Osteoblasts (OBs) contribute to the maintenance of bone homeostasis and their activity can be influenced by immune cells localized in bone lacunae. We investigated the expression of the chemokine receptors in isolated human OBs by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry, and report a novel finding, namely, that OBs express high levels of CXC chemokine receptor 3 (CXCR3) and 5 (CXCR5). Functional assays to evaluate CXCR3 and CXCR5 demonstrated that their ligands-CXCL10 and CXCL13, respectively-significantly induce the release of beta-N-acetylhexosaminidase, an enzyme involved in endochondral ossification and bone remodeling able to degrade important extracellular matrix components. Alkaline phosphatase activity, a useful index of matrix formation was also up-regulated by CXCL10 and CXCL13. However, OB activation by these ligands does not affect OB proliferation. Both Bordetella pertussis toxin and neutralizing anti-CXCR3/anti-CXCR5 monoclonal antibodies block CXCL10 and CXCL13 induction, respectively. We also demonstrated the expression of CXCL10 and CXCL13 in human bone tissue biopsies. These results indicate that both CXCR3/CXCL10 and CXCR5/CXCL13 receptor-ligand pairs may play an important role in OB activity through the specific up-regulation of two enzymes, which are involved in the bone remodeling process. Moreover, our data suggest that OBs may play a role in the modulation of bone formation through the combined action of these two enzymes.

BACKGROUND

The role of different subtypes of immune cells is still a matter of debate.

METHODS

We compared the prognostic relevance for metastasis-free survival (MFS) of a B-cell signature (BS), a T-cell signature (TS), and an immune checkpoint signature (CPS) in node-negative breast cancer (BC) using mRNA expression. Microarray-based gene-expression data were analyzed in six previously published cohorts of node-negative breast cancer patients not treated with adjuvant therapy (n = 824). The prognostic relevance of the individual immune markers was assessed using univariate analysis. The amount of independent prognostic information provided by each immune signature was then compared using a likelihood ratio statistic in the whole cohort as well as in different molecular subtypes.

RESULTS

Univariate Cox regression in the whole cohort revealed prognostic significance of CD4 (HR 0.66, CI 0.50-0.87, p = 0.004), CXCL13 (HR 0.86, CI 0.81-0.92, p < 0.001), CD20 (HR 0.76, CI 0.64-0.89, p = 0.001), IgκC (HR 0.81, CI 0.75-0.88, p < 0.001), and CTLA-4 (HR 0.67, CI 0.46-0.97, p = 0.032). Multivariate analyses of the immune signatures showed that both TS (p < 0.001) and BS (p < 0.001) showed a significant prognostic information in the whole cohort. After accounting for clinical-pathological variables, TS (p < 0.001), BS (p < 0.05), and CPS (p < 0.05) had an independent effect for MFS. In subgroup analyses, the prognostic effect of immune cells was most pronounced in HER2+ BC: BS as well as TS showed a strong association with MFS when included first in the model (p < 0.001).

CONCLUSIONS

Immune signatures provide subtype-specific additional prognostic information over clinical-pathological variables in node-negative breast cancer.

The thymus plays a primary role in early-onset Myasthenia Gravis (MG) mediated by anti-acetylcholine receptor (AChR) antibodies. As we recently showed an inflammatory and anti-viral signature in MG thymuses, we investigated in detail the contribution of interferon (IFN)-I and IFN-III subtypes in thymic changes associated with MG. We showed that IFN-I and IFN-III subtypes, but especially IFN-β, induced specifically α-AChR expression in thymic epithelial cells (TECs). We also demonstrated that IFN-β increased TEC death and the uptake of TEC proteins by dendritic cells. In parallel, we showed that IFN-β increased the expression of the chemokines CXCL13 and CCL21 by TECs and lymphatic endothelial cells, respectively. These two chemokines are involved in germinal center (GC) development and overexpressed in MG thymus with follicular hyperplasia. We also demonstrated that the B-cell activating factor (BAFF), which favors autoreactive B-cells, was overexpressed by TECs in MG thymus and was also induced by IFN-β in TEC cultures. Some of IFN-β effects were down-regulated when cell cultures were treated with glucocorticoids, a treatment widely used in MG patients that decreases the number of thymic GCs. Similar changes were observed in vivo. The injections of Poly(I:C) to C57BL/6 mice triggered a thymic overexpression of IFN-β and IFN-α2 associated with increased expressions of CXCL13, CCL21, BAFF, and favored the recruitment of B cells. These changes were not observed in the thymus of IFN-I receptor KO mice injected with Poly(I:C), even if IFN-β and IFN-α2 were overexpressed. Altogether, these results demonstrate that IFN-β could play a central role in thymic events leading to MG by triggering the overexpression of α-AChR probably leading to thymic DC autosensitization, the abnormal recruitment of peripheral cells and GC formation.

