Enhanced osteoclastogenesis in patients with MSMD due to impaired response to IFN-γ

概要

Enhanced osteoclastogenesis in patients with
MSMD due to impaired response to IFN-g
Miyuki Tsumura, PhD,a* Mizuka Miki, MD,a,b*à Yoko Mizoguchi, MD, PhD,a Osamu Hirata, MD, PhD,a,cà
Shiho Nishimura, MD, PhD,a,dà Moe Tamaura, MD, PhD,a,eà Reiko Kagawa, MD, PhD,a Seiichi Hayakawa, MD, PhD,a
Masao Kobayashi, MD, PhD,a,fৠand Satoshi Okada, MD, PhDa§
Hiroshima, Japan
Background: Patients with Mendelian susceptibility to
mycobacterial disease (MSMD) experience recurrent and/or
persistent infectious diseases associated with poorly virulent
mycobacteria. Multifocal osteomyelitis is among the
representative manifestations of MSMD. The frequency of
multifocal osteomyelitis is especially high in patients with
MSMD etiologies that impair cellular response to IFN-g, such
as IFN-gR1, IFN-gR2, or STAT1 deficiency.
Objectives: This study sought to characterize the mechanism
underlying multifocal osteomyelitis in MSMD.
Methods: GM colonies prepared from bone marrow
mononuclear cells from patients with autosomal dominant (AD)
IFN-gR1 deficiency, AD STAT1 deficiency, or STAT1 gain of
function (GOF) and from healthy controls were differentiated
into osteoclasts in the presence or absence of IFN-g. The
inhibitory effect of IFN-g on osteoclastogenesis was investigated
by quantitative PCR, immunoblotting, tartrate-resistant acid
phosphatase staining, and pit formation assays.
Results: Increased osteoclast numbers were identified by
examining the histopathology of osteomyelitis in patients with
AD IFN-gR1 deficiency or AD STAT1 deficiency. In the presence
of receptor activator of nuclear factor kappa-B ligand and
M-CSF, GM colonies from patients with AD IFN-gR1
deficiency, AD STAT1 deficiency, or STAT1 GOF differentiated
into osteoclasts, similar to GM colonies from healthy volunteers.
IFN-g concentration-dependent inhibition of osteoclast
formation was impaired in GM colonies from patients with AD

From athe Department of Pediatrics, Hiroshima University Graduate School of Biomedical Sciences, bthe Department of Pediatrics, Hiroshima Red Cross Hospital and
Atomic-bomb Survivors Hospital, cthe Hidamari Children Clinic, dthe Department
of Pediatrics, Hiroshima City Hiroshima Citizens Hospital, ethe Department of Pediatrics, Hiroshima-Nishi Medical Center, and fthe Japanese Red Cross, Chugoku-Shikoku Block Blood Center.
*These authors contributed equally to this work.
àCurrent affiliation.
§These authors contributed equally to this work.
This study was supported in part by Grants in Aid for Scientific Research from the Japan
Society for the Promotion of Science (grants 15K21189, 17K10112, and 20K08158 to
M.T.; 22591161 to M.K.; 16H05355 and 19H03620 to S.O.) and was supported in part
by the Practical Research Project for Rare/Intractable Diseases from Japan Agency for
Medical Research and Development (grants JP16ek0109179, JP19ek0109209, and
JP20ek0109480 to S.O.).
Disclosure of potential conflict of interest: The authors declare that they have no relevant
conflicts of interest.
Received for publication February 15, 2021; revised May 6, 2021; accepted for publication May 11, 2021.
Corresponding author: Satoshi Okada, MD, PhD, Department of Pediatrics, Hiroshima
University Graduate School of Biomedical and Health Sciences, 1-2-3 Kasumi,
Minami-ku, Hiroshima 734-8551, Japan. E-mail: sokada@hiroshima-u.ac.jp.
0091-6749/$36.00
Ó 2021 American Academy of Allergy, Asthma & Immunology
https://doi.org/10.1016/j.jaci.2021.05.018

IFN-gR1 deficiency or AD STAT1 deficiency, whereas it was
enhanced in GM colonies from patients with STAT1 GOF.
Conclusions: Osteoclast differentiation is increased in AD IFNgR1 deficiency and AD STAT1 deficiency due to an impaired
response to IFN-g, leading to excessive osteoclast proliferation
and, by inference, increased bone resorption in infected foci,
which may underlie multifocal osteomyelitis. (J Allergy Clin
Immunol 2021;nnn:nnn-nnn.)
Key words: Mendelian susceptibility to mycobacterial diseases,
MSMD,
STAT1,
IFN-gR1,
mycobacteria,
osteomyelitis,
osteoclastogenesis

