Publications by authors named "Larry S Schlesinger"

154 Publications

IFN signaling and neutrophil degranulation transcriptional signatures are induced during SARS-CoV-2 infection.

Commun Biol 2021 03 5;4(1):290. Epub 2021 Mar 5.

Department of Molecular Microbiology, Washington University in St. Louis, St. Louis, MO, USA.

SARS-CoV-2 virus has infected more than 92 million people worldwide resulting in the Coronavirus disease 2019 (COVID-19). Using a rhesus macaque model of SARS-CoV-2 infection, we have characterized the transcriptional signatures induced in the lungs of juvenile and old macaques following infection. Genes associated with Interferon (IFN) signaling, neutrophil degranulation and innate immune pathways are significantly induced in macaque infected lungs, while pathways associated with collagen formation are downregulated, as also seen in lungs of macaques with tuberculosis. In COVID-19, increasing age is a significant risk factor for poor prognosis and increased mortality. Type I IFN and Notch signaling pathways are significantly upregulated in lungs of juvenile infected macaques when compared with old infected macaques. These results are corroborated with increased peripheral neutrophil counts and neutrophil lymphocyte ratio in older individuals with COVID-19 disease. Together, our transcriptomic studies have delineated disease pathways that improve our understanding of the immunopathogenesis of COVID-19.
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http://dx.doi.org/10.1038/s42003-021-01829-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7935909PMC
March 2021

Responses to acute infection with SARS-CoV-2 in the lungs of rhesus macaques, baboons and marmosets.

Nat Microbiol 2021 01 18;6(1):73-86. Epub 2020 Dec 18.

Texas Biomedical Research Institute, San Antonio, TX, USA.

Non-human primate models will expedite therapeutics and vaccines for coronavirus disease 2019 (COVID-19) to clinical trials. Here, we compare acute severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in young and old rhesus macaques, baboons and old marmosets. Macaques had clinical signs of viral infection, mild to moderate pneumonitis and extra-pulmonary pathologies, and both age groups recovered in two weeks. Baboons had prolonged viral RNA shedding and substantially more lung inflammation compared with macaques. Inflammation in bronchoalveolar lavage was increased in old versus young baboons. Using techniques including computed tomography imaging, immunophenotyping, and alveolar/peripheral cytokine response and immunohistochemical analyses, we delineated cellular immune responses to SARS-CoV-2 infection in macaque and baboon lungs, including innate and adaptive immune cells and a prominent type-I interferon response. Macaques developed T-cell memory phenotypes/responses and bystander cytokine production. Old macaques had lower titres of SARS-CoV-2-specific IgG antibody levels compared with young macaques. Acute respiratory distress in macaques and baboons recapitulates the progression of COVID-19 in humans, making them suitable as models to test vaccines and therapies.
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http://dx.doi.org/10.1038/s41564-020-00841-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7890948PMC
January 2021

Lethality of SARS-CoV-2 infection in K18 human angiotensin-converting enzyme 2 transgenic mice.

Nat Commun 2020 11 30;11(1):6122. Epub 2020 Nov 30.

Texas Biomedical Research Institute, San Antonio, TX, 78227, USA.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.
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http://dx.doi.org/10.1038/s41467-020-19891-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7705712PMC
November 2020

GPR183 Regulates Interferons, Autophagy, and Bacterial Growth During Infection and Is Associated With TB Disease Severity.

Front Immunol 2020 6;11:601534. Epub 2020 Nov 6.

Translational Research Institute-Mater Research Institute, The University of Queensland, Brisbane, QLD, Australia.

Oxidized cholesterols have emerged as important signaling molecules of immune function, but little is known about the role of these oxysterols during mycobacterial infections. We found that expression of the oxysterol-receptor GPR183 was reduced in blood from patients with tuberculosis (TB) and type 2 diabetes (T2D) compared to TB patients without T2D and was associated with TB disease severity on chest x-ray. GPR183 activation by 7,25-dihydroxycholesterol (7,25-OHC) reduced growth of (Mtb) and BCG in primary human monocytes, an effect abrogated by the GPR183 antagonist GSK682753. Growth inhibition was associated with reduced IFN-β and IL-10 expression and enhanced autophagy. Mice lacking GPR183 had significantly increased lung Mtb burden and dysregulated IFNs during early infection. Together, our data demonstrate that GPR183 is an important regulator of intracellular mycobacterial growth and interferons during mycobacterial infection.
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http://dx.doi.org/10.3389/fimmu.2020.601534DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677584PMC
June 2021

Cy3-tilmanocept labeling of macrophages in joints of mice with antibody-induced arthritis and synovium of human patients with rheumatoid arthritis.

J Orthop Res 2021 04 15;39(4):821-830. Epub 2020 Dec 15.

Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio, USA.

γ-Tilmanocept ( Tc-tilmanocept) is a receptor-directed, radiolabeled tracer that is FDA-approved for guiding sentinel lymph node biopsy. Tilmanocept binds the C-type lectin mannose receptor (MR, CD206) on macrophages. In this study, nonradioactive, fluorescently-labeled Cy3-tilmanocept was used to detect CD206 mononuclear cells in the cartilage of mice with antibody-induced arthritis and in the synovial fluid and tissue of human subjects with rheumatoid arthritis (RA) for comparison with osteoarthritis (OA), and healthy volunteer (HV) controls. Murine arthritis was induced by injection of monoclonal anti-cartilage antibody followed by injection of Escherichia coli lipopolysaccharide. Post-arthritis development (7-11 days), the mice were injected intravenously with Cy3-tilmanocept followed by in vivo and ex vivo epifluorescence imaging. Two-photon imaging, immunofluorescence, and immunohistochemistry were used to identify articular and synovial macrophages (CD206, F4/80, and Cy3-tilmanocept binding) in murine tissues. Cy3-tilmanocept epifluorescence was present in arthritic knees and elbows of murine tissues; no radiographic changes were noted in the skeletons. However, inflammatory arthritic changes were apparent by histopathology and immunohistochemistry (F4/80), immunofluorescence (CD206) and Cy3-tilmanocept binding. In human RA synovial fluid, Cy3-tilmanocept staining correlated with CD206 /CD16 cells; negligible labeling was observed in OA samples. Cy3-tilmanocept colocalized with CD206 and staining was significantly higher in RA synovial tissue compared to OA or HV. Our results demonstrate that imaging with Cy3-tilmanocept can detect in vivo inflammatory, CD206 macrophages in an early arthritis animal model and in human RA patients. These data establish a novel tool for preclinical research of early arthritis and have implications for early RA detection and monitoring of therapeutic efficacy in humans.
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http://dx.doi.org/10.1002/jor.24900DOI Listing
April 2021

