ITIH4 alleviates OVA-induced asthma by regulating lung-gut microbiota

Abstract

Background: Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), a Type 2 acute phase protein, is critical for resolving inflammation and promoting tissue repair. While its role in chronic respiratory diseases is recognized, its effects on asthma remain unclear. This study investigated the effects of ITIH4 on the modulation of lung and gut microbiota, the attenuation of allergic inflammation, and the improvement of respiratory outcomes in an asthma mouse model.

Methods: Six-week-old male Balb/c mice were divided into five groups: control, ITIH4, ovalbumin (OVA), and two OVA + ITIH4 treatment groups at different doses. Lung function and oxygen saturation were measured, and bronchoalveolar lavage fluid (BALF) was analyzed for white blood cell counts and cytokines. Lung and gut microbiota were profiled using 16 S rRNA gene sequencing, and short-chain fatty acids (SCFAs) were measured using gas chromatography-mass spectrometry (GC-MS). Proteomic profiling of intestinal tissues was conducted to identify ITIH4-associated signaling pathways.

Results: ITIH4 administration significantly mitigated OVA-induced asthma symptoms by reducing weight loss, airway resistance, and tissue damping (p < 0.05). Histological analysis showed decreased airway wall thickening and lung injury scores (p < 0.05). ITIH4 also lowered BALF eosinophils and lymphocytes, IgE, and Th2 cytokines (IL-4, IL-5, and IL-13) (p < 0.05). ITIH4 treatment modulated microbiome composition, enriching Gram-positive taxa (Nocardioidaceae and Acholeplasmataceae) and depleting Gram-negative Helicobacteraceae (p < 0.05). SCFAs correlated with microbiome alterations, notably reduced 4-methylpentanoic acid levels (p < 0.05). Proteomic analysis revealed a dose-dependent activation of granzyme A signaling and suppression of metabolic and solute transport pathways.

Conclusions: ITIH4 ameliorates asthma symptoms by modulating lung and gut microbiota, dampening Th2-driven inflammation, and restoring mucosal immune balance. These findings support ITIH4 as a potential candidate for microbiome-targeted asthma therapy.

Keywords: Airway hyperresponsiveness; Inflammation; Microbiome; Short-chain fatty acid; Th2.

Fig. 1

ITIH4 attenuated OVA-induced weight loss and improves lung function in asthmatic mice. a Schematic diagram of experiment in which Balb/c mice of 6 weeks old were induced asthma by intraperitoneal administration of OVA followed by intranasal administration of ITIH4. b Body weight difference and exercise SpO2 difference in mice. c Resistance, airway resistance, elastance, tissue damping, and tissue elastance of mice followed methacholine challenges. *p < 0.05; ****p < 0.0001

Fig. 2

ITIH4 reduced airway wall thickening, lung injury, and inflammatory cell infiltration. a Airway wall thickness of OVA-induced asthma mice with ITIH4 administration. b Lung damage severity assessment by the k-means clustering algorithm. The image depicts regions (blue) that were classified as normal zones, active morphological remodeling (green) areas as mild damage zones, and places of maximum damage (red) defined as severe damage zones. c Total number of BALF white blood cell, lymphocyte, monocyte, eosinophil and neutrophil of OVA-induced asthma mice with ITIH4 administration. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Fig. 3

ITIH4 suppressed IgE and Th2 cytokines in serum, BALF, and intestinal tissues. a Serum IgE, b BALF TNF-α, c BALF IL-4, IL-5 and IL-13, and d intestine IL-4, IL-5 and IL-13 in OVA-induced asthma mice with ITIH4 administration. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Fig. 4

ITIH4 modulated lung microbiota composition and redistributes gram-positive/negative bacteria. a Alpha diversity analysis including Observed, Chao1, Shannon, and Simpson indexes. Beta diversity analysis of lung microbiome. b Gram-positive bacterial at the phylum level. c Gram-negative bacterial at the phylum level. d Brown-Brenn tissue gram stain (magnification 80X). The arrows showed Gram-positive bacteria surrounding alveolar regions in controls and OVA groups, and Gram-negative bacteria clustering in alveolar zones of ITIH4-treated lungs. * p < 0.05

Fig. 5

ITIH4 reshaped intestinal microbiota and reduces gram-negative bacterial abundance. a Alpha diversity analysis including Observed, Chao1, Shannon, and Simpson indexes. Beta diversity analysis of lung microbiome. b Gram-positive bacterial at the phylum level. c Gram-negative bacterial at the phylum level. d Brown-Brenn tissue gram stain (magnification 80X). The arrows showed Gram-negative bacteria were visualized predominantly along the intestinal mucosa in the ITIH4 groups, indicating compartmental redistribution possibly linked to inflammation control. * p < 0.05; ** p < 0.01; *** p < 0.001

Fig. 6

ITIH4 modulated SCFA levels and correlates with microbiome profiles in lung and gut. a Serum SCFAs of OVA-induced asthma mice with ITIH4 administration. b Correlation of SCFAs with lung microbiome at the family level. c Correlation of SCFAs with intestine microbiome at the family level. * p < 0.05; ** p < 0.01

Fig. 7

ITIH4 induced dose-dependent proteomic alterations in intestinal tissues. a The protein ratio distributions of low-dose and high-dose ITIH4 treatments in compared with untreated control. The red dash lines indicate the threshold of up- or down-regulation. b Overlapping of up- and down-regulated proteins in the high-dose and low-dose ITIH4 groups. c The enriched pathways with dose-dependent activation or suppression. Pathway name in blue is related to metabolism of proteins, in green for small molecule metabolism, and in purple for molecular transportations. Pathway with z-score > 0 indicates activation while z-score < 0 indicates suppression