Although much is described about the molecules involved in neutrophil migration from circulation into tissues, less is known about the molecular mechanisms that regulate neutrophil entry into lymph nodes (LNs) draining a local inflammatory site. In this study, we investigated neutrophil migration toward LNs in a context of inflammation induced by immunization of BALB/c mice with OVA emulsified in CFA. We demonstrated that neutrophils can enter LNs of OVA/CFA-immunized mice not only via lymphatic vessels but also from blood, across high endothelial venules. By adoptive transfer experiments, we showed that this influx was dependent on an inflammatory-state condition and previous neutrophil stimulation with OVA/anti-OVA immune complexes. Importantly, we have demonstrated that, in the migratory pattern to LNs, neutrophils used L-selectin and P-selectin glycoprotein ligand-1, macrophage-1 Ag and LFA-1 integrins, and CXCR4 to get access across high endothelial venules, whereas macrophage-1 Ag, LFA-1, and CXCR4 were involved in their trafficking through afferent lymphatics. Strikingly, we found that stimulation with immune complexes significantly upregulated the expression of sphingosine-1-phosphate receptor 4 on neutrophils, and that treatment with the sphingosine-1-phosphate agonist FTY720 altered neutrophil LN-homing ability. These findings summarized in this article disclose the molecular pattern that controls neutrophil recruitment to LNs.

Interferon-c priming is involved in the activation of arginase by oligodeoxinucleotides containing CpG motifs in murine macrophages IntroductionIt is well known that unmethylated cytosine guanine motifs contained in bacterial DNA or synthetic oligonucleotides CpG-DNA are powerful immunostimulatory molecules. Initially, CpG-DNA were believed to be predominantly pro-inflammatory molecules, stimulating a T helper 1 (Th1)-like response dominated by the release of interleukin (IL)-12 and interferon-c (IFN-c).1 However, in recent years there has been a spate of interest in some anti-inflammatory properties of CpG-DNA. Pulmonary inflammation decreased in response to lipopolysaccharide (LPS) after systemic exposure to CpG-DNA. 2 CpG-DNA were found to stimulate the production of IL-10, [3][4][5] increase the expression of indoleamine 2,3-dioxygenase (IDO), an enzyme linked to the suppression of T-cellmediated immunity, 6,7 and enhance the shedding of the tumour necrosis factor-a (TNF-a) receptor in macro-

Dendritic cells (DCs) play critical functions in the initiation of immune responses. Understanding their role in reactive arthritis (ReA) will help delineate the pathogenesis of this arthropathy. In early studies, we detected IL-12/23p40 deregulation in Yersinia entercolitica (Ye)-induced ReA in TNFRp55-deficient (TNFRp55-/-) mice. In this study, we assessed the contribution of DCs in this overproduction. First, greater levels of IL-12/23p40, IFN-γand IL-17A were confirmed in supernatants of lipopolysaccharide (LPS)-stimulated TNFRp55-/-splenocytes obtained on arthritis onset (day 14 after Ye infection). Later, DCs were identified as a precise source of IL-12/23p40 since increased frequency of splenic IL-12/23p40+DCs was detected in TNFRp55-/- mice. After robust in vivo amplification of DCs by injection of Fms-like tyrosine kinase 3-Ligand (Flt3L)-transfected BL16 melanoma, DCs were purified. These cells recapitulated the higher production of IL-12/23p40 under TNFRp55deficiency. In agreement with these results, TNFRp55-/- DCs promoted Th1 and Th17 programs by co-culture with WT CD4+lymphocytes. A mechanistic study demonstrated that JNK and p38 MAPK pathways are involved in IL-12/23p40 overproduction in purified TNFRp55-/- DCs as well as in the JAWS II cell line. This deregulation was once again attributed to TNFRp55 deficiency since CAY10500, a specific inhibitor of this pathway, compromised TNF-mediated IL-12/23p40 control in LPS-stimulated WT DCs. Simultaneously, this inhibition reduced IL-10 production, suggesting its role mediating IL-12/23p40 regulation by TNFRp55 pathway. These results provide experimental data on the existence of a TNFRp55-mediated anti-inflammatory circuit in DCs. Moreover, these cells may be considered as a novel target in the treatment of ReA.

