Comparative analysis of bronchial asthma and ulcerative colitis in a combined mouse model

Abstract

Background

Bronchial asthma (BA) and ulcerative colitis (UC) are chronic immune-mediated inflammatory diseases affecting the airway and intestinal mucosa, respectively. Increasing clinical evidence suggests a close association between BA and UC, with mutual aggravation of disease severity when both conditions coexist. However, the underlying mechanisms and suitable experimental models to investigate BA–UC comorbidity remain limited. This study aimed to establish a reliable murine model of comorbid BA–UC and to evaluate the bidirectional impact of airway and intestinal inflammation.

Methods

Forty specific-pathogen-free (SPF)-grade female C57BL/6 mice were randomly assigned to four groups: control, BA, UC, and comorbid BA–UC. BA was induced by intraperitoneal sensitization with ovalbumin (OVA) followed by repeated aerosolized OVA challenges, while UC was induced by intermittent administration of dextran sulfate sodium (DSS). The comorbid model was established by synchronously combining both protocols in the same animals. Pulmonary function, airway hyperresponsiveness, Disease Activity Index (DAI), body weight, quality-of-life scores, colon length, and histopathological changes in lung and colon tissues were assessed and compared among groups.

Results

Compared with the control group, mice in the BA, UC, and comorbid BA–UC groups exhibited significant abnormalities in pulmonary function parameters. Among all experimental groups, the comorbid BA–UC group showed the most pronounced decline in quality-of-life scores. Line graph analysis revealed significant differences in colon length shortening and body weight changes in the comorbid BA–UC group (P < 0.05), with colon shortening being significantly greater than that observed in the UC group alone (P < 0.05). Inflammatory cell infiltration was significantly increased in the BA and comorbid BA–UC groups compared with the UC group (P < 0.05). Although granuloma scores were higher in the BA and comorbid BA–UC groups than in the control group, these differences did not reach statistical significance. In contrast to controls, all disease groups (BA, UC, and comorbid BA–UC) exhibited significantly increased pathological injury scores.

Conclusion

The synchronous induction of BA and UC using OVA and DSS successfully establishes a stable and reproducible murine model of comorbid BA–UC. This model demonstrates that BA and UC mutually exacerbate airway and intestinal inflammation, supporting the existence of a bidirectional gut–lung immune interaction. The comorbid BA–UC model provides a valuable experimental platform for future mechanistic studies and the development of integrated therapeutic strategies targeting coexisting inflammatory diseases.

Introduction

Bronchial asthma (BA) is a chronic inflammatory disease of the airways characterized by airway hyperresponsiveness, reversible airflow limitation, and airway remodeling, clinically presenting with wheezing, dyspnea, cough, chest tightness, and sputum production. Despite advances in treatment strategies, BA remains a highly heterogeneous disease, frequently coexisting with other immune-mediated disorders, including allergic diseases, gastrointestinal inflammatory conditions, and systemic inflammatory syndromes (Nishida et al., 2022; Freuer, Linseisen & Meisinger, 2022). Such heterogeneity is a major contributor to poor disease control and variable therapeutic responses.

Increasing epidemiological and clinical evidence suggests a close association between BA and ulcerative colitis (UC) (Kuenzig et al., 2018b; Barbiellini Amidei et al., 2020). UC is a chronic inflammatory bowel disease primarily driven by T cell–mediated immune responses and characterized by persistent mucosal inflammation, epithelial barrier disruption, and infiltration of mononuclear immune cells. Notably, both BA and UC are immune-mediated inflammatory diseases that share overlapping immunopathological features, including dysregulated adaptive immunity, excessive cytokine production, and impaired mucosal barrier function (Jostins et al., 2012).

In our previous clinical observations, patients with BA exhibited a markedly higher prevalence and severity of UC compared with patients with other T cell–mediated immune disorders. Conversely, the presence of UC appeared to aggravate asthma symptoms and reduce treatment responsiveness. These findings suggest a potential bidirectional interaction between airway and intestinal inflammation, possibly mediated through systemic immune activation and the gut–lung immune axis (Jiang et al., 2025). However, the biological mechanisms underlying the coexistence and bidirectional aggravation of BA and UC have not yet been fully elucidated. Consequently, in-depth exploration of the pathogenic mechanisms of comorbid BA–UC may provide valuable insights for the prevention, treatment, and clinical management of BA.

To investigate the pathogenic interaction between BA and UC, the establishment of a reliable animal model that recapitulates the comorbid inflammatory phenotypes is essential. A combined model is vital for elucidating the complex immune crosstalk between airway and intestinal inflammation. However, existing studies largely focus on single-disease models, and mechanistically oriented comorbid BA–UC models are scarce. To address this gap, we intend to complete the establishment of comorbidities by simultaneously using the animal model modeling methods of BA and UC in the same mouse, and evaluate whether the model is established or not using lung function, Disease Activity Index (DAI), lung tissue, and colon tissue pathology, as well as further evaluate the differences between comorbidities and a single disease. Results showed that compared with BA-alone mice, the comorbid model exhibited more severe lung function impairment and airway hyperresponsiveness. Although colon shortening occurred in both UC and comorbid groups, pathological damage in colon tissue was exacerbated in the comorbid group. Histological analysis further revealed enhanced inflammatory cell infiltration in both lung and colon tissues, indicating a mutual aggravating effect between BA and UC. These findings not only provide preliminary evidence supporting the bidirectional promotion of BA and UC in comorbid conditions, but also confirm the feasibility of this integrated animal model for future mechanistic investigations and new therapeutic targets.

