IFN-λ resolves inflammation via suppression of neutrophil infiltration and IL-1β production

Abstract

The most studied biological role of type III interferons (IFNs) has so far been their antiviral activity, but their role in autoimmune and inflammatory diseases remains largely unexplored. Here, we show that treatment with IFN-λ2/IL-28A completely halts and reverses the development of collagen-induced arthritis (CIA) and discover cellular and molecular mechanisms of IL-28A antiinflammatory function. We demonstrate that treatment with IL-28A dramatically reduces numbers of proinflammatory IL-17-producing Th17 and γδ T cells in the joints and inguinal lymph nodes, without affecting T cell proliferative responses or levels of anticollagen antibodies. IL-28A exerts its antiinflammatory effect by restricting recruitment of IL-1b-expressing neutrophils, which are important for amplification of inflammation. We identify neutrophils as cells expressing high levels of IFN-λ receptor 1 (IFNLR1)-IL-28 receptor α (IL28RA) and targeted by IL-28A. Our data highlight neutrophils as contributors to the pathogenesis of autoimmune arthritis and present IFN-λs or agonists of IFNLR1-IL28RA as putative new therapeutics for neutrophil-driven inflammation.

Figure 1.

Treatment with IL-28A halts and reverses the development of CIA. (a) Schematic diagram showing treatment and harvest time points in the CIA model. (b) Comparison of clinical score and (c) paw swelling between arthritic mice treated with recombinant mouse IL-28A (0.4 mg/kg/d) or PBS control. The data are mean and SEM derived from eight mice treated with IL-28A and eight mice treated with PBS in a representative out of three CIA experiments. (d) Representative images of the tarsometatarsal joint of the hind limb showing areas of bone erosion (arrowhead), synovial thickening (asterisk), and leukocyte infiltration into joint space (arrow). The average histological scores of IL-28A–treated and control mice were 0.676 ± 0.964 and 1.933 ± 0.201, respectively. The data are mean and SEM using seven mice treated with IL-28A and eight mice treated with PBS from two independent CIA experiments. Statistical analysis was performed by two-tailed Mann-Whitney U test. (e) Comparison of clinical score between arthritic IL28RA^−/− mice and their littermate controls (WT). The data are mean and SEM derived from eight control mice and six IL-28RA^−/− mice from a representative out of three independent CIA experiments.

Figure 2.

