Infectious disease
Abstract
In view of emerging drug-resistant tuberculosis (TB), host directed adjunct therapies are urgently needed to improve treatment outcomes with currently available anti-TB therapies. One approach is to interfere with the formation of lipid-laden "foamy" macrophages in the host, as they provide a nutrient-rich host cell environment for Mycobacterium tuberculosis (Mtb). Here, we provide evidence that Wnt family member 6 (WNT6), a ligand of the evolutionarily conserved Wingless/Integrase 1 (WNT) signaling pathway, promotes foam cell formation by regulating key lipid metabolic genes including acetyl-CoA carboxylase-2 (ACC2) during pulmonary TB. Using genetic and pharmacological approaches, we demonstrated that lack of functional WNT6 or ACC2 significantly reduced intracellular triacylglycerol (TAG) levels and Mtb survival in macrophages. Moreover, treatment of Mtb-infected mice with a combination of a pharmacological ACC2 inhibitor and the anti-TB drug isoniazid (INH) reduced lung TAG and cytokine levels, as well as lung weights compared to treatment with INH alone. This combination also reduced Mtb bacterial numbers and the size of mononuclear cell infiltrates in livers of infected mice. In summary, our findings demonstrated that Mtb exploits WNT6/ACC2-induced storage of TAGs in macrophages to facilitate its intracellular survival, a finding opening new perspectives for host directed adjunctive treatment of pulmonary TB.
Authors
Julius Brandenburg, Sebastian Marwitz, Simone C. Tazoll, Franziska Waldow, Barbara Kalsdorf, Tim Vierbuchen, Thomas Scholzen, Annette Gross, Svenja Goldenbaum, Alexandra Hölscher, Martina Hein, Lara Linnemann, Maja Reimann, Andreas Kispert, Michael Leitges, Jan Rupp, Christoph Lange, Stefan Niemann, Jochen Behrends, Torsten Goldmann, Holger Heine, Ulrich E. Schaible, Christoph Hölscher, Dominik Schwudke, Norbert Reiling
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.
Abstract
MHC-E, a non-classical MHC molecule, restricted CD8 T-cell responses have been associated with protection in an SIV/rhesus macaque model. The biological relevance of HLA-E restricted CD8 T-cell responses in HIV infection however remains unknown. In this study, CD8 T cells responding to HIV-1 Gag peptides presented by HLA-E were analyzed. Using in-vitro assays, we observed HLA-E restricted T-cell responses to what we believe to be a newly identified subdominant Gag-KL9 as well as a well-described immuno-dominant Gag-KF11 epitope in T-cell lines derived from chronically HIV-infected patients and also primed from healthy donors. Blocking of the HLA-E/KF11 binding by the B7 signal peptide resulted in decreased CD8 T-cell responses. KF11 presented via HLA-E in HIV infected cells was recognized by antigen specific CD8 T cells. Importantly, bulk CD8 T cells obtained from HIV infected individuals recognized infected cells via HLA-E presentation. Ex-vivo analyses at the epitope level showed a higher responder frequency of HLA-E restricted responses to KF11 compared to KL9. Taken together, our findings of HLA-E restricted HIV specific immune responses offer intriguing and possibly paradigm shifting insights into factors that contribute to the immuno-dominance of CD8 T-cell responses in HIV infection.
Authors
Anju Bansal, Mika N. Gehre, Kai Qin, Sarah Sterrett, Ayub Ali, Ying Dang, Sojan Abraham, Margaret C. Costanzo, Leon A. Venegas, Jianming Tang, N. Manjunath, Mark A. Brockman, Otto O. Yang, June Kan-Mitchell, Paul A. Goepfert
Abstract
BACKGROUND SARS-CoV-2 plasma viremia has been associated with severe disease and death in COVID-19 in small-scale cohort studies. The mechanisms behind this association remain elusive.METHODS We evaluated the relationship between SARS-CoV-2 viremia, disease outcome, and inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using a quantitative reverse transcription PCR–based platform. Proteomic data were generated with Proximity Extension Assay using the Olink platform.RESULTS This study included 300 participants with nucleic acid test–confirmed COVID-19. Plasma SARS-CoV-2 viremia levels at the time of presentation predicted adverse disease outcomes, with an adjusted OR of 10.6 (95% CI 4.4–25.5, P < 0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and 3.9 (95% CI 1.5–10.1, P = 0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, and endothelium/vasculature, and alterations in coagulation pathways.CONCLUSION These results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes.FUNDING Mark and Lisa Schwartz; the National Institutes of Health (U19AI082630); the American Lung Association; the Executive Committee on Research at Massachusetts General Hospital; the Chan Zuckerberg Initiative; Arthur, Sandra, and Sarah Irving for the David P. Ryan, MD, Endowed Chair in Cancer Research; an EMBO Long-Term Fellowship (ALTF 486-2018); a Cancer Research Institute/Bristol Myers Squibb Fellowship (CRI2993); the Harvard Catalyst/Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, NIH awards UL1TR001102 and UL1TR002541-01); and by the Harvard University Center for AIDS Research (National Institute of Allergy and Infectious Diseases, 5P30AI060354).
