Once IL-36 cytokines are cleaved and fully activated, they bind to IL-1Rrp2 and transduce a complex signaling cascade that turns on an inflammatory response (48). In humans, IL-1Rrp2 is widely expressed in keratinocytes in the skin and the esophagus but poorly expressed in other organs like reproductive organs, lymph nodes or lungs (16). Various immune cells also express IL-1Rrp2 (49–52). One report showed that circulating DCs and monocytes, but not CD3⁺ T cells express IL-1Rrp2 on their surface and respond to IL-36 cytokines in vitro (49). A subsequent study showed that CD19⁺ B cells and CD8⁺ T cells express IL-1Rrp2 on their surface but CD4⁺ T cells expressed high amounts of IL-1Rrp2 in the cytoplasm (50). Human neutrophils do not express IL-1Rrp2 at baseline (51), but during chronic rhinosinusitis where IL-36 cytokines are locally upregulated, neutrophils were found to be the main cell type expressing IL-1Rrp2 in nasal polyps, but not in the bloodstream (51). This finding suggests that the microenvironment can induce the expression of IL-1Rrp2 in neutrophils that normally do not express this receptor. This hypothesis is supported by the fact that peripheral blood neutrophils, which do not express IL-1Rrp2 under basal conditions, expressed IL-1Rrp2 after incubation with the pro-inflammatory cytokines IL-6 and IL-1β (51). In mice, the expression of IL-1Rrp2 in immune cells follows a similar pattern as in humans with some notable differences. BMDCs, generated from bone marrow precursors in presence of GM-CSF, and CD4⁺ T cells express IL-1Rrp2 and can respond to IL-36 agonists in the absence of any other stimuli (52). Neutrophils and macrophages derived from the bone marrow (BMDMs), on the other hand, do not significantly express IL-1Rrp2 at baseline (52).

The molecular mechanism by which IL-36 cytokines bind to IL-1Rrp2 is similar to the mechanism described for IL-1/IL-1R (53). The IL-36 receptor heterodimer is comprised of IL1-Rrp2 and the accessory protein IL-1RAcP (54). When agonistic IL-36 cytokines bind to IL-1Rrp2 on the cell surface, IL-1RAcP is recruited to the IL-36 receptor-ligand complex (54). Once the complex is complete, TIR domains present within the intracellular region of IL-1Rrp2 and IL-1RAcP recruit MyD88 and IL-1R associated kinase (IRAK), which in turn trigger NF-κB and MAPK activation to promote the expression of pro-inflammatory genes ( Figure 4A ).

The anti-inflammatory cytokine, IL-36Ra (K _D =5.8nM) binds with a higher affinity to IL-1Rrp2 compared to N-terminally processed K⁶-IL-36α (K _D =480nM) and S¹⁸-IL-36γ (K _D =1800nM) (54). A previous study analyzed the affinity of the IL-36 cytokines that bind to the IL-1Rrp2/IL-1RAcP heterodimer. This study found that K⁶-IL-36α (K _D =0.021nM), R⁵-IL-36β (K _D =0.007nM) and S¹⁸-IL-36γ (K_D=0.147nM) bound to the IL-1Rrp2/IL-1RAcP heterodimer with higher affinity than V²-IL-36Ra (K_D=10.2nM) (14). From a biological standpoint, IL-36 agonists initially bind to IL-1Rrp2, promoting the recruitment of IL-1RAcP that leads to subsequent signaling. Whereas the first study showed that the dominant pathway of IL-36 activation is through agonist-ILRrp2 complex formation and subsequent recruitment of IL-1RAcP, IL-1RAcP does not bind IL-1Rrp2 when no agonist is present (54). In contrast to agonist IL-36 cytokines, the binding of IL-36Ra to IL-1Rrp2 does not result in the recruitment IL-1RAcP, thus preventing the induction of downstream signaling (54) ( Figure 4B ). While the Towne et al., 2011 findings show that the affinity of processed IL-36 agonists are in orders of magnitude higher than IL-36Ra, the findings focused on binding affinity to the heterodimer which is restricted to the agonists and not IL-36Ra. In other words, IL-36Ra does not bind the heterodimer but just binds to IL-1Rrp2.

