A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.

Short-term and 1-year outcome of patients' with borderline personality admitted to a short-term recovery-oriented residential service.

OBJECTIVES: Given the paucity of literature, this study investigated whether a prevention and recovery care (PARC) service supported recovery in patients with borderline personality disorder (BPD). METHOD: This retrospective study included patients with BPD who had their first (index) admission to North West PARC between 2011 and 2016. Patient medical records and the state-wide database were the sources of information. RESULTS: Of the 67 patients included, over 70% attended group activities. All patients achieved their recovery goals, either fully or partially. Compared to admission, the frequency of substance use and the Health of the Nation Outcome Scale (HoNOS) scores at discharge were significantly less. A significantly smaller number of patients needed inpatient treatment during the 12 months following their PARC admission. CONCLUSION: The PARC service appears to promote clinical and psychosocial recovery in patients with BPD.

Impact of Diabetes and Low Body Mass Index on Tuberculosis Treatment Outcomes.

BACKGROUND: Diabetes was identified as a tuberculosis (TB) risk factor mostly in retrospective studies with limited assessments of metabolic variables. The prospective Effects of Diabetes on Tuberculosis Severity study compared adults with pulmonary TB in Chennai, India, who were classified as having either diabetes or a normal glucose tolerance at enrollment. METHODS: Baseline TB severity, sputum conversion, and treatment outcomes (cure, failure, death, or loss to follow-up) were compared between groups with respect to glycemic status and body mass index (BMI). RESULTS: The cohort of 389 participants included 256 with diabetes and 133 with a normal glucose tolerance. Low BMIs (<18.5 kg/m2) were present in 99 (74.4%) of nondiabetic participants and 85 (33.2%) of those with diabetes. Among participants with normal or high BMIs, rates of cure, treatment failure, or death did not vary by glycemic status. Participants with low BMIs had the highest radiographic severity of disease, the longest time to sputum culture conversion, and the highest rates of treatment failure and death. Among participants with low BMIs, poorly controlled diabetes (glycohemoglobin [HbA1c] ≥8.0%) was unexpectedly associated with better TB treatment outcomes. A high visceral adiposity index was associated with adverse outcomes and, despite an overall correlation with HbA1c, was elevated in some low-BMI individuals with normal glucose tolerance. CONCLUSIONS: In this South Indian cohort, a low BMI was significantly associated with an increased risk for adverse TB treatment outcomes, while comorbid, poorly controlled diabetes lessened that risk. A high visceral adiposity index, either with or without dysglycemia, might reflect a novel TB susceptibility mechanism linked to adipose tissue dysfunction.

Identification of QS-21 as an Inflammasome-activating Molecular Component of Saponin Adjuvants.

Many immunostimulants act as vaccine adjuvants via activation of the innate immune system, although in many cases it is unclear which specific molecules contribute to the stimulatory activity. QS-21 is a defined, highly purified, and soluble saponin adjuvant currently used in licensed and exploratory vaccines, including vaccines against malaria, cancer, and HIV-1. However, little is known about the mechanisms of cellular activation induced by QS-21. We observed QS-21 to elicit caspase-1-dependent IL-1ß and IL-18 release in antigen-presenting cells such as macrophages and dendritic cells when co-stimulated with the TLR4-agonist adjuvant monophosphoryl lipid A. Furthermore, our data suggest that the ASC-NLRP3 inflammasome is responsible for QS-21-induced IL-1ß/IL-18 release. At higher concentrations, QS-21 induced macrophage and dendritic cell death in a caspase-1-, ASC-, and NLRP3-independent manner, whereas the presence of cholesterol rescued cell viability. A nanoparticulate adjuvant that contains QS-21 as part of a heterogeneous mixture of saponins also induced IL-1ß in an NLRP3-dependent manner. Interestingly, despite the role NLRP3 plays for cellular activation in vitro, NLRP3-deficient mice immunized with HIV-1 gp120 and QS-21 showed significantly higher levels of Th1 and Th2 antigen-specific T cell responses and increased IgG1 and IgG2c compared with wild type controls. Thus, we have identified QS-21 as a nonparticulate single molecular saponin that activates the NLRP3 inflammasome, but this signaling pathway may contribute to decreased antigen-specific responses in vivo.

