The mechanism of action of Spi-B in the transcriptional activation of the interferon-α4 gene.

Plasmacytoid dendritic cells (pDCs) are characterized by an exclusive expression of nucleic acid sensing Toll-like receptor 7 (TLR7) and TLR9, and production of high amounts of type I interferon (IFN) in response to TLR7/9 signaling. This function is crucial for both antiviral immunity and the pathogenesis of autoimmune diseases. An Ets family transcription factor, i.e., Spi-B (which is highly expressed in pDCs) is required for TLR7/9 signal-induced type I IFN production and can transactivate IFN-α promoter in synergy with IFN regulatory factor-7 (IRF-7). Herein, we analyzed how Spi-B contributes to the transactivation of the Ifna4 promoter. We performed deletion and/or mutational analyses of the Ifna4 promoter and an electrophoretic mobility shift assay (EMSA) and observed an Spi-B binding site in close proximity to the IRF-7 binding site. The EMSA results also showed that the binding of Spi-B to the double-stranded DNA probe potentiated the recruitment of IRF-7 to its binding site. We also observed that the association of Spi-B with transcriptional coactivator p300 was required for the Spi-B-induced synergistic enhancement of the Ifna4 promoter activity by Spi-B. These results clarify the molecular mechanism of action of Spi-B in the transcriptional activation of the Ifna4 promoter.

TNF-α augments RANKL-dependent intestinal M cell differentiation in enteroid cultures.

Microfold (M) cells are phagocytic intestinal epithelial cells in the follicle-associated epithelium of Peyer's patches that transport particulate antigens from the gut lumen into the subepithelial dome. Differentiation of M cells from epithelial stem cells in intestinal crypts requires the cytokine receptor activator of NF-κB ligand (RANKL) and the transcription factor Spi-B. We used three-dimensional enteroid cultures established with small intestinal crypts from mice as a model system to investigate signaling pathways involved in M cell differentiation and the influence of other cytokines on RANKL-induced M cell differentiation. Addition of RANKL to enteroids induced expression of multiple M cell-associated genes, including Spib, Ccl9 [chemokine (C-C motif) ligand 9], Tnfaip2 (TNF-α-induced protein 2), Anxa5 (annexin A5), and Marcksl1 (myristoylated alanine-rich protein kinase C substrate) in 1 day. The mature M cell marker glycoprotein 2 (Gp2) was strongly induced by 3 days and expressed by 11% of cells in enteroids. The noncanonical NF-κB pathway was required for RANKL-induced M cell differentiation in enteroids, as addition of RANKL to enteroids from mice with a null mutation in the mitogen-activated protein kinase kinase kinase 14 (Map3k14) gene encoding NF-κB-inducing kinase failed to induce M cell-associated genes. While the cytokine TNF-α alone had little, if any, effect on expression of M cell-associated genes, addition of TNF-α to RANKL consistently resulted in three- to sixfold higher levels of multiple M cell-associated genes than RANKL alone. One contributing mechanism is the rapid induction by TNF-α of Relb and Nfkb2 (NF-κB subunit 2), genes encoding the two subunits of the noncanonical NF-κB heterodimer. We conclude that endogenous activators of canonical NF-κB signaling present in the gut-associated lymphoid tissue microenvironment, including TNF-α, can play a supportive role in the RANKL-dependent differentiation of M cells in the follicle-associated epithelium.

Polydatin alleviates sepsis­induced acute lung injury via downregulation of Spi­B.