Mendelian susceptibility to mycobacterial disease (MSMD)
(Online Mendelian Inheritance in Man no. 209950) is a primary
immunodeficiency characterized by susceptibility to clinical
disease caused by intramacrophagic pathogens, such as BCG,
nontuberculous mycobacteria, or salmonella.1 To date, 11
MSMD-causing genes (IFNGR1, IFNGR2, STAT1, IL12B,
IL12RB1, ISG15, IRF8, TYK2, SPPL2A, CYBB, and IKBKG)
that are involved in IL-12/IFN-g immune responses have been reported.2-18 Patients with MSMD experience recurrent and/or
persistent infectious diseases associated with poorly virulent mycobacteria, such as BCG and nontuberculous mycobacteria.
Indeed, BCG disease after vaccination is frequently found in patients with MSMD.2 Multifocal osteomyelitis, sometimes with
confirmation of the presence of mycobacteria in biopsy specimens, is among the representative and specific manifestations
of MSMD.2,19 An x-ray examination of osteomyelitic regions
shows osteolytic changes occasionally surrounded by sclerotic lesions, indicating the presence of chronic osteomyelitis.20,21 Histopathological analysis of biopsy specimens generally shows
granuloma. Although the frequency is relatively low, the presence
of occasional acid-fast bacilli has been identified in granulomatous lesions in typical cases.20 Interestingly, the frequency of
multifocal osteomyelitis is especially high in patients with
MSMD due to an impaired response to IFN-g, such as that resulting from IFN-gR1, IFN-gR2, or STAT1 deficiency.2,19,22,23
Among these disorders, the frequency of bone involvement is
high in patients with autosomal dominant (AD) IFN-gR1 deficiency or AD STAT1 deficiency, whereas it is somehow relatively
low in patients with complete defect of IFN-g signaling due to
autosomal recessive (AR) IFN-gR1 complete deficiency or AR
complete STAT1 deficiency (Table I, and see Tables E1-E3 in
this article’s Online Repository at www.jacionline.org).19,22 In
addition, it is known that patients with AD IFN-gR1 deficiency
typically present with multifocal osteomyelitis predominantly
affecting the axial skeleton.19 On the other hand, salmonellosis
is relatively frequent in patients with a deficiency in IL-12Rb1
or IL-12p40, which are involved in both IL-12 and IL-23
1

2 TSUMURA ET AL

Abbreviations used
AD: Autosomal dominant
AR: Autosomal recessive
BM-MNCs: Bone marrow derived mononuclear cells
CNO: Chronic nonbacterial osteomyelitis
GOF: Gain of function
IRF: Interferon regulatory factor
MSMD: Mendelian susceptibility to mycobacterial diseases
NFATc1: Nuclear factor of activated T cells, cytoplasmic 1
RANKL: Receptor activator of nuclear factor kappa-B ligand
TRAP: Tartrate-resistant acid phosphatase

signaling.2,24,25 However, the frequency of bone involvement is
low in patients with these 2 disorders.
Among other functions, IFN-g is a cytokine that can inhibit
osteoclastogenesis and osteoclast bone resorption activity in
humans and mice.26-29 In this study, we examined the inhibitory
effect of IFN-g on receptor activator of nuclear factor kappa-B
ligand (RANKL)- and M-CSF–mediated osteoclast formation
with bone marrow–derived osteoclast precursor cells from patients with AD IFN-gR1 deficiency or AD STAT1 deficiency.
Based on the results of current study, we propose a possible
link between multifocal osteomyelitis found in patients with
MSMD and enhanced osteoclast differentiation due to the loss
of suppression triggered by IFN-g signaling.

METHODS
Patients
One patient with AD IFN-gR1 deficiency (patient 1: heterozygous
c.774delTCTA mutation in IFNGR1), 2 patients with AD STAT1 deficiency
(patient 2: heterozygous p.G250E mutation in STAT1, patient 3: heterozygous
p.Y701C mutation in STAT1), 1 patient with STAT1 gain of function (GOF)
(patient 4: heterozygous GOF mutation, p.R274Q, in STAT1), and healthy volunteers were enrolled in this study. All 3 patients with AD IFN-gR1 or AD
STAT1 deficiency included in this study had clinical episodes of multifocal
osteomyelitis. Detailed clinical records are available in previous reports.22,30-33 Briefly, patient 1 had a history of BCG lymphadenitis at the
age of 6 months. She developed multifocal osteomyelitis associated with
Mycobacterium avium infection at the age of 12 years.30 Patient 2 developed
multifocal osteomyelitis at the age of 2 years. BCG was suspected as a pathogen based on detection of the M tuberculosis complex by PCR, with positive
tuberculin skin test and negative QuantiFERON-TB2G (Qiagen, Hilden, Germany) test results.32 Patient 3 developed multifocal osteomyelitis at the age of
3 years.22 Although the pathogenic bacteria were not isolated, mycobacterial
infection was suspected because the clinical symptoms improved in response
to antimycobacterial drugs.

Immunohistochemical staining of bone marrow
biopsy sections
Immunohistochemical staining for tartrate-resistant acid phosphatase
(TRAP) using 3,3’-diaminobenzidine was performed on ethanol-fixed frozen
bone marrow biopsy sections. The sections were fixed with 95% ethanol at
room temperature for 10 minutes. To block activity of endogenous enzymes,
the sections were treated with 3% H2O2 at room temperature for 5 minutes. The
sections were then treated with Blocking One (Nacalai Tesque, Kyoto, Japan)
and incubated with anti-TRAP mouse monoclonal antibodies (1:500 dilution;
Leica Biosystems, Wetzlar, Germany) overnight at 48C. Peroxidase-conjugated
goat anti-mouse IgG (Nichrei, Tokyo, Japan) was then used as a secondary antibody. The sections were then treated with 3,3-diaminobenzidine-4HCl at room
temperature for 1 minute. Nuclei were then stained with Mayer’s hematoxylin

J ALLERGY CLIN IMMUNOL
nnn 2021

at room temperature for 1 minute. Images were acquired with a Keyence
BZ-9000 (Osaka, Japan) bright-field microscope. ...

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