Last updated: 2025-02-20

Checks: 7 0

Knit directory: paed-inflammation-CITEseq/

This reproducible R Markdown analysis was created with workflowr (version 1.7.1). The Checks tab describes the reproducibility checks that were applied when the results were created. The Past versions tab lists the development history.

R Markdown file: up-to-date

Great! Since the R Markdown file has been committed to the Git repository, you know the exact version of the code that produced these results.

Environment: empty

Great job! The global environment was empty. Objects defined in the global environment can affect the analysis in your R Markdown file in unknown ways. For reproduciblity it’s best to always run the code in an empty environment.

Seed: set.seed(20240216)

The command set.seed(20240216) was run prior to running the code in the R Markdown file. Setting a seed ensures that any results that rely on randomness, e.g. subsampling or permutations, are reproducible.

Session information: recorded

Great job! Recording the operating system, R version, and package versions is critical for reproducibility.

Cache: none

Nice! There were no cached chunks for this analysis, so you can be confident that you successfully produced the results during this run.

File paths: relative

Great job! Using relative paths to the files within your workflowr project makes it easier to run your code on other machines.

Repository version: 7654aa3

Great! You are using Git for version control. Tracking code development and connecting the code version to the results is critical for reproducibility.

The results in this page were generated with repository version 7654aa3. See the Past versions tab to see a history of the changes made to the R Markdown and HTML files.

Note that you need to be careful to ensure that all relevant files for the analysis have been committed to Git prior to generating the results (you can use wflow_publish or wflow_git_commit). workflowr only checks the R Markdown file, but you know if there are other scripts or data files that it depends on. Below is the status of the Git repository when the results were generated: 


Ignored files:
    Ignored:    .Rhistory
    Ignored:    .Rproj.user/
    Ignored:    analysis/obsolete/
    Ignored:    data/.DS_Store
    Ignored:    data/C133_Neeland_batch1/
    Ignored:    data/C133_Neeland_merged/
    Ignored:    output/dge_analysis/obsolete/
    Ignored:    renv/library/
    Ignored:    renv/staging/

Untracked files:
    Untracked:  analysis/16.2_Figure_3.Rmd
    Untracked:  analysis/16.3_Figure_4.Rmd
    Untracked:  analysis/16.4_Figure_5.Rmd
    Untracked:  analysis/16.5_Supplementary_Figure_ADTs.Rmd
    Untracked:  analysis/cellxgene_submission.Rmd
    Untracked:  analysis/cellxgene_submission.nb.html
    Untracked:  broad_markers_seurat.csv
    Untracked:  code/background_job.R
    Untracked:  code/reverse_modifier_severity_comparisons.sh
    Untracked:  data/.Rprofile
    Untracked:  data/.gitattributes
    Untracked:  data/.gitignore
    Untracked:  data/.renvignore
    Untracked:  data/Homo_sapiens.gene_info.gz
    Untracked:  data/analysis/
    Untracked:  data/cellxgene_cell_ontologies_ann_level_1.xlsx
    Untracked:  data/cellxgene_cell_ontologies_ann_level_3 (1).xlsx
    Untracked:  data/cellxgene_cell_ontologies_ann_level_3.xlsx
    Untracked:  data/code/
    Untracked:  data/data/
    Untracked:  data/homologs.rds
    Untracked:  data/intermediate_objects/CD4 T cells.CF_samples.fit.rds
    Untracked:  data/intermediate_objects/CD4 T cells.all_samples.fit.rds
    Untracked:  data/intermediate_objects/CD8 T cells.CF_samples.fit.rds
    Untracked:  data/intermediate_objects/CD8 T cells.all_samples.fit.rds
    Untracked:  data/intermediate_objects/DC cells.CF_samples.fit.rds
    Untracked:  data/intermediate_objects/DC cells.all_samples.fit.rds

