Tissue-Dependent Adaptations and Functions of Innate Lymphoid Cells

In mice, NK cells account for ~3% of cardiac immune cells, ILC2s for ~1.7%, and ILC1s for 0.2%, while ILC3 abundance is negligible ( 330 ). Compared to lung ILC2s, murine cardiac ILC2s had lower expression of ICOS, CD25, and Ki-67, and higher expression of Sca-1 and GATA3 ( 330 ). Only 2% of cardiac ILC2s in mouse were donor-derived after 2 months of parabiosis, indicating that ILC2s are a stable tissue-resident population in the heart ( 330 ). Cardiac-resident ILC2s respond to IL-33 but not IL-25, and a committed cardiac ILC2 precursor (ILC2p) in mice and humans exists in a quiescent state with the capability to differentiate into ILC2s in response to myocardial infarct or myocarditis ( 331 ). The existence of undifferentiated ILC2p within tissues has been observed before and suggests a role for this pool of precursors as a reservoir for ILCs to protect from tissue damage ( 332 ). Cardiac ILC activity has been implicated in several disease models ( Figure 10 ). ILCs in the heart. Limited studies have examined ILCs in the heart. In mice, IL-33 administration or CD27 + Sca-1 + fibroblast-derived IL-33 expands ILC2s in the pericardium, driving cardiac fibroblasts to secrete CCL-11/eotaxin, leading to the accumulation of eosinophils. In humans, CD127 + ILCs increase in the pericardial fluid during in cardiac disease, particularly during pericarditis. Separately, the expansion of ILC2s by IL-33 was cardioprotective after injury, reducing fibrosis. Within 12 hours of acute ST-segment elevation myocardial infraction (STEMI), ILC1 elevation peaks. Higher ILC1-associated IL-12, IL-18, IFN-γ, and TNF-α levels lead to a higher risk of major adverse cardiovascular events. Created with Biorender.org . Conflicting findings on ILC subset functions in atherosclerosis and coronary artery disease (CAD) have been reported. Selathurai et al. found murine NK cells were detrimental to atherosclerotic disease, increasing lesion size in a Perforin- and Granzyme B-dependent manner, while Nour-Eldine et al. found no effect of NK cells on lesion development using a distinct genetic depletion model, likely accounting for divergent findings ( 333 , 334 ). In humans, a study of acute ST-segment elevation myocardial infarction (STEMI) found elevated circulating ILC1s within 12 hours of symptom onset which produced more IL-12, IL-18, IFN-γ, and TNF-α, and were associated with a higher risk of major adverse cardiovascular events ( 335 ). In contrast, NK cells were reduced and have lower cytotoxicity in CAD patients, both in the case of stable angina and incidence of myocardial infarction or unstable disease ( 336 – 338 ). During follow-up, patients who failed to reconstitute their peripheral NK cells post myocardial infarction had higher levels of serum IL-6 and exhibited characteristics of metabolic syndrome, suggesting poor NK cell recovery corresponds with low-grade inflammation ( 336 ). Recovery of NK cells in CAD patients is potentially self-regulated, as apoptotic NK cells both respond to and produce FasL, which is elevated in serum and correlates with NK cell levels and apoptotic susceptibility ( 339 ). Increased proportions of CD56 bright NK cells were identified in carotid plaques compared to autologous peripheral blood, and greater NK cell infiltration corresponded with symptomatic versus asymptomatic CAD patients ( 340 ). Soluble B7-H6 levels of 250 pg/ml were detected in symptomatic patients but not in asymptomatic patients or healthy controls ( 340 ). Notably, B7-H6 can interact with NKp30, yet further studies are needed to directly assess B7-H6 and NK cell interactions in this context. Circulating NK cells from atherosclerotic patients had higher TIM-3 expression than healthy controls, with the greatest levels in those with unstable plaques ( 341 ). TIM-3 blockade reduced the death of NK cells cultured in TNF-α, suggesting that TIM-3 promotes cytokine-induced NK cell apoptosis in atherosclerosis ( 341 ). Whether NK cells are preferentially recruited to unstable carotid plaques, or functionally contribute to plaque destabilization requires additional study ( 340 ). ILC2s appear to have