Necrosis, netosis, and apoptosis in pulmonary tuberculosis and type-2 diabetes mellitus. Clues from the patient's serum.

Pulmonary tuberculosis (PTB) and type 2 diabetes mellitus (T2DM) are two inflammatory diseases whose pathology involves neutrophils (NEU) as key participants. Countless inflammatory elements produced at the lesion sites leak into the blood and are distributed systemically. The study aimed to investigate the effect of the serum of patients with PTB, T2DM, and PTB + T2DM on the cellular and nuclear morphology of healthy NEU. Monolayers of NEU were prepared and incubated with sera from PTB (n꓿ 10), T2DM (n꓿10), PTB + T2DM (n꓿ 10) patients, or sera from healthy people (n = 10). Monolayers were stained for histones, elastase, and myeloperoxidase for NETosis, annexin V for apoptosis, and Iris fuchsia for necrosis. Hoechst stain (DNA) was used to identify the nuclear alterations. Necrosis was the predominant alteration. Sera from PTB + T2DM were the most potent change inducers. Normal sera did not induce cell alterations. The blood of TBP and T2DM patients carries a myriad of abnormal elements that induce necrosis of NEU in normal people, thus reflecting what might occur in the neutrophils of the patients themselves. These findings reinforce the participation of NEU in the pathology of these diseases. Necrosis is expected to be the most frequent neutrophil-induced alteration in tuberculosis and diabetes mellitus.

A neutrophil-based test as an auxiliary tool for substantiating the diagnosis of bovine tuberculosis.

Background: Bovine tuberculosis (bTB) is still a prominent threat to animal health; lacking an efficient vaccine, other than BCG to get rid of tuberculosis, the most effective way for this is culling and slaughtering the infected animals. There are several cellular, serological, and molecular tests for the diagnosis of the disease but the most practical one at the field level is the double skin testing with bovine and aviary tuberculins. This is not a very specific test but is sensitive enough to identify most diseased animals; adjunct practical tests are desirable to strengthen the utility of skin tests. All lymphoid and myeloid cells participate, in diverse grades, in the immune response to tuberculosis with neutrophils playing an unintended pathologic role. The study aimed to investigate the response of neutrophils to agents present in the sera of tuberculous cows. Methods: We have developed a neutrophil-based test (N BT) to identify diseased cows within a herd suspected of having tuberculosis; a positive N BT correlates with a positive double skin test. In this test, healthy neutrophils are incubated with the sera of healthy or tuberculous cows for 3 and 6 h, and the nuclear morphologic changes are recorded and analyzed. Results: Sera from tuberculous but not from healthy cows induce nuclear alterations including pyknosis, swelling, apoptosis, and sometimes NETosis, in healthy neutrophils, and CFP 10 and ESAT 6 participate in the phenomenon. Conclusion: We propose the N BT as an auxiliary tool for substantiating the diagnosis of bTB reinforcing the PPD test outcome to help decide whether or not a cow should be sacrificed.

Sera from patients with tuberculosis increase the phagocytic-microbicidal activity of human neutrophils, and ESAT-6 is implicated in the phenomenon.

Background: It has been reported that sera from patients with active pulmonary tuberculosis (APT) induced nuclear changes in normal neutrophils that included pyknosis, swelling, apoptosis, and production of extracellular traps (NETs). Similar changes were observed with some sera from their household contacts but not with sera from healthy, unrelated individuals. It was suggested that those sera from household contacts that induced neutrophil nuclear changes might correspond to people with subclinical tuberculosis. Thus, our experimental approach might serve to identify individuals with early, ongoing disease. Methods: Nuclear changes in neutrophils were fully evident by 3 h of contact and beyond. Circulating mycobacterial antigens were the most likely candidates for this effect. We wanted to know whether the nuclear changes induced on neutrophils by the sera of APT patients would negatively affect the phagocytic/microbicidal ability of neutrophils exposed to APT sera for short periods. Results: We now provide evidence that short-term contact (30 min) with sera from patients with pulmonary tuberculosis increases several phagocytic parameters of normal neutrophils, including endocytosis, myeloperoxidase levels, production of free reactive oxygen species, phagolysosome fusion, and microbicidal activity on Staphylococcus aureus, with these effects not being observed with sera from healthy donors. We also give evidence that suggests that ESAT-6 and CFP-10 are involved in the phenomenon. Conclusion: We conclude that activation is a stage that precedes lethal nuclear changes in neutrophils and suggests that autologous neutrophils must circulate in an altered state in the APT patients, thus contributing to the pathology of the disease.

Sera from patients with active pulmonary tuberculosis and their household contacts induce nuclear changes in neutrophils.

BACKGROUND: Resident alveolar macrophages, dendritic cells, and immigrating neutrophils (NEU) are the first cells to contact Mycobacterium tuberculosis in the lung. These cells, and additional lymphoid cells in the developing granuloma, release a series of components that may concentrate in the serum and affect disease progression. PURPOSE: The aim of this study was to investigate the effect of the serum from tuberculosis (TB) patients and their household contacts (HHC) on the nuclear morphology of NEU. MATERIALS AND METHODS: NEU from healthy (HLT) people were incubated with sera from patients with active pulmonary TB, their HHC, and unrelated people. Changes in the nuclear morphology of NEU were analyzed by light and electron microscopy. RESULTS: Sera from patients with TB induced changes in the nuclear morphology of NEU that included pyknosis, swelling, apoptosis, and netosis in some cases. Sera from some HHC induced similar changes, while sera from HLT people had no significant effects. Bacteria did not appear to participate in this phenomenon because bacteremia is not a recognized feature of nonmiliary TB, and because sera from patients that induced nuclear changes maintained their effect after filtration through 0.22 µm membranes. Neither anti-mycobacterial antibodies, TNFα, IL-6, IFNγ, or IL-8 participated in the phenomenon. In contrast, soluble mycobacterial antigens were likely candidates, as small quantities of soluble M. tuberculosis antigens added to the sera of HLT people led to the induction of nuclear changes in NEU in a dose-dependent manner. CONCLUSION: These results might help to detect subclinical TB within HHC, thus leading to a recommendation of prophylactic treatment.

In vivo induction of neutrophil extracellular traps by Mycobacterium tuberculosis in a guinea pig model.

In 2004, a novel mechanism of cellular death, called 'NETosis', was described in neutrophils. This mechanism, different from necrosis and apoptosis, is characterized by the release of chromatin webs admixed with microbicidal granular proteins and peptides (NETs). NETs trap and kill a variety of microorganisms. Diverse microorganisms, including Mycobacterium tuberculosis, are NET inducers in vitro. The aim of this study was to examine whether M. tuberculosis can also induce NETs in vivo and if the NETs are bactericidal to the microorganism. Guinea pigs were intradermally inoculated with M. tuberculosis H37Rv, and the production of NETs was investigated at several time points thereafter. NETs were detected as early as 30 min post-inoculation and were clearly evident by 4 h post-inoculation. NETs produced in vivo contained DNA, myeloperoxidase, elastase, histones, ROS and acid-fast bacilli. Viable and heat-killed M. tuberculosis, as well as Mycobacterium bovis BCG were efficient NET inducers, as were unilamellar liposomes prepared with lipids from M. tuberculosis. In vitro, guinea pig neutrophils also produced NETs in response to M. tuberculosis. However, neither the in vivo nor the in vitro-produced NETs were able to kill M. tuberculosis. Nevertheless, in vivo, neutrophils might propitiate recruitment and activation of more efficient microbicidal cells.