BCG Provides Short-Term Protection from Experimental Cerebral Malaria in Mice.

Clinical and experimental evidence suggests that the tuberculosis vaccine BCG offers protection against unrelated pathogens including the malaria parasite. Cerebral malaria (CM) is the most severe complication associated with Plasmodium falciparum infection in humans and is responsible for most of the fatalities attributed to malaria. We investigated whether BCG protected C57BL/6 mice from P. berghei ANKA (PbA)-induced experimental CM (ECM). The majority of PbA-infected mice that were immunized with BCG showed prolonged survival without developing clinical symptoms of ECM. However, this protective effect waned over time and was associated with the recovery of viable BCG from liver and spleen. Intriguingly, BCG-mediated protection from ECM was not associated with a reduction in parasite burden, indicating that BCG immunization did not improve anti-parasite effector mechanisms. Instead, we found a significant reduction in pro-inflammatory mediators and CD8+ T cells in brains of BCG-vaccinated mice. Together these data suggest that brain recruitment of immune cells involved in the pathogenesis of ECM decreased after BCG vaccination. Understanding the mechanisms underlying the protective effects of BCG on PbA-induced ECM can provide a rationale for developing effective adjunctive therapies to reduce the risk of death and brain damage in CM.

Increased male susceptibility to Mycobacterium tuberculosis infection is associated with smaller B cell follicles in the lungs.

Tuberculosis prevalence is significantly higher among men than women. We have previously revealed an increased susceptibility of male C57BL/6 mice towards Mycobacterium tuberculosis (Mtb) H37Rv. In the current study, we confirm the male bias for infection with the Beijing strain HN878. Males succumbed to HN878 infection significantly earlier than females. In both models, premature death of males was associated with smaller B cell follicles in the lungs. Analysis of homeostatic chemokines and their receptors revealed differences between H37Rv and HN878 infected animals, indicating different immune requirements for follicle formation in both models. However, expression of IL-23, which is involved in long-term containment of Mtb and lymphoid follicle formation, was reduced in male compared to female lungs in both models. Our study reveals sex differences in the formation of B cell follicles in the Mtb infected lung and we propose that impaired follicle formation is responsible for accelerated disease progression in males.

Blocking IL-10 receptor signaling ameliorates Mycobacterium tuberculosis infection during influenza-induced exacerbation.

Epidemiological findings indicate that coinfection with influenza viruses is associated with an increased risk of death in patients suffering from tuberculosis but the underlying pathomechanisms are not well understood. In this study, we demonstrate that influenza A virus (IAV) coinfection rapidly impairs control of Mycobacterium tuberculosis (Mtb) in C57BL/6 mice. IAV coinfection was associated with significantly increased bacterial loads, reduced survival and a substantial modulation of innate and adaptive immune defenses including an impaired onset and development of Mtb-specific CD4+ T cell responses and the accumulation of macrophages with increased arginase-1 production in the lungs. Our findings strongly indicate that IAV coinfection compromises the host's ability to control Mtb infection via the production of IL-10 which was rapidly induced upon viral infection. The blockade of IL-10 receptor signaling reduced the bacterial load in coinfected mice to a level comparable with that in Mtb-only-infected animals. Taken together, our data suggest that IL-10 signaling constitutes a major pathway that enhances susceptibility to Mtb during concurrent IAV infection.

Author Correction: Sex differences in the C57BL/6 model of Mycobacterium tuberculosis infection.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Sex differences in the C57BL/6 model of Mycobacterium tuberculosis infection.

Globally, tuberculosis (Tb) notification data show a male-to-female ratio of 1.7 and higher, but the underlying reasons for the male bias remain elusive. Despite the well-known gender bias in human pulmonary Tb, a majority of experimental animal studies either do not separate and analyze data by sex or do not report the sex of their subjects at all. In the present study, we report increased male susceptibility in one of the most commonly used mouse models for Tb, C57BL/6 mice. Our study revealed that disease progression upon aerosol infection with Mycobacterium tuberculosis (Mtb) was accelerated in males resulting in increased morbidity and mortality compared to females. Elevated Mtb loads in males were associated with an early exaggerated pulmonary inflammatory response which likely was detrimental to the host, as reflected by exacerbated pathology and increased mortality. Our data emphasis the urgent need to include and separately analyze both sexes in future animal studies of Tb in order to appreciate the differences in immune responses and disease pathogenesis between males and females.

One Episode of Self-Resolving Plasmodium yoelii Infection Transiently Exacerbates Chronic Mycobacterium tuberculosis Infection.

