Objective
Systemic juvenile idiopathic arthritis (JIA) is associated with high levels of interleukin‐6 (IL‐6) in the serum and synovial fluid, and impairment of natural killer (NK) cell function is ...often observed. This study was undertaken to evaluate a possible link between these 2 biologic findings and whether they may be associated with the development of macrophage activation syndrome, a condition frequently observed in systemic JIA.
Methods
Splenocytes from wild‐type (WT) or IL‐6–transgenic (Tg) mice were evaluated for NK cell cytotoxicity using a 51Cr‐release assay. Numbers of NK cells and expression of perforin, granzyme B, CD69, and CD107a were evaluated by flow cytometry. Human peripheral blood mononuclear cells (PBMCs) isolated from healthy donors were treated with IL‐6 and cultured in the presence or absence of tocilizumab (TCZ), an IL‐6 receptor blocker. Human polyclonal NK cells from healthy donor PBMCs were evaluated for cell cytotoxicity and expression of perforin, granzyme B, and CD107a. PBMCs harvested from patients with systemic JIA during periods of active or inactive disease were left untreated or treated with IL‐6 in combination with soluble IL‐6 receptor and analyzed for the expression of perforin and granzyme B.
Results
Splenic NK cell cytotoxicity was reduced in IL‐6–Tg mice compared to WT mice. Levels of CD69 and CD107a showed no significant differences, whereas expression of perforin and granzyme B was impaired in NK cells from IL‐6–Tg mice. Exposure of human peripheral blood NK cells to IL‐6 led to reduced expression of perforin and granzyme B. Culturing human polyclonal NK cells in the presence of TCZ significantly increased cell cytotoxicity, and also increased expression of perforin and granzyme B. In patients with systemic JIA, a reduction in IL‐6 plasma levels during disease remission correlated with the rescue of perforin and granzyme B expression in NK cells from these patients.
Conclusion
In both mice and humans, IL‐6 down‐modulated the cytotoxic activity of NK cells. This decrease was associated with reduced perforin and granzyme B levels in the absence of altered granule exocytosis.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Macrophage activation syndrome (MAS) is a potentially fatal complication of rheumatic diseases. The condition is considered part of secondary hemophagocytic lymphohistiocytoses (HLH). There are ...similarities in genetic background, pathogenesis, and clinical and laboratory features with primary HLH (p-HLH). We describe findings in mouse models of secondary HLH, comparing them with models of p-HLH and the cellular and molecular mechanisms involved, and relate them to recent findings in patients with secondary HLH. A multilayer model is presented in which background inflammation, infections, and genetics all contribute in different proportions and in several ways. Once the "threshold" has been reached, inflammatory cytokines are the final effectors, independent of the interplay between different upstream pathogenic factors.
Nerve growth factor (NGF) levels are highly increased in inflamed tissues, but their role is unclear. We show that NGF is part of a regulatory loop in monocytes: inflammatory stimuli, while ...activating a proinflammatory response through TLRs, upregulate the expression of the NGF receptor TrkA. In turn, NGF, by binding to TrkA, interferes with TLR responses. In TLR-activated monocytes, NGF reduces inflammatory cytokine production (IL-1β, TNF-α, IL-6, and IL-8) while inducing the release of anti-inflammatory mediators (IL-10 and IL-1 receptor antagonist). NGF binding to TrkA affects TLR signaling, favoring pathways that mediate inhibition of inflammatory responses: it increases Akt phosphorylation, inhibits glycogen synthase kinase 3 activity, reduces IκB phosphorylation and p65 NF-κB translocation, and increases nuclear p50 NF-κB binding activity. Use of TrkA inhibitors in TLR-activated monocytes abolishes the effects of NGF on the activation of anti-inflammatory signaling pathways, thus increasing NF-κB pathway activation and inflammatory cytokine production while reducing IL-10 production. PBMC and mononuclear cells obtained from the synovial fluid of patients with juvenile idiopathic arthritis show marked downregulation of TrkA expression. In ex vivo experiments, the addition of NGF to LPS-activated juvenile idiopathic arthritis to both mononuclear cells from synovial fluid and PBMC fails to reduce the production of IL-6 that, in contrast, is observed in healthy donors. This suggests that defective TrkA expression may facilitate proinflammatory mechanisms, contributing to chronic tissue inflammation and damage. In conclusion, this study identifies a novel regulatory mechanism of inflammatory responses through NGF and its receptor TrkA, for which abnormality may have pathogenic implications for chronic inflammatory diseases.
