High-dimensional analysis of intestinal immune cells during helminth infection.

Single cell isolation from helminth-infected murine intestines has been notoriously difficult, due to the strong anti-parasite type 2 immune responses that drive mucus production, tissue remodeling and immune cell infiltration. Through the systematic optimization of a standard intestinal digestion protocol, we were able to successfully isolate millions of immune cells from the heavily infected duodenum. To validate that these cells gave an accurate representation of intestinal immune responses, we analyzed them using a high-dimensional spectral flow cytometry panel and confirmed our findings by confocal microscopy. Our cell isolation protocol and high-dimensional analysis allowed us to identify many known hallmarks of anti-parasite immune responses throughout the entire course of helminth infection and has the potential to accelerate single-cell discoveries of local helminth immune responses that have previously been unfeasible.

Introduction

Recent advances in single cell analysis have significantly increased our understanding of multiple diseases and cell types in different tissues (Svensson et al., 2018; Hwang et al., 2018). However, many of these technologies require single cell suspensions as an input, which limits our assessment of difficult-to-process tissues (Hwang et al., 2018; Nguyen et al., 2018; Chen et al., 2018). One prominent example is the intestine, which is at the center of many research questions that focus on nutrient uptake (Kiela and Ghishan, 2016), host-microbiome interactions (Belkaid and Hand, 2014; Tilg et al., 2019; Sekirov et al., 2010), local and systemic immune tolerance (Worbs et al., 2006; Harrison and Powrie, 2013; Whibley et al., 2019) and gastrointestinal diseases and infections (Mowat and Agace, 2014; Sell and Dolan, 2018; Fletcher et al., 2013; Hendrickson et al., 2002; Saleh and Elson, 2011), but represents a challenging tissue to digest (Weigmann et al., 2007; Reißig et al., 2014).

The standard digestion procedure to isolate intestinal immune cells located in the small intestinal lamina propria consists of three steps (Weigmann et al., 2007; Reißig et al., 2014; Scott et al., 2016; Esterházy et al., 2019) (Figure 1a). First the intestinal segment of interest is collected, opened longitudinally to remove its luminal content, washed and cut into small pieces. These pieces then undergo several wash steps with EDTA containing wash buffers at 37°C to remove the epithelial layer and make the lamina propria accessible for enzymatic digestion. Lastly, the tissue is enzymatically digested with collagenases (Collagenase VIII from Clostridium histolyticum being among the most popular) and later filtered to obtain a single cell suspension.

Figure 1.

Optimization of a standard intestinal digestion protocol for the heavily infected duodenum.

(a) Schematic of a general intestinal digestion protocol (created with biorender.com). (b) Digest of naïve and day 14 hr. polygyrus (Hp)-infected duodenal segments using the standard digestion protocol. (c) Intestinal cryosections stained with CD45-FITC (green) and DAPI (blue) from naïve and day 14 infected intestines. Scale bar = 100 µm (representative of >10 sections from 3 to 5 mice per group and two independent experiments). (d) Number of live cells isolated from naïve or day 14 infected duodenal segments during the systematic optimization of the standard digestion protocol. Further details can be found in Figure 1—figure supplement 1 and Figure 1—figure supplement 2 (n = 3–5 samples per group, combined data from at least two independent experiments; # depicts the digestion protocol that yielded comparable cell numbers between naïve and infected samples, all other protocols showed a significant difference to the naïve group when compared by ordinary one-way ANOVA followed by Holm-Sidak’s multiple comparisons test). (e) Digest of naïve and day 14 infected duodenal segments using the optimised Hp digestion protocol (#13). (f) Number of live cells isolated from naïve, day 7, day 14 and day 28 infected duodenal segments using the optimized Hp digestion protocol (n > 12 samples per group, combined data from at least three independent experiments; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group; ***p≤0.001). (g) Quantification of CD45+ cells present in the field of view (fov, 635.90µm x 635.90µm) in cryosections from the same timepoints (representative of >10 sections from 3 to 5 mice per group from two independent experiments; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group; **p≤0.01).

Dots plots of acquired events from day 14 hr. polygyrus-infected duodenal segments using digestion protocols #1–8 (representative of 3–5 samples per group from at least two independent experiments). Details for each digestion protocol are annotated. The gating strategy shows cells of interest, viability and CD45 staining.

Dots plots of acquired events from day 14 hr. polygyrus-infected duodenal segments using digestion protocols #9–14 (representative of 3–5 samples per group from at least two independent experiments). Details for each digestion protocol are annotated. The gating strategy shows cells of interest, viability and CD45 staining.

(a) H and E stained FFPE (Formalin fixed paraffin embedded) sections from naïve C57BL/6 and day 14 hr. polygyrus-infected C57BL/6 and Stat6ko mice. Scale bar = 100 µm (representative of >10 sections from 3 to 5 mice per group and two independent experiments). (b) Worm counts from day 14 infected C57BL/6 and Stat6ko mice (n = 5 mice per group, representative for two independent experiments; unpaired t-test). (c,d) Dots plots of acquired events and number of live cells isolated from naïve C57BL/6 and day 14 hr. polygyrus-infected C57BL/6 and Stat6ko mice using the standard digestion protocol (representative of 3–5 samples per group from two independent experiments; ordinary one-way ANOVA followed by Holm-Sidak’s multiple comparisons test compared to the naïve group; ***p≤0.001). Details for the standard digestion protocol are annotated. The gating strategy shows cells of interest, viability and CD45 staining.

(a) Comparison of Collagenase A digested and non-digested splenocytes stained for each surface marker used in our 23-color spectral flow cytometry panel. Gates and percentages of positively stained populations are shown (representative of two independent experiments). (b) Comparison of MFIs for populations shown in a (unpaired t-test; *p≤0.05, **p≤0.01, ***p≤0.001).

