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Treg versus Th17 lymphocyte lineages are cross-regulated by LIF versus IL-6 - PubMed

  • ️Thu Jan 01 2009

Treg versus Th17 lymphocyte lineages are cross-regulated by LIF versus IL-6

Wenda Gao et al. Cell Cycle. 2009.

Abstract

Within the immune system there is an exquisite ability to discriminate between "self" and "non-self" that is orchestrated by T lymphocytes. Discriminatory pathways guide differentiation of these lymphocytes into either regulatory (Treg) or effector (Teff) T cells, influenced by cues from the naïve T cell's immediate micro-environment as it responds to cognate antigen. Reciprocal pathways may lead to commitment of naïve T cells into either the protective tolerance-promoting Treg, or to the pro-inflammatory Th17 effector phenotype. Primary activation of CD4(+) lymphocytes stimulates their release of leukemia inhibitory factor (LIF), and Treg continue to release LIF in response to antigen, implying a role for LIF in tolerance. In contrast, interleukin- 6 (IL-6), although very closely related to LIF, promotes maturation of Th17 cells. Here we show that LIF and IL-6 behave as polar opposites in promoting commitment to the Treg and Th17 lineages. Unlike IL6, LIF supported expression of Foxp3, the Treg lineage transcription factor, and LIF opposed IL6 by suppressing IL-6-induced IL-17A protein release. In striking contrast, we found that IL6 effectively inhibited LIF signalling, repressing transcription of the LIF receptor gp190, and strongly inducing axotrophin/MARCH-7, a novel E3 ubitquitin ligase that we discovered to be active in degradation of gp190 protein. In vivo, anti-LIF treatment reduced donor-specific Treg in recipients of foreign spleen cells. Conversely, a single dose of biodegradable LIF nanoparticles, targeted to CD4, successfully manipulated the LIF/IL6 axis towards development of donor-specific Foxp3(+) Treg. The implications for therapy are profound, harnessing endogenous immune regulation by paracrine delivery of LIF to CD4(+) cells in vivo.

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Figures

Figure 1
Figure 1

LIF is polar opposite to IL-6 in T cell regulation. (A) CBA recipients of a fully mismatched BALB/c vascularised heart were rendered tolerant to their graft by CD4 plus CD8 blockade as described previously. In the same model, other recipients were allowed to reject their graft. Ex vivo stimulation of spleen cells from these in vivo primed allo-tolerant, or allo-rejected, mice used donor-type irradiated spleen cells, and LIF and IL-6 release were measured by ELISA. (B) FACS-sorted CD4+GFP+(Foxp3+) nTreg cells from Foxp3-GFP knockin mice were stimulated with anti-CD3 and anti-CD28 microbeads in the presence of IL-2 and TGFβ alone (open triangle—broken line), or plus LIF (open square—solid line) or IL-6 (closed circle—heavy solid line). Expression of Foxp3, gp190, IL-17A and RORt mRNA relative to control GAPDH was determined at indicated time points by real-time PCR. (C) FACS-sorted CD4+GFP− cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of TGFβ, TGFβ+LIF or TGFβ+IL-6 for 3 days. Fold changes in relative gene expression were derived by comparing LIF− (open bar) or IL-6 (closed bar) supplemented cultures to control (TGF only). (D) Amount of IL-17A secreted by Th17 cells in the absence (closed bar) or presence (open bar) of LIF. Representative ELISA data of three independent experiments (mean of duplicate wells ± s.d., * <0.01).

