Elsevier

Clinical Immunology

Volume 145, Issue 1, October 2012, Pages 69-76
Clinical Immunology

Brief Communication
Attenuated TLR4/MAPK signaling in monocytes from patients with CRMO results in impaired IL-10 expression

https://doi.org/10.1016/j.clim.2012.07.012Get rights and content

Abstract

Chronic recurrent multifocal osteomyelitis (CRMO) is an autoinflammatory bone disorder of unknown origin. We previously demonstrated that monocytes from CRMO patients fail to express the immune-modulatory cytokine interleukin‐10 (IL‐10) in a chromatin dependent manner. Here, we demonstrate that attenuated extracellular-signal regulated kinase (ERK)1 and 2 signaling in response to TLR4 activation results in failure to induce IL-10 expression in monocytes from CRMO patients. Attenuated ERK1/2 activation results in 1) reduced levels of Sp-1, a transcription factor that induces IL-10 expression in monocytes, and 2) impaired H3S10 phosphorylation of the IL10 promoter, an activating epigenetic mark. The pro-inflammatory cytokines tumor necrosis factor (TNF)α and IL-6 are not negatively affected, resulting in an imbalance towards pro-inflammatory cytokines. Thus, impaired ERK1/2 signaling with subsequently reduced Sp-1 expression and H3S10 phosphorylation of the IL10 promoter may centrally contribute to the pathophysiology of CRMO.

Highlights

► Attenuated TLR4-ERK1/2 signaling results in impaired Sp-1 recruitment to IL10. ► Disrupted TLR4-ERK1/2 signaling affects H3S10 phosphorylation of IL10. ► The pro-inflammatory cytokines TNFα and IL-6 are not affected.

Introduction

Chronic non-bacterial osteomyeltis (CNO) is an autoinflammatory disease of unknown origin. CNO covers a relatively wide clinical spectrum with asymptomatic mono-focal bone lesions and its most severe form chronic recurring multifocal osteomyelitis (CRMO) at the two ends of the spectrum. CRMO is characterized by sterile osteomyelitis that can generally affect any bone in the human body. However, metaphyses of long bones are predominantly affected, frequently showing symmetric involvement. Systemic symptoms, including malaise and low-grade fevers can occur [1], [2], [3], [4], [5], [6], [7], [8], [9].

We recently documented attenuated expression of the immune-regulatory cytokine interleukin (IL-)10 in monocytes from patients with CRMO in response to toll-like receptor (TLR)4 stimulation with LPS. IL-10 controls inflammation on multiple levels, including reduced antigen presentation by antigen-presenting cells, and the limitation of pro-inflammatory cytokine responses [10], [11]. Attenuated IL-10 expression can be caused by multiple factors, including disrupted transcription factor networks, genomic variation and epigenetic patterns [10], [11]. Reduced IL-10 expression results in chronic inflammation and autoimmune disorders in mouse models and humans [10], [11]. In CRMO, reduced IL-10 expression is caused by impaired recruitment of the transcription factor Sp-1 to a Sp-1 binding motif within the IL10 promoter, -636/-627 bp upstream of the transcriptional initiation site [10], [12]. We furthermore reported reduced phosphorylation of histone H3 serine 10 (H3S10P) in the same region [4]. H3S10 phosphorylation of the IL10 promoter has been demonstrated to be an activating epigenetic mark that plays a key role in inducing IL-10 expression in response to macrophage stimulation [13], [14]. Epigenetic mechanisms play a central role in the regulation of genes, including IL10. Epigenetic mechanisms regulate the gene expression capacities of cells and tissues without altering the underlying genomic sequence. Histone proteins organize the structure of DNA, thus influencing its accessibility to transcription factors and RNA polymerases. Post-transcriptional histone modifications define patterns defined as histone code and mediate chromatin compaction or decompaction. Specific histone modifications are mediated through enzymes that recognize histone tails and add or remove molecules. One of these modifications, phosphorylation of H3S10, has been reported to be essential for the activation of IL10 in monocytes and macrophages [15], [16], [17].

