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LGP2 ATP hydrolysis is required for enhanced MDA5-mediated IFN signaling. A, HEK293T cells were transfected with a 110 IFN-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5 or LGP2. MDA5 was transfected at a constant 25 ng of plasmid/well, whereas the amount of LGP2 transfected varied at 0.03, 0.16, 0.8, 4, 20, 100, and 500 ng. Following a 24-h transfection, cells were transfected with 5 g/ml of poly(I:C) (left) or infected with 3 pfu/cell EMCV (right) for 8 h before harvesting. At low concentrations LGP2 enhances MDA5- mediated IFN signaling, but at higher concentrations LGP2 functions as a negative regulator. B, HEK293T cells were transfected with a 110 IFN-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5, LGP2 MI, or LGP2 MIIa mutants. MDA5 was transfected at a constant 25 ng of plasmid/well, whereas the amount of LGP2 transfected varied at 0.03, 0.16, 0.8, 4, 20, 100, and 500 ng. Following a 24-h transfection, cells were transfected with 5 g/ml of poly(I:C) for 8 h before harvesting. Neither LGP2 MI nor LGP2 MIIa are able to enhance MDA5-mediated signaling. C, HEK293T cells were transfected with a 110 IFN-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5, LGP2, or LGP2 mutants. MDA5 was transfected at a constant 50 ng of plasmid/well, whereas the amount of LGP2 transfected was varied at 5 (white), 10 (gray), and 50 ng (black) of plasmid. Following a 24-h transfection, cells were infected with 3 pfu/cell EMCV or transfected with 5 g/ml of poly(I:C) for 8 h before harvesting. None of the ATP hydrolysis defective mutants are able to enhance MDA5-mediated signaling.  

LGP2 ATP hydrolysis is required for enhanced MDA5-mediated IFN signaling. A, HEK293T cells were transfected with a 110 IFN-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5 or LGP2. MDA5 was transfected at a constant 25 ng of plasmid/well, whereas the amount of LGP2 transfected varied at 0.03, 0.16, 0.8, 4, 20, 100, and 500 ng. Following a 24-h transfection, cells were transfected with 5 g/ml of poly(I:C) (left) or infected with 3 pfu/cell EMCV (right) for 8 h before harvesting. At low concentrations LGP2 enhances MDA5- mediated IFN signaling, but at higher concentrations LGP2 functions as a negative regulator. B, HEK293T cells were transfected with a 110 IFN-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5, LGP2 MI, or LGP2 MIIa mutants. MDA5 was transfected at a constant 25 ng of plasmid/well, whereas the amount of LGP2 transfected varied at 0.03, 0.16, 0.8, 4, 20, 100, and 500 ng. Following a 24-h transfection, cells were transfected with 5 g/ml of poly(I:C) for 8 h before harvesting. Neither LGP2 MI nor LGP2 MIIa are able to enhance MDA5-mediated signaling. C, HEK293T cells were transfected with a 110 IFN-luciferase reporter gene, control Renilla luciferase plasmid, and expression vectors for the indicated helicase proteins MDA5, LGP2, or LGP2 mutants. MDA5 was transfected at a constant 50 ng of plasmid/well, whereas the amount of LGP2 transfected was varied at 5 (white), 10 (gray), and 50 ng (black) of plasmid. Following a 24-h transfection, cells were infected with 3 pfu/cell EMCV or transfected with 5 g/ml of poly(I:C) for 8 h before harvesting. None of the ATP hydrolysis defective mutants are able to enhance MDA5-mediated signaling.  

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Article
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Laboratory of genetics and physiology 2 (LGP2) is a member of the RIG-I-like receptor family of cytoplasmic pattern recognition receptors that detect molecular signatures of virus infection and initiate antiviral signal transduction cascades. The ATP hydrolysis activity of LGP2 is essential for antiviral signaling, but it has been unclear how the e...

