[advances in immunology] advances in immunology volume 115 volume 115 || the immunobiology of il-27

CHAPTER 1

Advances in Immunology,ISSN 0065-2776, DOI: 10.1

Department of Pathobiolog

The Immunobiology of IL-27

Aisling O’Hara Hall, Jonathan S. Silver, and

Christopher A. Hunter

Contents 1. The Biology of IL-27: Subunits, Sources, Receptors, and

Volu016/

y, Sc

Signaling

me 115 # 2012B978-0-12-394299-9.00001-1 All righ

hool of Veterinary Medicine, Philadelphia, Pennsylvania, USA

Elsts

2

1.1.
D iscovery and characterization of IL-27 subunits
and receptors
2
1.2.
E BI3 and p28 subunits of IL-27 5
1.3.
R egulating the expression of IL-27 8
1.4.
T he IL-27 receptor 9
1.5.
IL -27 signaling and transcriptional mediators 14
1.6.
C omplexity and combinatorial biology of the
IL-27 system
15
2. P
roinflammatory Properties of IL-27 16
2.1.
IL -27 can promote TH1 responses 16
2.2.
IL -27 limits Foxp3þ regulatory T cell populations 17
2.3.
IL -27 enhances CD8þ T cell responses 18
2.4.
IL -27 and humoral responses 19
3. A
nti-Inflammatory Properties of IL-27 20
3.1.
IL -27 and type I responses 21
3.2.
IL -27 and type 2 responses 22
3.3.
IL -27 inhibits TH17 responses 23
3.4.
IL -27 promotes production of IL-10 25
4. T
he Role of IL-27 in Other Cell Types 26
4.1.
IL -27 and innate immunity 26
4.2.
IL -27 and non-hematopoietic cells 28
5. T
ranslational Implications of IL-27 29
5.1.
T argeting IL-27 pathways for therapies 29
5.2.
T he role of IL-27 in human disease 31
6. C
onclusions 32
evier Inc.reserved.

1

2 Aisling O’Hara Hall et al.

Ackn
owledgments 32
Refer
ences 33
Abstract Like many cytokines, IL-27 has pleiotropic properties that can limit

or enhance ongoing immune responses depending on context. Thus,

under certain circumstances, IL-27 can promote TH1 differentiation

and has been linked to the activation of CD8þ Tcells and enhanced

humoral responses. However, IL-27 also has potent inhibitory prop-

erties and mice that lack IL-27 mediated signaling develop exagger-

ated inflammatory responses in the context of infection or

autoimmunity. This chapter reviews in depth the biology of IL-27,

including the initial discovery, characterization, and signaling

mediated by IL-27 as well as more recent insights into the molecular

and cellular basis for its pleiotropic effects. Many of these advances

are relevant to human diseases and highlight the potential of

therapies that harness the regulatory properties of IL-27.

1. THE BIOLOGY OF IL-27: SUBUNITS, SOURCES, RECEPTORS,AND SIGNALING

IL-27 is a heterodimeric cytokine composed of two subunits, Epstein-Barrvirus-induced gene 3 (EBI3) and IL-27p28, which engages a receptor com-posed of gp130 and the IL-27Ra that activates Janus kinase (JAK)/signaltransducer and activator of transcription (STAT) and mitogen activatedprotein kinase (MAPK) signaling. There are a number of distinct structuralmotifs that characterize the receptor and cytokine subunits of IL-27 thathighlight its evolutionary relationship to other factors (IL-6, IL-12, and IL-23) that are central regulators of cell mediated and humoral responses.Indeed, early studies suggested that IL-27 was proinflammatory, but it isnow recognized that this factor can limit the intensity andduration of awidearray of adaptive responses (Table 1.1). The sections below describe theparallel studies that led to the definition of the IL-27 signaling cassette andour current understanding of how it functions in the immune system. Theprogress in these areas has now started to translate into the clinical situationand IL-27 has been implicated in the pathogenesis of a number of inflamma-tory conditions and its potential role as a therapeutic targetwill bediscussed.

1.1. Discovery and characterization of IL-27 subunitsand receptors

A comprehensive understanding of how the various components of theIL-27 system are integrated was a consequence of multiple discovery-based approaches that identified individual receptor or cytokine subunits

TABLE 1.1 The role of IL-27 during infection

Model Mouse Phenotype References

Influenza Mixed bone marrowchimeras with IL-

27Ra�/� mice, EBI3�/�

IL-27 is required for optimal CD8þ T cell effectorresponse.

Mayer et al. (2008),Sun et al. (2011)

Leishmania donovanii IL-27Ra�/� IL-27Ra�/� has enhanced control of the parasite

but increased liver pathology due to increased

TNF-a and IFN-g production.

Rosas et al. (2006)

Leishmania major IL-27Ra�/�, EBI3�/� Knockouts have initial TH1 defect and increased

parasite burden, but no TH1 defect or defect in

parasite control at later time-points.

Yoshida et al. (2001),

Zahn et al. (2005)

L. major IL-27Ra�/�, EBI3�/� Knockouts have increased TH2 response

responsible for early susceptibility.

Increased lesion size is associated with

increased TH17 responses. Normal TH1 and

pathogen clearance at later time-points.

Anderson et al.

(2009), Artis et al.

(2004a), Zahn

et al. (2005)

L. major IL-27Ra�/� Enhanced antigen presentation and TH1

differentiation by IL-27Ra�/� DCs.

Wang et al. (2007)

Listeria

monocytogenes

IL-27Ra�/� Defective TH1 and bacterial clearance, lowerIgG2a response.

Chen et al. (2000)

L. monocytogenes IL-27Ra�/� IL-27Ra�/� TH1 response is normal, but

decreased IL-10 production by CD4þ T cells.

Batten et al. (2008)

L. monocytogenes EBI3�/� TH1 response not impaired with increased

bacterial clearance and increased TH17

response.

Yang et al. (2008b)

(continued)

TABLE 1.1 (continued )


Mycobacterium bovis

BCG

IL-27Ra�/� No defect in IFN-g or bacterial clearance,

defective granuloma formation in IL-27Ra�/�.Yoshida et al. (2001)

Plasmodium bergheri IL-27Ra�/� CD4þ T cell-mediated liver pathology,

enhanced TH1 response, enhanced parasite

clearance in the absence of IL-27 signals.

Findlay et al. (2010)

Toxoplasma gondii Mixed bone marrow

chimeras with

IL-27Ra�/� mice

IL-27 is required for optimal CD8þ T cell effector

response.

Mayer et al. (2008)

T. gondii IL-27Ra�/� Acute lethal and pathological TH1 response in

the absence of IL-27Ra with enhanced IFN-gand parasite control.

Increased TH17 responses during chronic

toxoplasmic encephalitis

Stumhofer et al.

(2006), Villarino

et al. (2003)

Trichuris muris IL-27Ra�/� Enhanced expulsion of parasites, increased

production of TH2 cytokines, and increased

mast cell activity in the absence of IL-27Ra.

Artis et al. (2004b)

Trypanosoma cruzi IL-27Ra�/� Increased liver pathology and production of the

proinflammatory cytokines IFN-g, TNF-a, andIL-4.

Hamano et al. (2003)

The Immunobiology of IL-27 5

without an appreciation of how these distinct components were related.The section below attempts to place these events in a wider context andhighlights the reports that were critical to our current understanding ofhow this system functions as well as newer questions about the combina-torial biology that appears inherent to this system.

1.2. EBI3 and p28 subunits of IL-27

EBI3 was first identified in 1996 from a subtractive hybridization screen ofgenes expressed in Epstein-Barr virus (EBV) transformed B cell lines(Devergne et al., 1996). These initial studies revealed that EBI3 sharesstructural homology with other known members of the class I cytokinereceptor family, namely, IL-12p40, and the ciliary neurotrophic factorreceptor (CNTFR) (Fig. 1.1). Like these proteins, EBI3 contains no mem-brane anchoring motifs, suggesting that it is secreted, and two cytokinebinding domains containing WSXWS motifs characteristic of othercytokines in the hematopoietin receptor family (Devergne et al., 1996).Furthermore, when EBI3 was ectopically expressed in COS7 cells it accu-mulated in the endoplasmic reticulum and was not secreted as a mono-mer or homodimer (Devergne et al., 1997). This finding implied that EBI3would pair with another subunit for secretion, and since EBI3 is related toIL-12p40, the p35 subunit of IL-12 represented a likely candidate. Indeed,when co-expressed with p35, EBI3 could form a novel secreted IL12p35/EBI3 heterodimer (now named IL-35) (Collison et al., 2007; Devergne et al.,1997). The finding that the deletion of EBI3 was associated with alteredsusceptibility to oxazolone-induced colitis (Nieuwenhuis et al., 2002) sug-gested a role in immunity, but its function as a subunit of IL-27 was notapparent for several more years.

The eventual recognition that IL-27p28 is a partner for EBI3 was aconsequence of a computational approach, using the genomic databasesthat emerged in the late 1990s, to identify novel a-helical cytokines of theIL-6 family (Pflanz et al., 2002). One of the central structural features ofcytokines such as IL-6 and IL-12p35, that bind to type I cytokine receptors,is that they have long-chain four-helix bundle motifs and this featureformed the basis to identify IL-27p28. (Bazan, 1990a). Since IL-27p28 onits own was poorly secreted and had no obvious biological properties,these findings indicated that, like IL-12p35, it may need to partner with asoluble receptor-like molecule in order to be secreted (Pflanz et al., 2002).A number of candidate molecules (soluble IL-11 receptor, CLF-1, and IL-12p40) were tested for their ability to facilitate the secretion of p28, butonly EBI3 permitted efficient secretion (Pflanz et al., 2002). This hetero-dimeric protein was designated IL-27 and fusion proteins linking p28 toEBI3 allowed Kastelein and colleagues to define its biological propertiesand showed that IL-27 could synergize with IL-12 to promote proliferation

Four-helix bundle

cytokine

Cytokine receptor

homology domain

Immunoglobulin

domain

Fibronectin-like

domain

Humanin peptide

IL-12Rb2

IL-12Rb1-

IL-12p35p40

IL-23p19p40

IL-12Ra1

IL-23R

??

