Improved cytometric analysis of untouched lung leukocytes by enzymatic liquefaction of sputum samples

Immunophenotyping of sputum-resident leukocytes is a valuable approach for monitoring local immune responses in pulmonary infections [1,2,3,4] or allergic reactions [5], as well as airways diseases with autoinflammatory and/or autoimmune features [6,7,8,9,10,11,12]. The lung is a major immunological organ that harbors complex interactions between immune and structural cells which are essential to sustain the host homeostasis [13]. Thus, unraveling the cell types and functions that contribute to lung immunity has tremendous potential to better understand susceptibility to infections and aid in the definition of inflammatory stages in chronic airways diseases [14, 15]. These analyses require accurate identification and characterization of individual cell populations and the gold-standard technique to address them is flow cytometry. In spite of sputum samples being a valuable source of lung leukocytes from central airways (a region of the lung which is difficult to sample non-invasively) their cytometric analysis is hampered because they need to be liquefied prior to analysis. Moreover, sputum contains contaminating epithelial cells and variable amounts of debris which make the cytometric analysis challenging.

Standard protocols for liquefying respiratory samples are based on reducing disulfide bonds cross-linking mucins in the matrix (Fig. 1A) [16, 17]. The most frequently used reducing agent is DTT, despite its negative impact on cell functionality and detection of surface markers [18, 19]. Using mild buffer treatments (e.g., PBS) can circumvent these issues [20], but they require intense mechanical dispersion and do not liquefy highly viscous sputum samples, which jeopardizes the proper handling of cells. Therefore, developing better liquefying methods that preserve the cell integrity is still a challenge in flow cytometry of sputum samples.

Fig. 1figure 1

Schematic illustration of methods for liquefying respiratory samples and comparative cytometric analysis of sputum leukocytes. A DTT reduces disulfide bonds cross-linking mucins in the matrix, and the enzymatic method uses endogenous catalase from sputum samples for producing oxygen bubbles, which mechanically disrupts cross-linked mucins. B The enzymatic liquefaction of sputum samples improves the single-cell analysis of sputum-resident leukocytes by flow cytometry, ensures the cell viability and holds their activation status unaltered

In this manuscript we introduce an alternative approach for sample liquefaction that improves the cytometric analysis of sputum-resident leukocytes. It consists of using endogenous catalase enzymes for producing oxygen bubbles that mechanically disrupt respiratory samples (Fig. 1A). Catalase is a ubiquitous enzyme produced by commensal flora and opportunistic pathogens of the lungs [21] as well as by host immune cells in normal and pathological conditions [22,23,24,25,26]. We already validated this enzymatic method for detecting pathogens causing pulmonary infections [27], and for improving the detection of lung biomarkers [28,29,30]. Here it will be shown that it can be used to prepare sputum samples for cytometric analysis as well. Following our protocol for enzymatically liquefying respiratory samples the single-cell suspensions required for flow cytometry are obtained more rapidly and efficiently than with the traditional reducing procedure. Furthermore, the proposed enzymatic method could be used to address functional analysis of sputum-resident leukocytes, since it leaves the cells viable and widely untouched (Fig. 1B). The results shown here pave the way for a better profiling of pathological cellular states in lung leukocytes, which could improve the diagnosis and drive the development of targeted therapeutics in airways diseases.

Materials and methodsRespiratory samples collection

Forty-five expectorated sputum samples were collected from June 2021 to May 2022 by the Department of Microbiology from Son Espases University Hospital (Balearic Islands). The study was conducted according to the ethical guidelines of the 1975 Declaration of Helsinki. A microscopic screening was performed to determine their suitability for being included in the study. The inclusion criteria were; (i) sputum expectorated within last 24 h, (ii) a positive result in the Gram’s stain test, and (iii) the presence of ≥ 25 polymorphonuclear white blood cells and < 10 squamous epithelial cells per field (at 100 × total magnification). All samples were anonymized leftover specimens that, otherwise, would have been discarded. They were obtained in the clinical routine practice from patients with suspected pulmonary infection. Any leftovers were destroyed. For these reasons the institutional review board considered the study as minimal-risk research and waived the requirement for informed consent (Ethics and Scientific Committee approval with reference IB 4005/19-PI).

Liquefaction of sputum samples

120 mg of each sputum sample was weighed out in triplicate using a HR-150AZ analytical balance (e = 0.001 g) and collected in conical 15 mL polypropylene tubes. Then, sputum samples were liquefied following the gold standard method based on using dithiotreitol (DTT) as reducing agent, or our enzymatic method based on adding H2O2. Briefly, for the reducing method sputum samples were liquefied by adding 6.5 mM DTT (Invitrogen) in PBS at a 10:1 constant ratio (v/w) for 30 min at 37 ºC with 10-min intervals of vigorous vortex mixing. For the enzymatic method sputum samples were liquefied by adding 0.3 M H2O2 (Invitrogen) in PBS at a 10:1 constant ratio (v/w) for 60–120 s at room temperature (RT). Additionally, sputum samples treated with PBS at a 10:1 constant ratio (v/w) for 30 min at RT with 10-min intervals of vigorous vortex mixing were included as controls. 200 µL of liquefied sputum samples was collected in 1.8 mL cryotubes and kept at -20 °C until turbidimetry evaluation. The remaining solutions of liquefied sputum samples were subsequently washed by centrifugation at 1700 rpm for 5 min after adding 10 mL flow cytometry buffer (FCB); PBS supplemented with 2% heat inactivated fetal bovine serum (Sigma-Aldrich) and 1 mM ethylene diamine tetraacetic acid (EDTA, Sigma-Aldrich). After supernatants removal, cell pellets from liquefied sputum samples were resuspended in 1 mL FCB and then filtered through 50 µm cup-type cell strainers (Becton Dickinson) in 1.5 mL Eppendorf tubes. The resulting cell suspensions account for the cellular content of 100 mg of liquefied sputum.

