The neonatal intestinal epithelial cell line (IEC4.1) was a kind gift from Dr. Pingchang Yang (McMaster University, Hamilton, Canada). DMEM-F-12 culture media was supplemented with 10% fetal bovine serum (FBS) (Ambion, Austin, TX) and 1% penicillin/streptomycin (ThermoFisher Scientific, Waltham, MA). HCT-8 cells were purchased from ATCC (Manassas, VA). RPMI-1640 cell culture media was supplemented with 10% fetal bovine serum (FBS) (Ambion, Austin, TX) and 1% penicillin/streptomycin (ThermoFisher Scientific, Waltham, MA). Cells were grown in tissue culture flasks at 37°C in 5% CO₂ and allowed to reach 80% confluency before passage.

C57BL/6J mice were originally purchased from the Jackson Laboratory. Mice at the age of 5 days after birth were used for in vivo infection. Intestinal villus/crypt components from mice at 5 days after birth were isolated and cultured as described in our previous studies (20). Briefly, small intestines were opened longitudinally and washed with ice-cold Ca²⁺ and Mg²⁺ free PBS, then were cut into 1–2 mm fragments and washed with ice-cold Ca²⁺ and Mg²⁺ free PBS 3 times. The cut fragments were incubated in ice-cold 2 mM PBS/EDTA at 4°C for 30 min with gentle rotation followed by vigorous shake until the PBS solution was mostly opaque with dislodged crypt and villus particles. Large tissue fragments were removed through a 100-μm cell strainer (Becton-Dickinson Bioscience, Franklin Lakes, NJ). The pass through was centrifuged 150g for 5 min at 4°C and the pellet was collected as the intestinal epithelium. 2D monolayers derived from 3D enteroids were cultured as previously described (20, 21).

C. parvum oocysts harvested from calves inoculated with the Iowa strain originally obtained from Harley Moon at the National Animal Disease Center (Ames, IA) were purchased from a commercial source (Bunch Grass Farms, Deary, ID). Oocysts were purified using a modified ether extraction technique and then suspended in phosphate-buffered saline (PBS) and stored at 4°C. For in vitro, 2D monolayer ex vivo, and in vivo infection, oocysts were treated with 1% sodium hypochlorite on ice for 20 min and washed 3 times with Dulbecco’s modified Eagle medium (DMEM) culture media. All parasite preparations were tested using the Limulus amebocyte lysate gel formation test as previously described to exclude the possibility of contamination with lipopolysaccharides (22, 23).

For in vitro infection using IEC4.1 and HCT-8 cells, or ex vivo infection using 2D monolayers derived from 3D enteroids, infection was performed in culture medium containing viable C. parvum oocysts after treatment with 1% sodium hypochlorite (1:1 ratio of oocyst:cell). Cells were then cultured for 4 h at 37°C for attachment and invasion by the parasites. Cells were then washed with cell medium three times to remove free parasites. Cells were cultured for additional time periods as described.

The neonatal murine infection model of intestinal cryptosporidiosis was used for in vivo experiments (24–26). Neonates (5 days after birth) received C. parvum oocysts by oral gavage (10⁵ oocysts per mouse) to develop intestinal cryptosporidiosis. Control mice received an equal volume of PBS by oral gavage. At 24, 48, and 72 h after C. parvum oocysts or PBS administration, at least five animals were sacrificed per condition, and ileal intestinal tissue was collected for biochemical analyses.

For real-time PCR analysis, total RNA was isolated from cells with Trizol reagent (Applied Biosystems, Carlsbad, CA). An amount of 200 ng total RNA was reverse-transcribed using the iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA). Comparative real-time PCR was performed using the SYBR Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) on the Bio-Rad CFX96 Touch™ Real-Time PCR Detection Systemn (Bio-Rad, Hercules, CA). The sequences for all the primers are listed in Supplementary Table 1 . Normalization was performed using Gapdh. Relative expression was calculated using the comparative Ct (ΔΔCt) method.

