Type I interferon suppresses type II interferon-triggered human anti-mycobacterial responses

Abstract

Type I interferons (IFN-α and IFN-β) are important for protection against many viral infections, whereas type II interferon (IFN-γ) is essential for host defense against some bacterial and parasitic pathogens. Study of IFN responses in human leprosy revealed an inverse correlation between IFN-β and IFN-γ gene expression programs. IFN-γ and its downstream vitamin D-dependent antimicrobial genes were preferentially expressed in self-healing tuberculoid lesions and mediated antimicrobial activity against the pathogen Mycobacterium leprae in vitro. In contrast, IFN-β and its downstream genes, including interleukin-10 (IL-10), were induced in monocytes by M. leprae in vitro and preferentially expressed in disseminated and progressive lepromatous lesions. The IFN-γ-induced macrophage vitamin D-dependent antimicrobial peptide response was inhibited by IFN-β and by IL-10, suggesting that the differential production of IFNs contributes to protection versus pathogenesis in some human bacterial infections.

Fig. 1

IFN signatures in leprosy skin lesions. (A) Unsupervised principal component analysis (PCA) of L-lep, T-lep, and RR skin lesion gene expression profiles. Total three-dimensional PCA mapping as 81.5% of variance (PC1 = 69.5%, PC2 = 8.75%, and PC3 = 3.25%). (B) Unsupervised clustering analysis using differentially expressed genes between L-lep, T-lep, and RR (coefficient of variation ≥ 1.0). Individual squares represent the relative gene expression intensity of the given gene (rows) in a patient (columns), with red indicating an increase in expression and green a decrease. (C) Enrichment analysis of overlap between IFN-α–, IFN-β–, and IFNγ– specific genes (induced and repressed; limited to genes modulated by only one IFN family member) identified in healthy human PBMCs and L-lep– and T-lep–specific leprosy lesion transcripts (fold change ≥ 1.5 and P ≤ 0.05). Dotted lines indicate either the expected fold enrichment of one (top) or the hypergeometric enrichment P value of 0.05 (log P = 1.3, bottom). Hypergeometric analyses were performed to determine fold enrichment (observed/expected) and signed log enrichment P value (negative for deenriched). The Bonferroni multiple hypothesis test correction was applied for each group (n = 6). (D) IFN-β– and IFN-γ–specific gene voting summation scores were calculated for individual patient lesions in the leprosy subtypes of L-lep, T-lep, and RR.

Fig. 2

IFN-β is up-regulated in L-lep lesions. (A) Total mRNA was isolated from L-lep (n= 10), T-lep (n = 10), and RR (n = 10) skin lesions; and the IFN-β, IFNAR1, and IFN-γ mRNA levels were analyzed by quantitative PCR (qPCR). The levels of IFN-β, IFNAR1 and IFN-γ were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in the same tissue. Statistical significance was calculated by one-way ANOVA followed by the posttest, Newman-Keuls multiple comparison test for IFN-β and IFNR1, and Kruskal-Wallis followed by Dunn’s multiple comparison test for IFN-γ. **P ≤ 0.01; *P ≤ 0.05. (B) IFN-β and IFNAR1 expression were detected in leprosy lesions (T-lep, L-lep, and RR); one representative labeled section is shown out of at least five; scale bars, 40 µm. Original magnification, 100×. (Insets) Higher magnification of inflammatory infiltrate areas. Original magnification, 400×.

