The Gut Microbiome Modulates Body Temperature Both in Sepsis and Health.

Rationale: Among patients with sepsis, variation in temperature trajectories predicts clinical outcomes. In healthy individuals, normal body temperature is variable and has decreased consistently since the 1860s. The biologic underpinnings of this temperature variation in disease and health are unknown. Objectives: To establish and interrogate the role of the gut microbiome in calibrating body temperature. Methods: We performed a series of translational analyses and experiments to determine whether and how variation in gut microbiota explains variation in body temperature in sepsis and in health. We studied patient temperature trajectories using electronic medical record data. We characterized gut microbiota in hospitalized patients using 16S ribosomal RNA gene sequencing. We modeled sepsis using intraperitoneal LPS in mice and modulated the microbiome using antibiotics, germ-free, and gnotobiotic animals. Measurements and Main Results: Consistent with prior work, we identified four temperature trajectories in patients hospitalized with sepsis that predicted clinical outcomes. In a separate cohort of 116 hospitalized patients, we found that the composition of patients' gut microbiota at admission predicted their temperature trajectories. Compared with conventional mice, germ-free mice had reduced temperature loss during experimental sepsis. Among conventional mice, heterogeneity of temperature response in sepsis was strongly explained by variation in gut microbiota. Healthy germ-free and antibiotic-treated mice both had lower basal body temperatures compared with control animals. The Lachnospiraceae family was consistently associated with temperature trajectories in hospitalized patients, experimental sepsis, and antibiotic-treated mice. Conclusions: The gut microbiome is a key modulator of body temperature variation in both health and critical illness and is thus a major, understudied target for modulating physiologic heterogeneity in sepsis.

Plasma metabolomics and quantitative interstitial abnormalities in ever-smokers.

BACKGROUND: Quantitative interstitial abnormalities (QIA) are an automated computed tomography (CT) finding of early parenchymal lung disease, associated with worse lung function, reduced exercise capacity, increased respiratory symptoms, and death. The metabolomic perturbations associated with QIA are not well known. We sought to identify plasma metabolites associated with QIA in smokers. We also sought to identify shared and differentiating metabolomics features between QIA and emphysema, another smoking-related advanced radiographic abnormality. METHODS: In 928 former and current smokers in the Genetic Epidemiology of COPD cohort, we measured QIA and emphysema using an automated local density histogram method and generated metabolite profiles from plasma samples using liquid chromatography-mass spectrometry (Metabolon). We assessed the associations between metabolite levels and QIA using multivariable linear regression models adjusted for age, sex, body mass index, smoking status, pack-years, and inhaled corticosteroid use, at a Benjamini-Hochberg False Discovery Rate p-value of ≤ 0.05. Using multinomial regression models adjusted for these covariates, we assessed the associations between metabolite levels and the following CT phenotypes: QIA-predominant, emphysema-predominant, combined-predominant, and neither- predominant. Pathway enrichment analyses were performed using MetaboAnalyst. RESULTS: We found 85 metabolites significantly associated with QIA, with overrepresentation of the nicotinate and nicotinamide, histidine, starch and sucrose, pyrimidine, phosphatidylcholine, lysophospholipid, and sphingomyelin pathways. These included metabolites involved in inflammation and immune response, extracellular matrix remodeling, surfactant, and muscle cachexia. There were 75 metabolites significantly different between QIA-predominant and emphysema-predominant phenotypes, with overrepresentation of the phosphatidylethanolamine, nicotinate and nicotinamide, aminoacyl-tRNA, arginine, proline, alanine, aspartate, and glutamate pathways. CONCLUSIONS: Metabolomic correlates may lend insight to the biologic perturbations and pathways that underlie clinically meaningful quantitative CT measurements like QIA in smokers.

Vitamin D Insufficiency as a Risk Factor for Paclitaxel-Induced Peripheral Neuropathy in SWOG S0221.

