AOAH Antibody

What is AOAH and why is it important in immunological research?

AOAH (Acyloxyacyl hydrolase) is a host lipase that plays a critical role in the detoxification of lipopolysaccharide (LPS) by removing secondary fatty acyl chains from the lipid A moiety, essentially inactivating LPS in tissues . AOAH is particularly important in immunological research because it regulates endotoxin tolerance, a state of hyporesponsiveness that occurs after initial exposure to LPS. Without AOAH, fully acylated, bioactive LPS persists in tissues, keeping macrophages in a tolerant state and preventing restoration of macrophage homeostasis . AOAH also possesses phospholipase A2 (PLA2) activity that can generate neolipid antigens recognized by CD1a-restricted T cells, influencing inflammatory responses .

In which cell types is AOAH predominantly expressed and how can this inform antibody-based detection strategies?

AOAH is typically found at low levels in multiple circulating immune cells including neutrophils, monocytes, macrophages, immature dendritic cells, and NK cells . The enzyme can be proteolytically processed into both endocytic and secreted forms . For effective antibody-based detection, researchers should consider cell-specific expression patterns and potentially target both intracellular and secreted forms. In inflammatory conditions like psoriasis, AOAH expression is significantly upregulated in lesional skin , suggesting that experimental designs should account for baseline versus induced expression levels when developing detection protocols.

What are the key considerations when selecting anti-AOAH antibodies for experimental validation?

When selecting anti-AOAH antibodies, researchers should consider:

• Epitope specificity: Determine whether the antibody recognizes the active site, processed forms, or specific domains of AOAH

• Cross-reactivity: Validate that the antibody does not cross-react with other lipases or phospholipases

• Applications compatibility: Confirm suitability for desired applications (immunohistochemistry, flow cytometry, immunoprecipitation, Western blotting)

• Species reactivity: Ensure compatibility with the experimental model system

• Validation status: Prioritize antibodies validated in peer-reviewed studies for AOAH detection

• Neutralizing capabilities: Determine if the antibody can block AOAH's enzymatic activities, which is critical for functional studies

How can AOAH antibodies be effectively used in immunoprecipitation experiments to study AOAH functions?

AOAH antibodies can be used in immunoprecipitation (IP) experiments to isolate AOAH from complex biological samples and study its functions. A methodological approach involves:

• Sample preparation: Prepare cell lysates or tissue homogenates under conditions that preserve AOAH activity

• Pre-clearing: Pre-clear lysates with control beads to reduce non-specific binding

• Immunoprecipitation: Incubate samples with anti-AOAH antibody (1-5 μg/ml) followed by protein A/G beads

• Stringency control: Perform parallel IPs with isotype control antibodies

• Validation: Confirm immunoprecipitation efficiency by Western blotting

• Functional assessment: Measure the enzymatic activity of immunoprecipitated AOAH using LPS deacylation assays or PLA2 activity assays

This approach has been validated in studies examining AOAH-specific responses. When AOAH was immunoprecipitated with anti-human AOAH antibody, CD1a-reactive T-cell responses were attenuated, confirming AOAH-specific CD1a-reactive responses .

What protocols are recommended for using anti-AOAH antibodies in immunofluorescence studies of tissue samples?

For immunofluorescence detection of AOAH in tissue samples, the following protocol is recommended:

• Tissue preparation:

  • Fix tissues in 4% paraformaldehyde for 24 hours

  • Embed in paraffin or prepare frozen sections (8-10 μm thickness)

  • For paraffin sections, perform antigen retrieval using citrate buffer (pH 6.0)

• Blocking and permeabilization:

  • Block with 5% normal serum in PBS containing 0.1% Triton X-100 for 1 hour

  • For cell-specific co-staining, include antibodies against cell markers (CD68 for macrophages, CD11c for dendritic cells)

• Primary antibody incubation:

  • Apply anti-AOAH antibody (1:100-1:500 dilution) overnight at 4°C

  • Include appropriate isotype controls

• Detection and visualization:

  • Use fluorophore-conjugated secondary antibodies (1:500 dilution)

  • Counterstain nuclei with DAPI

  • Mount with anti-fade mounting medium

This approach has been successfully used to demonstrate differential AOAH expression in psoriatic lesions compared to healthy skin .

