C-terminal truncation of IFN-γ inhibits proinflammatory macrophage responses and is deficient in autoimmune disease

Abstract

Controlled macrophage differentiation and activation in the initiation and resolution of inflammation is crucial for averting progression to chronic inflammatory and autoimmune diseases. Here we show a negative feedback mechanism for proinflammatory IFN-γ activation of macrophages driven by macrophage-associated matrix metalloproteinase 12 (MMP12). Through C-terminal truncation of IFN-γ at 135Glu↓Leu136 the IFN-γ receptor-binding site was efficiently removed thereby reducing JAK-STAT1 signaling and IFN-γ activation of proinflammatory macrophages. In acute peritonitis this signature was absent in Mmp12 ^-/- mice and recapitulated in Mmp12 ^+/+ mice treated with a MMP12-specific inhibitor. Similarly, loss-of-MMP12 increases IFN-γ-dependent proinflammatory markers and iNOS⁺/MHC class II⁺ macrophage accumulation with worse lymphadenopathy, arthritic synovitis and lupus glomerulonephritis. In active human systemic lupus erythematosus, MMP12 levels were lower and IFN-γ higher compared to treated patients or healthy individuals. Hence, macrophage proteolytic truncation of IFN-γ attenuates classical activation of macrophages as a prelude for resolving inflammation.

Fig. 1

Human PBMC MMP12 and IFN-γ response gene mRNAs in SLE. a PMBC mRNA levels and b quantification of MMP12, C3, C4, C5, CD74, IFNA2, IFNB1, IFNG, IFNGR1, IFNGR2, ITGAM, ITGAX S100A8, S100A9, STAT1, and NOS2 mRNAs in SLE patients (n = 102) or healthy control subjects (n = 12) in the GSE11909 transcript dataset. Vertical bars are the mean values in all bean plots. c Comparison of these mRNA levels in PBMCs of drug-treated SLE patients (n = 40) or healthy control subjects (n = 32) in the GSE37356 mRNA dataset. Statistical significance in b and d was determined by calculating the q-values in a and in c. e Longitudinal comparison (31 time points) in 20 individual subjects of the fold changes of MMP12 and IFNG PBMC mRNA levels upon clinical deterioration (increasing SLEDAI in individual patients over time, n = 7 time points), or upon clinical improvement (unchanged or decreasing SLEDAI in individual patients over time, n = 24 time points). Patient information (ethnicity, gender, age, SLEDAI and drug treatment or not) from GSE11909 is in Supplementary Tables S4-S5. Statistical significance was determined by a two-tailed paired Student’s t-test: p = 3 × 10⁻³

Fig. 2

MMP12 cleaves IFN-γ removing the IFN-γ receptor-binding site. a Silver stained 15% SDS-PAGE analysis of in vitro cleavage of human (h) IFN-γ by 10 or 100 ng hMMP12 catalytic domain at 1:10 and 1:100 enzyme to substrate ratios, incubated over 18 h at 37 °C. Revealing C-terminal cleavage, N-terminal sequencing identified an intact N-terminus commencing at 1MQDPY both in IFN-γ and in the two cleavage products (red arrows). The MMP12-specific inhibitor, Rxp470.1, blocked IFN-γ cleavage and autocatalytic cleavage of MMP12 resulting in stabilized MMP12 protein levels over 18 h. Molecular weight marker positions are shown. b Q-TOF-MS analysis of IFN-γ cleavage reaction products revealed C-terminal cleavage first between 157Met↓Leu158 and then at 135Glu↓Leu136 (see Supplementary Fig. 4). The k_cat/K_M values calculated for each cleavage event in human and mouse (m) IFN-γ are shown. c Based on the crystal structures of the IFN-γ homodimer (pdb entry: 1HIG)^, , a secondary complex consisting of the IFN-γ dimer, two high-affinity IFN-γ receptor 1 molecules (IFNGR1(pdb entry: 1FG9); 29Val-Ser241; dark gray), and two low affinity IFN-γ receptor 2 chains (IFNGR2 (pdb entry: 1FYH); 30Leu-Thr237; light pink) were modeled. The IFN-γ C-terminal peptide (135Glu–158Leu) responsible for IFN-γ receptor interaction and signaling was modeled (green). The transmembrane peptide and JAK1/2 are shown in cartoon form. d Frontal view of the structured region of the IFN-γ homodimer with the C-terminal non-structured flexible region (from 146Ala to Gln166) cartooned in green. The two MMP12 cleavage sites are shown: 157Met↓Leu158 and 135Glu↓Leu136

