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Innate lymphoid cells in atherosclerosis
Author’s Accepted Manuscript Innate lymphoid cells in atherosclerosis Daniel Engelbertsen, Andrew H. Lichtman
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S0014-2999(17)30289-3 http://dx.doi.org/10.1016/j.ejphar.2017.04.030 EJP71187
To appear in: European Journal of Pharmacology Received date: 8 December 2016 Revised date: 23 March 2017 Accepted date: 20 April 2017 Cite this article as: Daniel Engelbertsen and Andrew H. Lichtman, Innate lymphoid cells in atherosclerosis, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.04.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Innate lymphoid cells in atherosclerosis
Daniel Engelbertsen*, Andrew H. Lichtman
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA
*
Corresponding Author: Daniel Engelbertsen, Department of Pathology, Brigham and Women's Hospital
and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. email: [email protected]
Abstract The family of innate lymphoid cells (ILCs) consisting of NK cells, lymphoid tissue inducer cells and the ‘helper’-like ILC subsets ILC1, ILC2 and ILC3 have been shown to have important roles in protection against microbes, regulation of inflammatory diseases and involved in allergic reactions. ILC1s produce IFN-γ upon stimulation with IL-12 and IL-18, ILC2s produce IL-5 and IL-13 responding to IL-33 and IL25 while ILC3s produce IL-17 and IL-22 after stimulation with IL-23 or IL-1. Although few studies have directly investigated the role for ILCs in atherosclerosis, several studies have investigated transcription
factors and cytokines shared by ILCs and T helper cells. In this review we summarize our current understanding of the role of ILC in atherosclerosis and discuss future directions.
Keywords: Innate lymphoid cells; atherosclerosis; inflammation; mouse models; cytokines
1. Introduction Leukocytes are subdivided into a wide variety of distinct cell lineages,each of which plays different roles in preventing infectious disease and promoting tissue homeostasis and barrier function. Innate lymphoid cells (ILCs) are a recently discovered addition to the expanding family of immune cells consisting of several distinct subsubsets. Experiments in mice have demonstrated that ILCs influence other cells by two main mechanisms: production of cytokines and other soluble factors or cytotoxic effector function. NK cells were once considered to be the only innate cell derived from the common lymphoid precursor lacking a T-cell receptor. The group of known innate lymphoid cells was expanded when lymphoid tissue inducer (LTi) cells, important for lymphoid tissue generation, were discovered in 1997 (Mebius et al.). LTi (now classified as an ILC3 subset) produces Lymphotoxin α/β and RANKL important for lymphoid organogenesis. (Walker et al.). Around 2010, cells producing IL-17 and IL-22 residing in gut and expressing natural cytotoxicity markers (NKp46 and NKp44), the transcription factor RORγT and the receptor for IL-7 (IL-7R) were described (Sanos et al.). Simultaneously, non-cytotoxic IL-7R+ ILCs were characterized that responded to Thymic stromal lymphopoietin (TSLP), IL-33 and IL-25 by the secretion of type 2 cytokines IL-5 and IL-13 (Moro et al.; Neill et al.; Price et al.). Reasons for the relatively late discovery of non-cytotoxic ILCs was (i) their relative scarcity in lymphoid tissue (<1% of all cells in spleen and blood) and (ii) the lack of cell surface markers specific for ILCs. This review will discuss the role of ILCs in atherosclerosis, evaluating recent findings and providing re-interpretations of past discoveries. Lineage tracing experiments have shown that non-cytotoxic ILCs (ILC1, ILC2 and ILC3) as well as NK cells are derived from common lymphocyte precursors in the bone marrow. A committed ILC/NK precursor expressing NFIL3 and α4β7+ can give rise to all ILC subsets: NK cells, ILC1, ILC2 and ILC3 (including LTi cells) (Geiger et al.; Klose et al.). Non-cytotoxic ILCs display a similar subdivision as CD4+ T cells: T-bet or EOMES-expressing ILC1, GATA3-expressing ILC2 and RORT expressing ILC3,
analogous to T helper (Th)1, Th2, and TH17 cells respectively (Walker et al.). Cytokine production is also related between T helper and ILC subsets: ILC1 cells produce IFN-γ, ILC2 cells produce IL-4, IL-5 and IL-13 and ILC3 cells produce GM-CSF, lymphotoxin-α/β, IL-17 and IL-22 (depending on ILC3 subset). Mature ILCs are defined by lack of expression of myeloid or lymphoid lineage markers (negative for CD11b, CD11c, Gr1, CD3 etc.) and by expression of IL-7R and CD90 (Thy1). It is clear that some of these lineages are plastic: ILC3s may under certain conditions transform to ILC1 (Klose et al., 2013) and human ILC2s can be induced to produce IFN- after IL-12 stimulation (Silver et al., 2016). Recent studies in mice have demonstrated that ILCs are predominantly resident cells that arise during early development and are maintained in tissues by local proliferation rather than being replenished by circulating precursors (Gasteiger et al.). Levels of circulating ILCs are high in human cord blood then decline during childhood reaching even lower levels in adults (Vely et al.). In adult mice and humans, most ILCs are found in epithelial barrier tissues, including skin, gut, and lungs, although they are also present in adipose tissues, joints, and various other tissue sites (Klose and Artis, 2016). Classical NK cells (Bjorkstrom et al.) and ILC3s (Doisne et al., 2015) are present relatively abundant in the uterus, but their precise role there is unknown. Unlike T cells, activation of cytokine production by ILCs is not contingent upon antigen recognition (ILCs do not express TCR) but rather is induced by integrating cytokine signals produced by myeloid or epithelial cells (Klose and Artis, 2016). IL-12 and IL-18 promote ILC1 production of IFN-γ, IL-33 and IL-25 promotes ILC2s production of type 2 cytokines while IL-23 and IL-1 trigger ILC3 cytokines. 2. ILCs and atherosclerosis To this date, few studies have directly investigated the role of ILCs in atherosclerosis. However, there are several studies performed in the ‘pre-ILC-era’ where the primary aim of the study was to study T cell responses but where ILCs most likely also were affected. We have summarized what we believe to be relevant studies in order to gain insight into possible contributions of ILCs in the pathogenesis of
