Paneth cells, the main antimicrobial peptide producers in the small intestine, constitutively secrete a broad spectrum of bactericidal proteins including defensins, cryptdin-related sequence peptides, and Lysozyme 1 (Bevins and Salzman, 2011; Clevers and Bevins, 2013; Sato et al., 2011; Adolph et al., 2013). Beyond regulating the microbiota through the secretion of antimicrobial peptides, Paneth cells play crucial roles in regulating intestinal stem cells during steady-state and stress conditions caused by starvation, aging, tissue damage, or inflammation (Sato et al., 2011; Hodin et al., 2011; Pentinmikko et al., 2019; Jones et al., 2019; Sato et al., 2009; Vaishnava et al., 2008; Rodríguez-Colman et al., 2017).

Mechanistic target of rapamycin (mTOR) senses and responds to nutrients, growth factors, and stress (Saxton and Sabatini, 2017). Paneth cell-expressed mTOR regulates stem cell self-renewal, as is particularly evident in models of intestinal recovery and caloric restriction (Yilmaz et al., 2012; Igarashi and Guarente, 2016), emphasizing a non-Paneth cell-autonomous role for mTOR in intestinal homeostasis, whereas Paneth cell-intrinsic roles for mTOR are not well defined. Important modulators of Paneth cell functions and survival are proinflammatory cytokines, especially IFN-g (Raetz et al., 2013; Farin et al., 2014). We and others have previously observed that IFN-γ is a pleiotropic cytokine that plays a central role in the induction of severe intestinal inflammation characterized by the selective loss of intestinal Paneth cells (Raetz et al., 2013; Farin et al., 2014; Burger et al., 2018). These observations raise the questions of how IFN-γ mediates Paneth cell loss, whether this cytokine is capable of inducing Paneth cell death, and whether direct or indirect effects of IFN-γ are involved in these processes.

Cellular responses to IFN-γ are mediated by its heterodimeric cell-surface receptor (IFN-γR), which is widely expressed by epithelial and immune cells (Bach et al., 1997; O’Neill et al., 2016). To examine whether the direct effects of IFN-γ on Paneth cells are responsible for Paneth cell loss during acute gastrointestinal infection, mice with an intestinal pan-epithelial cell- or Paneth cell-restricted deficiency in IFN-γRII were generated by crossing Vil1-Cre or Defa4IRES-Cre (Paneth cell-specific, PC-Cre) mice, respectively (Burger et al., 2018), with Infgr2 ^flox/flox mice (Tcyganov et al., 2021) and orally infected with Toxoplasma gondii. While wild-type (WT) animals (Cre-negative Infgr2 ^flox/flox mice) lost practically all detectable Paneth cells, as was evident in a quantitative analysis of the representative Paneth cell-specific transcripts for the antimicrobial peptides lysozyme 1 (Lyz1), defensin alpha 23 (Defa23), and Defa24 (Figure 1A, Figure 1—source data 1) and histological quantification of the loss of these cells (Figure 1B and C). Epithelial or Paneth cell-restricted IFN-γRII deficiency largely prevented the loss of Paneth cells (Figure 1A,C, Figure 1—source data 1). An additional analysis of Paneth cells through immunofluorescence-based detection of Lyz1-expressing cells at the base of the intestinal crypts further supported the finding that selective deletion of IFN-γRII in Paneth cells prevented their elimination during T. gondii infection (Figure 1D, Figure 1—source data 1). These results revealed that the direct effects of IFN-γ on Paneth cells are responsible for the rapid loss of these cells during acute microbial infection.

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Analysis of Paneth cells in the small intestines of WT, E-Ifngr2 KO, and PC- Ifngr2 KO infected orally with Toxoplasma gondii.

Figure 1A: qRT-PCR analysis of relative Lyz1, Defa23, and Defa24 expression measured in the small intestines of WT, E-Ifngr2 KO, and PC- Ifngr2 KO infected orally with 20 cysts per mouse of the ME49 T. gondii on day 7 after infection. Figure 1C: Quantification of Paneth cells per crypt in naïve and infected WT, E- Ifngr2 KO, and PC- Ifngr2 KO mice with 20 cysts of ME49 T. gondii on day 7. Figure 1F: numbers of PI⁺ puncta per crypt in the small intestines of WT, E-Ifngr2 KO, and PC- Ifngr2 KO infected orally with 20 cysts per mouse of the ME49 T. gondii on day 7 after infection.

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To examine whether Paneth cell death or degranulation results in the apparent disappearance of these cells in response to T. gondii-induced IFN-γ, in vivo labeling with propidium iodide (PI), which binds to DNA in dying cells, was next applied. In naïve mice, PI positivity was largely restricted to dying enterocytes located at the top of the villi, and we did not detect PI-positive Paneth cells, while acute infection with the parasite resulted in the selective acquisition of PI positivity by Paneth cells (Figure 1E and F and Figure 1—figure supplement 1A). A similar conclusion was reached with genetically labeled Paneth cells that allowed integrity analysis in vivo (Figure 1—figure supplement 1B). We observed that both membrane and nuclear reporters of Paneth cells lost fluorescence signals during T. gondii infection, strongly indicating that the parasitic infection caused the loss of Paneth cells via induction of cell death (Figure 1—figure supplement 1B and data not shown).

