Cells are sensitive to changes in their environment. For example, maintaining normal salt levels in the blood, also called tonicity, is essential for the health of individual cells and the organism as a whole. Tonicity controls the movement of water in and out of the cell: high levels of salt inside the cell draw water in, while high levels of salt outside the cell draw water out. If salt levels in the environment surrounding the cells become too high, too much water will be drawn out, causing the cells to shrink.

Changes in tonicity can cause the cell to become stressed. Initially, cells adapt to this stress by switching on sets of genes that help restore fluid balance and allow the cell to regain its normal shape and size. If the increase in tonicity exceeds tolerable stress levels and harms the cell, this initiates an inflammatory response which ultimately leads to cell death. However, it remained unclear how cells switch from adapting to responding with inflammation.

Now, Farabaugh et al. have used an experimental system which mimics high salt to identify the mechanism that allows cells to switch between these two responses. The experiments showed that when salt levels are too high, cells switch on a stress sensing protein called PACT, which activates another protein called PKR. When PACT was deleted from mouse cells, this led to a decrease in the activity of inflammatory genes, and prevented the cells from self-destructing.

Other proteins that are involved in the adaptive and inflammatory response are the NF-κB family of proteins and TonEBP. Farabaugh et al. found that under low intensity stress, when salt levels outside the cell are slightly too high, a family member of NF-κB works with TonEBP to switch on adaptive genes. But, if salt levels continue to rise, PACT activates and turns on PKR. This blocks the interaction between NF-κB and TonEBP, allowing another family member of NF-κB to interact with TonEBP instead. This switches the adaptive response off and the inflammatory response on.

There are many diseases that involve changes in tonicity, including diabetes, cancer, inflammatory bowel disease, and dry eye syndrome. Understanding the proteins involved in the adaptive and inflammatory response could lead to the development of drugs that help to protect cells from stress-induced damage.

The cellular response to increased extracellular osmolytes is a tightly regulated fundamental process that dictates the cell fate (Burg et al., 2007). Multiple signaling pathways are involved in orchestrating the hyperosmotic stress response, with the ultimate goal of maintaining a constant cell volume. Although the initial sensor of hyperosmolarity is unknown in mammalian cells, adaptation to hyperosmotic stress has been shown to be dependent on induction of genes by the transcription factor Tonicity Enhancer Binding Protein (TonEBP) (Burg et al., 2007). TonEBP translocates to the nucleus in response to an increase in osmolarity to regulate transcription of target genes associated with osmoadaptation (Miyakawa et al., 1999). These include chaperone proteins (heat shock protein 70 [HSP70, Hspa1a]; Woo et al., 2002), enzymes involved in biosynthesis of neutral osmolytes (aldose reductase [AR, Akr1b1]; Ferraris et al., 1994), and membrane proteins that transport organic osmolytes such as amino acids proline and alanine across the plasma membrane (sodium-coupled neutral amino acid transporter [SNAT2, Slc38a2]; Trama et al., 2002). The SNAT2-mediated arm of the adaptive response is also dependent on the membrane trafficking function of PP1/GADD34 (Krokowski et al., 2017).

When the degree of hyperosmotic stress becomes too high to overcome, the adaptive response fails. The failure to adapt will lead to an increase in inflammatory signaling, followed by apoptosis (Brocker et al., 2012). We have previously determined in MEFs that extracellular osmolarities of 500 and 600 mOsm are representative of low stress intensity/adaptive response and high stress intensity/non-adaptive response, respectively (Farabaugh et al., 2017). Inflammation is known to be regulated by many signaling pathways, including but not limited to NF-κB signaling (Liu et al., 2017). An increase in hyperosmolarity is associated with an increase in NF-κB activation (Németh et al., 2002) and inflammation (Müller et al., 2019), as well as production and release of multiple proinflammatory cytokines, including IL8 (10), TNFα (Lang et al., 2002), IL1β (Ip and Medzhitov, 2015), and IL6 (Igarashi et al., 2014). Apoptosis occurs following exposure to hyperosmolarity beyond which adaptation is possible, the limit of which can vary between cell types but is sharply defined within each (Burg et al., 2007). Apoptosis following hyperosmotic stress is characterized by depolarization of mitochondria leading to an increase in the Bax/Bcl-2 ratio (Michea et al., 2002), as well as sometimes by clustering and internalization of TNFα receptors (Rosette and Karin, 1996). This potentially contributes to a number of human diseases, such as dry eye syndrome (Lemp et al., 2011), diabetes (Stookey et al., 2004), and inflammatory bowel disease (Schwartz et al., 2009).

