Frederic Sierro

Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43

The immune system responds to pathogens by a variety of pattern recognition molecules such as the Toll-like receptors (TLRs), which promote recognition of dangerous foreign pathogens. However, recent evidence indicates that normal intestinal microbiota might also positively influence immune responses, and protect against the development of inflammatory diseases. One of these elements may be short-chain fatty acids (SCFAs), which are produced by fermentation of dietary fibre by intestinal microbiota. A feature of human ulcerative colitis and other colitic diseases is a change in ‘healthy’ microbiota such as Bifidobacterium and Bacteriodes, and a concurrent reduction in SCFAs. Moreover, increased intake of fermentable dietary fibre, or SCFAs, seems to be clinically beneficial in the treatment of colitis. SCFAs bind the G-protein-coupled receptor 43 (GPR43, also known as FFAR2), and here we show that SCFA–GPR43 interactions profoundly affect inflammatory responses. Stimulation of GPR43 by SCFAs was necessary for the normal resolution of certain inflammatory responses, because GPR43-deficient (Gpr43-/-) mice showed exacerbated or unresolving inflammation in models of colitis, arthritis and asthma. This seemed to relate to increased production of inflammatory mediators by Gpr43-/- immune cells, and increased immune cell recruitment. Germ-free mice, which are devoid of bacteria and express little or no SCFAs, showed a similar dysregulation of certain inflammatory responses. GPR43 binding of SCFAs potentially provides a molecular link between diet, gastrointestinal bacterial metabolism, and immune and inflammatory responses.

Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7

Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr7−/− mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr7−/− mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr7−/− heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4.

TRAF2 and TRAF3 Signal Adapters Act Cooperatively to Control the Maturation and Survival Signals Delivered to B Cells by the BAFF Receptor

Tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2 and TRAF3) were shown to function in a cooperative and nonredundant manner to suppress nuclear factor-kappaB2 (NF-kappaB2) activation, gene expression, and survival in mature B cells. In the absence of this suppressive activity, B cells developed independently of the obligatory B cell survival factor, BAFF (B cell-activating factor of the tumor necrosis factor family). However, deletion of either TRAF2 or TRAF3 from the T cell lineage did not promote T cell survival, despite causing extensive NF-kappaB2 activation. This constitutive, lineage-specific suppression of B cell survival by TRAF2 and TRAF3 determines the requirement for BAFF to sustain B cell development in vivo. Binding of BAFF to BAFF receptor reversed TRAF2-TRAF3-mediated suppression of B cell survival by triggering the depletion of TRAF3 protein. This process was TRAF2 dependent, revealing dual roles for TRAF2 in regulating B cell homeostasis.

The BAFF/APRIL System: An Important Player in Systemic Rheumatic Diseases

Many rheumatic diseases have an autoimmune basis, characterized by organ-specific inflammation and tissue destruction. Diseases such as rheumatoid arthritis, systemic lupus erythematosus or Sjögrens syndrome often associate with abnormal B cell function and the production of various autoantibodies. B cell activating factor belonging to the TNF family (BAFF) is a B cell survival factor essential for B cell maturation, but also contributes to autoimmunity when overexpressed in mice. In addition, elevated levels of BAFF have been detected in the serum of patients with various rheumatic diseases, suggesting a role for this factor in these pathologies. BAFF has additional functions that may be important in rheumatic diseases. For instance, excess BAFF leads to the expansion of a subset of B cells named marginal zone (MZ) B cells, a cell type able to activate naïve T cells. In addition, expansion of the MZ B cell population correlates with certain autoimmune diseases, and these cells have been detected in inflamed tissues in mice and humans. Recently, BAFF was shown to also stimulate T cell activation, an aspect that may also contribute to autoimmunity. Finally, BAFF has emerged as a potent survival factor for B cell lymphomas and as such may be involved in promoting B cell cancers. This result possibly offers an explanation for the occasional lymphoma complication observed in a subset of patients with certain rheumatic diseases, particularly Sjögrens syndrome. New elements about BAFF biology indicate that this factor may be involved in a wider range of pathologies than first anticipated, and inhibitors of this factor are likely to provide attractive new treatments for rheumatic diseases and B cell lymphomas.

Selection of human antibody fragments by phage display

Here, we describe a protocol for the selection of human antibody fragments using repertoires displayed on filamentous bacteriophage. Antigen-specific clones are enriched by binding to immobilized antigen, followed by elution and repropagation of phage. After multiple rounds of binding selection, specific clones are identified by ELISA. This article provides an overview of phage display and antibody technology, as well as detailed protocols for the immobilization of antigen, the selection of repertoires on purified or complex antigens and the identification of binders.