The homeostatic chemokine CXCL13 is preferentially produced in B-follicles and is crucial in the lymphoid organ development by attracting B-lymphocytes that express its selective receptor CXCR5. Follicular dendritic cells (FDCs) have been identified as the main cellular source of this chemokine in lymphoid organs. Recently, genome-wide approaches have suggested follicular CD4 T-helper cells (T(H)F) as additional CXCL13 producers in the germinal centre and the neoplastic counterpart of T(H)F (CD4+ tumour T-cells in angioimmunoblastic T-cell lymphoma) retains the capability of producing this chemokine. In contrast, no data are available on CXCL13 expression on FDC sarcoma (FDC-S) cells. By using multiple approaches, we investigated the expression of CXCL13 at mRNA and protein level in reactive and neoplastic FDCs. In reactive lymph nodes and tonsils, CXCL13 protein is mainly expressed by a subset of FDCs in B-cell follicles. CXCL13 is maintained during FDC transformation, since both dysplastic FDCs from 13 cases of Castleman's disease and neoplastic FDCs from ten cases of FDC-S strongly and diffusely express this chemokine. This observation was confirmed at mRNA level by using RT-PCR and in situ hybridization. Of note, no CXCL13 reactivity was observed in a cohort of epithelial and mesenchymal neoplasms potentially mimicking FDC-S. FDC-S are commonly associated with a dense intratumoural inflammatory infiltrate and immunohistochemistry showed that these lymphocytes express the CXCL13 receptor CXCR5 and are mainly of mantle zone B-cell derivation (IgD+ and TCL1+). In conclusion, this study demonstrates that CXCL13 is produced by dysplastic and neoplastic FDCs and can be instrumental in recruiting intratumoural CXCR5+ lymphocytes. In addition to the potential biological relevance of this expression, the use of reagents directed against CXCL13 can be useful to properly identify the origin of spindle cell and epithelioid neoplasms.

The CXC chemokine CXCL13, known as BCA-1 (B cell-attracting chemokine 1) or BLC (B-lymphocyte chemoattractant), has been identified as an efficacious attractant selective for B lymphocytes. The chemokine receptor BLR1 (Burkitt's lymphoma receptor 1)/CXCR5 expressed by all mature B cells has to date been identified as the only known receptor for BCA-1. As the loss of the BLR1/CXCR5 receptor is sufficient to disrupt organization of follicles in spleen and Peyer's patches, BCA-1 may act as a B cell homing chemokine. Nonetheless, BCA-1 has not been tested against all known chemokine receptors. In this study, we report that human BCA-1 competes with radiolabeled interferon gamma (IFN-gamma) inducible protein 10 (IP-10) for binding to the human CXCR3 receptor expressed in Ba/F3 and 293EBNA cell lines. Furthermore, human BCA-1 is an efficacious attractant for human CXCR3 transfected cells; BCA-1-induced chemotaxis is inhibited by a monoclonal antibody against human CXCR3. In these cells, as in human B lymphocytes expressing CXCR5, BCA-1 does not induce a calcium flux. Indeed, BCA-1 attenuates the calcium flux induced by IP-10. In addition, human BCA-1 is an agonist in stimulating GTP gamma S binding. Together these data suggest that human BCA-1 is a specific and functional G-protein-linked chemotactic ligand for the human CXCR3 receptor. The biological significance of this new finding is supported by our recent observation that human BCA-1 induces chemotaxis of activated T cells and the BCA-1-induced chemotaxis is inhibited by a monoclonal antibody against human CXCR3.

OBJECTIVE

Cardiac ageing involves the progressive development of cardiac fibrosis and diastolic dysfunction coordinated by MMP-9. Here, we report a cardiac ageing signature that encompasses macrophage pro-inflammatory signalling in the left ventricle (LV) and distinguishes biological from chronological ageing.

