RELM b Human, His

What is RELM-β and what are its biological functions?

RELM-β (Resistin-like molecule beta, also known as FIZZ2) is a member of the RELM family of secreted proteins characterized by conserved C-terminus cysteines. The RELM family consists of Resistin (FIZZ3), RELM-α (FIZZ1), RELM-β (FIZZ2), and RELM-γ (FIZZ4), with only Resistin and RELM-β being present in humans, whereas all four members exist in rodents .

RELM-β serves multiple critical biological functions:

• Antimicrobial activity: RELM-β functions as a bactericidal protein that selectively kills Gram-negative bacteria by forming size-selective pores that permeabilize bacterial membranes, thereby preventing bacteria from coming into close contact with host tissues .

• Intestinal barrier function: It is constitutively expressed in the colon by goblet cells and enterocytes and plays a crucial role in maintaining homeostasis of the colonic barrier and gastrointestinal innate immunity .

• Airways remodeling: RELM-β contributes to airway remodeling in asthmatic airways by increasing fibroblast proliferation and differentiation, resulting in extracellular matrix protein deposition .

• Immunomodulation: Research indicates RELM-β regulates expression of type III regenerating genes (REG3β and γ), which influence nuclear factor κB signaling pathways involved in immune responses .

How is RELM-β expressed in human tissues?

RELM-β demonstrates tissue-specific expression patterns that correlate with its diverse biological functions:

• Gastrointestinal tract: RELM-β is predominantly expressed in the colon by goblet cells and enterocytes, where it contributes to maintaining intestinal barrier function and regulating innate immunity . It is secreted from the intestinal surface into the mucus layer, providing a protective antimicrobial barrier.

• Respiratory system: Significant expression of RELM-β has been detected in the bronchial mucosa of asthmatic patients. Immunohistochemistry studies have demonstrated that in addition to epithelial cells, macrophages, fibroblasts, and vascular endothelial cells constitute the majority of cells expressing RELM-β in the bronchial submucosa .

• Expression during inflammation: RELM-β expression is significantly increased in the bronchial submucosa of human asthmatics compared to non-asthmatic controls, and its expression correlates positively with that of fibronectin and α-smooth muscle actin, suggesting its involvement in airway remodeling processes .

• Regulation of expression: While the detailed mechanisms regulating human RELM-β expression remain under investigation, studies in murine models indicate that its expression can be induced during active infections, particularly with intestinal helminths .

What methodological approaches are available for detecting RELM-β in experimental and clinical samples?

Several complementary approaches can be employed to detect and quantify RELM-β expression:

• Enzyme-Linked Immunosorbent Assay (ELISA): ELISA can be used to measure RELM-β concentrations in biological fluids such as bronchoalveolar lavage (BAL) fluid from asthmatics and controls . This approach provides quantitative data on protein levels but requires specific antibodies.

• Immunohistochemistry: This technique allows visualization of RELM-β protein expression in tissue sections, enabling identification of specific cell types expressing the protein. This method has been successfully employed to detect increased RELM-β expression in the bronchial submucosa of asthmatic patients .

• Real-time PCR: Quantitative PCR provides a sensitive method for measuring RELM-β mRNA expression levels in tissues or isolated cells. This technique has been used to monitor RELM-β gene expression during helminth infections in experimental models .

• Non-invasive techniques: A novel non-invasive method has been developed to detect RELM-β transcript in gut barrier analysis. This approach utilizes exfoliated intestinal cells released with feces to mirror immunological host responses corresponding to gut barrier changes during infection, offering a less invasive alternative to tissue biopsies .

What experimental approaches are optimal for studying RELM-β's bactericidal activity?

To effectively investigate RELM-β's bactericidal properties, researchers should consider the following methodological approaches:

• Bacterial membrane permeabilization assays: Since RELM-β kills Gram-negative bacteria by forming size-selective pores that permeabilize bacterial membranes, assays that measure membrane integrity are crucial. Fluorescent dye-based permeability assays using membrane-impermeable dyes can assess the formation of pores in bacterial membranes after RELM-β treatment .

• Bacterial killing assays: Quantitative bacterial killing assays comparing wild-type and RELM-β-deficient models can determine bactericidal efficacy. These should specifically focus on Gram-negative bacteria, as research has shown RELM-β's selectivity for this bacterial class .

