Interleukin-6 signaling drives fibrosis in unresolved inflammation

Abstract

Fibrosis in response to tissue damage or persistent inflammation is a pathological hallmark of many chronic degenerative diseases. By using a model of acute peritoneal inflammation, we have examined how repeated inflammatory activation promotes fibrotic tissue injury. In this context, fibrosis was strictly dependent on interleukin-6 (IL-6). Repeat inflammation induced IL-6-mediated T helper 1 (Th1) cell effector commitment and the emergence of STAT1 (signal transducer and activator of transcription-1) activity within the peritoneal membrane. Fibrosis was not observed in mice lacking interferon-γ (IFN-γ), STAT1, or RAG-1. Here, IFN-γ and STAT1 signaling disrupted the turnover of extracellular matrix by metalloproteases. Whereas IL-6-deficient mice resisted fibrosis, transfer of polarized Th1 cells or inhibition of MMP activity reversed this outcome. Thus, IL-6 causes compromised tissue repair by shifting acute inflammation into a more chronic profibrotic state through induction of Th1 cell responses as a consequence of recurrent inflammation.

Figure 1

Il6^−/− Mice Are Protected from the Development of Fibrosis after Repeated Inflammation (A) Submesothelial compact zone thickness was compared in peritoneal biopsies from PD patients with either no previous infection history or a defined infection history. Data are presented as box plots of the interquartile range (IQR). Lines extend from the box to the highest and lowest values, excluding outliers. The median value is represented by a thick line across each box. (B) WT or Il6^−/− mice were injected (i.p.) with SES at weekly intervals for 3 weeks (day 0–21) and left for a further 4 weeks until day 49 before histological analysis of the peritoneal membrane. Peritoneal membrane sections (5 μm) taken from SES-treated and age-matched control mice on day 49 were stained with hematoxylin and eosin and examined for submesothelial compact zone thickening (layer between the muscle and membrane surface). Representative fields are shown from two individual mice per group (×400 magnification). Scale bar represents 50 μM. Submesothelial compact zone (SMC) and muscle layers (M) are indicated on representative WT sections. (C) Fibrosis scores for WT and Il6^−/− mice are shown over the duration of the model. Values reflect the fold-change in submesothelial zone thickness compared to the WT control group at day 0 (n ≥ 3–12 per group, unpaired t test ^∗p < 0.05 compared to WT day 0, ^∗∗p < 0.001 compared to WT control day 49 and Il6^−/− 4×SES day 49). No significant difference in membrane thickening was observed in sections from WT control mice taken on day 0 and day 49. (D) Peritoneal membrane sections from day 49 were immunostained with antibodies against type-1 collagen. Representative fields are shown from two individual mice per group (×400 magnification). Scale bar represents 50 μm. See also Figure S1.

Figure 2

Changes in the Inflammatory Response after Repeated SES Challenge (A) EMSA for NF-κB and STAT signaling in nuclear extracts from the peritoneum of SES-challenged WT and Il6^−/− mice. Analysis of samples from the first (1×SES) and fourth (4×SES) episode of inflammation are shown. An NF1 probe was used as a loading control. (B) Supershift analysis of the STAT DNA-binding complex in nuclear extracts (3 hr sample) from WT mice. Analysis of STAT1, STAT3, and STAT5 is shown from 1×SES and 4×SES. The antibody-induced STAT3 supershift (SS) and loss of STAT1 binding to the probe (LB) are indicated by black and white arrows, respectively. (C) Immunoblot with phospho-specific antibodies for STAT1 and STAT3 in protein lysates from the peritoneal membranes of mice. Samples were obtained from 1×SES and 4×SES. In all cases, results are representative of lysates from three different mice per time point and genotype. (D) Detection of IFN-γ in lavage from challenged WT and Il6^−/− mice (mean ± SEM; n > 5 per time point). (E) qPCR of Irf1 and Isg15 in total RNA from the peritoneal membrane of challenged mice. Values are expressed relative to the WT baseline control and represent the mean for each genotype from three different mice per time. See also Figure S4.

Figure 3

Detection of Peritoneal IFN-γ-Expressing CD4⁺ T Cells Correspond with STAT1 Activation (A) WT, Ifng^−/−, and Rag1^−/− mice were repeatedly challenged with SES and the peritoneal membrane harvested at day 49. Serial sections were immunostained for type-1 collagen and counterstained with hematoxylin. Representative fields are shown from two individual mice per group (×400 magnification). (B) Fibrosis was scored by assessment of the fold-change in the submesothelial compact zone thickness compared to the control group at day 49 (n ≥ 4 per group unpaired t test; ^∗p < 0.05). (C) STAT activation at 3 hr post-SES injection was measured by EMSA of nuclear extracts prepared from the peritoneal membrane of WT, Ifng^−/−, and Rag1^−/− mice taken during 1×SES and 4×SES. Supershift analysis of nuclear extracts from 1×SES and 4×SES by specific anti-STAT antibodies. Data are representative of results from three mice. The antibody-induced STAT3 supershift (SS) and loss of STAT1 binding to the probe (LB) are indicated by black and white arrows, respectively. (D) Intracellular flow cytometry for IFN-γ-secreting CD4⁺ T cells in peritoneal lavage from SES-challenged WT and Il6^−/− mice. Lavage samples were isolated at 3 hr during 1×SES and 4×SES (n ≥ 4 per group, unpaired t test; ^∗p < 0.05 compared with the 1×SES WT group). (E) Peritoneal leukocytes obtained from WT mice (6 hr after SES) were stimulated ex vivo for a further 4 hr with SES in the presence of monensin. Intracellular flow cytometry for IFN-γ production in CD4⁺ and CD19⁺ lymphocytes is shown. See also Figure S5.

