Differentiation of Type 17 Mucosal-Associated Invariant T Cells in Circulation Contributes to the Severity of Sepsis.

Mucosal-associated invariant T (MAIT) cells are essential in defending against infection. Sepsis is a systemic inflammatory response to infection and a leading cause of death. The relationship between the overall competency of the host immune response and disease severity is not fully elucidated. This study identified a higher proportion of circulating MAIT17 with expression of IL-17A and retinoic acid receptor-related orphan receptor γt in patients with sepsis. The proportion of MAIT17 was correlated with the severity of sepsis. Single-cell RNA-sequencing analysis revealed an enhanced expression of lactate dehydrogenase A (LDHA) in MAIT17 in patients with sepsis. Cell-culture experiments demonstrated that phosphoinositide 3-kinase-LDHA signaling was required for retinoic acid receptor-related orphan receptor γt expression in MAIT17. Finally, the elevated levels of plasma IL-18 promoted the differentiation of circulating MAIT17 cells in sepsis. In summary, this study reveals a new role of circulating MAIT17 in promoting sepsis severity and suggests the phosphoinositide 3-kinase-LDHA signaling as a driving force in MAIT17 responses.

TNF deficiency dysregulates inflammatory cytokine production, leading to lung pathology and death during respiratory poxvirus infection.

Excessive tumor necrosis factor (TNF) is known to cause significant pathology. Paradoxically, deficiency in TNF (TNF-/-) also caused substantial pathology during respiratory ectromelia virus (ECTV) infection, a surrogate model for smallpox. TNF-/- mice succumbed to fulminant disease whereas wild-type mice, and those engineered to express only transmembrane TNF (mTNF), fully recovered. TNF deficiency did not affect viral load or leukocyte recruitment but caused severe lung pathology and excessive production of the cytokines interleukin (IL)-6, IL-10, transforming growth factor beta (TGF-ß), and interferon gamma (IFN-γ). Short-term blockade of these cytokines significantly reduced lung pathology in TNF-/- mice concomitant with induction of protein inhibitor of activated STAT3 (PIAS3) and/or suppressor of cytokine signaling 3 (SOCS3), factors that inhibit STAT3 activation. Consequently, inhibition of STAT3 activation with an inhibitor reduced lung pathology. Long-term neutralization of IL-6 or TGF-ß protected TNF-/- mice from an otherwise lethal infection. Thus, mTNF alone is necessary and sufficient to regulate lung inflammation but it has no direct antiviral activity against ECTV. The data indicate that targeting specific cytokines or cytokine-signaling pathways to reduce or ameliorate lung inflammation during respiratory viral infections is possible but that the timing and duration of the interventive measure are critical.

Alterations of subset and cytokine profile of peripheral T helper cells in PBMCs from Multiple Sclerosis patients or from individuals with MS risk SNPs near genes CYP27B1 and CYP24A1.

T helper cells play an important role in the aetiology of Multiple Sclerosis (MS). Vitamin D has an anti-inflammatory effect on T helper cells and can affect onset and pathogenesis of MS. Two genes of the metabolic Vitamin D pathway expressed by activated T helper (Th) cells have been identified as MS risk genes by genome-wide association studies, CYP27B1 (25(OH)D3 1-alpha-hydroxylase) and CYP24A1 (1,25(OH)2D3 24-alpha-hydroxylase). Therefore, we hypothesize that the MS risk alleles around gene CYP27B1 and CYP24A1 are associated with the altered inflammatory profile of peripheral Th cells in PBMCs both ex vivo and in vitro potentially influencing the pathogenesis of MS. PBMCs from MS patients (41 RRMS patients in their remitting stage and 4 SPMS patients) and 12 healthy controls were collected, subpopulation of Th cells in PBMCs and cytokine profile were tested by Flow cytometry and Cytometric Bead Array (CBA), respectively. MS risk SNPs were genotyped by allele-specific PCR analysis. Data were analysed using nonparametric tests and linear regression for adjusting multiple factors. The proportion of Th17.1, Th17 and Th1 cells were all associated with MS while the proportions of Th2 (significant) and Th17 (near significant) cells were correlated with the expanded disability scale score of MS patients. Additionally, we found a MS-specific dysregulation in the IL-6 and TNF production of Th cells in Concanavalin A-stimulated PBMCs. Furthermore, the risk allele rs2248359-C (near gene CYP24A1) showed a consistent inhibitory effect on the proportions of Th1 and Th17.1 cells, and the presence of the homozygous risk allele rs703842-AA (near gene CYP27B1) reduced the production of IL-2. In conclusion, both MS disease and its risk alleles near Vitamin D metabolism genes influence the inflammatory profile of T helper cells in PBMCs.