IFN-γ-dependent loss of Paneth cells occurs independently of canonical cell death

To dissect the cell death pathway responsible for Paneth cell loss during T. gondii infection, we performed experiments in which the role of apoptosis, which is known to be involved in intestinal cell death, was investigated via a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and the expression of cleaved Caspase 3, an essential mediator of apoptosis was also evaluated. We observed that Paneth cells did not acquire TUNEL- or cleaved Caspase 3-positivity on any of the examined days post infection, ruling out apoptosis as the major mechanism of Paneth cell death (Figure 2), even though it was previously reported that in some rare cases IFN-γ is capable to activate caspase-3/7-dependent pathway of cell death in Paneth cells (Eriguchi et al., 2018).

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In addition, inhibition of apoptosis by caspase inhibitor zVAD failed to prevent Paneth cell death (Figure 2—figure supplement 1). Importantly, zVAD treatment of the intestinal organoids prevented the loss of the stem cell-specific transcript, signifying a role for apoptosis in stem cell death caused by IFN-γ, and suggesting that in contrast to the intestinal stem cells (Kretzschmar and Clevers, 2019; Takashima et al., 2019), Paneth cells undergo a different mechanism of death in response to this cytokine (Figure 2—figure supplement 1). Two additional cell death pathways that are associated with inflammatory responses were next investigated. During pyroptosis, intracellular activation of inflammasomes and Caspase 1 or 11 results in rapid formation of plasma membrane pores and cell lysis (Broz et al., 2020; Place and Kanneganti, 2018). In agreement with the zVAD treated mice, the absence of Caspases 1 and 11 failed to prevent the loss of Paneth cells, further confirming no role for these caspases in Paneth cell death. The absence of the additional key proteins involved in pyroptosis, including NLRP3 and ASC (encoded by Pycard) (Orning et al., 2019), failed to prevent the death of Paneth cells, eliminating pyroptosis as the mediator of Paneth cell death during T. gondii infection (Figure 2—figure supplement 2A and B).

Similarly, we ruled out a role for necroptosis, a cell death mechanism that depends on RIPK1 and RIPK3, in Paneth cell death downstream of IFN-γ via analysis of mice deficient in RIPK3 (Figure 2—figure supplement 2C and D) and animals treated with necrostatin (Figure 2—figure supplement 2E), a potent and well-characterized inhibitor of RIPK1 (Choi et al., 2019). We observed that inactivation of RIPK1 or RIPK3 had no detectable effect on Paneth cell loss during T. gondii infection (Figure 2—figure supplement 2C–2E). Taken together, these data revealed that IFN-γ-dependent death in Paneth cells was independent of apoptosis, pyroptosis, and necroptosis (Figure 2, Figure 2—figure supplement 1, Figure 2—figure supplement 2).

T. gondii infection leads to IFN-γ-mediated inhibition of mTORC1 activity in Paneth cells

Infections result in cytokine-mediated metabolic reprogramming, which has been primarily studied in immune cell populations (O’Neill et al., 2016) . Among several soluble mediators, IFN-γ is capable of triggering profound metabolic changes in multiple cell types, including epithelial cells (Nava et al., 2010). One of the key sensors in cell metabolism is the mTORC1 kinase complex, which plays a critical role in integrating metabolism with signal transduction (Condon and Sabatini, 2019). In addition, mTORC1 has been strongly implicated in controlling proper Paneth cell functions (Yilmaz et al., 2012; Barron et al., 2017; Sampson et al., 2016). Tracking the activity of mTOR measured by ribosomal S6 protein phosphorylation (pS6^{Ser 240/244}) in the intestinal crypts revealed high level of mTOR activity in Paneth cells when compared to stem cells in the PC-Cre x TdTomato x Lgr5-GFP double reporter mice that allowed simultaneous detection of these cell types (Figure 3—figure supplement 1). These observations prompted us to examine if IFN-γ leads to Paneth cell death via mTORC1 inactivation. We first analyzed the phosphorylation of the ribosomal S6 protein, a well-characterized marker of mTORC1 activity in mice infected with T. gondii and treated with anti-IFN-γ therapy throughout infection. We observed a rapid loss of ribosomal S6 protein phosphorylation in Paneth cells as early as day 3 post infection (Figure 3A), and this loss preceded the loss of Paneth cells detected by immunofluorescence staining (Figure 3A) or quantitative analysis of Paneth cell-specific transcripts (Figure 3B, Figure 3—source data 1 and data not shown). Successful blockade of IFN-γ in vivo with a neutralizing antibody (Figure 3A and C) prevented the loss of ribosomal S6 protein phosphorylation in Paneth cells, which were instead retained throughout the course of the experiments (Figure 3A and B). These results reveal that IFN-γ plays a role in modulating mTORC1 activity in Paneth cells in vivo. Additionally, Paneth cells lacking IFN-γRII retained ribosomal protein S6 phosphorylation during T. gondii infection (Figure 3D and E, Figure 3—source data 1), further suggesting that direct effects of IFN-γ on Paneth cells result in the loss of mTORC1 activity in this cell type. In addition, we observed a moderate increase in mTORC signaling in anti-IFN-γ treated animals or in mice lacking IFN-γRII in Paneth cells (Figure 3A). These observations suggest that basal IFN-γ, most likely driven by the intestinal microbiota (Benson et al., 2009), has a negative impact on mTOR activity.

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Toxoplasma gondii infection leads to IFN-γ-mediated inhibition of mTORC1 activity in Paneth cells.

Figure 3B qRT-PCR analysis for the relative Lyz1 expression in small intestines of naïve and T. gondii-infected mice on days 3, 5, and 7 post infection. Figure 3C: qRT-PCR analysis for the relative IFN-γ and Irgm1 expression in small intestines of naïve and T. gondii-infected mice on days 3, 5, and 7 post infection. Figure 3E Mean fluorescent intensity of p-Ser ^240/244 channel in individual intestinal sections of T. gondii-infected WT, E-ifngr2 KO, and PC-ifngr2 KO mice on day 7 post infection.