Phagosome-regulated mTOR signalling during sarcoidosis granuloma biogenesis.

Eur Respir J 2021 Mar 18;57(3). Epub 2021 Mar 18.

Texas Biomedical Research Institute, San Antonio, TX, USA.

Introduction: Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis.

Methods: A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established human granuloma model wherein peripheral blood mononuclear cells were exposed to antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin).

Results: In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction.

Conclusions: Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.
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http://dx.doi.org/10.1183/13993003.02695-2020DOI Listing
March 2021

Current Sarcoidosis Models and the Importance of Focusing on the Granuloma.

Front Immunol 2020 4;11:1719. Epub 2020 Aug 4.

Division of Pulmonary, Allergy, Critical Care, and Sleep Medicine, The Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, United States.

The inability to effectively model sarcoidosis in the laboratory or in animals continues to hinder the discovery and translation of new, targeted treatments. The granuloma is the signature pathological hallmark of sarcoidosis, yet there are significant knowledge gaps that exist with regard to how granulomas form. Significant progress toward improved therapeutic and prognostic strategies in sarcoidosis hinges on tractable experimental models that recapitulate the process of granuloma formation in sarcoidosis and allow for mechanistic insights into the molecular events involved. Through its inherent representation of the complex genetics underpinning immune cell dysregulation in sarcoidosis, a recently developed human granuloma model holds promise in providing detailed mechanistic insight into sarcoidosis-specific disease regulating pathways at play during early stages of granuloma formation. The purpose of this review is to critically evaluate current sarcoidosis models and assess their potential to progress the field toward the goal of improved therapies in this disease. We conclude with the potential integrated use of preclinical models to accelerate progress toward identifying and testing new drugs and drug combinations that can be rapidly brought to clinical trials.
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http://dx.doi.org/10.3389/fimmu.2020.01719DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417311PMC
April 2021

IFN signaling and neutrophil degranulation transcriptional signatures are induced during SARS-CoV-2 infection.

bioRxiv 2020 Aug 6. Epub 2020 Aug 6.

Southwest National Primate Research Center, Texas Biomedical Research Institute, San Antonio, TX 78245.

The novel virus SARS-CoV-2 has infected more than 14 million people worldwide resulting in the Coronavirus disease 2019 (COVID-19). Limited information on the underlying immune mechanisms that drive disease or protection during COVID-19 severely hamper development of therapeutics and vaccines. Thus, the establishment of relevant animal models that mimic the pathobiology of the disease is urgent. Rhesus macaques infected with SARS-CoV-2 exhibit disease pathobiology similar to human COVID-19, thus serving as a relevant animal model. In the current study, we have characterized the transcriptional signatures induced in the lungs of juvenile and old rhesus macaques following SARS-CoV-2 infection. We show that genes associated with Interferon (IFN) signaling, neutrophil degranulation and innate immune pathways are significantly induced in macaque infected lungs, while pathways associated with collagen formation are downregulated. In COVID-19, increasing age is a significant risk factor for poor prognosis and increased mortality. We demonstrate that Type I IFN and Notch signaling pathways are significantly upregulated in lungs of juvenile infected macaques when compared with old infected macaques. These results are corroborated with increased peripheral neutrophil counts and neutrophil lymphocyte ratio in older individuals with COVID-19 disease. In contrast, pathways involving VEGF are downregulated in lungs of old infected macaques. Using samples from humans with SARS-CoV-2 infection and COVID-19, we validate a subset of our findings. Finally, neutrophil degranulation, innate immune system and IFN gamma signaling pathways are upregulated in both tuberculosis and COVID-19, two pulmonary diseases where neutrophils are associated with increased severity. Together, our transcriptomic studies have delineated disease pathways to improve our understanding of the immunopathogenesis of COVID-19 to facilitate the design of new therapeutics for COVID-19.
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http://dx.doi.org/10.1101/2020.08.06.239798DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418717PMC
August 2020

Ornithine-A urea cycle metabolite enhances autophagy and controls Mycobacterium tuberculosis infection.

Nat Commun 2020 07 15;11(1):3535. Epub 2020 Jul 15.

Department of Pulmonary Immunology, Center for Biomedical Research, University of Texas Health Center, Tyler, TX, 75708, USA.

Macrophages are professional phagocytes known to play a vital role in controlling Mycobacterium tuberculosis (Mtb) infection and disease progression. Here we compare Mtb growth in mouse alveolar (AMs), peritoneal (PMs), and liver (Kupffer cells; KCs) macrophages and in bone marrow-derived monocytes (BDMs). KCs restrict Mtb growth more efficiently than all other macrophages and monocytes despite equivalent infections through enhanced autophagy. A metabolomics comparison of Mtb-infected macrophages indicates that ornithine and imidazole are two top-scoring metabolites in Mtb-infected KCs and that acetylcholine is the top-scoring in Mtb-infected AMs. Ornithine, imidazole and atropine (acetylcholine inhibitor) inhibit Mtb growth in AMs. Ornithine enhances AMPK mediated autophagy whereas imidazole directly kills Mtb by reducing cytochrome P450 activity. Intranasal delivery of ornithine or imidazole or the two together restricts Mtb growth. Our study demonstrates that the metabolic differences between Mtb-infected AMs and KCs lead to differences in the restriction of Mtb growth.
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http://dx.doi.org/10.1038/s41467-020-17310-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363810PMC
July 2020

Challenges of Immune Response Diversity in the Human Population Concerning New Tuberculosis Diagnostics, Therapies, and Vaccines.

