Invariant natural killer T cells in lung diseases

Invariant natural killer T (iNKT) cells are a subset of T cells that are characterized by a restricted T-cell receptor (TCR) repertoire and a unique ability to recognize glycolipid antigens. These cells are found in all tissues, and evidence to date suggests that they play many immunological roles in both homeostasis and inflammatory conditions. The latter include lung inflammatory diseases such as asthma and infections: the roles of lung-resident iNKT cells in these diseases have been extensively researched. Here, we provide insights into the biology of iNKT cells in health and disease, with a particular focus on the role of pulmonary iNKT cells in airway inflammation and other lung diseases.


INTRODUCTION
NKT cells are a unique subset of T cells that share features of both conventional T cells and natural killer cells.Unlike conventional T cells, NKT cells express TCRs that recognize glycolipid antigens loaded on CD1d, which is a nonpolymorphic major histocompatibility complex (MHC) class I-like protein 1 .NKT cells are classified into two distinct subsets (types I and II) on the basis of their TCR repertoire.Type I NKT cells express semi-invariant TCRβ chains combined with an invariant TCRα chain (Vα14 chain in mice and Vα24 chain in humans); these cells are, therefore, also called iNKT cells.They respond particularly strongly to α-galactosyl ceramide (α-GalCer), a marine sponge-derived glycolipid.In contrast, type II NKT cells have diverse polyclonal TCR repertoires that recognize lipid antigens such as sulfatide 2 .Studies on type II NKT cells are very limited due to the rarity of these cells and their lack of clear surface markers 3 .In contrast, iNKT cells have been extensively studied in many contexts because they are relatively abundant and can be readily identified with α-GalCer-loaded CD1d tetramers.The remainder of this review will focus on iNKT cells.
iNKT cells are largely sessile cells that reside in most tissues, including nonlymphoid tissues such as the liver, lungs, intestine, urogenital tract, adipose tissue, and skin 4 .However, their frequency relative to other lymphocytes varies depending on the tissue 5,6 .Thus, in mice, the lymphocytes in the liver are dominated by iNKT cells (10-30% of all lymphocytes).iNKT-cell dominance is also observed in murine adipose tissue (2-8% of all lymphocytes, 8-12% of adipose T cells), lungs (5-10% of all lymphocytes), and spleen (1-3% of all lymphocytes) 6,7 .In humans, iNKT cells are much less frequent in the liver (0.05-1% of all lymphocytes) 8 and spleen (0.5-2% of all lymphocytes).However, they represent 10-25% of the T cells in adipose tissue, similar to their frequency in murine fat 9 .In contrast, the blood and thymus of both mice and humans contain low frequencies of iNKT cells (0.5-2%) 10 .
Although iNKT cells account for only ~1% percent of circulating T cells overall 10 , they often have a powerful immunological effect because of their abundant secretion of cytokines a few hours after activation of their TCR.Their cytokines include T-helper type-1 (Th1) cytokines, namely, interferon-gamma (IFN-γ) and tumornecrosis factor-α (TNF-α); T-helper type-2 (Th2) cytokines, namely, interleukin (IL)-4 and IL-13; T-helper type-17 (Th17) cytokines, namely, IL-17A and IL-22; and the regulatory cytokine IL-10 11 .These cytokines impact various immune cells, ranging from innate immune cells such as macrophages, dendritic cells (DCs), and natural killer cells to adaptive immune cells such as T and B cells.Thus, iNKT cells can regulate both innate and adaptive immune responses.These regulatory activities have been found to play a role in many diseases, including rheumatoid arthritis 12 , asthma 13 , tumors 14 , and infectious diseases 15 .

THYMIC DEVELOPMENT OF INKT CELLS
Similar to conventional T cells, iNKT cells develop in the thymus via four double-negative stages 16 that eventually lead to TCRα chain-expressing CD4 + CD8 + double-positive (DP) thymocytes.These cells then undergo positive selection with CD1dpresented glycolipids on DP thymocytes 17 .This differs from the development of conventional T cells, which are instead positively selected by MHC-presented peptides on thymic epithelial cells.iNKT-cell positive selection is driven by not only TCR activation but also costimulation signals from the signaling lymphocytic-activation molecule (SLAM) receptor on nascent iNKT cells; these signals induce the expression of the transcription factor early growth response protein 2 (Egr2), which is dispensable for conventional T-cell development 18 .Positively selected iNKT cells then enter unique development stages.The earliest is stage 0, which is characterized by CD24 expression.Stage 0 cells transition into the next stages, which can be defined differently depending on the proposed model.The linear maturation model, which is based on the expression of surface molecules such as CD24, CD44, and NK1.1, proposes that stages 1, 2, and 3 are characterized by CD24 lo CD44 hi NK1.1 − , CD24 lo CD44 lo NK1.1 − , and CD24 lo CD44 hi NK1.1 + phenotypes,