Previous reports document that TLR4 stimulation with lipopolysaccharide (LPS) induces mitogen-activated protein (MAP) kinase activation in monocytes and macrophages, including the p38 MAP kinase [13], [18], [19], the extracellular-signal regulated kinases (ERK) 1 and 2, and the c-Jun N-terminal kinase (JNK) pathway [20], [21]. Furthermore, ERK 1/2 are implicated in H3S10 phosphorylation in macrophages [13], [14]. Thus, we hypothesized that our recent observations contribute to the pathophysiology of CRMO.

In this report, we demonstrate that monocytes from CRMO patients fail to express IL-10 in response to TLR4 stimulation with LPS, while monocytes from patients with juvenile idiopathic arthritis (JIA) express IL-10. Attenuated IL-10 expression results in an increased TNFα/IL-10 ratio with an imbalance towards pro-inflammatory signals. Western blot analyses unveiled attenuated Sp-1 expression and reduced levels of ERK1 and 2 in monocytes from CRMO patients, while the p38 MAP kinase was not affected. Treatment of monocytes from healthy controls with ERK inhibitors resulted in a phenotype that was comparable to CRMO monocytes.

Section snippets

Patient and control samples

Peripheral blood mononuclear cells (PBMCs) were collected from CRMO patients by members of the Department of Pediatrics, University Children's Hospital Würzburg. Ethical approval for the study was obtained from the ethics commissions, Julius-Maximilian's University Würzburg and parents and/or patients gave written informed consent. An ethnically matched healthy control population and age and gender matched JIA patients were included in this study.

Monocyte isolation

PBMCs were collected from CRMO patients,

CRMO monocytes fail to produce IL-10 but not TNFα in response to stimulation with LPS, resulting in a disrupted TNFα/IL-10 ratio

We previously demonstrated that TLR4 signaling is involved in the dysregulation of myeloid-derived IL-10 expression in CRMO [4]. In order to establish our previous findings in more individuals with CRMO and in comparison to other autoimmune diseases (JIA), we investigated cytokine expression from resting monocytes and cells that were stimulated with LPS. We harvested monocytes from 8 CRMO patients, 8 JIA patients and 8 healthy controls after 24 h of LPS stimulation.

We and others furthermore

Discussion

CRMO is an autoinflammatory osteopathy of unknown origin. In a recent study, we demonstrated that the stimulation of TLR4 with LPS in monocytes from CRMO patients failed to induce Sp-1 recruitment to the IL10 promoter. Furthermore, the IL10 promoter failed to undergo activation-induced chromatin remodeling as assessed by H3S10 phosphorylation. TLR4 stimulation with LPS had previously been reported to activate mitogen-activated protein kinases (MAPK), including the p38 MAPK, the

Conclusions

CRMO is an autoinflammatory disorder that is characterized by aseptic bone inflammation. Monocytes from CRMO patients exhibit an increased ratio between the pro-inflammatory TFNα and the anti-inflammatory IL-10. A lack of activated ERK1/2 results in attenuated Sp-1 expression and impaired H3S10 phosphorylation of the IL10 promoter, resulting in reduced gene expression.

Further studies are warranted in order to target the molecular mechanisms that result in impaired ERK activation, chromatin

Conflict of interest statement

The authors S.R Hofmann, H. Morbach, T. Schwarz, A. Rösen-Wolff, H.J. Girschick and C.M. Hedrich declare no conflict of interest.

Acknowledgments

We thank all participating CRMO patients and their families as well as the JIA and healthy control individuals for supporting this study. We thank the staff of University Children's Hospital Würzburg for their support and patient care. The authors thank Diana Paul for excellent technical support. Work performed in the authors lab was supported by MeDDrive 2010 (to C.M.H.). The research of Sigrun R. Hofmann and Angela Rösen-Wolff are supported by the German Research Foundation (Klinische

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