BACKGROUND: Prior work suggests that patients with vitamin D insufficiency may have a higher risk of chemotherapy-induced peripheral neuropathy (CIPN) from paclitaxel. The objective of this study was to validate vitamin D insufficiency as a CIPN risk factor. METHODS: We used data and samples from the prospective phase III SWOG S0221 (ClinicalTrials.gov identifier: NCT00070564) trial that compared paclitaxel-containing chemotherapy regimens for early-stage breast cancer. We quantified pretreatment 25-hydroxy-vitamin D in banked serum samples using a liquid chromatography-tandem mass spectrometry targeted assay. We tested the association between vitamin D insufficiency (≤20 ng/mL) and grade ≥3 sensory CIPN via multiple logistic regression and then adjusted for self-reported race, age, body mass index, and paclitaxel schedule (randomization to weekly or every-2-week dosing). We also tested the direct effect of vitamin D deficiency on mechanical hypersensitivity in mice randomized to a regular or vitamin D-deficient diet. RESULTS: Of the 1,191 female patients in the analysis, 397 (33.3%) had pretreatment vitamin D insufficiency, and 195 (16.4%) developed grade ≥3 CIPN. Patients with vitamin D insufficiency had a higher incidence of grade ≥3 CIPN than those who had sufficient vitamin D (20.7% vs 14.2%; odds ratio [OR], 1.57; 95% CI, 1.14-2.15; P=.005). The association retained significance after adjusting for age and paclitaxel schedule (adjusted OR, 1.65; 95% CI, 1.18-2.30; P=.003) but not race (adjusted OR, 1.39; 95% CI, 0.98-1.97; P=.066). In the mouse experiments, the vitamin D-deficient diet caused mechanical hypersensitivity and sensitized mice to paclitaxel (both P<.05). CONCLUSIONS: Pretreatment vitamin D insufficiency is the first validated potentially modifiable predictive biomarker of CIPN from paclitaxel. Prospective trials are needed to determine whether vitamin D supplementation prevents CIPN and improves treatment outcomes in patients with breast and other cancer types.

Lung Microbiota and Metabolites Collectively Associate with Clinical Outcomes in Milder Stage Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is variable in its development. Lung microbiota and metabolites collectively may impact COPD pathophysiology, but relationships to clinical outcomes in milder disease are unclear. Objectives: Identify components of the lung microbiome and metabolome collectively associated with clinical markers in milder stage COPD. Methods: We analyzed paired microbiome and metabolomic data previously characterized from bronchoalveolar lavage fluid in 137 participants in the SPIROMICS (Subpopulations and Intermediate Outcome Measures in COPD Study), or (GOLD [Global Initiative for Chronic Obstructive Lung Disease Stage 0-2). Datasets used included 1) bacterial 16S rRNA gene sequencing; 2) untargeted metabolomics of the hydrophobic fraction, largely comprising lipids; and 3) targeted metabolomics for a panel of hydrophilic compounds previously implicated in mucoinflammation. We applied an integrative approach to select features and model 14 individual clinical variables representative of known associations with COPD trajectory (lung function, symptoms, and exacerbations). Measurements and Main Results: The majority of clinical measures associated with the lung microbiome and metabolome collectively in overall models (classification accuracies, >50%, P < 0.05 vs. chance). Lower lung function, COPD diagnosis, and greater symptoms associated positively with Streptococcus, Neisseria, and Veillonella, together with compounds from several classes (glycosphingolipids, glycerophospholipids, polyamines and xanthine, an adenosine metabolite). In contrast, several Prevotella members, together with adenosine, 5'-methylthioadenosine, sialic acid, tyrosine, and glutathione, associated with better lung function, absence of COPD, or less symptoms. Significant correlations were observed between specific metabolites and bacteria (Padj < 0.05). Conclusions: Components of the lung microbiome and metabolome in combination relate to outcome measures in milder COPD, highlighting their potential collaborative roles in disease pathogenesis.

Co-occurrence and metabolic biomarkers of sensory and motor subtypes of peripheral neuropathy from paclitaxel.