Impaired Recognition of Mycobacterium tuberculosis by Alveolar Macrophages From Diabetic Mice.

BACKGROUND: Diabetes mellitus is associated with increased tuberculosis risk and severity. We previously reported that tuberculosis susceptibility in diabetic mice results from a delay in innate immune response to inhaled Mycobacterium tuberculosis, leading to delayed adaptive immune priming and, consequently, a higher plateau lung bacterial burden and greater immune pathology. METHODS: We tested the capacity of alveolar macrophages from diabetic mice to phagocytose M. tuberculosis ex vivo and promote T-cell activation in vivo. RESULTS: Alveolar macrophages from diabetic mice had reduced expression of CD14 and macrophage receptor with collagenous structure (MARCO), which recognize the bacterial cell wall component trehalose 6,6'-dimycolate (TDM). Diabetic alveolar macrophages exhibited reduced phagocytosis of M. tuberculosis or TDM-coated latex beads. This alveolar macrophage phenotype was absent in peritoneal and bone marrow-derived macrophages. Transfer of infected alveolar macrophages from diabetic mice into nondiabetic recipients confirmed an intrinsic alveolar macrophage defect that hindered T-cell priming. The diabetic alveolar macrophage phenotype depended in part on expression of the receptor for advanced glycation end products. CONCLUSIONS: Reduced MARCO and CD14 expression contributes to defective sentinel function of alveolar macrophages, promoting tuberculosis susceptibility in diabetic hosts at a critical early step in the immune response to aerosol infection.

Chromatin decondensation and T cell hyperresponsiveness in diabetes-associated hyperglycemia.

Diabetes is linked to increased inflammation and susceptibility to certain infectious diseases including tuberculosis (TB). We previously reported that aerosol TB in mice with chronic (≥ 12 wk) hyperglycemia features increased bacterial load, overproduction of several cytokines, and increased immune pathology compared with normoglycemic controls. A similar phenotype exists in human patients with diabetes with TB. The mechanisms of increased T cell activation in diabetes are unknown. In the current study, we tested the hypothesis that hyperglycemia modifies the intrinsic responsiveness of naive T cells to TCR stimulation. Purified T cells from chronically hyperglycemic (HG) mice produced higher levels of Th1, Th2, and Th17 cytokines and proliferated more than T cells from normoglycemic controls after anti-CD3e or Ag stimulation. In this way, naive T cells from HG mice resembled Ag-experienced cells, although CD44 expression was not increased. Chromatin decondensation, another characteristic of Ag-experienced T cells, was increased in naive T cells from HG mice. That phenotype depended on expression of the receptor for advanced glycation end products and could be reversed by inhibiting p38 MAPK. Chromatin decondensation and hyperresponsiveness to TCR stimulation persisted following transfer of T cells from HG mice into normoglycemic mice. We propose that chronic hyperglycemia causes receptor for advanced glycation end products-mediated epigenetic modification of naive T cells leading to p38 MAPK-dependent chromatin decondensation. This preactivation state facilitates transcription factor access to DNA, increasing cytokine production and proliferation following TCR stimulation. This mechanism may contribute to pathological inflammation associated with diabetes and might offer a novel therapeutic target.

Trait stacking via targeted genome editing.

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.

Glycerol contributes to tuberculosis susceptibility in male mice with type 2 diabetes.

Diabetes mellitus increases risk for tuberculosis disease and adverse outcomes. Most people with both conditions have type 2 diabetes, but it is unknown if type 1 and type 2 diabetes have identical effects on tuberculosis susceptibility. Here we show that male mice receiving a high-fat diet and streptozotocin to model type 2 diabetes, have higher mortality, more lung pathology, and higher bacterial burden following Mycobacterium tuberculosis infection compared to mice treated with streptozotocin or high-fat diet alone. Type 2 diabetes model mice have elevated plasma glycerol, which is a preferred carbon source for M. tuberculosis. Infection studies with glycerol kinase mutant M. tuberculosis reveal that glycerol utilization contributes to the susceptibility of the type 2 diabetes mice. Hyperglycemia impairs protective immunity against M. tuberculosis in both forms of diabetes, but our data show that elevated glycerol contributes to an additional adverse effect uniquely relevant to type 2 diabetes.