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Context 1
... assays. MDA5 is constitutively active in this assay and can activate the IFN promoter when expressed, irrespective of infection with encephalomyocarditis virus (EMCV) or transfection with poly(I:C) (Fig. 4) (16, 25). Introducing low levels of wild type LGP2 is synergistic with MDA5 and results in a 2-fold increase in IFN promoter activity ( Fig. 4A and supplemental Fig. S3). Raising the level of LGP2 expression reduces the MDA5-de- pendent IFN promoter activity, consistent with the reported feedback inhibition effect of LGP2 expression (3-5). To test if ATP hydrolysis is required for synergistic MDA5-mediated IFN promoter activity, two LGP2 mutants were co-trans- fected at a range of plasmid ...
Context 2
... LGP2 MIIa lacks basal ATP hydrolysis, but retains RNA-stimulated ATP hydrolysis. Unlike wild type LGP2, nei- ther of these mutants is able to enhance MDA5-mediated sig- naling at low expression levels, but both display the inhibitory activity of LGP2 at higher expression levels ( Fig. 4B and supple- mental Fig. S3). Parallel analysis of all the LGP2 mutants defec- tive for ATP hydrolysis verified that none of the mutant pro- teins were able to enhance MDA5-mediated signaling (Fig. 4C), demonstrating that intact ATP hydrolysis is critical for LGP2 to stimulate MDA5 signal transduction ...
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... is able to enhance MDA5-mediated sig- naling at low expression levels, but both display the inhibitory activity of LGP2 at higher expression levels ( Fig. 4B and supple- mental Fig. S3). Parallel analysis of all the LGP2 mutants defec- tive for ATP hydrolysis verified that none of the mutant pro- teins were able to enhance MDA5-mediated signaling (Fig. 4C), demonstrating that intact ATP hydrolysis is critical for LGP2 to stimulate MDA5 signal transduction ...

Citations

... LGP2 competitively recognizes the same viral PAMP for the RIG-I and effects a downregulation, whereas the interaction of the LGP2 CTD and RNA facilitates the enhanced recognition of viral RNA by MDA5 (Rothenfusser et al., 2005;Li et al., 2009;Bruns et al., 2013;Childs et al., 2013;Reikine et al., 2014;Sanchez David et al., 2019;Wu et al., 2020). Molecular characterizations of RLRs have been undertaken in different teleost species (Table 1) and have shown that the domains are found to be conserved in these species. ...
Article
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The innate antiviral response through the RIG-1-like receptor pathway is oriented towards a key regulatory molecule, mitochondrial antiviral signaling protein (MAVS). Localized to the mitochondria, MAVS coordinates the interferons (IFN) production through regulated signaling cascades. Protein-protein interaction, post-translational modifications, and mitochondrial dynamics considerably regulate MAVS signaling on the mitochondrial membrane. This review gives insight into the inevitable role of MAVS in the innate antiviral immune response of fish.
... In contrast, LGP2, which binds dsRNA [139,140] but lacks CARDS, may sequester the dsRNA to antagonize RIG-I signaling [140][141][142][143]. However, LGP2 acts as a co-factor of MDA5 and promotes MDA5 signaling [54,139,[143][144][145][146][147][148]. ...
Article
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Influenza virus possesses an RNA genome of single-stranded, negative-sensed, and segmented configuration. Influenza virus causes an acute respiratory disease, commonly known as the "flu" in humans. In some individuals, flu can lead to pneumonia and acute respiratory distress syndrome. Influenza A virus (IAV) is the most significant because it causes recurring seasonal epidemics , occasional pandemics, and zoonotic outbreaks in human populations, globally. The host innate immune response to IAV infection plays a critical role in sensing, preventing, and clearing the infection as well as in flu disease pathology. Host cells sense IAV infection through multiple receptors and mechanisms, which culminate in the induction of a concerted innate antiviral response and the creation of an antiviral state, which inhibits and clears the infection from host cells. However, IAV antagonizes and escapes many steps of the innate antiviral response by different mechanisms. Herein, we review those host and viral mechanisms. This review covers most aspects of the host innate immune response, i.e., (1) the sensing of incoming virus particles, (2) the activation of downstream innate antiviral signaling pathways, (3) the expression of interferon-stimulated genes, (4) and viral antagonism and escape.
