Unique Action of Interleukin-18 on T Cells and Other Immune Cells

Interleukin (IL)-18 was originally discovered as a factor that enhances interferon (IFN)-γ production by anti-CD3-stimulated Th1 cells, particularly in association with IL-12. IL-12 is a cytokine that induces development of Th1 cells. IL-18 cannot induce Th1 cell development, but has the capacity to activate established Th1 cells to produce IFN-γ in the presence of IL-12. Thus, IL-18 is regarded as a proinflammatory cytokine that facilitates type 1 responses. However, in the absence of IL-12 but presence of IL-2, IL-18 stimulates natural killer cells, NKT cells, and even established Th1 cells to produce IL-3, IL-9, and IL-13. Thus, IL-18 also facilitates type 2 responses. This unique function of IL-18 contributes to infection-associated allergic diseases. Together with IL-3, IL-18 stimulates mast cells and basophils to produce IL-4, IL-13, and chemical mediators such as histamine. Thus, IL-18 also induces innate-type allergic inflammation. IL-18 belongs to the IL-1 family of cytokines, which share similar molecular structures, receptors structures, and signal transduction pathways. Nevertheless, IL-18 shows a unique function by binding to a specific receptor expressed on distinct types of cells. In this review article, I will focus on the unique features of IL-18 in lymphocytes, basophils, and mast cells, particularly in comparison with IL-33.

lipopolysaccharide (LPS) induces IL6 production in vivo. We found that there were at least two groups: an endotoxin shock susceptible group, characterized by high serum IL6 level with low serum LPS level, and an endotoxinresistant group, characrized by low serum IL6 level with high serum LPS level. These findings indicated the presence of certain limiting factors that determined the sensitivity of patients to endotoxin shock. I learned that priming with heatkilled Propionibacterium acnes (P. acnes), a Grampositive bacterium, or BCG increased the sen sitivity of animals to the lethal effect of LPS. Thus, with Tomohiro Yoshimoto, my longterm collaborator, I studied the mechanism for how P. acnes increase the responsiveness of mice to LPS. We found that P. acnes priming rendered mice highly susceptible to the lethal effect of LPS by enhanced production of IL1 and/ or tumor necrosis factorα (TNFα) as well as increased respon siveness to the stimulation with IL1 and/or TNFα.
After publishing these results (1) in 1992, I observed the very interesting phenomenon that P. acnesprimed BALB/c nu/nu mice were resistant to LPSinduced lethal shock, and instead most of them died of fulminant hepatitis through apoptosismediated hepatocytotoxicity. My colleagues, Haruki Okamura and Hiroko Tsutsui, demonstrated this severe liver injury was prevented by administration of a neutralizing antiIL18 antibody (2). These experiments were my first exposure to the unique action of IL18, which forms the longterm target of my investigations and the main theme of this manuscript. In this review, I will initially describe animal models of LPSinduced diseases, and then describe the actions of IL18 on T cells and other immune cells, as the major topic of the manuscript. Finally, I will compare the actions of IL18 and IL33 in various aspects. Pathological roles of IL18 in various diseases, including hepatic, metabolic, inflamma tory, allergic, and autoimmune diseases, are also documen ted in previous (3,4) and recent (5, 6) reviews.

Susceptibility to LPS-induced endotoxin Shock
Mice primed with P. acnes markedly increased production of IL1 and TNFα in response to LPS. Further more, these mice were highly susceptible to the lethal shockinducing effect of IL1 and/ or TNFα (1). We tried to identify the limiting cells for LPS sensitiv ity. As P. acnesprimed BALB/c nu/nu mice were resistant to LPS induced lethal shock, we examined the LPS susceptibility of these mice after recons titution with splenic T cells from wildtype mice (7). We found that BALB/c nu/nu mice reconstituted with T cells became highly susceptible to LPS shock after P. acnes treatment and systemic administration of P. acnes induced development of Th1 cells in wildtype mice as well as in BALB/c nu/nu mice reconstituted with splenic T cells (7). Furthermore, IL12p40 deficient mice or interferon (IFN)γdeficient mice were highly resistant to sequential treatment with P. acnes and LPS (7). Thus, IFNγproducing Th1 cells play an important role in determi ning host sensitivity to LPS shock (7).