atherosclerosis (Table 1). It is important to note that it is difficult to properly predict the how ILCs will respond to loss of certain factors. For example, mice deficient in T-bet and Rag1 exhibit colitis that is due to exaggerated ILC3 response and high levels of IL-17 (Powell et al.). However, given the current scarcity of mechanistic studies of ILCs in atherosclerosis, we believe that this is a useful approach. 2.1. ILC1 and atherosclerosis Many studies have demonstrated the link between Th1 immunity and atherosclerosis (Witztum and Lichtman). Mice lacking T-bet (Buono et al.), IFN- γ (Buono et al.) and IL-12(Davenport and Tipping) all display reduced plaque burden. These studies have been largely interpreted as proof of a role for Th1 immunity, but it still remains unclear the extent to which ILC1s (or other innate sources of IFN-γ) are influencing the phenotype observed in these mice. Recent studies have demonstrated that IL-7R+ ILC1s, but not EOMES+ cNK cells, are dependent on T-bet for development (Klose et al.). The role of cytotoxic NK cells in atherosclerosis is reviewed in this issue of European Journal of Pharmacology by Bobik and colleagues. In brief, there is evidence that ILC1 (including NK cells) may play a role in atherosclerosis. Depletion of NK cells using anti-Asialo-GM1 antibodies reduced atherosclerosis in apoe-/- mice (Selathurai et al.). Transfer of NK cells to Apoe−/−Rag2−/−Il2rg−/− mice increased plaque formation and the pathogenicity of NK cells was found to be contingent on cytotoxic activity rather than production of IFN-γ. Given the continued lack of defined auto-antigens in mouse models of atherosclerosis, it remains an intriguing possibility that innate sources of IFN-γ from cytotoxic (classical NK cells) and noncytotoxic ILC1 may significantly contribute to lesion development. Arguing against an important role for NK cells in cardiovascular disease, NK cells are found in very limited numbers in the human atherosclerotic plaques and only represent a minor fraction of all leukocytes found in lesions (Bobryshev and Lord; Millonig et al.).
2.2. ILC2 and atherosclerosis In a study predating the discovery of ILCs, mice lacking RORα developed larger aortic root fatty streak lesions when fed a high fat diet, compared to C57BL/6 controls (Mamontova et al., 1998), although the mice had abnormal lipoproteins and displayed ataxia. RORα was subsequently shown to be required for ILC2 development. ILC2s produce type 2 cytokines and are enriched in the lung and adipose tissue. Perry and colleges demonstrated that aortic CD90+ST2+Sca-1+cKit (CD117)+ ILC2s (termed natural helper cells) produce IL-5 to trigger B1 cell proliferation and production of natural IgM (Perry et al.). Our lab sorted CD90+IL-7Rα+CD25+ ILC2s from digested aortas and demonstrated production of type 2 cytokines from these cells after stimulation with PMA and ionomycin (Engelbertsen et al.). In both these studies ILC2s were predominantly located in aortic periadventitial adipose tissue but virtually absent in atherosclerotic lesions. There are yet no published reports of ILC2s in human atherosclerotic plaques. ILC2s are activated by IL-33, IL-25 and TSLP produced mainly by epithelial cells in response to injury or inflammation (Sonnenberg and Artis). All these three cytokines have been studied in the context of atherosclerosis. Treatment of mice with IL-33 reduced atherosclerosis while injections of soluble ST2 (the receptor for IL-33) increased atherosclerosis (Miller et al., 2008). The protective effect of IL-33 was lost when co-treating with IL-5 blocking antibodies. ILCs were not directly assessed by this study, but many others have demonstrated the potent ILC expanding effects of IL-33 (Sonnenberg and Artis). It seems likely that at least part of the anti-atherogenic effects demonstrated in this study is due to expansion of ILC2s. Similarly, Mantani et al. demonstrated that treatment of Apoe-/- mice with IL-25 reduced atherosclerosis (Mantani et al.). Treatment with IL-25 boosted ILC2 cell numbers and serum levels of ILC2 cytokines (IL-5, IL-9 and IL-13) and was associated with increased levels of anti-phosphorylcholine (PC) natural IgM. Notably, IL-5 deficient mice did not exhibit increased natural IgM upon IL-25 treatment. In another study, injecting ApoE null mice with TSLP reduced plaque formation and was associated with reduced macrophage infiltration and an increase in lesional regulatory T cells (ILCs were not investigated in this study) (Yu et al., 2013). Notably, injections of TSLP dramatically boosted IgM
levels suggesting that the ILC2-B1 cell axis is affected by the treatment. Binder et al. reported that transfer of IL-5-deficient bone marrow to Ldlr-/- recipients increased plaque formation relative to controls. This effect was associated with reduced natural IgM produced by B1 cells (Binder et al.). It is unclear to what extent the effect reflects impaired function of Th2 cells or ILC2s. Recent studies have demonstrated that serum levels of IL-5 are similar in T cell deficient Rag1-/- mice compared to wildtype controls, while ILC and T cell-deficient IL7Ra deficient mice do not exhibit measurable levels of serum IL-5 (Nussbaum et al.). Altogether, these studies indicate that the IL-33/IL-25/TSLP-ILC2-IL-5-IgM axis inhibits lesion development. Another key cytokine produced by ILC2s, but also by Th2 cells, is IL-13. Treatment of mice with rIL-13 increased plaque collagen deposition and polarized macrophages towards a “M2” like phenotype. Transfer of IL-13-deficient bone marrow increased lesion size and decreased M2 macrophage polarization (Cardilo-Reis et al.). It is unclear to what extent homeostatic or ‘hyperlipidemic’ IL-13 production is derived from ILC2s or Th2 cells and what cell type is responsible for the observed atheroprotective effect of IL-13. Recently, our lab investigated the effects of anti-CD90.2 mediated ILCs depletion or IL-2/anti-IL-2 (antiIL2 clone: JES6-1) mediated ILC expansion in Rag1-/-Ldlr-/- mice that lack adaptive immunity. The results were mixed: ILC depletion did not alter plaque progression while IL-2/anti-IL-2 treatment (expanding mainly ILC2 cells) reduced lesion burden (Engelbertsen et al.). Explaining part of the phenotype observed in ILC2 expanded mice, we observed a reduction in cholesterol accompanied with increased levels of triglycerides. Analysis of liver RNA revealed decreased transcription of Apob after ILC2 expansion, potentially explaining the reduced cholesterol levels in serum. IL-2/anti-IL-2 treatment also caused increased leukocyte recruitment to the liver which exhibited early signs of fibrosis. It is unclear whether the effects on cholesterol and lipid levels are due to physiologically relevant effects of ILC2 or just pathologic effects of chronic ILC2 activation leading to hepatic damage and scarring. Although IL-2/anti-IL-2 treatment was associated with dramatically elevated IL-5 levels and eosinophilia,
