IL-1O (interleukin-10) negatively regulates inflammation through a mechanism that blocks the expression of pro-inflammatory genes encoding cytokines, chemokines, cell-surface molecules and other molecules required for the full activation of the innate and adaptive immune responses. The signalling pathway used by the IL-10 receptor to generate the anti-inflammatory response requires STAT3 (signal transducer and activator of transcription 3) and is indirect. Thus STAT3 activates other genes whose task is to selectively control transcription of inflammatory targets. Here, I summarize current knowledge of the key features of IL-10 signalling and make predictions concerning the mechanism of IL-10 at the level of inflammatory genes. Understanding IL-10 signalling should be a gateway to the development of broadly acting anti-inflammatory agents.

Overview

Both the adaptive and innate arms of the mammalian immune response have developed multiple extrinsic and intrinsic mechanisms to regulate, inhibit and terminate responses to pathogens. Intrinsic inhibitory mechanisms include the regulation of signal transduction, for example by the action of phosphatases, ubiquitin ligases or negative feedback loops such as the elevation in IκB (inhibitory κB) synthesis to inhibit NF-κB (nuclear factor κB) signalling. Extrinsic mechanisms encompass external cues from one cell to another that lead to inhibitory activity. The subject of the present paper is the extrinsic pathway mediated by the inhibitory cytokine IL-10 (interleukin-10). IL-10 is an essential negative regulatory cytokine of myeloid-derived innate immune cells such as macrophages and dendritic cells that have been activated by pathogen-derived signals. IL-10 is required to regulate these cells in both acute and chronic immune and inflammatory responses through molecular mechanisms that are beginning to be understood.

Immune regulation and disease

Failure of immune regulation is common to many diseases. For example, chronic bacterial, parasitic and viral infections, autoimmune diseases and acute and chronic inflammation are all caused by, or associated with, inappropriate immune cell activation. Because of the clinical significance and breadth of inflammatory diseases, biological agents and drugs that target inflammation are a major component of the modern pharmacopoeia. For example, the use of anti-TNFα (tumour necrosis factor α) monoclonal antibodies, or soluble receptors to reduce the pro-inflammatory effects of TNFα in rheumatoid arthritis and Crohn's disease have been successful for large numbers of patients suffering from these inflammatory syndromes [1]. IL-10 is a strong anti-inflammatory cytokine, but why has it not been developed as a useful drug? The answer to this question lies in both the side effects and off-target effects of immunologically active cytokines administered systemically and in the complex biology of the IL-10 signal transduction system [2]. Although IL-10 itself is unlikely to be a useful anti-inflammatory agent, its signal transduction pathway should yield new information about anti-inflammatory signalling that can be exploited for future use.

Role of IL-10 at the organismal level

Both loss- and gain-of-function approaches in mouse models have been used to address the in vivo function of IL-10 in multiple homoeostatic and disease contexts [3]. Collectively, these studies reinforce the idea that the absence of IL-10 propels inflammatory responses forward, often to extremes that result in death. For example, mice lacking IL-10 are susceptible to challenge with bacterial endotoxin due to a massive systemic outpouring of pro-inflammatory cytokines including TNFα, IL-1α and IL-12 [4,5]. Similarly, IL-10-deficient mice infected with parasites that provoke acute inflammatory responses, such as Trypanosoma cruzi [6] and Toxoplasma gondii, are mortally challenged [7]. Death of the mice was due not to excessive parasite growth but instead to excessive inflammatory responses overproducing pro-inflammatory cytokines when challenged. In contrast, transgenic mice engineered to constitutively express IL-10 from macrophages are protected from endotoxin-induced shock and make less pro-inflammatory cytokines [8]. In these examples, IL-10 is predominantly produced by macrophages and dendritic cells and functions in an autocrine/paracrine way to reduce the amplitude of the inflammatory response.

In contrast with the acute inflammatory reactions described above, the function of IL-10 in chronic infections and inflammation has been more complex to dissect because the source of IL-10 is largely shifted to T-cells, and in particular, regulatory T-cell subsets [9]. The chronic (but not acute) inflammatory phenotypes in mice lacking IL-10 in all cells are recapitulated in mice lacking IL-10 only in T-cells, suggesting that T-cells are the major source of IL-10 in established inflammatory responses [10]. Therefore T-cell-mediated IL-10 production is critical in chronic inflammatory diseases.

