Regulation of B-1a cells: another novel function of the basic helix-loop-helix transcriptional regulator BHLHE41

The basic helix-loop-helix transcription factors e40 (BHLHE40) and e41 (BHLHE41) were originally discovered as regulators of the proliferation and differentiation of chondrocytes and nerve cells.^{1, 2, 3} Later studies showed that these transcriptional regulators participate in a variety of physiological processes for the maintenance of homeostasis, such as the regulation of endothelial cells,⁴ adipocytes,⁵ circadian rhythms,⁶ and tumor development.⁷ Interest in the significance of these transcriptional regulators in the immune response was spurred by the finding that the deletion of BHLHE40 leads to abnormal activation of T cells and B cells with resistance to activation induced cell death and accumulation of autoreactive T and B cells resulting in autoimmune diseases in aged animals, although the deletion of BHLHE40 does not affect the development and maturation of T and B cell compartments.⁸ However, these initial findings could contradict recent reports about the essential roles of these transcriptional regulators in the development of autoimmune diseases identified in experimental autoimmune disease models,^{9, 10} such as experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis. However, this can be explained by the potential roles of these transcriptional regulators in the maintenance of T regulatory cells (Tregs) through the maintenance and regulation of CD25 expression in cooperation with Runx1.¹¹ Recent studies also confirmed that these transcriptional regulators are expressed in T cells in a CD28 activation dependent manner and participate in the regulation of T cell cytokine production, including that of Interleukin 2 (IL-2), Interferon gamma (IFN-γ) and granulocyte-macrophage colony stimulating factor (GM-CSF).¹² BHLHE41 regulates IL-1β production, and IL-1β induced GM-CSF to produce Th17 cells with a pathological signature in the central nervous system that is dependent on BHLHE40.¹³ In cooperation with the transcription factor T-bet, BHLHE40 also regulates IFN-γ production by iNKT cells,¹⁴ providing immunity against tumor development. Furthermore, these transcriptional regulators may also play a role in the differentiation of Th2 cells by enhanced expression of CD25.^{15, 16} These studies have established the roles of these two transcriptional regulators in the immune response and the immunopathology mediated by T cells. This hints at the role of the BHLHE transcription factors in regulating B-1 cells, as these cells play a critical role in autoimmune diseases¹⁷ by producing natural IgM-based autoreactive antibodies in addition to participating in the innate immune defense against bacterial pathogens in the absence of pathogen-specific adaptive immunity. Recently, Kreslavsky et al.¹⁸ demonstrated that BHLHE41 alone and, to lesser extent, together with BHLHE40 regulates the development and maintenance of B-1a B cells.

B cells as mediators of humoral immunity play critical roles in protection against infection by producing pathogen-specific antibodies and in homeostasis by their regulatory mechanisms involving the production of different cytokines. B cells can now be divided into B-1 cells and B-2 cells based on their surface molecular markers, function, and developmental origin. B-1 cells originate predominantly from the hematopoietic stem cells of the fetal liver and yolk sac and have the capacity for maintenance through self-renewal during adulthood, although the hematopoietic stem cells of the bone marrow also replenish B-1 cells to a lesser extent. B-1 cells produce IgM-type autoreactive natural antibodies and, therefore, may play a role in autoimmune diseases in addition to providing protection against infection during the priming period of pathogen-specific adaptive immunity¹⁹ and the regulation of T regulatory cells.²⁰ B-1 cells were identified in the early 1990s as they expressed the common T cell surface marker Ly-1 in mouse but called leu-2 in humans, also called cluster of differentiation (CD) 5 while also expressing cell surface IgM and producing IgM-based natural antibodies. B-1 cells were further subdivided into the B-1a and B-1b subsets after the observation that certain B-1 cells do not express CD5 molecules, and these were named B-1b B cells. Although the existence of B-1 cells with unique developmental and functional characteristics was established in the murine system, their human counterparts were considered controversial. However, this has been resolved recently with the demonstration that human B-1 cells with unique surface markers (CD20+CD27+CD43+CD70−) are derived from Lin-CD34+CD38^lo stem cells from both the umbilical cord and adult peripheral blood.¹⁹ This has set the stage for detailed studies of B-1 cell development and function in both physiological and pathological settings. Initial studies of the transcriptional regulation of B-1 cells identified the critical roles of different transcription factors, including that of Arid3a, Oct2, and IkBNS, as well as the Lin28 miRNAs and the miRNA Let-7 axis, in the regulation of B-1 cell development.²¹ However, a comprehensive understanding of the transcriptional regulation of B-1 cells is lacking.