Treatment with IL-28A dramatically reduces proinflammatory IL-17–producing Th17 and γδ T cells in the joints. (a) Total numbers of Th17 and IL-17–producing γδ T cells in the hind paw arthritic joints of mice on the 10th day of treatment with IL-28A (0.4 mg/kg/day) or PBS control. The data are mean and SEM derived from eight mice treated with IL-28A and eight mice treated with PBS in a representative of three CIA experiments. Fold induction of (b) IL-17A, IL-1b, and IL-23a mRNA in dissociated cells from arthritic joints of mice treated with IL-28A or control. The data are mean and SEM derived from 19 mice treated with IL-28A and 19 mice treated with PBS from 3 independent CIA experiments. (c) Total numbers of CD4⁺ T cells and γδ T cells in the lymph nodes and blood of PBS and IL-28A–treated mice. The data are mean and SEM derived from 15 mice that developed arthritis, 8 treated with IL-28A, in a representative of three late CIA experiments. (d) Levels of IFN-γ, IL-1β, IL-6, and CXCL1 in the serum of mice on the 10th day of treatment with IL-28A or PBS control. The data are shown as mean with 95% confidence interval using 16 mice treated with IL-28A and 16 mice treated with PBS, from 3 independent late CIA experiments. (e) Proliferation of lymphocytes after 48-h stimulation with αCD3 or bovine CII. The data are mean and SEM derived from nine independent lymphocyte cultures from LNs of arthritic mice, four of which were treated with IL-28A. Statistical analysis was performed by one-tailed paired Student’s t test. (f) Levels of collagen-specific IgG1 and IgG2a in the serum. The data are mean and SEM using eight mice treated with IL-28A and eight mice treated with PBS, from a representative out of three independent late CIA experiments. (g) Total number of B cells in the lymph nodes. The data are mean and SEM derived from nine independent lymphocyte preparations from LNs of arthritic mice, four treated with IL-28A. Statistical analysis was performed by two-tailed Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Treatment with IL-28A restricts neutrophil infiltration into joint at the early stages of arthritis. (a) Total numbers of neutrophils, macrophages, B cells, CD4⁺ T cells, and γδ T cells in the hind paw arthritic joints of mice on day 4 of treatment with IL-28A or PBS control. The data are mean and SEM derived from five mice treated with IL-28A and five mice treated with PBS in a representative out of three CIA experiments. (b) Histology sections of arthritic joints after 10 d of treatment with IL-28A or PBS control stained for Ly6G. The mean number of positive cells per view over 10 views: 10.2 for mice treated with IL-28A and 37.41 for mice treated with PBS from 2 independent CIA experiments. (c) mRNA levels of neutrophil recruitment mediators in the arthritic joints of mice on day 4 of treatment with IL-28A or PBS control. The data are mean and SEM derived from five mice treated with IL-28A and five mice treated with PBS in a representative out of three CIA experiments. (d) Levels of IFN-γ, IL-1β, IL-6, and CXCL1 in the serum of mice on day 4 of treatment with IL-28A or PBS control. The data are shown as mean with 95% confidence interval using eight mice treated with IL-28A and eight mice treated with PBS from two independent early CIA experiments. Statistical analysis was performed by one-tailed Mann-Whitney U test. *, P < 0.05; **, P < 0.01.

Figure 4.

IL-28A targets neutrophils. Levels of pSTAT1 in Percoll-purified neutrophils from bone marrow (a) and total bone marrow (b) stimulated with IL-28A or IFN-β in vitro for 15 or 30 min. A representative of four independent experiments is shown. (c) mRNA levels of IL-28RA in a neutrophil enriched population of cells from mouse arthritic joints (CD11b⁺ nonadherent) compared with macrophages (CD11b⁺ adherent) and total cells isolated from the joint (all cells). Statistical analysis was performed by one-tailed Mann-Whitney U test.

Figure 5.

Treatment with IL-28A reduces neutrophil recruitment in the air pouch model of acute inflammation by limiting neutrophil migratory capacity. (a) Schematic timeline of the air pouch model showing subcutaneous air injections, treatment with IL-28A or PBS, and challenge with zymosan. (b) Total number of neutrophils per milliliter of wash recovered from the air pouches of mice that had been treated with IL-28A or PBS control before inflammatory challenge. The data are mean and SEM from 12 mice treated with IL-28A and 12 mice treated with PBS from 2 independent experiments. (c) Center-zeroed tracks of air pouch neutrophilic infiltrate in an EZ-Taxiscan migrating toward LTB4; scale is shown in micrometers. (d) The tracks presented in c were analyzed to show the Euclidean distance each cell traveled in a 45-min time period. Data shown are the average and SEM of three independent experiments, each including 9–20 movies per treatment. (e) Percentage of cells infiltrating into the air pouch staining positive for PI and Annexin V. The data are mean and SEM using 12 mice treated with IL-28A and 12 treated with PBS, from 2 independent experiments. (f) Percentage of neutrophils that were positive for pro–IL-1β and infiltrated into the air pouch space of mice. (g) Levels of IL-1β secreted into the air pouch after challenge with zymosan. (h) Percentage of neutrophils or macrophages lining the air pouch membrane that were positive for pro-IL-1β. (i) Expression of IL28RA mRNA in cells infiltrating into the air pouch space compared with cells lining the air pouch, as well as in Percoll-purified neutrophils. (f–i) The data are mean and SEM derived from six mice treated with IL-28A and six mice treated with PBS, in a representative out of two experiments. Statistical analysis was performed by two-tailed Mann-Whitney U test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.