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To formally examine whether IFN-γ alone is capable of impacting mTORC1 activity in Paneth cells, naïve mice were injected with recombinant IFN-γ. This treatment resulted in levels of circulating IFN-γ comparable to those seen in T. gondii-infected mice (Figure 4A, Figure 4—source data 1) and triggered the induction of the representative IFN-stimulated genes, including Cxcl10, Ido1, and Irgm3, in the small intestine (Figure 4C). We observed that IFN-γ treatment alone triggered Paneth cell disappearance (Figure 4B, Figure 4—source data 1) and resulted in a rapid loss of S6 ribosomal protein phosphorylation that preceded Paneth cell loss (Figure 4D).

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IFN-γ is sufficient for inhibition of mTORC1 activity in Paneth cells.

Figure 4A: the serum levels of IFN-γ were analyzed by ELISA in comparison to Toxoplasma gondii-infected mice on day 7 post infection. Figure 4B qRT-PCR analysis for the relative Defa24 and Lyz1 expression in small intestines of mice treated with recombinant IFN-γ. Figure 4F: The frequency and absolute numbers of mTOR+ Paneth cells in control and IFN-γ treated mice.

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Finally, phosphorylation of the mTOR kinase itself was severely compromised in Paneth cells during IFN-γ treatment (Figure 4E,F, Figure 4—source data 1). Taken together, these experimental approaches reveal that IFN-γ alone is sufficient to elicit profound inhibition of mTORC1 activity in Paneth cells that precedes their loss in vivo.

Paneth cell preservation is dependent on mTORC1 signaling

We next formally examined the requirements for an intact Paneth cell-intrinsic mTORC1 signaling pathway in the survival of these cells. To achieve this, mice with targeted deletion of the mTORC1 activator Raptor (PC-Raptor KO mice) or the mTOR kinase (PC-Mtor KO mice) in Paneth cells were generated. We observed that a Paneth cell-restricted deficiency in Raptor or mTOR resulted in nearly complete disappearance of these cells in naïve mice (Figure 5). This was evident in immunofluorescence analysis of crypts in the small intestine, which showed that Paneth cells were detected in WT mice but not in PC-Mtor or PC-Raptor KO mice (Figure 5A). Similarly, unbiased analysis of Paneth cell-specific transcripts revealed that both mTOR and Raptor1 were required for the presence of Paneth cells and functioned in a cell-intrinsic manner for Paneth cell maintenance (Figure 5B, Figure 5—source data 1). In addition to the genetic models, we observed that treatment of mice with the mTORC1 inhibitor rapamycin resulted in the rapid loss of Paneth cells (Figure 5C), which was also evidenced by reduced expression of Paneth cell-specific transcripts (Figure 5D, Figure 5—source data 1). When combined, our experiments reveal that Paneth cell-intrinsic mTORC1 activity is essential for the presence of Paneth cells.

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Paneth cells require intrinsic mTORC1 signaling.

Figure 5B qRT-PCR analysis of Lyz1, Defa23, and Defa24 relative expression in the small intestines of WT mice and mice lacking Raptor or mTOR in Paneth cells. Figure 5D qRT-PCR analysis for relative Lyz1, Defa23, and Defa24 expression in the small intestines in WT mice treated with rapamycin daily for 24 hr (D1) or 72 hr (D3). WT, wild-type.

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IFN-γ-mediated mitochondrial damage leads to mTORC1 inhibition in Paneth cells

The kinase mTOR integrates inputs from growth factor receptors, nutrient availability, and mitochondrial function (Saxton and Sabatini, 2017). In an effort to investigate whether mitochondrial alterations contribute to Paneth cell mTORC1 inhibition in response to IFN-γ, we first analyzed mitochondrial integrity in Paneth cells in vivo during T. gondii infection. Flow cytometric analysis revealed that by day 5 post infection, when Paneth cell loss is starting to become noticeable (Raetz et al., 2013), the remaining Paneth cells while retaining MitoTracker positivity, which allows identification of all mitochondria independent of their membrane potential (Figure 6A and B, Figure 6—source data 1), were significantly compromised by day 5 post infection in an IFN-γ-dependent manner, as assessed by MitoStatus staining, which detects functionally polarized mitochondria (Figure 6A,C). These results were in the full agreement with the previous electron microscopy experiments that revealed extensive damage and vacuolization of the mitochondria in Paneth cells during T. gondii infection (Raetz et al., 2013).

Figure 6

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IFN-γ-mediated mitochondrial damage accompanies mTORC1 inhibition in Paneth cells.

Figure 6B Flow cytometric quantification of mitostatus and mitotracker positive Paneth cells in naïve or Toxoplasma gondii-infected PC-Cre x Rosa-YFP reporter mice on day 5 post infection. Figure 6E: MitoStatus MFI of the individual intestinal organoids treated with IFN-γ in vitro for 24 hr. Figure 6F: qRT-PCR analysis for relative Defa23 expression in organoids stimulated with IFN-γ (200 ng/ml) for 24 hr. Figure 6H: qRT-PCR analysis for relative Defa23 and Olfm4 expression in PC-Cre x TdTomato organoids stimulated with IFN-γ(200 ng/ml), Rapamycin (500 nM) or FCCP (100 µM) for 6 hr. MFI, mean fluorescence intensity.

