IFN-λ suppresses intestinal inflammation by non-translational regulation of neutrophil function

Abstract

Interferon-λ (IFN-λ) is a central regulator of mucosal immunity; however, its signaling specificity relative to that of type I interferons is poorly defined. IFN-λ can induce antiviral interferon-stimulated genes (ISGs) in epithelia, while the effect of IFN-λ in non-epithelial cells remains unclear. Here we report that neutrophils responded to IFN-λ. We found that in addition to inducing ISG transcription, IFN-λ (but not IFN-β) specifically activated a translation-independent signaling pathway that diminished the production of reactive oxygen species and degranulation in neutrophils. In mice, IFN-λ was elicited by enteric viruses and acted on neutrophils to decrease oxidative stress and intestinal damage. Thus, IFN-λ acted as a unique immunomodulatory agent by modifying transcriptional and non-translational neutrophil responses, which might permit a controlled development of the inflammatory process.

Figure 1

Mouse and human neutrophils are responsive to IFN-λ. (a) qPCR analysis of Ifnlr1 mRNA (left) and Il10rb mRNA (right), which encode the two components of the IFN-λ receptor, in mouse bone-marrow-derived neutrophils (PMN), primary intestinal epithelial cells (IEC), splenic plasmacytoid DCs (pDC) and splenic conventional DCs (cDC) derived from wild-type (WT) or IFNLR1-deficient (Ifnlr1^−/−) mice (key); results are presented relative to those of the control gene Gapdh. ND, not detectable. (b) qPCR analysis of Viperin mRNA in neutrophils (left), plasmacytoid DCs (middle) or conventional DCs (right) left unstimulated (US) or stimulated for 3 h with IFN-λ or IFN-β (100 U/ml each) (key); results presented as in a. (c) qPCR analysis of Ifnlr1 in cells as in a, as well as T cells, B cells and peritoneal macrophages (pM0), left unstimulated or stimulated for 3 h with LPS (10 μg/ml) or TNF (100 ng/ml) (key). (d) qPCR analysis of Ifnlr1 (left) in human neutrophils left unstimulated or stimulated for 3 h with LPS (10 μg/ml), and of Viperin mRNA (right) in human neutrophils treated for 3 h with IFN-λ2 (100 U/ml) or IFN-β (100 U/ml) (horizontal axis) and left unstimulated or stimulated for 3 h with LPS (10 μg/ml) (key). (e) Immunoblot analysis of phosphorylated (p−) STAT1 and STAT3, and β-actin (loading control throughout), in neutrophils derived from the bone marrow of wild-type mice (top) or Ifnlr1^−/− mice (bottom) and stimulated for 0–60 min (above lanes) with IFN-λ or IFN-β (blots cropped to show bands of the appropriate molecular size). (f) NanoString analysis of gene expression (nCounter Mouse Inflammation v2 panel; genes modulated more than twofold, with a P value of > 0.05) in mouse neutrophils stimulated for 30 min, 1 h or 3 h (left margin) with IFN-λ or IFN-β (100 U/ml). Data are from one of three experiments (a; mean + s.e.m. of three biological replicates) or one experiment representative of three experiments (b,d; mean + s.e.m. of three biological replicates) or are representative of three independent experiments (c,e,f; mean + s.e.m. in c).