We have previously shown that NF-κB-dependent signaling is pro-apoptotic in high-intensity hyperosmotic stress, in part via induction of inducible nitric oxide synthetase (iNOS) (Farabaugh et al., 2017). The NF-κB transcription factor family consists of five subunits, three of which have transactivation activity (RelA/p65, Rel/c-Rel, and RelB) (www.bu.edu/nf-kb). These subunits are extensively post-translationally modified and can homodimerize or heterodimerize to form unique complexes that fine-tune different transcriptional programs (Oeckinghaus and Ghosh, 2009). NF-κB p65 has been previously shown to play a role in both hyperosmotic stress and inflammation (Liu et al., 2017; Németh et al., 2002). NF-κB p65 is phosphorylated and accumulates in the nucleus in hyperosmotic conditions, and induces the transcription of a number of proinflammatory target genes, such as Nos2 (Jia et al., 2013), Il6 (Farabaugh et al., 2017), and Tnfa (Iwata et al., 1999), as well as caspase 3/9 activation (Eisner et al., 2006). NF-κB p65 is necessary for initiation of the inflammatory response in response to LPS (McDonald et al., 1997; Hobbs et al., 2018), for the development of Th17 cells via induction of the transcription factor RORγ (Ruan et al., 2011a), and for pathology in many diseases with inflammatory components, including inflammatory bowel disease (Han et al., 2018; Schreiber et al., 1998) and dry eye syndrome (Tan et al., 2018; He et al., 2011). Although deletion of NF-κB p65 is embryonically lethal in mice, simultaneous deletion of Tnfa leads to survival, and demonstrates that NF-κB p65 plays a role in the prevention of TNF-induced toxicity during development (Doi et al., 1999). The role of NF-κB c-Rel has not been examined in hyperosmotic stress, though it has been shown to have both proinflammatory (de Jesús and Ramakrishnan, 2020; Liu et al., 2017) and developmental (Gilmore and Gerondakis, 2011) functions. c-Rel is required for transcription of many genes necessary for immune system function, including Irf4, which promotes proliferation of B cells (Grumont and Gerondakis, 2000), Bcl2l1 and Bcl2A1, which promote survival of B cells (Owyang et al., 2001), and Il21, which promotes development of Th17 cells (Chen et al., 2010). c-Rel-deficiency leads to altered phenotypes in many disease models: c-Rel KO mice are resistant to EAE, an autoimmune model of multiple sclerosis driven by Th17 cells (Chen et al., 2011); c-Rel KO mice fail to develop severe colitis induced by treatment with dextran sodium sulfate (DSS)(Luu et al., 2017), perhaps also as a result of loss of Th17 cell development (Chen et al., 2011); and overexpression of c-Rel has been shown to protect human islets from cell death mediated by the cell death protein PDCD4 via an increase in microRNA-21 expression (Ruan et al., 2011b).

We have shown that protein kinase R (PKR) promotes the nuclear localization of Serine-536 phosphorylated NF-κB family member p65, which leads to an increase in inflammatory gene induction and cell death (Farabaugh et al., 2017). PKR is activated under hyperosmotic stress (Farabaugh et al., 2017), and it has also been shown previously to regulate NF-κB signaling in inflammatory conditions, via the direct phosphorylation of the inhibitor of κB (IκB; Kumar et al., 1994) or interaction with the inhibitor of κB kinase (IKK; Ishii et al., 2001). PKR activation and downstream NF-κB signaling were also evident after high intensity hyperosmotic treatment with dextran sodium sulfate (DSS, 10%) in cell culture (Farabaugh et al., 2017). In contrast, we observed adaptation to low intensity hyperosmotic treatment (5% DSS)(Farabaugh et al., 2017). PKR consists of two repeated double-stranded RNA (dsRNA)-binding motifs, DRBM I and DRBM II, and a C-terminal kinase domain (Clemens and Elia, 1997). PKR phosphorylates Ser-51 of the eukaryotic translation initiation factor 2 α subunit (eIF2α), contributing to inhibition of global mRNA translation (Saelens et al., 2001; Bevilacqua et al., 2010). Although PKR is classically activated by the binding of viral double-stranded RNA (dsRNA) to the dsRNA-binding domain, we have shown that the binding of dsRNA is unnecessary for PKR activation and downstream eIF2α phosphorylation in hyperosmotic conditions (Farabaugh et al., 2017).