Authors
Yijia Li, Alexis M. Schneider, Arnav Mehta, Moshe Sade-Feldman, Kyle R. Kays, Matteo Gentili, Nicole C. Charland, Anna L.K. Gonye, Irena Gushterova, Hargun K. Khanna, Thomas J. LaSalle, Kendall M. Lavin-Parsons, Brendan M. Lilley, Carl L. Lodenstein, Kasidet Manakongtreecheep, Justin D. Margolin, Brenna N. McKaig, Blair A. Parry, Maricarmen Rojas-Lopez, Brian C. Russo, Nihaarika Sharma, Jessica Tantivit, Molly F. Thomas, James Regan, James P. Flynn, Alexandra-Chloé Villani, Nir Hacohen, Marcia B. Goldberg, Michael R. Filbin, Jonathan Z. Li
Abstract
Seasonal influenza vaccination elicits a diminished adaptive immune response in the elderly, and the mechanisms of immunosenescence are not fully understood. Using Ig-Seq, we found a marked increase with age in the prevalence of cross-reactive (CR) serum antibodies that recognize both the H1N1 (vaccine-H1) and H3N2 (vaccine-H3) components of an egg-produced split influenza vaccine. CR antibodies accounted for 73% ± 18% of the serum vaccine responses in a cohort of elderly donors, 65% ± 15% in late middle-aged donors, and only 13% ± 5% in persons under 35 years of age. The antibody response to non-HA antigens was boosted by vaccination. Recombinant expression of 19 vaccine-H1+H3 CR serum monoclonal antibodies (s-mAbs) revealed that they predominantly bound to non-HA influenza proteins. A sizable fraction of vaccine-H1+H3 CR s-mAbs recognized with high affinity the sulfated glycans, in particular sulfated type 2 N-acetyllactosamine (Galβ1-4GalNAcβ), which is found on egg-produced proteins and thus unlikely to contribute to protection against influenza infection in humans. Antibodies against sulfated glycans in egg-produced vaccine had been identified in animals but were not previously characterized in humans. Collectively, our results provide a quantitative basis for how repeated exposure to split influenza vaccine correlates with unintended focusing of serum antibody responses to non-HA antigens that may result in suboptimal immunity against influenza.
Authors
Jiwon Jung, Sophia T. Mundle, Irina V. Ustyugova, Andrew P. Horton, Daniel R. Boutz, Svetlana Pougatcheva, Ponraj Prabakaran, Jonathan R. McDaniel, Gregory R. King, Daechan Park, Maria D. Person, Congxi Ye, Bing Tan, Yuri Tanno, Jin Eyun Kim, Nicholas C. Curtis, Joshua DiNapoli, Simon Delagrave, Ted M. Ross, Gregory C. Ippolito, Harry Kleanthous, Jiwon Lee, George Georgiou
Abstract
To the Editor: Povysil G. et al. report that “rare loss-of-function (LOF) variants in type I interferon (IFN) immunity genes are not associated with severe COVID-19” (1). We disagree with the authors’ interpretation of our data and their own (2), for six reasons: 1) Only predicted LOF (pLOF) variants are relevant for comparison between the two studies, because, unlike us, these authors did not test variants experimentally. The relevant proportion in our data is therefore not 24/659=3.5%, but 9/659= 1.36%, whereas theirs is 1/713=0.14%. 2) Our definitions of ‘severe/critical’ patients are different: we defined critical disease as severity grades 6-10 of the WHO scale (3), whereas they restricted their recruitment to grades 7-10 (i.e., excluding patients on high-flow oxygen, considered in our study). Their cohort of ‘mild’ cases may therefore include ‘severe’ COVID-19 cases (grade 6), such as perhaps their ‘mild’ TLR3 pLOF carrier. 3) Their ‘controls’ are subjects from the general population, without depletion of COVID-19 genetic risk factors, whereas we used pauci-/asymptomatic infected subjects (grades 1-3) as ‘controls’. Consequently their power computation in Figure 1 is based on an incorrect hypothesis about the odds ratio, which would be expected to be lower when using general population controls (as they did), than when using pauci- and asymptomatic infected individuals (as we did). 4) The ethnic origin of the patients differs between the two studies: 58% of our 659 patients (and 8 of our 9 pLOF carriers) were European, versus only 10% of their 713 patients with severe disease (and their pLOF carrier is East Asian). 5) Age is a key factor neglected in their comparison: our sample was much younger (mean age: 51.8 years) than theirs (mean: 65.9 years), and seven of our nine pLOF carriers were < 60 years old. We performed a comparison stratified by age (<60/≥60 years), and no significant difference in pLOF proportion was found between the two studies, even ignoring the only patient carrying a pLOF they found (of unknown age): 7/458 in our sample vs. 0/192 in their sample (p=0.11, Fisher’s exact test) for patients <60 years old, and 2/201 vs. 0/521 (p=0.07) for patients ≥60 years old. 6) Finally, and crucially, the authors did not exclude patients with autoantibodies against type I IFN, which account for at least 10% of critical cases and are much more frequent in patients > 60 years of age, particularly men (4).