Materials & Methods

Animals and experimental design

Forty healthy specific-pathogen-free (SPF)-grade 8-week-old female C57BL/6 mice (18–22 g; Experimental Animal Center of Xinjiang Medical University) were randomly assigned to four groups: Control (n = 10), BA (n = 10), UC (n = 10), and Comorbid BA-UC (n = 10). The sample size was determined using G*Power 3.1 software based on pilot studies, with a 0.05 alpha error and 0.80 power to ensure statistical significance.

The Disease Activity Index (DAI) was chosen as the primary outcome measure for sample size determination. The DAI is a comprehensive scoring method that integrates multiple criteria, such as body weight loss, fecal consistency, and the presence of bloody stools, to objectively assess the severity of intestinal inflammation in UC models. In light of the study’s objective to explore the comorbid relationship between BA and UC, the DAI score was regarded as a crucial indicator for reflecting the progression and severity of UC in the comorbid model. Consequently, it served as the key metric for sample—size calculation to guarantee sufficient statistical power for detecting significant differences in disease activity among the experimental groups.

Housing and acclimatization

All mice were housed in a specific pathogen-free (SPF) environment with controlled temperature (22 ± 2 °C), humidity (50 ± 5%), and a 12-hour light/dark cycle, with free access to standard laboratory chow and sterile water throughout the experimental period. Before model establishment, mice were acclimatized for 7 days to minimize stress-related effects on the experimental results. Body weights were measured daily during the acclimatization period to ensure stable health status, and any mice showing signs of illness or abnormal behavior were excluded from the study.

Ethical statement

The experimental protocols were approved by the Institutional Animal Care and Use Committee of Xinjiang Medical University (Approval No. IACUC-20250702-12), and all procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

Experimental randomization, blinding, measurement standardization and Exclusion criteria

To reduce experimental bias, all measurements were performed following a standardized sequence across experimental sessions. Body weight was measured first each morning, followed by behavioral observation and subsequent sample collection, with fixed time intervals maintained between procedures to limit temporal variability.

Group allocation and outcome assessment were conducted in a blinded manner. One investigator was responsible for randomization and group assignment, while a second investigator—unaware of group identities—performed measurements, outcome evaluation, and data analysis.

Exclusion criteria: A total of 40 C57BL/6 mice were initially incorporated into the experiment, with matching for gender, age, and weight, fulfilling the reporting requirements for the “initial sample size” as specified in ARRIVE 2.0 guideline 8 (Experimental animals). The final sample size for analysis was n = 24 (each group n = 6). One mouse in the UC group and three mice in the BA—UC group died of enteritis and were excluded from the analysis due to non-anthropogenic factors. The tissue sub-set size was n = 16 (each group n = 4). Among the six surviving mice in each group, four exhibited intact lung or intestinal tissues that satisfied the quality criteria for immunohistochemical detection (absence of autolysis and section artifacts), whereas the remaining two were excluded from the sub-set analysis owing to tissue damage or uneven fixation. This constituted a technical screening rather than data exclusion, in line with the description of the “analysis sub-set” in the ARRIVE 2.0 guideline 9 (Sample size).

Euthanasia procedure

Mix 250 mg of Stresnil 50 with 2.5 ml of chlorpromazine hydrochloride (100 mg/ml), and dilute with 22.5 ml of sterile normal saline to form a mixed solution of Stresnil 50 (10 mg/ml) + chlorpromazine hydrochloride (10 mg/ml) as an anesthetic. All mice were anesthetized and then underwent pulmonary function tests, and blood was drawn from the orbital sinus. Finally, they were sacrificed by cervical dislocation. The mouse who shown severe weight loss exceeding 20% of the initial body weight, persistent severe diarrhea with bloody stools for more than three consecutive days, obvious respiratory distress characterized by labored breathing, cyanosis, or reduced activity, as well as any signs of severe pain or distress such as hunched posture, unresponsiveness to stimuli, or inability to eat and drink. If any of these criteria were met, the mice would be euthanized immediately to minimize suffering, following the same anesthesia and cervical dislocation procedure as described above. All euthanasia procedures were performed by trained personnel to ensure humane and consistent implementation, and detailed records were kept regarding the time, reason, and method of euthanasia for each animal.

Model establishment

Mouse model of BA

To establish the BA model, 20 µg of ovalbumin (OVA) and 2.25 mg of aluminum hydroxide were mixed in 200 µL phosphate-buffered saline (PBS) to create a sensitizing solution. Mice were sensitized by intraperitoneal injection of this mixture on days 0, 7, and 14. From day 15 to day 35, the mice were placed in a transparent, sealed container (30  ×  20  ×  15 cm) and exposed to daily 30 mg/mL OVA + PBS nebulization for 30 min each time. During this period, mice in the BA group had free access to normal drinking water. This protocol was followed for three consecutive weeks, after which the mice were sacrificed on day 35. (100 µL of 1% pentobarbital sodium solution was intraperitoneally injected to anesthetize the mice, carry out the execution using the cervical dislocation.)

Model success was determined by evaluating typical asthma symptoms, such as tachypnea, coughing, and reduced activity. Additional indicators of successful asthma modeling included increased airway resistance following acetylcholine stimulation, elevated levels of eosinophils in peripheral blood and bronchoalveolar lavage fluid (BALF), and increased OVA-specific IgE levels in serum, all of which confirmed airway inflammation typical of asthma.