IL-38, another cytokine from the IL-1 family, also binds to IL-36R and suppresses the pro-inflammatory activity of IL-36 cytokines through a similar mechanism as IL-36Ra (55) ( Figure 4B ). Although the crystal structure of IL-36R has not been resolved, biochemical and in silico approaches have provided important insights regarding IL-1Rrp2-ligand interactions (54, 56, 57). IL-1Rrp2 consist of three extracellular structural domains (D1-D3) and one intracellular TIR domain that triggers downstream signaling ( Figure 4C ). The three extracellular domains are part of the ligand-binding pocket, where D1 conforms to the upper part of the pocket, D2 the backside and D3 the bottom part (56). D2 of IL-1Rrp2 is also important for the interaction with IL-1RAcP (56). Disulfide bonds between Cysteine residues C²²/C¹⁰³, C¹⁴⁶/C¹⁹⁵ and C²⁴⁹/C³¹⁶ in D1, D2 and D3 respectively as well as Asparagine (N) linked glycosylation of IL-1Rrp2 at positions N⁴¹ (D1), N²³⁴, N²⁵⁰, N²⁶⁶, N²⁹⁹ (D3) of the extracellular domains are required for the intracellular signaling and trafficking to the cell surface of IL-1Rrp2 ( Figure 4C ). Molecular modeling predicted the relevance of these IL-1Rrp2 sites and confirmed biochemically after mutating each cysteine or asparagine to alanine. Each of these substitutions impaired IL-1Rrp2/IL-1RAcP signaling in response to agonist (56).

Different residues within the structural domains were found to be crucial for IL-36α, IL-36β and IL-36γ to bind and induce signaling via IL-1Rrp2/IL-1RAcP, suggesting that each IL-36 cytokine is recognized differently by IL-1Rrp2 (56). This hypothesis has been corroborated by testing the role of specific amino acids (N⁴¹, C⁴² and C¹¹⁸) of IL-1Rrp2 in response to each IL-36 cytokine (56). Whereas substitution of N⁴¹ by A impaired IL-1Rrp2-mediated signaling in response to the three pro-inflammatory IL-36 cytokines, C⁴² substitution highly impaired the response to IL-36α but did not affect the IL-36β response and only mildly affected the response to IL-36γ. Similarly, substitution of C¹¹⁸ highly affected IL-36α-responsiveness, but not IL-36γ responsiveness and only mildly affected IL-36β-responsiveness (56). Regarding why the binding of pro-inflammatory IL-36 cytokines to IL-1Rrp2 induces the recruitment of IL-1ARcP whereas the ligation of IL-36Ra to IL-1Rrp2 does not, one report identified the existence of two structural loops present in human IL-36γ – one between β-strands 11 and 12 and one between β-strands 4-5 – that appear to be involved in the recruitment of IL-1RAcP to the IL-1Rrp2/IL-36γ complex and the consequent induction of downstream signaling ( Figure 4D ) (57). Similar loops are also present within IL-36Ra, but these loops slightly differ in structure from those observed in IL-36γ ( Figure 4E ). Interestingly, the exchange of these loops between IL-36γ and IL-36Ra inverted the ability of these cytokines to bind IL-1RAcP (57), indicating that these structural regions are crucial for the recruitment of IL-1RAcP to the receptor-ligand complex. IL-36γ directly interacts with both IL-1Rrp2 and IL-1RAcP (56), and thus, IL-36γ may facilitate the recruitment of IL-1RAcP to the IL-36 receptor-ligand complex. However, molecular modeling and biochemical approaches also established a direct interaction between IL-1Rrp2 and IL-1RAcP in the absence of a ligand (56). This interaction involves aromatic residues stacking and electrostatic interactions between D2-D2 and D3-D3 interactions of both receptors (56) ( Figure 4C ).

