Analysis of intestinal epithelial cell responses to Cryptosporidium highlights the temporal effects of IFN-γ on parasite restriction

The production of IFN-γ is crucial for control of multiple enteric infections, but its impact on intestinal epithelial cells (IEC) is not well understood. Cryptosporidium parasites exclusively infect epithelial cells and the ability of interferons to activate the transcription factor STAT1 in IEC is required for parasite clearance. The use of single cell RNA sequencing to profile IEC during infection revealed induction of IFN-γ-dependent gene signatures that was comparable between uninfected and infected cells, and IEC expression of the IFN-γ receptor was required for parasite control. Unexpectedly, treatment of Ifng−/− mice with IFN-γ demonstrated the IEC response to this cytokine correlates with a delayed reduction in parasite burden but did not affect parasite development. These data sets provide insight into the impact of IFN-γ on IEC and suggest a model in which IFN-γ-mediated bystander activation of uninfected enterocytes is important for control of Cryptosporidium.


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In this study, the impact of IFN-γ signalling on IEC, and its role in restriction of C. parvum infection, were investigated.Single cell RNA-sequencing (scRNA-seq) revealed global infectioninduced alterations in the IEC compartment, which included widespread induction of IFN-γstimulated gene expression.Additional scRNA-seq and flow cytometry-based experiments demonstrated that infected IEC remain responsive to IFN-γ and that this response peaks 12-24h post-exposure.These kinetics correlated with in vivo and in vitro observations that the administration of IFN-γ led to a robust, but delayed, decrease in oocyst shedding.Together, these studies provide insight into the global impact of infection with Cryptosporidium on IEC and suggest a model in which IFN-γ-mediated bystander activation of uninfected enterocytes is important to limit the Cryptosporidium growth cycle within its host.

Single cell transcriptomic analysis of IEC responses during Cryptosporidium infection
To gain a global understanding on how Cryptosporidium infection impacts IEC in vivo, mice were infected with a mouse-adapted strain of C. parvum (maCp) that expresses mCherry and nanoluciferase (nLuc) (17) and scRNA-seq was performed on cells from the ileum.For these experiments, the epithelial fraction, which includes IEC and intraepithelial lymphocytes (IEL), was extracted from uninfected mice and those at 4-or 10-days post-infection (dpi); 4 dpi is a timepoint with detectable oocyst shedding and IFN-γ production while by day 10 infection is nearly resolved (17).Across samples a total of 24,302 cells were sequenced and after quality control there remained 5,411 cells from the uninfected sample, 7,309 from 4 dpi, and 5,290 from 10 dpi.Following data normalization, cells were partitioned into 24 clusters.Uniform Manifold Approximation and Projection (UMAP) visualization of these clusters was used to identify .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 November 16, 2023.; https://doi.org/10.1101/2023.11.14.567008 doi: bioRxiv preprint prominent Epcam + IEC (clusters 1-13) and Ptprc + (CD45 + ) IEL (clusters [14][15][16][17][18][19], which are shown in Fig 1A .Analysis of IEC clusters for cell type-specific gene expression revealed that the majority of cells sequenced were enterocytes (clusters 2-9), but also identified less common cell types such as intestinal stem cells/transit amplifying cells (cluster 1), goblet cells (cluster 10), tuft cells