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To further investigate the effects of IFN-γ on mitochondrial functions in Paneth cells, intestinal organoids were generated from WT mice and treated with recombinant IFN-γ. This in vitro system revealed that IFN-γ administration resulted in rapid mitochondrial depolarization, which was followed by the loss of Paneth cells (Figure 6D, F). These data suggest that IFN-γ is responsible for the compromised mitochondrial integrity in Paneth cells that leads to mTORC1 inactivation. To formally examine whether the mitochondrial changes downstream of IFN-γ are sufficient to trigger mTORC1 inhibition in Paneth cells, intestinal organoids were treated with FCCP, a potent uncoupler of oxidative phosphorylation in the mitochondria that disrupts ATP synthesis. We established that, similar to IFN-γ and rapamycin treatment, FCCP addition led to a rapid loss of ribosomal S6 phosphorylation (Figure 6G). These experiments revealed that disruption of mitochondrial function is sufficient to induce the loss of TORC1 activity and subsequent Paneth cell death (Figure 6G and H, Figure 6—source data 1). Importantly, rapamycin treatment of intestinal organoids did not result in loss of the inner mitochondrial membrane potential, indicating that mTORC1 functions downstream of mitochondrial functions in Paneth cells (Figure 6I).

Intestinal microbiota contributes to mTORC1 dysfunction during acute gastrointestinal infection

The intestinal microbiota has a profound effect on intestinal homeostasis (Hooper and Macpherson, 2010; Garrett et al., 2010; Belkaid and Harrison, 2017). We have previously observed that the presence of the microbiota is required for Paneth cell loss during acute infection with T. gondii (Raetz et al., 2013), even though the precise understanding of microbiota-mediated effects on Paneth cell death remains incomplete. To examine a potential role for the intestinal microbiota in modulating the IFN-γ-mediated inhibition of mTORC1 during acute T. gondii infection, we first analyzed mTOR activity in naïve microbiologically sterile (germ-free [GF]) mice. We observed that naïve germ-free mice exhibited Paneth cell mTOR activity similar to that in conventional (CONV) mice, as was evident from comparable staining for ribosomal protein S6 phosphorylation in the small intestine between CONV and GF mice (Figure 7, Figure 7—source data 1). Thus, the presence of the microbiota is not required for basal mTOR activity in Paneth cells. In contrast, while parasitic infection resulted in the rapid loss of Paneth cell mTOR activity in CONV mice, GF mice retained phosphorylated ribosomal protein S6 at all examined time points post infection (Figure 7B and C, and data not shown), strongly suggesting that the presence of the intestinal microbiota is required for the loss of mTOR activity in Paneth cells. As a result, Paneth cells were largely retained in GF mice, and only a mild loss of Paneth cells was observed in the GF mice compared with CONV mice (Figure 7).

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Microbiota is required for IFN-γ-mediated mTORC1 inhibition during Toxoplasma gondii infection.

Figure 7A: qRT-PCR analysis for relative expression of Lyz1, Defa23, and Defa24 measured in the small intestines of conventional (CONV) and germ-free (GF) C57Bl/6 mice infected orally with 20 cysts of the ME49 T. gondii (day 7 post infection). Figure 7C: MFI quantification of p-Ser^240/244 channel in individual intestinal sections of T. gondii-infected WT CONV and GF mice on day 7 post infection. MFI, mean fluorescence intensity; WT, wild-type.

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To test if microbiota-dependent loss of Paneth cells was due to the contribution of the intestinal bacteria to the potent IFN-γ responses caused by the parasitic infection (Figure 7—figure supplement 1A), GF mice were injected with the recombinant IFN-γ and Paneth cells were next analyzed in those mice. We observed that GF mice treated with rIFN-γ (Figure 7—figure supplement 1B) lost Paneth cell specific transcripts similarly to CV mice (Figure 7—figure supplement 1C), ruling out an intrinsic resistance of Paneth cells to IFN-γ in the absence of the intestinal bacteria. Thus, the reduced levels of IFN-γ seen in GF mice infected with T. gondii (Figure 7—figure supplement 1A) are responsible for the survival of Paneth cells in the absence of microbiota. Altogether, these data show that the microbiota is required for IFN-γ-mediated mTORC1 dysfunction and the subsequent death of Paneth cells during acute gastrointestinal infection.

IFN-γ is a pleotropic cytokine that is induced in response to all groups of pathogens including viruses, bacteria, and protozoan parasites (Schroder et al., 2004; Shtrichman and Samuel, 2001; Taylor et al., 2004). Much attention has been focused on IFN-γ induced host defense mechanisms; particularly in myeloid cells (Taylor et al., 2004; Shenoy et al., 2007; Yamamoto et al., 2012; Fabri et al., 2011; Boehm et al., 1997; Barrat et al., 2019). The major effects of IFN-γ include, but are not limited to, enhanced antigen processing and presentation (Matsuo et al., 2004; Büning et al., 2005; Gerosa et al., 2002), induction of antimicrobial peptides, and expression of immunity-related GTPases (IRGs) (Taylor et al., 2004; Boehm et al., 1998), and changes in immunometabolism that leads to the degradation of amino acids required by pathogens for intracellular growth (Yamamoto et al., 2012; Beatty et al., 1994; Dai et al., 1994). Surprisingly and despite an early realization that IFN-γ is also among the central mediators of the immunopathological responses (Panitch et al., 1987; Belnoue et al., 2008; Reinhardt et al., 2015), especially at the mucosal tissues (Obermeier et al., 1999; Heimesaat et al., 2006), relatively little is known regarding the pathological mechanisms mediated by this cytokine.

