B Lymphocyte Development in the Bursa of Fabricius of Young Broilers is Influenced by the Gut Microbiota

ABSTRACT Chickens have been used as a valuable and traditional model for studies on basic immunology. B lymphocytes were first identified in the bursa of Fabricius (BF) of broilers. The microbiota is important for immune system development and function. However, the effect of the microbiota on mediating B cell development and its regulatory mechanism is poorly elucidated. Here, we show that the gut microbiota is associated with the development of bursal B cells in young chickens. Changing patterns of both the alpha diversity and the expression of the B cell marker Bu-1α in the gut microbiota were related to the ages of chickens at different growth phases. Further correlation analysis revealed the marked correlation between the relative abundances of Intestinimonas, Bilophila, Parasutterella, Bacteroides, Helicobacter, Campylobacter, and Mucispirillum and the expression of Bu-1α. In antibiotic-treated chickens, BF and B cell development had aberrations as the relative abundance of the microbiota in early life decreased. These findings were consistent with Spearman’s correlation results. Single-cell transcriptome analysis indicated that the heterogeneity in the cellular composition and developmental trajectory of bursal B cells from antibiotic-treated chickens was large. We found a novel subpopulation of unnamed B cells and identified Taf1 as a new pivotal regulator of B cell lineage differentiation. Therefore, we provide novel insights into the regulatory role of the gut microbiota in B cell development in early life and the maturation of host humoral immunity. IMPORTANCE In this study, we used young broilers to investigate the relationship between their gut microbiota and bursal B cell development. We characterized the important variables, microbes, B cells, and immunoglobulins during the posthatch development of birds. We also identified several candidate taxa in the cecal contents associated with B cells. Our study provides a rich resource and cell-cell cross talk model supporting B cell differentiation from the bursa in vitro at single-cell resolution. Furthermore, we determined a new pivotal regulator (Taf1) of B cell differentiation. We believe that our study makes a significant contribution to the literature because our findings may elucidate the role of the gut microbiota in B cell differentiation. This study also serves as a basis for developing new strategies that modulate B cell differentiation to prevent diseases.

(2) How long for the antibiotic treatment? (3) Check 2.5 Microbiome sequencing data analysis, "The correlations between microbes and immunohistochemistry factors were calculated using the "Spearman" algorithm; significant correlations (P < 0.05) were selected, and the heatmap was drawn using the pheatmap (v1.0.12) package by R (v3.4.1)", is the pheatmap the right one? 3. In 3.2, "The pattern of changes in the gut microbiota and B cells in chickens of all ages was similar on different days", here the diversity (or other index) of gut microbiota and the development of B cells should be clear.

Much more relative elements should be cited in Discussion
Reviewer #2 (Comments for the Author): Comments to the Author The manuscript describes the relationship between gut microbiota and bursal B cell development. The topic will contribute to the field of poultry microbiology, and will be of interest in a broader research domain. The experimental and methodological of the study is rigorous, and the data is available. However, minor issues require attention before a recommendation can be made.

Introduction:
Line 54-56 The author want to discuss the relationship between the gut microbiota and bursal B cells in birds. However, the reference cited by author is redundancy. The author should rephrase or delete the sentence. Line 57-58 In this section the author emphasized the importance of gut microbiota in immune system. However, the literature has focused on human and mice, the author should provide more evidence that gut microbiota plays a critical role in poultry immune system regulation.

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