B-cell responses during primary and secondary dengue virus infections in humans.

Low-avidity serotype-cross-reactive antibodies are hypothesized to play a key role in triggering severe disease in patients with secondary dengue virus (DENV) infection. However, there is little systematic information about the frequency, avidity, and cross-reactivity of DENV-specific B cells in individuals experiencing primary instead of secondary infection. We compared DENV-specific B-cell responses in a cohort of Thai children with primary or secondary DENV infection. B cells specific for DENV precursor membrane protein, envelope (E) protein, and nonstructural protein 1 were detectable in immune peripheral blood mononuclear cells with the highest frequencies of DENV E-specific B cells detected in patients experiencing primary DENV-1 infections. DENV E-specific B cells were highly serotype-specific after primary DENV infections, whereas most E-specific B cells in patients with secondary infection were serotype-cross-reactive and secreted antibodies with higher avidity to heterologous DENV serotypes. Our data suggest that the minor populations of serotype-cross-reactive B cells generated by primary DENV infection are preferentially expanded during secondary DENV infection.

Maternal health after Ebola: unmet needs and barriers to healthcare in rural Sierra Leone.

Sierra Leone has the world's highest estimated maternal mortality. Following the 2014-16 Ebola outbreak, we described health outcomes and health-seeking behaviour amongst pregnant women to inform health policy. In October 2016-January 2017, we conducted a sequential mixed-methods study in urban and rural areas of Tonkolili District comprising: household survey targeting women who had given birth since onset of the Ebola outbreak; structured interviews at rural sites investigating maternal deaths and reporting; and in-depth interviews (IDIs) targeting mothers, community leaders and health workers. We selected 30 clusters in each area: by random GPS points (urban) and by random village selection stratified by population size (rural). We collected data on health-seeking behaviours, barriers to healthcare, childbirth and outcomes using structured questionnaires. IDIs exploring topics identified through the survey were conducted with a purposive sample and analysed thematically. We surveyed 608 women and conducted 29 structured and 72 IDIs. Barriers, including costs of healthcare and physical inaccessibility of healthcare facilities, delayed or prevented 90% [95% confidence interval (CI): 80-95] (rural) vs 59% (95% CI: 48-68) (urban) pregnant women from receiving healthcare. Despite a general preference for biomedical care, 48% of rural and 31% of urban women gave birth outside of a health facility; of those, just 4% and 34%, respectively received skilled assistance. Women expressed mistrust of healthcare workers (HCWs) primarily due to payment demanded for 'free' healthcare. HCWs described lack of pay and poor conditions precluding provision of quality care. Twenty percent of women reported labour complications. Twenty-eight percent of villages had materials to record maternal deaths. Pregnant women faced important barriers to care, particularly in rural areas, leading to high preventable mortality and morbidity. Women wanted to access healthcare, but services available were often costly, unreachable and poor quality. We recommend urgent interventions, including health promotion, free healthcare access and strengthening rural services to address barriers to maternal healthcare.

Multifunctional T-cell characteristics induced by a polyvalent DNA prime/protein boost human immunodeficiency virus type 1 vaccine regimen given to healthy adults are dependent on the route and dose of administration.

A phase I clinical vaccine study of a human immunodeficiency virus type 1 (HIV-1) vaccine regimen comprising a DNA prime formulation (5-valent env and monovalent gag) followed by a 5-valent Env protein boost for seronegative adults was previously shown to induce HIV-1-specific T cells and anti-Env antibodies capable of neutralizing cross-clade viral isolates. In light of these initial findings, we sought to more fully characterize the HIV-1-specific T cells by using polychromatic flow cytometry. Three groups of participants were vaccinated three times with 1.2 mg of DNA administered intradermally (i.d.; group A), 1.2 mg of DNA administered intramuscularly (i.m.; group B), or 7.2 mg of DNA administered i.m. (high-dose group C) each time. Each group subsequently received one or two doses of 0.375 mg each of the gp120 protein boost vaccine (i.m.). Env-specific CD4 T-cell responses were seen in the majority of participants; however, the kinetics of responses differed depending on the route of DNA administration. The high i.m. dose induced the responses of the greatest magnitude after the DNA vaccinations, while the i.d. group exhibited the responses of the least magnitude. Nevertheless, after the second protein boost, the magnitude of CD4 T-cell responses in the i.d. group was indistinguishable from those in the other two groups. After the DNA vaccinations and the first protein boost, a greater number of polyfunctional Env-specific CD4 T cells (those with > or = 2 functions) were seen in the high-dose group than in the other groups. Gag-specific CD4 T cells and Env-specific CD8 T cells were seen only in the high-dose group. These findings demonstrate that the route and dose of DNA vaccines significantly impact the quality of immune responses, yielding important information for future vaccine design.