Even though IL-36 cytokines are not directly chemotactic to inflammatory/immune cells, they strongly triggers pro-inflammatory cytokine/chemokine production via MAPK- and NF-κB-signaling in a variety of cells and tissues (58). As IL-1Rrp2 is highly expressed in keratinocytes, many studies investigating the biological effects of IL-36 cytokines have initially focused on their role during skin inflammatory disorders such as psoriasis and dermatitis. In keratinocytes, IL-36 cytokines enhance the production of a variety of pro-inflammatory cytokines and chemokines, including CXCL-8, IL-23A, IL-6, IL-8 and TNF-α, as well as antimicrobial peptides such as β-defensins 2 and 3, the human cathelicidin LL37 and the antimicrobial psoriasin S10047 (1, 59, 60) ( Figure 5A ). It has been shown that, in keratinocytes, IL-36 cytokines enhance their own production in an autocrine manner (35, 61). Interestingly, in keratinocytes, IL-36 cytokines also induce the production of IL-36Ra (59), which may be a crucial negative feedback mechanism to regulate the inflammatory effects of IL-36 cytokines.

Of particular interest is the observation that IL-36α and IL-36γ induce the expression of NF-κB inhibitor zeta (IκBζ) in keratinocytes (60). IκBζ is a transcription factor that regulates some NF-κB target genes in response to IL-36α and IL-36γ (60). In primary keratinocytes and HaCaT cells, IκBζ is essential for IL-36-induced inflammation, and in the context of IL-36 stimulation, its silencing downregulates several genes involved in a variety of processes, including neutrophil chemotaxis, IL-6 response factors, STAT phosphorylation, leukocyte adhesion and differentiation, T cell-activation, innate immunity and leukocyte activation (60) ( Figure 5A ). Moreover, it has been described that during injury, IL-36γ induces keratinocyte differentiation and proliferation through the induction of Reg3A (17) ( Figure 5A ).

Although IL-1Rrp2 expression is generally highest in the skin compared to other organs and tissues (16), IL-36 cytokines also exert important inflammatory effects in other tissues. In the intestine, it has been described that IL-36 cytokines induce and amplify the expression of the IL-23/IL-22 axis (62), as IL-36R^-/- mice with DSS-induced colitis present with reduced expression of IL-23 and IL-22 in the intestine compared to WT mice (62). Ex vivo analysis of colonic explants from DSS treated mice showed that IL-36γ induced the expression of IL-23 in DCs through a Notch2-dependent mechanism. The induction of IL-23 by IL-36γ was found to be mediated by NF-κB and promoted the recovery of colonic damage induced by DSS via the induction of IL-22 responses (62). This IL-36/IL-23/IL-22 axis has been recently proven to be important in host defense against the enteropathogenic bacteria Citrobacter rodentium (63). In this model of infection, IL-36γ induces the production of IL-22 in CD4⁺ T cells in the presence of DCs, a response that is mediated by the transcription factor Aryl hydrocarbon receptor (AhR) and by the production of IL-6 (63). Here, the activity of IL-36γ is protective against C. rodentium infection, as IL-36R^-/- mice not only showed impaired bacterial clearance and higher intestinal damage, but also impaired survival compared to WT mice (63).

Another major effect of IL-36 cytokines is the activation of the IL-23/IL-17A axis in the skin and in the kidney during inflammation and fibrosis (18, 64). In a model of psoriasiform dermatitis, IL-36 cytokines produced by DCs were shown to induce the recruitment of neutrophils and γδ T cells (18). In this model, lack of IL-1Rrp2 reduces the disease severity and at the same time reduces the production of IL-17A by γδ T cells (18). IL-17A^-/- and IL-23^-/- mice showed the same phenotype, suggesting that IL-36 cytokines contribute to the severity of experimental psoriasiform dermatitis by inducing the expression of IL-23 and IL-17A (18).