... These RNA features are commonly found in viral RNA genomes and replication intermediates generated during viral infections, and detecting these foreign RNAs is critical for activating the RIG-I / MDA5 signaling pathway for interferon-mediated antiviral response. LGP2, the smallest member of the RLR family, is pivotal in regulating the signaling pathway through positive and negative regulation of MDA5 and RIG-I, respectively (9)(10)(11)(12) . ...
... The presence of ATP had minimal effect on the K D values of both blunt-end and overhang RNAs (Figure 4 A-B, E, Supplemental Table S1). This contrasts with a previous report that suggested that ATP enhances the binding of LGP2 to RNA ( 10 ). ...
... Thus, the around 10-fold difference in K D values of the two RNAs is due to the difference in the k on rather than the k off . The rate measurements of LGP2 with the blunt-end RNA are consistent with previously published values obtained from single-molecule experiments ( 10 ). ...
Article
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The RIG-I family helicases, comprising RIG-I, MDA5 and LGP2, are cytoplasmic RNA sensors that trigger an antiviral immune response by specifically recognizing foreign RNAs. While LGP2 lacks the signaling domain necessary for immune activation, it plays a vital role in regulating the RIG-I/MDA5 signaling pathway. In this study, we investigate the mechanisms underlying this regulation by examining the oligomeric state, RNA binding specificity, and translocation activity of human LGP2 and the impact of ATPase activity. We show that LGP2, like RIG-I, prefers binding blunt-ended double-stranded (ds) RNAs over internal dsRNA regions or RNA overhangs and associates with blunt-ends faster than with overhangs. Unlike RIG-I, a 5′-triphosphate (5′ppp), Cap0, or Cap1 RNA-end does not influence LGP2’s RNA binding affinity. LGP2 hydrolyzes ATP in the presence of RNA but at a 5–10 fold slower rate than RIG-I. Nevertheless, LGP2 uses its ATPase activity to translocate and displace biotin-streptavidin interactions. This activity is significantly hindered by a methylated RNA patch, particularly on the 3′-strand, suggesting a 3′-strand tracking mechanism like RIG-I. The preference of LGP2 for blunt-end RNA binding, its insensitivity to Cap0/Cap1 modification, and its translocation/protein displacement ability have substantial implications for how LGP2 regulates the RNA sensing process by MDA5/RIG-I.
... The CTD provides the specificity of RNA recognition and binding mechanism. The LGP2 is a regulator of the signaling mechanism of RLR [10]. ...
Article
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Despite the efforts to develop the strategies of tuberculosis control, this disease still takes more than a million lives annually. The development of tuberculosis infection can be considered as an imbalance between the immune response of the host organism and the growth of Mycobacterium tuberculosis bacteria. In order to gain a foothold successfully in an infected organism, M. tuberculosis must overcome the mechanisms of innate immunity, including those that aim at the recognition of alien nucleic acids. RIG-I-like receptors (RLR) is a system of intracellular receptors (sensors of alien RNA), which is involved in the recognition of viruses and bacterial pathogens. The RIG-I, MDA5, and LGP2 receptors interact directly with RNA in the cell cytoplasm and trigger a cascade of interactions, which leads to the synthesis of type I interferons and proinflammatory cytokines. To date, it has been proven that the activation of RLR during tuberculosis infection is the most important component of innate immunity. Their obvious role in the activation of type I interferons (which, however, can be not only protective, but also negative for the immune system) was demonstrated. The review considers the latest data on the functioning of RLR in tuberculosis on the example of model organisms and humans.
... LGP2, arguably the most important MDA5 cofactor, promotes MDA5 filament formation on dsRNA (10,(38)(39)(40)(41). ...
... LGP2 has also been found incorporated within MDA5 filaments, accelerating MDA5 filament formation and resulting in more numerous, shorter filaments that retain signaling activity (40,41). The ATPase activity of LGP2 has been reported to promote dissociation of MDA5 from RΝΑ (41), but a separate study reported that ATP hydrolysis by LGP2 enhances LGP2 RNA binding and hence promotes MDA5 signaling (39). It remains unclear, therefore, whether LGP2 interacts directly with MDA5, or exactly how the RNA binding and ATPase activities of LGP2 contribute to sensitive and selective dsRNA recognition by MDA5. ...
