SIRPB1 Human

What is the basic structure and function of SIRPB1 in humans?

SIRPB1 encodes SIRPβ, a cell surface glycoprotein that belongs to both the immunoglobulin superfamily and the signal regulatory protein family. Structurally, SIRPβ contains three extracellular Ig-like domains, with a transmembrane region containing a basic amino acid side chain that facilitates interaction with the activating adaptor protein DAP12 .

Unlike its inhibitory counterpart SIRPα (which contains immunoreceptor tyrosine-based inhibitory motifs or ITIMs), SIRPβ functions as an activating receptor. When engaged, SIRPβ triggers DAP12 signaling through its immunoreceptor tyrosine-based activation motif (ITAM), leading to downstream signal transduction including Syk phosphorylation and activation of MAPK pathways . This ultimately enhances phagocytic activity in macrophages and promotes inflammation through cytokine production.

How is SIRPB1 expression regulated in different human tissues?

SIRPB1 expression is predominantly observed in cells of myeloid lineage, including monocytes, macrophages, and dendritic cells . Analysis of expression data from the GTEx database (1152 normal samples) and TCGA database (670 glioma samples) reveals tissue-specific regulation patterns .

Research indicates that SIRPB1 expression is significantly upregulated in inflammatory conditions. For instance, in Crohn's disease patients with active disease (A-CD), SIRPB1 expression is considerably elevated in ileocolonic tissue compared to both healthy individuals and patients with CD in remission (R-CD) . This suggests that inflammatory stimuli may regulate SIRPB1 expression.

Additionally, DAP12 expression appears to be crucial for the surface presentation of SIRPB1, indicating post-translational regulation of this protein . Abnormal expression has been documented in various pathological conditions, most notably in gliomas, where significantly higher levels of SIRPB1 expression have been found compared to normal brain tissue .

What signaling pathways does SIRPB1 activate in human immune cells?

SIRPB1 triggers several key signaling cascades when activated:

• DAP12-Syk Pathway: SIRPB1 stimulates tyrosine phosphorylation of its adaptor protein DAP12, which subsequently leads to Syk phosphorylation .

• MAPK Signaling: Activated Syk promotes mitogen-activated protein kinase (MAPK) pathway activation, contributing to cellular responses .

• Calcium Signaling: Research demonstrates that SIRPB1 activation leads to calcium signaling, particularly in myeloid-derived cells .

• NF-κB Pathway: SIRPB1 activation promotes NF-κB nuclear translocation and transcriptional activity, as shown in THP-1 cell experiments .

• Jak2-Akt Signaling: The frameshift variant in SIRPB1 associated with Crohn's disease induces tyrosine phosphorylation of not only Syk but also Akt and Jak2 .

These pathways collectively contribute to increased production of pro-inflammatory cytokines including IL-1β, TNF-α, and IL-6, as well as enhanced phagocytic activity in macrophages .

What role does SIRPB1 play in inflammatory bowel disease, particularly Crohn's disease?

SIRPB1 has been implicated as a susceptibility gene for Crohn's disease (CD), particularly in Han Chinese populations. A rare frameshift variant (c.1143_1144insG; p. Leu381_Leu382fs) in SIRPB1 was found to significantly increase the risk of developing CD (p = 0.03, OR 4.59, 95% CI 0.98–21.36) .

This gain-of-function frameshift mutation alters the amino acid sequence in both the transmembrane and cytoplasmic domains of the SIRPβ protein, leading to:

• Elevated expression of SIRPB1 at both mRNA and protein levels

• Increased activation of DAP12 and phosphorylation of Syk, Akt, and Jak2

• Enhanced activation of NF-κB in macrophages

• Increased synthesis of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6

Immunohistochemistry analysis of ileocolonic tissue revealed that CD patients carrying the SIRPB1 variant exhibited significantly higher levels of SIRPβ and its adaptor protein DAP12 compared to CD patients with wild-type SIRPB1. This suggests that the variant contributes to inflammation exacerbation in the intestinal tissue of CD patients .

How is SIRPB1 implicated in the pathogenesis of human gliomas?

SIRPB1 shows significantly elevated expression in human gliomas compared to normal brain tissue, and this overexpression correlates with poor patient survival . The relationship between SIRPB1 and glioma pathogenesis involves several mechanisms:

These findings indicate that glioma cells can be activated by macrophages via SIRPB1, subsequently reprogramming the tumor microenvironment in ways that promote tumor progression .

What techniques are recommended for studying SIRPB1 genetic variants in human populations?

Based on successful research approaches, the following methodologies are recommended for investigating SIRPB1 genetic variants:

• Whole Genome Sequencing (WGS): Useful for initial identification of variants. The frameshift variant in Han Chinese CD patients was initially identified through WGS .

