Microbiology
Abstract
IL-17A (IL-17), a driver of the inflammatory phase of fracture repair, is produced locally by several cell lineages including γδ T cells and Th17 cells. However, the origin and relevance for fracture repair of these T cells are unknown. Here we show that fractures rapidly expanded callus γδ T cells, which led to increased gut permeability by promoting systemic inflammation. When the microbiota contained the Th17 cell-inducing taxa segmented filamentous bacteria (SFB), activation of γδ T cells was followed by expansion of intestinal Th17 cells, their migration to the callus, and improvement of fracture repair. Mechanistically, fractures increased the S1P-receptor-1 (S1PR1) mediated egress of Th17 cells from the intestine and enhanced their homing to the callus through a CCL20 mediated mechanism. Fracture repair was impaired by deletion of γδ T cells, depletion of the microbiome by antibiotics, blockade of Th17 cell egress from the gut or antibody neutralization of Th17 cell influx into the callus. These findings demonstrated the relevance of the microbiome and T cell trafficking for fracture repair. Modifications of microbiome composition via Th17 cell-inducing bacteriotherapy and avoidance of broad-spectrum antibiotics may represent novel therapeutic strategies to optimize fracture healing.
Authors
Hamid Y. Dar, Daniel S. Perrien, Subhashis Pal, Andreea Stoica, Sasidhar Uppuganti, Jeffry S. Nyman, Rheinallt M. Jones, M. Neale Weitzmann, Roberto Pacifici
Abstract
Multiple Sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specifically causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin (ETX)-producing strains of C. perfringens within their gut microbiomes compared to healthy controls (HC). MS patient-derived isolates produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.
Authors
Yinghua Ma, David Sannino, Jennifer R. Linden, Sylvia Haigh, Baohua Zhao, John B. Grigg, Paul Zumbo, Friederike Dündar, Daniel J. Butler, Caterina P. Profaci, Kiel M. Telesford, Paige N. Winokur, Kareem R. Rumah, Susan A. Gauthier, Vincent A. Fischetti, Bruce A. McClane, Francisco A. Uzal, Lily Zexter, Michael Mazzucco, Richard Rudick, David Danko, Evan Balmuth, Nancy Nealon, Jai Perumal, Ulrike W. Kaunzner, Ilana L. Brito, Zhengming Chen, Jenny Z. Xiang, Doron Betel, Richard Daneman, Gregory F. Sonnenberg, Christopher E. Mason, Timothy Vartanian
Abstract
Induction of lipid-laden foamy macrophages is a cellular hallmark of tuberculosis (TB) disease, which involves transformation of infected phagolysomes from a site of killing into a nutrient-rich replicative niche. Here we show that a terpenyl nucleoside shed from Mycobacterium tuberculosis (Mtb), 1-tuberculosinyladenosine (1-TbAd), causes lysosomal maturation arrest and autophagy blockade, leading to lipid storage in M1 macrophages. Pure 1-TbAd, or infection with terpenyl nucleoside-producing Mtb, caused intralysosomal and peribacillary lipid storage patterns that match both the molecules and subcellular locations known in foamy macrophages. Lipidomics showed that 1-TbAd induced storage of triacylglycerides and cholesterylesters, and 1-TbAd increased Mtb growth under conditions of restricted lipid access in macrophages. Further, lipidomics dentified 1-TbAd induced lipid substrates that define Gaucher's disease, Wolman's disease and other inborn lysosomal storage diseases. These data identify genetic and molecular causes of Mtb-induced lysosomal failure, leading to successful testing of an gonist of TRPML1 calcium channels that reverses lipid storage in cells. These data establish the host-directed cellular functions of an orphan effector molecule that promotes survival in macrophages, providing both an upstream cause and detailed picture of lysosome failure in foamy macrophages.