Susceptibility to LPS-induced Liver injury
The liver has a potent immune system (3). It contains residen tial immunocompetent cells with selfrenewing ability, such as liver NK cells, extrathymically developed T cells, thymically developed CD4 + NKT cells, expressing CD4 and NK cell mar kers, and a limited Tcell antigen receptor repertoire, and Kupffer cells, tissue macrophages. With my longterm colleague Kiyoshi Matsui, I demonstrated that hepatic CD4 + NKT cells in non treated wildtype mice promptly produced large amounts of IL4 and IFNγ upon stimulation with immobilized antiCD3 in vitro (8). However, administration of heatkilled P. acnes in duced hepatic CD4 + NKT cells to increase IFNγ production, but decrease IL4 production upon antiCD3 stimulation in vitro (8). These effects were attributable to the action of IL12 from P. acneselicited Kupffer cells, suggesting a role for Kupffer cells in regulation of immune responses in the liver (3,8). As noted above, most mice sequentially treated with P. acnes and LPS developed lethal shock, while the surviving mice suffered from liver injury. Meanwhile, BALB/c nu/nu mice sequentially treated with P. acnes and LPS developed severe liver injury. However, this severe liver injury was prevented by administration of a neutralizing antiIL18 antibody (2). Furthermore, P. acnesprimed IL18 deficient mice did not develop liver injury upon LPS challenge (9,10). However, we found that administration of IL18induced liver injury in P. acnesprimed IL18deficient mice by inducing Fas ligand expression and TNFα production in hepatic NK cells (3,11). Based on these findings, we concluded that the develop ment of thymic T cells into Th1 cells and hepatic CD4 + NKT cells into predominant IFNγproducing cells was important for induction of LPSdriven endotoxin shock and LPSinduced liver injury in P. acnesprimed mice, respectively (7,8).

OveRview OF THe iL-18/iNTeRLeUKiN 18 ReCePTOR (iL-18R) SYSTeM
Interleukin18 was originally designated IFNγinducing fac tor (IGIF), because it was first identified through its capacity to induce IFNγ production by antiCD3stimulated Th1 cells (2,12).Okamura and colleagues discovered this activity in sera or liver extracts from mice sequentially treated with P. acnes and LPS (2,4). Based on the homology of its amino acid sequence to that of IL1β, and its shared βpleated sheet structure with IL1β (2), IL18 was classified into the IL1 family of cytokines (13,14). IL18 is produced as a biologically inactive precursor, proIL18, that is localized in the cytoplasm and requires proteolytic pro cessing for secretion as active IL18 (2)(3)(4). In collaboration with K. Kuida (Vertex, USA), S. Taniguchi (Shinsyu University, Japan), and J. Tschopp (University of Lausanne, Switzerland), we demonstrated that cleavage of proIL1β and proIL18 into mature IL1β and IL18, respectively, depended on the action of intracellular cysteine protease caspase1, produced in the NLRP3 inflammasome consisting of pattern recognition receptor NLRP3 (NACHTLRR and pyrin domaincontaining protein 3), adaptor molecule ASC (apoptosisassociated specklike protein contain ing a caspase recruitment domain), and procaspase1 (15)(16)(17)(18). However, we also found that Fas ligand treatment stimulated Wild-type mice or mice deficient for interleukin (IL)-18, MyD88, or TRIF were administered with heat-killed P. acnes and examined for their hepatic granuloma formation at day 7 after this treatment. Only P. acnes-primed MyD88 did not develop hepatic granuloma at day 7 after treatment, suggesting that P. acnes treatment induces hepatic granuloma in a MyD88-dependent manner, but TRIF-independent manner. Although TRIF-deficient mice normally developed hepatic granulomas after P. acnes treatment, they could not release IL-18 or develop liver injury, suggesting that LPS TRIF-dependently activated caspase-1 via NLRP3 inflammasome. And, resultant IL-18 induces liver injury by induction of interferon-γ, FasL, and tumor necrosis factor-α.
Fasexpressing Kupffer cells or macrophages to produce active IL18 in a caspase1independent manner, indicating the presence of some other caspasemediated pathways for IL18 secretion (11). A recent study revealed that Fas mediated noncanonical IL1β and IL18 maturation via caspase8 (19). In addition, IL18 can be activated in an inflammasomeindependent manner by proteases, such as proteinase 3 (20), chymase (21), and granzyme B (22).
Interleukin18dependent cell activation can be inhibited at least by two distinct molecules. One is the naturally occurring IL18binding protein (IL18BP) (27). Because IL18BP binds to IL18 with high affinity (400 pM), it can downregulate IL18 induced cell responses, such as IL18induced Th1 cell IFNγ production. Another inhibitor is the antiinflammatory cytokine IL37, a member of the IL1 family of cytokines (28). Although IL37 binds to IL18Rα with low affinity, the resulting complex inhibits recruitment of IL18Rβ, thereby abolishing signal trans duction via IL18R. Furthermore, this complex induces recruit ment of IL1R8, an orphan receptor of the IL1 family formerly known as SIGIRR, to form a tripartite complex (IL37/IL18Rα/ IL1R8), which does not bind MyD88, but instead induces anti inflammatory signal into the cell. Thus, IL18 activity is inhibited by these two distinct inhibitors (6).