The role of Interleukin(IL)-6 in the pathogenesis of joint and systemic inflammation in rheumatoid arthritis (RA) and systemic juvenile idiopathic arthritis (s-JIA) has been clearly demonstrated. ...However, the mechanisms by which IL-6 contributes to the pathogenesis are not completely understood. This study investigates whether IL-6 affects, alone or upon toll like receptor (TLR) ligand stimulation, the production of inflammatory cytokines and chemokines in human peripheral blood mononuclear cells (PBMCs), synovial fluid mononuclear cells from JIA patients (SFMCs) and fibroblast-like synoviocytes from rheumatoid arthritis patients (RA synoviocytes) and signalling pathways involved. PBMCs were pre-treated with IL-6 and soluble IL-6 Receptor (sIL-6R). SFMCs and RA synoviocytes were pre-treated with IL-6/sIL-6R or sIL-6R, alone or in combination with Tocilizumab (TCZ). Cells were stimulated with LPS, S100A8-9, poly(I-C), CpG, Pam2CSK4, MDP, IL-1β. Treatment of PBMCs with IL-6 induced production of TNF-α, CXCL8, and CCL2, but not IL-1β. Addition of IL-6 to the same cells after stimulation with poly(I-C), CpG, Pam2CSK4, and MDP induced a significant increase in IL-1β and CXCL8, but not TNF-α production compared with TLR ligands alone. This enhanced production of IL-1β and CXCL8 paralleled increased p65 NF-κB activation. In contrast, addition of IL-6 to PBMCs stimulated with LPS or S100A8-9 (TLR-4 ligands) led to reduction of IL-1β, TNF-α and CXCL8 with reduced p65 NF-κB activation. IL-6/IL-1β co-stimulation increased CXCL8, CCL2 and IL-6 production. Addition of IL-6 to SFMCs stimulated with LPS or S100A8 increased CXCL8, CCL2 and IL-1β production. Treatment of RA synoviocytes with sIL-6R increased IL-6, CXCL8 and CCL2 production, with increased STAT3 and p65 NF-κB phosphorylation. Our results suggest that IL-6 amplifies TLR-induced inflammatory response. This effect may be relevant in the presence of high IL-6 and sIL-6R levels, such as in arthritic joints in the context of stimulation by endogenous TLR ligands.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Aim of this study was to investigate the activation of the IFNγ pathway in the affected liver and in the blood of patients with secondary hemophagocytic lymphohistiocytosis (sHLH). To this purpose, ...the mRNA expression levels of IFNG and IFNγ-inducible genes as well as Tyrosine (701)-phosphorylated signal transducer and activator of transcription 1 (STAT1) protein levels were evaluated in the liver and in peripheral blood mononuclear cells (PBMCs) of three patients with sHLH with predominant liver involvement. The mRNA expression levels of IFNG and IFNγ-inducible genes were markedly higher in patient livers compared to control livers and to one disease control liver. Conversely, slight differences in the expression levels of Type I IFN-inducible genes and other classical inflammatory cytokine genes were found. Further supporting the activation of the IFNγ pathway, higher protein levels of phosphorylated and total STAT1 were detected in patient livers compared to control livers. When the expression of the same genes analysed in liver tissues was evaluated in PBMCs collected from 2 out of 3 patients before the liver biopsy, we found that mRNA levels of IFNγ-inducible genes were markedly increased. Accordingly, high circulating levels of IFNγ-inducible CXCL9 were observed in patients. Altogether, these data demonstrate the selective and marked up-regulation of the IFNγ pathway in the liver tissue and blood of patients with active sHLH. Finally, we show that measurement of circulating CXCL9 levels and evaluation of IFNγ-inducible gene expression levels in PBMCs may represent a new valid tool to better identify patients with suspected HLH with predominant liver involvement.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cystinosis is a rare autosomal recessive disorder caused by mutations in the
gene that encodes cystinosin, a ubiquitous lysosomal cystine/H
antiporter. The hallmark of the disease is progressive ...accumulation of cystine and cystine crystals in virtually all tissues. At the kidney level, human cystinosis is characterized by the development of renal Fanconi syndrome and progressive glomerular and interstitial damage leading to end-stage kidney disease in the second or third decade of life. The exact molecular mechanisms involved in the pathogenesis of renal disease in cystinosis are incompletely elucidated. We have previously shown upregulation of NLRP2 in human cystinotic proximal tubular epithelial cells and its role in promoting inflammatory and profibrotic responses. Herein, we have investigated the role of NLRP2