Another potential mechanism of PKR activation is via protein–protein binding of PKR and the PKR-activating protein (PACT)(Patel and Sen, 1998). PACT is a dsRNA-binding protein that can bind PKR at several domains including the two dsRNA-binding domains and the N-terminal portion of the kinase domain (Peters et al., 2001). PACT interaction with the kinase domain of PKR, inhibits its closed conformation and promotes an open, active conformation (Li et al., 2006). In addition to constitutive phosphorylations at Ser-18 and Ser-246, PACT can become additionally phosphorylated at Ser-287 in stress conditions such as arsenite treatment and oxidative stress, which leads to increased association with PKR and PKR activation (Peters et al., 2006). The involvement of PACT in hyperosmotic stress conditions has not been previously investigated.

Here, we show that hyperosmotic stress induces PACT-mediated PKR activation, which disrupts the association of NF-κB c-Rel with other Rel homology domain (RHD)-containing binding partners NF-κB p65 and TonEBP. The loss of NF-κB c-Rel/NF-κB p65 and NF-κB c-Rel/TonEBP heterodimers in hyperosmotic conditions leads to a reduction in osmoadaptive and pro-survival gene transcription and enhances TonEBP/NF-κB p65-dependent proinflammatory signaling (Figure 1A). We also show that differential gene expression programs based on hyperosmotic stress intensity persist across multiple cell types and treatments with various osmolytes, the results of which equate to a universal switch from adaptation to increased inflammatory signaling.

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In this study, we have shown that PACT-PKR interaction is necessary for PKR activation and the expression of inflammatory genes and cell death under hyperosmotic conditions. Our findings suggest a mechanism in which PACT-PKR signaling promotes proinflammatory gene expression in a stress intensity-dependent manner, via the modulation of interactions among members of the Rel-homology-domain family of transcription factors. Specifically, we show that in low intensity hyperosmotic stress, NF-κB subunit c-Rel forms complexes with Rel-homology domain transcription factor TonEBP. This interaction preserves adaptive signaling at low-intensity stress via the induced transcription of a key factor in the adaptive response, SNAT2, and its positive regulator, GADD34. The loss of these complexes at high-intensity stress may lead to increased TonEBP interaction with NF-κB p65 and subsequent increased expression of proinflammatory genes such as IL6 and Tnfrsf9. The conservation of this loss of adaptive (Slc38a2, Ppp1r15a) gene expression indicates conserved expression programs in response to hyperosmotic stress.

The mechanism of PKR activation by PACT is only partially understood. PACT is known to bind to PKR via the N-terminal dsRNA binding domains and the C-terminal kinase domain, though only binding of the kinase domain is necessary for subsequent PKR phosphorylation at Thr-451 and activation (Peters et al., 2001). Phosphorylation of PACT at Ser-246 and Ser-287 has been demonstrated to occur constitutively and in response to various cellular stresses such as thapsigargin, hydrogen peroxide, and sodium arsenite treatment, respectively (Ito et al., 1999). We show here that the same sites are likely phosphorylated under hyperosmotic conditions, and are necessary for the activation of PKR. PKR activation via PACT was previously reported in conditions of overexpression of PACT protein, treatment with actinomycin D, growth factor withdrawal (Marques et al., 2008), and endoplasmic reticulum (ER) stress (Singh et al., 2009). This PACT-mediated activation of PKR in ER stress has been shown to be required for apoptosis after tunicamycin treatment (Singh et al., 2009); similarly, PACT phosphorylation at Ser-287 was increased, and association with binding partner TRBP was decreased (Chukwurah and Patel, 2018), hinting at an additional mechanism of regulation. To our knowledge this is the first report of PACT-mediated PKR activation in hyperosmotic stress conditions. It is shown here that signaling along the PACT-PKR axis has pro-apoptotic functions during high-intensity hyperosmotic stress. Our findings are in agreement with a human disease state of dystonia, in which an identified PACT mutation was shown to increase association with PKR, correlating with increased apoptosis in patient-derived lymphoblasts (Vaughn et al., 2015).