MeCHANiSM FOR LPS-iNDUCeD LiveR iNJURY iN P. acnes-PRiMeD MiCe
Consistent with a previous report (29), wildtype mice primed with P. acnes developed dense granulomas in the liver. These mice also developed acute liver injury and elevated serum IL18 level after challenge with a sublethal dose of LPS (2)(3)(4)(5). Although P. acnesprimed IL18deficient mice exhibited dense granu lomas, similar to the liver of P. acnesprimed wildtype mice, they did not develop liver injury after LPS treatment (10,11). In contrast, MyD88deficient mice primed with P. acnes showed very poor hepatic granuloma formation and produced an undetectable level of IL18 upon LPS challenge (17). This failure to produce IL18 in response to LPS was not caused by a loss of potential of MyD88deficient Kupffer cells to produce IL18, because MyD88deficient Kupffer cells were able to secrete IL18 in response to LPS in vitro (30). Thus, P. acnes treatment induced hepatic granuloma formation in a MyD88dependent manner and LPS stimulated Kupffer cells to produce IL18 in a MyD88independent manner (Figure 1). Next, we examined the contribution of TRIF (TIR domaincontaining adapter indu cing IFNβ) to P. acnesinduced hepatic granuloma formation and LPSinduced IL18 secretion. In contrast to MyD88deficient mice, P. acnesprimed TRIFdeficient mice showed normal devel opment of hepatic dense granuloma, but did not release IL18 and, therefore, did not develop liver injury (17). Thus, we con cluded that P. acnes treatment induced hepatic granuloma for mation in a MyD88dependent manner and that subsequent LPS challenge induced caspase1 activation in a TRIFdependent manner in the NLRP3 inflammasome and induced IL18 release, eventually leading to liver injury (17) (Figure 1).

SeveRAL TOPiCS FOR THe UNiQUe FUNCTiONS OF iL-18 iFN-γ Production
Consistent with its original discovery as an IFNγinducing fac tor, IL18 can induce IFNγ production by natural killer (NK) cells and Th1 cells that express IL18R (2, 4) (Figure 2). However, IL18 also synergizes with IL12 to induce marked IFNγ pro duction by various cell types, including nonpolarized T cells, NKT cells, dendritic cells, macrophages, and B cells, through reciprocal induction of expression of their corresponding rece ptors (4). It is well known that B cells produce IgG1 and IgE when stimulated with antiCD40 and IL4. To our surprise, a combination of IL12 and IL18 inhibited IL4dependent IgG1 and IgE production, but enhanced IgG2a production by indu cing IFNγ production in B cells stimulated with IL12 and IL18 (31). Indeed, IL12stimulated B cells expressed IL18R and strongly produced IFNγ in response to IL18, particularly in association with IL12 (23). We also found that naïve Th cells stimulated with antigen (Ag) and IL12 or IL4 developed into IL18Rexpressing Th1 or ST2expressing Th2 cells, respectively (23,24,32). Thus, expression of IL18R and ST2 can be a conve nient cell marker for Th1 and Th2 cells, respectively.

