NOD1 promotes leukocyte clearance and limits inflammation in female mice during obesity-associated acute lung injury

Abstract

Obesity is associated with metabolic inflammation, which includes changes to innate immune responses relevant to acute lung injury. NOD1 is a cytosolic pattern recognition receptor involved in sensing bacterial peptidoglycan and has been linked to metabolic inflammation. However, its role in obesity-associated acute lung injury, especially in females, remains unclear. Here, we investigated the impact of NOD1 deficiency on pulmonary inflammation in female mice subjected to a high-fat diet and lipopolysaccharide-induced acute lung injury. Compared to wild-type controls, obese Nod1^-/- mice showed reduced leukocyte and neutrophil numbers in the bronchoalveolar lavage (BAL), but increased BAL levels of TNF-α, IL-1β, IL-6, IL-17A, and IL-22, suggesting impaired neutrophil clearance. In the lung tissue, NOD1 deficiency during obesity led to elevated neutrophil accumulation, increased myeloperoxidase activity, reduced CD163⁺ macrophages, and enhanced β-galactosidase activity. Gene expression analysis revealed upregulation of chemokines, adhesion molecules, and inflammasome components, alongside downregulation of M2 polarization markers. Additionally, obese Nod1^-/- mice showed higher NF-κB and ERK1/2 activation and lower p38 phosphorylation. These findings indicate that NOD1 regulates leukocyte dynamics, inflammation, and macrophage function in the obese lung. We identify NOD1 as a key protective modulator of pulmonary immune responses during acute lung injury under metabolic stress.

Keywords: NOD1; acute lung injury; inflammation; obesity.

FIGURE 1

Experimental design and metabolic characterization of WT (C57Bl/6N) and Nod1^−/− mice fed either a control or high‐fat diet. (a) Experimental design. (b) Body weight gain, food intake, relative perigonadal fat mass, serum levels of glucose, triglycerides, and total cholesterol. (c) Hepatic triglyceride content and representative images of liver stained with H&E. Data are presented as mean ± SD. Each dot represents one mouse (n = 5–8/group). Comparisons were made between WT and Nod1^−/− mice within each group (control or HFD) and between WT and WT or Nod1^−/− and Nod1^−/− using unpaired Student's t‐test. Numerical p values are shown in the figures for comparisons where p < 0.05. WT, wild type. Scale bar, 100 μM.

FIGURE 2

NOD1 deletion impairs neutrophil recruitment and enhances cytokine production in the bronchoalveolar lavage (BAL) during LPS‐induced acute lung injury and obesity. (a) Representative image of BAL fluid collected from LPS‐challenged mice stained with Wright‐Giemsa. (b) Total leukocyte, neutrophil, and mononuclear cell counts in the BAL. (c) Neutrophil myeloperoxidase (MPO) activity as a marker of activation. (d) Nitrite/arginase ratio in the BAL as an index of the inflammatory environment. (e) Levels of TNF‐α, IL‐1β, IL‐6, IL‐17A, and IL‐22 in the BAL fluid. Data are presented as mean ± SD. Each dot represents one mouse (n = 5–8/group). Comparisons were made between WT and Nod1^−/− mice within each group (control or HFD) using unpaired Student's t‐test. Numerical p values are shown in the figures for comparisons where p < 0.05. WT, wild type. Scale bar, 50 μM.

FIGURE 3

NOD1 deletion exacerbates lung inflammation during LPS‐induced acute lung injury and obesity. (a) Panoramic scan of entire lung sections at low magnification. This overview allows visualization of the overall distribution and extent of inflammatory changes across the lung. (b) Representative immunohistochemistry images showing neutrophil myeloperoxidase (MPO) and the M2 macrophage marker CD163 in lung sections. (c) Quantification of MPO protein expression in lung tissue by Western blot. (d) Neutrophil MPO enzymatic activity. (e) CD163‐positive area in lung parenchyma as assessed by immunohistochemistry. (f) Nitrite/arginase ratio in lung tissue. (g) Levels of TNF‐α, IL‐1β, IL‐6, and SA‐β‐galactosidase activity in lung homogenates. Data are presented as mean ± SD. Each dot represents one mouse (n = 5–8/group). Comparisons were made between WT and Nod1^−/− mice within each group (control or HFD) using unpaired Student's t‐test. Numerical p values are shown in the figures for comparisons where p < 0.05. WT, wild type. A = airway. V = vessel. Scale bar, 500 μM (a) and 50 μM (b). Protein ladder (L): 63 kDa (magenta) and 35 kDa (blue).

FIGURE 4

NOD1 deletion alters the pulmonary gene expression profile during LPS‐induced acute lung injury and obesity. Gene expression heatmap of genes related to chemokines, adhesion molecules, M1 and M2 macrophage polarization, inflammasome components, resolution and cell death pathways, tissue remodeling, and other pattern recognition receptors. Data are represented as a heat map showing group averages (n = 5–8 mice/group). Comparisons were made between WT and Nod1^−/− mice within each group (control or HFD) using unpaired Student's t‐test. Numerical p values are shown in the figures for comparisons where p < 0.05. WT, wild type. Genes shaded in gray represent >3‐fold expression change relative to controls. Gray was used intentionally to avoid distortion of the color scale for the remaining genes, which would have occurred due to extreme expression values.

FIGURE 5

NOD1 deletion enhances NF‐κB and ERK activation while suppressing p38 MAPK signaling in the lungs during LPS‐induced acute lung injury and obesity. Shown are the protein expression levels of phosphorylated and total forms of (a) NF‐κB p65, (b) p38 MAPK, (c) ERK1/2, and (d) JNK in lung tissue. Data represent the ratio of phosphorylated to total protein, as determined by Western blot. Data are presented as mean ± SD. Each dot represents one mouse (n = 5–8/group). Comparisons were made between WT and Nod1^−/− mice within each group (control or HFD) using unpaired Student's t‐test. Numerical p values are shown in the figures for comparisons where p < 0.05. WT, wild type. Protein ladder (L): 63 kDa (magenta), 48 kDa (green) and 35 kDa (blue).