Lung and general health effects of Toll-like receptor-4 (TLR4)-interacting SPA4 peptide

Abstract

Background: A surfactant protein-A-derived peptide, which we call SPA4 peptide (amino acids: GDFRYSDGTPVNYTNWYRGE), alleviates lung infection and inflammation. This study investigated the effects of intratracheally administered SPA4 peptide on systemic, lung, and health parameters in an outbred mouse strain, and in an intratracheal lipopolysaccharide (LPS) challenge model.

Methods: The outbred CD-1 mice were intratracheally administered with incremental doses of SPA4 peptide (0.625-10 μg/g body weight) once every 24 h, for 3 days. Mice left untreated and those treated with vehicle were included as controls. Mice were euthanized after 24 h of last administration of SPA4 peptide. In order to assess the biological activity of SPA4 peptide, C57BL6 mice were intratracheally challenged with 5 μg LPS/g body weight and treated with 50 μg SPA4 peptide via intratracheal route 1 h post LPS-challenge. Mice were euthanized after 4 h of LPS challenge. Signs of sickness and body weights were regularly monitored. At the time of necropsy, blood and major organs were harvested. Blood gas and electrolytes, serum biochemical profiles and SPA4 peptide-specific immunoglobulin G (IgG) antibody levels, and common lung injury markers (levels of total protein, albumin, and lactate, lactate dehydrogenase activity, and lung wet/dry weight ratios) were determined. Lung, liver, spleen, kidney, heart, and intestine were examined histologically. Differences in measured parameters were analyzed among study groups by analysis of variance test.

Results: The results demonstrated no signs of sickness or changes in body weight over 3 days of treatment with various doses of SPA4 peptide. It did not induce any major toxicity or IgG antibody response to SPA4 peptide. The SPA4 peptide treatment also did not affect blood gas, electrolytes, or serum biochemistry. There was no evidence of injury to the tissues and organs. However, the SPA4 peptide suppressed the LPS-induced lung inflammation.

Conclusions: These findings provide an initial toxicity profile of SPA4 peptide. Intratracheal administration of escalating doses of SPA4 peptide does not induce any significant toxicity at tissue and organ levels. However, treatment with a dose of 50 μg SPA4 peptide, comparable to 2.5 μg/g body weight, alleviates LPS-induced lung inflammation.

Keywords: Anti-inflammatory activity; Health effects; Pulmonary toxicology; Surfactant protein-A-derived peptide.

Fig. 1

Assessment of body weight in SPA4 peptide-treated CD-1 mice. The schedule of SPA4 peptide treatment and study design are illustrated in (a). Body weights were taken for each mouse after every 24 h. Results shown are from 11 to 15 mice per group (seven groups total) (b). No statistically significant difference was observed in body weights of mice over time and among different study groups (Two-way ANOVA followed by Tukey’s post hoc analysis)

Fig. 2

Levels of total lung protein, albumin, and lactate, and LDH activity. Lung tissue homogenates were subjected to the measurement of total protein by BCA assay (a), albumin by ELISA (b), lactate by a fluorometric assay (c), and LDH activity using a colorimetric method (d). The amounts of total protein, albumin, and lactate, and activity of LDH, were normalized with gram lung weight or total lung protein. Results shown are mean + SEM of measurements in lung tissue homogenates from 5 to 12 CD-1 mice per group (seven groups total). No statistically significant difference was observed in the measurement between the study groups (One-way ANOVA followed by Tukey’s post hoc analysis)

Fig. 3

Assessment of lung wet/dry weight ratio as an indicator of lung edema. Whole lungs harvested from CD-1 mice were weighed immediately (wet weight). After drying at 60°C for 48 h, lungs were weighed again. The ratios of lung wet/dry weights were calculated. Data shown are mean + SEM values from lungs of 3–5 mice per group (seven groups total). There was no statistically significant difference in the values among different study groups (One-way ANOVA followed by Tukey’s post hoc analysis)

