High-dimensional analysis of intestinal immune cells during helminth infection
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 .
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 ) . 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 . 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 ) . 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 .
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 . 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 . 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 . 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 . 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 ) . 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 . 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 . 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 . 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 . All spectral flow cytometry data sets presented in this study can be downloaded from flowrepository ( http://flowrepository . org/id/FR-FCM-Z28B ) .
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 .
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 .