Unstaged changes:
    Modified:   .gitignore
    Modified:   analysis/06.0_azimuth_annotation.Rmd
    Modified:   analysis/09.0_integrate_cluster_macro_cells.Rmd
    Modified:   analysis/13.1_DGE_analysis_macro-alveolar.Rmd
    Modified:   analysis/13.7_DGE_analysis_macro-proliferating.Rmd
    Modified:   analysis/15.0_proportions_analysis_ann_level_1.Rmd
    Deleted:    analysis/99.0_Figure_1.Rmd
    Deleted:    analysis/99.0_Figure_2.Rmd
    Deleted:    analysis/99.0_Figure_3.Rmd
    Deleted:    analysis/99.0_Figure_4.Rmd
    Deleted:    analysis/99.0_Figure_5.Rmd
    Deleted:    analysis/99.0_Supplementary_Figure_3-5.Rmd
    Deleted:    analysis/99.0_Supplementary_Figure_ADTs.Rmd
    Modified:   code/utility.R

Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.

These are the previous versions of the repository in which changes were made to the R Markdown (analysis/index.Rmd) and HTML (docs/index.html) files. If you’ve configured a remote Git repository (see ?wflow_git_remote), click on the hyperlinks in the table below to view the files as they were in that past version.

File Version Author Date Message
Rmd 7654aa3 Jovana Maksimovic 2025-02-20 wflow_publish("analysis/index.Rmd")
html 4da7e60 Jovana Maksimovic 2025-02-17 Build site.
Rmd 8570b01 Jovana Maksimovic 2025-02-17 wflow_publish("analysis/index.Rmd")
html 4c945a5 Jovana Maksimovic 2025-02-17 Build site.
Rmd 06eea5f Jovana Maksimovic 2025-02-17 wflow_publish("analysis/index.Rmd")
html 46deec5 Jovana Maksimovic 2025-02-17 Build site.
Rmd fe39889 Jovana Maksimovic 2025-02-17 wflow_publish("analysis/index.Rmd")
html 313a19b Jovana Maksimovic 2025-02-17 Build site.
Rmd feb99f9 Jovana Maksimovic 2025-02-17 wflow_publish("analysis/index.Rmd")
html 578fe51 Jovana Maksimovic 2025-02-17 Build site.
Rmd 3349c27 Jovana Maksimovic 2025-02-17 wflow_publish("analysis/index.Rmd")
html eee49c7 Jovana Maksimovic 2025-02-17 Build site.
Rmd f658337 Jovana Maksimovic 2025-02-17 wflow_publish("analysis/index.Rmd")
html 38ed75b Jovana Maksimovic 2025-01-08 Build site.
Rmd d981df6 Jovana Maksimovic 2025-01-08 wflow_publish("analysis/index.Rmd")
html 8fda8e1 Jovana Maksimovic 2025-01-07 Build site.
Rmd f997c86 Jovana Maksimovic 2025-01-07 wflow_publish("analysis/index.Rmd")
html 46af878 Jovana Maksimovic 2025-01-06 Build site.
Rmd e9817e4 Jovana Maksimovic 2025-01-06 wflow_publish("analysis/index.Rmd")
html ad7e033 Jovana Maksimovic 2024-12-24 Build site.
Rmd 0d7ad6c Jovana Maksimovic 2024-12-24 wflow_publish("analysis/index.Rmd")
html e879c15 Jovana Maksimovic 2024-12-05 Build site.
html 8224316 Jovana Maksimovic 2024-12-05 Build site.
Rmd e41ab7d Jovana Maksimovic 2024-12-05 wflow_publish("analysis/index.Rmd")
html e7e3228 Jovana Maksimovic 2024-12-05 Build site.
Rmd 208daae Jovana Maksimovic 2024-12-05 wflow_publish("analysis/index.Rmd")
html 8f1bb39 Jovana Maksimovic 2024-12-05 Build site.
Rmd 9826fd9 Jovana Maksimovic 2024-12-05 wflow_publish("analysis/index.Rmd")
html 6496315 Jovana Maksimovic 2024-12-05 Build site.
Rmd 2de0391 Jovana Maksimovic 2024-12-05 wflow_publish("analysis/index.Rmd")
html 66a77b8 Jovana Maksimovic 2024-12-05 Build site.
Rmd b827cd3 Jovana Maksimovic 2024-12-05 wflow_publish("analysis/index.Rmd")
html 58e67bc Jovana Maksimovic 2024-12-05 Build site.
Rmd 36374d2 Jovana Maksimovic 2024-12-05 wflow_publish("analysis/index.Rmd")
html c52df9b Jovana Maksimovic 2024-12-05 Build site.
Rmd ccdf2fe Jovana Maksimovic 2024-12-05 wflow_publish("analysis/index.Rmd")
html 551d1cd Jovana Maksimovic 2024-10-08 Build site.
Rmd 2f6af9a Jovana Maksimovic 2024-10-08 wflow_publish("analysis/index.Rmd")
html cc35a23 Jovana Maksimovic 2024-10-06 Build site.
Rmd 5d26d19 Jovana Maksimovic 2024-10-06 wflow_publish("analysis/index.Rmd")
html 08a6cad Jovana Maksimovic 2024-09-20 Build site.
Rmd d984571 Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html 940146e Jovana Maksimovic 2024-09-20 Build site.
Rmd a6a40cf Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html a424d2c Jovana Maksimovic 2024-09-20 Build site.