• Bacterial colonization studies: In vivo studies using RELM-β knockout mice can assess how RELM-β deficiency affects bacterial spatial distribution, particularly evaluating the presence of Proteobacteria in the inner mucus layer and their invasion of mucosal tissues .

• Recombinant protein studies: Comparing bactericidal activities of recombinant mouse and human RELM-β can reveal cross-species functional conservation. When using His-tagged RELM-β, researchers should confirm that the tag does not interfere with pore-forming activity .

• Structure-function analysis: Mutational studies targeting the conserved C-terminus cysteines can identify critical domains for bactericidal activity, as these regions are characteristic of the RELM family .

What are the key considerations when investigating RELM-β's role in airway remodeling?

When studying RELM-β's involvement in airway remodeling processes, researchers should consider these methodological approaches:

• Fibroblast-specific assays: Since RELM-β targets airways fibroblasts to effect remodeling, in vitro studies should include:

  • Proliferation assays to measure fibroblast proliferation rates after RELM-β exposure

  • Western blotting to detect activation of signaling pathways (particularly ERK1/2)

  • ELISA and real-time PCR to measure production of TGF-β1, TGF-β2, and extracellular matrix proteins

• Comparative models: Studies comparing wild-type and RELM-β-deficient mice in allergen exposure models provide valuable insights. For example, sensitization and challenge with Aspergillus fumigatus (Af) induces RELM-β release with a time course coincident with procollagen in airways .

• Correlation analysis: In human studies, analysis of the correlation between RELM-β expression and markers of airway remodeling (such as fibronectin and α-smooth muscle actin) provides evidence for RELM-β's role in this process .

• Comprehensive protein profiling: Assessment of RELM-β's effects on multiple extracellular matrix components is essential, including:

  • Collagen I

  • Fibronectin

  • Smooth muscle α-actin

  • Laminin α1

  • Hyaluronan and proteoglycan link protein 1 (Hapl1)

• Signaling pathway investigation: Studies should examine RELM-β's effects on ERK1/2 activation in lung fibroblasts, as this pathway has been implicated in the fibrotic response .

How can researchers effectively utilize mouse models to study human RELM-β function?

While mouse and human RELM-β share functional similarities, important considerations must be addressed when translating findings between species:

• Species-specific expression patterns: Researchers should note that humans express only two RELM family members (Resistin and RELM-β), while rodents express all four (Resistin, RELM-α, RELM-β, and RELM-γ) . This difference may impact functional redundancy and compensatory mechanisms.

• RELM-β knockout models: RELM-β-/- mice have been generated by homologous recombination using VelociGene technology, where the RELM-β gene is replaced by a reporter-selection cassette. These models show altered colonic mRNA transcripts and epithelial barrier function, demonstrating RELM-β's role in homeostasis .

• Cross-validation of bactericidal activity: Studies have shown that both mouse and human RELM-β selectively kill Gram-negative bacteria through similar membrane-permeabilizing mechanisms, suggesting functional conservation across species .

• Infection models: Mouse models of helminth infection reveal RELM-β induction patterns that may parallel human responses. Analysis of RELM-β expression during Heligmosomoides polygyrus infection shows dose-dependent responses that correlate with parasite burden .

• Microarray analysis: When comparing wild-type and RELM-β-/- mice, genome-wide expression profiling using platforms like Affymetrix GeneChip microarrays can identify downstream pathways affected by RELM-β deficiency .

• Validation in human samples: Findings from mouse models should be validated using human samples whenever possible, as demonstrated in studies comparing RELM-β expression in bronchial biopsies from asthmatic and non-asthmatic individuals .

What are the optimal methods for producing and purifying recombinant His-tagged human RELM-β?

For researchers working with recombinant His-tagged RELM-β, the following methodological considerations are critical:

• Expression systems: Selection of an appropriate expression system is crucial for producing functional recombinant RELM-β. Mammalian expression systems often yield protein with proper folding and post-translational modifications, which may be essential for RELM-β's bactericidal and signaling activities .

• Purification protocols: His-tagged RELM-β can be purified using immobilized metal affinity chromatography (IMAC). Care should be taken to optimize elution conditions to maintain protein activity while achieving high purity.

