Use of alternaria alternata and metabolites thereof in the prevention of neutrophilic asthma

Abstract

This invention discloses the application of *Alternaria alternata* and its metabolites in the prevention of neutrophilic asthma. ITS sequencing revealed that *Alternaria alternata* was the most abundant fungus in the gut of a mouse model of this disease, with an increasing trend. This confirmed that pre-administration of live *Alternaria alternata* or its metabolite oleic acid significantly reduced neutrophil infiltration in lung tissue, decreased IL-17A and KC levels, and alleviated airway inflammation and mucus hypersecretion. Mechanistically, *Alternaria alternata* and oleic acid inhibit the migration of Th17 / ILC3 cells to lung tissue by downregulating CXCL16 expression in lung tissue and reducing CXCR6 expression on the surface of ILC3 cells, thereby blocking the IL-17A-mediated inflammatory pathway. This effect depends on the metabolic activity of *Alternaria alternata* and is independent of gut bacteria. This invention provides a novel gut-lung axis-based microbial intervention strategy for neutrophilic asthma.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of biomedicine and microbial applications, and relates to the application of Alternaria alterniflora and its metabolites in the prevention of neutrophilic asthma. Background Technology

[0002] Bronchial asthma (asthma) is a heterogeneous disease characterized by airway inflammation, airway hyperresponsiveness, and airway remodeling. Based on the type of airway inflammation, asthma is classified into type 2 asthma and non-type 2 asthma. The main inflammatory cells in type 2 asthma include eosinophils, Th2 cells, and type 2 innate lymphocytes (ILC2); the main inflammatory cells in non-type 2 asthma include neutrophils, Th1 cells, and Th17 cells. Type 2 asthma patients generally experience effective symptom relief after receiving inhaled corticosteroids / long-acting β2 receptor agonists (ICS / LABA). For type 2 asthma that cannot be effectively controlled by ICS / LABA (severe asthma), various targeted biological drugs are currently available, such as monoclonal antibodies targeting interleukin (IL)-4, IL-5, and IL-13, which can achieve clinical cure. Compared with type 2 asthma, which is predominantly characterized by eosinophilic infiltration, non-type 2 asthma, which is predominantly characterized by neutrophilic infiltration, has a poor hormone response and very limited existing targeted drug treatment options, making it a current challenge in the clinical treatment of severe asthma.

[0003] The human microbiome plays a crucial role in the development of the immune system and tissue homeostasis. The gut is the organ with the most dense and widespread colonization of microorganisms (bacteria, fungi, protozoa, etc.). Gut microbiota dysbiosis not only leads to dysregulation of the intestinal immune response but also regulates the immune status of the distal lungs through the gut-lung axis, participating in the development and progression of respiratory diseases such as asthma. Similarly, lung inflammation can inversely disrupt the gut microbiota through immune cell migration and circulating inflammatory mediators, forming a bidirectional gut-lung communication network. The gut-lung axis mainly relies on circulating metabolites (short-chain fatty acids, SCFAs, etc.), migration of innate and adaptive immune cells, and lymphatic transport to transmit gut microenvironment signals to lung tissue, which is an important pathway for microbial regulation of lung inflammation.

[0004] Current research on the gut-lung axis largely focuses on gut bacteria. Animal experiments and some clinical studies have confirmed that oral probiotics such as Lactobacillus and Bifidobacterium can reduce airway inflammation and airway hyperresponsiveness in type 2 asthma by regulating Th1 / Th2 balance, inducing regulatory T cell differentiation, and promoting short-chain fatty acid production, thereby reducing the risk of acute asthma attacks. However, existing research reveals several limitations of this type of microbial intervention: First, the protective effect is highly strain-dependent, with significant differences in intervention effects among different strains of the same genus, and some strains even showing no protective effect, making it difficult to promote its application across strains; Second, most reported protective effects are based on OVA and house dust mite-induced type 2 asthma models; for neutrophilic severe asthma, there is a severe lack of animal and clinical evidence for probiotic intervention, and there is no clearly effective oral bacterial intervention regimen; Third, most studies only observe phenotypic improvement, and the complete cellular and molecular regulatory mechanisms are not fully elucidated, limiting the further translation and application of such preparations. Besides bacteria, the gut is also colonized by fungi. As an indispensable component of the gut microbiome, gut fungi participate in the regulation of host immune homeostasis. However, compared to gut bacteria, there are still many gaps in research on the regulation of lung inflammation through the gut-lung axis, especially in neutrophilic severe asthma. Previous studies have reported that the gut-symbiotic protozoan Tritrichomonas musculis (T.mu) can drive the migration of enterogenic ILC2 cells to the lungs through the metabolite succinate in conjunction with gut microbiota, exacerbating eosinophilic asthma. This further suggests that gut eukaryotic microorganisms can regulate the migration and recruitment of intrinsic lymphocytes in the lungs. However, whether gut fungi can regulate the migration of ILC3 / Th17 cells to the lungs and thus intervene in neutrophilic asthma has not yet been reported.

[0005] Currently, asthma prevention and treatment still primarily rely on chemical drugs, which suffer from significant side effects, poor targeting, and a high risk of drug resistance. Even the aforementioned biological agents have drawbacks such as high preparation costs, complex administration methods (mostly intravenous injection), and low patient compliance. Existing technologies do not disclose oral administration of *Alternaria* and its metabolite oleic acid for the prevention of neutrophilic asthma, nor do they offer any insights into using intestinal fungi to regulate the CXCL16 / CXCR6 signaling axis and inhibit ILC3 and Th17 cell lung migration to improve neutrophilic airway inflammation. Therefore, there is an urgent need in this field to develop low-cost, orally administered, and highly targeted microbial agents to alleviate neutrophil-infiltrated non-type 2 severe asthma. Summary of the Invention

[0006] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing the application of Alternaria alternata and its metabolites in the prevention of neutrophilic asthma.

[0007] The objective of this invention can be achieved through the following technical solutions:

[0008] The use of Alternaria in the preparation of oral medications for the prevention of neutrophilic asthma.

[0009] Preferably, the *Alternaria* strain is a live bacterium, and the dosage is 1 × 10⁻⁶. 7 CFU / day.

[0010] Preferably, the *Alternaria* species achieves this by downregulating CXCL16 expression in lung tissue and / or reducing CXCR6 receptor expression on the surface of ILC3 cells, thereby reducing the migration of Th17 cells and / or ILC3 cells to lung tissue.

[0011] Preferably, the Alternaria alternatae achieves this by reducing IL-17A and / or KC levels in lung tissue.

[0012] A pharmaceutical composition comprising an effective amount of Alternaria alternata and a pharmaceutically acceptable carrier.

[0013] Preferably, the Alternaria is a suspension of live bacterial spores.

[0014] The use of the pharmaceutical composition in the preparation of an oral medicine for the prevention of neutrophilic asthma.

[0015] Application of Alternaria metabolite oleic acid in the preparation of oral medications for the prevention of neutrophilic asthma.

[0016] The use of pharmaceutical compositions containing oleic acid in the preparation of oral medications for the prevention of neutrophilic asthma.

[0017] Preferably, the pharmaceutical composition comprises an effective amount of oleic acid and a pharmaceutically acceptable carrier.

[0018] To investigate the pathogenesis of neutrophilic asthma, the inventors analyzed the gut fungi of an asthmatic mouse model using ITS sequencing. They found that *Alternaria alternata* was the most abundant among the annotable fungi, suggesting that *Alternaria alternata* may be associated with the development of asthma, possibly participating in the exacerbation of the disease, or that *Alternaria alternata* may play a role in the negative feedback regulation of disease progression. To verify the role of *Alternaria alternata* in asthma, *Alternaria alternata* (a commercially available strain) was further isolated and cultured, and its spores were eluented and administered to mice by gavage for 2 weeks (1×10⁻⁶). 7 (CFU / day) was administered, and an HDM / LPS neutrophilic asthma model was subsequently constructed. The proportion and number of neutrophils in lung tissue were detected by flow cytometry. The results showed that, compared with the asthmatic group mice, the number of neutrophils in the lung tissue of asthmatic mice administered Alternaria via gavage was significantly reduced. Oleic acid, a metabolite of Alternaria, was identified in vitro using metabolomics. Gavage administration of oleic acid also significantly reduced neutrophil infiltration in the lung tissue of asthmatic mice.

[0019] Beneficial effects:

[0020] The core of neutrophilic asthma is the activation of the IL-17A inflammatory pathway, leading to massive neutrophil infiltration into the lungs. Oral administration of Alternaria alternata and its metabolite oleic acid may reduce Th17 / ILC3 migration to the lungs by regulating the CXCL16 / CXCR6 pathway, thereby downregulating IL-17A levels in lung tissue, reducing neutrophil infiltration, and ultimately alleviating lung inflammation. Attached Figure Description

[0021] Figure 1 ITS sequencing reveals dysbiosis of the fecal gut fungal community in mice with severe asthma.

[0022] (A) Schematic diagram of the HDM / LPS-based mouse model of severe asthma; (B) HE staining; (C) α diversity analysis; (D) β diversity analysis; (E) Stacked bar chart of the top 30 most abundant species; (F) Heatmap of the top 30 most abundant species.