GENERAL CHARACTERISTICS OF PULMONARY INKT CELLS
In the mouse lung, iNKT cells account for ~5% of the resident lymphocytes and localize in the interstitial space as well as the vasculature of the lungs 42 .All three iNKT-cell subsets are present in the lung.Interestingly, RNA sequencing analysis of iNKT cells in various tissues showed that pulmonary iNKT subsets share common characteristics in terms of their transcriptome profiles that distinguish them from iNKT cells in other tissues 6,43 .Specifically, all pulmonary iNKT cells, but not other iNKT cells, display high expression of AP-1, other bZIP family members, some NF-κB family members, CTLA-4, CD69, and Nur77 43 .It is likely that this unique transcriptome profile is driven by the homeostatic lung microenvironment since Lee et al. showed that the profile was unchanged by local infection or inflammation 6,43 .This notion is further supported by the fact that lung-resident mucosaassociated invariant T (MAIT) cells, γδ T cells, and alveolar macrophages also bear lung-specific signatures that are different from those of the corresponding cells in other tissues [44][45][46] .

LOCALIZATION OF PULMONARY INKT CELLS
After moving from the thymus into the circulation, iNKT cells accumulate in the lung microvasculature.When the lung tissue is stimulated by airborne antigens or infections that bear the glycolipid or microbial membrane component targets of iNKT cells, the iNKT cells extravasate into the interstitium and bronchiolo-alveolar spaces 42 .This is not observed in other organs; for example, the large numbers of iNKT cells in the liver remain within the microvasculature 40 .The trafficking of iNKT cells to the lung and their extravasation into the lung appears to be driven by (i) the chemokines that are generated by the lung tissue when it encounters airborne iNKT-cell ligands 42 and (ii) iNKT-cell expression of a specific chemokine-receptor profile 47 .The chemokines include thymus-and-activation-regulated chemokine (TARC, also known as CCL17), MIG/CXCL9, and BCA-1/CXCL13 41,48,49 , while the iNKT-cell chemokine receptors include CCR4, CCR9, and CXCR6 47 (Fig. 1a).CCR4, in particular, appears to play a crucial role in iNKTcell migration to the lungs and airways and the subsequent redistribution of iNKT cells within the lung.For example, aerosol administration of exogenous CCR4 ligand (TARC/CCL17) causes iNKT cells to promptly extravasate into the lung tissue 42 .Moreover, antibody-mediated neutralization of CCR4 or CCR4 deletion blocks iNKT cell migration to the lungs, thereby attenuating the airway hyperresponsiveness (AHR) induced in mice by pulmonary administration of antigen or αGalCer [47][48][49] .In addition, asthma patients have elevated levels of both CCR4 ligands (TARC/CCL17 and CCL22, which is also known as macrophage-derived chemokine) in their bronchoalveolar lavage fluid (BALF) [50][51][52][53] .Similarly, mice that are deficient in one component of another key pulmonary iNKT-cell chemokine/chemokine-receptor combination, namely, CXCR6 and its ligand CXCL16, bear significantly fewer iNKT cells within the intravascular compartment of the lungs 40,42 .Similar observations have been made for CCR9, the third pulmonary iNKT-cell chemokine receptor [54][55][56] .
Notably, intravital imaging of the lung tissue showed that neutrophils play a key role in the α-GalCeror Streptococcus pneumoniae infection-induced migration of pulmonary iNKT cells from the vasculature into the interstitium; specifically, neutrophils produce CCL17, which guides the migration of iNKT cells through the lung tissue.Antigen-presenting cells (APCs), including monocyte-derived DCs, also play an important role in iNKT-cell migration: they promote neutrophil extravasation into the lung and present antigen to iNKT cells, thereby halting further migration of these cells 57 (Fig. 1a).