Rmd ec7a1e7 Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html 3ae6d3a Jovana Maksimovic 2024-09-20 Build site.
Rmd ef32d0e Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html b4b0451 Jovana Maksimovic 2024-09-20 Build site.
Rmd 96ba6ad Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html cdc811c Jovana Maksimovic 2024-09-20 Build site.
Rmd ebde854 Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html 409ab83 Jovana Maksimovic 2024-09-20 Build site.
Rmd e8a0431 Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html 965bc3a Jovana Maksimovic 2024-09-20 Build site.
Rmd ad95c63 Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html b0e1d6a Jovana Maksimovic 2024-09-20 Build site.
Rmd 485c7da Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html eb4219f Jovana Maksimovic 2024-09-20 Build site.
Rmd 073c6c8 Jovana Maksimovic 2024-09-20 wflow_publish("analysis/index.Rmd")
html d3d3208 Jovana Maksimovic 2024-09-10 Build site.
Rmd d6becda Jovana Maksimovic 2024-09-10 wflow_publish("analysis/index.Rmd")
html a2bdb1a Jovana Maksimovic 2024-09-09 Build site.
Rmd 1220c10 Jovana Maksimovic 2024-09-09 wflow_publish("analysis/index.Rmd")
html b8aa02b Jovana Maksimovic 2024-09-09 Build site.
Rmd 20d5234 Jovana Maksimovic 2024-09-09 wflow_publish("analysis/index.Rmd")
html 9f3cc9c Jovana Maksimovic 2024-09-05 Build site.
Rmd 89fab0f Jovana Maksimovic 2024-09-05 wflow_publish("analysis/index.Rmd")
html e8f4078 Jovana Maksimovic 2024-08-09 Build site.
Rmd 67d23a6 Jovana Maksimovic 2024-08-09 wflow_publish("analysis/index.Rmd")
html 0a984c7 Jovana Maksimovic 2024-08-09 Build site.
Rmd 409ecff Jovana Maksimovic 2024-08-09 wflow_publish("analysis/index.Rmd")
html 0588973 Jovana Maksimovic 2024-08-06 Build site.
Rmd 4ed52fb Jovana Maksimovic 2024-08-06 wflow_publish("analysis/index.Rmd")
html 839ebca Jovana Maksimovic 2024-08-06 Build site.
Rmd 61a99c6 Jovana Maksimovic 2024-08-06 wflow_publish("analysis/index.Rmd")
html fa2ea71 Jovana Maksimovic 2024-07-05 Build site.
Rmd ee9d775 Jovana Maksimovic 2024-07-05 wflow_publish(c("analysis/index.Rmd", "analysis/11.0_manual_annotations_t_cells_decontx.Rmd"))
html 0b24220 Jovana Maksimovic 2024-07-05 Build site.
Rmd 256e8cf Jovana Maksimovic 2024-07-05 wflow_publish(c("analysis/index.Rmd", "analysis/12.0_manual_annotations_other_cells.Rmd"))
html 4d7d217 Jovana Maksimovic 2024-07-04 Build site.
Rmd 13a1d06 Jovana Maksimovic 2024-07-04 wflow_publish(c("analysis/13.0_DGE_analysis_macro-alveolar_cells_CF-only-samples.Rmd",
html b1c0edc Jovana Maksimovic 2024-06-28 Build site.
Rmd 14a581d Jovana Maksimovic 2024-06-28 wflow_publish("analysis/index.Rmd")
html c51f4fb Jovana Maksimovic 2024-06-24 Build site.
Rmd 351975f Jovana Maksimovic 2024-06-24 wflow_publish("analysis/index.Rmd")
html d1785d7 Jovana Maksimovic 2024-05-20 Build site.
Rmd d178efc Jovana Maksimovic 2024-05-20 wflow_publish("analysis/index.Rmd")
html facb05f Jovana Maksimovic 2024-04-27 Build site.
Rmd 2c5e5ac Jovana Maksimovic 2024-04-27 wflow_publish(c("analysis/index.Rmd", "analysis/09.1_integrate_cluster_macro_cells_decontx.Rmd"))
html 048f0f3 Jovana Maksimovic 2024-03-20 Build site.
Rmd 6f4600b Jovana Maksimovic 2024-03-20 wflow_publish(c("analysis/index.Rmd", "analysis/integrate_cluster"))
html fb50017 Jovana Maksimovic 2024-03-19 Build site.
Rmd 3e9ce40 Jovana Maksimovic 2024-03-19 wflow_publish(c("analysis/index.Rmd", "analysis/integrate"))
html 7701592 Jovana Maksimovic 2024-02-26 Build site.
Rmd e6e53e9 Jovana Maksimovic 2024-02-26 wflow_publish(c("analysis/index.Rmd"))
html ecfde3d Jovana Maksimovic 2024-02-26 Build site.
Rmd 025ceae Jovana Maksimovic 2024-02-26 wflow_publish(c("analysis/index.Rmd", "analysis/01*"))
html e719a2c Jovana Maksimovic 2024-02-20 Build site.
Rmd 20cce23 Jovana Maksimovic 2024-02-20 wflow_publish("analysis/index.Rmd")
html 7b431a0 Jovana Maksimovic 2024-02-20 Build site.
Rmd 9f3012a Jovana Maksimovic 2024-02-20 wflow_publish("analysis/index.Rmd")
Rmd c1716bb Jovana Maksimovic 2024-02-16 Start workflowr project.

