[This corrects the article DOI: 10.1371/journal.pone.0169687.].

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare, pediatric, autoinflammatory disease characterized by bone pain due to sterile osteomyelitis, and is often accompanied by psoriasis or inflammatory bowel disease. There are two syndromic forms of CRMO, Majeed syndrome and DIRA, for which the genetic cause is known. However, for the majority of cases of CRMO, the genetic basis is unknown. Via whole-exome sequencing, we detected a homozygous mutation in the filamin-binding domain of FBLIM1 in an affected child with consanguineous parents. Microarray analysis of bone marrow macrophages from the CRMO murine model (cmo) determined that the Fblim1 ortholog is the most differentially expressed gene, downregulated over 20-fold in the cmo mouse. We sequenced FBLIM1 in 96 CRMO subjects and found a second proband with a novel frameshift mutation in exon 6 and a rare regulatory variant. In SaOS2 cells, overexpressing the regulatory mutation showed the flanking region acts as an enhancer, and the mutation ablates enhancer activity. Our data implicate FBLIM1 in the pathogenesis of sterile bone inflammation and our findings suggest CRMO is a disorder of chronic inflammation and imbalanced bone remodeling.

Polymorphonuclear leukocytes (PMN) achieve an intermediate or primed state of activation following stimulation with certain agonists. Primed PMN have enhanced responsiveness to subsequent stimuli, which can be beneficial in eliminating microbes but may cause host tissue damage in certain disease contexts, including sepsis. As PMN priming by TLR4 agonists is well described, we hypothesized that ligation of TLR2/1 or TLR2/6 would prime PMN. Surprisingly, PMNfrom only a subset of donors were primed in response to the TLR2/1 agonist, Pam(3)CSK(4), although PMN from all donors were primed by the TLR2/6 agonist, FSL-1. Priming responses included generation of intracellular and extracellular reactive oxygen species, MAPK phosphorylation, integrin activation, secondary granule exocytosis, and cytokine secretion. Genotyping studies revealed that PMN responsiveness to Pam3CSK4 was enhanced by a common single-nucleotide polymorphism (SNP) in TLR1 (rs5743618). Notably, PMN from donors with the SNP had higher surface levels of TLR1 and were demonstrated to have enhanced association of TLR1 with the endoplasmic reticulum chaperone gp96. We analyzed TLR1 genotypes in a pediatric sepsis database and found that patients with sepsis or septic shock who had a positive blood culture and were homozygous for the SNP associated with neutrophil priming had prolonged pediatric intensive care unit length of stay. We conclude that this TLR1 SNP leads to excessive PMN priming in response to cell stimulation. Based on our finding that septic children with this SNP had longer pediatric intensive care unit stays, we speculate that this SNP results in hyperinflammation in diseases such as sepsis.

To the Editor: Majeed syndrome (OMIM #609628) is a syndromic form of chronic recurrent multifocal osteomyelitis (CRMO) that presents with early onset, severe CRMO, and a microcytic dyserythropoietic anemia of variable severity. Onset in all reported cases has been in the first 2 years of life1,2,3. A minority of patients develop a neutrophilic dermatosis. The disease is due to mutations in LPIN2 , which encodes LIPIN2, a phosphatidate phosphatase important in lipid metabolism2,3,4,5,6. Here we present the case histories of 2 male cousins of Indian heritage with Majeed syndrome. Both born to parents with consanguineous marriages, the proband presented with typical Majeed syndrome (onset of CRMO by 2 yrs of age, significant microcytic dyserythropoietic anemia, and failure to thrive), but the cousin had later onset, milder disease more reminiscent of nonsyndromic CRMO (onset at 8 yrs of age and minimal anemia). Both were found to be homozygous for the same novel LPIN2 mutation. Our report details the natural history of the disease in these 2 boys, expands the at-risk ethnic and racial group of Majeed syndrome, and demonstrates that the disease can present … Address correspondence to Dr. P.J. Ferguson, Department of Pediatrics, University of Iowa Carver College of Medicine, 200 Hawkins Drive, Iowa City, Iowa 52242, USA. E-mail: polly-ferguson{at}uiowa.edu

SignificanceChronic recurrent multifocal osteomyelitis (CRMO) is a human disorder of the innate immune system that causes bone inflammation that mimics infectious osteomyelitis. There is a spontaneous mutant mouse model of the disease that is caused by homozygous mutations in the genePstpip2. Our studies show that bone inflammation in this model is mediated by the cytokine IL-1β, but that the disease is independent of the nucleotide-binding domain, leucine-rich repeat-containing family, pyrin domain-containing 3 (NLRP3) inflammasome and caspase-1, which is different from most other IL-1–mediated disorders. Further, we implicate neutrophils and neutrophil serine proteases in disease pathogenesis. These data provide a rationale for directly targeting the IL-1 receptor or IL-1β as a therapeutic strategy in CRMO.

