Biology of IL-27 and its Role in the Host Immunity against Mycobacterium Tuberculosis

Abstract

IL-27, a heterodimeric cytokine of IL-12 family, regulates both innate and adaptive immunity largely via Jak-Stat signaling. IL-27 can induce IFN-γ and inflammatory mediators from T lymphocytes and innate immune cells. IL-27 has unique anti-inflammatory properties via both Tr1 cells dependent and independent mechanisms. Here the role and biology of IL-27 in innate and adaptive immunity are summarized, with special interest with immunity against Mycobacterium tuberculosis.

Introduction

Tuberculosis remains a leading cause of death worldwide which afflicts approximately one-third of the world's population and claims a death toll around 1.2 million annually ¹.

Mycobacterium tuberculosis (Mtb) is an unusual facultative intracellular pathogen which multiplies within macrophage ². Macrophages are crucial for the innate and adaptive immune response to Mtb because of their potent antimicrobicidal activities, antigens-presenting abilities, secretion of inflammatory mediators such as the IL-12 family of cytokines ^{3, 4}, and their role in granuloma formation to control the pathogen ^5-7. However, Mtb can persist within granuloma lifelong without clinical symptoms ^{3, 4, 8}. In-depth understanding of the underlying molecular mechanisms can inspire better drug design and treatment of TB.

The interleukin-12 (IL-12) family, including IL-12, IL-23, IL-27 and IL-35, are important for TB pathogenesis and control, which can regulate the Th1 response ^{9, 10}.

Interleukin-27 (IL27) was first discovered in 2002 as a new member of the IL-12 cytokine family ¹¹. It is a heterodimer, comprised of p28 and EBI3 subunits that are structurally similar to the p35 and p40 subunits of IL-12 ¹².

IL-27 is largely secreted from activated antigen-presenting cells (APCs) such as macrophages and Dendritic cells (DCs) ^13-15 (Figure 1). The receptor of IL-27 consists of two subunits: IL-27Rα and gp130. Both subunits are essential ^16-18 for activating Janus Kinase (Jak) and Transcription factor (Stat). Specifically, Stat1and Stat 3 are predominant mediators of IL-27 effects (figure 2) ^{19, 20}. IL-27 can mediate Th1 cells differentiation and proliferation ^{17, 18}. IL-27 has potent antitumor activities by activation of cytotoxic T lymphocytes CD8+ T cells ^21-25, Natural killer (NK) cells ^26-28, NK T cells ²⁸ and anti-angiogenic factors ^{29, 30}. However, IL-27R signaling was demonstrated to be involved in the potent antagonizing of Th1, Th2 and Th17 inflammatory responses ^31-33 and agonist of Tr1 cell response ^{34, 35}. IL-27 can ameliorate symptoms of autoimmune diseases in preclinical studies ^{33, 36, 37}.

Figure 1.

Signaling involved in IL-27 expression. IL-27 is largely produced by Antigen-presenting cells (APCs) such as Dendritic cells (DCs) and macrophages upon stimulation with TLRs agonists, IFN-α, IFN-γ or microbial infections. It consists of two subunits (p28/EBI3) which are expressed independently. TLR2, TLR4 and TLR9-associated MyD88 can induce EBI3 expression through the binding of NF-𝛋B subunits (p50/p65) and PU.1 to the EBI3 promoter. TLR4-associated MyD88 induces p28 expression through binding of NF-𝛋B-c-Rel and AP-1/c-Fos to the p28 promoter, TLR4-associated TRIF induces p28 expression by binding of IRF3 to the p28 promoter. The IFN-α and IFN-γ induces p28 expression through the binding of IRF3 and IRF8 to the p28 promoter, respectively. In addition, IFN-γ-mediated IL-27 instead of IL-27p28 gene expression is positively regulated by the C-Jun N-terminal kinases (JNK), mitogen-activated protein kinases (MAPKs) and the phosphoinositide 3-kinase (PI3K).

Figure 2.