cardioprotective functions based on mouse models. ILC2s protect from cardiac fibrosis and are enhanced by exogenous IL-33, producing AREG and BMP-7 to support cardioprotective responses to injury ( 342 ). Expansion of ILC2s reduces atherosclerosis severity and lesion size, while genetic ablation ( Staggerer/Rora Flox -CD127 Cre ) exacerbates disease ( 343 , 344 ). Notably, protection is IL-5 and IL-13-dependent, recruiting eosinophils and polarizing macrophages towards an anti-inflammatory phenotype ( 342 , 343 ). Further, pericardial and cardiac ILC2s expand early post-experimental myocardial infarction, peaking at day 3 before returning to homeostatic levels, while the absence of ILC2s impairs cardiac remodeling and results in larger areas of scarring ( 345 ). Together, this supports a role for cardiac-resident ILC2s in directing repair pathways in response to injury. ILC expansion has been observed in patients with pericarditis ( 346 ). In contrast to cardioprotective findings above, ILC2s have been implicated in pericarditis pathology. Exogenous IL-33 expanded murine pericardial ILC2s, driving cardiac fibroblasts to secrete CCL11/eotaxin-1 and recruit eosinophils, initiating pericarditis ( 346 ). Pericardial fluid from humans revealed an elevated frequency of CD127 + ILCs in patients with cardiac disease versus controls, indicating that ILCs are also involved in human pericardial pathology ( 346 ). In an opposing role, NK cell depletion led to greater inflammation and fibrosis of the heart, dependent on NK cell-mediated prevention of eosinophilic infiltration ( 347 ). A possible cross-regulation of NK cells and ILC2s during cardiac inflammation should be investigated. Sex differences in mortality and morbidity of Coxsackievirus B3 (CVB3) viral myocarditis may also reflect sex-based regulation of NK cell function. Male mice experience greater morbidity and mortality from myocarditis following CVB3 infection, with increased IFN-γ + NK cell infiltration in cardiac tissue ( 348 ). Ovariectomized or sexually immature female mice show similar susceptibility to infection-triggered myocarditis when compared to male mice, while estrogen-treated male mice had ameliorated myocarditis ( 348 ). CVB3-stimulated NK cells cultured with estrogen down-regulated T-bet expression and consequently had reduced IFN-γ production, indicating that regulation of T-bet expression by estrogen might underlie the decreased IFN-γ + NK cell infiltration in female mice and contribute to sex differences in myocarditis, in line with prior reports of hormonal regulation of other ILC subsets ( 100 , 348 ).

Innate lymphoid cells (ILCs) orchestrate immune responses to signals such as cytokines, alarmins, neuropeptides and hormones, interacting with hematopoietic and non-hematopoietic cells alike. ILCs lack rearranged antigen receptors and while predominantly tissue-resident, are also observed in circulation and secondary lymphoid tissues where they exhibit distinct spatial and temporal functions ( 1 ). Outside of roles in immunity, ILCs have key roles in maintaining tissue homeostasis, promoting tissue repair, and regulating inflammation. Via crosstalk with parenchymal cells, ILCs are also involved in processes previously thought to lack immune system influence, such as thermal regulation, neuronal signal transduction, circadian rhythms, and tissue remodeling ( 2 – 6 ). The regulation of both immune functions and tissue-specific processes by ILCs highlights the importance of understanding how they respond and function within tissue niches, and conversely how ILC biology is controlled by the microenvironment in which they reside. Development of ILCs in non-lymphoid tissues occurs when circulating ILC progenitors seed tissue niches, and requires the expression of local survival factors including IL-7 and thymic stromal lymphopoietin (TSLP) ( 7 , 8 ). Differentiated ILCs express signature cytokines and transcription factors that parallel CD4 + and CD8 + T cells in both humans and mice ( Figure 1 ) ( 6 , 8 ), and can be broadly categorized as cytotoxic (NK cells) or non-cytotoxic ‘helper’ ILCs. Human NK cells express TBET and Eomesodermin (EOMES), release IFN-γ and TNF-α and are