1. Research update on
SJIA and MAS
SJIA Family Education Day
July 22, 2017
Grant Schulert, MD PhD
Assistant Professor of Pediatrics
Cincinnati Children’s Hospital Medical Center

2. Key Questions
• What is the cause of systemic JIA?
• What happens during MAS, and why do
only some patients get MAS?

3. Autoimmune
Disease (1900s)
Adaptive immunity
High-titer autoantibodies
and/or autoreactive,
antigen-specific T cells
Destructive immune
response to self
antigens
Lupus, rheumatoid
arthritis
Autoinflammatory
Disease (late 1990s)
Innate immunity
NO high-titer autoantibodies
or autoreactive, antigen-
specific T cells
Episodes of seemingly
unprovoked inflammation
that sometimes results in
tissue destruction
Periodic fever syndromes
(FMF, TRAPs, etc);
Systemic JIA?

4. Complex diseases: (many) genes
and the environment
Source: CDC

5. Genetics and systemic JIA
• Systemic JIA is rarely
familial* although
twins/siblings with
disease have been
described
• Higher incidence in some
ethnic groups (Asia,
Northern vs Southern
Europe) suggests shared
genetic risk

6. DNA Sequence Variation in the
Human Genome
• In general, each human’s
genome is >99.5% identical to
any other humans
• But, that means up to 10-20
million bases of differences
– Mostly single nucleotide
changes
• Nomenclature:
– Single nucleotide polymorphism
(SNP) – change present in at
least 1% of population
– Rare variant – present in less
than 1% of population

7. SNPs and genome-wide
association studies (GWAS)
• GWAS are large-scale studies to
identify associations between SNPs
and phenotypes (diseases)
• Compare SNP prevalence between
thousands of patients and controls
• SNPs that are associated with disease
are considered to mark genomic
regions that may influence risk of
disease
• Results: Manhattan plots, where height
of peak reflects strength of association

8. GWAS in (non-systemic) JIA
• Examined 2816
patients with JIA
oligoarticular or RF-
negative polyarticular
JIA (poligos) compared
to 13000 controls
• Used Immunochip –
123,000 SNPs with
minor allele frequency
>1%
Nature Genetics 45, 664–669 (2013)

9. GWAS in (non-systemic) JIA
Nature Genetics 45, 664–669 (2013)

10. GWAS in (non-systemic) JIA
• Highest single SNP in
HLA-DQB1 region, 2%
in controls vs. 12% in
patients (OR=6.01,
p=3.14x10-174)
• Other identified genes
with known functions in
immunity and roles in
other autoimmune
diseases

11. GWAS in systemic JIA
• Large, multinational collaborative led by Dr.
Michael Ombrello at NIH
• Examined 982 children with systemic JIA
compared to ~9000 controls
– 770 SJIA patients stratified into 9 geographically
defined, ancestrally matched case-control collections
• 27-35K SNPs in the MHC region, overall 4-6million
genotypes or imputed SNPs
Proc Natl Acad Sci USA (2015) 112: 15970

12. GWAS in systemic JIA
Proc Natl Acad Sci USA (2015) 112: 15970
Ann Rheum Dis (2016)

13. GWAS in systemic JIA
• Clearly identified the
MHC locus as a
significant (if weaker)
bona fide susceptibility
region for systemic JIA
in multiple populations
• Top SNP: HLA-
DRB1*11, OR 2.6
(p=2.8x10-17)
Proc Natl Acad Sci USA (2015) 112: 15970

14. GWAS in systemic JIA
• In addition, 24 genetic
regions with suggestive
association with systemic
JIA
• None previously associated
with any rheumatic
diseases
• No evidence for shared
genetic architecture
between systemic JIA and
other JIA subtypes
Ann Rheum Dis (2016)

15. Key Questions
• What is the cause of systemic JIA?
• What happens during MAS, and why do
only some patients get MAS?

16. MΦ
Proposed Pathogenic Mechanism of Macrophage
Activation Syndrome
CD8+
IFN-γ
GM-CSF
MΦ
MΦ
MΦ
MΦ
MΦMΦ
MΦ
Mo
TNF-α
IL-6
IL-1
IL-18
M-CSF
Hemostatic
Tissue Factor
sCD163
sCD8
sIL-2Rα
CD8+
CD8+
CD8+
CD8+
CD8+
CD8+
CD8+
CD8+
CD8+
CD8+
CD8+
MΦ
Cytokine
storm

17. IFNgamma and MAS/HLH
• IFNg believed to be key
driver of inflammation in
familial HLH
– High levels in patients
– IFNg blockade protective in
mice
– Promising early results in
clinical trial of anti-IFNg
monoclonal antibody
• Does IFNg have similar
central role in MAS?
Hemophagocytic macrophages in MAS