ACTIVATION OF PULMONARY INKT CELLS
iNKT cells are most often activated by recognizing glycolipid antigens presented on CD1d 58 (Fig. 1b).While many immune cells express CD1d on their surface, DCs are the most potent APCs in terms of glycolipid:CD1d-induced iNKT-cell activation 59,60 .While the most potent glycolipid identified to date is α-GalCer 61 , several studies have shown that iNKT cells can also recognize glycolipids from various microorganisms 62 .For example, the transfer of Sphingomonas-pulsed DCs into mice successfully activated lungresident iNKT cells; the stimulatory ligands were found to be αgalacturonosylceramide and α-glucuronosylceramide 63 .Moreover, mycobacterium-derived phosphatidylinositol mannoside can activate iNKT cells 64 .Thus, despite the restricted TCR repertoire of iNKT cells, they may recognize a wider range of glycolipid antigens than initially thought.
Another common mechanism by which iNKT cells are activated is the signaling induced by cytokines 65 from surrounding immune cells, particularly DCs 66 (Fig. 1b).For example, when DCs are activated by the engagement of Toll-like receptors (TLRs) on their surface, they produce IL-12, which successfully activates pulmonary iNKT cells even when CD1d is absent 67 .The importance of this mechanism is supported by the constitutive expression of the IL-12 receptor of iNKT cells and the fact that they rapidly upregulate their STAT4 expression when they encounter IL-12.Similarly, IL-18 and type-1 interferon produced by DCs can also induce pulmonary iNKT cells to secrete their own cytokines [68][69][70] .
Finally, iNKT cells can be activated by their own innate immune sensors, including TLR4.Flow cytometry and confocal microscopy analyses show that iNKT cells express TLR4 on their surface and in their endosomes and that simultaneously stimulating TCR and TLR4 on iNKT cells increases their expression of IFN-γ.Notably, this dual stimulation also decreases iNKT-cell expression of IL-4, which suggests that the TLR4 signaling pathway skews iNKT cells toward type-1 immune responses.Notably, this regulatory skewing appears to be particularly important for several lung diseases.For example, we showed that while the adoptive transfer of LPStreated iNKT cells (LPS stimulates TLR4) suppresses pulmonary fibrosis, it worsens hypersensitivity pneumonitis 71 .
Taken together, these studies suggest that pulmonary iNKT cells can be activated via multiple mechanisms, although the mechanism that plays the most prominent role in lung health and disease remains to be determined.These observations also suggest that pulmonary iNKT cells may be readily activated in pathological environments that bear iNKT-cell antigens, cytokines from other immune cells, and/or TLR-activating molecules.Indeed, it is likely that such environments activate pulmonary iNKT cells via several or all of these mechanisms.Further improving our understanding of these mechanisms is important for enhancing our understanding of pulmonary iNKT cells.

FUNCTIONS OF PULMONARY INKT CELLS iNKT cells exert their immune effects in vivo via several mechanisms.
The most prominent involves iNKT-cell secretion of cytokines and other soluble factors 65 .Consequently, most studies on iNKT-cell functions have focused on this mechanism.Given that multiple iNKTcell subsets exist (i.e., iNKT1, iNKT2, iNKT17, and iNKT10), iNKT cells are considered multipotent cytokine-secreting cells.This ability to secrete powerful cytokines with opposing properties also suggests that these cells can regulate the cytokine production of neighboring immune cells in various ways, thus controlling the entire cytokine milieu (Fig. 1b).Indeed, there are many cases of this in the literature.For example, iNKT cells secrete granulocyte-macrophage colonystimulating factor (GM-CSF), which promotes macrophage production of IL-1β.Conversely, they also express IL-4, which promotes M2macrophage differentiation; this inhibits macrophage secretion of IL-1β in vitro 11 .Another example is that the IL-4 secreted by iNKT cells can suppress the IFN-γ production of neutrophils in the respiratory tract 72 .Conversely, iNKT cells can themselves secrete IFN-γ, which in turn regulates neutrophil production of IL-10 and C5a in a sepsis model 73 .Thus, iNKT cells have the clear potential to balance immune responses via their versatile secretion of powerful cytokines.
Indeed, the cytokine production of iNKT cells has been shown to facilitate or even play a critical role in immune diseases such as autoimmunity, allergy, infection, and cancer 6,65,[74][75][76] .More specifically, the cytokine production of iNKT cells has been implicated in numerous lung conditions, including allergic asthma 77 , mycobacterium infection 78 , viral infection 79 , chronic obstructive pulmonary disease (COPD) 80 , pulmonary fibrosis 81 , hypersensitivity pneumonitis 72 , and immune complex-induced lung injury 82 .Of the many soluble factors that pulmonary iNKT cells can secrete, IL-4 and IFN-γ appear to play particularly important roles in respiratory tract diseases 83,84 .Other important soluble factors include IL-17, IL-22, and IL-13, which regulate the activities of neighboring cells such as helper T cells 85 , lung epithelial cells 86 , DCs 13,87 , and macrophages 88 .The roles of these factors in lung disease are detailed further below (Fig. 1b).
Other mechanisms by which iNKT cells exert immune effects include cytotoxicity and cell-to-cell contact-mediated immune regulation 89 .However, research on the roles of these mechanisms in lung diseases is lacking.
It should be noted here that iNKT cells can play both beneficial roles, such as protecting the lungs from tuberculosis (TB) 90 , and detrimental roles, such as driving COPD by producing IL-17 91 .Further research on the roles of pulmonary iNKT cells in respiratory tract diseases is likely to be particularly valuable because it may promote the development of novel therapeutic targets.