The clearest example of the importance of IL-10 production from regulatory T-cells comes from the chronic Leishmania infection model where administration of neutralizing antibodies to the IL-10R (IL-10 receptor) is sufficient to eliminate residual numbers of parasites [11]. These results suggest that IL-10 is the key factor in ability of parasites to persist because of the anti-inflammatory effects on macrophages, the host cell of the parasite. Superficially, it would appear that IL-10 is deleterious to the ability of the host to eliminate a chronic infection. However, once IL-10 signalling was ablated and the parasites cleared, a loss of parasite-specific T-cells occurred: these mice were easily re-infected with Leishmania [11]. The Leishmania model encapsulates the idea, at least for some pathogens, that maintaining a chronic infection is preferable to fulminant infection.

Overview of the biological effect of IL-10 at the level of the cell

Shortly after the discovery of IL-10, several groups showed that IL-10 had an inhibitory effect on cytokine and chemokine production from activated macrophages [1214]. In these experimental systems, variants of which are used today, macrophages or dendritic cells are activated with Toll-like receptor agonists (typically LPS) in the presence or absence of IL-10. IL-10 inhibits the production of numerous inflammatory mediators, but not to the same extents or with the same kinetics. For example, IL-10 almost completely inhibits IL-12 production but is a weaker inhibitor of TNFα. The basis for these differences lies in the differential effects of IL-10 on inflammatory transcription (discussed below).

Many contradictory and confusing results have been published to account for the IL-10 mechanism and have recently been debated [3,1518]. However, a number of recent findings have addressed several outstanding issues concerning the IL-10 mechanism. First, an important source of IL-10 in the assays used to probe IL-10 function is macrophages or dendritic cells themselves. Indeed, autocrine/paracrine IL-10 production from LPS-stimulated bone marrow-derived macrophages can be prodigious and inhibit macrophage cytokine and chemokine production in the absence of exogenously added IL-10. For this reason, we have restricted our studies of the IL-10 mechanism to the use of macrophages from IL-10-deficient mice where IL-10 can be added back without concern for background IL-10 production [19]. Secondly, microarray analysis comparing LPS-activated macrophages treated in the presence or absence of IL-10 revealed that IL-10 inhibited approx. 10–15% of the LPS-induced gene pool [19]. Within the ‘inhibited’ pool are the historically recognized IL-10 targets (TNFα, IL-12p40, IL-1α, IL-1β, IL-6 etc.). However, most genes induced by LPS are unaffected by IL-10. Some genes are even synergistically induced [19]. These results suggest an extraordinary conclusion: that IL-10 selectively inhibits inflammatory gene expression, targeting subsets of genes induced by powerful pro-inflammatory stimuli while leaving the expression of other induced genes unaffected. The clearest interpretation of these results is that IL-10 does not act via a general cellular mechanism (i.e. inhibiting basal transcription or splicing) but is instead highly selective in its effects. Thirdly, the effects of inhibitory effects of IL-10 are mediated predominantly at the transcriptional level and require new protein expression [20]. Finally, activation of STAT3 (signal transducer and activator of transcription 3) is essential for all the known effects of IL-10 [19,21,22]. Therefore IL-10 induces STAT3, which then activates the expression of one or more genes that are then required to selectively block sets of inflammatory genes (Figure 1).

Model to reflect the current state of understanding of STAT3 within the IL-10 signalling pathway

The IL-10 receptor and initial signal transduction mechanism

Expression of the IL-10R is confined to myeloid lineage cells and is most robustly expressed in macrophages and dendritic cells, accounting for the cell-specific effects of IL-10 [17]. The IL-10R is composed of two chains: the IL-10Rα chain that is responsible for docking STAT3 and the IL-10Rβ chain that is a shared subunit with other members of the class II cytokine receptor family [23]. The exact functions of the IL-10Rβ chain in signal transduction are unknown. It is reasonable to speculate that this chain plays a structural or chaperone function to ensure the correct function of the IL-10Rα subunit or the other family members. The IL-10Rα chain has two tyrosine residues that once phosphorylated by JAK1 (Janus kinase 1; the only known JAK family member to have an obligate role in IL-10R function) serve as docking sites for STAT3.

