Researchers discovered that m6A RNA methylation plays a critical role in how intestinal epithelial cells respond to IFN-γ signaling to fight parasitic infections. The study reveals that this epigenetic modification enhances cell-intrinsic antiparasitic defenses, suggesting m6A methylation as a potential therapeutic target for improving parasite resistance at the cellular level.
N6-methyladenosine (m6A) RNA methylation is one of the most prevalent reversible post-transcriptional RNA modifications and has been recognized as a crucial regulator of host immune responses. Intestinal epithelial cells (IECs) constitute an important component of gastrointestinal mucosal immunity. Interferons (IFNs) play a central role in maintaining intestinal homeostasis, and m6A methylation status influences IFN-mediated cell-intrinsic defense. In this study, we investigated the potential role of m6A RNA modifications in IFN-γ-stimulated IEC-intrinsic defense. We observed significant alterations in the topology of the m6A mRNA methylome in murine IECs following IFN-γ stimulation. A subset of IFN-γ-stimulated immune gene transcripts exhibited increased m6A RNA methylation, including several members of the immunity-related GTPase family M (IRGM) genes. In addition, IFN-γ-responsive long non-coding RNAs may modulate the m6A methylation levels of multiple IFN-γ-stimulated immune transcripts. Enhanced m6A methylation of the Irgm2/3 transcripts was associated with strengthened cell-intrinsic defense against infection by the protozoan parasite Cryptosporidium. Notably, Cryptosporidium infection altered the host m6A mRNA methylome in IECs, thereby counteracting the IFN-γ-mediated defense response. Although the RNA levels of Irgm2/3 genes were upregulated, their m6A RNA methylation levels and protein expression were reduced in infected cells. This effect was associated with host delivery of dsRNAs derived from Cryptosporidium parvum virus 1, a virus harbored in the parasite. Collectively, our findings suggest that m6A methylation of RNA transcripts enhances IFN-γ-mediated IEC-intrinsic antiparasitic defense, while Cryptosporidium has evolved mechanisms to evade this response by suppressing m6A RNA methylation of IFN-γ-stimulated immune genes.
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Cell-intrinsic defense refers to the ability of an individual host cell to autonomously eliminate invading microbes, a process that is especially important for controlling intracellular pathogens. The cytokine IFN-γ is a key regulator of epithelial cell–intrinsic defense. RNA modifications have emerged in recent years as a critical component of cell biology, including the regulation of host innate immune defense. The most common RNA modification in mRNA is N6 methyladenosine (m6A). The obligate intracellular parasite Cryptosporidium establishes an intracellular but extracytoplasmic infection by forming a membrane-bound vacuole on the apical surface of intestinal epithelial cells (IECs). Because infection is largely restricted to IECs, cell–intrinsic defense mechanisms—particularly those induced by IFN-γ—are essential for clearing the parasite. In this study, we investigated the role of m⁶A-mediated RNA methylation in regulating the IFN-γ–induced, cell-intrinsic anti-Cryptosporidium response in IECs. Our findings indicate that m⁶A-mediated post-transcriptional gene regulation is a key determinant of IFN-γ–induced epithelial antiparasitic defense. We further show that C. parvum has evolved mechanisms to evade host immunity by suppressing m⁶A methylation of host RNAs. These results provide new mechanistic insights into IEC-intrinsic immunity and may inform the development of novel therapeutic strategies.
Citation: Pok C, Gong A-Y, Graham ML, Wang S, Deng S, Sharmin Z, et al. (2026) m6A RNA methylation modulates IFN-γ-stimulated intestinal epithelial cell-intrinsic antiparasitic defense. PLoS Pathog 22(7): e1014442. https://doi.org/10.1371/journal.ppat.1014442
Editor: Tracey J. Lamb, University of Utah, UNITED STATES OF AMERICA
Received: January 5, 2026; Accepted: July 3, 2026; Published: July 20, 2026
Data Availability: All relevant data are within this manuscript, associated supporting information files, or in public repositories. The MeRIP-seq data are deposited in the NCBI database under the accession numbers SRR38251195-SRR38251206. The RNA-Seq data are deposited in the NCBI database under the accession number GSE245591.