grouped into CD56 dim CD16 + or CD56 bright CD16 - NK cells. CD56 dim CD16 + NK cells express killer cell immunoglobulin-like receptors (KIRs) and exhibit profound cytotoxic potential ( 6 , 8 ). CD56 bright CD16 - NK cells lack KIR expression but are superior producers of IFN-γ and TNF-α ( 9 , 10 ). NK cells discriminate between self and non-self or altered-self and function in anti-viral and anti-tumor immunity similar to CD8 + cytotoxic T cells ( 6 , 8 ). ‘Helper’ ILC (hILCs) are non-cytotoxic and are classified based on function and development into Group 1 (ILC1s), Group 2 (ILC2s), Group 3 (ILC3s) as well as Lymphoid Tissue inducer LTi cells ( 6 ). ILC1s produce IFN-γ in a TBET-dependent but EOMES-independent manner ( 6 ). ILC2s express GATA-3 and RORα and secrete interleukin (IL)-4, IL-5, IL-9, IL-13 and Amphiregulin (AREG), aiding in anti-parasite immunity or the promotion of allergic responses ( 6 ). ILC3s rely on the transcription factor RORC and produce IL-22, IL-17, and GM-CSF ( 6 ). ILC3s include subsets which express natural cytotoxicity receptors (NCRs) NKp44 (human) and NKp46 (mouse and human). LTi cells express ILC3-associated transcription factors and cytokines but also express surface Lymphotoxin (sLT) ( 11 ). Further, ILCs with immunosuppressive activity have been identified in cancer, intestinal inflammation, allergy, autoimmunity and ischemia reperfusion injury ( 12 – 18 ). These include both NK-like ILCs, IL-10 producing ILC2s (ILC2 10 ) and ID3 + regulatory ILCs [reviewed in Jegatheeswaran et al. ( 19 )]. Despite growing appreciation of ILCs with regulatory functions, their development and function are poorly characterized, particularly in humans. Common phenotypic markers of mice (top) and human (bottom) ILCs and their common cytokine expression profiles. Cytotoxic Natural Killer cells can be subdivided into two major subsets based on surface marker expression in both mice and humans. In mice, NK cells are subdivided into two subsets based on CD27 and CD11b expression: CD27 high CD11b - immature NK cells and mature CD27 low CD11b + NK cells. In humans, CD56 bright CD16 - and CD56 dim CD16 + are generally used to identify immature and mature NK cells in blood. However, tissue NK cells often display a CD56 bright CD16 - phenotype. ILC1s, ILC2s, and ILC3s are classified based on surface marker and transcription factor expression profiles that parallel CD4 + T helper subsets. ILC3s are further subdivided into natural cytotoxicity receptor (NCR) + and NCR - subsets. Created with Biorender.org . Mouse studies identified central roles for ILCs in regulating tissue homeostasis, repair and remodeling, transforming our understanding of cellular interactions between immune cells and the tissues in which they reside. Across tissue microenvironments, ILCs adapt and acquire distinct phenotypes and functional properties ( Figure 2 ). While ILC subsets have important functions within these tissues, dysregulation of ILC numbers and functions is associated with diverse human pathologies including arthritis, diabetes, psoriasis, asthma, and inflammatory bowel disease [reviewed in ( 20 )], highlighting the need to identify how local tissue factors promote or inhibit inflammatory ILC responses. Within this review, we explore NK cell and hILC biology across different tissues in health and disease, highlighting evidence of similarities between human and mouse ILC function where data is available. We summarize current understanding of organ-specific functions of ILCs, focusing on their contributions to tissue homeostasis, host-defense, and inflammatory disease progression across the body. Body-wide distribution and surface phenotypes of human ILCs. Surface marker expression of CD56 dim NK cells (teal), CD56 bright NK cells (dark blue), ILC1s (purple), ILC2s (green), NCR - ILC3s (red) and NCR + ILC3s (pink) in nervous system, lung, heart, liver, kidney, gut, reproductive system, adipose tissue, and skin. ILC subsets that have not yet been identified during steady state visualized in lighter color. Represented selection of ILC markers is based on the consistent use of these markers across multiple independent studies. Created with Biorender.org .