For gene silencing, the small interfering RNA (siRNA) duplexes for mouse Nostrill were synthesized using Integrated DNA Technologies. The siRNA sequences are listed in Supplementary Table 1 . Cells were treated with siRNAs (final concentration, 60 nM) using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. For Nostrill overexpression, Nostrill cDNA was amplified through PCR, inserted into the PTarget (Promega, Madison, WI) expression vector to generate PTarget-Nostrill, and subsequently sequenced. According to the manufacturer’s protocol, cells were transfected with plasmid DNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Quantitative RT-PCR was used to determine the significant alteration of each target gene.

Formaldehyde crosslinking RIP was performed as described (27). Briefly, lysates were precleaned with 20 μl of PBS washed Magna ChIP Protein A + G Magnetic Beads (Millipore, Burlington, MA). The precleaned lysate (250 μl) was then diluted with the whole cell extract buffer (250 μl), mixed with the specific antibody-coated beads, and incubated with rotation at 4 °C for 4 h, followed by 4 times washing with the whole cell extract buffer containing protease and RNase inhibitors. The collected immunoprecipitated RNP complexes and input were digested in RNA PK Buffer pH 7.0 (100 mM NaCl, 10 mM TrisCl pH 7.0,1 mM EDTA, 0.5% SDS) with addition of 10 μg Proteinase K and incubated at 50°C for 45 min with end-to-end shaking at 400 rpm. Formaldehyde cross-links were reversed by incubation at 65°C with rotation for 4 h. RNA was extracted from these samples using Trizol according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA) and treated with DNA-free DNase Treatment & Removal I kit according to the manufacturer’s protocol (Ambion, Austin, TX). The presence of RNA was measured by qRT-PCR as described above. Gene-specific PCR primer pairs are listed in Supplementary Table 1 . The following antibodies were used for RIP analysis: anti-NF-κB p50 (Santa Cruz, Dallas, TX), anti-NF-κB p65 (Santa Cruz, Dallas, TX), normal mouse IgG (Santa Cruz, Dallas, TX).

ChIP assays were performed as described previously (28, 29). Briefly, cells were fixed with 1% formaldehyde for 10 min, collected in ice-cold PBS, and resuspended in an SDS lysis buffer. Genomic DNA was then sheared to lengths ranging from 200 to 1000 bp by sonication. Cell extracts were incubated with either anti-NF-κB p65 (Santa Cruz, Dallas, TX), anti-H3K4me3 (Cell Signaling Technology, Danvers, MA), anti-RNA Polymerase 2 (Millipore, Burlington, MA), or normal mouse IgG (Santa Cruz, Dallas, TX) overnight at 4°C, followed by precipitation with protein G-agarose beads. The immunoprecipitates were sequentially washed once with a low-salt buffer, once with a high-salt buffer, once with an LiCl buffer, and twice with a Tris buffer. The DNA–protein complex was eluted, and proteins were then digested with proteinase K for 1 h at 45°C. The DNA was detected by qRT-PCR analysis as described above and analyzed using the fold enrichment method. Gene-specific PCR primer pairs are listed in Supplementary Table 1 .

ChIRP analysis was performed as previously reported (19, 27). Briefly, a pool of tiling oligonucleotide probes with affinity specific to the Nostrill sequence was used and glutaraldehyde cross-linked for chromatin isolation. The sequences for each probe are listed in Supplementary Table 1 ; probe 1, 3, 5, and 7 are mixed as the probe pool Odd and probe 2, 4, 6, and 8 as the probe pool Even. The DNA sequences of the chromatin isolates were confirmed and quantified by qRT-PCR using the same primer sets covering the gene promoter regions of interest as for ChIP analysis. A pool of oligo probes for LacZ were served as controls. The percent input method was used to normalize the ChIRP data.

Data are expressed as mean values and error bars represent standard error of the mean (SEM). Student T test with Bonferroni’s correction or one-way ANOVA followed by Tukey-Kramer post hoc tests were performed where appropriate. For determination of significant differences between percents and for multiple comparisons between culture conditions, two-way ANOVA followed by Tukey-Kramer multiple analyses post hoc tests were used. p values < 0.05 were considered statistically significant.