We and others have previously reported that IFN-γ is responsible for severe intestinal inflammation characterized by the rapid loss of Paneth cells, a highly specialized type of secretory epithelial cell located in the small intestine that produce large quantities of antimicrobial peptides including α-defensins and lysozyme (Raetz et al., 2013; Farin et al., 2014). Paneth cell-derived antimicrobial peptides regulate the composition of the microbiota and are critical for host defense against enteric pathogens (; Clevers and Bevins, 2013; Vaishnava et al., 2008). While we and others were able to demonstrate that IFN-γ is required for the loss of Paneth cells, whether the direct or indirect effects of this cytokine on Paneth cells causes their disappearance remained unknown. Furthermore, the precise knowledge of the mechanism leading to IFN-γ-mediated Paneth cell death is unknown. This is a significant gap as both experimental and clinical data strongly suggest a link between high IFN-γ levels, impaired Paneth cell function, and inflammatory intestinal disorders (Fais et al., 1991; Matsuoka et al., 2004; Krausgruber et al., 2016). Furthermore, a crucial role for IFN-γ in driving Paneth cell-dependent intestinal inflammation has been established in the mouse models with compromised homeostasis functions controlled by Paneth cell-restricted autophagy (Burger et al., 2018; Grizotte-Lake and Vaishnava, 2018; Matsuzawa-Ishimoto et al., 2017). Direct gene targeting in Paneth cells revealed that compromised cell-intrinsic autophagic machinery enhanced the susceptibility of Paneth cells to proinflammatory cytokines and caused an exaggerated or even lethal intestinal pathology (Burger et al., 2018). Thus, in order to understand the mechanism linking IFN-γ to intestinal inflammation, it is essential to understand how IFN-γ triggers the loss of Paneth cells.

In this work, our in vivo experimental data established a mTORC1-dependent mechanism of Paneth cell death in direct response to IFN-γ. The current work revealed that IFN-γ alone was sufficient for mitochondrial damage in Paneth cells. Although apoptosis, necrosis, and pyroptosis can be triggered by mitochondrial damage (Green, 2019), these mechanisms of cell death were not detected in Paneth cells. Furthermore, the possible roles for pyroptosis and necroptosis in IFN-γ-mediated Paneth cell death were ruled out by definitive mouse models deficient in those cell death pathways. Instead, we uncovered that a loss of mTORC1 activity downstream of IFN-γ signaling was responsible for Paneth cell death in vivo and in vitro. The results from our work revealed that impaired mitochondrial function caused by IFN-γ is at least in part responsible for mTOR inhibition. This process alone is sufficient to inhibit mTOR in Paneth cells, but cannot exclude other IFN-γ induced pathways that may play a role in mTOR inhibition.

Mouse genetic models with Paneth cell-specific targeting of mTOR and Raptor formally confirmed that intact mTORC1 holds an essential role in the presence of Paneth cells. Overall, our results here established that the direct effects of IFN-γ on Paneth cells caused impaired mitochondrial respiration in Paneth cells that was responsible for mTORC1 inactivation. Given the crucial role for IFN-γ, which is a cytokine frequently associated with the development of inflammatory bowel disease and compromised Paneth cell functions, the identified mechanisms of mTORC1-dependent Paneth cell death downstream of IFN-γ may provide promising novel approaches for treating intestinal inflammation.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Acceptance summary:

Araujo and colleagues report on the role of IFNγ in the inhibition of mTOR pathway and induction of Paneth cell death. The authors use multiple gene deficient mouse strains, in combination with Toxoplasma infection, to systematically define the Paneth-cell autonomous role of IFNγ and mTOR. The investigators also make use of a series of KOs for genes encoding key components of distinct cell death modalities, such as necroptosis and pyroptosis to indicate that none of the known pathways o cell death are involved in this novel finding. The genetic models are complemented with pharmacological approaches, such as inhibitors of cell death pathways or mTOR signaling and the use of intestinal organoids to further probe the mechanism leading to Paneth cell death. Taken together the data clearly demonstrate a role for IFNγ in promoting the death of intestinal Paneth cells via a mTOR dependent pathway.

Decision letter after peer review:

Thank you for submitting your article "IFN-γ mediates Paneth cell death via suppression of mTOR" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, including Nicola L Harris as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Wendy Garrett as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Gillian Coakley (Reviewer #2); Carla V Rothlin (Reviewer #4).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

Paneth cells produce antimicrobials and growth factors that protect and promote the small intestinal stem cell niche. The authors previously demonstrated that IFN-g produced during Toxoplasma gondii infection mediates loss of Paneth cells, a key event that affects intestinal barrier function in this and other disease models. In this study, the authors take advantage of a previously characterized aDefensin4-IRES-Cre mouse to make Paneth cell-specific KOs to investigate cell-intrinsic mechanisms. They demonstrate that IFN-g acts directly on Paneth cells during T. gondii infection and induces an atypical cell death. IFN-g was found to mediate this effect through mitochondrial damage that leads to mTOR inhibition. They also demonstrate that loss of mTOR activity in Paneth cells is dependent on the microbiota. Thus, by using T. gondii infection as a model to study immunopathology, the authors identify a new Paneth cell-intrinsic role for mTOR in preventing a type of cytokine-induced cell death.

The above results provide important insight into the toxic effect of IFN-g. The genetic techniques clearly establish mTOR within Paneth cells as an important target for this cytokine that has been implicated in many inflammatory settings.

