DHRS9 Antibody

What is DHRS9 and why is it significant for immunology research?

DHRS9 (dehydrogenase/reductase 9) is a retinol dehydrogenase belonging to the short-chain dehydrogenase/reductase (SDR) family of NAD(P)(H)-dependent oxidoreductases. It has emerged as a robust marker of human regulatory macrophages (Mregs), making it particularly valuable for immunology research. DHRS9 expression is relatively specific to Mregs within the spectrum of monocyte-derived macrophage polarization states, allowing researchers to positively identify this cell population . The significance of DHRS9 lies in its stability as a marker even after inflammatory stimulation with lipopolysaccharide (LPS), which indicates that certain aspects of the Mreg phenotype remain stable despite repolarizing signals . This characteristic makes DHRS9 particularly valuable for tracking Mregs in experimental and potentially therapeutic contexts, especially in transplantation immunology where these cells show promise as adjunct immunosuppressive therapy .

How does DHRS9 expression distinguish Mregs from other macrophage populations?

DHRS9 expression provides a distinctive molecular signature that differentiates Mregs from other macrophage polarization states. Quantitative PCR analysis has demonstrated that high-level expression of DHRS9 mRNA is restricted to Mregs within comparative panels of macrophage types . At the protein level, immunoblotting using anti-DHRS9 antibodies confirms that DHRS9 protein expression uniquely identifies Mregs among comparator macrophages .

Furthermore, DHRS9 expression distinguishes Mregs from other tolerogenic monocyte-derived cells being developed as therapeutic products, including tolerogenic dendritic cells (Tol-DC), rapamycin-treated dendritic cells (Rapa-DC), IL-10 conditioned dendritic cells (DC-10), and prostaglandin E2-induced myeloid-derived suppressor cells . This distinction is observable at both mRNA and protein levels, with consistently higher DHRS9 expression in Mregs than in these related cell types .

Can DHRS9 be detected in tissue-resident macrophage populations?

Yes, DHRS9-expressing macrophages have been identified in human tissues. Immunohistochemical staining using the anti-DHRS9 monoclonal antibody (clone 3C6) has revealed a subpopulation of DHRS9-expressing cells with typical macrophage morphology in human spleen sections . These cells appear to be most prevalent in the subcapsular red pulp of the spleen . This finding suggests that DHRS9 expression by in vitro generated Mregs is not merely an artifact of cell culture conditions but may reflect a naturally occurring macrophage phenotype.

While these splenic DHRS9-positive macrophages cannot definitively be considered the physiological counterpart of in vitro derived human Mregs, their existence suggests potential biological relevance of DHRS9 expression in tissue-resident macrophage populations . This observation opens avenues for further research into the relationship between in vitro generated Mregs and naturally occurring regulatory macrophage populations.

What methods are available for detecting DHRS9 expression in experimental samples?

Multiple complementary techniques can be employed to detect DHRS9 expression, each with specific advantages for different experimental questions. The primary methods validated in the research literature include:

Table 1: DHRS9 Primer Sequences for qPCR

For optimal results, DHRS9 signals should be normalized against GAPDH mRNA expression when performing qPCR analysis . PCR specificity should be confirmed by sequencing of amplicons to ensure target-specific amplification .

How can DHRS9 expression be analyzed by flow cytometry for single-cell characterization?

Flow cytometric analysis of DHRS9 expression provides valuable information about expression patterns at the single-cell level. A standardized protocol for DHRS9 detection by flow cytometry involves:

• Surface staining performed at 4°C in staining buffer (DPBS/1% BSA/0.02% NaN3/10% FcR-block)

• Incubation for 60 minutes using directly-conjugated antibodies at saturating concentrations

• Exclusion of dead cells by 7-AAD staining

• Data acquisition using appropriate cytometer (e.g., Calibur cytometer) and analysis software (e.g., FlowJo)

Flow cytometric analysis has revealed that DHRS9 signal is continuously distributed within Mreg populations, indicating that expression is not restricted to a subset of cells but is a characteristic of the entire Mreg population . This technique demonstrates that DHRS9 expression is significantly greater in Mregs than in either resting macrophages or IFN-γ-stimulated macrophages .

What are the recommended protocols for immunoprecipitation and Western blotting of DHRS9?