This site presents the code and results of the analyses described in the pre-print: “Single-cell atlas of BAL from preschool cystic fibrosis reveals key inflammatory pathways modified by ivacaftor in the early life lung”.

All the code and results of this analysis are available from GitHub at https://github.com/Oshlack/paediatric-cf-inflammation-citeseq. To reproduce the complete analysis follow the instructions on the getting started page.

Abstract

Aberrant inflammation and structural lung damage occurs early in life for people with cystic fibrosis (CF). Even in the era of CFTR modulators, anti-inflammatory therapy may still be needed to prevent establishment, and lifelong consequences, of bronchiectasis. In this study, we integrated transcriptome-wide single-cell RNA sequencing data, highly multiplexed surface protein expression, and functional pathway analysis to comprehensively investigate inflammatory cell responses in 37 BAL samples from children with CF and 8 BAL samples from non-CF controls. We show widespread dysregulation of macrophage function in the preschool CF lung, including alterations in pathways associated with SARS-COV-1 and -2 responses, TNF/IFN signalling and cholesterol homeostasis, that were further altered by the early development of bronchiectasis. We showed that the CFTR modulator ivacaftor restores some of these macrophage-related functional deficits and reduces expression of pathways associated with neutrophil infiltration, however the modulator lumacaftor/ivacaftor was not associated with any detectable change in response. This work represents the most comprehensive, multi-omic single-cell analysis of bronchoalveolar lavage from preschool children and the results inform the future development of anti-inflammatory therapy for children with CF.

Authors

Jovana Maksimovic1,2,9*, Shivanthan Shanthikumar 2,3,4*, George Howitt1,9, Gunjan Dixit1,9, Peter F Hickey5,6, William Ho5, Casey J.A. Anttila5, Daniel V. Brown5, Anne Senabouth7,8, Dominik Kaczorowski7,8, Daniela Amann-Zalcenstein5,6, Joseph E. Powell7,8, Sarath C. Ranganathan2,3,4, Alicia Oshlack1,9,10, Melanie R. Neeland2,3#.