Front Cell Infect Microbiol 2020 8;10:139. Epub 2020 Apr 8.

Host-Pathogen Interaction Program, Texas Biomedical Research Institute, San Antonio, TX, United States.

Universal approaches to the prevention and treatment of human diseases fail to take into account profound immune diversity resulting from genetic variations across populations. Personalized or precision medicine takes into account individual lifestyle, environment, and biology (genetics and immune status) and is being adopted in several disease intervention strategies such as cancer and heart disease. However, its application in infectious diseases, particularly global diseases such as tuberculosis (TB), is far more complex and in a state of infancy. Here, we discuss the impact of human genetic variations on immune responses and how they relate to failures seen in current TB diagnostic, therapy, and vaccine approaches across populations. We offer our perspective on the challenges and potential for more refined approaches going forward.
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http://dx.doi.org/10.3389/fcimb.2020.00139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156588PMC
June 2021

Identification of an Increased Alveolar Macrophage Subpopulation in Old Mice That Displays Unique Inflammatory Characteristics and Is Permissive to Infection.

J Immunol 2019 10 11;203(8):2252-2264. Epub 2019 Sep 11.

Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH 43210; and.

The elderly population is more susceptible to pulmonary infections, including tuberculosis. In this article, we characterize the impact of aging on the phenotype of mouse alveolar macrophages (AMs) and their response to Uninfected AMs were isolated from bronchoalveolar lavage of young (3 mo) and old (18 mo) C57BL/6 mice. AMs from old mice expressed higher mRNA levels of CCL2, IFN-β, IL-10, IL-12p40, TNF-α, and MIF than young mice, and old mice contained higher levels of CCL2, IL-1β, IFN-β, and MIF in their alveolar lining fluid. We identified two distinct AM subpopulations, a major CD11c CD11b population and a minor CD11c CD11b population; the latter was significantly increased in old mice (4-fold). Expression of CD206, TLR2, CD16/CD32, MHC class II, and CD86 was higher in CD11c CD11b AMs, and these cells expressed monocytic markers Ly6C, CX3CR1, and CD115, suggesting monocytic origin. Sorted CD11c CD11b AMs from old mice expressed higher mRNA levels of CCL2, IL-1β, and IL-6, whereas CD11c CD11b AMs expressed higher mRNA levels of immune-regulatory cytokines IFN-β and IL-10. CD11c CD11b AMs phagocytosed significantly more , which expressed higher RNA levels of genes required for survival. Our studies identify two distinct AM populations in old mice: a resident population and an increased CD11c CD11b AM subpopulation expressing monocytic markers, a unique inflammatory signature, and enhanced phagocytosis and survival when compared with resident CD11c CD11b AMs, which are more immune regulatory in nature.
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http://dx.doi.org/10.4049/jimmunol.1900495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783358PMC
October 2019

Targeting innate immunity for tuberculosis vaccination.

J Clin Invest 2019 09;129(9):3482-3491

Department of Internal Medicine and Radboud Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, Netherlands.

Vaccine development against tuberculosis (TB) is based on the induction of adaptive immune responses endowed with long-term memory against mycobacterial antigens. Memory B and T cells initiate a rapid and robust immune response upon encounter with Mycobacterium tuberculosis, thus achieving long-lasting protection against infection. Recent studies have shown, however, that innate immune cell populations such as myeloid cells and NK cells also undergo functional adaptation after infection or vaccination, a de facto innate immune memory that is also termed trained immunity. Experimental and epidemiological data have shown that induction of trained immunity contributes to the beneficial heterologous effects of vaccines such as bacille Calmette-Guérin (BCG), the licensed TB vaccine. Moreover, increasing evidence argues that trained immunity also contributes to the anti-TB effects of BCG vaccination. An interaction among immunological signals, metabolic rewiring, and epigenetic reprogramming underlies the molecular mechanisms mediating trained immunity in myeloid cells and their bone marrow progenitors. Future studies are warranted to explore the untapped potential of trained immunity to develop a future generation of TB vaccines that would combine innate and adaptive immune memory induction.
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http://dx.doi.org/10.1172/JCI128877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6715374PMC
September 2019

Induction of autophagy through CLEC4E in combination with TLR4: an innovative strategy to restrict the survival of .

Autophagy 2020 06 8;16(6):1021-1043. Epub 2019 Sep 8.

Immunology Division, CSIR-Institute of Microbial Technology , Chandigarh, India.

Host-directed therapies are gaining considerable impetus because of the emergence of drug-resistant strains of pathogens due to antibiotic therapy. Therefore, there is an urgent need to exploit alternative and novel strategies directed at host molecules to successfully restrict infections. The C-type lectin receptor CLEC4E and Toll-like receptor TLR4 expressed by host cells are among the first line of defense in encountering pathogens. Therefore, we exploited signaling of macrophages through CLEC4E in association with TLR4 agonists (C.T) to control the growth of (). We observed significant improvement in host immunity and reduced bacterial load in the lungs of infected mice and guinea pigs treated with C.T agonists. Further, intracellular killing of was achieved with a 10-fold lower dose of isoniazid or rifampicin in conjunction with C.T than the drugs alone. C.T activated MYD88, PtdIns3K, STAT1 and RELA/NFKB, increased lysosome biogenesis, decreased and gene expression and enhanced macroautophagy/autophagy. Macrophages from autophagy-deficient ( knockout or knockdown) mice showed elevated survival of . The present findings also unveiled the novel role of CLEC4E in inducing autophagy through MYD88, which is required for control of growth. This study suggests a unique immunotherapeutic approach involving CLEC4E in conjunction with TLR4 to restrict the survival of through autophagy.