18. 1 Number (percentile); 2 Median (IQR); 3 At least one time during disease course
4 Complete remission of MAS/HLH episode after treatment according to the judgment of the investigator
5 Clinical inactive disease according to Wallace Criteria
PATIENTS
NUMBER OF SAMPLES
ACTIVE
DISEASE
INACTIVE
DISEASE
sec-HLH n=14 11 114
Gender, Female1 5 (36)
Age at disease onset (years)2 8.6 (4.1 – 12.9)
sJIA n=54 48 355
Gender, Female1 26 (48)
Age at disease onset (years)2 7.9 (4.6 – 13.6)
sJIA with MAS3 n=27 20 204
Gender, Female1 13 (48)
Age at disease onset
(years)2
9.4 (4.8 – 13.8)
sJIA without MAS n=27 28 155
Gender, Female1 13 (48)
Age at disease onset
(years)2
7.3 (4.1 – 12.1)
Patients’ samples collected at OPBG in Roma, IGG in Genova, and CCHMC in
Cincinnati
Bracaglia et al (in revision

19. Active sHLH
(n=11)
Active MAS
(n=20)
Active sJIA
No MAS
(n=28)
Inactive sJIA
(n=35)
IL-6 11.4
(3.2-49.3)
22.9
(5.5-45.6)
20.3
(5.9-54.9)
3.2
(3.2-7.9)
TNFa 27.6
(10.8-49.2)
14.7
(7.1-33.1)
10
(5.3-15.0)
9.4
(6.1-18.5)
IFNg 34.7
(23.9-170.1)
15.4
(5.1-52.6)
4.9
(3.2-8.6)
4.2
(3.2-9.3)
CXCL9 33598
(3083-127687)
13392
(2163-35452)
837
(471-2505)
901
(466-1213)
CXCL10 4420
(799-8226)
1612
(425-4309)
307
(199-694)
235
(172-407)
CXCL11 1327
(189-2000)
565
(198-1007)
122
(62-197)
111
(63-187)
*p<0.01 MAS vs Active sJIA
Bracaglia, et al. Ann Rheum Dis 2016

20. Serum IFNγ and IFN-induced
chemokines in MAS versus active SJIA
Bracaglia, et al. Ann Rheum Dis 2016

21. IFNg and IFN-induced chemokines
and laboratory features of MAS
Bracaglia, et al. Ann Rheum Dis 2016

22. IFNg, IL-18 and MAS
• IL-18 is a key
proinflammatory cytokine
– Inflammasome-mediated
production in myeloid cells
– Key driver of IFNg
production by CD8 cells
and NK cells
• IL-18 hypersecretion 
recurrent MAS
– NLRC4 gain-of-function
mutations (Canna et al)
Nature Immunology (2012) 13:115–117

23. High IL-18 subset of SJIA patients
develop MAS
• Cytokine levels during
active disease can
define two subsets of
SJIA patients:
– High IL-18/IL-6 – high
risk of MAS (green)
– Low IL-18/IL-6 – older,
more arthritis, no MAS
(n=33)
Clinical Immunology 160 (2015) 277–281

24. Cytolytic Defects in FHLH
Cytolytic cells cause destruction of target cells by
delivering granules that contain proteins such as
perforin and granzymes1
Recognition of target
Polarization of the
cytoskeleton
Movement of cytolytic
granules along MTs to the
site of contact
Degranulation and release
of contents
Delivery of content of
granules to target cell aided
by Perforin
24
Apoptosis
Target Cell
Effector Cell
(NK Cell)
Granzyme B
Fusion
Priming
Docking
Munc13-4
Munc18-2
Syntaxin 11
Rab27a
Nucleus
Cytolytic
Granule
Perforin
MTOC
Cytolytic Pathway1,2
Granzymes

25. HLH mutations associated with MAS
• Several reports have
identified rare mutations in
cytolytic pathway genes in
patients with MAS
– PRF1: Vastert et al (2010),
Unal et al (2013), Schulert et
al (2015)
– UNC13D: Hazen et al
(2008), Zhang et al (2008)
• Could there be a genetic
predisposition to MAS in
some patients with systemic
JIA?

26. Genetics of MAS in systemic JIA
• Trio-based whole exome sequencing performed for 14
patients with SJIA and MAS and unaffected parents
– Comparison: 29 patients with SJIA and no history of MAS
• Primary analysis – variants in HLH-associated genes
– PRF1, MUNC13-4, Syntaxin 11, STXBP2, LYST, Rab27A
• Secondary analysis
– Identification of other candidate genes / rare variants
Kaufman et al (2014) Arthritis Rheum

27. Kaufman et al (2014) Arthritis Rheum
Top IPA category of all identified genes:
“Cellular assembly and organization” (p<3.1 x 10-5)
5/14
36%

28. Current model for MAS in systemic
JIA

29. Some answers, many new
questions …
• How do genetic and environmental factors
interact to cause systemic JIA?
• Can cytokine-targeted therapy treat (anti-
IFNg, anti-IL-18) MAS?
• Can genetic or cytokine testing predict risk for
MAS?