IL-35p35EBI3

IL-27Ra

EBI3

gp130

IL-27p28

CLC/CLF

gp130

CNTF-Ra

LIF-RCLCCLF

CLC/CNTFR

gp130

CNTF-Ra

LIF-RCLCCNTFR

gp130

IL-27RaIL-6Ra

p28CLF

CLF/p28

gp130

IL-6Ra

IL-6

gp130

CNTF-RaIL-27Ra

HumaninHumanin

gp130

CNTF-Ra

LIF-R

CNTFCNTF

gp130 family IL-12 family

FIGURE 1.1 Structural relationship of IL-27/IL-27R to the different gp130/IL-12 family of cytokines. The schematic cytokine and receptor

complexes for IL-27, IL-6, Humanin, ciliary neurotrophic factor (CNTF), cardiotrophin-like cytokine (CLC), CLC/CNTFR, cytokine-like factor

(CLF)/p28, IL-12, IL-23, and IL-35 are shown. IL-27 is a heterodimer consisting of Epstein-Barr virus-induced gene 3 (EBI3) and IL-27p28. IL-27p28 is

a four-helix bundle cytokine (yellow barrels) and resembles other helical cytokines such as IL-6, CLC, IL-12p35, and IL-23p19. EBI3 has an

immunoglobulin domain (diamonds) and cytokine receptor homology domain (see legend), and WSXWS motifs (yellow lines) like IL-12/IL-

23p40 and has homology to the soluble receptors IL-6Ra, CNTFR, and CLF. The heterodimeric IL-27 receptor consists of IL-27Ra (WSX-1, TCCR)

and glycoprotein 130 (gp130), both of which are involved in signal transduction; this is in contrast to the IL-6Ra, which lacks intrinsic kinase

activity. IL-27Ra has structural homology to gp130 and IL-12Rb2. Each of the related heterodimeric cytokine receptors consists of one or more

subunits with an extracellular cytokine binding domain with WSXWS motifs and fibronectin-like domains (see legend). IL-27Ra is a shared

receptor subunit of the Humanin and CLF/p28 receptors. EBI3 and IL-12p35 form IL-35.


of naı̈ve CD4þ T cells and the production of IFN-g from NK cells andCD4þ T cells (Fig. 1.2) (Pflanz et al., 2002). In the same study, these authorsdemonstrated that the orphan cytokine receptorWSX1 could bind to IL-27and was required for the effect of IL-27 and their subsequent workrevealed that gp130 constituted the second subunit of the IL-27 receptorcomplex (Pflanz et al., 2004).

T-bet ≠T-bet ≠ T-bet ≠≠c-Maf

GATA3RORa, RORgt

FIGURE 1.2 The IL-27 receptor is expressed by a variety of different cell types and can

exert pro- and anti-inflammatory effects. (A) In CD4þ and CD8þ T cells, as well as in

natural killer (NK) cells, IL-27 promotes the expression of the transcription factor T-bet

and the cytokines IL-10 and IFN-g. IL-27 also limits production of IL-17 by CD4þ T cells

and NK cells. In response to IL-27, B cell subsets increase their proliferation and antibody

production. (B) In mast cells and eosinophils IL-27 can promote proinflammatory

responses by increasing expression of IL-1, TNF-a, and IL-6; however, in neutrophils, IL-27

limits cytokine secretion of IL-6 and IL-12p40. (C) The effects of IL-27 on antigen

presenting cells are less well characterized; however, IL-27 can exert anti-inflammatory

effects on macrophage and dendritic cells by decreasing their production of TNF-a and

IL-12p40 while promoting the production of IL-10. However, in monocytes, IL-27

negatively regulates the expression of IL-10 in response to inflammatory stimuli and

following LPS stimulation, IL-27 can promote increased expression of IL-6 and TNF-a.


1.3. Regulating the expression of IL-27

With the description of the pairing of EBI3 and IL-27p28, interest centeredon what cell types were likely sources of this heterodimer. Initial studiesthat identified EBI3 as being induced in response to EBV was an indica-tion that microbial stimuli were likely to be involved. Early surveys ofexpression patterns indicated that it was restricted to myeloid cells andexpressed highly in LPS activated DCs, monocytes and macrophages(Hashimoto et al., 2000; Liu et al., 2007; Smits et al., 2004; Veckman et al.,2004; Wirtz et al., 2005). The observation that other cell types includingplasma cells, endothelial cells, microglia, placental trophoblasts, anduterine NK cells can express IL-27p28 and EBI3 (Devergne et al., 1996;Larousserie et al., 2004; Sonobe et al., 2005; Zhang et al., 2003) impliesthat IL-27 may have a broad role in immune regulation in specializedenvironments such as the brain and uterus.

Consistent with the idea that microbial stimuli can induce IL-27, anumber of different TLR agonists (LPS, poly (I:C), CPG, and Gram-negative and -positive bacteria) are able to induce EBI3 and IL-27p28mRNA expression in human and mouse antigen presenting cells(Hashimoto et al., 2000; Liu et al., 2007; Smits et al., 2004; Veckman et al.,2004; Wirtz et al., 2005). Although the role of Gram-positive bacteria inpromoting EBI3 expression is debated (Schuetze et al., 2005), it is clear thatLPS is a potent inducer of EBI3 and IL-27p28 and its effects are furtherenhanced by the presence of IFN-g (Sonobe et al., 2005). The pathwaystriggered by IFN-g and LPS are distinct but facilitate the sustained expres-sion of IL-27, which is greatly abrogated in MyD88 and NF-kB p50deficient mice (Liu et al., 2007; Wirtz et al., 2005). In response to TLR4signals, NF-kB binds to the IL-27p28 promoter, and although c-Rel (anNF-kB family member) was shown to associate with this NF-kB bindingsite, c-Rel is only partially required for the LPS-induced effects (Liu et al.,2007). Following NF-kB activation, the synergistic effects of IFN-g in thissystem are attributed to interferon response fragment-1 (IRF-1) and IRF-8 (also known as interferon consensus sequence-binding protein) bindingIRF response elements within the IL-27p28 promoter (Zhang et al., 2010).These studies have led to a model in which early NF-kB signals initiatethe transcription of IL-27p28, but sustained production requires othertranscription factors such as IRF-1 and IRF-8. It should be noted thatstimuli that utilize the adapter molecule MyD88 could also promoteIL-27p28 expression through the activation of the MAPK induced tran-scription factor AP-1. This is illustrated by reports in which incubation ofhuman and mouse macrophages with Mycobacterium tuberculosis resultedin MyD88 dependent recruitment of AP-1, which trans-activated theIL-27p28 promoter (Zhang et al., 2011).


MyD88-independent pathways can also trigger the production of type Iinterferons which promote IL-27 expression. Thus, in response to TLR4and TLR3, signals through the adaptor Toll/IL-1R-related domain-containing adaptor-inducing IFN (TRIF) (Molle et al., 2007) activatesIRF3 and IRF7 (Fitzgerald et al., 2003), and the expression of IL-27p28and EBI3 is increased. Cells that lack TRIF cannot produce robust amountsof either IL-27p28 or EBI3 mRNA in response to TLR4 ligands (Molle et al.,2007). While IRF3 is recruited to the promoter region and is required forexpression of IL-27p28, it is not required for the expression of EBI3 (Molleet al., 2007, 2010). The promoter region of EBI3 does contain a consensussequence for IRF7 binding (Wirtz et al., 2005), but its role in promoting EBI3expression is unknown. Similar to the synergistic effects of IFN-g onMyD88 dependent signals, IFN-a/b can amplify IL-27p28 expression byactivating IRF1 and further enhance IL-27p28 expression through theactivation of the STAT1/STAT2/IRF9 (ISGF3) complex (Molle et al.,2010; Pirhonen et al., 2007). It has been known for some time that IFN-bcan promote the expression of IL-27 (Remoli et al., 2007; van Seventer et al.,2002) and that, while IFN-b can promote IL-27p28 and EBI3, it can alsoblock the expression of IL-12p40 and IL-23p19 from DCs, depending onthe context of the IFN-b signals (Nagai et al., 2007). This observation is ofclinical interest as type I interferon therapy has been used to ameliorate theinflammatory response during experimental autoimmune encephalitis(EAE) and multiple sclerosis (MS) (Brod et al., 1995; Knobler et al., 1984).While one mechanism by which this is thought to occur is due to suppres-sion of IL-12, another potential result of such treatment could be increasedIL-27 which could contribute to the anti-inflammatory effects of this treat-ment regimen (Guo et al., 2008; Karp, 2000; Sweeney et al., 2011).

Despite advances in our understanding of the types of microbial andhost signals that control the production of IL-27, there is still a limitedappreciation of the cell biology that allows the p28 and EBI3 subunits todimerize and be secreted. One of the difficulties in studying this aspect ofthe biology of IL-27 stems from the paucity of reagents to detect theheterodimer, and new approaches are needed to define the relative con-tribution of cell specific expression of IL-27 to inflammatory responsesand immune homeostasis. This would be facilitated by the developmentof more sensitive antibody reagents, cytokine reporters, and/or floxedalleles of EBI3 and p28 to allow lineage specific deletion of these factors.

1.4. The IL-27 receptor

WSX-1 is a transmembrane protein that represents the alpha chain of theIL-27R (IL-27Ra) and was first identified from a human infant braincDNA library in 1998 using screening approaches to identify receptors


with homology to glycoprotein 130 (gp130) (Sprecher et al., 1998). Not onlydoes WSX-1 have significant amino acid similarity with gp130, but it alsocontains theWSXWS sequencemotif characteristic of class I cytokine recep-tors. Subsequent work that focused on cytokine receptors on immune cellscloned this receptor (termed T-cell cytokine receptor, TCCR) and noted thehomology to gp130 and IL-12Rb2 (Chen et al., 2000). These class I cytokinereceptors are characterized by an extracellular cytokine binding domain ofapproximately 200 amino acids that consists of four N-terminal cysteines,three fibronectin type III domains, and the highly conservedWSXWSmotifnear the C-terminus (Bazan, 1990b). In contrast to other related cytokinereceptor a chains such as IL-6Ra or IL-11Ra, which lack intrinsic tyrosinekinase activity and the ability to transduce signals but confer receptorspecificity (Yamasaki et al., 1988), the cytoplasmic domain of IL-27Ra has aBox 1 motif that associates with JAK1 and JAK2 and is important for signaltransduction (Sprecher et al., 1998).