Roquin-2 shares functions with its paralog Roquin-1 in the repression of mRNAs controlling T follicular helper cells and systemic inflammation.

Accumulation of T follicular helper (Tfh) cells and proinflammatory cytokines drive autoantibody-mediated diseases. The RNA-binding protein Roquin-1 (Rc3h1) represses the inducible costimulator ICOS and interferon-γ (IFN-γ) in T cells to prevent Tfh cell accumulation. Unlike Rc3h1(san) mice with a mutation in the ROQ domain of Roquin-1, mice lacking the protein, paradoxically do not display increased Tfh cells. Here we have analyzed mice with mutations that eliminate the RING domain from Roquin-1 or its paralog, Roquin-2 (Rc3h2). RING or ROQ mutations both disrupted Icos mRNA regulation by Roquin-1, but, unlike the ROQ mutant that still occupied mRNA-regulating stress granules, RING-deficient Roquin-1 failed to localize to stress granules and allowed Roquin-2 to compensate in the repression of ICOS and Tfh cells. These paralogs also targeted tumor necrosis factor (TNF) in nonlymphoid cells, ameliorating autoantibody-induced arthritis. The Roquin family emerges as a posttranscriptional brake in the adaptive and innate phases of antibody responses.

Absence of TNF Leads to Alternative Activation in Peritoneal Macrophages in Experimental Listeria Monocytogenes Infection.

Macrophages are crucial effectors of innate immunity against the pathogenic bacterium Listeria monocytogenes. The pro-inflammatory cytokine tumour necrosis factor-α (TNF) has been shown to be crucial for resistance to L. monocytogenes and mice deficient in TNF signalling succumb quickly after infection. However, the mechanisms underlying TNF-mediated defence against L. monocytogenes infection have not been completely elucidated. Here, we demonstrate that TNF concurrently functions to support a pro-inflammatory M1 phenotype while actively blocking macrophage polarization to the M2 phenotype. Compared to WT mice, peritoneal macrophages in TNF-deficient mice inoculated with L. monocytogenes respond with M2 polarization by upregulating Arg1. Consistently, TNF blockade in vitro resulted in M2 polarization in peritoneal macrophages during L. monocytogenes infection. Additionally, TNF promotes the transition from M2 to M1 polarization in peritoneal macrophages. Further investigation of peritoneal macrophage polarization suggested the NF-κB pathway is involved in the TNF-dependent M2 to M1 shift. Conversely, treatment of peritoneal macrophage with a PPARγ agonist blunted the expression of M1 genes induced by TNF and reduced NF-κB signalling pathway activation. Competing signalling mechanisms therefore play an essential role in the ability of peritoneal macrophage to resolve L. monocytogenes infections with TNF playing an essential role in driving M1 polarization.Abbreviations: LPM: large peritoneal macrophage; SPM: small peritoneal macrophage; LLO: listeriolysin O; iNOS: inducible nitric oxide synthase; DCs: dendritic cells.

Angiotensin II Type 2 Receptor Modulates Synovial Macrophage Polarization by Inhibiting GRK2 Membrane Translocation in a Rat Model of Collagen-Induced Arthritis.

