Benchmarking differential expression, imputation and quantification methods for proteomics data.

Data analysis is a critical part of quantitative proteomics studies in interpreting biological questions. Numerous computational tools for protein quantification, imputation and differential expression (DE) analysis were generated in the past decade and the search for optimal tools is still going on. Moreover, due to the rapid development of RNA sequencing (RNA-seq) technology, a vast number of DE analysis methods were created for that purpose. The applicability of these newly developed RNA-seq-oriented tools to proteomics data remains in doubt. In order to benchmark these analysis methods, a proteomics dataset consisting of proteins derived from humans, yeast and drosophila, in defined ratios, was generated in this study. Based on this dataset, DE analysis tools, including microarray- and RNA-seq-based ones, imputation algorithms and protein quantification methods were compared and benchmarked. Furthermore, applying these approaches to two public datasets showed that RNA-seq-based DE tools achieved higher accuracy (ACC) in identifying DEPs. This study provides useful guidelines for analyzing quantitative proteomics datasets. All the methods used in this study were integrated into the Perseus software, version 2.0.3.0, which is available at https://www.maxquant.org/perseus.

Marginal Zone B Cells Assist With Neutrophil Accumulation to Fight Against Systemic Staphylococcus aureus Infection.

In addition to regulating immune responses by producing antibodies that confer humoral immunity, B cells can also affect these responses by producing cytokines. How B cells participate in the clearance of pathogenic infections via functions other than the production of pathogen-specific antibodies is still largely unknown. Marginal zone (MZ) B cells can quickly respond to bacterial invasion by providing the initial round of antibodies. After a bloodborne bacterial infection, neutrophils promptly migrate to the MZ. However, the mechanisms regulating neutrophil accumulation in the MZ during the initial phase of infection also remain obscure. Here, we found that MZ B cell-deficient mice are more susceptible to systemic Staphylococcus aureus (S. aureus) infection compared with wildtype mice. The expression levels of interleukin (IL)-6 and CXCL1/CXCL2 in MZ B cells increased significantly in mice at 3-4 h after infection with S. aureus, then decreased at 24 h post-infection. After systemic S. aureus infection, splenic neutrophils express increased CXCR2 levels. Our results from confocal microscopy imaging of thick-section staining demonstrate that neutrophils in wildtype mice form cell clusters and are in close contact with MZ B cells at 3 h post-infection. This neutrophil cluster formation shortly after infection was diminished in both MZ B cell-deficient mice and IL-6-deficient mice. Blocking the action of CXCL1/CXCL2 by injecting anti-CXCL1 and anti-CXCL2 antibodies 1 h before S. aureus infection significantly suppressed the recruitment of neutrophils to the MZ at 3 h post-infection. Compared with peptidoglycan stimulation alone, peptidoglycan stimulation with neutrophil co-culture further enhanced MZ B-cell activation and differentiation. Using a Förster resonance energy transfer by fluorescence lifetime imaging (FLIM-FRET) analysis, we observed evidence of a direct interaction between neutrophils and MZ B cells after peptidoglycan stimulation. Furthermore, neutrophil depletion in mice resulted in a reduced production of S. aureus-specific immunoglobulin (Ig)M at 24 h post-infection. Together, our results demonstrate that MZ B cells regulate the rapid neutrophil swarming into the spleen during the early phase of systemic S. aureus infection. Interaction with neutrophils assists MZ B cells with their differentiation into IgM-secreting cells and contributes to the clearance of systemic bacterial infections.

HLA-B27-mediated activation of TNAP phosphatase promotes pathogenic syndesmophyte formation in ankylosing spondylitis.

