Trp'ing tuberculosis.

IFN-γ-activated macrophages attempt to limit pathogen growth through tryptophan starvation. Zhang et al. demonstrate that Mycobacterium tuberculosis responds to this threat by synthesizing its own tryptophan. Genetic and chemical inhibition of this pathway reduces mycobacterial survival, revealing a new immune-dependent drug target for tuberculosis.

ARL5b inhibits human rhinovirus 16 propagation and impairs macrophage-mediated bacterial clearance.

Human rhinovirus is the most frequently isolated virus during severe exacerbations of chronic respiratory diseases, like chronic obstructive pulmonary disease. In this disease, alveolar macrophages display significantly diminished phagocytic functions that could be associated with bacterial superinfections. However, how human rhinovirus affects the functions of macrophages is largely unknown. Macrophages treated with HRV16 demonstrate deficient bacteria-killing activity, impaired phagolysosome biogenesis, and altered intracellular compartments. Using RNA sequencing, we identify the small GTPase ARL5b to be upregulated by the virus in primary human macrophages. Importantly, depletion of ARL5b rescues bacterial clearance and localization of endosomal markers in macrophages upon HRV16 exposure. In permissive cells, depletion of ARL5b increases the secretion of HRV16 virions. Thus, we identify ARL5b as a novel regulator of intracellular trafficking dynamics and phagolysosomal biogenesis in macrophages and as a restriction factor of HRV16 in permissive cells.

Triggering MSR1 promotes JNK-mediated inflammation in IL-4-activated macrophages.

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4-activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4-activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization.

The ability of Interleukin-10 to negate haemozoin-related pro-inflammatory effects has the potential to restore impaired macrophage function associated with malaria infection.

BACKGROUND: Although pro-inflammatory cytokines are involved in the clearance of Plasmodium falciparum during the early stages of the infection, increased levels of these cytokines have been implicated in the pathogenesis of severe malaria. Amongst various parasite-derived inducers of inflammation, the malarial pigment haemozoin (Hz), which accumulates in monocytes, macrophages and other immune cells during infection, has been shown to significantly contribute to dysregulation of the normal inflammatory cascades. METHODS: The direct effect of Hz-loading on cytokine production by monocytes and the indirect effect of Hz on cytokine production by myeloid cells was investigated during acute malaria and convalescence using archived plasma samples from studies investigating P. falciparum malaria pathogenesis in Malawian subjects. Further, the possible inhibitory effect of IL-10 on Hz-loaded cells was examined, and the proportion of cytokine-producing T-cells and monocytes during acute malaria and in convalescence was characterized. RESULTS: Hz contributed towards an increase in the production of inflammatory cytokines, such as Interferon Gamma (IFN-γ), Tumor Necrosis Factor (TNF) and Interleukin 2 (IL-2) by various cells. In contrast, the cytokine IL-10 was observed to have a dose-dependent suppressive effect on the production of TNF among other cytokines. Cerebral malaria (CM) was characterized by impaired monocyte functions, which normalized in convalescence. CM was also characterized by reduced levels of IFN-γ-producing T cell subsets, and reduced expression of immune recognition receptors HLA-DR and CD 86, which also normalized in convalescence. However, CM and other clinical malaria groups were characterized by significantly higher plasma levels of pro-inflammatory cytokines than healthy controls, implicating anti-inflammatory cytokines in balancing the immune response. CONCLUSIONS: Acute CM was characterized by elevated plasma levels of pro-inflammatory cytokines and chemokines but lower proportions of cytokine-producing T-cells and monocytes that normalize during convalescence. IL-10 is also shown to have the potential to indirectly prevent excessive inflammation. Cytokine production dysregulated by the accumulation of Hz appears to impair the balance of the immune response to malaria and exacerbates pathology.

Inhibition of the lncRNA SAF drives activation of apoptotic effector caspases in HIV-1-infected human macrophages.

