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Am J Respir Crit Care Med
Am J Respir Crit Care Med
ajrccm
American Journal of Respiratory and Critical Care Medicine
1073-449X
1535-4970
American Thoracic Society

202312-2279LE
10.1164/rccm.202312-2279LE
Correspondence
Single-Cell RNA Sequencing Shows that Circulating Monocytes Enriched in IFN Signaling Are Associated with Nontuberculous Mycobacteria Pulmonary Disease in Cystic Fibrosis
https://orcid.org/0000-0001-9159-1632
Lorè Nicola I. 1 *
Gramegna Andrea 2 3 *
https://orcid.org/0000-0003-1103-7688
de Pretis Stefano 5 *
https://orcid.org/0000-0003-0376-1342
Di Marco Federico 1
https://orcid.org/0000-0001-8695-649X
Giannese Francesca 5
Saliu Fabio 1
https://orcid.org/0009-0006-4553-2221
Oneto Caterina 5
Contarini Martina 3
Cariani Lisa 4
Blasi Francesco 2 3 ‡
https://orcid.org/0000-0001-6415-1535
Cirillo Daniela M. 1 ‡
1 Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Ospedale San Raffaele, Milan, Italy;
2 Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy;
3 Respiratory Unit and Adult Cystic Fibrosis Center and
4 SC Clinical Pathology, Laboratory of Microbiology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; and
5 Center for Omics Sciences, IRCCS San Raffaele Institute, Milan, Italy
Correspondence and requests for reprints should be addressed to Nicola I. Lorè, Ph.D., Emerging Bacterial Pathogens Unit, Division of Immunology, Transplantation and Infectious Diseases, IRCCS Ospedale San Raffaele, Via Olgettina, 60, 20132 Milan, Italy. Email: lore.nicolaivan@hsr.it.
* Co–first authors.

‡ Co–last authors.

18 6 2024
15 9 2024
18 6 2024
210 6 834837
Copyright © 2024 by the American Thoracic Society
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0. For commercial usage and reprints, please e-mail Diane Gern (dgern@thoracic.org).

Fondazione per la Ricerca sulla Fibrosi Cistica 10.13039/501100008385 FFC#20/2020 FFC#7/2022 Ministero della Salute 10.13039/501100003196 GR-2021-12374157
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pmcTo the Editor:

Nontuberculous mycobacteria (NTM) infection can promote progressive lung function decline and damage, a condition known as NTM pulmonary disease (NTM-PD). People with an underlying respiratory disease, such as people with cystic fibrosis (pwCFs) or bronchiectasis, are at a higher risk of developing a progressive NTM-PD that is normally associated with poorer clinical outcomes and a possible contraindication to lung transplant (1–3). Mycobacterium abscessus complex (MABSC) is a common cause of infections in pwCFs (4–7). In CF, it is difficult to distinguish whether the worsening of clinical and radiological endpoints is attributable to other concomitant chronic bacterial infections or a direct role of NTM. Therefore, new tools that can diagnose NTM-PD and aid in the clinical management of pwCFs are among the most relevant unmet needs in translational research.

Reliable immune signatures, such as complex circulating transcriptional profiles in specific cell subsets, could define the different pulmonary disease stages and improve decision-making processes associated with mycobacterial persistence, as in the case of NTM pulmonary disease by the MABSC. Here, we performed the first single-cell (sc) transcriptome characterization of circulating peripheral blood mononuclear cells (PBMCs) in pwCFs with MABSC infections.

We enrolled a cohort of 10 consecutive adults with CF followed at the Adult CF Center of Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca’ Granda Ospedale Maggiore Policlinico (Milan, Italy; BioMol-MA study protocol 23/INT/2021) and stratified them into three groups: 1) group CF included individuals with no history of positive sputum cultures for NTM, 2) group MABSC included individuals who had two or more positive sputum cultures for MABSC but met no clinical or radiological criteria for NTM-PD in the previous 12 months (1, 8), and 3) group MABSC-PD included individuals with two or more positive sputum cultures for MABSC and clinical and radiological deterioration associated with NTM-PD. All patients had diagnostic criteria for CF according to current recommendations (9) and were NTM treatment–naive. Further clinical characteristics are reported in Table 1.

Table 1. Clinical Characteristics of Subjects Included in the Study

Characteristic	CF (n = 4)	MABSC (n = 3)	MABSC-PD (n = 3)	
Mean age ± SD, yr	40.5 ± 11.15	40 ± 5.5	51.25 ± 20.59	
Sex	 	 	 	
 Male	1 (25%)	2 (66.6%)	0	
 Female	3 (75%)	1 (33.3%)	3 (100%)	
Mutation background	 	 	 	
 ⩾1 Phe508del variant	3 (75%)	1 (33.3%)	3 (75%)	
 No F508del variant	1 (25%)	2 (66.6%)	1 (25%)	
Median FEV1 ± SD, % predicted	65.5 ± 15.15	76 ± 18.24	76 ± 10.4	
Mean BMI ± SD, kg/m2	21.44 ± 0.78	22.77 ± 2.29	19.98 ± 0.76	
NTM microbiology	 	 	 	
 MABSC	No	3 (100%)	3 (100%)	
 Other NTM	No	No	No	
Chronic Pseudomonas aeruginosa infection	2 (50%)	2 (66.6%)	3 (100%)	
Chronic Staphylococcus aureus infection	2 (50%)	1 (33.3%)	3 (100%)	
Pancreatic exocrine insufficiency	1 (25%)	1 (33.3%)	0	
Definition of abbreviations: BMI = body mass index; CF = cystic fibrosis; MABSC = Mycobacterium abscessus complex; NTM = nontuberculous mycobacteria; PD = pulmonary disease; SD = standard deviation.