PURPOSE: Chemotherapy-induced peripheral neuropathy (CIPN) is the major treatment-limiting toxicity of paclitaxel, which predominantly presents as sensory symptoms, with motor symptoms in some patients. Differentiating CIPN into subtypes has been recommended to direct CIPN research. The objective of this study was to investigate whether sensory and motor CIPN are distinct subtypes with different predictive biomarkers in patients with breast cancer receiving paclitaxel. METHODS: Data were from a prospective cohort of 60 patients with breast cancer receiving up to 12 weekly infusions of 80 mg/m2 paclitaxel (NCT02338115). European Organisation for Research and Treatment of Cancer Quality of Life questionnaire CIPN20 was used to evaluate CIPN. Clusters of the time course of sensory (CIPNS), motor (CIPNM), and the difference between sensory and motor (CIPNS-CIPNM) were identified using k-means clustering on principal component scores. Predictive metabolomic biomarkers of maximum CIPNS and CIPNM were investigated using linear regressions adjusted for baseline CIPN, paclitaxel pharmacokinetics, and body mass index. RESULTS: More sensory than motor CIPN was found (CIPNS change: mean = 10.8, ranged [-3.3, 52.1]; CIPNM change: mean = 3.5, range: [-7.5, 35.0]). Three groups were identified with No CIPN, Mixed CIPN, and Sensory-dominant CIPN (maximum CIPNS: mean = 12.7 vs. 40.9 vs. 74.3, p < 0.001; maximum CIPNM: mean = 5.4 vs. 25.5 vs. 36.1, p < 0.001; average CIPNS-CIPNM: mean = 2.8 vs. 5.8 vs. 24.9, p < 0.001). Biomarkers of motor CIPN were similar to previously identified biomarkers of sensory CIPN, including lower serum histidine (p = 0.029). CONCLUSION: Our findings suggest that sensory and motor CIPN co-occur and may not have differentiating metabolic biomarkers. These findings need to be validated in larger cohorts of patients treated with paclitaxel and other neurotoxic agents to determine the optimal approach to predict, prevent, and treat CIPN and improve patients' outcomes.

Functional lower airways genomic profiling of the microbiome to capture active microbial metabolism.

BACKGROUND: Microbiome studies of the lower airways based on bacterial 16S rRNA gene sequencing assess microbial community structure but can only infer functional characteristics. Microbial products, such as short-chain fatty acids (SCFAs), in the lower airways have significant impact on the host's immune tone. Thus, functional approaches to the analyses of the microbiome are necessary. METHODS: Here we used upper and lower airway samples from a research bronchoscopy smoker cohort. In addition, we validated our results in an experimental mouse model. We extended our microbiota characterisation beyond 16S rRNA gene sequencing with the use of whole-genome shotgun (WGS) and RNA metatranscriptome sequencing. SCFAs were also measured in lower airway samples and correlated with each of the sequencing datasets. In the mouse model, 16S rRNA gene and RNA metatranscriptome sequencing were performed. RESULTS: Functional evaluations of the lower airway microbiota using inferred metagenome, WGS and metatranscriptome data were dissimilar. Comparison with measured levels of SCFAs shows that the inferred metagenome from the 16S rRNA gene sequencing data was poorly correlated, while better correlations were noted when SCFA levels were compared with WGS and metatranscriptome data. Modelling lower airway aspiration with oral commensals in a mouse model showed that the metatranscriptome most efficiently captures transient active microbial metabolism, which was overestimated by 16S rRNA gene sequencing. CONCLUSIONS: Functional characterisation of the lower airway microbiota through metatranscriptome data identifies metabolically active organisms capable of producing metabolites with immunomodulatory capacity, such as SCFAs.

Serum citrullinated histone H3 concentrations differentiate patients with septic verses non-septic shock and correlate with disease severity.