Mouse model of UC

To induce UC, 2.5% dextran sulfate sodium (DSS) (MW 36,000–50,000) was prepared in drinking water. Mice were allowed to drink this DSS solution for seven consecutive days (days 1–7), followed by a 14-day rest period (days 8–21) with normal water. DSS was reintroduced for seven consecutive days from days 22–28, followed by another 7-day rest period (days 29–35), the daily intake of the DSS solution was monitored to ensure proper ingestion by the mice. Similarly, water intake in the UC group was not restricted during the experimental period to maintain normal drinking behavior. There were two cycles in total, and the death was performed painlessly on the 35th day.

The successful establishment of the UC model was evaluated prior to euthanasia. Mice exhibited progressive body-weight loss, reduced activity, and lethargy, accompanied by changes in fecal consistency from formed stools to loose or bloody stools. These clinical manifestations were incorporated into the Disease Activity Index (DAI), which includes body-weight loss, stool consistency, and fecal occult or gross blood scores. In addition, colonoscopy revealed colon shortening, increased tissue fragility, and a roughened mucosal surface, while clinical assessment further indicated the presence of intestinal inflammation. Together with the characteristic histopathological features observed after DSS treatment, these findings confirmed the successful establishment of the ulcerative colitis model.

Mouse model of comorbid BA-UC

For each mouse, a sensitizing solution was prepared by thoroughly mixing 20 µg OVA and 2.25 mg aluminum hydroxide in 200 µL PBS. Mice received intraperitoneal injections on days 0,7, and 14. To induce colitis, mice were allowed free access to DSS-containing drinking water for seven consecutive days (days 1–7). Beginning on day 15, mice were placed in a transparent sealed chamber (30  × 20  ×  15 cm) and challenged daily by ultrasonic nebulization with 30 mg/mL OVA in PBS for 30 min. OVA aerosol exposure continued until day 35. During this period, DSS drinking water was reintroduced from days 22–28, followed by a recovery phase with normal water from days 29–35. Mice were humanely euthanized on day 35.

Model establishment was evaluated prior to euthanasia. Mice exhibited combined features of asthma and colitis, including airway inflammation manifested by tachypnea, reduced activity, enhanced airway hyperresponsiveness, increased eosinophil proportions in BALF, and elevated serum OVA-specific IgE levels. Concurrently, colitis-related manifestations were observed, such as body-weight loss, loose or bloody stools, increased DAI scores, and colon shortening. The presence of both respiratory and intestinal inflammatory phenotypes was considered indicative of successful establishment of the comorbid BA–UC model.

Control group

Each mouse received intraperitoneal injections of an equal volume of normal saline on days 0, 7, and 14. During the experimental period, mice had free access to standard drinking water. All mice were humanely euthanized on day 35.

Outcome measurements

Pulmonary function testing

Pulmonary function was assessed 24 h after the final OVA challenge. Respiratory rate (f), tidal volume (TVb), minute ventilation (MVb), peak inspiratory and expiratory flow rates (PIFb and PEFb), inspiratory time (Ti), expiratory time (Te), and expiratory flow rate at 50% of tidal volume (EF50) were measured in all mice using a biological function experimental system.

Airway reactivity assessment

Airway reactivity was assessed 24 h after the final OVA challenge. Mice were anesthetized by intraperitoneal injection of 100 µL of 1% pentobarbital sodium, placed supine, and intubated. The animals were then positioned in the nebulization chamber of the Buxco mouse pulmonary function system, with the nebulization tubing connected. Mechanical ventilation was set at a frequency of 150 breaths/min, a tidal volume of 0.3 mL, and a positive end-expiratory pressure of four cmH₂O (one cmH₂O = 0.098 kPa).

Acetylcholine was administered via nebulization at increasing concentrations (0, 3.125, 6.25, 12.5, and 25 g/L). Each concentration was nebulized for 3 min, during which airway resistance was automatically recorded. The system was flushed with PBS between doses. Airway reactivity was evaluated using enhanced pause (Penh) and dynamic compliance (Cdyn). A baseline respiratory curve was recorded prior to acetylcholine administration, followed by measurements after each dose.

General condition monitoring and disease activity assessment

During the experimental period, the general condition of the mice was monitored to evaluate disease progression and overall health status. Parameters including mental state, fur luster, activity level, food intake, survival, and body weight were assessed throughout the study. Body weight was recorded every 2–3 days, while stool consistency and the presence of blood were examined and documented daily for DAI scoring.

Assessment of general condition and quality of life

Mice were observed every 2–3 days for mental state, locomotor activity, responsiveness to external stimuli, and feeding and drinking behavior. A quality-of-life score was assigned according to predefined criteria (Table 1).

Table 1. Disease activity index scoring criteria.

Daily observations include the softness of the stool and the presence of blood. The DAI is also scored, with higher scores indicating a higher level of disease activity and a more severe condition.

Weight loss rate	Fecal viscosity	Constipation/bloody stool	Score
–	Normal	Normal	0
1%–5%	Loose	Constipation	1
6%–10%	Loose	Constipation	2
11%–15%	Loose	Constipation	3
>15%	Diarrhea	Bloody stool	4

DAI

The DAI was calculated based on three parameters: percentage of body-weight loss, stool consistency, and fecal occult or gross blood. Daily scores were recorded and statistically analyzed to reflect the severity of intestinal inflammation (Table 2).

Table 2. Quality of life scoring criteria.

Every day observe the mental state, fur luster, activity, and diet of the mice. The lower the score, the poorer the survival quality of the mice.