RESULTS

Young (6-9 months), middle-aged (12-15 months), old (18-24 months), and senescent (26-34 months) mice of both C57BL/6J wild type (WT) and MMP-9 null were evaluated. Using an identified inflammatory pattern, we were able to define individual mice based on their biological, rather than chronological, age. Bcl6, Ccl24, and Il4 were the strongest inflammatory markers of the cardiac ageing signature. The decline in early-to-late LV filling ratio was most strongly predicted by Bcl6, Il1r1, Ccl24, Crp, and Cxcl13 patterns, whereas LV wall thickness was most predicted by Abcf1, Tollip, Scye1, and Mif patterns. With age, there was a linear increase in cardiac M1 macrophages and a decrease in cardiac M2 macrophages in WT mice; of which, both were prevented by MMP-9 deletion. In vitro, MMP-9 directly activated young macrophage polarization to an M1/M2 mid-transition state.

CONCLUSIONS

Our results define the cardiac ageing inflammatory signature and assign MMP-9 roles in mediating the inflammaging profile by indirectly and directly modifying macrophage polarization. Our results explain early mechanisms that stimulate ageing-induced cardiac fibrosis and diastolic dysfunction.

CXCR5 and/or CXCL13 expression is elevated in certain carcinomas and lymphomas. To determine if these factors are involved in progression of non-small cell lung cancer (LuCa), we evaluated their expression in patients with various forms of this disease. Lung biopsies from patients with non-neoplastic cells (n=8), squamous cell carcinoma (SCC; n=24), or adenocarcinoma (AC; n=54) were stained for CXCR5. Histopathological analysis of these samples showed significantly higher expression of CXCR5 (p<0.001) in carcinomas (i.e., SCCs and ACs) relative to non‑neoplastic lung tissue. Nuclear and membrane CXCR5 intensities were highest in ACs, with median values of 185 and 130, respectively, followed by SCCs with median values of 170 and 110, respectively. The lowest nuclear and membrane expressions of CXCR5 were found in non-neoplastic tissues, having median values of 142 and 90, respectively. Sera from SCC patients (n=17), AC patients (n=14), and healthy controls (n=9) were tested for the presence of CXCL13. Serum CXCL13 levels in LuCa patients were higher than in healthy controls. CXCR5 expression in cell lines of human non-small cell lung carcinoma (NCI-H1915) and small cell lung carcinoma (SW-1271) were evaluated by flow cytometry. CXCR5 expression was higher in NCI-H1915 cells relative to SW-1271 cells. The functional significance of CXCR5 expression was tested in a migration assay. In response to CXCL13, more NCI-H1915 cells migrated than SW-1271 cells. These findings suggest that the CXCR5‑CXCL13 axis influences LuCa progression. After validation in larger patient groups, CXCR5 and CXCL13 may prove useful as biomarkers for LuCa. Correspondingly, blockade of this axis could serve as an effective therapy for LuCa.

Whole-genome sequencing has identified highly prevalent somatic mutations including MYD88, CXCR4, and ARID1A in Waldenström macroglobulinemia (WM). The impact of these and other somatic mutations on transcriptional regulation in WM remains to be clarified. We performed next-generation transcriptional profiling in 57 WM patients and compared findings to healthy donor B cells. Compared with healthy donors, WM patient samples showed greatly enhanced expression of the VDJ recombination genes DNTT, RAG1, and RAG2, but not AICDA Genes related to CXCR4 signaling were also upregulated and included CXCR4, CXCL12, and VCAM1 regardless of CXCR4 mutation status, indicating a potential role for CXCR4 signaling in all WM patients. The WM transcriptional profile was equally dissimilar to healthy memory B cells and circulating B cells likely due increased differentiation rather than cellular origin. The profile for CXCR4 mutations corresponded to diminished B-cell differentiation and suppression of tumor suppressors upregulated by MYD88 mutations in a manner associated with the suppression of TLR4 signaling relative to those mutated for MYD88 alone. Promoter methylation studies of top findings failed to explain this suppressive effect but identified aberrant methylation patterns in MYD88 wild-type patients. CXCR4 and MYD88 transcription were negatively correlated, demonstrated allele-specific transcription bias, and, along with CXCL13, were associated with bone marrow disease involvement. Distinct gene expression profiles for patients with wild-type MYD88, mutated ARID1A, familial predisposition to WM, chr6q deletions, chr3q amplifications, and trisomy 4 are also described. The findings provide novel insights into the molecular pathogenesis and opportunities for targeted therapeutic strategies for WM.