Adipocytes are critical regulators of energy and glucose homeostasis. They are a heterogeneous population of cells, comprising energy-storing white adipocytes, thermogenic brown adipocytes that express uncoupling protein-1 (UCP-1) to dissipate energy as heat, as well as beige adipocytes that reside within white adipose tissue (WAT) and upregulate UCP-1 in response to environmental cues ( 349 ). Beige adipocyte accumulation protects from insulin resistance, and regulation of the beiging process has become an attractive therapeutic target for metabolic dysregulation and type 2 diabetes ( 350 ). ILC2s are the dominant hILC subtype identified in adipose tissue, with phenotypic variation across different murine adipose compartments: ILC2s in para-aortic adipose tissue have an inflammatory phenotype defined by IL-25 responsiveness and high KLRG1 expression, whereas peri-gonadal adipose ILC2s are IL-33 responsive, expressing ST2 ( 343 , 351 ). Intriguingly, mouse ILC2s highly express bone morphogenetic protein ( Bmp)2 and Bmp7 , which promote adipocyte differentiation, while ILC-deficient mice have more CD34 + PDGFRα + precursor adipocytes, supporting a role for ILCs in adipogenesis ( 352 ). Under homeostatic conditions, ILC2s orchestrate immune responses in adipose tissues by recruiting eosinophils, promoting alternative activation of macrophages, and regulating beiging, glucose catabolism, and insulin sensitivity in adipose tissues ( Figure 11A ) ( 353 , 354 ). In support of this, depletion of IL-5 and IL-13-producing cells (mainly ILC2s) corresponded to a reduction of eosinophils and Arg-1 + adipose macrophages in visceral adipose tissue ( 355 ). ILC2s also promote Treg responses through ICOSL and OX40L co-stimulation in adipose tissues, critical for supporting insulin sensitivity ( 356 – 358 ). ILCs in adipose tissue. (A) Using mostly mouse models, ILC2s are linked to adipogenesis, where they maintain homeostasis and prevent obesity by promoting adipose beiging. ILC2s are sustained by IL-33 from adipose stem and progenitor cells (ASPCs) and required stromal cell interactions via ICAM-1 and LFA-1. The resulting release of IL-4, IL-13 by ILC2s promotes eosinophil recruitment via stromal cell-derived eotaxin (CCL11). IL-33 stimulates ILC2-produced methionine-enkephalin (MetEnk), an endogenous opioid-like peptide that promotes adipose beiging. (B) Both obese humans and mice, have reduced ILC2s in the white adipose tissue due to diet-driven impairment of IL-33 production by ASPCs. The infiltration of IFN-γ-producing ILCs actively represses cytokine release by ILC2s and propagates ILC2 inhibition through via PD-1 and macrophage-expressed PD-L1. A unique subpopulation of CSF1R + IL16Rα + NK cells and increased ILC1 abundance positively correlates with glucose intolerance and insulin resistance. Overall, reduction of adipose ILC2s fosters metabolic dysfunction, insulin resistance and obesity. Created with Biorender.org . Mouse studies support that ILC2s can directly promote adipose beiging, supporting homeostasis and preventing obesity ( 5 , 359 , 360 ). ILC2s are sustained by IL-33 from adipose stem and progenitor cells (ASPCs) ( 360 ). Stromal ICAM-1 interactions with LFA-1 on ILC2s promotes ILC2 activation and proliferation in adipose tissue, while IL-4/IL-13 expression by ILC2s induces eotaxin (CCL11) expression in stromal cells, supporting eosinophil recruitment ( 361 ). Peritoneal IL-33 administration expands adipocyte precursors and promotes beige lineage commitment in an ILC2-dependent manner, as effects are abolished in the absence of ILC2s and when adipocyte precursors are not receptive to IL-4/IL-13 signaling ( Il4ra fl/fl Pdgfra Cre ) ( 359 ). An alternative mechanism of ILC2-dependent beiging of mouse adipose tissue was