1 Computational Biology Program, Peter MacCallum Cancer Centre, Parkville, VIC, Australia

2 Respiratory Diseases, Murdoch Children’s Research Institute, Parkville, VIC, Australia

3 Department of Paediatrics, University of Melbourne, Parkville, VIC, Australia

4 Respiratory and Sleep Medicine, Royal Children’s Hospital, Parkville, VIC, Australia

5 Advanced Genomics Facility, Advanced Technology and Biology Division, WEHI, Parkville, VIC, Australia

6 Department of Medical Biology, University of Melbourne, Parkville, VIC, Australia

7 Garvan-Weizmann Centre for Cellular Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia

8 UNSW Cellular Genomics Futures Institute, University of New South Wales, Sydney, NSW, Australia

9 Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, VIC, Australia

10 School of Mathematics and Statistics, University of Melbourne, Parkville, VIC, Australia

*Contributed equally

Analysis Overview

Follow the links below to view the different parts of the analysis.

Differential Expression Analysis between Conditions

  1. Differential expression analysis of macrophages
    1. Macrophages
    2. Alveolar macrophages
    3. APOC2+ macrophages
    4. CCL macrophages
    5. IFI21 macrophages
    6. Lipid macrophages
    7. Monocyte-derived macrophages
    8. Proliferating macrophages
  1. Differential expression analysis of T/NK and rare cells
    1. CD4 T-cells
    2. CD8 T-cells
    3. DC cells
  1. Analyse cell types proportions
    1. Annotation level 1
    2. Annotation level 3, non macro. cells
    3. Annotation level 3, macro. cells

Licenses

The code in this analysis is covered by the MIT license and the written content on this website is covered by a Creative Commons CC-BY license.

Version Information

R version: R version 4.3.3 (2024-02-29)

Bioconductor version: 3.18


sessionInfo()
R version 4.3.3 (2024-02-29)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 22.04.4 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3 
LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.20.so;  LAPACK version 3.10.0

locale:
 [1] LC_CTYPE=en_AU.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_AU.UTF-8        LC_COLLATE=en_AU.UTF-8    
 [5] LC_MONETARY=en_AU.UTF-8    LC_MESSAGES=en_AU.UTF-8   
 [7] LC_PAPER=en_AU.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_AU.UTF-8 LC_IDENTIFICATION=C       

time zone: Etc/UTC
tzcode source: system (glibc)

attached base packages:
[1] stats     graphics  grDevices datasets  utils     methods   base     

other attached packages:
[1] workflowr_1.7.1

loaded via a namespace (and not attached):
 [1] jsonlite_1.8.8      compiler_4.3.3      BiocManager_1.30.22
 [4] renv_1.0.3          promises_1.2.1      Rcpp_1.0.12        
 [7] stringr_1.5.1       git2r_0.33.0        callr_3.7.3        
[10] later_1.3.2         jquerylib_0.1.4     yaml_2.3.8         
[13] fastmap_1.1.1       R6_2.5.1            knitr_1.45         
[16] tibble_3.2.1        rprojroot_2.0.4     bslib_0.6.1        
[19] pillar_1.9.0        rlang_1.1.4         utf8_1.2.4         
[22] cachem_1.0.8        stringi_1.8.3       httpuv_1.6.14      
[25] xfun_0.42           getPass_0.2-4       fs_1.6.5           
[28] sass_0.4.9          cli_3.6.3           magrittr_2.0.3     
[31] ps_1.7.6            digest_0.6.34       processx_3.8.3     
[34] rstudioapi_0.15.0   lifecycle_1.0.4     vctrs_0.6.5        
[37] evaluate_0.23       glue_1.8.0          whisker_0.4.1      
[40] fansi_1.0.6         rmarkdown_2.25      httr_1.4.7         
[43] tools_4.3.3         pkgconfig_2.0.3     htmltools_0.5.8.1