We have previously reported genome-wide RNA transcriptome analysis of C. parvum-infected IEC4.1 cells, a transformed but non-tumorigenic neonatal mouse intestinal epithelial cell line (30). Nostrill was additionally identified as being strongly upregulated during genome-wide RNA transcriptome analysis of murine microglia stimulated with LPS, and we have published data on the role of Nostrill in regulating the expression of iNOS in the microglial inflammatory response (19). In response to C. parvum infection, many lncRNAs displayed differential expression as previously identified by microarray analysis, including Nostrill (2.47-fold) (17). Nostrill, or NR_126553, is transcribed from the 2500002B13Rik gene locus on chromosome 8 between the Sap30 and Hmgb2 gene loci ( Supplementary Figure 1 ) (31). Using qRT-PCR, we confirmed the upregulation of Nostrill in IEC4.1 cells 24 h following C. parvum infection at 2.94-fold, p < 0.01 compared to uninfected controls ( Figure 1A ). Nostrill did not display upregulation prior to the 24 h timepoint but remained significantly elevated 48 h post-infection, albeit at a lower level than the 24 h timepoint ( Figure 1A ). To verify biological relevance of Nostrill induction in C. parvum infection in vivo, we utilized a neonatal murine model of intestinal cryptosporidiosis via oral administration of the parasite ( Figure 1B ) to examine expression of Nostrill in the intestinal epithelium (24–26). Nostrill was significantly upregulated 24 h post-infection in the intestinal epithelium, however, its expression returned to basal levels by the 48 h timepoint ( Figure 1C ). An ex vivo infection model employing 2D enteroid monolayers derived 3D enteroids originating from the neonatal mouse ileum ( Figure 1D ) was utilized to ensure the significant upregulation of Nostrill observed in vivo was due to differential expression in the cells of the intestinal epithelium. Infection of 2D enteroid monolayers does not require microinjection of C. parvum as infection of 3D enteroids does (33). C. parvum infection of 2D epithelial monolayers displayed a 2.78-fold significant increase in Nostrill expression at 24 h post-infection ( Figure 1E ), consistent with in vitro and in vivo results (20, 21). We then investigated the response of Nostrill in IEC4.1 cells to known inflammatory mediators, including IFN-γ, LPS, and TNF-α ( Figure 1F ). Nostrill displayed significant upregulation in response to IFN-γ, LPS, and TNF-α at 24 h post-stimulation in response to all three stimuli, providing additional evidence that Nostrill may function broadly in the inflammatory response in different cell types and in response to numerous stimuli ( Figure 1F ) (19). No significant differential expression of Nostrill was observed at earlier timepoints ( Figure 1F ). Initial characterization of the intracellular localization of Nostrill was performed using nuclear and cytoplasmic extraction with the nuclear marker U2 and the cytoplasmic marker Rps14 ( Figure 1G ). Utilizing this technique, Nostrill displayed a preference for nuclear localization and its localization did not change significantly in C. parvum-infected cells compared to uninfected controls ( Figure 1G ). Together, this data demonstrates that the lncRNA Nostrill is induced in response to C. parvum infection and more broadly in response to stimulation with inflammatory mediators and demonstrates a preference for nuclear localization intracellularly.

