Co-infection with Toxoplasma gondii leads to a loss of resistance in Heligmosomoides bakeri trickle-infected mice due to ineffective granulomas

The intestinal roundworm Heligmosomoides bakeri causes chronic infection in susceptible (C57Bl/6) mice; however, repeat (trickle) infection confers immunity and facilitates worm clearance. We previously showed that this acquired immunity is associated with a strong Th2 response, notably the enhanced production of intestinal granulomas. Here we demonstrate that elevated proportions of IgG1-bound eosinophils and macrophages are observed around the developing tissue worms of trickle-infected female C57Bl/6 mice compared to bolus infected animals. Levels of IgG2c, IgA or IgE were not detected in the granulomas. Increased proportions of SiglecF+ and CD206+ cells, but not Ly6G+ and/or NK1.1+ cells, were also found in the granulomas of trickle-infected mice. However, in the natural world rather than the laboratory setting, immune environments are more nuanced. We examined the impact of a mixed immune environment on trickle infection-induced immunity, using a pre-infection with Toxoplasma gondii. The mixed immune environment resulted in fewer and smaller granulomas with a lack of IgG -bound cells as well as reduced proportions of SiglecF+ and CD206+ cells, measured by immunofluorescence and flow cytometry. This was associated with a higher worm burden in the co-infected animals. Our data confirm the importance of intestinal granulomas and parasite-specific antibody for parasite clearance. They highlight why it may be more difficult to clear worms in the field than in the laboratory. AUTHOR’S SUMMARY Despite decades of research on intestinal parasitic worms, we are still unable to clearly point to why so many people (approximately 1.8 billion) and most livestock/wild animals are infected with these parasites. We have made progress in understanding how the immune system responds to parasitic worms, and how these parasites manipulate our immune system. However, identifying effective clearance mechanisms is complex and context dependent. We have used models of trickle infection (multiple low doses of parasites) and co-infection (two intestinal parasites) to simulate how people/animals get infected in the real world. Using these models, we have confirmed the host/parasite interface (the granuloma) within the intestinal tissue to be key in determining the host’s ability to clear worms. The lack of specific immune cells and antibodies within the granuloma was associated with chronic infection. Our results help explain why intestinal parasitic worms are so prevalent and why it may be difficult to clear worms in natural settings.

149 data demonstrating that in susceptible mice, trickle infection results in reduced worm 150 burdens. However, we were also able to show that this reduction was associated with 151 increased levels of antibodies bound to tissue larvae and a specific gene expression 152 signature in the granulomas. All other correlates of Th2 immunity measured between 185 186 H. polygyrus antigen was prepared by collecting live adult worms from 14-day infected 187 mice using modified Baerman's apparatus. Worms were washed multiple times and 188 homogenized in PBS using a glass homogenizer. The resulting solution was centrifuged 189 (13, 000 g, 10 minutes, 4 0 C) and the supernatant filtered (0.2μm filter, Nalgene). The 190 protein concentration was calculated using the Bradford assay. The antigen was stored 191 at 15 mg/ml at -80 0 C.

192
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 214 antigen or 2 μg/ml concanavalin A (Sigma) at 37 0 C with 5% CO 2 . Supernatants were 215 collected for cytokine measurements. Measurements for antigen specific production 216 were not included in the analysis unless cytokine production was observed in the wells 217 with concanavalin A stimulation.  229 surface (with its mucus) was gently scraped using a glass slide. Scrapings were weighed, 230 added to 500 μl lysis buffer (10 μM tris HCl, 0.025% sodium azide, 1% tween 80, 0.02% 231 phenylmethylsulfonyl fluoride) with one complete protease inhibitor tablet (Roche 232 diagnostics GmbH, Germany) and homogenized using a bead beater (40 seconds at 233 speed 6 using the Fast-prep-24 bead beater, MP biomedical). The homogenate was 234 centrifuged at 11, 000 g at 4 o C for one hour. Supernatants were collected and used fresh 235 or stored at -80 o C.
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint    (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 258 ethanol and 70% ethanol for 5 minutes. Slides were incubated in 2% sodium borohydride 259 (VWR, BDH4604) in PBS for 40 minutes at RT to remove auto fluorescence. Antigens were 260 retrieved using 2.5% trypsin (Thermo scientific, 15090046) in 0.1% HEPES buffer, 261 incubated at 37 0 C for 25 minutes. Blocking steps were performed following PBS washes.
262 All samples were blocked with starting block (Thermo Scientific, 37578) for 1 hour at RT 263 and rat/rabbit/goat serum for 30 minutes at RT. Following blocking steps, slides were (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 280 hybridization assay were normalized with NanostringNorm (2) using the negative control 281 probes, positive control probes and housekeeping genes Eif2b4, Polr1b, and Edc3. Of the 282 20 housekeeping genes included in the assay Eif2b4, Polr1b, and Edc3 were the only ones   (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 324 larvae in low frequent doses to C57Bl/6 mice changed their susceptibility to infection (Fig.   325 1D).    (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 367 no difference in levels between bolus-and trickle-infected groups at any post-infection 368 time point (Fig. 3A & B).

