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Overweight and obesity have been shown to significantly affect brain structures and size. Obesity has been associated with cerebral atrophy, alteration of brain functions, including cognitive impairement, and psychiatric diseases such as depression. Given the importance of lipids in the structure of the brain, here, by using 47 mice fed a high fat diet (HFD) with 60% calories from fat (40% saturated fatty acids) and 20% calories from carbohydrates and age-matched control animals on a normal chow diet, we examined the effects of HFD and diet-induced obesity on the brain lipidome. Using a targeted liquid chromatography mass spectrometry analysis and a non-targeted mass spectrometry MALDI imaging approach, we show that the relative concentration of most lipids, in particular brain phospholipids, is modified by diet-induced obesity (+ 40%of body weight). Use of a non-targeted MALDI-MS imaging approach further allowed define cerebral regions of interest (ROI) involved in eating behavior and changes in their lipid profile. Principal component analysis (PCA) of the obese/chow lipidome revealed persistence of some of the changes in the brain lipidome of obese animals even after their switch to chow feeding and associated weight loss. Altogether, these data reveal that HFD feeding rapidly modifies the murine brain lipidome. Some of these HFD-induced changes persist even after weight loss, implying that some brain sequelae caused by diet-induced obesity are irreversible.

A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals as well as the inherent caveats of in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo granuloma culture technique, wherein mature, fully organized granulomas are microdissected and maintained in three-dimensional culture. This approach, in which granulomas retain key bacterial and host characteristics, enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation. Through mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We describe a method to transfect isolated granulomas, enabling genetic manipulation. In addition, we provide proof-of-concept for host-directed small molecule screens, identifying PKC signaling as an important regulator of granuloma macrophage organization.

This study presents a comprehensive lesion-centric analysis of disease progression in the rabbit model of tuberculosis, showing immunopathology and lesion heterogeneity similar to human and nonhuman primates. In this model, pyrazinamide sterilizes necrotic lesions, where persistent bacterial populations reside., In the 1970s, inclusion of pyrazinamide (PZA) in the drug regimen of tuberculosis (TB) patients for the first 2 mo achieved a drastic reduction of therapy duration. Until now, however, the mechanisms underlying PZA’s unique contribution to efficacy have remained controversial, and animal efficacy data vary across species. To understand how PZA kills bacterial populations present in critical lung lesion compartments, we first characterized a rabbit model of active TB, showing striking similarities in lesion types and fates to nonhuman primate models deemed the most appropriate surrogates of human TB. We next employed this model with lesion-centric molecular and bacteriology readouts to demonstrate that PZA exhibits potent activity against Mycobacterium tuberculosis residing in difficult-to-sterilize necrotic lesions. Our data also indicate that PZA is slow acting, suggesting that PZA administration beyond the first 2 mo may accelerate the cure. In conclusion, we provide a pharmacodynamic explanation for PZA’s treatment-shortening effect and deliver new tools to dissect the contribution of immune response versus drug at the lesion level.

The length and complexity of tuberculosis (TB) therapy, as well as the propensity ofMycobacterium tuberculosisto develop drug resistance, are major barriers to global TB control efforts.M. tuberculosisis known to have the ability to enter into a drug-tolerant state, which may explain many of these impediments to TB treatment. We have identified a novel mechanism of genetically encoded but rapidly reversible drug-tolerance inM. tuberculosiscaused by transient frameshift mutations in a homopolymeric tract (HT) of seven cytosines (7C) in theglpKgene. Inactivating frameshift mutations associated with the 7C HT inglpKproduce small colonies that exhibit heritable multi-drug increases in minimal inhibitory concentrations and decreases in drug-dependent killing; however, reversion back to a fully drug-susceptible large-colony phenotype occurs rapidly through the introduction of additional insertions or deletions in the sameglpKHT region. These reversible frameshift mutations in the 7C HT ofM. tuberculosis glpKoccur in clinical isolates, accumulate inM. tuberculosisinfected mice with further accumulation during drug treatment, and exhibit a reversible transcriptional profile including induction ofdosRandsigHand repression ofkstRregulons, similar to that observed in otherin vitromodels ofM. tuberculosistolerance. These results suggest that GlpK phase variation may contribute to drug-tolerance, treatment failure and relapse in human TB. Drugs effective against phase-variantM. tuberculosismay hasten TB treatment and improve cure rates.SIGNIFICANCEThe ability ofM. tuberculosisto survive during prolonged treatment has been attributed to either transient stress responses or fixed heritable drug-resistance producing mutations. We show that phase-variation in theM. tuberculosis glpKgene represents a third type of resistance mechanism. The ability of theseglpKmutants to grow slowly and then rapidly revert suggests that these transiently-heritable changes may also explain how a hidden population of drug-tolerant bacteria develops during TB treatment. As a genetically trackable cause of drug-tolerance,M. tuberculosis glpKmutants provides a unique opportunity to study these phenomena at a cellular and mechanistic level. These mutants could also be used for developing drugs that target tolerant populations, leading to more rapid and effective TB treatments.

Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of “universal” drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone., Fluoroquinolones represent the pillar of multidrug-resistant tuberculosis (MDR-TB) treatment, with moxifloxacin, levofloxacin, or gatifloxacin being prescribed to MDR-TB patients. Recently, several clinical trials of “universal” drug regimens, aiming to treat drug-susceptible and drug-resistant TB, have included a fluoroquinolone. In the absence of clinical data comparing their side-by-side efficacies in controlled MDR-TB trials, a pharmacological rationale is needed to guide the selection of the most efficacious fluoroquinolone. The present studies were designed to test the hypothesis that fluoroquinolone concentrations (pharmacokinetics) and activity (pharmacodynamics) at the site of infection are better predictors of efficacy than the plasma concentrations and potency measured in standard growth inhibition assays and are better suited to determinations of whether one of the fluoroquinolones outperforms the others in rabbits with active TB. We first measured the penetration of these fluoroquinolones in lung lesion compartments, and their potency against bacterial populations that reside in each compartment, to compute lesion-centric pharmacokinetic-pharmacodynamic (PK/PD) parameters. PK modeling methods were used to quantify drug penetration from plasma to tissues at human-equivalent doses. On the basis of these metrics, moxifloxacin emerged with a clear advantage, whereas plasma-based PK/PD favored levofloxacin (the ranges of the plasma AUC/MIC ratio [i.e., the area under the concentration-time curve over 24 h in the steady state divided by the MIC] are 46 to 86 for moxifloxacin and 74 to 258 for levofloxacin). A comparative efficacy trial in the rabbit model of active TB demonstrated the superiority of moxifloxacin in reducing bacterial burden at the lesion level and in sterilizing cellular and necrotic lesions. Collectively, these results show that PK/PD data obtained at the site of infection represent an adequate predictor of drug efficacy against TB and constitute the baseline required to explore synergies, antagonism, and drug-drug interactions in fluoroquinolone-containing regimens.

Understanding the distribution patterns of antibiotics at the site of infection is paramount to selecting adequate drug regimens and developing new antibiotics. Tuberculosis (TB) lung lesions are made of various immune cell types, some of which harbor persistent forms of the pathogen, Mycobacterium tuberculosis. By combining high resolution MALDI MSI with histology staining and quantitative image analysis in rabbits with active TB, we have mapped the distribution of a fluoroquinolone at high resolution, and identified the immune-pathological factors driving its heterogeneous penetration within TB lesions, in relation to where bacteria reside. We find that macrophage content, distance from lesion border and extent of necrosis drive the uneven fluoroquinolone penetration. Preferential uptake in macrophages and foamy macrophages, where persistent bacilli reside, compared to other immune cells present in TB granulomas, was recapitulated in vitro using primary human cells. A nonlinear modeling approach was developed to help predict the observed drug behavior in TB lesions. This work constitutes a methodological advance for the co-localization of drugs and infectious agents at high spatial resolution in diseased tissues, which can be applied to other diseases with complex immunopathology.

