DHRS9 Human

What is DHRS9 and what protein family does it belong to?

DHRS9 (also known as SDR9C4) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. Specifically, it belongs to the SDR9C subfamily, which is the largest subfamily of SDRs with eight human members and at least 13 mouse homologs . The additional mouse homologs likely appeared due to multiple rounds of lineage-specific gene duplications . DHRS9 encodes a protein that exhibits various enzymatic activities, including all-trans-retinol dehydrogenase activity, alcohol dehydrogenase activity, and other reductase functions . The protein has a molecular weight of approximately 35 kDa as confirmed by immunoprecipitation and MALDI-MS sequencing studies .

What is the tissue distribution of DHRS9 in humans?

Human DHRS9 mRNA has a broad tissue distribution pattern . Expression analysis shows that DHRS9 is found in various tissues and is frequently altered during tissue inflammation, carcinogenesis, metabolic disorders, and obesity . Immunohistochemistry has identified a subpopulation of DHRS9-expressing human splenic macrophages . The wide distribution suggests DHRS9 plays roles in multiple physiological processes, though its expression is particularly notable in certain immune cell populations, especially regulatory macrophages.

How does DHRS9 function as a marker for human regulatory macrophages?

DHRS9 has been identified as a robust and specific marker for human regulatory macrophages (Mregs) . Expression of DHRS9 at both mRNA and protein levels effectively discriminates human Mregs from other polarized macrophage states and from other tolerogenic monocyte-derived cells. Unlike other markers that are defined by their absence in Mregs (such as CD14−/low, CD16−/low, TLR2−, and CD163−/low), DHRS9 serves as a positive marker, which is particularly valuable for identification and characterization purposes .

What are the most effective methods for detecting DHRS9 expression in research samples?

Multiple complementary techniques have been validated for detecting DHRS9 expression:

Flow Cytometry: Intracellular staining using PE-conjugated anti-DHRS9 mAb clone 3C6 has been optimized for single-cell analysis of DHRS9 expression . The protocol typically involves staining at 4°C in buffer containing DPBS/1% BSA/0.02% NaN3/10% FcR-block for 60 minutes with saturating antibody concentrations. Dead cells can be excluded using 7-AAD staining .

Quantitative PCR (qPCR): DHRS9 mRNA expression can be reliably detected using specific primers. Recommended protocol includes using SuperScript-III for reverse-transcription reactions and FastStart DNA Master SYBR Green I kit for qPCR with a real-time PCR system . DHRS9 signals should be normalized against housekeeping genes like GAPDH.

Western Blotting: Both custom-made and commercial antibodies have been effective for detecting DHRS9 protein expression. A rabbit polyclonal antibody generated against the N-terminal epitope (CTDPENVKRTAQWVKNQVGEKG) of DHRS9 has been successfully used, as well as commercial antibodies like clone 3C6 from Abnova and ab98155 from Abcam .

How can researchers optimize DHRS9 primer design for qPCR experiments?

Researchers should consider the following primer specifications for optimal DHRS9 detection:

PCR specificity should be confirmed by sequencing of amplicons . When designing primers, researchers should ensure they span exon-exon junctions to prevent amplification of genomic DNA. For optimal results, DHRS9 signals should be normalized against stable reference genes such as GAPDH . Additionally, verification of primer efficiency through standard curve analysis is recommended before experimental use.

What are the technical considerations for analyzing DHRS9 expression in different cell types?

When analyzing DHRS9 expression across different cell types, several technical considerations should be addressed:

• Appropriate controls: Include both positive controls (verified Mregs) and negative controls (other macrophage types) to establish expression baselines .

• Cell preparation: For monocyte-derived cells, standardize isolation and culture conditions as DHRS9 expression increases gradually during in vitro development from CD14+ monocytes .

• Stimulation timing: Consider that DHRS9 expression is enhanced by IFN-γ treatment (typically on day 6 of culture) but is not extinguished by LPS stimulation (100 ng/mL for 24 hours) .

• Single-cell analysis: Flow cytometry reveals that DHRS9 signal is continuously distributed in Mreg populations rather than restricted to a subset, important for gating strategies .

• Species considerations: Importantly, Dhrs9 is not an informative marker of mouse Mregs, so species-specific protocols must be developed for comparative studies .

How does DHRS9 expression distinguish Mregs from other tolerogenic cell types?

DHRS9 expression effectively distinguishes human Mregs from other tolerogenic monocyte-derived antigen-presenting cells currently under development as cell-based immunotherapies. Comparative analysis has shown that DHRS9 mRNA expression is significantly higher in Mregs than in:

• Immature monocyte-derived dendritic cells (mo-DC)

• Tolerogenic DC (Tol-DC)

• Rapamycin-treated DC (Rapa-DC)

• IL-10 conditioned DC (DC-10)

• PGE2-induced myeloid-derived suppressor cells

At the protein level, DHRS9 expression is consistently greater in Mregs than in any of these comparator monocyte-derived cells . This selective expression pattern makes DHRS9 a valuable marker for identifying and isolating Mregs in mixed cell populations and for monitoring the purity of Mreg preparations intended for therapeutic applications.