• Reconstitution procedures: As indicated in product instructions, recombinant RELM-β should be reconstituted by gently pipetting the recommended solution down the sides of the vial rather than vortexing. For prolonged storage, dilution to working aliquots in a 0.1% BSA solution, storage at -80°C, and avoiding repeated freeze-thaw cycles are recommended .

• Functional validation: After purification, recombinant His-tagged RELM-β should be validated for its known biological activities, including:

  • Bactericidal activity against Gram-negative bacteria

  • Ability to stimulate fibroblast proliferation

  • Induction of extracellular matrix protein production

• Storage considerations: Proper storage conditions are essential for maintaining RELM-β activity. Centrifuge vials before opening, and for prolonged storage, prepare aliquots in BSA-containing buffer and store at -80°C to avoid repeated freeze-thaw cycles .

How can RELM-β expression be used as a biomarker in gastrointestinal research?

RELM-β expression analysis offers promising applications as a biomarker in gastrointestinal research:

• Non-invasive diagnostic approach: The detection of RELM-β transcript in exfoliated intestinal cells from fecal samples provides a novel non-invasive technique to investigate immunological host responses representing gut barrier changes during infection. This method eliminates the need for invasive tissue sampling procedures .

• Helminth infection indicator: RELM-β expression has been shown to reflect both the intensity and type of helminth infection. Linear regression analysis demonstrates that RELM-β gene expression correlates with worm burden in mouse models, while in wild house mice, RELM-β expression is significantly elevated by either the presence or species richness of helminths .

• Monitoring therapeutic efficacy: Changes in RELM-β expression during primary and secondary helminth infections can serve as an indicator of immune response development. Studies in BALB/c mice infected with H. polygyrus and subsequently treated with pyrantel pamoate for worm clearance showed distinct RELM-β expression patterns during reinfection .

• Inflammatory bowel disease research: Since RELM-β plays a central role in the regulation of susceptibility to colonic inflammation, its expression patterns may provide insights into inflammatory bowel disease pathogenesis and treatment responses .

• Methodological considerations:

  • All relative gene expression analysis should be compared to housekeeping genes such as β-actin using standardized cDNA amounts (e.g., 10ng)

  • Statistical analysis should employ appropriate tests such as Mann-Whitney U Test, multiple t-tests with correction for multiple comparisons, or linear regression models depending on the experimental design

How should researchers address contradictory data regarding RELM-β function in different tissues?

When faced with apparently contradictory findings regarding RELM-β functions across different tissues or experimental systems, researchers should consider:

• Tissue-specific context: RELM-β demonstrates different functions in different tissues - bactericidal activity in the intestine , barrier function maintenance in the colon , and promotion of fibrosis in airways . These apparently contradictory roles likely reflect tissue-specific microenvironments and cellular contexts.

• Experimental design harmonization:

  • Standardize protein preparation methods to ensure consistent activity

  • Use multiple complementary assays to validate findings

  • Include both positive and negative controls in all experiments

  • Consider the impact of tags (such as His-tag) on protein function

• Integrated analysis approach: Employ systems biology approaches to integrate seemingly contradictory data. Microarray analysis comparing wild-type and RELM-β-/- mice can identify broader regulatory networks affected by RELM-β, potentially explaining tissue-specific differences .

• Functional validation across species: Cross-validate findings between mouse models and human samples whenever possible. While mouse and human RELM-β share bactericidal functions against Gram-negative bacteria , other functions may show species-specific differences.

• Signaling pathway mapping: Investigate whether tissue-specific effects of RELM-β are mediated through different downstream signaling pathways. For example, RELM-β activates ERK1/2 signaling in lung fibroblasts , but may engage different pathways in intestinal epithelial cells.

What are the current gaps in RELM-β research and recommended future directions?

Based on the current literature, several significant knowledge gaps and promising research directions emerge:

• Structural biology: Detailed structural analysis of RELM-β, particularly focused on understanding the molecular mechanism of pore formation in bacterial membranes, would advance our understanding of its bactericidal function .

• Receptor identification: The specific cell surface receptors through which RELM-β mediates its effects on fibroblasts and other cell types remain poorly characterized. Receptor identification would provide new targets for modulating RELM-β activity in disease contexts .

