Application of CD74 as biomarker in preparation of kit for evaluating or diagnosing bullous pemphigus

Abstract

The invention discloses application of D74 as a biomarker in preparation of a kit for evaluating or diagnosing bullous pemphigus. The CD74 expression of the skin lesion part of a to-be-detected subject and the CD74 expression of the corresponding position of the normal skin are compared, bullous pemphigus is positive if the CD74 at the skin lesion part is linearly deposited along the true epidermis junction, and the normal tissue is not positively stained. A BP characteristic gene module is identified by adopting a multi-omics integration analysis strategy, and it is found that the CD74 expression level is remarkably related to the eosinophilic granulocyte infiltration degree; the potential of the aptamer as a diagnostic biomarker is verified in patient tissues through immunohistochemistry, the detection sensitivity is 100%, the specificity is 83.3%, the positive predictive value is 78.9%, and the accuracy is 89.7%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomarkers, in particular to the application of CD74 as a biomarker in the preparation of a kit for evaluating or diagnosing bullous pemphigoid. BACKGROUND

[0002] Bullous pemphigoid (BP) is the most common autoimmune subepidermal bullous disease, and its incidence has increased significantly with population aging. Epidemiological data shows that the annual incidence in Europe has risen to 30-47 cases per million, and the one-year mortality rate of hospitalized patients is as high as 20%, mainly due to infectious complications and glucocorticoid-related adverse reactions. This serious situation highlights the urgency of exploring precise diagnosis and treatment strategies.

[0003] The typical pathological feature of BP is epidermal-dermal separation mediated by anti-BP180 / BP230 autoantibodies. Although these antibodies are valuable in diagnosis, their serum titers have no reliable correlation with disease activity. Current diagnosis relies on invasive skin biopsy and direct immunofluorescence detection with high technical requirements, and treatment still uses systemic glucocorticoids as the first-line regimen, with about 27%-53% of patients showing treatment resistance.

[0004] More and more studies have revealed that type II immune response plays a core role in the pathogenesis of BP. Local Th2 cytokines (IL-4, IL-5, IL-13) are significantly enriched in BP lesions, accompanied by eosinophil infiltration and elevated serum IgE levels. Eosinophils not only directly mediate blister formation by releasing toxic granule proteins, but also may amplify Th2 immune response through a positive feedback loop. However, the key molecular mechanisms driving eosinophil recruitment and activation are still unclear.

[0005] CD74 (HLA class II histocompatibility antigen gamma chain; HLA-DR antigens-associated invariant chain) is a type II transmembrane protein, which has dual functions in immune regulation as a multifunctional receptor molecule: it is a chaperone protein of MHC class II molecules and a signal transduction receptor of macrophage migration inhibitory factor (MIF). Recent studies have shown that when MIF binds to CD74, it triggers downstream signaling pathways, such as activation of NF-κB and ERK MAP kinase, promoting cell proliferation, migration and survival, while inhibiting apoptosis. MIF mRNA and protein levels are found to be elevated in activated Th2 cells, while eosinophils store preformed MIF protein and secrete large amounts of MIF upon stimulation. Studies have found that MIF is involved in multiple aspects of eosinophil physiology, including differentiation, survival, activation and migration. CD4 T cells and eosinophils express CD74 and CXCR4. CD74 also interacts with molecules such as CD44, CXCR2 and CXCR4 to form complexes, enhancing inflammatory and immune regulation responses, suggesting that CD74 may be a key molecular hub connecting autoantibody-mediated immune activation and effector cell recruitment.

[0006] There is no public report on the association of CD74 with bullous pemphigoid. SUMMARY

[0007] The present application discloses the application of CD74 as a biomarker in the preparation of a kit for the diagnosis and prognosis of bullous pemphigoid.

[0008] In another aspect of the present application, the application of a CD74 detection reagent in the preparation of a kit for evaluating or diagnosing bullous pemphigoid is proposed, wherein the CD74 detection reagent is a reagent for detecting the expression level of CD74 in a biological sample.