PURPOSE: Microbial infection stimulates neutrophil/macrophage/monocyte extracellular trap formation, which leads to the release of citrullinated histone H3 (CitH3) catalyzed by peptidylarginine deiminase (PAD) 2 and 4. Understanding these molecular mechanisms in the pathogenesis of septic shock will be an important next step for developing novel diagnostic and treatment modalities. We sought to determine the expression of CitH3 in patients with septic shock, and to correlate CitH3 levels with PAD2/PAD4 and clinically relevant outcomes. METHODS: Levels of CitH3 were measured in serum samples of 160 critically ill patients with septic and non-septic shock, and healthy volunteers. Analyses of clinical and laboratory characteristics of patients were conducted. RESULTS: Levels of circulating CitH3 at enrollment were significantly increased in septic shock patients (n = 102) compared to patients hospitalized with non-infectious shock (NIC) (n = 32, p < 0.0001). The area under the curve (95% CI) for distinguishing septic shock from NIC using CitH3 was 0.76 (0.65-0.86). CitH3 was positively correlated with PAD2 and PAD4 concentrations and Sequential Organ Failure Assessment Scores [total score (r = 0.36, p < 0.0001)]. The serum levels of CitH3 at 24 h (p < 0.01) and 48 h (p < 0.05) were significantly higher in the septic patients that did not survive. CONCLUSION: CitH3 is increased in patients with septic shock. Its serum concentrations correlate with disease severity and prognosis, which may yield vital insights into the pathophysiology of sepsis.

Untargeted Metabolomics Differentiates l-Carnitine Treated Septic Shock 1-Year Survivors and Nonsurvivors.

l-Carnitine is a candidate therapeutic for the treatment of septic shock, a condition that carries a ≥40% mortality. Responsiveness to l-carnitine may hinge on unique metabolic profiles that are not evident from the clinical phenotype. To define these profiles, we performed an untargeted metabolomic analysis of serum from 21 male sepsis patients enrolled in a placebo-controlled l-carnitine clinical trial. Although treatment with l-carnitine is known to induce changes in the sepsis metabolome, we found a distinct set of metabolites that differentiated 1-year survivors from nonsurvivors. Following feature alignment, we employed a new and innovative data reduction strategy followed by false discovery correction, and identified 63 metabolites that differentiated carnitine-treated 1-year survivors versus nonsurvivors. Following identification by MS/MS and database search, several metabolite markers of vascular inflammation were determined to be prominently elevated in the carnitine-treated nonsurvivor cohort, including fibrinopeptide A, allysine, and histamine. While preliminary, these results corroborate that metabolic profiles may be useful to differentiate l-carnitine treatment responsiveness. Furthermore, these data show that the metabolic signature of l-carnitine-treated nonsurvivors is associated with a severity of illness (e.g., vascular inflammation) that is not routinely clinically detected.

Inkjet-printed micro-calibration standards for ultraquantitative Raman spectral cytometry.

Herein we report the development of a cytometric analysis platform for measuring the contents of individual cells in absolute (picogram) scales; this study represents the first report of Raman-based quantitation of the absolute mass - or the total amount - of multiple endogenous biomolecules within single-cells. To enable ultraquantitative calibration, we engineered single-cell-sized micro-calibration standards of known composition by inkjet-printer deposition of biomolecular components in microarrays across the surface of silicon chips. We demonstrate clinical feasibility by characterizing the compositional phenotype of human skin fibroblast and porcine alveolar macrophage cell populations in the respective contexts of Niemann-Pick disease and drug-induced phospholipidosis: two types of lipid storage disorders. We envision this microanalytical platform as the foundation for many future biomedical applications, ranging from diagnostic assays to pathological analysis to advanced pharmaco/toxicokinetic research studies.

Pharmacometabolomics reveals a role for histidine, phenylalanine, and threonine in the development of paclitaxel-induced peripheral neuropathy.