Figure 4

SES Promotes the IL-6-Dependent Expansion of Th1 Cells In Vitro (A) Peritoneal monocytic cells were recovered by lavage from WT mice and stimulated with SES in culture overnight. Cell-free conditioned media from these cultures (SES-CM) were used to stimulated CFSE-labeled naive T cells under anti-CD3 and anti-CD28 costimulation. After 4 days culture, IFN-γ production was monitored in proliferating CD4⁺ T cells. Representative data are shown for cells treated with media alone (-), costimulatory antibodies alone (-SES), or in combination with SES-CM (+SES). (B) ELISA quantification of IL-6, sIL-6R, and IFN-γ in SES-CM (mean ± SEM; n = 3 for IL-6 and IFN-γ, n = 6 for sIL-6R; values below the limit of detection [+ T cells cultured for 4 days under anti-CD3 and anti-CD28 costimulation in the presence or absence of SES-CM. Data are shown for T cells derived from WT and Cd126^−/− mice. (D) Relative quantification of IFN-γ-secreting CD4⁺ T cells in all experimental repeats (n = 5 WT and n = 3 Cd126^−/− mice per group). (E) Comparable analysis of IFN-γ, IL-4, and IL-9 in T cell cultures from WT and Cd126^−/− mice. See also Figure S6i.

Figure 5

IL-6 Modulation of Th1 Cell Activity In Vivo (A) SES-induced peritoneal inflammation was initiated in Il6^−/− mice. Local IL-6 (trans) signaling was reconstituted via administration (i.p.) of 1 μg/mouse HDS or control PBS at 0 hr (same time as SES challenge), 24 hr, and 48 hr. After 72 hr, the peritoneal infiltrate was recovered by lavage and CD4⁺ T cells examined by flow cytometry. The proportion of CD4⁺ T cells displaying a Th1 and Th17 effector cell phenotype was determined by intracellular flow cytometry for IFN-γ and IL-17A (n = 8 mice per treatment, p < 0.05). (B) Intracellular flow cytometry of IFN-γ and IL-6 production by naive CD4⁺ T cells from WT mice that had been activated for 4 days in the presence or absence of SES-CM from peritoneal monocytic cells. The relative quantification of cells releasing IL-6 or IFN-γ is presented from all experiments (n = 3, p < 0.05). (C) WT Th1 cells expanded ex vivo under costimulation with SES-CM. These Th1 cells (0.5–1.0 × 10⁶) were adoptively transferred (i.p.) into Cd126^−/− mice together with SES. Peritoneal lavage were recovered (3 hr) and IFN-γ quantified by ELISA (mean ± SEM from four separate mice). Values are compared against mice receiving freshly sorted naive CD4⁺ T cells (Th0). (D) Immunoblot of STAT1 activation in peritoneal membranes from SES-challenged Cd126^−/− mice receiving ex vivo expanded Th1 cells or Th0 cells. Data are shown for each individual adoptive transfer (n = 4). See also Figure S6ii.

Figure 6

Peritoneal Fibrosis Is Linked to Stromal STAT1 Activity and Th1 Cells (A) WT and Stat1^−/− mice were repeatedly challenged with SES. Peritoneal membranes were harvested at day 49 and sections scored for fibrosis. Sections are compared against age-matched control mice as before (unpaired t test, ^∗p < 0.05). (B) Naive CD4⁺ T cells were committed in vitro to Th1 cells with SES-CM. The proportion of IFN-γ⁺CD4⁺ cells was determined by flow cytometry and used to calculate the number of T cells for transfer into Il6^−/− mice. 0.5–1.0 × 10⁶ Th1 cells or naive CD4⁺ T cells (Th0) were coadministered to mice with SES (i.p.) on day 0, 7, 14, and 21. Peritoneal fibrosis was assessed on day 49 as before (unpaired t test, ^∗p < 0.05). See also Figure S7i.

Figure 7

IFN-γ-STAT1 Activity Regulates Matrix Metalloproteinase Expression (A and B) ELISA quantification of MMP-3 and TIMP-1 in peritoneal lavage fluid from WT and Ifng^−/− (A) or Stat1^−/− (B) mice. (C) WT and Il6^−/− were injected with SES at four weekly intervals. At day 43, day 45, and day 47, Il6^−/− mice received drinking water containing the collagenase-specific MMP inhibitor Ro32-355 (12.5 mg/50 ml). Groups of WT and Il6^−/− mice received the ethanol vehicle alone. At day 49, the peritoneal membrane was harvested and fibrosis scored as before. (D) Growth-arrested human peritoneal mesothelial cells (HPMCs) were treated with medium alone (control), IL-1β (100 pg/ml), IFN-γ (100 U/ml), or IL-1β in combination with IFN-γ for up to 72 hr. Cell-free supernatants were analyzed for MMP-3 or TIMP-1 by ELISA. (E) HPMCs were transfected with empty control or constitutive STAT1 (STAT1-C) containing plasmid vectors overnight and stimulated with IL-1β (100 pg/ml) for 24 hr. Cell-free supernatants were analyzed for MMP-3 or TIMP-1 by ELISA. See also Figure S7ii.