Abbreviations: 3MA: 3 methyladenine; AO: acridine orange; Atg5: autophagy related 5; AVOs: acidic vesicular organelles; BECN1: beclin 1, autophagy related; BMDMs: bone marrow derived macrophages; bw: body weight; C.T: agonists of CLEC4E (C/TDB) and TLR4 (T/ultra-pure-LPS); CFU: colony forming unit; CLEC4E/Mincle: C-type lectin domain family 4, member e; CLR: c-type lectin receptor; INH: isoniazid; LAMP1: lysosomal-associated membrane protein 1; Mφ: infected C.T stimulated macrophages; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MDC: monodansylcadaverine; MTOR: mechanistic target of rapamycin kinase; MYD88: myeloid differentiation primary response 88; NFKB: nuclear factor of kappa light polypeptide gene enhance in B cells; NLR: NOD (nucleotide-binding oligomerization domain)-like receptors; PFA: paraformaldehyde; PPD: purified protein derivative; PtdIns3K: class III phosphatidylinositol 3-kinase; RELA: v-rel reticuloendotheliosis viral oncogene homolog A (avian); RIF: rifampicin; RLR: retinoic acid-inducible gene-I-like receptors; TDB: trehalose-6,6´-dibehenate; TLR4: toll-like receptor 4; Ultra-pure-LPS: ultra-pure lipopolysaccharide-EK; V-ATPase: vacuolar-type H ATPase.
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http://dx.doi.org/10.1080/15548627.2019.1658436DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7469444PMC
June 2020

Mycobacterium tuberculosis and macrophage nuclear receptors: What we do and don't know.

Tuberculosis (Edinb) 2019 05 25;116S:S98-S106. Epub 2019 Apr 25.

Texas Biomedical Research Institute, San Antonio, TX, USA. Electronic address:

Nuclear receptors (NRs) are ligand-activated transcription factors that are expressed in a wide variety of cells and play a major role in lipid signaling. NRs are key regulators of immune and metabolic functions in macrophages and are linked to macrophage responses to microbial pathogens. Pathogens are also known to induce the expression of specific NRs to promote their own survival. In this review, we focus on the NRs recently shown to influence macrophage responses to Mycobacterium tuberculosis (M.tb), a significant cause of morbidity and mortality worldwide. We provide an overview of NR-controlled transcriptional activity and regulation of macrophage activation. We also discuss in detail the contribution of specific NRs to macrophage responses to M.tb, including influence on macrophage phenotype, cell signaling, and cellular metabolism. We pay particular attention to PPARγ since it is required for differentiation of alveolar macrophages, an important niche for M.tb, and its role during M.tb infection is becoming increasingly appreciated. Research into NRs and M.tb is still in its early stages, therefore continuing to advance our understanding of the complex interactions between M.tb and macrophage NRs may reveal the potential of NRs as pharmacological targets for the treatment of tuberculosis.
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http://dx.doi.org/10.1016/j.tube.2019.04.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6750772PMC
May 2019

In Vitro Granuloma Models of Tuberculosis: Potential and Challenges.

J Infect Dis 2019 05;219(12):1858-1866

Texas Biomedical Research Institute, San Antonio, Texas.

Despite intensive research efforts, several fundamental disease processes for tuberculosis (TB) remain poorly understood. A central enigma is that host immunity is necessary to control disease yet promotes transmission by causing lung immunopathology. Our inability to distinguish these processes makes it challenging to design rational novel interventions. Elucidating basic immune mechanisms likely requires both in vivo and in vitro analyses, since Mycobacterium tuberculosis is a highly specialized human pathogen. The classic immune response is the TB granuloma organized in three dimensions within extracellular matrix. Several groups are developing cell culture granuloma models. In January 2018, NIAID convened a workshop, entitled "3-D Human in vitro TB Granuloma Model" to advance the field. Here, we summarize the arguments for developing advanced TB cell culture models and critically review those currently available. We discuss how integrating complementary approaches, specifically organoids and mathematical modeling, can maximize progress, and conclude by discussing future challenges and opportunities.
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http://dx.doi.org/10.1093/infdis/jiz020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534193PMC
May 2019

The Lung Mucosa Environment in the Elderly Increases Host Susceptibility to Mycobacterium tuberculosis Infection.

J Infect Dis 2019 07;220(3):514-523

Texas Biomedical Research Institute, San Antonio.

As we age, there is an increased risk for the development of tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infection. Few studies consider that age-associated changes in the alveolar lining fluid (ALF) may increase susceptibility by altering soluble mediators of innate immunity. We assessed the impact of adult or elderly human ALF during Mtb infection in vitro and in vivo. We identified amplification of pro-oxidative and proinflammatory pathways in elderly ALF and decreased binding capability of surfactant-associated surfactant protein A (SP-A) and surfactant protein D (SP-D) to Mtb. Human macrophages infected with elderly ALF-exposed Mtb had reduced control and fewer phagosome-lysosome fusion events, which was reversed when elderly ALF was replenished with functional SP-A/SP-D. In vivo, exposure to elderly ALF exacerbated Mtb infection in young mice. Our studies demonstrate how the pulmonary environment changes as we age and suggest that Mtb may benefit from declining host defenses in the lung mucosa of the elderly.
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http://dx.doi.org/10.1093/infdis/jiz138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6603975PMC
July 2019

Macrophage nuclear receptors: Emerging key players in infectious diseases.