[0023] Figure 2 Inhaling Alternaria does not worsen airway inflammation in asthma.

[0024] (A) Schematic diagram of inhalation of Alternaria and severe asthma model; (B) Classification and counting of BALF cells in mice; (C) ELISA detection of IL-17A expression level in mouse lung tissue; (D) ELISA detection of IL-1β expression level in mouse lung tissue; (E) HE staining and PAS staining of mouse lung tissue.

[0025] Figure 3 Intestinal colonization by Alternaria alternata relieves airway inflammation in severe asthma.

[0026] (A) Morphology of Alternaria alternata spores; (B) Schematic diagram of intestinal Alternaria alternata colonization and severe asthma modeling; (C) ELISA detection of serum IgE level; (D) HE staining and PAS staining of mouse lung tissue; (E) Classification and counting of mouse BALF cells; (F) ELISA detection of KC expression level in BALF; (G) Flow cytometry detection of neutrophil proportion and number in mouse lung tissue; (H) ELISA detection of IL-17A, KC, IL-1β, IL-6, and TNF-α expression levels in lung tissue.

[0027] Figure 4 Intestinal Alternaria targets the IL-17A axis in Th17 / ILC3 cells in the lungs to alleviate neutrophilic asthma.

[0028] (A) Flow cytometry detection of Th17 cells (CD4+) in mouse lung tissue + IL-17A + ) occupying CD45 +Cell proportion; (B) Flow cytometry detection of ILC3 cells in mouse lung tissue (lin - CD127 + RORγt + (C) Proportion of Th17 cells (CD45+) in mouse lung tissue as a percentage of CD45+ cells; + IL-17A + (D) Immunofluorescence detection of mouse lung tissue ILC3 cells (CD3) - RORγt + (E) Schematic diagram of in vivo experimental modeling of IL-17A neutralizing antibody; (F) HE staining and PAS staining of mouse lung tissue; (GI) Flow cytometry detection of the proportion and number of neutrophils in mouse lung tissue; (J) ELISA detection of IL-17A expression level in lung tissue; (K) ELISA detection of KC expression level in lung tissue.

[0029] Figure 5 Intestinal Alternaria drives gut-lung migration of Th17 / ILC3 cells via the CXCR6 / CXCL16 axis.

[0030] (A) ELISA was used to detect the expression level of CXCL16 in mouse lung tissue; (B) A severe asthma model was established by administering PBS or Alternaria alternata via gavage to mice for 2 weeks, and the expression level of CXCR6 on the surface of Th17 cells in mouse lung tissue was detected by flow cytometry; (C) A severe asthma model was established by administering PBS or Alternaria alternata via gavage to mice for 2 weeks, and the expression level of CXCR6 on the surface of ILC3 cells in mouse lung tissue was detected by flow cytometry; (D) After administering PBS or Alternaria alternata via gavage to mice for 2 weeks, the expression level of Th17 cells (CD4+) in mouse intestinal tissue was detected by flow cytometry. + IL-17A + ) occupy CD4 + Cell percentage; (E) After mice were administered PBS or Alternaria via gavage for 2 weeks, flow cytometry was used to detect ILC3 cells (lin) in mouse lung tissue. - CD127 + RORγt + ) - CD127 + Cell ratio.

[0031] Figure 6 Intestinal Alternaria reduces neutrophil infiltration in mouse lung tissue independently of gut bacteria.

[0032] (A) Schematic diagram of mouse experimental grouping and Alternaria alternata gavage for severe asthma modeling in Example 10; (B) HE and PAS staining of mouse lung tissue; (C) Flow cytometry analysis of neutrophils in mouse lung tissue; (D) Neutrophils (CD11b + Ly6G +) occupying CD45 + Cell proportion; (E) Neutrophils (CD11b + Ly6G + Absolute count.

[0033] Figure 7 The protective effect of *Alternaria alternata* depends on its metabolic activity.

[0034] (A) Schematic diagram of mouse experimental grouping and Alternaria gavage and severe asthma modeling; (B) ELISA detection of IL-17A expression level in mouse lung tissue; (C) ELISA detection of KC expression level in mouse lung tissue; (D) Non-targeted metabolomics detection of differential metabolites of live and heat-inactivated Alternaria.

[0035] Figure 8 The role of oleic acid in alleviating severe neutrophilic asthma

[0036] (A) Schematic diagram of mouse experimental grouping and oleic acid gavage for severe asthma modeling; (B) HE and PAS staining of mouse lung tissue; (C) Flow cytometry analysis of neutrophils in mouse lung tissue; (D) Neutrophils (CD11b + Ly6G + ) occupying CD45 + Cell proportion; (E) Neutrophils (CD11b + Ly6G + (F) Absolute count; (G) ELISA detection of IL-17A expression level in mouse lung tissue; (H) ELISA detection of KC expression level in mouse lung tissue; (F) ELISA detection of CXCL16 expression level in mouse lung tissue. Detailed Implementation

[0037] Example 1: Preparation of a mouse model of neutrophilic severe asthma

[0038] A schematic diagram of the severe asthma mouse model constructed using HDM / LPS is shown below. Figure 1The modeling method is referenced in Zhang X, Yang C, Wang M, et al. MMP-12 blockade in B lymphocytes inhibits the formation of inducible BALTs and alleviates neutrophilic allergic airway inflammation. IntImmunopharmacol. 2025;167:115706. doi:10.1016 / j.intimp.2025.115706. Ten 6-8 week old female wild-type C57BL / 6J mice were randomly divided into two groups: a control group and an asthma group, with 5 mice in each group. Mice were anesthetized with isoflurane by inhalation. A 35 μl solution of 10 μg LPS (Sigma Aldrich, L4391) and 25 μg HDM (Greerlabs, XPB91D3A25) was prepared in PBS. This 35 μl LPS / HDM mixture was administered via the airway on days 0, 1, and 2 to sensitize the mice. The sensitization method involved airway coughing; one person held the mouse's nose with forceps and pulled out the base of the tongue, while another person slowly dripped the drug along the posterior pharyngeal wall using a pipette. A coughing sound indicated successful administration. For eight consecutive days (days 7-14), 25 μg / 35 μl HDM was administered via the airway to challenge the mice. The control group received an equal volume of PBS via airway infusion. On day 15, paraffin sections of mouse lung tissue were prepared and stained with hematoxylin and eosin (HE) to reveal inflammatory cell infiltration in the lungs.

[0039] The results showed a significant increase in inflammatory cell infiltration in the lung tissue of asthmatic mice, indicating that the neutrophilic severe asthma model was successfully established. Figure 1 B).

[0040] Example 2: Fecal ITS Sequencing and Analysis in Mice with Neutrophilic Severe Asthma

[0041] After establishing a severe asthma model in mice, two fecal pellets from control mice and model mice were collected at the same time point. Genomic DNA was extracted by Bestway Biotechnology using standard methods. DNA concentration and purity were determined using a Nanodrop nucleic acid detector, and DNA integrity was assessed by agarose gel electrophoresis. PCR amplification was performed using barcode-specific primers and high-fidelity DNA polymerase targeting the fungal ITS1–ITS2 region, with strict control of the amplification cycle number and a negative control included. The amplified products underwent end repair, 3′ A addition, Illumina adapter ligation, and purification. Fragment selection was performed by 2% agarose gel electrophoresis to construct an amplicon library. Qualified libraries with a concentration ≥2 nM and unique index sequences were diluted, mixed, and sequenced at 2×250 pairs using the Illumina NovaSeq platform. The raw paired-end sequencing sequences underwent sample splitting, quality control filtering, and overlapping region splicing. Amplicon sequence variants (ASVs) were obtained using the DADA2 workflow and flattened. Subsequent studies will include α-diversity, β-diversity, species composition, differential abundance, correlation analysis, and fungal function analysis.

[0042] The results showed no significant differences in gut fungal α- and β-diversity between the two groups of mice. Figure 1 (C, D). However, at the genus level, among the fungi with high abundance in the feces of both groups of mice, *Alternaria* showed an increasing trend in abundance in the feces of mice in the severe asthma group, excluding unidentified fungi. In contrast, the abundance of *Eurotium*, *Malassezia*, and *Aspergillus* showed a decreasing trend in the severe asthma group. Figure 1 E, F).

[0043] Example 3: Effects of Alternaria inhalation on asthma phenotype

[0044] Alternaria destruens inhalation: 100 μl of Alternaria destruens (purchased from BioBio Biotechnology Co., Ltd., bio-85418, identified as Alternaria destruens by ITS sequencing) was inoculated onto a PDA plate and cultured at 25°C for 5–7 days. After colony maturation, spores were eluted with autoclaved PBS to prepare a spore suspension. The spores were observed and photographed under an optical microscope to confirm the morphology as Alternaria destruens (…). Figure 3 A). Counting. Experimental mice were randomly divided into three groups: a healthy control group, a simple asthma group, and an Alt. inhalation + asthma group, with five mice in each group. Subsequently, mice in the third group were anesthetized by inhalation of isoflurane and then given 1×10 mmol / L of isoflurane via their airway. 4 CFU of Alternaria alternata. Healthy controls and asthma-only groups were given equal volumes of PBS.

