Caspase-1 Regulates Monocyte-to-macrophage Differentiation And The Involved Mechanisms

Macrophage is an important part of the mononuclear phagocyte system (MPS),which plays critical roles in both innate and adaptive immunity. Macrophages are derived from monocytes, in situ proliferation of macrophages and/or primitive yolk sac macrophages. Monocytes-derived macrophages are the main source of tissue macrophages in vivo. In steady state or inflammatory state, circulating monocytes are able to differentiate into macrophages in response to microenvironmental factors.Dysregulation of monocyte-to-macrophage differentiation is tightly related to various diseases, such as autoimmune and inflammatory diseases, metabolic diseases and tumors. Thus, the monocyte-to-macrophage differentiation process and the involved molecular mechanisms remain a subject of extensive investigation.Caspase-1 is a member of the caspase family of proteins, which is relevant to inflammation and essential for regulating macrophage function. For example,caspase-1-/- macrophages show defective release of IL-1? and IL-18. Caspase-1 has been defined as an essential regulator of macrophage M1/M2 polarization. Caspase-1 is also implicated in the control of cell differentiation. Inflammasome-mediated caspase-1 activation controls adipocyte and Th17 cell differentiation via IL-1? signaling. However, the potential roles of caspase-1 in regulating monocyte-to-macrophage differentiation remain largely unknown.Considering the broad biological functions of caspase-1 and its central role in monocytes and macrophages, we speculate that caspase-1 may play important roles in modulating macrophage differentiation. In this study, monocyte-to-macrophage differentiation was studied using in vitro models with THP-1 and U937 cells and peripheral blood mononuclear cells (PBMCs). We investigated the regulatory effect of caspase-1 and the mechanisms thereof, and the main results were shown as follows:1. We employed PMA to successfully induce macrophage differentiation of THP-1 cells and U937. The monocyte-to-macrophage differentiation process could be divided into two stages (early stages and late stages) by analysis of the cell morphology, cell surface makers and phagocytic ability.2. Caspase-1 was constitutively expressed in THP-1 and U937 cells. However, the activity of caspase-1 was gradually increased along with the differentiation process.Activation of caspase-1 was also demonstrated in M-CSF- or GM-CSF-induced macrophage differentiation of PBMCs. Further analysis of the mRNA levels of inflammasome components showed that activation of caspase-1 was associated with inflammasome assembly.3. The caspase-1 specific inhibitor WEHD could inhibit PMA induced expression of CD11b and CD14 in a dose-dependent manner,which occurred mainly at the late stages. The phagocytic ability was also impaired by WEHD treatment. WEHD could also obviously suppress M-CSF induced macrophage differentiation of PBMCs.4. Caspase-1 knockdown caused inhibition of caspase-1 activity, and could significantly inhibit the expression of CD11b and CD 14 and the phagocytic ability.Caspase-1 was downregulated in acute monocytic leukemia (AML-M5b), indicating a close association between caspase-1 downregulation and accumulation of monocytes in AML-M5b.5. Based on these findings,we tried to explore the molecular mechanisms involved in caspase-1-mediated monocyte-to-macrophage differentiation:(1) PPARy was upregulated during the early stages,while markedly downregulated at the late stages. Caspase-1 inhibitor or knockdown could block the downreglation of PPARy. However, the mRNA level was not affected. We further demonstrated the downregulation of PPARy expression by caspase-1 activation by reconstitution of NLRP3 inflammasome in HEK293T cells, which was not due to caspase-1-mediated cleavage.(2) During the early stages, PPAR? expression and transcriptional activity were increased, which induced cell cycle arrest via modulation of cyclin D1 and p21.During the late stages, PPARy ligand troglitazone could inhibit caspase-1 activity and promoted accumulation of PPARy, which resulted in inhibition of NF-?B activation and monocyte-to-macrophage differentiation program.(3) We also studied the potential involvement of IL-1? and IL-18 in monocyte-to-macrophage differentiation. The results from washing away, adding exogenous cytokines or their neutralizing antibodies showed that the differentiation program was independent of IL-1? and IL-18. IL-1? and IL-33 were not released and not implicated in the regulation of differentiation program.6. By use of orthotopic breast cancer model, we found that caspase-1 specific inhibitor YVAD could facilitate tumor growth via increased accumulation of MDSCs,indicating that caspase-1 activation could regulate MDSCs differentiation. However,the TAMs were not affected by YVAD treatment.Our findings have defined caspase-1 as an important regulator of monocyte-to-macrophage differentiation. Caspase-1 could promote the differentiation program by fine-tuning the expression of PPAR? at the late stages. PPARy played distinct roles in macrophage differentiation at the early and late stages. The tight link between caspase-1 and AML-M5, and the role of caspase-1 activation in MDSCs differentiation in tumor microenvironment, revealed that caspase-1 might be a useful target for treating leukemia and other malignancies.