Tenofovir attenuates cytokine storm and bronchiolar damage in a mouse model of bleomycin-induced acute lung injury

Categories: Disease & Virus

August 28, 2025

Ethics statement

All animal experiments were conducted in accordance with the guidelines of the European Parliament and Council Directive 2010/63/EU and the Spanish Royal Decree 53/2013 on the protection of animals used for scientific purposes. All procedures were reviewed and approved by the CEAA/CIBIR Bioethics Committee (Ref. 00860–2023/028740). In addition, all the study was carried out in compliance with the ARRIVE guidelines (https://arriveguidelines.org).

Animals and BLM-induced acute lung injury mouse model

A total of 64 eight-week-old C57BL/6J mice (Charles River, France), both male and female, were randomly assigned to four experimental groups (n = 8 per sex per group): (i) oral TDF administered in drinking water from day 0 to day 10, followed by oropharyngeal aspiration (OA) of saline on day 7 (TDF); (ii) oral TDF in drinking water from day 0 to day 10, followed by OA of bleomycin on day 7 (TDF-BLM); (iii) regular drinking water throughout the experiment, followed by OA of saline on day 7 (SAL); and (iv) regular drinking water throughout the experiment, followed by OA of bleomycin on day 7 (BLM). Animals were euthanized 72 h after OA (day 10), and serum and lung tissues were collected for analysis. The animals were randomly assigned to receive or not receive TDF (Fig. 1A). TDF was administered orally to mice for 10 days (D0-D10); water consumption was calculated as 5 mL per day for each mouse¹⁴. The average TDF dosage was 100 mg/kg/d¹⁴. One week later (week 9) (D7 post-TDF treatment), they were randomized again and exposed to BLM by OA to induce acute lung injury. The choice of timing was based on prevention studies conducted in people living with HIV (PLWH) in which, after 7 days of treatment with TDF/FTC, protection against HIV infection was observed¹⁵.

BLM (10 mg/kg) or SAL OA was delivered to the oropharyngeal cavity of mice under isoflurane anaesthesia, while the tongue was gently pulled out with forceps. Simultaneously, the nares were blocked to prevent obligated nasal breathing and forced BLM inhalation¹⁶. The mice were observed daily, weighed and all observations were recorded. Around 72 h later (D10), the OA of BLM or SAL mice were euthanized.

Serum and Bronchoalveolar lavage fluid (BALF) obtention, quantification of BALF, and lung collection

Animals were euthanized in accordance with institutional guidelines via intraperitoneal injection of sodium pentobarbital (Dolethal, 100 mg/kg in 10 mL/kg saline), followed by exsanguination. The high dose ensured rapid and humane euthanasia while minimizing distress. To preserve lung integrity for histological and biochemical analyses, thoracic procedures were avoided prior to euthanasia. Serum was obtained by centrifugation at 3000 x g for 10 min at 4 °C and stored at −80 °C until use in ELISA analyses. Bronchoalveolar lavage was performed twice (0.8 Ml phosphate-buffered saline, PBS, per wash) to collect BALF. After lavage, the lungs, including the trachea, were excised and weighed. BALF was centrifuged at 500 × g for 5 min and total protein concentration in BALF supernatants was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). Total cell counts were determined with a hemocytometer, and differential cell counts were performed on May-Grünwald/Giemsa (Sigma-Aldrich, St), counting a minimum of 300 cells per slide in a blinded manner. Cells were identified as macrophages, lymphocytes, or neutrophils based on standard morphological criteria. The right lung lobes were dissected and snap-frozen for quantitative PCR (qPCR) analysis, while the left lungs were inflated with formalin, post-fixed by immersion in formalin for 8–10 h, dehydrated, embedded in paraffin, and sectioned into 3 μm slices for histopathology and immunohistochemistry.

Histopathological and immunostaining assessments

Slides were stained with haematoxylin and eosin (H&E), observed, and photographed using a light microscope (Nikon Instruments Inc., Melville, NY). Inflamed lung areas were identified by darker H&E-stained foci and the presence of inflammatory cells, mainly lymphocytes. Two sections at different levels from six left lungs per group and sex of mice were evaluated. Lung injure score were evaluated by an expert pathologist in a blinded fashion.