Signaling by type I cytokines normally involves a heterodimeric receptorandwith the identification of IL-27 and IL-27Ra as its partner it was quicklyrecognized that gp130 was the additional receptor component (Pflanz et al.,2004). Gp130 is a signal-transducing receptor that is utilized by at least eightdifferent cytokines, including IL-6, IL-11, leukemia inhibitory factor (LIF),oncostatin-M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1(CT-1), and cardiotrophin-like-cytokine (CLC) (Fig. 1.1) (Lupardus et al.,2011). Gp130 is present on hematopoietic and non-hematopoietic cells, andits expression can vary depending on the cell’s activation status (Anderssonet al., 1978; Betz and Muller, 1998; Saito et al., 1992; Taga and Kishimoto,1997; Wang et al., 1998).

The initial studies that identified IL-27Ra reported that its mRNA washighly expressed in lymphoid tissues such as the spleen, lymph node, andthymus, indicating a broad role for this receptor in immune function(Chen et al., 2000; Sprecher et al., 1998). Indeed, the use of IL-27Ra specificantibodies demonstrated that this receptor is expressed by T and B cells aswell as NK cells (Villarino et al., 2005). In murine systems, the IL-27Ra isnot readily apparent on accessory cells, but there are data that support thenotion that macrophages or DCs are responsive to IL-27, and this isreadily detected in human macrophages (Fig. 1.2C) (Chen et al., 2000;Holscher et al., 2005; Kalliolias et al., 2010a; Yoshida et al., 2001). HumanB cell subsets have also been analyzed for their expression of gp130 andIL-27Ra protein and naive (IgDþ CD38�) and memory (IgD� CD38�) Bcells express the highest levels of the IL-27 receptor (Boumendjel et al.,2006; Larousserie et al., 2006). Germinal center (GC) (IgD� CD38þ) B cellsalso express low levels of these receptor subunits, and there is evidencethat plasma cells express IL-27Ra (Cocco et al., 2011; Larousserie et al.,2006). Consistent with these reports, stimulation of B cells through the Bcell receptor or through CD40 ligation can upregulate gp130 and IL-27Ra,


suggesting that IL-27 can affect a variety of B cell subsets (Larousserieet al., 2006).

IL-27Ra is also expressed on a variety of human and mouse tumor celllines such as HeLa cells, B16 melanoma, and Lewis Lung Carcinoma andis upregulated on cancer cells from patients with acute myeloid leukemia(Dibra et al., 2009a; Pradhan et al., 2007). However, the role of IL-27Raexpression in cancer biology is complex, with reports that attribute pro-and anti-tumor activity to IL-27 (Table 1.2) (Dibra et al., 2009b, 2011;Pradhan et al., 2007; Yoshimoto et al., 2008). For example, the effects ofIL-27Ra expression on tumors have been associated with expression ofMHC class I-related chain A (MICA) which is a ligand for NKG2D, anactivating receptor expressed on NK cells, which promotes cytotoxicity(Dibra et al., 2009a). In contrast, work from the same group has alsoshown that IL-27Ra expression on different tumor cell lines inhibitedeffector responses and promoted tumor growth (Dibra et al., 2011). Onepossible explanation for this observation is that IL-27Ra may act as ahomodimeric receptor and, via its Box 1 motif, can activate the JAK/STAT pathway leading to the transformation of myeloid cells (Pradhanet al., 2007). However, studies with B16 melanoma demonstrated thattumor expression of IL-27Ra mediated tumor growth inhibition, andthese effects were IL-27 dependent (Yoshimoto et al., 2008). The studiesdescribed above appear contradictory, with IL-27 promoting growth orkilling of tumors, but it seems likely that these effects are contextspecific and could be shaped by the individual tumor microenviron-ment and how the cancer cells have evolved to evade the immunesystem.

Like gp130, the levels of the IL-27Ra chain are altered in response toactivation (Betz and Muller, 1998). Thus, while naı̈ve CD4þ T cells areresponsive to IL-27, they express basal levels of the IL-27Ra receptor butfollowing TCR engagement these levels are markedly increased. In con-trast, resting NK cells express high levels of the IL-27Ra but activationleads to downregulation of this receptor (Villarino et al., 2005). As formany cytokines, a change in the expression of the receptor subunits is oneway to mediate responsiveness and to influence the amplitude and dura-tion of the signals. Although it is clear that there is a dynamic regulationof IL-27Ra, our understanding of how this impacts on the biologicalproperties of IL-27 remains poor. Nonetheless, the controlled expressionof cytokine-specific alpha chains (such as IL-6Ra, etc.) is one mechanismto regulate responses to gp130-family cytokines. Moreover, it is notablethat the pattern of IL-27Ra expression correlates with the biological effectsof IL-27 on T cells, where IL-27 is required to negatively regulateresponses of activated CD4þ T cells in multiple settings (discussedbelow). Thus, the regulated expression of IL-27Ra is a critical variable inthe role of this cytokine during inflammation and additional studies

TABLE 1.2 The role of IL-27 in mouse models of cancer and autoimmunity


Colitis (T cell

transfer model)

IL-27Ra�/� IL-27 limits Treg populations, IL-27Ra�/�

transferred T cells induce less severe disease.

Cox et al. (2011)

Proteoglycan-

induced arthritis

IL-27Ra�/� IL-27 promotes TH1-mediated pathology.

Reduced IFN-g and anti-PG specific antibody in

the absence of IL-27Ra.

Cao et al. (2008)

Cancer Tumor over-

expresses IL-27

IL-27 promotes NK cell and CTL responses, and

tumor regression.

Chiyo et al. (2004, 2005),

Hisada et al. (2004),

Salcedo et al. (2004),

Shinozaki et al. (2009)

Cecal ligation andpuncture model

of sepsis

EBI3�/� IL-27 limits neutrophil recruitment and bacterialclearance.

Laroni et al. (2011), Wirtzet al. (2006)

Experimental

autoimmune

encephalitis

(EAE)

IL-27Ra�/�, p28�/� IL-27R/IL-27 knockouts have more severe EAE,

increased TH17 responses, and decreased IL-10

production.

Batten et al. (2006), Diveu

et al. (2009)

EAE IL-27Ra�/� IL-27Ra�/� TH1 response normal and decreased

IL-10 production by CD4þ T cells.


MRL/lpr lupus IL-27Ra transgenic Overexpression of IL-27Ra ameliorated disease and

limited autoantibody production.

Sugiyama et al. (2008)

MRL/lpr lupus IL-27Ra�/� Increased TH2-type skin inflammation. Kido et al. (2011)

OVA-airway

inflammation

IL-27Ra�/� IL-27Ra�/� has increased airway responsiveness

and goblet cell hyperplasia, exacerbated TH1 and

TH2 responses, and increased serum IgE levels.

NKT cell-derived IL-27 limits TH2 responses.

Fujita et al. (2009),

Miyazaki et al. (2005)

OVA-hapten

immunization

IL-27Ra�/� IL-27Ra�/� has decreased IL-21 production by

CD4þ T cells, less germinal center B cells, and

less hapten-specific antibody production.


Pristane-induced

lupus

IL-27Ra�/� IL-27Ra�/� has less severe disease, diminished B

cell responses, and less autoantibodies.


Scurfy-like disease IL-27p28 and EBI3

double

transgenic

Transgenic mice lack peripheral Treg due to

suppression of IL-2 production by

overexpression of IL-27

Tait Wojno et al. (2011)

Streptozotocin-

induced type

2 diabetes

IL-27Ra�/�, EBI3�/

�Hyperglycemia and pancreatic islet inflammation

are increased in the absence of IL-27/IL-27

signals.

Fujimoto et al. (2011)


examining the regulation of receptor expression should provide insightinto the biological activities of IL-27 in vivo.

1.5. IL-27 signaling and transcriptional mediators

Upon ligation of its receptor, IL-27 induces a signal transduction cascadethat engages the JAK/STAT pathway as well as MAPK signaling. Thelatter events have not been extensively studied but are characterized bythe activation of p38 MAPK and ERK1/2 (Owaki et al., 2006). In humannaı̈ve CD4þ T cells, the activation of MAPK induces c-Myc and theexpression of cyclins D2, D3, cyclin A, and the cyclin dependent kinase(CDK) 4, a process that was shown to be regulated by the IL-27-mediatedinduction of Pim-1 (Charlot-Rabiega et al., 2011). These events have beenmost closely associated with the ability of IL-27 to promote T cell prolif-eration and the transition from the G0/G1 to S phase. The capacity ofIL-27 to activate the JAK/STAT pathway in lymphocytes has been exten-sively studied and the pathways induced by IL-27 resemble those propa-gated by IL-6. So, although IL-27 shares properties with IL-6—such asshared receptor subunits and the ability to signal through STAT1 andSTAT3—there are also distinct effects of these two cytokines. These maybe explained by differences in signaling or by how cells integrate thesesignals depending on activation status or through the regulation of recep-tor expression. As mentioned earlier, the alpha chain of the IL-6 receptordoes not have the capacity to signal, but gp130 and IL-27Ra containmultiple motifs that are important for binding to JAKs (Sprecher et al.,1998). Early studies demonstrated that IL-27Rawas associated with JAK1(Takeda et al., 2003), whereas gp130 is associated with JAK1, JAK2, andtyrosine kinase 2 (TYK2) (Lutticken et al., 1994; Narazaki et al., 1994; Stahlet al., 1994). Indeed, stimulation of naı̈ve CD4þ T cells with IL-27 inducesthe activation of JAK1, JAK2, and TYK2 (Kamiya et al., 2004) and themarked phosphorylation of STAT 1, 3, and 5 (Kamiya et al., 2004; Lucaset al., 2003; Villarino et al., 2003). Subsequent studies on T helper celldifferentiation revealed that the ability of IL-27 to activate STAT1 pro-moted T cell expression of the transcription factor T-bet which is asso-ciated with the development of a TH1 response (Hibbert et al., 2003;Kamiya et al., 2004; Owaki et al., 2005). Additional insights into thesignaling pathways utilized by IL-27 include the ability to activateSTAT3, which is required for the expression of c-Maf, a transcriptionfactor associated with the ability of IL-27 to promote IL-10 production(Pot et al., 2009; Xu et al., 2009).