Dots plots of Collagenase A digested lamina propria cells from naïve C57BL/6 mice stained with Zombie NIR, CD45, CD4, ki67, FoxP3 and RORγt using four different commercial intracellular staining kits following the respective manufacturers’ instructions (representative of two independent experiments). The eBioscience FoxP3/Transcription Factor Staining Buffer showed the best separation of our cell populations of interest and was used henceforth.

Figure 1—figure supplement 1.

Modification of a standard intestinal digestion protocol to isolate single cells from heavily infected duodenal segments.

Dots plots of acquired events from day 14 hr. polygyrus-infected duodenal segments using digestion protocols #1–8 (representative of 3–5 samples per group from at least two independent experiments). Details for each digestion protocol are annotated. The gating strategy shows cells of interest, viability and CD45 staining.

Figure 1—figure supplement 2.

Further optimization of a single cell isolation protocol from heavily infected duodenal segments based on Collagenase A digestion.

Dots plots of acquired events from day 14 hr. polygyrus-infected duodenal segments using digestion protocols #9–14 (representative of 3–5 samples per group from at least two independent experiments). Details for each digestion protocol are annotated. The gating strategy shows cells of interest, viability and CD45 staining.

While this method results in a high cell yield from steady state intestines, the isolation of cells from severely infected segments remains challenging (Reißig et al., 2014; Scott et al., 2016; Webster et al., 2020) (Figure 1b). One of the most prominent examples are infections with intestinal helminths, which represent over 50% of all parasitic infections in human and livestock populations (McSorley and Maizels, 2012; Hotez et al., 2008; Jourdan et al., 2018; Jackson et al., 2009). This has limited our analysis of anti-parasite immune responses to imaging approaches, phenotypical observations in transgenic mouse strains or the assessment of secondary locations like the draining lymph nodes, blood or spleen (McSorley and Maizels, 2012; Maizels and McSorley, 2016; Mishra et al., 2014), which might only partially reflect local immunity. As helminth infections are strongly linked to chronic impairments that affect nutrition availability (Koski and Scott, 2001; Crompton and Nesheim, 2002); memory, cognition and physical development (Ezeamama et al., 2005; Pabalan et al., 2018; Nokes et al., 1992); changes in the microbiota (Gause and Maizels, 2016; Ramanan et al., 2016) and modulation of local and systemic immunity (Mishra et al., 2014; Maizels et al., 2009), an optimized digestion protocol is needed to further investigate the infected intestinal tissue.

Results and discussion

Difficulties with intestinal digests during helminth infection have been associated with a strong anti-parasite type 2 immune response that drives mucus production (Hashimoto et al., 2009; von Moltke et al., 2016), alters the epithelium (Gerbe et al., 2016; Howitt et al., 2016), induces immune cell infiltration (Inclan-Rico and Siracusa, 2018) and causes tissue remodelling (Motran et al., 2018; Boyett and Hsieh, 2014) (Figure 1c). In order to investigate a model of both acute and chronic helminth infection, we infected C57BL/6 mice with Heligmosomoides polygyrus bakeri (also known as Heligmosomoides bakeri; Behnke and Harris, 2010), a naturally occurring rodent parasite with an exclusive intestinal life cycle (Monroy and Enriquez, 1992; Reynolds et al., 2012). Infective L3 larvae penetrate the intestinal tissue of the duodenum within 24 hr of ingestion, undergo larval development in the muscularis externa and return to the lumen within 10 days post infection, where the adult worms mate and develop a chronic infection in C57BL/6 mice (Reynolds et al., 2012; Smith et al., 2016). The peak of acute immunity is usually studied around day 14 post infection and we focused on this time point and the heavily infected duodenum (Filbey et al., 2014; Elliott et al., 2008), to optimize our digestion protocol.

In order to develop a digestion protocol for heavily infected intestines, we followed a systematic approach and optimized each step of the standard intestinal digestion protocol. First, we modified the EDTA wash steps to remove the increased amount of mucus but did not observe an improvement in cell yield (Figure 1d and Figure 1—figure supplement 1; digestion protocols #1, 2, 6). This was followed by testing a variety of collagenases that have been reported for intestinal digests, as we hypothesized that the intestinal remodeling that occurred during helminth infection could negatively impact the digestion procedure. Indeed, we found that Collagenase A from Clostridium histolyticum (Figure 1d and Figure 1—figure supplement 1; digestion protocols #7, 8), but not Collagenase VIII, Collagenase D, Dispase or Liberase TM (Figure 1d and Figure 1—figure supplement 1; digestion protocols #3–5), showed an increase in cell yield when used in conjunction with the standard digestion protocol.

To further optimize the protocol, we focused on Collagenase A-based digestion and increased and modified the wash steps and observed a further increase in cell yield (Figure 1d and Figure 1—figure supplement 2; digestion protocols #9–12). Importantly, strong vortexing after each wash step significantly improved the outcome of digestion (Figure 1d and Figure 1—figure supplement 2; digestion protocol #13), suggesting that the epithelium is harder to remove in helminth-infected tissues. Indeed, observations from Stat6ko mice confirmed that the physiological changes that impair the intestinal digest using the standard protocol, were all linked to type two immune responses, as intestines from infected Stat6ko mice could readily be digested (Figure 1—figure supplement 3). We also assessed intra-epithelial cells in the EDTA wash, but could not detect any CD45+ cells in preparations from infected animals, emphasizing that our protocol should only be used to isolate lamina propria cells. Several intestinal cell isolation protocols also utilize a final gradient centrifugation step to further isolate immune cells (Weigmann et al., 2007; Esterházy et al., 2019). However, in our hands this resulted in a dramatic drop in cell yield and was therefore omitted (Figure 1d and Figure 1—figure supplement 2; digestion protocol #14). Our optimized lamina propria cell isolation protocol for H. polygyrus-infected intestines thus included three 10 min 2 mM EDTA wash steps (each followed by vigorous vortexing) and a 30 min digest with 1 mg/ml Collagenase A, 20% FCS and 0.05 mg/ml DNase (see Appendix 1 for step-by-step instructions).

Figure 1—figure supplement 3.