• Sanger Sequencing for Validation: After PCR amplification, WGS-identified candidate variants should be verified using Sanger sequencing. Standard PCR procedures can be conducted on systems like the Applied Biosystems Dual 96-Well GeneAmp PCR System 9,700 .

• MassARRAY for Large-scale Validation: For validation in larger cohorts, MassARRAY technology provides an efficient platform. In the SIRPB1 CD study, this technique was used to analyze 381 probands with IBD and 381 unrelated control individuals .

• Statistical Analysis: Fisher's exact testing with statistical significance set at p < 0.05 is commonly used to assess the association of variants with disease. For replication analyses, researchers should consider multiple testing correction to strengthen findings .

• Software Tools: Primer3 software for PCR primer design and Vector NTI for variant analyses have proven effective in SIRPB1 research .

When conducting genetic association studies for SIRPB1, researchers should be aware that population-specific effects may exist, as demonstrated by the frameshift variant found in Han Chinese patients. Validation across diverse populations is advisable for any newly identified variants .

What are the optimal in vitro models for investigating SIRPB1 functional mechanisms?

Several effective in vitro models have been utilized to study SIRPB1 function:

• THP-1 Cell Line: This human monocytic cell line derived from acute monocytic leukemia has proven valuable for SIRPB1 research. THP-1 cells can be differentiated into macrophage-like cells using phorbol 12-myristate 13-acetate (PMA; 100 ng/mL for 48h) .

• CRISPR/Cas9 Knockout Models: Generation of SIRPB1 knockout THP-1 cell lines using CRISPR/Cas9 technology provides an excellent system for loss-of-function studies .

• Ectopic Expression Systems: Plasmids carrying either wild-type or mutant SIRPB1 can be transfected into appropriate cell lines to study gain-of-function effects .

• Co-culture Systems: Co-cultures of macrophages (with or without SIRPB1 manipulation) and disease-relevant cells (such as glioma cells) allow for the study of intercellular interactions mediated by SIRPB1 .

• Luciferase Reporter Assays: These provide quantitative assessment of NF-κB transcriptional activity downstream of SIRPB1 signaling .

For functional readouts, researchers should consider:

• Western blotting to assess phosphorylation of downstream signals (Syk, Akt, Jak2)

• ELISA for measuring cytokine production (IL-1β, IL-6, TNF-α)

• Immunohistochemistry for tissue expression analysis

How can researchers effectively measure SIRPB1-mediated inflammatory responses?

To comprehensively evaluate SIRPB1-mediated inflammatory responses, researchers should employ a multi-faceted approach:

• Cytokine Production Analysis:

  • ELISA assays to measure secreted pro-inflammatory cytokines (IL-1β, TNF-α, IL-6, IL-8) in cell culture supernatants

  • qRT-PCR for quantifying cytokine mRNA expression levels

  • Multiplex cytokine assays for simultaneously measuring multiple inflammatory mediators

• Signaling Pathway Activation:

  • Western blotting to assess phosphorylation status of key signaling molecules:

    • p-Syk (indicator of proximal signaling)

    • p-Akt and p-Jak2 (downstream effectors)

    • NF-κB pathway components

  • Immunoprecipitation to detect protein-protein interactions between SIRPB1 and DAP12

• Transcriptional Activity Assessment:

  • Luciferase reporter assays specifically designed for NF-κB activity measurement

  • Chromatin immunoprecipitation (ChIP) to identify transcription factor binding to inflammatory gene promoters

• Functional Cellular Assays:

  • Phagocytosis assays (as SIRPB1 enhances phagocytic activity in macrophages)

  • Migration/chemotaxis assays

  • Flow cytometry to assess activation markers on immune cells

• Inhibitor Studies:

  • SYK inhibitors (such as GS9973) to confirm the dependency of inflammatory responses on SYK signaling

  • Other pathway-specific inhibitors to dissect the relative contribution of different cascades

How can SIRPB1 variants be incorporated into disease risk prediction models?

SIRPB1 variants, particularly those with established functional consequences, can be valuable components of disease risk prediction models:

• Genotype-Phenotype Correlation Analysis: For the SIRPB1 frameshift variant (c.1143_1144insG; p. Leu381_Leu382fs) in Crohn's disease, carriers showed distinct clinical characteristics. Researchers should carefully document:

  • Disease location (e.g., ileocolonic inflammation)

  • Disease behavior (penetrating vs. non-stricturing/non-penetrating)

  • Age of onset and disease duration

  • Response to therapy

• Multivariate Models: Integrate SIRPB1 variant status with other established risk factors:

  • For Crohn's disease: NOD2/CARD15 variants, ATG16L1 variants, family history

  • For gliomas: IDH mutation status, 1p/19q co-deletion, MGMT promoter methylation

• Nomogram Development: For gliomas, a nomogram incorporating SIRPB1 expression along with additional clinicopathological variables showed high prediction accuracy for patient outcomes. Similar approaches could be developed for other SIRPB1-associated diseases .

