Mucosal epithelia represent the frontline of multicellular organisms, and they are continuously exposed to several types of stress. Pathogens and environmental stress (thermal, oxidative, and chemical) can result in cell damage and loss of tissue function. To control inflammation and promote tissue repair, different mechanisms for the detection and elimination of stressed cells have evolved, including activation of the immune system.

NKG2D is a C-type lectin-like activating/co-stimulatory receptor expressed by innate and adaptive lymphocytes, such as natural killer (NK) cells, CD8⁺ αβ T cells, γδ T cells, and iNKT cells. Engagement of NKG2D triggers the cytolytic function of effector CD8⁺ T cells independently of TCR recognition in some circumstances (1–4) while directly activates the effector functions of NK cells, namely, cytolytic granule release and IFNγ secretion. Thus, expression of NKG2D ligands is strictly linked to the immunosurveillance of stressed cells by innate lymphoid cells, especially NK cells (5–7). In humans, NKG2D ligands are MICA and MICB (MHC class I chain-related proteins A and B), encoded by genes in the MHC region, and ULBP1-6 (UL16-binding proteins), with the encoding genes located on chromosome 6. Murine NKG2D ligands include five different isoforms of RAE-1 (retinoic acid early inducible-1), MULT-1 (murine ULBP-like transcript-1), and three different isoforms of H60 (histocompatibility 60) (8). These molecules are present at low or undetectable levels on normal cells (9, 10) but are rapidly induced upon cellular stress and are frequently upregulated in virus-infected and neoplastic cells (11–13). Even if it is known that the promoter region of the MICA gene contains a heat-shock element able to respond to cellular stress (14), only recently the molecular mechanisms driving NKG2D ligand expression during cellular stress have more deeply been investigated (15).

Expression of NKG2D ligands has been shown in tissues from patients with chronic inflammatory diseases, including rheumatoid arthritis (16, 17), type 1 diabetes (18), and atherosclerosis (19); much less is known regarding NKG2D ligand expression and relevance in mucosal epithelia during both normal and stress conditions.

This review focuses on different conditions that are able to modulate NKG2D ligands on epithelial cells, and, in particular, on the role of NKG2D/NKG2D ligands in controlling the homeostasis of the gut and lung epithelia during inflammation. Recent findings link toll-like receptor (TLR) signaling to NKG2D ligand expression, and we can now think of epithelial and immune cells as an integrated system able to deal promptly with environmental stress.

Among the pattern recognition receptors, TLRs play a key role in innate immunity, serving as first line sensors of structurally conserved bacterial and viral components, the so-called pathogen-associated molecular patterns. It is clear that TLRs can be triggered also by endogenous ligands, such as nucleic acids released by necrotic cells and matrix components generated during tissue injury, collectively called damage-associated molecular patterns. As TLR engagement leads to the production of inflammatory cytokines and chemokines, which contribute to local inflammation and leukocyte recruitment, the recent findings showing that TLR signaling is able to modulate the NKG2D axis and thus lymphocyte activation are of great interest (Table 1).

Rotaviruses are causative of intestinal alterations leading to diarrhea, and it has been reported that in mice viral dsRNA from rotavirus genome induces severe intestinal injury via TLR3 activation (20). Indeed, upon stimulation, TLR3 forces intestinal epithelial cells (IECs) to express both IL-15 and RAE-1, which promote mucosal damage by activating intraepithelial lymphocytes (IELs), in particular CD8αα⁺ T cells, engaging their NKG2D receptor (21). In this scenario, Tada and colleagues have shown that probiotics belonging to Lactobacillus strains are able to reduce the levels of IL-15 and RAE-1, and at the same time to increase the level of IL-10 in the intestine, thus performing immunomodulatory activities (22) (Figure 1). Interestingly, the NKG2D axis is modulated in an opposite way by TLR9 during Salmonella typhimurium infection. S. typhimurium is an important food-derived pathogen and unmethylated CpG-containing DNA from S. typhimurium is recognized by TLR9 (23). TLR9 triggering promotes the accumulation of IkBα, resulting in strong inhibition of the NF-kB pathway and thus controlling intestinal tissue inflammation. On the contrary, in TLR9-deficient mice, TLR9 signal deficiency releases its inhibition on NF-kB and leads to pro-IL-1β expression in IECs. In addition, lack of TLR9 signal causes activation of NLRP3 inflammasomes, resulting in pro-IL-1β processing and IL-1β secretion. Secreted IL-1β acts in an autocrine way stimulating IECs to expose NKG2D ligands (namely, RAE-1, MULT-1, and H60) and with a paracrine mechanism induces the expression of NKG2D on IELs (24). This positive loop augments the susceptibility of IECs to the cytotoxicity of IELs leading to the breaking of the epithelial barrier and to the spread of S. typhimurium infection (Figure 1).

TLR-dependent NKG2D ligand expression has also been observed in mouse kidney during renal ischemia–reperfusion injury (25). HMGB1 is a DNA-binding protein showing inflammatory function after ischemia–reperfusion injury by binding TLR4 (26–28). Chen and colleagues have reported that HMGB1 can induce RAE-1 and MULT-1 upregulation on injured kidney cells through a TLR4/MyD88-dependent signaling (25), suggesting a contribution of the NKG2D axis during tissue damage after ischemia. Accordingly, NK cell depletion has been demonstrated to ameliorate kidney damage following ischemia, with NK cells having a direct role in killing tubular epithelial cells via perforin secretion (29).