[0045] Severe asthma modeling: A 35 μl solution of 10 μg LPS and 25 μg HDM was prepared by dissolving in PBS. Two weeks after inhalation of Alternaria alternata / PBS, designated as day 0, mice were anesthetized with isoflurane inhalation. The mice were then sensitized by coughing after inhaling the 35 μl LPS / HDM mixture via the airway. From day 7 to 14, 25 μg / 35 μl HDM was administered intratracheally for 8 consecutive days to challenge the model. The healthy control group received an equal volume of PBS. On day 15, the following procedures were performed:

[0046] 1. BALF collection

[0047] After anesthetizing the mice, they were fixed to the operating table, and the skin and tissue of their necks were cut open. The trachea was bluntly dissected to fully expose it. A small hole was gently punctured in the trachea, and a tracheal cannula was inserted, ensuring it was secured with surgical sutures to prevent dislodgement. 0.5 ml of cold PBS containing 1 mM EDTA was slowly aspirated three times and injected into the trachea. After waiting 5 seconds, the PBS was slowly withdrawn, with a recovery rate >80%. The collected BALF was placed on ice and centrifuged at 500 g, 4 °C for 5 minutes. The supernatant was aliquoted and stored at -80 °C. The cell pellet was resuspended in 200 μl of PBS, and cell classification and counting were performed using a hematology analyzer.

[0048] 2. Mouse lung tissue sampling

[0049] (1) After BALF irrigation, the thoracic cavity and abdominal cavity of the mouse were cut open to expose the inferior vena cava and the inferior vena cava was clamped.

[0050] (2) Irrigate the right ventricle with PBS to remove blood from the pulmonary circulation. Stop irrigating when the lung lobes turn white and separate the lung lobes with forceps.

[0051] (3) The upper right lung was placed at 4 ℃ for flow cytometry detection of neutrophils. The lower right lobe was fixed overnight with 4% paraformaldehyde. The remaining lobes were placed at -80 ℃ for later use.

[0052] 3. Staining of lung tissue pathological sections

[0053] (1) Preparation of lung tissue sections:

[0054] 1) Dehydration: The lung tissue fixed with 4% paraformaldehyde was taken out, washed three times with PBS, and then gradually placed in 70%, 85%, 95%, and 100% ethanol for dehydration.

[0055] 2) Clearing: The dehydrated lung tissue was placed in xylene solution for clearing treatment, and this was repeated twice.

[0056] 3) The xylene-treated lung tissue was embedded in molten liquid paraffin.

[0057] 4) Cool the paraffin-embedded lung tissue, shape it, and then remove it.

[0058] 5) Sectioning: Use a paraffin microtome to cut lung tissue sections, with a thickness of approximately 5-8 μm.

[0059] 6) Baking: Place the slices from the previous step on a glass slide and dry at 60 ℃ for later use.

[0060] (2) H&E staining

[0061] 1) Section preparation: Immerse in xylene I and II for 15 min each to dewax, then immerse in anhydrous ethanol I and II for 5 min each, and finally immerse in 95%, 90%, 80%, and 70% ethanol for 3 min each to dehydrate; rinse for 3 min.

[0062] 2) Immerse the treated tissue sections in hematoxylin staining solution for 5 min, rinse with distilled water, differentiate in 1% hydrochloric acid alcohol solution for a few seconds, rinse, then blue again in 0.6% ammonia solution, and rinse.

[0063] 3) Immerse the treated sections in eosin staining solution for about 2 minutes, then rinse with distilled water.

[0064] 4) First, soak in 95% ethanol I and 95% ethanol II for 3 minutes each, then soak in anhydrous ethanol I and II for 3 minutes each to dehydrate, then soak in xylene I and II for 3 minutes each to clear, air dry and seal with neutral resin.

[0065] 5) Observe and take photos.

[0066] (3) PAS staining

[0067] 1) Soak the slices in amylase solution for 20 minutes, then rinse.

[0068] 2) Immerse in 0.5% periodic acid solution for oxidation treatment for 10 minutes, then rinse again.

[0069] 3) Immerse in Schiff's reagent for 20 minutes, then rinse with distilled water for 5-10 minutes until the lung tissue turns pink.

[0070] 4) Soak the slices in hematoxylin stain for 5 minutes, then rinse again.

[0071] 5) Disintegrate in 1% hydrochloric acid-alcohol solution for a few seconds, then rinse again.

[0072] 6) Perform dehydration and clearing treatment according to the H&E staining steps, air dry, mount with neutral resin, and cover with a fixed slide.

[0073] 7) Observe and take photos.

[0074] 4. ELISA detection of IL-1β and IL-17A cytokine levels in lung tissue homogenate

[0075] The following steps use IL-1β as an example:

[0076] (1) On the first day, prepare 1× capture antibody solution, add 100 μl to each well of the ELISA plate, and incubate overnight at 4 ℃, generally for 16-18 hours.

[0077] (2) On the second day, all materials were brought to room temperature. An appropriate amount of washing buffer (PBST) was prepared.

[0078] (3) Remove the microplate, discard the liquid, add 300 μl of PBST washing buffer to each well, wash 4 times, 1 min each time, and pat dry.

[0079] (4) Prepare 1× Assay Diluent A solution, add 200 μl to each well, cover and incubate on a shaker at room temperature for 1 hour.

[0080] (5) During the sealing period, prepare the standard and the sample. Dilute the standard according to the instructions, thaw the sample and dilute it to the appropriate concentration.

[0081] (6) Discard the liquid, add 300 μl of PBST washing buffer to each well, wash 4 times, 1 min each time, and pat dry. Add 100 μl of standard or sample to each well, and set up 2 replicates for each sample. Cover and incubate on a shaker at room temperature for 2 hours.

[0082] (7) Discard the solution, add 300 μl of PBST washing buffer to each well, wash 4 times, 1 min each time, and pat dry. Prepare the detection antibody solution, add 100 μl to each well, cover, and incubate on a shaker at room temperature for 1 hour.

[0083] (8) Discard the solution, add 300 μl of PBST washing buffer to each well, wash 4 times, 1 min each time, and pat dry. Prepare HRP antibody solution, add 100 μl to each well, cover, and incubate on a shaker at room temperature for 30 min.

[0084] (9) Discard the liquid, add 300 μl of PBST washing solution to each well, wash 5 times, each time for more than 1 min, and pat dry.

[0085] (10) Add 100 μl of TMB colorimetric solution to each well and incubate at room temperature in the dark for 20 min, paying attention to color changes during the process.

[0086] (11) Add 100 μl of stop solution to each well.

[0087] (12) Detect absorbance at 450 nm-570 nm within 10 min after termination, plot the standard curve and calculate the sample concentration.

[0088] Results and conclusions:

[0089] BALF inflammatory cell count results ( Figure 2 B) showed that, compared with the control group, the total white blood cell count and neutrophil count in the BALF of the SA group mice were significantly increased, and the eosinophil count showed an increasing trend. The neutrophil count in the BALF of the Alternaria inhalation + SA group was not significantly different from that in the SA group, suggesting that Alternaria inhalation does not affect the number of inflammatory cells in the BALF of asthmatic mice.

[0090] Results of lung tissue homogenate inflammatory factor detection ( Figure 2 (C, D) shows that, compared with the SA group mice, the levels of IL-1β and IL-17A in the lung tissue of the Alternaria inhalation + SA group were not significantly different from those in the SA group, suggesting that Alternaria inhalation does not affect the secretion of pro-inflammatory cytokines in the lung tissue of mice with severe asthma.

[0091] HE staining of lung tissue showed that, compared with the control group, the SA group mice had a large number of inflammatory cell infiltrations around the airways and in the lung interstitium; the inflammatory cell infiltration in the Alternaria inhalation + SA group was not significantly different from that in the SA group. PAS staining showed that, compared with the control group, the PAS positive staining of the airway epithelium of the SA group mice was significantly enhanced, suggesting goblet cell proliferation and increased mucus secretion; the PAS positive staining of the airway in the Alternaria inhalation + SA group was not significantly different from that in the SA group. Figure 2 E). This indicates that Alternaria inhalation does not affect the severe asthma phenotype in mice.

[0092] Example 4: Effects of Alternaria gavage on asthma phenotype

[0093] Alternaria destruens gavage: 100 μl of Alternaria destruens (purchased from BioBio Biotechnology Co., Ltd., bio-85418, identified as Alternaria destruens by ITS sequencing) was inoculated onto a PDA plate and cultured at 25°C for 5–7 days. After colony maturation, spores were eluted with autoclaved PBS to prepare a spore suspension. The spores were observed and photographed under an optical microscope to confirm the morphology as Alternaria destruens. Figure 3 A). Counting. Experimental mice were randomly divided into three groups: a healthy control group, a simple asthma group, and an Alt. + asthma group, with 5-6 mice in each group. Subsequently, they were counted at a rate of 1×10⁻⁶. 7 Group 3 mice were administered CFU / day via gavage for 2 weeks. The healthy control group and the asthma-only group were administered an equal volume of PBS via gavage.