METABOLISM OF PULMONARY INKT CELLS
Although little is known about the metabolism of pulmonary iNKT cells, it can be inferred by examining the metabolic properties of peripheral iNKT cells and the metabolic environment of the lung.Before we discuss these points, we will first describe what is known about the metabolism of conventional T cells.These cells first engage in glycolytic metabolism, which is induced by PI3K-Akt signals from the pre-TCR and Notch1 during β-selection in the thymus 92 .Thereafter, mature naïve quiescent T cells in the periphery primarily utilize oxidative phosphorylation (OXPHOS) or fatty acid oxidation (FAO) to generate ATP; thus, fatty acids are their main energy source.However, once T cells are activated, they switch to aerobic glycolysis 93 for ATP production and primarily use glucose as their fuel source.While this is less efficient than glycolysis, this switch provides the rapid energy needed for effector T-cell long-term survival, proliferation, cytokine secretion, and migration to sites of inflammation 92,94 .
Although the proliferation of developing iNKT cells appears to rely on glucose utilization, glycolysis has been suggested to exert a negative effect on the function of iNKT cells since glucose uptake and Glut1 expression are higher in immature iNKT cells present in the thymus than in mature iNKT cells 34,95 .Glucose uptake and Glut1 expression are attenuated in mature iNKT cells and upregulated when there are defects in developmental factors of iNKT cells, including Keap1 or PLZF 34,96,97 .Furthermore, iNKT cells are more susceptible to modifications in the function of the mitochondrial electron transport chain than conventional T cells.The crucial role of mitochondrial metabolism in the development and function of iNKT cells is exerted through the regulation of TCR/IL-15 signaling and NFAT activity 98 .
Peripheral iNKT cells bear memory T-cell features such as high cell surface expression of CD44 and retention of antigen specificity after maturation and antigen exposure 99 .Moreover, these cells are in a ready-to-respond state that allows them to produce cytokines within hours, even when they are only stimulated by interleukins such as IL-12 and IL-18 100 .Thus, peripheral iNKT cells are similar to memory T cells in that their metabolism allows rapid responsiveness.However, while conventional effector/memory, CD4 + T cells use glucose for glycolysis, which leads to lactate production, iNKT cells metabolize glucose via the pentose phosphate pathway and OXPHOS, and these pathways are essential for their survival, proliferation, and cytokine production 96 .This is supported by the fact that iNKT cells have higher ATP levels than CD4 + T cells both before and after activation 96 .Moreover, peripheral iNKT cells display lower glucose uptake than conventional CD4 + T cells due to the inhibitory effect of PLZF on glycolysis 96 .Finally, although both CD4 + T and iNKT cells require glutamine to proliferate, CD4 + T cells need glucose to expand optimally, whereas iNKT cells depend on fatty acid metabolism 96 .
The metabolism of iNKT cells in the lungs may be shaped by the metabolic environment in the lung.This environment is determined by several dynamic and complex factors, including mucus and microbacterial components.The mucus is produced by the airway epithelium.Since it is a rich source of nutrients for bacteria, it can affect the metabolic activity of these microorganisms 101 .Moreover, microbacterial components such as LPS and other bacterial byproducts can create a complex metabolic environment that alters the local pH, oxygen levels, and nutrient availability.This can affect the metabolism and consequent energy production and cellular behavior of not only the microorganisms but also the host cells 102 , including local iNKT cells.It seems likely that pulmonary iNKT cells are characterized by a specific metabolomic profile that promotes their use of metabolic pathways such as fatty acid metabolism.
iNKT-cell responses are now known to be highly dependent on their synthesis of lipids.For example, compared to conventional T cells, iNKT cells express higher levels of PPAR-γ, a master regulator of lipid metabolism.They also increase their cholesterol synthesis after activation, which is needed for their TCR signaling, proliferation, and production of IFN-γ.Interestingly, however, blocking cholesterol synthesis only slightly diminished the production of IL-4 by activated iNKT cells; rather, glucose appeared to be more critical for IL-4 production by iNKT cells 103 .The influence of lipid biosynthesis on iNKT-cell function is highlighted by the fact that iNKT cells skew toward an iNKT1 phenotype.

INKT CELLS IN PULMONARY DISEASES
Despite their relatively low numbers in the lungs, pulmonary iNKT cells appear to play vital roles in host defense against microorganisms.This role involves them patrolling the lumen of the pulmonary vessels and the interstitial tissue until the lung tissue is injured by infection and emits danger signals.Such signals cause iNKT cells to migrate to the injured site, which contains glycolipid antigens, and induce iNKT cells to elicit early host defense mechanisms.However, pulmonary iNKT cells can also participate in the pathogenesis of various lung diseases 104 , either via direct pathogenic effects of their cytokines or more indirectly via regulation of neighboring immune-cell subset functions 105 .The triggers that generate pathogenic iNKT-cell activity are generally the same as those that initiate protective iNKT-cell responses, namely, glycolipid antigens and/or the surrounding cytokine milieu.Below, we will summarize what is known about the role of iNKT cells in the four most common lung diseases (asthma, mycobacterium infection, viral infection, and COPD) as well as several more minor pulmonary diseases.