Authors
Melissa Bedard, Sanne van der Niet, Elliott M. Bernard, Gregory H. Babunovic, Tan-Yun Cheng, Beren Aylan, Anita E. Grootemaat, Sahadevan Raman, Laure Botella, Eri Ishikawa, Mary P. O'Sullivan, Seonadh O'Leary, Jacob A. Mayfield, Jeffrey Buter, Adriaan J. Minnaard, Sarah M. Fortune, Leon O. Murphy, Daniel S. Ory, Joseph Keane, Sho Yamasaki, Maximiliano Gabriel Gutierrez, Nicole van der Wel, D. Branch Moody
Abstract
The alternative sigma factor RpoS in Borrelia burgdorferi (Bb), the Lyme disease pathogen, is responsible for programmatic positive and negative gene regulation essential for the spirochete’s dual-host enzootic cycle. RpoS is expressed during tick-to-mammal transmission and throughout mammalian infection. Although the mammalian-phase RpoS regulon is well described, its counterpart during the transmission blood meal is unknown. Here, we used Bb-specific transcript enrichment by TBDCapSeq to compare the transcriptomes of wild-type and ΔrpoS Bb in engorged nymphs and following mammalian host-adaptation within dialysis membrane chambers. TBDCapSeq revealed dramatic changes in the contours of the RpoS regulon within ticks and mammals and further confirmed that RpoS-mediated repression is specific to the mammalian-phase of Bb’s enzootic cycle. We also provide evidence that RpoS-dependent gene regulation, including repression of tick-phase genes, is required for persistence in mice. Comparative transcriptomics of engineered Bb strains revealed that BosR, a non-canonical Fur family regulator, and the c-di-GMP effector PlzA reciprocally regulate RpoS function. BosR is required for RpoS-mediated transcription activation and repression in addition to its well-defined role promoting RpoN-dependent transcription of rpoS. During transmission, liganded-PlzA antagonizes RpoS-mediated repression, presumably acting through BosR.
Authors
André A. Grassmann, Rafal Tokarz, Caroline Golino, Melissa A. McLain, Ashley M. Groshong, Justin D. Radolf, Melissa J. Caimano
Abstract
A close association with its vertebrate and tick hosts allows Borrelia burgdorferi, the bacterium responsible for Lyme disease, to eliminate many metabolic pathways and instead scavenge key nutrients from the host. A lipid-defined culture medium was developed to demonstrate that exogenous lipids are an essential nutrient of B. burgdorferi, which can accumulate intact phospholipids from its environment to support growth. Antibody responses to host phospholipids were studied in mice and humans using an antiphospholipid ELISA. Several of these environmentally acquired phospholipids including phosphatidylserine and phosphatidic acid, as well as borrelial phosphatidylcholine, are the targets of antibodies that arose early in infection in the mouse model. Patients with acute infections demonstrated antibody responses to the same lipids. The elevation of antiphospholipid antibodies predicted early infection with better sensitivity than did the standardized 2-tier tests currently used in diagnosis. Sera obtained from patients with Lyme disease before and after antibiotic therapy showed declining antiphospholipid titers after treatment. Further study will be required to determine whether these antibodies have utility in early diagnosis of Lyme disease, tracking of the response to therapy, and diagnosis of reinfection, areas in which current standardized tests are inadequate.
Authors
Peter J. Gwynne, Luke H. Clendenen, Siu-Ping Turk, Adriana R. Marques, Linden T. Hu
Abstract
The gut microbiome shapes local and systemic immunity. The liver is presumed to be a protected sterile site. As such, a hepatic microbiome has not been examined. Here, we showed a liver microbiome in mice and humans that is distinct from the gut and is enriched in Proteobacteria. It undergoes dynamic alterations with age and is influenced by the environment and host physiology. Fecal microbial transfer experiments revealed that the liver microbiome is populated from the gut in a highly selective manner. Hepatic immunity is dependent on the microbiome, specifically Bacteroidetes species. Targeting Bacteroidetes with oral antibiotics reduced hepatic immune cells by ~90%, prevented APC maturation, and mitigated adaptive immunity. Mechanistically, our findings are consistent with presentation of Bacteroidetes-derived glycosphingolipids to NKT cells promoting CCL5 signaling, which drives hepatic leukocyte expansion and activation, among other possible host-microbe interactions. Collectively, we reveal a microbial – glycosphingolipid – NKT – CCL5 axis that underlies hepatic immunity.