PLoS Pathog 2019 03 21;15(3):e1007585. Epub 2019 Mar 21.

Texas Biomedical Research Institute, San Antonio, Texas, United States of America.

Nuclear receptors (NRs) are ligand-activated transcription factors that are expressed in a variety of cells, including macrophages. For decades, NRs have been therapeutic targets because their activity can be pharmacologically modulated by specific ligands and small molecule inhibitors. NRs regulate a variety of processes, including those intersecting metabolic and immune functions, and have been studied in regard to various autoimmune diseases. However, the complex roles of NRs in host response to infection are only recently being investigated. The NRs peroxisome proliferator-activated receptor γ (PPARγ) and liver X receptors (LXRs) have been most studied in the context of infectious diseases; however, recent work has also linked xenobiotic pregnane X receptors (PXRs), vitamin D receptor (VDR), REV-ERBα, the nuclear receptor 4A (NR4A) family, farnesoid X receptors (FXRs), and estrogen-related receptors (ERRs) to macrophage responses to pathogens. Pharmacological inhibition or antagonism of certain NRs can greatly influence overall disease outcome, and NRs that are protective against some diseases can lead to susceptibility to others. Targeting NRs as a novel host-directed treatment approach to infectious diseases appears to be a viable option, considering that these transcription factors play a pivotal role in macrophage lipid metabolism, cholesterol efflux, inflammatory responses, apoptosis, and production of antimicrobial byproducts. In the current review, we discuss recent findings concerning the role of NRs in infectious diseases with an emphasis on PPARγ and LXR, the two most studied. We also highlight newer work on the activity of emerging NRs during infection.
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http://dx.doi.org/10.1371/journal.ppat.1007585DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6428245PMC
March 2019

l-Arginine Synthesis from l-Citrulline in Myeloid Cells Drives Host Defense against Mycobacteria In Vivo.

J Immunol 2019 03 1;202(6):1747-1754. Epub 2019 Feb 1.

Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229;

Immunonutrition as a therapeutic approach is rapidly gaining interest in the fight against infection. Targeting l-arginine metabolism is intriguing, considering this amino acid is the substrate for antimicrobial NO production by macrophages. The importance of l-arginine during infection is supported by the finding that inhibiting its synthesis from its precursor l-citrulline blunts host defense. During the first few weeks following pulmonary mycobacterial infection, we found a drastic increase in l-citrulline in the lung, even though serum concentrations were unaltered. This correlated with increased gene expression of the l-citrulline-generating (i.e., iNOS) and l-citrulline-using (i.e., Ass1) enzymes in key myeloid populations. Eliminating l-arginine synthesis from l-citrulline in myeloid cells via conditional deletion of either or resulted in increased bacillus Calmette-Guérin and HR burden in the lungs compared with controls. Our data illustrate the necessity of l-citrulline metabolism for myeloid defense against mycobacterial infection and highlight the potential for host-directed therapy against mycobacterial disease targeting this nutrient and/or its metabolic pathway.
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http://dx.doi.org/10.4049/jimmunol.1801569DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6401247PMC
March 2019

Protein Interaction Mapping Identifies RBBP6 as a Negative Regulator of Ebola Virus Replication.

Cell 2018 12;175(7):1917-1930.e13

Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA 94158, USA; Quantitative Biosciences Institute, University of California, San Francisco, San Francisco, CA 94158, USA; J. David Gladstone Institutes, San Francisco, CA 94158, USA. Electronic address:

Ebola virus (EBOV) infection often results in fatal illness in humans, yet little is known about how EBOV usurps host pathways during infection. To address this, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 194 high-confidence EBOV-human PPIs, including one between the viral transcription regulator VP30 and the host ubiquitin ligase RBBP6. Domain mapping identified a 23 amino acid region within RBBP6 that binds to VP30. A crystal structure of the VP30-RBBP6 peptide complex revealed that RBBP6 mimics the viral nucleoprotein (NP) binding to the same interface of VP30. Knockdown of endogenous RBBP6 stimulated viral transcription and increased EBOV replication, whereas overexpression of either RBBP6 or the peptide strongly inhibited both. These results demonstrate the therapeutic potential of biologics that target this interface and identify additional PPIs that may be leveraged for novel therapeutic strategies.
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http://dx.doi.org/10.1016/j.cell.2018.08.044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6366944PMC
December 2018

Diabetes screen during tuberculosis contact investigations highlights opportunity for new diabetes diagnosis and reveals metabolic differences between ethnic groups.

Tuberculosis (Edinb) 2018 12 18;113:10-18. Epub 2018 Aug 18.

DST-NRF Centre of Excellence for Biomedical Tuberculosis Research, South African Medical Research Council Centre for Tuberculosis Research, Division of Molecular Biology and Human Genetics, Faculty of Medicine and Health Sciences, Stellenbosch University, Cape Town, South Africa; Mater Research Institute - The University of Queensland, Translational Research Institute, Brisbane, QLD, Australia. Electronic address:

Type 2 diabetes (T2D) is a prevalent risk factor for tuberculosis (TB), but most studies on TB-T2D have focused on TB patients, been limited to one community, and shown a variable impact of T2D on TB risk or treatment outcomes. We conducted a cross-sectional assessment of sociodemographic and metabolic factors in adult TB contacts with T2D (versus no T2D), from the Texas-Mexico border to study Hispanics, and in Cape Town to study South African Coloured ethnicities. The prevalence of T2D was 30.2% in Texas-Mexico and 17.4% in South Africa, with new diagnosis in 34.4% and 43.9%, respectively. Contacts with T2D differed between ethnicities, with higher smoking, hormonal contraceptive use and cholesterol levels in South Africa, and higher obesity in Texas-Mexico (p < 0.05). PCA analysis revealed striking differences between ethnicities in the relationships between factors defining T2D and dyslipidemias. Our findings suggest that screening for new T2D in adult TB contacts is effective to identify new T2D patients at risk for TB. Furthermore, studies aimed at predicting individual TB risk in T2D patients, should take into account the heterogeneity in dyslipidemias that are likely to modify the estimates of TB risk or adverse treatment outcomes that are generally attributed to T2D alone.
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http://dx.doi.org/10.1016/j.tube.2018.08.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284235PMC
December 2018

AR-13 reduces antibiotic-resistant bacterial burden in cystic fibrosis phagocytes and improves cystic fibrosis transmembrane conductance regulator function.

