Brief CommunicationAttenuated TLR4/MAPK signaling in monocytes from patients with CRMO results in impaired IL-10 expression
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
References (38)
- et al.
Chronic recurrent multifocal osteomyelitis and pustulosis palmoplantaris
J. Pediatr.
(1978) - et al.
Chronic non-bacterial osteomyelitis is associated with impaired Sp1 signaling, reduced IL10 promoter phosphorylation, and reduced myeloid IL-10 expression
Clin. Immunol.
(2011) - et al.
Chronic recurrent multifocal osteomyelitis: clinical outcomes after more than five years of follow-up
J. Pediatr.
(2002) - et al.
Biological properties and regulation of IL-10 related cytokines and their contribution to autoimmune disease and tissue injury
Clin. Immunol.
(2012) - et al.
Epigenetic mechanisms in systemic lupus erythematosus and other autoimmune diseases
Trends Mol. Med.
(2011) - et al.
The p38 mitogen-activated kinase pathway regulates the human interleukin-10 promoter via the activation of Sp1 transcription factor in lipopolysaccharide-stimulated human macrophages
J. Biol. Chem.
(2001) - et al.
LPS induction of gene expression in human monocytes
Cell. Signal.
(2001) - et al.
A new autosomal dominant disorder of pyogenic sterile arthritis, pyoderma gangrenosum, and acne: PAPA syndrome
Mayo Clin. Proc.
(1997) - et al.
Pyrin activates the ASC pyroptosome in response to engagement by autoinflammatory PSTPIP1 mutants
Mol. Cell
(2007) - et al.
Functional diversity and regulation of different interleukin-1 receptor-associated kinase (IRAK) family members
Mol. Cell
(2003)
Chronic recurrent multifocal osteomyelitis in children: diagnostic value of histopathology and microbial testing
Hum. Pathol.
Subacute and chronic “symmetrical” osteomyelitis
Ann. Radiol. (Paris)
Chronic non-bacterial osteomyelitis in children
Ann. Rheum. Dis.
Chronic recurrent multifocal osteomyelitis
Semin. Musculoskelet. Radiol.
Association of chronic non-bacterial osteomyelitis with Crohn's disease but not with CARD15 gene variants
Rheumatol. Int.
Chronic recurrent multifocal osteomyelitis in children
Pediatr. Infect. Dis. J.
Chronic recurrent multifocal osteomyelitis: what is it and how should it be treated?
Nat. Clin. Pract. Rheumatol.
Cell type-specific regulation of IL-10 expression in inflammation and disease
Immunol. Res.
IL-10 gene expression is controlled by the transcription factors Sp1 and Sp3
J. Immunol.
Cited by (83)
Gathering expert consensus to inform a proposed trial in chronic nonbacterial osteomyelitis (CNO)
2023, Clinical ImmunologyChronic Nonbacterial Osteomyelitis: Insights into Pathogenesis, Assessment, and Treatment
2021, Rheumatic Disease Clinics of North AmericaChronic nonbacterial osteomyelitis (CNO) and chronic recurrent multifocal osteomyelitis (CRMO)
2021, Journal of Translational AutoimmunityCitation Excerpt :Recently reported observations from basic and early translational studies delivered a number of potential future treatment targets in CNO/CRMO. Altered activation of protein kinases [33,60] in CNO/CRMO patients may deliver arguments for future therapeutic targeting of these enzymes (e.g. Src kinase inhibition in patients with FGR mutations) [96]. The involvement of mast cells in bone inflammation may result in trials testing mast cell inhibitors in CNO/CRMO to control disease [109].
Chronic non-bacterial osteomyelitis and autoinflammatory bone diseases
2020, Clinical Immunology