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. 2020 Sep;8(17):e14553.
doi: 10.14814/phy2.14553.

Recombinant adiponectin protects the newborn rat lung from lipopolysaccharide-induced inflammatory injury

Julijana Ivanovska  1 , Na-Young Cindy Kang  1 , Nikola Ivanovski  1 , Avita Nagy  2 , Jaques Belik  1 , Estelle B Gauda  1
Affiliations

Recombinant adiponectin protects the newborn rat lung from lipopolysaccharide-induced inflammatory injury

Julijana Ivanovska et al. Physiol Rep. 2020 Sep.

Abstract

Preterm infants are at high risk for developing bronchopulmonary dysplasia and pulmonary hypertension from inflammatory lung injury. In adult models, adiponectin (APN)-an adipocyte-derived hormone-protects the lung from inflammatory injury and pulmonary vascular remodeling. Cord blood APN levels in premature infants born < 26 weeks gestation are 5% of the level in infants born at term. We previously reported the expression profile of APN and its receptors in neonatal rat lung homogenates during the first 3 weeks of postnatal development. Here, we characterize the expression profile of APN and its receptors in specific lung cells and the effects of exogenous recombinant APN (rAPN) on lipopolysaccharide-(LPS)-induced cytokine and chemokine production in total lung homogenates and specific lung cells. In vitro, rAPN added to primary cultures of pulmonary artery smooth muscle cells attenuated the expression of LPS-induced pro-inflammatory cytokines while increasing the expression of anti-inflammatory cytokines. In vivo, intraperitoneal rAPN (2 mg/kg), given 4 hr prior to intrapharyngeal administration of LPS (5 mg/kg) to newborn rats at postnatal day 4, significantly reduced gene and protein expression of the pro-inflammatory cytokine IL-1ß and reduced protein expression of the chemokines monocyte chemoattractant protein (MCP-1) and macrophage inflammatory protein-1 alpha (MIP-1α) in the lung. LPS-induced histopathological changes in the lung were also decreased. Moreover, rAPN given 20 hr after intrapharyngeal LPS had a similar effect on lung inflammation. These findings suggest a role for APN in protecting the lung from inflammation during early stages of lung development.

Keywords: adiponectin; adiponectin receptors 1 and 2; bronchopulmonary dysplasia; chemokines; cytokines; neonatal rat.

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Conflict of interest statement

The authors have no conflict of interest to declare.