Intestines from H. polygyrus-infected Stat6ko mice can be digested with the standard cell isolation protocol.

(a) H and E stained FFPE (Formalin fixed paraffin embedded) sections from naïve C57BL/6 and day 14 hr. polygyrus-infected C57BL/6 and Stat6ko mice. Scale bar = 100 µm (representative of >10 sections from 3 to 5 mice per group and two independent experiments). (b) Worm counts from day 14 infected C57BL/6 and Stat6ko mice (n = 5 mice per group, representative for two independent experiments; unpaired t-test). (c,d) Dots plots of acquired events and number of live cells isolated from naïve C57BL/6 and day 14 hr. polygyrus-infected C57BL/6 and Stat6ko mice using the standard digestion protocol (representative of 3–5 samples per group from two independent experiments; ordinary one-way ANOVA followed by Holm-Sidak’s multiple comparisons test compared to the naïve group; ***p≤0.001). Details for the standard digestion protocol are annotated. The gating strategy shows cells of interest, viability and CD45 staining.

When we compared intestinal digests from naïve animals using the standard or optimized cell isolation protocol, we observed highly comparable outcomes (Figure 1d; digestion protocols #1 and 13). Both digestion protocols resulted in a cell yield of 3–6 million live cells per duodenum with 70–80% viability and 20–30% frequency of CD45+ cells. To assess the effectiveness of our digestion protocol during the different stages of H. polygyrus infection, we harvested the duodenum from naïve C57BL/6 mice and at day 7, day 14 and day 28 of H. polygyrus infection, which represented time points of larval development in the muscularis externa, as well as acute and chronic adult worm infection, respectively. We observed that samples from all time points could be successfully digested using our optimized digestion protocol and that duodenal digests from 14- and 28 days post infection yielded 3–6 million live cells per sample (Figure 1e,f). We furthermore observed a consistent doubling of the cell count to 8–11 million live cells per duodenum at day seven post infection and observed a similar trend when we quantified CD45+ cells in cryosections from these time points (Figure 1f,g).

To understand these differences and validate whether our protocol was suitable for subsequent single cell analysis and immunophenotyping, we characterized the isolated cells further using a 23-color spectral flow cytometry panel that incorporated many of the hallmark surface and intracellular markers for type two immune responses that have been associated with helminth infections (Maizels and McSorley, 2016; Reynolds et al., 2012) (see Supplementary file 1 for details regarding markers, fluorophores, clones and staining concentrations used). Our staining panel was designed to identify both innate and adaptive immune cell populations and allowed us to assess eosinophils, neutrophils, different subsets of monocytes, macrophages and dendritic cells, as well as the three main populations of innate lymphoid cells (ILC1, ILC2, and ILC3) and effector T cells populations (Th1, Th2, Th17), T regulatory cells and B cells as well as their proliferation through ki67 expression within the same panel.

To guarantee optimal staining conditions, we tested our optimized digestion protocol on splenocytes and compared digested to non-digested cells, as collagenase digests can negatively affect surface epitope integrity. While we observed a reduction in the MFIs of several markers (namely Ly6G, MHCII, CD45 and CD127), all positive stained cell populations could be clearly identified (Figure 1—figure supplement 4). Isolated intestinal lamina propria cells also proved a challenge for intracellular staining, as different commercial intracellular staining kits significantly affected the cellular, but not debris, scatter profiles and varied in the resolution of intracellularly antibody staining (Figure 1—figure supplement 5). In our hands, the eBioscience FoxP3/Transcription Factor Staining Buffer Set yielded the best results and was used henceforth.

Figure 1—figure supplement 4.

Assessment of epitope integrity of digested and non-digested splenocytes.

(a) Comparison of Collagenase A digested and non-digested splenocytes stained for each surface marker used in our 23-color spectral flow cytometry panel. Gates and percentages of positively stained populations are shown (representative of two independent experiments). (b) Comparison of MFIs for populations shown in a (unpaired t-test; *p≤0.05, **p≤0.01, ***p≤0.001).

Figure 1—figure supplement 5.

Comparison of different commercial intracellular staining kits on digested lamina propria cells.

Dots plots of Collagenase A digested lamina propria cells from naïve C57BL/6 mice stained with Zombie NIR, CD45, CD4, ki67, FoxP3 and RORγt using four different commercial intracellular staining kits following the respective manufacturers’ instructions (representative of two independent experiments). The eBioscience FoxP3/Transcription Factor Staining Buffer showed the best separation of our cell populations of interest and was used henceforth.

We isolated immune cells from the three main stages of H. polygyrus infection (day 7, day 14 and day 28), representing larval development, as well as acute and chronic worm infection and used a combination of high-dimensional analysis tools and manual gating strategies to assess changes within each immune cell population (Figure 2a and Figure 2—figure supplement 1 and Figure 2—figure supplement 2). In line with previous findings (Inclan-Rico and Siracusa, 2018), we observed a strong infiltration of immune cells such as neutrophils and monocytes at day 7 post infection, which we verified by confocal microscopy and were primarily localized around the developing larvae explaining the increase in total cell number at this timepoint (Figure 2a–c and Figure 2—figure supplement 3). At later time points this inflammatory response receded, which was likely linked to the worms exiting the intestinal tissue and inhabiting the lumen. Peak expression of RELMα in resident macrophages, which is a hallmark for their alternative activation and wound repair responses (Esser-von Bieren et al., 2013; Krljanac et al., 2019), was observed at day 14 and was again localized within the granulomas (Figure 2b,c and Figure 2—figure supplement 3). While type two innate lymphoid cells did not increase in frequency over time, ki67 expression increased, suggesting cell proliferation and activation (Figure 2d), as previously described (von Moltke et al., 2016; Schneider et al., 2018). GATA3+ Th2 cells, important drivers of type two immunity (Reynolds et al., 2012; Mohrs et al., 2005), were detected throughout all stages of infection, increased in frequency over time and showed high ki67 expression (Figure 2b). Interestingly, ki67 expression strongly decreased at day 28 post infection for all cell types analyzed (Figure 2d and Figure 2—figure supplement 4), which could be linked to the strong immunomodulatory properties reported during chronic worm infection (Maizels and McSorley, 2016; Grainger et al., 2010) (Figure 2e).