Figure 2

IFN-λ suppresses the tissue-damaging function of neutrophils in a non-translational and non-transcriptional manner. (a) Kinetic analysis of the release of superoxide (respiratory burst) from bone-marrow-derived neutrophils and left untreated (UT) or treated with various combinations (key) of stimulation with TNF (left; 100 ng/ml) or LPS (middle; 10 μg/ml) and treatment with IFN-λ (100 U/ml), assessed as the oxidized form of cytochrome C and presented as the concentration of superoxide ion (O·) per 1 × 10⁶ cells (left and middle). Right, inhibition of ROS production in neutrophils treated for 30 min with LPS or TNF (horizontal axis) in the presence of IFN-λ or IFN-β (key; 100 U/ml each), relative to ROS production in neutrophils treated for 30 min with LPS or TNF alone. (b) Kinetic analysis of the respiratory burst in human neutrophils left untreated or treated with various combinations (key) of stimulation with TNF (100 ng/ml) and treatment with IFN-λ2 (100 U/ml), presented as in a (left), and inhibition of ROS production in neutrophils treated for 30 min with TNF in the presence of IFN-λ2 or IFN-β (horizontal axis), relative to ROS production in neutrophils treated for 30 min with TNF alone (right). (c) Inhibition of MMP-9 activity, assessing degranulation in supernatants of wild-type and IFNLR1-deficient neutrophils (key) treated with IFN-λ plus various concentrations (horizontal axis) of LPS, presented relative to that of wild type cells treated with LPS alone (left), and electrophoretic analysis of proteolytic activity (zymography) of supernatants of wild-type and IFNLR1-deficient neutrophils (left margin) left untreated or treated with IFN-λ (above gels) and left unstimulated or stimulated with LPS (above lanes) (right). (d) Kinetic analysis of the respiratory burst in neutrophils treated with cycloheximide (10 μg/ml) and stimulated as in a (key), with results presented as in a (left), and inhibition of ROS production in neutrophils treated with TNF in the presence of IFN-λ with (CHX) or without (−) cycloheximide (horizontal axis), relative to ROS production by neutrophils treated with TNF only (right). (e) Inhibition of ROS production in neutrophils obtained from wild-type (WT) mice, STAT1-deficient (Stat1^−/−) mice or LysM^creStat3^fl/fl (Stat3^−/−) mice, in which STAT3 is deleted in myeloid cells, and treated with TNF in the presence of IFN-λ as in a; results presented as in a. (f,g) Kinetic analysis of the respiratory burst in mouse neutrophils treated for 0–30 min (horizontal axes) with Py6 (50 nM) (f) or HBC (10 μM) (g) and stimulated as in a, with results presented as in a (top), and inhibition of ROS production by neutrophils treated with TNF in the presence of IFN-λ with or without (UT) Py6 (f) or HBC (g), relative to ROS production by neutrophils treated with TNF only, assessed and presented as in a (bottom). (h) Immunoblot analysis of AKT phosphorylated at Ser473 (p-AKT 473) or Thr308 (p-AKT 308) and total AKT, phosphorylated and total STAT1, and p38 phosphorylated at Thr180 and Tyr182 (p-p38) (left margin), in mouse neutrophils treated for 30 min with IFN-λ (+) or not (−) in the presence (+) or absence (−) of HBC (10 μM), and stimulated for 30 min with LPS (10 μg/ml) (+) or not (−) (blots cropped as in Fig. 1e). NS, not significant (P > 0.05); *P < 0.05, **P < 0.01 and ***P < 0.001 (unpaired two tailed t-test). Data are representative of (a (left and middle), b,d (left), f,g (top), c (right), h) or from (a,b,d (right), f,g (bottom), c (left), e) three independent experiments (mean + s.e.m.).

Figure 3

IFN-λ protects mice from DSS-induced colitis and diminishes oxidative stress. (a–d) Colitis progression and severity in wild-type and IFNLR1-deficient mice (key) co-housed for 2 weeks and then left untreated (UT) or treated with 2.5% DSS in the drinking water for 7 d (DSS), assessed as body weight relative to initial weight, set as 100% (a), disease activity index (DAI) (b), colon length (c) and histology (d). Original magnification (d), ×4 (left) or ×10 (right). (e) qPCR analysis of Hmox1 and Sod2 (genes induced in response to oxidative stress), assessing oxidative damage in colonic tissue obtained from wild-type and IFNLR1-deficient mice (key) at day 7 after the administration of DSS (as in a); results are presented relative to those of Gapdh. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 (two-way ANOVA (a–c) or nonparametric two-tailed t-test (d,e)). Data are pooled from three independent experiments (a,c; mean + s.e.m. of 20 mice per group) or are from one experiment representative of three independent experiments (b,d,e; mean + s.e.m. of 5 mice per group).

Figure 4

The protective effect of IFN-λ in DSS-induced colitis is independent of the genetic background or strain-specific microbiota. Colitis in wild-type mice left treated with various combinations (keys and axes) of 2.5% DSS in their drinking water (DSS) or not (− DSS) and daily intraperitoneal injection of antibody to IFN-λ (α-IFN-λ) (10 g/kg) or not (no Ab), assessed as body weight (a), colon length (b), histopathological score (c) and histology (d). Scale bars (d), 500 μm. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-way ANOVA (a,b) or nonparametric two-tailed t-test (c)). Data are from one experiment representative of three independent experiments (mean + s.e.m. of five mice in a–c).

