Effects of STING stimulation on macrophages: STING agonists polarize into “classically” or “alternatively” activated macrophages?

ABSTRACT

Stimulator of interferon genes (STING) was defined as an important molecule for promoting antitumor immunity through mediating type I interferon (IFN) production by sensing its ligands such as cyclic GMP-AMP (cGAMP). Our recent study indeed demonstrated that intratumoral injection of cGAMP showed effective antitumor responses via accumulating activated macrophages in the tumor microenvironment in a STING-dependent manner. Because the antitumor effect of cGAMP was abrogated when macrophages were depleted, the existence of the activated macrophages in the tumor site would be important for effective antitumor immune responses. Macrophages show phenotypic diversity and plasticity and are categorized into several groups by stimulation factors, e.g. IFN-γ and IL-4 for M1 and M2 macrophages, respectively. However, the impact of STING stimulation on the macrophage activation status remains to be evaluated. Here we summarize the complex polarized status of macrophages and the signaling cascade triggered by STING stimulation and also discuss the impact of STING signaling on the macrophage activation status for future directions.

Introduction

STING, which is a four-transmembrane protein localized in the endoplasmic reticulum (ER) and mitochondria-associated ER membrane, plays an important role of an adaptor in inducing type I IFNs following sensing of cyclic dinucleotides (CDN), i.e., bacteria-derived c-di-GMP and c-di-AMP and cyclic GMP-AMP (cGAMP) generated from intracellularly located viral or host DNAs including necrotic tumor cells by cGAMP synthase (cGAS).^1,2

Macrophages are usually divided into two categories: (1) M1 or classically and (2) M2 or alternatively activated macrophages due to the type of stimulant factors.³ M1 macrophages are generally stimulated with IFN-γ and/or LPS, which activate JAK-STAT1 signaling pathway to provide an anti-tumor phenotype with production of nitric oxide (NO) and pro-inflammatory cytokines such as TNF-α, IL-6, and IL-12. Whereas, M2 macrophages are involved in tissue repair and immunosuppressive functions and polarized by IL-4, IL-13, and other factors.^3–5 With regard to the widespread use of terms and definitions of macrophage as above, a common framework for nomenclature of macrophage status is recently proposed.⁶

STING signaling pathway and its contributions to antitumor immunity

STING activates several transcription factors such as NF-κB, IRF3, IRF7, and STAT6 to enhance inflammatory responses after receiving STING ligands. NF-κB and IRF3 induce type I IFNs to exert a potent antiviral immunity.¹ IRF7 is reported to contribute to the efficacy of DNA vaccines by inducing potent cytokine production in a STING-dependent manner.⁷ Whereas, phosphorylated STAT6 by STING activation results in production of CCL2 and CCL20 to enhance antiviral innate immunity.⁸ STING was initially identified as a key molecule for protecting hosts from bacterial and virus infections.^9,10 More recently, a role of STING in the field of antitumor immunity has been assessed and discussed since it was demonstrated that STING contributes to antitumor immunity as a spontaneous trigger of type I IFNs in the tumor microenvironment and a therapeutic target for enhancing cancer immunotherapy.^11–14 Although the main producer of STING-triggered type I IFNs in the tumor microenvironment still remains controversial, CD11c⁺ dendritic cells, CD11b⁺ myeloid cells, and endothelial cells are proposed as a sourse of type I IFNs via STING activation.^11,15,16 Based on these findings in several mouse models, STING ligands are regarded as one of the promising immune adjuvants for promoting antitumor immune responses.^17,18 Especially, intratumoral injections of CDNs effectively enhance production of type I IFNs and migration of CD8⁺ T-cells, and thereby suppressing tumor growth although there may be some difficulties in direct injection into the tumor site.¹⁷ Furthermore, we found that CD11b⁺Ly6C^high macrophages are recruited in the tumor site in a STING-dependent manner after intrartumoral injection of cGAMP and show inflammatory phenotypes, i.e. induction of TNF-α, type I IFNs, and T-cell-attracting chemokines. Because depletion of the macrophages by using clodronate liposome abrogated the antitumor effect of STING activation, it is suggested that STING-triggered macrophages would contribute to antitumor immunity.¹⁹ It, however, remains to be determined which signaling cascade is activated in the macrophages stimulated with STING agonists like cGAMP.

Alternatively activated and polarized macrophages in STAT6 signaling pathway

M2 macrophages are further classified into three forms based on the activation and polarization status, which are referred to as M2a, M2b, and M2c macrophages.²⁰ M2a macrophages induced by IL-4 or IL-13 are so-called “alternatively activated macrophages” and produce IL-10 and IL-1 receptor antagonist. M2b macrophages produce high amounts of IL-10 and low levels of IL-12 and are induced by combined immune complexes and TLR/IL-1R ligands. M2c macrophages are polarized by IL-10, glucocorticoid, or secosteroid hormones and characterized by high production of IL-10 and TGF-β with deactivated status.²¹

M2-polarized macrophages in the tumor site, referred to as tumor-associated macrophages (TAMs),^22,23 suppress antitumor activity of immune cells and accelerate tumor progression via producing TGF-β and IL-10 and promoting angiogenesis, respectively.²⁴ Stimulation with IL-4 and IL-13 induces M2 polarization via STAT6 activation and translocation, enhancing expression of several transcription factors such as peroxisome proliferator activated receptors (PPARs), PPARδ and PPARγ and PPARγ-coactivator-1β (PGC-1β). PPARδ is required for expression of arginase-1, which is one of the critical molecules in suppressing antitumor immunity²⁵ and is also reported to be induced by IL-6/STAT3 signaling cascade.²⁶ When arginine is starved in the tumor microenvironment by TAMs, NO production by M1 macrophages is reduced and MHC class II expression is down-regulated in antigen-presenting cells including macrophages, leading to suppress the immune responses to tumors.^26,27 In addition to increasing arginase-1 expression, PPARδ plays a role in maintaining expression of M2 macrophage-related molecules such as resistin-like α, mannose receptor, chitinase 3-like 3, and programmed cell death 1 ligand 2 in response to IL-4/STAT6 signals.²⁵ In contrast, PPARγ is involved in transcriptional regulation of lipolysis, fatty acid uptake, and β-oxidation of fatty acids, which are associated with maturation of alternatively activated status in macrophages.²⁸ Furthermore, PGC-1β enhances maturation of anti-inflammatory alternatively activated macrophages as a coactivator of STAT6.²⁹ STAT6 signaling cascade plays a critical role in polarizing macrophages into M2 phenotype via cooperating with the relevant transcription factors which STAT6 promoted itself. Thus, because STAT6 is phosphorylated by STING activation, STING ligands would also polarize macrophages into M2 phenotype as well as IL-4 and IL-13.

Conclusions: Effects of STING signaling on macrophage phenotype

STING activates both IRF3 and IRF7 and STAT6 for inducing type I IFNs and promoting inflammatory responses, respectively after receiving CDNs. It is therefore expected that STING-triggered macrophages show “classically” and “alternatively” activated status. This raises the question of how much STING signaling polarizes macrophages into M1 and/or M2 phenotypes. In our previous study, STING-triggered tumor-infiltrating macrophages showed more M1-like phenotype compared with TAMs.¹⁹ However, TAMs are polarized M2 phenotype and a number of factors will be involved in the activation status of macrophages in the tumor microenvironment. Simply designed experiments in vitro, therefore, are required to answer the above question, helping us to understand how STING activation impacts on the macrophages in tumor-bearing host when STING ligands are used as an immune adjuvant in antitumor immunotherapy.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.