Authors
Qian Zhang, Aurélie Cobat, Paul Bastard, Luigi D. Notarangelo, Helen C. Su, Laurent Abel, Jean-Laurent Casanova
Abstract
The authors reply: We appreciate the interest of Dr. Zhang and colleagues in our manuscript. The main difference between our publication and that of Zhang et al. (1), was that we assessed all rare predicted loss-of-function variants (pLOFs) meeting the same criteria in cases and controls, which is a well-established paradigm in the field (2). On the other hand, Zhang et al. included specific variants which were experimentally confirmed only in cases, but not controls, precluding a valid case-control comparison. We matched patients as closely as possible to the previous study, and the inclusion of more severe cases (WHO grades 7-10) should only strengthen the signal against population controls. The use of population controls is standard in such settings and has minimal impact on power, because only a small proportion of individuals exposed to SARS-Cov-2 develop severe disease (3). Additionally, for the pLOF model we report adequate power even for an odds ratio of 5.5, which is considerably lower than the one reported by Zhang et al. We tested the same dominant model as Zhang et al., even though LOF variants in these genes have only been reported to cause disease under recessive inheritance (4). We have serious concerns about confounding by ancestry in the analysis by Zhang et al. in which the pLOF carriers were mostly European, but functionally validated missense variants were found in various nationalities from Asia, Europe, Latin America, and the Middle East. Because the rates of pLOFs vary considerably across populations, adjusting for only 3 principal components of ancestry in rare-variant association tests of multi-ethnic cohorts does not provide adequate control for population structure. While we noted that age differences may contribute to the discrepancies between the two studies, Zhang et al. do not discuss the role of age in the interpretation of their results stating: “Inborn errors of TLR3- and IRF7-dependent type I IFN immunity at eight loci were found in as many as 23 patients (3.5%) of various ages (17 to 77 years) and ancestries (various nationalities from Asia, Europe, Latin America, and the Middle East) and in patients of both sexes.” We also note that the patients with autoantibodies were not excluded from the primary analysis by Zhang et al., but this was done only in the post-hoc analysis. Most importantly, our negative findings are in full agreement with the recently published independent study of 586,157 individuals, including 20,952 cases of COVID-19 (4,928 hospitalized and 1,304 requiring ventilation or resulting in death) (5). There were no significant associations with any of the 13 candidate genes examined either individually or in aggregate, or when comparisons included all hospitalized cases or only the most severe cases. Indeed, none of the associations displayed even marginal significance. Therefore, consistent with our study, these findings do not support substantial contributions of inborn errors in type I IFN immunity to COVID-19 severity. These negative results underscore the importance of proper study design, selection of appropriate genetic models, adequate control for genetic ancestry, and adherence to unbiased methods for genetic discovery rather than focusing only on a candidate biological pathway.
Authors
Gundula Povysil, Guillaume Butler-Laporte, Ali G. Gharavi, J. Brent Richards, David B. Goldstein, Krzysztof Kiryluk
Cytomegalovirus mediates expansion of IL-15-responsive innate-memory cells with SIV killing function
Abstract
Inter-individual immune variability is driven predominantly by environmental factors including exposure to chronic infectious agents such as cytomegalovirus (CMV). We investigated the effects of rhesus CMV (RhCMV) on composition and function of the immune system in young macaques. Within months of infection, RhCMV was associated with impressive changes in antigen presenting cells, T cells, and NK cells — and marked expansion of innate-memory CD8+ T cells. These cells express high levels of NKG2A/C and the IL-2- and IL-15-receptor beta chain, CD122. IL-15 was sufficient to drive differentiation of the cells in vitro and in vivo. Expanded NKG2A/C+CD122+CD8+ T cells in RhCMV-infected macaques, but not their NKG2-negative counterparts, were endowed with cytotoxicity against class I-deficient K562 targets and prompt IFN-ɣ production in response to stimulation with IL-12 and IL-18. Because RhCMV clone 68-1 forms the viral backbone of RhCMV-vectored SIV vaccines, we also investigated immune changes following administration of RhCMV 68-1-vectored SIV vaccines. These vaccines led to impressive expansion of NKG2A/C+CD8+ T cells with capacity to inhibit SIV replication ex vivo. Thus, CMV infection and CMV-vectored vaccination drive expansion of functional innate-like CD8 cells via host IL-15 production, suggesting that innate-memory expansion could be achieved by other vaccine platforms expressing IL-15.