Regulatory role of IL-27 in the immune response against Mycobacterium tuberculosis. (A) IL-27 induced by Mtb infection modulates macrophage response. IL-27 inhibits autophagy by inducing negative regulator factors of autophagy mTOR and Mcl-1 through PI3K/AKT and PI3K, respectively. IL-27 induces IL-10 production through Stat1/Stat3, which in turn blocks phagosomal maturation. It also suppresses TNF-α and IL-12 via Stat3 and both cytokines required for augmenting IFN-γ production by macrophages. In addition, IL-27 targeting NF-kB to inhibit IL-18 mediated IFN-γ production. Suppression of IFN-γ led to down-regulation of V-ATPase and CD63 and Capethsin D (CD) and subsequently suppression of phagosomal acidification. (B) IL-27 induced IL-10-producing type 1 regulatory T cells (Tr1) cell via Stat1/Stat3 and AhR/c-Maf pathway, which in turn suppress Th1 and Th17 cells. IL-27 directly inhibits Th17 cells by inhibiting ROR-γ expression and IL-6 signaling.

The expression level of IL-27 in granuloma suggested a role in tuberculosis ³⁸. The elevated level of IL-27 in tuberculous pleural fluid suggested a potential biomarker for tuberculous pleurisy diagnosis ^{39, 40}. IL-27 level might be manipulated to benefit pathogen ^41-43.

Molecular characteristics of IL-27

IL27 is a heterodimeric cytokine consisting of p28 and Epstein-Barr virus induced gene 3 (EBI3) subunits ^{16, 44}. EBI3 expression is high in human B lymphoblast cell lines transformed in vitro by EBV, activated APCs and placental syncytiotrophoblasts ^{16, 45}. EBI3 is a 34-kDa glycoprotein similar to the p40 subunits of IL-12 ⁴⁶. The EBI3 reversibly binds to IL-12p35-related subunit, namely p28, to form heterodimeric cytokine IL-27^{46, 47}. The connection between p28 and EBI3 is labile and these subunits can be secreted independently ¹⁶. EBI3 is also capable of binding to the IL-12p35 to form IL-35 ⁴⁸. The human p28 gene encodes a 24.5kDa polypeptide ¹⁶. The p28 is structurally similar to an IL-6/IL-12 family, composed of a long chain of four α helix bundle named A-D from the N terminus to the C terminus⁴⁹. The polypeptide loop connecting the p28 C and D α helices contains a stretch of polyglutamic acids (poly-E) unique among helical cytokines and is highly conserved ⁵⁰. The p28 alone can suppress IL-27 mediated Th1 responses ⁵¹ and IL-6 mediated signaling ⁵². These data suggest a regulatory role of p28 in IL-27-mediated immune response.

A polymorphism (-964A>G) in the p28 promoter has been noted in certain diseases such as asthma ⁵³, inflammatory bowel diseases ⁵⁴, chronic obstructive pulmonary disease⁵⁵, and epithelial ovarian cancer ⁵⁶, which seems to be associated with an increase in disease susceptibility. However, the effects of SNPs on the expression of IL-27 remain elusive.

IL-27 is mainly produced by activating APCs such as DCs and macrophages (figure 1). Macrophages-stimulated with TLRs agonists, (polyinosinic: polycytidylic acid (poly (I:C)), Lipopolysaccharide (LPS), or R848 can induce both subunits of IL-27 ¹³. It has been reported that p28 production is completely dependent on the TLR4-associated myeloid differentiation factor 88 (MyD88) mediated pathway and partially dependent on NF-κBc-Rel transcription factor ¹⁴. MyD88 also regulates p28 expression through binding of AP-1/c-Fos to the p28 promoter in both human and mouse macrophages. However, the binding of c-Fos to the p28 promoter can be blocked by overexpression of p38 MAPK ⁵⁷. In addition, TLR4 can induce the expression of p28 subunit through activating the TIR domain-containing adaptor inducing IFN-β (TRIF) and IFN regulatory factor 3 (IRF3) pathways ⁵⁸. TLR2, TLR4, and TLR9-associated MyD88 are required for the induction of EBI3 expression through binding of NF-kB subunits (p50/p65) and PU.1 to the EBI3 promoter ⁵⁹.

IFN-alpha can promote the production of IL-27 by enhancing the expression and binding of IRF-1 to the IFN-stimulated response element (ISRE) in the p28 gene promoter ¹³. Likewise, IFN-γ induced IRF-8 expressions can upregulate p28 gene transcription in synergy with IRF-1 ¹⁵. IFN-γ can selectively induce IL-27 expression via activating the C-Jun N-terminal kinases (JNK), MAPKs and the phosphoinositide-3-kinase (PI3K) signaling in primary human monocyte⁶⁰. Many molecular details of the negative regulation of IL-27 expression remain to be determined.

