Beneficial effects of CCL8 inhibition at lipopolysaccharide-induced lung injury

Abstract

CCL8 (MCP-2) is a chemoattractive cytokine associated with various immune-related pathologies. Recent studies show that CCL8 is significantly stimulated during acute respiratory distress syndrome in severely ill patients with COVID-19, making the inhibition of CCL8 activity a promising treatment. Lipopolysaccharide (LPS)-induced lung injury was evaluated in mice using a neutralizing antibody (1G3E5) against human CCL8. Pharmacokinetic studies indicated that following IP administration, 1G3E5 was sustained at higher levels and for a longer period compared to IV administration. CCL8 expression in the lungs was not enhanced by LPS, but CCR2 and CCR5 receptors were significantly stimulated. 1G3E5-mediated inhibition of CCL8 was associated with the reduction of pulmonary inflammation and suppression of various pro-inflammatory cytokines. These results point to a previously unrecognized, permissive role for CCL8 in mediating cytokine induction and ultimately sustaining inflammation. Disruption of CCL8 activity may provide a strategy for mitigating pulmonary inflammation during lung injury when related to abnormal cytokine induction.

Keywords: Biological sciences; Immunology; Immunology specialty.

Graphical abstract

Figure 1

Effect of CCL8 genetic ablation in LPS-induced pulmonary inflammation in mice Animals received 2.5 mg LPS via a nebulizer and were sacrificed 6 hrs later. (A) Representative microphotographs of H&E-stained sections of lungs of wt and CCL8KO mice that received LPS showing the development of hyaline membranes (yellow arrows), the presence of neutrophils (red arrows), and the formation of proteinaceous debris in the alveolar spaces (blue arrows) (scale bar: 25 uM). (B) Scoring of inflammation was evaluated histologically in wt (n = 5) and CCL8KO (n = 8) mice. Side by side dot plot is shown. p value is indicated (Mann-Whitney U test). (C) Expression of IL1β, IL6, and TNFα in the lungs of wt (n = 5) and CCL8KO mice (n = 7) following LPS administration as described. Results were normalized versus GAPDH expression, expressed in arbitrary units (AU), and analyzed by unpaired t-test. Data are represented as mean ± SEM. P values are indicated.

Figure 2

Neutralizing activity of 1G3E5 against CCL8-induced migration (A) Alignment between human CCL8 and either mouse (Mus) or deer mouse (Peromyscus maniculatus) CCL8. Homologies are shown. The antigenic peptide is indicated by the dashed blue square. (B) Anti-CCL8 antibody 1G3E5 inhibits RAW macrophage migration that is induced by either human or Peromyscus CCL8. RAW cells migrated in the presence of hCCL8 or pCCL8 at 10 ng/mL alone or combined with 1G3E5 at 1.5 ug/mL. Results are expressed as mean ± SEM (n = 4) and analyzed by unpaired t-test. ∗, p < 0.05; ∗∗∗, p < 0.001.

Figure 3

Effect of 1G3E5 in LPS-induced pulmonary inflammation in deer mice (P. maniculatus) Animals received 2.5 mg LPS via a nebulizer and were sacrificed 6 hrs later. (A) Representative microphotographs of H&E-stained sections of lungs of animals that received LPS showing the development of hyaline membranes (yellow arrows), the presence of neutrophils (red arrows), and the formation of proteinaceous debris in the alveolar spaces (blue arrows). These histological findings were abolished in the group that received LPS+1G3E5 (scale bar: 25 uM). (B) Scoring of inflammation evaluated histologically in animals that received saline (n = 3), 1G3E5 at 2.5 mg/kg (n = 3), LPS (n = 5), or 1G3E5 at 2.5 mg/kg plus LPS (n = 5). Results were analyzed by the Kruskal-Wallis test. P value is indicated.