blockade of IL-5, which also reversed the eosinophilia, did not alter lesion size or lipid levels. It is important to note that although we observed a predominant ILC2 type response after treatment with IL2/anti-IL-2, CD25 is also expressed on certain subsets of ILC3 (Dumoutier et al.) and ILC1 cells (Gasteiger et al.). Anti-CD90.2 antibody treatment was efficient in depleting ILCs in lymphoid organs but did not fully deplete tissue ILCs (as has been reported by others (Roediger et al.)), complicating the interpretation of the study. Moreover, the lack of adaptive immunity in the Rag1-/-Ldlr-/- mouse obscures potential effects of ILC2s interacting with B cells or even T cells, as they have recently been proposed to perform class II MHC restricted antigen presentation and promote Th2 responses (Oliphant et al.). Further, the lack of B and T-cells in Rag-deficient mice may affect ILCs due to decreased competition for ILC stimulating cytokines like IL-7 and IL-2. In summary, activation of ILC2s is associated with reduced atherosclerotic burden and mice lacking ILC2 effector cytokines display increased atherosclerosis. Most studies are in line with type 2 immunity being atheroprotective, however it is unclear to what extent ILC2s are important in themselves and by what mechanisms they affect atherogenesis. Notably, ILC2s have also been shown important in regulation of adipose tissue homeostasis, promoting ‘beiging’ of white adipose tissue and limiting obesity (Brestoff et al.). In summary, current knowledge points to three mechanisms by which ILC2s (may) reduce atherosclerosis: (i) the IL-33/IL-25/TSLP-ILC2-IL-5-IgM axis, (ii) modulation of lipid homeostasis and (iii) modulation of lesional macrophages. 2.3. ILC3 and atherosclerosis ILC3s produce a range of cytokines that is shared with Th17 cells: IL-17, IL-22 and GM-CSF. The proinflammatory properties of Th17 cells in other immune diseases (experimental autoimmune encephalitis (Langrish et al., 2005), psoriasis (Elloso et al., 2012) and rheumatoid arthritis (Lubberts, 2008)) have prompted investigations of Th17 cytokines in the context of atherosclerosis. However, Th17 cytokines are not only produced by Th17 cells but also by innate cells including γδ T cells, mucosal-associated
invariant T cells (MAIT cells) and ILC3s. T cells and ILC3s reside in mucosal tissue and skin where they maintain barrier function through production of IL-17 and IL-22, sharing many effector functions with Th17 cells. Genetic deletion of IL-17 or the IL-17RA receptor in the Ldlr-/- resulted in reduced atherosclerotic lesion formation (Butcher et al.). However, other studies have indicated opposite effects of IL-17 on atherosclerosis. For example, there was increased atherosclerosis and reduced lesional vascular smooth muscle cells and type I collagen in Apoe-/-Il17A-/- mice compared to Apoe-/- controls (Danzaki et al., 2012). Another study showed decreased atherosclerosis after IL-17 treatment and increased atherosclerosis after blocking anti-IL-17 antibody injections (Taleb et al.). IL-22 acts on nonhematopoietic cells such as epithelial cells to promote barrier function and limit microbial invasion by increased production of defensins but has also been implicated in inflammatory skin diseases such as psoriasis. Rattik and coworkers demonstrated that Apoe-/-Il22-/- mice displayed reduced atherosclerosis and plaque collagen deposition compared to Apoe-/- controls (Rattik et al., 2015). This effect was attributed to effects on vascular smooth muscle cells that were found to express the IL-22 receptor. Overall, studies investigating the impact ILC3-associated cytokines on atherosclerosis have been inconsistent. Furthermore, the anatomical localization of ILC3s to mucosal tissue puts into question its relevance in atherosclerosis. The presence of ILC3s in murine and human atherosclerotic lesions remains uncertain, indicating that these cells are not present at high frequencies. We did not observe IL-17 production from either CD25+ or CD25- ILCs (lineage-CD90+IL-7Rα+) isolated from atherosclerotic mouse aortas (Engelbertsen et al.) 3. ILC mouse models: Complexity vs. Clarity Studying ILCs presents the researcher with a specific set of obstacles. As an example, ILC depletion using anti-CD90.2 antibodies is only possible in immunodeficient mice, as T cells also express CD90. Moreover, these antibodies are not efficient in depleting tissue resident ILCs. Mice deficient in important ILC transcription factors (GATA-3, T-bet, RORγT, TOX, Id3) also display defects in other leukocyte