How can flow cytometry with anti-AOAH antibodies be optimized to detect intracellular AOAH in different immune cell populations?

Optimizing flow cytometry for intracellular AOAH detection requires:

• Cell isolation and surface staining:

  • Isolate cells of interest (peripheral blood mononuclear cells, alveolar macrophages, etc.)

  • Stain with viability dye and surface markers to identify cell populations

  • For alveolar macrophages, include CD45, F4/80, CD11c, and Siglec-F markers

• Fixation and permeabilization:

  • Fix cells with 2% paraformaldehyde for 10 minutes

  • Permeabilize with 0.1% saponin or commercially available permeabilization buffers

• Intracellular staining:

  • Block Fc receptors to reduce non-specific binding

  • Incubate with fluorophore-conjugated anti-AOAH antibody or unconjugated primary antibody followed by fluorophore-conjugated secondary antibody

  • Include fluorescence minus one (FMO) and isotype controls

• Analysis considerations:

  • Use multiparameter gating strategies to identify specific cell populations

  • Compare AOAH expression levels between resting and activated states

  • Consider the influence of inflammatory stimuli on AOAH expression

This approach allows for quantitative assessment of AOAH expression across immune cell subsets and can reveal cell-specific regulation under different conditions.

How can researchers use AOAH antibodies to investigate the gut-lung axis in pulmonary defense studies?

Investigating AOAH's role in the gut-lung axis requires strategic use of AOAH antibodies in multi-tissue experimental designs:

• Experimental design approach:

  • Compare wildtype and AOAH-deficient (Aoah-/-) mouse models

  • Manipulate gut microbiota using antibiotics (e.g., neomycin) or direct LPS administration

  • Perform pulmonary challenge with bacterial pathogens (e.g., Pseudomonas aeruginosa)

  • Collect tissues from intestine, circulation, and lungs for parallel analysis

• AOAH antibody applications:

  • Immunohistochemistry to visualize AOAH distribution across tissues

  • Western blotting to quantify AOAH protein levels

  • Flow cytometry to analyze cell-specific AOAH expression

• Functional correlation with:

  • LPS deacylation activity in intestinal and lung samples

  • Alveolar macrophage phagocytic capacity assessment

  • Cytokine production measurements in bronchoalveolar lavage fluid

This research approach has revealed that AOAH regulates pulmonary mucosal immunity partly by inactivating LPS in the gut, as AOAH-deficient mice showed greater susceptibility to pulmonary Pseudomonas aeruginosa infection, with alveolar macrophages demonstrating hyporesponsiveness to innate stimulation and reduced phagocytic activity .

What are the recommended techniques for simultaneously assessing AOAH expression and alveolar macrophage function in pulmonary defense models?

To assess AOAH expression and alveolar macrophage function simultaneously:

• Isolation of alveolar macrophages:

  • Perform bronchoalveolar lavage (BAL) with cold PBS (5 × 1 ml)

  • Process BAL fluid by centrifugation and resuspend cells

  • Confirm macrophage purity by flow cytometry (CD45+F4/80+CD11c+Siglec-F+)

• AOAH expression assessment:

  • Quantify AOAH protein by Western blotting with specific anti-AOAH antibodies

  • Perform intracellular flow cytometry for cell-specific AOAH detection

  • Assess AOAH mRNA expression by qRT-PCR

• Functional assays (parallel samples):

  • Phagocytosis assay: Incubate AMs with fluorescent E. coli particles

  • Cytokine production: Measure TNF-α, KC, and MIP-2 mRNA and protein production following LPS stimulation

  • Expression of negative regulators: Assess SOCS-1, A20, IRAK-M, and SHIP expression levels

• Data integration:

  • Correlate AOAH expression levels with functional parameters

  • Compare results between wildtype and AOAH-deficient models

  • Assess the impact of interventions (antibiotics, LPS administration)

Studies using these techniques have demonstrated that AOAH-deficient alveolar macrophages have reduced responsiveness to LPS stimulation, decreased phagocytic capacity, and altered expression of negative regulators like SOCS-1 .