using a mouse model of cystinosis in which we have confirmed upregulation of
in the renal parenchyma. Our studies show that double knock out
animals exhibit delayed development of Fanconi syndrome and kidney tissue damage. Specifically, we observed at 4-6 months of age that animals had less glucosuria and calciuria and markedly preserved renal tissue, as assessed by significantly lower levels of inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis. Also, the mRNA expression of some inflammatory mediators (
and
) and the rate of apoptosis were significantly decreased in 4-6-month old kidneys harvested from
mice compared to those obtained from
mice. At 12-14 months of age, renal histological was markedly altered in both genetic models, although double KO animals had lower degree of polyuria and low molecular weight proteinuria and decreased mRNA expression levels of
and
. Altogether, these data indicate that Nlrp2 is a potential pharmacological target for delaying progression of kidney disease in cystinosis.
Haploinsufficiency of A20 (HA20) is an inflammatory disease caused by mutations in the
gene classically presenting with Behcet's-like disease. A20 acts as an inhibitor of inflammation through its ...effect on NF-kB pathway. Here we describe four consanguineous patients (three sisters and their mother) with a predominantly autoimmune phenotype, including thyroiditis, type I diabetes, hemolytic anemia and chronic polyarthritis. All patients had recurrent oral ulcers, with only 1 patient presenting also recurrent fever episodes, as a classical autoinflammatory feature. Next generation sequencing identified a novel heterozygous frameshift mutation (p.His577Alafs*95) that causes a premature stop codon in the zinc finger domain of A20, leading to a putative haploinsufficiency of the protein. Functional analyses confirmed the pathogenicity of the mutation. The variant was associated with decreased levels of A20 in blood cells. Accordingly,
lipopolysaccharide (LPS)-stimulated patients' peripheral blood mononuclear cells (PBMCs) showed higher levels of p65 NF-kB phosphorylation, as well as increased production of the proinflammatory cytokines IL-1β, IL-6 and TNF-α. Moreover, in agreement with recent observations, demonstrating a role for A20 in inhibiting STAT1 and IFNγ pathways, markedly higher circulating levels of the two IFNγ-inducible chemokines CXCL9 and CXCL10 were detected in all patients. Supporting the findings of a hyperactivation of IFNγ signaling pathway in HA20 patients, patients' monocytes showed higher levels of STAT1 without stimulation, as well as higher phosphorylated (active) STAT1 levels following IFNγ stimulation. In conclusion, our study show that in the clinical spectrum of HA20 autoimmune features may predominate over autoinflammatory features and demonstrate, from a molecular point of view, the involvement of A20 in modulating not only the NF-kB, but also the IFNγ pathway.
We describe a 2 year old boy with two previously undescribed frameshift mutations in the interferon (IFN)α/β receptor 2 (
IFNAR2
) gene presenting with hemophagocytic lymphohistiocytosis (HLH) ...following measles-mumps-rubella vaccination. Functional analyses show the absence of response to type I IFN in the patient’s cells, as revealed by the lack of phosphorylation of STAT1 and the lack of induction of interferon-stimulated genes upon
ex vivo
stimulation with IFNα. HLH has been reported in patients with inborn errors of type I IFN-mediated immune responses following vaccination with live-attenuated viruses. The relation between HLH and defective type I IFN-mediated responses is unclear. We show that in patient’s natural killer (NK) cells stimulated with IFNα the expected increase in degranulation and inhibition of IFNγ production were affected. These data support a role for NK cell function dysregulation and lack of inhibition of IFNγ production as contributors to the development of HLH in patients with impaired type I IFN signaling.
