In contrast to mammals, early B cell differentiation and diversification of the antibody repertoire in chickens do not take place in the bone marrow but in a specialized gut associated lymphoid tissue (GALT), the bursa of Fabricius. During embryonic development, B cell precursors migrate to the bursa anlage, where they proliferate and diversify their B cell receptor repertoire. Around hatch these diversified B cells start to emigrate from the bursa of Fabricius and populate peripheral lymphoid organs, but very little is known how the migratory processes are regulated. As CXCL12 (syn. SDF-1) and CXCR4 were shown to be essential for the control of B cell migration during the development of lymphoid tissues in mammals, we analyzed expression and function of this chemokine/chemokine-receptor pair in the chicken bursa. We found a strong variation of mRNA abundance of CXCL12 and CXCR4 in different stages of bursa development, with high abundance of CXCL12 mRNA in the bursa anlage at embryonic day 10 (ED10). In situ hybridization demonstrated disseminated CXCL12 expression in the early bursa anlage, which condensed in the developing follicles and was mainly restricted to the follicle cortex post-hatch. Flow cytometric analysis detected CXCR4 protein already on early B cell stages, increasing during bursal development. Post-hatch, a subpopulation with the hallmarks of emigrating B cells became detectable, which had lower CXCR4 expression, suggesting that downregulation of CXCR4 is necessary to leave the CXCL12-high bursal environment. In vivo blockade of CXCR4 using AMD3100 at the time of B cell precursor immigration strongly inhibited follicle development, demonstrating that CXCL12 attracts pre-bursal B cells into the bursal anlage. Altogether, we show that CXCL12 and its receptor CXCR4 are important for both populating the bursa with B cells and emigration of mature B cells into the periphery post hatch, and that CXCR4 function in primary B cell organs is conserved between mammals and birds.
Antibody producing B-cells are an essential component of the immune system. In contrast to human and mice where B-cells develop in the bone marrow, chicken B-cells develop in defined stages in the bursa of Fabricius, a gut associated lymphoid tissue. In order to gain a better understanding of critical biological processes like immigration of B-cell precursors into the bursa anlage, their differentiation and final emigration from the bursa we analyzed the proteome dynamics of this organ during embryonic and posthatch development. Samples were taken from four representative developmental stages (embryonic day (ED) 10, ED18, day 2, and day 28) and compared in an extensive 2D DIGE approach comprising six biological replicates per time point. Cluster analysis and PCA demonstrated high reliability and reproducibility of the obtained data set and revealed distinctive proteome profiles for the selected time points, which precisely reflect the differentiation processes. One hundred fifty three protein spots with significantly different intensities were identified by MS. We detected alterations in the abundance of several proteins assigned to retinoic acid metabolism (e.g. retinal-binding protein 5) and the actin-cytoskeleton (e.g. vinculin and gelsolin). By immunohistochemistry, desmin was identified as stromal cell protein associated with the maturation of B-cell follicles. Strongest protein expression difference (10.8-fold) was observed for chloride intracellular channel 2. This protein was thus far not associated with B-cell biology but our data suggest an important function in bursa B-cell development.
Marek’s disease virus (MDV) is an alphaherpesvirus that causes immunosuppression and deadly lymphoma in chickens. Lymphoid organs play a central role in MDV infection in animals. B-cells in the bursa of Fabricius facilitate high levels of MDV replication and contribute to dissemination at early stages of infection. Several studies investigated host responses in bursal tissue of MDV-infected chickens; however, the cellular responses specifically in bursal B-cells has never been investigated. We took advantage of our recently established in vitro infection system to decipher the cellular responses of bursal B-cells to infection with a very virulent MDV strain. Here, we demonstrate that MDV infection extends the survival of bursal B-cells in culture. Microarray analyses revealed that most cytokine/cytokine-receptor-, cell cycle- and apoptosis-associated genes are significantly down-regulated in these cells. Further functional assays validated these strong effects of MDV infections on cell cycle progression and thus, B-cell proliferation. In addition, we confirmed that MDV infections protect B-cells from apoptosis and trigger an accumulation of the autophagy marker Lc3-II. Taken together, our data indicate that MDV-infected bursal B-cells show hallmarks of a senescence-like phenotype, leading to a prolonged B-cell survival. This study provides an in-depth analysis of bursal B-cell responses to MDV infection and important insights into how the virus extends the survival of these cells.
Outbreaks of avian influenza in poultry and humans are a global public health concern. Aquatic birds are the primary natural reservoir of avian influenza viruses (AIVs), and strikingly, AIVs mainly cause asymptomatic or mild infection in these species.
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