How can anti-AOAH antibodies be used to study the role of AOAH in psoriasis pathogenesis?

Investigating AOAH's role in psoriasis pathogenesis requires a multifaceted approach using anti-AOAH antibodies:

• Tissue expression analysis:

  • Compare AOAH expression in lesional psoriatic skin versus healthy skin using immunofluorescence with anti-AOAH antibodies

  • Perform dual staining to identify AOAH-expressing cell types (neutrophils, dendritic cells)

  • Quantify expression levels using digital image analysis

• Functional mechanistic studies:

  • Isolate CD1a-reactive T cells from patients with psoriasis and healthy controls

  • Use anti-AOAH antibodies to neutralize AOAH activity in co-culture experiments

  • Measure IFN-γ and IL-22 production in response to AOAH using ELISpot assays

  • Block CD1a with anti-CD1a antibodies as a specificity control

• Phospholipase activity assessment:

  • Measure PLA2 activity of AOAH using colorimetric detection of thiol release from diheptanoyl thio-PC (for sPLA2) and arachidonoyl thio-PC (for cPLA2)

  • Test PLA2 inhibitors (e.g., manoalide) to confirm specificity

Research using these approaches has demonstrated that AOAH is highly expressed in psoriatic lesions compared to healthy skin and that its PLA2 activity leads to activation of CD1a auto-reactive T cells, resulting in elevated IL-22 production in patients with psoriasis .

What experimental design can distinguish between the LPS-detoxifying and phospholipase functions of AOAH in inflammatory conditions?

To distinguish between AOAH's dual functions in inflammatory conditions:

• Parallel enzymatic activity assays:

  • LPS deacylation: Measure AOAH's ability to detoxify LPS using a pH-controlled buffer system at pH 5.0 and 7.5

  • PLA2 activity: Assess phospholipase activity using specific substrates for sPLA2 and cPLA2

  • Include specific inhibitors for each pathway

• Selective inhibition approach:

  • Use PLA2 inhibitors (manoalide, oleyloxyethyl phosphorylcholine) to block phospholipase activity

  • Employ site-directed mutagenesis to create AOAH variants with selective functional impairments

  • Utilize neutralizing antibodies targeting different functional domains

• Functional readouts:

  • LPS pathway: Measure IL-6 production by PMA-differentiated THP-1 cells in response to AOAH-treated versus untreated LPS

  • PLA2 pathway: Assess CD1a-dependent T cell activation and cytokine production

• Analysis in disease models:

  • Compare the relative contribution of each function in different inflammatory conditions

  • Correlate with disease severity markers

This experimental design has been used to demonstrate that LPS deacylation by AOAH significantly diminishes the ability of LPS to stimulate IL-6 production in THP-1 cells, while the PLA2 activity separately activates CD1a-reactive T cells to produce IFN-γ and IL-22 .

How can researchers develop and validate neutralizing anti-AOAH antibodies for functional studies?