Th2 Cytokine Production by Mast Cells and Basophils Stimulated with iL-18
In 1989, Marshall Plaut and Bill Paul reported in Nature that, upon crosslinkage of FcεR1 with Ag/IgE complex, mast cells, and basophils produce Th2 cytokines, including IL4 and IL13 (33). Thus, I was interested to know whether mast cells and baso phils also had the potential to produce IFNγ after stimulation with IL12 and IL18. I discussed this matter with Bill, and he said "I am very interested in what will happen. " Thus, Tomohiro and I started collaboration with Bill. We found that basophils and mast cells derived by culture of bone marrow cells with IL3 for 10 days expressed the IL18Rα chain and produced large amounts of IL4 and IL13 in response to stimulation with IL3 and IL18 (34). These were unexpected results, but turned out to be very important findings. To our disappointment, however, mast cells and basophils never produced IFNγ in response to various combinations of IL3, IL18, and IL12 (34). As the com bination of IL18 and IL3 stimulated basophils and mast cells to produce histamine and Th2 cytokines, we speculated that IL18 could induce allergic inflammation without assistance from the Ag/IgE complex. Thus, we reported a new aspect of IL18 as an inducer of Th2 cytokine production from basophils and mast cells in 1999 (34) (Figure 3). Later, I became interested in the capacity of basophils to produce IL4 upon crosslinkage of FcεR1 with Ag/IgE complex. Surprisingly, we detected expression of MHC class II molecules on basophils (35). Thus, we examined the capacity of basophils pulsed with Ag/IgE complex to induce deve lopment of naïve Th cells into Th2 cells. We found that basophils had the capacity to induce development of Th2 cells (35). Although we were still unable to determine the physiological role of baso phils as APCs, we believe that further studies will demonstrate such an activity in basophils.

innate-Type Allergic inflammation
After publication of the paper on Th2 cytokine production by basophils and mast cells stimulated with IL3 and IL18, I specu lated that IL18 may have the potential to induce IL4 production by CD4 + T cells and/or CD4 + NKT cells. I found that injection of a mixture of IL12 and IL18 increased serum IgE levels in helminthinfected IFNγdeficient mice. Most surprisingly, daily administration of IL18 in particular with IL2 induced a marked increase in serum IgE levels in a CD4 + T cell and IL4/IL4R/ STAT6dependent manner (36). Furthermore, CD4 + NKT cells stimulated with IL2 and IL18 increased their CD40 ligand expression and IL4 production. In addition, these activated CD4 + NKT cells induced development of B cells into IgG1 and IgEproducing cells. Consistent with these findings, transgenic mice overexpressing human caspase1 in keratinocytes, estab lished by Hitoshi Mizutani (Mie University), produced IL18 and IgE in their sera, and also spontaneously developed atopic derma titis (AD)like skin lesions (37). Disruption of STAT6, required for IL4 signal transduction, abolished IgE production without affecting the skin manifestations. In contrast, disruption of IL18 in caspase1 transgenic mice diminished their chronic derma titis almost completely, although they still produced significant amounts of IgE. Thus, overproduction of IL18 by keratinocytes induced ADlike skin lesions even in the absence of IgE and IgG1 (37). Based on these results, we designated this IL18induced allergic inflammation an innatetype allergic inflammation.

Nakanishi
Unique Action of IL-18 Frontiers in Immunology | www.frontiersin.org April 2018 | Volume 9 | Article 763 In the presence of IL2, but absence of IL12, IL18 stimulated NK cells, CD4 + NKT cells, and splenic CD4 + T cells to produce IL3, IL9, and IL13 (26, 36) (Figure 2). Because IL3 and IL9 induce mucosal mastocytosis, we examined whether the animals developed mucosal mastocytosis after treatment with IL2 and IL18. We found that C57BL/6 mice pretreated with IL18 and IL2 developed mucosal mastocytosis with high levels of serum mMCP1, an activation marker of MMC, and became able to promptly expel the intestinal nematode Strongyloides venezu elensis. Thus, IL18 is important for expulsion of intestinal nema todes by induction of mucosal mastocytosis, and we published these results in J Exp Med (38).