We isolated human PBMCs from 7 ml of blood using density gradient centrifugation and created a standardized scRNA sequencing workflow for the collected PBMCs. Single cells were immediately encapsulated within 4 hours of the blood drawing and barcoded using the 10X Chromium Controller platform (10X Genomics) and Chromium Single Cell 3′ Reagent Kit (v3.1 Chemistry; 10X Genomics). The cell viability determined with trypan blue staining was >90%. Reads were then assigned to genes, and counts were extracted using featureCount (version 1.6.4) (10). Cells with a feature count lower than 200 or comprising >25% mitochondrial reads were filtered out. Genes expressed in fewer than five cells were excluded from the analysis. The analysis was conducted using the R package Seurat (version 4.1.0) (11). We profiled a total of 55,904 PBMCs (median of 5,570 cells per sample, median of 18,063 cells per group; accession GSE234785 and https://github.com/Allen13x/MABSc_PBMC_scRNAseq.git). We identified the following 10 distinct cell populations based on known transcriptomic markers (11) (Figure 1A): CD4+ naive T cells, CD14+ monocytes, CD8+ effector cells, natural killer (NK) cells, CD8 naive T cells, CD16+ monocytes, B cells, CD4+ effector cells, platelets, and dendritic cells. In terms of different cell types or cellular abundance, we did not observe cellular variation among pwCFs with different risks of MABSC-PD (Figure 1B). Our findings indicated that pwCFs with different risks of M. abscessus lung disease displayed similar cellular PBMC profiles.

Figure 1. The landscape of single-cell RNA sequencing of peripheral blood mononuclear cells in people with cystic fibrosis (pwCFs) with different Mycobacterium abscessus lung disease stages. Peripheral blood mononuclear cells (PBMCs) were isolated from adult pwCFs who were categorized into three groups based on their infection status and clinical stability: pwCFs with no history of nontuberculous mycobacteria (NTM) detection and in clinically stable condition (CF group), pwCFs colonized with NTM (Mycobacterium abscessus complex [MABSC] group), and pwCFs with a clinical diagnosis of pulmonary disease (MABSC-PD group). (A) Uniform Manifold Approximation and Projection (UMAP) visualization of PBMCs from 10 pwCFs at different MABSC disease stages (each dot represents a single cell, and cells are labeled by cell type and percentage of PBMC cellular composition among different cell types) and dot plot depicting the expression of the top three canonical marker genes associated with PBMCs. (B) Proportions of each cell type in each sample. (C) Dot plot showing the number of differentially expressed genes (DEGs) obtained from DEG analysis among groups (x-axis; e.g., MABSC-PD vs. MABSC) in each cell cluster/cell type (y-axis) (log2 fold change ⩾0.25 and Bonferroni-adjusted P < 0.001). Dot size represents the number of DEGs for upregulated (red) and downregulated (blue) genes. (D) Heat map of CD14+ transcriptional profiles of a unique RNA signature consisting of 47 upregulated (log2 fold change ⩾0.25 and adjusted P < 0.001) in the MABSC-PD group versus the CF group and the MABSC-PD group versus the MABSC group. (E) Dot plot of Gene Ontology (GO) term enrichment showing selected enriched pathways within the 47 genes related to the RNA signature in the CD14+ monocyte cluster. The overall ranking of the selected GO terms is indicated in brackets in each GO term. Colors indicate the adjusted P value, and the dot size is proportional to the number of DEGs in the given pathway (F) UMAP visualization of CF, MABSC, and MABSC-PD groups color-coded (blue, low; red, high) based on RNA signature score (scored with 47 genes) in each cell type and in the monocyte subcluster (CD14+ and CD16+ monocytes). (G) Dot plot representing the expression-weighted cell type enrichment results to infer cell type activity from bulk tissue transcriptomes. Dot size represents enrichment z-scores for upregulated (red) and downregulated (blue) genes for the genes of different cell types of our dataset (12). Results are reported for the differential gene expression analysis performed including all NTM species (all) or only M. abscessus. DC = dendritic cell; NK = natural killer.