Fig. 3

MMP12 decreases IFN-γ-activated macrophage markers in acute peritonitis. a ELISA of IFN-γ protein levels in peritoneal exudate of male Mmp12^+/+ B10.RIII (n = 20) and Mmp12^–/– B10.RIII (n = 20) mice at days 0–4 after induction of peritonitis (n = 4 for each genotype for each time point, N = 2) expressed as the mean ± s.d. There was no IFN-γ quantified in healthy peritoneum in the absence of inflammation on day 0. Statistical significance was determined by two-tailed unpaired Student’s t-test: *p < 0.05. b 10% SDS-PAGE western blot analysis of IFN-γ, MMP12, and iNOS proteins in primary peritoneal macrophages harvested daily from Mmp12^+/+ (n = 20) and Mmp12^–/– (n = 20) B10.RIII mice (N = 2). Tubulin, loading control. c Cellular ROS levels in primary peritoneal macrophages were quantified by calculating the mean fluorescence intensity after treatment with 2,-7-dichlorofluorescein diacetate (DCF) (n = 20 for each genotype, n = 4 for each time point, N = 2). Data were normalized to day 1 Mmp12^+/+ B10.RIII macrophages and expressed as fold differences. Error bars, s.d. Statistical significance was determined by a two-tailed unpaired Student’s t-test: ***p < 0.005. d 10% SDS-PAGE western blot analysis of markers characteristic for macrophage activation by IFN-γ (MHCII, S100A8, and S100A9) and STAT1, or by IL-4 (CD36) and STAT6 in Mmp12^+/+ and Mmp12^–/– B10.RIII mouse macrophages harvested daily (n = 20 for each genotype, n = 4 for each time point, N = 2). Tubulin, loading control. Molecular weight marker positions in all blots are as shown

Fig. 4

MMP12 reduces IFN-γ signaling and responses in macrophages. a Mouse RAW264.7 cells were treated for 15 min or 24 h with PBS, 20 ng/mL mouse IFN-γ, 20 ng/mL mouse IFN-γ pre-incubated with 2 ng/mL mouse MMP12 (37 °C, 18 h), 2 ng/mL mouse MMP12 alone, or 30 ng/mL mouse IL-4 (n = 4, N = 2). After 10% SDS-PAGE, cell lysates were western blotted for pSTAT1-Y701 and STAT1 proteins (Supplementary Fig. 7b). b Western blot of iNOS protein in RAW264.7 cells treated as above for 24 h (n = 4, N = 2). ROS levels were quantified by calculating the mean fluorescence intensity after treatment with 2-,7-dichlorofluorescein diacetate (DCF)(n = 4, N = 2). Data were normalized to PBS-treated cells and expressed as fold differences. Error bars denote s.d. Statistical significance was determined by a two-tailed unpaired Student’s t-test: *p < 0.05; **p < 0.01; ***p < 0.005. c, d Western blotting, quantification, and statistical analyses of pSTAT1-Y701, STAT1, and iNOS protein in human THP-1 cells as described for a and b (n = 4, N = 2) (see also Supplementary Fig. 7a). Tubulin and molecular weight marker positions are shown. e Representative images and f phagocytic index of THP-1 macrophages incubated for 24 h with PBS, 30 ng/mL IL-4, 20 ng/mL IFN-γ, or 20 ng/mL IFN-γ pretreated with 2 ng/mL human MMP12 (37 °C, 18 h), or 2 ng/mL MMP12 alone, and then incubated with serum-coated fluorescent 2-μm microparticles. Scale bars, 20 μm. The phagocytic index was quantified from the number of beads per cell (5–30 cells per field) in each of 20 fields (n = 3, N = 2). Data were normalized to PBS-treated THP-1 macrophages and the means expressed as fold differences. Error bars, s.d. Statistical significance was determined by a two-tailed unpaired Student’s t-test: ***p < 0.005. g MMP12 mRNA analysis of PMA-matured THP-1 cells treated with PBS, IFN-γ, or IL-4 (n = 3, N =2). A-values were normalized to IFN-γ-induced THP-1 cell mean values and expressed as fold differences. Error bars, s.d. Statistical significance was determined by a two-tailed unpaired Student’s t-test: NS not significant difference; ***p = 1 × 10⁻⁵