Malaria and tuberculosis (Tb) are two of the main causes of death from infectious diseases globally. The pathogenic agents, Plasmodium parasites and Mycobacterium tuberculosis, are co-endemic in many regions in the world, however, compared to other co-infections like HIV/Tb or helminth/Tb, malaria/Tb has been given less attention both in clinical and immunological studies. Due to the lack of sufficient human data, the impact of malaria on Tb and vice versa is difficult to estimate but co-infections are likely to occur very frequently. Due to its immunomodulatory properties malaria might be an underestimated risk factor for latent or active Tb patients particularly in high-endemic malaria settings were people experience reinfections very frequently. In the present study, we used the non-lethal strain of Plasmodium yoelii to investigate, how one episode of self-resolving malaria impact on a chronic M. tuberculosis infection. P. yoelii co-infection resulted in exacerbation of Tb disease as demonstrated by increased pathology and cellular infiltration of the lungs which coincided with elevated levels of pro- and anti-inflammatory mediators. T cell responses were not impaired in co-infected mice but enhanced and likely contributed to increased cytokine production. We found a slight but statistically significant increase in M. tuberculosis burden in co-infected animals and increased lung CFU was positively correlated with elevated levels of TNFα but not IL-10. Infection with P. yoelii induced the recruitment of a CD11c(+) population into lungs and spleens of M. tuberculosis infected mice. CD11c(+) cells isolated from P. yoelii infected spleens promoted survival and growth of M. tuberculosis in vitro. 170 days after P. yoelii infection changes in immunopathology and cellular immune responses were no longer apparent while M. tuberculosis numbers were still slightly higher in lungs, but not in spleens of co-infected mice. In conclusion, one episode of P. yoelii co-infection transiently exacerbated disease severity but had no long-term consequences on disease progression and survival of M. tuberculosis infected mice.

Mycobacterium tuberculosis Coinfection Has No Impact on Plasmodium berghei ANKA-Induced Experimental Cerebral Malaria in C57BL/6 Mice.

Cerebral malaria (CM) is the most severe complication of human infection with Plasmodium falciparum. The mechanisms predisposing to CM are still not fully understood. Proinflammatory immune responses are required for the control of blood-stage malaria infection but are also implicated in the pathogenesis of CM. A fine balance between pro- and anti-inflammatory immune responses is required for parasite clearance without the induction of host pathology. The most accepted experimental model to study human CM is Plasmodium berghei ANKA (PbANKA) infection in C57BL/6 mice that leads to the development of a complex neurological syndrome which shares many characteristics with the human disease. We applied this model to study the outcome of PbANKA infection in mice previously infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Tuberculosis is coendemic with malaria in large regions in the tropics, and mycobacteria have been reported to confer some degree of unspecific protection against rodent Plasmodium parasites in experimental coinfection models. We found that concomitant M. tuberculosis infection did not change the clinical course of PbANKA-induced experimental cerebral malaria (ECM) in C57BL/6 mice. The immunological environments in spleen and brain did not differ between singly infected and coinfected animals; instead, the overall cytokine and T cell responses in coinfected mice were comparable to those in animals solely infected with PbANKA. Our data suggest that M. tuberculosis coinfection is not able to change the outcome of PbANKA-induced disease, most likely because the inflammatory response induced by the parasite rapidly dominates in mice previously infected with M. tuberculosis.

Lysosomal phospholipase A2: a novel player in host immunity to Mycobacterium tuberculosis.

Phospholipases catalyze the cleavage of membrane phospholipids into smaller bioactive molecules. The lysosomal phospholipase A2 (LPLA2 ) is specifically expressed in macrophages. LPLA2 gene deletion in mice causes lysosomal phospholipid accumulation in tissue macrophages leading to phospholipidosis. This phenotype becomes most prominent in alveolar macrophages where LPLA2 contributes to surfactant phospholipid degradation. High expression of LPLA2 in alveolar macrophages prompted us to investigate its role in host immunity against the respiratory pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis. Here we report that adaptive immune responses to M. tuberculosis were impaired in LPLA2 deficient mice. Upon aerosol infection with M. tuberculosis, LPLA2 deficient mice showed enhanced mycobacterial counts but less lung immunopathology and pulmonary inflammatory responses. Compromised T-cell priming in the lymph nodes was associated with impaired pulmonary T-cell recruitment and activation. Together with reduced Th1 type cytokine production, these results indicate that LPLA2 is indispensable for the induction of adaptive T-cell immunity to M. tuberculosis. Taken together, we identified an unexpected and novel function of a lysosomal phospholipid-degrading enzyme.

An experimental model to study tuberculosis-malaria coinfection upon natural transmission of Mycobacterium tuberculosis and Plasmodium berghei.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

Natural transmission of Plasmodium berghei exacerbates chronic tuberculosis in an experimental co-infection model.