[0094] Severe asthma modeling: 10 μg LPS and 25 μg HDM were dissolved in PBS to prepare a 35 μl solution. Day 0 was defined as the day after the end of Alternaria / PBS gavage. On days 0, 1, and 2, mice were anesthetized by inhalation of isoflurane and then sensitized by coughing with a 35 μl LPS / HDM mixture via the airway. From days 7 to 14, 25 μg / 35 μl HDM was administered via the airway for 8 consecutive days to challenge the mice. The healthy control group received an equal volume of PBS. On day 15, the following procedures were performed:

[0095] 1. Serum sample preparation

[0096] After anesthetizing mice with isoflurane inhalation, one eyeball was gently grasped and enucleated using sterile ophthalmic forceps. The mouse was then quickly inverted to allow blood from the orbital venous plexus to flow out naturally, and the blood was collected in a pre-heparinized or anticoagulant-free centrifuge tube. After incubating at room temperature for 2 hours to allow the blood to clot naturally, the tube was centrifuged at 3000 r / min for 15 min at 4°C. The supernatant serum was separated and stored at −80°C for later use.

[0097] 2. BALF collection

[0098] After anesthetizing the mice, they were fixed to the operating table, and the skin and tissue of their necks were cut open. The trachea was fully exposed by blunt dissection. A small hole was gently punctured in the trachea, and a tracheal cannula was inserted, ensuring it was secured with surgical sutures to prevent dislodgement. 0.5 ml of cold PBS containing 1 mM EDTA was slowly aspirated three times and injected into the trachea. After waiting 5 seconds, the PBS was slowly withdrawn, with a recovery rate >80%. The collected BALF was placed on ice and centrifuged at 500 g, 4 °C for 5 minutes. The supernatant was aliquoted and stored at -80 °C. The cell pellet was resuspended in 200 μl of PBS, and cell classification and counting were performed using a hematology analyzer.

[0099] 3. Mouse lung tissue sampling

[0100] (1) After BALF irrigation, the thoracic cavity and abdominal cavity of the mouse were cut open to expose the inferior vena cava and the inferior vena cava was clamped.

[0101] (2) Irrigate the right ventricle with PBS to remove blood from the pulmonary circulation. Stop irrigating when the lung lobes turn white and separate the lung lobes with forceps.

[0102] (3) The upper right lung was placed at 4 ℃ for flow cytometry detection of neutrophils. The lower right lobe was fixed overnight with 4% paraformaldehyde. The remaining lobes were placed at -80 ℃ for later use.

[0103] 4. Staining of lung tissue pathological sections

[0104] (1) Preparation of lung tissue sections:

[0105] 1) Dehydration: The lung tissue fixed with 4% paraformaldehyde was taken out, washed three times with PBS, and then gradually placed in 70%, 85%, 95%, and 100% ethanol for dehydration.

[0106] 2) Clearing: The dehydrated lung tissue was placed in xylene solution for clearing treatment, and this was repeated twice.

[0107] 3) The xylene-treated lung tissue was embedded in molten liquid paraffin.

[0108] 4) Cool the paraffin-embedded lung tissue, shape it, and then remove it.

[0109] 5) Sectioning: Use a paraffin microtome to cut lung tissue sections, with a thickness of approximately 5-8 μm.

[0110] 6) Baking: Place the slices from the previous step on a glass slide and dry at 60 ℃ for later use.

[0111] (2) H&E staining

[0112] 1) Section preparation: Immerse in xylene I and II for 15 min each to dewax, then immerse in anhydrous ethanol I and II for 5 min each, and finally immerse in 95%, 90%, 80%, and 70% ethanol for 3 min each to dehydrate; rinse for 3 min.

[0113] 2) Immerse the treated tissue sections in hematoxylin staining solution for 5 min, rinse with distilled water, differentiate in 1% hydrochloric acid alcohol solution for a few seconds, rinse, then blue again in 0.6% ammonia solution, and rinse.

[0114] 3) Immerse the treated sections in eosin staining solution for about 2 minutes, then rinse with distilled water.

[0115] 4) First, soak in 95% ethanol I and 95% ethanol II for 3 minutes each, then soak in anhydrous ethanol I and II for 3 minutes each to dehydrate, then soak in xylene I and II for 3 minutes each to clear, air dry and seal with neutral resin.

[0116] 5) Observe and take photos.

[0117] (3) PAS staining

[0118] 1) Soak the slices in amylase solution for 20 minutes, then rinse.

[0119] 2) Immerse in 0.5% periodic acid solution for oxidation treatment for 10 minutes, then rinse again.

[0120] 3) Immerse in Schiff's reagent for 20 minutes, then rinse with distilled water for 5-10 minutes until the lung tissue turns pink.

[0121] 4) Soak the slices in hematoxylin stain for 5 minutes, then rinse again.

[0122] 5) Disintegrate in 1% hydrochloric acid-alcohol solution for a few seconds, then rinse again.

[0123] 6) Perform dehydration and clearing treatment according to the H&E staining steps, air dry, mount with neutral resin, and cover with a fixed slide.

[0124] 7) Observe and take photos.

[0125] 5. Serum total IgE level detection

[0126] Serum IgE concentration was detected using a mouse total IgE ELISA kit, strictly following the kit instructions: standard wells, blank wells, and sample wells were set up. Different concentrations of standard were added to the standard wells, and 100 μL of the test serum (diluted 1:20) was added to each sample well. The mixture was incubated at room temperature for 2 h. After discarding the solution, biotin-labeled anti-IgE antibody was added, and the mixture was incubated at room temperature for 1 h. After washing, streptavidin-HRP was added, and the mixture was incubated at room temperature in the dark for 30 min. After washing, substrate chromogenic solution was added, and the reaction was incubated at room temperature in the dark for 15 min. The reaction was terminated by adding stop solution. The absorbance (OD) value was measured at 450 nm using a microplate reader, and the serum IgE concentration was calculated based on the standard curve.

[0127] 6. ELISA was used to detect the KC content in BALF and the levels of cytokines such as IL-1β, IL-17A, IL-6, TNF-α, and KC in lung tissue homogenates.

[0128] The following steps use IL-1β as an example:

[0129] (1) On the first day, prepare 1× capture antibody solution, add 100 μl to each well of the ELISA plate, and incubate overnight at 4 ℃, generally for 16-18 hours.

[0130] (2) On the second day, all materials were brought to room temperature. An appropriate amount of washing buffer (PBST) was prepared.

[0131] (3) Remove the microplate, discard the liquid, add 300 μl of PBST washing buffer to each well, wash 4 times, 1 min each time, and pat dry.

[0132] (4) Prepare 1× Assay Diluent A solution, add 200 μl to each well, cover and incubate on a shaker at room temperature for 1 hour.

[0133] (5) During the sealing period, prepare the standard and the sample. Dilute the standard according to the instructions, thaw the sample and dilute it to the appropriate concentration.

[0134] (6) Discard the liquid, add 300 μl of PBST washing buffer to each well, wash 4 times, 1 min each time, and pat dry. Add 100 μl of standard or sample to each well, and set up 2 replicates for each sample. Cover and incubate on a shaker at room temperature for 2 hours.

[0135] (7) Discard the solution, add 300 μl of PBST washing buffer to each well, wash 4 times, 1 min each time, and pat dry. Prepare the detection antibody solution, add 100 μl to each well, cover, and incubate on a shaker at room temperature for 1 hour.

[0136] (8) Discard the solution, add 300 μl of PBST washing buffer to each well, wash 4 times, 1 min each time, and pat dry. Prepare HRP antibody solution, add 100 μl to each well, cover, and incubate on a shaker at room temperature for 30 min.

[0137] (9) Discard the liquid, add 300 μl of PBST washing solution to each well, wash 5 times, each time for more than 1 min, and pat dry.

[0138] (10) Add 100 μl of TMB colorimetric solution to each well and incubate at room temperature in the dark for 20 min, paying attention to color changes during the process.

[0139] (11) Add 100 μl of stop solution to each well.

[0140] (12) Detect absorbance at 450 nm-570 nm within 10 min after termination, plot the standard curve and calculate the sample concentration.

[0141] 7. Preparation of single-cell suspension from mouse lung tissue

[0142] Preparation of digestion solution: First, tear the lung into small pieces with tweezers and put them into 15 ml centrifuge tubes. Add 5 ml of 1640 culture medium, 50 μl of 100 mg / ml collagenase IV and 3.5 μl of 10 mg / ml DNase I (working concentration = 7 μg / ml) to each tube. Digest at 37℃ for 30 min, with intermittent pipetting during digestion.

[0143] After digestion, the cells were passed through a 70 μm cell sieve. The cell suspension was centrifuged at 500 g at 4 ℃ for 5 min. If necessary, the cells were lysed with 1 ml of erythrocyte lysis buffer for 2-3 min to terminate the process. Cell counting was then performed.

[0144] 8. Flow cytometry detection of neutrophils in mouse lung tissue

[0145] Single-cell suspensions of mouse lung tissue were obtained using the method described above, and the cell surface markers detected were as follows: CD45, CD11b, and Ly6G.

[0146] (1) Centrifuge at 500 g, 4 ℃, for 5 min and discard the supernatant.