Fig. 3

IL-10 is increased in L-lep lesions and is induced by IFN-β and M. leprae in vitro. (A) Total mRNA was isolated from L-lep (n = 10), T-lep (n = 10), and RR (n = 10) skin lesions, and IL-10 mRNA levels were analyzed by qPCR. The levels of IL-10 were normalized to GAPDH levels in the same tissue. Statistical significance was calculated by ANOVA followed by Newman-Keulsmultiple comparison test. ***P ≤ 0.001; **P ≤ 0.01. (B) IL-10 expression in leprosy lesions (T-lep, L-lep, and RR); one representative labeled section is shown out of at least five individuals; scale bars, 40 µm. Original magnification, 200×. (Insets) Higher magnification of inflammatory infiltrate area. Original magnification, 400×. (C) Colocalization of IFN-β (green) and IL-10 (red) in the inflammatory infiltrate of L-lep lesions. Data are representative of three individual L-lep samples; arrows indicate colocalization of the two cytokines. (D) Human monocytes were stimulated with live M. leprae (mLEP) or sonicated mLEP. After 6 hours, qPCR was performed for detection of IFN-β (FC, fold change); supernatants were collected after 24 hours for detection of IFN-β by enzyme-linked immunosorbent assay. Data are represented as mean ± SEM, n = 7. Statistical significance was calculated by two-tailed Student’s t test. **P ≤ 0.01; *P ≤ 0.05. (E) Monocytes were stimulated with live mLEP or sonicated mLEP for 24 hours, and IL-10 protein levels were detected. Data are represented as mean ± SEM, n = 7. Statistical significance was calculated by two-tailed Student’s t test. *P ≤ 0.05. (F) Human monocytes were stimulated with mLEP sonicated alone or in combination with either human IFNAR2 antibody or isotype control for 24 hours, and IL-10 protein levels were detected. Data are represented as mean ± SEM, n = 4. Left graph shows the levels of IL-10 subtracted from media (average ± 64.5 pg/ml), and right graph shows the percentage of inhibition of IL-10 levels. Statistical significance was calculated by one-way ANOVA, and comparison between two groups was confirmed by the posttest and Newman-Keuls multiple comparison test. ***P ≤ 0.001; ** P ≤ 0.01. IgG2a, immunoglobulin γ2a.

Fig. 4

IFN-β and IL-10 inhibit antimicrobial pathway induced by IFN-γ. (A) Distribution of CYP27B1 and VDR mRNA by microarray analysis of L-lep (n = 6), T-lep (n = 10), and RR (n = 7) skin lesions shown in arbitrary units (AU). Statistical significance was calculated for CYP27B1 by ANOVA followed by Newman-Keuls multiple comparison test and, for VDR, by Kruskal-Wallis followed by Dunn’s multiple comparison test. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05. (B) Human monocytes were treated with IL-10, IFN-γ, and IFN-γ plus IL-10 for 48 hours followed by incubation with radiolabeled metabolite cholecalciferol (D3), 25(OH)D₃ for 5 hours, and the ability to convert 25(OH)D₃ to 1,25(OH)₂D₃ was measured by HLPC. Enzymatic conversion data are represented as mean ± SEM and show three different donors, each studied in triplicate. Statistical significance was calculated by one-way ANOVA repeated measures test followed by Newman-Keuls multiple comparison test. *P ≤ 0.05. (C) Human monocytes were stimulated with IFN-γ alone or in combination with IFN-β and IL-10, antibody against human IL-10 was added in the monocyte culture in combination with IFN-γ + IFN-β for 24 hours, and the cathelicidin (Cath) and DEFB4 mRNA levels were detected by qPCR. Data are represented as mean ± SEM, n = 7. Statistical significance was calculated by two-tailed Student’s t test. *P ≤ 0.05. (D) Human monocytes were pretreated with IFN-γ and then infected with M. leprae at a multiplicity of infection of 10:1 overnight. After infection, cells were treated with IFN-γ alone or in combination with VDR antagonist (VAZ). Viability of mLEP was calculated by the ratio of bacterial 16S RNA and DNA (RLEP) detected by qPCR, and percent increase or decrease relative to no treatment (media) was determined. Data are represented as mean ± SEM, n = 5. Statistical significance was calculated by two-tailed Student’s t test. *P ≤ 0.05. (E) Human monocytes were pretreated and infected as described in (D). After infection, cells were treated with IFN-γ alone or in combination with IFN-β or IL-10; in some instances, human IL-10 antibody was added to the culture in combination with IFN-γ plus IFN-β for 4 days. Viability of M. leprae was calculated as described in (D). Data are represented as mean ± SEM, n = 10. Statistical significance was calculated by two-tailed Student’s t test. *P ≤ 0.05.