J Cyst Fibros 2019 09 23;18(5):622-629. Epub 2018 Oct 23.

Center for Microbial Pathogenesis, The Research Institute at Nationwide Children's Hospital, Columbus, OH, United States; Division of Pulmonary Medicine, Nationwide Children's Hospital, Columbus, OH, United States. Electronic address:

Background: There are no effective treatments for Burkholderia cenocepacia in patients with cystic fibrosis (CF) due to bacterial multi-drug resistance and defective host killing. We demonstrated that decreased bacterial killing in CF is caused by reduced macrophage autophagy due to defective cystic fibrosis transmembrane conductance regulator (CFTR) function. AR-12 is a small molecule autophagy inducer that kills intracellular pathogens such as Francisella. We evaluated the efficacy of AR-12 and a new analogue AR-13 in reducing bacterial burden in CF phagocytes.

Methods: Human CF and non-CF peripheral blood monocyte-derived macrophages, neutrophils, and nasal epithelial cells were exposed to CF bacterial strains in conjunction with treatment with antibiotics and/or AR compounds.

Results: AR-13 and not AR-12 had growth inhibition on B. cenocepacia and methicillin-resistantStaphylococcus aureus (MRSA) in media alone. There was a 99% reduction in MRSA in CF macrophages, 71% reduction in Pseudomonas aeruginosa in CF neutrophils, and 70% reduction in non-CF neutrophils using AR-13. Conversely, there was no reduction in B. cenocepacia in infected CF and non-CF macrophages using AR-13 alone, but AR-13 and antibiotics synergistically reduced B. cenocepacia in CF macrophages. AR-13 improved autophagy in CF macrophages and CF patient-derived epithelial cells, and increased CFTR protein expression and channel function in CF epithelial cells.

Conclusions: The novel AR-12 analogue AR-13, in combination with antibiotics, reduced antibiotic-resistant bacterial burden in CF phagocytes, which correlated with increased autophagy and CFTR expression. AR-13 is a novel therapeutic for patients infected with B. cenocepacia and other resistant organisms that lack effective therapies.
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http://dx.doi.org/10.1016/j.jcf.2018.10.010DOI Listing
September 2019

IL-13-regulated Macrophage Polarization during Granuloma Formation in an In Vitro Human Sarcoidosis Model.

Am J Respir Cell Mol Biol 2019 01;60(1):84-95

6 Texas Biomedical Research Institute, San Antonio, Texas.

The mechanisms underlying abnormal granuloma formation in patients with sarcoidosis are complex and remain poorly understood. A novel in vitro human granuloma model was used to determine the molecular mechanisms of granuloma genesis in patients with sarcoidosis in response to putative disease-causing mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis and from normal, disease-free control subjects were incubated for 7 days with purified protein derivative-coated polystyrene beads. Molecular responses, as reflected by differential expression of genes, extracellular cytokine patterns, and cell surface receptor expression, were analyzed. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared with human lung and mediastinal lymph node gene expression profiles. Compared with identically treated PBMCs of control subjects (n = 5), purified protein derivative-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Ingenuity Pathway Analysis of differential expression gene patterns identified molecular pathways that are primarily regulated by IL-13, which promotes alternatively activated (M2) macrophage polarization. M2 polarization was further demonstrated by immunohistochemistry performed on the in vitro sarcoidosis granuloma-like structures. IL-13-regulated gene pathways were confirmed in human sarcoidosis lung and mediastinal lymph node tissues. The in vitro human sarcoidosis granuloma model provides novel insights into early granuloma formation, particularly IL-13 regulation of molecular networks that regulate M2 macrophage polarization. M2 macrophages are predisposed to aggregation and multinucleated giant cell formation, which are characteristic features of sarcoidosis granulomas. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).
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http://dx.doi.org/10.1165/rcmb.2018-0053OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6348723PMC
January 2019

Deletion of PPARγ in lung macrophages provides an immunoprotective response against M. tuberculosis infection in mice.

Tuberculosis (Edinb) 2018 07 21;111:170-177. Epub 2018 Jun 21.

Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, USA. Electronic address:

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear transcription factor belonging to the superfamily of ligand-activated nuclear receptors. It is activated by diverse endogenous lipid metabolites as well as by exogenous ligands such as the thiazolidinediones. It regulates cellular metabolism, proliferation, differentiation, and inflammation, the latter in part through trans-repression of pro-inflammatory cytokines. PPARγ is highly expressed in alternatively activated alveolar macrophages (AMs), a primary host cell for airborne Mycobacterium tuberculosis (M.tb). Our previous in vitro study identified the importance of PPARγ activation through the mannose receptor (CD206) on human macrophages in enabling M. tb growth. The aim of the current study was to investigate the role of PPARγ in vivo during M. tb infection using a macrophage-specific PPARγ knock out mouse model with special emphasis on the lung environment. Our data show that the absence of PPARγ in lung macrophages reduces the growth of virulent M. tb, enhances pro-inflammatory cytokines and reduces granulomatous infiltration. These findings demonstrate that PPARγ activation, which down-regulates macrophage pro-inflammatory responses, impacts the lung's response to M. tb infection, thereby supporting PPARγ's role in tuberculosis (TB) pathogenesis.
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http://dx.doi.org/10.1016/j.tube.2018.06.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6481684PMC
July 2018

PPARγ is critical for Mycobacterium tuberculosis induction of Mcl-1 and limitation of human macrophage apoptosis.