IFN-γ signals to IEC to promote control of Cryptosporidium infection
As an unbiased measure to investigate the IEC pathways that are altered during infection, differential gene expression analysis was applied to identify the top 100 marker genes that distinguish the enterocyte and goblet cell clusters (S1 Table ).While there is some overlap of gene expression between enterocytes, this approach highlighted distinct transcriptional profiles of each  ), processes in which IFN-γ may also play a role (24).Together, these analyses emphasize that Cryptosporidium infection results in the generation and expansion of a subset of IEC The scRNA-seq analysis described above examines the global impact of Cryptosporidium on IEC responses but does not distinguish uninfected and infected cells.To determine which IEC were infected, the reads from this data set were aligned to the C. parvum genome and 390 infected cells (that contained parasite transcripts) were identified 4 dpi ( To assess if the transcriptional data sets described above correlate with translation of IFNγ-target genes, M1Red mice were utilized, in which the Irgm1 promoter drives dsRed2 gene expression and provides a way to detect cells that are responsive to type I, II and III IFNs (26).As such, M1Red mice infected with maCp provide the opportunity to concurrently monitor infection (mCherry + ) and response to IFN-γ (RFP + ).WT and M1Red mice were infected with maCp and reporter induction was analyzed 5 dpi, a time point when type I IFN is not detected and type III IFN has returned to baseline (15).In these experiments, approximately 20% of IEC from infected M1Red mice were RFP + (Fig 3G and 3H) and a higher proportion of RFP + IEC were infected compared to RFP -IEC (Fig 3I).In addition, a comparison of surface expression of MHC-I and MHC-II, canonical IFN-γ response genes (27,28), highlighted that these proteins are upregulated during infection and their expression is highest on IEC from infected mice (S4B and S4C Fig).
However, in infected M1Red mice, the expression of these proteins was highest on RFP + mCherry + cells (Fig 3J -3M), which suggests that infected cells are most likely to be exposed to high levels of IFN-γ.
To directly test whether IEC infected with Cryptosporidium are responsive to IFN-γ, uninfected and infected Ifng -/-mice (5 dpi) were treated with PBS or IFN-γ and 12 hours later IEC were extracted for scRNA-seq.In initial studies, when Ifng -/-mice were treated with a single dose of 1μg or 5μg of murine IFN-γ 6h prior to infection, the 5μg dose resulted in parasite suppression treatment does not appear to preferentially impact asexual, male, or female parasites.

IFN-γ treatment promotes delayed control of Cryptosporidium infection
Since treatment with IFN-γ elicited a robust transcriptional response in IEC within 12 hours, experiments were performed to study the dynamics of IFN-γ-mediated parasite control.To determine how quickly IFN-γ restricts Cryptosporidium in situ, the impact of a single dose of IFNγ on parasite burden in tissue and feces was analyzed.Ifng -/-mice were infected for 5 days with maCp and treated with PBS or IFN-γ.Mice from each group were sacrificed at 6h, 12h, or 24h post-treatment and ileal punch biopsies and feces were assayed for parasite burden.In the control group, parasite burden increased from the time of injection (T0) through the remainder of the time course (Fig 5A and 5B).IFN-γ-treated mice showed similar parasite burden to controls at 6h and 12h but a significant decrease in parasite burden occurred between 12h and 24h (Fig 5A and 5B).
Flow cytometric analysis of the percentage of infected IEC, based on detection of mCherry, demonstrated that it took 24h to observe a reduced frequency of mCherry + IEC (Fig 5C and 5D).
Thus, despite an in vivo half-life of 1.1 minutes (30), a single treatment with IFN-γ elicited a transient, but delayed, reduction in Cryptosporidium burden.
In other experimental settings the ability of IFN-γ to activate macrophages to limit pathogen replication does not require extended pre-incubation with IFN-γ and transcriptional responses are rapid (2).Thus, one interpretation of these kinetics is that the response of IEC to IFN-γ may be delayed.Indeed, when naïve M1Red mice were treated with a single dose of 5μg of