As NF-κB is a master regulator of the inflammatory response and has been previously shown to control the expression of Nostrill in response to inflammatory stimuli (19), we investigated whether inhibition of NF-κB influences the C. parvum-mediated induction of Nostrill in intestinal epithelial cells. Two different NF-κB inhibitors, SC-514 and JSH-23, were used to query the dependence of Nostrill expression on NF-κB signaling in a dose-dependent manner. C. parvum infection significantly increased Nostrill expression 1.76-fold compared to uninfected controls ( Figure 2A ). Both SC-514 (100 µM) and JSH-23 (30 μM) inhibited significant upregulation of Nostrill in C. parvum-infected cells compared to uninfected controls, while lower doses of NF-κB inhibitors failed to inhibit C. parvum-induced Nostrill upregulation ( Figure 2A ). Cxcl2, a known NF-κB response gene, served as a positive control to demonstrate effective inhibition of NF-κB signaling ( Figure 2B ) (34). Without NF-κB inhibition, Cxcl2 showed a 2.73-fold significant upregulation after C. parvum infection ( Figure 2B ). At higher doses, both SC-514 (100 μM) and JSH-23 (30 μM) successfully inhibited induction of Cxcl2 in response to C. parvum infection ( Figure 2B ). Pretreatment with NF-κB inhibitors at lower doses failed to inhibit upregulation of Cxcl2 following C. parvum infection ( Figure 2B ). We additionally confirmed the impact of NF-κB signaling on C. parvum infection burden as previously reported (11). Pretreatment with the NF-κB inhibitors SC-514 (100 µM) and JSH-23 (30 μM) followed by infection of IEC4.1 cells with C. parvum resulted in a significant increase in parasite infection burden as assessed by qRT-PCR ( Figure 2C ). These data suggest that optimal antiparasitic defense and induction of the lncRNA Nostrill are NF-κB dependent in C. parvum infection of intestinal epithelial cells.

To determine whether Nostrill may contribute to anti-C. parvum defense, we infected IEC4.1 cells after knockdown or overexpression of Nostrill then measured the impact on parasite infection burden at different timepoints. The RNAi approach was utilized for Nostrill knockdown and the siRNA targeting Nostrill successfully reduced the level of Nostrill in uninfected and C. parvum-infected intestinal epithelial cells compared to a scrambled siRNA ( Supplementary Figure 2 ). Complementarily, overexpression was performed via transfection with the pTargeT Mammalian Expression Vector containing a cloned insert of Nostrill and resulted in significant induction of Nostrill expression compared to empty vector control in both uninfected and infected cells ( Supplementary Figure 2 ). We began by assessing the impact of Nostrill expression on C. parvum attachment/invasion into IEC4.1 cells by measuring parasite burden 4 h after infection (35). Neither knockdown nor overexpression of Nostrill significantly affected C. parvum infection burden after parasite attachment/invasion ( Figures 3A, B ). We then measured C. parvum burden 24 h after infection and found that knockdown of Nostrill significantly increased infection burden compared to scrambled siRNA controls ( Figure 3C ). Conversely, overexpression of Nostrill significantly decreased C. parvum burden at 24 h post-infection compared to empty vector controls ( Figure 3D ). Indirect immunofluorescence microscopy was additionally utilized to detect infection burden in IEC4.1 cells. In accordance with results observed by qRT-PCR, knockdown of Nostrill resulted in a significant 1.93-fold increase in C. parvum infection burden ( Figure 1E ), while Nostrill overexpression caused a significant 0.31-fold decrease in C. parvum infection burden ( Figure 1F ).

We additionally employed the 2D intestinal monolayer model to further examine the impact of Nostrill on C. parvum infection burden. Successful knockdown or overexpression of Nostrill was observed ( Supplementary Figure 2 ). Similar to the results seen in IEC4.1 cells, knockdown of Nostrill significantly increased the infection burden of C. parvum 24 h post-exposure compared to scrambled siRNA controls ( Figure 3G ), while overexpression of Nostrill resulted in a significant decrease in C. parvum infection burden compared to empty vector controls ( Figure 3H ). Taken together, the above data suggests that induction of the lncRNA Nostrill contributes to epithelial defense against C. parvum infection.

