J Pathol Transl Med.  2022 Mar;56(2):81-91. 10.4132/jptm.2021.12.27.

Blocking Toll-like receptor 9 attenuates bleomycin-induced pulmonary injury

• Alzahrani B
• Gaballa MMS
• Tantawy AA
• Moussa MA
• Shoulah SA
• Elshafae SM

• KMID: 2527192
• DOI: http://doi.org/10.4132/jptm.2021.12.27

Abstract

Background
Acute respiratory distress syndrome (ARDS) is one of the most common complications in coronavirus disease 2019 patients suffering from acute lung injury (ALI). In ARDS, marked distortion of pulmonary architecture has been reported. The pulmonary lesions in ARDS include hemodynamic derangements (such as alveolar edema and hemorrhage), vascular and bronchiolar damage, interstitial inflammatory cellular aggregations, and eventually fibrosis. Bleomycin induces ARDS-representative pulmonary damage in mice and rats; therefore, we used bleomycin model mice in our study. Recently, Toll-like receptor 9 (TLR9) was implicated in the development of ARDS and ALI.
Methods
In this study, we evaluated the efficiency of a TLR9 blocker (ODN2088) on bleomycin-induced pulmonary damage. We measured the apoptosis rate, inflammatory reaction, and fibroplasia in bleomycin- and bleomycin + ODN2088-treated mice.
Results
Our results showed a significant amelioration in bleomycin-induced damage to pulmonary architecture following ODN2088 treatment. A marked decrease in pulmonary epithelial and endothelial apoptosis rate as measured by cleaved caspase-3 expression, inflammatory reaction as indicated by tumor necrosis factor α expression, and pulmonary fibrosis as demonstrated by Van Gieson staining and α-smooth muscle actin immunohistochemistry were observed following ODN2088 treatment.
Conclusions
All these findings indicate that blocking downstream TLR9 signaling could be beneficial in prevention or mitigation of ARDS through hemodynamic derangements, inflammation, apoptosis, and fibrosis.

Keyword

TLR9; ODN2088; Bleomycin; Acute respiratory distress syndrome; Fibrosis

Figure

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  Fig. 1. Representative lung photomicrographs in control- (A), ODN2088- (B), bleomycin- (C–L), and bleomycin + ODN2088 (BLEO/ODN2088)–treated mice (M–O). (A) Normal respiratory bronchioli and alveoli in the control group. (B) Mild hyperplasia of alveolar pneumocytes in the lung of an ODN2088-treated mouse. (C–L) Lung sections of mice treated with bleomycin, showing focal to confluent, moderate, to severe aggregation of inflammatory cells in the interalveolar septa (C–E), hyperplastic bronchiolar epithelia (F), peribronchial inflammatory aggregations and necrosis of bronchiolar epithelia (G, H, arrowhead), peribronchiolar fibroplasia (I, arrow), perivascular (arrow) inflammatory cellular infiltration and intra-alveolar hemorrhage (J), thrombosis in a pulmonary blood vessel (K, arrowhead), and pleural thickening (L). (M–O) Lungs of BLEO/ODN2088-treated mice showing necrosis in a few bronchiolar epithelia (M), small focal inflammatory aggregation in the interstitial space (N), and congested interalveolar blood capillaries (O).

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  Fig. 2. Photomicrographs (A–C) and statistical analysis (D) of Van Gieson staining of pulmonary tissues in bleomycin- (A, B) and bleomycin+ODN2088 (BLEO/ODN2088)–treated mice (C). Data in the bar graph (D) represent mean±standard deviation, and significant differences (p≤.05) are indicated by an asterisk.

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  Fig. 3. Immunohistochemical photomicrographs (A–C) and statistical analysis (D) of α-smooth muscle actin (α-SMA) expression in the pulmonary tissues of bleomycin- (A, B) and bleomycin+ODN2088 (BLEO/ODN2088)–treated mice (C). Data in the bar graph (D) represent mean±standard deviation, and significant differences (p≤.05) are indicated by an asterisk.

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  Fig. 4. Cleaved caspase-3 (CC3) immunohistochemical photomicrographs (A–C) and statistical analysis of CC3-positive cells (D) in the pulmonary section of bleomycin- and bleomycin+ODN2088 (BLEO/ODN2088)–treated mice. Data in the bar graphs (D) represent mean±standard deviation, and significant differences (p<.001) are indicated by four asterisks.

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  Fig. 5. Tumor necrosis factor α (TNF-α) immunohistochemical photomicrographs (A–C) and analysis of TNF-α positivity (D) in the lung sections of bleomycin- and bleomycin+ODN2088 (BLEO/ODN2088)–treated mice. Data in the bar graph (D) represent mean±standard deviation, and significant differences (p≤.05) are indicated by an asterisk.

Reference

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