Assessment content	Score
The fur is shiny and smooth; the body is well-proportioned and moves nimbly; the eyes are bright and expressive; the response is quick.	4
The fur is smooth, shiny, or slightly dull; the movement is relatively flexible or slightly sluggish; the eyes are bright, and the response is sensitive.	3
Hair is messy and dark, or the person is physically weak or thin; movement is slow, and the mental state is poor.	2
The coat is messy; the physique is weak or thin; the movements are extremely slow and difficult; the mental state is extremely poor, dull, and motionless.	1
Death.	0

Sample collection and tissue processing

For histopathological analysis, four mice were randomly selected from each experimental group. Tissue processing and histological scoring were performed by an investigator blinded to the group allocation.

Blood, bronchoalveolar lavage fluid, and lung tissue

After the airway reactivity test on day 35, all mice were anesthetized, and blood samples were collected from the orbital sinus. Bronchoalveolar lavage was then performed, followed by collection of the BALF and dissection of right lung tissues.

Serum: two mL of blood was collected from the orbital sinus of each mouse, allowed to stand for 30 min, and then centrifuged at 2,000 r/min for 10 min to collect the supernatant.

Bronchoalveolar lavage fluid (left lung): After completing the airway reactivity test, the left lung was ligated with a tracheal tube, and one mL of normal saline was slowly instilled into the right lung via the tracheal cannula. The lavage fluid was withdrawn three times, achieving a recovery rate of over 80%. The lavage fluid was centrifuged (4 °C, 1,000 r/min, 10 min), and the supernatant was stored at −80 °C for cytokine analysis. The pellet was resuspended in five mL of Hank’s solution, and the total cell count was determined under a microscope. The cell concentration was adjusted to 2  ×  10⁶/mL for slide preparation and Giemsa staining.

Right lung tissue: The unflushed two-thirds of the right lung was dissected into several pieces and placed in cryovials for storage at −80 °C for subsequent protein analysis. The unflushed remaining one-third was cut into several pieces fixed in 4% paraformaldehyde for 12 h, then paraffin-embedded and sectioned. Some sections were stained with hematoxylin and eosin (H&E).

Colon tissue:

Approximately eight cm of colon (from the ileocecal junction to the anal verge) was collected, longitudinally opened, and the fecal contents gently removed prior to macroscopic examination. The tissue was then divided into two portions: one was fixed in 4% paraformaldehyde at room temperature, and the other was snap-frozen in liquid nitrogen and stored at −80 °C.

Statistical analysis

Statistical analyses were performed using SPSS version 23.0. Data conforming to a normal distribution are presented as mean ± standard deviation (SD). Comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA). When a significant overall effect was detected, post hoc pairwise comparisons were performed using the least significant difference (LSD) test for homogeneous variances or Dunnett’s T3 test for heterogeneous variances.

Data that did not meet the assumption of normality are expressed as median with interquartile range [M (P25–P75)]. Multiple-group comparisons were carried out using the Kruskal–Wallis H test, and pairwise comparisons were performed using the Kruskal–Wallis one-way ANOVA (k samples) procedure. Given the multiple inter-group comparisons performed in this study, the potential inflation of type I error was taken into consideration. To mitigate this risk, a stepwise analytical strategy was applied, whereby post hoc pairwise comparisons were conducted only after a statistically significant overall test. In addition, more conservative multiple-comparison correction methods, such as Bonferroni or Tukey adjustments, were considered at the statistical design stage. All graphical representations were generated using GraphPad Prism version 5.0.

Results

Comorbid BA–UC mice exhibit deteriorated general condition

During the modeling period, mice in the control group remained active, maintained normal appetite, and showed steady weight gain. In contrast, mice subjected to BA induction by aerosol sensitization or airway instillation displayed reduced activity, decreased appetite, and absence of weight gain. These mice also exhibited lethargy, coughing, and delayed responses to external stimuli. All mice in each group were sacrificed 35 days after model establishment.

UC exacerbates pulmonary function impairment in BA mice

Pulmonary function tests revealed significant functional impairment in mice with BA and comorbid BA–UC compared with controls. Indicators including Ti, Te, TVb, EF50, PIF, PEF, respiratory frequency (f), and minute ventilation (MVb) were all significantly altered in the BA and comorbid groups (P < 0.001 for all).

Compared with the UC group, pulmonary dysfunction was more pronounced in BA and comorbid BA–UC mice. Furthermore, lung function impairment in the comorbid BA–UC group was significantly more severe than in the BA-only group (P < 0.05). Notably, TI was also significantly reduced in the UC group compared with controls (P = 0.021), suggesting that intestinal inflammation alone may induce a certain degree of bronchial functional impairment (Table 3, Fig. 1).

Table 3. Pulmonary function data statistical analysis.

This table presents the differences among the control group, BA group, UC group, and BA-UC group of mice in eight indicators of lung function. BA-UC group has the worse lung function.

Group	f (breaths/min)	TVb (mL)	Mvb (mL)	PIFb (mL/s)	PEFb (mL/s)	Ti (ms)	Te (ms)	EF50 (mL/s)
WT-Control	120.50 ± 3.21	3.75 ± 0.40	165.17 ± 4.60	8.22 ± 1.08	8.67 ± 0.18	74.74 ± 3.02	144.72 ± 7.20	5.39 ± 0.30
WT-UC	119.83 ± 2.56a	3.78 ± 0.40	160.32 ± 1.84	8.20 ± 0.41	8.53 ± 0.20	68.69 ± 4.00a	142.08 ± 2.18	5.03 ± 0.43
WT-BA	148.17 ± 2.86a,b	2.79 ± 0.37a,b	129.50 ± 5.99a,b	5.09 ± 0.24a,b	7.10 ± 0.22a,b	33.77 ± 4.17a,b	102.68 ± 7.40a,b	3.02 ± 0.35a,b
WT-BA combined UC	153.00 ± 2.97a,b,c	2.21 ± 0.33a,b,c	82.81 ± 8.29a,b,c	4.43 ± 0.45a,b	6.28 ± 0.45a,b,c	26.30 ± 5.22a,b,c	78.15 ± 8.78a,b,c	2.88 ± 0.25a,b

Notes.