Asthma
Asthma is a common respiratory disease that affects millions of people worldwide 104 .Its cardinal feature is AHR, but there are several distinct forms of asthma that are underpinned by disparate pathogenic pathways 106,107 .The most common form is allergic asthma, which is triggered by allergens and is characterized by Th2-immune responses, eosinophil infiltration, high IgE levels, and AHR 108 .Another important endotype is nonallergic asthma, which is Th2-independent and characterized by lung/airway neutrophil infiltration, Th17-immune responses, and AHR 109 .
Studies in mice [110][111][112] and nonhuman primates 113 show that iNKT cells can directly trigger the development of asthma.For example, intranasal administration of α-GalCer 114 or Sphingomonas-derived glycolipids in mice induces AHR 54 .Moreover, IL-4 and particularly IL-13 from iNKT cells are key drivers of allergic asthma 83 : IL-4 facilitates the overall Th2 response in allergic lungs, while IL-13 acts as a direct pathogenic factor 83 by inducing lung epithelial cell contraction 115 .The triggers for IL-4/IL-10 secretion by iNKT cells are IL-25, thymic stromal lymphopoietin (TSLP), and IL-33: these so-called 'alarmin' cytokines are released by injured lung cells, can directly activate iNKT cells and are abundant in early asthmatic lungs 116 .Similarly, iNKT cells help drive the development of Th17-mediated asthma.While less is known about the role of iNKT cells 117,118 , it has been shown that intranasal administration of α-GalCer causes pulmonary CD4 − NK1.1 − iNKT cells to secrete IL-17 119 , which promotes airway neutrophilia and AHR.This was also observed when a more physiological model of IL-25-dependent AHR was employed 120 (Fig. 2).
Our recent study 13 on the role of iNKT cells in murine allergic asthma models induced by ovalbumin (OVA) and house-dust mite (HDM) suggests that iNKT cells also promote the development of allergic asthma by enhancing the migration of XCR1-expressing type-1 conventional DCs (cDC1s) into asthmatic lungs.This migration appears to be driven by iNKT-cell production of X-Cmotif chemokine ligand 1 (XCL1).This ligand may be specific for cDC1s since they are the only immune-cell subset that appears to express the receptor for XCL1 (XCR1).The importance of this iNKT cell-cDC1 relationship was demonstrated by the fact that XCL1knockout (KO), XCR1-KO, and Jα18-KO (which lack iNKT cells) mice failed to develop AHR and Th2-immune responses, which was associated with diminished cDC1 infiltration into the lungs.This iNKT cell-cDC1 relationship was confirmed by adoptively transferring wild-type (WT) or XCL1-deficient iNKT cells into Jα18-KO mice: the WT cells, but not the XCL1-deficient cells, induced AHR and cDC1 migration into the lungs.Similarly, adoptive transfer of WT cDC1s into XCL1-KO, XCR1-KO, or Jα18-KO mice induced AHR.Notably, we also showed that once cDC1s entered the lungs, they activated the Th2 responses of CD4 + T cells 13 .This is interesting because cDC1s are generally thought to regulate CD8 + T-cell responses 121 .Our finding is supported by Nakano et al., who also showed that cDC1s can regulate the Th2 responses of CD4 + T cells in allergic asthma 122 .Thus, iNKT cells play critical roles in the murine asthma model by not only generating directly pathogenic cytokines but also recruiting key cells that then evoke the pathogenic activities of Th2 T cells (Fig. 2).
It should be noted that there is some controversy about the importance of iNKT cells in murine asthma models 123 because several studies have shown that KO of Jα18 or CD1d (and therefore iNKT cells) has no effect on the development of asthma in commonly used murine models (the OVA and HDM models) 124,125 .This may reflect differences in microbiota between animal facilities since germ-free mice show increased pulmonary iNKT-cell numbers 126 .However, the role of iNKT cells in asthma is even more controversial in humans.While Akbari et al. showed that the BALF of patients with moderate to severe asthma had very high frequencies of iNKT cells (60% of CD3 + cells) 83 , other groups observed frequencies of only ~2%.These disparities may reflect improper flow cytometric analysis, nonspecific binding of the CD1d tetramers used, or differences in patient cohorts.As an aside, the BALF of childhood asthma patients bears increased iNKT-cell numbers 127 , which suggests that iNKT cells may participate in juvenile asthma.Overall, it seems possible that iNKT cells promote asthma, although this role may only emerge in specific conditions.

