Loss-of-function OGFRL1 variants identified in autosomal recessive cherubism families.

Cherubism (OMIM 118400) is a rare craniofacial disorder in children characterized by destructive jawbone expansion due to the growth of inflammatory fibrous lesions. Our previous studies have shown that gain-of-function mutations in SH3 domain-binding protein 2 (SH3BP2) are responsible for cherubism and that a knock-in mouse model for cherubism recapitulates the features of cherubism, such as increased osteoclast formation and jawbone destruction. To date, SH3BP2 is the only gene identified to be responsible for cherubism. Since not all patients clinically diagnosed with cherubism had mutations in SH3BP2, we hypothesized that there may be novel cherubism genes and that these genes may play a role in jawbone homeostasis. Here, using whole exome sequencing, we identified homozygous loss-of-function variants in the opioid growth factor receptor like 1 (OGFRL1) gene in 2 independent autosomal recessive cherubism families from Syria and India. The newly identified pathogenic homozygous variants were not reported in any variant databases, suggesting that OGFRL1 is a novel gene responsible for cherubism. Single cell analysis of mouse jawbone tissue revealed that Ogfrl1 is highly expressed in myeloid lineage cells. We generated OGFRL1 knockout mice and mice carrying the Syrian frameshift mutation to understand the in vivo role of OGFRL1. However, neither mouse model recapitulated human cherubism or the phenotypes exhibited by SH3BP2 cherubism mice under physiological and periodontitis conditions. Unlike bone marrow-derived M-CSF-dependent macrophages (BMMs) carrying the SH3BP2 cherubism mutation, BMMs lacking OGFRL1 or carrying the Syrian mutation showed no difference in TNF-ɑ mRNA induction by LPS or TNF-ɑ compared to WT BMMs. Osteoclast formation induced by RANKL was also comparable. These results suggest that the loss-of-function effects of OGFRL1 in humans differ from those in mice and highlight the fact that mice are not always an ideal model for studying rare craniofacial bone disorders.

Second-Generation SYK Inhibitor Entospletinib Ameliorates Fully Established Inflammation and Bone Destruction in the Cherubism Mouse Model.

Cherubism is a craniofacial disorder characterized by maxillary and mandibular bone destruction. Gain-of-function mutations in the SH3-domain binding protein 2 (SH3BP2) are responsible for the excessive bone resorption caused by fibrous inflammatory lesions. A homozygous knock-in (KI) mouse model for cherubism (Sh3bp2KI/KI ) develops autoinflammation resulting in systemic bone destruction. Although administration of the TNF-α blocker etanercept to neonatal Sh3bp2KI/KI mice prevented the disease onset, this therapy was not effective for adult Sh3bp2KI/KI mice or human cherubism patients who already had lesions. Because genetic ablation of spleen tyrosine kinase (SYK) in myeloid cells rescues Sh3bp2KI/KI mice from inflammation, we examined whether SYK inhibitor administration can improve fully developed cherubism symptoms in adult Sh3bp2KI/KI mice. Entospletinib (GS-9973) was intraperitoneally injected into 10-week-old Sh3bp2KI/KI mice every day for 6 weeks. Treatment with GS-9973 improved facial swelling and histomorphometric analysis of lung and liver tissue showed that GS-9973 administration significantly reduced inflammatory infiltrates associated with decreased levels of serum TNF-α. Micro-computed tomography (µCT) analysis showed that GS-9973 treatment reduced bone erosion in mandibles, calvariae, and ankle and elbow joints of Sh3bp2KI/KI mice compared to Sh3bp2KI/KI mice treated with dimethyl sulfoxide (DMSO). Taken together, the results demonstrate that administration of the SYK inhibitor ameliorates an already established cherubism phenotype in mice, suggesting that pharmacological inhibition of SYK may be a treatment option for cherubism patients with active disease progression. © 2018 American Society for Bone and Mineral Research.

Increased susceptibility to Staphylococcus aureus colonization of the skin of the NOA mouse: a potentially useful animal model for evaluating antiseptic effects.