Foam cells are lipid-laden macrophages that contribute to the inflammation and tissue damage associated with many chronic inflammatory disorders. Although foam cell biogenesis has been extensively studied in atherosclerosis, how these cells form during a chronic infectious disease such as tuberculosis is unknown. Here we report that, unlike the cholesterol-laden cells of atherosclerosis, foam cells in tuberculous lung lesions accumulate triglycerides. Consequently, the biogenesis of foam cells varies with the underlying disease. In vitro mechanistic studies showed that triglyceride accumulation in human macrophages infected with Mycobacterium tuberculosis is mediated by TNF receptor signaling through downstream activation of the caspase cascade and the mammalian target of rapamycin complex 1 (mTORC1). These features are distinct from the known biogenesis of atherogenic foam cells and establish a new paradigm for non-atherogenic foam cell formation. Moreover, they reveal novel targets for disease-specific pharmacological interventions against maladaptive macrophage responses.

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

MALDI mass-spectrometry imaging (MALDI-MSI) is a technique capable of the label-free identification and visualization of analytes in tissue sections. We have previously applied MALDI-MSI to the study of the spatial distribution of tuberculosis (TB) drugs in necrotic lung granulomas characteristic of pulmonary TB disease, revealing heterogeneous and often suboptimal drug distributions. To investigate the impact of differential drug distributions at sites of infection, we sought to image mycobacterial biomarkers to coregister drugs and bacteria in lesion sections. The traditional method of visualizing Mycobacterium tuberculosis inside lesions is acid-fast staining and microscopy. Directly analyzing and visualizing mycobacteria-specific lipid markers by MALDI-MSI provides detailed molecular information on bacterial distributions within granulomas, complementary to high-spatial-resolution staining and microscopy approaches. Moreover, spatial monitoring of molecular changes occurring in bacteria during granuloma development can potentially contribute to a greater understanding of pulmonary-TB pathogenesis. In this study, we developed a MALDI-MSI method to detect and visualize specific glycolipids of mycobacteria within TB lesions. The biomarker signal correlated well with the bacteria visualized by IHC and acid-fast staining. This observation was seen in samples collected from multiple animal models. Although individual bacteria could not be visualized because of the limit of spatial resolution (50 μm), bacterial clusters were clearly detected and heterogeneously distributed throughout lesions. The ability to visualize drugs, metabolites, and bacterial biomarkers by MALDI-MSI enabled direct colocalization of drugs with specific bacterial target populations (identifiable by distinct metabolic markers). Future applications include assessing drug activity in lesions by visualizing drug-mediated lipid changes and other drug-induced mycobacterial metabolic responses.

Tuberculosis (TB) recently became the leading infectious cause of death in adults, while attempts to shorten therapy have largely failed. Dormancy, persistence, and drug tolerance are among the factors driving the long therapy duration. Assays to measure in situ drug susceptibility of Mycobacterium tuberculosis bacteria in pulmonary lesions are needed if we are to discover new fast-acting regimens and address the global TB threat. Here we take a first step toward this goal and describe an ex vivo assay developed to measure the cidal activity of anti-TB drugs against M. tuberculosis bacilli present in cavity caseum obtained from rabbits with active TB. We show that caseum M. tuberculosis bacilli are largely nonreplicating, maintain viability over the course of the assay, and exhibit extreme tolerance to many first- and second-line TB drugs. Among the drugs tested, only the rifamycins fully sterilized caseum. A similar trend of phenotypic drug resistance was observed in the hypoxia- and starvation-induced nonreplicating models, but with notable qualitative and quantitative differences: (i) caseum M. tuberculosis exhibits higher drug tolerance than nonreplicating M. tuberculosis in the Wayne and Loebel models, and (ii) pyrazinamide is cidal in caseum but has no detectable activity in these classic nonreplicating assays. Thus, ex vivo caseum constitutes a unique tool to evaluate drug potency against slowly replicating or nonreplicating bacilli in their native caseous environment. Intracaseum cidal concentrations can now be related to the concentrations achieved in the necrotic foci of granulomas and cavities to establish correlations between clinical outcome and lesion-centered pharmacokinetics-pharmacodynamics (PK-PD) parameters.