PURPOSE: Approximately 25% of breast cancer patients experience treatment delays or discontinuation due to paclitaxel-induced peripheral neuropathy (PN). Currently, there are no predictive biomarkers of PN. Pharmacometabolomics is an informative tool for biomarker discovery of drug toxicity. We conducted a secondary whole blood pharmacometabolomics analysis to assess the association between pretreatment metabolome, early treatment-induced metabolic changes, and the development of PN. METHODS: Whole blood samples were collected pre-treatment (BL), just before the end of the first paclitaxel infusion (EOI), and 24 h after the first infusion (24H) from sixty patients with breast cancer receiving (80 mg/m2) weekly treatment. Neuropathy was assessed at BL and prior to each infusion using the sensory subscale (CIPN8) of the EORTC CIPN20 questionnaire. Blood metabolites were quantified from 1-D-1H-nuclear magnetic resonance spectra using Chenomx® software. Metabolite concentrations were normalized in preparation for Pearson correlation and one-way repeated measures ANOVA with multiple comparisons corrected by false discovery rate (FDR). RESULTS: Pretreatment histidine, phenylalanine, and threonine concentrations were inversely associated with maximum change in CIPN8 (ΔCIPN8) (p < 0.02; FDR ≤ 25%). Paclitaxel caused a significant change in concentrations of 2-hydroxybutyrate, 3-hydroxybutyrate, pyruvate, o-acetylcarnitine, and several amino acids from BL to EOI and/or 24H (p < 0.05; FDR ≤ 25%), although these changes were not associated with ΔCIPN8. CONCLUSIONS: Whole blood metabolomics is a feasible approach to identify potential biomarker candidates of paclitaxel-induced PN. The findings suggest that pretreatment concentrations of histidine, phenylalanine, and threonine may be predictive of the severity of future PN and paclitaxel-induced metabolic changes may be related to disruption of energy homeostasis.

Associations of the plasma lipidome with mortality in the acute respiratory distress syndrome: a longitudinal cohort study.

BACKGROUND: It is unknown if the plasma lipidome is a useful tool for improving our understanding of the acute respiratory distress syndrome (ARDS). Therefore, we measured the plasma lipidome of individuals with ARDS at two time-points to determine if changes in the plasma lipidome distinguished survivors from non-survivors. We hypothesized that both the absolute concentration and change in concentration over time of plasma lipids are associated with 28-day mortality in this population. METHODS: Samples for this longitudinal observational cohort study were collected at multiple tertiary-care academic medical centers as part of a previous multicenter clinical trial. A mass spectrometry shot-gun lipidomic assay was used to quantify the lipidome in plasma samples from 30 individuals. Samples from two different days were analyzed for each subject. After removing lipids with a coefficient of variation > 30%, differences between cohorts were identified using repeated measures analysis of variance. The false discovery rate was used to adjust for multiple comparisons. Relationships between significant compounds were explored using hierarchical clustering of the Pearson correlation coefficients and the magnitude of these relationships was described using receiver operating characteristic curves. RESULTS: The mass spectrometry assay reliably measured 359 lipids. After adjusting for multiple comparisons, 90 compounds differed between survivors and non-survivors. Survivors had higher levels for each of these lipids except for five membrane lipids. Glycerolipids, particularly those containing polyunsaturated fatty acid side-chains, represented many of the lipids with higher concentrations in survivors. The change in lipid concentration over time did not differ between survivors and non-survivors. CONCLUSIONS: The concentration of multiple plasma lipids is associated with mortality in this group of critically ill patients with ARDS. Absolute lipid levels provided more information than the change in concentration over time. These findings support future research aimed at integrating lipidomics into critical care medicine.

An Expandable Mechanopharmaceutical Device (3): a Versatile Raman Spectral Cytometry Approach to Study the Drug Cargo Capacity of Individual Macrophages.

