No abstract

Initiation of the adaptive immune response to Mycobacterium tuberculosis occurs in the lung-draining mediastinal lymph node, and requires transport of M. tuberculosis by migratory dendritic cells (DCs) to the local lymph node. The previously-published observations that: 1) neutrophils are a transiently prominent population of M. tuberculosis-infected cells in the lungs early in infection; and 2) that the peak of infected neutrophils immediately precedes the peak of infected DCs in the lungs, prompted us to characterize the role of neutrophils in the initiation of adaptive immune responses to M. tuberculosis. We found that, although depletion of neutrophils in vivo increased the frequency of M. tuberculosis infected DCs in the lungs, it decreased trafficking of DCs to the mediastinal lymph node. This resulted in delayed activation (CD69 expression) and proliferation of naïve M. tuberculosis Ag85B-specific CD4 T cells in the mediastinal lymph node. To further characterize the role for neutrophils in DC-migration we used a Transwell chemotaxis system and found that DCs that were directly infected by M. tuberculosis migrated poorly in response to CCL19, an agonist for the chemokine receptor CCR7. In contrast, DCs that had acquired M. tuberculosis through uptake of infected neutrophils exhibited unimpaired migration. These results reveal a mechanism wherein neutrophils promote adaptive immune responses to M. tuberculosis by delivering M. tuberculosis to DCs in a form that make DCs more effective initiators of naïve CD4 T cell activation. These observations provide insight into a mechanism for neutrophils to facilitate initiation of adaptive immune responses in tuberculosis.

Lysosomal membrane permeabilization (LMP) is emerging as an important regulator of cell apoptosis. Human neutrophils are highly granulated phagocytes, which respond to pathogens by exhibiting increased production of reactive oxygen species (ROS) and lysosomal degranulation. In a previous study, we observed that intracellular, nonphagosomal generation of ROS triggered by adherent bacteria induced ROS-dependent neutrophil apoptosis, whereas intraphagosomal production of ROS during phagocytosis had no effect. In the present study, we measured lysosomal membrane stability and leakage in human neutrophils and found that adherent, noningested, Type 1-fimbriated Escherichia coli bacteria induced LMP rapidly in neutrophils. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium markedly blocked the early LMP and apoptosis in neutrophils stimulated with Type 1-fimbriated bacteria but had no effect on the late LMP seen in spontaneously apoptotic neutrophils. The induced lysosomal destabilization triggered cleavage of the proapoptotic Bcl-2 protein Bid, followed by a decrease in the antiapoptotic protein Mcl-1. Involvement of LMP in initiation of apoptosis is supported by the following observations: Bid cleavage and the concomitant drop in mitochondrial membrane potential required activation of cysteine-cathepsins but not caspases, and the differential effects of inhibitors of cysteine-cathepsins and cathepsin D on apoptosis coincided with their ability to inhibit Bid cleavage in activated neutrophils. Together, these results indicate that in microbe-induced apoptosis in neutrophils, ROS-dependent LMP represents an early event in initiation of the intrinsic apoptotic pathway, which is followed by Bid cleavage, mitochondrial damage, and caspase activation.

Summary Mycobacterium tuberculosis promotes its replication by inhibiting the apoptosis of infected macrophages. A proapoptotic M. tuberculosis mutant lacking nuoG, a subunit of the type I NADH dehydrogenase complex, exhibits attenuated growth in vivo, indicating that this virulence mechanism is essential. We show that M. tuberculosis also suppresses neutrophil apoptosis. Compared to wild-type, the nuoG mutant spread to a larger number of lung phagocytic cells. Consistent with the shorter lifespan of infected neutrophils, infection with the nuoG mutant resulted in fewer bacteria per infected neutrophil, accelerated bacterial acquisition by dendritic cells, earlier trafficking of these dendritic cells to lymph nodes, and faster CD4 T cell priming. Neutrophil depletion abrogated accelerated CD4 T cell priming by the nuoG mutant, suggesting that inhibiting neutrophil apoptosis delays adaptive immunity in tuberculosis. Thus, pathogen modulation of apoptosis is beneficial at multiple levels, and enhancing phagocyte apoptosis promotes CD4 as well as CD8 T cell responses.

Objective. NALP3, ASC, and TUCAN are components of the NALP3 inflammasome, which triggers caspase 1-mediated interleukin-1␤ (IL-1␤) release. Activating mutations in the gene encoding NALP3 (NLRP3) have recently been linked to familial periodic fever syndromes. We undertook this study to determine whether a patient with arthritis and antibiotic-resistant fever carried mutations in the genes encoding the NALP3 inflammasome.Methods. Genetic analysis of NLRP3 and the gene encoding TUCAN (CARD-8) was performed on genomic DNA from the patient and from a population-based collection of DNA (806 subjects). For in vitro studies of IL-1␤ production and caspase 1 activity, blood was obtained from the patient at different time points after administration of anakinra, an IL-1 receptor antagonist, as well as from 5 healthy age-and sex-matched control subjects.Results. Mutation analysis of the patient's genes encoding NALP3, ASC, and TUCAN revealed variations in the NLRP3 (Q705K) and CARD-8 (C10X) genes. The allele frequencies of these single-nucleotide polymorphisms (SNPs) in the population were 6.5% and 34%, respectively. The elevated activity of caspase 1 and the high levels of IL-1␤ measured in samples from the patient returned to normal levels after treatment with anakinra.Conclusion. Our results indicate that the patient's symptoms were due to elevated levels of IL-1␤, since treatment with anakinra effectively abolished the symptoms. The compound SNPs may explain the increased IL-1␤ levels and inflammatory symptoms observed, but further studies are needed to reveal a functional relationship. The prevalence of the polymorphisms (4% of the population carry both SNPs) in the general population may suggest a genetic predisposition for common inflammatory disorders.