Endogenous Uteroglobin as Intrinsic Anti-inflammatory Signal Modulates Monocyte and Macrophage Subsets Distribution Upon Sepsis Induced Lung Injury

Abstract

Sepsis is a serious clinical condition which can cause life-threatening organ dysfunction, and has limited therapeutic options. The paradigm of limiting excessive inflammation and promoting anti-inflammatory responses is a simplified concept. Yet, the absence of intrinsic anti-inflammatory signaling at the early stage of an infection can lead to an exaggerated activation of immune cells, including monocytes and macrophages. There is emerging evidence that endogenous molecules control those mechanisms. Here we aimed to identify and describe the dynamic changes in monocyte and macrophage subsets and lung damage in CL57BL/6N mice undergoing blunt chest trauma with subsequent cecal ligation and puncture. We showed that early an increase in systemic and activated Ly6C⁺CD11b⁺CD45⁺Ly6G^- monocytes was paralleled by their increased emigration into lungs. The ratio of pro-inflammatory Ly6C^highCD11b⁺CD45⁺Ly6G^- to patrolling Ly6C^lowCD11b⁺CD45⁺Ly6G^- monocytes significantly increased in blood, lungs and bronchoalveolar lavage fluid (BALF) suggesting an early transition to inflammatory phenotypes during early sepsis development. Similar to monocytes, the level of pro-inflammatory Ly6C^highCD45⁺F4/80⁺ macrophages increased in lungs and BALF, while tissue repairing Ly6C^lowCD45⁺F4/80⁺ macrophages declined in BALF. Levels of inflammatory mediators TNF-α and MCP-1 in blood and RAGE in lungs and BALF were elevated, and besides their boosting of inflammation via the recruitment of cells, they may promote monocyte and macrophage polarization, respectively, toward the pro-inflammatory phenotype. Neutralization of uteroglobin increased pro-inflammatory cytokine levels, activation of inflammatory phenotypes and their recruitment to lungs; concurrent with increased pulmonary damage in septic mice. In in vitro experiments, the influence of uteroglobin on monocyte functions including migratory behavior, TGF-β1 expression, cytotoxicity and viability were proven. These results highlight an important role of endogenous uteroglobin as intrinsic anti-inflammatory signal upon sepsis-induced early lung injury, which modules the early monocyte/macrophages driven inflammation.

Short summary: Blunt chest injury is the third largest cause of death following major trauma, and ongoing excessive pro-inflammatory immune response entails high risk for the development of secondary complications, such as sepsis, with limited therapeutic options. In murine double hit trauma consisting of thoracic trauma and subsequent cecal ligation and puncture, we investigated the cytokine profile, pulmonary epithelial integrity and phenotypic shift of patrolling Ly6C^lowCD11b⁺CD45⁺Ly6G^- monocytes and Ly6C^lowCD45⁺F4/80⁺ macrophages to pro-inflammatory Ly6C^highCD11b⁺CD45⁺Ly6G^- monocytes and Ly6C^highCD45⁺F4/80⁺ cells in blood, lungs and bronchoalveolar lavage fluid (BALF). Pro-inflammatory mediators and phenotypes were elevated and uteroglobin neutralization led to further increase. Enhanced total protein levels in BALF suggests leakage of respiratory epithelium. In vitro, uteroglobin inhibited the migratory capacity of monocytes and the TGF-β1 expression without affecting the viability. These results highlight an important role of endogenous uteroglobin as an intrinsic anti-inflammatory signal upon sepsis-induced early lung injury, which modulates the early monocyte/macrophages driven inflammation.

Keywords: CC16; CLP; acute lung injury; chest injury; macrophages; monocytes; sepsis; uteroglobin.

Figure 1

Impact of CC16 neutralization on expression levels of pro-inflammatory mediators (A–D) and the total pulmonary protein amount (E) following thoracic trauma (TxT) with cecal ligation and puncture (CLP). Plasma levels of TNF (A) and MCP-1 (B), and RAGE protein levels in lungs (C) and BALF (D) were measured. Total protein amount was determined in BALF. Data are represented as box-whisker plot and min to max, *p < 0.05 vs. control.

Figure 2

Representative gating strategy for the flow cytometric analyses and evaluation of different monocyte subsets in whole blood (A,B) and macrophage subsets in lungs (C,D) as dot plot analyses is shown. (A,C) are showing the gating upon staining with isotype control antibodies, while (B,D) show results from the staining with specific antibodies as described in the material and method section.

Figure 3

Impact of CC16 neutralization on distribution of monocyte subsets following thoracic trauma (TxT) with cecal ligation and puncture (CLP). Activated monocyte count (Ly6C⁺) as well the pro-inflammatory (Ly6C^hi) and patrolling (Ly6C^lo) subsets and the ratio between them were assessed in blood (A–D), lungs (E–H) and BALF (I–L) by flow cytometry. Data are represented as box-whisker plot and min to max, *p < 0.05 vs. control.

Figure 4

Impact of CC16 neutralization on distribution of macrophage subsets following thoracic trauma (TxT) with cecal ligation and puncture (CLP). Amount of macrophages (Ly6C⁺F4/80⁺) as well as of the pro-inflammatory (Ly6C^hi) and patrolling (Ly6C^lo) subsets was assessed in blood (A–C), lungs (D–F) and BALF (G–I) by flow cytometry. Data are represented as box-whisker plot and min to max, *p < 0.05 vs. control.

Figure 5

Representative gating strategy for the flow cytometric analysis and evaluation of isolated CD14⁺ monocytes with regard to their TGF-β1 expression as dot plot analysis is shown. (A) is showing representative isotype staining controls, while (B) shows representative gating after staining with specific antibodies. (C–F) show effects of serum samples obtained from healthy volunteers (HV) or trauma patients (TP) and CC16 on CD14⁺ monocytes behavior. CD14⁺ monocytes isolated from HV were incubated with sera obtained from HV or TP. Cell culture medium was applied alone (control) or with/without prior intervention/neutralization with either CC16 antibody (aCC16) or IgG control antibody (IgG) in sera from HV or TP. Migratory capacity toward MCP-1 (C), TGF-β1 expression (D), and the impact of sera on monocyte cytotoxicity (E) and viability (F) were evaluated. Data are represented as box-whisker plot and min to max, *p < 0.05 vs. indicated.