Ankylosing spondylitis (AS) is a type of axial inflammation. Over time, some patients develop spinal ankylosis and permanent disability; however, current treatment strategies cannot arrest syndesmophyte formation completely. Here, we used mesenchymal stem cells (MSCs) from AS patients (AS MSCs) within the enthesis involved in spinal ankylosis to delineate that the HLA-B27-mediated spliced X-box-binding protein 1 (sXBP1)/retinoic acid receptor-ß (RARB)/tissue-nonspecific alkaline phosphatase (TNAP) axis accelerated the mineralization of AS MSCs, which was independent of Runt-related transcription factor 2 (Runx2). An animal model mimicking AS pathological bony appositions was established by implantation of AS MSCs into the lumbar spine of NOD-SCID mice. We found that TNAP inhibitors, including levamisole and pamidronate, inhibited AS MSC mineralization in vitro and blocked bony appositions in vivo. Furthermore, we demonstrated that the serum bone-specific TNAP (BAP) level was a potential prognostic biomarker to predict AS patients with a high risk for radiographic progression. Our study highlights the importance of the HLA-B27-mediated activation of the sXBP1/RARB/TNAP axis in AS syndesmophyte pathogenesis and provides a new strategy for the diagnosis and prevention of radiographic progression of AS.

Blimp-1 Contributes to the Development and Function of Regulatory B Cells.

Regulatory B cells (Bregs) are a B cell subset that plays a suppressive role in immune responses. The CD19+CD1dhiCD5+ Bregs that can execute regulatory functions via secreting IL-10 are defined as B10 cells. Bregs suppress autoimmune and inflammatory diseases, whereas they exacerbate infectious diseases caused by bacteria, viruses, or parasites. Notably, the molecular mechanisms regulating the development and functions of Bregs are still largely unknown. Furthermore, the biological impact of Bregs in fungal infection has not yet been demonstrated. Here, we compared the gene expression profiles of IL-10-producing and -non-producing mouse splenic B cells stimulated with lipopolysaccharide (LPS) or anti-CD40 antibody. Blimp-1, a transcription factor known to be critical for plasma cell differentiation, was found to be enriched in the IL-10-producing B cells. The frequency of Blimp-1+ B10 cells was increased in LPS-treated mice and in isolated B10 cells that were stimulated with LPS. Surprisingly, B cell-specific Blimp-1 knockout (Cko) mice, generated by CD19 promoter driven Cre recombinase-dependent deletion of Prdm1 (gene encoding Blimp-1), showed higher frequencies of B10 cells both in the steady state and following injection with LPS, as compared with control littermates. However, B10 cells lacking Blimp-1 failed to efficiently suppress the proliferation of naïve CD4+ T cells primed with anti-CD3 and anti-CD28 antibodies. B10 cells can be stimulated for further differentiation into plasmablasts, and a subset of plasmablasts express IL-10. We found that B10 cells from Cko mice failed to generate both IL-10-non-producing and IL-10-producing plasmablasts. Mechanistically, we found that Blimp-1 can directly suppress Il-10, whereas, in the presence of activated STAT3, Blimp-1 works together with activated STAT3 to upregulate Il-10. Moreover, we also found that B10 cells improve the clearance of Candida albicans infection but worsen the infection mortality. Notably, a lack of Blimp-1 in B10 cells did not change these effects of adoptively transferred B10 cells on fungal infections. Together, our data show that Blimp-1 regulates the generation, differentiation, and IL-10 production of Bregs.

The KDM4A/KDM4C/NF-κB and WDR5 epigenetic cascade regulates the activation of B cells.

T follicular helper (Tfh) cell-derived signals promote activation and proliferation of antigen-primed B cells. It remains unclear whether epigenetic regulation is involved in the B cell responses to Tfh cell-derived signals. Here, we demonstrate that Tfh cell-mimicking signals induce the expression of histone demethylases KDM4A and KDM4C, and the concomitant global down-regulation of their substrates, H3K9me3/me2, in B cells. Depletion of KDM4A and KDM4C potentiates B cell activation and proliferation in response to Tfh cell-derived signals. ChIP-seq and de novo motif analysis reveals NF-κB p65 as a binding partner of KDM4A and KDM4C. Their co-targeting to Wdr5, a MLL complex member promoting H3K4 methylation, up-regulates cell cycle inhibitors Cdkn2c and Cdkn3. Thus, Tfh cell-derived signals trigger KDM4A/KDM4C - WDR5 - Cdkn2c/Cdkn3 cascade in vitro, an epigenetic mechanism regulating proper proliferation of activated B cells. This pathway is dysregulated in B cells from systemic lupus erythematosus patients and may represent a pathological link.

Uncovering MicroRNA Regulatory Hubs that Modulate Plasma Cell Differentiation.