[0009] Among them, the biological sample is the tissue of the lesion of the subject ex vivo.

[0010] In one embodiment, the expression level of CD74 is detected by immunohistochemical staining.

[0011] Specifically, by comparing the CD74 expression of the lesion and the corresponding position of the normal skin of the subject to be tested, wherein the linear deposition of CD74 along the true epidermal junction is positive for bullous pemphigoid, and no positive staining is found in normal tissue.

[0012] The application further provides application of CD74 as a biomarker in preparation of a kit for evaluating the prognosis effect of bullous pemphigoid, wherein the kit evaluates disease progression, recurrence risk or treatment response by detecting the expression level of CD74 in a biological sample.

[0013] The application adopts a multi-omics integrated analysis strategy to identify BP characteristic gene modules, finds that the expression level of CD74 is significantly related to the degree of eosinophil infiltration, and verifies the potential of CD74 as a diagnostic biomarker in patient tissues through immunohistochemistry. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 It is a box plot before standardization of the expression matrix of the data set; Figure 2 It is a box plot after standardization of the expression matrix of the data set, wherein the above samples are 8 normal samples and 17 BP patient samples obtained from a public database, HD-001 is the first normal sample, HD-002 is the second normal sample, and so on, and similarly, BP-001 is the first BP patient sample, and BP-002 is the second BP patient sample; Figure 3 It is a display of 174 differentially expressed genes (DEGs) in a volcano plot; Figure 4 It is a display of the first 20 DEGs in a heat map; Figure 5 It is a GO enrichment analysis of differentially expressed genes (DEGs); Figure 6 It is a KEGG pathway enrichment analysis result of DEGs; Figure 7 It is the three most significant pathways in the GO enrichment analysis and the expression profiles of the related genes; Figure 8 It is a display of genes participating in the first 10 pathways in KEGG analysis; Figure 9 It is a heat map of the enrichment distribution of 28 immune cells among samples (the color gradient represents the standardized enrichment score); Figure 10 It is a difference in immune cell infiltration between the BP group and the normal control group (*P<0.05, **P<0.01, two-tailed t test); Figures 11-12 It is a cross analysis of differentially expressed genes and ImmPort immune gene sets, wherein, Figure 11 It is 42 up-regulated immune-related genes, Figure 12 It is 36 down-regulated genes; Figure 13 It is a GO enrichment analysis showing significantly related immune biological processes; Figure 14 KEGG pathway analysis reveals key immune signaling pathways; Figure 15 Heatmap shows the expression pattern of core genes involved in the top 3 GO pathways; Figure 16 GO enrichment heatmap visualizes the comprehensive analysis results; Figure 17 ROC curve confirms the diagnostic value of CD74 for BP (AUC = 0.886); Figure 18 GSEA analysis shows significant enrichment of antigen processing and presentation pathway in CD74 high expression samples (P.adj = 0.0002453); Figure 19 CD74 protein interaction network constructed by STRING database; Figure 20 Expression distribution of CD74 in BP patient and normal skin tissue, wherein, A-B is CD74 along the linear deposition of the true epidermal junction in BP patient lesions; C, D, I is no CD74 positive staining in normal skin tissue; E, F is positive staining in PV patient lesions; G, H is negative staining in PV patient lesions; Figure 21 CD74 expression characteristics and its clinical relevance; wherein, (A) comparison of CD74 expression level in BP patient (n = 15) and normal control (n = 13) skin tissue (P < 0.05, t test); (B) correlation of CD74 expression with local eosinophil infiltration density (n = 14) (r = 0.78, P < 0.001, Spearman correlation analysis); (C) CD74 expression and BPDAI score (n = 15); (D) serum CRP level (n = 15), (E) IgG deposition intensity (n = 14), (F) BP180 antibody level (n = 11) and (G) local eosinophil number in HE section of BP patient (n = 14) have no significant correlation (all P > 0.05). DETAILED DESCRIPTION

[0015] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, in which:

[0016] The test methods and analysis methods involved in the following examples are as follows: (I) Microarray data and immune-related gene set Raw gene expression data of BP patients and healthy controls were obtained from GEO database (GSE280220; https: / / www.ncbi.nlm.nih.gov / geo / ). This dataset contains 17 BP patients skin samples from the affected area, i.e. the lesion, and 8 healthy control skin samples. The immune-related gene set was derived from ImmPort database (https: / / www.immport.org / ).

