B7H3 ameliorates LPS-induced acute lung injury via attenuation of neutrophil migration and infiltration

Abstract

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are characterized by an excessive inflammatory response within the lungs and severely impaired gas exchange resulting from alveolar-capillary barrier disruption and pulmonary edema. The costimulatory protein B7H3 functions as both a costimulator and coinhibitor to regulate the adaptive and innate immune response, thus participating in the development of microbial sepsis and pneumococcal meningitis. However, it is unclear whether B7H3 exerts a beneficial or detrimental role during ALI. In the present study we examined the impact of B7H3 on pulmonary inflammatory response, polymorphonuclear neutrophil (PMN) influx, and lung tissue damage in a murine model of lipopolysaccharide (LPS)-induced direct ALI. Treatment with B7H3 protected mice against LPS-induced ALI, with significantly attenuated pulmonary PMN infiltration, decreased lung myeloperoxidase (MPO) activity, reduced bronchoalveolar lavage fluid (BALF) protein content, and ameliorated lung pathological changes. In addition, B7H3 significantly diminished LPS-stimulated PMN chemoattractant CXCL2 production by inhibiting NF-κB p65 phosphorylation, and substantially attenuated LPS-induced PMN chemotaxis and transendothelial migration by down-regulating CXCR2 and Mac-1 expression. These results demonstrate that B7H3 substantially ameliorates LPS-induced ALI and this protection afforded by B7H3 is predominantly associated with its inhibitory effect on pulmonary PMN migration and infiltration.

Figure 1. B7H3 attenuates LPS-induced PMN infiltration and lung tissue damage.

Male Balb/c mice were randomized into PBS, B7H3, LPS, and LPS+B7H3 groups (n = 12 per group). BALF leukocyte (A) and PMN (B) counts, lung MPO activity (C), and BALF protein concentrations (D) were assessed 24 hrs after LPS inhalation as described in the Methods. Intranasal instillation of LPS (20 μg/mouse) resulted in significantly increased BALF leukocyte numbers, PMN counts, and protein concentrations as well as lung MPO activity, which were markedly attenuated by B7H3 treatment. Data are mean ± SD. *p < 0.05, **p < 0.01 versus PBS group; ^≠p < 0.05, ^≠≠p < 0.01 versus LPS group.

Figure 2. B7H3 ameliorates LPS-induced lung pathological alterations.

Male Balb/c mice were randomized into PBS, B7H3, LPS, and LPS+B7H3 groups (n = 12 per group). Lung histological changes were assessed 24 hrs after LPS inhalation as described in the Methods. Representative images of haematoxylin and eosin stained sections of lung tissues for PBS (A), B7H3 (B), LPS (C), and LPS+B7H3 (D) are shown, indicating that treatment with B7H3 strongly ameliorates LPS-induced lung edema, haemorrhage, alveolar collapse, and PMN infiltration.

Figure 3. B7-H3 down-regulates LPS-stimulated CXCL2, but not TNF-α, IL-1β, and IL-6, expression and release.

Male Balb/c mice were randomized into PBS, B7H3, LPS, and LPS+B7H3 groups (n = 12 per group). Lung mRNA (A) and protein (B) expression and BALF levels (C) of TNF-α, IL-1β, IL-6, and CXCL2 were measured 24 hrs after LPS inhalation as described in the Methods. Remarkably, treatment with B7H3 down-regulated LPS-stimulated chemokine CXCL2 expression in the lung and attenuated its release into the BALF. Data are mean ± SD. *p < 0.05, **p < 0.01 versus PBS group; ^≠p < 0.05, ^≠≠p < 0.01 versus LPS group.

Figure 4. The effect of B7-H3 on PMN apoptosis and ROS production.

(A,B) PMN apoptosis in BALF collected 24 hrs after LPS inhalation (n = 12 per group) or PMN apoptosis in vitro at indicated time points after PMNs treated with PBS, B7H3, LPS, and LPS+B7H3 (n = 8 per group) were assessed as described in the Methods. Data are mean ± SD. (C,D) ROS production was determined after PMNs treated with LPS and LPS+B7H3 or PMA and PMA+B7H3 as described in the Methods. B7H3 had no effect on PMN apoptosis both in vitro and in vivo, but significantly diminished LPS-induced ROS generation in PMNs. Data are mean ± SD of seven to eight independent experiments and each experiment was conducted in duplicate. *p < 0.05, **p < 0.01 versus PBS group; ^≠p < 0.05, ^≠≠p < 0.01 versus LPS group.

Figure 5. B7H3 attenuates LPS-induced PMN chemotaxis and transendothelial migration by down-regulating CXCR2 and Mac-1 expression.

PMN chemotaxis toward CXCL2 (A) and migration across the MPEC monolayer (D) were assessed as described in the Methods. Surface expression of CXCR2 (B,C) and Mac-1 (E,F) on PMNs was detected by FACScan analysis. The addition of B7H3 significantly attenuated LPS-induced PMN chemotaxis and transendothelial migration toward the chemoattractants CXCL2 and fMLP, which are closely associated with the inhibitory effect of B7H3 on LPS-induced up-regulation of CXCR2 and Mac-1 expression. Data are mean ± SD of six to eight independent experiments and each experiment was conducted in duplicate. *p < 0.05, **p < 0.01 versus PBS group; ^≠p < 0.05 versus LPS group.

Figure 6. B7H3 inhibits LPS-stimulated MAMϕ CXCL2 expression by attenuating NF-κB p65 phosphorylation.

MAMϕs were incubated with PBS, B7H3, LPS, and LPS+B7H3 for various time periods. CXCL2 mRNA (A) and protein (B) expression was assessed as described in the Methods. Intracellular expression of phophorylated NF-κB p65 (C) and MAPK p38 (D) was detected by FACScan analysis. B7H3 strongly down-regulated LPS-stimulated CXCL2 expression in MAMϕs at both mRNA and protein levels, and this correlates with the attenuation of LPS-induced phosphorylation of NF-κB p65, but not MAPK p38, by B7H3. Data are mean ± SD of six to eight independent experiments and each experiment was conducted in duplicate. **p < 0.01 versus PBS group; ^≠p < 0.05, ^≠≠p < 0.01 versus LPS group.