For immunoprecipitation and Western blotting of DHRS9, the following methodological approach is recommended:

• Immunoprecipitation:

  • Use Protein A/G sepharose to immunoprecipitate the target antigen

  • For specific recognition of DHRS9, mouse monoclonal antibody ASOT1 has been validated

  • The immunoprecipitated protein can be verified by MALDI-MS sequencing

• Western Blotting:

  • Follow conventional gel electrophoresis and immunoblotting methods

  • Anti-DHRS9 antibodies validated for Western blotting include:
    a) Custom-made rabbit polyclonal antibody against the N-terminal epitope (CTDPENVKRTAQWVKNQVGEKG) of DHRS9
    b) Commercial mouse monoclonal antibody (clone 3C6, Abnova)
    c) Commercial rabbit polyclonal antibody (ab98155, Abcam)

  • The expected molecular weight of DHRS9 is approximately 35 kDa

This approach has successfully demonstrated that DHRS9 protein expression is unique to Mregs within panels of comparator human macrophages and is maintained even after LPS stimulation .

How does DHRS9 expression change during Mreg development and in response to stimulation?

DHRS9 expression follows a specific pattern during Mreg development and responds differentially to various stimuli. The temporal dynamics and response patterns include:

• Developmental pattern: DHRS9 expression increases steadily throughout the transition of CD14+ monocytes to Mregs in culture .

• Effect of cytokines:

  • IFN-γ treatment on day 6 of culture further enhances DHRS9 expression in developing Mregs .

  • Treating resting macrophages with IL-4, plate-bound immunoglobulin, dexamethasone, IFN-γ, or LPS + IFN-γ does not induce DHRS9 expression to the same level as seen in Mregs .

• Stability under inflammatory conditions: Stimulating fully developed Mregs with 100 ng/mL lipopolysaccharide (LPS) for 24 hours does not extinguish DHRS9 expression, indicating phenotypic stability under inflammatory conditions .

This expression pattern suggests that DHRS9 expression is a regulated process during Mreg development and is maintained as a stable feature of the Mreg phenotype even under inflammatory conditions. Researchers hypothesize that undefined components of serum may influence DHRS9 expression during in vitro development of Mregs from monocytes .

What is the relationship between DHRS9 expression and retinoid metabolism in macrophages?

The functional implications of DHRS9 expression in human Mregs remain incompletely defined, but several lines of evidence suggest connections to retinoid metabolism:

• DHRS9 is classified as a retinol dehydrogenase of the short-chain dehydrogenase/reductase (SDR) family of NAD(P)(H)-dependent oxidoreductases .

• While Mregs express retinoid metabolizing enzymes (including DHRS9), direct evidence that Mregs generate retinoic acid (RA) is lacking .

• Certain tissue-resident macrophage populations, particularly in the gut, suppress T cell reactions and induce regulatory T cells through production of retinoic acid (RA) .

• It is speculated that ex vivo generated human Mregs may act through similar retinoic acid-dependent pathways when administered to patients, though this requires further investigation .

The investigation of DHRS9's role in retinoid metabolism in macrophages represents an important area for future research, potentially linking this marker to the functional immunoregulatory properties of Mregs.

What differences exist between human and mouse Mregs regarding DHRS9 expression?

An important species difference has been identified regarding DHRS9 as a marker of regulatory macrophages:

• DHRS9 is a robust marker for human Mregs, with high specificity and stability across various experimental conditions .

• In contrast, Dhrs9 was found not to be an informative marker of mouse Mregs .

This species difference highlights the importance of selecting appropriate markers when working with regulatory macrophages in different model systems. The lack of Dhrs9 as a marker in mouse Mregs suggests potential differences in the molecular programming or metabolic pathways between human and mouse regulatory macrophages. Researchers working with murine models should be aware of this distinction and utilize alternative markers appropriate for mouse Mregs.

What controls should be included when validating anti-DHRS9 antibodies for experimental use?

Proper validation of anti-DHRS9 antibodies is essential for experimental reliability. The following control measures are recommended:

• Antibody cross-validation: Confirm antibody specificity by demonstrating that multiple antibodies recognize the same protein. For example, research has shown that ASOT1 monoclonal antibody, a custom-made rabbit polyclonal antibody, and the commercial antibody clone 3C6 (Abnova) all recognize the same ~35 kDa protein identified as DHRS9 .

• Immunoprecipitation and sequencing: Verify the identity of the recognized protein by immunoprecipitation followed by MALDI-MS sequencing .