• Therapeutic potential exploration:

  • Investigation of RELM-β as a novel antibiotic against Gram-negative bacteria

  • Development of RELM-β antagonists for treating airway remodeling in asthma

  • Exploration of recombinant RELM-β for treating inflammatory bowel conditions

• Interaction with microbiome: Further studies on how RELM-β shapes the intestinal microbiome composition and how microbiome changes feedback to regulate RELM-β expression would provide insights into host-microbe interactions .

• Comparative genomics: More comprehensive analysis of the evolutionary conservation of RELM family members across species could reveal insights into their fundamental biological functions and tissue-specific adaptations .

• Clinical correlations: Larger clinical studies correlating RELM-β expression with disease severity, progression, and treatment response in conditions such as asthma and inflammatory bowel disease would establish its value as a biomarker .

What are common technical challenges when working with recombinant His-tagged RELM-β?

Researchers working with recombinant His-tagged RELM-β may encounter several technical challenges:

• Protein stability issues: RELM-β contains multiple cysteine residues that form disulfide bonds critical for its structure and function. Improper handling during reconstitution may disrupt these bonds and compromise activity.

  • Solution: Follow strict reconstitution protocols, avoiding vortexing and using gentle pipetting techniques

  • For prolonged storage, prepare aliquots in 0.1% BSA solution, store at -80°C, and avoid repeated freeze-thaw cycles

• His-tag interference with function: The presence of a His-tag may potentially alter protein folding or activity in certain experimental contexts.

  • Solution: Include appropriate controls comparing tagged and untagged versions of the protein when possible

  • Consider tag removal using specific proteases when necessary for functional studies

• Concentration determination: Accurate determination of active protein concentration is critical for reproducible experiments.

  • Solution: Use multiple protein quantification methods (BCA, Bradford, UV absorbance)

  • Validate activity using dose-response curves in functional assays

• Endotoxin contamination: Bacterial expression systems may introduce endotoxin contamination, which can confound immunological studies.

  • Solution: Use endotoxin-free purification systems or include endotoxin removal steps

  • Test final preparations with Limulus Amebocyte Lysate (LAL) assay to confirm endotoxin levels

• Batch-to-batch variability: Different batches of recombinant protein may show variable activity.

  • Solution: Establish internal standards and normalization procedures

  • Maintain detailed records of protein production, purification, and functional validation

How can researchers optimize experimental design for studying RELM-β's effects on airways remodeling?

To maximize the reliability and translational value of studies investigating RELM-β's role in airways remodeling:

• Comprehensive cell panel: Include multiple relevant cell types in experimental designs:

  • Primary human lung fibroblasts from both asthmatic and non-asthmatic donors

  • Bronchial epithelial cells

  • Vascular endothelial cells

  • Macrophages
    These cell types have been identified as expressing RELM-β in the bronchial submucosa

• Dose-response relationships: Establish clear dose-response relationships for RELM-β effects on:

  • Fibroblast proliferation

  • Production of extracellular matrix proteins (collagen I, fibronectin, laminin α1)

  • Expression of smooth muscle α-actin

  • Production of TGF-β1 and TGF-β2

• Signaling pathway validation: Confirm ERK1/2 pathway activation using:

  • Western blotting for phosphorylated ERK1/2

  • Specific ERK1/2 inhibitors to demonstrate causality

  • Complementary approaches such as siRNA knockdown of pathway components

• Translation between models: Design experiments that facilitate translation between:

  • In vitro cell culture systems

  • Mouse models of allergic airway disease

  • Human clinical samples
    This approach strengthens the physiological relevance of findings

• Statistical considerations:

  • Power analyses to determine appropriate sample sizes

  • Multiple comparison corrections when testing multiple outcomes

  • Mixed effects models to account for donor-to-donor variability in primary cells

  • Linear regression models to identify significant correlations between RELM-β expression and remodeling markers

How should researchers interpret changes in RELM-β expression in different experimental contexts?

Proper interpretation of RELM-β expression data requires consideration of several factors:

• Tissue-specific baseline expression: RELM-β is constitutively expressed in certain tissues (e.g., colon) but may be induced in others (e.g., airways during asthmatic inflammation) . This baseline variation must be considered when interpreting fold-changes.

