Roles of programmed death-1 and muscle innate lymphoid cell-derived interleukin 13 in sepsis-induced intensive care unit-acquired weakness.

BACKGROUND: Intensive care unit-acquired weakness (ICU-AW) is a syndrome characterized by a long-term muscle weakness often observed in sepsis-surviving patients during the chronic phase. Although ICU-AW is independently associated with increased mortality, effective therapies have yet to be established. Programmed death-1 (PD-1) inhibitors have attracted attention as potential treatments for reversing immune exhaustion in sepsis; however, its impact on ICU-AW remains to be elucidated. Here, we study how PD-1 deficiency affects sepsis-induced skeletal muscle dysfunction in a preclinical sepsis model. METHODS: Chronic sepsis model was developed by treating wild-type (WT) and PD-1 knockout (KO) mice with caecal slurry, followed by resuscitation with antibiotics and saline. Mice were euthanized on days 15-17. Body weights, muscle weights, and limb muscle strengths were measured. Interleukin 13 (IL-13) and PD-1 expressions were examined by flow cytometry. Messenger RNA (mRNA) expressions of slow-twitch muscles were measured by reverse transcription and quantitative polymerase chain reaction (RT-qPCR). In an in vitro study, C2C12 myotubes were treated with lipopolysaccharide (LPS) and recombinant IL-13 followed by gene expression measurements. RESULTS: WT septic mice exhibited decreased muscle weight (quadriceps, P < 0.01; gastrocnemius, P < 0.05; and tibialis anterior, P < 0.01) and long-term muscle weakness (P < 0.0001), whereas PD-1 KO septic mice did not exhibit any reduction in muscle weights and strengths. Slow-twitch specific mRNAs, including myoglobin (Mb), troponin I type 1 (Tnni1), and myosin heavy chain 7 (Myh7) were decreased in WT skeletal muscle (Mb, P < 0.0001; Tnni1, P < 0.05; and Myh7, P < 0.05) after sepsis induction, but mRNA expressions of Tnni1 and Myh7 were increased in PD-1 KO septic mice (Mb, not significant; Tnni1, P < 0.0001; and Myh7, P < 0.05). Treatment of C2C12 myotube cells with LPS decreased the expression of slow-twitch mRNAs, which was restored by IL-13 (Mb, P < 0.0001; Tnni1, P < 0.001; and Myh7, P < 0.05). IL-13 production was significantly higher in ILC2s compared to T cells in skeletal muscle (P < 0.05). IL-13-producing ILC2s in skeletal muscle were examined and found to increase in PD-1 KO septic mice, compared with WT septic mice (P < 0.05). ILC2-derived IL-13 was increased by PD-1 KO septic mice and thought to protect the muscles from experimental ICU-AW. CONCLUSIONS: Long-term muscle weakness in experimental ICU-AW was ameliorated in PD-1 KO mice. ILC2-derived IL-13 production in skeletal muscles was increased in PD-1 KO mice, thereby suggesting that IL-13 alleviates muscle weakness during sepsis. This study demonstrates the effects of PD-1 blockade in preserving muscle strength during sepsis through an increase in ILC2-derived IL-13 and may be an attractive therapeutic target for sepsis-induced ICU-AW.

Group 2 innate lymphoid cells and their surrounding environment.

Since the discovery of group 2 innate lymphoid cells (ILC2s) in 2010, subsequent studies have revealed their developmental pathways, mechanisms of activation and regulation, and immunological roles in tissue homeostasis and tissue-specific diseases in various organs. Although ILC2s are known to express tissue-specific features depending on where they reside, how the surrounding environment affects the functions of ILC2s remains to be fully elucidated. Recent histologic analyses revealed that ILC2s resides in specific perivascular regions in peripheral tissues with their function being controlled by the surrounding cells via cytokines, lipid mediators, neurotransmitters, and cell-cell interactions through surface molecules. This review summarizes the interactions between ILC2s and surrounding cells, including epithelial cells, neurons, immune cells, and mesenchymal cells, with the objective of promoting the development of novel diagnostic and therapeutic methods for ILC2-related diseases.

Metabolic regulation by prostaglandin E2 impairs lung group 2 innate lymphoid cell responses.

BACKGROUND: Group 2 innate lymphoid cells (ILC2s) play a critical role in asthma pathogenesis. Non-steroidal anti-inflammatory drug (NSAID)-exacerbated respiratory disease (NERD) is associated with reduced signaling via EP2, a receptor for prostaglandin E2 (PGE2 ). However, the respective roles for the PGE2 receptors EP2 and EP4 (both share same downstream signaling) in the regulation of lung ILC2 responses has yet been deciphered. METHODS: The roles of PGE2 receptors EP2 and EP4 on ILC2-mediated lung inflammation were investigated using genetically modified mouse lines and pharmacological approaches in IL-33-induced lung allergy model. The effects of PGE2 receptors and downstream signals on ILC2 metabolic activation and effector function were examined using in vitro cell cultures. RESULTS: Deficiency of EP2 rather than EP4 augments IL-33-induced mouse lung ILC2 responses and eosinophilic inflammation in vivo. In contrast, exogenous agonism of EP4 and EP2 or inhibition of phosphodiesterase markedly restricts IL-33-induced lung ILC2 responses. Mechanistically, PGE2 directly suppresses IL-33-dependent ILC2 activation through the EP2/EP4-cAMP pathway, which downregulates STAT5 and MYC pathway gene expression and ILC2 energy metabolism. Blocking glycolysis diminishes IL-33-dependent ILC2 responses in mice where endogenous PG synthesis or EP2 signaling is blocked but not in mice with intact PGE2 -EP2 signaling. CONCLUSION: We have defined a mechanism for optimal suppression of mouse lung ILC2 responses by endogenous PGE2 -EP2 signaling which underpins the clinical findings of defective EP2 signaling in patients with NERD. Our findings also indicate that exogenously targeting the PGE2 -EP4-cAMP and energy metabolic pathways may provide novel opportunities for treating the ILC2-initiated lung inflammation in asthma and NERD.