Interestingly, IL-36α expression is also increased in chronic kidney disease in patients with renal tubulointerstitial lesions (64). In a mouse model of unilateral ureteral obstruction that mimics the renal tubulointerstitial lesions observed in humans, IL-36α activated the IL-23/IL-17 axis, and by doing so, IL-36α amplified inflammation and the development of renal lesions (64). Besides upregulating Th17 response, IL-36 cytokines have been implicated in the induction of Th1 and Th9 responses. One study has shown that IL-36 cytokines directly induce proliferation and secretion of IL-2 in naïve CD4⁺ T cells and IL-36 cytokines act synergically with IL-12 to drive the acquisition of a Th1 phenotype characterized by the production of IFN-γ (65). Moreover, IL-36 cytokines can induce a Th9 response in T cells, which is characterized by the production of IL-9 and inhibits the proliferation of FoxP3⁺ regulatory T cells. Whereas the induction of IL-9 expression by IL-36γ was found to be dependent on IL-2/STAT5 and IL-4/STAT6 signaling, the inhibition of regulatory T cells was mediated through the impairment of the Foxp3 locus acetylation. In conjunction, both effects contributed to colonic inflammation in an experimental oxazolone-induced colitis (66). Finally, in vitro analyses have determined that IL-36γ triggered IFN-γ production in CD8⁺ T cells previously stimulated with anti-CD3 (67) ( Figure 5B ).

IL-36 cytokines exert direct effects in different myeloid cells, inducing several inflammatory processes, such as pro-inflammatory cytokine production (49, 52), DC maturation (49, 52), antimicrobial peptide production, and inflammasome activation in macrophages (64, 68). The induction of pro-inflammatory cytokines by IL-36 cytokines has been described in human monocytes and DCs, in which IL-36α, IL-36β and IL-36γ upregulated IL-6 at the transcript and protein level (49). In mice, IL-36α, IL-36β and IL-36γ induced the production of IL-6, IL-12p40, IL-12p70, CCL11, CCL4, TNF-α and G-CSF in BMDCs (52). IL-36 cytokines also induced DC maturation and increased the expression of the surface marker CD83 and the co-stimulatory molecule CD86, which are required for T cell activation (49, 52) ( Figure 5C ). In macrophages, IL-36γ induced the activation of NLRP3 inflammasome through TLR4/MyD88 pathway (64) and increased the production of the antimicrobial peptides cathelicidin and β-defensin 2 in a dose-dependent manner (68, 69). In addition, IL-36γ was shown to modulate M1 macrophage polarization (70, 71) ( Figure 5D ). As discussed above, IL-1Rrp2 has been found to be expressed on the surface of neutrophils and B cells (50, 51). However, little is known about the effect of IL-36 cytokines on the function of these cells ( Figures 5E, F ). Since neutrophil proteases cleave and activate IL-36 cytokines, it is speculated that neutrophils may do so to enhance their own activation and antimicrobial activity. However, this has not been elucidated to date.

The Gram-positive pathogen Spn is the most common cause of community acquired pneumonia worldwide (79) and poses a major threat to public health given its ability to cause pneumococcal invasive diseases and the emergence of multi-drug resistant clones and serotypes not covered in the current vaccines (80–82). Spn has several virulence factors on its surface that impair the action of the host complement system and antibodies, and it also possesses a polysaccharide capsule that allows it to prevent phagocytosis by macrophages and neutrophils (83, 84). During Spn infection, IL-36γ – but not IL-36α or IL-36β – is rapidly produced in the lungs by macrophages, which packages IL-36γ into microparticles and exosomes for its secretion to the extracellular space (22, 75).