Microscopy for cell counting and viability

Cell density from liquefied sputum suspensions was adjusted for appropriate cell counting in a Neubauer chamber (BlauBrand). 10 µL of previously adjusted cell suspensions in FCB was added to 10 µL 0.4% Trypan blue solution (Sigma-Aldrich), then two counting grids of the Neubauer chamber were filled and the viable/dead cell counts were performed in duplicate in a bright field light microscope (Leica). Cell counts were calculated as follows; [Sputum cells·mg−1 = A (cell count mean of two grids) × B (cell density dilution factor) × 2 (Trypan blue dilution factor) × 10 (v/w liquefaction factor)].

Flow cytometryCell surface staining protocol

Cell suspensions from liquefied sputum samples were centrifuged at 1500 rpm for 5 min, and then a cell membrane staining protocol was conducted. Briefly, cell pellets were resuspended with 100 µL FcR blocking solution (FCB with 1 µg·mL−1 human IgG from Abcam) and incubated on ice for 15 min. Next, the following mixture of fluorochrome-conjugated antibodies was added immediately without washing; mouse anti-human CD63-PCy7 (IgG1k, clone H5C6 from Becton Dickinson), mouse anti-human HLA-DR-APC/Fire 810 (IgG2ak, clone L243), mouse anti-human CD45-APC (IgG1k, clone 2D1), mouse anti-human CD11b-FITC (IgG1k, clone ICRF44), mouse anti-human CD206-PE (IgG1k, clone 15–2) and mouse anti-human CD16-PCy5 (IgG1k, clone 3G8), all purchased from Biolegend. Then, 2 µL of DAPI solution (Becton Dickinson) previously diluted 1:20 in FCB was added and cell suspensions were placed on ice for 20 min. After incubation, cells were washed once by adding 1 mL FCB and centrifuged for 5 min at 1500 rpm. The mouse IgG1k-PCy7 isotype control (clone P3.6.2.8.1 from Becton Dickinson) was included in order to evaluate the non-specific binding of fluorescent IgG antibodies. Finally, stained cells were resuspended with 300 µL FCB and acquired on a BD FACSVerse flow cytometer (Becton Dickinson) equipped with 488, 640 and 405 nm lasers. Fluorescence compensation was performed by acquiring single-stained leukocytes for each fluorochrome and using the automated compensation tool in FACSuit software v1.0.5.3841 (Becton Dickinson). All flow cytometry data was subsequently analyzed with FlowJo software v10.6 (Becton Dickinson). Cellular autofluorescence measurements (see Table S1) were performed in DAPI‾ viable cells following the abovementioned protocol without conducting the blockade of FcR nor the addition of fluorochrome-conjugated antibodies.

Phenotyping and activation status of sputum leukocytes after liquefaction

Cells were gated in a FSC-H/FSC-A dot-plot to eliminate doublets and viable cells were selected by excluding DAPI dye (Fig. 4A and B). Then, total CD45High leukocytes were gated in a SSC-A/CD45 dot-plot (Fig. 4C). Granulocytes and lymphocytes were selected in a subsequent gate by excluding CD206 fluorescence (macrophages and dendritic cells marker) (Fig. 4D). Next, in a SSC-A/CD11b dot-plot SSCLowCD11bLow/‾ total lymphocytes and SSCHigh/+CD11b+ total granulocytes were identified (Fig. 4E). Then, granulocytes were plotted in a CD16/CD11b dot-plot and two gates were defined, one for the selection of CD16HighCD11b+ mature neutrophils and another one including CD16LowCD11b+ neutrophils and CD16‾CD11b+ eosinophils (Fig. 4F). Finally, eosinophils were definitely identified as SSCHigh cells in a subsequent SSC-A/FSC-A dot-plot (Fig. 4G). Frequency of total lymphocytes was referred to CD45HighCD206‾leukocytes and frequencies of CD16HighCD11b+ mature neutrophils and CD16‾CD11b+SSCHigh eosinophils were referred to total granulocytes. The median fluorescence intensity (MFI) of HLA-DR (activation marker), CD63 (degranulation marker) and CD11b (leukocyte adhesion marker) was evaluated in order to analyze the impact of the liquefaction methods on the activation status of sputum leukocytes subpopulations (histograms in Fig. 4E-G). A minimum of 100 events in each gate were analyzed in order to ensure the accuracy of the data.

Turbidimetry

One hundred μL of all liquefied sputum samples was placed in 96-well ELISA plates (Thermo Fisher Scientific). Then, the absorbance at 800 nm was measured using a PowerWave HT plate reader (Biotek). The absorbance was evaluated in triplicate with 30 s of gentle manual shaking between determinations, in order to evaluate the impact of the degree of liquefaction on the variability of the measurement. The transmittance was calculated as the inverse of the absorbance at 800 nm.

Statistical analysis

Statistical analysis was performed using the GraphPad Prism software (San Diego, USA). Kruskall-Wallis and Mann–Whitney tests were used, respectively, to compare differences between three liquefying procedures (the proposed enzymatic method, the traditional reducing procedure and PBS control) and two groups of derived data (ratios treatment/PBS). A p value < 0.05 was considered statistically significant.

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