Figure 2.

Spectral flow cytometric analysis of isolated intestinal immune cells during the course of H. polygyrus infection.

(a) FlowSOM (top) and manual (bottom) analysis of live CD45+ cells isolated from naïve, day 7, day 14 and day 28 infected duodenal segments stained with 23 surface and intracellular antibodies and gated as described in Figure 2—figure supplement 1 (n = 3–8 samples per group, combined data from two independent experiments). (b) Quantification of different innate immune cell populations during the course of H. polygyrus infection (mean ± s.e.m.; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group; *p≤0.05, ***p≤0.001). (c) Representative images from intestinal cryosections stained with Ly6G-PECF594 (orange) and DAPI (blue) from naïve and day 7 infected duodenal segments (top) or stained with RELMα-APC (red) and DAPI (blue) from naïve, day 7 and day 14 infected duodenal segments (bottom). Scale bar = 100 µm (representative of >10 sections from 3 to 5 mice per group and two independent experiments). (d) Proportions of ILC and CD4 T cell populations and their expression of the proliferation marker ki67 during the course of infection (mean ± s.e.m.; 2-way ANOVA followed by Dunnett’s multiple comparisons test compared to each of the naïve groups (stacked bar graphs) or compared to the combined naïve group (line graphs); **p≤0.01, ***p≤0.001). (e) Schematic of H. polygyrus development, location and associated immune responses during the course of infection.

Contour plots of Collagenase A digested lamina propria cells from day 7 hr. polygyrus-infected C57BL/6 mice stained with our 23-color spectral flow cytometry panel. The gating strategy was used to identify innate and adaptive immune cells that have been associated with helminth infection and used for manual analysis of all stages of infection.

(a) FlowSOM analysis of live CD45+ cells isolated from naïve, day 7, day 14 and day 28 hr. polygyrus-infected duodenal segments stained with 23 surface and intracellular antibodies and gated as described in Figure 2—figure supplement 6 (n = 3–8 samples per group, combined data from two independent experiments). (b) Range of expression of each antibody for the combined FlowSOM analysis shown in a). Due to the uptake of Zombie NIR by eosinophils, only live CD45+ Siglec F- cells are shown.

Representative images from intestinal cryosections stained with CD45-FITC (green), RELMα-APC (red), CD64-PE (white), Ly6G-PECF594 (orange) and DAPI (blue) from naïve, day 7, day 14 and day 28 hr. polygyrus-infected duodenal segments and their quantification per field of view (fov, 635.90µmx635.90µm). Scale bar = 100 µm (representative of >10 sections from 3 to 5 mice per group from two independent experiments; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group; **p≤0.01, ***p≤0.001).

Analysis of live CD45+ cells (a), B cells (b), CD4 T cells (c), Dendritic cells (d) and CD64+ cells (e) isolated from naïve, day 7, day 14 and day 28 infected H. polygyrus duodenal segments as gated in Figure 2—figure supplement 6 (n = 3–8 samples per group, combined data from two independent experiments; mean ± s.e.m.; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group (bar graphs), 2-way ANOVA followed by Dunnett’s multiple comparisons test compared to each of the naïve groups (stacked bar graphs) or compared to the combined naïve group (line graphs); *p≤0.05, **p≤0.01, ***p≤0.001).

Representative images from intestinal cryosections stained with CD45-FITC, B220-PECF594 (red), Siglec F- PECF594 (yellow), CD3-PE (magenta), CD4-APC (green), CD64-PE (cyan) and DAPI (blue) from naïve (a) or day 14 hr. polygyrus infected (b) duodenal segments. Scale bar = 100 µm (representative of >10 sections from 3 to 5 mice per group from two independent experiments). (c) Quantification of CD45+, B220+, eosinophils (counted as non-epithelial Siglec F+ cells), CD3+ CD4+, CD3+ CD4- and CD64+ cells per villi section per mm2 (>15 villi from >10 sections from 3 to 5 mice per group from two independent experiments were quantified; unpaired t-test or 2-way ANOVA followed by Sidak’s multiple comparisons test compared to each of the naïve groups; **p≤0.01, ***p≤0.001). d, Comparison of immune cell frequencies detected by confocal microscopy (CM, as shown in c) or spectral flow cytometry (SFC, as shown in Figure 1a) from naïve or day 14 infected duodenal segments (mean ± s.e.m.; 2-way ANOVA followed by Sidak’s multiple comparisons test comparing detected cell frequencies between technologies or between naïve and infected segments; **p≤0.01, ***p≤0.001).

Contour plots of digested duodenal draining mesenteric lymph node cells from day 7 hr. polygyrus-infected C57BL/6 mice stained with our 23-color spectral flow cytometry panel. The gating strategy was used to identify innate and adaptive immune cells that have been associated with helminth infection and used for manual analysis of all stages of infection.

(a) FlowSOM (top) and manual (bottom) analysis of live CD45+ duodenum draining mesenteric lymph node cells from naïve, day 7, day 14 and day 28 hr. polygyrus-infected C57BL/6 mice stained with 23 surface and intracellular antibodies and gated as described in Figure 2—figure supplement 6 (n = 2–9 samples per group, combined data from two independent experiments). Number and percentage of live cells (b) CD45+ cells (c) B cells (d), innate immune cells (e,f) ILCs (g) T cells (h) and CD4 T cells (i) are shown (combined data from two independent experiments; mean ± s.e.m.; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group (bar graphs), 2-way ANOVA followed by Dunnett’s multiple comparisons test compared to each of the naïve groups (stacked bar graphs) or compared to the combined naïve group (line graphs); *p≤0.05, **p≤0.01, ***p≤0.001).