... binding ATP hydrolysis by LGP2 had been reported to enhance binding of LGP2 to RNA and thus promote MDA5 signaling (39), but a subsequent study reported that the ATPase activity of LGP2 promoted dissociation of MDA5 from RΝΑ (41). We applied negativestain electron microscopy (ns-EM) and biochemical assays to clarify the role of ATP hydrolysis by LGP2 in RNA binding and MDA5 filament formation. ...
Preprint
Cytosolic long double-stranded RNA (dsRNA), among the most potent proinflammatory signals, is recognized by MDA5. MDA5 binds dsRNA cooperatively, forming helical filaments. ATP hydrolysis by MDA5 fulfills a proofreading function by promoting dissociation of shorter endogenous dsRNAs from MDA5 while allowing longer viral dsRNAs to remain bound leading to activation of interferon-β responses. Here, we show that adjacent MDA5 subunits in MDA5-dsRNA filaments hydrolyze ATP cooperatively, inducing cooperative filament disassembly. This amplifies the RNA footprint expansion that accompanies each round of ATP hydrolysis and allows MDA5 to displace tightly bound proteins from dsRNA. Our electron microscopy and biochemical assays show that LGP2 binds to dsRNA at internal binding sites through noncooperative ATP hydrolysis. Unlike MDA5, LGP2 has low nucleic acid selectivity and can hydrolyze GTP and CTP as well as ATP. Binding of LGP2 to dsRNA promotes nucleation of MDA5 filament assembly resulting in shorter filaments. Molecular modeling of the MDA5-LGP2 interface suggests that MDA5 interacts with dsRNA stem-bound rather than end-bound LGP2. We conclude that NTPase-dependent binding of LGP2 to internal sites on dsRNA increases the number and signaling output of MDA5-dsRNA complexes. Our work identifies novel molecular mechanisms contributing the selectivity and sensitivity of cytosolic dsRNA sensing. KEY POINTS Cooperative ATP hydrolysis in MDA5 filaments confers selectivity for dsRNA and displaces other proteins from RNA Noncooperative NTP hydrolysis by LGP2 induces binding to internal RNA sites with low selectivity RNA stem-bound LGP2 nucleates assembly of MDA5 signaling complexes on a broader set of RNA ligands
... The RLR family consists of three homologous helicases, including RIG-I (Retinoic acid-Inducible Gene I), MDA5 (Melanoma Differentiation-Associated factor 5), and LGP2 (Laboratory of Genetics and Physiology 2) (13,14). They are responsible for viral double-stranded RNA (dsRNA) recognition and ATP hydrolyzation, while the ability for signaling activation differs (15). Two RLRs, RIG-I and MDA5, have been extensively studied and exerted their activity by interacting with downstream adaptor MAVS via their caspase activation recruitment domains (CARDs). ...
... DExDc and HELICc domains contain RNA binding and helicase superfamily ATP binding domains. It was reported that LGP2 ATP hydrolysis promotes dsRNA recognition and enhances MDA5mediated IFN signaling (15). Accordingly, it is the possible reason why the two regions restrained PRRSV replication. ...
Article
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LGP2, a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), shows higher-affinity binding to RNA and work synergism with RIG-I or MDA5. However, LGP2 has divergent responses to different viruses, which remains controversial in antiviral immune responses. Here, we present the detailed process of LGP2 in positively regulating the anti-PRRSV response. Upon PRRSV infection, LGP2 was prone to bind to MDA5 and enhanced MDA5 signaling, manifesting the enrichment of viral RNA on MDA5 and the activation of downstream IRF3 and NF-κB, which results in increased proinflammatory cytokines and type I interferon expression, ultimately inhibiting PRRSV at the early stage of infection. Moreover, PRRSV Nsp1 and Nsp2 interacted with LGP2 via ubiquitin-proteasome pathways, thus blocking LGP2-mediated immune response. This research helps us understand the host recognition and innate antiviral response to PRRSV infection by neglected pattern recognition receptors, which sheds light on the detailed mechanism of virus-host interaction.