subsets including T cells. A potential problem in using Rag-deficient mice to study ILCs has emerged by the surprising finding that Rag proteins are expressed during ILC development and have functions unrelated V-D-J recombinase activity (Karo et al., 2014). Thus Rag-deficient ILCs may be intrinsically abnormal. To solve these issues, mouse models where a certain subset of ILC loses its effector function (i.e. ILC2 specific IL-5 knockout) or is depleted (ILC2 specific knockout mice) would represent the best option. Recently, an ILC2-specific knockout mouse was described where RORα is deleted in cells expressing IL7R (Rorαfl/sg Il7r-Cre) (Oliphant et al.). IL-7R is expressed by T cells and ILCs but the authors claimed no effect on T cells, although others have reported defects in Th17 cells in Rorα-/- mice (Yang et al.). As more sophisticated mouse models enter the scene it is becoming clear that they will be in the form of double or triple knock-out/transgene mice. Further complicating the logistics of such studies, experimental atherosclerosis models has classically required a hyperlipidemic background such as Apoe-/or Ldlr-/-, demanding another knockout/transgene to be introduced. We believe that models of inducible atherosclerosis, for example injection of adeno-associated virus encoding a mutant form of proprotein convertase subtilisin/kexin type 9 (PCSK9) that efficiently cause LDLr degradation and subsequent hypercholesterolemia (Bjorklund et al.) are needed in order to facilitate studies of ILCs in atherosclerosis. Bone marrow transfer of ILC-deficient bone marrow into Ldlr-/- mice may be possible for some cases, but it is not clear to what extent bone marrow-derived ILCs outcompetes host ILCs which may or may not be eliminated by irradiation. 4. Translational aspects and future directions A recent study has raised questions about the role of ILCs in humans. Patients with severe combined immunodeficiency (SCID) due to inherited deficiency of either IL-2Rγc or JAK3 were found to lack both T cells and ILCs, whereas RAG-1-deficient SCID patients lacked T cells but had an intact ILC compartment (Vely et al.). Patients with both forms of SCID were treated with hematopoietic stem cell
transfer (HSCT) that reconstituted the T cell compartment but failed to reconstitute circulating or tissue ILCs in the in the patients with IL-2Rγc or JAK3 deficiency. When comparing incidence of disease in HSCT-treated SCID patients during follow-up (7-39 years), IL-2Rγc or JAK3 deficient SCID patients (‘ILC deficient’ patients) did not exhibit any increase in inflammatory disease or opportunistic infections compared to ILC-sufficient SCID patients. Although this study was not powered or designed to estimate the risk of cardiovascular disease in patients lacking ILCs, it is striking that ILCs were redundant. Importantly, this study does not preclude the possibility of ILC deregulation or chronic activation being pro-inflammatory and causing disease. What therapeutic targets are conceivable? Globally boosting ILCs may be associated with serious side effects. The risk of inducing type 2 related immunopathology (e.g. asthma and allergies) argue against this notion as ILC2s are increased in blood (Bartemes et al.) and sputum (Smith et al.) from allergic asthma patients and enriched in skin lesions of atopic patients (Kim et al.). Conversely, reducing ILC1 and ILC3 may reduce barrier function and lead to opportunistic infections or intestinal disease. The more attractive but for now elusive goal, is to selectively enhance ILC2 responses and/or decrease ILC1 and 3 responses in the arterial wall. 5. Conclusions The discovery of ILCs has prompted reevaluation of previous work intended to investigate the role of T cell subsets. In this review we have summarized such studies, along with the very few papers designed to directly assess the role of ILCs in atherosclerosis. In summary, promotion of a type 2 immune response, associated with ILC2s, appear to be consistently related with reduced atherosclerosis. Future studies are required to demonstrate the potential role of ILC2s in mediating this effect. ILC1s produce cytokines related to progression of atherosclerosis making these cells potential culprits and targets for further investigations. ILC3-related cytokines have mostly been associated with atheroprogression but the
relevance of this population remains unclear. Determining the presence (or absence) of these cells in the human atherosclerotic plaque remains an important objective. Acknowledgements The authors were supported by a postdoctoral fellowship from the Swedish Research Council (D.E.) and grants from NIH R01 HL087282 (A.H.L.).
Table 1. Innate lymphoid cells in atherosclerosis Effect on ILCs
Effect on other
Effect on
Reference
leukocytes
atherosclerosis
Impaired Th1/NK
Reduced
effector function
atherosclerosis
N/A
Increased
Selathurai et
atherosclerosis
al
Loss of Th1 cells,
Reduced
Buono et al
cytotoxic T cells
atherosclerosis
Reduced ILC1
Impaired
Reduced
Davenport et
activation1
Th1/NK/cytotoxic T
atherosclerosis
al
Miller et al
ILC1-related Ifn-/-Ldlr-/-
Impaired ILC1 effector function
NK cell transfer to Apoe
−/−
−/−
Rag2
1
N/A −/−
Il2rg
Buono et al
mice Tbet-/- Ldlr-/Apoe-/-Il12-/-
ILC1 deficiency1
cells function ILC2-related IL-33 treatment of
Increased levels
Increased Th2
Reduced
Apoe-/ -mice1
of ILC2s1
immunity
atherosclerosis
IL-25 treatment of
Increased levels
Increased Th2
Reduced
Mantani et
of ILC2s
immunity, increased
atherosclerosis
al
Increased Tregs,
Reduced
Yu et al
reduced plaque
atherosclerosis
-/-
Apoe mice
IgM production TSLP treatment of -/-
Apoe mice
Increased levels of ILC2s
1
macrophages Il5-/- BMT to Ldlr-/-
IL-13 treatment of Ldlr
Impaired ILC2
Impaired IgM
Increased
effector function1
production
atherosclerosis
N/A
Macrophages and B
Increased plaque
Cardilo-Reis
cells
collagen
et al
Impaired ILC2
Macrophage and B
Increased
Cardilo-Reis
effector function1
cells
atherosclerosis
et al
-/-
Il13-/- BMT to Ldlr-/-
IL-2/anti-IL-2 treatment of Rag1-/-Ldlr-/- mice
Expansion of ILC2
Eosinophilia, CD25 ILC3/ILC1
+
Binder et al
Reduced atherosclerosis
Engelbertsen 2
et al
ILC3-related Ldlr-/-Il17ra-/-
Impaired ILC3
Impaired Th17 effector
Reduced
function
atherosclerosis
Impaired ILC3
Impaired Th17 effector
Reduced
effector function1
function
atherosclerosis
N/A
Reduced T cell
Reduced
recruitment to lesions
atherosclerosis
Impaired ILC3
Impaired Th17 effector
Increased
Danzaki et
effector function1
function
atherosclerosis
al
Impaired ILC3
Impaired Th17 effector
Reduced
Rattik et al
function
atherosclerosis
N/A
No net effect on
Engelbertsen
atherosclerosis
et al
effector function -/-
Ldlr Il17
-/-
IL-17 treatment of
1
-/-
Ldlr mice -/-
Apoe Il17a
-/-
Apoe-/-Il22-/-
effector function
1
Butcher et al
Butcher et al
Taleb et al
Pan ILC Anti-CD90.2 mAb treatment of
Depletion of +
CD90 ILCs
Rag1-/-Ldlr-/- mice 1
ILCs were not investigated in these studies. The assumed effect on ILCs listed above has been inferred
from work by other investigators using similar knockout mice or treatments. BMT: bone marrow transfer.