^avs. WT-control, P < 0.05.

^bvs. WT-UC, P < 0.05.

^cvs. WT-BA, P < 0.05.

Figure 1. Bar chart of pulmonary function indicators.

This graph shows the comparison among the control group, the BA group, the UC group, and the BA-UC group in terms of the eight indicators of lung function. Data are presented as mean ± SD (n = 6). Statistical tests were performed using the one-way ANOVA test followed by the LSD test. ***P < 0.001.

Airway hyperresponsiveness is aggravated in comorbid BA–UC mice

Airway reactivity tests demonstrated that Penh and Cdyn values were significantly elevated in BA and comorbid BA–UC mice compared with control and UC groups (P < 0.001 for all), indicating enhanced airway hyperresponsiveness and increased susceptibility to airway constriction.

Compared with BA mice, only the Penh value was further increased in comorbid BA–UC mice (P < 0.05), while Cdyn showed no significant difference between the two groups. These results suggest that intestinal inflammation may selectively influence certain indices of airway obstruction rather than uniformly exacerbating airway reactivity (Table 4, Fig. 2).

Table 4. Airway reactivity measurement.

This table presents the differences in airway reactivity indicators among the control group, the BA group, the UC group, and the BA-UC group of mice. The BA-UC group of mice had higher airway sensitivity.

Group	Penh	Cdyn
WT-control	0.78 ± 0.05	0.53 ± 0.03
WT-UC	0.70 ± 0.08a	0.54 ± 0.06
WT-BA	1.27 ± 0.05a,b	1.22 ± 0.06a,b
WT-BA combined UC	1.46 ± 0.07a,b,c	1.44 ± 0.06a,b

Notes.

^avs. WT-control, P < 0.05.

^bWT- vs. WT-UC, P < 0.05.

^cvs. WT-BA, P < 0.05.

Figure 2. Airway responsiveness measurement data.

This figure shows the comparison of airway responsiveness among the control group, the BA group, the UC group, and the BA-UC group.

BA modulates colonic inflammation severity in UC mice

Colon length and colon index were used to evaluate intestinal injury across groups. Compared with the BA group, mice in the UC group exhibited significant colon shortening and increased colon index (P < 0.0001 and P = 0.012). Similar alterations were observed in the comorbid BA–UC group when compared with control and BA groups (P < 0.05).

Interestingly, colon shortening was significantly more severe in comorbid BA–UC mice than in UC mice alone (P = 0.003), whereas no significant difference in colon index was observed between these two groups. These findings suggest that the coexistence of BA may partially modify the extent of intestinal inflammatory infiltration in UC mice (Table 5, Fig. 3).

Table 5. Length and weight of the colon.

This table presents the differences in the length and colon index of the colon tissues among the control group, BA group,UC group, and BA-UC group of mice.

Group	Length (cm)	Colon index (%)
WT-Control	7.823 ± 0.633	0.949 ± 0.097
WT-BA	6.788 ± 0.698a	1.260 ± 0.210a,b
WT-UC	4.680 ± 0.831a,b	1.590 ± 0.228a
WT-BA combined UC	3.227 ± 1.058a,b,c	1.213 ± 0.369a,b

Notes.

^avs. WT-control, P < 0.05.

^bvs. WT-UC, P < 0.05.

^cvs. WT-BA, P < 0.05.

Figure 3. Length and weight of the colon.

This graph shows the comparison among the control group, the BA group, the UC group, and the BA-UC group in terms of colon index and colon length. Data are presented as mean ± SD (n = 6). Statistical tests were performed using the one-way ANOVA test followed by the LSD test. *P < 0.05, ***P < 0.001.

Comorbid BA–UC markedly impairs quality of life in mice

Body weight changes, DAI, and quality-of-life scores were used to assess overall disease burden. Weight loss was primarily associated with intestinal inflammation and was most pronounced in the comorbid BA–UC group compared with other groups (P < 0.05).

DAI scores were significantly higher in UC and comorbid BA–UC mice than in control and BA mice (P < 0.05), reflecting increased severity of diarrhea and bloody stools. No significant difference in DAI was observed between UC and comorbid BA–UC groups. Consistently, quality-of-life scores declined most markedly in comorbid BA–UC mice (P < 0.05), indicating a cumulative negative impact of concurrent respiratory and intestinal inflammation (Fig. 4).

Figure 4. Mouse quality of life score.

The comparison situation among the control group, the BA group, the uc group, and the BA-uc group in the three indicators of quality of life is shown through a line graph. (A) Score for weight, (B) DAI, and (C) survival index.

Comorbid BA–UC enhances inflammatory injury in lung tissue

Histopathological changes in lung tissue

H&E staining revealed well-organized alveolar structures with minimal inflammatory cell infiltration in control mice. In contrast, lung tissues from BA, UC, and comorbid BA–UC mice exhibited varying degrees of edema, alveolar wall thickening, and infiltration of neutrophils and lymphocytes.