PSTPIP1 is a cytoskeletal adaptor and F-BAR protein that has been implicated in autoinflammatory disease, most notably in the PAPA syndrome: pyogenic sterile arthritis, pyoderma gangrenosum, and acne. However, the mechanism by which PSTPIP1 regulates the actin cytoskeleton and contributes to disease pathogenesis remains elusive. Here, we show that endogenous PSTPIP1 negatively regulates macrophage podosome organization and matrix degradation. We identify a novel PSTPIP1-R405C mutation in a patient presenting with aggressive pyoderma gangrenosum. Identification of this mutation reveals that PSTPIP1 regulates the balance of podosomes and filopodia in macrophages. The PSTPIP1-R405C mutation is in the SRC homology 3 (SH3) domain and impairs Wiskott-Aldrich syndrome protein (WASP) binding, but it does not affect interaction with protein-tyrosine phosphatase (PTP)-PEST. Accordingly, WASP inhibition reverses the elevated F-actin content, filopodia formation, and matrix degradation induced by PSTPIP1-R405C. Our results uncover a novel role for PSTPIP1 and WASP in orchestrating different types of actin-based protrusions. Our findings implicate the cytoskeletal regulatory functions of PSTPIP1 in the pathogenesis of pyoderma gangrenosum and suggest that the cytoskeleton is a rational target for therapeutic intervention in autoinflammatory disease.

Dynamic regulation of the actin cytoskeleton and cell migration is essential for cellular immunity, because leukocytes travel long distances between tissues to perform their effector functions. Indeed, immunodeficiency syndromes, including Wiskott-Aldrich syndrome, leukocyte adhesion deficiency, and warts-hypogammaglobulinemiainfections-myelokathexis syndrome, are secondary to defects in the cytoskeleton and motility of leukocytes. Colchicine, which inhibits microtubule polymerization, is used to treat inflammatory conditions, and several other compounds that target cell motility are in development as immunomodulators, which indicates the importance of regulating the cytoskeleton to control immunity and inflammation. Conversely, neutrophils from patients with the autoinflammatory disease, neonatal onset multisystem inflammatory disease/ Muckle-Wells syndrome, have impaired chemotaxis, which suggests that altered leukocyte migration may also promote a proinflammatory state. While altered leukocyte motility has been established as a cause of immunodeficiency, the role of cytoskeletal dysregulation and altered motility in inflammation and tissue damage remains poorly characterized. Proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1) is a cytoskeleton-associated adaptor and F-BAR domain– containing protein that is linked to PAPA syndrome, the human inflammatory disease consisting of pyogenic sterile arthritis, pyoderma gangrenosum, and acne. A hallmark of pyoderma gangrenosum is extensive tissue damage of unclear etiology. PAPA syndrome is considered to be an autoinflammatory disease because the adaptor function of PSTPIP1 links it to the inflammasome component, pyrin, to regulate interleukin-1b activity. However, patients with PAPA syndrome and pyoderma gangrenosum are often resistant to treatment with antiinflammatory agents that block tumor necrosis factor alpha and interleukin-1b signaling. Additionally, a mouse model of PAPA syndrome indicated that PSTPIP1 did not regulate the inflammasome in vivo, suggesting the interesting possibility that other PSTPIP1 adaptor functions are important in the pathogenesis of PAPA syndrome. The PSTPIP1 interacting partners, Wiskott-Aldrich syndrome protein (WASP) and protein-tyrosine phosphatase (PTP)-PEST (Protein Tyrosine Phosphatase, Non-Receptor Type 12 [PTPN12]), are important regulators of the cytoskeleton and cell migration, suggesting that PSTPIP1mutations could contribute to disease through these pathways.WASP is a key activator of actin-related protein2/3 (Arp2/3), and it plays an important role in nucleating new F-actin

