Regulation of B-1A Cells: Another Novel Function of the Basic Helix-Loop-Helix Transcriptional Regulator BHLHE41

RESEARCH HIGHLIGHT

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

Buka Samten

Cellular & Molecular Immunology (2017) 14, 802–804; doi:10.1038/cmi.2017.75; published online 4 September 2017

The basic helix-loop-helix transcription of autoimmune diseases identified in factors in regulating B-1 cells, as these factors e40 (BHLHE40) and e41 experimental autoimmune disease cells play a critical role in autoimmune (BHLHE41) were originally discovered models,9,10 such as experimental auto- diseases17 by producing natural as regulators of the proliferation and immune encephalomyelitis (EAE), the IgM-based autoreactive antibodies in differentiation of chondrocytes and nerve animal model for multiple sclerosis. addition to participating in the innate cells.1–3 Later studies showed that these However, this can be explained by the immune defense against bacterial patho- transcriptional regulators participate in a potential roles of these transcriptional gens in the absence of pathogen-specific variety of physiological processes for the regulators in the maintenance of T adaptive immunity. Recently, Kreslavsky maintenance of homeostasis, such as regulatory cells (Tregs) through the et al.18 demonstrated that BHLHE41 the regulation of endothelial cells,4 maintenance and regulation of CD25 alone and, to lesser extent, together with adipocytes,5 circadian rhythms,6 and expression in cooperation with BHLHE40 regulates the development tumor development.7 Interest in the Runx1.11 Recent studies also confirmed and maintenance of B-1a B cells. significance of these transcriptional reg- that these transcriptional regulators are B cells as mediators of humoral ulators in the immune response was expressed in T cells in a CD28 activation immunity play critical roles in protection spurred by the finding that the deletion dependent manner and participate in the against infection by producing pathogen- of BHLHE40 leads to abnormal activa- regulation of T cell cytokine production, specific antibodies and in homeostasis by tion of T cells and B cells with resistance including that of Interleukin 2 (IL-2), their regulatory mechanisms involving to activation induced cell death and Interferon gamma (IFN-ɤ)andthe production of different cytokines. accumulation of autoreactive T and B granulocyte-macrophage colony stimu- B cells can now be divided into cells resulting in autoimmune diseases in lating factor (GM-CSF).12 BHLHE41 B-1 cells and B-2 cells based on their aged animals, although the deletion of regulates IL-1β production, and IL-1β surface molecular markers, function, and BHLHE40 does not affect the develop- induced GM-CSF to produce Th17 cells developmental origin. B-1 cells originate ment and maturation of T and B cell with a pathological signature in the predominantly from the hematopoietic compartments.8 However, these initial central nervous system that is dependent stem cells of the fetal liver and yolk sac findings could contradict recent reports on BHLHE40.13 In cooperation with the and have the capacity for maintenance about the essential roles of these tran- transcription factor T-bet, BHLHE40 through self-renewal during adulthood, scriptional regulators in the development also regulates IFN-ɤ production by iNKT although the hematopoietic stem cells of cells,14 providing immunity against the bone marrow also replenish B-1 cells tumor development. Furthermore, these to a lesser extent. B-1 cells produce IgM- The Department of Pulmonary Immunology, the transcriptional regulators may also play a type autoreactive natural antibodies and, University of Texas Health Science Center, Tyler, role in the differentiation of Th2 cells by therefore, may play a role in autoim- TX 75708, USA enhanced expression of CD25.15,16 These mune diseases in addition to providing Correspondence: Dr B Samten, MD, MS, The Department of Pulmonary Immunology, The Uni- studies have established the roles of these protection against infection during the versity of Texas Health Science Center at Tyler, two transcriptional regulators in the priming period of pathogen-specific 11937 US Hwy 271, Tyler, TX 75708-3154, USA. immune response and the immuno- adaptive immunity19 and the regulation E-mail: [email protected] pathology mediated by T cells. This hints of T regulatory cells.20 B-1 cells were Received: 29 June 2017; Accepted: 30 June fi 2017 at the role of the BHLHE transcription identi ed in the early 1990s as they Regulation of B-1 B Cells by Bhlhe41 B Samten