Fig. 5

Altered macrophage markers in rheumatoid arthritis in Mmp12^–/– mice. a Hind ankle widths of Mmp12^+/+ and Mmp12^–/– male B10.RIII mice (n = 18 and 20, respectively, for each time point) after onset of collagen-induced arthritis (day 0), means ± s.e.m. Mann–Whitney t-test: *p < 0.05, **p < 0.01, ***p < 0.005. b Histopathology of hind ankles after H&E staining (day 18) Mmp12^+/+ (n = 3) and Mmp12^–/– (n = 3). Bone destruction (p < 6 × 10⁻⁵), pannus formation (NS not significant), synovial hyperplasia (p < 3 × 10⁻⁵), and subsynovial inflammation (p < 7 × 10⁻⁴) were quantified as a histopathological score (p < 9 × 10⁻⁴, means ± s.d.), two-tailed unpaired Student’s t-test: *p < 0.05, **p < 0.01. c Representative images of H&E, IFN-γ, iNOS, MHCII, and CD36 immunostaining of hind ankle joints of Mmp12^+/+ (n = 3) and Mmp12^–/– (n = 3) mice. Here and in g: N NETs; C cartilage; S synovial space; arrowheads, high antibody or H&E staining; scale bars, 100 μm. d Immunostaining quantification of Mmp12^+/+ versus Mmp12^–/– male B10.RIII mice hind ankle joints for IFN-γ (p < 2 × 10⁻⁵), iNOS (p < 1 × 10⁻³), MHCII (p < 2 × 10⁻⁵), and CD36 (p < 2 × 10⁻²) (means ± s.d.), two-tailed unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.005. e Ninety-day-female MRL/lpr mice were injected with CFA (day 0). Hind ankle size was measured in Mmp12^+/+and Mmp12^–/– MRL/lpr mice (n = 30 and 28, respectively, for each time point) (means ± s.d.), two-tailed unpaired Student’s t-test: ***p < 0.005. f Histopathology of H&E stained-hind ankle joints of Mmp12^+/+ (n = 3) and Mmp12^–/– (n = 3) MRL/lpr (day 25). Bone destruction (n.s.), pannus formation (p < 0.01), synovial hyperplasia (p < 3 × 10⁻²), and subsynovial inflammation (p < 3 × 10⁻²) were quantified as the histopathological score (p < 6 × 10⁻³) (means ± s.d.), two-tailed unpaired Student’s t-test: *p < 0.05, **p < 0.01. g Representative images of H&E, IFN-γ, iNOS, MHCII, and CD36 immunostaining of hind ankle joints of Mmp12^+/+ and Mmp12^–/– MRL/lpr female mice. BM bone marrow. h Immunostaining intensities of Mmp12^+/+ and Mmp12^–/– mouse hind ankle joints for IFN-γ (p < 2 × 10⁻⁵), iNOS (p < 1 × 10⁻³), MHCII (p < 4 × 10⁻⁴), and CD36 (p < 4 × 10⁻⁴) (means ± s.d.), two-tailed unpaired Student’s t-test: ***p < 0.005

Fig. 6

Mmp12^–/– mouse mortality and IFN-γ macrophages numbers in SLE. a Following CFA injection, the size of the superficial cervical lymph nodes of Mmp12^+/+ and Mmp12^–/– MRL/lpr (n = 16 and 12, respectively, for each time point) female mice were measured and expressed as means ± s.d. Two-tailed unpaired Student’s t-test: ***p < 0.005. b Kaplan–Meier curves showing mortality rates of female Mmp12^+/+ MRL/lpr (n = 16) and Mmp12^–/– (n = 12) mice. Two-tailed unpaired Student’s t-test: p < 2 × 10⁻¹⁰, (see Supplementary Fig. 9c, d for survival data). c Representative images of superficial cervical lymph nodes (white arrows) immunostained for IFN-γ, iNOS, MHCII, and CD36 from Mmp12^+/+ (n = 3) and Mmp12^–/– (n = 3) female MRL/lpr mice at their humane end points (Mmp12^+/+ (day 112) and Mmp12^–/– (day 98)). Scale bars, 100 μm. d Quantification of immunostaining intensities of Mmp12^+/+ (n = 3) versus Mmp12^–/– (n = 3) MRL/lpr mice superficial cervical lymph nodes for IFN-γ (p < 9 × 10⁻⁶), iNOS (p < 2 × 10⁻⁷), MHCII (p < 1 × 10⁻²), and CD36 (p < 2 × 10⁻²) expressed as the mean ± s.d. Two-tailed unpaired Student’s t-test: *p < 0.05, **p < 0.01, ***p < 0.005. e Superficial cervical lymph nodes were analyzed by western blotting for macrophages markers of IFN-γ activation (iNOS, MHCII, S100A8, and S100A9) and STAT1, as well as for IL-4 induced CD36 and STAT6. Two biological replicates analyses from Mmp12^+/+ (n = 8) and Mmp12^–/– (n = 8) MRL/lpr mice, are shown. Red arrows indicate lower molecular forms of IFN-γ. f Mean kidney weights and g mean histological scores of activity and chronicity indexes from Mmp12^+/+ (n = 11) and Mmp12^–/– MRL/lpr (n = 15) mice were measured at their humane end points. Error bars, s.d. Two-tailed unpaired Student’s t-test: *p < 0.05, ***p < 0.005. Quantification of immunostaining in h glomeruli and i in glomeruli and interstitium of Mmp12^+/+ (n = 3) and Mmp12^–/– MRL/lpr (n = 3) mice at their humane end points for IFN-γ (p < 2 × 10⁻¹²), MHCII (p < 7 × 10⁻¹⁷), and CD36 (p < 2 × 10⁻⁶). Quantification is expressed as means ± s.d. Two-tailed unpaired Student’s t-test: ***p < 0.005. j Representative images of H&E, IFN-γ, MHCII, and CD36 immunostaining of the renal glomeruli sections of Mmp12^+/+ and Mmp12^–/– female MRL/lpr mice; arrowheads, strong IFN-γ staining. Scale bars, 100 μm