Compared with BA mice, lung tissue damage was milder in UC mice, whereas inflammatory cell infiltration was most pronounced in comorbid BA–UC mice. Notably, inflammatory changes were also observed in the lungs of UC mice, indicating potential systemic inflammatory involvement across immune-mediated diseases (Fig. 5).

Figure 5. H&E staining of mouse lung tissue (400x).

The comparison of lung tissue pathology among the control group, the BA group, the UC group, and the BA-UC group. (A) Control (B) BA (C) UC (D) BA-UC.

Quantitative assessment of pulmonary inflammatory injury

Inflammatory cell infiltration scores were significantly elevated in BA, UC, and comorbid BA–UC groups compared with controls (P < 0.05), with no significant differences among disease groups. Granuloma scores showed a non-significant increase in BA and comorbid BA–UC mice. Given that BA primarily affects the airways, granuloma formation was rare across all disease models, suggesting that pulmonary injury was predominantly characterized by inflammatory cell infiltration rather than granulomatous lesions (Table 6, Fig. 6).

Table 6. Pathological injury scores of lung tissues.

This table presents the differences in the pathological damage scores of lung tissues among the control group, the BA group, the UC group, and the BA-UC group of mice. The granuloma score in the BA and comorbid BA- UC groups increased, but the difference was not significant.

Group	Granuloma score	Inflammatory cell infiltration score
WT-Control	0.00 ± 0.00	0.50 ± 0.58
WT-BA	1.25 ± 1.26	2.75 ± 0.96a,c
WT-UC	0.00 ± 0.00	1.75 ± 0.50a
WT-BA combined UC	1.25 ± 1.26	3.25 ± 0.50a,c

Notes.

^avs. WT-control, P < 0.05.

^bvs. WT-UC, P < 0.05.

^cvs. WT-BA, P < 0.05.

Figure 6. Bar chart of inflammation injury score of mouse lung tissue.

The comparison of inflammatory damage in lung tissues among the control group, the BA group, the UC group, and the BA-UC group in ganglion score and Inflammatory cell infiltration degree. Data are presented as mean ± SD (n = 4). Statistical tests were performed using the one-way ANOVA test followed by the LSD test. *P < 0.05, ***P < 0.001.

Quantitative analysis of inflammatory cells in BALF

After the airway reactivity test was completed, the BALFs of left lung were collected. Total inflammatory cell, eosinophils, neutrophils, lymphocytes, and macrophages were significantly elevated in BA, UC, and comorbid BA–UC groups compared with controls, with a more pronounced increase observed in the comorbid group (P < 0.05), suggesting that the comorbid BA–UC groups had more inflammatory cell aggregation than the BA or UC group, as shown in (Table 7 and Fig. 7). Notably, the ulcerative colitis group also showed an increase in inflammatory cells in BALF compared with the control group, indicating that inflammatory cell aggregation also occurred in the airway mucosa of the UC group.

Table 7. Cell classification count data in BALF.

This table presents the differences in the inflammatory cells of the BALFs among the control group, the BA group, the UC group, and the BA-UC group of mice. The comorbid BA–UC group has more inflammatory cell aggregation than the BA or UC group.

Group	Total cell (×105/ml)	Eosinophils (×105/ml)	Neutrophils (×105/ml)	Lymphocytes (×105/ml)	Macrophages (×105/ml)
WT-Control	1.23 ± 0.29	0.01 ± 0.00	0.13 ± 0.03	0.10 ± 0.01	0.28 ± 0.01
WT-UC	3.57 ± 0.12a	0.82 ± 0.01a	0.31 ± 0.01a	0.51 ± 0.031a	0.83 ± 0.05a
WT-BA	4.80 ± 0.32a,b	1.21 ± 0.18a,b	0.37 ± 0.05a,b	0.54 ± 0.051a	0.93 ± 0.17a
WT-BA combined UC	5.52 ± 0.38a,b,c	1.54 ± 0.19a,b,c	0.45 ± 0.05a,b,c	0.60 ± 0.041a,b,c	1.21 ± 0.06a,b,c

Notes.

^avs. WT-control, P < 0.05.

^bvs. WT-UC, P < 0.05.

^cvs. WT-BA, P < 0.05.

Figure 7. The bar chart of inflammatory cells in BALF.

The comparison of inflammatory cells in BALF among the control group, the BA group, the UC group, and the BA-UC group. Data are presented as mean ± SD (n = 6). Statistical tests were performed using the one-way ANOVA test followed by the LSD test. ***p < 0.001.

Comorbid BA aggravates colonic mucosal injury in UC mice

Histopathological changes in colon tissue

In control mice, the colonic wall structure was intact, the intestinal mucosa was regular in shape, the glands were arranged neatly and minimal inflammatory infiltration. In BA mice, a slight increase in inflammatory cells was observed in the mucosal interstitium without obvious structural disruption.

In contrast, UC and comorbid BA–UC mice exhibited disorganized colonic architecture, irregular gland arrangement, mucosal edema, epithelial shedding, and marked inflammatory infiltration in the lamina propria. The severity of mucosal injury was greater in comorbid BA–UC mice than in UC mice alone (Fig. 8).

Figure 8. H&E staining of mouse colon tissue (400x).

The comparison of colon tissue pathology among the control group, the BA group, the UC group, and the BA-UC group. (A) Control. (B) BA. (C) UC. (D) BA combined UC.