Given the significant impact of Nostrill on C. parvum infection burden in the intestinal epithelium, we next sought to answer whether this effect may be due to modulation of C. parvum-induced defense gene expression by the lncRNA Nostrill. Knowing that lncRNAs have been observed to modulate target gene expression, both in cis and in trans, we began by evaluating the impact of Nostrill knockdown or overexpression on C. parvum-induced expression of the neighboring genes, Sap30 and Hmgb2. C. parvum infection in scrambled siRNA controls resulted in a downregulation of Sap30 expression compared to uninfected cells, although this effect was non-significant ( Figure 4A ). Knockdown of Nostrill did not significantly affect Sap30 expression in uninfected cells but caused a significant decrease in Sap30 expression in C. parvum infected cells ( Figure 4A ). Overexpression of Nostrill did not significantly impact Sap30 expression ( Figure 4A ). C. parvum infection did not significantly affect Hmgb2 expression in the scrambled siRNA or empty vector controls ( Figure 4B ). Neither Nostrill knockdown nor overexpression caused a significant difference in levels of Hmgb2 mRNA ( Figure 4B ). Together, this indicated that Nostrill has little impact on transcriptional regulation in cis. We then investigated whether Nostrill impacts the transcription of iNOS as previously observed in microglia (19). Knockdown of Nostrill in C. parvum infected cells demonstrated a significant decrease in iNOS expression compared to both uninfected and infected scrambled siRNA controls ( Figure 4C ). Overexpression of Nostrill produced no significant alterations in iNOS expression in intestinal epithelial cells in response to C. parvum infection ( Figure 4C ).

Given previous data on the importance of interferon-mediated epithelial anti-C. parvum defense (12, 13), we next investigated whether Nostrill may be involved in regulation of the interferon response via modulation of interferon regulatory transcription factor (IRF) family members. Induction of Irf3 was not observed following C. parvum infection in the scrambled siRNA or Nostrill knockdown samples ( Figure 4D ). However, Nostrill knockdown caused a significant increase in Irf3 expression in uninfected cells compared to scrambled siRNA control. Interestingly, C. parvum infection induced Irf3 expression 1.46-fold and 1.54-fold in empty vector and Nostrill overexpression conditions, respectively. The overexpression of Nostrill did not significantly affect Irf3 expression in uninfected cells ( Figure 4D ). Irf9 was significantly induced by C. parvum infection to 1.50-fold and 1.76-fold in both scrambled siRNA and empty vector conditions, respectively ( Figure 4E ). Knockdown of Nostrill in uninfected cells resulted in a significant 0.71-fold decrease in Irf9 expression, while knockdown of Nostrill in C. parvum-infected cells resulted in Irf9 expression not significantly different from uninfected scrambled siRNA controls, however, this was not significantly different from scrambled siRNA C. parvum-infected cells ( Figure 4E ). Overexpression of Nostrill had no significant effect on Irf9 expression compared to empty vector controls in uninfected of C. parvum-infected cells ( Figure 4E ). Irf7 expression was significantly induced by C. parvum exposure to 2.14-fold and 2.78-fold in both scrambled siRNA and empty vector conditions, respectively ( Figure 4F ). Knockdown of Nostrill in uninfected cells resulted in significantly decreased expression of Irf7 to 0.67-fold of uninfected scrambled control. Significant Irf7 induction (1.41-fold) was observed in C. parvum infected cells with Nostrill knockdown, however, there was a significant reduction in Irf7 expression in the siRNA-Nostrill C. parvum infected cells compared to siRNA-Control C. parvum infected cells ( Figure 4F ). Conversely, Nostrill overexpression resulted in significantly increased Irf7 induction in C. parvum infected cells ( Figure 4F ). Interestingly, Nostrill overexpression alone in uninfected cells demonstrated a significant 2.09-fold increase in Irf7 mRNA expression compared to uninfected empty vector controls ( Figure 4F ). The above data suggest Nostrill induction is involved in the transcriptional regulation of Irf7 in trans in intestinal epithelial cells following C. parvum infection.