While the main events downstream of IL-27R signaling are describedabove, there is less known about the mechanisms that temper IL-27signaling. SOCS proteins have a prominent role in providing negativefeedback to many type I cytokines and represent obvious candidates to be


involved in this process. For gp130, the activation of the STAT pathwaysleads to increased expression of SOCS3 that can then bind to SH2 domainsin gp130 and so prevent further association with JAKs (Nicholson et al.,2000). This mechanism also appears relevant to IL-27, as IL-27 can induceSOCS proteins (Villarino et al., 2007) and T cells frommice in which gp130lacks the ability to interact with SOCS3 have a sustained pattern of STATactivation when stimulated with IL-27 (J.S. Silver, unpublished observa-tions). Nevertheless, even in this setting, IL-27 signaling is eventuallydownregulated but the additional mechanisms that underlie these eventshave not been explored.

1.6. Complexity and combinatorial biology of the IL-27 system

The sections above summarize our current understanding of the compo-sition of the IL-27 signaling cassette, but even the initial studies providedhints that there are additional levels of complexity in this system. Theobservation that the p28 and EBI3 subunits of IL-27 can be secretedindependently from one another suggested that they had properties dis-tinct from their functions as IL-27 subunits (Batten and Ghilardi, 2007;Devergne et al., 1996; Pflanz et al., 2002; Stumhofer et al., 2010). Thus, theearly report that EBI3 could partner with p35 (Devergne et al., 1997) hasnow been linked to the cytokine IL-35, and regulatory T cell biology(Collison et al., 2007, 2009; Niedbala et al., 2007). However, there areconflicting reports as to whether human regulatory T cells express IL-35(Bardel et al., 2008; Collison et al., 2010), and the signals downstream of IL-35, including the constituents of the IL-35 receptor are at this point poorlyunderstood. Other heterodimers consisting of EBI3 or p28 have also beenshown to form in vitro; IL-23p19 can bind to EBI3 (Kastelein et al., 2007),and IL-27p28 has been reported to associate with cytokine-like factor(CLF), and signal through a tripartite receptor composed of gp130,IL-27Ra, and IL-6Ra (Crabe et al., 2009). Although the in vivo significanceof these novel complexes is unclear, they present potential complicationsfor the interpretation of studies with IL-27 and IL-27Ra knockout animals.

As mentioned earlier, the pairing of the soluble IL-6Ra with IL-6structurally resembles the heterodimeric cytokines IL-12, IL-23, andIL-27, and it has been proposed that these heterodimeric cytokinesco-evolved from an ancestral IL-6 like cytokine (Trinchieri, 2003). Conse-quently, the well-characterized interactions of IL-6 with its receptors canbe used to infer other areas that might be relevant to the biology of IL-27.For example, while the IL-6Ra chain is expressed on the surface of manycell types, the ability to generate a soluble version through alternativesplicing or enzymatic cleavage of the membrane bound form allows itto pair with free IL-6, and this heterodimer can bind to gp130 anddirectly activate the downstream signaling cascades (Lust et al., 1992;


Novick et al., 1989). This process is referred to as trans-signaling and evenin the steady state, there are basal levels of circulating soluble IL-6 recep-tor in the serum and synovial fluid, which are elevated during inflamma-tion (Desgeorges et al., 1997). Relevant to this review, there are reports ofan alternatively spliced form of IL-27Ra that gives rise to a soluble versionof this alpha chain of the IL-27R (Hashimoto et al., 2009). While this hasbeen linked to forming a complex with gp130 and CNTFR, whether this isinvolved in trans-signaling has not been addressed. Similarly, manycytokine systems include natural receptor antagonists, and there is asoluble form of gp130 that antagonizes the process of IL-6-mediatedtrans-signaling, but which does not inhibit conventional IL-6 or IL-27signaling (Scheller et al., 2005). Work from our group has revealed animmunoregulatory role for IL-27p28 independently of EBI3, whereby itcan bind with low affinity to the Ig-like domain of gp130 and antagonizethe ability of IL-6, IL-11, and IL-27 to signal (Stumhofer et al., 2010). Thus,analogous to the IL-1Ra and IL-4Ra, the IL-27 p28 subunit may also have arole as a low-affinity receptor antagonist, and its possible role in humandisease is discussed below. Taken together, an understanding of the uniquebiological properties of IL-27 and its components is emerging as well as anappreciation of the combinatorial biology that is apparent in this familythat indicate its potential to impact many aspects of the immune system.

2. PROINFLAMMATORY PROPERTIES OF IL-27

2.1. IL-27 can promote TH1 responses

Before it was recognized as the receptor for IL-27, two separate groupsgenerated mice that lacked the IL-27Ra (WSX-1, TCCR) and, based on itssimilarity to other immune receptors, screened these mice for alteredimmune phenotypes (Chen et al., 2000; Yoshida et al., 2001). Although noobvious immune defects were noticed in mice that lacked IL-27Ra, whenthey were challenged with intracellular pathogens they were reported tobe more susceptible to Listeria monocytogenes and Leishmania major as aconsequence of defects in TH1 immunity (Table 1.1). The discovery thatIL-27Rawas the receptor for IL-27 and that IL-27 can enhance the prolifer-ation of naı̈ve CD4þ T cells and the production of IFN-g (Pflanz et al.,2002), supported the idea that signaling through the IL-27Ra was proin-flammatory. A molecular basis for these events was provided by studiesin which IL-27 was found to promote STAT1 activation, and the transcrip-tion factor T-bet which induces the expression of the IL-12Rb2 therebysensitizing the T cell to be responsive to the signals that facilitate TH1development (Hibbert et al., 2003; Lucas et al., 2003; Takeda et al., 2003).Similarly, IL-27 also mediates the expression of the adhesion molecules


ICAM-1/LFA-1, downstream of STAT1, which also aids the differentia-tion of TH1 cells (Morishima et al., 2010; Owaki et al., 2006).

With a growing appreciation of the broad immunoregulatory effects ofIL-27 the interpretation of some of the earlier in vivo studies has beenrevisited. Thus, while initial reports suggested that the TCCR deficientmice were susceptible to L. monocytogenes, there is now a consensus thatthey are, in fact, better able to control this pathogen (Batten et al., 2008;Yang et al., 2008a). Furthermore, with an appreciation that IL-27 inhibitsTH2 responses, the reduced TH1 response observed during L. majorinfection appears to be a secondary consequence of enhanced TH2responses observed in the absence of IL-27 (Artis et al., 2004a). This isdiscussed in more detail below, but the complex cross regulation of TH1and TH2 responses can make it difficult to equate altered IFN-g responsesobserved in the IL-27Ra deficient mice with the ability of IL-27 to promoteCD4þ TH1 responses. However, one example is provided by a model ofproteoglycan (PG)-induced arthritis where IL-27 is associated with thedevelopment of a pathological TH1 response (Cao et al., 2008). In thisexperimental system, T cell production of IFN-g is required for diseaseprogression and loss of the IL-27Ra leads to decreased IFN-g and reduceddisease. This is also a system in which anti-PG Abs are important for thedevelopment of arthritis and the IL-27Ra�/�mice also have a reduction inPG-specific IgG2a. Indeed, IL-27 could promote IgG2a indirectly throughthe production of IFN-g (Monteyne et al., 1993), and directly through theactivation of STAT1 and T-bet (Yoshimoto et al., 2004). Nevertheless, thesections below highlight the studies in which the effects of IL-27 havebeen most concretely linked to promoting cell mediated and humoralresponses.

2.2. IL-27 limits Foxp3þ regulatory T cell populations

It has been proposed that one proinflammatory property of IL-27 is itsability to antagonize the differentiation of Foxp3þ regulatory T cell (Treg)populations. Multiple groups have reported that when T cells were dif-ferentiated under inducible Treg differentiation conditions (TGF-b, IL-2)in the presence of IL-27, there was a marked decrease in the frequency ofTreg that were generated (Cox et al., 2011; Huber et al., 2008; Neufert et al.,2007; Stumhofer et al., 2006). Furthermore, in the CD45RBhi transfer modelof colitis, IL-27Ra�/� T cells more readily acquired Foxp3 and had lesssevere disease compared to WT CD45RBhi recipients (Cox et al., 2011).Studies by Cox and Ghilardi also demonstrated that in an oral tolerancemodel where mice are fed OVA, transferred OVA specific CD4þ T cellsfrom IL-27Ra�/� mice had an increase in their ability to express Foxp3,suggesting that in these experimental settings IL-27 could limit Tregpopulations in vivo. Interestingly, mice which have been engineered to


overexpress IL-27p28 and EBI3 have a profound defect in the peripheralhomeostasis of Treg and succumb to a scurfy-like immunoproliferativedisease. However, this appears to be an indirect effect of the ability of IL-27 to limit production of IL-2 (Tait Wojno et al., 2011). The interpretation ofthis body of work is complicated by the limited amount of in vivo evidencethat this occurs during normal inflammatory processes. For instance, atsteady state, mice deficient in IL-27 or the IL-27Ra do not have increasednumbers or frequency of Treg, and it is unclear at this point if IL-27Raknockout mice have altered Treg homeostasis. However, given thebroad anti-inflammatory properties of IL-27 and its ability to drive theexpression of IL-10, discussed below, the capacity of IL-27 to directly limitTreg activaties appears incongruous and suggests additional studies arerequired to understand where this property of IL-27 is most biologicallyrelevant.