The chronic inflammatory autoimmune disease rheumatoid arthritis (RA) is characterized by an infiltration of activated proinflammatory immune cells into the joint that is accompanied by an overproduction of various mediators, leading to destruction of cartilage and bone erosion. Angiotensin II type 2 receptor (AT2R) is involved in antioxidative, anti-inflammatory, and antifibrotic responses. Synovial macrophages (SMs) are a type of tissue macrophages that are derived from bone marrow cells. SMs plays a central role in synovial regional immunization, which is significantly increased in both collagen-induced mice with arthritis mice and RA patients. AT2R activation caused a reversal of the polarization of SMs in the joint from the proinflammatory M1 SM to the tolerogenic, benign M2 SM. In consequence, this switch resulted in an attenuated form of the joint pathology in a rat model of collagen-induced arthritis. These results were mechanistically linked to the observation that GRK2 was translocated into cytoplasm, and ERK1/2 and NF-κB activation were inhibited. These findings open the way to a new therapeutic approach using an activation of AT2R to subvert joint inflammation in RA.

CP-25 alleviates antigen-induced experimental Sjögren's syndrome in mice by inhibiting JAK1-STAT1/2-CXCL13 signaling and interfering with B-cell migration.

The etiology of primary Sjögren's syndrome (pSS) remains unknown, and there is no complete curative drug. In this study, we treated a mouse model of the submandibular gland (SG) protein-immunized experimental Sjögren's syndrome (ESS) with paeoniflorin-6'-O-benzene sulfonate (termed CP-25) to evaluate the potential therapeutic effects of CP-25. Through in vivo experiments, we found that CP-25 increased saliva flow, alleviated the salivary gland indexes, and improved tissue integrity in the ESS model. The viability of splenocytes and B-lymphocyte migration from spleen were reduced in ESS mice. Furthermore, CP-25 decreased the expression of IgG antibodies, anti-SSA and anti-SSB antibodies and modulated the levels of cytokines in the serum of SS mice. The numbers of total B lymphocytes, plasma cells (PCs), and memory B cells diminished in the salivary gland. Increased expression of the JAK1-STAT1-CXCL13 axis and IFNα was found in human tissue isolated from pSS patients. In vitro, after stimulation with IFNα, the levels of CXCL13 mRNA and CXCL13 in human salivary gland epithelial cells (HSGEC) increased, while CP-25 counteracted the secretion of CXCL13 and downregulated the expression of CXCL13. IFN-α activated the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which was negatively regulated by additional CP-25. As a consequence, B-cell migration was downregulated in coculture with IFN-α-stimulated HSGEC. Taken together, this study demonstrated that the therapeutic effects of CP-25 were associated with the inhibition of the JAK1-STAT1/2-CXCL13 signaling pathway in HSGEC, which impedes the migration of B cells into the salivary gland. We identified the underlying mechanisms of the therapeutic effect of CP-25 and provided an experimental foundation for CP-25 as a potential drug in the treatment of the human autoimmune disorder pSS.

CC chemokine receptor 6 (CCR6) in the pathogenesis of systemic lupus erythematosus.

The CC chemokine receptor 6 (CCR6) and its sole chemokine ligand, CCL20, are an intriguing pair that have been implicated in a growing number of inflammatory, autoimmune and malignant disease processes. Recent observations have also highlighted this chemokine axis in the regulation of humoral immune responses. Through this review article, we explore the emerging links of CCR6-CCL20 with an archetypal autoimmune disease of humoral dysregulation: systemic lupus erythematosus (SLE). CCR6 is expressed prominently on several immune cells involved in the pathogenesis of SLE, such as dendritic cells and T-helper 17 cells. CCR6's expression is correlated with disease activity and serological markers of disease severity, suggesting a possible role in disease pathogenesis. However, there are numerous holes in our understanding of the functions of CCR6 and CCL20, and future studies are required to determine if there are any diagnostic, prognostic or monitoring roles for these important molecules.

TNF May Negatively Regulate Phagocytosis of Devil Facial Tumour Disease Cells by Activated Macrophages.

