Abstract
Calreticulin (CALR) has anti-tumor effects by increasing dendritic cell maturation and tumor antigen presentation. However, whether CALR affects macrophages and modulates progression of acute respiratory distress syndrome/acute lung injury (ARDS/ALI) remains unknown. In this study, we discovered that CALR protein was highly expressed in the mice with LPS-induced ALI and CALR expression level was positively correlated to the severity of ALI. Commercial anti-CALR antibody (aCALR) can neutralize recombinant CALR (rCALR) and suppress the expression of TNF-alpha and IL-6 in the rCALR-treated macrophages. Blocking CALR activity by intraperitoneal (i.p.) administration of aCALR significantly suppressed ALI, accompanied with lower total cell counts, neutrophil and T cell infiltration in bronchoalveolar lavage (BAL) and lung tissues. The expression of CXCL15, IL-6, IL-1beta, TNF-alpha, and CALR were significantly reduced, in association with more polarization of Siglec F+CD206+M2 subtype macrophages in the aCALR-treated mice. Pre-depletion of circulating monocytes did not abolish the aCALR-mediated suppression of ALI. Further analysis in bone marrow-derived macrophages (BMDMs) showed that aCALR suppressed the expression of CD80, IL-6, IL-1beta, IL-18, NLRP3, and p-p38 MAPK; but enhanced the expression of CD206 and IL-10. In addition, we observed more expression and phosphorylation of STAT6 in the aCALR-treated BMDM. Lack of STAT6 resulted in comparable and slightly higher expression of CALR, TNF-alpha and IL-6 in the aCALR-treated STAT6-/- BMDMs than the untreated cells. Therefore, we conclude that CALR is a novel biomarker in the evaluation of ALI. Blocking CALR activity by aCALR effectively suppressed ALI independent of circulating monocytes. Siglec F+CD206+M2 subtype macrophages and p38 MAPK/STAT6 signaling pathway played important role in the immune regulation of aCALR. Blocking CALR activity is a promising therapeutic approach in the treatment of ARDS/ALI.
Highlights
Acute respiratory distress syndrome (ARDS) is a severe inflammatory lung disease with severe hypoxemia, diffuse bilateral pulmonary infiltrates with mortality rate up to 36–52% [1, 2]
The CALR was located in cytoplasm at low level, but mostly located onto cell membrane after cells were activated by LPS (Figure 1A, lower panel)
The results revealed that co-treatment with anti-CALR antibody (aCALR) can effectively suppress the expression of p-p38 MAPK and NLRP3, as shown by moderately reduced ratio of p-p38/p38, p-p38/GAPDH, and NLRP3/GAPDH in WT bone marrow-derived macrophages (BMDMs)
Summary
Acute respiratory distress syndrome (ARDS) is a severe inflammatory lung disease with severe hypoxemia, diffuse bilateral pulmonary infiltrates with mortality rate up to 36–52% [1, 2]. Macrophages are activated and release a large amount of pro-inflammatory cytokines and mediators that effectively attract neutrophils and T lymphocytes into the lung tissues, contributing to the pathogenesis of ARDS and murine acute lung injury (ALI), an ARDS mouse model. Modulation of macrophage activation and polarization is an effective therapeutic approach in the treatment of ARDS/ALI. Our previous research revealed that antioxidant resveratrol can attenuate ALI through polarization of alternatively activated macrophage or M2 cells [8]. Because STAT3 and SOCS3 forms a negative feedback loop in the lung inflammation of ALI, loss of SOCS3 can activate STAT3, induces more lung inflammation, and resulting in the resistant of ALI mouse model to resveratrol therapy [9, 10]
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