Funding: This work was supported by funding from the National Institutes of Health (AI116323, AI136877, and AI177188) to XMC. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Center for Research Resources or the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Cell-intrinsic immunity (also called cell-autonomous immunity) is the ability of a host cell to eliminate an invasive/intracellular infectious agent at the cellular level. It serves as the first line of host defense against intracellular pathogens [1]. Intestinal epithelial cells (IECs) possess the necessary molecular machinery to mount a cell-intrinsic defense response, constituting an important component of gastrointestinal mucosal immunity. Ligation of their pattern recognition receptors leads to the upregulation of antimicrobial factors, secretion of cytokines and chemokines, and conditioning of immune cells for direct antimicrobial action or instruction of adaptive immune responses [2]. On the other hand, IECs serve as targets of mucosal immune mediators released from immune cells residing at the gastrointestinal mucosa [3]. One group of such cytokines is the family of interferons (IFNs). The IFN family can be classified into three main types: type I (e.g., IFN-α and IFN-β), type II (IFN-γ), and type III (IFN-λ family) [4]. Growing evidence supports the essential role of IFN-γ in antibacterial and antiparasitic immunity, whereas type I and type III IFNs are key to antiviral immunity [5, 6]. The canonical IFN-γ signaling utilizes the JAK/STAT signaling to activate STAT1, resulting in the formation and nuclear translocation of active STAT1 homodimers and transcription of IFN-stimulated genes (ISGs) [7, 8]. Many ISGs are primary effectors of the innate immune response, such as Mx1/MxA, the interferon-Induced proteins with tetratricopeptide repeats (IFIT) family, and the immunity-related GTPase family M (IRGM) genes [9–11]. The protein products of these genes can target different stages of a pathogen’s life cycle [9– 11], regulate autophagy formation [12–14], and have been identified as cornerstones of IFN-γ-mediated responses to intracellular pathogens [15].
RNA modifications have gained traction in the past decade as a crucial element in eukaryotic biological processes. Among the most prevalent modifications occurring in approximately 25% of transcripts at the genome level in eukaryotes is m6A methylation [16]. N6-methyladenosine (m6A) is a post-transcriptional modification that can influence aspects of RNA metabolism, such as mRNA degradation, RNA splicing, mRNA stabilization, and translation efficiency [17]. m6A dynamics and functions are executed by three groups of proteins: methyltransferases, demethylases, and m6A-binding proteins [18]. m6A is installed on RNA molecules by the methyltransferase complex, consisting of methyltransferase-like 3 (METTL3) and METTL14 and their cofactor the WT1 Associated Protein [19]. Structural studies revealed that METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding platform [20, 21]. m6A is mainly removed by the AlkB homolog H5 and fat mass and obesity-associated protein [22]. The m6A modification regulates RNA splicing, translocation, stability, and translation [23– 25]. It has been well-recognized as a crucial regulator in T cell homeostasis, inflammation, and immune response [24, 25]. Selectively altering m6A levels, along with other immunotherapies, may be effective management strategies for a variety of immunological diseases.
Cryptosporidium, a coccidian parasite and an NIAID Category B priority pathogen, infects the gastrointestinal epithelium and is a leading cause of infectious diarrhea and diarrheal-related death in children worldwide [26]. The infection can also cause a life-threatening diarrheal disease in AIDS patients [27]. Cryptosporidium attaches to the apical membrane surface of epithelial cells and forms an intracellular but extracytoplasmic vacuole in which the organism remains [28]. Thus, cell-intrinsic defense is critical to the host’s defense against Cryptosporidium infection [29], providing an ideal model to explore intestinal epithelial cell-intrinsic immunity [30]. IFN-γ is key to epithelial cell-intrinsic anti-Cryptosporidium defense [31].