Numerous reports have described histological changes in the gut associated with
Cryptosporidium in experimental and natural models of infection that highlight increased intestinal stem cell turnover, crypt deepening and epithelial cell hyperplasia along the villus (14,17,34).
The data presented here leverage scRNA-seq to gain a better understanding of how Cryptosporidium infection and IFN-γ signalling impact infected and uninfected IEC.Perhaps the most prominent changes associated with infection were the changes in mid-villus enterocytes, consistent with the reported hyperplasia of these cells.Increases in enterocyte frequency have also been observed using scRNA-seq during other intracellular infections (rotavirus and Salmonella) (35,36), while helminth infection induced goblet cell and tuft cell expansion (35), which illustrates infection-specific alterations to IEC populations.Differential gene expression and cluster analysis of enterocytes during Cryptosporidium infection identified broad induction of immune response genes, in particular those involved in antigen processing and presentation and response to IFN-γ.
Since IEC express MHC-I and MHC-II, this is consistent with the idea that IFN-γ stimulation of IEC promotes cognate interactions between CD4 + and CD8 + T cells and epithelial cells required for parasite control (5).Indeed, a recent report highlighted that during infection with SFB, the ability of CD4 + T cells to promote IEC turnover was dependent on IEC expression of MHC-II (37).The ability to genetically modify Cryptosporidium to express model antigens (38) will likely facilitate similar studies in this system.There is currently a limited appreciation of the mechanisms used by IFN-γ to promote control of Cryptosporidium (5).The identification of IFN-γ-stimulated genes expressed in infected cells, many of which are associated with anti-microbial activity in other model systems (39,40), provides a candidate list of effectors for additional studies.From this list, only IRGM1 and IRGM3 have been linked to control of Cryptosporidium (17).Many of these genes are also upregulated in other systems where scRNA-seq has been used to study IEC responses.For example, during rotavirus infection there is upregulation of type I IFN response genes (many of which are also regulated by IFN-γ) in IEC (36) and expression of the anti-bacterial genes Reg3b and Reg3g are increased Salmonella-infected mice (35).The latter study also identified elevated expression of Nlrp6 during Salmonella infection (35), which is upregulated in the scRNA-seq data presented here (S1 Table ) and is a key initiator of the production of IL-18 required for innate immunity to Cryptosporidium (41).At present, it remains a challenge to readily distinguish conserved IEC responses to inflammation associated with the production of IFNs versus those that are specific to different classes of pathogen.
IFN-γ has a prominent role in resistance to many intracellular bacterial and parasitic infections and this cytokine can activate haemopoietic and non-haemopoietic cells to limit pathogen replication (2).This is typically a rapid process in which a short pre-exposure, or even simultaneous addition of IFN-γ and pathogen is sufficient to restrict intracellular growth.Thus, previous studies with the M1Red reporter mice highlighted that following treatment with IFN-γ, high reporter activity is induced in monocytes after 6 hours and returns to baseline by 24 hours (26).In contrast, in the studies presented here the administration of IFN-γ prior to or during infection limits Cryptosporidium burden, but requires between 12 and 24h, which correlates with maximal expression of the M1Red RFP reporter in IEC.These in vivo kinetics of IEC responsiveness to IFN-γ and the 12h Cryptosporidium life cycle (10) imply that for an IEC to restrict parasite replication, it needs to encounter IFN-γ prior to infection.The observation that a single IFN-γ treatment prior to infection with Cryptosporidium delayed the onset of oocyst shedding further indicates that IFN-γ acts on uninfected IEC to limit parasite spread within the host.These data do not distinguish whether IFN-γ promotes death and shedding of infected cells, leads to direct death of the parasite or prevents the parasite from completion of its replication cycle and causes it to remain within the host cell until the IEC is shed into the lumen.The restriction of Cryptosporidium replication in IFN-γ-stimulated ALI monolayers suggests this system will enable formal testing of these possibilities, as similar studies in transformed cell lines are limited by the failure to support the complete parasite life cycle (11).
It has been proposed that the rapid replication cycle of Cryptosporidium represents a response to the rapid turnover of IEC (10).It is also possible that the ability to invade, replicate and lyse IEC within 12h also provides a window for infected cells to evade restriction downstream of IFN-γ.There is precedence for a delayed responsiveness to IFN-γ in other cell types; while macrophages can be rapidly activated with IFN-γ to limit the replication of T. gondii, neurons must be pre-incubated with IFN-γ for 24h for parasite control (42,43).In this context, low basal expression of STAT1 causes neurons to be comparatively "slow" to respond to IFN-γ, with maximal expression of Stat1 and other IFN-γ-inducible genes occurring 24-48h post-stimulation (42,44).Since IFN-γ can also cause cell death, delayed responsiveness to IFN-γ observed with IEC may be a host protective mechanism that reflects the immune tolerant state of the small intestine and could indicate a higher threshold of activation for IEC compared to other immune cells.A broad theme in host-pathogen interactions is that the ability of microbes to disrupt relevant host immune pathways is important for pathogen success.Consistent with this idea, multiple intracellular pathogens have effectors that target the anti-microbial activities of IFN-γ.
For example, T. gondii (a relative of Cryptosporidium) and rotavirus have effectors that disrupt IFN-γ-and STAT1-dependent transcription and signalling, and the function of antimicrobial effectors (45)(46)(47)(48).Since IFN-γ is critical for resistance to Cryptosporidium, we originally considered that infected cells might be hypo-responsive to the effects of IFN-γ.However, the data presented here indicate that infected cells remain responsive to IFN-γ, although this does not preclude the possibility that Cryptosporidium disrupts other aspects of IFN-γ-dependent immunity.
An additional consideration may be the potential impact of parasite species and host specificity on immune evasion strategies.Different strains of T. gondii show dramatic differences in their ability to antagonize IFN-γ-mediated GBP recruitment to the parasitophorous vacuole (49).Recent studies on the population structure of Cryptosporidium revealed adaptation to humans as a host and it seems likely that this was dependent on the development of species-specific immune evasion strategies (49)(50)(51).As such, the ability to compare Cryptosporidium species and strains that differ in virulence may provide new insights into host-specific interference with IFN-γ signalling while the ability to conduct genetic crosses between strains (52) offers the opportunity to uncover parasite genes and proteins that underlie these differences.

