Sphingosine Kinase 2 Expression in CD11b+ Macrophages Negatively Regulates cGASSTING Activity and Resolves Lung Injury

Abstract

Lung macrophages (MΦ) are important in triggering the immune response and thereby resolve acute lung injury (ALI) by sensing pathogens through pattern recognizing receptors including TLRs. However, the role of macrophages in resolving inflammation and lung injury is less well understood. Herein, we used transgenic mice expressing human diphtheria toxin receptor (DTR) under the control of Cd11b‐promoter (DTR) mice to assess their specific role in regulating lung inflammatory injury. Administration of diphtheria toxin (DT) in DTR mice selectively depletes CD11b cells by causing their apoptosis. We first assessed alteration in lung macrophages (gated using CD11b+, CD11c+, Gr1−, CD64+) using LyzM‐GFP mice (green myeloid cells) following lung injury. Flow cytometry analysis showed a significant decline in CD11C+/CD11b+ MΦ at the peak of lung injury at 4h while these cells increased in proportion during resolution at 24h and 48h respectively. Next, we administered DT after LPS‐induced lung injury which only depleted CD11b+ MΦ by 50% in DTR mice at 24 h without inducing neutropenia. Loss of Cd11b+ MΦ did not alter the magnitude of LPS‐induced injury in DTR mice but markedly impaired resolution of injury. Survival curve of cecal ligation puncture and high dose of LPS in DTR mice also indicated that CD11b+ MΦ are critical during resolution of lung injury from sepsis. The recognition of endogenous DNA released during cell death is a major mechanism to activate innate immune responses. Cyclic GMP‐AMP (cGAMP) synthase (cGAS) is a critical sensor of cytosolic dsDNA and stimulator of interferon gene (STING) in the endoplasmic reticulum which leads to induction of IFNb and downstream inflammatory signaling. Intriguingly, we found that loss of CD11b MΦ augmented IFN‐β production by LPS in addition to canonical NF‐kB mediated inflammatory signaling in DTR mice. Further, we also observed that LPS administration in DTR mice induced TBK1 phosphorylation indicating STING activation. Sphingosine‐1‐phosphate (S1P) generated by the action of sphingosine kinases (SPHK1 and SPHK2) plays a critical role in dampening inflammation and thereby lung injury. Therefore, we addressed if altered SPHK1 or SPHK2 expression in CD11b MΦ was responsible for maladaptive inflammatory response in DTR mice. Transplantation of SPHK2 or SPHK1 bone marrow into lethally irradiated wild type (WT) mice indicated that SPHK2 expression in hematopoietic cells play a critical role in resolving lung injury. Thus, we adoptively transferred WT or SPHK2−/−CD11b+ bone marrow cells in DTR mice following LPS and DT challenge. We observed that WT‐CD11b cells resolved inflammatory injury in DTR mice while CD11b cells lacking SPHK2 could not. Moreover, SPHK2 null bone marrow derived cells showed increased IFN‐β production by LPS in association with increase in TBK1‐phosphorylation. Thus, these data demonstrated that following lung injury SPHK2 is activated in CD11b MΦ to suppress STING activity and thereby dampens IFN‐β generation leading to resolution of ALI.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.