[0147] (2) Resuspend the cells in 100 μl PBS, add 1 μl of live / dead dye (1:1000 dilution), and incubate at room temperature in the dark for 15-30 min.

[0148] (3) Prepare flow cytometry buffer (1% BSA). Wash twice with 1 ml of flow cytometry buffer and resuspend in 100 μl of flow cytometry buffer.

[0149] (4) Add 0.5 μl of CD16 / 32 antibody and mix thoroughly. Incubate at room temperature in the dark for 20 min.

[0150] (5) Add flow cytometry antibodies against anti-CD45, anti-CD11b and anti-Ly6G, and incubate at 4 °C in the dark for 1 hour.

[0151] (6) Add 1 ml of flow cytometry buffer, centrifuge at 500 g, 4 ℃, 5 min, and wash twice.

[0152] (7) Resuspend in 180 μl of flow cytometry buffer, add 20 μl of beads, and wait for instrument detection.

[0153] Results and conclusions:

[0154] like Figure 3 As shown in Figure C, compared with the control group, the serum total IgE level of mice in the SA group (model group) was significantly increased, while the serum IgE level in the Alternaria+SA group showed a decreasing trend compared with the SA group.

[0155] HE staining of lung tissue showed that, compared with the control group, the SA group mice had a large number of inflammatory cell infiltrations around the airways and in the lung interstitium; the inflammatory cell infiltration in the Alternaria+SA group was significantly reduced compared with the SA group. PAS staining showed that, compared with the control group, the SA group mice had significantly enhanced PAS positive staining in the airway epithelium, indicating goblet cell proliferation and increased mucus secretion; the Alternaria+SA group had weakened PAS positive staining in the airway compared with the SA group, indicating reduced mucus secretion. Figure 3 D).

[0156] BALF inflammatory cell count results ( Figure 3 E) showed that, compared with the control group, the total number of BALF cells in the SA group mice was significantly increased, and the number of neutrophils and eosinophils also showed an increasing trend. The number of neutrophils in BALF in the Alternaria+SA group showed a decreasing trend compared with the SA group, suggesting that Alternaria gavage may reduce airway neutrophil infiltration in mice with severe asthma.

[0157] BALF inflammatory factor test results ( Figure 3F) showed that, compared with the control group, the KC level in the BALF of the SA group mice was significantly increased, and the KC level in the BALF of the Alternaria+SA group was significantly decreased compared with the SA group, suggesting that Alternaria gavage can inhibit the secretion of pro-inflammatory factors in the airways of mice with severe asthma.

[0158] Lung tissue flow cytometry results ( Figure 3 G) showed that, compared with the control group, the number of CD11b⁺Ly6G⁺ neutrophils in the lung tissue of mice in the SA group was significantly increased; the number of neutrophils in the lung tissue of the Alternaria+SA group was significantly reduced compared with the SA group.

[0159] Results of lung tissue homogenate inflammatory factor detection ( Figure 3 H) showed that, compared with the SA group mice, the levels of KC and IL-17A in the lung tissue of the Alternaria+SA group were significantly lower, suggesting that Alternaria gavage can inhibit the secretion of pro-inflammatory cytokines in the lung tissue of mice with severe asthma.

[0160] Example 5: Flow cytometry detection of Th17 / ILC3 cell ratio in lung tissue of severely asthmatic mice after gavage administration of Alternaria alternata.

[0161] 1. Flow cytometry detection of Th17 cells in mouse lung tissue:

[0162] Experimental mice were randomly divided into three groups: a healthy control group, a simple asthma group, and an Alt. + asthma group, with 4-5 mice in each group. Alternaria administration, mouse modeling, and lung tissue collection were the same as in Example 4.

[0163] The method for preparing single-cell suspensions of mouse lung tissue is the same as in Example 4. The cell markers detected are as follows: CD45, CD4, and IL-17A.

[0164] (1) Centrifuge at 500 g, 4 ℃, for 5 min and discard the supernatant.

[0165] (2) Resuspend the cells in 100 μl PBS, add 1 μl of live / dead dye (1:1000 dilution), and incubate at room temperature in the dark for 15-30 min.

[0166] (3) Prepare flow cytometry buffer (1% BSA). Wash twice with 1 ml of flow cytometry buffer and resuspend in 100 μl of flow cytometry buffer.

[0167] (4) Add 0.5 μl of CD16 / 32 antibody and mix thoroughly. Incubate at room temperature in the dark for 20 min.

[0168] (5) Add anti-CD45 and anti-CD4 flow cytometry antibodies to each tube and incubate at 4 °C in the dark for 1 hour.

[0169] (6) Add 1 ml of flow cytometry buffer, centrifuge at 500 g, 4 ℃, 5 min, and wash twice.

[0170] (7) Prepare the flow cytometry membrane rupture solution in the following ratio: solution A: solution B = 1:3.

[0171] (8) Add 1 ml of fixative membrane-breaking solution to each tube to resuspend the cells, and break the membrane at 4 ℃ in the dark for 1 hour.

[0172] (9) Centrifuge at 500 g, 4 ℃, 5 min, discard the supernatant, wash twice with 1 ml of membrane breaking solution, and resuspend with 100 μl of membrane breaking solution.

[0173] (10) Add anti-IL-17A flow cytometry antibody and incubate at 4 °C in the dark for 1 hour.

[0174] (11) Wash twice with 1 ml of membrane breaking wash buffer, resuspend in 200 μl of flow cytometry buffer, and wait for instrument detection.

[0175] 2. Flow cytometry detection of ILC3 cells in mouse lung tissue:

[0176] The mouse modeling and lung tissue collection were the same as in Example 4.

[0177] The method for preparing single-cell suspensions of mouse lung tissue is the same as in Example 4. The cell markers detected are as follows: CD45, Lineage, CD127, and RORγt.

[0178] (1) Centrifuge at 500 g, 4 ℃, for 5 min and discard the supernatant.

[0179] (2) Resuspend the cells in 100 μl PBS, add 1 μl of live / dead dye (1:1000 dilution), and incubate at room temperature in the dark for 15-30 min.

[0180] (3) Prepare flow cytometry buffer (1% BSA). Wash twice with 1 ml of flow cytometry buffer and resuspend in 100 μl of flow cytometry buffer.

[0181] (4) Add 0.5 μl of CD16 / 32 antibody and mix thoroughly. Incubate at room temperature in the dark for 20 min.

[0182] (5) Add flow cytometry antibodies against anti-CD45, anti-Lineage, and anti-CD127 to each tube and incubate at 4 °C in the dark for 1 hour.

[0183] (6) Add 1 ml of flow cytometry buffer, centrifuge at 500 g, 4 ℃, 5 min, and wash twice.

[0184] (7) Prepare the flow cytometry membrane rupture solution in the following ratio: solution A: solution B = 1:3.

[0185] (8) Add 1 ml of fixative membrane-breaking solution to each tube to resuspend the cells, and break the membrane at 4 ℃ in the dark for 1 hour.

[0186] (9) Centrifuge at 500 g, 4 ℃, 5 min, discard the supernatant, wash twice with 1 ml of membrane breaking solution, and resuspend with 100 μl of membrane breaking solution.

[0187] (10) Add anti-RORγt flow cytometry antibody and incubate at 4 °C in the dark for 1 hour.

[0188] (11) Wash twice with 1 ml of membrane breaking wash buffer, resuspend in 200 μl of flow cytometry buffer, and wait for instrument detection.

[0189] The results showed that, compared with the SA group, the proportions of Th17 cells and ILC3 cells in the lung tissue of severely asthmatic mice administered Alternaria via gavage were significantly reduced. Figure 4 A, B). This suggests that oral administration of Alternaria alternata significantly reduces the infiltration of Th17 and ILC3 cells in lung tissue.

[0190] Example 6: Immunofluorescence detection of Th17 / ILC3 cell count in lung tissue of severely asthmatic mice after gavage administration of Alternaria alternata.

[0191] The experimental mice were randomly divided into three groups: a healthy control group, a simple asthma group, and an Alt. + asthma group, with 4-5 mice in each group. The mouse modeling and lung tissue collection were the same as in Example 4.

[0192] 1. Preparation of paraffin sections of lung tissue

[0193] (1) Dehydration: After cleaning, the lung tissue was placed in 70%, 80%, 95% and 100% ethanol in sequence for dehydration.

[0194] (2) Transparent: Soak in xylene for 30 minutes each time, repeat twice.

[0195] (3) Paraffin embedding: The tissue was immersed in molten paraffin at about 60 °C for 1 hour, and this was repeated twice. After the paraffin solidified, it was cut into thin sections with a thickness of 3-5 μm.

[0196] (4) Drying: Dry the cut slices at 60 ℃.

[0197] (5) Dewaxing and rehydration: After dewaxing the slide in xylene, it is then rehydrated in a gradient of 100%, 95%, 80%, and 70% alcohol and then rinsed before use.

[0198] 2. Immunofluorescence staining

[0199] (1) Cut a 4 μm thick section from the paraffin-embedded lung tissue, lay it flat on a glass slide, and perform dewaxing and rehydration treatment. Finally, rinse the glass slide with distilled water.