Quantitative assessment of colonic inflammatory injury

Histopathological scores confirmed significantly more severe colonic injury in BA, UC, and comorbid BA–UC groups compared with controls (P < 0.05). Colonic damage was significantly greater in UC and comorbid BA–UC mice than in BA mice (P < 0.05). Moreover, comorbid BA–UC mice exhibited significantly higher injury scores than UC mice, indicating that concurrent BA further aggravates intestinal mucosal inflammation (Table 8, Fig. 9).

Table 8. Pathological injury scores of the colon tissues.

This table presents the differences in the inflammatory injury scores of the colon tissues among the control group, the BA group, the UC group, and the BA-UC group of mice.The comorbid BA–UC mice exhibited significantly higher injury scores than UC mice.

Group	Score
WT-Control	0.25 ± 0.50
WT-BA	1.75 ± 0.50a
WT-UC	2.75 ± 0.96a,c
WT-BA combined UC	4.25 ± 0.50a,cb

Notes.

^avs. WT-control, P < 0.05.

^bvs. WT-UC, P < 0.05.

^cvs. WT-BA, P < 0.05.

Figure 9. Bar chart of colonic tissue pathological injury score.

The comparison among the control group, the BA group, the UC group, and the BA-UC group in terms of the score for intestinal tissue inflammatory damage. Data are presented as mean ± SD (n = 4). Statistical tests were performed using the one-way ANOVA test followed by the LSD test. **P < 0.01, ***P < 0.001.

Discussion

In this study, we successfully established murine models of BA, UC, and comorbid BA–UC, and systematically compared their physiological, functional, and pathological characteristics. The results demonstrate that BA and UC do not exist as isolated inflammatory conditions but instead exhibit bidirectional interactions that aggravate both airway and intestinal inflammation when coexisting in the same host.

Validation and characteristics of the BA and UC models

The BA model established using OVA sensitization and challenge displayed classical features of allergic airway inflammation, including impaired pulmonary function, enhanced airway hyperresponsiveness, increased inflammatory cells in bronchoalveolar lavage fluid, and inflammatory infiltration in lung tissue. These findings are consistent with previously reported murine BA models (Oliveira et al., 2023; Kianmeher, Ghorani & Boskabady, 2016) andconfirm the reliability of the modeling strategy.

Similarly, the UC model induced by intermittent DSS (Mizoguchi, 2012; Lu et al., 2023) administration reproduced typical manifestations of colitis, such as diarrhea, hematochezia, body weight loss, elevated disease activity index, colon shortening, and marked inflammatory cell infiltration in colonic tissue. These pathological and clinical features are widely accepted indicators of successful UC model establishment.

Importantly, the comorbid BA–UC model simultaneously exhibited hallmark features of both diseases, providing a stable in vivo platform for investigating the interaction between airway and intestinal inflammation.

Intestinal inflammation contributes to airway inflammatory changes

One notable finding of this study is that UC alone induced detectable inflammatory alterations in the lung. Although pulmonary function and airway reactivity in UC mice showed only mild changes compared with controls, inflammatory cell counts in BALF and histopathological analysis revealed increased pulmonary inflammatory infiltration. This suggests that intestinal inflammation can influence the airway immune environment even in the absence of overt airway dysfunction.

This phenomenon may be explained by the concept of the “common mucosal immune system.” The respiratory and intestinal tracts share similar epithelial barrier structures, embryological origins, and mucosal immune mechanisms. Inflammatory mediators, immune cells, or microbial-derived signals generated during intestinal inflammation may circulate systemically and modulate immune responses in the airway. Similar observations have been reported in previous studies (Hammad & Lambrecht, 2021; Liu et al., 2021), supporting the existence of gut–lung immune crosstalk.

UC exacerbates airway dysfunction in BA mice

When UC coexisted with BA, airway dysfunction and hyperresponsiveness were further aggravated compared with BA alone. The comorbid BA–UC mice exhibited more severe impairment in pulmonary function parameters and elevated airway reactivity indices, indicating that intestinal inflammation can amplify the severity of allergic airway disease.

Interestingly, lung histopathology in comorbid BA–UC mice was dominated by inflammatory cell infiltration rather than structural destruction, which is consistent with the pathological characteristics of BA, a disease primarily targeting the airway rather than the alveolar parenchyma. These findings suggest that UC may enhance airway inflammation primarily by intensifying immune cell recruitment rather than inducing additional structural lung damage.

Collectively, these results indicate that intestinal inflammation promotes airway inflammation and functional impairment in BA, thereby accelerating disease progression.

Airway inflammation aggravates colonic injury in UC mice

Conversely, BA also exerted a promoting effect on intestinal inflammation. Although BA mice did not exhibit significant changes in colon length or gross morphology, histopathological examination revealed inflammatory damage in colonic tissues, suggesting subclinical intestinal involvement during airway inflammation.

In the comorbid BA–UC group, colonic injury was more severe than in UC alone, as evidenced by greater colon shortening and higher pathological injury scores. These findings indicate that airway inflammation can exacerbate intestinal mucosal damage, potentially through systemic immune activation or shared inflammatory pathways.

Thus, BA and UC appear to mutually reinforce each other’s inflammatory processes, leading to a more severe disease phenotype when both conditions coexist.

Mechanistic insights into BA–UC comorbidity

Recent work has further strengthened the experimental basis for asthma–colitis crosstalk along the lung–gut axis. In particular, a 2025 study established a combined house dust mite (HDM)-induced asthma and DSS-induced colitis mouse model and identified IL-17A as a key mediator of pulmonary–intestinal immune interactions (Wu et al., 2025). Importantly, neutralization of IL-17A attenuated inflammatory injury in both the lung and the colon, highlighting a mechanistic link between Th17-associated responses and bidirectional mucosal inflammation.