... iScience 25, 104821, August 19, 2022 13 iScience Article inhibitors of RIG-I and MDA5 signaling (Bruns et al., 2013(Bruns et al., , 2014Childs et al., 2013). Correlating with the expression characteristics of LGP2, these data suggested that cellular LGP2 expression level might determine its function switch in RLR signaling (Bruns et al., 2013(Bruns et al., , 2014Childs et al., 2013;Pippig et al., 2009). ...
... iScience 25, 104821, August 19, 2022 13 iScience Article inhibitors of RIG-I and MDA5 signaling (Bruns et al., 2013(Bruns et al., , 2014Childs et al., 2013). Correlating with the expression characteristics of LGP2, these data suggested that cellular LGP2 expression level might determine its function switch in RLR signaling (Bruns et al., 2013(Bruns et al., , 2014Childs et al., 2013;Pippig et al., 2009). However, we provide solid evidence that zebrafish LGP2, either at lower levels or at higher levels, shows dual effects on the IFN response triggered by SVCV (Figure 4). ...
... Similar to human LGP2, zebrafish LGP2 triggers IFN response with the requirement of MDA5. It is well documented that mammalian LGP2 coordinates fiber formation and subsequent activation of MDA5 (Bruns et al., 2013(Bruns et al., , 2014Duic et al., 2020;Esser-Nobis et al., 2020). These results suggest that during the early stage of viral infection, zebrafish LGP2 might facilitate MDA5 activation for the onset of host IFN response. ...
Article
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In mammals, LGP2 is the enigmatic RLR family member, being initially believed as an inhibitor of RLR-triggered IFN response but subsequently as an activator of MDA5 signaling and an inhibitor of RIG-I signaling. The contradiction happens to fish LGP2. Here, we generate a lgp2 loss-of-function (lgp2lof/lof) zebrafish mutant, which is highly susceptible to SVCV infection, displaying an initially decreased activation of IFN response and a following increased one. Mechanistically, zebrafish LGP2 functions as the essential activator of IFN response dependently of MDA5 at the early stage of viral infection, but as a negative regulator by impairing mRNA levels of tbk1 and ikki at the late stage of viral infection. The function switch of LGP2 is related to cellular IFN production during viral infection. Our data demonstrate that zebrafish LGP2 is a key homeostatic regulator of IFN response and thus essential for zebrafish survival against SVCV infection.
... RIG-I and LGP2 are physiologically found in an auto-repressed state and are activated by the presence of immunostimulatory RNAs that induce their conformational change leading to the binding with ATP (Kowalinski et al., 2011;Bruns et al., 2013). Compared to RIG-I and LGP2, MDA5 shows a more open structural conformation even in the absence of RNA ligands (Berke and Modis, 2012;Brisse and Ly, 2019). ...
Article
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Glioblastoma (GB) is an incurable form of brain malignancy in an adult with a median survival of less than 15 months. The current standard of care, which consists of surgical resection, radiotherapy, and chemotherapy with temozolomide, has been unsuccessful due to an extensive inter- and intra-tumoral genetic and molecular heterogeneity. This aspect represents a serious obstacle for developing alternative therapeutic options for GB. In the last years, immunotherapy has emerged as an effective treatment for a wide range of cancers and several trials have evaluated its effects in GB patients. Unfortunately, clinical outcomes were disappointing particularly because of the presence of tumor immunosuppressive microenvironment. Recently, anti-cancer approaches aimed to improve the expression and the activity of RIG-I-like receptors (RLRs) have emerged. These innovative therapeutic strategies attempt to stimulate both innate and adaptive immune responses against tumor antigens and to promote the apoptosis of cancer cells. Indeed, RLRs are important mediators of the innate immune system by triggering the type I interferon (IFN) response upon recognition of immunostimulatory RNAs. In this mini-review, we discuss the functions of RLRs family members in the control of immune response and we focus on the potential clinical application of RLRs agonists as a promising strategy for GB therapy.