The gut system represents a peculiar challenge for the immune system, as it is continuously exposed to commensal bacteria and food antigens. IECs constitutively express MICA and MICB, even if at low levels (14, 30), while much less is known regarding ULBP proteins. In physiological conditions, this expression does not lead to an immune response, but during chronic inflammatory diseases, MICA/B-expressing cells become target of IELs, namely, intraepithelial NK cells, γδ T cells, and CD8⁺ αβ T cells. Although with different etiology, both Crohn’s disease and celiac disease are characterized by chronic inflammation. This type of stress strongly relies on the relationship between the environment and the immune system.

The respiratory tract is often under attack of environmental pathogens and is equipped with different populations of NKG2D-expressing lymphocytes. Bronchial airway epithelial cells constitutively express MICA, MICB, and ULBP1–4 transcripts, while cell surface expression is largely absent in normal conditions. However, upon oxidative stress, NKG2D ligands become visible at the protein level on the cellular membrane, with a molecular mechanism based on the ERK pathway (59). The capability of stressed airway epithelial cells to rapidly express NKG2D ligands suggests that they are able to directly engage and activate the immune system. This finding has been confirmed in mice with lung Pseudomonas aeruginosa infection. Acute infection stimulates the alveolar epithelium to express RAE-1, and epithelial cell death (likely due to the activation of cytotoxic NKG2D-bearing lymphocytes) is associated with increased bacterial clearance (60). Pulmonary P. aeruginosa infection is also associated with increased levels of IL-1β, TNF-α, and IFN-γ, which may be another effect of NKG2D engagement. Thus, NKG2D ligand expression leads to a competent host response that can be blocked with NKG2D-specific antibody (60).

The NKG2D system plays an important role in lung homeostasis also in the event of physicochemical stress, as demonstrated in chronic obstructive pulmonary disease (COPD). COPD is a severe form of airway epithelium inflammation largely due to cigarette smoke exposure. Cigarette smoke is known to induce necrotic and apoptotic cell death with concomitant nucleic acid release (61). This leads to TLR3/7/9 activation and, with an unknown mechanism, to RAE-1 expression on mucosal airway epithelial cells. The consequent inflammation and NKG2D-driven cytotoxicity is responsible for further tissue damage, worsening the initial cigarette smoke exposure. NK cells become hyperresponsive breaking the balance between injury and repair (62–64). Notably, NKG2D ligand overexpression in transgenic mice is sufficient to induce pulmonary emphysema, strongly suggesting that NKG2D/NKG2D ligand axis via cytotoxic lymphocyte activation plays a major role in alveolar epithelial injury (65). The strong relationship between NKG2D ligand expression and COPD has been further confirmed by the finding of enhanced MICA expression in the airway epithelium specimens from smokers (65).

Expression of NKG2D ligands can be achieved in several circumstances, ranging from viral and bacterial infections to physiochemical stress (Table 1). This versatility is needed to face a plethora of stressful stimuli with common pathways leading to the involvement of the immune system. As a result, intracellular insults are associated with intercellular responses, with great advantage for the whole organism. However, only recently the molecular mechanisms underlining these processes have been investigated, and many questions remain unanswered:

Which ligand is displayed? Is this choice stress type dependent?

Indeed, both NKG2D and NKG2D ligand genes are highly polymorphic. This polymorphism has been associated with host–pathogen coevolution, with great relevance for viral infections, but functional consequences have also been described during hematopoietic transplantation and cancer immunosurveillance (48, 66, 67). The impact of this variability in the context of stress response and inflammation still needs to be fully elucidated.

Another important point to be considered is the strict polarity of epithelial tissues. In the gut, the apical side of the epithelial layer displays different functions from the basolateral side, where IELs are located. Human ULBP1–3 and murine RAE-1 are anchored to the cell membrane via a glycosylphosphatidylinositol molecule and thus are supposed to be trapped in the lipid raft-enriched apical side of epithelial cells (Figure 1), while MICA presents a basolateral-targeting motif in his structure (68). Hence, MICA expression is prone to a rapid recognition by NKG2D-bearing IELs, while ULBP1–3 and murine RAE-1 become available only in the event of epithelial polarity breaking, as result of infection or autoimmunity (69). Remarkably, MICA allele*008, due to its truncated cytoplasmic tail, lacks the basolateral-targeting motif and changes its localization, leading to hypothesize a different NKG2D engagement (68, 69). Furthermore, it is now clear that NKG2D ligand transcript and even protein expression does not always mean cell surface exposure, with obvious functional implications (70, 71).

Great interest is growing regarding the role of the microbiome in human pathophysiology, as commensal bacteria can establish an immune regulatory milieu in the intestine (72, 73). The articles here reported suggest that, beside the production of TGF-β and IL-10, modulation of the NKG2D axis is a key function in this context. Indeed, it is now emerging that the NKG2D receptor and NKG2D ligands not only function as stress sensor molecules but also play a pivotal role in shaping innate and adaptive lymphocyte populations, thus contributing to the homeostatic regulation of the mucosal immune system.

FA searched for literature articles; conceived and wrote the manuscript. ANS conceived and wrote the manuscript. ALS, CC, and GS revised and critically contributed to the manuscript drafting. FA, ALS, CC, GS, and ANS approved the final version of the manuscript.

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