• Temporal dynamics: RELM-β expression shows distinct temporal patterns during infections and inflammatory responses. In helminth infection models, RELM-β expression varies with infection stage and parasite burden, necessitating time-course analyses rather than single time-point measurements .

• Correlation with functional outcomes: Changes in RELM-β expression should be correlated with functional outcomes, such as:

  • Bacterial presence in the inner mucus layer in intestinal studies

  • Extracellular matrix deposition in airway remodeling studies

  • Epithelial barrier function in colonic homeostasis studies

• Analysis methods:

  • For quantitative PCR data: Use appropriate housekeeping genes (e.g., β-actin) and standard amounts of cDNA (e.g., 10ng)

  • For microarray data: Apply robust normalization methods such as the Robust MultiArray Analysis algorithm

  • For wild animal studies: Consider confounding variables through appropriate statistical models (e.g., linear regression accounting for co-infections)

What benchmarks should be used to evaluate the quality of recombinant His-tagged human RELM-β preparations?

To ensure consistent experimental results, researchers should establish quality control benchmarks:

These benchmarks should be established for each laboratory's reference standard and used to evaluate new preparations before experimental use.

How might understanding RELM-β's dual roles in inflammation and infection inform therapeutic strategies?

RELM-β's diverse functions offer multiple potential therapeutic applications:

• Antimicrobial therapies: RELM-β's selective bactericidal activity against Gram-negative bacteria suggests potential development as a novel antimicrobial agent, particularly for intestinal infections. Its mechanism of forming size-selective pores in bacterial membranes represents a distinct mode of action compared to conventional antibiotics .

• Inflammatory bowel disease interventions: RELM-β's critical role in maintaining colonic barrier function and regulating innate immunity makes it a potential target for IBD therapies. Both RELM-β supplementation and inhibition strategies might be considered depending on the specific disease context and RELM-β expression patterns .

• Asthma treatment approaches: In asthmatic airways, RELM-β contributes to remodeling by increasing fibroblast proliferation and extracellular matrix deposition. Inhibiting RELM-β or its downstream signaling pathways (particularly ERK1/2) might reduce pathological airway remodeling in chronic asthma .

• Dual-targeting strategies: The most sophisticated therapeutic approaches might leverage RELM-β's dual roles - for example, modulating its expression or activity to maintain beneficial antimicrobial functions while limiting detrimental pro-fibrotic effects in specific tissues .

• Biomarker development: RELM-β expression levels could serve as biomarkers for:

  • Intestinal barrier function and helminth infection burden

  • Airways remodeling in asthma

  • Inflammatory processes in IBD

• Personalized medicine approaches: Given RELM-β's multiple roles, therapeutic strategies might need to be tailored based on individual patients' RELM-β expression patterns and functional genetic variants.

What considerations should inform the design of clinical studies investigating RELM-β as a biomarker?

For researchers planning clinical studies to evaluate RELM-β as a biomarker:

• Sample collection standardization:

  • For intestinal studies: Standardize fecal sample collection, storage, and processing protocols to ensure reliable detection of RELM-β in exfoliated intestinal cells

  • For respiratory studies: Establish consistent bronchoalveolar lavage fluid collection and processing methods

  • For tissue biopsies: Develop uniform immunohistochemistry protocols to quantify RELM-β expression

• Control group definition: Careful definition of control groups is essential, considering:

  • Age and sex matching

  • Absence of relevant comorbidities

  • Medication status (particularly immunomodulatory drugs)

  • For intestinal studies, consideration of helminth infection status

• Statistical analysis approaches:

  • Power calculations based on preliminary data to determine appropriate sample sizes

  • Multivariate analysis to account for confounding variables

  • Receiver operating characteristic (ROC) curve analysis to determine optimal cut-off values for diagnostic applications

  • Longitudinal sampling and mixed-effects models to capture temporal dynamics

• Correlation with established markers: Validate RELM-β as a biomarker by correlating with:

  • Established markers of disease activity (e.g., FEV1 in asthma, fecal calprotectin in IBD)

  • Clinical outcomes and disease progression

  • Response to therapeutic interventions

• Technical validation: Ensure technical validation of RELM-β detection methods:

  • Assess inter-laboratory reproducibility

  • Determine assay sensitivity and specificity

  • Evaluate stability of RELM-β in clinical samples under different storage conditions

  • Establish reference ranges in healthy populations