Introduction: Macrophage phagocytosis of pathogens and tumour cells is an important early event in protection against infectious disease and cancer. As tumour necrosis factor α (TNF) is an important cytokine in macrophage activation, we investigated the involvement of TNF in macrophage phagocytosis of tumour cells. Methods: We used Devil Facial Tumour Disease (DFTD) cancer cells as the target tumour cells. The Tasmanian devil (Sarcophilus harrisii) population is threatened by the transmissible DFTD. Using DFTD cells provided the opportunity to determine if these cells can be phagocytosed and investigate requirement for TNF. As effector cells, bone marrow derived macrophages (BMDMs), generated from C57BL/6 wild type (B6.WT) and C57BL/6 TNF-/- (B6.TNF-/-) mice were used. Phagocytosis of DFTD cells was investigated by confocal microscopy and flow cytometry. Results: DFTD cells were consistently phagocytosed by B6.WT and B6.TNF-/- BMDMs with similar efficiency in vitro. Consequently the DFTD cells are not resistant to phagocytosis. Following activation by exposure to IFNγ and LPS or LPS alone, B6.TNF-/- BMDMs had higher phagocytic efficiency and lower nitric oxide (NO) production compared to wild-type controls. In addition, NO seems to be unlikely to be the involved in phagocytosis efficiency in IFNγ and LPS activated B6.TNF-/- macrophages and consequences thereof. Conclusion: Our results indicate that TNF is not required for IFNγ and LPS or LPS alone activation of macrophage phagocytosis. TNF may negatively regulate macrophage phagocytosis of tumour cells.

MicroRNA-31 Negatively Regulates Interleukin-34 Expression In Vitro.

Interleukin-34 (IL-34) is a recently discovered cytokine that promotes tissue macrophage maturation and differentiation. We previously found that 1α,25-Dihydroxyvitamin D3 up-regulated IL-34 expression in SH-SY5Y neural cells. However, whether microRNA regulates IL-34 expression is not completely clear. By using on-line TargetScan and MiRanda software, we found that there was only one conserved microRNA-31 (miR-31) binding site in the 3' untranslated region (3'UTR) of IL-34 mRNA. Intriguingly, using qPCR we demonstrated that miR-31 levels were negatively correlated to IL-34 mRNA levels in different cell lines. By examining the effect of miR-31 on IL-34 3' UTR reporter luciferase activity and on IL-34 mRNA and argonaute RISC catalytic component 2 (AGO2) binding, it was found that miR-31 bound directly to IL-34 3'UTR and regulated the post-transcriptional expression of IL-34 in MGC-803 cells. Moreover, a miR-31 mimic significantly reduced IL-34 expression levels while a miR-31 inhibitor up-regulated IL-34 expression in KYSE-45 and HT-29 cells. Taken together, these results show that miR-31 negatively regulates IL-34 expression by directly binding to the IL-34 3' UTR in vitro.

Voltage-gated sodium channel Nav1.5 promotes proliferation, migration and invasion of oral squamous cell carcinoma.

The protein voltage-gated sodium channel Nav1.5 is highly upregulated in various types of cancer and, in general, promotes cancer cell invasiveness and metastatic progression. A previous study found that Nav1.5 was highly expressed in poorly differentiated oral squamous cell carcinoma (OSCC). However, whether Nav1.5 enhances invasiveness and metastasis of OSCC are still unknown. In this study, we found that Nav1.5 was highly expressed in OSCC cell lines compared with normal oral keratinocyte HOK cell line by using western blot analysis. CCK-8 assay results revealed that downregulation of Nav1.5 expression by its specific siRNA reduced proliferation of OSCC HSC-3 cells. Moreover, transwell assay results showed Nav1.5 knockdown significantly inhibited migration and invasion of HSC-3 cells. Meanwhile, qRT-PCR and western blot analysis results showed that epidermal growth factor (EGF) induced Nav1.5 expression in a time- and dose-dependent manner. In addition, EGF promoted proliferation, migration and invasion of HSC-3 cells. Importantly, the Nav1.5 inhibitor tetrodotoxin significantly inhibited the proliferation of HSC-3 cells and impeded the migration and invasion of HSC-3 cells. Furthermore, it was found that siRNA-mediated knockdown of Nav1.5 also lessened the proliferation of HSC-3 cells and blocked the migration and invasion of HSC-3 cells. Taken together, these results indicate that Nav1.5 is involved in the progression of OSCC and Nav1.5 promotes the proliferation, migration and invasion of OSCC cells.