Sepsis-induced acute lung injury (ALI) is related to the dysregulation of inflammatory responses. Polydatin supplement was reported to exhibit anti-inflammatory effects in several diseases. The present study aimed to investigate the role of polydatin in sepsis-induced ALI. A cecum ligation and puncture (CLP)-induced mouse ALI model was established first and the pathological changes of lung tissues were assessed using hematoxylin and eosin staining. Meanwhile, to mimic sepsis-induced ALI in vitro, pulmonary microvascular endothelial cells (PMVECs) were treated with lipopolysaccharide (LPS). Pro-inflammatory cytokines levels were measured in lung tissues and PMVECs using ELISA. Reverse transcription-quantitative PCR was used to measure the mRNA levels of Spi-B in lung tissues and PMVECs. Moreover, the expression levels of Spi-B, p-PI3K, p-Akt, and p-NF-κB in lung tissues and PMVECs were determined using western blotting. The data revealed that polydatin attenuated CLP-induced lung injury and inhibited sepsis-induced inflammatory responses in mice. Furthermore, polydatin significantly inhibited the expression of Spi-B, p-PI3K, p-Akt, and p-NF-κB in lung tissues of mice subjected to CLP-induced ALI, while this phenomenon was reversed through Spi-B overexpression. Consistently, the anti-inflammatory effect of polydatin was abolished by Spi-B overexpression. Taken together, the current findings revealed that polydatin alleviated sepsis-induced ALI via the downregulation of Spi-B.

Spi-B alleviates food allergy by securing mucosal barrier and immune tolerance in the intestine.

Food allergy is a type I allergic reaction induced by mast cells and is mainly activated by allergen-specific immunoglobulin (Ig)E. Spi-B is an E26-transformation-specific (Ets) family transcription factor essential for the differentiation and functional maturation of several immune cell subsets, including mast cells. However, the possible involvement of Spi-B in food allergy remains unclear. In this study, we found that Spi-B-deficient mice were highly susceptible to food allergy to ovalbumin (OVA), as indicated by the exacerbation of diarrhea and elevation of serum IgE levels. These pathological changes were associated with enhanced mast cell infiltration into the intestinal lamina propria. Activation of mast cells in the intestinal mucosa was observed in Spib -/- mice, even under physiological conditions. Accordingly, Spi-B deficiency increased the translocation of fluorescently labeled dextran from the lumen to the serum, suggesting increased intestinal permeability in Spib -/- mice. Moreover, Spib -/- mice showed defects in oral tolerance induction to OVA. These data illustrate that Spi-B suppresses the development of food allergies by controlling the activation of intestinal mast cells and by inducing immune tolerance to food allergens.

Spi-B Promotes the Recruitment of Tumor-Associated Macrophages via Enhancing CCL4 Expression in Lung Cancer.

Tumor immune escape plays a critical role in malignant tumor progression and leads to the failure of anticancer immunotherapy. Spi-B, a lymphocyte lineage-specific Ets transcription factor, participates in mesenchymal invasion and favors metastasis in human lung cancer. However, the mechanism through which Spi-B regulates the tumor immune environment has not been elucidated. In this study, we demonstrated that Spi-B enhanced the infiltration of tumor-associated macrophages (TAMs) in the tumor microenvironment using subcutaneous mouse models and clinical samples of human lung cancer. Spi-B overexpression increased the expression of TAM polarization- and recruitment-related genes, including CCL4. Moreover, deleting CCL4 inhibited the ability of Spi-B promoting macrophage infiltration. These data suggest that Spi-B promotes the recruitment of TAMs to the tumor microenvironment via upregulating CCL4 expression, which contributes to the progression of lung cancer.

Opposing Roles for the Related ETS-Family Transcription Factors Spi-B and Spi-C in Regulating B Cell Differentiation and Function.