Fig. 4

Histological evaluation of H&E-stained sections of tissues (lung, heart, kidney, liver, spleen, and intestine) harvested from SPA4 peptide-treated, vehicle-treated, and untreated CD-1 mice. Major organs were harvested from mice at the time of necropsy and fixed in 10% buffered-formalin overnight, dehydrated, embedded in paraffin, sectioned, and stained with H&E. Tissue sections were evaluated for any evidence of tissue damage and inflammation by board-certified veterinary pathologists. Representative photomicrographs are shown from respective tissue section for each study group (11–15 mice per group, seven groups total) revealing normal histology. Atypical findings in isolated specimens (shown as*) from one mouse in selected study groups are shown, which are not related to the dose of SPA4 peptide or study design

Fig. 5

Serum biochemical profile of CD-1 mice treated with escalating doses of SPA4 peptide. Fresh blood harvested from mice in different groups were clotted at room temperature and subjected to separation of serum by centrifugation. Serum samples from 6 to 12 mice per group (seven groups total) were then analyzed for biochemical profile, specifically, ALP (a), AST (b), ALT (c), creatine kinase (d), albumin (e), total bilirubin (f), total protein (g), globulin (h), BUN (i), cholesterol (j), glucose (k), and bile acids (l). One-way ANOVA followed by Tukey’s post hoc analysis was used for statistical analysis of data from different study groups

Fig. 6

SPA4 peptide-specific IgG levels in serum samples harvested from SPA4 peptide-treated, vehicle-treated, and untreated CD-1 mice. Diluted mouse serum samples [1:10 (a), 1:100 (b), and 1:1000 (c)] were subjected to ELISA for detection of SPA4 peptide-specific IgG levels. The ∆OD405 readings for positive control wells are shown within the figure. The SEM bars smaller than the size of the symbol are not visible in the chart. There was no statistically significant difference in the ∆OD405 readings among different study groups (One-way ANOVA followed by Tukey’s post hoc analysis)

Fig. 7

Effect of SPA4 peptide on LPS-induced lung edema and reactivity in an intratracheal LPS challenge model in C57BL6 mice. Wet weights of lungs were noted at the time of necropsy (a). Ratios of lung wet/dry weight (b), μg total protein/g lung wet weight (c), and index for the symptoms of sickness (d). Mean + SEM values of measurements are from 8 to 19 mice per group (lung wet weight), 3–15 mice per group (lung wet/dry weight ratio), 14–15 mice per group (μg protein/g lung wet weight), or 12–29 mice per group (symptoms of sickness) included in separate experiments. The p values (* p < 0.05, **** p < 0.0001) were determined by ANOVA with Tukey’s post-hoc analysis

Fig. 8

Assessment of inflammation in H&E-stained lung tissue sections of LPS-challenged and SPA4 peptide-treated C57BL6 mice. Representative photomicrographs of H&E-stained lung tissue sections of mice included in five separate experiments. The photomicrographs were taken using a 10X and 40X objective (a). Low and high magnification photomicrographs demonstrate differences in the semi-quantitative grading criteria, including the extent of peribronchial/bronchiolar infiltrates, severity of peribronchial/bronchiolar infiltrates, extent of alveolar infiltrates, and severity of alveolar septal and luminal neutrophilic infiltrates. The pulmonary inflammation scores (Mean + SEM) for mice in each group are shown as bar chart (b). Inflammation scores for individual representative images are indicated within (a). The number of cell nuclei counted in five randomly selected regions per lung tissue section (c). Total number of intercepts counted on horizontal and vertical transverses in five randomly selected regions per lung tissue section. The cell nuclei and number of intercepts were counted using the STEPanizer program (d). Mean + SEM values are shown within each figure for different types of measurements of lung inflammation. The p values (* p < 0.05, ** p < 0.005, **** p < 0.0001) were determined by ANOVA with Tukey’s post-hoc analysis