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. 2014 May;184(5):1268-73.
doi: 10.1016/j.ajpath.2014.01.006. Epub 2014 Mar 4.

The impact of IFN-γ receptor on SLPI expression in active tuberculosis: association with disease severity

Nancy L Tateosian  1 , Virginia Pasquinelli  2 , Rodrigo E Hernández Del Pino  2 , Nella Ambrosi  1 , Diego Guerrieri  1 , Sigifredo Pedraza-Sánchez  3 , Natalia Santucci  4 , Luciano D'Attilio  4 , Joaquín Pellegrini  2 , María A Araujo-Solis  5 , Rosa M Musella  6 , Domingo J Palmero  6 , Rogelio Hernandez-Pando  7 , Verónica E Garcia  2 , H Eduardo Chuluyan  8
Affiliations

The impact of IFN-γ receptor on SLPI expression in active tuberculosis: association with disease severity

Nancy L Tateosian et al. Am J Pathol. 2014 May.

Abstract

Interferon (IFN)-γ displays a critical role in tuberculosis (TB), modulating the innate and adaptive immune responses. Previously, we reported that secretory leukocyte protease inhibitor (SLPI) is a pattern recognition receptor with anti-mycobacterial activity against Mycobacterium tuberculosis (Mtb). Herein, we determined whether IFN-γ modulated the levels of SLPI in TB patients. Plasma levels of SLPI and IFN-γ were studied in healthy donors (HDs) and TB patients. Peripheral blood mononuclear cells from HDs and patients with TB or defective IFN-γ receptor 1* were stimulated with Mtb antigen and SLPI, and IFN-γR expression levels were measured. Both SLPI and IFN-γ were significantly enhanced in plasma from those with TB compared with HDs. A direct association between SLPI levels and the severity of TB was detected. In addition, Mtb antigen stimulation decreased the SLPI produced by peripheral blood mononuclear cells from HDs, but not from TB or IFN-γR patients. Neutralization of IFN-γ reversed the inhibition of SLPI induced by Mtb antigen in HDs, but not in TB patients. Furthermore, recombinant IFN-γ was unable to modify the expression of SLPI in TB patients. Finally, IFN-γR expression was lower in TB compared with HD peripheral blood mononuclear cells. These results show that Mtb-induced IFN-γ down-modulated SLPI levels by signaling through the IFN-γR in HDs. This inhibitory mechanism was not observed in TB, probably because of the low expression of IFN-γR detected in these individuals.

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Figures

Figure 1
Figure 1
Plasma levels of SLPI in HDs and TB patients. A: Plasmatic SLPI levels. SLPI levels were analyzed by sandwich ELISA in plasma from HDs (n = 23) and TB patients (n = 29). B: Association between plasma SLPI levels and disease severity. HD data from A were plot as subjects who had been in contact with TB patients (HCs) and those who had not been in contact with patients (HDs). Patients with TB were also classified as mild, moderate, and severe. Student’s t-test (A) or analysis of variance post hoc Dunnett’s multiple comparisons test (B) was used. ∗P < 0.05, between severe and mild TB patients; ∗∗P < 0.01 (A); ††P < 0.01 between severe TB patients and HDs or HCs (B).
Figure 2
Figure 2
Correlation between the levels of IFN-γ and SLPI in HDs and TB patients. IFN-γ plasma levels in HD and TB patients were determined. A: ELISA results for plasmatic IFN-γ levels from HDs (n = 15) and TB patients (n = 19). B: Correlation between the levels of SLPI and IFN-γ in the plasma of HDs (white circles; n = 15) and TB patients (black circles; n = 19). Student’s t-test (A) and Pearson correlation coefficient (B). ∗P < 0.05.
Figure 3
Figure 3
Effect of IFN-γ on SLPI levels. A: PBMCs from HDs and TB patients were cultured for 48 hours in the presence or absence of 10 μg/mL Mtb-Ag, ±7.5 ng/mL rIFN-γ, and ±15 μg/mL blocking monoclonal antibody against IFN-γ (30 minutes) or 15 μg/mL isotype control. B: PBMCs from IFNGR1∗ patients and age-matched HDs were cultured for 5 days in the presence or absence of Mtb-Ag. A and B: After culture, medium was removed and cell-free supernatants were collected and assayed for SLPI. Data are expressed as the means ± SEM in HDs (n = 6) and TB patients (n = 6) (A) and in HDs (n = 4) and IFNGR1∗ patients (n = 2) (B). Analysis of variance post hoc Dunnett’s multiple comparisons test. C and D: Real-time PCR for IFN-γR expression on PBMCs. C: PBMCs were obtained from patients with TB classified as mild (n = 6), moderate (n = 6), and severe (n = 6) and HD subjects (n = 6). IFN-γR expression was determined by quantitative real-time PCR. Values are represented as fold of increase using the comparative method for relative quantification. Expression of IFN-γR was calculated as follows (a comparative method for relative quantification after normalization to GAPDH expression): Foldincrease=2−ΔΔCt, where ΔCt=[Ct(IFN-γR)−Ct(GAPDH)] and ΔΔCt=[ΔCtTB−ΔCtHD]. Mann-Whitney test. C: Significant differences between mild and severe TB patients. D: PBMCs from patients with TB (n = 9) and HDs (n = 11) were cultured by 16, 24, or 48 hours in the presence or absence of Mtb-Ag. Cells were harvested, and IFN-γR expression was determined as in C. Expression of IFN-γR was calculated as follows (a comparative method for relative quantification after normalization to GAPDH expression): Foldincrease=2−ΔΔCt, where ΔCt=[Ct(IFN-γR)−Ct(GAPDH)] and ΔΔCt = [ΔCtstimulated−ΔCtunstimulated]. Unpaired t-test, for significant differences between HDs and TB patients at 16 hours and for significant differences between 16 and 48 hours in TB patients. ∗P < 0.05 (A–D); ††P < 0.01, for significant differences between 16 and 48 hours in HDs (D).
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