[0017] (B) Skin sample collection This study was approved by the Medical Research Ethics Committee of the Second Affiliated Hospital of Anhui Medical University (Approval No: YX2024-231) and all sections were derived from old samples taken from patients. In the skin paraffin-embedded tissue samples, 15 BP patients were taken from the lesion tissue with bullae, itching, redness, etc., derived from 2 upper limbs, 4 lower limbs, 4 trunks, 1 foot, 1 hip, and 3 skin tissues with no specific site recorded, 11 PV patients' lesion tissues were derived from 5 trunks, 1 upper limb, 2 lower limbs, and 3 skin tissues with no specific site recorded. 13 normal skin tissues were derived from 8 heads, 3 feet, 1 trunk, and 1 skin tissue with no specific site recorded. HE sections were derived from the Department of Pathology of the Second Affiliated Hospital of Anhui Medical University, including 15 BP patients and 24 controls (13 normal skin, 11 PV).

[0018] (3) Immunohistochemical staining 1) The removed skin tissue was fixed in 4% paraformaldehyde, paraffin-embedded and cut into 5 μm sections; 2) Paraffin section deparaffinization: place the sections in environmental deparaffinization solution I for 10 min, environmental deparaffinization solution II for 10 min, environmental deparaffinization solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and distilled water for washing; 3) Antigen repair: heat repair the antigen for 4 h, during which time the buffer should be prevented from excessive evaporation, and the slice should not be dried. After natural cooling, place the slide in PBS (PH 7.4) and shake for 3 times, 5 min each time, on a decolorizing shaker; 4) Endogenous peroxidase blocking: place the sections in 3% hydrogen peroxide solution, incubate at room temperature for 25 min in the dark, and place the slide in PBS (PH 7.4) and shake for 3 times, 5 min each time, on a decolorizing shaker; 5) Serum blocking: add 3% BSA in the histological circle to evenly cover the tissue, and block at room temperature for 30 min; 6) First antibody: Gently shake off the blocking solution, add anti-CD74 primary antibody (Cell Signaling Technology, #77274T, 1:500) to the slices, and incubate the slices in a wet box at 4°C overnight; 7) Second antibody: Place the slides in PBS (pH 7.4) on a decolorizing shaker and shake for 3 times, 5 min each time. After gently shaking off the slices, add HRP-labeled goat anti-rabbit IgG to cover the tissue in the circle, and incubate at room temperature for 50 min; 8) DAB color development: Place the slides in PBS (pH 7.4) on a decolorizing shaker and shake for 3 times, 5 min each time. After gently shaking off the slices, add freshly prepared DAB color developing solution in the circle, control the color developing time under a microscope, and the positive is brownish yellow. Rinse the slices with tap water to stop the color development; 9) Nuclear counterstaining: Hematoxylin counterstain for about 3 min, rinse with tap water, differentiate with hematoxylin differentiation solution for a few seconds, rinse with tap water, and return to blue with hematoxylin return blue solution, and rinse with running water; 10) Dehydration and mounting: Place the slices in 75% alcohol for 5 min, 85% alcohol for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, n-butanol for 5 min, and xylene I for 5 min. Dehydrate and clear the slices, and mount them with mounting medium.

[0019] 11) Use Pannoramic MIDI digital slide scanner (3DHISTECH) to obtain images, and analyze with ImageJ software.

[0020] (4) Differential gene screening The expression data (GSE280220) of 17 BP lesions and 8 healthy skin samples were standardized using the R package "limma". The differential expression gene screening criteria were corrected P value <0.05 and |log2FC|>0.5. Heatmap, volcano plot and boxplot were drawn using R packages "heatmap" and "ggplot2" for visualization.