Losartan suppresses the inflammatory response in collagen-induced arthritis by inhibiting the MAPK and NF-κB pathways in B and T cells.

The angiotensin II type 1 receptor (AT1R) antagonist losartan has been confirmed to have a moderate anti-inflammatory effect in vitro and in vivo. However, how it affects immune cells in Rheumatoid Arthritis (RA) is still unknown. We found that in human synovial tissues, AT1R is significantly expressed on T cells and B cells. Treatment with losartan (15 mg/kg) alone and in combination with a low dose of methotrexate (MTX 0.25 mg/kg/3 days) significantly suppressed the progression of CIA. Secondary paw swelling, joint destruction and the presence of pro-inflammatory cytokines (TNF-α and IFN-γ) in the serum were alleviated after treatment. The therapeutic effects of losartan were based on reduced T-cell and B-cell activation, specifically by decreased cell vitality and pro-inflammatory cytokine production. In addition, losartan combined with a low dose of MTX achieved a similar therapeutic effect, while protecting liver and kidney from MTX damage. Mechanistically, losartan inhibits the production of pro-inflammatory mediators, reduces the phosphorylation of p38, ERK, and p65, p50 nuclear transposition in T cells and B cells. Phosphorylation of JNK is not affected by losartan in the CIA rat model. losartan can be used as an effective RA treatment, which exhibits anti-arthritic effects potentially through down-regulating the phosphorylation of p38, ERK and signaling through NF-κB. While achieving similar anti-rheumatic effects, a combination therapy of losartan with a low dose of MTX, can protect from liver and renal damage caused by giving a high dose of MTX.

NLRP3 inflammasome in colitis and colitis-associated colorectal cancer.

A low level of inflammation is an integral part of the balance between the immune system and the microbiota in the high antigen environment of the gastrointestinal tract and maintains homeostasis. A failure of this balance can lead to chronic intestinal inflammation and increase the chances to develop colorectal cancer significantly. The underlying mechanisms that link inflammation and carcinogenesis are not clear but the molecular platforms of the inflammasomes have been implicated. Inflammasomes are molecule complexes that are assembled in response to microbial components or cellular danger signals and facilitate the production of bioactive pro-inflammatory cytokines. One inflammasome in particular, NLRP3, has been analysed extensively in its contribution to colitis and has been shown to be associated with the development of colitis-associated colorectal cancer. This review will summarise the role of NLRP3 in intestinal inflammation, discuss some of the triggers of inflammation in the gastrointestinal tract such as diet and introduce some opportunities to use this inflammasome as therapeutic target for the treatment of colitis and colitis-associated colorectal cancer.

Roles of SAMHD1 in antiviral defense, autoimmunity and cancer.

The enzyme, sterile α motif and histidine-aspartic acid domain-containing protein 1 (SAMHD1) diminishes infection of human immunodeficiency virus type 1 (HIV-1) by hydrolyzing intracellular deoxynucleotide triphosphates (dNTPs) in myeloid cells and resting CD4+ T cells. This dNTP degradation reduces the dNTP concentration to a level insufficient for viral cDNA synthesis, thereby inhibiting retroviral replication. This antiviral enzymatic activity can be inhibited by viral protein X (Vpx). The HIV-2/SIV Vpx causes degradation of SAMHD1, thus interfering with the SAMHD1-mediated restriction of retroviral replication. Recently, SAMHD1 has been suggested to restrict HIV-1 infection by directly digesting genomic HIV-1 RNA through a still controversial RNase activity. Here, we summarize the current knowledge about structure, antiviral mechanisms, intracellular localization, interferon-regulated expression of SAMHD1. We also describe SAMHD1-deficient animal models and an antiviral drug on the basis of disrupting proteasomal degradation of SAMHD1. In addition, the possible roles of SAMHD1 in regulating innate immune sensing, Aicardi-Goutières syndrome and cancer are discussed in this review.

