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, 139 (1), 281-91

Human Risk Allele HLA-DRB1*0405 Predisposes Class II Transgenic Ab0 NOD Mice to Autoimmune Pancreatitis

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Human Risk Allele HLA-DRB1*0405 Predisposes Class II Transgenic Ab0 NOD Mice to Autoimmune Pancreatitis

Tobias L Freitag et al. Gastroenterology.

Abstract

Background & aims: Autoimmune pancreatitis (AIP) underlies 5%-11% of cases of chronic pancreatitis. An association between AIP and the human leukocyte antigen (HLA)-DRB1*0405/DQB1*0401 haplotype has been reported, but linkage disequilibrium has precluded the identification of predisposing HLA gene(s). We studied the role of single HLA genes in the development of AIP in transgenic mice.

Methods: CD4(+) T-cell-negative I-Abeta chain(-/-) (Ab0) mice develop AIP spontaneously, likely due to dysregulation of CD8(+) T- cell responses. We generated Ab0 nonobese diabetic (NOD) mice transgenic for HLA-DR*0405, leading to rescue of CD4(+) T cells; we compared their susceptibility to AIP with HLA-DQ8 or HLA-DR*0401 (single) transgenic, or HLA-DR*0405/DQ8 (double) transgenic mice.

Results: CD4(+) T-cell-competent HLA-DR*0405 transgenic Ab0 NOD mice develop AIP with high prevalence after sublethal irradiation and adoptive transfer of CD90(+) T cells, leading to complete pancreatic atrophy. HLA-DR*0405 transgenic mice can also develop unprovoked AIP, whereas HLA-DR*0401, HLA-DQ8, and HLA-DR*0405/DQ8 transgenic Ab0 NOD controls all remained normal, even after irradiation and adoptive transfer of CD90(+) T cells. Pancreas histology in HLA-DR*0405 transgenic mice was characterized by destructive infiltration of the exocrine tissue with CD4(+) and CD8(+) T cells, B cells, and macrophages. Mice with complete pancreatic atrophy lost weight, developed fat stools, and had reduced levels of serum lipase activity.

Conclusions: Because HLA-DR*0405 expression fails to protect mice from AIP, the HLA-DRB1*0405 allele appears to be an important risk factor for AIP on the HLA-DRB1*0405/DQB1*0401 haplotype. This humanized mouse model should be useful for studying immunopathogenesis, diagnostic markers, and therapy of human AIP.