Fig. 7

IFN-γ epitope antibody staining of human lupus nephritis biopsies. a Left, amino acid sequences (underlined) of human IFN-γ peptides used to raise and affinity-purify rabbit anti–N-terminal, C-terminal-1, and C-terminal-2 IFN-γ epitope antibodies. Right, western blot analysis of human IFN-γ after time-dependent cleavage to 1080 min by human MMP12. Note: disappearance of the C-terminal epitopes as MMP12 cleavage proceeds. Molecular weight marker positions in all blots are as shown. b Immunostaining of human kidney biopsies using anti–N-terminal, C-terminal-2, C-terminal-1, and MMP12 antibodies; and staining with hematoxylin and eosin (H&E), Trichrome, Jones, and PAS of kidney biopsies of healthy (n = 5), lupus nephritis at Grades III-(A) (n = 3) and IV-(A) (n = 2) as diagnosed in Supplementary Fig. 11. Scale bar, 100 μm. Original magnification, ×400. c Quantification of immunostaining intensities in healthy (n = 5) and lupus nephritis (n = 5) kidney biopsies (additional data are included in Supplementary Fig. 11) and expressed as means ± s.d. Statistical significance was determined by a two-tailed unpaired Student’s t-test: ***p < 0.005

Fig. 8

Prolonged IFN-γ signaling in Mmp12^–/– primary peritoneal macrophages. Western blot analysis for pSTAT1-Y701 and STAT1 in primary peritoneal macrophages harvested from individual (a, e) Mmp12^+/+ B10.RIII (n = 5 for each time point) and (b, d) Mmp12^–/– B10.RIII (n = 4 for each time point) mice 4 days after induction of peritonitis. Cells were treated with 20 ng/mL of IFN-γ for 0–1080 min. c Ratios of pSTAT1-Y701 to STAT1 protein levels were determined after densitometry quantification of the western blots. The data are expressed as fold differences in the ratio of the means for Mmp12^+/+ (n = 5 for each time point) and Mmp12^–/– (n = 4 for each time point) B10.RIII mice. d Rescue of Mmp12^–/– peritoneal macrophages with recombinant mouse MMP12 protein (1:100) incubated for the times shown (n = 4 for each time point). e Mmp12^+/+ B10.RIII macrophages were incubated for 30 min with 100 nm specific MMP12 inhibitor Rxp470.1 before addition of 20 ng/mL IFN-γ (n = 4 for each time point). f Western blot analysis of IFN-γ, MMP12, iNOS, MHCII, S100A8, S100A9, STAT1, CD36, and STAT6 proteins in primary peritoneal macrophages harvested from Mmp12^+/+ (n = 4) and Mmp12^–/– (n = 4) B10.RIII mice at day 4 post-intraperitoneal injection with vehicle; and Mmp12^+/+ B10.RIII mice treated daily with 5 mg/kg Rxp470.1 (n = 4) for 4 days during the induction of peritonitis. Actin or tubulin loading controls and molecular weight marker positions in all blots are as shown