Role of STAT3 in the anti-inflammatory response

As noted above, overwhelming evidence shows STAT3 is the only obligate factor required for IL-10-mediated anti-inflammatory signalling. The obligate requirement for STAT3 in IL-10R signalling raises a vexing problem: STAT3 is the central signalling molecule for many cytokine receptors including the IL-6R. How is the specificity generated between receptors and why is anti-inflammatory signalling apparently unique to the IL-10R? This question has yet to be adequately addressed but it seems that negative regulation of some cytokine receptors prevents their ability to activate anti-inflammatory signalling. Macrophages lacking SOCS3 (suppressor of cytokine signalling 3), an inhibitor of IL-6 signalling, activate an anti-inflammatory signal when stimulated with IL-6 in the presence of LPS [24]. The interpretation of these results centred on the longevity of STAT3 phosphorylation in the absence of SOCS3: the proposed mechanism was that SOCS3 prevented the IL-6 from making the (IL-10-like) anti-inflammatory signal because it terminated STAT3 signalling on a shorter time frame relative to STAT3 activated by IL-10 [24]. Resolution of this issue will require structure–function analysis of multiple cytokine receptors introduced into primary macrophages.

How does IL-10 selectively inhibit inflammatory gene transcription?

Activation of IL-10R signalling, via STAT3, activates gene expression of the executioner of the anti-inflammatory response, a molecule(s) yet to be identified. As noted above, this process acts predominantly at the level of transcription. Several models can accommodate these results (Figure 2). First, IL-10 could active a transcriptional repressor that inhibits the function of key pro-inflammatory transcription factors such as NF-κB. The problem with this idea is that IL-10 has no effect on NF-κB activation (in the cytoplasm) nor does it have any effects on other signalling pathways important to inflammatory gene expression (e.g. mitogen-activated protein kinase p38 phosphorylation) [15,18,20]. However, the hypothetical repressor could function at the level of specific NF-κB-regulated genes and leave others unaffected, depending on gene-specific cues. Secondly, IL-10 could induce the expression of factors that modify chromatin. Gene-specific silencing at the chromatin level could be one mechanism of inhibiting transcription. Finally, IL-10 could induce the modification (e.g. phosphorylation and dephosphorylation) or degradation (e.g. STAT3-mediated induction of an E3 ligase that targeted a pro-inflammatory transcription factor) or sequestration of key pro-inflammatory transcriptional mediators. At this stage, and especially in the absence of the identity of the STAT3-regulated genes that encode the anti-inflammatory mediator, each of these possibilities has merit.

Model for the effects of IL-10 on pro-inflammatory transcription

Figure 2
Model for the effects of IL-10 on pro-inflammatory transcription

The induced inhibitor ‘X’ could function via transcriptional repression, post-translational modifying transcription factors, modifying chromatin or sequestration of transcription factors away from active pro-inflammatory promoters.

Figure 2
Model for the effects of IL-10 on pro-inflammatory transcription

The induced inhibitor ‘X’ could function via transcriptional repression, post-translational modifying transcription factors, modifying chromatin or sequestration of transcription factors away from active pro-inflammatory promoters.

Perspectives

Based on our present level of understanding, it is possible to make predictions that can guide future efforts to understand and exploit the IL-10 signalling. First, we suspect that IL-10 signalling is not unique to the IL-10R or activated macrophages. We hypothesize that the IL-10 signalling mechanism is used by other receptors in other contexts. For example, mice that lack STAT3 only in endothelial cells are highly susceptible to endotoxin-induced shock [25]. Since the IL-10R is not expressed in endothelial cells, another STAT3-dependent mechanism must operate in these cells to protect them from the deleterious effects of LPS. Perhaps another IL-10-like molecule signals through a similar mechanism in these cells? Similarly, STAT3 has potent anti-inflammatory effects in non-myeloid cells [2629]. Therefore we expect that the anti-inflammatory response is general and not restricted to the IL-10R, but rather found in multiple biological contexts.