PURPOSE: To improve cytometric phenotyping abilities and better understand cell populations with high interindividual variability, a novel Raman-based microanalysis was developed to characterize macrophages on the basis of chemical composition, specifically to measure and characterize intracellular drug distribution and phase separation in relation to endogenous cellular biomolecules. METHODS: The microanalysis was developed for the commercially-available WiTec alpha300R confocal Raman microscope. Alveolar macrophages were isolated and incubated in the presence of pharmaceutical compounds nilotinib, chloroquine, or etravirine. A Raman data processing algorithm was specifically developed to acquire the Raman signals emitted from single-cells and calculate the signal contributions from each of the major molecular components present in cell samples. RESULTS: Our methodology enabled analysis of the most abundant biochemicals present in typical eukaryotic cells and clearly identified "foamy" lipid-laden macrophages throughout cell populations, indicating feasibility for cellular lipid content analysis in the context of different diseases. Single-cell imaging revealed differences in intracellular distribution behavior for each drug; nilotinib underwent phase separation and self-aggregation while chloroquine and etravirine accumulated primarily via lipid partitioning. CONCLUSIONS: This methodology establishes a versatile cytometric analysis of drug cargo loading in macrophages requiring small numbers of cells with foreseeable applications in toxicology, disease pathology, and drug discovery.

An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver.

PURPOSE: Drug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage. METHODS: Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity. RESULTS: Granuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals. CONCLUSIONS: Granuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering "organoids" -an induced, specialized sub-compartment- to limit tissue damage.

An Expandable Mechanopharmaceutical Device (1): Measuring the Cargo Capacity of Macrophages in a Living Organism.

PURPOSE: Clofazimine (CFZ) is an FDA-approved, poorly soluble small molecule drug that precipitates as crystal-like drug inclusions (CLDIs) which accumulate in acidic cytoplasmic organelles of macrophages. In this study, we considered CLDIs as an expandable mechanopharmaceutical device, to study how macrophages respond to an increasingly massive load of endophagolysosomal cargo. METHODS: First, we experimentally tested how the accumulation of CFZ in CLDIs impacted different immune cell subpopulations of different organs. Second, to further investigate the mechanism of CLDI formation, we asked whether specific accumulation of CFZ hydrochloride crystals in lysosomes could be explained as a passive, thermodynamic equilibrium phenomenon. A cellular pharmacokinetic model was constructed, simulating CFZ accumulation driven by pH-dependent ion trapping of the protonated drug in the acidic lysosomes, followed by the precipitation of CFZ hydrochloride salt via a common ion effect caused by high chloride concentrations. RESULTS: While lower loads of CFZ were mostly accommodated in lung macrophages, increased CFZ loading was accompanied by organ-specific changes in macrophage numbers, size and intracellular membrane architecture, maximizing the cargo storage capabilities. With increasing loads, the total cargo mass and concentrations of CFZ in different organs diverged, while that of individual macrophages converged. The simulation results support the notion that the proton and chloride ion concentrations of macrophage lysosomes are sufficient to drive the massive, cell type-selective accumulation and growth of CFZ hydrochloride biocrystals. CONCLUSION: CLDIs effectively function as an expandable mechanopharmaceutical device, revealing the coordinated response of the macrophage population to an increasingly massive, whole-organism endophagolysosomal cargo load.

Clofazimine Biocrystal Accumulation in Macrophages Upregulates Interleukin 1 Receptor Antagonist Production To Induce a Systemic Anti-Inflammatory State.

Clofazimine (CFZ) is a poorly soluble antibiotic and anti-inflammatory drug indicated for the treatment of leprosy. In spite of its therapeutic value, CFZ therapy is accompanied by the formation of drug biocrystals that accumulate within resident tissue macrophages, without obvious toxicological manifestations. Therefore, to specifically elucidate the off-target consequences of drug bioaccumulation in macrophages, we compared the level of inflammasome activation in CFZ-accumulating organs (spleen, liver and lung) in mice after 2 and 8 weeks of CFZ treatment when the drug exists in soluble and insoluble (biocrystalline) forms, respectively. Surprisingly, the results showed a drastic reduction in caspase 1 and interleukin-1ß (IL-1ß) cleavage in the livers of mice treated with CFZ for 8 weeks (8-week-CFZ-treated mice) compared to 2-week-CFZ-treated and control mice, which was accompanied by a 3-fold increase in hepatic IL-1 receptor antagonist (IL-1RA) production and a 21-fold increase in serum IL-1RA levels. In the lung and spleen, IL-1ß cleavage and tumor necrosis factor alpha expression were unaffected by soluble or biocrystal CFZ forms. Functionally, there was a drastic reduction of carrageenan- and lipopolysaccharide-induced inflammation in the footpads and lungs, respectively, of 8-week-CFZ-treated mice. This immunomodulatory activity of CFZ biocrystal accumulation was attributable to the upregulation of IL-1RA, since CFZ accumulation had minimal effect in IL-1RA knockout mice or 2-week-CFZ-treated mice. In conclusion, CFZ accumulation and biocrystal formation in resident tissue macrophages profoundly altered the host's immune system and prompted an IL-1RA-dependent, systemic anti-inflammatory response.