Mycobacterium infection
TB remains a serious public health concern worldwide: approximately 2 million people die per year from this disease 128 .This reflects the high contagiousness of the pathogenic Mycobacterium species (M.tuberculosis, M. bovis, M. microti, and M. africanum ) and the poor protection generated by the Bacillus Calmette-Guerin vaccine in adults 130 .
role of iNKT cells in TB has been studied in murine models.iNKT cells in infected mice are activated by not only CD1dpresented mycobacterial antigen 131 but also cytokines in the environment such as IL-12 and IL-18 132 : the resulting activated iNKT cells help protect the mice from the infection 78 .This role is partly mediated by the GM-CSF secretion of iNKT cells: this activates cDC1s, which in turn promote the antimicrobial activities of CD8 + T cells 133 .Another mechanism is that iNKT cells facilitate the priming of T cells against TB infection: treatment of TB-infected mice with α-GalCer increases the numbers of TB antigen-specific IFN-γ-producing T cells in the lung 134 .Finally, it is possible that iNKT cells exert their protective abilities in TB by releasing IFN-γ and engaging in cytotoxicity: when iNKT cells from M. tuberculosis or M. bovisinfected mice were cocultured in vitro with infected macrophages, the iNKT cells secreted IFN-γ and killed the macrophages 132 (Fig. 3a).However, this function remains to be validated in vivo.
With regard to humans, the iNKT cells in the sputum of TB patients bear activated phenotypes and secrete high IFN-γ levels 135 .However, their numbers are significantly decreased.Interestingly, these cells express high levels of PD-1 on their surface and have a tendency to undergo apoptosis.Indeed, when Fig. 2 Roles of pulmonary iNKT cells in asthma pathogenesis.Inhaled allergens or pollutants cause lung epithelial cells to release alarmins and dendritic cells (DCs) to present the antigens contained in these environmental insults.Both the alarmins and the DCs then activate iNKT cells.Activated iNKT cells subsequently secrete cytokines that modulate the function of various immune cells or cause airway smooth muscle constriction.In particular, iNKT cells promote the recruitment and lung retention of XCR1-expressing cDC1s by secreting the chemokine XCL1.The XCR1 + cDC1s subsequently amplify the type-2 immune responses during asthma.these cells were treated in vitro with a PD-1 blocker, the iNKT-cell frequency rose 136 .Moreover, the decreased iNKT-cell numbers in the blood of humans normalize after TB is eliminated by treatment 137 .Notably, cocultured human iNKT cells can also kill M. tuberculosis-infected macrophages in vitro 138 .This supports the murine in vitro study above that suggests iNKT cells can protect the host from TB infection via cytotoxicity 132 .
These preclinical and clinical studies suggest that iNKT cells can protect against TB infection.Thus, cell-based therapies with iNKT cells may be useful for TB patients 139 .This is supported by a study showing that when iNKT cells were activated exogenously with α-GalCer and then transferred into mice, the mice were protected from lethal TB infection 140 .Another possibility is to incorporate α-GalCer into the Bacillus Calmette-Guerin vaccine: this strategy has been shown to enhance the overall immune response of TB-infected mice 134 .

Viral infections
Influenza A virus.Influenza A virus (IAV) is another common cause of respiratory tract disease.It is especially prominent during winter and can cause severe and sometimes fatal lung damage.It can also increase host susceptibility to secondary bacterial infections 141 .
Pulmonary iNKT cells protect mice from the H1N1 and H3N2 IAV strains since iNKT cell deficiency (Jα18-KO and CD1d-KO) resulted in more severe bronchopneumonia, greater weight loss, and shorter time to death 142,143 .Moreover, treatment with exogenous α-GalCer before IAV infection reduces infection-induced weight loss and increases CD8 + T-and NK-cell responses to IAV infection; the latter effects are due to IFN-γ secreted by iNKT cells 144 (Fig. 3b).There are also several other mechanisms by which iNKT cells protect the host from IAV.First, they downregulate myeloidderived suppressor cells, which inhibit the CD8 + T-cell responses that limit IAV replication during moderate H1N1 infection 143 .Second, they promote the maturation of DCs and their migration to draining lymph nodes, which enhances CD8 + T-cell responses during severe H3N2 infection 142 .Third, iNKT cells protect the host from excessive lung damage by producing IL-22, which may strengthen the epithelial barrier integrity of host lungs 86 .Fourth, iNKT cells reduce the local levels of MCP-1.This decrease downregulates the accumulation of inflammatory monocytes in the lungs, which diminishes the overall damage to the lung tissue.This, in turn, promotes the survival of mice infected with a highly pathogenic strain of IAV 88 .Thus, iNKT cells help block the deleterious effects of IAV infection by both promoting anti-viral responses and protecting the lung tissue of the host (Fig. 3b).