Mice
C57BL/6J mice (stock #000664), Ifng -/-(stock #002287), Vil1-Cre (stock #004586) and Ifngr1 fl/fl mice (stock #025394) were purchased from Jackson Laboratory and maintained in-house.Stat1 fl/fl mice were generated as previously described (53) and maintained in-house.M1Red mice (26) were provided by Dr. Gregory Taylor (Duke University) and were maintained in-house.In-house breeding was performed to obtain all Cre-lox combinations.Mice used in this study were males or females ranging from 6 to 11 weeks.No differences were observed in infection burden between male and female mice.All mice were age matched within individual experiments.All protocols for animal care were approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania (Protocol #806292).

Parasites and Infection
Transgenic C. parvum expressing nanoluciferase and mCherry ( 17) is propagated by orally infecting Ifng -/-mice.Oocysts are purified from fecal collections of infected mice using sucrose flotation followed by a cesium chloride gradient, as previously described (14).Mice were infected with 1×10 4 -5×10 4 oocysts by oral gavage, diluted in a final volume of 100μL of PBS.To measure parasite burden in intestinal tissue, 5 mm biopsy punches were taken from the ileum and suspended in 1mL lysis buffer (50 mM tris HCl (pH 7.6), 2 mM DTT, 2 mM EDTA, 10% glycerol, and 1% TritonX in ddH 2 O).
To quantify fecal oocyst shedding, 20mg fecal material was suspended in 1mL lysis buffer.
Samples were shaken with glass beads for 5 minutes, then combined in a 1:1 ratio with Nano-Glo® Luciferase solution (Promega, Ref N1150).A Promega GloMax plate reader was used to measure luminescence in technical triplicate for each sample.For tracking parasite burden over time, data are pooled cage-to-cage comparisons representing the average fecal parasite burden in each cage.
The numbers of mice per experimental group and experimental replicates are provided in the figure legends and no data sets were excluded.Mice were randomly selected for experimental use and no blinding strategies were employed.
For ALI monolayer experiments, C. parvum (IOWA II strain, purchased from Bunchgrass Farms in Dreary, ID) was excysted prior to infection.Oocysts were pelleted and resuspended in 25% bleach in PBS on ice for 5 minutes and washed three times with PBS.Oocysts were then resuspended in 0.2 mM taurocholate (Sigma) in PBS for 10 minutes at 15°C, washed three times, and resuspended in organoid growth medium for infections.For some experiments, sporozoites were excysted and heat-inactivated at 95°C for 5 minutes.

