Neutrophil extracellular traps contribute to the pathogenesis of leprosy type 2 reactions - PubMed (original) (raw)

. 2019 Sep 10;13(9):e0007368.

doi: 10.1371/journal.pntd.0007368. eCollection 2019 Sep.

André Alves Dias 1, José Augusto da Costa Nery 2, Alice de Miranda Machado 2, Helen Ferreira 2, Thais Fernanda Rodrigues 1, João Pedro Sousa Santos 1, Natalia Rocha Nadaes 3, Euzenir Nunes Sarno 2, Elvira Maria Saraiva 3, Verônica Schmitz 2, Maria Cristina Vidal Pessolani 1

Affiliations

Neutrophil extracellular traps contribute to the pathogenesis of leprosy type 2 reactions

Camila Oliveira da Silva et al. PLoS Negl Trop Dis. 2019.

Abstract

Up to 50% of patients with the multibacillary form of leprosy are expected to develop acute systemic inflammatory episodes known as type 2 reactions (T2R), thus aggravating their clinical status. Thalidomide rapidly improves T2R symptoms. But, due to its restricted use worldwide, novel alternative therapies are urgently needed. The T2R triggering mechanisms and immune-inflammatory pathways involved in its pathology remain ill defined. In a recent report, we defined the recognition of nucleic acids by TLR9 as a major innate immunity pathway that is activated during T2R. DNA recognition has been described as a major inflammatory pathway in several autoimmune diseases, and neutrophil DNA extracellular traps (NETs) have been shown to be a prime source of endogenous DNA. Considering that neutrophil abundance is a marked characteristic of T2R lesions, the objective of this study was to investigate NETs production in T2R patients based on the hypothesis that the excessive NETs formation would play a major role in T2R pathogenesis. Abundant NETs were found in T2R skin lesions, and increased spontaneous NETs formation was observed in T2R peripheral neutrophils. Both the M. leprae whole-cell sonicate and the CpG-Hlp complex, mimicking a mycobacterial TLR9 ligand, were able to induce NETs production in vitro. Moreover, TLR9 expression was shown to be higher in T2R neutrophils, suggesting that DNA recognition via TLR9 may be one of the pathways triggering this process during T2R. Finally, treatment of T2R patients with thalidomide for 7 consecutive days resulted in a decrease in all of the evaluated in vivo and ex vivo NETosis parameters. Altogether, our findings shed light on the pathogenesis of T2R, which, it is hoped, will contribute to the emergence of novel alternative therapies and the identification of prognostic reactional markers in the near future.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1

Fig 1. NETs are abundant in T2R skin lesions.

(A) Histology of cutaneous lesions from T2R and T2Rthal patients stained by H&E. The arrows indicate the presence of neutrophils. Representative images of 3 patients (400x). (B) Skin lesions of T2R and T2Rthal patients were processed for immunostaining of NETs (MPO, green; histone H1, red; and DNA, blue). The arrows indicate the presence of NETs characterized by filamentous structures stained for these 3 macromolecules. The images are representative of 3 patients. Scale bar: 20 μm.

Fig 2

Fig 2. Circulating NETs components are higher in T2R patients, which decrease after thalidomide treatment.

Patient serum samples were obtained; and the levels of the DNA-histone (A, B) and DNA-MPO (C, D) complexes were quantified by ELISA. DNA-MPO complex levels are expressed as percentages values above the blank. (B, D) Follow-up of the complexes serum levels during thalidomide treatment. Outliers were removed from analysis. Box plots show median, interquartile range, sample minimum, and maximum. Each dot represents a donor. **P<0.01 (Mann Whitney test). (E) Spearman’s correlation between DNA-histone and DNA-MPO complexes per patient (n = 36, r = 0.5247, P = 0.001).

Fig 3

Fig 3. T2R neutrophils show spontaneous ex vivo NETs production, which decreases with thalidomide treatment.

Neutrophils from LL/BL, T2R, and T2Rthal patients were isolated and incubated in medium alone for 90 min. (A) Immunostaining of NETs components (MPO, green; histone, red; and DNA, blue). Arrows indicate the presence of NETs. Images are representative of 3 LL/BL, 4 T2R and 4 T2Rthal. Scale bar: 20 μm. (B) The percentage of NETs-releasing cells from 5 fields captured from 3 LL/BL, 4 T2R and 4 T2Rthal patients is shown. (C) Neutrophils from LL/BL, T2R, and T2Rthal patients were incubated for 90 min and DNA release was measured by picogreen. Box plots show median, interquartile range, sample minimum, and maximum. Each dot represents a donor. (D) Comparison of patients who developed T2R during MDT versus those with reactional episodes before or after MDT. (E) Follow-up of DNA levels released after 7 days of thalidomide treatment. Dots in red are patients who developed T2R during MDT. *P<0.05 (Mann Whitney test).

Fig 4

Fig 4. M. leprae induces NETs formation in vitro.

Neutrophils from LL/BL, T2R, and T2Rthal patients were isolated and stimulated or not with M. leprae whole-cell sonicate (MLWCS) for 90 min. (A) Immunostaining of NETs components (MPO, green; histone, red; and DNA, blue). Arrows indicate the presence of NETs. Images are representative of 3 LL/BL, 4 T2R, and 4 T2Rthal. Scale bar: 20 μm. (B) DNA release was measured in culture supernatants by picogreen. Box plots show median, interquartile range, sample minimum, and maximum. Each dot represents a donor. **P<0.01 (Mann Whitney test).

Fig 5

Fig 5. CpG-Hlp, a mycobacterial TLR9 ligand, induces NETs formation in vitro.

Neutrophils from LL/BL, T2R, and T2Rthal patients were isolated and stimulated or not with CpG-Hlp complex for 90 min. (A) Immunostaining of NETs components (MPO, green; histone, red; and DNA, blue). Arrows indicate the presence of NETs. Images are representative of 3 LL/BL, 4 T2R, and 4 T2Rthal. Scale bar: 20 μm. (B) DNA release was measured in culture supernatants with picogreen. Box plots show median, interquartile range, sample minimum, and maximum. Each dot represents a donor. *P<0.05 and **P<0.01 (Mann Whitney test).

Fig 6

Fig 6. T2R patient neutrophils express higher levels of TLR9, which decrease after thalidomide treatment.

(A) Neutrophils of LL/BL, T2R and T2Rthal patients were isolated and ex vivo TLR9 expression levels were quantified by flow cytometry. *P<0.05 (Kruskal-Wallis test). (B) Follow-up of TLR9 expression in T2R patients after 7 days of thalidomide treatment. Box plots show median, interquartile range, sample minimum, and maximum. Each dot represents a donor. **P<0.01 (Wilcoxon test). (C) Spearman’s correlation between DNA-MPO complex and TLR9 expression per patient (n = 14, r = 0.789, P = 0.0013). (D) Spearman’s correlation between DNA-histone complex and TLR9 expression per patient (n = 14, r = 0.4813, P = 0.0840).

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