• Risk Stratification: Calculate odds ratios (OR) for specific variants, as seen with the frameshift variant in CD (OR 4.59, 95% CI 0.98–21.36), to determine the magnitude of risk conferred .

• Ethnic-Specific Considerations: As demonstrated by the Han Chinese CD association study, SIRPB1 variants may have population-specific effects. Risk models should be validated in diverse populations .

What are the potential therapeutic implications of targeting SIRPB1 in human diseases?

Given its role in inflammatory signaling and disease pathogenesis, SIRPB1 represents a promising therapeutic target with several potential approaches:

• Antibody-Based Therapies:

  • Blocking antibodies against SIRPB1 could inhibit its interaction with ligands

  • Non-activating antibodies that bind SIRPB1 without triggering downstream signaling

  • Research suggests that specific antibodies can modulate SIRPB1 activation status

• Small Molecule Inhibitors:

  • SYK inhibitors (e.g., GS9973) have shown promise in blocking SIRPB1-mediated effects in glioma models

  • DAP12-targeting compounds could potentially disrupt the SIRPB1-DAP12 interaction

  • Inhibitors of downstream pathways (MAPK, NF-κB) might mitigate SIRPB1-induced inflammation

• Gene Therapy Approaches:

  • CRISPR/Cas9 editing to correct pathogenic variants like the frameshift mutation in CD

  • RNA interference to downregulate SIRPB1 expression in relevant cell types

• Cell-Based Therapies:

  • Ex vivo modification of macrophages to modulate SIRPB1 expression or function

  • For gliomas, reprogramming tumor-associated macrophages through SIRPB1 modulation

• Combination Therapies:

  • Targeting SIRPB1 alongside other inflammatory mediators

  • In cancer contexts, combining SIRPB1 inhibition with conventional therapies to modify the tumor microenvironment

The development of SIRPB1-targeted therapies would benefit from further characterization of its exact role in different disease contexts and identification of patient subgroups most likely to respond to such interventions.

How should researchers design experiments to investigate SIRPB1 expression in human tissue samples?

When analyzing SIRPB1 expression in human tissues, consider these methodological approaches:

• Tissue Collection and Processing:

  • For immunohistochemistry (IHC): 4 μm sections of formalin-fixed paraffin-embedded (FFPE) tissue samples are recommended

  • Fresh-frozen tissues may be preferable for RNA-based analyses

  • When studying intestinal inflammation, samples from both inflamed and non-inflamed regions should be collected

• IHC Protocol Optimization:

  • Automated staining systems (e.g., BenchMark XT) provide consistent results

  • Validate antibody specificity using appropriate positive and negative controls

  • Consider multiplex IHC to simultaneously visualize SIRPB1 with DAP12 and downstream markers

• RNA Expression Analysis:

  • qRT-PCR with carefully validated primers specific for SIRPB1

  • RNA-seq for comprehensive transcriptomic profiling

  • Microarray analysis, as used in studies analyzing GEO datasets (GSE75214, GSE36807, GSE59071)

• Bioinformatic Approaches:

  • Use tools like GEO2R for analyzing publicly available gene expression data

  • Apply appropriate normalization methods accounting for tissue-specific expression patterns

  • Consider cell type deconvolution approaches, as SIRPB1 is predominantly expressed in myeloid cells

• Experimental Controls:

  • Include tissue samples from healthy individuals, patients in disease remission, and those with active disease

  • For cancer studies, incorporate matched tumor and adjacent normal tissue samples

• Validation Across Methods:

  • Confirm protein expression (IHC) findings with mRNA expression data

  • Use multiple antibodies targeting different epitopes when possible

What are the critical considerations when interpreting SIRPB1 functional assay results?

Researchers should be aware of several factors that can influence the interpretation of SIRPB1 functional studies:

• Cell Type Specificity:

  • SIRPB1 function may vary between different myeloid cell populations

  • Effects observed in THP-1 cells might not fully recapitulate primary human macrophages

  • Expression pattern differences between monocytes, macrophages, and dendritic cells should be considered

• DAP12 Dependency:

  • SIRPB1 function is intimately linked to DAP12 expression

  • Always assess DAP12 levels when interpreting SIRPB1 functional data

  • Consider the ratio of SIRPB1 to DAP12 rather than absolute SIRPB1 expression alone

• Activation Status:

  • Determine whether your experimental system is evaluating basal or stimulated SIRPB1 activity

  • Different activation methods (antibody crosslinking, ligand stimulation) may yield different results

  • Pre-stimulation with LPS or other TLR ligands can alter SIRPB1-mediated responses

• Pathway Crosstalk:

  • SIRPB1 signaling intersects with multiple pathways (MAPK, NF-κB, etc.)