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S0014-2999(17)30289-3 http://dx.doi.org/10.1016/j.ejphar.2017.04.030 EJP71187
To appear in: European Journal of Pharmacology Received date: 8 December 2016 Revised date: 23 March 2017 Accepted date: 20 April 2017 Cite this article as: Daniel Engelbertsen and Andrew H. Lichtman, Innate lymphoid cells in atherosclerosis, European Journal of Pharmacology, http://dx.doi.org/10.1016/j.ejphar.2017.04.030 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Innate lymphoid cells in atherosclerosis
Daniel Engelbertsen*, Andrew H. Lichtman
Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA
*
Corresponding Author: Daniel Engelbertsen, Department of Pathology, Brigham and Women's Hospital
and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA. email: [email protected]
Abstract The family of innate lymphoid cells (ILCs) consisting of NK cells, lymphoid tissue inducer cells and the ‘helper’-like ILC subsets ILC1, ILC2 and ILC3 have been shown to have important roles in protection against microbes, regulation of inflammatory diseases and involved in allergic reactions. ILC1s produce IFN-γ upon stimulation with IL-12 and IL-18, ILC2s produce IL-5 and IL-13 responding to IL-33 and IL25 while ILC3s produce IL-17 and IL-22 after stimulation with IL-23 or IL-1. Although few studies have directly investigated the role for ILCs in atherosclerosis, several studies have investigated transcription
factors and cytokines shared by ILCs and T helper cells. In this review we summarize our current understanding of the role of ILC in atherosclerosis and discuss future directions.
Keywords: Innate lymphoid cells; atherosclerosis; inflammation; mouse models; cytokines
1. Introduction Leukocytes are subdivided into a wide variety of distinct cell lineages,each of which plays different roles in preventing infectious disease and promoting tissue homeostasis and barrier function. Innate lymphoid cells (ILCs) are a recently discovered addition to the expanding family of immune cells consisting of several distinct subsubsets. Experiments in mice have demonstrated that ILCs influence other cells by two main mechanisms: production of cytokines and other soluble factors or cytotoxic effector function. NK cells were once considered to be the only innate cell derived from the common lymphoid precursor lacking a T-cell receptor. The group of known innate lymphoid cells was expanded when lymphoid tissue inducer (LTi) cells, important for lymphoid tissue generation, were discovered in 1997 (Mebius et al.). LTi (now classified as an ILC3 subset) produces Lymphotoxin α/β and RANKL important for lymphoid organogenesis. (Walker et al.). Around 2010, cells producing IL-17 and IL-22 residing in gut and expressing natural cytotoxicity markers (NKp46 and NKp44), the transcription factor RORγT and the receptor for IL-7 (IL-7R) were described (Sanos et al.). Simultaneously, non-cytotoxic IL-7R+ ILCs were characterized that responded to Thymic stromal lymphopoietin (TSLP), IL-33 and IL-25 by the secretion of type 2 cytokines IL-5 and IL-13 (Moro et al.; Neill et al.; Price et al.). Reasons for the relatively late discovery of non-cytotoxic ILCs was (i) their relative scarcity in lymphoid tissue (<1% of all cells in spleen and blood) and (ii) the lack of cell surface markers specific for ILCs. This review will discuss the role of ILCs in atherosclerosis, evaluating recent findings and providing re-interpretations of past discoveries. Lineage tracing experiments have shown that non-cytotoxic ILCs (ILC1, ILC2 and ILC3) as well as NK cells are derived from common lymphocyte precursors in the bone marrow. A committed ILC/NK precursor expressing NFIL3 and α4β7+ can give rise to all ILC subsets: NK cells, ILC1, ILC2 and ILC3 (including LTi cells) (Geiger et al.; Klose et al.). Non-cytotoxic ILCs display a similar subdivision as CD4+ T cells: T-bet or EOMES-expressing ILC1, GATA3-expressing ILC2 and RORT expressing ILC3,
analogous to T helper (Th)1, Th2, and TH17 cells respectively (Walker et al.). Cytokine production is also related between T helper and ILC subsets: ILC1 cells produce IFN-γ, ILC2 cells produce IL-4, IL-5 and IL-13 and ILC3 cells produce GM-CSF, lymphotoxin-α/β, IL-17 and IL-22 (depending on ILC3 subset). Mature ILCs are defined by lack of expression of myeloid or lymphoid lineage markers (negative for CD11b, CD11c, Gr1, CD3 etc.) and by expression of IL-7R and CD90 (Thy1). It is clear that some of these lineages are plastic: ILC3s may under certain conditions transform to ILC1 (Klose et al., 2013) and human ILC2s can be induced to produce IFN- after IL-12 stimulation (Silver et al., 2016). Recent studies in mice have demonstrated that ILCs are predominantly resident cells that arise during early development and are maintained in tissues by local proliferation rather than being replenished by circulating precursors (Gasteiger et al.). Levels of circulating ILCs are high in human cord blood then decline during childhood reaching even lower levels in adults (Vely et al.). In adult mice and humans, most ILCs are found in epithelial barrier tissues, including skin, gut, and lungs, although they are also present in adipose tissues, joints, and various other tissue sites (Klose and Artis, 2016). Classical NK cells (Bjorkstrom et al.) and ILC3s (Doisne et al., 2015) are present relatively abundant in the uterus, but their precise role there is unknown. Unlike T cells, activation of cytokine production by ILCs is not contingent upon antigen recognition (ILCs do not express TCR) but rather is induced by integrating cytokine signals produced by myeloid or epithelial cells (Klose and Artis, 2016). IL-12 and IL-18 promote ILC1 production of IFN-γ, IL-33 and IL-25 promotes ILC2s production of type 2 cytokines while IL-23 and IL-1 trigger ILC3 cytokines. 2. ILCs and atherosclerosis To this date, few studies have directly investigated the role of ILCs in atherosclerosis. However, there are several studies performed in the ‘pre-ILC-era’ where the primary aim of the study was to study T cell responses but where ILCs most likely also were affected. We have summarized what we believe to be relevant studies in order to gain insight into possible contributions of ILCs in the pathogenesis of
