M1 and M2 Macrophages Play Different Roles in the Pathogenesis of Acute Graft-Versus-Host Disease Post-Allotransplant By Modulating Immune Microenvironment

Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Macrophages (MΦs) are an important immune population that are essential for disease pathogenesis. MΦs are now commonly classified as either M1, which produce pro-inflammatory cytokines, or M2, which produce anti-inflammatory cytokines. Our recent study reported that patients who received an allograft with a higher M1/M2 ratio exhibited a higher incidence of grade 2–4 aGVHD(BMT, 2019). Moreover, an aberrant M1/M2 polarization was found in the colon of aGVHD mice, and regulating the polarization of MΦs cultured from aGVHD patients towards M2 phenotype modulated T cells favoring a type 2 response in vitro(Sci China Life Sci, 2020). However, the primary subsets and function of MΦs in aGVHD patients, and the precise roles of different MΦ subsets in the development of aGVHD remain to be elucidated.
Aims: To determine the subsets and function of MΦs in primary peripheral blood(PB) of aGVHD patients. Moreover, to investigate whether M1 and M2 had different effects on the development of aGVHD, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT.

Methods: In this prospective case-control study, a total of 20 patients with aGVHD and 20 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. MΦ subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. M1 and M2 were identified as CD14⁺CCR2⁺CD68⁺ and CD14⁺CX3CR1⁺CD163⁺, respectively. In order to determine the function of MΦs in patients with aGVHD and non-aGVHD, the phagocytosis was analyzed using a DiI-AcLDL assay. The protein expressions for the costimulatory molecules and the cytokine production of MΦs were measured by flow cytometry. To further investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of MΦs. Subsequently, to explore the role of different subsets of MΦs in the development of aGVHD, M1 and M2 were infused into aGVHD mice, respectively. Mice were monitored for survival, weight, and aGVHD score. Histological scores of tissues from aGVHD target organs (liver, intestine, spleen and skin) were evaluated by HE staining.

Results: When compared with non-aGVHD patients, MΦs in primary PB of aGVHD patients were polarized towards pro-inflammatory M1, characterized by an elevated proportion of M1 and a reduced proportion of M2. Furthermore, MΦs isolated from aGVHD patients exhibited lower phagocytic function, higher expression of TNF-α and IL-6 and higher expression of costimulatory molecules CD80 and CD86. Consistent with the increased activated MΦs from aGVHD patients, the mRNA levels of genes involved in the pro-inflammatory M1 polarization, antigen presenting and promoting the activation of T cells pathway were substantially elevated in MΦs of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vivo infusion with pro-inflammatory M1 aggravated aGVHD response by modulating immune microenvironment of spleen and liver in mice, characterized by enhanced effector function of T cell, including elevated percentages of Tc1, Th1 and Th17 as well as increased proliferation of T cells from spleen and liver. By contrast, infusion with anti-inflammatory M2 alleviated aGVHD through down-regulating the activity of T cells derived from aGVHD mice, characterized by decreased proliferation and decreased percentages of Tc1, Th1 and Th17.

Summary/Conclusion: The current study demonstrated that the primary MΦs in aGVHD patients preferentially polarize into pro-inflammatory M1. Moreover, M1 aggravate aGVHD whereas M2 ameliorate aGVHD by differently modulating immune microenvironment. Although further validation is required, modulating the polarization state of MΦs promises to be a novel therapeutic target for aGVHD patients after allo-HSCT.