Triggering ubiquitination of IFNAR1 protects tissues from inflammatory injury

Abstract

Type 1 interferons (IFN) protect the host against viruses by engaging a cognate receptor (consisting of IFNAR1/IFNAR2 chains) and inducing downstream signaling and gene expression. However, inflammatory stimuli can trigger IFNAR1 ubiquitination and downregulation thereby attenuating IFN effects in vitro. The significance of this paradoxical regulation is unknown. Presented here results demonstrate that inability to stimulate IFNAR1 ubiquitination in the Ifnar1(SA) knock-in mice renders them highly susceptible to numerous inflammatory syndromes including acute and chronic pancreatitis, and autoimmune and toxic hepatitis. Ifnar1(SA) mice (or their bone marrow-receiving wild type animals) display persistent immune infiltration of inflamed tissues, extensive damage and gravely inadequate tissue regeneration. Pharmacologic stimulation of IFNAR1 ubiquitination is protective against from toxic hepatitis and fulminant generalized inflammation in wild type but not Ifnar1(SA) mice. These results suggest that endogenous mechanisms that trigger IFNAR1 ubiquitination for limiting the inflammation-induced tissue damage can be purposely mimicked for therapeutic benefits.

Figure 1

Acute pancreatitis is exacerbated in Ifnar1SA mice incapable of stimulating IFNAR1 ubiquitination.

1. Experimental pancreatitis induced after injection of caerulein assessed by amylase activity levels in plasma from indicated mice at indicated time points; (*P < 0.05 compared to Ifnar1^+/+).

2. H'E staining of pancreata obtained from mice at the indicated times after caerulein injections. Black and blue arrows point to the regions of acinar degeneration and leukocyte infiltration (respectively). The size bar (here and thereafter) is 200 μm. Additional pictures from other animals in similar or greater magnification as well as quantification of acinar tissue loss are shown in Supplementary Figs 3, 4 and 5.

3. IHC analysis of activated p-p38 kinase in pancreata from panel B.

4. Immunoblotting analyses of IFNAR1 immunoprecipitated from the whole pancreatic tissue lysates from indicated mice harvested at 2 days after caerulein or saline injections was carried out using the indicated antibodies. Analyses of activated p38 kinase as well as STAT1 and PKR proteins in whole tissue lysates (WTL) are also shown. Total levels of p38 kinase are depicted as a loading control.

Source data are available for this figure.

Figure 2

Inflammatory stimuli fail to downregulate IFNAR1 in the Ifnar1^SA mice.

1. FACS analysis of the cell surface IFNAR1 levels in peripheral blood leukocytes (PBL) or peritoneal leukocytes (PL) from indicated mice harvested at 3 h after injection of either saline (Sal, red) or LPS (blue). Ig, antibody isotype control (gray).

2. Analysis of relative Ifnar1 mRNA levels in PBL treated as in panel A (n = 3 for each group).

3. ELISA analyses of the levels of indicated cytokines (in ng/ml) in blood plasma of indicated mice (n = 3 for each genotype) treated with saline or LPS for 12 h; nd, levels below 40 pg/ml.

Figure 3

An aberrant cytokine balance and inflammatory monocyte recruitment to inflamed pancreatic tissues from Ifnar1^SA mice.

1. Fold induction of mRNA of TNFα, IL10, CXCL1, CCL2 and CCL5 (normalized per β-actin mRNA) in pancreata from indicated mice (n = 3 for each genotype) harvested 3 days after caerulein injections. Normalized mRNA levels in mice that received saline were assigned a value of 1.0. *P < 0.05 in comparison with wild type mice.

2. Immunofluorescent analysis of pancreata from indicated mice (harvested 7 days after caerulein injections) was carried out on frozen sections using anti-CD45 antibody.

3. Immunofluorescent analysis of indicated pancreata processed as in B using indicated antibodies. Positive and negative controls for the specificity of these antibodies are shown in Supplementary Fig 10.

4. Immunofluorescent co-localization analysis of indicated pancreata processed as described in B.

Figure 4

Inability to stimulate IFNAR1 ubiquitination augments and prolongs tissue damage and inhibits tissue regeneration under conditions of chronic pancreatitis.