atherosclerosis (Table 1). It is important to note that it is difficult to properly predict the how ILCs will respond to loss of certain factors. For example, mice deficient in T-bet and Rag1 exhibit colitis that is due to exaggerated ILC3 response and high levels of IL-17 (Powell et al.). However, given the current scarcity of mechanistic studies of ILCs in atherosclerosis, we believe that this is a useful approach. 2.1. ILC1 and atherosclerosis Many studies have demonstrated the link between Th1 immunity and atherosclerosis (Witztum and Lichtman). Mice lacking T-bet (Buono et al.), IFN- γ (Buono et al.) and IL-12(Davenport and Tipping) all display reduced plaque burden. These studies have been largely interpreted as proof of a role for Th1 immunity, but it still remains unclear the extent to which ILC1s (or other innate sources of IFN-γ) are influencing the phenotype observed in these mice. Recent studies have demonstrated that IL-7R+ ILC1s, but not EOMES+ cNK cells, are dependent on T-bet for development (Klose et al.). The role of cytotoxic NK cells in atherosclerosis is reviewed in this issue of European Journal of Pharmacology by Bobik and colleagues. In brief, there is evidence that ILC1 (including NK cells) may play a role in atherosclerosis. Depletion of NK cells using anti-Asialo-GM1 antibodies reduced atherosclerosis in apoe-/- mice (Selathurai et al.). Transfer of NK cells to Apoe−/−Rag2−/−Il2rg−/− mice increased plaque formation and the pathogenicity of NK cells was found to be contingent on cytotoxic activity rather than production of IFN-γ. Given the continued lack of defined auto-antigens in mouse models of atherosclerosis, it remains an intriguing possibility that innate sources of IFN-γ from cytotoxic (classical NK cells) and noncytotoxic ILC1 may significantly contribute to lesion development. Arguing against an important role for NK cells in cardiovascular disease, NK cells are found in very limited numbers in the human atherosclerotic plaques and only represent a minor fraction of all leukocytes found in lesions (Bobryshev and Lord; Millonig et al.).
2.2. ILC2 and atherosclerosis In a study predating the discovery of ILCs, mice lacking RORα developed larger aortic root fatty streak lesions when fed a high fat diet, compared to C57BL/6 controls (Mamontova et al., 1998), although the mice had abnormal lipoproteins and displayed ataxia. RORα was subsequently shown to be required for ILC2 development. ILC2s produce type 2 cytokines and are enriched in the lung and adipose tissue. Perry and colleges demonstrated that aortic CD90+ST2+Sca-1+cKit (CD117)+ ILC2s (termed natural helper cells) produce IL-5 to trigger B1 cell proliferation and production of natural IgM (Perry et al.). Our lab sorted CD90+IL-7Rα+CD25+ ILC2s from digested aortas and demonstrated production of type 2 cytokines from these cells after stimulation with PMA and ionomycin (Engelbertsen et al.). In both these studies ILC2s were predominantly located in aortic periadventitial adipose tissue but virtually absent in atherosclerotic lesions. There are yet no published reports of ILC2s in human atherosclerotic plaques. ILC2s are activated by IL-33, IL-25 and TSLP produced mainly by epithelial cells in response to injury or inflammation (Sonnenberg and Artis). All these three cytokines have been studied in the context of atherosclerosis. Treatment of mice with IL-33 reduced atherosclerosis while injections of soluble ST2 (the receptor for IL-33) increased atherosclerosis (Miller et al., 2008). The protective effect of IL-33 was lost when co-treating with IL-5 blocking antibodies. ILCs were not directly assessed by this study, but many others have demonstrated the potent ILC expanding effects of IL-33 (Sonnenberg and Artis). It seems likely that at least part of the anti-atherogenic effects demonstrated in this study is due to expansion of ILC2s. Similarly, Mantani et al. demonstrated that treatment of Apoe-/- mice with IL-25 reduced atherosclerosis (Mantani et al.). Treatment with IL-25 boosted ILC2 cell numbers and serum levels of ILC2 cytokines (IL-5, IL-9 and IL-13) and was associated with increased levels of anti-phosphorylcholine (PC) natural IgM. Notably, IL-5 deficient mice did not exhibit increased natural IgM upon IL-25 treatment. In another study, injecting ApoE null mice with TSLP reduced plaque formation and was associated with reduced macrophage infiltration and an increase in lesional regulatory T cells (ILCs were not investigated in this study) (Yu et al., 2013). Notably, injections of TSLP dramatically boosted IgM
levels suggesting that the ILC2-B1 cell axis is affected by the treatment. Binder et al. reported that transfer of IL-5-deficient bone marrow to Ldlr-/- recipients increased plaque formation relative to controls. This effect was associated with reduced natural IgM produced by B1 cells (Binder et al.). It is unclear to what extent the effect reflects impaired function of Th2 cells or ILC2s. Recent studies have demonstrated that serum levels of IL-5 are similar in T cell deficient Rag1-/- mice compared to wildtype controls, while ILC and T cell-deficient IL7Ra deficient mice do not exhibit measurable levels of serum IL-5 (Nussbaum et al.). Altogether, these studies indicate that the IL-33/IL-25/TSLP-ILC2-IL-5-IgM axis inhibits lesion development. Another key cytokine produced by ILC2s, but also by Th2 cells, is IL-13. Treatment of mice with rIL-13 increased plaque collagen deposition and polarized macrophages towards a “M2” like phenotype. Transfer of IL-13-deficient bone marrow increased lesion size and decreased M2 macrophage polarization (Cardilo-Reis et al.). It is unclear to what extent homeostatic or ‘hyperlipidemic’ IL-13 production is derived from ILC2s or Th2 cells and what cell type is responsible for the observed atheroprotective effect of IL-13. Recently, our lab investigated the effects of anti-CD90.2 mediated ILCs depletion or IL-2/anti-IL-2 (antiIL2 clone: JES6-1) mediated ILC expansion in Rag1-/-Ldlr-/- mice that lack adaptive immunity. The results were mixed: ILC depletion did not alter plaque progression while IL-2/anti-IL-2 treatment (expanding mainly ILC2 cells) reduced lesion burden (Engelbertsen et al.). Explaining part of the phenotype observed in ILC2 expanded mice, we observed a reduction in cholesterol accompanied with increased levels of triglycerides. Analysis of liver RNA revealed decreased transcription of Apob after ILC2 expansion, potentially explaining the reduced cholesterol levels in serum. IL-2/anti-IL-2 treatment also caused increased leukocyte recruitment to the liver which exhibited early signs of fibrosis. It is unclear whether the effects on cholesterol and lipid levels are due to physiologically relevant effects of ILC2 or just pathologic effects of chronic ILC2 activation leading to hepatic damage and scarring. Although IL-2/anti-IL-2 treatment was associated with dramatically elevated IL-5 levels and eosinophilia,