Essential revisions:

A) Regarding the reporting of a novel method of cell death, named "interferonoptosis",

All reviewers agreed that the study falls short from identifying the type of cell death modality involved and that this is a major weakness. While the knowledge that neither apoptosis, necroptosis or pyroptosis appear to account for this type of death is relevant, the results included in this manuscript are not sufficient to coin a new type of cell death (tentatively named as interferonoptosis by the authors). The mechanism involved in inducing the Paneth cell death should be explored in more detail (see notes on further experimental work as outlined below). Alternatively, the authors may choose to perform a subset of these experiments in addition to substantiality 'toning down' their language' regarding the cell death pathway involved and providing a clear discussion of what conclusions can and cannot be drawn from the existing dataset.

– A further confirmation of the ability of IFNγ to directly signal to paneth cells and activated mTORC1 signaling should be provided by adding IFNγ to organoids from the reporter mice using the same assay as described in SFIg4.

– To substantiate their findings that IFN-mediated Paneth cell death is caspase-3 independent, it would be important to address the published findings by Eriguchi et al., (2017) – 10.1172/jci.insight.121886, which reports that "IFN-γ induces Paneth cell death through a caspase-3/7-dependent pathway", again using an enteroid-based system.

– Do the authors know whether they successfully inhibited RIPK1 following administration of necrostatin in Figure S3E? This is potentially of great interest because RIPK1 lies upstream of both apoptosis and necroptosis, and thus, this experiment provides additional support for their conclusion that the cell death modality represents something other than these two forms, which can interconvert. (If technically challenging in vivo, the authors may consider using the organoids to demonstrate a lack of a role for RIPK1).

– What leads to the inhibition of mTOR downstream of IFN γ receptor? This should be tested as multiple mechanisms can lead to inhibition of mTOR. For example, is this event downstream of AMPK, amino acid starvation? Does TSC have a role?

– The assessment of the pathway cannot be limited to measurements p-S6Ser240/244 and should include other markers such as 4E-BP1 phosphorylation. p-S6Ser240/244 staining appears inconsistent across panels. Please see Figure 4A and Figure S6B.

– Given the role of mTOR, mitochondrial damage and the vacuolization detected by EM, is it possible that Paneth cells undergo a type of autophagy-dependent cell death. This is particularly intriguing given the prior report from the same authors on the induction of autophagy by IFNγ in Paneth cells (Burger et al., CHM 2018). The relationship between these events should be explored. Is this dependent on ULK1, ATG13? Is mitophagy selectively being triggered? Ultrastructural changes are important in defining cell death and figure 5C lacks the inclusion of a non-infected control.

– How does IFN γ induced cell death compares to the cell death triggered by other cytokines, such as TNF?

B) Regarding the impact of the microbiota on mTORC and Paneth cells:

The authors also show that intestinal microbiota contributes to mTOR dysfunction and Paneth cell death. However the data shown in Figure S6D is not called out in the results and this should be corrected. Is the reason microbiota depletion rescues mTOR activity because the bacteria are necessary for IFN-g production during T. gondii infection? Please clarify this point in the manuscript. It should be noted that degranulation of Paneth cells is not stimulated in germ-free mice. Thus, Paneth cells might be more resilient to mTOR inhibition in germ-free mice. A more quantitative approach for the assessment of mTOR signaling in SPF versus WT mice across time, including earlier time points than 7 days post infection (see Figure S6) and additional markers of mTOR signaling is needed.

Essential revisions:

A) Regarding the reporting of a novel method of cell death, named "interferonoptosis",

All reviewers agreed that the study falls short from identifying the type of cell death modality involved and that this is a major weakness. While the knowledge that neither apoptosis, necroptosis or pyroptosis appear to account for this type of death is relevant, the results included in this manuscript are not sufficient to coin a new type of cell death (tentatively named as interferonoptosis by the authors). The mechanism involved in inducing the Paneth cell death should be explored in more detail (see notes on further experimental work as outlined below). Alternatively, the authors may choose to perform a subset of these experiments in addition to substantiality 'toning down' their language' regarding the cell death pathway involved and providing a clear discussion of what conclusions can and cannot be drawn from the existing dataset.

We fully agree with this criticism. We have now avoided the usage of ‘interferonoptosis’ entirely and instead use precise terminology such as “Paneth cell preservation is dependent on mTORC1 signaling”.

We also performed a subset of the requested experiments for clarification of several important issues raised by the referees in combination with the suggested ‘toning down’ of the claim regarding a novel mechanism of cell death.

– A further confirmation of the ability of IFNγ to directly signal to paneth cells and activated mTORC1 signaling should be provided by adding IFNγ to organoids from the reporter mice using the same assay as described in SFIg4.

The requested experiment was included in our original submission (Figure 5h, now Figure 6g).

As expected, IFN-γ largely eliminated Paneth cells and terminated mTOR signaling indirectly measured by the analysis of P-S6^Ser240/244 (Figure 6g)

– To substantiate their findings that IFN-mediated Paneth cell death is caspase-3 independent, it would be important to address the published findings by Eriguchi et al., (2017) – 10.1172/jci.insight.121886, which reports that "IFN-γ induces Paneth cell death through a caspase-3/7-dependent pathway", again using an enteroid-based system.

We would like to thank the reviewers and editors for this question. We now discuss this mechanism in our submission. Specifically, we state that:

“We observed that Paneth cells did not acquire TUNEL- or cleaved Caspase 3-positivity on any of the examined days post infection, ruling out apoptosis as the major mechanism of Paneth cell death (Figure 2), even though it was previously reported that in some rare cases IFN-γ is capable to activate caspase-3/7-dependent pathway of cell death in Paneth cells (19).”