We show here that PKR activation affects the nuclear accumulation of Rel homology domain transcription factors. The exact nature of this mechanism is still unknown. PKR has been shown to directly phosphorylate the inhibitor of κB protein IκBα in vitro and in vivo (Kumar et al., 1994), although a loss of PKR does not affect hyperosmotic stress-induced phosphorylation of p65 at Ser-536 (Farabaugh et al., 2017). Additionally, the exact mechanism by which PKR signaling affects specific NF-κB dimers in response to hyperosmotic stress remains unclear. It is possible that a subset of NF-κB complexes in the cytoplasm are affected by PACT-PKR signaling via additional interacting or intermediary proteins, and that this subset translocates to the nucleus in response to hyperosmotic stress response. The identification of these complexes and intermediaries will remain for future studies.

PACT when not bound to PKR appears to have some effect on the TonEBP-dependent osmoadaptive gene expression program. Although SNAT2 mRNA is still induced at low stress intensity in PACT-deficient cells, it is induced to a lesser degree (Figure 1E), indicating that PACT may be important for osmoadaptation. The mechanisms of the osmoadaptive action of PACT independent of PKR are not known. Because other osmoadaptive mRNAs, such as Slc6a6 and Akr1b1 also show reduced induction in PACT-deficient MEFs compared to wild-type MEFs (Figure 1—figure supplement 2A), it is suggested that PACT depletion may indirectly inhibit the TonEBP-dependent osmoadaptive program. In agreement with the PACT/PKR signaling inhibiting osmoadaptive gene expression, we also found increased-half lives of Slc38a2 and Ppp1r15a mRNAs during osmoadaptation in PACT-deficient cells (Figure 1—figure supplement 2B). Similar data were obtained in PKR deficient cells (data not shown). In contrast to the regulation of the half-life of Slc38a2 and Ppp1r15a mRNAs by PACT availability, the half-life of the Akr1b1 mRNA was not altered (Figure 1—figure supplement 2B). These data suggest that some undetectable levels of PACT-mediated PKR activation plays a role in reducing mRNA stability of the two osmoadaptive genes, potentially via interaction with the PACT-binding partner TRBP (Chukwurah and Patel, 2018).

We have shown that the nuclear accumulation of TonEBP is not sufficient to induce transcription of certain osmoadaptive genes in hyperosmotic stress conditions. We show that TonEBP directly forms a complex with NF-κB c-Rel, and that transcription of the osmoadaptive genes Slc38a2 and Ppp1r15a is dependent on NF-κB family transcription factor c-Rel. As both expression of SNAT2 and the NF-κB c-Rel/TonEBP complex are present at low-intensity stress and lost at high-intensity stress, it follows that Slc38a2 transcription may result from binding of the NF-κB c-Rel/TonEBP complex to the Slc38a2 promoter. Still outstanding is definitive proof that TonEBP/NF-κB c-Rel complex nuclear accumulation promotes DNA binding and increased transcription, as these aspects can be regulated separately via IκB degradation and re-synthesis (O'Dea and Hoffmann, 2010). It is possible that localized DNA characteristics dictate the binding of NF-κB c-Rel/TonEBP complexes to specific gene promoters (Samee et al., 2019), or that activation of transcription of these genes is dependent on unique interacting transcription-enhancing proteins rather than DNA binding (Lee et al., 2016). In-depth analysis of the binding sites of Rel-homology domain transcription factor heterodimers will need to be performed on genes responsive to NF-κB c-Rel/TonEBP complexes to determine what differences may exist between these and classical tonicity-responsive enhancer sites (TonE sites) known to respond to TonEBP homodimers (Stroud et al., 2002).

TonEBP target genes that have been described in the literature include those necessary for osmoadaptation, such as Slc38a2, Akr1b1, and Slc6a6, as well as several that are involved in inflammation, such as Nos2 (Lee et al., 2016), Ccl2 and Ptgs2 (Buxadé et al., 2012). The combination of TonEBP and NF-κB p65 has been shown to induce transcription of only these inflammatory genes, and not osmoadaptive TonEBP targets (Johnson et al., 2017). Nos2 gene expression was shown to be dependent on the presence of NF-κB p65 (Farabaugh et al., 2017), and hyperamplification of its expression correlated with increased levels of nuclear NF-κB p65, as both were increased upon loss of NF-κB c-Rel. This may indicate that controlling the availability of p65 is a critical factor in the amplification of proinflammatory signaling during hyperosmotic stress. Although iNOS was not regulated by TonEBP in response to hyperosmotic stress, other proinflammatory NF-κB targets were induced, including Il6 and Tnfrsf9 (Figure 5D). It has been shown that while Nos2 induction is dependent on STAT1 and IRF1 signaling in response to cytokine stimulation, IL6 secretion is independent of this signaling pathway in mouse fibroblasts (Samardzic et al., 2001); it is likely that elements in the promoters of these genes exist that lead to differential expression in unique cellular contexts dependent on cell type or stress stimulant.