Possible connection between intestinal tuft cells, ILC2s and obesity.

Intestinal tuft cells (TCs) are defined as chemosensory cells that can "taste" danger and induce immune responses. They play a critical role in gastrointestinal parasite invasion, inflammatory bowel diseases and high-fat diet-induced obesity. Intestinal IL-25, the unique product of TCs, is a key activator of type 2 immunity, especially to promote group 2 innate lymphoid cells (ILC2s) to secret IL-13. Then the IL-13 mainly promotes intestinal stem cell (ISCs) proliferation into TCs and goblet cells. This pathway formulates the circuit in the intestine. This paper focuses on the potential role of the intestinal TC, ILC2 and their circuit in obesity-induced intestinal damage, and discussion on further study and the potential therapeutic target in obesity.

The NF-κB Transcription Factor c-Rel Modulates Group 2 Innate Lymphoid Cell Effector Functions and Drives Allergic Airway Inflammation.

Group 2 innate lymphoid cells (ILC2s) play a key role in the initiation and orchestration of early type 2 immune responses. Upon tissue damage, ILC2s are activated by alarmins such as IL-33 and rapidly secrete large amounts of type 2 signature cytokines. ILC2 activation is governed by a network of transcriptional regulators including nuclear factor (NF)-κB family transcription factors. While it is known that activating IL-33 receptor signaling results in downstream NF-κB activation, the underlying molecular mechanisms remain elusive. Here, we found that the NF-κB subunit c-Rel is required to mount effective innate pulmonary type 2 immune responses. IL-33-mediated activation of ILC2s in vitro as well as in vivo was found to induce c-Rel mRNA and protein expression. In addition, we demonstrate that IL-33-mediated activation of ILC2s leads to nuclear translocation of c-Rel in pulmonary ILC2s. Although c-Rel was found to be a critical mediator of innate pulmonary type 2 immune responses, ILC2-intrinsic deficiency of c-Rel did not have an impact on the developmental capacity of ILC2s nor affected homeostatic numbers of lung-resident ILC2s at steady state. Moreover, we demonstrate that ILC2-intrinsic deficiency of c-Rel alters the capacity of ILC2s to upregulate the expression of ICOSL and OX40L, key stimulatory receptors, and the expression of type 2 signature cytokines IL-5, IL-9, IL-13, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Collectively, our data using Rel-/- mice suggest that c-Rel promotes acute ILC2-driven allergic airway inflammation and suggest that c-Rel may contribute to the pathophysiology of ILC2-mediated allergic airway disease. It thereby represents a promising target for the treatment of allergic asthma, and evaluating the effect of established c-Rel inhibitors in this context would be of great clinical interest.

Orchestration between ILC2s and Th2 cells in shaping type 2 immune responses.

The type 2 immune response is critical for host defense against large parasites such as helminths. On the other hand, dysregulation of the type 2 immune response may cause immunopathological conditions, including asthma, atopic dermatitis, rhinitis, and anaphylaxis. Thus, a balanced type 2 immune response must be achieved to mount effective protection against invading pathogens while avoiding immunopathology. The classical model of type 2 immunity mainly involves the differentiation of type 2 T helper (Th2) cells and the production of distinct type 2 cytokines, including interleukin-4 (IL-4), IL-5, and IL-13. Group 2 innate lymphoid cells (ILC2s) were recently recognized as another important source of type 2 cytokines. Although eosinophils, mast cells, and basophils can also express type 2 cytokines and participate in type 2 immune responses to various degrees, the production of type 2 cytokines by the lymphoid lineages, Th2 cells, and ILC2s in particular is the central event during the type 2 immune response. In this review, we discuss recent advances in our understanding of how ILC2s and Th2 cells orchestrate type 2 immune responses through direct and indirect interactions.

Isolation and Identification of Group 2 Innate Lymphoid Cells in Settings of Type 2 Inflammation.

The recent discovery of innate lymphoid cells (ILCs), a major source of canonical T cell cytokines, has prompted significant interest into understanding the role of these novel cells in immune responses. Unlike T cells, ILCs lack antigen receptors, instead receiving activation from locally derived tissue signals. Group 2 ILCs (ILC2s), which express the genes encoding GATA-3, interleukin (IL)-4, IL-5, IL-9, and IL-13, are distributed throughout nonlymphoid tissues. Although ILC2s lack antigen receptors, there are considerable similarities that they share with their Th2 cell counterparts, including receptors, secreted signals, and key transcription factors. Here we describe a method of isolating ILC2s for analysis from peripheral tissues of the mouse. The approach consists of digesting and mechanically dissociating harvested organs followed by staining with fluorescently labeled antibodies for flow cytometry or cell sorting. We suggest panels of antibodies for each tissue that can be used as positive and negative markers to selectively separate ILC2s from other cells, and we demonstrate marker specificity with example cells from a "cytokine reporter" mouse strain.