Severe COVID-19 is associated with fungal colonization of the nasopharynx and potent induction of IL-17 responses in the nasal epithelium

Recent case reports and epidemiological data suggest fungal infections represent an under-appreciated complication among people with severe COVID-19. However, the frequency of fungal colonization in patients with COVID-19 and associations with specific immune responses in the airways remain incompletely defined. We previously generated a single-cell RNA-sequencing (scRNA-seq) dataset characterizing the upper respiratory microenvironment during COVID-19, and mapped the relationship between disease severity and the local behavior of nasal epithelial cells and infiltrating immune cells. Our study, in agreement with findings from related human cohorts, demonstrated that a profound deficiency in host immunity, particularly in type I and type III interferon signaling in the upper respiratory tract, is associated with rapid progression to severe disease and worse clinical outcomes. We have now performed further analysis of this cohort and identified a subset of participants with severe COVID-19 and concurrent detection of Candida species-derived transcripts within samples collected from the nasopharynx and trachea. Here, we present the clinical characteristics of these individuals, including confirmatory diagnostic testing demonstrating elevated serum (1, 3)-β-D-glucan and/or confirmed fungal culture of the predicted pathogen. Using matched single-cell transcriptomic profiles of these individuals’ respiratory mucosa, we identify epithelial immune signatures suggestive of IL-17 stimulation and anti-fungal immunity. Further, we observe significant expression of anti-fungal inflammatory cascades in the nasal and tracheal epithelium of all participants who went on to develop severe COVID-19, even among participants without detectable genetic material from fungal pathogens. Together, our data suggests that IL-17 stimulation – in part driven by Candida colonization – and blunted type I/III interferon signaling represents a common feature of severe COVID-19 infection. ### Competing Interest Statement A.K.S. reports compensation for consulting and/or SAB membership from Merck, Honeycomb Biotechnologies, Cellarity, Repertoire Immune Medicines, Hovione, Ochre Bio, Third Rock Ventures, FL82, Empress Therapeutics, Senda Biosciences, IntrECate Biotherapeutics, Relation Therapeutics, and Dahlia Biosciences. J.O.M. reports compensation for consulting services with Cellarity and Hovione. ### Funding Statement This project has been made possible in part by grant number 2020-216949 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation to A.K.S. and J.O.M.. C.G.K.Z. was supported by award number T32GM007753 and T32GM144273 from the National Institute of General Medical Sciences. J.O.M is a New York Stem Cell Foundation - Robertson Investigator. J.O.M was supported by the Richard and Susan Smith Family Foundation, the AGA Research Foundation's AGA-Takeda Pharmaceuticals Research Scholar Award in IBD - AGA2020-13-01, the HDDC Pilot and Feasibility P30 DK034854, the Food Allergy Science Initiative, the Leona M. and Harry B. Helmsley Charitable Trust, The Pew Charitable Trusts Biomedical Scholars, The Broad Next Generation Award, The Chan Zuckerberg Initiative Pediatric Networks, The Mathers Foundation, The New York Stem Cell Foundation, and The Manton Foundation Cell Discovery Network at Boston Children's Hospital. B.H.H. was supported by DK122532-01A1, NIH; 12019PG-CD002, The Leona M. and Harry B. Helmsley Charitable Trust; SRA #54518, and Crohn's and Colitis Foundation. A.K.S. was supported by the Bill and Melinda Gates Foundation, Sloan Fellowship in Chemistry, the NIH (5U24AI118672), and the Ragon Institute of MGH, MIT and Harvard. This work was supported in part by the Division of Intramural Research of the NIAID. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB of the University of Mississippi Medical Center gave ethical approval for this work under IRB#2020-0065. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data analyzed in this study is publicly available from prior manuscripts. Aligned and annotated scRNA-seq data can be downloaded via the Single Cell Portal, study SCP1289 (https://singlecell.broadinstitute.org/single\_cell/study/SCP1289). Aligned TPM-normalized RNA-seq data can be downloaded via the Single Cell Portal, study SCP822 (https://singlecell.broadinstitute.org/single\_cell/study/SCP822).