Figures

FIGURE 1
FIGURE 1
APN, AdipoR1, and AdipoR2 protein expression in lung cells in vitro. Fibroblast (a1–3), epithelial cells (b1–3), and pulmonary artery smooth muscle cells (c1–3) were isolated from lungs of Sprague‐Dawley rats at PND4 and PND7, grown in culture, homogenized, and assayed for protein expression with western blot (WB). The specificity of each cell type was preliminarily determined by WB detection of H‐caldesmon for PASMCs, EpCAM for epithelial cells, and pro‐collagen for fibroblasts (data not shown). APN, AdipoR1, AdipoR2 protein was normalized to β‐actin. Representative immunoblots are under each graph. Bars represent mean ± SEM. *p < .05 by t‐test compared to PND4. All cell experiments were done in triplicate
FIGURE 2
FIGURE 2
Representative photomicrographs of immunocytochemistry showing the co‐expression of APN, AdipoR1, or AdipoR2 with α‐actin in PASMC, in vitro. APN (a), AdipoR1 (b), and AdipoR2 (c) are depicted as red in the cytoplasm; α‐actin, a marker for smooth muscle, is depicted as green. Nuclear localization was visualized by DAPI, blue nuclei. Merged photomicrographs show co‐localized expression (yellow) of the protein of interest with α‐actin. All images were captured with confocal microscopy
FIGURE 3
FIGURE 3
Effect of rAPN on LPS‐induced cytokine expression in PASMCs. To determine the effect of rAPN on LPS‐induced expression in PASMCs during development, PASMCs from PND4 and PND7 rat pups were treated with rAPN (20 µg/ml) or with LPS (3 µg/ml) + rAPN (20 µg/ml) (a–c) in culture for 48 hr (n = 4 per group); afterward the cells were homogenized and protein extracted from western blot (WB). Immunoblots were normalized to β‐actin. Bars represent mean ± SEM.*p < .05 by one‐way ANOVA followed by post hoc analyses using Holm–Sidak test compared to all other groups; #p < .05 compared to corresponding control groups. This experiment was repeated four times
FIGURE 4
FIGURE 4
Effect of rAPN on the expression of lung mRNA and protein levels for inflammatory cytokines in response to aspiration of LPS, in vivo. PND4 rat pups were treated with rAPN (2 mg/kg) or saline by IP injections and, after 4 hr, LPS (5 mg/kg) or saline was administered to the posterior pharynx (intrapharyngeal, IPh). After 24 hr, mRNA (left panel) or protein levels (right panel) were measured in lung homogenates for IL‐6 (a and b), TNF‐α (c and d), IL‐1β (e and f), and IL‐10 (g and h) (n = 4 per group). Change in relative mRNA expression was calculated by 2ΔCt method normalized to β‐actin mRNA expression. Immunoblots were normalized to β‐actin protein expression. Bars represent mean ± SEM.*p < .05 by one‐way ANOVA followed by post hoc analyses using Holm–Sidak test compared to certain other groups. # represents p < .05 compared to all other groups. ¥ represents p < .05 compared to saline + LPS group. ‡ represents p < .05 compared to saline and saline + LPS groups
FIGURE 5
FIGURE 5
Effect of rAPN on the expression of lung mRNA and protein levels for inflammatory chemokines in response to aspiration of LPS, in vivo. PND4 pups were treated with rAPN (2 mg/kg) or saline by IP injections, and after 4 hr, LPS (5 mg/kg) or saline were administered by IPh route. After 24 hr, lungs were collected and processed for qPCR and western blot (WB). mRNA relative expression of MCP‐1 and MIP‐1α (a, c); and protein content of MCP‐1, MIP‐1α, and IL‐8 (b, d, e) was measured (n = 4 per group). mRNA relative expression change was calculated by 2ΔCt method normalized to β‐actin mRNA expression. Immunoblots were normalized to β‐actin and representative immunoblots under each graph show two contiguous lanes for each group. Bars represent mean ± SEM by one‐way ANOVA followed by post hoc analyses using Holm–Sidak test. * represents p < .05 compared to all other groups and # represents p < .05 compared to saline IP + LPS IPh group
FIGURE 6
FIGURE 6
Effect of rAPN on LPS‐induced expression of inflammatory cell markers. PND4 rat pups were treated with rAPN (2 mg/kg) or saline by IP injections and, after 4 hr, LPS (5 mg/kg) or saline was administered by IPh route. After 24 hr, CD68 (a), E‐selectin (b), CD45 (c), and CD16 (d) protein levels were measured in lung homogenates by western blot (WB; n = 4 per group). Bars represent mean ± SEM. * represents p < .05 by one‐way ANOVA followed by post hoc analyses using Holm–Sidak test compared to saline group. # represents p < .05 compared to rAPN/saline group and saline/LPS group. ¥ represents p < .05 compared to rAPN/saline and saline/LPS. ‡ represents p < .05 compared to all other groups
FIGURE 7
FIGURE 7
rAPN given prior to LPS decreases lung histopathology. Representative photomicrographs of lung sections from animals treated with saline/LPS (top and bottom left hand), rAPN/LPS and bottom (top right hand), rAPN/saline (middle left hand), saline/saline (middle right hand). PND4 rat pups were treated with rAPN (2 mg/kg) or saline by IP injections and, after 4 hr LPS (5 mg/kg) or saline was administered IPh. Twenty‐four hours later, animals were given an additional dose of saline or rAPN IP, and 48 hr later lungs were inflated, paraffin‐embedded, and stained with H&E (n = 4 per group). LPS‐exposed animals had thickened alveolar septa and cellular infiltrate in the interstitium of the lung that was not observed in the animals treated with rAPN/LPS or the other two treatment groups (rAPN/saline; saline/saline control). Bottom images are magnification of insets in top panel
FIGURE 8
FIGURE 8
Effect of rAPN on LPS‐induced lung histopathology score. Total lung histopathology scores were determined for PND4 rats who were given saline or rAPN IP 4 hr before, and 24 hr after IPh LPS (prevention protocol) or given rAPN 20 hr and 48 after IPh LPS (rescue protocol). The lung was removed and processed for histology 72 hr after initial LPS exposure in both protocols. rAPN given before LPS reduced the histopathology score while rAPN given 20 hr after LPS did not. Bars represent mean ± SD one‐way ANOVA followed by post hoc analyses using Tukey's test to compare scores between groups in the prevention protocol and rescue protocol separately. * represents p < .02 saline versus saline + LPS and rAPN alone. + represents p = .003 versus saline + LPS in the prevention protocol; N = 4 in each group. Four treatment groups were in the prevention protocol while only three treatment groups were in the rescue protocol
FIGURE 9
FIGURE 9
Effect of rAPN given after aspiration of LPS on mRNA and protein expression for inflammatory cytokines, in vivo, rescue. PND4 rats were given LPS (5 mg/kg) or saline IPh followed by rAPN (2 mg/kg) or saline by IP injections 20 hr after LPS. (n = 4 per group). Six hours after rAPN and 24 hr after LPS was given, lungs were removed and processed for mRNA (left panel) or protein (right panel) detection for IL‐6 (a and b), TNF‐α (c and d), IL‐1β (e and f), and IL‐10 (g and h). Change in relative mRNA expression was calculated by 2ΔCt method normalized to β‐actin mRNA expression. Immunoblots were normalized to β‐actin protein expression. Bars represent mean ± SEM. One‐way ANOVA followed by post hoc analyses using Holm–Sidak test *p < .05 compared to other groups; ¥ represents p < .05 compared to saline/saline and LPS/rAPN treatment groups for TNF‐α and IL‐10; # represents p < .05 compared to saline/saline and LPS/rAPN treatment groups for IL‐1β
FIGURE 10
FIGURE 10
Effect of the rAPN when given as rescue treatment on LPS‐induced lung chemokine production, in vivo. PND4 rats were given LPS (5 mg/kg) or saline IPh followed by rAPN (2 mg/kg) or saline by IP injections 20 hr after LPS (n = 4 per group). Six hours after rAPN was given, lungs were removed and processed for mRNA (left panel) or protein (right panel) detection of MCP‐1 (a and b) and MIP‐1α (c and d). Change in relative mRNA expression was calculated by 2ΔCt method normalized to β‐actin mRNA expression. Immunoblots were normalized to β‐actin protein expression. Bars represent mean ± SEM. One‐way ANOVA followed by post hoc analyses using Holm–Sidak test. * represents p < .05 compared to other groups
FIGURE 11
FIGURE 11
Schematic diagram summarizing the proposed downstream effects of rAPN on LPS‐induced lung inflammation during the saccular stage of lung development. rAPN blocks upregulation of inflammatory cytokines and chemokines, activation of neutrophils, macrophages, endothelial, epithelial, pulmonary artery smooth muscle cells in the lung, ultimately blocking endothelial leak and alveolar damage, and vascular remodeling. HMW—rAPN is high molecular weight—recombinant adiponectin; PASMC is pulmonary artery smooth muscle cells.
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