Authors
Joshua C. Leinwand, Bidisha Paul, Ruonan Chen, Fangxi Xu, Maria A. Sierra, Madan M. Paluru, Sumant Nanduri, Carolina G. Alcantara Hirsch, Sorin A.A. Shadaloey, Fan Yang, Salma A. Adam, Qianhao Li, Michelle Bandel, Inderdeep Gakhal, Lara Appiah, Yuqi Guo, Mridula Vardhan, Zia J. Flaminio, Emilie R. Grodman, Ari Mermelstein, Wei Wang, Brian Diskin, Berk Aykut, Mohammed Khan, Gregor Werba, Smruti Pushalkar, Mia McKinstry, Zachary Kluger, Jaimie J. Park, Brandon Hsieh, Kristen Dancel-Manning, Feng-Xia Liang, James S. Park, Anjana Saxena, Xin Li, Neil D. Theise, Deepak Saxena, George Miller
Abstract
BACKGROUND. Bacterial vaginosis (BV) causes genital inflammation and increases HIV risk, while a vaginal microbiota dominated by Lactobacillus species is associated with immune quiescence and relative HIV protection. BV treatment reduces genital inflammation, but it is unclear whether this is driven by a decrease in BV-associated bacteria or an increase in Lactobacillus. METHODS. To evaluate the short-term impact of standard BV treatment on genital immunology and the vaginal microbiota, vaginal swabs were collected immediately before and after metronidazole treatment for BV and analyzed with multiplex ELISA, metagenomic sequencing, and quantitative polymerase chain reaction. RESULTS. Topical metronidazole treatment rapidly reduced vaginal levels of proinflammatory cytokines, chemokines, and soluble immune markers of epithelial barrier disruption. Although the vaginal microbiota shifted to dominance by L. iners or L. jensenii, this proportional shift was primarily driven by a 2-4 log10 fold reduction in BV-associated bacteria absolute abundance; BV treatment induced no change in the absolute abundance of L. crispatus or L. iners, and only minor (<1 log10 fold) increases in L. gasseri and L. jensenii that were not independently associated with reduced inflammation in multivariable models. CONCLUSION. The genital immune benefits that are associated with Lactobacillus dominance following BV treatment were not directly attributable to an absolute increase in lactobacilli, but rather to the loss of BV-associated bacteria. TRAIL REGISTRATION. Participants were recruited as part of a randomized controlled trial (NCT02766023) from 2016 to 2020. FUNDING. Canadian Institutes of Health Research (PJT-156123) and the National Institute of Allergy and Infectious Diseases (HHSN2722013000141 and HHSN27200007).
Authors
Eric Armstrong, Anke Hemmerling, Steve Miller, Kerianne E. Burke, Sara J. Newmann, Sheldon R. Morris, Hilary Reno, Sanja Huibner, Maria Kulikova, Rachel Liu, Emily D. Crawford, Gloria R. Castañeda, Nico Nagelkerke, Bryan Coburn, Craig R. Cohen, Rupert Kaul
Abstract
Mycobacterium tuberculosis (M. tuberculosis) causes an enormous burden of disease worldwide. As a central aspect of its pathogenesis, M. tuberculosis grows in macrophages, and host and microbe influence each other’s metabolism. To define the metabolic impact of M. tuberculosis infection, we performed global metabolic profiling of M. tuberculosis–infected macrophages. M. tuberculosis induced metabolic hallmarks of inflammatory macrophages and a prominent signature of cholesterol metabolism. We found that infected macrophages accumulate cholestenone, a mycobacterial-derived, oxidized derivative of cholesterol. We demonstrated that the accumulation of cholestenone in infected macrophages depended on the M. tuberculosis enzyme 3β-hydroxysteroid dehydrogenase (3β-Hsd) and correlated with pathogen burden. Because cholestenone is not a substantial human metabolite, we hypothesized it might be diagnostic of M. tuberculosis infection in clinical samples. Indeed, in 2 geographically distinct cohorts, sputum cholestenone levels distinguished subjects with tuberculosis (TB) from TB-negative controls who presented with TB-like symptoms. We also found country-specific detection of cholestenone in plasma samples from M. tuberculosis–infected subjects. While cholestenone was previously thought to be an intermediate required for cholesterol degradation by M. tuberculosis, we found that M. tuberculosis can utilize cholesterol for growth without making cholestenone. Thus, the accumulation of cholestenone in clinical samples suggests it has an alternative role in pathogenesis and could be a clinically useful biomarker of TB infection.