The production of IL-36γ has been shown to be essential for host defense against acute pulmonary Spn infection in mice, as both genetic IL-36γ deficiency and antibody-mediated depletion of IL-36γ are associated with impaired lung bacterial clearance, increased bacterial dissemination, and reduced survival (75). Interestingly, IL-36γ deficiency did not affect neutrophil recruitment to the lungs, but instead, diminished the local production of key pro-inflammatory cytokines, including IFN-γ (75), which is well-known to activate macrophages and to enhance their ability to kill bacteria (85). Consistently, ex vivo stimulated macrophages derived from IL-36γ^-/- and IL-36R^-/- mice exhibited an impaired response to Spn that was characterized by a reduced phagocytic ability and reduced expression of Nos2 (75), which encodes iNOS that is part of the killing machinery of macrophages (86). Importantly, treatment of IL-36γ^-/- mice with IL-36γ-loaded microparticles restored the production of IFN-γ, IL-12 and IL-23 in the lungs and improved lung Spn clearance (75). These observations indicate that IL-36γ is essential for the induction of key pro-inflammatory cytokines, including the Th1 cytokine IFN-γ, suggesting that IL-36γ is a key regulator of macrophage activation during Spn infection (75).

A similar role for IL-36γ has been described during pulmonary infection with the Gram-negative bacterium Kpn. The polysaccharide capsule of Kpn is crucial for enabling the bacteria to evade phagocytosis and escape clearance by macrophages and neutrophils (87, 88). As during Spn infection, the absence of IL-36γ during acute pulmonary Kpn infection leads to impaired bacterial clearance in the lungs, higher bacterial dissemination to the bloodstream and increased mortality. The impaired host defense in IL-36γ^-/- mice was characterized by impaired production of type-1 cytokines, such as IL-12, IL-23 and IFN-γ at 48 hours post infection (75).

IL-36 cytokines have also been shown to be important in host defense against infection with the Gram-negative bacterium Legionella pneumophila (Lpn). Lpn is the cause of Legionnaires’ disease, which can cause a severe form of pneumonia that is increasing in incidence in the United States and Europe, with an associated mortality of around 10% (89–91). A key characteristic of Lpn is the secretion of virulence factors via a type 4 secretion system. These secreted virulence factors prevent phagosome maturation and acidification, which allows Lpn to escape phagocytic killing by macrophages (92). In a murine model of lung Lpn infection, the expression of IL-36α and IL-36γ, but not IL-36β, is rapidly induced in the lungs following infection (71). In contrast to Spn and Kpn infection, where exclusively IL-36γ exerted the main protective effect, during Lpn infection, the combined effect of IL-36α and IL-36γ were required to protect the host and promote survival (71). The lack of IL-36 signaling was associated with reduced production of chemokines and pro-inflammatory cytokines, leading to impaired recruitment of neutrophils and monocytes into the broncho-alveolar space and to impaired lung Lpn clearance (71). In addition, ex vivo studies performed in alveolar macrophages showed that IL-36 signaling is crucial for macrophage polarization during Lpn infection, as macrophages lacking IL-1Rrp2 showed reduced expression of iNOS (M1 marker), increased expression of Arg-1 (M2 marker) and reduced production of pro-inflammatory cytokines, including IL-1β, TNF-α and IFN-γ (71).

The protective role of IL-36 cytokines has also been described in an experimental model of lung BCG infection and in Mtb infection in vivo and in vitro (65, 68, 69, 76). In the case of BCG infection, IL36R^-/- mice showed similar host survival (76), but resulted in excessive recruitment of immune cells and excessive lung inflammation compared to WT mice (65, 76). Moreover, isolated splenocytes from IL-36R^-/- mice produced lower amounts of pro-inflammatory cytokines involved in type 1 responses, such as IFN-γ, IL-6 and TNF-α after stimulation with BCG-antigen or viable bacterium in vitro (65). Finally, IL-36β was shown to act synergistically with IL-2 and IL-12 to promote Th1 polarization in vitro and to also enhance Th1 immune response in vivo during BCG infection (65).