369
370 We also measured an increase in parasite specific IgG1 over time, as has previously been 371 reported [23]. In BALB/c mice, at both days 21 and 28 post-infection, titers were above 372 10^4 (Fig. 3C), while in C57Bl/6 mice, despite an increase in titers, these remained below 373 10^4 at all time points (Fig. 3D). However, again, no differences were observed between  (Fig. 4A, left panel).
387 However, this decrease was only apparent in trickle infected animals by day 21 post-388 infection (approximate 12% difference between the trickle-and bolus-infected groups).
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 389 At day 28 post-infection, the difference between bolus-and trickle-infected groups 390 disappeared (approximate 3% difference between the trickle and bolus infected 391 groups). We also measured mucus production indirectly through intestinal tissue

410
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made 413 levels in the small intestine (Fig. 4C). Intestinal IgA levels did not differ between bolus-414 or trickle-infected mice over the course of infection (Fig. 4C, right panel). Interestingly,

course of infection in C56BL/6 mice.
428 429 Since few differences were recorded between trickle-and bolus-infected mice in the 430 small intestine, we focused on the host/parasite interface, the intestinal granuloma.

431
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 432 Granulomas are a characteristic response to intestinal roundworms [14]. In response to 433 H. polygyrus., they are round, opaque hard structures protruding from the small 434 intestinal wall, easily identifiable and quantifiable using a dissection microscope (Fig.   435 5A). We measured these structures in both C56Bl/6 and BALB/c mice over the course of 436 infection (days 7, 14 , 21 and 28 days post-infection). We found that, BALB/c mice, both 437 trickle-and bolus-infected, had consistently high granuloma numbers over the first 438 three time points (granuloma numbers >45, Fig. 5B). At day 28 post-infection, numbers 439 dropped below this. In C57Bl/6 mice, a similar pattern was observed for the bolus-440 infected mice: at days 7 and 14, granuloma numbers were high, and >45, at days 21 and 441 28, granuloma numbers were reduced to ~17 (Fig. 5C). However, in trickle-infected 442 C57Bl/6 mice, granulomas remained > 45 for the entire time course (Fig. 5C). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 476 the 46 genes were highly downregulated, and were associated with all functional 477 categories. At day 21 post-infection, 75 genes were upregulated in infected vs. naïve 478 (NvT and NvB) (Fig. 8A, Fig. S2): 12 involved in cell migration, 12 in chemokine signalling, 479 17 in ECM, 14 as growth factors, 10 in metabolism and 15 in lymphocyte activation.
480 Forty-one of these 75 genes were highly upregulated; these are involved in attracting 481 macrophages and eosinophils (e. g. chil4, chil3, Serpine 1, ccl7, cxcl3, cxcr4), as well as 482 ECM remodelling (e. g. mmp12, col1a2, cma1, Arg1). Twelve genes were downregulated (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 498 involved in cytokine signaling. No genes were highly upregulated, all were expressed 499 below 7-fold and were associated with many different functions. The three most highly 500 upregulated in the bolus infected animals (cxcr3, cxcr4, ptgdr) are involved in eosinophil 501 recruitment and Th1 immunity. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 520 degrees. Since we found no differences in the expression of Fc receptor signalling genes 521 within the granuloma between bolus and trickle-infected groups, we measured the 522 accumulation of IgG1, IgG2c, IgE and IgA antibodies using immunofluorescence, both 523 within the intestine and focusing on the host parasite interface within the granulomas 524 ( Fig. 9-10). First, we found that high levels of IgG1 in the serum at day 21 post-infection 525 (Fig. 3D) correlated with the presence of IgG1 in granulomas in both bolus-and trickle-526 infected mice (Fig. S3A). At day 7, where antigen specific serum IgG1 could not be 527 detected (Fig. 3D), IgG1 levels were minimal and/or absent (Fig. S3B).