COVID-19.
Only a few studies have assessed the role of iNKT cells in the recent pandemic caused by SARS-CoV-2 infection.One recent study reported that patients with severe COVID-19, but not patients with mild COVID-19, demonstrate a tenfold reduction in pulmonary iNKT-cell frequencies 145 .another group found that COVID-19 patients and healthy individuals did not differ markedly in iNKT-cell frequencies 146 .Another study showed that COVID-19-mediated pneumonia was associated with elevated eosinophil and neutrophil numbers in the sputum and BALF and that these numbers correlated positively with the numbers of iNKT cells in peripheral blood and BALF.This suggests that iNKT cells could play a pathogenic role in COVID-19 147 .Finally, hematopoietic stem cell (HSC)-engineered iNKT cells, which were differentiated from TCR-engineered HSCs, efficiently killed SARS-CoV-2-infected cells in vitro 148 .Thus, iNKT cells may help protect the host from SARS-CoV-2 infection by killing infected cells.These observations together suggest that iNKT cells are involved in COVID-19-related immune responses.However, whether their role is primarily protective or pathogenic remains to be determined.
Chronic obstructive pulmonary disorder (COPD).The frequency and severity of COPD has steadily increased over the last few decades, and COPD now affects more than 200 million people worldwide and is the fourth leading cause of death 149 .COPD is characterized by lung tissue emphysema, respiratory bronchiolitis, and eventual chronic bronchitis 150 .
Key risk factors for COPD are cigarette smoke and industrial pollutants 150 .Several studies suggest that iNKT cells play pathogenic roles in COPD.First, the murine cigarette smoke model of COPD features increased pulmonary iNKT-cell numbers.Second, both Jα18-KO and CD1d-KO mice are resistant to forming COPD based on exposure to cigarette smoke 80 .Third, repeated intranasal administration of α-GalCer can induce COPD, and this is ameliorated when an anti-IL-4 antibody is coadministered 80 .Fourth, consistent with the mouse COPD model, COPD patients have greater iNKT-cell numbers in their peripheral blood and sputum than healthy individuals 151 .Fifth, iNKT cells from COPD patients produce high levels of IFN-γ and IL-17A that can activate cigarette smoke extract-exposed lung DCs or airway epithelial cells in vitro 91 .Sixth, iNKT-cell deficiency ameliorates symptoms in the Sendai virus infection model of COPD.This is mediated by iNKTcell downregulation of the numbers of IL-13 + macrophages, which are critical COPD regulators because IL-13 derived from these cells directly induces goblet cell metaplasia, AHR, and mucus production.This mechanism may also participate in human COPD since the lungs of COPD patients demonstrate elevated numbers of not only iNKT cells but also IL-13 + CD68 + macrophages 152 (Fig. 3c).Thus, it is likely that iNKT cells promote COPD pathogenesis, possibly by direct and/or indirect interactions with IL-13 + macrophages.
Pulmonary fibrosis, hypersensitivity pneumonia, and immune complex-induced lung injury Our studies suggest that pulmonary iNKT cells also play critical protective or pathogenic roles in pulmonary fibrosis, hypersensitivity pneumonitis, and immune complex-induced lung injury, as detailed below.
Pulmonary fibrosis.Pulmonary fibrosis is characterized by progressive scarring of the lungs and the eventual development of lung interstitial fibrosis.This results in progressive shortness of breath 153 .Since this disease is caused by excessive extracellular matrix (ECM) production by fibroblasts in the lungs 154 , the recruitment, proliferation, and ECM production of fibroblasts have been extensively studied.At present, it is thought that these fibroblast activities are largely driven by transforming growth factor beta (TGF-β), which is a potent profibrotic mediator 155 .
Two studies suggest that iNKT cells can play protective roles in pulmonary fibrosis by downregulating TGF-β expression 81,156 .First, we found that CD1d-KO mice developed more severe bleomycin-induced pulmonary fibrosis than WT mice, as shown by their worse lung histology, higher hydroxyproline levels, and greater mortality 64 .These severe effects were associated with higher TGF-β levels and lower IFN-γ levels in the lungs.Moreover, the adoptive transfer of WT iNKT cells into CD1d-KO mice not only ameliorated the severity of the disease in these mice but also restored the IFN-γ levels in the lung while concomitantly reducing the TGF-β levels.Interestingly, when bleomycin-treated BALF cells were treated in vitro with recombinant IFN-γ, their production of TGF-β dropped markedly.These findings suggest that IFN-γ-secreting iNKT cells help protect against pulmonary fibrosis by downregulating TGF-β1 expression in the fibrotic lung 81 .Our findings are supported by Grabarz et al., who observed that when WT mice were injected intratracheally with bleomycin and intraperitoneally with α-GalCer on the same day, pulmonary fibrosis was less severe.This was associated with lower IL-4 levels in the lung and lower expression of arginase-1 by neighboring alveolar macrophages.These findings suggest that iNKT cells may protect mice from pulmonary fibrosis by suppressing lung production of IL-4, which inhibits M2-macrophage polarization 156 .This is significant because the recruitment of monocytes to the lung and their conversion into type-2 cytokine-and TGF-β-secreting M2 macrophages drive the overall progression of pulmonary fibrosis 157 .Thus, iNKT cells protect mice from pulmonary fibrosis by (i) producing IFN-γ, thereby downregulating lung TGF-β levels and (ii) decreasing lung IL-4 levels, thereby inhibiting M2-macrophage activity (Fig. 3d).
Hypersensitivity pneumonitis.Hypersensitivity pneumonitis is caused by repetitive exposure to inhaled environmental antigens that provoke a hyperreactive immune response.This response induces inflammation of the alveoli and bronchioles and often leads to other interstitial lung diseases 158 .There is little in the literature about the roles of iNKT cells in hypersensitivity pneumonitis.However, we found that CD1d-KO mice were more susceptible to Saccharopolyspora rectivirgula-induced hypersensitivity pneumonitis than WT mice and that this was associated with elevated IFN-γ levels in the lung.IFN-γ was mainly produced by Gr-1 + neutrophils and played an important pathogenic role since blocking IFN-γ or depleting Gr-1 + neutrophils attenuated hypersensitivity pneumonitisassociated inflammation in CD1d-KO mice.Additional experiments then showed that the production of IFN-γ by Gr-1 + neutrophils in hypersensitivity pneumonitis was impaired by IL-4 produced by iNKT cells: adoptive transfer of IFN-γ-deficient, but not IL-4-deficient, iNKT cells downregulated hypersensitivity pneumonitis-related inflammation in CD1d-KO mice 72 .
Immune complex-induced lung injury.IgM or IgG immune complexes (ICs) are critical regulators of the immune system.However, they can cause acute respiratory distress syndrome (ARDS) or acute lung injury (ALI) due to unwanted inflammation 159 .We showed that CD1d-KO and Jα18-KO mice are less susceptible to developing ALI in the chicken egg albumin-and anti-chicken egg albumin IgG-induced IC-ALI model than WT mice.Additional experiments showed that the IC-ALI in WT mice was induced by iNKT cells, whose secretion of IFN-γ and macrophage inflammatory protein-1α (MIP-1α) caused neighboring alveolar macrophages and DCs to secrete proinflammatory cytokines.In particular, adoptive transfer of WT iNKT cells into CD1d-KO mice generated the IC-ALI seen in WT mice, but this was not observed when IFN-γ-deficient, MIP-1α-deficient, or FcγR-deficient iNKT cells were transferred 82 .
Altogether, these studies show that along with their crucial roles in common respiratory tract diseases, pulmonary iNKT cells are also important in many other respiratory tract diseases.These findings demonstrate the importance of these cells in governing overall lung immunity.