Authors
Gema Méndez-Lagares, Ning Chin, W.L. William Chang, Jaewon Lee, Míriam Rosás-Umbert, Hung T. Kieu, David Merriam, Wenze Lu, Sungjin Kim, Lourdes Adamson, Christian Brander, Paul A. Luciw, Peter A. Barry, Dennis J. Hartigan-O’Connor
Abstract
BACKGROUND. Matrix metalloproteinases (MMPs) are implicated as key regulators of tissue destruction in tuberculosis (TB) and may be a target for host-directed therapy. Here, we conducted a Phase 2 randomized, double-blind, placebo-controlled trial investigating doxycycline, a licensed broad spectrum MMP inhibitor, in pulmonary TB patients. METHODS. Thirty pulmonary TB patients were enrolled within 7 days of initiating anti-TB treatment and randomly assigned to receive either doxycycline 100 mg or placebo twice a day for 14 days in addition to standard care. RESULTS. There were significant changes in the host transcriptome, and suppression of systemic and respiratory markers of tissue destruction with the doxycycline intervention. Whole blood RNA-sequencing demonstrated that doxycycline accelerated restoration of dysregulated gene expression patterns in TB towards normality, with more rapid down-regulation of type I and II interferon and innate immune response genes and concurrent up-regulation of B-cell modules relative to placebo. The effects persisted for 6 weeks after doxycycline was discontinued, concurrent with suppression of plasma MMP-1. In respiratory samples, doxycycline reduced MMP-1, -8, -9, -12 and -13 concentrations, suppressed type I collagen and elastin destruction, and reduced pulmonary cavity volume despite unchanged sputum Mycobacterium tuberculosis loads between the study arms. Two weeks of adjunctive doxycycline with standard anti-TB treatment was well-tolerated, with no serious adverse events related to doxycycline. CONCLUSION. These data demonstrate that adjunctive doxycycline with standard anti-TB treatment suppresses pathological MMPs in pulmonary tuberculosis patients, and suggest that larger studies on adjunctive doxycycline to limit immunopathology in TB are merited.
Authors
Qing Hao Miow, Andres F. Vallejo, Yu Wang, Jia Mei Hong, Chen Bai, Felicia S.W. Teo, Alvin Dingyuan Wang, Hong Rong Loh, Tuan Zea Tan, Ying Ding, Hoi Wah She, Suay Hong Gan, Nicholas I. Paton, Josephine Lum, Alicia Tay, Cynthia B.E. Chee, Paul A. Tambyah, Marta E. Polak, Yee Tang Wang, Amit Singhal, Paul Elkington, Jon S. Friedland, Catherine W.M. Ong
Abstract
Tuberculosis (TB) is a persistent global pandemic and standard treatment has not changed for thirty years. Mycobacterium tuberculosis (Mtb) has undergone prolonged co-evolution with humans, and patients can control Mtb even after extensive infection, demonstrating the fine balance between protective and pathological host responses within infected granulomas. We hypothesised that whole transcriptome analysis of human TB granulomas isolated by laser capture microdissection could identify therapeutic targets, and that comparison with a non-infectious granulomatous disease, sarcoidosis, would identify disease-specific pathological mechanisms. Bioinformatic analysis of RNAseq data identified numerous shared pathways between TB and sarcoidosis lymph nodes, and also specific clusters demonstrating TB results from a dysregulated inflammatory immune response. To translate these insights, we compared three primary human cell culture models at the whole transcriptome level, and demonstrated that the 3D collagen granuloma model most closely reflected human TB disease. We investigated shared signaling pathways with human disease and identified twelve intracellular enzymes as potential therapeutic targets. Sphingosine kinase 1 inhibition controlled Mtb growth, concurrently reducing intracellular pH in infected monocytes and suppressing inflammatory mediator secretion. Immunohistochemical staining confirmed that sphingosine kinase 1 is expressed in human lung TB granulomas, and therefore represents a host therapeutic target to improve TB outcomes.
Authors
Michaela T. Reichmann, Liku B. Tezera, Andres F. Vallejo, Milica Vukmirovic, Rui Xiao, James Reynolds, Sanjay Jogai, Susan Wilson, Ben Marshall, Mark G. Jones, Alasdair Leslie, Jeanine M. D'Armiento, Naftali Kaminski, Marta E. Polak, Paul Elkington
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ISSN: 0021-9738 (print), 1558-8238 (online)