To counteract host defense, Cryptosporidium has evolved mechanisms to effectively dysregulate the IFN-γ signaling pathway [32]; thus, it can survive host immune attack during the early stages of infection [33, 34]. The Cryptosporidium parvum virus 1 (CSpV1), a non-enveloped RNA virus of the Partitiviridae family, infects C. parvum and other Cryptosporidium spp. including C. hominis [35]. Its genome comprises two distinct double-stranded RNAs (dsRNAs), sized 1,836 bp (CSpV1-dsRdRp) and 1,510 bp (CSpV1-dsCA) [36]. The CSpV1-dsRNAs are not 5’-capped at either end and unlikely to be 3’-polyadenylylated [37]. CSpV1 is likely to be regularly transmitted only by intracellular routes, as it lacks the machinery for cell entry [38]. Importantly, the fecundity of C. parvum is correlated with its intracellular CSpV1 levels [39]. In our recent studies [40], we demonstrated that CSpV1-dsRNAs are present within infected host cells and are associated with attenuation of IFN-γ–mediated antiparasitic defense in IECs.
In this study, we demonstrated that IFN-γ stimulation causes significant alterations in the RNA m6A landscape of murine IECs, involving Mettl3/14 and long non-coding RNAs (lncRNAs). Increased m6A methylation of Irgm2/3 RNAs following IFN-γ stimulation is associated with enhanced cell-intrinsic defense against Cryptosporidium infection. In contrast, Cryptosporidium infection is associated with reduced m6A methylation of Irgm2/3 RNAs in infected IECs, potentially mediated by parasite-derived CSpV1-dsRNAs, coinciding with attenuation of IFN-γ-stimulated epithelial cell-intrinsic defense. Overall, our findings suggest that m6A-mediated post-transcriptional gene regulation may be a key determinant of IFN-γ-mediated epithelial antiparasitic defense, and that C. parvum may, at least in part, evade host immunity by suppressing m6A methylation of host RNAs.
We first examined the landscape of the m6A mRNA methylome in IECs upon IFN-γ stimulation by performing m6A methylated RNA immunoprecipitation sequence (MeRIP-seq). IEC4.1 cells, a transformed but non-tumorigenic intestinal epithelial cell line derived from neonatal mice (5–7 days old) [41], were treated with IFN-γ (10 ng/ml) for 6 h. Total mRNA was then collected and processed for MeRIP-seq as previously reported [42]. We compared the abundance and distribution of m6A peaks on mRNAs between untreated control and IFN-γ-treated cells. IFN-γ stimulation resulted in significant alterations in m6A peaks across 721 mRNAs in the transcriptome, including 590 with increased m6A peaks and 131 with decreased m6A peaks (Fig 1A-1C and S1 Table). The top 50 mRNAs with significant increase or decrease in their m6A peaks are shown in Fig 1A. The complete distribution of m6A sites, regions, and corresponding mRNAs is provided in S1 Table. Among these m6A sites, the majority were in the intronic regions (37.6% at 320 sites), distal regions (33.8% at 287 sites), and promoter regions (16.2% at 138 sites) (Fig 1B, 1C and S2 Table). The remaining peaks were distributed across the exonic (non-coding sequence) regions (7.9% at 67 sites), 3’UTR (3.4% at 29 sites), 5’UTR (0.7% at 6 sites), and downstream regions (0.4% at 3 sites) (Fig 1B, 1C and S2 Table). More sites with increased m6A methylation than with deceased m6A methylation were detected across these regions (Fig 1C). No significant difference in m6A peak alterations was observed between the 5’UTR and 3’UTR regions (Fig 1C). Motif analysis identified the top most enriched sequence motifs associated with newly emerged and lost m6A peaks in IEC4.1 cells following IFN-γ stimulation (Fig 1D). All sequencing data were generated in accordance with MIAME guidelines and were deposited in the NCBI database (with the NCBI accession numbers: SRR38251195 - SRR38251206).