2.3. IL-27 enhances CD8þ T cell responses

Similar to the effect on CD4þ T cells in vitro, in mouse and human CD8þ

T cells, IL-27 induces the activation of pSTATs 1–5 and increases prolifer-ation, T-bet expression, IFN-g production, and IL-12Rb2 expression(Fig. 1.2A) (Morishima et al., 2005, 2010; Schneider et al., 2011). Addition-ally, IL-27 promotes cytotoxic T lymphocyte (CTL) responses by upregu-lating the expression of perforin, granzyme, and the specific lysis of targetcells (Morishima et al., 2005; Schneider et al., 2011). The efficacy of IL-27 inpromoting CD8þ T cell effector function has been best illustrated by anumber of in vivo studies where cancer cell lines engineered to expressIL-27 promote tumor specific CTL responses, tumor regression, and insome cases, complete remission with memory responses to subsequentchallenge (Table 1.2) (Chiyo et al., 2004, 2005; Hisada et al., 2004; Salcedoet al., 2004; Shinozaki et al., 2009). In models of colon carcinoma andneuroblastoma, the ability of IL-27 to promote tumor regression wasfound to be dependent on CD8þ T cells, but not NK or CD4þ T cells(Hisada et al., 2004; Salcedo et al., 2004). The efficacy of IL-27 in the regres-sion of colon carcinoma was also shown to be IFN-g and T-bet dependentbut surprisingly independent of STAT4 signals, indicating a potential rolefor IL-27 that is distinct from that of IL-12 (Hisada et al., 2004).

The role of IL-27 in driving CD8þ T cell responses in autoimmune andinfectious disease settings is less well characterized. Initial studiesshowed that, in the absence of IL-27Ra, CD8þ T cell responses are notdefective during acute toxoplasmosis, or infection with Trypanosoma cruzi,influenza, or malaria (Findlay et al., 2010; Hamano et al., 2003; Sun et al.,2011; Villarino et al., 2003). However, a subsequent report, using a mixedchimeric approach to study the CD8þ T cell response concluded that inmice infected with Toxoplasma gondii or influenza, the expression of the


IL-27Ra was critical for the induction of T-bet and IFN-g (Mayer et al.,2008; Sun et al., 2011). This apparent discrepancy may be due to indirecteffects of the global IL-27Ra deficiency versus those that are revealed in acompetitive setting that is engineered using mixed chimeras. There is alsoa role for IL-27 in driving effector CD8þ T cell responses during influenza,where IL-27 receptor but not IFN-g receptor expression is required foroptimal effector function, T-bet expression, and presence of IFN-gþ CD8þ

T cells in the lungs of infected animals (Mayer et al., 2008). These studiessuggest that IL-27 does play a part in shaping the CD8þ T cell response;however, it has not been extensively studied in other well-characterizedmodels of CTL responses such as LCMV and L. monocytogenes, and itremains to be determined if it is critical in the acquisition of effectorfunctions in these settings. In addition, the impact of reports that linkIL-27 to the production of IL-10 and the related cytokine IL-21, both ofwhich are CD8þ T cell growth factors, have not been explored in thecontext of CTL responses during infection or cancer.

2.4. IL-27 and humoral responses

Although B cells were first identified as a source of EBI3 these cells arealso responsive to IL-27, and B cell subsets have differential expression ofIL-27Ra and gp130 (Boumendjel et al., 2006; Yoshimoto et al., 2004). Thedifferentiation of naı̈ve IgMþ IgDþ B cells into class switched antibody-secreting cells following B cell receptor stimulation and CD40 ligation is aprocess that is shaped by factors such as IL-4, IL-6, IL-21 IFN-g, andlymphotoxin a. Indeed, in vitro, IL-27 has been reported to promoteIgG2a secretion by mouse B cells (Yoshimoto et al., 2004), but IgG1 pro-duction by human B cells (Boumendjel et al., 2006). Regardless, it shouldbe noted that the effects of IL-27 on class switching are modest whencompared to the effects of other cytokines such as IFN-g and IL-4. Never-theless, some of the direct effects of IL-27 on B cells have been addressedby studies in which polyclonal stimulation of naı̈ve and GC B cells in thepresence of IL-27 increase their proliferation, but IL-27 does not seem topromote formation of memory B cells (Boumendjel et al., 2006; Charlot-Rabiega et al., 2011; Larousserie et al., 2006). IL-27 can also upregulate Bcell expression of costimulatory molecules such as ICAM-1, and CD86 inaddition to increasing Fas/CD95 expression and modulating chemokinereceptor expression (Cocco et al., 2011; Larousserie et al., 2006). Consistentwith these reports, the lack of IL-27Ra has been associated with altered Bcell responses in a number of experimental settings. For instance, follow-ing infection with L. monocytogenes, IL-27Ra�/� mice have diminishedIgG2a responses compared to wild-typemice (Chen et al., 2000). Similarly,in the PG-induced model of arthritis, IL-27Ra�/� mice have diminishedPG-specific IgG2a, as discussed previously (Cao et al., 2008). Furthermore,


following airway OVA challenge, IL-27Ra�/� mice have exacerbated IgEproduction (Miyazaki et al., 2005). These studies suggest a potential rolefor IL-27 in the regulation of class switched antibody responses.

It is not clear from the studies described above whether the alteredhumoral responses observed in the absence of IL-27 are a consequence ofdirect or indirect effects of IL-27 on B cells. Indeed, IL-27 can induce CD4þ

T cells to produce IL-21, which promotes B cell expression of Blimp-1 andBcl-6 and is critical for plasma cell differentiation and B cell function(Ozaki et al., 2002, 2004; Pot et al., 2009,). Ghilardi and colleagues investi-gated the role of IL-27 in the development of T follicular helper (TFH) cellsand TFH cell-dependent B cell responses in vitro and also in vivo inresponse to multiple immunizations with OVA conjugated to the haptenTNP. They reported that while the differentiation of TFH did not requireIL-27, this cytokine did stimulate their production of IL-21 and TFHsurvival was increased. The in vivo production of IL-21 by CD4þ T cellsappeared to be dependent on IL-27, and in IL-27Ra�/� mice, this IL-21expression was compromised in an OVA immunization model (Battenet al., 2010). Following immunization, these mice also displayed dimin-ished GC (B220þ GL-7þ Fasþ) B cell responses, decreased class switchedantibodies and lower levels of hapten-specific antibody production(Batten et al., 2010). Furthermore, in a pristane-induced lupus model,characterized by the development of anti-double stranded DNA autoan-tibodies, the absence of IL-27R expression resulted in less severe pathol-ogy (Batten et al., 2010). It should be noted that in other models usingIL-27Ra�/� mice, such as in the studies using the OVA airway inflamma-tion model (Miyazaki et al., 2005), there is no defect in class switchedantibody production, suggesting that IL-27 is not critical for the GCreaction. Moreover, there are studies in which the overexpression of IL-27Ra in the MRL/lpr mouse model of lupus ameliorated autoantibodyresponses (Sugiyama et al., 2008), suggesting that IL-27Ra expression canantagonize antibody production. Taken together, these studies demon-strate that IL-27 is a key regulator of B cell responses through its ability toact directly on multiple B cell subsets as well as its effects on CD4þ T cells.Given the ability of IL-27 to influence class-switching and TFH develop-ment, this cytokine may be useful as an adjuvant of vaccine-inducedhumoral immunity, although this hypothesis has yet to be directly tested.

3. ANTI-INFLAMMATORY PROPERTIES OF IL-27

While early reports focused on the ability of IL-27 to promote TH1 immu-nity, subsequent studies, primarily using parasitic systems, revealed theimmune suppressive effects of IL-27. This section summarizes that early


work and how these studies led to our current understanding of themechanisms utilized by IL-27 to limit an array of inflammatory responses.

3.1. IL-27 and type I responses

The sections above highlight the proinflammatory properties of IL-27 inpromoting TH1 activity, CTL responses, inhibiting Tregs, and promotinghumoral immunity. Based on these data, one would predict that theabsence of IL-27 signaling would lead to a failure to generate type Iresponses and humoral immunity, and that Treg responses would beenhanced, resulting in a decreased ability to control pathogens. However,there is now a preponderance of in vivo evidence that this is not the case(Table 1.1). Studies with an array of parasitic (T. gondii, Leishmania donova-nii, T. cruzi, Plasmodium bergheri) (Artis et al., 2004a; Findlay et al., 2010;Hamano et al., 2003; Rosas et al., 2006; Villarino et al., 2003), bacterial (M.tuberculosis) (Holscher et al., 2005; Robinson and Nau, 2008), viral (influ-enza) (Sun et al., 2011), and autoimmune models of inflammation(Table 1.2) (lupus (Igawa et al., 2009; Shimizu et al., 2005; Sugiyama et al.,2008), colitis (Sasaoka et al., 2011; Troy et al., 2009), asthma (Dokmeci et al.,2011;Miyazaki et al., 2005; Shimanoe et al., 2009; Yoshimoto et al., 2007),MS(Batten et al., 2006; Diveu et al., 2009; Fitzgerald et al., 2007), and hepatitis(Frank et al., 2010)) show that IL-27 is a critical negative regulator of thepathology associated with these models.

The ability of IL-27 to limit TH1 responses is highlighted by studieswhich showed that IL-27Ra�/� mice infected with the protozoan parasiteT. gondii developed a lethal CD4þ T cell-mediated immune-pathology(Villarino et al., 2003). Although IL-27Ra�/� mice infected with T. gondiisuccumbed within 2 weeks of challenge, they efficiently controlled para-site replication, but actually had enhanced CD8þ and CD4þ T cellresponses that were associated with increased production of IFN-g. Exa-cerbated TH1 responses and disease pathology are characteristic of otherstudies with the IL-27Ra�/� mice such as those following infection withT. cruzi, P. bergheri, and L. donovanii (Findlay et al., 2010; Hamano et al.,2003; Rosas et al., 2006). Initial evidence of the ability of IL-27 to limitmultiple classes of T helper cells came from studies with T. cruzi. In thismodel, IL-27Ra�/� mice infected with T. cruzi developed enhanced TH1and TH2 activities, and the elevated IL-4 contributes to the higher parasiteburden in these mice. In contrast, the heightened TH1 activity in thesemice resulted in severe immune pathology, and exacerbated IFN-g pro-duction contributes to liver injury and lethality (Hamano et al., 2003).Recent studies looking at the role of IL-27 during infection with P. bergherireveal that IL-27 can prevent CD4þ T cell-mediated pathology, which ischaracterized by elevated IFN-g, IL-17, and TNF-a (Findlay et al., 2010).Similar findings have also been observed following infection of IL-27Ra�/�


animals withM. tuberculosis, L. major and L. donovanii (Anderson et al., 2009;Holscher et al., 2005; Rosas et al., 2006).