Authors
Pallavi Chandra, Héloise Coullon, Mansi Agarwal, Charles W. Goss, Jennifer A. Philips
Abstract
Tuberculous (TB) meningitis is the most severe form of TB, requiring 12 months of multidrug treatment for cure, and is associated with high morbidity and mortality. High-dose rifampin (35 mg/kg/day) is safe and improves the bactericidal activity of the standard-dose (10 mg/kg/day) rifampin-containing TB regimen in pulmonary TB. However, there are conflicting clinical data regarding its benefit for TB meningitis, where outcomes may also be associated with intracerebral inflammation. In this study, we conducted cross-species studies in mice and rabbits, demonstrating that an intensified high-dose rifampin-containing regimen has significantly improved bactericidal activity for TB meningitis over the first-line, standard-dose rifampin regimen, without an increase in intracerebral inflammation. Positron emission tomography in live animals demonstrated spatially compartmentalized, lesion-specific pathology, with post-mortem analyses showing discordant brain tissue and cerebrospinal fluid rifampin levels and inflammatory markers. Longitudinal multimodal imaging in the same cohort of animals during TB treatment as well as imaging studies in two cohorts of TB patients demonstrated that spatiotemporal changes in localized blood-brain barrier disruption in TB meningitis are an important driver of rifampin brain exposure. These data provide unique insights into the mechanisms underlying high-dose rifampin in TB meningitis with important implications for developing new antibiotic treatments for infections.
Authors
Camilo A. Ruiz-Bedoya, Filipa Mota, Elizabeth W. Tucker, Farina J. Mahmud, Maria I. Reyes-Mantilla, Clara Erice, Melissa Bahr, Kelly Flavahan, Patricia De Jesus, John Kim, Catherine A. Foss, Charles A. Peloquin, Dima A. Hammoud, Alvaro A. Ordonez, Carlos A. Pardo, Sanjay K. Jain
Abstract
Macrophages deploy numerous strategies to combat invasion by microbes. One tactic is to restrict acquisition of diverse nutrients including trace metals, a process termed nutritional immunity. Intracellular pathogens adapt to a resource poor environment by marshalling mechanisms to harvest nutrients. Carbon acquisition is crucial for pathogen survival; compounds that reduce availability are a potential strategy to control intracellular replication. Treatment of macrophages with the glucose analog, 2-deoxy-D-glucose (2-DG), armed phagocytes to eliminate the intracellular fungal pathogen Histoplasma capsulatum in vitro and in vivo. Killing did not rely on altering access to carbon-containing molecules, or changes in ATP, ER stress, or autophagy. Unexpectedly, 2-DG undermined import of exogenous zinc into macrophages decreasing the quantity of cytosolic and phagosomal zinc. The fungus perished as a result of zinc starvation. This change in metal ingress was not ascribed to a defect in a single importer; rather, there was a collective impairment in transporter activity. This undescribed effect promotes the antifungal machinery of macrophages and expands the complexity of 2-DG activities far beyond manipulating glycolysis. Mechanistic metabolic studies employing 2-DG will have to consider its effect on zinc transport. Our preclinical data support consideration of this agent as a possible adjunctive therapy for histoplasmosis.
Authors
Diego C.P. Rossi, Julio A. Landero Figueroa, William R. Buesing, Kathleen Candor, Logan T. Blancett, Heather M. Evans, Rena Lenchitz, Bradford L. Crowther, Waleed Elsegeiny, Peter R. Williamson, Jan Rupp, George S. Deepe Jr.
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ISSN: 0021-9738 (print), 1558-8238 (online)