Several cell types such as human peripheral blood mononuclear cells (PBMCs), human monocytes-derived macrophages, THP-1 macrophages, murine bone marrow-derived macrophages and, to a lesser extent, A549 human lung epithelial cell line, produced IL-36γ in response to live Mtb in vitro. This IL-36γ production was shown to depend on recognition of Mtb PAMPs via TLR2 and TLR4 and subsequent signaling via MyD88. In addition, inflammasome-dependent production of active IL-1β and IL-18 was shown to further enhance late-stage IL-36γ production in response to Mtb (68). In Mtb-infected macrophages, IL-36γ induces the activation of Liver X Receptor (LXR) (69), which actively contribute to Mtb immunity in mice by improving bacterial clearance and reducing lung inflammation (93). Mechanistically, LXR induction by IL-36γ triggered the production of antimicrobial peptides such as Cathelicidin, β-defensin 1 and β-defensin 2 (68, 69), improving Mtb clearance by macrophages. These in vitro findings suggest a protective role of the LXR/IL-36γ axis in response to Mtb. However, in vivo mouse models demonstrated that the lack of IL-36 signaling did not significantly affect host defense, lung inflammation or host survival during a pulmonary Mtb infection. This finding indicates that IL-36 signaling possess a limited contribution in the immunity against Mtb in the lungs (76).

In contrast to the beneficial effects of IL-36 cytokines in host defense against the majority of the reported bacterial infections in the lungs thus far, the effects of IL-36 cytokines are detrimental during pulmonary infection with Pa (77). Pa is a Gram-negative pathogenic bacterium that causes acute lower respiratory tract infections in the ICU (94) and chronic infections in cystic fibrosis (CF) patients. Pa pneumonia is characterized by excessive lung inflammation as a consequence of the large number of toxins and virulent factors produced and released by this pathogen to the alveolar lumen (95). Murine alveolar macrophages and alveolar epithelial cells rapidly produce high levels of IL-36α and IL-36γ within the first 24 hours following Pa infection (41, 77). The early production of IL-36γ, but not IL-36α, induces excessive production of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-17 (77). IL-36γ^-/- and IL-36R^-/- mice display reduced pro-inflammatory cytokine production, reduced lung damage, and improved bacterial clearance following Pa infection compared to WT mice, suggesting that the excessive inflammation caused by IL-36γ during PA infection impairs bacterial clearance and exacerbates lung injury (77). Consistent with this finding, IL-36γ^-/- and IL-36R^-/- mice showed increased survival compared to WT mice (77). Mechanistically, IL-36γ induced the expression of COX-2 and the production of prostaglandin E2 (PGE2) in alveolar macrophages, which impaired the clearance of Pa by these cells (77). This observation shows that IL-36γ impairs bacterial clearance in a PGE2-dependent manner and is in line with previous studies that describe the immunosuppressive properties of PGE2 in the context of Pa infection (96, 97). Despite this observation, it is unclear why IL-36γ production is detrimental during acute lung Pa infection. One possibility is that Pa secretes proteases such as LasB, known to trigger endogenous host neutrophil proteases such as NE and CatG, that may result in excessive cleavage and activation of IL-36γ during Pa infection leading to uncontrolled inflammation and negatively impacting host survival (41). Further studies analyzing the role of IL-36γ examining clinical isolates with high and low proteolytic activity would provide useful insights regarding this hypothesis.

IL-36 cytokines are also important players in the lung immune response against influenza virus, although their exact function in this context remains uncertain. While one study indicates that IL-36 signaling (IL-36R^-/- mice) exerts detrimental effects for the host and increases mortality following infection (78), a later study indicates that IL-36γ enhances host survival (70). Using IL-36R^-/- mice, the first study showed that IL-36 signaling exerts a pathogenic response during pulmonary influenza virus infection, which is characterized by severe lung injury and higher mortality compared to WT mice and IL-36γ^-/- mice, (78). Absence of IL-36 signaling improved host survival and reduced the infiltration of neutrophils and monocytes/macrophages, reduced the production of pro-inflammatory cytokines involved in neutrophilic inflammation such as IL-6, IL-17A, CXCL-1 at day 6 post infection and limited lung inflammation and injury. Interestingly, IL-36R^-/- mice showed increased viral burden in the lungs, indicating that the increased resistance of IL-36R^-/- mice to influenza virus is determined by their ability to temper the immune response and prevent lung injury rather than due to enhanced viral clearance (78). This study did not detect a survival difference between WT and IL-36γ^-/- mice and suggested that the detrimental effects of IL-36 signaling in influenza virus are driven by IL-36α or IL-36β and not IL-36γ.