[0021] (5) Differential gene functional enrichment analysis To explore the function of differential genes in biological processes (BP), molecular functions (MF), cellular components (CC), and immune infiltration, GO and KEGG enrichment analysis was performed using the R package "clusterProfiler". P value <0.05 was statistically significant difference.

[0022] (6) Hub gene identification and ROC analysis The differentially expressed genes were intersected with the immune-related gene set, and 78 immune-related differentially expressed genes were obtained. GO enrichment analysis was performed on these genes, and hub genes were determined according to their simultaneous participation in the top 10 pathways. ROC curves were drawn to evaluate their diagnostic performance.

[0023] (7) Protein-protein interaction analysis The protein-protein interaction network of CD74 was constructed using the STRING database (https: / / cn.string-db.org / ). The distribution of CD74 in the skin was predicted using the Human Protein Atlas database (https: / / www.proteinatlas.org / ENSG00000019582-CD74 / tissue / skin).

[0024] (8) Statistical analysis Statistical analysis was performed using R software (version 4.4.2). Student's t-test was used to compare differences between groups, and P<0.05 was considered statistically significant. Spearman rank correlation analysis was used to assess the correlation between measurement data, with a significance level set at P<0.05 (two-sided).

[0025] Example 1 Identification and functional enrichment analysis of differentially expressed genes in BP lesions.

[0026] To explore the differentially expressed genes (DEGs) in BP lesions, we downloaded the original gene expression data from the GSE280220 dataset in the GEO database. After collecting data from 17 BP patients and 8 healthy individuals, we standardized the expression matrix of the dataset. The trend of the box plot was almost a straight line ( Figure 1 and Figure 2 ). Next, we used the threshold of corrected P<0.05 and |logFC|>0.5 to screen the DEGs in the GSE280220 comprehensive dataset. Subsequently, a total of 174 DEGs were identified, including 101 up-regulated DEGs and 73 down-regulated DEGs. The DEGs were displayed in a volcano plot ( Figure 3 ), and the top 20 DEGs were shown in a heatmap ( Figure 4 ).

[0027] GO and KEGG enrichment analysis was then performed using the R “clusterprofiler” package to explore the potential biological functions of the differentially expressed genes (DEGs). The results showed that the enriched GOs were related to adaptive immune responses based on immune receptor body cell recombination, leukocyte-mediated immunity, leukocyte intercellular adhesion, positive regulation of cell activation, regulation of T cell activation (biological process, BP); membrane, plasma membrane lateral, secretory granule membrane and membrane raft, membrane microdomain (cellular component, CC); cytokine receptor binding, immune receptor activity, cytokine activity, cytokine receptor activity, and DNA-binding transcriptional repression activity (molecular function, MF) (Table 2). Figure 5 The top 3 pathways involved in GO enrichment genes were displayed (Table 3). Figure 7 According to KEGG enrichment, the differentially expressed genes played an important role in cytokine-cytokine receptor interaction, tuberculosis, chemokine signaling pathway, Th17 cell differentiation, hepatitis B, and osteoclast differentiation (Table 4). Figure 6 The KEGG analysis results were displayed in the form of a heatmap, and no genes participating in the top 10 pathways were found (Figure 2). Figure 8

[0028] Example 2: Analysis of differentially expressed genes and identification of immune infiltrate-related differentially expressed genes.

[0029] Single-sample gene set enrichment analysis (ssGSEA) performed by the GSVA R package showed that compared with the control group, various immune cell subgroups were significantly enriched in bullous pemphigoid (BP) lesions (Table 5). Figure 9 , Figure 10 The infiltration score of lymphocyte subgroups in the BP microenvironment was significantly increased, including activated and memory CD8+ T cells, activated CD4+ T cells, follicular helper T cells, γδ T cells (P = 0.002), and type 1 / 17 helper T cells. As a supplement to the adaptive immune features, we also observed significant enrichment of innate immune effector cells, including activated B cells, natural killer cells, and activated dendritic cells. By cross-analyzing BP-related differentially expressed genes with the ImmPort immune group database (1,509 immune-related genes), we identified 78 immune infiltration-related differentially expressed genes, including 42 up-regulated genes and 36 down-regulated genes (Table 6). Figure 11 , Figure 12 This synergistic expression pattern of immune activation genes confirms that BP is a disease characterized by excessive activity of the adaptive and innate immune systems in the skin microenvironment.