IEC 4.1 cells were treated with IFN-γ (10 ng/mL, 6 h). Total RNAs were collected and processed for m6A methylated RNA immunoprecipitation sequence (MeRIP-seq). (A) IFN-γ stimulation resulted in significant alterations in m6A peaks of mRNAs in the transcriptome. Bar is a schematic representation of the number of mRNAs with either unchanged or altered m6A levels after IFN-γ treatment. Heat map depicts top 50 mRNAs with increased or decreased m6A peaks. (B) Pie chart depicting overall distribution of m6A methylation sites across genomic transcript regions. RefSeq-based coordinates from MeRIP-Seq were converted to genomic coordinates using biomaRt. ChIPseeker was used to assign m6A peaks to genomic regions. (C) Bar graph depicting gain and loss of m6A methylation sites within each transcript region, determined by fold change status. Genomic annotations were derived from ChIPseeker. (D) Motif enrichment analysis showing the top three sequence motifs associated with newly emerged and lost m6A peaks in IEC4.1 cells following IFN-γ stimulation. (E) Gene ontology (GO) analysis depicting biological processes of mRNAs with increased or decreased m6A peaks in IFN-γ-treated-IEC 4.1 cells. p values were calculated based on Kolmogorov-Smirnov test and adjusted by Benjamini-Hochberg method. Data from A-E were derived from three biological replicates for each group (untreated and IFN-γ treated).
https://doi.org/10.1371/journal.ppat.1014442.g001
Gene ontology analysis of mRNAs with altered m6A peaks identified a broad range of enriched pathways among both newly gained and lost m6A methylation sites. These include immune-related pathways, RNA splicing and translation, mitochondrion functions, and cell proliferation (Fig 1E and S3 Table). Immune-related genes include Gbp5, Zbp1, Irgm3, Gbp6, and Irgm2. Genes involved in RNA translation and splicing include Larp1, Pnn, Zfp36, Bcl3, and Zrsr2. Genes associated with mitochondrion functions include Slc35f6, Gper1, Mmp9, and Triap1. Cell proliferation-related genes included Batf, Batf3, Tgfa, Has2, and B4galt1 (Fig 1E and S3 Table).
We also took a portion of the mRNA isolated from untreated and IFN-γ-treated IEC4.1 cells, as described above, for whole genome RNA sequencing (RNA-Seq) analysis. Consistent with previous studies [43], numerous genes were found to be either upregulated or downregulated following IFN-γ stimulation (Fig 2A and S4 Table). The top 30 induced genes are shown in Fig 2A, and a complete list of differentially expressed genes is provided in S4 Table. Among the upregulated genes are immune-related genes (Gbp2, Irgm3, Irgm2, and Zbp1), stress-responsive genes (Psmb9 and H3c1), and metabolism-related genes (Ido2, and Parp14) (Fig 2B and S5 Table). All sequencing data were generated in accordance with MIAME guidelines and were deposited in the NCBI database (with the GEO accession numbers: GSE245591).
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Interestingly, comparison of genes with altered RNA expression and those with changes in m6A RNA methylation in IFN-γ-treated cells revealed only a small degree of overlap (Fig 2C and S6 Table). Following IFN-γ stimulation, only 8.2% of genes exhibited both upregulated expression and increased RNA m6A levels, while 1.5% showed both downregulated expression and decreased RNA m6A levels (Fig 2C and S6 Table). The majority of genes with altered RNA m6A levels did not display significant changes in expression following IFN-γ treatment. Representative overlapping genes include Irgm3, Irgm2, Gbp2b, Zbp1, and Batf2 (Fig 2C and S6 Table). Moreover, gene ontology analysis of the 553 genes with both altered expression and m6A methylation revealed board biological processes, including cell adhesion, metabolism, and immune responses (Fig 2D and S7 Table). The IFN-γ-induced increase in RNA m6A levels in selected genes, and its association with RNA expression, was further validated by quantitative PCR (qPCR) in IEC4.1 cells (Fig 2E) and in 2D mouse intestinal epithelial monolayers (Fig 2F).