– Do the authors know whether they successfully inhibited RIPK1 following administration of necrostatin in Figure S3E? This is potentially of great interest because RIPK1 lies upstream of both apoptosis and necroptosis, and thus, this experiment provides additional support for their conclusion that the cell death modality represents something other than these two forms, which can interconvert. (If technically challenging in vivo, the authors may consider using the organoids to demonstrate a lack of a role for RIPK1).

We would like to thank all the reviewers for this very valuable comment. We are confident that RIPK1 was successfully blocked based on the following experiments:

1. We have adopted a protocol for in vivo administration of necrostatin from Immunity 35, 908–918, December 23, 2011 (RIP Kinase-Dependent Necrosis Drives Lethal Systemic Inflammatory Response Syndrome). In agreement with the previous publication (Figure 5), necrostatin administration in vivo completely prevented TNF-induced mortality in mice. We have not included those technical experiments as the purpose of those experiments was to validate the commercial reagents and establish an experimental protocol in the lab.

While it is theoretically possible that we failed to completely inhibit RIPK1 in Paneth cells, we hope the reviewers would agree that even partial RIPK1 inhibition should prevent some Paneth cell death if this pathway plays a significant role in IFN-γ mediated Paneth cell death. However, we observed no effect of necrostatin on Paneth cell death.

2. We have attempted to analyze RIPK1 in sort-purified Paneth cells in response to T. gondii infection (on day 6 post infection prior a complete loss of Paneth cells). We have included the data for the reviewers’ benefit in Author response image 1. As evident from two independent experiments, while a potent STAT-1 phosphorylation was observed in sort-purified Paneth cells in response to T. gondii infection, we could not detect a phosphorylated form of RIPK1 in those cells. Please note that while this may look like a trivial experiment, it required multiple mice to purified sufficient numbers of Paneth cells (200,000) from T. gondii infected mice on day 6 post infection due to their substantial loss. We fully acknowledge that WB is a crude approach to analyze the kinase activity of RIPK1, and we hope that these additional data satisfies the reviewers’ technical concern as they revealed that Paneth cells responded to IFN-γ (phospho-STAT-1), but did not demonstrate potent or at least easily detectable RIPK1 activation.

Author response image 1

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– What leads to the inhibition of mTOR downstream of IFN γ receptor? This should be tested as multiple mechanisms can lead to inhibition of mTOR. For example, is this event downstream of AMPK, amino acid starvation? Does TSC have a role?

The results from our manuscript revealed that impaired mitochondrial function caused by IFN-γ is at least in part responsible for mTOR inhibition. This process alone is sufficient to inhibit mTOR in Paneth cells (now Figure 6G). We also fully acknowledge that other pathways may play a role in mTOR inhibition. Specifically, we state that:

“The results from our work revealed that impaired mitochondrial function caused by IFN-γ is at least in part responsible for mTOR inhibition. This process alone is sufficient to inhibit mTOR in Paneth cells, but cannot exclude other IFN-γ induced pathways that may play a role in mTOR inhibition”

We have analyzed several other possible pathways, especially IFN-γ induced amino acid starvation by analyzing iNOS knockout mice or by blocking Trp metabolism, but those experiments revealed that IFN-γ induced iNOS played no role in Paneth cell loss. We have not included those negative data as they would further dilute the main message of the manuscript (IFN-γ mediated mitochondrial dysfunction is responsible for mTOR inactivation and Paneth cell death).

We appreciate the suggestion regarding TSC as the TSC complex may mediate nutrient signaling upstream of mTOR to regulate Paneth cell functions and survival. We are in the process of generating Paneth cell specific TSC KO mice using Tsc2 flox mice (Jax #027458). These mice were cryopreserved, but we took the recommendation seriously and acquired those mice as we anticipate a significant Paneth cell dysfunction in the absence Tsc2 as TSC1/2 antagonizes the mTOR signaling pathway. We hope nevertheless that the reviewers would agree that this analysis is beyond the scope of this submission and may reveal ‘gain of function’ phenotype in Paneth cells.

– The assessment of the pathway cannot be limited to measurements p-S6Ser240/244 and should include other markers such as 4E-BP1 phosphorylation. p-S6Ser240/244 staining appears inconsistent across panels. Please see Figure 4A and Figure S6B.

We do not conclude that the mTOR pathway is involved in Paneth cells death based on the measurements of p-S6Ser^240/244. Instead, the major evidence comes from the genetic data (now Figure 5A) that formally confirmed that the mTOR pathway is essential for Paneth cells. We strongly believe that the genetic data fully support our claims. We would like to stress that p-S6Ser^240/244 is a common readout for mTOR activity and the measurements of p-S6Ser^240/244 were perform to analyze mTOR activity without any statement regarding the downstream events associated with this particular mTOR function in Paneth cells. In addition, we also analyzed phospho-mTOR Ser²⁴⁴⁸ (Figure 4e, 4f).

We respectfully disagree that there is an inconsistency across the panels shown in Figure 4A (now Figure 5A) and Figure S6B (now Figure 7B). Both panels revealed high intensity for p-S6 protein in Paneth cells detected in both conventional (CV) and germ-free (GF) mice. The staining pattern of p-S6 protein in epithelial cells is distinct between GF and CV mice; however, this difference is significant in that the variation is not an experimental inconsistency, but most likely due to a physiological response to microbiota. We hope the reviewers would agree that the analysis of microbiota-dependent mTOR activation in enterocytes (not Paneth cells) is beyond the scope of this submission, even though it is an interesting research direction.

