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).
Qian Zhang, Aurélie Cobat, Paul Bastard, Luigi D. Notarangelo, Helen C. Su, Laurent Abel, Jean-Laurent Casanova
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.
Gundula Povysil, Guillaume Butler-Laporte, Ali G. Gharavi, J. Brent Richards, David B. Goldstein, Krzysztof Kiryluk
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.
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
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.
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
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.
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
After extensive exposure to Mycobacterium tuberculosis (Mtb), most individuals acquire latent Mtb infection (LTBI) defined by a positive tuberculin skin test (TST) or interferon-γ release assay (IGRA). To identify mechanisms of resistance to Mtb infection, we compared transcriptional profiles from highly-exposed contacts who resist TST/IGRA conversion (resisters, RSTRs) and controls with LTBI using RNAseq. Gene sets related to carbon metabolism and free fatty acid (FFA) transcriptional responses enriched across two independent cohorts suggesting RSTR and LTBI monocytes have distinct activation states. We compared intracellular Mtb replication in macrophages treated with FFAs and found that palmitic acid (PA), but not oleic acid (OA), enhanced Mtb intracellular growth. This PA activity correlated with its inhibition of pro-inflammatory cytokines in Mtb-infected cells. Mtb growth restriction in PA-treated macrophages was restored by activation of AMP kinase (AMPK), a central host metabolic regulator known to be inhibited by PA. Finally, we genotyped AMPK variants and found seven SNPs in PRKAG2, which encodes the AMPKγ subunit, that strongly associated with RSTR status. Taken together, RSTR and LTBI phenotypes are distinguished by FFA transcriptional programs and by genetic variation in a central metabolic regulator, which suggests immunometabolic pathways regulate TST/IGRA conversion.
Jason D. Simmons, Phu T. Van, Catherine M. Stein, Violet Chihota, Thobani Ntshiqa, Pholo Maenetje, Glenna J. Peterson, Anthony Reynolds, Penelope Benchek, Kavindhran Velen, Katherine L. Fielding, Alison D. Grant, Andrew D. Graustein, Felicia K. Nguyen, Chetan Seshadri, Raphael Gottardo, Harriet Mayanja-Kizza, Robert S. Wallis, Gavin Churchyard, W. Henry Boom, Thomas R. Hawn
Chronic hepatitis B (CHB) infection is rarely eradicated by current antiviral nucleos(t)ide analogues. We found that α2,6-biantennary sialoglycans of HBV surface antigen (HBsAg) bound human SIGLEC-3 (CD33) by IP and ELISA, and the binding affinity between SIGLEC-3 and α2,6-biantennary sialoglycans was determined by biolayer interferometry (equilibrium dissociation constant [KD]: 1.95 × 10–10 ± 0.21 × 10–10 M). Moreover, HBV activated SIGLEC-3 on myeloid cells and induced immunosuppression by stimulating immunoreceptor tyrosine-based inhibitory motif phosphorylation and SHP-1/-2 recruitment via α2,6-biantennary sialoglycans on HBsAg. An antagonistic anti–SIGLEC-3 mAb reversed this effect and enhanced cytokine production in response to TLR-7 agonist GS-9620 in PBMCs from CHB patients. Moreover, anti–SIGLEC-3 mAb alone was able to upregulate the expression of molecules involved in antigen presentation, such as CD80, CD86, CD40, MHC-I, MHC-II, and PD-L1 in CD14+ cells. Furthermore, SIGLEC-3 SNP rs12459419 C, which expressed a higher amount of SIGLEC-3, was associated with increased risk of hepatocellular carcinoma (HCC) in CHB patients (HR: 1.256, 95% CI: 1.027–1.535, P = 0.0266). Thus, blockade of SIGLEC-3 is a promising strategy to reactivate host immunity to HBV and lower the incidence of HCC in the CHB patient population.