1. Amylase activity in serum from indicated mice injected with caerulein (50 μg/kg, 5 days a week for indicated amount of weeks).

2. H'E staining of pancreata from mice that received injections of saline or caerulein for 4 weeks as described in panel A. Arrows point to the areas of restored tissue (white) and chronic tissue damage manifested in continuous edema (black), and fibrotic tissue replacing acinar epithelium (blue).

3. Acinar tissue damage in indicated pancreata from mice described in panel B was quantified as% of acinar tissue loss compared to saline-treated animals. *P < 0.05.

4. H'E staining of pancreata from indicated caerulein-treated mice (treated as in panel B) is depicted at different magnification to highlight tissue degeneration and leukocytic infiltrates. Arrows indicate areas described in panel B.

Figure 5

Deficient ubiquitination of IFNAR1 sensitizes mice to experimental autoimmune and toxic hepatitis.

1. Immunoblotting analyses of IFNAR1 immunoprecipitated from the liver lysates from indicated mice harvested at 10 h after treatment with ConA (or saline as a control) or 48 h after treatment with CCl₄ (or oil as a control) was carried out using the indicated antibodies. Analyses of activated p38 kinase as well as STAT1 and PKR proteins in whole tissue lysates (WTL) are also shown. Total levels of p38 kinase are depicted as a loading control.

2. Activity of alanine (ALT) and aspartate (AST) aminotransferases in blood plasma from wild type or Ifnar1^SA mice (n = 5 for each group) treated as indicated. *P < 0.05 compared to wild type.

3. H'E staining of liver tissue from mice described in panel B. Arrows point to hepatic lesions and concurrent areas of leukocyte infiltration.

4. IHC analysis of activated p-p38 kinase in liver tissues from panel C.

Source data are available for this figure.

Figure 6

Elimination of IFNAR1 in bone marrow-derived cells protects inflamed tissues from excessive damage.

1. Amylase activity levels in serum from chimeric mice after indicated bone marrow transplantation (donor recipient, n = 4 for each group) following caerulein injections (*P < 0.05). +/+, Ifnar1^+/+; SA, Ifnar1^SA.

2. H'E staining of pancreata obtained from chimeric mice described in panel A.

3. Activity of alanine (ALT) and aspartate (AST) aminotransferases from serum of chimeric mice after indicated bone marrow transplantation (donor recipient, n = 3 for each group) and injection of CCl₄ or oil (as a vehicle control). *P < 0.05.

4. H'E staining of liver tissue from mice described in panel C.

Figure 7

Pharmacologic induction of IFNAR1 ubiquitination attenuates the severity of toxic hepatitis and septic shock.

1. Immunoblotting analyses of immunoprecipitated IFNAR1 and whole tissue lysates (WTL) from the spleens of mice injected or not with LPA (10 mg/kg; 50 min prior to harvesting) was carried out using the indicated antibodies.

2. FACS analysis of IFNAR1 cell surface levels in splenocytes from indicated mice harvested at 3 h after treatment of either saline or LPA (20 μg/ml). Right panel depicts relative Ifnar1 mRNA levels analyzed under these conditions (n = 3 for each group).

3. Immunoblotting analysis of STAT1 phosphorylation and levels in splenocytes from Ifna1r^+/+ and Ifnar1^SA mice treated for 3 h with either saline or LPA (20 μg/ml) and then isolated and treated in vitro with IFNβ (1000 U/ml for 30 min) where indicated.

4. AST activity in plasma from Ifnar1^+/+ or Ifnar1^SA mice collected 24 h after the injection of acetaminophen (APAP, 150 mg/kg, i.p.). Where indicated, mice were also injected with LPA (i.v., 10 mg/kg, n = 4 mice for treatment and control groups) or anti-IFNAR1 neutralizing antibody (i.v., 100 μg, n = 3 mice for treatment and control groups) at 0, 8 and 16 h after APAP treatment).

5. H'E staining of lungs from indicated mice treated with saline or LPS for 12 h.

6. Kaplan-Meier analysis of survival of indicated mice treated with LPS (10.5 mg/kg) without or with LPA (10 mg/kg).

Source data are available for this figure.