Using genome-wide approaches, we studied the microRNA (miRNA) expression profile during human plasma cell (PC) differentiation induced by stimulation of human blood B cells with T follicular helper cell-dependent signals. Combining the profiles of differentially expressed genes in PC differentiation with gene ontology (GO) analysis revealed that a significant group of genes involved in the transcription factor (TF) activity was preferentially changed. We thus focused on studying the effects of differentially expressed miRNAs on several key TFs in PC differentiation. Cohorts of differentially expressed miRNAs cooperating as miRNA hubs were predicted and validated to modulate key TFs, including a down-regulated miRNA hub containing miR-101-3p, -125b-5p, and -223-3p contributing to induction of PRDM1 as well as an up-regulated miRNA hub containing miR-34a-5p, -148a-3p, and -183-5p suppressing BCL6, BACH2, and FOXP1. Induced expression of NF-κB and PRDM1 during PC differentiation controlled the expression of up- and down-regulated miRNA hubs, respectively. Co-expression of miR-101-3p, -125b-5p, and -223-3p in stimulated B cells showed synergistic effects on inhibition of PC formation, which can be rescued by re-introduction of PRDM1. Together, we catalogue the complex roadmap of miRNAs and their functional interplay in collaboratively directing PC differentiation.

Transcription factor ABF-1 suppresses plasma cell differentiation but facilitates memory B cell formation.

Ag-primed B cells that result from an immune response can form either memory B cells or Ab-secreting plasma cells; however, the molecular machinery that controls this cellular fate is poorly understood. In this study, we show that activated B cell factor-1 (ABF-1), which encodes a basic helix-loop-helix transcriptional repressor, participates in this regulation. ABF-1 was prevalently expressed in purified memory B cells and induced by T follicular helper cell-mediated signals. ABF-1 expression declined by the direct repression of B lymphocyte-induced maturation protein-1 during differentiation. Ectopic expression of ABF-1 reduced the formation of Ab-secreting cells in an in vitro differentiation system of human memory B cells. Accordingly, knockdown of ABF-1 potentiates the formation of Ab-secreting cells. A transgenic mouse that expresses inducible ABF-1 in a B cell-specific manner was generated to demonstrate that the formation of germinal center and memory B cells was augmented by induced ABF-1 in an immune response, whereas the Ag-specific plasma cell response was dampened. This effect was associated with the ability of ABF-1 to limit cell proliferation. Together, our results demonstrate that ABF-1 facilitates formation of memory B cells but prevents plasma cell differentiation.

Suppression of the SOX2 neural effector gene by PRDM1 promotes human germ cell fate in embryonic stem cells.

The mechanisms of transcriptional regulation underlying human primordial germ cell (PGC) differentiation are largely unknown. The transcriptional repressor Prdm1/Blimp-1 is known to play a critical role in controlling germ cell specification in mice. Here, we show that PRDM1 is expressed in developing human gonads and contributes to the determination of germline versus neural fate in early development. We show that knockdown of PRDM1 in human embryonic stem cells (hESCs) impairs germline potential and upregulates neural genes. Conversely, ectopic expression of PRDM1 in hESCs promotes the generation of cells that exhibit phenotypic and transcriptomic features of early PGCs. Furthermore, PRDM1 suppresses transcription of SOX2. Overexpression of SOX2 in hESCs under conditions favoring germline differentiation skews cell fate from the germline to the neural lineage. Collectively, our results demonstrate that PRDM1 serves as a molecular switch to modulate the divergence of neural or germline fates through repression of SOX2 during human development.

Inducible deletion of the Blimp-1 gene in adult epidermis causes granulocyte-dominated chronic skin inflammation in mice.