CCR6/CCL20 chemokine axis in human immunodeficiency virus immunity and pathogenesis.

Recent studies in human immunodeficiency virus (HIV) have garnered interest for the role of CC chemokine receptor 6 (CCR6) and its known ligands, CC chemokine ligand 20 (CCL20) and human ß-defensins, in viral entry, dissemination and antiviral immunity. Several studies have suggested that CCR6 may also act as a weak co-receptor of HIV entry, in addition to the canonical CXC chemokine receptor 4 (CXCR4) and CCR5. However, the pathogenic significance has yet to be demonstrated as the observations for preferential infection of CD4+CCR6+ over CD4+CCR6- T cells appear to be independent of CCR6 expression. This indicates means for preferential infection other than CCR6 co-receptor use. Attention has also turned to the inadvertent role of the CCR6/CCL20 axis in attracting key immune cells, including TH17 cells and dendritic cells, to sites of infection and propagating the virus to other sites of the body. This review article will summarize the latest evidence that the CCR6/CCL20 chemokine axis is playing an important role in HIV pathogenesis and immunity. Further work with in vivo studies is needed to establish the biological and, hence, therapeutic significance of these findings.

Early CCR6 expression on B cells modulates germinal centre kinetics and efficient antibody responses.

The CC-chemokine receptor 6 (CCR6) can be detected on naive and activated B cells. Counterintuitively, its absence accelerates the appearance of germinal centres (GCs) and increases the production of low-affinity antibodies. The detailed mechanism of CCR6 function during the humoral response has remained elusive, but previously we identified a distinct CCR6high B-cell population in vivo early after antigenic challenge. In this study, we defined this population specifically as early, activated pre-GC B cells. In accordance, we show that CCR6 is upregulated rapidly within hours on the protein or mRNA level after activation in vitro. In addition, only activated B cells migrated specifically towards CCL20, the specific ligand for CCR6. Lack of CCR6 increased the dark zone/light zone ratio of GC and led to decreased antigen-specific IgG1 and IgG2a antibody generation in a B-cell intrinsic manner in mixed bone marrow chimeras. In contrast, antigen-specific IgM responses were normal. Hence, CCR6 negatively regulates entry of activated, antigen-specific pre-GC B cells into the GC reaction.

CCX-CKR deficiency alters thymic stroma impairing thymocyte development and promoting autoimmunity.

The atypical chemokine receptor CCX-CKR regulates bioavailability of CCL19, CCL21, and CCL25, homeostatic chemokines that play crucial roles in thymic lymphopoiesis. Deletion of CCX-CKR results in accelerated experimental autoimmunity induced by immunization. Here we show that CCX-CKR deletion also increases incidence of a spontaneous Sjögren's syndrome-like pathology, characterized by lymphocytic infiltrates in salivary glands and liver of CCX-CKR(-/-) mice, suggestive of a defect in self-tolerance when CCX-CKR is deleted. This prompted detailed examination of the thymus in CCX-CKR(-/-) mice. Negatively selected mature SP cells were less abundant in CCX-CKR(-/-) thymi, yet expansion of both DP and immature SP cells was apparent. Deletion of CCX-CKR also profoundly reduced proportions of DN3 thymocyte precursors and caused DN2 cells to accumulate within the medulla. These effects are likely driven by alterations in thymic stroma as CCX-CKR(-/-) mice have fewer cTECs per thymocyte, and cTECs express the highest level of CCX-CKR in the thymus. A profound decrease in CCL25 within the thymic cortex was observed in CCX-CKR(-/-) thymi, likely accounting for their defects in thymocyte distribution and frequency. These findings identify a novel role for CCX-CKR in regulating cTEC biology, which promotes optimal thymocyte development and selection important for self-tolerant adaptive immunity.