To investigate the activation of the IFNγ signaling pathway in monocytes of patients with secondary hemophagocytic lymphohistiocytosis (sHLH)/macrophage activation syndrome (MAS) and to evaluate ...whether levels of phosphorylated STAT1 represent a biomarker for the identification of patients at early stages of the disease.
Fresh whole blood samples from pediatric patients with active sHLH/MAS, not receiving (n=10) and receiving glucocorticoids (n=14) at time of sampling, were prospectively collected. As disease control groups, patients with active systemic juvenile idiopathic arthritis (sJIA) without MAS, patients with sHLH/MAS in remission and patients with other rheumatic diseases were also sampled. Whole blood cells were left unstimulated or stimulated with increasing concentrations of IFNγ for 10 minutes and the intracellular Tyrosine (701)-phosphorylated STAT1 (pSTAT1) levels were evaluated in monocytes by flow cytometry.
Monocytes from untreated sHLH/MAS patients showed significantly higher basal levels of pSTAT1 compared to those observed in monocytes from glucocorticoid-treated sHLH/MAS patients and from all the other disease controls. In addition, a significant increase in responsiveness to IFNγ, as assessed by increased levels of pSTAT1 following
stimulation, was observed in monocytes from untreated sHLH/MAS patients. pSTAT1 levels in monocytes distinguished patients with sHLH/MAS not treated with glucocorticoids from patients with active sJIA or with other rheumatic diseases AUC, 0.93; 95% confidence interval 0.85-1.00, p<0.001. Statistically significant correlations between
mRNA levels in whole blood cells, circulating IFNγ levels and pSTAT1 levels in sHLH/MAS monocytes were found.
Our data demonstrating higher basal levels of pSTAT1 as well as a hyperreactivity to IFNγ stimulation in monocytes from patients with sHLH/MAS point to perturbations in the activation of downstream IFNγ signaling pathway as a contributor to the hyperinflammation occurring in these patients. Finally, the observation that glucocorticoids affect pSTAT1 levels
, makes it difficult to consider the measurement of pSTAT1 levels as a biomarker to identify patients at early stages of sHLH/MAS in clinical practice.
We have recently provided new evidence for a role of p75NTR receptor and its preferential ligand proNGF in amplifying inflammatory responses in synovial mononuclear cells of chronic arthritis ...patients. In the present study, to better investigate how activation of the p75NTR/proNGF axis impacts synovial inflammation, we have studied the effects of proNGF on fibroblast-like synoviocytes (FLS), which play a central role in modulating local immune responses and in activating pro-inflammatory pathways. Using single cell RNA sequencing in synovial tissues from active and treatment-naïve rheumatoid arthritis (RA) patients, we demonstrated that p75NTR and sortilin, which form a high affinity receptor complex for proNGF, are highly expressed in PRG4
lining and THY1
COL1A1
sublining fibroblast clusters in RA synovia but decreased in RA patients in sustained clinical remission. In
experiments we found that FLS from rheumatoid arthritis patients (RA-FLS) retained
a markedly higher expression of p75NTR and sortilin than FLS from osteoarthritis patients (OA-FLS). Inflammatory stimuli further up-regulated p75NTR expression and induced endogenous production of proNGF in RA-FLS, leading to an autocrine activation of the proNGF/p75NTR pathway that results in an increased release of pro-inflammatory cytokines. Our data on the inhibition of p75NTR receptor, which reduced the release of IL-1β, IL-6 and TNF-α, further confirmed the key role of p75NTR activation in regulating inflammatory cytokine production. In a set of
experiments, we used RA-FLS and cultured them in the presence of synovial fluids obtained from arthritis patients that, as we demonstrated, are characterized by a high concentration of proNGF. Our data show that the high levels of proNGF present in inflamed synovial fluids induced pro-inflammatory cytokine production by RA-FLS. The blocking of NGF binding to p75NTR using specific inhibitors led instead to the disruption of this pro-inflammatory loop, reducing activation of the p38 and JNK intracellular pathways and decreasing inflammatory cytokine production. Overall, our data demonstrate that an active proNGF/p75NTR axis promotes pro-inflammatory responses in synovial fibroblasts, thereby contributing to chronic synovial inflammation, and point to the possible use of p75NTR inhibitors as a novel therapeutic approach in chronic arthritis.
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