IL-27 signaling pathways

IL-27R is indispensable for the IL-27 signaling. IL-27R consists of the class I cytokine receptor family (TCCR) (also known as WSX-1) and gp130 ^16-18. IL-27R is expressed in various cell types including naïve T cells, NK cells, activated B cells, monocytes, dendritic cells, activated endothelial cells, and mast cells ⁶¹. IL-27 induced intracellular signaling involves phosphorylation of different isoforms of Jak and Stat, which varies with immune cell types: Jak1, Jak2, Tyrosine kinase 2 (Tyk2), Stat1, 2, 3, 4 and Stat5 in naïve CD4 T cells ⁶², Jak-1, Stat1, Stat-3 and Stat5 in NK cells^{61, 63} , Stat1, Stat3, and NF-κB activation in monocytes ²⁰, and Stat-3 in mast cells ⁶⁴. Interestingly, the effect of the IL-27 can be both pro-inflammatory and anti-inflammatory via same Stat1/Stat3 signaling.

The pro-inflammatory role of IL-27 as key inflammatory mediator for Th1 differentiation and IFN-γ production has been intensively explored ^{16, 63, 65}. Stimulation of Th0 in the presence of IL-27 induces the expression of the key signature Th1 cytokine IFN-γ via up-regulating of the transcription factor T-bet and IL-12Rβ2 chain which is essential for responding to IL-12 and the differentiation of a Th1 phenotype ^{61, 62, 65}. This effect of IL-27 on Th cell depends on multiple transcription factors such as Stat-1 and Stat-3 ^{61, 62, 65}. In vivo studies demonstrated the role of IL-27 in Th1 responses. In this context, it was shown that WSX-1-/- mice were more susceptible to Leishmania major infection and impaired IFN-γ production ¹⁷. Similarly, reduced Th1 responses and IgG2a production was demonstrated in WSX-1-deficient mice infected with Listeria monocytogenes ¹⁸. Moreover, it was shown that IL-27R signaling is vital in vivo for the IFN-γ production by CD8+ T cells ⁶⁶. IL-27 can enhance the proliferation of naïve CD8+ T cells and IFN-γ and granzyme B production dependent on Stat1 and Stat3 ²².

IL-27 can stimulate human monocyte to express TLR4 through activation of Stat-3, and NF-κB which subsequently respond to LPS-inducing IL-6, TNF-α, MIP-1α, and MIP-1β expression ⁶⁷. IL-27 increased the production of nitric oxide from peritoneal macrophages via activation of Stat-1, NF-κB and MAPKs ⁶⁸. IL-27 also enhanced monocyte derived dendritic cells (moDCs) to express IL-27, IL-8, CXCL10, chemokine receptor (CCR1), IFN-stimulated genes, IRF8 and other genes involved in antigen presentation ⁶⁹. IL-27 negatively regulated Tr1 cells by induction of metallothioneins (MTs) which in turn reduced stat1 and stat3 phosphorylation resulting in impaired IL-10 production ⁷⁰. Taken together, IL-27 positively regulates both innate and adaptive immune responses.

IL-27 can negatively modulate inflammatory processes. Improved control of Leishmania donovani correlates with massive inflammatory responses were reported in IL-27R-deficient mice ⁷¹. Similarly, WSX-1-/- mice infected with Toxoplasma gondii or Trypanosoma cruzi generated robust IFN-γ responses and developed lethal T cell-mediated inflammation ^{31, 72}. Greater effector and memory CD4+ T cells responses were noted in IL27R lacking mice challenged by Plasmodium berghei ⁷³.

The negative feedback of IL-27 largely depends on the induction of type 1 regulatory T cells (Tr1). IL-27 can induce Tr1 cells via various mechanisms including activation of Stat1, Stat3 ^75-76, upregulation of Blimp1 ⁷⁷, aryl hydrocarbon receptor (AhR), transcription factor c-Maf, inducible T cell costimulator (ICOS), and IL-21 production, which is indispensable for the expansion and maintenance of Tr1 cells ^{34, 35}. Furthermore, the anti-inflammatory effect of IL-27 needs more than the induction of Tr1 cells. IL-27R-/- CD4+ T cells produce more IL-2 than wild-type during in vitro differentiation. The addition of recombinant IL-27 suppressed the expression of IL-2 both transcriptionally and translationally ⁷⁸. IL-27 suppresses CD28-mediated IL-2 production by Stat 1 which in turn induces the expression of the suppressor of cytokine signaling 3 (SOCS3) ⁷⁹. Moreover, it was observed that WSX-1-deficient macrophages are more efficient in inducing IFN-γ and IL-17A production by CD4+ T cells than control. IL-27 can activate stat1 which in turn suppresses cyclooxygenase (COX) expression and followed by reducing prostaglandin (PG2) secretion, which can affect the CD4+ T cell responses ⁸⁰.