In contrast, another study reported that IL-36γ^-/- mice display a higher mortality following an acute pulmonary influenza infection and impaired viral clearance at day 6 post infection, suggesting that IL-36γ is required for an effective host defense against influenza virus (70). Interestingly, whereas no major differences in innate cell infiltration to the lungs between IL-36γ^-/- and WT mice were identified during the first 6 days of infection, a surprising difference was that IL-36γ^-/- mice showed massive apoptosis of alveolar macrophages, leading to a rapid reduction of alveolar macrophages in the lungs of these mice (70). The increased apoptosis of alveolar macrophages observed in IL-36γ^-/- mice was thought to be due to enhanced M2 macrophage polarization, as macrophages from IL-36γ^-/- mice showed higher expression of M2 markers such as CD206, CD200R in the surface, as well as Retnla transcript. Importantly, the transfer of WT alveolar macrophages to IL-36γ^-/- mice improved the survival of these mice in response to influenza to a similar level as in WT mice. Overall, these findings suggest that IL-36γ modulates alveolar macrophage polarization, thereby enhancing macrophage survival and host defense during influenza infection (70). Taken together, these two studies show that IL-36 signaling has a major role in host defense and lung inflammation during influenza infection, although the specific role of each IL-36 cytokine in host defense and lung inflammation, their differential cellular source and how their kinetic production influences the host antiviral response remains to fully understood.

The role of IL-36 cytokines in the lungs is not restricted to infectious diseases. Studies have described a potentially important role of the IL-36 pathway in asthma (98, 99), idiopathic pulmonary fibrosis (21, 100), chronic obstructive pulmonary disease (101, 102), cystic fibrosis (103) and cancer (104). During asthma, the levels of the anti-inflammatory IL36Ra in the serum and sputum of asthmatic patients are reduced compared to healthy controls (98). In vitro data from the same study showed that IL36Ra inhibits the production of various cytokines known to be important drivers of asthmatic inflammation, such as IL-1β, IL-6, TNF-α and IL-17A in PMBCs and in sputum mononuclear cells (SMNCs) isolated from asthmatic patients pre-stimulated with LPS, that are presumably induced by IL-36 cytokines (98). In addition, using an experimental asthma model of OVA-sensitized mice, it was established that intravenous administration of recombinant IL-36Ra (rIL-36Ra) alleviated the severity of experimental asthma by reducing airway hyper-responsiveness, alveolar inflammation and eosinophil, neutrophil, macrophage, and lymphocyte recruitment. The protective effects of rIL-36Ra were not observed in IL-36R^-/- mice, showing that the mechanism by which rIL-36Ra protected the lungs in sensitized mice was through the IL-36 receptor pathway (98).

The in vivo administration of the anti-inflammatory cytokine IL-38, which also inhibits IL-36R-signaling (55), was shown to reduce the severity of house dust mite (HDM)-induced asthma (99). Specifically, IL-38 administration decreased airway hyper-responsiveness of HDM-treated mice to baseline levels, reduced lung inflammation, BALF eosinophilic recruitment and limited the production of IL-4, IL-5, IL-6 in the lungs. Importantly, IL-38 administration also inhibited the proliferation of Th2 and Th17 cells, prevented the recruitment of ILC2 cells and promoted the proliferation of Treg cells (99). IL-36γ has been found to be upregulated during HDM-induced asthma, triggering a strong inflammatory response in the lungs of sensitized mice (74).

