Impact of pulmonary African trypanosomes on the immunology and function of the lung

Approximately 20% of sleeping sickness patients exhibit respiratory complications, however, with a largely unknown role of the parasite. Here we show that tsetse fly-transmitted Trypanosoma brucei parasites rapidly and permanently colonize the lungs and occupy the extravascular spaces surrounding the blood vessels of the alveoli and bronchi. They are present as nests of multiplying parasites exhibiting close interactions with collagen and active secretion of extracellular vesicles. The local immune response shows a substantial increase of monocytes, macrophages, dendritic cells and γδ and activated αβ T cells and a later influx of neutrophils. Interestingly, parasite presence results in a significant reduction of B cells, eosinophils and natural killer cells. T. brucei infected mice show no infection-associated pulmonary dysfunction, mirroring the limited pulmonary clinical complications during sleeping sickness. However, the substantial reduction of the various immune cells may render individuals more susceptible to opportunistic infections, as evident by a co-infection experiment with respiratory syncytial virus. Collectively, these observations provide insights into a largely overlooked target organ, and may trigger new diagnostic and supportive therapeutic approaches for sleeping sickness.


Statistics
For all statistical analyses, confirm that the following items are present in the figure legend, table legend, main text, or Methods section.
n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement A statement on whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one-or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section.
A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted

Give P values as exact values whenever suitable.
For Bayesian analysis, information on the choice of priors and Markov chain Monte Carlo settings For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes Estimates of effect sizes (e.g. Cohen's d, Pearson's r), indicating how they were calculated Our web collection on statistics for biologists contains articles on many of the points above.

Software and code
Policy information about availability of computer code Data collection LivingImage v4.3.1, AnalySIS iTEM software (Olympus), nSolver 4.0 (NanoString) Data analysis Omics Playground v2.8.5, GraphPad Prism 7 and 9, FlowLogic 8.6, LivingImage v4.3.1, nSolver 4.0 (NanoString) For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

Data
Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy The authors declare that the data underlying the findings of this study are available within the paper and its Supplementary  All studies must disclose on these points even when the disclosure is negative.

Sample size
For all in vivo work 3-5 animals have been used in each group, time point and independent experiment. Sample size information is provided in the methods section (animals and statistics section) and in the figure legends of the corresponding experiments.
For the descriptive microscopy of the infected lung tissue and the nCounter transcript analysis 3 mice per group was chosen as sufficient based on our previous experience with transcriptomics and the observed variation in the infection model. For the flow cytometric analysis, the required amount of mice per group was estimated using the G-power software package considering an ANOVA repeated measures, between factors analysis was performed to compare immune cells per milliliter obtained by flow cytometry of three groups (non-infected, 10 dpi and 21 dpi). The means and SD used in the effect size calculation are based on an experiment performed with 3 mice/group. The total sample size was estimated to be 9, resulting in 3 mice per group and time point. For the lung function measurements, the exact amount of mice per group was calculated using the G-power software package. An ANOVA repeated measures, between factors analysis was performed to compare airway resistance obtained by flexiVent analysis of three groups (non-infected, 10 dpi and 21 dpi) with nine measurements. The means and SD used in the effect size calculation are based on an experiment performed with 3 mice/group. The total sample size was estimated to be 15 resulting in 5 mice per group and time point. This is in accordance with the sample size reported in literature of Flexivent and breath analysis measurements (Kottom et al., 2022; https://doi.org/10.1007/ s40268-022-00389-0).
Data exclusions In the flexivent experiment, 1 mouse of each group (NI, 10 dpi and 21 dpi) was excluded due to errors with the experimental setup (which led to an accumulation of fluid into the lungs) which did not allow us to acquire a complete lung function profile of these mice.

Replication
Microscopy experiments were performed in 1 independent experiment. Findings were considered relevant only if they occurred in all tested animals. nCounter analysis was performed once on 3 independent mice as well as 2 different timepoints. All flow cytometry experiments were performed in at least 2 independent repeats (except for the gamma delta T cells which was performed once as part of the rebuttal process).
Randomization Animals were allocated in experimental groups based on simple randomization.

Blinding
Blinding was not executed for this study since it consists of a characterization of a disease phenotype and thus considered not subject to subjectivity of the researcher.

Reporting for specific materials, systems and methods
We require information from authors about some types of materials, experimental systems and methods used in many studies. Here, indicate whether each material, system or method listed is relevant to your study. If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.

Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals C57BL/6JRj mice were used, all female between 6-8 weeks at the start of infection. Male and female tsetse flies (Glossina morsitans morsitans) were used in this study. Newly emerged flies were infected and used after 3-4 weeks post infection for natural transmission to the mouse host. Infected flies were used throughout their entire lifespan.

Wild animals
No wild animals were used in this study.
Field-collected samples No field-collected samples were used in this study.

Ethics oversight
Ethical Committee (ECD) of the University of Antwerp. Ethical approval: UA-ECD 2017-04 and 2022-55 Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology Sample preparation
To generate single-cell suspensions from perfused lungs, the Mouse Lung Dissociation Kit (Miltenyi Biotec) was used. Briefly, resected lung lobes were transferred into gentleMACSTM C tubes (Miltenyi Biotec) containing 2.4 mL of the enzyme mix (Miltenyi Biotec). After running a first lung-specific gentleMACSTM program, samples were incubated for 30 min in a 37°C water bath, whilst shaken every 5 min, followed by a second lung-specific gentleMACSTM program. Next, cell suspensions were transferred into Falcon tubes and centrifuged for 10 min at 300×g (4°C), and pellets were resuspended in 3 mL ammonium-chloride-potassium (ACK) buffer (0.15 M NH4Cl, 1.0 mM KHCO3, 0.1 mM Na2EDTA) for a 7 min erythrocyte lysis at RT. Pellets were recovered after a centrifugation step at 300×g (4°C), resuspended in PBS + 0.2% bovine serum albumin (BSA) buffer and filtered through a 100 μm filter (Miltenyi Biotec). Cell suspensions were counted using a KOVA® counting chamber. Cell suspensions (2 × 107/mL) were treated with FcɣR-blocking agent (anti-CD16/32, clone 2.4G2, BD Biosciences)

March 2021
for 15 min in PBS + 0.2% BSA buffer. Next, cells were incubated for 20 min at 4°C with a mix of fluorescent conjugated antimouse antibodies at optimized concentrations (Table S1). DAPI Staining Solution (Miltenyi Biotec) was used to assess viability.
Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.