversed by increasing histone acetylation using sodium butyrate. Patterns of histone H3 modifications in CXCR3⁺CCR4⁻ and CXCR3⁻CCR4⁺ CD4⁺ T‐cell subsets from adult blood matched those in cells cultured under polarizing conditions in vitro. Our data show that high‐level lineage‐independent induction of CCR4 can occur following T‐cell activation without accessibility‐associated changes in histone H3, but that without such changes expression is transient rather than persistent.
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