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease characterized by the progressive loss of lung function. Interestingly, IL-38 is highly expressed in type II pneumocytes in patients with IPF but not in healthy controls (100). Moreover, normal human lung fibroblasts express IL-1Rrp2 at baseline and produce several pro-inflammatory cytokines such as IL-8, CXCL3, G-CSF and CCL20 in response to IL-36γ (21) ( Figure 5G ). Currently, it is not clear which factors induce IL-38 expression in IPF patients and what the role of this cytokine is in IPF progression. However, as lung fibroblasts respond to IL-36γ and IL-1Rrp2 neutralization has protective effects during intestinal fibrosis (105), it can be speculated that the IL-36 pathway may be a major pathway during lung fibrosis.

Chronic obstructive pulmonary disease (COPD) is a chronic lung inflammatory disease characterized by airflow obstruction. Promising data regarding the role of IL-36 cytokines in the development and severity of COPD has emerged in the last years. Studies have demonstrated that normal human bronchial epithelial cells and primary human bronchial epithelial cells express high amounts of IL-36α, IL-36β, IL-36γ and IL-36Ra after being treated with cigarette smoke components – a causative agent of COPD – in vitro (101, 102). Moreover, smokers with and without COPD presented with elevated levels of IL-36α, and IL-36γ in plasma and BALF at baseline, although no differences in IL-36 cytokines levels were found between non-COPD smokers and COPD smokers (102). Another study showed that IL-36α, and IL-36γ were increased in the sputum of neutrophilic COPD patients, compared to eosinophilic COPD, and were associated with the amount of neutrophils in the sputum (106). Despite the emergence of novel data regarding the role of IL-36 cytokines in COPD, more studies are needed to accurately understand the possible pathogenic role of IL-36 cytokines during COPD.

Cystic fibrosis (CF) is a chronic lung disorder caused by genetic mutations leading to functional defects in the cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel that regulates the flow of chloride ions in epithelial cells (107). CF is characterized by the excessive production of sticky mucus and lung inflammation caused by neutrophilic inflammation and chronic bacterial infection (107). The role of IL-36 cytokines in lung inflammation during CF remains relatively unexplored. However, CF patients usually present with a neutrophil-predominant inflammatory response in sputum supernatants, and massive release of neutrophil elastase (108), and therefore it is highly possible that IL-36 cytokines play a role in lung inflammation and injury in CF. An RNA-seq analysis of bronchial brushings obtained from CF patients and healthy controls showed that IL-36γ gene expression is upregulated in the airways of CF patients (4-fold) compared to healthy controls (103). The expression of IL-36α did not differ between CF patients and healthy controls, while the expression of IL-36β and the anti-inflammatory IL-36Ra were downregulated in CF patients (103). These data suggest that, during CF, IL-36γ but no other members of the IL-36 family may be contributing to the pro-inflammatory environment. Additional studies are needed to corroborate this hypothesis.

A potential protective role of IL-36 signaling has been identified in different types of cancer in mice and humans. In mice, IL-36γ has been shown to promote a Th1 response in a tumor model of B16 cells (67). This anti-tumor response elicited by IL-36γ was characterized by increased production of IFN-γ by CD4⁺ cells, CD8⁺ cells, NK cells and γδ T cells, which ultimately resulted in the reduction of tumor size and higher survival (67). In humans, IL-36α expression in the liver of hepatocellular carcinoma patients was found to be negatively correlated with tumor size and vascularization, while IL-36α expression was positively correlated with CD8⁺ tumor-infiltrating lymphocytes and patient survival (109). Finally, the production of IL-36γ was found to be reduced in late stages of lung squamous cell carcinoma compared to early stages (67). Although more research is needed to elucidate the exact role of IL-36 signaling during lung cancer, altogether these data suggest that IL-36 cytokines may trigger a pro-inflammatory response in the tumor microenvironment, exhibiting anti-tumoral effects in a context where pro-inflammatory signals versus immunosuppressive mechanisms determine tumor elimination or survival.