RNA m6A methylation is primarily mediated by the Mettl3-Mettl14 complex in most eukaryotes [44]. We therefore hypothesized that Mettl3/14 physically associates with target mRNAs to mediate m6A methylation. To test this, we performed RIP-Seq using antibodies against Mettl3 and Mettl14 to identify RNAs interacting with these proteins in IEC4.1 cells following IFN-γ treatment. Differential enrichment was analyzed using edgeR, enabling statistical assessment in single-sample comparisons. Our analysis revealed that anti-Mett3 and anti-Mettl14 immunoprecipitation pulled down a similar subset of mRNAs from IFN-γ-treated cells (Fig 3A and S8 Table). Many of these mRNAs overlapped with these exhibiting increased m6A methylation following IFN-γ treatment (Fig 3B and S9 Table). The association of Mettl3/14 with selected IFN-γ-induced mRNAs with increased m6A methylation (e.g., Irgm3 and Irgm2) was further validated by RNA immunoprecipitation-qPCR (RIP-qPCR) analysis (Fig 3C).
IEC 4.1 cells were treated with IFN-γ (10 ng/mL, 4 h). Total RNAs were collected and processed for RIP-Seq using antibodies against Mettl3 an Mettl14. (A) Volcano plot illustrating differential mRNA profiles from RIP-seq with anti-Mettl3 or RIP-seq with anti-Mettl14 of IFN-γ treated-IEC 4.1 cells vs untreated cells. (B) Venn diagram depicting mRNAs associated with Mettl3 and with an altered m6A methylation level in cells following IFN-γ stimulation. Overlay of mRNAs represents these that were enriched with anti-Mettl3 and an increased m6A level, or with a decreased association with Mettl3 and a decreased m6A level. Data (in A and B) were derived from sequencing of a single biological replicate for each group (untreated and IFN-γ treated). (C) IFN-γ-induced association of Irgm2/3 mRNAs with the Mettl3/14 complex in IEC4.1 cells and 2D mouse intestinal epithelial monolayers by RIP-qPCR. IEC4.1 cells and 2D mouse intestinal epithelial monolayers were exposed to IFN-γ (10 ng/mL) for 4 h and total RNA was collected. Association of Irgm2 and Irgm3 mRNAs with the Mettl3/14 complex was assessed by RIP-qPCR with anti-Mettl3 or anti-Mettl14. Data are presented as the mean ± standard deviation from three independent experiments and analyzed by Student’s t test; * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001.
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RIP-Seq using antibodies against Mettl3 and Mettl14 revealed that many lncRNAs were pulled down from IFN-γ-treated cells (Fig 4A and S10 Table). LncRNAs are known to regulate a wide range of cellular functions [45]. RNA-Seq analysis of IEC4.1 cells following IFN-γ stimulation showed significant alterations in the lncRNA expression profile, including 59 upregulated and 41 downregulated lncRNAs (Fig 4C and S11 Table). The top 20 upregulated and downregulated lncRNAs are listed in S12 and S13 Tables. Notably, several lncRNAs identified in the immunoprecipitation with Mettl3 and Mettl14 antibodies were also upregulated in IFN-γ-treated cells, including Gm12216, Gm20599, Au020206, and 4933412E12Rik (Fig 4D and S12 Table). Their association with the Mettl3 protein was further validated by RIP-qPCR (Fig 4E).