The mechanism by which IL-27 limits TH1 responses to intracellularpathogens is not completely understood, especially since early studiesindicated a role for promoting TH1 immunity. However, in the studieswith T. gondii, it was clear that depletion of CD4þ T cells could rescue acutelethality in these mice, suggesting that IL-27 directly limits TH1 cells(Villarino et al., 2003). Consequently, the suppressive role of IL-27 in vivohas been attributed to some of the pleiotropic anti-inflammatory proper-ties of IL-27, such as its ability to limit IL-2 or IFN-g production by CD4þ

T cells and promote T cell expression of the potent anti-inflammatorycytokine IL-10 (Anderson et al., 2009; Batten et al., 2006; Diveu et al., 2009;El-behi et al., 2009; Murugaiyan et al., 2009; Stumhofer et al., 2006; Villarinoet al., 2006, 2010; Yoshimura et al., 2006). It is also worth noting that theinhibitory effects of IL-27 on T cells could be indirect. Studies withM. tuberculosis suggest that IL-27Ra deficient macrophages in this modelproduce more IL-12p40, IL-6, and TNF-a which could manifest as exacer-bated TH1 responses (Holscher et al., 2001, 2005; Robinson andNau, 2008).

Another example of the anti-inflammatory properties of IL-27 isillustrated by studies in the MRL/lpr mouse model of systemic lupuserythematosus (SLE). These mice, which have defective apoptosis(Watanabe-Fukunaga et al., 1992), develop disease characterized by anti-DNA antibodies and mixed T helper response including TH1, TH2, andTH17 cells (reviewed in Fairhurst et al., 2006; Shin et al., 2011).MRL/lprmicetransgenic for the IL-27Ra have less severe disease characterized by reducedglomerulonephritis, decreased production of IFN-g and IL-4, lower levels ofanti-dsDNA antibodies, and less severe skin inflammation, associated withincreased survival compared to nontransgenic animals (Sugiyama et al.,2008). Furthermore, the lack of IL-27 signals in MRL/lpr mice results inexacerbated disease characterized by more severe skin lesions resemblinghuman SLE disease (Kido et al., 2011). These examples illustrate the broadand sometimes synergistic anti-inflammatory properties of IL-27 duringTH1 responses but much of the mechanism by which this occurs is stillunknown and may differ depending on the inflammatory context. None-theless, in othermodels such as TH2 and TH17 immunity it is apparent thatIL-27 can exert its effects in a more direct fashion, described below.

3.2. IL-27 and type 2 responses

One of the earliest studies with the IL-27Ra deficient mice challengedthemwith L. major, a parasite that requires type I immunity for protection.The course of disease in these mice was characterized by a markedincrease in the TH2 response, reduced production of IFN-g, and an earlybut transient inability to control this infection (Yoshida et al., 2001; Zahn


et al., 2005). One interpretation of these findings was that IL-27 wasimportant for the development of TH1 immunity to this parasite. Animportant characteristic of this model system is that while IFN-g isrequired to limit this parasite, TH2 cells dominate during the earlyphase of this infection. As noted earlier, the complex relationship betweenTH1 and TH2 cells canmake it difficult to interpret these early studies andadditional studies suggested that the initial failure of these mice to controlL. major was not because IL-27 was required for the genesis of TH1 cellsbut rather that the IL-27Ra�/� mice were unable to downregulate theearly TH2 response (Anderson et al., 2009; Artis et al., 2004a). Thus, whenIL-27Ra�/� mice were treated with a-IL-4 antibodies prior to infectionwith L. major, they developed normal parasite specific TH1 cells andcontrol of this infection. Thus, these studies demonstrate that the earlysusceptibility in these mice was not due to a defect in TH1 immunity, butrather a consequence of unrestrained TH2 responses.

In the setting of other TH2-centered experimental systems, IL-27 hasalso emerged as a critical negative regulator and IL-27-deficient mice haveenhanced protective immunity to helminth infection (Artis et al., 2004b)and increased IL-4 production in response to T. cruzi (Hamano et al.,2003). During the induction of experimental asthma, IL-27 can also playa protective role by limiting TH2 responses (Dokmeci et al., 2011; Fujitaet al., 2009; Miyazaki et al., 2005; Yoshimoto et al., 2007). This effect hasbeen linked to the ability of invariant NKT cells to produce IL-27 tonegatively regulate TH2 responses (Fujita et al., 2009). Finally, adminis-tration of IL-27 during experimental asthma ameliorated disease, suggest-ing a novel therapeutic approach for the treatment of T cell-mediatedautoimmunity (Miyazaki et al., 2005; Yoshimoto et al., 2007). The molecu-lar basis of the effect of IL-27 on developing TH2 responses are revealedby studies which showed that IL-27 antagonized expression of the masterregulator of TH2 responses, GATA3, and so provided a mechanisticinsight into these effects (Lucas et al., 2003).

3.3. IL-27 inhibits TH17 responses

The ability of IL-27 to attenuate T cell differentiation is not limited to TH1or TH2 responses, as multiple reports have shown that IL-27 also mod-ulates TH17 activities (Anderson et al., 2009; Diveu et al., 2009; El-behiet al., 2009; Fitzgerald et al., 2007; Liu and Rohowsky-Kochan, 2011;Murugaiyan et al., 2009; Sasaoka et al., 2011; Stumhofer et al., 2006; Troyet al., 2009; Villarino et al., 2010; Yang et al., 2008b; Yoshimura et al., 2006).TH17 cells, characterized by their production of IL-17 and IL-22, areassociated with protective immunity to a number of extracellular patho-gens such as Candida albicans. Most notably, however, TH17 cells arecausally associated with in a number of autoimmune disease states such


as in rheumatoid arthritis (characterized by proinflammatory autoreac-tive TH17 cells (Nakae et al., 2003), inflammatory bowel disease (TH17cells can induce colitis (Elson et al., 2007)), and MS (TH17 cells areassociated with increased EAE scores (Komiyama et al., 2006)), alsoreviewed in Korn et al., 2009).

Following infection with L. major, L. monocytogenes, or during chronictoxoplasmosis in IL-27Ra�/� or EBI3�/� mice, exacerbated disease hasbeen associated with the increased presence of TH17 cells (Anderson et al.,2009; Stumhofer et al., 2006; Yang et al., 2008a). Of particular relevance,these studies have identified a role for IL-27 in limiting the expansion ofTH17 cells during chronic inflammation, and highlight the opposing rolesof IL-6 and IL-27 on the generation of TH17 cells: IL-6 promotes TH17development whereas IL-27 inhibits IL-17 production (Batten et al., 2006;Fitzgerald et al., 2007; Stumhofer et al., 2006). Notably, IL-27p28- or WSX1-deficient mice develop more severe EAE, associated with enhanced TH17responses, demonstrating the significance of this pathway in diseasedevelopment (Batten et al., 2006; Diveu et al., 2009; Fitzgerald et al., 2007).

The mechanistic basis underlying the inhibitory effects of IL-27 duringTH17 responses are not entirely clear, but several studies have providedsome insights into these events. Thus, during in vitro TH17 cultures(a-CD3, a-CD28, IL-6, and TGF-b), IL-27 can block the production ofIL-17 from CD4þ T cells, in a STAT1 and partially STAT3 dependentmanner (Stumhofer et al., 2006). This is independent of the ability ofIL-27 to upregulate T-bet, IL-10, and Socs3 which are facets of theanti-inflammatory properties of IL-27 in other systems (Liu andRohowsky-Kochan, 2011; Stumhofer et al., 2006; Yoshimura et al., 2006).However, in the absence of STAT1 signals there may be a role for T-bet indirectly inhibiting TH17 responses, as evidenced by recent studies byVillarino et al. (2010). Furthermore, during in vitro polarization of TH17cells, IL-27 can directly inhibit the expression of RORa and RORgt, twotranscription factors associated with TH17 development and inhibit pro-duction of IL-22 a cytokine which is important for TH17 effector function(Diveu et al., 2009; El-behi et al., 2009; Yang et al., 2008a).

IL-27 can inhibit the de novo differentiation of TH17 cells althoughthere is some debate as to whether IL-27 can also limit fully differentiatedTH17 cells. Some of the disparities in these studies may be due to thedifferences between mouse and human TH17 cells or the sources of thememory cell populations (El-behi et al., 2009; Liu and Rohowsky-Kochan,2011). For instance, in the human studies, IL-27 was capable of inhibitingproduction of IL-17 by polyclonal CD45ROþ memory cells and when itwas present during the induction phase of TH17 polarizing cultures (Liuand Rohowsky-Kochan, 2011). Similarly, during toxoplasmic encephali-tis, IL-27 can block IL-17 secretion by effector CD4þ and CD8þ T cellsisolated from the CNS of infected mice (Stumhofer et al., 2006). However,in an assay where the memory cells were isolated from mice with EAE,


IL-27 could not inhibit IL-17 production (El-behi et al., 2009). Thus,while IL-27 may be a promising therapeutic target for the treatment ofpathological TH17 effectors, fully understanding the effects of IL-27 ondeveloping and existing pathogenic TH17 responses will be critical indetermining how to tailor its use therapeutically.