Figures

Figure 1
Figure 1. Clinical and macroscopic changes in HLA-DR*0405 transgenic Ab0 NOD mice
a, b) Progressive loss of a) body weight and b) pancreas weight in HLA-DR*0405 transgenic vs. a) HLA-DR*0405/DQ8 transgenic, or b) HLA-DQ8 transgenic Ab0 NOD mice sublethally irradiated and transferred with CD90+ T cells from syngenic donor mice. Mice were monitored for 9–21 weeks post-treatment (n=6–10, weight- and sex-matched groups). c) Pancreas weights in 4 groups of HLA-DR*0405 Ab0 NOD mice (male vs. female, n=7–8) treated with sublethal irradiation and adoptive CD4+ T cell transfer, compared to animals that were left untreated. Mice were sacrificed after 18 weeks. d) Pancreas weights in sublethally irradiated normal NOD mice (n=11) analyzed 9 weeks post-treatment, or in untreated Ab0 NOD mice (n=21) at 35 weeks of age (***p<0.001, **p<0.01, *p<0.05).
Figure 1
Figure 1. Clinical and macroscopic changes in HLA-DR*0405 transgenic Ab0 NOD mice
a, b) Progressive loss of a) body weight and b) pancreas weight in HLA-DR*0405 transgenic vs. a) HLA-DR*0405/DQ8 transgenic, or b) HLA-DQ8 transgenic Ab0 NOD mice sublethally irradiated and transferred with CD90+ T cells from syngenic donor mice. Mice were monitored for 9–21 weeks post-treatment (n=6–10, weight- and sex-matched groups). c) Pancreas weights in 4 groups of HLA-DR*0405 Ab0 NOD mice (male vs. female, n=7–8) treated with sublethal irradiation and adoptive CD4+ T cell transfer, compared to animals that were left untreated. Mice were sacrificed after 18 weeks. d) Pancreas weights in sublethally irradiated normal NOD mice (n=11) analyzed 9 weeks post-treatment, or in untreated Ab0 NOD mice (n=21) at 35 weeks of age (***p<0.001, **p<0.01, *p<0.05).
Figure 2
Figure 2. Histological changes in the pancreas of HLA-DR*0405 transgenic Ab0 NOD mice (I)
a–d) Severe pancreatic atrophy characterized by extensive loss of acinar cell mass and replacement by degenerative fat, scattered infiltrates of lymphocytes and fibrosis (examples taken from HLA-DR* 0405 transgenic mice sacrificed 20 weeks after sublethal irradiation and adoptive CD90+ T cell transfer). a, b) H/E staining demonstating loss of zymogen granules in acinar cells and formation of ductular structures (→), indicating acinar-to-ductal metaplasia. Note preservation of the islets of Langerhans (←; original magn. a) 100×, b) 200×). c, d) Sirius red staining demonstrating variable degrees of fibrosis (mainly periductal, perivascular) in 2 samples with severe atrophy (magn. 200×).
Figure 3
Figure 3. Histological changes in the pancreas of HLA-DR*0405 transgenic Ab0 NOD mice (II)
a) H/E staining demonstrating periductal and perivascular mononuclear cell infiltration of the pancreas as an early finding in HLA-DR*0405 transgenic mice (magn. 200×). b, c) Fulminant AIP in HLA-DR* 0405 transgenic mice at 8 weeks post-treatment, characterized by multifocal, destructive invasion of the acinar tissue with lymphocytes, macrophages and few plasma cells (H/E staining; magn. b) 100×, c) 200×). d) Sirius red staining revealing collagen deposition in infiltrated lobuli in fulminant pancreatitis (representative sample, magn. 200×).
Figure 4
Figure 4. Immunohistochemical characterization of pancreatic inflammatory infiltrates in HLA-DR*0405 transgenic Ab0 NOD mice
a–e) Immunostainings of pancreas from HLA-DR*0405 mice with fulminant pancreatitis demonstrate inflammatory infiltrates consisting mainly of a) CD3+ T cells, b) B220+ B cells and c) Mac-2+ macrophages. Within the T cell compartment, the d) CD4+ and e) CD8+ subpopulations are equally represented. Stainings performed on a–c) paraffin-embedded or d, e) frozen tissue, using DAB chromogen and hematoxylin for counterstaining (original magn. 200×).
Figure 5
Figure 5. Histological scores for pancreatic atrophy and inflammatory cell infiltration in HLA-DR*0405 transgenic Ab0 NOD mice
Scores indicating area involved (percentage) in whole organ sections. a) Pancreatic atrophy in HLA-DR*0405 transgenic NOD mice, but not in HLA-DQ8 NOD mice (sublethally irradiated and transfered with CD90+ T cells from syngenic donor mice), monitored for 9–19 weeks post-treatment (n=7–8, weight- and sex-matched groups; **p<0.01, *p<0.05). b) Spontaneous development of pancreatic atrophy in untreated HLA-DR*0405 transgenic mice after 18 weeks of study. HLA-DR*0405 transgenic mice treated with sublethal irradiation and adoptive CD4+ T cell transfer are more severely affected than untreated mice (n.s.), while HLA-DR*0401 transgenic controls monitored for 9 weeks post-treatment do not show any signs of disease (***p<0.001, *p<0.05). c) Close correlation of pancreatic atrophy with inflammatory cell infiltration in samples with atrophy scores ranging from 0–70% (***r=0.96). In advanced stages (>70%; dotted line), infiltration with inflammatory cells diminishes.
Figure 5
Figure 5. Histological scores for pancreatic atrophy and inflammatory cell infiltration in HLA-DR*0405 transgenic Ab0 NOD mice
Scores indicating area involved (percentage) in whole organ sections. a) Pancreatic atrophy in HLA-DR*0405 transgenic NOD mice, but not in HLA-DQ8 NOD mice (sublethally irradiated and transfered with CD90+ T cells from syngenic donor mice), monitored for 9–19 weeks post-treatment (n=7–8, weight- and sex-matched groups; **p<0.01, *p<0.05). b) Spontaneous development of pancreatic atrophy in untreated HLA-DR*0405 transgenic mice after 18 weeks of study. HLA-DR*0405 transgenic mice treated with sublethal irradiation and adoptive CD4+ T cell transfer are more severely affected than untreated mice (n.s.), while HLA-DR*0401 transgenic controls monitored for 9 weeks post-treatment do not show any signs of disease (***p<0.001, *p<0.05). c) Close correlation of pancreatic atrophy with inflammatory cell infiltration in samples with atrophy scores ranging from 0–70% (***r=0.96). In advanced stages (>70%; dotted line), infiltration with inflammatory cells diminishes.
Figure 6
Figure 6. Extrapancreatic pathology in HLA-DR*0405 transgenic Ab0 NOD mice
a, b) Pneumonitis characterized by perivascular and peribronchial mononuclear cell infiltration, but also by infiltration of the alveoli with foamy macrophages (→), predominantly in the periphery of the organ (original magn. a) 100×, b) 400×; example taken from an untreated HLA-DR*0405 transgenic mouse, age 26 weeks at sacrifice).
Figure 7
Figure 7. Functional changes in HLA-DR*0405 transgenic Ab0 NOD mice
a) Decrease of serum lipase activity with the progression to pancreatic atrophy (**r=−0.47). Significant reductions in serum lipase activity in groups of HLA-DR*0405 transgenic mice with 100% or 40–60% vs. 0% loss of acinar tissue (**p<0.01 and *p<0.05; data expressed as lipase activity relative to the mean of mice with 0% tissue loss). b) Increased stool fat concentrations in HLA-DR*0405 transgenic mice with complete pancreatic atrophy (100%) vs. mice with either 0% or 40–60% loss of acinar tissue (*p<0.05; data expressed as fat concentration relative to mean for normal wildtype controls), demonstrating fat maldigestion due to pancreatic insufficiency in end stage disease.
Figure 7
Figure 7. Functional changes in HLA-DR*0405 transgenic Ab0 NOD mice
a) Decrease of serum lipase activity with the progression to pancreatic atrophy (**r=−0.47). Significant reductions in serum lipase activity in groups of HLA-DR*0405 transgenic mice with 100% or 40–60% vs. 0% loss of acinar tissue (**p<0.01 and *p<0.05; data expressed as lipase activity relative to the mean of mice with 0% tissue loss). b) Increased stool fat concentrations in HLA-DR*0405 transgenic mice with complete pancreatic atrophy (100%) vs. mice with either 0% or 40–60% loss of acinar tissue (*p<0.05; data expressed as fat concentration relative to mean for normal wildtype controls), demonstrating fat maldigestion due to pancreatic insufficiency in end stage disease.

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