Serum sST2/ miR-223 as a Dual Biomarker for Disease Activity & Clinical Efficacy of SLIT in HDM-Induced Allergic Rhinitis

Soluble suppression of tumorigenicity 2 (sST2), a decoy receptor of interleukin-33 (IL-33), involved in allergic inflammation. Objectives: This study explored serum sST2 as a biomarker for disease activity and for predicting clinical response to sublingual immunotherapy (SLIT) in patients with house dust mite (HDM) induced AR

1st stage: A case-control study conducted over 3 months at the Department of Medical Microbiology and Immunology, Faculty of Medicine, Zagazig University, in collaboration with the Allergy Unit at Zagazig University Hospitals from February to May 2025. 2nd stage an interventional study (quasi experimental) for (MSAR patients): conducted over 6 months.

Study Population, Inclusion and Exclusion Criteria Eligible participants were 14 years or older with a diagnosis of HDM-induced AR and its Impact on Asthma (ARIA) guidelines . Patients were required to have persistent moderate to severe symptoms for at least two years and be willing to initiate and maintain SLIT for six months. Exclusion criteria included the presence of autoimmune or chronic inflammatory conditions, severe asthma or other pulmonary disease, systemic corticosteroid or immunosuppressive therapy within four weeks prior to enrollment, pregnancy or lactation, and refusal to provide informed consent. Healthy, age- and sex-matched individuals without a history of allergic disease and with negative skin prick test (SPT) results were recruited as controls.

Sample size:

The sample size was calculated using open epi info according to the following mean sST2 level among AR cases versus control was 19.8+_7.5 versus 14.3+_6.9 (ref.) at power of study 80%, ci 95%, case: control 1:1, The minimum required sample size was 27 participants per group. However, we were able to recruit 54 participants in each group, all of whom were included in the final analysis. Participants were selected using a simple random sampling technique according to the predefined inclusion criteria.

• Clinical and Laboratory Procedures
• Baseline Assessment All participants underwent a detailed clinical assessment, including demographic data, duration of disease, body mass index (BMI), and previous treatment history.
• Symptom Scoring Symptom severity was evaluated using the TNSS, which includes four parameters-rhinorrhea, nasal congestion, nasal itching, and sneezing-each rated on a 0 to 4 scale, for a maximum score of 16. A 10-cm Visual Analogue Scale (VAS) was also used to quantify overall symptom burden.
• Skin Prick Testing (SPT) SPT was performed using standardized allergen extracts, including Dermatophagoides pteronyssinus and D. farinae. Histamine and saline served as positive and negative controls, respectively. A wheal diameter of ≥3 mm compared to the negative control was considered positive.
• Immunological marker and sST2 measurement Venous blood samples were collected from all participants at baseline to determine serum eosinophils, total immunoglobulin E (IgE) levels, and house dust mite-specific HDM-IgE (kU/l) measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's protocol (Chemus Bioscience, USA).

sST2 concentrations were quantified using a commercial ELISA kit (Elabscience, Cat No. E-EL-H6082). For patients undergoing SLIT, additional serum samples were collected after six months of therapy. All measurements were performed in duplicate. Laboratory personnel conducting the assays were blinded to the clinical information and sampling time.

• Detection of microRNA-223 Expression level via quantitative polymerase chain reaction (qRT-PCR): MicroRNA was extracted by commercial miRNeasy Serum/Plasma Kits (Qiagen, Germany), according to the manufacturer's instructions. After extraction, microRNA was reversibly transcribed by reverse transcription kits (miScript II RT Kit, Qiagen, Germany) according to the manufacturer's instructions. Then, it was incubated in dry bath block (Rocker Sahara 320 dry bath heat block, Rocker Scientific, Taiwan), initially at 37°C for 60 min, followed by a second incubation at 95°C for 5 min to inactivate miScript RT. After that, the reaction mixture was stored at -80°C.Quantitative determination of mature miR-223 was fulfilled by target-specific miScript Primer Assays using commercial kits (miScript SYBR Green real-time PCR Kit, Qiagen, Germany), according to the manufacturer's instructions. Human RNU6B (RNU6-2) was used as a house keeping gene. After the initial activation step of HotStar Taq DNA Polymerase fo 15 min at 95°C, the real-time PCR reactions were run for 40 cycles of each of a denaturation for 15 s at 95 ºC, annealing for 30 s at 55°C, and extension for 30 s at 70°C. The Stratagene Mx3005P platform software (Agilent Technologies, USA) was used to figure out the threshold cycle (Ct) value. The delta-delta Ct method formula was utilized to estimate the fold changes of miR-223 expression .

• Intervention stage for (MSAR patients) Sublingual Immunotherapy (SLIT) Protocol Patients received standardized daily SLIT containing HDM extracts for a period of six months. Administration began under clinical supervision, and patients were monitored monthly for adherence and adverse effects. The therapeutic response was evaluated after six months based on a composite outcome: a reduction of at least 30% in the combined symptom and medication score, improvement in TNSS and VAS scores, and a decrease in serum sST2 levels. Patients meeting these criteria were classified as responders; those not meeting the threshold were considered non-responders.

Inclusion criteria

• Eligible participants were 14 years or older with a diagnosis of HDM-induced AR
• Patients were required to have persistent moderate to severe symptoms for at least two years

Exclusion criteria

• The presence of autoimmune or chronic inflammatory conditions, severe asthma or other pulmonary disease, systemic corticosteroid or immunosuppressive therapy within four weeks prior to enrollment
• Pregnancy
• lactation
• Refusal to provide informed consent