Long noncoding RNAs (lncRNAs) impart significant regulatory functions in a diverse array of biological pathways and manipulation of these RNAs provides an important avenue to modulate such pathways, particularly in disease. Our knowledge about lncRNAs' role in determination of cellular fate during HIV-1 infection remains sparse. Here, we have identified the impact of the lncRNA SAF in regulating apoptotic effector caspases in macrophages, a long-lived cellular reservoir of HIV-1, that are largely immune to virus-induced cell death. Expression of SAF is significantly up-regulated in HIV-1-infected human monocyte-derived macrophages (MDM) compared with bystander and virus-nonexposed cells. A similar enhancement in SAF RNA expression is also detected in the HIV-1-infected airway macrophages obtained by bronchoalveolar lavage of HIV-1-infected individuals. Down-regulation of SAF with siRNA treatment increases caspase-3/7 activity levels in virus-infected MDMs. This induction of apoptotic caspases occurs exclusively in HIV-1-infected macrophages and not in bystander cells, leading to a significant reduction in HIV-1 replication and overall viral burden in the macrophage culture. This study identifies targeting of the lncRNA SAF as a potential means to specifically induce cell death in HIV-1-infected macrophages.

Parental Enrollment Decision-Making for a Neonatal Clinical Trial.

OBJECTIVE: To describe the parental experience of recruitment and assess differences between parents who participated and those who declined to enroll in a neonatal clinical trial. STUDY DESIGN: This was a survey conducted at 12 US neonatal intensive care units of parents of infants who enrolled in the High-dose Erythropoietin for Asphyxia and encephaLopathy (HEAL) trial or who were eligible but declined enrollment. Questions assessed 6 factors of the parental experience of recruitment: (1) interactions with research staff; (2) the consent experience; (3) perceptions of the study; (4) decisional conflict; (5) reasons for/against participation; and (6) timing of making the enrollment decision. RESULTS: In total, 269 of 387 eligible parents, including 183 of 242 (75.6%) of those who enrolled their children in HEAL and 86 of 145 (59.3%) parents who declined to enroll their children in HEAL, were included in analysis. Parents who declined to enroll more preferred to be approached by clinical team members rather than by research team members (72.9% vs 49.2%, P = .005). Enrolled parents more frequently reported positive initial impressions (54.9% vs 10.5%, P < .001). Many parents in both groups made their decision early in the recruitment process. Considerations of reasons for/against participation differed by enrollment status. CONCLUSIONS: Understanding how parents experience recruitment, and how this differs by enrollment status, may help researchers improve recruitment processes for families and increase enrollment. The parental experience of recruitment varied by enrollment status. These findings can guide future work aiming to inform optimal recruitment strategies for neonatal clinical trials.

Foamy macrophages and the progression of the human tuberculosis granuloma.

The progression of tuberculosis from a latent, subclinical infection to active disease that culminates in the transmission of infectious bacilli is determined locally at the level of the granuloma. This progression takes place even in the face of a robust immune response that, although it contains infection, is unable to eliminate the bacterium. The factors or environmental conditions that influence this progression remain to be determined. Recent advances have indicated that pathogen-induced dysregulation of host lipid synthesis and sequestration serves a critical role in this transition. The foamy macrophage seems to be a key participant in both sustaining persistent bacteria and contributing to the tissue pathology that leads to cavitation and the release of infectious bacilli.

Exploitation of Synthetic mRNA To Drive Immune Effector Cell Recruitment and Functional Reprogramming In Vivo.

Therapeutic strategies based on in vitro-transcribed mRNA (IVT) are attractive because they avoid the permanent signature of genomic integration that is associated with DNA-based therapy and result in the transient production of proteins of interest. To date, IVT has mainly been used in vaccination protocols to generate immune responses to foreign Ags. In this "proof-of-principle" study, we explore a strategy of combinatorial IVT to recruit and reprogram immune effector cells to acquire divergent biological functions in mice in vivo. First, we demonstrate that synthetic mRNA encoding CCL3 is able to recruit murine monocytes in a nonprogrammed state, exhibiting neither bactericidal nor tissue-repairing properties. However, upon addition of either Ifn-γ mRNA or Il-4 mRNA, we successfully polarized these cells to adopt either M1 or M2 macrophage activation phenotypes. This cellular reprogramming was demonstrated through increased expression of known surface markers and through the differential modulation of NADPH oxidase activity, or the superoxide burst. Our study demonstrates how IVT strategies can be combined to recruit and reprogram immune effector cells that have the capacity to fulfill complex biological tasks in vivo.