B lymphocyte-induced maturation protein-1 (Blimp-1) is a transcriptional repressor important for the differentiation and function of several types of immune cells. Because skin serves as a physical barrier and acts as an immune sentinel, we investigated whether Blimp-1 is involved in epidermal immune function. We show that Blimp-1 expression is reduced in skin lesions of some human eczema samples and in stimulated primary keratinocytes. Epidermal-specific deletion of PR domain containing 1, with ZNF domain (Prdm1), the gene encoding Blimp-1, in adult mice caused spontaneously inflamed skin characterized by massive dermal infiltration of neutrophils/macrophages and development of chronic inflammation associated with higher levels of cytokines/chemokines, including granulocyte colony-stimulating factor (G-CSF), and enhanced myelopoiesis in bone marrow. Deletion of Prdm1 in the epidermis of adult mice also led to stronger inflammatory reactions in a tape-stripping test and in a disease model of contact dermatitis. The elevated G-CSF produced by keratinocytes after deletion of Prdm1 in vitro was mediated by the transcriptional activation of FBJ osteosarcoma oncogene (Fos) and fos-like antigen 1 (Fosl1). Systemic increases in G-CSF contributed to the inflammatory responses, because deletion of the G-CSF gene [colony stimulating factor 3, (Csf3)] prevented neutrophilia and partially ameliorated the inflamed skin in Prdm1-deficient mice. Our findings indicate a previously unreported function for Blimp-1 in restraining steady-state epidermal barrier immunity.

SUMOylation of Blimp-1 is critical for plasma cell differentiation.

Transcriptional repressor B lymphocyte-induced maturation protein-1 (Blimp-1) is a master regulator of plasma cell differentiation. Here we show that Blimp-1 is covalently modified by SUMO1 at lysine 816, a modification mediated by SUMO E3 ligase PIAS1. Mutation of Blimp-1 lysine 816 reduces transcriptional repression--correlating with a reduced interaction with a histone deacetylase, HDAC2--and impairs differentiation of antibody-secreting cells. Thus, the SUMO pathway critically regulates Blimp-1 function during plasma cell differentiation.

Curcumin upregulates insulin-like growth factor binding protein-5 (IGFBP-5) and C/EBPalpha during oral cancer suppression.

Curcumin is a common food ingredient derived from the plant Curcuma longa and is a potent drug against tumorigenesis. Both insulin-like growth factor binding protein-5 (IGFBP-5) and CCAAT/enhancer-binding protein alpha (C/EBPalpha) are suppressors of head and neck carcinogenesis. We identified curcumin as an inducer of IGFBP-5 expression in multiple types of oral keratinocytes; furthermore, curcumin induces IGFBP-5 promoter activity in SAS oral cancer cells. Promoter deletion mapping identified a region (nt -71 to nt -59 relative to the transcription start site) as containing a C/EBPalpha-binding element that is indispensable for curcumin-mediated IGFBP-5 upregulation. Chromatin immunoprecipitation assays revealed that in vivo binding of C/EBPalpha to this region was remarkably increased in the presence of curcumin. Curcumin increased nuclear C/EBPalpha expression and IGFBP-5 expression through p38 activation and this was abrogated by SB203580 treatment. Furthermore, MKK6 expression activated p38 and C/EBPalpha, increasing IGFBP-5 promoter activity and expression. Finally, curcumin-induced IGFBP-5 expression is associated with the suppression of xenograft tumorigenesis in mice due to oral cancer cells. We conclude that curcumin activates p38, which, in turn, activates the C/EBPalpha transactivator by interacting with binding elements in the IGFBP-5 promoter. The consequential upregulation of C/EBPalpha and IGFBP-5 by curcumin is crucial to the suppression of oral carcinogenesis.

Galectin-1 promotes immunoglobulin production during plasma cell differentiation.

Galectin-1, a beta-galactoside-binding soluble lectin, has been implicated in regulating immune system homeostasis. We investigated the function of galectin-1 in plasma cell differentiation and found that it is induced in primary murine and human differentiating B cells. B lymphocyte-induced maturation protein-1 (Blimp-1), a master regulator for plasma cell differentiation, was necessary and sufficient to induce galectin-1 expression. Notably, ectopic expression of galectin-1 in mature B cells increased Ig mu-chain transcript levels as well as the overall level of Ig production. This function of galectin-1 was dependent on binding to cell surface glycosylated counter receptors, as a galectin-1 mutant deficient in beta-galactoside binding showed diminished ability to promote Ig production. Extracellular galectin-1 bound more significantly to mature B cells than to plasma cells. Lastly, we found that the sugar compound N-acetyllactosamine blocked the binding of galectin-1 to murine splenic B cells and inhibited their differentiation. Taken together, these data are the first to demonstrate a role for galectin-1 in promoting Ig production during plasma cell differentiation.