Missing in metastasis (MIM) is a member of newly emerged inverse Bin-Amphiphysin-Rvs (BAR) domain protein family and a putative metastasis suppressor. Although reduced MIM expression has been associated with bladder, breast and gastric cancers, evidence for the role of MIM in tumor progression remains scarce and controversial. Herein we characterized a MIM knockout mouse strain and observed that MIM-deficient mice often developed enlarged spleens. Autopsy and histological analysis revealed that nearly 78% of MIM(-/-) mice developed tumors with features similar to diffuse large B lymphoma during a period from 1 to 2 years. MIM(-/-) mice also exhibited abnormal distribution of B cells in lymphoid organs with decrease in the spleen but increase in the bone marrow and the peripheral blood. Furthermore, the bone marrow of MIM(-/-) mice contained a higher percentage of pre-B2 cells but fewer immature B-cells than wild-type mice. In response to CXCL13, a B-cell chemokine released from splenic stromal cells, MIM-deficient B-cells did not undergo chemotaxis or morphological changes in response to the chemokine and also did not internalize CXCR5, the receptor of CXCL13. Microarray analyses demonstrated that MIM is the only member of the I-BAR domain family that was highly expressed in human B cells. However, low or absent MIM expression was common in either primary B-cell malignancies or established B-cell acute lymphocytic leukemia or lymphomas. Thus, our data demonstrate for the first time an important role for MIM in B-cell development and suggest that predisposition of MIM-null mice to lymphomagenesis may involve aberrant interactions between B lineage cells and the lymphoid microenvironment.

BACKGROUND

Cerebrospinal fluid (CSF) biomarkers have been suggested to predict multiple sclerosis (MS) after clinically isolated syndromes, but studies investigating long-term prognosis are needed.

OBJECTIVE

To assess the predictive ability of CSF biomarkers with regard to MS development and long-term disability after optic neuritis (ON).

METHODS

Eighty-six patients with ON as a first demyelinating event were included retrospectively. Magnetic resonance imaging (MRI), CSF leukocytes, immunoglobulin G index and oligoclonal bands were registered. CSF levels of chitinase-3-like-1, osteopontin, neurofilament light-chain, myelin basic protein, CCL2, CXCL10, CXCL13 and matrix metalloproteinase-9 were measured by enzyme-linked immunosorbent assay. Patients were followed up after 13.6 (range 9.6-19.4) years and 81.4% were examined, including Expanded Disability Status Scale and MS functional composite evaluation. 18.6% were interviewed by phone. Cox regression, multiple regression and Spearman correlation analyses were used.

RESULTS

Forty-six (53.5%) developed clinically definite MS (CDMS) during follow-up. In a multivariate model MRI (p=0.0001), chitinase 3-like 1 (p=0.0033) and age (p=0.0194) combined predicted CDMS best. Neurofilament light-chain predicted long-term disability by the multiple sclerosis severity scale (p=0.0111) and nine-hole-peg-test (p=0.0202). Chitinase-3-like-1 predicted long-term cognitive impairment by the paced auditory serial addition test (p=0.0150).

CONCLUSIONS

Neurofilament light-chain and chitinase-3-like-1 were significant predictors of long-term physical and cognitive disability. Furthermore, chitinase-3-like-1 predicted CDMS development. Thus, these molecules hold promise as clinically valuable biomarkers after ON as a first demyelinating event.

Follicular helper T-cells (Tfh cells) are a subset of CD4(+) T-cells that are essential for normal production of high affinity antibodies. Tfh cells characteristically produce IL21 and IL4 and show high expression of surface markers CXCR5, ICOS, PDCD1 (PD-1) and the chemokine CXCL13. In this review we will focus on the emerging links between Tfh cells and subtypes of T-cell non-Hodgkin lymphoma: angioimmunoblastic T-cell lymphoma (AITL) and ~20% of peripheral T-cell lymphoma not otherwise specified (PTCL-NOS) have surface marker features of Tfh cells and share a spectrum of genetic abnormalities. The recurrent genetic abnormalities associated with AITL include mutations in epigenetic modifiers such as TET2 and DNMT3A and the motility and adhesion gene, RHOA, is mutated in up to 70% of cases. ~20% of PTCL-NOS demonstrate RHOA mutations and have other characteristics suggesting an origin in Tfh cells. The recognition that specific genetic and surface markers are associated with malignant Tfh cells suggests that the next few years will bring major changes in diagnostic and treatment possibilities. For example, antibodies against IL21, PDCD1 and ICOS are already in clinical trials for autoimmune disease or other malignancies and antibodies against CXCL13 are in pre-clinical development.