Matrix metalloproteinase inhibitors enhance the efficacy of frontline drugs against Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) remains a grave threat to world health with emerging drug resistant strains. One prominent feature of Mtb infection is the extensive reprogramming of host tissue at the site of infection. Here we report that inhibition of matrix metalloproteinase (MMP) activity by a panel of small molecule inhibitors enhances the in vivo potency of the frontline TB drugs isoniazid (INH) and rifampicin (RIF). Inhibition of MMP activity leads to an increase in pericyte-covered blood vessel numbers and appears to stabilize the integrity of the infected lung tissue. In treated mice, we observe an increased delivery and/or retention of frontline TB drugs in the infected lungs, resulting in enhanced drug efficacy. These findings indicate that targeting Mtb-induced host tissue remodeling can increase therapeutic efficacy and could enhance the effectiveness of current drug regimens.

Cellular Microbiology: The metabolic interface between host cell and pathogen.

Cellular Microbiology has benefited greatly from the use of immortalized cell lines as host cells for tissue culture models of infection. However, these cells lack many important characteristics of the different cell lineages that are found in vivo. This deficiency is particularly true of macrophages that we now know derive from several distinct ontogenic lineages. This perspective discusses these challenges and possible approaches to overcome them.

Correction: Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.

[This corrects the article DOI: 10.1371/journal.ppat.1006389.].

Mycobacterium tuberculosis arrests host cycle at the G1/S transition to establish long term infection.

Signals modulating the production of Mycobacterium tuberculosis (Mtb) virulence factors essential for establishing long-term persistent infection are unknown. The WhiB3 redox regulator is known to regulate the production of Mtb virulence factors, however the mechanisms of this modulation are unknown. To advance our understanding of the mechanisms involved in WhiB3 regulation, we performed Mtb in vitro, intraphagosomal and infected host expression analyses. Our Mtb expression analyses in conjunction with extracellular flux analyses demonstrated that WhiB3 maintains bioenergetic homeostasis in response to available carbon sources found in vivo to establish Mtb infection. Our infected host expression analysis indicated that WhiB3 is involved in regulation of the host cell cycle. Detailed cell-cycle analysis revealed that Mtb infection inhibited the macrophage G1/S transition, and polyketides under WhiB3 control arrested the macrophages in the G0-G1 phase. Notably, infection with the Mtb whiB3 mutant or polyketide mutants had little effect on the macrophage cell cycle and emulated the uninfected cells. This suggests that polyketides regulated by Mtb WhiB3 are responsible for the cell cycle arrest observed in macrophages infected with the wild type Mtb. Thus, our findings demonstrate that Mtb WhiB3 maintains bioenergetic homeostasis to produce polyketide and lipid cyclomodulins that target the host cell cycle. This is a new mechanism whereby Mtb modulates the immune system by altering the host cell cycle to promote long-term persistence. This new knowledge could serve as the foundation for new host-directed therapeutic discovery efforts that target the host cell cycle.

Trans-species communication in the Mycobacterium tuberculosis-infected macrophage.

Much of the infection cycle of Mycobacterium tuberculosis (Mtb) is spent within its host cell, the macrophage. As a consequence of the chronic, enduring nature of the infection, this cell-cell interaction has become highly intimate, and the bacterium has evolved to detect, react to, and manipulate the evolving, immune-modulated phenotype of its host. In this review, we discuss the nature of the endosomal/lysosomal continuum, the characterization of the bacterium's transcriptional responses during the infection cycle, and the dominant environmental cues that shape this response. We also discuss how the metabolism of both cells is modulated by the infection and the impact that this has on the progression of the granuloma. Finally, we detail how these transcriptional responses can be exploited to construct reporter bacterial strains to probe the temporal and spatial environmental shifts experienced by Mtb during the course of experimental infections. These reporter strains provide new insights into the fitness of Mtb under immune- and drug-mediated pressure.

Novel inhibitors of cholesterol degradation in Mycobacterium tuberculosis reveal how the bacterium's metabolism is constrained by the intracellular environment.