Developing and validating neutralizing anti-AOAH antibodies involves:

• Antigen design and immunization:

  • Identify functional domains based on crystal structure analysis

  • Generate recombinant AOAH or functional domain fragments

  • Immunize animals with purified antigens using appropriate adjuvants

  • Consider multiple species to increase diversity of antibody repertoire

• Screening strategy:

  • Initial screening by ELISA for binding to recombinant AOAH

  • Secondary functional screening using:

    • LPS deacylation inhibition assay

    • PLA2 activity inhibition assay

    • CD1a-restricted T cell activation inhibition assay

• Validation methodology:

  • Confirmation of specificity through immunoprecipitation followed by mass spectrometry

  • Dose-dependent inhibition of enzymatic activities

  • Cell-based assays demonstrating functional neutralization

  • Comparison with known PLA2 inhibitors as positive controls

• Application testing:

  • Validate in relevant disease models (e.g., psoriasis, pulmonary infection)

  • Confirm target engagement in complex biological samples

  • Assess off-target effects on related lipases

Research has demonstrated that removing AOAH protein through immunoprecipitation with anti-human AOAH antibody attenuates AOAH-specific IL-22 production by CD1a autoreactive T-cell clones, supporting the specificity of AOAH-mediated effects .

What are the current challenges and solutions in studying AOAH expression in tissue-resident macrophage populations?

Studying AOAH in tissue-resident macrophages presents several challenges with corresponding solutions:

These approaches can address the complexity of studying AOAH in tissue-resident macrophages, such as alveolar macrophages, which demonstrate altered functional states in AOAH-deficient models, including reduced responsiveness to LPS stimulation and decreased phagocytic capacity .

How can researchers investigate the mechanistic relationship between AOAH expression and CD1a-mediated T cell activation in inflammatory diseases?

Investigating the AOAH-CD1a-T cell axis requires sophisticated experimental approaches:

• Advanced co-culture systems:

  • Develop three-cell co-culture systems with:

    • CD1a-expressing antigen-presenting cells (APCs)

    • AOAH-expressing cells or recombinant AOAH

    • CD1a-reactive T cell clones or primary T cells

  • Use transwell systems to distinguish direct vs. indirect effects

• Lipid antigen identification:

  • Perform lipidomics analysis before and after AOAH treatment

  • Isolate CD1a-binding lipids from cell membranes after AOAH exposure

  • Use synthetic lipid libraries to identify specific CD1a-presented neolipid antigens

• Molecular manipulation approaches:

  • Create AOAH knockdown/knockout in relevant cell types using CRISPR-Cas9

  • Develop cell lines with controlled expression of wildtype vs. mutant AOAH

  • Generate CD1a variants with altered lipid-binding properties

• In vivo models:

  • Develop humanized mouse models expressing human CD1a and AOAH

  • Create tissue-specific conditional AOAH knockout models

  • Perform adoptive transfer of CD1a-reactive T cells

This research approach has revealed that AOAH's PLA2 activity generates neolipid antigens that activate circulating CD1a-restricted T cells, leading to production of IFN-γ and IL-22, with elevated IL-22 production observed in patients with psoriasis .

How can researchers address contradictory findings regarding the pro-inflammatory versus anti-inflammatory roles of AOAH?

Addressing contradictions in AOAH function requires:

• Experimental design considerations:

  • Standardize AOAH activity measurements across studies

  • Control for varying experimental conditions (pH, temperature, cofactors)

  • Account for cell type-specific responses

  • Distinguish acute versus chronic effects

• Context-dependent analysis:

  • Compare AOAH functions in different tissues (gut vs. lung vs. skin)

  • Assess time-dependent effects following inflammatory stimuli

  • Consider the influence of microbiome composition

• Dual function resolution:

  • Design experiments that simultaneously measure:

    • Anti-inflammatory effects: LPS detoxification, prevention of endotoxin tolerance

    • Pro-inflammatory effects: Generation of lipid antigens, CD1a-T cell activation

  • Quantify the relative contribution of each pathway in different conditions

• Integrative approach:

  • Use systems biology approaches to model competing pathways

  • Perform transcriptomics/proteomics on AOAH-deficient vs. wildtype models

  • Develop mathematical models of AOAH activity in different tissue microenvironments

Research has shown that AOAH can have seemingly opposing roles: preventing endotoxin tolerance in alveolar macrophages (promoting bacterial clearance) while also generating lipid antigens that activate pro-inflammatory T cells in skin inflammation .