B lymphocytes can be triggered in lymph nodes by nonopsonized antigens (Ag), potentially in their native form. However, the mechanisms that promote encounter of B lymphocytes with unprocessed antigens in lymph nodes are still elusive. We show here that antigens are detected in B cells in the draining lymph nodes of mice injected with live, but not fixed, dendritic cells (DCs) loaded with antigens. This highlights active processes in DCs to promote Ag transfer to B lymphocytes. In addition, antigen-loaded DCs found in the draining lymph node were CD103+. Using 3 different model Ag, we then show that immature DCs efficiently take up Ag by macropinocytosis and store the internalized material in late endocytic compartments. We find that DCs have a unique ability to release antigens from these compartments in the extracellular medium, which is controlled by Rab27. B cells take up the regurgitated Ag and the chemokine CXCL13, essential to attract B cells in lymph nodes, enhances this transfer. Our results reveal a unique property of DCs to regurgitate unprocessed Ag that could play an important role in B-cell activation.

OBJECTIVE

To characterise global chemokine expression in systemic sclerosis (SSc) skin in order to better understand the relationship between chemokine expression and vascular inflammation in this disease.

METHODS

We investigated chemokine mRNA expression in the skin through quantitative PCR analysis comparing patients with diffuse cutaneous (dcSSc) or limited cutaneous (lcSSc) disease with healthy controls. We tested correlations between the most regulated chemokines and vascular inflammation and macrophage recruitment. CCL19 expression was examined in human primary immune cells treated with innate immune activators.

RESULTS

The chemokines, CCL18, CCL19 and CXCL13, were upregulated in dcSSc skin, and CCL18 in lcSSc skin. Expression of CCL19 in dcSSc skin correlated with markers of vascular inflammation and macrophage recruitment. Immunofluorescence data showed CCL19 colocalisation with CD163 macrophages in dcSSc skin. In vitro studies on human primary cells demonstrated that CCL19 expression was induced after toll-like receptor activation of peripheral blood mononuclear cells and separated populations of CD14 monocytes.

CONCLUSIONS

CCL18, CCL19 and CXCL13-chemoattractants for macrophage and T cell recruitment-were three of six chemokines with the highest expression in dcSSc skin. Increased CCL19 expression in the skin suggests a role for CCL19 in the recruitment of immune cells to the peripheral tissue. Induction of CCL19 in macrophages but not structural cells indicates a role for skin-resident or recruited immune cells in perivascular inflammation. This study demonstrates that CCL19 is a sensitive marker for the perivascular inflammation and immune cell recruitment seen in dcSSc skin disease.

B lymphocyte chemoattractant (BLC/CXCL13) is ectopically and highly expressed in the target organs such as the thymus and kidney in aged (NZB x NZW)F1 (BWF1) mice, a murine model for SLE. Ectopic expression of BLC/CXCL13 was attributed to mature myeloid DCs infiltrating in the target organs. DCs were also increased in the peripheral blood in aged BWF1 mice and differentiated into BLC/CXCL13-producing DCs in the presence of TNF-alpha or IL-1beta, but not IFN-alpha or IFN-gamma. BLC/CXCL13 expression in mature myeloid DCs was confirmed in bone marrow derived-DCs generated in vitro in the presence of GM-CSF and TNF-alpha. B1 cells expressed higher level of CXCR5 and migrated towards BLC/CXCL13 much better than B2 cells. B1 cells failed to home to the peritoneal cavity and preferentially recruited to the target organs in aged BWF1 mice developing lupus nephritis. B1, but not B2 cells possessed a potent antigen presenting activity in allo-MLR and activated autologous thymic CD4 T cells in the presence of IL-2. CXCR5+ CD4 T cells were also increased in aged BWF1 mice and they enhanced IgG production by B1 cells in vitro. These results suggest a possible involvement of aberrant B1 cell trafficking in activation of autoreactive CD4 T cells and autoantibody production in the target organs during the development of lupus, providing a new insight for the pathogenesis of B1 cells in lupus.

Inflammation is an obligatory attempt of the immune system to protect the host from infections. However, unregulated synthesis of proinflammatory products can have detrimental effects. Although mechanisms that lead to inflammation are well appreciated, those that restrain it are not adequately understood. Creating macrophage-specific L13a-knockout mice, we report that depletion of ribosomal protein L13a abrogates the endogenous translation control of several chemokines in macrophages. Upon LPS-induced endotoxemia, these animals displayed symptoms of severe inflammation caused by widespread infiltration of macrophages in major organs causing tissue injury and reduced survival rates. Macrophages from these knockout animals show unregulated expression of several chemokines (e.g., CXCL13, CCL22, CCL8, and CCR3). These macrophages failed to show L13a-dependent RNA binding complex formation on target mRNAs. In addition, increased polyribosomal abundance of these mRNAs shows a defect in translation control in the macrophages. Thus, to our knowledge, our studies provide the first evidence of an essential extraribosomal function of ribosomal protein L13a in resolving physiological inflammation in a mammalian host.