Human populations are rarely exposed to one pathogen alone. Particularly in high incidence regions such as sub-Saharan Africa, concurrent infections with more than one pathogen represent a widely underappreciated public health problem. Two of the world's most notorious killers, malaria and tuberculosis, are co-endemic in impoverished populations in the tropics. However, interactions between both infections in a co-infected individual have not been studied in detail. Both pathogens have a major impact on the lung as the prime target organ for aerogenic Mycobacterium tuberculosis and the site for one of the main complications in severe malaria, malaria-associated acute respiratory distress syndrome (MA-ARDS). In order to study the ramifications caused by both infections within the same host we established an experimental mouse model of co-infection between Mycobacterium tuberculosis and Plasmodium berghei NK65, a recently described model for MA-ARDS. Our study provides evidence that malaria-induced immune responses impair host resistance to Mycobacterium tuberculosis. Using the natural routes of infection, we observed that co-infection exacerbated chronic tuberculosis while rendering mice less refractory to Plasmodium. Co-infected animals presented with enhanced inflammatory immune responses as reflected by exacerbated leukocyte infiltrates, tissue pathology and hypercytokinemia accompanied by altered T-cell responses. Our results--demonstrating striking changes in the immune regulation by co-infection with Plasmodium and Mycobacterium--are highly relevant for the medical management of both infections in humans.

Interferon gamma activated macrophages kill mycobacteria by nitric oxide induced apoptosis.

Mycobacterium tuberculosis is an intracellular pathogen of macrophages and escapes the macrophages' bactericidal effectors by interfering with phagosome-lysosome fusion. IFN-γ activation renders the macrophages capable of killing intracellular mycobacteria by overcoming the phagosome maturation block, nutrient deprivation and exposure to microbicidal effectors including nitric oxide (NO). While the importance about NO for the control of mycobacterial infection in murine macrophages is well documented, the underlying mechanism has not been revealed yet. In this study we show that IFN-γ induced apoptosis in mycobacteria-infected macrophages, which was strictly dependent on NO. Subsequently, NO-mediated apoptosis resulted in the killing of intracellular mycobacteria independent of autophagy. In fact, killing of mycobacteria was susceptible to the autophagy inhibitor 3-methyladenine (3-MA). However, 3-MA also suppressed NO production, which is an important off-target effect to be considered in autophagy studies using 3-MA. Inhibition of caspase 3/7 activation, as well as NO production, abolished apoptosis and elimination of mycobacteria by IFN-γ activated macrophages. In line with the finding that drug-induced apoptosis kills intracellular mycobacteria in the absence of NO, we identified NO-mediated apoptosis as a new defense mechanism of activated macrophages against M. tuberculosis.

A role for IL-18 in protective immunity against Mycobacterium tuberculosis.

Tuberculosis remains the most hazardous bacterial infection worldwide. The causative agent, Mycobacterium tuberculosis, is a facultative intracellular pathogen of resting MPhi. IFN-gamma secreted by natural killer, CD4 Th 1 and CD8 T cells upon instruction by IL-12 and -18 activates MPhi to restrict mycobacterial growth. Production of both cytokines is induced by TLR signalling in DC and MPhi. Mice deficient for the TLR adaptor, MyD88, are highly susceptible to M. tuberculosis infection. Shared usage of MyD88 by signalling cascades for TLR and receptors for IL-1 and IL-18 prompted us to revisit the role of IL-18 during experimental infection with M. tuberculosis. We show that mice deficient for IL-18 and MyD88 but not for IL-18 receptor promptly succumbed to M. tuberculosis infection in contrast to WT or TLR-2/-4 double KO mice indicating that lack of IL-18 contributes to the high susceptibility of MyD88 KO mice to M. tuberculosis. Without IL-18, the protective Th1 response was decreased and hence, mycobacterial propagation was favoured. Neutrophil-driven lung immunopathology concomitant with unrestrained growth of tubercle bacilli are most likely responsible for the premature death of IL-18 KO mice. Thus, IL-18 plays a decisive role in protective immunity against tuberculosis.

Innate immunity in tuberculosis: myths and truth.

Tuberculosis is the most important bacterial infection world wide. The causative agent, Mycobacterium tuberculosis survives and proliferates within macrophages. Immune mediators such as interferon gamma (IFN-gamma) and tumour necrosis factor alpha (TNF-alpha) activate macrophages and promote bacterial killing. IFN-gamma is predominantly secreted by innate cells (mainly natural killer (NK) cells) and by T cells upon instruction by interleukin 12 (IL-12) and IL-18. These cytokines are primarily produced by dendritic cells and macrophages in response to Toll-like receptor (TLR) signalling interaction with tubercle bacilli. These signals also induce pro-inflammatory cytokines (including IL-1beta and TNF-alpha), chemokines and defensins. The inflammatory environment further recruits innate effector cells such as macrophages, polymorphonuclear neutrophils (PMN) and NK cells to the infectious foci. This eventually leads to the downstream establishment of acquired T cell immunity which appears to be protective in more than 90% of infected individuals. Robust innate immune activation is considered an essential prerequisite for protective immunity and vaccine efficacy. However, data published so far provide a muddled view of the functional importance of innate immunity in tuberculosis. Here we critically discuss certain aspects of innate immunity, namely PMN, TLRs and NK cells, as characterised in tuberculosis to date, and their contribution to protection and pathology.