Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk.

Tuberculosis remains second only to HIV/AIDS as the leading cause of mortality worldwide due to a single infectious agent. Despite chemotherapy, the global tuberculosis epidemic has intensified because of HIV co-infection, the lack of an effective vaccine and the emergence of multi-drug-resistant bacteria. Alternative host-directed strategies could be exploited to improve treatment efficacy and outcome, contain drug-resistant strains and reduce disease severity and mortality. The innate inflammatory response elicited by Mycobacterium tuberculosis (Mtb) represents a logical host target. Here we demonstrate that interleukin-1 (IL-1) confers host resistance through the induction of eicosanoids that limit excessive type I interferon (IFN) production and foster bacterial containment. We further show that, in infected mice and patients, reduced IL-1 responses and/or excessive type I IFN induction are linked to an eicosanoid imbalance associated with disease exacerbation. Host-directed immunotherapy with clinically approved drugs that augment prostaglandin E2 levels in these settings prevented acute mortality of Mtb-infected mice. Thus, IL-1 and type I IFNs represent two major counter-regulatory classes of inflammatory cytokines that control the outcome of Mtb infection and are functionally linked via eicosanoids. Our findings establish proof of concept for host-directed treatment strategies that manipulate the host eicosanoid network and represent feasible alternatives to conventional chemotherapy.

DEPRESSION MEDIATES THE RELATION OF INSOMNIA SEVERITY WITH SUICIDE RISK IN THREE CLINICAL SAMPLES OF U.S. MILITARY PERSONNEL.

BACKGROUND: A growing body of empirical research suggests insomnia severity is directly related to suicide ideation, attempts, and death in nonmilitary samples, even when controlling for depression and other suicide risk factors. Few studies have explored this relationship in U.S. military personnel. METHODS: The present study entailed secondary data analyses examining the associations of insomnia severity with suicide ideation and attempts in three clinical samples: Air Force psychiatric outpatients (n = 158), recently discharged Army psychiatric inpatients (n = 168), and Army psychiatric outpatients (n = 54). Participants completed the Beck Scale for Suicide Ideation, the Beck Depression Inventory-II or Patient Health Questionnaire-9, the Insomnia Severity Index, and the Posttraumatic Stress Disorder Checklist at baseline; two samples also completed these measures during follow-up. RESULTS: Sleep disturbance was associated with concurrent (ß's > 0.21; P's < 0.059) and prospective (ß's > 0.39; P's < 0.001) suicide ideation in all three samples. When adjusting for age, gender, depression, and posttraumatic stress, insomnia severity was no longer directly associated with suicide ideation either concurrently (ß's < 0.19; P's > 0.200) or prospectively (ß's < 0.26; P's > 0.063), but depression was (ß's > 0.22; P's < 0.012). Results of a latent difference score mediation model indicated that depression mediated the relation of insomnia severity with suicide ideation. CONCLUSIONS: Across three clinical samples of military personnel, depression explained the relationship between insomnia severity and suicide risk.

Pathogenic mycobacterium upregulates cholesterol 25-hydroxylase to promote granuloma development via foam cell formation.

Pathogenic mycobacteria orchestrate the complex cell populations known as granuloma that is the hallmark of tuberculosis. Foam cells, a lipid-rich cell-type, are considered critical for granuloma formation; however, the causative factor in foam cell formation remains unclear. Atherosclerosis is a chronic inflammatory disease characterized by the abundant accumulation of lipid-laden-macrophage-derived foam cells during which cholesterol 25-hydroxylase (CH25H) is crucial in foam cell formation. Here, we show that M. marinum (Mm), a relative of M. tuberculosis, induces foam cell formation, leading to granuloma development following CH25H upregulation. Moreover, the Mm-driven increase in CH25H expression is associated with the presence of phthiocerol dimycocerosate, a determinant for Mm virulence and integrity. CH25H-null mice showed decreased foam cell formation and attenuated pathology. Atorvastatin, a recommended first-line lipid-lowering drug, promoted the elimination of M. marinum and concomitantly reduced CH25H production. These results define a previously unknown role for CH25H in controlling macrophage-derived foam cell formation and Tuberculosis pathology.

IQGAP1 domesticates macrophages to favor mycobacteria survival via modulating NF-κB signal and augmenting VEGF secretion.

Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), still ranks among the leading causes of annual human death by infectious disease. Mtb has developed several strategies to survive for years at a time within the host despite the presence of a robust immune response, including manipulating the progression of the inflammatory response and forming granulomatous lesions. Here we demonstrate that IQGAP1, a highly conserved scaffolding protein, compartmentalizes and coordinates multiple signaling pathways in macrophages infected with Mycobacterium marinum (Mm or M.marinum), the closest relative of Mtb. Upregulated IQGAP1 ultimately suppresses TNF-α production by repressing the MKK3 signal and reducing NF-κBp65 translocation, deactivating the p38MAPK pathway. Accordingly, IQGAP1 silencing and overexpression significantly alter p38MAPK activity by modulating the production of phosphorylated MKK3 during mycobacterial infection. Pharmacological inhibition of IQGAP1-associated microtubule assembly not only alleviates tissue damage caused by M.marinum infection but also significantly decreases the production of VEGF-a critical player for granuloma-associated angiogenesis during pathogenic mycobacterial infection. Similarly, IQGAP1 silencing in Mm-infected macrophages diminishes VEGF production, while IQGAP1 overexpression upregulates VEGF. Our data indicate that mycobacteria induce IQGAP1 to hijack NF-κBp65 activation, preventing the expression of proinflammatory cytokines as well as promoting VEGF production during infection and granuloma formation. Thus, therapies targeting host IQGAP1 may be a promising strategy for treating tuberculosis, particularly in drug-resistant diseases.

Automated quantitative assay of fibrosis characteristics in tuberculosis granulomas.

Introduction: Granulomas, the pathological hallmark of Mycobacterium tuberculosis (Mtb) infection, are formed by different cell populations. Across various stages of tuberculosis conditions, most granulomas are classical caseous granulomas. They are composed of a necrotic center surrounded by multilayers of histocytes, with the outermost layer encircled by fibrosis. Although fibrosis characterizes the architecture of granulomas, little is known about the detailed parameters of fibrosis during this process. Methods: In this study, samples were collected from patients with tuberculosis (spanning 16 organ types), and Mtb-infected marmosets and fibrotic collagen were characterized by second harmonic generation (SHG)/two-photon excited fluorescence (TPEF) microscopy using a stain-free, fully automated analysis program. Results: Histopathological examination revealed that most granulomas share common features, including necrosis, solitary and compact structure, and especially the presence of multinuclear giant cells. Masson's trichrome staining showed that different granuloma types have varying degrees of fibrosis. SHG imaging uncovered a higher proportion (4%~13%) of aggregated collagens than of disseminated type collagens (2%~5%) in granulomas from matched tissues. Furthermore, most of the aggregated collagen presented as short and thick clusters (200~620 µm), unlike the long and thick (200~300 µm) disseminated collagens within the matched tissues. Matrix metalloproteinase-9, which is involved in fibrosis and granuloma formation, was strongly expressed in the granulomas in different tissues. Discussion: Our data illustrated that different tuberculosis granulomas have some degree of fibrosis in which collagen strings are short and thick. Moreover, this study revealed that the SHG imaging program could contribute to uncovering the fibrosis characteristics of tuberculosis granulomas.

E3 Ligase FBXW7 Facilitates Mycobacterium Immune Evasion by Modulating TNF-α Expression.

Tumor necrosis factor alpha (TNF-α) is a crucial factor in the control of Mycobacterium tuberculosis (Mtb) infection. Pathogenic mycobacteria can inhibit and/or regulate host cell TNF-α production in a variety of ways to evade antituberculosis (anti-TB) immunity as well as facilitate immune escape. However, the mechanisms by which TNF-α expression in host cells is modulated to the benefit of mycobacteria is still an interesting topic and needs further study. Here, we report that macrophages infected with Mycobacterium marinum (Mm)-a close relative of Mtb-upregulated the expression of E3 ubiquitin ligase FBXW7. Specific silencing FBXW7 with small interfering RNA (siRNA) significantly elevates TNF-α expression and eventually promotes the elimination of intracellular bacteria. In turn, overexpression of FBXW7 in Raw264.7 macrophages markedly decreased TNF-α production. Furthermore, partial inhibition of FBXW7 in an Mm-infected murine model significantly reduced TNF-α tissue content, alleviated tissue damage as well as reduced the bacterial load of mouse tails. Finally, FBXW7 could decrease TNF-α in a K63-linked ubiquitin signaling dependent manner. Taken together, our study uncovered a previously unknown role of FBXW7 in regulating TNF-α dynamics during mycobacterial infection, which provides new insights into understanding the role of FBXW7 in anti-tuberculosis immunity and its related clinical significance.

Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates.

Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow the targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in four animal models of disease: mouse, guinea pig, rabbit, and nonhuman primate. Following infusion of pimonidazole into animals with established infections, lung tissues from the guinea pig, rabbit, and nonhuman primate showed discrete areas of pimonidazole adduct formation surrounding necrotic and caseous regions of pulmonary granulomas by immunohistochemical staining. This labeling could be substantially reduced by housing the animal under an atmosphere of 95% O(2). Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3-mm) pulmonary lesions were severely hypoxic (1.6 +/- 0.7 mm Hg). Finally, metronidazole, which has potent bactericidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total-lung bacterial burdens in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions.

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

The Mycobacterium tuberculosis ß-oxidation genes echA5 and fadB3 are dispensable for growth in vitro and in vivo.

There are several lines of evidence pointing towards the importance of ß-oxidation in host survival of Mycobacterium tuberculosis including enormous gene redundancy for this process; approximately 100 genes are annotated as ß-oxidation genes for the five biochemical reactions that break down fatty acids into acetyl-CoA. Although most of these genes are predicted to be non-essential, two of the genes (echA5 and fadB3) are annotated as essential for growth in vitro, and therefore could be considered as putative drug targets. However, here we report the construction of echA5 and fadB3 null mutants confirming they are non-essential. No significant difference in growth between the mutant and parent strains was observed in either standard Middlebrook medium or in minimal medium supplemented with various carbon sources. Macrophage survival and mouse infection studies also showed no significant difference between the mutant and parent strains. Therefore, we conclude that these genes are dispensable for growth in vitro and in vivo.

Interleukin (IL)-15 and IL-2 reciprocally regulate expression of the chemokine receptor CX3CR1 through selective NFAT1- and NFAT2-dependent mechanisms.

We have recently reported that interleukin (IL)-15 and IL-2, which signal through IL-2Rbetagamma, oppositely regulate expression of the proinflammatory chemokine receptor CX3CR1. Here we delineate molecular mechanisms responsible for this paradox. By using a luciferase reporter plasmid, we identified a 433-bp region spanning the major transcriptional start point of human CX3CR1 that, when expressed in human peripheral blood mononuclear cells (PBMCs), possessed strong constitutive promoter activity. IL-2 and IL-15 treatment increased and abolished this activity, respectively, mimicking their effects on endogenous CX3CR1. IL-2 and IL-15 have been reported to also have opposite effects on the immunoregulatory transcription factor NFAT (nuclear factor of activated T cells), and the 433-bp region contains a kappaB-like NFAT site. The effects of IL-15 and IL-2 on both CX3CR1 reporter activity and endogenous CX3CR1 transcription in PBMCs were abolished by the NFAT inhibitors cyclosporin A and VIVIT. Moreover, mutation of the kappaB-like NFAT sequence markedly attenuated IL-2 and IL-15 modulation of CX3CR1 promoter-reporter activity in PBMCs. Furthermore, chromatin immunoprecipitation revealed that IL-15 promoted specific recruitment of NFAT1 but not NFAT2 to the CX3CR1 promoter, whereas IL-2 had the converse effect. This appears to be relevant in vivo because mouse CX3CR1 mRNA was expressed in both PBMCs and splenocytes from NFAT1-/- mice injected with recombinant IL-15 but was undetectable in cells from IL-15-injected NFAT1+/+ BALB/c mice; as predicted, IL-2 up-regulated cx3cr1 in both mouse strains to a similar extent. Thus, by pharmacologic, genetic, and biochemical criteria in vitro and in vivo, our results suggest that IL-15 and IL-2 oppositely regulate CX3CR1 gene expression by differentially recruiting NFAT1 and NFAT2 to a kappaB-like NFAT site within the CX3CR1 promoter. We propose that expression of CX3CR1 and possibly other immunoregulatory genes may be determined in part by the balance of NFAT1 and NFAT2 activity in leukocytes.