proposed by Brestoff et al. who found IL-33-stimulated ILC2s produce methionine-enkephalin (MetEnk), an endogenous opioid-like peptide which induces WAT beiging ( 5 ). Overall, these studies demonstrate that ILC2-dependent eosinophil-derived IL-4 and ILC2-derived IL-13 and/or MetEnk directly promote murine adipocyte precursor proliferation and beige lineage commitment ( 5 , 359 ). Group 1 ILCs are largely resident in murine adipose tissue ( 362 ). Although their homeostatic role is poorly understood, ILC1s regulate adipose macrophage homeostasis ( 363 ). Alternatively activated macrophages scavenge potentially cytotoxic molecules released during adipose tissue remodeling and upregulate stress ligands (i.e. Rae-1) at steady state, and their selective depletion by adipose type 1 ILCs prevents stress-induced inflammation in macrophages during homeostatic tissue remodeling ( 363 ). Dysregulation of the immune environment associated with obesity can lead to metabolic dysfunction and insulin resistance, driving type 2 diabetes (T2D) ( 364 ). ILC dysregulation has been implicated in obesity ( Figure 11B ). In obese humans and mice, ILC2s are reduced in WAT, possibly due to reduced IL-33 production by ASPCs in response to high fat diet ( 5 , 360 , 365 ). High PD-1 expression on ILC2s reduced IL-5 and IL-13 production, an effect partially rescued by macrophage depletion, suggesting PD-1/PD-L1 interactions between ILC2s and macrophages dampens ILC2 function in obese conditions in mice ( 365 ). Additionally, adipocyte-derived soluble ST2 is induced by obesity and interrupts IL-33 signaling, impairing ILC2 homeostasis ( 366 ). Infiltration of IFN-γ-producing cells also contributes to reduced ILC2 abundance and function, as IFN-γ directly represses ILC2s and counteracts IL-33 ( 357 ). Obese mice fed a high fat diet had adipose tissue-specific IL-12-dependent accumulation of ILC1s with elevated IFN-γ production, resulting in insulin resistance and glucose intolerance ( 362 , 367 ). Interestingly, CD56 dim CD16 – ILCs accumulating during obesity have reduced cytotoxicity, a potential secondary mechanism contributing to macrophage accumulation and glucose intolerance ( 363 ). Wensveen et al. demonstrated NKp46 on adipose-resident mouse NK cells may regulate this effect ( 368 ). High fat diet-induced obesity triggered the expression of NCR1 ligands on adipocytes which promoted local NK cell proliferation and production of IFN-γ, inducing the differentiation of pro-inflammatory macrophages and promoting insulin resistance ( 368 ). Wang et al. further found that the ILC1 IFN-γ-dependent expansion of pro-inflammatory macrophages exacerbated adipose fibrosis by promoting TGF-β1 and pro-fibrotic programs in macrophages, resulting in higher collagen deposition ( 367 ). In agreement with murine models, circulating and adipose ILC1s are increased in obese patients, especially those with T2D, and the abundance of ILC1s positively correlates with measures of glucose intolerance and insulin resistance ( 367 ). A unique subpopulation of CSF1R + IL6Rα + NK cells is expanded in human and murine obesity ( 369 ). Selective depletion of this subset ( Csf1r -loxSTOPlox-DTR x Ncr Cre ) resulted in decreased weight gain, better glucose tolerance, and insulin responsiveness in mice fed a high fat diet ( 369 ). Further, the expression of RORγt, lymphotoxin and IL-22 all elevated weight gain and adipose tissue size, paralleling findings that IL-22 from Th17 cells exacerbates inflammation in obesity ( 370 , 371 ). The regulation of metabolism by intestinal ILC3s suggests a gut-adipose axis that remains to be explored. Overall, ILC2s mediate adipose homeostasis and are dysregulated in obesity, while ILC1s and potentially ILC3s have a role in exacerbating inflammation.