IL-27 was also reported to suppress Th2 cells development and their cytokines production by downregulation of Gata3 and upregulation of T-beta in differentiated Th2 cells ³². In addition to suppress both Th1 and Th2 cells, IL-27 can inhibit the development of Th17 cells. Consistently, it was demonstrated that IL-27 can inhibit Th17 cell development through various mechanisms including the suppression of IL-6 signaling mediated IL-17 production ³³, retinoid-related orphan receptor γ (RORγ) expression (Th17-specific transcription factor) ³⁶, and also via induction of the IL-10 production ⁸¹, and programmed death ligand 1 (PD-L1) on naïve T cells ³⁷. IL-27 upregulates the expression of Blimp1 in pre-committed Th17 cells and acquired a Tr-1-like phenotype characterized by the production of IL-10 and IFN-γ ⁷⁷.

More recently, it was recognized that IL-27 can inhibit the development of Th9 cells and limit its related encephalitis by mechanism partly depending on Stat-1 ⁸². Taken together, IL-27 induces an immunosuppressive effect by both Tr1 cells dependent and independent mechanisms. However, the contribution of IL-27 in the induction of IL-35-producing T regulatory cells remains unknown.

Role of the IL-27 in innate and adaptive immune response to Mtb infection

The innate immunity plays a decisive role in the early clearance of Mtb via the recognition of the antigen and subsequent induction of pro-inflammatory cytokines and antimicrobial peptides ⁸³. The production of IL-27 in response to Mtb infection modulates macrophage responses ^41-43. The combination of IL-12 and anti-IL-27R can limit Mtb growth via upregulation of pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-18^{41, 84}. However, the molecular mechanisms by which IL-27 negatively regulated macrophages during Mtb infections remain unclear. It was demonstrated that IL-27R singling in mice peritoneal macrophages suppresses IL-12 and TNF-α production by Stat3 ⁴³. IL-27 can antagonize IL-18 signaling in human macrophages challenged with Mtb by inhibiting the expression of the IL-18 receptor beta-chain and IL-18R downstream signaling component NF-kB ⁴². IL-18 and IL-12, synergistically induced IFN-γ production by human macrophages and promoted the killing of Mtb ⁸⁴. It has been shown that IL-27 can inhibit the expression of the phagosomal vacuolar ATPase (V-ATPase) and lysosomal integrated membrane protein-1 (CD63), resulted in the suppression of phagosomal acidification and cathepsin D maturation ⁸⁵. IL-27 signaling is necessary for the IL-10 production by macrophages through enhancing the activity and binding of Stat1/Stat3 to the IL-10 promoter ⁸⁶. Recently, it was found that IL-10 induced by Mtb during macrophage infection can manipulate phagosomal maturation to enhance its own intracellular growth ⁸⁷. Most recently a study demonstrated that IL-27 suppresses IFN-γ mediated autophagy in human macrophages infected with Mtb by inducing autophagy negative regulatory factors mTOR and Mcl-1 through JAK/PI3-K/Akt and PI3-K pathway, respectively ⁸⁸. In brief, Mtb can subvert the normal bactericidal function of macrophages by inducing IL-27 (Figure 2A, Table 1).

Table 1.

The consequence of IL-27 signaling in tuberculosis

Immune cells involved	Consequence of IL-27 signaling	References
Macrophages	Inhibits the production of pro-inflammatory cytokines	43-42
	Induces the production of anti-inflammatory cytokine.	86
	Inhibits phagosomal acidification.	85, 87
	Suppresses the autophagy formation.	88
T cells	Suppresses Th1 cell responses.	43, 89-90.
	Induces the IL-10 producing Tr1 cells, promotes proliferation and maintenance of Tr1 cells.	34-35, 75-77, 94.
	Suppresses the Th17 cell development.	33, 36, 37, 81.