If the reviewer pointed that there were some variations for UEA staining, we would like to clarify that UEA detects both Paneth cells and mucus-producing Goblet cells. We see no variations for the detection of Paneth cells via UEA staining. As for the mucus detection, it is fully expected that mucus staining varies across the mice as it is very sensitive to the fixation protocol.

– Given the role of mTOR, mitochondrial damage and the vacuolization detected by EM, is it possible that Paneth cells undergo a type of autophagy-dependent cell death. This is particularly intriguing given the prior report from the same authors on the induction of autophagy by IFNγ in Paneth cells (Burger et al., CHM 2018). The relationship between these events should be explored. Is this dependent on ULK1, ATG13? Is mitophagy selectively being triggered? Ultrastructural changes are important in defining cell death and figure 5C lacks the inclusion of a non-infected control.

We have previously extensively tested this hypothesis (Burger et al., CHM 2018) and in our previous publication we have specifically state that:

“We initially hypothesized that additional induction of IFN-γ during the mucosal response to T. gondii leads to excessive autophagy activation and unintentional Paneth cell elimination. Contrary to our hypothesis, the experimental analysis of T. gondii-infected PC-Atg5 KO and E-Atg5 KO mice revealed that elimination of Atg5 only in Paneth cells or in all enterocytes did not protect Paneth cells from IFN-γ-dependent cell death. Instead, we revealed that the lack of Atg5 resulted in profound intestinal immunopathology characterized by near complete elimination of all epithelial cells, including Paneth cells.”

We believe the previous results from my lab (Burger et al., CHM 2018) and an independent study from Kevin Maloy (Cell Host and Microbe, Volume 23, Issue 2, 14 February 2018) along with the detailed analysis of the above mentioned publications by Mayara Grizotte-Lake and Shipra Vaishnava (CHM, 2018) have already largely addressed the raised question.

We initially included the electron microscopy experiments (previous Figure 5C) as an additional illustration for the new data shown in Figure 5 (now Figure 6). As the detailed ultrastructural changes in Paneth cells during T. gondii infection has been previously characterized by our lab (Nature Immunology, 2013 Feb;14(2):136-42), we now refer to those results instead of republishing the repetitive EM data.

Specifically, we deleted Figure 5C and instead state that ‘These results were in the full agreement with the previous electron microscopy experiments that revealed extensive damage and vacuolization of the mitochondria in Paneth cells during T. gondii infection

– How does IFN γ induced cell death compares to the cell death triggered by other cytokines, such as TNF?

This is both a very interesting and independent question. As suggested by all reviewers and editors, we chose to tone down the language since only a subset of the experiments were performed. In this work we have addressed the question of how IFN-γ ‘kills’ Paneth cells. We were able to reveal that this is a direct and mTOR-dependent mechanism. It is not trivial to compare it with the TNF-mediated mechanism as while this pathway is much better studied in general, the correct experiments would require development of additional in vivo models to study the effects of TNF on Paneth cells only (Paneth cell specific TNFRI KO mice as well as PC-specific TNFRII KO mice).

B) Regarding the impact of the microbiota on mTORC and Paneth cells:

The authors also show that intestinal microbiota contributes to mTOR dysfunction and Paneth cell death. However the data shown in Figure S6D is not called out in the results and this should be corrected. Is the reason microbiota depletion rescues mTOR activity because the bacteria are necessary for IFN-g production during T. gondii infection? Please clarify this point in the manuscript. It should be noted that degranulation of Paneth cells is not stimulated in germ-free mice. Thus, Paneth cells might be more resilient to mTOR inhibition in germ-free mice. A more quantitative approach for the assessment of mTOR signaling in SPF versus WT mice across time, including earlier time points than 7 days post infection (see Figure S6) and additional markers of mTOR signaling is needed.

We apologize for not being clear in our message. We only claim that the reduced levels of IFN-γ seen in GF mice (now Figure 7 – supplement 1) are responsible for the survival of Paneth cells in the absence of microbiota (Figure 7). These results are consistent with our previous publications (Cell Host Microbe. 2009; 6 187-196) in which we revealed that microbiota boosts IFN-γ during T. gondii infection.

We have now performed an additional experiment of injection of rIFN-γ in GF mice. We observed that GF mice treated with rIFN-γ loose PC similarly to CV mice (Figure 7 – supplement 1C). We hope that this addressed the reviewer’s concern and we again apologize for not delivering this simple message in our original submission.

Author details

1. Alessandra Araujo

   Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7568-074X

2. Alexandra Safronova

   Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, United States

   Contribution

   Conceptualization, Data curation, Investigation, Methodology, Software, Validation

   Competing interests

   No competing interests declared

3. Elise Burger

   Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Validation

   Competing interests

   No competing interests declared

4. Américo López-Yglesias

   Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6797-2179

5. Shilpi Giri

   Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, United States

   Contribution

   Dr. Giri contributed to Fig 7 of the revised manuscript. All authors agreed with Dr. Giri inclusion and place in the author list, Investigation, Methodology

   Competing interests

   No competing interests declared

6. Ellie T Camanzo

   Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

7. Andrew T Martin

   Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

8. Sergei Grivennikov

   1. Department of Medicine and Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, United States
   2. Cancer Prevention and Control Program, Fox Chase Cancer Center, Philadelphia, United States

   Contribution

   Methodology, Resources

   Competing interests

   No competing interests declared

9. Felix Yarovinsky

   Center for Vaccine Biology and Immunology, Department of Microbiology and Immunology, University of Rochester Medical Center, New York, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing - original draft, Writing - review and editing

   For correspondence

   felix_yarovinsky@URMC.Rochester.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-5825-8002

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