Secondly, it should be possible to engineer any cytokine receptor to produce an anti-inflammatory response. Since STAT3 is the only known obligate factor involved in the anti-inflammatory signalling pathway, any receptor engineered to activate STAT3 in a way that mimics the IL-10R should be sufficient to make an IL-10 response.

Finally, it should be possible to isolate the executioners of the anti-inflammatory response by modifying the conditions previously used to screen for IL-10-induced genes. Each of these screens has yet to isolate the key STAT3-dependent genes whose products control the anti-inflammatory response [18]. Once isolated, these genes should prove to be essential components of IL-10 signalling and be potential targets for the development of drugs that can control branches of inflammation in the same way as IL-10.

Control of Immune Responses: A Focus Topic at BioScience2006, held at SECC Glasgow, U.K., 23–27 July 2006. Edited by B. Foxwell (Imperial College London, U.K.), G. Graham (Glasgow, U.K.), R. Nibbs (Glasgow, U.K.) and S. Ward (Bath, U.K.).

Abbreviations

     
  • IL-10

    interleukin-10

  •  
  • IL-10R

    IL-10 receptor

  •  
  • JAK

    Janus kinase

  •  
  • LPS

    lipopolysaccharide

  •  
  • NF-κB

    nuclear factor κB

  •  
  • SOCS3

    suppressor of cytokine signalling 3

  •  
  • STAT3

    signal transducer and activator of transcription 3

  •  
  • TNFα

    tumour necrosis factor α

The research in our laboratory is supported by grants from the National Institutes of Health (Bethesda, MD, U.S.A.), the Sandler Program for Asthma Research, Cancer Center CORE grant P30 CA 21765 and the American Lebanese Syrian Associated Charities.