A Role for Low Density Lipoprotein Receptor-Related Protein 1 in the Cellular Uptake of Tissue Plasminogen Activator in the Lungs.

PURPOSE: To gain knowledge of lung clearance mechanisms of inhaled tissue plasminogen activator (tPA). METHODS: Using an in vivo mouse model and ex vivo murine whole organ cell suspensions, we examined the capability of the lungs to utilize LRP1 receptor-mediated endocytosis (RME) for the uptake of exogenous tPA with and without an LRP1 inhibitor, receptor associated protein (RAP), and quantitatively compared it to the liver. We also used a novel imaging technique to assess the amount LRP1 in sections of mouse liver and lung. RESULTS: Following intratracheal administration, tPA concentrations in the bronchoalveolar lavage fluid (BALF) declined over time following two-compartment pharmacokinetics suggestive of a RME clearance mechanism. Ex vivo studies showed that lung and liver cells are similarly capable of tPA uptake via LRP1 RME which was reduced by ~50% by RAP. The comparable lung and liver uptake of tPA is likely due to equivalent amounts of LRP1 of which there was an abundance in the alveolar epithelium. CONCLUSIONS: Our findings indicate that LRP1 RME is a candidate clearance mechanism for inhaled tPA which has implications for the development of safe and effective dosing regimens of inhaled tPA for the treatment of plastic bronchitis and other fibrin-inflammatory airway diseases in which inhaled tPA may have utility.

A far-red fluorescent probe for flow cytometry and image-based functional studies of xenobiotic sequestering macrophages.

Clofazimine (CFZ) is an optically active, red-colored chemotherapeutic agent that is FDA approved for the treatment of leprosy and is on the World Health Organization's list of essential medications. Interestingly, CFZ massively accumulates in macrophages where it forms crystal-like drug inclusions (CLDIs) after oral administration of the drug in animals and humans. The analysis of the fluorescence spectra of CLDIs formed by resident tissue macrophages revealed that CFZ, when accumulated as CLDIs, undergoes a red shift in fluorescence excitation (from Ex: 540-570 to 560-600 nm) and emission (Em: 560-580 to 640-700 nm) signal relative to the soluble and free-base crystal forms of CFZ. Using epifluorescence microscopy, CLDI(+) cells could be identified, relative to CLDI(-) cells, based on a >3-fold increment in mean fluorescence signal at excitation 640 nm and emission at 670 nm. Similarly, CLDI(+) cells could be identified by flow cytometry, based on a >100-fold increment in mean fluorescence signal using excitation lasers at 640 nm and emission detectors >600 nm. CLDI's fluorescence excitation and emission was orthogonal to that of cell viability dyes such as propidium iodide and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), cellular staining dyes such as Hoechst 33342 (nucleus) and FM 1-43 (plasma membrane), as well as many other fluorescently tagged antibodies used for immunophenotyping analyses. In vivo, >85% of CLDI(+) cells in the peritoneal exudate were F4/80(+) macrophages and >97% of CLDI(+) cells in the alveolar exudate were CD11c(+). Most importantly, the viability of cells was minimally affected by the presence of CLDIs. Accordingly, these results establish that CFZ fluorescence in CLDIs is suitable for quantitative flow cytometric phenotyping analysis and functional studies of xenobiotic sequestering macrophages.