To determine whether gene expression profiles may be differently regulated in the characterized PBMCs from pwCFs at different stages of NTM lung infection, we performed a differential gene expression analysis (Wilcoxon rank sum test). The overall counts of differentially expressed genes (DEGs; with absolute log2 fold change ⩾0.25 and adjusted P < 0.001) among different cellular clusters and groups are shown in Figure 1C. The CD14+ monocyte cluster in the MABSC-PD group displayed the highest number of upregulated genes among cellular clusters compared with the CF or MABSC group. Within the same comparisons, downregulated genes were mostly related to CD8+ T effector and NK cellular clusters (Figure 1C).

Then, we focused on CD14+ transcripts specifically upregulated in the MABSC-PD group (i.e., MABSC-PD vs. MABSC and MABSC-PD vs. CF) and determined a cell type–specific RNA signature characterized by 47 upregulated genes (Figure 1D) (adjusted P < 0.001 and absolute log2 fold change >0.25), mainly enriched with IFN-related Gene Ontology terms (Figure 1E). Moreover, we investigated how the expression of the upregulated gene signature in the CD14+ monocyte cluster (Figure 1D) may vary across cell types by calculating a CD14+ signature score based on the expression of the signature genes in each cell (Figure 1F). Using this approach, we were able to demonstrate that the CD14+ signature’s overall score expression is localized to the CD14+ monocyte cluster and is higher in the MABSC-PD group than in the CF and MABSC groups (Figure 1F). The same approach was applied to the CD16+ monocyte cluster, which revealed a small set of 18 altered genes. However, the expression of these genes was not specific to the CD16+ cluster.

Next, to validate the cellular phenotypes associated with our transcriptomic profiles within PBMCs, we exploited a publicly accessible whole-blood dataset (bulk RNA sequencing) derived from a cohort of pwCFs (GSE205161), with a particular focus on subjects positive for M. abscessus (i.e., MABSC) and Mycobacterium avium complex (MAC). For each NTM species, we selected from the bulk dataset the 250 most significantly up- and downregulated genes identified from the comparison between pwCFs with NTM colonization and NTM-PD (Figure 1G). These genes were then subjected to the expression-weighted cell type enrichment method using our single-cell dataset as a reference for defining cell types (12). The analysis revealed that the upregulated genes in MABSC cases (MABSC-PD, n = 5; MABSC, n = 6) were significantly enriched for monocyte-specific genes, including CD14+ and CD16+ subsets (Figure 1G). In addition, we confirmed decreased gene expression profiles in the T cell cellular clusters, including CD4+ naive T cell, CD4+ effector cell, and CD8 naive T cell clusters, although no enrichment was observed in CD8+ effector or NK cells. The same analysis conducted for MAC cases (MAC-PD, n = 7; MAC, n = 15 cases) revealed distinct enriched cell types, suggesting that pulmonary disease caused by MABSC or MAC may trigger different immune responses depending on the underlying pathogenic species. Finally, Gene Set Enrichment Analysis confirmed the enrichment of the IFN signaling term among positively regulated genes in the publicly available bulk RNA dataset comparing MABSC-PD (n = 5) versus MABSC (n = 6) groups (normalized enrichment score, 2.41; adjusted P = 9.98 × 10–13).

In conclusion, our data demonstrate the effectiveness of scRNA sequencing technologies and provide new insights in characterizing the circulating immune profile for the stratification of pwCFs with MABSC colonization or active MABSC-PD in CF. Here, we directly characterized the gene expression patterns related to IFN signaling in circulating monocytes, with a specific focus on the CD14+ monocyte cluster. Overall, the development of circulating transcriptomic profiling at the single-cell level for tailored identification of pwCFs with NTM infection holds promise for the identification of novel diagnostic signatures or therapeutic cellular targets to improve the quality of life of people with NTM-PD.

Supported by Fondazione per la Ricerca sulla Fibrosi Cistica grants FFC#7/2022 to N.I.L. and L.C. (with the contribution of “Delegazione FFC Ricerca di Milano” and “Delegazione FFC Ricerca di Olbia”) and FFC#23/2020 to N.I.L. (with the contribution of “Delegazione FFC Ricerca di Olbia”, “Kymos Srl SB”, “Antonio Guadagnin & Figlio Srl”, “Gruppo di sostegno FFC Ricerca di Martinsicuro Teramo”, “LIFC Toscana Onlus” and “Delegazione FFC Ricerca di Cecina e Rosignano”) and Ministero della Salute grant GR-2021-12374157 to N.I.L., S.d.P., and M.C.

Author Contributions: N.I.L. and A.G. conceptualized the study. N.I.L. acquired funding and supervised the study. N.I.L., A.G., F.G., F.S., M.C., and L.C. performed sample collection, phenotyping, and processing protocols. A.G. facilitated sample collection infrastructure. F.G., C.O., and N.I.L. performed single-cell barcoding library construction. Data were processed, curated, and visualized by F.D.M. under the supervision of S.d.P. and analyzed by F.D.M. and S.d.P. The manuscript was drafted by N.I.L., S.d.P., F.D.M., A.G., M.C., L.C., F.B., and D.M.C. and was reviewed and edited by all other authors.

Originally Published in Press as DOI: 10.1164/rccm.202312-2279LE on June 18, 2024

Author disclosures are available with the text of this letter at www.atsjournals.org.
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