Multiple parallels and differences between murine and human ILCs exist. Their evolutionarily conserved transcriptional programs and functional similarity emphasizes their importance across multiple distinct phylogenetic branches. However, a better understanding of homologies and analogies in their surface receptor expression and function are needed to inform conserved mechanisms underlying responses to infections, inflammation and malignancies. This will be of particular interest to enhance our understanding NK cell biology, where receptors regulating NK cells responses differ between mouse and human, but different receptors often perform similar function. The distinct living conditions of mice and humans require conserved and specified adaptations of organs and tissues to environmental triggers. ILCs as regulators of tissue homeostasis adapt to these species-specific environments. Shared and differing microbiota within humans and mice may explain conserved and distinct functions of ILCs across these organisms. Gnotobiotic technologies, humanized mice, knock-out mouse models or adoptive transfer experiments are suitable to investigate these differences but bring their own pitfalls. Beyond these challenges, a key obstacle for the study of ILCs in situ is the scarcity of ILC-specific models available to tease out cell-specific or even organ-specific functions. Adding to this, ILCs typically function in cellular networks to influence the outcome of a given immune response. To properly understand their function and tease out any redundancy, more systems-based approaches are needed, particularly in humans ( 295 , 372 , 373 ). Other roadblocks in understanding the role of various ILCs in homeostasis and disease are studies designed only to link the presence or absence of ILCs with disease outcome. This is especially evident in reports of NK cell function. Reporting expansion or reduction of NK cells as having a protective or detrimental effect assumes a homogeneous function of NK cells. While classically, NK cell function has been cytotoxic and inflammatory, NK cells have also been cast in an immunoregulatory role where they dampen an immune response ( 19 ). Additionally, identifying NK cells as CD3 - CD56 + does not rule out other non-cytotoxic ILC1s and ILC3s that can share CD56 expression, and does not address the large degree of heterogeneity within NK cells ( 374 ). Untangling the identity and functional capacity of distinct CD3 - CD56 + populations may help to clarify contradictory findings. The plasticity of ILCs makes definitively assigning them as “good” or “bad” quite problematic. Sometimes ILCs with identical or similar surface phenotype may be functionally distinct ( 206 ). While this may be context dependent, ILC plasticity may be partially responsible for conflicting reports of their function in disease. Future studies should consider assessing the functional role of ILC subsets correlated with ILC transcriptional and epigenetic profiles to identify mechanisms underlying distinct ILC functions and whether some level of ‘trained’ immunity contributes to differing findings. Additionally, it remains unclear if the tissue-specific functions of ILCs are due in part to the existence of specific subsets of ILCs that home to their niche, or instead these different functions are a directly due to microenvironment signals leading to niche adaptation. It is also entirely possible that both cases are true and contribute to establishing tissue-specific ILC functions. Moving forward, the characterization of tissue-specific networks and niches for ILCs will transform our understanding of ILC functions and underlying mechanisms controlling their tissue adaptations.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

JMM was supported by Queen Elizabeth II, Dick and Peggy Sharpe, and Peterborough KM Hunter Scholarships. LN was supported by an Ontario Graduate Scholarship and a NSERC-PGS award. AM's research program is supported by Canadian Institutes for Health Research (CIHR, 388337) and Natural Sciences and Engineering Research Council of Canada (NSERC, RGPIN-2019-04521), and is a Tier 2 Canadian Research Chair in Mucosal Immunology. SQC's research program is supported by funding from CIHR (168960 and 169084) and a NSERC (RGPIN-2021-03672), and is a Medicine By Design Investigator (Canada First Research Excellence Fund).

All authors contributed to conceptualization, topic curation, writing and editing of the manuscript. Figures were designed by LN, with input from JM, AM, and SC. All authors contributed to the article and approved the submitted version.