Ablation of IL-27, either by disruption of the IL-27R gene in mice or by an antibody blockade of the IL-27R, implicates an important role of IL-27 in adaptive immunity against mycobacteria infections. WSX-1-deficient mice have impaired IFN-γ production and granuloma formation when challenged with M. bovis BCG ⁸⁹. However, there are no differences in liver pathology and bacterial load between mutant and wild type mice ⁸⁹. Significant elevation of IL-27 throughout the infanthood and neutralization of IL-27 in neonatal macrophages improved control of bacterial replication ⁹⁰. Furthermore, blockade of IL-27 during incubation with the M. bovis (BCG) augmented the IFN-γ production by allogeneic CD4+ T cells ⁹⁰. Similarly, it has been demonstrated that improved control of Mtb growth in the lungs results from increased production of pro-inflammatory cytokines in WSX-1-knockout mice ⁴³. These results suggest that IL-27 negatively regulated Th1 response (Figure 2B, Table 1), however, the underlying mechanisms remain elusive.

More recently, it was recognized that IL-27 positively regulates IL-10 producing Tr1 cells during chronic inflammation ^{34, 35, 75-77} (Figure 2B, Table 1). It has been demonstrated that IL-10 suppressed the immune response to Mtb infections without immunopathology observed in both C57BL/6 and CBA/J mice ^{91, 92}. Moreover, it was shown that IL-10-deficient mice were more resistant to M. bovis (BCG) challenge in comparison to control mice ⁹³. Blocking of IL-10R in CBA/J mice improved M. bovis (BCG) evoked protection against Mtb characterized by enhanced Th1 and Th17 responses and increased IFN-γ and IL-17A production in the mice lungs ⁹⁴. Taken together, these data indicate IL-27 can modulate immune response against mycobacteria by inducing IL-10.

IL-27 directly mediated the suppression of the development of Th17 cells in chronic inflammation models ^{33, 36, 37, 81} (Figure 2). Th7 cells induce T cell chemokines (CXCL9, CXCL10 and CXCL11) in the lungs of infected mice to recruit IFN-γ-producing CD4+ T cells to limit Mtb growth ⁹⁵. BCG-induced Th17 cells which can downregulate IL-10 and generate Th1 immune response resulted in protection upon Mtb challenge ⁹⁶. IL-17-deficient mice were more susceptible to mycobacteria, evidenced by the impaired granulomas formation and decreased IFN-γ production ⁹⁷. Vaccination will elicit Th17 cells response associated with increased pro-inflammatory cytokines, granulocytes infiltration and severe lung damage in mice ⁹⁸. The higher amount of Th17 cells and related cytokines in active tuberculosis patients implicated a role in immunopathogenesis ^{99, 100}.

As an exceptional successful silent killer, Mtb can manipulate the host signaling to persist and reactivate at opportune occasion. Knowledge about the emerging player, namely IL-27, will inform better countermeasures against this hideous threat of global health public.

Abbreviations

IL

Interleukin

IL-27Rα

Interleukin-27 receptor alpha

TCCR

class I cytokine receptor family

gp130

glycoprotein 130

Mtb

Mycobacterium tuberculosis

TIR

Toll-Interleukin receptor

AhR

aryl hydrocarbon receptor

EBI3

Epstein-Barr virus induced gene 3

SNPs

single nucleated polymorphisms

DCs

Dendritic cells

moDCs

monocyte derived dendritic cells

Th cell

T helper cells

CTL

cytotoxic T lymphocyte

NK cells

Natural Killer cells

Tr1 cells

type 1 regulatory T cells

Tr35 cells

T regulatory type 35 cells

KO

Knockout

APCs

antigen-presenting cells

TLRs

Toll-Like Receptors

IFN

Interferon

Jak

Janus Kinase

Stat

Transcription factor

Tyk

Tyrosine kinase

NF-κB

nuclear factor-kappa beta

MAPK

mitogen activated protein kinase

MyD88

myeloid differentiation factor 88

IRF

IFN regulatory factor

JNK

C-Jun N-terminal kinases

PI3K

phosphoinositide 3-kinase

ICAM-1

intercellular adhesion molecule-1

c-Maf

transcription factor c-Maf

ICOS

inducible T cell costimulator

MIP

Macrophage inflammatory protein

SOCS

Suppressor of cytokine signaling

CD

cathepsin D

mTOR

mammalian target of rapamycin

PI3 K

phosphatidylinositol-3-kinase class I

Mcl-1

Myeloid cell leukemia 1.