(a,b) Frequency of B cells (identified as CD45+ CD19+ CD3- CD64- MHCII+ cells), CD4 T cells (identified as CD45+ CD19- CD3+ CD4+ CD64- MHCII- cells) and macrophages (identified as CD45+ CD19- CD3- CD64+ MHCII+ cells) from naïve or day 14 hr. polygyrus-infected C57BL/6 mice in CD45 enriched single cell suspensions (a) and after cell sorting for each individual population (b) (representative of 3–5 samples from three independent experiments). (c,d,e) RNA quality report from the Agilent TapeStation showing the gel image, electropherogram and reported RIN numbers from RNA extracted from 5,000 cells per population (representative of 2–3 samples from three independent experiments).

Figure 2—figure supplement 1.

Gating strategy for duodenal lamina propria cells.

Contour plots of Collagenase A digested lamina propria cells from day 7 hr. polygyrus-infected C57BL/6 mice stained with our 23-color spectral flow cytometry panel. The gating strategy was used to identify innate and adaptive immune cells that have been associated with helminth infection and used for manual analysis of all stages of infection.

Figure 2—figure supplement 2.

FlowSOM analysis of duodenal lamina propria cells.

(a) FlowSOM analysis of live CD45+ cells isolated from naïve, day 7, day 14 and day 28 hr. polygyrus-infected duodenal segments stained with 23 surface and intracellular antibodies and gated as described in Figure 2—figure supplement 6 (n = 3–8 samples per group, combined data from two independent experiments). (b) Range of expression of each antibody for the combined FlowSOM analysis shown in a). Due to the uptake of Zombie NIR by eosinophils, only live CD45+ Siglec F- cells are shown.

Figure 2—figure supplement 3.

Intestinal cryosections highlight infiltration of innate immune cells within larval granulomas.

Representative images from intestinal cryosections stained with CD45-FITC (green), RELMα-APC (red), CD64-PE (white), Ly6G-PECF594 (orange) and DAPI (blue) from naïve, day 7, day 14 and day 28 hr. polygyrus-infected duodenal segments and their quantification per field of view (fov, 635.90µmx635.90µm). Scale bar = 100 µm (representative of >10 sections from 3 to 5 mice per group from two independent experiments; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group; **p≤0.01, ***p≤0.001).

Figure 2—figure supplement 4.

Analysis of isolated intestinal immune cells during the course of H. polygyrus infection.

Analysis of live CD45+ cells (a), B cells (b), CD4 T cells (c), Dendritic cells (d) and CD64+ cells (e) isolated from naïve, day 7, day 14 and day 28 infected H. polygyrus duodenal segments as gated in Figure 2—figure supplement 6 (n = 3–8 samples per group, combined data from two independent experiments; mean ± s.e.m.; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group (bar graphs), 2-way ANOVA followed by Dunnett’s multiple comparisons test compared to each of the naïve groups (stacked bar graphs) or compared to the combined naïve group (line graphs); *p≤0.05, **p≤0.01, ***p≤0.001).

Figure 2—figure supplement 6.

Gating strategy for mesenteric lymph node cells.

Contour plots of digested duodenal draining mesenteric lymph node cells from day 7 hr. polygyrus-infected C57BL/6 mice stained with our 23-color spectral flow cytometry panel. The gating strategy was used to identify innate and adaptive immune cells that have been associated with helminth infection and used for manual analysis of all stages of infection.

To validate that our cell isolation protocol resulted in an accurate representation of intestinal immune responses, we quantified B220+, Siglec F+, CD3+ CD4+, CD3+ CD4- and CD64+ cells using confocal microscopy (Figure 2—figure supplement 5a–c) and compared their frequency to our spectral flow cytometry data. We observed that the frequencies of B220+, CD3+ CD4+, and CD64+ cells were highly comparable between confocal microscopy and spectral flow cytometry, while Siglec F+ cells were overrepresented in our spectral flow cytometry data and CD3+ CD4- cells were underrepresented. However, changes within immune cell populations at the peak of H. polygyrus infection were faithfully reported by both confocal microscopy and spectral flow cytometry (Figure 2—figure supplement 5d), emphasizing that cell ratios defined by single cell analysis need to be carefully validated within the tissue before conclusions are drawn.

Figure 2—figure supplement 5.

Quantification of intestinal immune cells detected by confocal microscopy or spectral flow cytometry.

Representative images from intestinal cryosections stained with CD45-FITC, B220-PECF594 (red), Siglec F- PECF594 (yellow), CD3-PE (magenta), CD4-APC (green), CD64-PE (cyan) and DAPI (blue) from naïve (a) or day 14 hr. polygyrus infected (b) duodenal segments. Scale bar = 100 µm (representative of >10 sections from 3 to 5 mice per group from two independent experiments). (c) Quantification of CD45+, B220+, eosinophils (counted as non-epithelial Siglec F+ cells), CD3+ CD4+, CD3+ CD4- and CD64+ cells per villi section per mm2 (>15 villi from >10 sections from 3 to 5 mice per group from two independent experiments were quantified; unpaired t-test or 2-way ANOVA followed by Sidak’s multiple comparisons test compared to each of the naïve groups; **p≤0.01, ***p≤0.001). d, Comparison of immune cell frequencies detected by confocal microscopy (CM, as shown in c) or spectral flow cytometry (SFC, as shown in Figure 1a) from naïve or day 14 infected duodenal segments (mean ± s.e.m.; 2-way ANOVA followed by Sidak’s multiple comparisons test comparing detected cell frequencies between technologies or between naïve and infected segments; **p≤0.01, ***p≤0.001).

Another important conclusion from our analysis was that the strong inflammatory immune responses that we had observed during H. polygyrus development in the muscularis externa, were specific to the infected tissue and were not observed to the same extent in the draining lymph nodes. Furthermore, ratios of ILC populations and T helper subsets were also strikingly different between the lamina propria and the draining lymph nodes at steady state, as were their proliferation kinetics and changes in proportion throughout the course of H. polygyrus infection (Figure 2—figure supplement 6 and Figure 2—figure supplement 7).

