Selective CB2 Receptor Agonist, HU-308, Reduces Systemic Inflammation in Endotoxin Model of Pneumonia-Induced Acute Lung Injury

Abstract

Acute respiratory distress syndrome (ARDS) and sepsis are risk factors contributing to mortality in patients with pneumonia. In ARDS, also termed acute lung injury (ALI), pulmonary immune responses lead to excessive pro-inflammatory cytokine release and aberrant alveolar neutrophil infiltration. Systemic spread of cytokines is associated with systemic complications including sepsis, multi-organ failure, and death. Thus, dampening pro-inflammatory cytokine release is a viable strategy to improve outcome. Activation of cannabinoid type II receptor (CB₂) has been shown to reduce cytokine release in various in vivo and in vitro studies. Herein, we investigated the effect of HU-308, a specific CB₂ agonist, on systemic and pulmonary inflammation in a model of pneumonia-induced ALI. C57Bl/6 mice received intranasal endotoxin or saline, followed by intravenous HU-308, dexamethasone, or vehicle. ALI was scored by histology and plasma levels of select inflammatory mediators were assessed by Luminex assay. Intravital microscopy (IVM) was performed to assess leukocyte adhesion and capillary perfusion in intestinal and pulmonary microcirculation. HU-308 and dexamethasone attenuated LPS-induced cytokine release and intestinal microcirculatory impairment. HU-308 modestly reduced ALI score, while dexamethasone abolished it. These results suggest administration of HU-308 can reduce systemic inflammation without suppressing pulmonary immune response in pneumonia-induced ALI and systemic inflammation.

Keywords: ARDS; acute lung injury; cannabinoid type II receptor (CB2); cytokines; inflammation; intravital microscopy; microcirculation.

Figure 1

Intranasal administration of LPS from P. aeruginosa induces ALI at 4, 6 h post-induction. (A) Histopathological lung scores. (B) Representative fields of view (FOV) from naïve group. (C) Representative FOV from control (CON) group at 2, 4, and 6 h timepoints. (D) Representative FOV from LPS group at 2, 4, and 6 h timepoints. Whiskers show minimum and maximum values, boxes represent 25–75% data ranges, and horizontal lines within boxes represent medians. Statistically significant differences were determined via Mann–Whitney tests between LPS and control groups at each timepoint and are depicted as ** p ≤ 0.01.

Figure 2

Systemic upregulation of cytokines following ALI. Plasma levels of (A) IL-6, (B) IL-10, (C) CXCL2, and (D) TNFα were elevated at 4 and 6 h post-induction. Whiskers show minimum and maximum values, boxes represent 25–75% data ranges, and horizontal lines within boxes represent medians. Statistically significant differences were determined via Mann–Whitney tests between LPS and control groups at each timepoint and are depicted as * p ≤ 0.05, ** p ≤ 0.01.

Figure 3

Impact of treatment on ALI-induced systemic cytokine release. Plasma levels of (A) IL-6, (B) IL-10, (C) CXCL2, (D) TNFα, (E) sICAM-1, and (F) CXCL1 were reduced following administration of HU-308 or DEX. Data are expressed as mean ± SD. Statistically significant differences were determined via one-way ANOVA with Dunnett’s multiple comparisons test and are depicted as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Figure 4

Impact of LPS and treatment on intestinal leukocyte activation. Representative images of ileal submucosal (A) V1 and (B) V3 venules in con + veh mice, and (C,D) lps + veh mice. Red arrows denote adherent leukocytes. Analysis of leukocyte adhesion in (E) V1 and (F) V3 venules. Analysis of leukocyte rolling in (G) V1 and (H) V3 venules. Data are expressed as mean ± SD. Statistically significant differences were determined via one-way ANOVA with Dunnett’s multiple comparisons test and are depicted as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Figure 5

Impact of LPS and treatment on intestinal capillary perfusion. Representative images of ileal (A) muscularis and (B) mucosal capillaries in con + veh mice, and (C,D) LPS + veh mice. Analysis of FCD in (E) muscularis and (F) mucosal layers. Data are expressed as mean ± SD. Statistically significant differences were determined via one-way ANOVA with Dunnett’s multiple comparisons test and are depicted as * p ≤ 0.05, *** p ≤ 0.001.

Figure 6

Impact of treatment on LPS-induced lung histopathology. Representative FOV of (A) con + veh and LPS + (B) veh, (C) HU-308, and (D) DEX mice. (E) Histopathological lung scores. Data are expressed as mean ± SD. Statistically significant differences were determined via one-way ANOVA with Dunn’s multiple comparisons test and are depicted as * p ≤ 0.05, **** p ≤ 0.0001.

Figure 7

Impact of LPS and treatment on leukocyte activation in pulmonary venules and arterioles. Representative images of pulmonary venules in (A) con + veh mice and (B) LPS + veh mice. Analysis of leukocyte (C) rolling and (D) adhesion in pulmonary venules. Representative images of pulmonary arterioles in (E) con + veh mice and (F) LPS + veh mice. Analysis of leukocyte (G) rolling and (H) adhesion in pulmonary arterioles. Red arrows denote adherent leukocytes. Data are expressed as mean ± SD. Statistically significant differences were determined via one-way ANOVA with Dunnett’s multiple comparisons test and are depicted as * p ≤ 0.05, ** p ≤ 0.01.

Figure 8

Impact of LPS-induced ALI and treatment on pulmonary capillaries. Representative images of leukocyte adhesion in capillary ROI in (A) con + veh mice and (B) LPS + veh mice. (C) Analysis of leukocyte adhesion in capillary ROI. (D) Analysis of FCD in capillary ROI. Representative images of FCD in capillary ROI in (E) con + veh mice and (F) LPS + veh mice. Data are expressed as mean ± SD. Statistically significant differences were determined via one-way ANOVA with Dunnett’s multiple comparisons test and are depicted as ** p ≤ 0.01.