PLoS Pathog 2018 06 21;14(6):e1007100. Epub 2018 Jun 21.

Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States of America.

Peroxisome proliferator-activated receptor (PPAR)γ is a global transcriptional regulator associated with anti-inflammatory actions. It is highly expressed in alveolar macrophages (AMs), which are unable to clear the intracellular pathogen Mycobacterium tuberculosis (M.tb). Although M.tb infection induces PPARγ in human macrophages, which contributes to M.tb growth, the mechanisms underlying this are largely unknown. We undertook NanoString gene expression analysis to identify novel PPARγ effectors that condition macrophages to be more susceptible to M.tb infection. This revealed several genes that are differentially regulated in response to PPARγ silencing during M.tb infection, including the Bcl-2 family members Bax (pro-apoptotic) and Mcl-1 (pro-survival). Apoptosis is an important defense mechanism that prevents the growth of intracellular microbes, including M.tb, but is limited by virulent M.tb. This suggested that M.tb differentially regulates Mcl-1 and Bax expression through PPARγ to limit apoptosis. In support of this, gene and protein expression analysis revealed that Mcl-1 expression is driven by PPARγ during M.tb infection in human macrophages. Further, 15-lipoxygenase (15-LOX) is critical for PPARγ activity and Mcl-1 expression. We also determined that PPARγ and 15-LOX regulate macrophage apoptosis during M.tb infection, and that pre-clinical therapeutics that inhibit Mcl-1 activity significantly limit M.tb intracellular growth in both human macrophages and an in vitro TB granuloma model. In conclusion, identification of the novel PPARγ effector Mcl-1 has determined PPARγ and 15-LOX are critical regulators of apoptosis during M.tb infection and new potential targets for host-directed therapy for M.tb.
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http://dx.doi.org/10.1371/journal.ppat.1007100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013021PMC
June 2018

AmpliSeq transcriptome analysis of human alveolar and monocyte-derived macrophages over time in response to Mycobacterium tuberculosis infection.

PLoS One 2018 30;13(5):e0198221. Epub 2018 May 30.

Center for Pharmacogenomics, Department of Cancer Biology and Genetics, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America.

Human alveolar macrophages (HAM) are primary bacterial niche and immune response cells during Mycobacterium tuberculosis (M.tb) infection, and human blood monocyte-derived macrophages (MDM) are a model for investigating M.tb-macrophage interactions. Here, we use a targeted RNA-Seq method to measure transcriptome-wide changes in RNA expression patterns of freshly obtained HAM (used within 6 h) and 6 day cultured MDM upon M.tb infection over time (2, 24 and 72 h), in both uninfected and infected cells from three donors each. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. AmpliSeqTM yields highly precise and reproducible gene expression profiles (R2 >0.99). Taking advantage of AmpliSeq's reproducibility, we establish well-defined quantitative RNA expression patterns of HAM versus MDM, including significant M.tb-inducible genes, in networks and pathways that differ in part between MDM and HAM. A similar number of expressed genes are detected at all time-points between uninfected MDM and HAM, in common pathways including inflammatory and immune functions, but canonical pathway differences also exist. In particular, at 2 h, multiple genes relevant to the immune response are preferentially expressed in either uninfected HAM or MDM, while the HAM RNA profiles approximate MDM profiles over time in culture, highlighting the unique RNA expression profile of freshly obtained HAM. MDM demonstrate a greater transcriptional response than HAM upon M.tb infection, with 2 to >10 times more genes up- or down-regulated. The results identify key genes involved in cellular responses to M.tb in two different human macrophage types. Follow-up bioinformatics analysis indicates that approximately 30% of response genes have expression quantitative trait loci (eQTLs in GTEx), common DNA variants that can influence host gene expression susceptibility or resistance to M.tb, illustrated with the TREM1 gene cluster and IL-10.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198221PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5976201PMC
November 2018

Complement Receptor 3-Mediated Inhibition of Inflammasome Priming by Ras GTPase-Activating Protein During Phagocytosis by Human Mononuclear Phagocytes.

Front Immunol 2018 26;9:561. Epub 2018 Mar 26.

Center for Microbial Interface Biology, Department of Microbial Infection and Immunity, The Ohio State University, Columbus, OH, United States.

is a remarkably infectious facultative intracellular bacterium of macrophages that causes tularemia. Early evasion of host immune responses contributes to the success of as a pathogen. entry into human monocytes and macrophages is mediated by the major phagocytic receptor, complement receptor 3 (CR3, CD11b/CD18). We recently determined that despite a significant increase in macrophage uptake following C3 opsonization of the virulent Type A spp. Schu S4, this phagocytic pathway results in limited pro-inflammatory cytokine production. Notably, MAP kinase/ERK activation is suppressed immediately during C3-opsonized Schu S4-CR3 phagocytosis. A mathematical model of CR3-TLR2 crosstalk predicted early involvement of Ras GTPase-activating protein (RasGAP) in immune suppression by CR3. Here, we link CR3-mediated uptake of opsonized Schu S4 by human monocytes and macrophages with inhibition of early signal 1 inflammasome activation, evidenced by limited caspase-1 cleavage and IL-18 release. This inhibition is due to increased RasGAP activity, leading to a reduction in the Ras-ERK signaling cascade upstream of the early inflammasome activation event. Thus, our data uncover a novel signaling pathway mediated by CR3 following engagement of opsonized virulent to limit inflammasome activation in human phagocytic cells, thereby contributing to evasion of the host innate immune system.
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http://dx.doi.org/10.3389/fimmu.2018.00561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5879101PMC
May 2019

Use of a leukocyte-targeted peptide probe as a potential tracer for imaging the tuberculosis granuloma.