Figure 2—figure supplement 7.

Spectral flow cytometric analysis of mesenteric lymph node cells during the course of H. polygyrus infection.

(a) FlowSOM (top) and manual (bottom) analysis of live CD45+ duodenum draining mesenteric lymph node cells from naïve, day 7, day 14 and day 28 hr. polygyrus-infected C57BL/6 mice stained with 23 surface and intracellular antibodies and gated as described in Figure 2—figure supplement 6 (n = 2–9 samples per group, combined data from two independent experiments). Number and percentage of live cells (b) CD45+ cells (c) B cells (d), innate immune cells (e,f) ILCs (g) T cells (h) and CD4 T cells (i) are shown (combined data from two independent experiments; mean ± s.e.m.; Kruskal-Wallis followed by Dunn’s multiple comparisons test compared to the naïve group (bar graphs), 2-way ANOVA followed by Dunnett’s multiple comparisons test compared to each of the naïve groups (stacked bar graphs) or compared to the combined naïve group (line graphs); *p≤0.05, **p≤0.01, ***p≤0.001).

To highlight the potential of our protocol for future studies that utilize current single-cell analysis tools, such as single cell RNA sequencing, we assessed the RNA quality of purified B cells, CD4 T cells and macrophages isolated from naïve or day 14 hr. polygyrus-infected mice (Figure 2—figure supplement 8a,b). Our RNA quality analysis using the Agilent TapeStation resulted in high RIN numbers (range 6.8–10 for naïve and 7.0–10 for day 14 hr. polygyrus samples) (Figure 2—figure supplement 8e). However, no clear separation of the 18S and 28S peaks could be observed on the gel image or the electropherograms (Figure 2—figure supplement 8c,d). While the RIN numbers might not have been correctly calculated, and a technical optimization of the TapeStation protocol might be necessary, no RNA degradation was observed in naïve and day 14 hr. polygyrus samples, suggesting that the extraction of high-quality RNA is feasible from both naïve and day 14 hr. polygyrus-infected mice using our cell isolation protocol.

Figure 2—figure supplement 8.

Assessment of RNA quality of sorted intestinal immune cells from naïve and H. polygyrus-infected mice.

(a,b) Frequency of B cells (identified as CD45+ CD19+ CD3- CD64- MHCII+ cells), CD4 T cells (identified as CD45+ CD19- CD3+ CD4+ CD64- MHCII- cells) and macrophages (identified as CD45+ CD19- CD3- CD64+ MHCII+ cells) from naïve or day 14 hr. polygyrus-infected C57BL/6 mice in CD45 enriched single cell suspensions (a) and after cell sorting for each individual population (b) (representative of 3–5 samples from three independent experiments). (c,d,e) RNA quality report from the Agilent TapeStation showing the gel image, electropherogram and reported RIN numbers from RNA extracted from 5,000 cells per population (representative of 2–3 samples from three independent experiments).

Thus, our cell isolation protocol and high-dimensional analysis allowed us to characterize many known hallmarks of innate and adaptive anti-parasite immune responses throughout the entire course of helminth infection. We were able to validate these changes using confocal microscopy and while we could observe differences in the reported cell ratios, changes between naïve and infected samples were faithfully reported by both approaches.

Importantly, many of these changes were only observed locally, highlighting the requirement for good cell isolation techniques to investigate intestinal responses against helminths directly.

In addition to flow cytometric immunophenotyping, we were also able to extract high-quality RNA from cells isolated with our protocol, which could accelerate single-cell discoveries of local helminth immune responses through current single-cell analysis tools, such as single cell RNA sequencing, which has previously been unfeasible.

Materials and methods

Ethics statement

All animal experiments were carried out at the Malaghan Institute of Medical Research, were approved by the Victoria University of Wellington Animal Ethics Committee (permit 24432) and carried out according to institutional guidelines.

Mice

C57BL/6 and Stat6ko (B6.129S2(C)-Stat6tm1Gru/J) mice were imported from The Jackson Laboratory and bred at the Malaghan Institute of Medical Research, Wellington, New Zealand. Mice were housed under specific pathogen free conditions and age-matched female adult animals were used in each experiment.

Heligmosomoides polygyrus infection

The H. polygyrus life cycle was maintained as previously described (Johnston et al., 2015). For experimental infections, mice were infected with 200 L3 larvae by oral gavage at 6–8 weeks of age and intestines and draining lymph nodes were harvested at the indicated time points. Adult worm burden was quantified by mounting opened intestines inside a 50 ml falcon filled with PBS. After 3 hr at 37°C, worms were collected from the bottom of the tube and counted under a microscope.

Cell isolation

Lamina propria cells were isolated from the first 8 cm of intestine according to isolation protocols described in this manuscript. Optimal digestion was achieved when intestinal segments were excised, cleaned and cut into small pieces. Samples were then washed with 2 mM EDTA/HBSS (Gibco) three times for 10 min at 37° C and 200 rpm in a shaking incubator, followed by three pulse vortexing steps at 2500 rpm (maximum speed) for 3 s after each incubation. After the final EDTA wash step, samples were digested in 10 ml RPMI (Gibco) containing 20% FBS (Gibco), 1 mg/ml Collagenase A (Roche #10103578001, 0.223 U/mg solid) and 0.05 mg/ml DNAse (Roche #10104159001, 2916 Kunitz units/mgL) for 30 min at 37°C and 200 rpm in a shaking incubator, with vigorous manual shaking every 5 min. Digestion was quenched with FACS buffer and samples were passed through a 100 µm and 40 µm cell strainer to obtain a single cell suspension. An illustrated step-by-step protocol describing the procedure can be found in Appendix 1.

Individual duodenum draining mesenteric lymph nodes were identified as the most proximal lymph nodes of the mesenteric lymph node chain (Esterházy et al., 2019; Mayer et al., 2017), and were digested with 100 µg/mL Liberase TL and 100 µg/mL DNase I (both from Roche, Germany) for 30 min at 37°C and passed through a 70 µm cell strainer.

