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.
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 antiparasite 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 eLife digest Parasitic worms known as helminths represent an important health problem in large parts of Africa, South America and Asia. Once their larvae enter the body, they head to the gut where they mature into adults and start laying eggs. In areas with poor sanitation, these may then get passed on to other individuals. To defend the body, the immune system sends large numbers of immune cells to the gut, but it usually struggles to eliminate the parasites. Without deworming medication, the infection can last for many years.
Scientists study helminth infections in the laboratory by using worms that naturally infect mice. Understanding exactly how the immune system responds to the infection is essential to grasp why it fails to clear the worms. However, it is difficult to extract immune cells from an infected gut, as the infection creates strong local responses -such as an intense 'slime' production to try to flush out the worms.
The standard procedure to obtain immune cells from the gut consists of three steps: collecting a gut segment and washing it, stripping away the surface layers with chemicals, and finally using enzymes to digest the tissues, which are then filtered to obtain individual cells. However, this protocol is not able to extract cells during infection. Ferrer-Font et al. therefore methodically refined every step of this method, and finally succeeded in obtaining millions of immune cells from infected guts.
For the first time, these cells could then be studied and identified using a new technology called spectral flow cytometry. Over 40 immune cell types were followed throughout the course of infection, revealing that many 'first responders' immune cells were recruited to the gut early on, when the worms were still larvae. However, these cells disappeared once the worms developed into adults. These findings were confirmed by microscopy, which also showed that the first responder cells were found around the developing larvae, likely attacking them. When the adult worms developed, these cells were replaced by other immune cells, which also decreased the longer the worms were present in the gut.
This new extraction process established by Ferrer-Font et al. can also be paired with other technologies that can, for example, reveal which genes are turned on in individual cells. This could help map out exactly how the body fights helminth infections, and how to improve this response. The method could also be useful to extract immune cells from the gut in other challenging scenarios, such food allergies or inflammatory bowel disorders.       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 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 1figure 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).
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 23color 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 1figure 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.
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 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.
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).
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. 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.

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.

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 mm and 40 mm 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 mg/mL Liberase TL and 100 mg/mL DNase I (both from Roche, Germany) for 30 min at 37˚C and passed through a 70 mm cell strainer.

Conventional and spectral flow cytometry
For conventional flow cytometry cells were resuspended in 0.5 ml of 20 mg/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 mL 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 mg/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. Flow-SOM 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.

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).