Fluorescent Imaging
Following infection or treatment with IFN-γ, mice were sacrificed and tissue from the distal third of the small intestine was flushed with ice-cold PBS.Tissue was opened laterally, rinsed again in ice-cold PBS, "swiss-rolled" and fixed for 1 hour in 2% paraformaldehyde in PBS at room temperature.Swiss rolls were transferred to 30% sucrose in PBS overnight at room temperature, followed by embedding in optimal cutting temperature (OCT) compound and storage at -80°C.
Tissues were sectioned, permeabilized for 45 minutes in hydration buffer (1% BSA and 0.1% Triton-x in PBS) and blocked for 45 minutes in saturation buffer (10% BSA and 0.1% Triton-x in PBS).Antibodies and stains included AF647-conjugated CD45 (BioLegend, clone 30-F11), polyclonal rabbit anti-MUC2 [C3] C-term (GeneTex), AF488-conjugated goat anti-rabbit IgG (H+L) (Invitrogen) and AF647-conjugated Phalloidin (Invitrogen) for F actin.Slides were stained with unconjugated primary antibodies diluted in hydration buffer for 2 hours, washed three times for 5 minutes in hydration buffer and stained for fluorescent antibodies or Phalloidin diluted in hydration buffer for 1 hour and 15 minutes.Nuclei were stained with Hoechst diluted in hydration buffer for 30 minutes, slides were washed three times for 5 minutes with hydration buffer, and mounted using fluorogel (Electron Microscopy Science) mounting medium.All incubations were performed in a dark, humid chamber at room temperature.Slides were imaged with a Leica DM6000 Widefield microscope, with 3-5 representative images taken per section, and analyzed using Fiji software or Imaris software (Oxford Instruments).

Single cell RNA sequencing
For scRNA-seq experiments, two mice were used for each group, and cells from both mice were pooled prior to sequencing.IEC/IEL single cell suspensions were prepared as above and dead cells were removed using the Dead Cell Removal Kit (Miltenyi Biotec) using LS columns (Miltenyi Biotec), per the manufacturer's instructions.We note that the extraction protocol used preferentially isolates IEC/IEL on the villus, while crypt-derived cells represent a smaller fraction of the single cell suspension.GEM encapsulation, reverse transcription, cleanup, and cDNA library preparation were done per manufacturer's instructions using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics), Chromium Controller (10X Genomics) and C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (Bio-Rad).Fragment sizes and concentrations were quantified using High Sensitivity D5000 and High Sensitivity D1000 ScreenTape and TapeStation 4200 system (Agilent).Final library concentration was confirmed with Qubit 1X dsDNA HS Assay Kit and Qubit 3 (Invitrogen).Libraries were sequenced on a NextSeq 500 or NextSeq 2000 (Illumina).
Cell Ranger v7.0.0 was used to process sequencing reads and build a reference genome for C.
parvum Iowa II (VEuPathDB, release 46).Processed reads from each sample were aligned to this reference genome or to the Mus musculus genome (GRCm38) to generate filtered feature-barcode matrices.

Analysis of mouse scRNA-seq data
Filtered feature matrices were imported into R to make a Seurat object for each sample (54).
Samples within each experiment were merged and filtered to remove empty cells (< 100 features), doublets (> 10,000 UMI) and cells with a high frequency of mitochondrial genes (>40%).Data were normalized and scaled, followed by principal component and jackstraw analyses to determine the dimensionality of the dataset and UMAP dimensional reduction.Differential gene expression analysis was performed using the FindAllMarkers function of Seurat on the IEC clusters.To identify pathways enriched within marker genes, functional enrichment clustering on the top 100 marker genes for each IEC cluster was performed using the Database for Annotation, Visualization and Integrated Discovery (55,56).Pseudotime trajectory analysis was performed using monocle3 (57)(58)(59).Single-sample GSEA was performed using escape (60).