Conventional and spectral flow cytometry

For conventional flow cytometry cells were resuspended in 0.5 ml of 20 µg/ml DNase containing FACS buffer, stained with DAPI to identify dead cells, filtered and analyzed using a BD LSRFortessa SORP flow cytometer. For spectral flow cytometry, intestinal and lymph node samples were washed in 200 μL FACS buffer and incubated with Zombie NIR Fixable Viability dye (Biolegend) for 15 min at room temperature. After washing, cells were incubated with Fc block (clone 2.4G2, affinity purified from hybridoma culture supernatant) for 10 min followed by the incubation of surface antibodies (see Supplementary file 1) for 25 min at 4°C in the presence of 20 μg/ml DNase and Brilliant Buffer Plus (BD Biosciences). Cells were fixed and permeabilized with the FoxP3/Transcription Factor Staining Buffer Set (eBioscience) according to manufacturer’s instructions and incubated with intracellular antibodies (see Supplementary file 1) for 45 min at 4°C. Cells were then resuspended in FACS buffer, filtered, and analyzed on a 3-laser Aurora spectral flow cytometer (Cytek Biosciences).

Data analysis

FCS files were manually analyzed using FlowJo (v10.6, Tree Star) or evaluated with high-dimensional data analysis tools using Cytobank (v7.2, Cytobank Inc). After compensation correction in FlowJo, single, live, CD45+ events were imported into Cytobank and transformed to arcsinh scales. FlowSOM analysis was performed on 1,200,000 concatenated lamina propria and 1,000,000 concatenated lymph node cells, with an equal distribution of samples. Different cluster analyses were performed and 121 clusters were identified as the most representative for both data sets.

Imaging

For histological sections, 5 µm FFPE (Formalin fixed paraffin embedded) sections were stained using a standard H and E protocol (Jacobson et al., 2008) and visualized using a BX51 microscope (Olympus) equipped with a 10X NA 0.3 objective. For confocal microscopy, samples were processed and stained using a standard immunofluorescence protocol (Schmidt et al., 2019). Briefly, 1 cm long pieces of intestine were fixed in 4% PFA for 1 hr, incubated in 20% sucrose overnight and rinsed in PBS. Samples were then snap-frozen in OCT compound (Tissue-Tek) using a Stand-Alone Gentle Jane Snap-freezing system (Leica Biosystems). Cryosections of 7 µm were blocked with Fc Block (clone 2.4G2, affinity purified from hybridoma culture supernatant) for 1 hr and stained with CD45-FITC (clone 30-F11, Biolegend), CD64-PE (clone X54-5/7.1, Biolegend), Ly6G-PECF594 (clone 1A8, Biolegend), RELMα-APC (clone DSBRELM, eBioscience), B220-PECF594 (clone RA3-6B2, BD Biosciences), CD3-PE (clone 145–2 C11, eBioscience) or CD4-APC (clone RM4-5, BD Biosciences) for 1 hr. For nuclear staining, sections were incubated with DAPI (2 µg/ml) for 10 min. Images were taken with an inverted IX 83 microscope equipped with a FV1200 confocal head (Olympus) using a 20X, N.A 0.75 objective. Images were acquired using the FV10-ASW software (v4.2b, Olympus) and analyzed with ImageJ (Schindelin et al., 2012) (v1.52n). Image quantification analysis per field of view was performed using CellProfiler (Lamprecht et al., 2007) (v3.1.8) and based on the spatial co-expression of immune cell markers and DAPI-positive nuclei. Cell quantification per villi section per mm (Hwang et al., 2018) was based on manual selection of the villi and electronic quantification of the area and positively stained cells.

Cell sorting, RNA extraction and RNA quality assessment

Single cell suspensions from naïve or day 14 hr. polygyrus-infected C57BL/6 mice were stained with CD45-BUV395 (clone 30-F11, BD Biosciences), CD64-Al647 (clone X54-5/7.1, Biolegend), MHCII-PE (clone M5/114.15.2, BD Biosciences), CD19-BB515 (clone 1D3, BD Biosciences), CD3-BV605 (clone 17A2, Biolegend), CD4-Pac Blue (clone RM4-5, BD Biosciences) and DAPI. 700,000 CD45+ cells were sorted into FACS buffer using a BD Influx cell sorter (BD Biosciences) followed by purification of B cells, CD4 T cells and macrophages. 5,000 cells of each population were sorted into 100 µl RNA lysis buffer (Zymo Research) and stored at −80C. RNA was extracted using the Quick-RNA MicroPrep Kit (Zymo Research) and its quality assessed using the High Sensitivity RNA Screen Tape (Agilent) and a 4150 TapeStation System (Agilent) according to the manufacturer’s instructions.

Statistical analysis

Experimental group sizes ranging from 3 to 5 animals were chosen to ensure that a two-fold difference between means could be detected with a power of at least 80%. Prism 6 Software (GraphPad) was used to calculate the s.e.m. and the statistical differences between groups and samples for each data set as detailed in the corresponding figure legends, with p≤0.05 being considered as significant.

Source data

All spectral flow cytometry data sets presented in this study can be downloaded from flowrepository (http://flowrepository.org/id/FR-FCM-Z28B).

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

Thank you for submitting your article "Single-cell analysis of intestinal immune cells during helminth infection" for consideration by eLife. Your article has been reviewed by three peer reviewers, including Nicola L Harris as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Satyajit Rath as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: John Grainger (Reviewer #2); Lisa Reynolds (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

All reviewers agree that the manuscript by Ferrer-Font et al. has the potential to accelerate discovery in the area of local helminth immune responses by reporting what is a significant methodological advance for the field. This advance, which the authors have enabled through careful comparison of a large variety of digestion techniques, will enable others in the field to investigate the intestinal responses against helminths directly, enabling important experimental tools, including single cell sequencing analysis, to be enacted where such approaches were previously unfeasible.