In this report, we identified the existence of NF-κB c-Rel in complexes with either TonEBP or NF-κB p65 in MEFs. Fibroblasts in particular can be an appropriate model system because not only are they present in all tissues, but they can act as sentinels that can produce inflammatory factors such as Toll-like receptors and cytokines in response to environmental stresses, such as in defense against microbial invasion (Bautista-Hernández et al., 2017). This study shows that subsets of genes that share functionality in osmoadaptation and proinflammatory signaling can be targets of specific Rel-homology domain complexes in MEFs, such as TonEBP/NF-κB c-Rel or TonEBP/NF-κB p65, respectively. These complexes were disrupted under high intensity stress conditions in a PACT/PKR-mediated manner. At this high intensity of stress, osmoadaptive genes were not induced, suggesting a duality of TonEBP function. Our data suggest that this duality of function is mediated via formation of complexes with partners, NF-κB p65 or NF-κB c-Rel. It seems plausible that in osmoadaptive conditions, competition between NF-κB c-Rel and NF-κB p65 binding to TonEBP can act as a rheostat between NF-κB c-Rel-mediated osmoadaptive signaling and NF-κB p65-mediated proinflammatory signaling. The existing literature shows anecdotal evidence of NF-κB subunits serving different functions in various cellular contexts: NF-κB c-Rel can have opposing functions to NF-κB p65 in neurons, where NF-κB c-Rel promotes neuronal survival while NF-κB p65 promotes neuronal cell death (Pizzi et al., 2002); or c-Rel repressing p65-mediated expression of HIV protein LTR and IL-2Rα (Doerre et al., 1993); or c-Rel-driven TRAIL-induced apoptosis rescued by overexpression of p65 (Chen et al., 2003). NF-κB subunits can also serve redundant functions; the loss of some individual NF-κB family members can be compensated for by other NF-κB family members, evidenced by deficiency of peripheral lymphoid cells as a result of loss of pro-survival proteins A1 and Bcl-2 in NF-κB p65/NF-κB c-Rel/TNF knockout mice, which is not present in either NF-κB p65/TNF or NF-κB c-Rel knockout mice (Grossmann et al., 2000). This is also consistent with genes being targets of specific NF-κB family members only in certain cell types, such as HEK cells having predominantly NF-κB p65/p50 dimers while NF-κB c-Rel is functionally negligible (O'Dea and Hoffmann, 2010), or B cells, in which NF-κB c-Rel/p50 is the predominant complex (Sen, 2006). The function of these NF-κB c-Rel/TonEBP complexes identified here need to be examined in specific cell types and conditions to be able to directly compare their function with that of other NF-κB complexes.

We have shown here that in high-intensity hyperosmotic stress conditions, osmoadaptation is terminated. However, KEGG pathway analysis of our RNA-seq data in high-intensity stress showed that the most significantly upregulated pathway at high-intensity stress is associated with the ribosome (Figure 4—figure supplement 1B), specifically genes that encode ribosomal proteins. It is possible that this increase in transcription is an attempt to recover translation capacity from the sustained reduction in protein synthesis associated with continued stress (Bevilacqua et al., 2010). This trend is consistent with the hypothesis that mRNAs transcribed upon high-intensity hyperosmotic stress exposure will be translated upon recovery of cap-dependent protein synthesis upon recovery to iso-osmolarity. Future studies will determine if the cellular response to severe stress involves alternative adaptation mechanisms to return to iso-osmolar conditions.