CONCLUDING REMARKS
This review sought to provide a broad perspective on pulmonary iNKT cells and their roles in lung diseases that hopefully aid further research on these cells.While it is clear that these cells are important for protection from lung infections and play important roles in the pathogenesis of many lung diseases, there are still many missing links.It is increasingly evident that overall immunity is driven by very complex and dynamic interactions between a wide variety of immune cells.Given the cytokine multipotency of iNKT cells and their multiple functions, it is likely that further research on the interrelationships between pulmonary iNKT cells and surrounding immune cells will be fruitful in terms of improving our understanding of the immune mechanisms that protect and harm the lungs.

Fig. 1
Fig. 1 Distribution of pulmonary iNKT cells and their interactions with other cells in the lung.a Migration of iNKT cells to the lungs.After developing in the thymus, iNKT cells express CXCR6, a tissue localization molecule, and migrate to the CXCL16-expressing periphery.iNKT cells accumulate in the lumen of the lung microvasculature and then enter the lung tissue when neutrophils in the lung interstitium secrete the CCR4 ligands CCL17/22 and CXCL9/13.The neutrophils, therefore, guide the iNKT cells to the source of lung injury in the interstitium.The monocyte-derived DCs (moDCs) in this area present glycolipid antigens from the lung injury source to the iNKT cells, which become activated and then remain in the interstitium.b Crosstalk between iNKT cells and other cells in the lung.iNKT cells are activated by antigens expressed by lung antigen-presenting cells (APCs), such as MoDCs, by cytokines from other cells (such as IL-12/18), and by TLR ligands (e.g., LPS).The activated iNKT cells then secrete a variety of cytokines that regulate the function of many types of neighboring cells.

Fig. 3
Fig. 3 Role of pulmonary iNKT cells in various lung diseases.a In mycobacterium infection, cDC1s activate pulmonary iNKT cells by secreting IL-12 and IL-18 and by presenting antigen on CD1d.The iNKT cells then reciprocally stimulate the cDC1s via GM-CSF, which activates the cytotoxic CD8 + T cells that eliminate the infected cells and clear the bacteria.This activity is further supported by the IFN-γ produced by the iNKT cells.b During lung viral infections, pulmonary iNKT cells produce IFN-γ, which downregulates myeloid-derived suppressor cells (MDSCs), which would otherwise suppress CD8 + T cells.Moreover, iNKT cells promote the maturation of DCs and their migration to draining lymph nodes by IFN-γ, which enhances CD8 + T-cell responses.Finally, iNKT cells protect the host by increasing epithelial cell integrity by producing IL-22.c COPD pathogenesis is promoted by IL-4-secreting iNKT cells.IL-4 generates IL-13-secreting macrophages and activates T cells, which in turn secrete many cytokines, chemokines, and growth factors that directly induce fibrosis, alveolar wall destruction, and mucus hypersecretion.d Pulmonary fibrosis is inhibited by iNKT cells.The mechanism involves iNKT-cell expression of IFN-γ, which suppresses the production of the master profibrotic regulator TGF-β in the lung.This blocks the extracellular matrix (ECM) production that is responsible for pulmonary fibrosis.Another mechanism involves downregulating lung expression of IL-4, which inhibits the polarization of lung macrophages to the M2 phenotype.This phenotype plays an important role in fibrosis by producing type-2 cytokines and large amounts of TGF-β.