[0200] (2) Antigen repair was performed using citric acid repair solution at a high temperature of 100 °C.

[0201] (3) Block endogenous peroxidase in brain tissue with 3% hydrogen peroxide, incubate at room temperature in the dark for 25 minutes, and wash with PBS 3 times.

[0202] (4) Shake off the PBS and permeate with 0.1% Triton X-100 for 5 min.

[0203] (5) Rinse three times with PBS, then block with 10% goat serum for 30 min.

[0204] (6) Remove the blocking solution, dilute the primary antibodies against anti-CD4 and anti-IL-17A to an appropriate concentration and add them to the sample to cover the sample, which is used to label Th17 cells; dilute the primary antibodies against anti-CD3 and anti-RORγt to an appropriate concentration and add them to the sample to cover the sample, which is used to label ILC3 cells, and incubate overnight at 4°C.

[0205] (7) After washing off the primary antibody, select a fluorescent secondary antibody that matches the primary antibody, dilute it to a suitable concentration, and incubate at 37°C for 1 hour.

[0206] (8) After cleaning, add DAPI to cover the tissue area and incubate at room temperature in the dark for 10 minutes.

[0207] (9) Microscopic observation results.

[0208] The results showed that, compared with the SA group, the lung tissue of severely asthmatic mice administered Alternaria via gavage contained Th17 cells (CD4+). + IL-17 + ) and ILC3 cells (CD3 - RORγt + The number of all decreased significantly. Figure 4 C, D).

[0209] Example 7: In vivo experiment of IL-17A neutralizing antibody

[0210] Alternaria alternata oral gavage: 100 μl of Alternaria alternata (purchased from Bio-85418, Bio-85418) was inoculated onto a PDA plate and cultured at 25°C for 5–7 days. After colony maturation, spores were eluted with autoclaved PBS to prepare a spore suspension. The spores were observed and photographed under an optical microscope to confirm the morphology as Alternaria alternata (…). Figure 2A). Counting. Experimental mice were randomly divided into four groups: asthma group, Alt. + asthma group, asthma + IL-17A neutralizing antibody group, and Alt. + asthma + IL-17A neutralizing antibody group, with six mice in each group. Subsequently, at a rate of 1×10⁻⁶... 7 Wild-type (WT) mice were administered CFU / day via gavage for 2 weeks. The control group was administered an equal volume of PBS via gavage.

[0211] Severe asthma modeling: 10 μg LPS and 25 μg HDM were dissolved in PBS to prepare a 35 μl system solution. The day after the end of Alternaria gavage was recorded as day 0. On days 0, 1, and 2, 35 μl of the LPS and HDM mixture was administered intratracheally to induce sensitization. From days 7 to 14, 25 μg / 35 μl of HDM was administered intratracheally for 8 consecutive days to induce provocation.

[0212] Throughout the modeling process, each mouse was administered 50 μg of IL-17A neutralizing antibody or the corresponding isotype control antibody via intraperitoneal injection twice a week. After modeling was completed, the following procedures were performed:

[0213] 1. Mouse lung tissue sampling

[0214] Same as Example 4.

[0215] 2. Flow cytometry detection of neutrophils in mouse lung tissue

[0216] Same as Example 4.

[0217] 3. ELISA detection of cytokine levels such as IL-17A and KC in lung tissue homogenate

[0218] Same as Example 4.

[0219] 4. Staining of lung tissue pathological sections

[0220] Same as Example 4.

[0221] Results and conclusions:

[0222] Results of HE staining and PAS staining of lung tissue ( Figure 4 F) showed that the SA group mice had significant peri-inflammatory cell infiltration and goblet cell mucus secretion. The above pathological changes in the Alternaria+SA group were significantly reduced compared with the SA group. However, there was no significant difference in the degree of airway inflammation and mucus secretion between the SA+IL-17A NAb group and the Alternaria+SA+IL-17A NAb group.

[0223] Lung tissue flow cytometry results ( Figure 4GI showed that, compared with the SA group, the number of CD11b⁺Ly6G⁺ neutrophils in the lung tissue of mice in the Alternaria+SA group was significantly reduced. After intervention with IL-17A neutralizing antibody, the proportion and number of neutrophils in the lung tissue of mice in both the SA+IL-17A NAb group and the Alternaria+SA+IL-17A NAb group were at a low level, and there was no statistically significant difference between the two groups.

[0224] Results of lung tissue inflammatory factor detection ( Figure 4 JK) showed that, compared with the SA group, the levels of IL-17A and neutrophil chemokine KC in the lung tissue of mice in the Alternaria+SA group were significantly reduced; however, after intervention with IL-17A neutralizing antibody, there was no significant difference in IL-17A and KC levels between the SA+IL-17A NAb group and the Alternaria+SA+IL-17A NAb group.

[0225] The above results indicate that gavage administration of Alternaria alternata significantly reduced airway inflammation and mucus hypersecretion in mice with neutrophilic severe asthma and decreased neutrophil infiltration in lung tissue. Intervention with IL-17A neutralizing antibody eliminated the inhibitory effect of Alternaria alternata on neutrophil infiltration, suggesting that the effect of Alternaria alternata in reducing neutrophil infiltration in lung tissue of mice with severe asthma mainly depends on the regulation of the IL-17A signaling pathway.

[0226] Example 8: ELISA detection of CXCL16 expression level in lung tissue of severely asthmatic mice after gavage administration of Alternaria alternata.

[0227] The experimental mice were randomly divided into three groups: a healthy control group, a simple asthma group, and an Alt. + asthma group, with 5-6 mice in each group. The modeling and lung tissue collection were performed in the same manner as in Example 4, strictly following the instructions.

[0228] The results showed that, compared with the SA group, the expression level of CXCL16 in the lung tissue of severely asthmatic mice administered Alternaria via gavage was significantly decreased. Figure 5 A).

[0229] Example 9: Flow cytometry detection of CXCR6 receptor expression levels on the surface of Th17 and ILC3 cells in mouse lung tissue

[0230] Experimental mice were randomly divided into three groups: a healthy control group, a simple asthma group, and an Alt. + asthma group, with six mice in each group. The mouse model preparation was the same as in Example 4. Lung tissue collection and single-cell suspension preparation were also the same as in Example 4.

[0231] 1. Flow cytometry detection of CXCR6 expression in Th17 cells of mouse lung tissue:

[0232] The cell markers detected were: CD45, CD4, IL-17A, and CXCR6.

[0233] (1) Centrifuge at 500 g, 4 ℃, for 5 min and discard the supernatant.

[0234] (2) Resuspend the cells in 100 μl PBS, add 1 μl of live / dead dye (1:1000 dilution), and incubate at room temperature in the dark for 15-30 min.

[0235] (3) Prepare flow cytometry buffer (1% BSA). Wash twice with 1 ml of flow cytometry buffer and resuspend in 100 μl of flow cytometry buffer.

[0236] (4) Add 0.5 μl of CD16 / 32 antibody and mix thoroughly. Incubate at room temperature in the dark for 20 min.

[0237] (5) Add flow cytometry antibodies against anti-CD45, anti-CD4, and anti-CXCR6 to each tube and incubate at 4 °C in the dark for 1 hour.

[0238] (6) Add 1 ml of flow cytometry buffer, centrifuge at 500 g, 4 ℃, 5 min, and wash twice.

[0239] (7) Prepare the flow cytometry membrane rupture solution in the following ratio: solution A: solution B = 1:3.

[0240] (8) Add 1 ml of fixative membrane-breaking solution to each tube to resuspend the cells, and break the membrane at 4 ℃ in the dark for 1 hour.

[0241] (9) Centrifuge at 500 g, 4 ℃, 5 min, discard the supernatant, wash twice with 1 ml of membrane breaking solution, and resuspend with 100 μl of membrane breaking solution.

[0242] (10) Add anti-IL-17A flow cytometry antibody and incubate at 4 °C in the dark for 1 hour.

[0243] (11) Wash twice with 1 ml of membrane breaking wash buffer, resuspend in 200 μl of flow cytometry buffer, and wait for instrument detection.

[0244] 2. Flow cytometry detection of CXCR6 expression in ILC3 cells of severely asthmatic mice after gavage administration of Alternaria alternata:

[0245] The cell markers detected were: CD45, Lineage, CD127, RORγt, and CXCR6.

[0246] (1) Centrifuge at 500 g, 4 ℃, for 5 min and discard the supernatant.

[0247] (2) Resuspend the cells in 100 μl PBS, add 1 μl of live / dead dye (1:1000 dilution), and incubate at room temperature in the dark for 15-30 min.

[0248] (3) Prepare flow cytometry buffer (1% BSA). Wash twice with 1 ml of flow cytometry buffer and resuspend in 100 μl of flow cytometry buffer.

[0249] (4) Add 0.5 μl of CD16 / 32 antibody and mix thoroughly. Incubate at room temperature in the dark for 20 min.

[0250] (5) Add flow cytometry antibodies against anti-CD45, anti-Lineage, anti-CD127, and anti-CXCR6 to each tube and incubate at 4°C in the dark for 1 hour.

[0251] (6) Add 1 ml of flow cytometry buffer, centrifuge at 500 g, 4 ℃, 5 min, and wash twice.