To better understand whether transcriptional regulation of Irf7 by Nostrill may be contributing to anti-C. parvum defense in intestinal epithelial cells, we used RNAi to knockdown Irf7 and examine the effect on parasite burden. Successful knockdown of Irf7 by the targeting siRNA is demonstrated by a significant 0.59-fold reduction in expression of Irf7 mRNA ( Figure 5A ). Knockdown of Irf7 resulted in a significant increase in C. parvum infection burden compared to infected scrambled siRNA controls ( Figure 5A ). Given the importance of NF-κB signaling in initiation of epithelial defense against C. parvum, and the presence of putative NF-κB binding sites in the promoter region of Irf7 identified by database search using TFBIND (https://tfbind.hgc.jp/), we asked whether induction of Irf7 in response to C. parvum infection is NF-κB dependent. Irf7 expression significantly increased 3.00-fold after C. parvum infection in control cells ( Figure 5B ). The use of NF-κB inhibitors SC-514 (100 µM) and JSH-23 (30 µM) resulted in the absence of C. parvum-induced Irf7 upregulation ( Figure 5B ), demonstrating that Irf7 is dependent on NF-κB signaling for its induction in intestinal epithelial cells in response to C. parvum.

To determine whether the transcriptional regulation of Irf7 by Nostrill is specific to C. parvum infection or is potentially present broadly in response to inflammatory stimuli, we investigated the impact of Nostrill knockdown or overexpression on Irf7 expression in response to stimulation of intestinal epithelial cells with IFN-γ. Successful knockdown or overexpression of Nostrill was observed ( Figure S2 ). Stimulation with IFN-γ resulted in a more robust induction of Irf7 when compared with C. parvum infection in Figure 4F , but similar trends were observed ( Figure 5C ). Stimulation with IFN-γ resulted in a significant 9.57-fold or 24.88-fold increase in Irf7 expression compared to control cells in the scrambled siRNA or empty vector conditions, respectively ( Figure 5C ). Knockdown of Nostrill in unstimulated cells demonstrated a significant 0.56-fold decrease in Irf7 expression compared to unstimulated siRNA-Control. Knockdown of Nostrill in IFN-γ-stimulated cells resulted in a significant decrease in levels of Irf7 mRNA compared to IFN-γ-stimulated siRNA-Control cells ( Figure 5C ). Conversely, overexpression of Nostrill in unstimulated cells showed a significant 5.35-fold increase in Irf7 expression compared to unstimulated empty vector control ( Figure 5C ). Overexpression of Nostrill enhanced the IFN-γ-induced Irf7 upregulation and demonstrated a synergistic effect with a significant increase in Irf7 when compared to IFN-γ-stimulated PTarget-Empty cells ( Figure 5C ). Taken together, these data suggest that NF-κB dependent Irf7 induction participates in epithelial defense against C. parvum and that the transcriptional regulation of Irf7 by Nostrill is not restricted to C. parvum infection.

Given that Nostrill has been previously found to physically associate with NF-κB p65 (19) and that Irf7 expression in response to C. parvum infection was observed to be NF-κB dependent, we sought to further explore whether an interaction between NF-κB and Nostrill may be present in the regulation of Irf7 gene transactivation in the intestinal epithelium after C. parvum infection. We began by investigating potential physical interactions between Nostrill and the NF-κB subunits p50 and p65 through RIP assays. A significant amount of Nostrill was detected in the anti-p65 immunoprecipitates from both uninfected and C. parvum-infected cells ( Figure 6A ). Interestingly, there was a significant 2.68-fold increase in Nostrill detected in anti-p65 C. parvum-infected immunoprecipitates compared to uninfected cells ( Figure 6A ). An increase of Nostrill in anti-p50 immunoprecipitates was seen in both infected and uninfected cells, however this increase was not significant. No significant differences in uninfected or infected cells were observed in the Actin controls using anti-p50 or anti-p65 compared to non-specific IgG. ChIRP was performed next to determine whether Nostrill may occupy genomic sites in the Irf7 gene promoter and potentially serve as a “guide” to enhance the binding of NF-κB p65. Two pools of biotinylated tiling oligonucleotide probes specific to Nostrill were used to purify chromatin fragments which were identified via PCR with five sets of primers in the Irf7 promoter region ( Figure 6B ). After C. parvum infection, significant increases in Nostrill occupancy were detected in sets 1, 2, and 5 ( Figure 6C ). However, only set 5 showed a significant increase in Nostrill occupancy in both the even and odd pools of probes, providing stronger evidence for a true interaction between Nostrill and the Irf7 promoter region at this site ( Figure 6C ). No significant difference in the Actin control gene was observed in C. parvum-infected cells compared with uninfected cells.