However, it does not offer, in its present form, any major new biological insight. Thus all three reviewers strongly recommended it is re-submitted as a tools and resources manuscript.

They also agreed the manuscripts main point – to provide a tool to allow single cell analysis – needs to be substantiated further by addressing the following points in a revision:

Essential revisions:

Experiment 1) The reviewers agree that a more quantitative comparison of their flow results to histology would be very valuable to confirm how representative their flow results are in reflecting cell types present in the tissue. It would be sufficient to provide this for day 14 (using specific markers for eosinophils, CD4+ or CD4- lymphoid cells, B cells and macrophages). The reviewers acknowledge that no digestion protocol will be able to perfectly preserve cell ratios present in the tissue, but it would be valuable for the reader to have this information and to clearly state in the manuscript the caveats of using single cell analysis to describe cell ratios. Please note that quantitative graphs (cell counts or pixel area if positive staining per mm2 of tissue, using a set number of villi and tissue sections) should be provided.

Experiment 2) To substantiate the claim that this techniques will be useful for single cell analysis it would be useful for the reader to know whether it can be employed for single cell RNA seq experiments. In the regard the authors should provide an indication of RNA quality able to be obtained from distinct cell populations harvested from the day 14 post-infection digest. Note that the reviewers acknowledge high quality RNA is difficult to obtain from neutrophils and eosinophils, thus it would be sufficient if the authors provided data for the other major cell subsets namely macrophages (CD64+MHCII+), T cells and B cells.

Experiment 3) I would be very useful if the authors collected the cells released from the epithelial layer (intra-epithelial cells) during the EDTA and vortexing stages and subjected these to flow cytometric analysis of major CD45+ cells collected including markers, at a minimum, for classical IEL populations and for eosinophils. Although not essential the tissues for this experiment would be available from the same animals as used for exp 2 above (day 14 cells samples would be sufficient) and the information would greatly strengthen the manuscript.

Essential revisions:

Experiment 1) The reviewers agree that a more quantitative comparison of their flow results to histology would be very valuable to confirm how representative their flow results are in reflecting cell types present in the tissue. It would be sufficient to provide this for day 14 (using specific markers for eosinophils, CD4+ or CD4- lymphoid cells, B cells and macrophages). The reviewers acknowledge that no digestion protocol will be able to perfectly preserve cell ratios present in the tissue, but it would be valuable for the reader to have this information and to clearly state in the manuscript the caveats of using single cell analysis to describe cell ratios. Please note that quantitative graphs (cell counts or pixel area if positive staining per mm2 of tissue, using a set number of villi and tissue sections) should be provided.

We performed additional experiments to address this comment. As suggested by the reviewers, we stained duodenal sections from naïve and day 14 infected mice for B cells, eosinophils, macrophages and CD3+ CD4+ and CD3+ CD4- T cells and quantified cell counts per villi per mm2 and as a percentage of CD45+ cells. When we compared these frequencies to our spectral flow cytometry data we observed that the frequencies of B220+, CD3+ CD4+, and CD64+ cells were highly comparable between confocal microscopy and spectral flow cytometry, while Siglec F+ cells were overrepresented in our spectral flow cytometry data and CD3+ CD4- cells were underrepresented. However, changes within immune cell populations at the peak of H. polygyrus infection were faithfully reported by both confocal microscopy and spectral flow cytometry, reminding us that cell ratios defined by single cell analysis need to be carefully validated within the tissue before conclusions are drawn. We have incorporated these findings into the main text (Results and Discussion, paragraph eight) and presented the data in a new supplementary figure (Figure 2—figure supplement 5).

Experiment 2) To substantiate the claim that this techniques will be useful for single cell analysis it would be useful for the reader to know whether it can be employed for single cell RNA seq experiments. In the regard the authors should provide an indication of RNA quality able to be obtained from distinct cell populations harvested from the day 14 post-infection digest. Note that the reviewers acknowledge high quality RNA is difficult to obtain from neutrophils and eosinophils, thus it would be sufficient if the authors provided data for the other major cell subsets namely macrophages (CD64+MHCII+), T cells and B cells.

We performed additional experiments to address this comment. As suggested by the reviewers, we sorted 5,000 B cells, CD4 T cells and macrophages from naïve or day 14 H. polygyrus infected mice, extracted their RNA and assessed RNA quality using the Agilent TapeStation. Our RNA quality analysis resulted in high RIN numbers (range 6.8-10 for naïve and 7.0-10 for day 14 H. polygyrus samples). However, no clear separation of the 18S and 28S peaks could be observed on the gel image or the electropherograms (Figure 2—figure supplement 8C,D). While the RIN numbers might not have been correctly calculated, and a technical optimization of the TapeStation protocol might be necessary, no RNA degradation was observed in naïve and day 14 H. polygyrus samples, suggesting that the extraction of high-quality RNA is feasible from both naïve and day 14 H. polygyrus infected mice using our cell isolation protocol. We have incorporated these findings into the main text and presented the data in a new supplementary figure (Figure 2—figure supplement 8). We also got in touch with Agilent to get advice on how to improve the separation of the 18S and 28S peaks, but despite lengthy discussions and several trials no solution was immediately available.

Experiment 3) I would be very useful if the authors collected the cells released from the epithelial layer (intra-epithelial cells) during the EDTA and vortexing stages and subjected these to flow cytometric analysis of major CD45+ cells collected including markers, at a minimum, for classical IEL populations and for eosinophils. Although not essential the tissues for this experiment would be available from the same animals as used for exp 2 above (day 14 cells samples would be sufficient) and the information would greatly strengthen the manuscript.

We have repeatedly analysed the EDTA wash preparations and while we can easily detect CD45+ cells, eosinophils and many different populations of IELs from naïve animals, no CD45+ cell were detected in preparations from infected animals. We have mentioned this finding in the main text and clarified that our protocol should only be used to isolate lamina propria cells from infected intestines, as we believe that a completely new protocol needs to be optimised to obtain intra-epithelial cells from infected intestines, which is beyond the scope of this study.