BACKGROUND AND OBJECTIVE:Majeed syndrome is an autosomal recessive disorder characterised by the triad of chronic recurrent multifocal osteomyelitis, congenital dyserythropoietic anaemia and a neutrophilic dermatosis that is caused by mutations in LPIN2. Long-term outcome is poor. This is the first report detailing the treatment of Majeed syndrome with biological agents and demonstrates clinical improvement with IL-1blockade. METHODS:We describe the clinical presentation, genetic analysis, cytokine profiles and response to biological therapy in two brothers with Majeed syndrome. RESULTS:Both boys were homozygous for a novel 2-base pair deletion in LPIN2 (c.1312_1313delCT; p.Leu438fs+16X), confirming the diagnosis. Their bone disease and anaemia were refractory to treatment with corticosteroids. Both siblings had elevated proinflammatory cytokines in their serum, including tumour necrosis factor α (TNF-α), however a trial of the TNF inhibitor etanercept resulted in no improvement. IL-1 inhibition with either a recombinant IL-1 receptor antagonist (anakinra) or an anti-IL-1β antibody (canakinumab) resulted in dramatic clinical and laboratory improvement. CONCLUSIONS:The differential response to treatment with TNF-α or IL-1 blocking agents sheds light into disease pathogenesis; it supports the hypothesis that Majeed syndrome is an IL-1β dependent autoinflammatory disorder, and further underscores the importance of IL-1 in sterile bone inflammation.

We describe a boy who developed autoinflammatory (chronic sterile multifocal osteomyelitis) and autoimmune (autoimmune cytopenias; vitiligo) phenotypes who subsequently developed disseminated granulomatous disease. Whole exome sequencing revealed homozygous RAG1 mutations thus expanding the spectrum of combined immunodeficiency with autoimmunity and granuloma that can occur with RAG deficiency.

We performed whole-exome sequencing of a family with autosomal dominant Dandy-Walker malformation and occipital cephaloceles and detected a mutation in the extracellular matrix (ECM) protein-encoding gene NID1. In a second family, protein interaction network analysis identified a mutation in LAMC1, which encodes a NID1-binding partner. Structural modeling of the NID1-LAMC1 complex demonstrated that each mutation disrupts the interaction. These findings implicate the ECM in the pathogenesis of Dandy-Walker spectrum disorders.

BACKGROUNDAutoinflammatory diseases manifest inflammation without evidence of infection, high-titer autoantibodies, or autoreactive T cells. We report a disorder caused by mutations of IL1RN, which encodes the interleukin-1-receptor antagonist, with prominent involvement of skin and bone.METHODSWe studied nine children from six families who had neonatal onset of sterile multifocal osteomyelitis, periostitis, and pustulosis. Response to empirical treatment with the recombinant interleukin-1-receptor antagonist anakinra in the first patient prompted us to test for the presence of mutations and changes in proteins and their function in interleukin-1-pathway genes including IL1RN.RESULTSWe identified homozygous mutations of IL1RN in nine affected children, from one family from Newfoundland, Canada, three families from the Netherlands, and one consanguineous family from Lebanon. A nonconsanguineous patient from Puerto Rico was homozygous for a genomic deletion that includes IL1RN and five other interleukin-1-family members. At least three of the mutations are founder mutations; heterozygous carriers were asymptomatic, with no cytokine abnormalities in vitro. The IL1RN mutations resulted in a truncated protein that is not secreted, thereby rendering cells hyperresponsive to interleukin-1 beta stimulation. Patients treated with anakinra responded rapidly.CONCLUSIONSWe propose the term deficiency of the interleukin-1-receptor antagonist, or DIRA, to denote this autosomal recessive autoinflammatory disease caused by mutations affecting IL1RN. The absence of interleukin-1-receptor antagonist allows unopposed action of interleukin-1, resulting in life-threatening systemic inflammation with skin and bone involvement. (ClinicalTrials.gov number, NCT00059748.)

SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis) is an inflammatory disorder of the bone, skin, and joints. We describe a family with multiple affected members who segregate a SAPHO syndrome-like phenotype, and we report the results of neutrophil studies and candidate gene analysis. We obtained written informed consent and a family history and reviewed medical records. We collected DNA and sequenced candidate genes, and we performed functional studies on neutrophils isolated from the proband and her mother. The pedigree segregated chronic osteomyelitis and cutaneous inflammation in a pattern that suggested an autosomal-dominant disorder. No coding sequence mutations were detected in PSTPIP1, PSTPIP2, LPIN2, SH3BP2, or NCF4. Analysis of neutrophil function in the proband, including nitroblue tetrazolium tests, myeloperoxidase assays, neutrophil chemotaxis, and neutrophil chemotaxis assays, revealed no identifiable abnormalities. However, an abnormality in the luminol, but not the isoluminol, respiratory burst assays following stimulation with phorbol myristate acetate (PMA) was detected in neutrophils isolated from the affected proband. Internal oxidant production was also reduced in the proband and her mother when neutrophils were treated with fMLP with or without platelet-activating factor, PMA alone, or tumor necrosis factor alpha alone. This family segregates a disorder characterized by chronic inflammation of the skin and bone. Functional differences in neutrophils exist between affected individuals and controls. The biologic significance of this defect remains unknown. Identification of the gene defect will help identify an immunologic pathway that, when dysregulated, causes inflammation of the skin and bone.