• Comparative analysis across cell types: Demonstrate differential expression across known positive and negative cell populations. Anti-DHRS9 antibodies should show strong reactivity with Mregs but minimal reactivity with other polarized macrophage types .

• Nucleic acid correlation: Correlate protein detection with mRNA expression data from qPCR to confirm specificity of detection .

• Protocol validation: For each application (Western blotting, flow cytometry, immunohistochemistry), optimize and validate specific protocols with appropriate positive and negative controls .

These validation steps ensure that experimental findings related to DHRS9 expression are reliable and reproducible across different detection methods.

What factors in cell culture conditions might affect DHRS9 expression in developing Mregs?

Several factors in the cell culture environment can influence DHRS9 expression during Mreg development:

Researchers investigating DHRS9 as a marker should standardize these culture conditions to ensure reproducible results and consider how variations might affect experimental outcomes.

How can techniques for DHRS9 detection be optimized for tissue section analysis?

For optimal detection of DHRS9-expressing cells in tissue sections, researchers should consider the following methodological approaches:

• Antibody selection: The monoclonal antibody clone 3C6 has been validated for immunohistochemical staining of DHRS9 in fixed tissue specimens .

• Protocol standardization: Follow established immunohistochemical protocols, and have stained sections evaluated by a qualified clinical histopathologist to ensure accurate interpretation .

• Positive controls: Include known DHRS9-positive tissues as controls. For example, stratified squamous epithelium of human skin has been reported to express DHRS9 and can serve as a positive control .

• Tissue selection: Based on trafficking studies of administered Mregs, spleen, liver, and bone marrow are promising tissues for detecting DHRS9-positive cells . In human spleen sections, DHRS9-expressing cells with macrophage morphology are most prevalent in the subcapsular red pulp .

• Correlation with other markers: Consider co-staining for other macrophage markers to further characterize DHRS9-positive cells in tissue contexts.

Optimization of these parameters will facilitate the identification and characterization of DHRS9-expressing cells in various tissue environments, potentially revealing physiological counterparts to in vitro generated Mregs.

How can DHRS9 antibodies be utilized in transplantation immunology research?

DHRS9 antibodies offer valuable tools for transplantation immunology research in several contexts:

• Monitoring therapeutic Mregs: As human Mregs emerge as promising cell-based adjunct immunosuppressive therapy in solid organ transplantation, DHRS9 antibodies provide a means to track these cells after administration to patients .

• Trafficking studies: Previous research has tracked ex vivo generated, radio-labeled allogeneic human Mregs after intravenous administration to prospective kidney transplant recipients. DHRS9 antibodies can complement such studies by identifying these cells in tissue sections from spleen, liver, and bone marrow, where administered Mregs have been shown to accumulate .

• Quality control: DHRS9 detection can serve as a quality control marker for Mreg preparation before clinical use, ensuring the generation of phenotypically appropriate cells .

• Mechanistic studies: By identifying DHRS9-expressing cells in transplant recipients, researchers can investigate the mechanisms through which Mregs exert their immunoregulatory effects, potentially revealing interactions with other immune cell populations .

These applications highlight the utility of DHRS9 antibodies in advancing understanding of Mreg-based immunomodulatory approaches in transplantation.

What research questions remain unresolved regarding DHRS9 function in regulatory macrophages?

Despite progress in identifying DHRS9 as a marker of human Mregs, several important questions remain unresolved:

• Functional significance: The functional implications of DHRS9 expression in human Mregs are not fully defined. While DHRS9 is classified as a retinol dehydrogenase, direct evidence that Mregs generate retinoic acid through DHRS9 activity is lacking .

• Regulatory mechanisms: The precise conditions and molecular pathways leading to DHRS9 expression in human Mregs remain incompletely defined, though glucocorticoids and IFN-γ appear to enhance expression to some degree .

• Serum components: Researchers hypothesize that undefined components of serum influence DHRS9 expression during in vitro development of Mregs from monocytes. Identifying these factors could enhance understanding of Mreg development .

• Physiological counterparts: While DHRS9-expressing macrophages have been identified in human spleen, the relationship between these naturally occurring cells and in vitro generated Mregs requires further investigation .

• Species differences: The lack of Dhrs9 as a marker in mouse Mregs raises questions about species differences in regulatory macrophage biology and the evolutionary conservation of DHRS9 functions .

Addressing these questions will deepen understanding of DHRS9's role in macrophage biology and potentially reveal new approaches to therapeutic manipulation of macrophage function.