References

References
1
Feldmann
M.
Maini
R.N.
Nat. Med.
2003
, vol. 
9
 (pg. 
1245
-
1250
)
2
Asadullah
K.
Sterry
W.
Volk
H.D.
Pharmacol. Rev.
2003
, vol. 
55
 (pg. 
241
-
269
)
3
Murray
P.J.
Curr. Opin. Pharmacol.
2006
, vol. 
6
 (pg. 
379
-
386
)
4
Berg
D.J.
Kuhn
R.
Rajewsky
K.
Muller
W.
Menon
S.
Davidson
N.
Grunig
G.
Rennick
D.
J. Clin. Invest.
1995
, vol. 
96
 (pg. 
2339
-
2347
)
5
Howard
M.
Muchamuel
T.
Andrade
S.
Menon
S.
J. Exp. Med.
1993
, vol. 
177
 (pg. 
1205
-
1208
)
6
Hunter
C.A.
Ellis-Neyes
L.A.
Slifer
T.
Kanaly
S.
Grunig
G.
Fort
M.
Rennick
D.
Araujo
F.G.
J. Immunol.
1997
, vol. 
158
 (pg. 
3311
-
3316
)
7
Gazzinelli
R.T.
Wysocka
M.
Hieny
S.
Scharton-Kersten
T.
Cheever
A.
Kuhn
R.
Muller
W.
Trinchieri
G.
Sher
A.
J. Immunol.
1996
, vol. 
157
 (pg. 
798
-
805
)
8
Lang
R.
Rutschman
R.L.
Greaves
D.R.
Murray
P.J.
J. Immunol.
2002
, vol. 
168
 (pg. 
3402
-
3411
)
9
O'Garra
A.
Vieira
P.L.
Vieira
P.
Goldfeld
A.E.
J. Clin. Invest.
2004
, vol. 
114
 (pg. 
1372
-
1378
)
10
Roers
A.
Siewe
L.
Strittmatter
E.
Deckert
M.
Schluter
D.
Stenzel
W.
Gruber
A.D.
Krieg
T.
Rajewsky
K.
Muller
W.
J. Exp. Med.
2004
, vol. 
200
 (pg. 
1289
-
1297
)
11
Belkaid
Y.
Piccirillo
C.A.
Mendez
S.
Shevach
E.M.
Sacks
D.L.
Nature
2002
, vol. 
420
 (pg. 
502
-
507
)
12
Bogdan
C.
Vodovotz
Y.
Nathan
C.
J. Exp. Med.
1991
, vol. 
174
 (pg. 
1549
-
1555
)
13
de Waal Malefyt
R.
Abrams
J.
Bennett
B.
Figdor
C.G.
de Vries
J.E.
J. Exp. Med.
1991
, vol. 
174
 (pg. 
1209
-
1220
)
14
Fiorentino
D.F.
Zlotnik
A.
Vieira
P.
Mosmann
T.R.
Howard
M.
Moore
K.W.
O'Garra
A.
J. Immunol.
1991
, vol. 
146
 (pg. 
3444
-
3451
)
15
Donnelly
R.P.
Dickensheets
H.
Finbloom
D.S.
J. Interferon Cytokine Res.
1999
, vol. 
19
 (pg. 
563
-
573
)
16
Lang
R.
Immunobiology
2005
, vol. 
210
 (pg. 
63
-
76
)
17
Moore
K.W.
de Waal Malefyt
R.
Coffman
R.L.
O'Garra
A.
Annu. Rev. Immunol.
2001
, vol. 
19
 (pg. 
683
-
765
)
18
Williams
L.M.
Ricchetti
G.
Sarma
U.
Smallie
T.
Foxwell
B.M.
Immunology
2004
, vol. 
113
 (pg. 
281
-
292
)
19
Lang
R.
Patel
D.
Morris
J.J.
Rutschman
R.L.
Murray
P.J.
J. Immunol.
2002
, vol. 
169
 (pg. 
2253
-
2263
)
20
Murray
P.J.
Proc. Natl. Acad. Sci. U.S.A.
2005
, vol. 
102
 (pg. 
8686
-
8691
)
21
Takeda
K.
Clausen
B.E.
Kaisho
T.
Tsujimura
T.
Terada
N.
Forster
I.
Akira
S.
Immunity
1999
, vol. 
10
 (pg. 
39
-
49
)
22
Williams
L.
Bradley
L.
Smith
A.
Foxwell
B.
J. Immunol.
2004
, vol. 
172
 (pg. 
567
-
576
)
23
Donnelly
R.P.
Sheikh
F.
Kotenko
S.V.
Dickensheets
H.
J. Leukocyte Biol.
2004
, vol. 
76
 (pg. 
314
-
321
)
24
Yasukawa
H.
Ohishi
M.
Mori
H.
Murakami
M.
Chinen
T.
Aki
D.
Hanada
T.
Takeda
K.
Akira
S.
Hoshijima
M.
, et al. 
Nat. Immunol.
2003
, vol. 
4
 (pg. 
551
-
556
)
25
Kano
A.
Wolfgang
M.J.
Gao
Q.
Jacoby
J.
Chai
G.X.
Hansen
W.
Iwamoto
Y.
Pober
J.S.
Flavell
R.A.
Fu
X.Y.
J. Exp. Med.
2003
, vol. 
198
 (pg. 
1517
-
1525
)
26
Burdelya
L.
Kujawski
M.
Niu
G.
Zhong
B.
Wang
T.
Zhang
S.
Kortylewski
M.
Shain
K.
Kay
H.
Djeu
J.
, et al. 
J. Immunol.
2005
, vol. 
174
 (pg. 
3925
-
3931
)
27
Kortylewski
M.
Kujawski
M.
Wang
T.
Wei
S.
Zhang
S.
Pilon-Thomas
S.
Niu
G.
Kay
H.
Mule
J.
Kerr
W.G.
, et al. 
Nat. Med.
2005
, vol. 
11
 (pg. 
1314
-
1321
)
28
Radaeva
S.
Sun
R.
Pan
H.N.
Hong
F.
Gao
B.
Hepatology
2004
, vol. 
39
 (pg. 
1332
-
1342
)
29
Wang
T.
Niu
G.
Kortylewski
M.
Burdelya
L.
Shain
K.
Zhang
S.
Bhattacharya
R.
Gabrilovich
D.
Heller
R.
Coppola
D.
, et al. 
Nat. Med.
2004
, vol. 
10
 (pg. 
48
-
54
)