803 expressed the common T cell surface prenatal precursor cells than in adult repertoire. Transgenic expression of pre- marker Ly-1 in mouse but called leu-2 bone marrow precursors. BHLHE41 was arranged VH12 and Vk4 genes did not in humans, also called cluster of differ- induced in both B-1 cells and follicular rescue the altered phenotype and devel- entiation (CD) 5 while also expressing (B-2) B cells in response to B cell opmental deficiencies of B-1a cells in cell surface IgM and producing IgM- stimulation, implying that BHLHE41 Bhlhe41 and Bhlhe40 dko mice, suggest- based natural antibodies. B-1 cells were may also be induced in B-1 cells during ing that an altered BCR repertoire was further subdivided into the B-1a and development due to the self-reactive responsible for the altered B-1a cell B-1b subsets after the observation that nature of this cell population. These data phenotype but not the cause of the certain B-1 cells do not express CD5 together established that BHLHE41 is a defects in the development and self- molecules, and these were named B-1b B B-1 B-cell-specific transcription factor. As renewal of B-1a cells. Determination of cells. Although the existence of B-1 cells B-1 B cells have a unique developmental the function of BHLHE41 in B-1a cells by with unique developmental and func- origin and process, BHLHE41 and its Chip-Seq and deep RNA-seq demon- tional characteristics was established in close homolog BHLHE40 were investi- strated that BHLHE41 targets 5509 genes the murine system, their human coun- gated for their roles in the development with the common E-box motif. Further terparts were considered controversial. of B-1 cells in two different experimental detailed analysis identified 41 directly However, this has been resolved settings. Bhlhe41 gene knockout (ko) activated and 90 directly repressed gene recently with the demonstration that mice demonstrated significantly reduced targets of BHLHE41 with gene repression human B-1 cells with unique surface B-1a cells in the spleen and peritoneal being the dominant feature, suggesting markers (CD20+CD27+CD43+CD70 − ) cavities compared to wild-type mice, with that BHLHE41 behaves as transcriptional are derived from Lin-CD34+CD38lo even the residual developed B-1a cells repressor in B-1a cells. B-1a cells lacking stem cells from both the umbilical cord showing reduced expression of CD5 and BHLHE41 exhibit reduced activation in and adult peripheral blood.19 This has set elevated expression of the B cell asso- response to BCR specific stimulation, and the stage for detailed studies of B-1 cell ciated molecule B220 in these mice. In this is not due to the general anergic development and function in both phy- contrast, B-1b cells were not affected by nature of these cells, as they respond to siological and pathological settings. the lack of BHLHE40. These changes anti-IgM stimulation with intracellular Initial studies of the transcriptional reg- were even more pronounced in Bhlhe41 calcium accumulation at levels compar- ulation of B-1 cells identified the critical and Bhlhe40 double knockout (dko) able with wild-type cells. The cause of roles of different transcription factors, mice. However, the deletion of Bhlhe40 reduced activation of Bhlhe41 ko B-1a including that of Arid3a, Oct2, and affected the development of B-1 cells to a cells is probably reduced IgM expression IkBNS, as well as the Lin28 miRNAs lesser degree. This was further confirmed and increased expression of repressors of and the miRNA Let-7 axis, in the regula- in studies involving chimeric mice gen- BCR signaling, such as the phosphatases tion of B-1 cell development.21 However, erated by mixing fetal livers of wild-type specific for mitogen activated protein a comprehensive understanding of the anddkoandtheirintroduction into kinases, due to the lack of BHLHE41 transcriptional regulation of B-1 cells is lethally irradiated Rag2 ko mouse in and BHLHE40. Reduced B-1a cell num- lacking. adoptive transfer experiments and subse- bers in dko mice are not due to the In a study designed to elucidate the quently following the B cell lineage cells. reduced proliferation of this cell popula- mechanisms of transcriptional regulation The fetal liver cells of dko mice did not tion in response to stimulation, as the in B-1 cells, Kreslavsky et al.18 reported produce mature B-1a cells. Lack of proliferation marker Ki67 was increased that BHLHE41 is essential for the devel- Bhlhe41 and 40 did not affect the pro- in different cell cycle stages. This is also opment and function of B-1a B cells. As a genitors but blocked transitional B-1a cell true of the mixed fetal liver chimera first step, Kreslavsky et al. identified development. Taken together, these find- mouse and is probably due to de- BHLHE41 as the most highly expressed ings support the essential roles of repression of the cell cycle regulation gene in B-1 cells following a comparison BHLHE41 and, to a lesser extent, genes; these genes have been shown to of differentially expressed genes in B-1 BHLHE40 in B-1a cell development. be directly repressed by BHLHE41 and and B-2 cells isolated from mouse spleen Bhlhe41 and Bhlhe40 dko also signifi- BHLHE40. This discrepancy between by RNA-Seq analysis. This was further cantly affected the B-1a cell repertoire. highly proliferating cells and their confirmed by the B-1 cell specificexpres- Changes were concentrated in the vari- reduced final cell numbers was resolved sion of BHLHE41 regulatory element able segments of both the heavy and light by the finding that B-1a cells with the controlled human CD2 molecules in a chains of Ig molecules with complete loss Bhlhe41 and Bhlhe40 dko lost their reporter mouse model. Interestingly, of Ighv12-3 and Igkv4-91mRNA, which capacity for self-renewal due to apoptotic BHLHE41 was also highly expressed in encode for a phosphatidylcholine (PtC) cell death. Further analysis identified theimmatureBcellsisolatedfromthe receptor that is preferentially expressed reduced IL-5 receptor expression in fetal and neonatal liver compared to the by 10% of the B-1a cells. These changes Bhlhe41 and Bhlhe40 dko B-1a cells, immature B cells from the bone marrow, in the BCR repertoire are due to defi- which could be responsible for the which is consistent with B-1 cell devel- ciencies in selection but not rearrange- reduced self-renewal capacity of opment predominantly in the fetal and ments that generate the B-1a BCR B-1a cells.

Cellular & Molecular Immunology Regulation of B-1 B Cells by Bhlhe41 B Samten

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Cellular & Molecular Immunology