The novel PACT-PKR signaling pathway expanded upon here will aid our understanding of potential mechanisms involved in hyperactivation of proinflammatory conditions. It is important to note that the influence of PACT-mediated PKR activation on proinflammatory gene expression is only observed in high-intensity stress. It remains to be determined whether drugs that inhibit PKR activation, including C16 (Xiao et al., 2016), or compounds that disrupt PACT/PKR-mediated signaling, such as luteolin (Dabo et al., 2017), will be effective in treating diseases with inflammatory components, such as IBD (Schwartz et al., 2009), or diseases with a hyperosmotic component, such as dry eye syndrome (Lemp et al., 2011). This signaling pathway may also be relevant in certain cancers, such as breast cancer and glioblastoma; inhibition of PKR has been proposed to sensitize Trastuzumab-resistant HER2+ breast cancer to increase efficacy of Trastuzumab in preventing tumorigenesis (Darini et al., 2019), and inhibition of NF-κB signaling or iNOS inhibition have been demonstrated to slow growth of glioblastoma stem cells (Eyler et al., 2011; Friedmann-Morvinski et al., 2016). Proliferation and function of other stem cells, such as hematopoietic stem/progenitor cells and mesenchymal stem cells, are also regulated by PKR activation and inflammatory conditions (Crop et al., 2010; Liu et al., 2013), and may be relevant model systems to further the exploration of the PACT-PKR signaling pathway.

Sequencing data have been deposited in GEO under accession code GSE138692.

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Author details

1. Kenneth T Farabaugh

   Department of Pharmacology, Case Western Reserve University, Cleveland, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9591-0466

2. Dawid Krokowski

   1. Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States
   2. Department of Molecular Biology, Maria Curie-Sklodowska University, Lublin, Poland

   Contribution

   Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Bo-Jhih Guan

   Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

   Contribution

   Validation, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Zhaofeng Gao

   Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

   Contribution

   Conceptualization, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Project administration

   Competing interests

   No competing interests declared

5. Xing-Huang Gao

   Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

   Contribution

   Data curation, Software, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0720-3690

6. Jing Wu

   Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

   Contribution

   Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

7. Raul Jobava

   Department of Biochemistry, Case Western Reserve University, Cleveland, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration

   Competing interests

   No competing interests declared

8. Greeshma Ray

   Department of Inflammation and Immunity, Cleveland Clinic Foundation, Cleveland, United States

   Contribution

   Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration

   Competing interests

   No competing interests declared

9. Tristan J de Jesus

   Department of Pathology, Case Western Reserve University, Cleveland, United States

   Contribution

   Conceptualization, Resources, Data curation, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration

   Competing interests

   No competing interests declared

10. Massimiliano G Bianchi

    Department of Medicine and Surgery, Universita degli Studi di Parma, Parma, Italy

    Contribution

    Data curation, Formal analysis, Investigation, Methodology

    Competing interests

    No competing interests declared

11. Evelyn Chukwurah

    Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

    Contribution

    Data curation

    Competing interests

    No competing interests declared

12. Ovidio Bussolati

    Department of Medicine and Surgery, Universita degli Studi di Parma, Parma, Italy

    Contribution

    Data curation, Formal analysis, Investigation, Methodology

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-4301-2939

13. Michael Kilberg

    Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, United States

    Contribution

    Resources

    Competing interests

    No competing interests declared

14. David A Buchner

    1. Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States
    2. Department of Biochemistry, Case Western Reserve University, Cleveland, United States

    Contribution

    Data curation, Formal analysis

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-3920-4871

15. Ganes C Sen

    Department of Inflammation and Immunity, Cleveland Clinic Foundation, Cleveland, United States

    Contribution

    Data curation, Investigation, Methodology

    Competing interests

    No competing interests declared

16. Calvin Cotton

    Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, United States

    Contribution

    Resources

    Competing interests

    No competing interests declared

17. Christine McDonald

    Department of Inflammation and Immunity, Cleveland Clinic Foundation, Cleveland, United States

    Contribution

    Formal analysis

    Competing interests

    No competing interests declared

18. Michelle Longworth

    Department of Inflammation and Immunity, Cleveland Clinic Foundation, Cleveland, United States

    Contribution

    Data curation, Formal analysis, Investigation, Methodology

    Competing interests

    No competing interests declared

19. Parameswaran Ramakrishnan

    Department of Pathology, Case Western Reserve University, Cleveland, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration

    For correspondence

    pxr150@case.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-1314-827X

20. Maria Hatzoglou

    Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, United States

    Contribution

    Conceptualization, Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Visualization, Methodology, Project administration

    For correspondence

    mxh8@case.edu

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2037-1231

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