In a study designed to elucidate the mechanisms of transcriptional regulation in B-1 cells, Kreslavsky et al.¹⁸ reported that BHLHE41 is essential for the development and function of B-1a B cells. As a first step, Kreslavsky et al. identified BHLHE41 as the most highly expressed gene in B-1 cells following a comparison of differentially expressed genes in B-1 and B-2 cells isolated from mouse spleen by RNA-Seq analysis. This was further confirmed by the B-1 cell specific expression of BHLHE41 regulatory element controlled human CD2 molecules in a reporter mouse model. Interestingly, BHLHE41 was also highly expressed in the immature B cells isolated from the fetal and neonatal liver compared to the immature B cells from the bone marrow, which is consistent with B-1 cell development predominantly in the fetal and prenatal precursor cells than in adult bone marrow precursors. BHLHE41 was induced in both B-1 cells and follicular (B-2) B cells in response to B cell stimulation, implying that BHLHE41 may also be induced in B-1 cells during development due to the self-reactive nature of this cell population. These data together established that BHLHE41 is a B-1 B-cell-specific transcription factor. As B-1 B cells have a unique developmental origin and process, BHLHE41 and its close homolog BHLHE40 were investigated for their roles in the development of B-1 cells in two different experimental settings. Bhlhe41 gene knockout (ko) mice demonstrated significantly reduced B-1a cells in the spleen and peritoneal cavities compared to wild-type mice, with even the residual developed B-1a cells showing reduced expression of CD5 and elevated expression of the B cell associated molecule B220 in these mice. In contrast, B-1b cells were not affected by the lack of BHLHE40. These changes were even more pronounced in Bhlhe41 and Bhlhe40 double knockout (dko) mice. However, the deletion of Bhlhe40 affected the development of B-1 cells to a lesser degree. This was further confirmed in studies involving chimeric mice generated by mixing fetal livers of wild-type and dko and their introduction into lethally irradiated Rag2 ko mouse in adoptive transfer experiments and subsequently following the B cell lineage cells. The fetal liver cells of dko mice did not produce mature B-1a cells. Lack of Bhlhe41 and 40 did not affect the progenitors but blocked transitional B-1a cell development. Taken together, these findings support the essential roles of BHLHE41 and, to a lesser extent, BHLHE40 in B-1a cell development. Bhlhe41 and Bhlhe40 dko also significantly affected the B-1a cell repertoire. Changes were concentrated in the variable segments of both the heavy and light chains of Ig molecules with complete loss of Ighv12-3 and Igkv4-91mRNA, which encode for a phosphatidylcholine (PtC) receptor that is preferentially expressed by 10% of the B-1a cells. These changes in the BCR repertoire are due to deficiencies in selection but not rearrangements that generate the B-1a BCR repertoire. Transgenic expression of prearranged VH12 and Vk4 genes did not rescue the altered phenotype and developmental deficiencies of B-1a cells in Bhlhe41 and Bhlhe40 dko mice, suggesting that an altered BCR repertoire was responsible for the altered B-1a cell phenotype but not the cause of the defects in the development and self-renewal of B-1a cells. Determination of the function of BHLHE41 in B-1a cells by Chip-Seq and deep RNA-seq demonstrated that BHLHE41 targets 5509 genes with the common E-box motif. Further detailed analysis identified 41 directly activated and 90 directly repressed gene targets of BHLHE41 with gene repression being the dominant feature, suggesting that BHLHE41 behaves as transcriptional repressor in B-1a cells. B-1a cells lacking BHLHE41 exhibit reduced activation in response to BCR specific stimulation, and this is not due to the general anergic nature of these cells, as they respond to anti-IgM stimulation with intracellular calcium accumulation at levels comparable with wild-type cells. The cause of reduced activation of Bhlhe41 ko B-1a cells is probably reduced IgM expression and increased expression of repressors of BCR signaling, such as the phosphatases specific for mitogen activated protein kinases, due to the lack of BHLHE41 and BHLHE40. Reduced B-1a cell numbers in dko mice are not due to the reduced proliferation of this cell population in response to stimulation, as the proliferation marker Ki67 was increased in different cell cycle stages. This is also true of the mixed fetal liver chimera mouse and is probably due to de-repression of the cell cycle regulation genes; these genes have been shown to be directly repressed by BHLHE41 and BHLHE40. This discrepancy between highly proliferating cells and their reduced final cell numbers was resolved by the finding that B-1a cells with the Bhlhe41 and Bhlhe40 dko lost their capacity for self-renewal due to apoptotic cell death. Further analysis identified reduced IL-5 receptor expression in Bhlhe41 and Bhlhe40 dko B-1a cells, which could be responsible for the reduced self-renewal capacity of B-1a cells.

In conclusion, this study established the functional significance of BHLHE41 and, to a lesser extent, BHLHE40 in B-1 cell development, maturation, and maintenance. The physiological significance of these two transcriptional repressors in the immune response against bacterial infection and that in autoimmune diseases, the two major functions of B-1a cells,²² has not yet been determined and should be further explored.