  • Use pathway-specific inhibitors to delineate which effects are directly SIRPB1-dependent

  • Consider compensatory mechanisms that may mask phenotypes in knockout models

• Variant-Specific Effects:

  • Different SIRPB1 variants (e.g., the frameshift variant in CD) may have distinct functional consequences

  • Compare mutant vs. wild-type in the same experimental system

  • Examine both expression level changes and functional alterations for each variant

• Temporal Dynamics:

  • SIRPB1-mediated responses may evolve over time

  • Design time-course experiments to capture both early (signaling) and late (gene expression) events

  • Consider persistence vs. resolution of inflammatory responses

What are the most promising unexplored areas for SIRPB1 research in human diseases?

Several promising research directions could significantly advance our understanding of SIRPB1 biology:

• Broader Disease Associations:

  • While SIRPB1 has been implicated in Crohn's disease and gliomas, its role in other inflammatory and neoplastic conditions remains largely unexplored

  • Systematic analysis across autoimmune disorders, other cancers, and neuroinflammatory conditions is warranted

• Ligand Identification:

  • Unlike SIRPα, which binds CD47, the physiological ligands for SIRPB1 remain poorly characterized

  • Identification of these ligands would provide crucial insights into SIRPB1 activation in different tissues

• Structural Biology:

  • Detailed structural analysis of SIRPB1, particularly examining how the frameshift variant alters protein conformation

  • Crystal structures of SIRPB1-DAP12 complexes could inform targeted therapeutic design

• Single-Cell Analysis:

  • Characterizing SIRPB1 expression and function at single-cell resolution in various tissues

  • Delineating cell-specific roles in complex inflammatory environments

  • Spatial transcriptomics to understand SIRPB1-expressing cells in their tissue context

• In Vivo Models:

  • Development of SIRPB1 knockout or knock-in mice to study systemic effects

  • Humanized mouse models expressing human SIRPB1 variants

  • Patient-derived xenograft models for cancer studies

• Epigenetic Regulation:

  • Investigation of how SIRPB1 expression is regulated at the epigenetic level in different pathological states

  • Identification of transcription factors controlling SIRPB1 expression

• Therapeutic Development:

  • Design and testing of specific SIRPB1 inhibitors or modulators

  • Development of cell-specific delivery systems targeting SIRPB1-expressing macrophages in disease tissues

How might conflicting data on SIRPB1 function be reconciled in the scientific literature?

When confronted with seemingly contradictory findings about SIRPB1, researchers should consider these approaches to reconciliation:

• Cell Type and Context Dependency:

  • SIRPB1 may have divergent functions in different cell types or tissue environments

  • Systematically compare experimental conditions across studies, noting cell types, activation states, and environmental factors

  • Design experiments explicitly testing SIRPB1 function across multiple cell types in parallel

• Species-Specific Differences:

  • SIRPB1 biology may differ between humans and model organisms

  • Mouse studies may not fully recapitulate human SIRPB1 function due to evolutionary divergence

  • When possible, confirm key findings in human primary cells or tissue samples

• Isoform Variations:

  • Multiple SIRPB1 isoforms may exist with distinct functions

  • Clearly define which isoform is being studied and use isoform-specific reagents

  • Catalog expression patterns of different isoforms across tissues and disease states

• Technical Considerations:

  • Antibody specificity issues could lead to discrepant results

  • Validate key reagents across multiple experimental platforms

  • Consider whether differing methods of activation (antibody crosslinking vs. physiological ligands) explain contradictory outcomes

• Genetic Background Effects:

  • The frameshift variant in SIRPB1 was identified in Han Chinese CD patients and might have different penetrance in other populations

  • Consider how genetic background might modify SIRPB1 function when integrating data from diverse studies

• Disease Stage and Severity:

  • SIRPB1's role may evolve during disease progression

  • Active vs. remission phases in inflammatory conditions might associate with different SIRPB1 functions

  • Carefully document and compare disease stages across conflicting studies

• Integrated Multi-Omics Approaches:

  • Combine genomic, transcriptomic, proteomic, and functional data to develop a more complete understanding

  • Meta-analysis of existing datasets may reveal patterns not apparent in individual studies