529
To study the host/parasite interface, we set up a trickle (larvae) model (Fig. 9A), in 530 which granulomas containing larvae (acute granulomas) and granulomas where larvae 531 had either escaped or been killed (chronic granulomas) could be observed. Using this 532 model, we found a high concentration of IgG1 at the host parasite interface at both day 533 14 and day 21 (Fig. 9B) post-infection in acute granulomas. However, in both trickle and 534 bolus infected animals, levels of IgG1 were similar in chronic granulomas (Fig. 9C). We 535 could not detect any IgG2c, IgE or IgA within acute granulomas of trickle (larvae) 536 infected mice (Fig. 10A). This was despite observing IgG2c in intestinal tissue infected by 537 C. rodentium (Fig. S4), IgE in the lamina propria of infected mice (Fig. 10B) and IgA in the 538 lamina propria and Peyer's patches of infected mice (Fig. 10C).

539
. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made  (Fig. 5) as well as higher levels of Th2 cytokines (Fig. 2) and 550 parasite specific antibodies (Fig. 3) compared to susceptible C57Bl/6 mice. These 551 differences are thought to contribute to the BALB/c resistance phenotype.
552 Using our trickle model, we observed a resistance phenotype in the C57Bl/6 mice as 553 opposed to the susceptible phenotype of bolus infected animals (Fig. 1). The improved 554 immune response we observed is likely due to the continuous stimulation of the host 555 immune system by multiple low doses of larvae resulting in a greater number of more 556 effective granulomas (better responsive myeloid cells and the presence of IgG1 557 antibodies, Fig. 8 & 9) and ultimately fewer adult worms (Fig. 1). Unlike differences 558 observed between resistant BALB/c and susceptible C57Bl/6 mice, we found no 559 significant differences between the systemic (serum antibody and spleen/MLN cytokine 560 response, Fig. 2 & 3) or tissue-wide (intestinal tissue physiological, cytokine and 561 antibody responses, Fig. 4) immune responses of bolus and trickle infected C57Bl/6 . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 562 mice. Only when studying the granulomas themselves, at the host parasite interface, 563 were important differences observed (Fig. 8 & 9). . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint 584 In the bolus-infected mice at day 7 post-infection, a number of genes were 585 downregulated compared to trickle-infected animals (Fig. 7B). These genes are 586 associated with growth factors, as well as TLR and cytokine signalling, in dendritic cells. 597 At day 21, the two most highly expressed genes in the trickle-infected mice are involved 598 in tissue remodelling (Adamts4: 124 fold, Osm: 49 fold, [72]). Adamts12 (7 fold increase, 599 (5)), Adamts3 (6 fold increase, (6)), Hdac5 (3 fold increase, [73]), Smad2 (2 fold increase 600 [74]), and Socs 3 (2 fold increase, [75]), were also upregulated and are involved in tissue 601 remodelling. The expression for all these genes is significantly higher compared to both 602 day 21 and day 7 bolus-infected mice. As such, the difference observed is not due to the 603 difference in 'age' of the granulomas, with the bolus day 21 granulomas being 'older' 604 than the trickle day 21 granulomas. Interestingly, to our knowledge, the 2 most highly 605 expressed genes (Adamts4 and Osm) have not been associated with helminth infection . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020.

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. CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint . CC-BY 4.0 International license available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprint this version posted December 17, 2020. ; https://doi.org/10.1101/2020.12.17.423220 doi: bioRxiv preprint