3.4. IL-27 promotes production of IL-10

In addition to the broad anti-inflammatory effects of IL-27 in directly limit-ing TH2 and TH17 effector cells, it also supports the in vitro and in vivogeneration ofCD4þ andCD8þT cells andNKcells thatmake IL-10 (Awasthiet al., 2007; Fitzgerald et al., 2007; Laroni et al., 2011; Stumhofer et al., 2007).IL-10 is a potent anti-inflammatory cytokine, with a critical role in limitingimmune pathology (reviewed in Ouyang et al., 2011). Studies with proto-zoan parasite models such as Plasmodium spp., T. cruzi, and T. gondii exem-plify the requirement for IL-10 production during infection (Findlay et al.,2010; Gazzinelli et al., 1996; Hunter et al., 1997; Jankovic et al., 2007; Neyeret al., 1997; Wilson et al., 2005, and reviewed in Couper et al., 2008). Whilethere are many sources of IL-10, CD4þ T cells are a key factor in this processand studies from Alan Sher’s group highlighted the critical role for T cellderived IL-10 in limiting infection-induced pathology caused by T. gondii,but the signals that promote their production of IL-10were unclear. The firstindication that IL-27 was involved in the regulation of IL-10 was providedby studies in IL-27Ra�/� mice with toxoplasmic encephalitis or EAE. Inthese models, the enhanced pathology observed in the absence of IL-27 alsocorrelated with reduced levels of IL-10 (Fitzgerald et al., 2007; Stumhoferet al., 2007). These findings suggest a role for IL-27 in this process and in vitrostudies established that IL-27was able to directly promote T cell (TH1, TH2,TH17, Treg) production of IL-10 (Stumhofer et al., 2007). These observationswere part of a series of studies that solidified a role for IL-27 in this processusing different experimental approaches (Awasthi et al., 2007; Fitzgeraldet al., 2007; Stumhofer et al., 2007). In the EAE studies, Fitzgerald, Rostami,and colleagues used an adoptive transfer model to show that normallyencephalitogenic T cells failed to induce disease when treated with IL-27(Fitzgerald et al., 2007). This was found to be IL-10 dependent, as IL-10knockout T cells treated with IL-27 induced disease (Fitzgerald et al.,2007). Studies from Awasthi, Kuchroo, and Weiner demonstrated thatDCs exposed to inducible Treg became tolerogenic, and were a potentsource of IL-27 that promoted the expression of IL-10 by CD4þ T cells(Awasthi et al., 2007). It should be noted that IL-6 in combination withTGF-b can also promote the production of IL-10 from CD4þ T cells(McGeachy et al., 2007; Stumhofer et al., 2007) and that TGF-b in additionto IL-27 can augment IL-10 production by CD4þ T cells (Stumhofer et al.,2007). These IL-27-induced regulatory cells (‘‘TR1’’ cells) express IL-10, andare distinct from Treg in that they do not express the transcription factor


Foxp3 reviewed in Pot et al. (2010). Whether these cells represent a distinctlineage of T helper cells or are a population that transiently expresses IL-10,similar to effector cells, remains to be determined. Nonetheless, onemecha-nism reported to promote the development of TR1 cells has been shown tobe indirect, through Foxp3þ Treg, and their ability to induce a regulatorysubset of DC that make IL-27, that in turn can promote TR1 cells (Awasthiet al., 2007).

In order to understand the mechanism by which IL-27 induced IL-10in CD4þ T cells, initial studies examined the role of STAT1 and STAT3 inthis process and suggested that both transcription factors were involvedin the ability of IL-27 to induce IL-10 in CD4þ T cells (Stumhofer et al.,2007). However, the role for these signaling molecules when TGF-b waspresent was not examined and recent studies illustrate that there are TGF-b dependent and independent pathways that can promote the develop-ment of TR1 cells. Thus, when TGF-b is absent, the differentiation of TR1cells in the presence of IL-27 is STAT1 dependent (Xu et al., 2009). Incontrast, when TGF-b is present, STAT1 is not required for the IL-27mediated induction of IL-10 (Stumhofer et al., 2007). Understandingthese events is further complicated by reports that IL-27 induces expres-sion of the ligand-activated transcription factor aryl hydrocarbon receptor(Ahr) and the transcription factor c-Maf, both of which trans-activate theIL-10 and IL-21 promoters (Apetoh et al., 2010). Interestingly, IL-21 hasemerged as an important cofactor for IL-10. Both IL-6 and IL-27 can inducethe production of IL-21 by CD4þ T cells, which acts as an autocrine growthfactor that is necessary for the expansion of TR1 cells (Pot et al., 2009).

One area that these studies highlight is the relationship between IL-6and IL-27 in promoting T cell production of IL-10. Although IL-6 may giverise to a population of cells that resemble IL-27-induced TR1 cells,whether they have redundant or complementary roles in vivo remains tobe determined. Nonetheless, IL-27 contributes to the development of IL-10 producing CD4þ T cell populations during EAE (Fitzgerald et al., 2007),or following infection with L. major (Anderson et al., 2009), T. gondii(Stumhofer et al., 2007), or L. monocytogenes (Batten et al., 2008). As thestudies using IL-27Ra�/� mice suggest non-redundant roles for IL-27exist, it remains to be determined if there is an in vivo role for IL-6 inthis process independently of IL-27.

4. THE ROLE OF IL-27 IN OTHER CELL TYPES

4.1. IL-27 and innate immunity

While the majority of studies on IL-27 have focused on its role in theregulation of adaptive immunity, this cytokine is part of an evolutionaryconserved pathway that has a prominent role in innate immunity. For


example, Drosophila and Caenorhabditis elegans have gp130 orthologuesand downstream JAK/STAT signaling associated with resistance to infec-tion, although no orthologues of the IL-27 or IL-27R subunits have yetbeen described in non-vertebrates (Huising, 2006). Nonetheless, innatecells are the major sources of IL-27 (described earlier) and multiple innatepopulations express gp130 and IL-27Ra. This includes NK and mast cellsas well as eosinophils and macrophages (Artis et al., 2004b; Pflanz et al.,2004; Villarino et al., 2005), and these cell types show a range of biologicalresponses to IL-27. For example, IL-27 has been linked to the ability ofhuman eosinophils to produce cytokines and chemokines (Hu et al., 2010).However, the role of IL-27 in mast cell biology is not clear. Pflanz andcolleagues demonstrated that human primary mast cells respond to IL-27,initiate STAT3 activation, and turn on the transcription of proinflamma-tory cytokines such as IL-1a, IL-1b, and TNF-a, but did not enhance Fcreceptor-mediated degranulation (Pflanz et al., 2004). Furthermore, in amodel of passive cutaneous anaphylaxis, IL-27Ra�/� mice have elevatedmast cell protease activity (Artis et al., 2004b), suggesting that IL-27 maylimit mast cell mediated inflammation. In contrast, recent studies havehighlighted that although mouse mast cells express IL-27Ra, they arelargely unresponsive to this cytokine, a finding that was attributed toincomplete glycosylation of gp130 and retention within the cytoplasm(Traum et al., 2011).

For other granulocytes, elevated neutrophil activity has been noted inthe absence of IL-27. Following cecal ligation and puncture, IL-27�/�miceare more resistant to sepsis caused by the leakage of bacteria into theperitoneal cavity, and this phenotype is associated with accelerated neu-trophil recruitment and reduced bacterial loads (Wirtz et al., 2006). Thesedata sets are consonant with the idea that the absence of IL-27 allowsneutrophils to reach the critical concentration that is essential for thecontrol of bacterial growth (Li et al., 2004). Regardless, it has been difficultto distinguish whether these enhanced granulocyte responses observed indifferent models were simply a secondary consequence of altered inflam-matory responses or directly limited by IL-27. For example, in the settingof innate immunity, IL-27 is an antagonist of NK cell production of IL-17(Passos et al., 2010), a cytokine that promotes neutrophil mobilization andcould contribute to the increased neutrophil response observed inIL-27Ra�/� animals.

In terms of macrophages and DC, there is experimental evidence forthe ability of IL-27 to inhibit murine DC during leishmaniasis (Wang et al.,2007), and studies with human DC describe the ability of IL-27 to promoteDC expression of B7-H1 (PD-L1), a molecule that provides suppressivesignals to T cells (Karakhanova et al., 2011). While there are reports thatresting mouse macrophages are not responsive to IL-27 (Kalliolias andIvashkiv, 2008), others have found that IL-27 inhibits the ability of murine


macrophages to produce IL-12 and TNF-a (Holscher et al., 2005) and canpromote macrophage production of IL-10 (Iyer et al., 2010). Whether theability of IL-10 to antagonize IL-12 production is part of an autocrine loopthat allows IL-27 to directly regulate accessory cell function has not beenaddressed.

In human macrophages, IL-27 can inhibit their responsiveness toproinflammatory cytokines such as IL-1 and TNF-a by down-regulatingexpression of their cognate receptors (Kalliolias et al., 2010a). In humanmonocytes, a different paradigm is emerging and Ivashkiv and colleagueshave found that in these cells, IL-27 signaling is associated with a proin-flammatory signature (Kalliolias and Ivashkiv, 2008). Consistent with thisactivity, IL-27 has been linked to the induction of type I IFNs that caninhibit the replication of HIV in macrophages. IL-27 can also inhibit theability of CD14þ monocyte precursors to generate osteoclasts (Kallioliaset al., 2010b) an activity that may be dependent on the production of type IIFNs (Greenwell-Wild et al., 2009; Imamichi et al., 2008; Kurihara andRoodman, 1990). Clearly, the studies described in this section highlightthe impact of IL-27 on innate populations, but dissecting cell intrinsiceffects can be difficult. The ability to use lineage specific deletions of theIL-27R in different innate populations will facilitate a better dissection ofthe effects of IL-27 on these innate cells in vivo.

4.2. IL-27 and non-hematopoietic cells

Although most of the studies examining the functions of IL-27 have beencentered on hematopoietic cells, there is also evidence that IL-27 can haveeffects on non-hematopoietic populations such as epithelial cells, fibro-blasts and keratinocytes. In the cancer literature, there are studiesshowing that IL-27Ra is expressed on epithelial tumors derived fromthe colon, breast and melanocytes, and that in certain settings, IL-27 canhave direct effects on these cells (Dibra et al., 2009a, 2011; Yoshimoto et al.,2008). In the setting of systemic sclerosis (SSc), IL-27 is hypothesized toplay a role in disease development because SSc patients express elevatedlevels of IL-27Ra in fibroblasts and IL-27 promotes fibroblast proliferationand the production of collagen (Yoshizaki et al., 2011). Another studyreported that during chronic eczema, human keratinocytes expressIL-27 and respond to IL-27 which triggers increased MHC Class I expres-sion and the production of the chemokine CXCL10 (Wittmann et al., 2009).IL-27 can also play a beneficial role in maintaining barrier function as wellas stimulating intestinal epithelial cells to express the scavenger receptorDMBT1 that can bind to a variety of Gram-positive and -negative bacteriaand act as an antimicrobial peptide (Diegelmann et al., 2011). Takentogether, the studies described in this section illustrate some of theeffects of IL-27—potentially beneficial or detrimental to the host—on


non-hematopoietic cells and thus need to be taken into considerationwhen examining the role of IL-27 in different settings.