Mycobacterium tuberculosis (Mtb) relies on a specialized set of metabolic pathways to support growth in macrophages. By conducting an extensive, unbiased chemical screen to identify small molecules that inhibit Mtb metabolism within macrophages, we identified a significant number of novel compounds that limit Mtb growth in macrophages and in medium containing cholesterol as the principle carbon source. Based on this observation, we developed a chemical-rescue strategy to identify compounds that target metabolic enzymes involved in cholesterol metabolism. This approach identified two compounds that inhibit the HsaAB enzyme complex, which is required for complete degradation of the cholesterol A/B rings. The strategy also identified an inhibitor of PrpC, the 2-methylcitrate synthase, which is required for assimilation of cholesterol-derived propionyl-CoA into the TCA cycle. These chemical probes represent new classes of inhibitors with novel modes of action, and target metabolic pathways required to support growth of Mtb in its host cell. The screen also revealed a structurally-diverse set of compounds that target additional stage(s) of cholesterol utilization. Mutants resistant to this class of compounds are defective in the bacterial adenylate cyclase Rv1625/Cya. These data implicate cyclic-AMP (cAMP) in regulating cholesterol utilization in Mtb, and are consistent with published reports indicating that propionate metabolism is regulated by cAMP levels. Intriguingly, reversal of the cholesterol-dependent growth inhibition caused by this subset of compounds could be achieved by supplementing the media with acetate, but not with glucose, indicating that Mtb is subject to a unique form of metabolic constraint induced by the presence of cholesterol.

The ins and outs of the Mycobacterium tuberculosis-containing vacuole.

The past few years have seen publication of reports from several groups documenting the escape of Mycobacterium tuberculosis (Mtb) from its intracellular vacuole to access the cytosol. The major questions addressed in these publications are the mechanism(s) underlying this process, the frequency of its occurrence and, most importantly, the biological significance of this phenomenon to bacterial survival, growth and virulence. I believe that the first two questions are moving towards resolution, but questions relating to biological context have yet to be answered fully. In this viewpoint article, I will try to convince the readers why escape from the vacuole in no way diminishes the significance of Mtb's intravacuolar survival mechanisms and why, as a lab, we continue to focus the majority of our efforts on the 'bug in the bag'.

2-N-Arylthiazole inhibitors of Mycobacterium tuberculosis.

To develop agents for the treatment of infections caused by Mycobacterium tuberculosis, a novel phenotypic screen was undertaken that identified a series of 2-N-aryl thiazole-based inhibitors of intracellular Mycobacterium tuberculosis. Analogs were optimized to improve potency against an attenuated BSL2 H37Ra laboratory strain cultivated in human macrophage cells in vitro. The insertion of a carboxylic acid functionality resulted in compounds that retained potency and greatly improved microsomal stability. However, the strong potency trends we observed in the attenuated H37Ra strain were inconsistent with the potency observed for virulent strains in vitro and in vivo.

Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses.

8E5/LAV cells harbor a single HIV provirus, and are used frequently to generate standards for HIV genome quantification. Using flow cytometry-based in situ mRNA hybridization validated by qPCR, we find that different batches of 8E5 cells contain varying numbers of cells lacking viral mRNA and/or viral genomes. These findings raise concerns for studies employing 8E5 cells for quantitation, and highlight the value of mRNA FISH and flow cytometry in the detection and enumeration of HIV-positive cells.

Exploitation of Mycobacterium tuberculosis reporter strains to probe the impact of vaccination at sites of infection.

Mycobacterium tuberculosis (Mtb) remains a major public health problem, with an effective vaccine continuing to prove elusive. Progress in vaccination strategies has been hampered by a lack of appreciation of the bacterium's response to dynamic changes in the host immune environment. Here, we utilize reporter Mtb strains that respond to specific host immune stresses such as hypoxia and nitric oxide (hspX'::GFP), and phagosomal maturation (rv2390c'::GFP), to investigate vaccine-induced alterations in the environmental niche during experimental murine infections. While vaccination undoubtedly decreased bacterial burden, we found that it also appeared to accelerate Mtb's adoption of a phenotype better equipped to survive in its host. We subsequently utilized a novel replication reporter strain of Mtb to demonstrate that, in addition to these alterations in host stress response, there is a decreased percentage of actively replicating Mtb in vaccinated hosts. This observation was supported by the differential sensitivity of recovered bacteria to the front-line drug isoniazid. Our study documents the natural history of the impact that vaccination has on Mtb's physiology and replication and highlights the value of reporter Mtb strains for probing heterogeneous Mtb populations in the context of a complex, whole animal model.