(A) Volcano plot depicting lncRNAs associated with the Mettl3/14 complex in IFN-γ-treated IEC4.1 cells. Cells were exposed to IFN-γ (10 ng/mL) for 4 h and RNA was collected for RIP-seq with anti-Mettl3 or RIP-seq with anti-Mettl14. Data were derived from sequencing analysis of a single biological replicate for each group (untreated and IFN-γ treated). Differential expression statistics were computed using edgeR enabling statistical testing in single-sample comparisons. (B) A volcano plot showing lncRNA expression profiles in IEC4.1 cells following IFN-γ stimulation. Cells were treated with IFN-γ (10 ng/mL) for 4 h and total RNAs were collected for RNA-Seq. Data were derived from sequencing of three biological replicates for each group (untreated and IFN-γ treated). (C) Venn diagram depicting lncRNAs with an altered expression levels and associated with the Mettl3/14 complex in IEC4.1 cells following IFN-γ stimulation. Overlay of lncRNAs represents these that were induced with an enriched association with the Mettl3/14 complex, or that were suppressed with a decreased association with Mettl3/14, in cells following IFN-γ stimulation. (D) Table listing top 5 lncRNAs with an increased expression level and enriched with the Mettl3/14 complex in IEC4.1 cells following IFN-γ stimulation. (E) Validation of IFN-γ-induced enrichment of the top 5 lncRNAs with the Mettl3/14 complex in IEC4.1 cells by RIP-qPCR. Cells were exposed to IFN-γ (10 ng/mL) for 4 h and total RNA was collected. Association of selected lncRNAs with the Mettl3/14 complex was assessed by RIP-qPCR with anti-Mettl3 or anti-Mettl14. Several unrelated lncRNAs, including Dancr, Snhg4, and NR_038009, were used as negative controls. (F) IFN-γ-induced enrichment of Nostrill with the Mettl3/14 complex in IEC4.1 cells by RIP-qPCR. Cells were exposed to IFN-γ (10 ng/mL) for 4 h followed by RIP-qPCR for Nostrill enrichment using anti-Mettl3 or anti-Mettl14. U90926 and NR_045064 were used as negative controls. (G) Knockdown of Nostrill partially blocked IFN-γ-induced m 6A methylation of Irgm3 and Irgm2 mRNAs in IEC4.1 cells. Cells were transfected with si_Nostrill or si_Control (24 h), then treated with IFN-γ (10 ng/mL, 6h), followed by RIP-qPCR for Irgm3 and Irgm2 using anti-m6A. Data in (E, F, and G) are presented as the mean ± standard deviation from three independent experiments and analyzed by Student’s t test; * p < 0.05, ** p < 0.01, ***p < 0.001, **** p < 0.0001.
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We previously demonstrated that induction of the lncRNA Nostrill promotes the expression of Irgm3 and iNos in IECs in response to IFN-γ stimulation [43]. We next investigated whether Nostrill is specifically involved in Mettl3/14-mediated m6A methylation of Irgm2/3 following IFN-γ stimulation. RIP-qPCR using anti-Mettl3/14 antibodies confirmed the presence of Nostrill, but not the two unrelated control lncRNAs U90926 and NR_045064, in immunoprecipitates from IFN-γ–treated IEC4.1 cells (Fig 4F). Interestingly, knockdown of Nostrill using siRNA (siR_Nostrill) reduced IFN-γ-induced m6A methylation of Irgm2/3 (Fig 4G). Although Nostrill did not reach statistical significance in the Mettl3/Mettl14 RIP-Seq dataset, this may reflect the limited statistical power of single-sample comparisons and the possibility that not all associated lncRNAs were captured.
We next investigated the effects of inhibiting m6A methylation on IFN-γ-stimulated cell-intrinsic antimicrobial defense. To this end, we employed an infection model of IECs with Cryptosporidium, a protozoan parasite that infects the gastrointestinal epithelium and other mucosal surfaces in humans and is an important cause of diarrheal disease in young children and AIDS patients [46]. This parasite infects IECs and resides within a specialized intracellular but extracytoplasmic vacuole [47]. IFN-γ-stimulated intrinsic immunity represents a frontline host defense against Cryptosporidium infection [48]. Consistent with previous studies [43, 49], we found that treatment of IEC4.1 cells with IFN-γ enhanced cell-intrinsic defense against Cryptosporidium infection, as evidenced by a decreased infection burden in the IFN-γ-treated cells compared with untreated controls (Fig 5A). Knockout of Mettl3 in IEC4.1 cells (IEC4.1-Mettl3-/- cells) using a CRISPR/Cas9 approach (S2 Fig) partially impaired IFN-γ-mediated cell-intrinsic defense, as indicated by a higher infection burden in IFN-γ-treated IEC4.1-Mettl3-/- cells compared with IFN-γ-treated wild-type cells (Fig 5A and 5B). Consistent with our previous findings [43], treatment of IEC4.1 cells with siR_Nostrill partially inhibited IFN-γ-mediated anti-Cryptosporidium defense.