5. TRANSLATIONAL IMPLICATIONS OF IL-27

5.1. Targeting IL-27 pathways for therapies

With the initial studies linking IL-27 to the development of TH1responses, it made sense that neutralizing IL-27 might be a useful strategyto limit inflammatory conditions associated with increased production ofIFN-g. Alternatively, the properties of IL-27 which promote TH1responses might be useful as part of an adjuvant to promote cell mediatedimmunity, and as discussed earlier, there is now a literature that supportsits use in cancer vaccines. Nonetheless, despite a better understanding ofthe role of endogenous IL-27 as an inhibitor of inflammation in manydisease settings, questions remain about which therapeutic approachesfor IL-27 are most likely to be successful. For example, IL-12 and IL-27have proven to be effective in similar mouse models of cancer, but inhumans, IL-12 has been associated with severe toxicity leading to a halt inclinical trials of IL-12 (Cohen, 1995; Leonard et al., 1997; Marshall, 1995).To date, there are no reports of toxicity caused by treatment of mice withIL-27 and so IL-27 may represent an attractive alternative to IL-12 as atherapeutic in the setting of cancer. Indeed, studies in which DNA mini-circles that express IL-27 in combination with IL-2 were given to micebearing neuroblastoma resulted in tumor regression, suggesting that IL-27 could be utilized in the clinical setting (Salcedo et al., 2009). Further-more, treatment with IL-27 can also attenuate collagen-induced arthritis(CIA) (Niedbala et al., 2008), and local delivery of an adenovirus expres-sing IL-27 into the ankles of mice with CIA also resulted in improveddisease scores (Pickens et al., 2011). There are also reports that the use ofblocking reagent, such as the IL-27R-Fc fusion protein, can limit diseaseprocesses, such as peritoneal sepsis (Wirtz et al., 2006). While the potentialapplication of these findings to the clinical setting are promising, it isimperative to consider the possible negative side-effects of altering levelsof bioactive IL-27. For instance, long-term ablation of IL-27 signals mayalso yield unwanted effects such as autoimmunity. Alternatively, admin-istration of IL-27, while beneficial in limiting pathology, in other contextscould limit protective immune responses.

In the context of murine models of EAE, several reports have identi-fied a role for IL-27 in limiting disease (Fitzgerald et al., 2007; Guo et al.,2008; Wang et al., 2008). In these experimental systems, IL-27 has beenshown to contribute to the efficacy of type I IFN therapy, a treatment thatis used in human patients to treat MS, but which is associated with


significant side effects (Biggioggero et al., 2010; Sweeney et al., 2011).Although type I interferons can promote the production of IL-27, it wassomewhat surprising that the therapeutic effects of type I interferontreatment were dependent on the induction of IL-27p28 (Guo et al., 2008;Shinohara et al., 2008). Thus, these findings suggest that the clinicalefficacy of IFN-b in patients with MS may be attributed to its ability toinduce IL-27 (or the IL-27p28 monomer). Therefore, the use of IL-27 mayrepresent an alternative strategy to manage this condition, without theside effects associated with the type I IFNs. Indeed, when others haveutilized a format that allows sustained delivery of IL-27, such as throughosmotic pumps, delayed the onset of EAE, and amelioration of estab-lished EAE was observed (El-behi et al., 2009; Fitzgerald et al., 2007).Similarly, the use of DNA mini-circles to force transient high levels ofIL-27 completely blocked the development of EAE (Stumhofer et al., 2010).

While IL-27 has not been used in any human therapies to date, thestudies above illustrate the potential impact of IL-27 in clinical settings.One consideration for the use of any cytokine treatment is the relativelyshort half lives of these proteins. In our experience treatment naı̈ve micewith IL-27 has had no adverse effects on the immune system (Tait Wojnoet al., 2011). In contrast, while the strategies described above, using tran-sient or local expression of IL-27, have shown the remarkable therapeuticpotential of IL-27, transgenic overexpression of IL-27 results in severedisease associated with defects in myelopoesis and/or the loss of Tregpopulations (Seita et al., 2008; Wojno et al., 2011). While these latter modelsmay not relate directly to the clinical use of IL-27, they do illustrate thecomplex biology of IL-27, and as for many cytokines, the challenge is findoptimal ways to deliver IL-27 to the appropriate cellular or tissue targets.For instance, the ability of IL-27 to inhibit inflammatory pathways asso-ciated with the development of inflammatory bowel disease (IBD) andCrohn’s disease (CD) means that strategies to deliver IL-27 in the gutmight be a viable therapy. Indeed, Durum and colleagues (personalcommunication) have used the Gram-positive bacteria Lactococcus lactisto transiently express IL-27 in the gut in a model of colitis and foundremarkable protective effects.

The use of nanoparticles to deliver IL-21 to T cells has been highlightedrecently (Stephan et al., 2010), and development of similar approacheswith IL-27 may provide platforms that allow very specific cell popula-tions to be targeted. Cytokine ‘‘engineering,’’ involving modifying thestructure or route of delivery, represents another potential approach touse IL-27 therapeutically and there is precedent for this strategy with IL-6.The fusion of IL-6 with its soluble receptor results in a ‘‘hyperkine’’ whichis capable of binding to gp130 and propagating signals that are a log foldmore potent than IL-6 (Rakemann, 1999). Thus, one potential option is toengineer novel forms of IL-27 that can preferentially interact with different


elements of the IL-27R, but whether a similar strategy to that usedwith IL-6 would be applicable to IL-17 is not known. One study utilizing a mutantform of IL-27, in which the p28 subunit could not interact with gp130,revealed that this mutant protein acted as a receptor antagonist andlimited TH1 mediated liver damage (Rousseau et al., 2010). A parallelstudy demonstrated that IL-27p28 could act as a receptor antagonist ofgp130 and transgenic expression of this subunit limited humoralresponses (Stumhofer et al., 2010). Regardless, while the crystal structuresof the interactions of IL-6 with gp130 and the IL-6Ra provide a templatefor the likely interactions of IL-27 with its receptor subunits, there is stilla need for additional structure function studies that would informthe development of altered versions of IL-27 that could act as receptoragonists or antagonists.

5.2. The role of IL-27 in human disease

Given the broad roles that cytokines play in shaping all aspects of innateand adaptive immunity, there has been a concerted effort toward identi-fying cytokine and cytokine receptor single nucleotide polymorphisms(SNPs) associated with human disease. For instance, polymorphisms inIL-10 have been strongly linked to susceptibility to lupus, asthma, andarthritis reviewed in Hollegaard and Bidwell (2006). Until recently, therewas a paucity of information about whether IL-27 or the IL-27R wasassociated with human disease, but several studies have highlightedpolymorphisms in IL-27p28 that are associated with autoimmunity. Oneof the first SNPs identified in IL-27p28 linked to disease (g.-964A > G)was associated with susceptibility to asthma and increased IgE and eosin-ophilia (Chae et al., 2007). This same polymorphism in IL-27 has also beenassociated with susceptibility to chronic obstructive pulmonary disease(COPD) and IBD (Huang et al., 2008; Li et al., 2009). Another studyutilizing genome-wide association studies and high-density SNP analysisidentified two loci near the IL-27p28 gene that were in linkage disequilib-rium (rs8049439 and rs1968752) with the onset of CD and ulcerative colitis(UC). These studies showed that the A allele of rs1968752 was statisticallyassociated with decreased mRNA expression of IL-27 in lymphoblasticcell lines and colonic epithelium taken from patients with early-onset CDand UC, thus providing evidence that IL-27 may play a protective role inpreventing these autoimmune diseases (Imielinski et al., 2009). However,the recent findings which illustrate that IL-27p28 can act as a receptorantagonist, may complicate understanding how polymorphisms in thisgene impact human disease. While the mechanisms by which these dis-ease states are affected by polymorphisms in IL-27p28 have yet to bedetermined, studies examining the role of STATs in human disease maybe informative. For instance, a gain-of-function mutation in STAT1


enhanced the ability of IL-27 to suppress TH17 responses, which in turnwas associated with reduced production of IL-17 and increased suscepti-bility to fungal pathogens (Liu et al., 2011; van de Veerdonk et al., 2011).Furthermore, polymorphisms in STAT3 are associated with Crohn’s dis-ease and these patients may provide insights into the role of IL-27 in thisdisease (Franke et al., 2008).

6. CONCLUSIONS

It has been more than 15 years since the identification of IL-27Ra andEBI3, and in that time there has been tremendous progress in understand-ing the biology of IL-27 and an appreciation of its contradictory nature inpromoting or inhibiting inflammatory processes. As highlighted through-out this review, major questions remain about identifying relevantsources of IL-27 and defining how its production is regulated in vivo.An appreciation of these events would in turn impact on understandingwhether endogenous IL-27 influences the most proximal events in theinitiation of T cell responses or simply acts as a modulator of effector andregulatory T cell populations. This work also relates to larger questions ofhow immune cells in different states of activation can interpret similarsignals (such as those provided by IL-27 vs. IL-6) to provide diverseoutcomes. The effects of IL-27 on cells other than T cells, such as B cells,mast cells, and non-hematopoietic cells have been less extensively stud-ied, but it is clear that IL-27 does modulate these populations. Thus, it isimportant to contemplate these effects when considering the use of IL-27as a therapy. In the same vein, JAK/STAT inhibitors are being developed,ostensibly to block proinflammatory effects of STAT signaling (O’Sheaet al., 2011). The studies that described the ability of IL-27 to limit TH2 andTH17 responses, and promote the production of IL-10, and data fromhuman patients with gain-of-function STAT1 mutations, highlight thatthese pathways are also a component of critical regulatory pathways thatare essential for limiting immune responses (Liu et al., 2011). Thus, there isthe possibility that JAK/STAT inhibitors will interfere with endogenousinhibitory activities of IL-27, and the clinical trials with these compoundshave significant potential to further inform us about the biology of IL-27.

ACKNOWLEDGMENTS

This work is supported by in part by NIH grant CAH: AI42334 and AOH: 5T32AI055428-09.


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