Background Intra-abdominal candidiasis (IAC) is a prominent invasive fungal infection associated with high mortality. Prompt antifungal therapy and source control are crucial for successful treatment. Echinocandin antifungal drugs are first-line agents. Yet, their clinical effectiveness is highly variable with known potential for breakthrough resistance, and little is known about drug exposure at the site of infection. Using matrix-assisted desorption/ionization (MALDI) mass spectrometry imaging as well as standard analytical techniques, we investigated the spatial and quantitative distribution in tissue lesions for two echinocandin drugs, micafungin and CD101, in a clinically relevant IAC mouse model. Methods Female 6–8 week old CD1 mice weighing 18–22 g were infected intraperitoneally (IP) with 1 × 107 CFU of C. albicans SC5314 mixed with sterile stool matrix. Single IP doses of CD101 at 5 or 20 mg/kg (equivalent to humanized therapeutic dose) or micafungin at 5 mg/kg (therapeutic dose) were administered to mice at day 3 post-inoculation. Mice were sacrificed at just before antifungal treatment (n= 1), and at 1, 3, 6, 24, and 48 hours post-dose (n = 3 per group per time point). Liver and kidney lesions were collected for MALDI imaging. Laser capture microdissection (LCM) followed by liquid chromatography coupled tandem mass spectrometry (LC/MS-MS) was applied to 6 and 24 hours samples for drug exposure measurement. In a separate experiment, mice were treated with 2 or 3 doses of micafungin (5 mg/kg), or a single dose of CD101 (20 mg/kg). Drug accumulation was analyzed at 48 and 72hours post the first dose. Results Drug accumulation within lesions was observed with both drugs at their humanized therapeutic dose. However, micafungin, even at steady-state, failed to approach the mutant prevention concentration (MPC) (16 µg/mL) of the infecting strain. CD101 demonstrated extensive penetration into the lesions after a single dose administration and persisted in lesions at above MPC level of 29.7 µg/mL at 72 hours postdose. Conclusion These findings indicate that current echinocandin drugs may be limited by penetration at the site of infection, which have implications for clinical outcomes and emergence of resistance in patients with IAC. Disclosures C. J. Clancy, Merck: Received research funding, Research support; Astellas: Received research funding, Research support; Cidara: Received research funding, Research support; Astellas: Scientific Advisor, Advisory board; Merck: Scientific Advisor, Advisory board; Cidara: Scientific Advisor, Advisory board; Medicines Company: Scientific Advisor, Advisory board; D. Perlin, Cidara: Research Contractor and Scientific Advisor, Research grant; Amplyx: Research Contractor and Scientific Advisor, Research grant; Matinas: Scientific Advisor, Research support; Scynexis: Research Contractor and Scientific Advisor, Research grant; Merck: Research Contractor, Research grant; Astellas: Research Contractor, Research grant

Clinical trials and practice have shown that ethambutol is an important component of the first-line tuberculosis (TB) regime. This contrasts the drug's rather modest potency and lack of activity against nongrowing persister mycobacteria. The standard plasma-based pharmacokinetic-pharmacodynamic profile of ethambutol suggests that the drug may be of limited clinical value. Here, we hypothesized that this apparent contradiction may be explained by favorable penetration of the drug into TB lesions. First, we utilized novel in vitro lesion pharmacokinetic assays and predicted good penetration of the drug into lesions. We then employed mass spectrometry imaging and laser capture microdissection coupled to liquid chromatography and tandem mass spectrometry (LCM and LC/MS-MS, respectively) to show that ethambutol, indeed, accumulates in diseased tissues and penetrates the major human-like lesion types represented in the rabbit model of TB disease with a lesion-to-plasma exposure ratio ranging from 9 to 12. In addition, ethambutol exhibits slow but sustained passive diffusion into caseum to reach concentrations markedly higher than those measured in plasma at steady state. The results explain why ethambutol has retained its place in the first-line regimen, validate our in vitro lesion penetration assays, and demonstrate the critical importance of effective lesion penetration for anti-TB drugs. Our findings suggest that in vitro and in vivo lesion penetration evaluation should be included in TB drug discovery programs. Finally, this is the first time that LCM with LC-MS/MS has been used to quantify a small molecule at high spatial resolution in infected tissues, a method that can easily be extended to other infectious diseases.