Figure 5

IFN-λ protects mice from DSS-induced colitis by inhibiting neutrophil function. (a–c) Colitis progression and severity in WT→WT and IFNLR1-KO→WT chimeras treated with 2.5% DSS in the drinking water for 7 d (or not (UT), in a), assessed as colon length (a), disease activity index (b) and histology (c). (d) qPCR analysis of Hmox1 and Sod2 in intestinal epithelial cells from mice as in a–c; results presented relative to those of Gapdh. (e–g) Colitis progression and severity in MRP8^creIfnlr1^fl/fl and Ifnlr1^fl/fl littermates treated with 2.5% DSS in the drinking water for 7 d (or not (UT), in e), assessed as colon length (e), disease activity index (f) and histology (g). (h) qPCR analysis of Hmox1 and Sod2 in intestinal epithelial cells from mice as in e–g, presented as in d. Original magnification (c,g), ×10. *P < 0.05, **P < 0.01,***P < 0.001 and ****P < 0.0001 (two-way ANOVA (a,e) or nonparametric two-tailed t-test (b,d,f,h)). Data are representative of three experiments (mean + s.e.m. in a,b,d,e,f,h).

Figure 6

The protective effect of IFN-λ in DSS-induced colitis is independent of IFNLR1 expression in epithelial cells. (a–d) Colitis progression and severity in WT→WT and WT→IFNLR1-KO chimeras co-housed for 2 weeks and then treated with 2.5% DSS in the drinking water for 7 d (or not (UT), in b), assessed as body weight (a), colon length (b), disease activity index at day 7 (c) and histology (d). (e,f) qPCR analysis of Hmox1 (e) and Sod2 (f) in intestinal epithelial cells at day 7 in mice as in a–d; results presented relative to those of Gapdh. (g) Oxidation of DNA, assessed as 8-oxoguanine (8-oxog) (per ng of DNA) at day 7 in the colon of mice as in a–d. (h–j) Colitis progression and severity in Vil^creIfnlr1^fl/fl and Ifnlr1^fl/fl littermates treated with 2.5% DSS in the drinking water for 7 d (or not (UT), in h), assessed as colon length (h), disease activity index at day 7 (i) and histology (j). (k) qPCR analysis of Hmox1 and Sod2 in intestinal epithelial cells at day 7 in mice as in h–j, presented as in e,f. Original magnification (d,j), ×10. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-way ANOVA (a,b,h) or nonparametric two-tailed t-test (c,e,f,g,I,k)). Data are from one experiment representative of three experiments (a–g; mean + s.e.m. of eight (WT→WT) or four (WT→IFNLR1 KO) mice per group in a–c,e,f) or one experiment representative of three independent experiments (h–k; mean + s.e.m. of five mice per group in h,i,k).

Figure 7

Enteric-virus-induced IFN-λ protects mice from DSS-induced colitis by diminishing oxidative stress. (a–e) Disease progression in wild-type and IFNLR1-deficient mice treated intragastrically with an AV drug ‘cocktail’ (+ AV) or PBS (control) for 10 d before the administration of 2.5% DSS in the drinking water, evaluated as weight loss (a), disease activity index (b), colon length (c), histopathological score (d) and histology (e). (f–i) Disease progression in wild-type mice treated with AV drugs or PBS, plus DSS (as in a), and given intraperitoneal injection of PBS (+ no IFN) or mouse recombinant IFN-λ to which polyethylene glycol was attached (+ IFN-λ; 4 mg per kg body weight) for 7 d, evaluated by histology (e), weight loss (f), disease activity index (g), colon length (h) and histopathological score (i). Original magnification (e), ×4 (top row) or ×10 (bottom row). *P < 0.05, **P < 0.01 and ***P < 0.001 (two-way ANOVA). Data are from one experiment representative of three independent experiments (mean + s.e.m. of eight mice per group in a–d,f–i).

Figure 8

IFN-λ displays a superior ability in suppressing DSS-induced colitis in the absence of enteric viruses, by diminishing oxidative stress. (a–d) Colitis development in wild-type mice treated intragastrically with a ‘cocktail’ of AV drugs or PBS for 10 d before the administration of 2.5% DSS in the drinking water, as well as no interferon (no IFN), mouse recombinant IFN-λ to which polyethylene glycol was attached (IFN-λ) or recombinant IFN-β (IFN-β) (200 U per gram body weight per day of either), evaluated as colon length (a), disease activity (b), weight at day 7, relative to initial weight (c), and histology (d). (e,f) qPCR analysis of Tnf in the total colon (e) and of Hmox1 in intestinal epithelial cells (measuring oxidative damage) (f) in mice as in a–d. (g) NanoString analysis of gene expression (nCounter Mouse Inflammation v2 panel; genes that generated >500 counts per treatment) in mRNA (100 ng) purified from total colon extracts of mice as in a–d. *P < 0.05, **P < 0.01 and ***P < 0.001 (two-way ANOVA.). Data are from one of three independent experiments (mean + s.e.m. of five mice per group in a–c,e,f).