TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells.

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-κB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human ß-defensin-2 (hßD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-κB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.

TLR-9 Plays a Role in Mycobacterium leprae-Induced Innate Immune Activation of A549 Alveolar Epithelial Cells

The respiratory tract is considered the main port of entry of Mycobacterium leprae, the causative agent of leprosy. However, the great majority of individuals exposed to the leprosy bacillus will never manifest the disease due to their capacity to develop protective immunity. Besides acting as a physical barrier, airway epithelium cells are recognized as key players by initiating a local innate immune response that orchestrates subsequent adaptive immunity to control airborne infections. However, to date, studies exploring the interaction of M. leprae with the respiratory epithelium have been scarce. In this work, the capacity of M. leprae to immune activate human alveolar epithelial cells was investigated, demonstrating that M. leprae-infected A549 cells secrete significantly increased IL-8 that is dependent on NF-kB activation. M. leprae was also able to induce IL-8 production in human primary nasal epithelial cells. M. leprae-treated A549 cells also showed higher expression levels of human b-defensin-2 (hbD-2), MCP-1, MHC-II and the co-stimulatory molecule CD80. Furthermore, the TLR-9 antagonist inhibited both the secretion of IL-8 and NF-kB activation in response to M. leprae, indicating that bacterial DNA sensing by this Toll-like receptor constitutes an important innate immune pathway activated by the pathogen. Finally, evidence is presented suggesting that extracellular DNA molecules anchored to Hlp, a histone-like protein present on the M. leprae surface, constitute major TLR-9 ligands triggering this pathway. The ability of M. leprae to immune activate respiratory epithelial cells herein demonstrated may represent a very early event during infection that could possibly be essential to the generation of a protective response.(AU)

Erythema Nodosum Leprosum Neutrophil Subset Expressing IL-10R1 Transmigrates into Skin Lesions and Responds to IL-10.

Erythema nodosum leprosum (ENL) is an inflammatory complication in leprosy. Yet, the involvement of ENL neutrophils in the inflammatory response against Mycobacterium leprae remains poorly explored. Our primary aim was to investigate the utility of the surface expression of neutrophil IL-10R1 as an ENL biomarker and, secondarily, to evaluate whether leprosy or healthy M. leprae-stimulated neutrophils produce cytokines and are able to respond to IL-10. We, in this study, describe a subpopulation of circulating neutrophils of ENL patients that exclusively expressed IL-10R1, providing evidence that IL-10R1+ neutrophils are present in ENL lesions. It was also found that ENL neutrophils, but not those of nonreactional leprosy controls, were able to secret detectable levels of TNF ex vivo and the addition of IL-10 blocked TNF release. It was likewise observed that M. leprae-stimulated, healthy neutrophils expressed IL-10R1 in vitro, and ENL-linked cytokines were released by M. leprae-cultured neutrophils in vitro. Moreover, consistent with the presence of a fully functional IL-10R, the addition of IL-10 prevented the release of M. leprae-induced cytokines. Most importantly, dead M. leprae revealed its superior capacity to induce CCL4 and IL-8 in primary neutrophils over live Mycobacterium, suggesting that M. leprae may hamper the inflammatory machinery as an immune escape mechanism.

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

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.

Neutrophils in Leprosy.

Leprosy is an infectious disease caused by the intracellular bacillus Mycobacterium leprae that mainly affects the skin and peripheral nerves. One of the most intriguing aspects of leprosy is the diversity of its clinical forms. Paucibacillary patients are characterized as having less than five skin lesions and rare bacilli while the lesions in multibacillary patients are disseminated with voluminous bacilli. The chronic course of leprosy is often interrupted by acute episodes of an inflammatory immunological response classified as either reversal reaction or erythema nodosum leprosum (ENL). Although ENL is considered a neutrophilic immune-complex mediated condition, little is known about the direct role of neutrophils in ENL and leprosy disease overall. Recent studies have shown a renewed interest in neutrophilic biology. One of the most interesting recent discoveries was that the neutrophilic population is not homogeneous. Neutrophilic polarization leads to divergent phenotypes (e.g., a pro- and antitumor profile) that are dynamic subpopulations with distinct phenotypical and functional abilities. Moreover, there is emerging evidence indicating that neutrophils expressing CD64 favor systemic inflammation during ENL. In the present review, neutrophilic involvement in leprosy is discussed with a particular focus on ENL and the potential of neutrophils as clinical biomarkers and therapeutic targets.

Elevated Pentraxin-3 Concentrations in Patients With Leprosy: Potential Biomarker of Erythema Nodosum Leprosum.

Background: Leprosy, the leading infectious cause of disability worldwide, remains a major public health challenge in the most severely affected countries despite the sharp decline in new cases in recent years. The search for biomarkers is essential to achieve a better understanding of the molecular and cellular mechanisms underlying the disease. Methods: Pentraxin-3 (PTX3) analyses of sera from 87 leprosy patients with or without reactions were conducted via enzyme-linked immunosorbent assay. In situ identification of PTX3 in skin lesion was confirmed by quantitative reverse-transcription polymerase chain reaction, immunohistochemistry, and immunofluorescence assays. Results: We found that PTX3 serum levels were higher in multibacillary patients when evaluated before the onset of acute erythema nodosum leprosum (ENL) and persistently elevated during reaction. Thalidomide treatment reduced PTX3 in the serum 7 days after starting treatment. In situ analyses have also demonstrated enhancement of PTX3 in ENL lesions and showed that treatment with thalidomide reduced its expression and the prominent neutrophilic infiltrate, a hallmark of the disease. Conclusions: In summary, our study provides in vivo evidence that PTX3 is enhanced during ENL but not in reversal reaction and provides a new molecular target in ENL pathogenesis.