Th1 CeLLS PRODUCe iFN-γ AND iL-13 iN ReSPONSe TO Ag AND iL-18
It is well established that IL18 increases IFNγ production by antiCD3stimulated Th1 cells, particularly in association with IL12 (2, 4). Furthermore, endogenous IL18 is required for host defense against intracellular microbes, such as Listeria mono cy togenes, Cryptococcus neoformans, and Leishmania major, because IL18induced IFNγ activated the infected macrophages suffi ciently to kill these pathogens (4,39,40). However, we had not examined the possibility that IL18stimulated Th1 cells can pro duce Th2 cytokines. Thus, we stimulated established ovalbumin (OVA)specific Th1 cells with OVA and/or IL18 and found that OVA plus IL18stimulated Th1 cells produce both a Th1 (IFNγ) and Th2 cytokines (IL9, IL13) (41) and additional IL2 stimula tion enhanced production of Th2 cytokines (Figure 2).
Next, we examined whether IL18 acts on memory Th1 cells to induce airway inflammation and airway hyperresponsiveness (AHR) in naïve host mice. In 2002, Nobuki Hayashi and Bill Paul developed a method to establish both resting Th1 and Th2 memory cells (42). Nobuki performed a wonderful study after coming back to my laboratory from the LI. To avoid a background response of hostderived T cells, he administered newly polar ized OVAspecific Th1 or Th2 cells into naïve mice and allowed them to adopt a resting memory phenotypy in vivo. Intranasal administration of OVA induced airway inflammation and AHR only in mice that received Th2 cells (41). However, mice that received Th1 cells developed airway inflammation and AHR after intranasal administration of both OVA and IL18 (41). Th1 cells stimulated with OVA and IL18 became harmful cells, which we designated "super Th1 cells, " that produced IFNγ and IL13, the combination of which induced difficult bronchial asthma (41). Nobuki further demonstrated that naïve mice having resting Th1 memory cells developed severe bronchial asthma in response to nasal administration of OVA plus LPS instead of IL18. He also revealed that endogenous IL18 from LPSstimulated bronchial epithelial cells was responsible for inducing severe bron chial asthma. He published these results in 2007 (43). This promi nent feature of IL18 can explain the mechanism for infection associated allergic diseases (44) (Figure 2).
Intriguingly, after several rounds of stimulation with Ag, IL2 plus IL18, Agspecific Th1 cells were found to differentiate from cells producing both IL13 and IFNγ into cells producing IL13, but little IFNγ. My colleague Masakiyo Nakahira verified that GATA3 was essential for induction of IL13 in Th1 cells after stimulation of these cells with Ag, IL2, and IL18 (45). Thus, IL18 has the potential to induce plasticity of established Th1 cells (41,(43)(44)(45) (Figure 2).
We found that mast cells and basophils express both IL18R and IL33R and produce IL4 and IL13, when stimulated with IL3 plus IL18 or with IL33, respectively (34,49) (Figure 3). Therefore, IL18 and IL33 have very similar effects on mast cells and basophils. Moreover, IL18 and IL33 show similar pathological effects on the lungs. Nasal administration of IL2 and IL18 induced AHR, pulmonary eosinophilia, and goblet cell hyperplasia in wildtype mice, but not in Rag2deficient mice (50) (Figure 4). However, nasal administration of IL33 induced the same changes in both wildtype mice and Rag2deficient mice (49) (Figure 4). Thus, IL2 plus IL18 induced these pulmonary changes in a NKT celldependent manner, while IL33 treat ment induced the same changes in a NKT cellindependent and innate celldependent manner (Figure 4). Moro et al. (51) and Neill et al. (52) showed that natural helper cells (NH cells) or nuo cytes, currently designated group 2 innate cells (ILC2s), express IL33R, and produce IL5 and IL13 in response to IL33. As natural killer (NK)T cells constitutively express IL-18R, intranasal administration of IL-18 into wild-type mice, but not into Rag2Ko mice induced bronchial asthma by induction of IL-4 and IL-13 from NKT cells. In contrast, intranasal administration of IL-33 into wild-type mice and Rag2Ko mice equally induce bronchial asthma, because Rag2Ko mice are equipped with ILC2 which express IL-33R and produce IL-13 in response to  hyperplasia by producing IL5 and IL13 in a Tcellindependent manner (53). Thus, IL33 plays an important role in the induc tion of ILC2dependent allergic diseases. Furthermore, he found that infection with the intestinal nematode S. venezuelensis, which transiently migrates into the lungs, increased the number of IL33producing alveolar epithelial type II cells in the lungs of wildtype mice and Rag2deficient mice (53). Thus, both types of mice infected with S. venezuelensis developed eosinophilic inflammation and goblet cell hyperplasia in their lungs (Loeffler syndrome) (53). Therefore, IL33 production and release in the lungs is very important for induction of pulmonary eosinophilic inflammation during nematode infection (53)(54)(55).

AUTHOR CONTRiBUTiONS
The author confirms being the sole contributor of this work and approved it for publication.

ACKNOwLeDgMeNTS
The author expresses his sincere gratitude to Drs. William Paul and Tadamitsu Kishimoto for their great help in all stages of his research. The author also thanks all members of the Department of Immunology and Medical Zoology, Hyogo College of Medi cine, and all of his collaborators inside and outside of Japan.

ReFeReNCeS
My longterm colleague Koubun Yasuda revealed the mech anism for how IL33 induced the above pulmonary changes in the absence of acquired immunity. He showed that IL33 tre atment increased the number of ILC2s and that the IL33 activatedILC2s induced pulmonary eosinophilia and goblet cell