Figure 2
Figure 2

Both gp190 and IL-6 are linked to axotrophin. (A) Probability plots of CD4+ cells analysed by flow cytometry for expression of gp190 before and after stimulation of mouse spleen cells with plate-bound anti-CD3 and soluble anti-CD28. Domains of gp190 negative, gp190 low, and gp190 high expression are delineated. (a–c) show wild-type mouse CD4+ cells. (a) 0 h, (b) 48 h, (c) 72 h. (d) shows axotrophin null mouse spleen cells at 72 h. At 48 h the axotrophin null CD4+ cells showed equivalent expression of gp190 to those in (c). (B) IP western of activated mouse spleen cells from axotrophin null and wild-type littermates. The two lanes are directly comparable and differ only in the absence of axotrophin in lane 1. The scan is from the same multilane blot; the cropped lanes were treated identically and no image enhancement used. Gel load indicates the loading well; gp190 degr. indicates gp190 degradation products; IgG indicates reactivity of the secondary goat anti-rabbit HRP antibody with rabbit anti-gp190 in the sample. (C) FACS-sorted CD4+GFP− cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 for 3 days in the presence or absence of TGFβ, LIF or IL-6 as indicated: the effect of cytokine on axotrophin transcript levels was measured relative to GAPDH. (D) FACS analysis of gp190 hi CD4+ mouse spleen cells after stimulation with anti-CD3/anti-CD28, in the presence or absence of either LIF, or IL-6.

Figure 3
Figure 3

LIF expands the Foxp3+ cell population. (A) Schematic of the nanoparticle construction0. Soluble LIF is trapped within the biodegradable matrix of poly (lactic-co-glycolic acid) (PLGA) prepared from FDA-approved materials. Avidin is incorporated at the particle surface, permitting attachment of biotinylated antibody and thus antibody-mediated targeting. The matrix is impermeable to enzyme, and degrades slowly providing a vehicle for sustained paracrine delivery of LIF. (B) FACS-sorted CD4+GFP cells were stimulated with plate-bound anti-CD3, soluble anti-CD28 and increasing doses of TGFβ in the presence of empty nanobeads or nanobeads that were loaded with either LIF, or IL-6 (50 µg nanobeads per 0.5 ml culture medium). Induced expression of Foxp3-GFP at 72 h is shown on the x-axis. (C) In vivo local delivery of LIF expands antigen-specific nTreg cells. DBA/2 splenocytes (DST) were incubated for 15 minutes with anti-CD4 conjugated empty-, or LIF-nanobeads, and infused (107 cells/mouse, i.v.) into BALB/c Foxp3-GFP mice (n = 3 per group). Host lymph node cells were harvested 5 days later, and ratios of GFP+ vs. GFP− cells were calculated in the donor specific Vβ6+ (black) or Vβ8+ (grey) CD4+ T cell compartments (mean ± s.d.).

Figure 4
Figure 4

Blocking LIF in vivo reduced donor-specific Foxp3+ cells. Using the DST model, anti-LIF antibody was given i.p. to the BALB/c Foxp3-GFP recipients (n = 3 per group) at a dose of 150 µg on days 0, 1, 2 post grafting. Five days after DST, host lymphocytes were harvested and CD4+GFP+ cells enumerated by flow cytometry. The anti-LIF therapy resulted in specific inhibition of the expansion of antigen-specific Vβ6+ Treg cells in spleen (A) and lymph nodes (B). * <0.05, compared to DST alone or DST plus control IgG.

Figure 5
Figure 5

Schematic model of LIF versus IL-6 cross-regulation for Treg versus Th17 lineage differentiation. Naive CD4+ T cells, when stimulated by cognate antigen through the T cell receptor (TCR), release endogenous LIF. This model predicts that, where TCR stimulation is weak or attenuated (A) released LIF induces further LIF transcription in addition to gp190 transcription, setting up an autocrine loop for LIF signaling where feedback regulation includes low level proteasomal degradation of gp190 protein primed by the E-3 ubiquitin ligase axotrophin. LIF signaling augments expression of Foxp3 leading to Treg-type epigenetic profiling for stable antigen-specific tolerance. (B) proposes that, where TCR stimulation occurs in the presence of IL-6, then suppression of gp190 by IL-6, acting to inhibit gp190 transcription and also massively induce axotrophin transcription, prevents a LIF autocrine loop becoming established, resulting in failure to establish a Foxp3-linked Treg epigenetic profile. Instead, IL-6 induces RORγt to drive Th17 lineage development. (C) predicts that, by providing exogenous LIF to the T cell micro-environment, LIF-induced gp190 expression is sufficiently maintained as to permit sustained LIF signaling and so supports Treg expansion. The model anticipates counter-balancing effects that create a rheostat control mechanism, sensitive to micro-environmental cues including inflammatory mediators (e.g., IL-6), or conversely to sources of LIF (e.g., local mast cells).

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