[0252] (7) Prepare the flow cytometry membrane rupture solution in the following ratio: solution A: solution B = 1:3.

[0253] (8) Add 1 ml of fixative membrane-breaking solution to each tube to resuspend the cells, and break the membrane at 4 ℃ in the dark for 1 hour.

[0254] (9) Centrifuge at 500 g, 4 ℃, 5 min, discard the supernatant, wash twice with 1 ml of membrane breaking solution, and resuspend with 100 μl of membrane breaking solution.

[0255] (10) Add anti-RORγt flow cytometry antibody and incubate at 4 °C in the dark for 1 hour.

[0256] (11) Wash twice with 1 ml of membrane breaking wash buffer, resuspend in 200 μl of flow cytometry buffer, and wait for instrument detection.

[0257] The results showed that, compared with the SA group, gavage administration of Alternaria alterniflora did not affect the fluorescence intensity of the CXCR6 receptor on the surface of Th17 cells, but it could reduce the fluorescence intensity of the CXCR6 receptor on the surface of ILC3 cells. Figure 5 (B, C) This suggests that Alternaria may reduce the migration of Th17 cells and / or ILC3 cells to lung tissue by downregulating CXCL16 expression in lung tissue and / or reducing CXCR6 receptor expression on the surface of ILC3 cells.

[0258] Example 10: Flow cytometry detection of Th17 and ILC3 cells in mouse intestinal tissue after gavage administration of Alternaria alternata.

[0259] Grouping:

[0260] Ten SPF-grade female B6 mice aged 6-8 weeks with similar weights were randomly divided into two groups: a PBS control group (n=5) and an Alternaria spore gavage group (n=5).

[0261] 1. Gavage administration of *Alternaria alternata*: *Alternaria alternata* was cultured in an incubator at 28°C for 5–7 days. After the colonies matured, the spores were washed away with autoclaved PBS to prepare a spore suspension. Subsequently, wild-type (WT) mice were administered the suspension by gavage at a dose of 1 × 10^7 spores / day for 2 weeks.

[0262] 2. Mouse intestinal tissue sampling:

[0263] Mice were anesthetized with sodium pentobarbital and then euthanized by cervical dislocation. The abdominal cavity was opened, and all intestinal tissue was separated and removed. The tissue was repeatedly rinsed in PBS until no feces were present, while adipose tissue was also removed.

[0264] 3. Flow cytometry analysis of the number and proportion of Th17 cells and ILC3 cells in mouse intestinal tissue:

[0265] (1) Preparation of single-cell suspension of mouse intestinal tissue

[0266] 1) Preparation of digestive juice: First, cut the intestinal tissue into small pieces with scissors. Prepare the digestive juice as before. Digest at 37 ℃ for 30 min, and blow air intermittently during the process.

[0267] 2) After digestion, the cells were passed through a 70 μm cell sieve, and the cell suspension was centrifuged at 500 g at 4 ℃ for 5 min.

[0268] 3) Cell counting.

[0269] (2) Flow cytometry (detection of Th17 cells in intestinal tissue)

[0270] 1) Single-cell suspensions of mouse intestinal tissue were obtained according to the above method. The cell surface markers detected were as follows: CD45, CD4, IL-17A.

[0271] 2) Centrifuge at 500 g, 4 ℃, for 5 min, and discard the supernatant completely.

[0272] 3) Resuspend the cells in 100 μl PBS, add 1 μl of live / dead dye (1:10 dilution), and incubate at room temperature in the dark for 15-30 min.

[0273] 4) Prepare flow cytometry buffer (1% BSA). Wash twice with 1 ml of flow cytometry buffer, and resuspend in 100 μl of flow cytometry buffer.

[0274] 5) Add 0.5 μl of CD16 / 32 antibody, mix thoroughly, and incubate at room temperature in the dark for 20 min.

[0275] 6) Add flow cytometry antibodies against anti-CD45 and anti-CD4, and incubate at 4 °C in the dark for 1 hour.

[0276] 7) Add 1 ml of flow cytometry buffer, centrifuge at 500 g, 4 ℃, for 5 min, and wash twice.

[0277] 8) Resuspend the cells in 200 μl of membrane-breaking fixative (1:3 dilution) and break the membrane at 4 ℃ for 1 hour.

[0278] 9) Add 1 ml of membrane breaking washing buffer (prepared to 1× in advance), centrifuge at 500 g, 4 ℃, for 5 min, and wash twice.

[0279] 10) Resuspend in 100 μl of flow cytometry buffer, add anti-IL-17A flow cytometry antibody, and incubate at 4 °C in the dark for 1 hour.

[0280] 11) Add 1 ml of membrane breaking wash buffer, centrifuge at 500 g, 4 ℃, 5 min, and wash twice.

[0281] 12) Resuspend the cells in 180 μl of flow cytometry buffer, add 20 μl of counting microspheres, and wait for instrumental analysis.

[0282] (3) Flow cytometry (detection of ILC3 cells in intestinal tissue)

[0283] 1) Single-cell suspensions of mouse lung tissue were obtained using the method described above. The cell surface markers detected were as follows: CD45, Lineage, CD127, and RORγt.

[0284] 2) Centrifuge at 500 g, 4 ℃, for 5 min, and discard the supernatant completely.

[0285] 3) Resuspend the cells in 100 μl PBS, add 1 μl of live / dead dye (1:10 dilution), and incubate at room temperature in the dark for 15-30 min.

[0286] 4) Prepare flow cytometry buffer (1% BSA). Wash twice with 1 ml of flow cytometry buffer, and resuspend in 100 μl of flow cytometry buffer.

[0287] 5) Add 0.5 μl of CD16 / 32 antibody, mix thoroughly, and incubate at room temperature in the dark for 20 min.

[0288] 6) Add flow cytometry antibodies against anti-CD45, anti-Lineage, and anti-CD127, and incubate at 4 °C in the dark for 1 hour.

[0289] 7) Add 1 ml of flow cytometry buffer, centrifuge at 500 g, 4 ℃, for 5 min, and wash twice.

[0290] 8) Resuspend the cells in 200 μl of membrane-breaking fixative (diluted 1:3) and break the membrane at 4 ℃ for 1 hour.

[0291] 9) Add 1 ml of membrane breaking washing buffer (prepared to 1× in advance), centrifuge at 500 g, 4 ℃, for 5 min, and wash twice.

[0292] 10) Resuspend in 100 μl of flow cytometry buffer, add anti-RORγt flow cytometry antibody, and incubate at 4 °C in the dark for 1 hour.

[0293] 11) Add 1 ml of membrane breaking wash buffer, centrifuge at 500 g, 4 ℃, for 5 min, and wash twice.

[0294] 12) Resuspend the cells in 180 μl of flow cytometry buffer, add 20 μl of counting microspheres, and wait for instrumental analysis.

[0295] Flow cytometry results showed that, compared with the PBS group, the proportions of Th17 cells and ILC3 cells in the intestinal tissue of mice administered Alternaria via gavage were significantly reduced. Figure 5 (D, E). This suggests that *Alternaria* bacteria may regulate local intestinal immune function.

[0296] Example 11: After clearing intestinal bacteria, Alternaria was administered by gavage to establish a mouse model of neutrophilic severe asthma, and the phenotypic changes of asthma were investigated.

[0297] The experimental mice were randomly divided into 4 groups: healthy control group, asthma group, Alt. + asthma group, and ABX + Alt. + asthma group, with 4-6 mice in each group.

[0298] 1. Prepare antibiotic solution: Neomycin 1mg / ml, Streptomycin 1mg / ml, Bacitracin 1mg / ml, ABX+Alt.+ Asthma group mice drink the solution for one week.

[0299] 2. Gavage administration of *Alternaria alternata*: *Alternaria alternata* was cultured in an incubator at 28°C for 5–7 days. After colony maturation, spores were washed away with autoclaved PBS to prepare a spore suspension. Subsequently, mice in the Alt.+asthma group and the ABX+Alt.+asthma group were administered the suspension by gavage at a dose of 1×10^7 spores / day for 2 weeks.

[0300] 3. Severe asthma modeling: 10 μg LPS and 25 μg HDM were dissolved in PBS to prepare a 35 μl system solution. On days 0, 1, and 2, 35 μl of the LPS and HDM mixture solution was administered intratracheally to induce sensitization. On days 7-14, 25 μg / 35 μl of HDM was administered intratracheally for 8 consecutive days to induce provocation.

[0301] 4. Mouse lung tissue sampling

[0302] Same as Example 4.

[0303] 5. Flow cytometry analysis of the number and proportion of neutrophils in mouse lung tissue

[0304] Same as Example 4.

[0305] Results and conclusions:

[0306] HE staining and PAS staining results of lung tissue (Figure 6B) showed that the SA group mice had significant peri-infiltration of inflammatory cells, proliferation of goblet cells in the airways, and increased mucus secretion; the above pathological changes in the Alternaria+SA group were significantly reduced compared with the SA group; while the ABX+Alternaria+SA group mice showed pathological changes that were not significantly different from those in the Alternaria+SA group.