In line with these findings, our comorbid OVA+DSS model demonstrated concomitant aggravation of airway dysfunction and colonic pathology, supporting the concept that bronchial asthma and ulcerative colitis share interconnected pathogenic mechanisms rather than representing coincidental coexistence. Based on functional, histopathological, and clinical observations, several complementary mechanisms may underlie the reciprocal amplification of airway and intestinal inflammation.

First, systemic spillover of inflammatory mediators likely plays a central role. In UC, epithelial barrier disruption facilitates the release of pro-inflammatory cytokines and damage-associated molecular patterns into the circulation, which may prime immune responses in distal organs such as the lung (Ahmadi et al., 2025). Consistent with this notion, UC mice in our study exhibited pulmonary inflammatory infiltration and mild functional impairment despite the absence of direct airway allergen exposure, suggesting that intestinal inflammation alone can induce lung immune alterations. Conversely, allergic airway inflammation in BA elevate circulating Th2-associated mediators (Brusselle & Koppelman, 2022; Fahy, 2015), which in turn may exacerbate colonic inflammation, providing a plausible explanation for the worsened pathology observed in comorbid BA–UC mice.

Second, shared T helper cell–mediated immune dysregulation may link inflammation across the gut–lung axis. While BA is classically Th2-dominant and UC exhibits mixed Th2/Th17 features, cytokines from these subsets exert overlapping pro-inflammatory effects on mucosal tissues (Habib, Pasha & Tang, 2022; Kobayashi et al., 2020; Neurath, 2019). Simultaneous activation of airway and intestinal immune compartments may expand pools of activated effector T cells with enhanced migratory capacity, facilitating immune cell redistribution between organs. The increased inflammatory cell infiltration observed in both lung and colon tissues in comorbid mice supports this mechanism. Notably, Th17-related cytokines such as IL-17A have been implicated in both airway hyperresponsiveness and intestinal epithelial injury, suggesting synergistic amplification of inflammation.

Third, dysfunction of the mucosal barrier represents a shared pathogenic mechanism. DSS-induced intestinal barrier disruption may facilitate the translocation of microbial products, while gut microbiota dysbiosis functions both as a trigger and a consequence of pulmonary inflammation (Ludka-Gaulke et al., 2018; Tavakoli et al., 2021). Patients with asthma may be predisposed to colitis and other gastrointestinal disorders through cytokine-mediated inflammatory responses and alterations in the microbiome (Yu, 2024; Varricchi et al., 2025; Oliva & McGowan, 2024). The more severe histopathological damage observed in both organs of comorbid mice further supports the existence of a vicious cycle, in which barrier impairment at one mucosal site increases susceptibility at another (Song et al., 2024; Ojha et al., 2018; Kuenzig et al., 2018a; Lynch et al., 2014; Ludka-Gaulke et al., 2018).

Finally, neuroimmune and stress-related pathways may further contribute to disease amplification. Chronic inflammation in either the lung or intestine can activate autonomic and enteric nervous system pathways, leading to altered neuropeptide release and stress hormone signaling. These neuroimmune mediators can modulate immune cell activity and vascular permeability at distant mucosal sites (Tavakoli et al., 2021; Yu, 2024). The pronounced deterioration in general condition and quality-of-life scores observed in comorbid BA–UC mice may reflect the cumulative burden of systemic inflammation and neuroimmune dysregulation, further amplifying disease severity.

Limitations and future perspectives

This study has several limitations. First, the relatively small sample size may limit the statistical power of certain analyses, and larger cohorts are needed to validate the findings. Second, only a single time point was assessed, preventing dynamic evaluation of disease progression and the temporal sequence of BA–UC interactions. Third, the present study focused primarily on inflammatory cells and histopathological changes, while the underlying molecular mechanisms—such as key cytokines, signaling pathways, and immune cell subsets—were not explored in depth. Finally, as an induced animal model, the comorbid BA–UC model may not fully recapitulate the complexity of human disease, and caution is warranted when extrapolating these findings to clinical settings.

Future studies incorporating longitudinal observation, expanded immunological profiling, and mechanistic analyses will be essential to further elucidate the pathophysiological basis of BA–UC comorbidity.

Conclusions

The synchronous induction of BA and UC using OVA and DSS provides a reliable and practical murine model of comorbid BA–UC. Our findings demonstrate that BA and UC exert synergistic effects on airway and intestinal inflammation, respectively, leading to aggravated disease severity. This comorbid model offers a valuable experimental platform for further investigation into the mechanisms underlying gut–lung immune interactions and may provide insights for the development of integrated therapeutic strategies.

Supplemental Information

Supplemental Information 1. The breeding status of mice.

The state of the mouse when it first enters the cage.

Supplemental Information 8. Quality of life score.

All the quality life scores of the mice were recorded at different time points.

Supplemental Information 9. Colon scores.

All the weights, colon lengths and colon indices of the mice were recorded during the dissection.

Supplemental Information 10. Lung of HE staining.

HE staining images of lung tissues from different groups of mice.

Supplemental Information 11. HE staining of colon.

HE staining images of colon tissues from different groups of mice

Funding Statement

This work was funded by the Tianshan medical and health talents training program of Xinjiang Uygur Autonomous Region (TSYC202301B136) and the Tianshan innovation team program of Xinjiang Uygur Autonomous Region (2022D14006). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.