[0030] Example 3: Screening of hub genes and functional features of hub genes.

[0031] ​​​1) Screening of hub genes Functional enrichment analysis of 78 differentially expressed genes related to immune infiltration showed that they were significantly enriched in processes such as immune response regulation and cytokine production. Figure 13 KEGG analysis mainly involves antigen processing and presentation and chemokine signaling pathways. Figure 14 Key genes in the first three GO pathways are as follows ( Figure 15 As shown in the image. To identify immune-related pivot genes in BP, we visualized candidate gene expression patterns using a heatmap. Figure 16 CD74 was present in all of the top 10 pathways, suggesting its core role. Further evaluation of the diagnostic efficacy of CD74 using ROC curves (GSE280220 dataset) showed an AUC of 0.886. Figure 17 This confirms its high specificity for BP. KEGG and GSEA analyses together showed that samples with high CD74 expression were significantly enriched in antigen processing and presentation-related pathways (P.adj=0.0002453; Figure 18 This study revealed that CD74 participates in the pathogenesis of BP by enhancing immune cell activation.

[0032] 2) Functional characteristics of the pivot gene CD74 By integrating proteomics database analysis, we systematically elucidated the biological functions of CD74 (HLA class II antigen γ chain, ENSP00000009530). The protein encoded by this gene plays a central role in the MHC class II antigen presentation pathway: ① Molecular chaperone function: stabilizing newly synthesized MHC class II α / β heterodimers, guiding their transport from the endoplasmic reticulum to the endosome / lysosome system, and promoting antigen peptide loading (STRING interaction score > 0.9); ② Cell surface receptor function: binding to the pro-inflammatory factor MIF, activating downstream signaling pathways (…). Figure 19 ).

[0033] Example 4: Validation of CD74 in clinical skin tissue.

[0034] Immunohistochemical results showed that in 15 patients with BP and 13 normal controls, linear deposition of CD74 along the dermal-epidermal junction was observed at the BP lesion site. Figure 20 A, B), while no positive staining was observed in normal tissue (A, B), Figure 20 C, D, I). Positive staining of PV lesions ( Figure 20 E, F), negative staining of PV lesions ( Figure 20 G, H). Of the 11 PV cases, 4 were positive and 7 were negative (Table 1).

[0035] To evaluate the diagnostic value of CD74 immunohistochemistry in BP, we analyzed the sensitivity, specificity, positive predictive value, and accuracy. In 11 PV cases, 4 were positive and 7 were negative (Table 1). According to Table 1, the diagnostic performance of CD74 in BP, sensitivity = true positive / (true positive + false negative), reflecting the ability of CD74 to detect BP (true positive rate). Specificity = true negative / (false positive + true negative), reflecting the ability of CD74 to exclude non-BP (true negative rate), positive predictive value = true positive / (true positive + false positive), reflecting the probability that a positive result of CD74 is true BP), accuracy = true positive + true negative / total number of cases. The results showed that the sensitivity was 100%, the specificity was 83.3%, the positive predictive value was 78.9%, and the accuracy was 89.7%. Fisher's exact test showed that the positive rate of the BP group was significantly higher than that of the control group (100% vs 16.7%, P < 0.001). The sensitivity (100%) and high specificity (83.3%) of the test for BP suggest that it can be used as an effective screening tool for BP. Semi-quantitative analysis of immunohistochemistry (i.e., using imageJ to quantify the average staining area as a numerical value) showed that the average staining area of CD74 in the BP group was significantly higher than that in the normal control group (P < 0.05) Figure 21 A).