[0307] Flow cytometry results showed that, compared with the SA group, the infiltration of CD11b⁺Ly6G⁺ neutrophils in the lung tissue of mice in the Alternaria+SA group was significantly reduced; while the level of neutrophil infiltration in the lung tissue of mice in the ABX+Alternaria+SA group was not significantly different from that in the Alternaria+SA group. Figure 6 CE).

[0308] The above results indicate that even after clearing intestinal bacteria, gavage administration of Alternaria alternatae can still reduce neutrophil recruitment in mouse lung tissue, suggesting that the protective effect of Alternaria alternatae may not depend on intestinal bacteria.

[0309] Example 12: A mouse model of severe neutrophilic asthma was established by gavage administration of live / heat-inactivated Alternaria alternatae, and the phenotypic changes of asthma were investigated.

[0310] The experimental mice were randomly divided into 4 groups: healthy control group, asthma group, Alt.+asthma group, and HK-Alt.+asthma group, with 4-6 mice in each group.

[0311] 1. Live / heat-inactivated Alternaria alternata via gavage: Alternaria alternata is cultured in a 28°C incubator for 5–7 days until colonies mature. Spores are then washed away with autoclaved PBS to prepare a spore suspension. The spore suspension is divided into two tubes, one of which is inactivated at 95°C for 10 minutes. Subsequently, the suspension is administered via gavage at a concentration of 1×10⁻⁶ mg / L. 7 Wild-type (WT) mice were administered a dose of 1 spore / day by gavage for 2 weeks.

[0312] 2. Severe asthma modeling: 10 μg LPS and 25 μg HDM were dissolved in PBS to prepare a 35 μl system solution. On days 0, 1, and 2, 35 μl of the LPS and HDM mixture solution was administered intratracheally to induce sensitization. On days 7-14, 25 μg / 35 μl of HDM was administered intratracheally for 8 consecutive days to induce provocation.

[0313] 3. Mouse lung tissue sampling:

[0314] Same as Example 4.

[0315] 4. ELISA detection of IL-17A and KC concentrations in mouse lung tissue

[0316] Same as Example 4.

[0317] The results show that ( Figure 7 Compared with the SA group, after gavage administration of live Alternaria, the levels of IL-17A and KC in the lung tissue of mice were significantly reduced, while after gavage administration of inactivated Alternaria, there was no significant change in the levels of IL-17A and KC in the lung tissue of mice, indicating that the protective effect of Alternaria depends on its metabolic activity.

[0318] Example 13: Non-targeted metabolomics detection of Alternaria metabolites

[0319] Grouping:

[0320] Alternaria spores were washed with 5 ml of sterile PBS in vitro and divided into two tubes, each adjusted to approximately 1 × 10^8 spores / ml. After centrifugation at 500 g for 5 min, the supernatant was discarded. One tube was left untreated, and the other was inactivated at 95 °C for 10 min.

[0321] 1. Metabolite extraction:

[0322] (1) Weigh 25 mg of sample into an EP tube at low temperature, add 2 homogenization beads, and add 500 μL of extraction solution (methanol:acetonitrile:water = 2:2:1 (V / V)). The extraction solution contains an isotope-labeled internal standard.

[0323] (2) Vortex mixing for 30 s;

[0324] (3) Homogenize in a homogenizer (35Hz, 4min), then transfer to an ice water bath and sonicate for 5min. Repeat this step 3 times.

[0325] (4) Let stand at -40 ℃ for 1 h;

[0326] (5) Centrifuge the sample at 4 ℃, 12000 rpm (centrifugal force 13800 (×g), radius 8.6cm) for 15 min;

[0327] (6) Collect the supernatant into a sample vial for instrumental analysis;

[0328] 2. On-machine testing:

[0329] For polar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UHPLC) with a Waters ACQUITY UPLC BEH Amide (2.1 mm × 100 mm, 1.7 μm) column for chromatographic separation of the target compounds. Phase A of the HPLC was aqueous, containing 25 mmol / L ammonium acetate and 25 mmol / L ammonia; Phase B was acetonitrile. Sample tray temperature: 4 ℃; injection volume: 2 μL.

[0330] For nonpolar metabolites, this project used a Vanquish (Thermo Fisher Scientific) ultra-high performance liquid chromatograph (UHPLC) with a Phenomenex Kinetex C18 (2.1 mm × 100 mm, 2.6 μm) column for chromatographic separation of target compounds. Phase A of the HPLC was aqueous containing 0.01% acetic acid, and Phase B was isopropanol:acetonitrile (1:1, v / v). Sample tray temperature: 4 ℃, injection volume: 2 μL.

[0331] The Orbitrap Exploris 120 mass spectrometer can perform primary and secondary mass spectrometry data acquisition under the control of software (Xcalibur, version 4.4, Thermo). Detailed parameters are as follows: Sheath gas flow rate: 50 Arb, Aux gas flow rate: 15 Arb, Capillary temperature: 320 ℃, Full ms resolution: 60000, MS / MS resolution: 15000, Collision energy: SNCE 20 / 30 / 40, SprayVoltage: 3.8 kV (positive) or -3.4 kV (negative).

[0332] 3. Offline data processing:

[0333] The raw data was converted into mzXML format using ProteoWizard software (V3.0.24054), and then metabolite identification was performed using a collaboratively developed R package. The database used was BiotreeDB (V3.0), and then visualization analysis was performed using a self-developed R package.

[0334] The results showed that, compared with inactivated Alternaria, the content of long-chain fatty acids such as oleic acid, palmitoleic acid, and α-linolenic acid in live Alternaria was significantly increased. Figure 7(D) This suggests that Alternaria may regulate airway inflammation in severe asthma through its metabolites.

[0335] Example 14: In vivo intervention experiment of Alternaria metabolites

[0336] The experimental mice were randomly divided into 3 groups: healthy control group, asthma group, and oleic acid + asthma group, with 7-8 mice in each group.

[0337] 1. Oleic acid was administered to wild-type (WT) mice by gavage at a dose of 1 mg / day / mouse for 2 weeks, while the control group was given the same volume of PBS.

[0338] 2. Severe asthma modeling: 10 μg LPS and 25 μg HDM were dissolved in PBS to prepare a 35 μl system solution. On days 0, 1, and 2, 35 μl of the LPS and HDM mixture solution was administered intratracheally to induce sensitization. On days 7-14, 25 μg / 35 μl of HDM was administered intratracheally for 8 consecutive days to induce provocation.

[0339] 3. Mouse lung tissue sampling and pathological staining

[0340] Same as Example 4.

[0341] 4. Flow cytometry analysis of the number and proportion of neutrophils in mouse lung tissue

[0342] Same as Example 4.

[0343] 5. ELISA detection of cytokine levels such as IL-17A, KC, and CXCL16 in mouse lung tissue.

[0344] Same as Example 4.

[0345] Results and conclusions:

[0346] HE and PAS staining results of lung tissue showed that, compared with the SA group mice, mice with severe asthma treated with oleic acid had reduced inflammatory cell infiltration in lung tissue and reduced airway mucus secretion. Figure 8 B).

[0347] Flow cytometry results showed that, compared with the SA group mice, the number of neutrophils in the lung tissue of severe asthmatic mice given oleic acid was significantly reduced. Figure 8 CE).

[0348] ELISA results showed that, compared with the SA group, gavage administration of oleic acid significantly reduced the levels of IL-17A / KC / CXCL16 cytokines in the lung tissue of mice with severe asthma. Figure 8 FH).

[0349] The above results indicate that oleic acid intervention can significantly reduce airway inflammation in mice with neutrophilic severe asthma, reduce neutrophil infiltration and chemokine secretion in lung tissue, suggesting that oleic acid may exert a protective effect against severe asthma by inhibiting the IL-17A-related inflammatory pathway.

Claims

1. Alternaria ( Alternaria Its application in the preparation of oral medications for the prevention of neutrophilic asthma.

2. The application according to claim 1, characterized in that, The Alternariae strain was a live bacterium, and the dosage was 1×10⁻⁶. 7 CFU / day.

3. The application according to claim 1, characterized in that, The Alternaria species achieves this by downregulating CXCL16 expression in lung tissue and / or reducing CXCR6 receptor expression on the surface of ILC3 cells, thereby reducing the migration of Th17 cells and / or ILC3 cells to lung tissue.

4. The application according to claim 1, characterized in that, The Alternaria species achieves this by reducing IL-17A and / or KC levels in lung tissue.

5. A pharmaceutical composition, characterized in that, It contains an effective amount of Alternaria alternata and a pharmaceutically acceptable carrier.

6. The pharmaceutical composition according to claim 5, characterized in that, The Alternaria species is a suspension of live bacterial spores.

7. The use of the pharmaceutical composition of claim 5 in the preparation of an oral medicament for the prevention of neutrophilic asthma.

8. Application of Alternaria metabolite oleic acid in the preparation of oral medications for the prevention of neutrophilic asthma.

9. The use of pharmaceutical compositions containing oleic acid in the preparation of oral medications for the prevention of neutrophilic asthma.

10. The application according to claim 9, characterized in that, The pharmaceutical composition comprises an effective amount of oleic acid and a pharmaceutically acceptable carrier.

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