Generation of specific antibodies during an immune response to infection or vaccination depends on the ability to rapidly and accurately select clones of antibody-secreting B lymphocytes for expansion. Antigen-specific B cell clones undergo the cell fate decision to differentiate into antibody-secreting plasma cells, memory B cells, or germinal center B cells. The E26-transformation-specific (ETS) transcription factors Spi-B and Spi-C are important regulators of B cell development and function. Spi-B is expressed throughout B cell development and is downregulated upon plasma cell differentiation. Spi-C is highly related to Spi-B and has similar DNA-binding specificity. Heterozygosity for Spic rescues B cell development and B cell proliferation defects observed in Spi-B knockout mice. In this study, we show that heterozygosity for Spic rescued defective IgG1 secondary antibody responses in Spib-/- mice. Plasma cell differentiation was accelerated in Spib-/- B cells. Gene expression, ChIP-seq, and reporter gene analysis showed that Spi-B and Spi-C differentially regulated Bach2, encoding a key regulator of plasma cell and memory B cell differentiation. These results suggest that Spi-B and Spi-C oppose the function of one another to regulate B cell differentiation and function.

Janus Kinase Mutations in Mice Lacking PU.1 and Spi-B Drive B Cell Leukemia through Reactive Oxygen Species-Induced DNA Damage.

Precursor B cell acute lymphoblastic leukemia (B-ALL) is caused by genetic lesions in developing B cells that function as drivers for the accumulation of additional mutations in an evolutionary selection process. We investigated secondary drivers of leukemogenesis in a mouse model of B-ALL driven by PU.1/Spi-B deletion (Mb1-CreΔPB). Whole-exome-sequencing analysis revealed recurrent mutations in Jak3 (encoding Janus kinase 3), Jak1, and Ikzf3 (encoding Aiolos). Mutations with a high variant-allele frequency (VAF) were dominated by C→T transition mutations that were compatible with activation-induced cytidine deaminase, whereas the majority of mutations, with a low VAF, were dominated by C→A transversions associated with 8-oxoguanine DNA damage caused by reactive oxygen species (ROS). The Janus kinase (JAK) inhibitor ruxolitinib delayed leukemia onset, reduced ROS and ROS-induced gene expression signatures, and altered ROS-induced mutational signatures. These results reveal that JAK mutations can alter the course of leukemia clonal evolution through ROS-induced DNA damage.

Intestinal M cells.

We have an enormous number of commensal bacteria in our intestine, moreover, the foods that we ingest and the water we drink is sometimes contaminated with pathogenic microorganisms. The intestinal epithelium is always exposed to such microbes, friend or foe, so to contain them our gut is equipped with specialized gut-associated lymphoid tissue (GALT), literally the largest peripheral lymphoid tissue in the body. GALT is the intestinal immune inductive site composed of lymphoid follicles such as Peyer's patches. M cells are a subset of intestinal epithelial cells (IECs) residing in the region of the epithelium covering GALT lymphoid follicles. Although the vast majority of IEC function to absorb nutrients from the intestine, M cells are highly specialized to take up intestinal microbial antigens and deliver them to GALT for efficient mucosal as well as systemic immune responses. I will discuss recent advances in our understanding of the molecular mechanisms of M-cell differentiation and functions.

The Mechanisms of M-cell Differentiation.

Intestinal M (microfold or membranous) cells are an enigmatic lineage of intestinal epithelial cells that initiate mucosal immune responses through the uptake and transcytosis of luminal antigens. Due to their rarity, the mechanisms of M-cell function and differentiation are poorly understood. To overcome this problem, experimental strategies to enrich for M-cells have been established. Transcriptome analyses have provided valuable insight, especially on the receptors for antigen uptake, and such studies have broadened our knowledge of M-cell function. In another line of investigation, we and others have begun to dissect the molecular pathways of M-cell differentiation. Among them, receptor activator of NF-κB ligand (RANKL) has been identified as an essential factor for M-cell differentiation. We have focused on the M-cell inducible activity of RANKL and have been able to observe temporal transitions during M-cell differentiation by using in vivo ectopic M-cell differentiation induced by exogenous RANKL treatment. We have found that the ets-family transcription factor Spi-B is essential for functional maturation of M cells. In the absence of Spi-B, the immune response to Salmonella Typhimurium is severely impaired, suggesting that M cells are important for maintaining intestinal homeostasis.