Analysis of C. parvum scRNA-seq data
Filtered feature matrices were imported into R to make a Seurat object for each sample (54).
Samples within each experiment were merged and filtered to remove empty cells (< 100 features), doublets (> 1,200 features or nCount > 4,000) and cells with greater than 60% of ribosomal genes (cgd2_1372, cgd2_1373, cgd3_665, cgd3_666, and cgd3_667).Cells that met these criteria were identified as infected in the mouse genome-aligned data sets.Data were normalized and variable features identified.Next, data were integrated with previously published scRNA-seq datasets from HCT-8 cells and Ifng -/-mice infected with C. parvum and sequenced at various time points post-  (29).Data were scaled, followed by principal component and jackstraw analyses to determine the dimensionality of the dataset and UMAP dimensional reduction.Male and female gene signatures were generated using previously defined gene lists (S2 Table) (29).
Mouse spheroids were collected from Matrigel domes and dissociated in 2 mL TrypLE (Fisher Scientific) for 5 minutes in a 37°C water bath.Dissociated organoid cells were counted and transwells were seeded with 5x10 4 IEC, with organoid growth medium present in both top and bottom compartments.Media was replenished every 3 days and on day 7 the medium in the top compartment was removed to establish the air-liquid interface.ALI monolayers were used for stimulation or infection 3 days after top media removal.

Stimulation and infection of ALI monolayers
Once ALI monolayers were established, wells were stimulated with cells 250ng/mL recombinant murine IFN-γ (Peprotech) in the bottom compartment for 24h or with media alone.After 24h of stimulation, cytokine and media were removed, bottom compartments were washed with PBS, and fresh growth medium was added.ALI monolayers were infected by incubating 2×10 5

(cluster 11 ) 8 (
Fig).Monocle3 trajectory analysis was used to order enterocyte and goblet cell clusters in cluster (S2A Fig and S1 Table).Use of the Database for Annotation, Visualization and Integrated Discovery (DAVID) identified significantly enriched functional annotations that were separated into five categories: Cell Structure, Cellular Processes, Immune Response, Mitochondria, and Nutrient/Ion Transport (S1 Table).Genes annotated with functions in nutrient and ion transport were expressed across clusters 3-5 and 7-9, which suggests these pathways are not differentially regulated during infection (S2E Fig).Intestinal stem cells and transit amplifying cells (cluster 1) showed enhanced expression of genes related to mitochondria and energy production (S2C Fig), which has been previously associated with homeostatic cell turnover within this niche (22).The goblet cell cluster stood out as being enriched for genes related to the unfolded protein response (S2B Fig), a cellular response associated with mucus secretion (23).Immune response pathways related to antigen processing and presentation and IFN-γ responsiveness were upregulated in enterocyte clusters 3, 4, and 8, which are induced 4 dpi (hereafter referred to as IFN-γ-stimulated gene [ISG] enterocytes) (Fig 2A).Additional functional annotations enriched in these ISG enterocytes included genes related to the actin cytoskeleton and cellular organization (S2D Fig and S1 Table Fig 3A).No infection was detected in the stem cells/transit amplifying populations but rather parasites were detected predominantly in the ISG enterocytes and goblet cells (Fig 3A and 3B).In a separate experiment, the use of fluorescence microscopy confirmed the localization of parasites mid-villus and the presence of infected goblet cells (Fig 3C and 3D).Interestingly, the percentage of infected cells within each IEC cluster correlated with the IFN-γ response score (S4A Fig) although uninfected and infected IEC showed comparable ISG upregulation (Fig 3E).Similarly, ssGSEA analysis demonstrated comparable enrichment of the IFN-γ response gene set in uninfected and infected cells (Fig 3F).

