Fluoroquinolone Efficacy against Tuberculosis Is Driven by Penetration into Lesions and Activity against Resident Bacterial Populations.

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.

Toll-like Receptor 2 Prevents Neutrophil-Driven Immunopathology during Infection with Mycobacterium tuberculosis by Curtailing CXCL5 Production.

The W-Beijing strain family is globally distributed and is associated with multidrug-resistant tuberculosis (TB) and treatment failure. Therefore, in this study, we examined the contribution of Toll-like receptor 2 (TLR2) to host resistance against Mycobacterium tuberculosis HN878, a clinical isolate belonging to the W-Beijing family. We show that TLR2 knockout (TLR2KO) mice infected with M. tuberculosis HN878 exhibit increased bacterial burden and are unable to control tissue-damaging, pulmonary neutrophilic inflammation. Consistent with a critical role for CXCL5 in regulating neutrophil influx, expression of epithelial cell-derived CXCL5 is significantly enhanced in TLR2KO mice prior to their divergence from wild-type (WT) mice in M. tuberculosis replication and neutrophilic inflammation. Depletion of neutrophils in TLR2KO mice by targeting Ly6G reverts lung inflammation and bacterial burden to levels comparable to those of WT mice. Together, the results establish that TLR2 controls neutrophil-driven immunopathology during infection with M. tuberculosis HN878 infection, likely by curtailing CXCL5 production.

Comparing efficacies of moxifloxacin, levofloxacin and gatifloxacin in tuberculosis granulomas using a multi-scale systems pharmacology approach.

Granulomas are complex lung lesions that are the hallmark of tuberculosis (TB). Understanding antibiotic dynamics within lung granulomas will be vital to improving and shortening the long course of TB treatment. Three fluoroquinolones (FQs) are commonly prescribed as part of multi-drug resistant TB therapy: moxifloxacin (MXF), levofloxacin (LVX) or gatifloxacin (GFX). To date, insufficient data are available to support selection of one FQ over another, or to show that these drugs are clinically equivalent. To predict the efficacy of MXF, LVX and GFX at a single granuloma level, we integrate computational modeling with experimental datasets into a single mechanistic framework, GranSim. GranSim is a hybrid agent-based computational model that simulates granuloma formation and function, FQ plasma and tissue pharmacokinetics and pharmacodynamics and is based on extensive in vitro and in vivo data. We treat in silico granulomas with recommended daily doses of each FQ and compare efficacy by multiple metrics: bacterial load, sterilization rates, early bactericidal activity and efficacy under non-compliance and treatment interruption. GranSim reproduces in vivo plasma pharmacokinetics, spatial and temporal tissue pharmacokinetics and in vitro pharmacodynamics of these FQs. We predict that MXF kills intracellular bacteria more quickly than LVX and GFX due in part to a higher cellular accumulation ratio. We also show that all three FQs struggle to sterilize non-replicating bacteria residing in caseum. This is due to modest drug concentrations inside caseum and high inhibitory concentrations for this bacterial subpopulation. MXF and LVX have higher granuloma sterilization rates compared to GFX; and MXF performs better in a simulated non-compliance or treatment interruption scenario. We conclude that MXF has a small but potentially clinically significant advantage over LVX, as well as LVX over GFX. We illustrate how a systems pharmacology approach combining experimental and computational methods can guide antibiotic selection for TB.

Ethambutol Partitioning in Tuberculous Pulmonary Lesions Explains Its Clinical Efficacy.

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.

Divergent Functions of TLR2 on Hematopoietic and Nonhematopoietic Cells during Chronic Mycobacterium tuberculosis Infection.

We have reported that TLR2 is crucial for host resistance against chronic Mycobacterium tuberculosis infection; however, which cell types are key players in this response remain unknown. This led us to decipher the relative contribution of TLR2 on nonhematopoietic and hematopoietic cells in resistance against chronic M. tuberculosis infection in mice infected with M. tuberculosis Erdman. Consistent with our previous report, at 8 wk of infection, TLR2 knockout (TLR2KO)→TLR2KO bone marrow chimeric mice exhibited increased bacterial burden, disorganized accumulation of lymphocytes and mononuclear cells, and extensive pulmonary immunopathology compared with wild-type (WT)→WT chimeric mice. Bacterial burden and pulmonary immunopathology of chimeric mice lacking TLR2 in the hematopoietic compartment (TLR2KO→WT) was comparable to TLR2KO mice. In contrast, chimeric mice deficient in TLR2 in the nonhematopoietic compartment (WT→TLR2KO) exhibited a marked attenuation in granulomatous inflammation compared with WT mice. Although the latter mice did not exhibit improved pulmonary bacterial control, significant reductions in bacterial burden in the draining lymph nodes, spleen, and liver were observed. These findings establish that the TLR2-mediated hematopoietic response promotes stable control of pulmonary bacterial burden and granuloma integrity, whereas TLR2 signaling on nonhematopoietic cells may partly facilitate granulomatous inflammation and bacterial dissemination.

Vaccine-mediated immunity to experimental Mycobacterium tuberculosis is not impaired in the absence of Toll-like receptor 9.

Accumulating evidence indicates that inflammatory signals required for maximizing effector T cell generation have opposing effects on the development of memory T cell precursors. Toll-like receptor (TLR)2, and TLR9 significantly contribute to the inflammatory milieu and therefore in this study we examined whether the absence of TLR9 alone or the combined absence of TLR2 and TLR9 would affect vaccine-mediated immunity to Mtb. We found that TLR9KO and TLR2/9DKO mice vaccinated with a live Mtb auxotroph, akin to vaccinated WT mice, exhibited early control of Mtb growth in the lungs compared to their naïve counterparts. The granulomatous response, IFNγ production and cellular recruitment to the lungs were also similar in all the vaccinated groups of mice. These findings indicate that there is minimal contribution from TLR2 and TLR9 in generating memory immunity to Mtb with live vaccines. Defining the innate milieu that can drive maximal memory T cell generation with a tuberculosis vaccine needs further inquiry.

Duality of lipid mediators in host response against Mycobacterium tuberculosis: good cop, bad cop.

Lipid mediators play an important role in infection- and tissue injury-driven inflammatory responses and in the subsequent inhibition and resolution of the response. Here, we discuss recent findings that substantiate how Mycobacterium tuberculosis promotes its survival in the host by dysregulation of lipid mediator balance. By inhibiting prostaglandin E2 (PGE2) and enhancing lipoxin production, M. tuberculosis induces necrotic death of the macrophage, an environment that favors its growth. These new findings provide opportunities for developing and repurposing therapeutics to modulate lipid mediator balance and enhance M. tuberculosis growth restriction.