blockade of IL-5, which also reversed the eosinophilia, did not alter lesion size or lipid levels. It is important to note that although we observed a predominant ILC2 type response after treatment with IL2/anti-IL-2, CD25 is also expressed on certain subsets of ILC3 (Dumoutier et al.) and ILC1 cells (Gasteiger et al.). Anti-CD90.2 antibody treatment was efficient in depleting ILCs in lymphoid organs but did not fully deplete tissue ILCs (as has been reported by others (Roediger et al.)), complicating the interpretation of the study. Moreover, the lack of adaptive immunity in the Rag1-/-Ldlr-/- mouse obscures potential effects of ILC2s interacting with B cells or even T cells, as they have recently been proposed to perform class II MHC restricted antigen presentation and promote Th2 responses (Oliphant et al.). Further, the lack of B and T-cells in Rag-deficient mice may affect ILCs due to decreased competition for ILC stimulating cytokines like IL-7 and IL-2. In summary, activation of ILC2s is associated with reduced atherosclerotic burden and mice lacking ILC2 effector cytokines display increased atherosclerosis. Most studies are in line with type 2 immunity being atheroprotective, however it is unclear to what extent ILC2s are important in themselves and by what mechanisms they affect atherogenesis. Notably, ILC2s have also been shown important in regulation of adipose tissue homeostasis, promoting ‘beiging’ of white adipose tissue and limiting obesity (Brestoff et al.). In summary, current knowledge points to three mechanisms by which ILC2s (may) reduce atherosclerosis: (i) the IL-33/IL-25/TSLP-ILC2-IL-5-IgM axis, (ii) modulation of lipid homeostasis and (iii) modulation of lesional macrophages. 2.3. ILC3 and atherosclerosis ILC3s produce a range of cytokines that is shared with Th17 cells: IL-17, IL-22 and GM-CSF. The proinflammatory properties of Th17 cells in other immune diseases (experimental autoimmune encephalitis (Langrish et al., 2005), psoriasis (Elloso et al., 2012) and rheumatoid arthritis (Lubberts, 2008)) have prompted investigations of Th17 cytokines in the context of atherosclerosis. However, Th17 cytokines are not only produced by Th17 cells but also by innate cells including γδ T cells, mucosal-associated
invariant T cells (MAIT cells) and ILC3s. T cells and ILC3s reside in mucosal tissue and skin where they maintain barrier function through production of IL-17 and IL-22, sharing many effector functions with Th17 cells. Genetic deletion of IL-17 or the IL-17RA receptor in the Ldlr-/- resulted in reduced atherosclerotic lesion formation (Butcher et al.). However, other studies have indicated opposite effects of IL-17 on atherosclerosis. For example, there was increased atherosclerosis and reduced lesional vascular smooth muscle cells and type I collagen in Apoe-/-Il17A-/- mice compared to Apoe-/- controls (Danzaki et al., 2012). Another study showed decreased atherosclerosis after IL-17 treatment and increased atherosclerosis after blocking anti-IL-17 antibody injections (Taleb et al.). IL-22 acts on nonhematopoietic cells such as epithelial cells to promote barrier function and limit microbial invasion by increased production of defensins but has also been implicated in inflammatory skin diseases such as psoriasis. Rattik and coworkers demonstrated that Apoe-/-Il22-/- mice displayed reduced atherosclerosis and plaque collagen deposition compared to Apoe-/- controls (Rattik et al., 2015). This effect was attributed to effects on vascular smooth muscle cells that were found to express the IL-22 receptor. Overall, studies investigating the impact ILC3-associated cytokines on atherosclerosis have been inconsistent. Furthermore, the anatomical localization of ILC3s to mucosal tissue puts into question its relevance in atherosclerosis. The presence of ILC3s in murine and human atherosclerotic lesions remains uncertain, indicating that these cells are not present at high frequencies. We did not observe IL-17 production from either CD25+ or CD25- ILCs (lineage-CD90+IL-7Rα+) isolated from atherosclerotic mouse aortas (Engelbertsen et al.) 3. ILC mouse models: Complexity vs. Clarity Studying ILCs presents the researcher with a specific set of obstacles. As an example, ILC depletion using anti-CD90.2 antibodies is only possible in immunodeficient mice, as T cells also express CD90. Moreover, these antibodies are not efficient in depleting tissue resident ILCs. Mice deficient in important ILC transcription factors (GATA-3, T-bet, RORγT, TOX, Id3) also display defects in other leukocyte