Accurate stage determination is crucial in the choice of treatment for patients suffering from sleeping sickness, also known as human African trypanosomiasis (HAT). Current staging methods, based on the counting of white blood cells (WBC) and the detection of parasites in the cerebrospinal fluid (CSF) have limited accuracy. We hypothesized that immune mediators reliable for staging T. b. gambiense HAT could also be used to stratify T. b. rhodesiense patients, the less common form of HAT.A population comprising 85 T. b. rhodesiense patients, 14 stage 1 (S1) and 71 stage 2 (S2) enrolled in Malawi and Uganda, was investigated. The CSF levels of IgM, MMP-9, CXCL13, CXCL10, ICAM-1, VCAM-1, neopterin and B2MG were measured and their staging performances evaluated using receiver operating characteristic (ROC) analyses.IgM, MMP-9 and CXCL13 were the most accurate markers for stage determination (partial AUC 88%, 86% and 85%, respectively). The combination in panels of three molecules comprising CXCL13-CXCL10-MMP-9 or CXCL13-CXCL10-IgM significantly increased their staging ability to partial AUC 94% (p value < 0.01).The present study highlighted new potential markers for stage determination of T. b. rhodesiense patients. Further investigations are needed to better evaluate these molecules, alone or in panels, as alternatives to WBC to make reliable choice of treatment.

BACKGROUND

Sleeping sickness, or human African trypanosomiasis (HAT), is a protozoan disease that affects rural communities in sub-Saharan Africa. Determination of the disease stage, essential for correct treatment, represents a key issue in the management of patients. In the present study we evaluated the potential of CXCL10, CXCL13, ICAM-1, VCAM-1, MMP-9, B2MG, neopterin and IgM to complement current methods for staging Trypanosoma brucei gambiense patients.

RESULTS

Five hundred and twelve T. b. gambiense HAT patients originated from Angola, Chad and the Democratic Republic of the Congo (D.R.C.). Their classification as stage 2 (S2) was based on the number of white blood cells (WBC) (>5/µL) or presence of parasites in the cerebrospinal fluid (CSF). The CSF concentration of the eight markers was first measured on a training cohort encompassing 100 patients (44 S1 and 56 S2). IgM and neopterin were the best in discriminating between the two stages of disease with 86.4% and 84.1% specificity respectively, at 100% sensitivity. When a validation cohort (412 patients) was tested, neopterin (14.3 nmol/L) correctly classified 88% of S1 and S2 patients, confirming its high staging power. On this second cohort, neopterin also predicted both the presence of parasites, and of neurological signs, with the same ability as IgM and WBC, the current reference for staging.

CONCLUSIONS

This study has demonstrated that neopterin is an excellent biomarker for staging T. b. gambiense HAT patients. A rapid diagnostic test for detecting this metabolite in CSF could help in more accurate stage determination.

Streptozotocin (STZ), a glucosamine-nitrosourea compound, has potent genotoxic effects on pancreatic β-cells and is frequently used to induce diabetes in experimental animals. Glucagon-like peptide-1 (GLP-1) has β-cell protective effects and is known to preserve β-cells from STZ treatment. In this study, we analyzed the mechanisms of STZ-induced diabetes and GLP-1-mediated β-cell protection in STZ-treated mice. At 1 week after multiple low-dose STZ administrations, pancreatic β-cells showed impaired insulin expression, while maintaining expression of nuclear Nkx6.1. This was accompanied by significant upregulation of p53-responsive genes in islets, including a mediator of cell cycle arrest, p21 (also known as Waf1 and Cip1). STZ treatment also suppressed expression of a wide range of genes linked with key β-cell functions or diabetes development, such as G6pc2, Slc2a2 (Glut2), Slc30a8, Neurod1, Ucn3, Gad1, Isl1, Foxa2, Vdr, Pdx1, Fkbp1b and Abcc8, suggesting global β-cell defects in STZ-treated islets. The Tmem229B, Prss53 and Ttc28 genes were highly expressed in untreated islets and strongly suppressed by STZ, suggesting their potential roles in β-cell function. When a pancreas-targeted adeno-associated virus (AAV) vector was employed for long-term Glp-1 gene delivery, pancreatic GLP-1 expression protected mice from STZ-induced diabetes through preservation of the β-cell mass. Despite its potent β-cell protective effects, however, pancreatic GLP-1 overexpression showed limited effects on the global gene expression profiles in the islets. Network analysis identified the programmed-cell-death-associated pathways as the most relevant network in Glp-1 gene therapy. Upon pancreatic GLP-1 expression, upregulation of Cxcl13 and Nptx2 was observed in STZ-damaged islets, but not in untreated normal islets. Given the pro-β-cell-survival effects of Cxcl12 (Sdf-1) in inducing GLP-1 production in α-cells, pancreatic GLP-1-mediated Cxcl13 induction might also play a crucial role in maintaining the integrity of β-cells in damaged islets.