for 24 -
48 hours (S5A Fig) and this dose was used in subsequent studies.It should be noted that a similar suppression was observed if IFN-γ was dosed multiple times either before or after infection (S5B and S5C Fig).In addition, treatment of Stat1 ∆IEC mice confirmed that restriction of infection was dependent on IFN-γ signalling to IEC (S5D Fig).This treatment regime controls the timing of IEC exposure to IFN-γ and permits direct comparison of gene expression between uninfected and infected cells.A total of 42,521 cells were sequenced and after quality control there were 6,263 cells from the uninfected sample, 5,967 from the uninfected + IFN-γ sample, 6,666 from the infected sample and 5,479 from the infected + IFN-γ sample.Analysis of these data identified 14 IEC clusters (Fig 4A), including enterocytes, goblet cells, and additional IEC sub-types as above (S6A-S6F Fig 6).Infected cells were identified by the presence of parasite transcripts and analysis of their distribution largely overlapped between PBS-and IFN-γ-treated mice (Fig 4B) and correlated with the size of that cluster in each sample (Fig 4C and S6H Fig).Next, based on the earlier profile, heatmaps of select IFN-γ-stimulated genes were generated for each sample.For the uninfected group, IFN-γ treatment resulted in the induction of several Gbp and Irg genes (S7A and S7B Fig).In the infected Ifng -/-mice, the induction of Irg genes was detectable in a small proportion of cells (S7C Fig) but ISG induction was most prominent after IFN-γ treatment (Fig 4D).This observation suggests there may be an infection-derived signal that "primes" IEC for IFN-γ responsiveness.Regardless, this IFN-γ-mediated induction of this gene set was comparable between uninfected and infected cells (Fig 4D and 4E) and ssGSEA analysis demonstrated nearly identical enrichment of the IFN-γ response hallmark between uninfected and infected cells (Fig 4F).These data sets indicate that infected cells remain responsive to IFN-γ.To determine whether IFN-γ treatment impacts parasite growth or development, a recent scRNA-seq analysis of the Cryptosporidium life cycle (29) was utilized.Data were aligned to the C. parvum genome and after quality control slightly more parasites were recovered from the PBStreated sample than the IFN-γ-treated sample (3,632 versus 2,676, respectively).To analyze parasite life stage distribution, these data were integrated with the sequencing data sets from the Cryptosporidium single cell atlas (29) for cluster analysis (S8A-S8C Fig).The UMAP projections show that both infected and infected + IFN-γ samples contain parasites distributed across clusters representing asexual (1-10, green clusters), male (11-13, blue clusters), and female (14-19, pink clusters) parasites (Fig 4G and 4H and S8D and S8E Fig).The normalized distribution of infected cells across parasite clusters was similar in either sample (Fig 4I), which indicates that IFN-γ -γ, the use of flow cytometry and fluorescence microscopy revealed that in Epcam + IEC there was modest induction of RFP at 12h post-treatment and by 24h approximately half of all IEC expressed RFP before declining at 48h post-treatment (S9A-S9E Fig).Thus, the IEC response to IFN-γ stimulation in vivo peaks 24h post-stimulation.To directly analyze whether IEC are primed by IFN-γ to control Cryptosporidium, organoids derived from the ilea of WT mice were used to generate IEC in an air-liquid interface (ALI) (31, 32) which support the Cryptosporidium life cycle (33).When these cultures were infected with C. parvum, qPCR revealed increasing amounts of parasite genomic DNA at 24h to 48h post-infection (which represents 2-4 cycles of replication, lysis and reinfection), with minimal background detected in cultures incubated with heat-inactivated parasites (S9F Fig).When these ALI monolayers were cultured with IFN-γ for 24h, cytokine was removed, and then infected with C. parvum there was no change in parasite burden at 24h post-infection, but there was a significant decrease in C. parvum gDNA equivalents at 48h post-infection (Fig 5E).Thus, prolonged exposure to IFN-γ primes IEC to activate cell-intrinsic mechanisms of parasite control.