mTORC2/Akt activation in adipocytes is required for adipose tissue inflammation in tuberculosis.

BACKGROUND: Mycobacterium tuberculosis has co-evolved with the human host, adapting to exploit the immune system for persistence and transmission. While immunity to tuberculosis (TB) has been intensively studied in the lung and lymphoid system, little is known about the participation of adipose tissues and non-immune cells in the host-pathogen interaction during this systemic disease. METHODS: C57BL/6J mice were aerosol infected with M. tuberculosis Erdman and presence of the bacteria and the fitness of the white and brown adipose tissues, liver and skeletal muscle were studied compared to uninfected mice. FINDINGS: M. tuberculosis infection in mice stimulated immune cell infiltration in visceral, and brown adipose tissue. Despite the absence of detectable bacterial dissemination to fat tissues, adipocytes produced localized pro-inflammatory signals that disrupted adipocyte lipid metabolism, resulting in adipocyte hypertrophy. Paradoxically, this resulted in increased insulin sensitivity and systemic glucose tolerance. Adipose tissue inflammation and enhanced glucose tolerance also developed in obese mice after aerosol M. tuberculosis infection. We found that infection induced adipose tissue Akt signaling, while inhibition of the Akt activator mTORC2 in adipocytes reversed TB-associated adipose tissue inflammation and cell hypertrophy. INTERPRETATION: Our study reveals a systemic response to aerosol M. tuberculosis infection that regulates adipose tissue lipid homeostasis through mTORC2/Akt signaling in adipocytes. Adipose tissue inflammation in TB is not simply a passive infiltration with leukocytes but requires the mechanistic participation of adipocyte signals.

Persistent inflammation during anti-tuberculosis treatment with diabetes comorbidity.

Diabetes mellitus (DM) increases risk for pulmonary tuberculosis (TB) and adverse treatment outcomes. Systemic hyper-inflammation is characteristic in people with TB and concurrent DM (TBDM) at baseline, but the impact of TB treatment on this pattern has not been determined. We measured 17 plasma cytokines and growth factors in longitudinal cohorts of Indian and Brazilian pulmonary TB patients with or without DM. Principal component analysis revealed virtually complete separation of TBDM from TB individuals in both cohorts at baseline, with hyper-inflammation in TBDM that continued through treatment completion at six months. By one year after treatment completion, there was substantial convergence of mediator levels between groups within the India cohort. Non-resolving systemic inflammation in TBDM comorbidity could reflect delayed lesion sterilization or non-resolving sterile inflammation. Either mechanism portends unfavorable long-term outcomes including risk for recurrent TB and for damaging immune pathology.

Systems Immunology of Diabetes-Tuberculosis Comorbidity Reveals Signatures of Disease Complications.

Comorbid diabetes mellitus (DM) increases tuberculosis (TB) risk and adverse outcomes but the pathological interactions between DM and TB remain incompletely understood. We performed an integrative analysis of whole blood gene expression and plasma analytes, comparing South Indian TB patients with and without DM to diabetic and non-diabetic controls without TB. Luminex assay of plasma cytokines and growth factors delineated a distinct biosignature in comorbid TBDM in this cohort. Transcriptional profiling revealed elements in common with published TB signatures from cohorts that excluded DM. Neutrophil count correlated with the molecular degree of perturbation, especially in TBDM patients. Body mass index and HDL cholesterol were negatively correlated with molecular degree of perturbation. Diabetic complication pathways including several pathways linked to epigenetic reprogramming were activated in TBDM above levels observed with DM alone. Our data provide a rationale for trials of host-directed therapies in TBDM, targeting neutrophilic inflammation and diabetic complication pathways to address the greater morbidity and mortality associated with this increasingly prevalent dual burden of communicable and non-communicable diseases.