Inducible BALT (iBALT) is associated with immune responses to respiratory infections as well as with local pathology derived from chronic inflammatory lung diseases. In this study, we assessed the role of oncostatin M (OSM) in B cell activation and iBALT formation in mouse lungs. We found that C57BL/6 mice responded to an endotracheally administered adenovirus vector expressing mouse OSM, with marked iBALT formation, increased cytokine (IL-4, IL-5, IL-6, IL-10, TNF-α, and IL-12), and chemokine (CXCL13, CCL20, CCL21, eotaxin-2, KC, and MCP-1) production as well as inflammatory cell accumulation in the airways. B cells, T cells, and dendritic cells were also recruited to the lung, where many displayed an activated phenotype. Mice treated with control adenovirus vector (Addl70) were not affected. Interestingly, IL-6 was required for inflammatory responses in the airways and for the expression of most cytokines and chemokines. However, iBALT formation and lymphocyte recruitment to the lung tissue occurred independently of IL-6 and STAT6 as assessed in gene-deficient mice. Collectively, these results support the ability of OSM to induce B cell activation and iBALT formation independently of IL-6 and highlight a role for IL-6 downstream of OSM in the induction of pulmonary inflammation.

BACKGROUND

The level of CXCL13 is a cerebrospinal fluid (CSF) biomarker for acute Lyme neuroborreliosis (LNB) with a high sensitivity. As the concentration rapidly declines during antibiotic therapy CXCL13 can also be used as a follow-up parameter. However, CXCL13 is not yet in use as a routine parameter due to concerns about the specificity.

OBJECTIVE

The sensitivity, specificity and predictive value of CXCL13 in the clinical routine work-up of suspected LNB was analyzed.

METHODS

Since July 2010 the CSF of all patients (n = 204) with suspected acute LNB was not only analyzed for the routine parameters (i.e. pleocytosis and intrathecal production of Borrelia-specific antibodies, AI) but also for CXCL13. In cases of incongruent findings, a follow-up puncture after antibiotic therapy was carried out. The cut-off level for acute LNB was set at 250 pg/ml.

RESULTS

This study included 179 patients who were not pretreated with antibiotics. Of these patients 15 suffered from definite LNB, 3 had a probable LNB and all had a CXCL13 value above the cut-off level. Only 2 of the 161 patients with a non-LNB diagnosis (both with a lymphoma) had a CXCL13 value in the CSF higher than 250 pg/ml. Especially noteworthy were two patients without pleocytosis in the CSF but with CXCL13 levels above the cut-off level in whom LNB could be confirmed in the follow-up CSF analysis.

CONCLUSIONS

The biomarker CXCL13 has a higher sensitivity (100 % vs. 87 %) with a specificity (99 %) comparable with the established diagnostic markers for LNB, e.g. CSF pleocytosis and Borrelia-AI in the investigated patient population. The negative predictive value of CXCL13 is 100 %. Therefore, a normal CXCL13 level virtually excludes LNB. In the clinical routine CXCL13 is a valuable and practical diagnostic marker for LNB and can even detect an acute LNB in patients without CSF pleocytosis.

Here, we investigated a role of CCL2 on the increased susceptibility of severely burned mice to Enterococcus faecalis translocation. After inoculation of Mphi from MLMphi of normal mice, 80% of the SCIDbgMN mice orally infected with the lethal dose of E. faecalis survived, and all mice inoculated with MLMphi from thermally injured mice died. At this time, SCIDbgMN mice inoculated with MLMphi from thermally injured CCL2(-/-) mice were shown to be resistant (90% survival). M1Mphi were not induced by E. faecalis antigen in cultures of MLMphi from thermally injured wild-type mice, and MLMphi from thermally injured CCL2(-/-) mice converted to M1Mphi after the antigen stimulation. MLMphi from wild-type mice 2 days postburn injury possessed M2a- and M2cMphi properties, and those from mice 7-21 days postburn injury carried M2bMphi properties. However, MLMphi from thermally injured CCL2(-/-) mice did not show any typical properties for M2a- or M2cMphi. CCL17 and CXCL13 (biomarkers for M2a- and M2cMphi), but not CCL1 (a biomarker of M2bMphi), were produced by MLMphi from thermally injured CCL2(-/-) mice treated with rCCL2. These results indicate that CCL2 converts resident MLMphi to M2a- and M2cMphi, detected early after burn injury, and decreases host antibacterial innate immunity against sepsis stemming from oral E. faecalis infection.