Tuberculosis (Edinb) 2018 01 8;108:201-210. Epub 2018 Jan 8.

Department of Microbial Infection and Immunity, Center for Microbial Interface Biology, 793 Biomedical Research Tower, 460 W. 12th Avenue, The Ohio State University Wexner Medical Center, Columbus, OH 43210, USA; Texas Biomedical Research Institute, 7620 NW Loop 410, San Antonio, TX 78227, USA. Electronic address:

Granulomas are the histopathologic hallmark of tuberculosis (TB), both in latency and active disease. Diagnostic and therapeutic strategies that specifically target granulomas have not been developed. Our objective is to develop a probe for imaging relevant immune cell populations infiltrating the granuloma. We report the binding specificity of Cyanine 3 (Cy3)-labeled cFLFLFK-PEG to human leukocytes and cellular constituents within a human in vitro granuloma model. We also report use of the probe in in vivo studies using a mouse model of lung granulomatous inflammation. We found that the probe preferentially binds human neutrophils and macrophages in human granuloma structures. Inhibition studies showed that peptide binding to human neutrophils is mediated by the receptor formyl peptide receptor 1 (FPR1). Imaging the distribution of intravenously administered cFLFLFK-PEG-Cy3 in the mouse model revealed probe accumulation within granulomatous inflammatory responses in the lung. Further characterization revealed that the probe preferentially associated with neutrophils and cells of the monocyte/macrophage lineage. As there is no current clinical diagnostic imaging tool that specifically targets granulomas, the use of this probe in the context of latent and active TB may provide a unique advantage over current clinical imaging probes. We anticipate that utilizing a FPR1-targeted radiopharmaceutical analog of cFLFLFK in preclinical imaging studies may greatly contribute to our understanding of granuloma influx patterns and the biological roles and consequences of FPR1-expressing cells in contributing to disease pathogenesis.
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http://dx.doi.org/10.1016/j.tube.2018.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6014630PMC
January 2018

Mycobacterium abscessus Smooth and Rough Morphotypes Form Antimicrobial-Tolerant Biofilm Phenotypes but Are Killed by Acetic Acid.

Antimicrob Agents Chemother 2018 03 23;62(3). Epub 2018 Feb 23.

Department of Microbial Infection and Immunity, The Ohio State University College of Medicine, Columbus, Ohio, USA

has emerged as an important pathogen in people with chronic inflammatory lung diseases such as cystic fibrosis, and recent reports suggest that it may be transmissible by fomites. exhibits two major colony morphology variants: a smooth morphotype ( ) and a rough morphotype ( ). Biofilm formation, prolonged intracellular survival, and colony variant diversity can each contribute to the persistence of and other bacterial pathogens in chronic pulmonary diseases. A prevailing paradigm of chronic infection is that is a noninvasive, biofilm-forming, persistent phenotype and an invasive phenotype that is unable to form biofilms. We show that is hyperaggregative and forms biofilm-like aggregates, which, like biofilm aggregates, are significantly more tolerant than planktonic variants to acidic pHs, hydrogen peroxide (HO), and treatment with amikacin or azithromycin. We further show that both variants are recalcitrant to antibiotic treatment inside human macrophage-like cells and that is more refractory than to azithromycin. Our results indicate that biofilm-like aggregation and protracted intracellular survival may each contribute to the persistence of this problematic pathogen in the face of antimicrobial agents regardless of morphotype. Biofilms of each variant are rapidly killed, however, by acetic acid, which may help to prevent local fomite transmission.
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http://dx.doi.org/10.1128/AAC.01782-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5826145PMC
March 2018

O-Mannosylation of Proteins Enables Yeast Survival at Mammalian Body Temperatures.

mBio 2018 01 2;9(1). Epub 2018 Jan 2.

Department of Microbiology, Ohio State University, Columbus, Ohio, USA

The ability to grow at mammalian body temperatures is critical for pathogen infection of humans. For the thermally dimorphic fungal pathogen , elevated temperature is required for differentiation of mycelia or conidia into yeast cells, a step critical for invasion and replication within phagocytic immune cells. Posttranslational glycosylation of extracellular proteins characterizes factors produced by the pathogenic yeast cells but not those of avirulent mycelia, correlating glycosylation with infection. yeast cells lacking the Pmt1 and Pmt2 protein mannosyltransferases, which catalyze O-linked mannosylation of proteins, are severely attenuated during infection of mammalian hosts. Cells lacking Pmt2 have altered surface characteristics that increase recognition of yeast cells by the macrophage mannose receptor and reduce recognition by the β-glucan receptor Dectin-1. Despite these changes, yeast cells lacking these factors still associate with and survive within phagocytes. Depletion of macrophages or neutrophils does not recover the virulence of the mutant yeast cells. We show that yeast cells lacking Pmt functions are more sensitive to thermal stress and consequently are unable to productively infect mice, even in the absence of fever. Treatment of mice with cyclophosphamide reduces the normal core body temperature of mice, and this decrease is sufficient to restore the infectivity of O-mannosylation-deficient yeast cells. These findings demonstrate that O-mannosylation of proteins increases the thermotolerance of yeast cells, which facilitates infection of mammalian hosts. For dimorphic fungal pathogens, mammalian body temperature can have contrasting roles. Mammalian body temperature induces differentiation of the fungal pathogen into a pathogenic state characterized by infection of host phagocytes. On the other hand, elevated temperatures represent a significant barrier to infection by many microbes. By functionally characterizing cells lacking O-linked mannosylation enzymes, we show that protein mannosylation confers thermotolerance on , enabling infection of mammalian hosts.
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http://dx.doi.org/10.1128/mBio.02121-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750402PMC
January 2018