Host sirtuin 1 regulates mycobacterial immunopathogenesis and represents a therapeutic target against tuberculosis.

Mycobacterium tuberculosis (Mtb) executes a plethora of immune-evasive mechanisms, which contribute to its pathogenesis, limited efficacy of current therapy, and the emergence of drug-resistant strains. This has led to resurgence in attempts to develop new therapeutic strategies/targets against tuberculosis (TB). We show that Mtb down-regulates sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent deacetylase, in monocytes/macrophages, TB animal models, and TB patients with active disease. Activation of SIRT1 reduced intracellular growth of drug-susceptible and drug-resistant strains of Mtb and induced phagosome-lysosome fusion and autophagy in a SIRT1-dependent manner. SIRT1 activation dampened Mtb-mediated persistent inflammatory responses via deacetylation of RelA/p65, leading to impaired binding of RelA/p65 on the promoter of inflammatory genes. In Mtb-infected mice, the use of SIRT1 activators ameliorated lung pathology, reduced chronic inflammation, and enhanced efficacy of anti-TB drug. Mass cytometry-based high-dimensional analysis revealed that SIRT1 activation mediated modulation of lung myeloid cells in Mtb-infected mice. Myeloid cell-specific SIRT1 knockout mice display increased inflammatory responses and susceptibility to Mtb infection. Collectively, these results provide a link between SIRT1 activation and TB pathogenesis and indicate a potential of SIRT1 activators in designing an effective and clinically relevant host-directed therapies for TB.

Defects in early cell recruitment contribute to the increased susceptibility to respiratory Klebsiella pneumoniae infection in diabetic mice.

Diabetes is associated with increased susceptibility to Klebsiella pneumoniae and poor prognosis with infection. We demonstrate accelerated mortality in mice with streptozotocin-induced diabetes following tracheal instillation of K. pneumoniae. Diabetic mice recruited fewer granulocytes to the alveolar airspace and had reduced early production of CXCL1, CXCL2, IL-1ß and TNF-α following tracheal instillation of K. pneumoniae-lipopolysaccharide. Additionally, TLR2 and TIRAP expression following K. pneumoniae-lipopolysaccharide exposure was decreased in hyperglycemic mice. These findings indicate that impaired innate sensing and failure to rapidly recruit granulocytes to the site of infection is a mechanism for diabetic susceptibility to respiratory K. pneumoniae infection.

High Prevalence and Heterogeneity of Diabetes in Patients With TB in South India: A Report from the Effects of Diabetes on Tuberculosis Severity (EDOTS) Study.

BACKGROUND: Previous studies reported an association of diabetes mellitus (DM) with TB susceptibility. Many studies were retrospective, had weak diagnostic criteria for DM, and did not assess other comorbidities. The Effects of Diabetes on Tuberculosis Severity (EDOTS) study is addressing these limitations with a longitudinal comparison of patients with TB who are classified as diabetic or normoglycemic according to World Health Organization criteria. We report interim findings after enrolling 159 of a planned 300 subjects. METHODS: A cohort study of patients with TB in South India with DM or normoglycemia defined by oral glucose tolerance test (OGTT) and fasting glucose. Glycohemoglobin (HbA1c), serum creatinine, lipids, and 25-hydroxyvitamin D were measured at enrollment. Patients were monitored monthly during TB treatment, and HbA1c measurement was repeated after 3 months. RESULTS: Of 209 eligible patients, 113 (54.1%) were classified as diabetic, 44 (21.0%) with impaired glucose tolerance, and 52 (24.9%) as normoglycemic. More patients with diabetes were detected by OGTT than by HbA1c. Diabetes was a newly received diagnosis for 37 (32.7%) in the DM group, and their median HbA1c (6.8%) was significantly lower than in those with previously diagnosed DM (HbA1c, 10.4%). Among 129 patients monitored for 3 months, HbA1c declined in all groups, with the greatest difference in patients with a newly received diagnosis of DM. CONCLUSIONS: Early EDOTS study results reveal a strikingly high prevalence of glycemic disorders in South Indian patients with pulmonary TB and unexpected heterogeneity within the patient population with diabetes and TB. This glycemic control heterogeneity has implications for the TB-DM interaction and the interpretation of TB studies relying exclusively on HbA1c to define diabetic status.