Toll-like receptor 5- and lymphotoxin β receptor-dependent epithelial Ccl20 expression involves the same NF-κB binding site but distinct NF-κB pathways and dynamics

Canonical and alternative NF-kappaB pathways depend on distinct NF-kappaB members and regulate expression of different gene subset in inflammatory and steady state conditions, respectively. In intestinal epithelial cells, both pathways control the transcription of the gene coding the CCL20 chemokine. Lymphotoxin beta receptor (LTbetaR) mediates long lasting CCL20 expression whereas Toll-like receptor 5 (TLR5) signals promote inducible and transient activation. Here, we investigated whether the regulation of ccl20 expression involves different promoter sites and NF-kappaB molecules in response to TLR5 and LTbetaR stimulation. In epithelial cells, both stimulation required the same promoter regions, especially the NF-kappaB binding site but involved different NF-kappaB isoforms: p65/p50 and p52/RelB, for TLR5 and LTbetaR-dependent activation, respectively. The dynamic of activation and interaction with CCL20-specific NF-kappaB site correlated with gene transcription. Similar Ccl20 expression and NF-kappaB activation was found in the small intestine of mice stimulated with TLR5 and LTbetaR agonists. In summary, different NF-kappaB pathways modulate CCL20 transcription by operating on the same NF-kappaB binding site in the same cell type.

Intrinsic Molecular Factors Cause Aberrant Expansion of the Splenic Marginal Zone B Cell Population in Nonobese Diabetic Mice

Marginal zone (MZ) B cells are an innate-like population that oscillates between MZ and follicular areas of the splenic white pulp. Differentiation of B cells into the MZ subset is governed by BCR signal strength and specificity, NF-κB activation through the B cell–activating factor belonging to the TNF family (BAFF) receptor, Notch2 signaling, and migration signals mediated by chemokine, integrin, and sphingosine-1-phosphate receptors. An imbalance in splenic B cell development resulting in expansion of the MZ subset has been associated with autoimmune pathogenesis in various murine models. One example is the NOD inbred mouse strain, in which MZ B cell expansion has been linked to development of type 1 diabetes and Sjögren’s syndrome. However, the cause of MZ B cell expansion in this strain remains poorly understood. We have determined that increased MZ B cell development in NOD mice is independent of T cell autoimmunity, BCR specificity, BCR signal strength, and increased exposure to BAFF. Rather, mixed bone marrow chimeras showed that the factor(s) responsible for expansion of the NOD MZ subset is B cell intrinsic. Analysis of microarray expression data indicated that NOD MZ and precursor transitional 2-MZ subsets were particularly dysregulated for genes controlling cellular trafficking, including Apoe, Ccbp2, Cxcr7, Lgals1, Pla2g7, Rgs13, S1pr3, Spn, Bid, Cd55, Prf1, and Tlr3. Furthermore, these B cell subsets exhibited an increased steady state dwell time within splenic MZ areas. Our data therefore reveal that precursors of mature B cells in NOD mice exhibit an altered migration set point, allowing increased occupation of the MZ, a niche favoring MZ B cell differentiation.

Y1 signalling has a critical role in allergic airway inflammation

Asthma affects 300 million people worldwide, yet the mechanism behind this pathology has only been partially elucidated. The documented connection between psychological stress and airway inflammation strongly suggests the involvement of the nervous system and its secreted mediators, including neuropeptides, on allergic respiratory disease. In this study, we show that neuropeptide Y (NPY), a prominent neurotransmitter, which release is strongly upregulated during stress, exacerbates allergic airway inflammation (AAI) in mice, via its Y1 receptor. Our data indicate that the development of AAI was associated with elevated NPY expression in the lung and that lack of NPY‐mediated signalling in NPYKO mice or its Y1 receptor in Y1KO mice significantly improved AAI. In vivo, eosinophilia in the bronchoalveolar fluid as well as circulating immunoglobulin E in response to AAI, were significantly reduced in NPY‐ and Y1‐deficient compared with wild‐type mice. These changes correlated with a blunting of the Th2 immune profile that is characteristic for AAI, as shown by the decreased release of interleukin‐5 during ex vivo re‐stimulation of T cells isolated from the thoracic draining lymph nodes of NPY‐ or Y1‐deficient mice subjected to AAI. Taken together this study demonstrates that signalling through Y1‐receptors emerges as a critical pathway for the development of airway inflammation and as such potentially opens novel avenues for therapeutic intervention in asthma.

Reduction of ARNT in myeloid cells causes immune suppression and delayed wound healing

Aryl hydrocarbon receptor nuclear translocator (ARNT) is a transcription factor that binds to partners to mediate responses to environmental signals. To investigate its role in the innate immune system, floxed ARNT mice were bred with lysozyme M-Cre recombinase animals to generate lysozyme M-ARNT (LAR) mice with reduced ARNT expression. Myeloid cells of LAR mice had altered mRNA expression and delayed wound healing. Interestingly, when the animals were rendered diabetic, the difference in wound healing between the LAR mice and their littermate controls was no longer present, suggesting that decreased myeloid cell ARNT function may be an important factor in impaired wound healing in diabetes. Deferoxamine (DFO) improves wound healing by increasing hypoxia-inducible factors, which require ARNT for function. DFO was not effective in wounds of LAR mice, again suggesting that myeloid cells are important for normal wound healing and for the full benefit of DFO. These findings suggest that myeloid ARNT is important for immune function and wound healing. Increasing ARNT and, more specifically, myeloid ARNT may be a therapeutic strategy to improve wound healing.