Mycoplasma gallisepticum infection in chickens leads to tracheitis, airsacculitis, poor feed conversion and reduced egg production, resulting in considerable economic hardship on the poultry industry. The chemokines and cytokines responsible for recruitment, activation and proliferation of leukocytes in affected tissues have not been described. In the current study, chemokine and cytokine gene expression profiles were investigated in tracheas of chickens inoculated with M. gallisepticum strains R(low) (pathogenic) and GT5 (attenuated) at days 1, 4 and 8 post-inoculation. Expression of lymphotactin mRNA was higher in R(low)-inoculated chickens than GT5- or PBS-inoculated chickens, while CXCL13/BCA1 mRNA expression level was higher in both GT5- or R(low)-inoculated chickens than in PBS-inoculated controls on day 1 post-inoculation. However, both R(low) and GT5 strains induced a down-regulation in mRNA expression of CCL20, IL-1beta, IL-8 and IL-12p40 genes, with CCL20 and IL-12 mRNA levels remaining lower on days 4 and 8 post-inoculation. On day 4, R(low)-inoculated chickens exhibited significantly higher tracheal lesion scores and higher levels of lymphotactin, CXCL13, CXCL14, RANTES, MIP-1beta, IL-1beta and IFN-gamma mRNA compared to PBS-inoculated controls. The mRNA levels of these genes were also higher in R(low)-inoculated chickens that had moderate to severe tracheal lesion scores on day 8 post-inoculation. These results reflect the importance of lymphocyte and monocyte chemotactic factors in the development of tracheal lesions in chickens inoculated with M. gallisepticum strain R(low). Our data also suggest that M. gallisepticum may modulate the host response causing dramatic decreases in CCL20, IL-8 and IL-12 mRNA levels in GT5- or R(low)-inoculated chickens as early as one day post-inoculation.

BACKGROUND

The chemotactic signals regulating cell trafficking in the herpes simplex virus type 1 (HSV-1) infected cornea are well documented, however, those in the cornea-associated tissues, such as the trigeminal ganglion (TG) and draining lymph nodes (LNs), are largely unknown.

OBJECTIVE

To examine chemokine expression and subsequent cell infiltration in the HSV-1 infected cornea and its associated tissues.

METHODS

Eight-week-old female BALB/c mice were infected with 10 mu l HSV-1 (CHR3 strain: 5 x 106 PFU/ml) by corneal scarification. Total RNAs were extracted from the corneas, TGs, and LNs at pre-inoculation, 3 days post-inoculation (P.I.) and 7 days P.I. The mRNA for 28 different chemokines in the extracts was amplified by RT-PCR. Infiltrating cells were identified by immunohistochemistry.

RESULTS

After the HSV-1 infection, the corneal stroma became edematous by infiltrated cells under the eroded epithelium. The TG and LNs were markedly swollen. The cornea was infiltrated with granulocytes and CD11b+ cells at 3 days P.I., followed by CD4+ and CD8+ T cells at 12 days P.I. In the TG, CD11b+ cells, but no granulocytes, infiltrated throughout the observation period. T cells migrated into the TG earlier than into the cornea. Gene expressions of neutrophil-attracting chemokines (CXCL1, 2, 3, and 5) increased in the cornea, but they did not enhance in the TG or LNs. On the other hand, gene expressions of chemokines which attract CD11b+ cells such as CCL2, 8, 7, 12, CCL3, 4, and CCL5, increased in the cornea and TG with its peak at 3 days P.I. Gene expressions of chemokines those work on T cells and B cells, such as CCL19, CCL21, CXCL9, CXCL13, CXCL10, XCL1, and CXCL16, were up-regulated and peaked at 3 days P.I. in the cornea and in the TG. Thus, pattern of chemokine gene expression was similar in the cornea and in the TG. On the contrary, gene expressions of chemokines in the draining LNs affecting CD11b+ cells and T cells were temporarily down-regulated.

CONCLUSIONS

Upon HSV-1 infection, dynamic gene expression of chemokines was observed not only in the inoculated cornea but also in its associated tissues.