Isolation of bacteria from wet skin lesions was attempted using Naruto Research Institute Otsuka Atrichia (NOA) mice, which develop such lesions spontaneously at a high rate. As a result, Staphylococcus aureus was demonstrated to have colonized the wet skin lesions at high density. In addition, the isolated S. aureus was found to be similar to the strain of S. aureus thought to colonize the eczematous lesions seen in humans with atopic dermatitis. Furthermore, a survey of the S. aureus colonization status of NOA mice with no wet skin lesions confirmed colonization at higher density than in HR-1 mice as control, indicating that the skin of the NOA mouse has the novel characteristic of increased susceptibility to S. aureus colonization. Thus, by using changes in S. aureus counts as an index, the NOA mouse can be expected to serve as a useful animal model for evaluating the effects of topical antiseptics. The antiseptic effects of an ointment and a lotion containing chlorhexidine gluconate were confirmed using this animal model.

Development of dry skin in the NOA mouse under individual housing conditions: a potentially useful animal model for evaluating moisturizing effects.

In a previous study, we reported the development of grossly observable dry skin in all of the Naruto Research Institute Otsuka Atrichia (NOA) mice that were housed individually. In the present study, dermal physiological function tests were conducted and the usefulness of this dry skin model for evaluating the efficacy of topical moisturizers was assessed. As a result, we have confirmed a marked reduction in the water content of the stratum corneum in these animals. Therefore, the development of dry skin in the NOA mouse strain under individual housing conditions may be expected to serve as a useful animal model for evaluating topical moisturizers. Specifically, the water content of the stratum corneum was restored in proportion to the oil content of the ointment base used to treat the animals, and the moisturizing effects of urea were confirmed in animals treated with urea-containing ointment. In addition, when the animals that had been housed individually were returned to group housing conditions, the water content of the stratum corneum was restored, with a corresponding improvement in dry skin. This finding suggests that socio-psychological factors are involved in the etiology of dry skin in individually housed NOA mice.

Effects of housing conditions on the development of wet skin lesions in the NOA mouse.

The effects of housing on the onset time and prevalence of wet skin lesions were investigated in NOA mice, which spontaneously develop these lesions at a high rate. Wet skin lesions developed earliest in mice that were housed individually. For mice that were housed in groups, the lesions developed earlier in mice with non-littermate group housing than in mice with littermate group housing. The prevalence of lesions was in the following order: individual housing > non-littermate group housing > littermate group housing. These results suggest that socio-psychological factors are involved in the etiology of wet skin lesions in the NOA mouse. Under individual housing conditions, two other novel characters of the NOA mouse were also observed, specifically, development of dry skin and wet skin lesions at the tail root. These characteristics developed early and with high prevalence and were easily observed on external examination. Therefore, these novel characteristics observed in NOA mice are potential markers of the psychological state of the animals.

Strong interaction between human herpesvirus 6 and peripheral blood monocytes/macrophages during acute infection.

Human herpesvirus 6 (HHV-6) encodes a viral chemokine and chemokine receptors that may modify the functions of monocytes/macrophages (MO/M phi) during productive HHV-6 infection. The interactions between HHV-6 and MO/M phi during acute infection, however, remain poorly understood. In this study, we investigated the tropism of HHV-6 in peripheral blood mononuclear cells (PBMCs) during acute infection. We detected 637 +/- 273 copies of viral DNA in 10(4) MO/M phi. in contrast, in 10(4) CD4+ T cells, which have been reported to be viral carriers during the acute infection of HHV-6, we found only 115 +/- 42 copies of viral DNA. Consistent with these data, virus was isolated from MO/M phi an order of magnitude more frequently than from CD4+ T cells. Viral mRNA U79/80, which indicates viral replication, was detectable in the MO/M phi. In addition, the mRNAs that encode viral chemokine receptors U12 and U51, which may modify the function of MO/M phi, were expressed in the cells. Therefore, productively infected MO/M phi may be the dominant cell population that is responsible for HHV-6 viremia during acute HHV-6 infection. The strong interaction of HHV-6 with MO/M phi may be partly responsible for the pathogenesis of this virus.