[0036] We further analyzed the correlation between CD74 immunohistochemistry results and eosinophil infiltration, BPDAI score, BP180 antibody titer, CRP and IgG immunofluorescence, and local tissue eosinophil infiltration in BP samples. The results of CD74 immunohistochemistry in BP samples were semi-quantitatively analyzed, and the correlation between the measured data was evaluated by Spearman rank correlation analysis of R, with a significance level of P < 0.05 (two-tailed). CD74 was significantly positively correlated with systemic eosinophils, but not statistically correlated with CRP, BPDAI score, BP180 antibody titer, local tissue eosinophil infiltration, or IgG Figure 21 B-E).

[0037] Table 1. Diagnostic performance of CD74 in BP

[0038] Table 2. Clinical characteristics of 15 BP patients enrolled

[0039] In summary, the present application first reveals that CD74 is specifically expressed in BP lesions with a diagnostic sensitivity of 100% (95% CI 92.1-100%) and a significant positive correlation between the expression intensity and local eosinophil infiltration density (r = 0.78, P < 0.001). Unlike existing systemic markers (such as BP180-IgG), the tissue expression of CD74 has significant spatial specificity. The lack of correlation between CD74 and BPDAI score, BP180 antibody concentration (P > 0.05) suggests that CD74 is more likely to reflect the destruction of the local immune microenvironment homeostasis rather than the overall burden of the disease. This feature makes it particularly suitable for two clinical scenarios: (1) early differential diagnosis of atypical lesions, especially when serological tests are at the critical value; (2) guiding the precise application of IL-4 / IL-13 targeted therapy. Recent clinical trials have confirmed that the degree of eosinophil infiltration can predict the response to dupilumab treatment. The data further suggest that patients with high CD74 expression may benefit from additional joint blockade of the MIF-CD74 axis.

[0040] CD74 is consistently and highly expressed in BP lesions, with excellent screening sensitivity, effectively excluding non-BP cases; its expression intensity is synchronized with blood eosinophil infiltration, but it has no significant correlation with the number of local skin eosinophil infiltration (Spearman p = 0.12, P = 0.679), suggesting that CD74 is not directly driving eosinophil chemotaxis. CD74 may be indirectly involved in local immune amplification loops of eosinophil chemotaxis or antigen presentation. CD74 levels also lack correlation with CRP, IgG, BPDAI score, and serum BP180 antibody titer, which may indicate that CD74 mainly reflects the skin microenvironment rather than the systemic acute phase response, highlighting its value as a tissue-specific marker.

[0041] The present application provides a method for using CD74 as a biomarker in the preparation of a kit for the diagnosis and prognosis of bullous pemphigoid. There are many methods and approaches to achieve this technical solution, and the above description is only the preferred embodiment of the present application. It should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, several improvements and refinements can be made, which should also be considered within the scope of protection of the present application. The components not explicitly described in the present embodiment can be implemented using existing technology.

Claims

1. Application of CD74 as a biomarker in the preparation of kits for the evaluation or diagnosis of bullous pemphigoid.

2. The application of CD74 detection reagents in the preparation of kits for assessing or diagnosing bullous pemphigoid, wherein, The CD74 detection reagent is used to detect the expression level of CD74 in biological samples.

3. The application according to claim 2, characterized in that, The biological sample is tissue from the skin lesion site of the subject ex vivo.

4. The application according to claim 2, characterized in that, The expression level of CD74 was detected by immunohistochemical staining.

5. The application according to claim 2, characterized in that, By comparing the CD74 expression at corresponding locations on the skin lesions and normal skin of the test subjects, it was found that the CD74 in the lesions showed linear deposition along the dermal-epidermal junction, indicating bullous pemphigoid, while no positive staining was observed in normal tissue.

6. The application of CD74 as a biomarker in the preparation of a kit for evaluating the prognostic effect of bullous pemphigoid, characterized in that, The kit assesses disease progression, relapse risk, or treatment response by detecting the expression level of CD74 in biological samples.

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