The relationship between CCR6 and its binding partners: does the CCR6-CCL20 axis have to be extended?

Chemokines and their receptors are vital for the trafficking of immune cells. In an orchestrated fashion, up- and down-regulation of chemokines and their receptors contribute to both immune system homeostasis as well as inflammation. The CC chemokine, CCL20 and its cognate receptor, CCR6, are described as one of the few chemokine-receptor pairs that show exclusivity. In our review, we analyze observations which indicate that CCR6 does not have CCL20 as an exclusive ligand as once appreciated. For example, attempts to study the pair, utilizing mainly CCR6-deficient mice, are confounded by a family of non-chemokine ligands known as ß-defensins that can bind to CCR6 and potentially can activate the cell. Therefore, a review of the activities of other potential binding partners of CCR6 is essential for interpretation of the current literature on this matter and for an understanding of their involvement in basic immunology and pathology.

CCR6 and CCL20: emerging players in the pathogenesis of rheumatoid arthritis.

The therapeutic targeting of pro-inflammatory TNF with neutralising biological anti-TNF agents, often in combination with other disease-modifying anti-rheumatic drugs, such as the purine synthesis inhibitor methotrexate has been the first major break-through in the treatment of chronic inflammatory diseases in decades. There are however, side effects and disadvantages of these treatments, such as general immunosuppression as well as therapy resistance in a large proportion of patients. This evokes the wish for other, more specialised forms of treatments. The targeting of chemokines and their receptors to disrupt cell movement specifically has been seen as a promising avenue of therapy for a considerable time. We will discuss one particular chemokine and its receptor, the C-C chemokine ligand CCL20 and the C-C chemokine receptor CCR6, and summarise its genetic and biological role in rheumatoid arthritis (RA). CCR6 has been associated with RA in genome-wide association studies and has been shown to be an interesting candidate for a therapeutic approach, considering its and CCL20's expression patterns within the tissue as well as the immune system.

CCR6 supports migration and differentiation of a subset of DN1 early thymocyte progenitors but is not required for thymic nTreg development.

T-cell selection and development occurs as precursor cells journey through the thymus and interact with stromal cells residing in distinct microenvironments. Although the chemokines CCL19, CCL21, CCL25 and CXCL12 are known to have major roles in intrathymic migration of thymocytes and thymocyte precursors, the significance of other chemokines such as CCL20, which is also expressed in the thymus, is unknown. This is of particular interest given that the thymus is the location of development of the natural regulatory T-cell (nTreg) population and that the CCL20 receptor CCR6 has an important role in peripheral tolerance via control of Treg cell migration. However, whether the CCL20/CCR6 axis has a role in the formation or migration of nTregs in the thymus is unknown. In this study, we addressed this by analyzing expression of CCR6/CCL20 within the thymus and assessing their role in thymocyte development using Ccr6(-/-) mice. CCL20 is predominately expressed in the thymic medulla and CCR6 expression is restricted to nTregs and a subset of early thymocyte progenitor double-negative 1 (DN1) cells (CD4(-)CD8(-)CD25(-)CD44(+)CD117(+)). Ex vivo chemotaxis assays indicated that these two subsets were apparently the sole subsets of thymocytes responsive to CCL20. The data indicate that nTreg frequencies and localization are unperturbed by deletion of Ccr6. However, in Ccr6(-/-) thymi, reduced frequencies of DN2 and DN3 cells, the thymocyte progenitor subsets that follow the DN1 stage, were apparent. Together, these data indicate that CCR6 has a supplementary role in coordination of early thymocyte precursor migration events important for normal subsequent thymocyte precursor development, but is not required for normal nTreg development.