Tsung-Yu Tsai, Ming-Ting Huang, Pei-Shan Sung, Cheng-Yuan Peng, Mi-Hua Tao, Hwai-I Yang, Wei-Chiao Chang, An-Suei Yang, Chung-Ming Yu, Ya-Ping Lin, Ching-Yu Bau, Chih-Jen Huang, Mei-Hung Pan, Chung-Yi Wu, Chwan-Deng Hsiao, Yi-Hung Yeh, Shiteng Duan, James C Paulson, Shie-Liang Hsieh
A recent report found that rare predicted loss-of-function (pLOF) variants across 13 candidate genes in TLR3- and IRF7-dependent type I IFN pathways explain up to 3.5% of severe COVID-19 cases. We performed whole-exome or whole-genome sequencing of 1,934 COVID-19 cases (713 with severe and 1,221 with mild disease) and 15,251 ancestry-matched population controls across four independent COVID-19 biobanks. We then tested if rare pLOF variants in these 13 genes were associated with severe COVID-19. We identified only one rare pLOF mutation across these genes amongst 713 cases with severe COVID-19 and observed no enrichment of pLOFs in severe cases compared to population controls or mild COVID-19 cases. We find no evidence of association of rare loss-of-function variants in the proposed 13 candidate genes with severe COVID-19 outcomes.
Gundula Povysil, Guillaume Butler-Laporte, Ning Shang, Chen Wang, Atlas Khan, Manal Alaamery, Tomoko Nakanishi, Sirui Zhou, Vincenzo Forgetta, Robert J. M. Eveleigh, Mathieu Bourgey, Naveed Aziz, Steven J.M. Jones, Bartha Knoppers, Stephen W. Scherer, Lisa J. Strug, Pierre Lepage, Jiannis Ragoussis, Guillaume Bourque, Jahad Alghamdi, Nora Aljawini, Nour Albesher, Hani M. Al-Afghani, Bader Alghamdi, Mansour S. Almutair, Ebrahim Sabri Mahmoud, Leen Abu-Safieh, Hadeel El Bardisy, Fawz S. Al Harthi, Abdulraheem Alshareef, Bandar Ali Suliman, Saleh A. Alqahtani, Abdulaziz Almalik, May M. Alrashed, Salam Massadeh, Vincent Mooser, Mark Lathrop, Mohamed Fawzy, Yaseen M. Arabi, Hamdi Mbarek, Chadi Saad, Wadha Al-Muftah, Junghyun Jung, Serghei Mangul, Radja Badji, Asma Al Thani, Said I. Ismail, Ali G. Gharavi, Malak S. Abedalthagafi, J Brent Richards, David B. Goldstein, Krzysztof Kiryluk
The upper respiratory tract is compromised in the early period of COVID-19, but SARS-CoV-2 tropism at the cellular level is not fully defined. Unlike recent single cell RNA-sequencing analyses indicating uniformly low mRNA expression of SARS-CoV-2 entry-related host molecules in all nasal epithelial cells, we show that the protein levels are relatively high and their localizations are restricted to the apical side of multiciliated epithelial cells. In addition, we provide evidence in COVID-19 patients that SARS-CoV-2 is massively detected and replicated within the multiciliated cells. We observed these findings during the early stage of COVID-19, when infected ciliated cells are rapidly replaced by differentiating precursor cells. Moreover, our analyses reveal that SARS-CoV-2 cellular tropism is restricted to the nasal ciliated versus oral squamous epithelium. These results imply that targeting ciliated cells of the nasal epithelium during the early stage of COVID-19 could be an ideal strategy to prevent SARS-CoV-2 propagation.
Ji Hoon Ahn, JungMo Kim, Seon Pyo Hong, Sung Yong Choi, Myung Jin Yang, Young Seok Ju, Young Tae Kim, Ho Min Kim, MD Tazikur Rahman, Man Ki Chung, Sang Duk Hong, Hosung Bae, Chang-Seop Lee, Gou Young Koh
Sepsis survivors exhibit impaired responsiveness to antigen (Ag) challenge associated with increased mortality from infection. The contribution of follicular dendritic cells (FDCs) in the impaired humoral response in sepsis-surviving mice is investigated in this study. We demonstrated that mice subjected to sepsis from cecal ligation and puncture (CLP) have reduced NP-specific high-affinity class-switched Ig antibodies compared to sham control mice following immunization with the T-dependent Ag, NP-CGG. NP-specific germinal center (GC) B cells in CLP mice exhibited reduced TNFα and AID mRNA expression compared to sham mice. CLP mice showed a reduction in FDC clusters, a reduced binding of immune complexes on FDCs, and reduced mRNA expression of CR2, ICAM-1, VCAM-1, FcγRIIB, TNFR1, IKK2 and LTbR compared to sham mice. Adoptive transfer studies showed there was no B cell-intrinsic defect. In summary, our data suggest that the reduced Ag-specific antibody response in CLP mice is secondary to a disruption in FDC and GC B cell function.
Minakshi Rana, Andrea La Bella, Rivka Lederman, Bruce T. Volpe, Barbara Sherry, Betty Diamond
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