... LGP2 comprises three domains: N-terminal helicase ATP binding domain (ATP bind), helicase C-terminal domain (CTER) and C-terminal regulatory domain (CTR) with the conserved helicase motifs I, II and III in the helicase ATP binding domain. These motifs I, II, III are implicated in ATPase hydrolysis activity of LGP2, whereas the K654 site in CTR is involved in dsRNA binding (42,43). To dissect the roles of individual domains of porcine LGP2 in the regulation of RIG-I and MDA5 signaling, the each LGP2 domains and domain deletion mutants were produced ( Figure 8A). ...
... Importantly, we found porcine LGP2 positively regulates porcine MDA5 as well as RIG-I activity. Positive regulation of MDA5 activation by physiological low concentration of LGP2 was widely accepted (15,45,46) and the biochemical and molecular mechanism recently became clear (17,18,43,47): MDA5 binds dsRNA weakly and the filament formation on long dsRNA chain is slow and unstable. On the contrary, LGP2 binds dsRNA with the highest affinity among three RLRs, regardless of 5'-PPP and RNA length, but not forming filaments (18,40,48). ...
... For LGP2, ATP hydrolysis enables it to efficiently engage diverse dsRNA species, which promotes MDA5 recognition of dsRNA, shorter filament formation and activation (47). In this case, both ATP hydrolysis and RNA binding of LGP2 are required for positive regulation of MDA5 (18,43). However, the roles of ATP hydrolysis in RLR activation and antiviral signaling are very complex. ...
Article
Full-text available
The RLRs play critical roles in sensing and fighting viral infections especially RNA virus infections. Despite the extensive studies on RLRs in humans and mice, there is a lack of systemic investigation of livestock animal RLRs. In this study, we characterized the porcine RLR members RIG-I, MDA5 and LGP2. Compared with their human counterparts, porcine RIG-I and MDA5 exhibited similar signaling activity to distinct dsRNA and viruses, via similar and cooperative recognitions. Porcine LGP2, without signaling activity, was found to positively regulate porcine RIG-I and MDA5 in transfected porcine alveolar macrophages (PAMs), gene knockout PAMs and PK-15 cells. Mechanistically, LGP2 interacts with RIG-I and MDA5 upon cell activation, and promotes the binding of dsRNA ligand by MDA5 as well as RIG-I. Accordingly, porcine LGP2 exerted broad antiviral functions. Intriguingly, we found that porcine LGP2 mutants with defects in ATPase and/or dsRNA binding present constitutive activity which are likely through RIG-I and MDA5. Our work provided significant insights into porcine innate immunity, species specificity and immune biology.
... Major classes of PRRs include Toll-like receptors (TLRs), C-type lectin receptors, nucleotide-binding oligomerization domain-like receptors, retinoic-acid-inducible gene-I-like receptors, and receptors for advanced glycation end products (RAGE) [27]. PRRs are comprised of both cell membrane and cytoplasmic receptors that can collectively recognize microbial peptidoglycans, lipopolysaccharides, glucans, phospholipids, high mobility group box (HMGB) proteins [28], S100 family of proteins, nucleic acids [29][30][31], glycoproteins and glycolipids [32], flagellin [33], glycosylated end products [28,34], and oxidation/nitrosylation products associated with cellular injury [27,[35][36][37]. Upon recognition of these molecular patterns, PRRs initiate downstream signaling events that induce a host-protective response [36]. ...
Article
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Implementation of protocolized surveillance, diagnosis, and management of septic patients, and of surgical sepsis patients in particular, is shown to result in significantly increased numbers of patients surviving their initial hospitalization. Currently, most surgical sepsis patients will rapidly recover from sepsis; however, many patients will not rapidly recover, but instead will go on to develop chronic critical illness (CCI) and experience dismal long-term outcomes. The elderly and comorbid patient is highly susceptible to death or CCI after sepsis. Here, we review aspects of the Persistent Inflammation, Immunosuppression, and Catabolism Syndrome (PICS) endotype to explain the underlying pathobiology of a dysregulated immune system in sepsis survivors who develop CCI; then, we explore targets for immunomodulatory therapy.