CC10 and Pro-SPC antibodies were used to quantify CC10⁺ and SPC⁺ cells in the epithelial area, while Cd4 antibody was employed to evaluate pulmonary lymphocyte recruitment associated with TDF treatment. For immunostaining, paraffin-embedded sections were dewaxed and rehydrated using standard methods. Antigen retrieval was performed by immersing slides in a boiling solution (EnVision FLEX High pH GV804) for 17 min, followed by cooling in the same buffer for 30 min. Peroxidase inhibition and blocking were performed using the Novolink Polymer Detection System (Leica Biosystems, Nussloch, Germany) according to the manufacturer’s instructions. For immunohistochemical, slides were incubated at overnight at 4 °C with a primary antibody against Cd4⁺ (1:130; clone EPR19514; Abcam, Cambridge, UK). Sections were incubated with the Novolink Polymer Detection System (RE7150-K; Leica Biosystems, Nussloch, Germany) for 30 min, developed with 3,3′-diaminobenzidine (DAB) chromogen (K3468; Agilent Dako, Santa Clara, CA, USA), and photodocumented using a Leica DM6000B microscope equipped with a K/3 C camera (Leica Microsystems). Cd4⁺ staining in male mice was quantified in five different lung areas from two sections per animal (n = 5 male mice per group) in a randomized way. For fluorescence immunostaining, slides were incubated overnight at 4 °C with primary antibodies against CC10 (1:100; clone E-11; cat. no. sc-365992; Santa Cruz Biotech. Inc., Dallas, TX) and Pro-SPC (1:100; clone M-20; cat. no. sc-7706; Santa Cruz Biotech. Inc). After washing, sections were incubated for 1 h with secondary antibodies: Alexa Fluor Donkey anti-mouse 555 and anti-goat 633, both from Santa Cruz Biotech, Inc. The slides were subsequently washed, mounted in VECTASHIELD mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA), and examined using a confocal microscope (Stellaris 8 Leica Microsystems). Quantification of CC10⁺ and Pro-SPC⁺ staining areas in the terminal bronchiole epithelium was performed with Leica Application Site LAS X software (Leica Microsystems) on 6–14 terminal bronchioles per animal (n = 6 animals per group) in a randomized manner.

Lung injury score

The acute lung injury (ALI) score was calculated as described Matute-Bello et al.¹⁷. This score system includes five histological features: (A) neutrophils in the alveolar space; (B) neutrophils in the interstitial space; (C) hyaline membranes formation; (D) proteinaceous debris filling the airspace; and (E) alveolar septal thickening. Each item was assigned a score of 0, 1, or 2 based on the severity of lung injury. The final ALI score was determined using the following formula: [(20 × A) + (14 × B) + (7 × C) + (7 × D) + (2 × E)] divided by the number of fields multiplied by 100, leading to a score between zero (no lung injury) and one (severe injury) (n = 4 animals per group). Histological samples were reviewed in a blinded manner to ensure unbiased evaluation.

RNA isolation, reverse transcription and gene expression quantification via qPCR

The inferior right lung lobes were homogenized in TRIzol (15596026 Invitrogen, Carlsbad, CA, USA), and total RNA was extracted and purified using the RNA RNeasy Mini Kit (74104 Qiagen, Valencia, CA, USA). RNA samples were treated with DNase I (AM2222 Ambiom™) according to the manufacturer’s instructions. cDNA was synthesized from 1 µg of total RNA using the SuperScript III First-Strand Synthesis kit (18080051 Invitrogen) in a total volume of 30 µL following the manufacturer’s protocol. qPCR amplification was performed using SybrGreen (RR420A Takara Bio Inc., Shiga, Japan) and specific primers as follows: Glyceraldehide 3-phoshate dehydrogenase (Gadph), sense: 5´-CATGTTCCAGTATGACTCCACTC-3´, antisense: 5´-GGCCTCACCCCATTTGATGT-3´. Interleukin-6 (Il6), sense: 5´-ATGGATGCTACCAAACTGGAT-3´ and antisense: 5´-TGAAGGACTCTGGCTTTGTCT-3´. Tumor necrosis factor- α (Tnfα), sense 5´-ACGGCATGGATCTCAAAGAC-3´ and antisense 5´-AGATAGCAAATCGGCTGACG-3´. Interleukin-1 beta (Il1β), sense: 5´-CTGAACTCAACTGTGAAATGCCA-3´ and antisense: 5′-AAAGGTTTGGAAGCAGCCCT-3´. Interleukin-10 (Il10), sense: 5´-ATAACTGCACCCACTTCCCA-3´ and antisense: 5´-GGGCATCACTTCTACCAGGT-3´. Transforming growth factor- beta 1 (Tgfβ1), sense: 5´-GCAACATGTGGAACTCTACCAGAA-3´ and antisense: 5´-GACGTCAAAAGACAGCCACTCA-3´. Angiotensin I converting enzyme (peptidyl-dipeptidase A) (Ace), sense: 5´-TGAGAAAAGCACGGAGGTATCC − 3´ and antisense: 5´-AGAGTTTTGAAAGTTGCTCACATCA-3´. Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), sense: 5´-GGATACCTACCCTTCCTACATCAGC-3´ and antisense: 5´-CTACCCCACATATCACCAAGCA-3´. Amplification and detection of specific products were performed using the QuantStudio™ 5 system (Applied Biosystems, Foster City, CA, USA). All reactions were carried out in duplicate for each sample. Relative biomarker expression levels were calculated using the ΔΔCt method, normalized to the housekeeping gene Gapdh, as per the manufacturer’s recommendations.

Sandwich enzyme linked immunosorbent assay (ELISA)

Serum IL6 levels were measured using a mouse ELISA kit (M6000B R&D Systems). This procedure was performed according to the manufacturer’s instructions.

Statistics

All data were analyzed using Graph Pad Prism 8 (Graphd Pad Software, Inc. San Diego, CA, USA) and are expressed as mean ± standard error of the mean. Normality was assessed using the Shapiro-Wilk tests, and homogeneity of variance was evaluated with Levene’s tests. Depending on data distribution, group differences were analyzed using either the Mann-Whitney U test for nonparametric data or Student’s t-test for parametric data. A p-value < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

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