Recombinant adenovirus vector vaccine induces stronger cytotoxic T-cell responses than recombinant vaccinia virus vector, plasmid DNA, or a combination of these.

The efficiency of prime-boost vaccinations on the induction of T-cell responses to Sin Nombre virus nucleocapsid protein expressed by recombinant vaccinia virus, replication-deficient adenovirus, and plasmid DNA in mice was quantitated by the number of epitope-specific interferon-gamma-producing T cells and cytotoxic T-lymphocyte activity induced. In prime-boost immunizations, all combinations that included the recombinant adenovirus induced a much higher number of epitope-specific interferon-gamma-producing T cells than did other combinations. A single immunization of the recombinant adenovirus was able to induce similarly high levels of epitope-specific interferon-gamma-producing cells, despite the fact that the recombinant adenovirus produces less amount of the Sin Nombre virus nucleocapsid protein.

Differential adjuvant activities of TLR7 and TLR9 agonists inversely correlate with nitric oxide and PGE2 production.

Activation of different pattern recognition receptors causes distinct profiles of innate immune responses, which in turn dictate the adaptive immune response. We found that mice had higher CD4+ T cell expansion to an immunogen, ovalbumin, when coadministered with CpG than with CL097 in vivo. To account for this differential adjuvanticity, we assessed the activities of CpG and CL097 on antigen-specific CD4+ T cell expansion in vitro using an OT-II CD4+ T cell/bone marrow-derived dendritic cell (DC) co-culture system. Unexpectedly, ovalbumin-stimulated expansion of OT-II CD4+ T cells in vitro was potently suppressed by both TLR agonists, with CL097 being stronger than CpG. The suppression was synergistically reversed by co-inhibition of cyclooxygenases 1 and 2, and inducible nitric oxide (NO) synthase. In addition, stimulation of OT-II CD4+ T cell/DC cultures with CL097 induced higher levels of CD4+ T cell death than stimulation with CpG, and this CD4+ T cell turnover was reversed by NO and PGE2 inhibition. Consistently, the co-cultures stimulated with CL097 produced higher levels of prostaglandin E2 (PGE2) and NO than stimulation with CpG. CL097 induced higher PGE2 production in DC cultures and higher IFN-γ in the OT-II CD4+ T cell/DC cultures, accounting for the high levels of PGE2 and NO. This study demonstrates that the adjuvant activities of immunostimulatory molecules may be determined by differential induction of negative regulators, including NO and PGE2 suppressing clonal expansion and promoting cell death of CD4+ T cells.

Bacillary replication and macrophage necrosis are determinants of neutrophil recruitment in tuberculosis.

We previously determined that burst size necrosis is the chief mode of mononuclear cell death in the lungs of mice with tuberculosis. The present study explored the link between infection-induced necrosis of mononuclear phagocytes and neutrophil accumulation in the lungs of mice challenged with one of four Mycobacterium tuberculosis strains of increasing virulence (RvΔphoPR mutant, H37Ra, H37Rv and Erdman). At all time points studied, Erdman produced the highest bacterial load and the highest proportion and number of M. tuberculosis-infected neutrophils. These parameters, and the proportion of TUNEL-positive cells, tracked with virulence across all strains tested. Differences in neutrophil infection were not reflected by levels of chemoattractant cytokines in bronchoalveolar lavage fluid, while interferon-γ (reported to suppress neutrophil trafficking to the lung in tuberculosis) was highest in Erdman-infected mice. Treating Erdman-infected mice with ethambutol decreased the proportion of mononuclear phagocytes with high bacterial burden and the ratio of infected neutrophils to infected mononuclear cells in a dose-dependent manner. We propose that faster replicating M. tuberculosis strains cause more necrosis which in turn promotes neutrophil recruitment. Neutrophils infected with M. tuberculosis constitute a biomarker for poorly controlled bacterial replication, infection-induced mononuclear cell death, and increased severity of immune pathology in tuberculosis.