subsets including T cells. A potential problem in using Rag-deficient mice to study ILCs has emerged by the surprising finding that Rag proteins are expressed during ILC development and have functions unrelated V-D-J recombinase activity (Karo et al., 2014). Thus Rag-deficient ILCs may be intrinsically abnormal. To solve these issues, mouse models where a certain subset of ILC loses its effector function (i.e. ILC2 specific IL-5 knockout) or is depleted (ILC2 specific knockout mice) would represent the best option. Recently, an ILC2-specific knockout mouse was described where RORα is deleted in cells expressing IL7R (Rorαfl/sg Il7r-Cre) (Oliphant et al.). IL-7R is expressed by T cells and ILCs but the authors claimed no effect on T cells, although others have reported defects in Th17 cells in Rorα-/- mice (Yang et al.). As more sophisticated mouse models enter the scene it is becoming clear that they will be in the form of double or triple knock-out/transgene mice. Further complicating the logistics of such studies, experimental atherosclerosis models has classically required a hyperlipidemic background such as Apoe-/or Ldlr-/-, demanding another knockout/transgene to be introduced. We believe that models of inducible atherosclerosis, for example injection of adeno-associated virus encoding a mutant form of proprotein convertase subtilisin/kexin type 9 (PCSK9) that efficiently cause LDLr degradation and subsequent hypercholesterolemia (Bjorklund et al.) are needed in order to facilitate studies of ILCs in atherosclerosis. Bone marrow transfer of ILC-deficient bone marrow into Ldlr-/- mice may be possible for some cases, but it is not clear to what extent bone marrow-derived ILCs outcompetes host ILCs which may or may not be eliminated by irradiation. 4. Translational aspects and future directions A recent study has raised questions about the role of ILCs in humans. Patients with severe combined immunodeficiency (SCID) due to inherited deficiency of either IL-2Rγc or JAK3 were found to lack both T cells and ILCs, whereas RAG-1-deficient SCID patients lacked T cells but had an intact ILC compartment (Vely et al.). Patients with both forms of SCID were treated with hematopoietic stem cell
transfer (HSCT) that reconstituted the T cell compartment but failed to reconstitute circulating or tissue ILCs in the in the patients with IL-2Rγc or JAK3 deficiency. When comparing incidence of disease in HSCT-treated SCID patients during follow-up (7-39 years), IL-2Rγc or JAK3 deficient SCID patients (‘ILC deficient’ patients) did not exhibit any increase in inflammatory disease or opportunistic infections compared to ILC-sufficient SCID patients. Although this study was not powered or designed to estimate the risk of cardiovascular disease in patients lacking ILCs, it is striking that ILCs were redundant. Importantly, this study does not preclude the possibility of ILC deregulation or chronic activation being pro-inflammatory and causing disease. What therapeutic targets are conceivable? Globally boosting ILCs may be associated with serious side effects. The risk of inducing type 2 related immunopathology (e.g. asthma and allergies) argue against this notion as ILC2s are increased in blood (Bartemes et al.) and sputum (Smith et al.) from allergic asthma patients and enriched in skin lesions of atopic patients (Kim et al.). Conversely, reducing ILC1 and ILC3 may reduce barrier function and lead to opportunistic infections or intestinal disease. The more attractive but for now elusive goal, is to selectively enhance ILC2 responses and/or decrease ILC1 and 3 responses in the arterial wall. 5. Conclusions The discovery of ILCs has prompted reevaluation of previous work intended to investigate the role of T cell subsets. In this review we have summarized such studies, along with the very few papers designed to directly assess the role of ILCs in atherosclerosis. In summary, promotion of a type 2 immune response, associated with ILC2s, appear to be consistently related with reduced atherosclerosis. Future studies are required to demonstrate the potential role of ILC2s in mediating this effect. ILC1s produce cytokines related to progression of atherosclerosis making these cells potential culprits and targets for further investigations. ILC3-related cytokines have mostly been associated with atheroprogression but the
relevance of this population remains unclear. Determining the presence (or absence) of these cells in the human atherosclerotic plaque remains an important objective. Acknowledgements The authors were supported by a postdoctoral fellowship from the Swedish Research Council (D.E.) and grants from NIH R01 HL087282 (A.H.L.).
Table 1. Innate lymphoid cells in atherosclerosis Effect on ILCs
Effect on other
Effect on
Reference
leukocytes
atherosclerosis
Impaired Th1/NK
Reduced
effector function
atherosclerosis
N/A
Increased
Selathurai et
atherosclerosis
al
Loss of Th1 cells,
Reduced
Buono et al
cytotoxic T cells
atherosclerosis
Reduced ILC1
Impaired
Reduced
Davenport et
activation1
Th1/NK/cytotoxic T
atherosclerosis
al
Miller et al
ILC1-related Ifn-/-Ldlr-/-
Impaired ILC1 effector function
NK cell transfer to Apoe
−/−
−/−
Rag2
1
N/A −/−
Il2rg
Buono et al
mice Tbet-/- Ldlr-/Apoe-/-Il12-/-
ILC1 deficiency1
cells function ILC2-related IL-33 treatment of
Increased levels
Increased Th2
Reduced
Apoe-/ -mice1
of ILC2s1
immunity
atherosclerosis
IL-25 treatment of
Increased levels
Increased Th2
Reduced
Mantani et
of ILC2s
immunity, increased
atherosclerosis
al
Increased Tregs,
Reduced
Yu et al
reduced plaque
atherosclerosis
-/-
Apoe mice
IgM production TSLP treatment of -/-
Apoe mice
Increased levels of ILC2s
1
macrophages Il5-/- BMT to Ldlr-/-
IL-13 treatment of Ldlr
Impaired ILC2
Impaired IgM
Increased
effector function1
production
atherosclerosis
N/A
Macrophages and B
Increased plaque
Cardilo-Reis
cells
collagen
et al
Impaired ILC2
Macrophage and B
Increased
Cardilo-Reis
effector function1
cells
atherosclerosis
et al
-/-
Il13-/- BMT to Ldlr-/-
IL-2/anti-IL-2 treatment of Rag1-/-Ldlr-/- mice
Expansion of ILC2
Eosinophilia, CD25 ILC3/ILC1
+
Binder et al
Reduced atherosclerosis
Engelbertsen 2
et al
ILC3-related Ldlr-/-Il17ra-/-
Impaired ILC3
Impaired Th17 effector
Reduced
function
atherosclerosis
Impaired ILC3
Impaired Th17 effector
Reduced
effector function1
function
atherosclerosis
N/A
Reduced T cell
Reduced
recruitment to lesions
atherosclerosis
Impaired ILC3
Impaired Th17 effector
Increased
Danzaki et
effector function1
function
atherosclerosis
al
Impaired ILC3
Impaired Th17 effector
Reduced
Rattik et al
function
atherosclerosis
N/A
No net effect on
Engelbertsen
atherosclerosis
et al
effector function -/-
Ldlr Il17
-/-
IL-17 treatment of
1
-/-
Ldlr mice -/-
Apoe Il17a
-/-
Apoe-/-Il22-/-
effector function
1
Butcher et al
Butcher et al
Taleb et al
Pan ILC Anti-CD90.2 mAb treatment of
Depletion of +
CD90 ILCs
Rag1-/-Ldlr-/- mice 1
ILCs were not investigated in these studies. The assumed effect on ILCs listed above has been inferred
from work by other investigators using similar knockout mice or treatments. BMT: bone marrow transfer.
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