Johns Hopkins Medicine researchers say they have identified molecular pathways controlled by the protein RhoA in club cells — a specialized population of epithelial cells that exclusively lines the inner and outer surfaces of the lung’s small airways, with known roles in immune regulation and airway repair — during allergen-induced airway inflammation. Through a series of translational mouse and cell culture experiments, the research team has demonstrated how RhoA can be selectively targeted to potentially prevent asthma-related complications.
A report on the group’s mouse and cell culture experiments, supported by the National Institutes of Health and published Feb. 1 in the Journal of Allergy and Clinical Immunology , offers new details on a potential cell-specific therapeutic target for allergic airway diseases, including asthma and allergic rhinitis .
Asthma is a chronic, inflammatory lung disease that causes a narrowing of the small airways in the lungs and symptoms such as coughing, wheezing, chest tightness and shortness of breath. According to the Centers for Disease Control and Prevention , it affects 8.2% of adults and children in the United States. Experts do not know why asthma develops; however, understanding the molecular mechanisms responsible for airway inflammation can reveal asthma’s root causes and lead to treatments capable of stopping or slowing its progression.
Peisong Gao, M.D., Ph.D., a professor of medicine at the Johns Hopkins University School of Medicine and corresponding author of the study, says expression of the protein RhoA, a molecular switch that regulates cell-to-cell interactions, has previously been linked to worsening asthma. But, research from his lab has found that removing it can worsen airway inflammation in some lung cell populations.
Gao and his team set out to understand how RhoA operates across different lung cell types by focusing on its presence and role in club cells. The lung’s epithelium is the first point of contact for foreign matter, such as allergens, microbes, viruses or pollutants that are known to trigger inflammation and asthma.
For their experiments, Gao’s team created a RhoA “knockout” mouse model, meaning an organism that does not express the RhoA gene or create its protein in its club cells, and compared them to a control group of mice with asthma that retained RhoA expression. Both groups of mice had cockroach allergen-induced asthma. The researchers conducted four primary experiments to understand how RhoA affected lung tissue, cells and molecular pathways.
In their first experiment, the researchers compared airway inflammation between allergen-exposed RhoA-deficient mice and control mice. They observed fewer immune cells and inflammatory cytokines (molecules secreted by immune cells to coordinate the body’s immune response to foreign pathogens) in the RhoA-deficient mice, which, Gao’s team believed demonstrated a potential connection between RhoA expression and airway inflammation.
Next, the researchers grew and compared 3D lung cell cultures from both mouse groups. The air-liquid interface cell cultures were designed to replicate the lung epithelial barrier’s structural, molecular and physiological features after allergen exposure. Unlike the control group, RhoA-deficient cultures retained typical epithelial barrier integrity and function. In people with asthma, epithelial barrier dysfunction contributes to the progressive worsening of symptoms and tissue damage that occurs over time. Gao’s team says the findings affirm a potential clinical role for RhoA in allergic airway diseases.
To identify the immune cell populations controlled by RhoA expression, the researchers used flow cytometry — a technique that uses lasers to detect and differentiate cells based on their chemical and physical properties — and discovered that the number of interstitial macrophages, a major immune cell population exclusively found in the lung’s connective tissue, was significantly reduced in the RhoA-deficient lung tissue. This information, the researchers say, allowed them to identify gene pathways regulated by RhoA that would help them target and selectively control immune cell activity without directly targeting RhoA itself.
Finally, the researchers used bulk and single-cell RNA-sequencing (RNA-seq) to identify immune pathways and cytokines controlled by RhoA expression that may be responsible for increasing the number of interstitial macrophages after RhoA amplifies airway inflammation.
Specifically, Gao’s team found CCL24, a cytokine that promotes immune cell activity and inflammation, was suppressed in RhoA-deficient cells and that treating allergen-exposed control mice with a CCL24-neutralizing antibody stopped airway inflammation.
“As the prevalence of asthma continues to increase, our findings, especially identification of the RhoA-controlled CCL24 as a key inflammation mediator in club cells, are critical because they provide a list of potential mediators that could be targeted for future asthma studies or as future asthma treatments,” says Gao.
Gao and his team plan on conducting additional experiments to explore how CCL24 mediates lung immune activity to control allergic airway inflammation.
The study was supported by National Institutes of Health grants 1R01AI153331 and R01AI141642.
The authors declared no conflicts of interest under Johns Hopkins University School of Medicine policies.
Alongside first author Wei Tu, the other researchers who contributed to the study include Martin Alphonse, Jingsi Chen, Peisong Gao, Baishakhi Ghosh, Wenjing Gu, Xinyue Hu, Hongpeng Jia, Jennifer Lin, Liyuan Liu, Yinchun Shen, Venkataramana Sidhaye, Mei Wan, Rongjun Wan, Maolan Wu, Shaobing Xie and Yan Zhang.
DOI: 10.1016/j.jaci.2026.01.016
Journal of Allergy and Clinical Immunology