Figure 4

Effect of 1G3E5 in cytokine expression in deer mice Expression of IL1β, IL6, TNFα, and CCL8 in the lungs of deer mice that received saline (n = 7), 1G3E5 at 2.5 mg/kg (n = 7), LPS (n = 8), and 1G3E5 (n = 8) at 2.5 mg/kg plus LPS. Expression was normalized versus GAPDH expression and expressed in arbitrary units (AU). Comparisons between all groups were performed by ANOVA, and the results are expressed as mean ± SEM. Significance is indicated as follows: #, p < 0.05; ###, p < 0.001. Comparisons between siblings in the groups that received LPS alone or combined with 1G3E5 were performed by Wilcoxon non-parametric test. Significance was indicated as follows: ∗, p < 0.05; ∗∗, p < 0.01. Inlets in dashed black ovals show the individual expression levels in the siblings that received LPS or LPS plus 1G3E5. The mating number of parents is indicated. Results were analyzed by paired t-test, and p values are indicated.

Figure 5

Effect of LPS and 1G3E5 in CCL8 receptors’ expression in deer mice (A) Expression of CCR1, 2, 3, 5, and 8 in the lungs of deer mice that received saline (n = 7), 1G3E5 at 2.5 mg/kg (n = 7), LPS (n = 8), and 1G3E5 (n = 8) at 2.5 mg/kg plus LPS. Expression was normalized versus GAPDH expression and expressed in arbitrary units (AU). Comparisons between all groups were performed by ANOVA, and the results are expressed as mean ± SEM. Significance vs ctr is indicated as follows: ∗, p < 0.05; ∗∗, p < 0.01, ∗∗∗∗, p < 0.00001. (B and C) The heatmap correlation matrix depicts the degree of co-expression between chemokine receptors, CCL8, and pro-inflammatory cytokines in sibling pairs of deer mice that received LPS alone (B) or combined with 1G3E5 (C). Correlation coefficients are indicated.

Figure 6

Long-term effects of 1G3E5 in LPS-induced lung inflammation in deer mice (P. maniculatus) Animals received LPS (2.5 mg/5 mL saline) via nebulizer for three consecutive days and were sacrificed 4 weeks later. 1G3E5 was administered following the administration of LPS. Microphotographs show representative H&E-stained sections of the lungs at which prominent mononuclear inflammatory infiltration and thickening of the alveolar septa are seen in the animals that received LPS alone or combined with non-specific IgG (scale bar: 25 μM). In the animals that received 1G3E5, the histopathology caused by LPS was abolished. Results are shown in the lower right panel and analyzed by Kruskal Wallis test. p value is indicated. Bar diagram shows the density of inflammation scores for individual animals that represent sibling pairs and received LPS in combination with either non-specific IgG or 1G3E5. The results were analyzed by Wilcoxon signed rank test, and P-value is shown.

Figure 7

Trichrome staining (blue) for collagen Representative images of trichrome-stained sections of lungs from the LPS + IgG and LPS+1G3E5 groups (scale bar: 25 uM) highlight the presence of fibrosis in the LPS + IgG as opposed to the LPS+1G3E5 group. Sections from 2 littermates are shown corresponding to mating #2094 (from Figure 6). Positivity consistent with fibrosis is indicated by yellow arrows.

Figure 8

Effect of 1G3E5 administered after the induction of LPS-induced pulmonary inflammation in deer mice (P. maniculatus) Animals received 2.5 mg LPS via a nebulizer and 24 h later received either IgG or 1G3E5. Animals were sacrificed 24 h later and lung histology was evaluated. (A) Representative microphotographs of H&E-stained sections of lungs of animals that received LPS + IgG or LPS+1G3E5 (scale bar: 25 uM). (B) Scoring of inflammation was evaluated histologically (n = 6 per group). Results were analyzed by the Mann Whitney U test.

Figure 9

Mean serum concentration-time profiles of 1G3E5 following a single (2.5 mg/kg) intravenous (i.v.) and intraperitoneal (i.p.) injection, n = 6 per data point