ChIP was next used to examine coordinated occupancy of NF-κB p65 at the Irf7 promoter region. There was a 2.84-fold significant increase in the occupancy of NF-κB p65 at set 5 of the Irf7 promoter region after C. parvum infection compared to uninfected scrambled siRNA controls ( Figure 6D ). Knockdown of Nostrill significantly reduced the C. parvum-induced NF-κB p65 occupancy at the set 5 region of the Irf7 gene promoter, suggesting that Nostrill contributes to the occupancy of NF-κB p65 at the Irf7 promoter ( Figure 6D ). Given that histone modifications are necessary for gene transcription, we queried whether Nostrill could impact H3K4 trimethylation, an activating histone modification. Significant increases in H3K4me3 were detected at the Irf7 gene promoter at PCR primer sets 1, 4, and 5 after C. parvum infection in scrambled siRNA samples ( Figure 6E ). Knockdown of Nostrill significantly reduced H3K4me3 levels at set 1 and 5 after C. parvum exposure compared to infected scrambled siRNA controls ( Figure 6E ). Lastly, we performed ChIP to investigate the role of Nostrill in occupancy of RNA polymerase II (Pol2) at the Irf7 promoter region after C. parvum infection. Significant increases in Pol2 occupancy at primer sets 1, 2, 4, and 5 were observed in scrambled siRNA controls after C. parvum infection ( Figure 6F ). Knockdown of Nostrill significantly reduced Pol2 occupancy at sites 1 and 5 in C. parvum-infected cells compared to scrambled siRNA controls ( Figure 6F ). Taken together, the above data suggest Nostrill physically interacts with NF-κB p65 and the Irf7 gene promoter at the set 5 region to participate in the induction of Irf7 transcription in the intestinal epithelium in response to C. parvum infection.

LncRNAs often display low primary sequence conservation between species (36, 37), however, lncRNAs have been observed to be expressed from syntenic coding gene loci and it has been suggested this positional conservation may indicate functional commonality (38, 39). We identified 3 positionally conserved human lncRNAs localized by the Sap30 and Hmgb2 gene loci, ENST00000609153.2, ENST00000651702.2, and ENST00000658397.1 ( Figure S3 ). Interestingly, upregulation of all three positionally conserved human lncRNAs was observed in cultured human epithelial HCT-8 cells following exposure to C. parvum at varying timepoints during infection ( Figure 7A ). Induction of positionally conserved orthologs was also observed in the FHs 74 Int human small intestine epithelial cell line after C. parvum infection and LPS stimulation ( Supplementary Figure 4 ). To determine the potential of these positionally conserved lncRNAs in anti-C. parvum defense, we knocked down their expression utilizing the RNAi approach and measured the impact on parasite burden, similar to the experiments in Figure 3 . Successful knockdown compared to a scrambled siRNA was confirmed ( Supplementary Figure 2 ). Knockdown of both ENST00000609153.2 and ENST00000651702.2 significantly increased parasite burden as measured by Cp18s and CpHsp70 compared to scrambled siRNA control ( Figure 7B ). However, only knockdown of ENST00000609153.2 significantly impacted the infection burden as measured by Cpv, the viral symbiont carried by C. parvum ( Figure 7B ). Knockdown of ENST00000658397.1 had no impact on C. parvum infection burden. We next sought to characterize the upstream regulation of ENST00000609153.2 induction and potential similarities to Nostrill by performing experiments with inhibition of the NF-κB signaling pathway. NF-κB inhibition by both SC-514 (100 µM)and JSH-23 (30 µM) abolished the induction of ENST00000609153.2 by C. parvum infection observed in DMSO control samples ( Figure 7C ). Together, these preliminary results suggest ENST00000609153.2 may function as a putative human ortholog of Nostrill, whose induction is orchestrated through NF-κB signaling and contributes to epithelial defense against C. parvum. Further studies are needed to evaluate the underlying molecular mechanism of ENST00000609153.2 in anti-C. parvum defense.