Rheumatoid arthritis (RA) is characterized by the presence of anti-citrullinated peptide antibodies (ACPAs) and neutrophils infiltrating the synovial fluid (SF) of the affected joints. The aim of this work was to analyze whether the presence of ACPAs in SF is associated with neutrophil infiltration and with their phenotype in the inflamed joints of RA patients. We found that in the presence of ACPAs, the number of synovial neutrophils correlated with severe disease activity. The SF were divided according to synovial ACPA levels in negative- (<25 U/mL), low- (25-200 U/mL) and high level (˃200 U/mL; ACPA ). We observed that IL-6, IL-17, and IL-8 were significantly elevated in ACPA SF and that IL-8 levels correlated positively with neutrophil counts and with worse clinical manifestations. Additionally, in vitro incubation of neutrophils with ACPA SF resulted in an increased ROS production and extracellular DNA release compared to neutrophils incubated with ACPA-negative SF. These exacerbated effector functions were associated with a fraction of ICAM-1-positive neutrophils, which were induced by ACPA SF. Likewise, in in vivo, we could also detect this subset among neutrophils present in ACPA SF. In conclusion, the data presented here shed light on the role of SF-ACPAs as inductors of a proinflammatory profile in neutrophils.

The ability of synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) to induce both stimulatory and counter-regulatory responses offers novel opportunities for using these molecules as immunomodulatory agents in different therapeutic strategies. Here, we investigated the potential of CpG-ODN to activate the arginase (ARG) enzyme in vivo and focused on the consequences of this activation in T-cell proliferation. Challenging mice subcutaneously with CpG-ODN emulsified in incomplete Freund's adjuvant (IFA) induced ARG and reduced T-cell proliferation associated with CD3f chain downregulation. Interestingly, impaired T-cell expansion correlated with elevated levels of CD11b + Gr1 + myeloid cells localized near T-cell areas in the spleen. In addition, purified CD11b + cells obtained from the spleen of CpG-ODN+IFA-treated mice exhibited increased ARG activity and ARG I expression along with an augmented [ 3 H]-L-arginine uptake. CD11b + myeloid cells significantly suppressed T-cell proliferation and CD3f chain expression induced by a polyclonal stimulus. Furthermore, these effects could be recovered by the addition of excess L-arginine or by treatment of CD11b + cells with a specific ARG inhibitor. This study provides a novel evidence that CpG-ODN+IFA are able to induce splenic CD11b + cells with ARG activity, with this population being responsible for the impaired T-cell proliferation observed after the treatment with CpG-ODN+IFA. These results underscore a key role of CpG-ODN on ARG activity in vivo and add support to the growing body of evidence in favor of a counter-regulatory role for CpG-ODN in an immune response.

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Objectives Serum autoantibodies are pivotal for the early detection of systemic autoimmune rheumatic diseases such as Systemic Sclerosis (SSc) and Poly/Dermatomyositis (PM/DM), and in some cases are associated with organ complications such as interstitial lung disease (ILD). A paradigmatic example is provided by the autoantibody against the Eukaryotic Initiation Factor 2B (eIF2B) that has been recently detected in SSc. Methods Sera from 118 patients with SSc, 8 Poly/Dermatomyositis, 2 overlap SSc/Polymyositis, 4 undifferentiated connective tissue disease-UCTD and 3 healthy controls were tested first by indirect immunofluorescence for anti-nuclear antibodies-ANA pattern. Further, we employed prot ein-radioimmunoprecipitation (IP) and IP- Western Blot for the detection and confirmation of anti-eIF2B antibodies. Serum findings were further correlated with the clinical features of patients. Results We identified 3 SSc cases (2.5%) positive for anti-eIF2B antibodies while this autoantibody was not detected in control sera. Using protein-IP all three patients manifested the 38kD protein which is the antigenic target of anti-eIF2B antibodies, and this was associated with a cytoplasmic pattern at indirect immunofluorescence. The presence of anti-eIF2B was associated with ILD and a diffuse SSc variant, in one case in association with anti-Scl70/topoI. Conclusions Our data confirm that a small subgroup (2.5%) of patients with SSc have detectable anti-eIF2B with cytoplasmic-positive staining at immunofluorescence and this reactivity is associated with ILD.

As we age, the homeostatic function of many systems in the body, such as the immune function declines, which in turn contributes to augment susceptibility to disease. Here we describe that challenging aged mice with synthetic oligodeoxynucleotides containing unmethylated cytosine guanine motifs (CpG-ODN) emulsified in incomplete Freund's adjuvant (IFA), (CpG-ODN+IFA) an inflammatory stimulus, led to the expansion of CD11b+Gr1+ myeloid cells with augmented expression of CD124 and CD31. These myeloid cells lasted longer in the spleen of aged mice than in their younger counterparts after CpG-ODN+IFA treatment and were capable of suppressing T cell proliferative response by arginase induction. Myeloid cells from aged CpG-ODN+IFA-treated mice presented increased arginase-1 expression and enzyme activity. In addition, we found a different requirement of cytokines for arginase induction according to mice age. In myeloid cells from young treated mice, arginase-1 expression and activity is induced by the presence of each IL-4 or IL-6 in their extracellular medium, unlike myeloid cells from aged treated mice which need the presence of both IL-4 and IL-6 together for arginase induction and suppressor function.