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High-dimensional Analysis of Intestinal Immune Cells During Helminth Infection

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High-dimensional Analysis of Intestinal Immune Cells During Helminth Infection

Laura Ferrer-Font et al. Elife.

Abstract

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.

Keywords: H. polygyrus; cell isolation; helminth infection; immunology; inflammation; intestinal immune cells; mouse; type 2 immunity.

Plain Language Summary

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.

Conflict of interest statement

LF, PM, PH, AS, SC, KP, IH, FR, Gl, JM No competing interests declared

Figures

Figure 1.
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).
Figure 1—figure supplement 1.
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.
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.
Figure 1—figure supplement 3.
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.
Figure 1—figure supplement 4.
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.
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.
Figure 2.
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.
Figure 2—figure supplement 1.
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.
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.
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.
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 5.
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).
Figure 2—figure supplement 6.
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.
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).
Figure 2—figure supplement 8.
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).
Appendix 1—figure 1.
Appendix 1—figure 1.. Illustrations of the step-by-step lamina propria cell isolation protocol for H. polygyrus-infected intestines.
Pictures illustrating the different steps required for the lamina propria single-cell isolation from H. polygyrus-infected intestines are shown.

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