Mice were administered 1μg or 5μg of recombinant murine IFN-γ (Peprotech, catalog #315-05) diluted in a final volume of 200μL of PBS and injected intraperitoneally.Control mice were administered 200μL of PBS.Flow CytometryMice were euthanized and the ileum (distal third of the small intestine) was harvested.Connective tissue and Peyer's patchers were removed, tissue was opened laterally, rinsed vigorously in icecold PBS and collected on ice into Hank's Balanced Salt Solution (HBSS) with 5% heatinactivated fetal bovine serum (FBS) and 10mM HEPES.Single-cell suspensions of the IEC/IEL layer were prepared by transferring tissue to HBSS with 5% FBS, 5mM EDTA and 1mM DTT and shaking at 37 °C for 25 minutes, followed by two 1-minute washes in HBSS with 2mM EDTA and 10mM HEPES.Cell pellets were then resuspended and passed sequentially through 70μm and 40μm filters.Cells were stained with Ghost Dye Violet 510 viability dye (Tonbo; catalog #13-0870-T500) and surface stained with the following antibodies in an appropriate combination of fluorochromes: CD45.2 (BioLegend, clone 104), Epcam (BioLegend, clone G8.8), MHC-I (BioLegend, clone AF6-88.5), and MHC-II (BioLegend, clone M5/114.15.2).For experiments with the fluorescent reporters RFP or mCherry, samples were not fixed prior to analysis.Otherwise, samples were fixed on ice for 10 minutes in 2% PFA, washed, and resuspend in PBS with 2% bovine serum albumin prior to analysis.Data were collected on a LSR Fortessa (BD Biosciences) or a FACSymphony A3 (BD Biosciences) and analyzed with FlowJo v10 software (BD Life Sciences).
excysted C. parvum oocysts in the top compartment for 3h at 37°C/5% CO 2 , after which the infection media was removed.Each condition was performed in triplicate.Genomic DNA extraction and measurement of parasite burdenDNA was collected from transwells using the QIAamp DNA Mini Kit (Qiagen) at 24h or 48h postinfection.Cells were lysed by scraping into 100 μL Buffer ATL and 20 μL Proteinase K per sample and incubated overnight at 56°C prior to column purification.DNA was eluted in 100 μL Buffer AE and diluted 1:10 in H 2 O.For qPCR, 2μL of this dilution was used as template in a reaction with SsoAdvanced Universal SYBR Green Supermix (BioRad) per the manufacturer's instructions.Primers targeting C. parvum GAPDH are as follows: forward primer, 5'-CGG ATG GCC ATA CCT GTG AG-3'; reverse primer, 5'-GAA GAT GCG CTG GGA ACA AC-3'.A standard curve for C. parvum genomic DNA was generated by extracting DNA from a known quantity of C. parvum oocysts as above and creating a dilution series for qPCR.Reactions were performed on QuantStudio 5 System qPCR machine.Genomic DNA equivalents were determined by generating a standard curve for average Ct versus oocyst number in Excel (Microsoft).StatisticsData were analyzed using GraphPad Prism 9 software.Specific tests for determining statistical significance are indicated in the figure legends.p-values of <0.05 were considered statistically significant.

Cryptosporidium-infected cells remain responsive to IFN-γ
were combined to generate an IFN-γ response score, which is calculated by subtraction of the expression of a control gene set (randomly selected genes that are expressed comparably between clusters) from the expression of the seven ISGs.Positive IFN-γ response scores reflect upregulation of ISGs, scores near 0 indicate expression that is not significantly different than the control gene set, and negative values reflect little to no expression in the cluster.In uninfected mice, these genes are largely silent while at 4 dpi infection there was increased IFN-γ-response score in goblet cells and ISG enterocytes (i.e.clusters 3, 4 and 8) which had largely resolved 10 dpi (Fig 2B and S2F Fig).A recent study identified that a set of canonical IFN-γ-stimulated genes were upregulated in mice 2 dpi with C. parvum (18) and these were also enhanced in our data sets (S2G Fig).Furthermore, when single-sample gene (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