What factors regulate DHRS9 expression in human macrophages?

Several factors influence DHRS9 expression in human macrophages:

• Differentiation stage: DHRS9 expression increases steadily throughout the transition of monocytes to Mregs in culture .

• Cytokine stimulation: IFN-γ treatment on day 6 of culture further upregulates DHRS9 expression in developing Mregs .

• Polarization conditions: Treatment of resting macrophages with IL-4, plate-bound Ig, dexamethasone, IFN-γ, or LPS + IFN-γ induces some DHRS9 expression, but not to the same level as in Mregs .

• Stability under inflammatory conditions: Unlike many macrophage markers that change with activation, DHRS9 expression is not diminished after stimulation with 100 ng/mL LPS for 24 hours, indicating it is a stable marker .

Microarray analysis of 29 differently stimulated human monocyte-derived macrophages revealed that more than twofold upregulation of DHRS9 was elicited by treatment with either 1 μM dexamethasone or 200 IU/mL IFN-γ . Conversely, 14 treatment conditions led to more than twofold downregulation of DHRS9 expression, demonstrating complex regulatory mechanisms .

What is the enzymatic function of DHRS9 in oxylipin metabolism?

Human DHRS9 exhibits robust oxidative activity toward various bioactive oxylipins, playing multiple roles during inflammation and immune responses. Specifically, DHRS9 shows activity toward oxylipins with hydroxyl groups located at:

• Carbons C9 and C13 of octadecanoids

• C12 and C15 carbons of eicosanoids

• C14 carbon of docosanoids

DHRS9 is also active toward the lipid inflammatory mediator dihydroxylated Leukotriene B4 . This enzymatic activity suggests that DHRS9 participates in the termination of inflammatory responses by metabolizing bioactive lipid mediators. The substrate specificity of DHRS9 appears to be evolutionarily conserved, indicating fundamental roles in mammalian immune regulation .

How does human DHRS9 differ from its rodent counterparts?

Important differences exist between human DHRS9 and its rodent homologs:

• Marker utility: While DHRS9 is a robust marker for human Mregs, Dhrs9 is not an informative marker for mouse Mregs, limiting direct translational research .

• Evolutionary expansion: The mouse genome contains additional Dhrs9 homologs that likely appeared through lineage-specific gene duplications, resulting in at least 13 mouse homologs compared to 8 human SDR9C family members .

• Orthology relationships: Despite these differences, core enzymatic functions appear conserved across species, with both human DHRS9 and rodent Dhrs9 exhibiting activity toward similar substrates .

For comparative studies, researchers should note these species differences when designing experiments and interpreting results. The table below shows some of the ortholog relationships:

What methodological approaches are most effective for studying DHRS9 across species?

When conducting comparative studies of DHRS9 across species, researchers should consider these methodological approaches:

• Protein expression systems: The baculovirus expression system in Sf9 cells has been successfully used to express human DHRS9 for kinetic analysis .

• Cross-reactivity testing: Antibodies raised against human DHRS9 should be carefully tested for cross-reactivity with rodent homologs before use in comparative studies.

• Functional assays: Enzymatic activity assays may be more informative than expression patterns when comparing across species, given the conservation of catalytic functions .

• Comparative genomics: Analysis of promoter regions and regulatory elements can help explain species-specific expression patterns and responses to stimuli.

• Evolutionary analysis: Phylogenetic approaches can clarify the relationships between multiple homologs in rodents and their relationship to human DHRS9 .

How is DHRS9 being applied in cell-based immunotherapies?

DHRS9 has emerged as an important marker in the development of human regulatory macrophages (Mregs) as a cell-based adjunct immunosuppressive therapy for solid organ transplant recipients . Specific applications include:

• Quality control: DHRS9 expression serves as a reliable quality control marker for confirming the identity and purity of human Mreg preparations intended for therapeutic use .

• Stability assessment: The stability of DHRS9 expression after inflammatory stimulation makes it valuable for predicting the in vivo functionality of therapeutic Mregs after administration .

• Monitoring: DHRS9 detection can be used to track the persistence and distribution of administered Mregs in patients.

• Standardization: As a specific marker, DHRS9 helps standardize Mreg production protocols across different manufacturing facilities, ensuring consistent therapeutic products.

This application of DHRS9 as a marker represents a significant advance in the field of cellular immunotherapy, particularly for transplantation medicine where Mregs show promise as adjunct immunosuppressive agents .

What is the relationship between DHRS9 expression and disease states?

DHRS9 expression is frequently altered in various pathological conditions:

• Inflammation: DHRS9 expression changes during tissue inflammation, potentially reflecting alterations in macrophage polarization states .

• Cancer: Changes in DHRS9 expression have been observed during carcinogenesis, suggesting potential roles in tumor immunology .

• Metabolic disorders: DHRS9 expression is altered in metabolic disorders and obesity, indicating possible involvement in metabolic inflammation .

The precise mechanisms and consequences of these expression changes remain areas of active investigation. The broad tissue distribution of DHRS9 and its enzymatic activities toward lipid mediators of inflammation suggest that alterations in its expression may contribute to disease pathogenesis through effects on inflammatory resolution and immune regulation .