Dual function of fungi-derived cytokines in inflammatory bowel diseases: protection or inflammation

Abstract Inflammatory bowel disease (IBD) is an immune-mediated inflammatory condition involving both the innate and adaptive immune systems. Recently, the role of intestinal fungal flora and their downstream immune pathways has been highlighted in the pathogenesis of IBD. Cytokines as primary immune mediators require a delicate balance for maintaining intestinal homeostasis. Although most cytokines have a predictable role in either amplifying or attenuating inflammation in IBD, a few cytokines have shown a dual function in the inflammatory state of the intestine. Some of these dual-faced cytokines are also involved in mucosal anti-microbial defense pathways, particularly against intestinal fungal residents. Here, we reviewed the role of these cytokines in IBD pathogenesis to achieve a better understanding of the fungal interactions in the development of IBD.


Introduction
Inflammatory bowel disease (IBD), which mainly includes Crohn's disease (CD) and ulcerative colitis (UC), is a chronic multifactorial disorder that places a considerable burden on the global health system [1,2].IBD is generally defined as an excessive inflammatory response to intestinal microbiota in a genetically susceptible individual [3].Fungi, as the second most prevalent residents of the intestine, are described to interfere with IBD pathogenesis [4].An increased risk of IBD is observed in the presence of distinct genetic variations in fungal recognition receptors, leading to aberrant immune responses against fungal species and further cytokine production [5].
Cytokines govern key cellular processes in the intestine including cell death, proliferation, and inflammatory responses to pathogens.They constantly mediate the crosstalk between the immune and the epithelial cells even under homeostatic conditions [6].IBD, as an immune-mediated inflammatory condition, alters the expression of a substantial number of cytokines [7,8].The altered cytokine network could result in either susceptibility or resistance to IBD.Therefore, recent biological therapies have targeted particular cytokines for better disease control; however, some of these potent biologic agents failed to induce remission [9].Despite cytokines with a predictable role in either amplifying or attenuating inflammation of IBD, a few cytokines showed a dual function in the inflammatory state of the intestine [10].The effect of these cytokines on IBD seems to be time-and dosedependent, requiring a delicate intestinal homeostasis [11].
On the other hand, some of these dual-faced cytokines are also involved in mucosal anti-microbial defense against fungal pathogens [12].Commensal gut fungi mainly act via the Dectinspleen tyrosine kinase-caspase recruitment domain 9 signaling pathway and produce a wide range of cytokines [13].These interactions result in protection or tolerance against fungal species and strengthen the role of an imbalanced intestinal fungal community in the development of IBD.Some studies have even indicated that distinct fungal species could initiate inflammatory or anti-inflammatory cascades in IBD [4,14].These specific cytokines include interleukin (IL)-1, IL-4, IL-6, IL-10, IL-13, IL-17, IL-18, IL-22, IL-23, IL-33, IL-35, tumor necrosis factor-alpha (TNF-a), and interferon-gamma (IFN-c) [10,11,15,16].Since the mechanisms by which intestinal fungi affect IBD are not clear, we aimed to review these cytokines to achieve a better understanding of their role in IBD development and their probable interactions against intestinal fungi.

Cytokine network in IBD
In an inflamed intestinal mucosa, IL-1, IL-18, IL-6, and IL-23 are produced by intestinal epithelial cells, innate lymphoid cells, and phagocytes.Of these cytokines, IL-1, IL-18, and IL-6 promote the survival and proliferation of intestinal epithelial cells, thereby maintaining the intestinal barrier [10].Simultaneously, in the opposite direction, IL-1b induces T helper (Th) 1 and Th17 differentiation [17] and IL-18 disrupts goblet cell maturation and function [18].In colitis models, both IL-1b and IL-18 generally Activated Th2 cells induce IL-4 and IL-13 cytokines; IL-13 is released from natural killer T cells as well [40].IL-13 and IL-4 can prompt intestinal fibrosis by down-regulating matrix metalloproteinase synthesis in fibroblasts, which has led to collagen accumulation in the intestinal tissue of in vitro and in vivo experimental models of CD and colitis [41,42].This effect promotes wound healing and reduces inflammation in the reparatory phase of intestinal inflammation.However, unresolved inflammation results in chronic inflammation, uncontrolled tissue remodeling, and ultimately tissue fibrosis [43].Furthermore, IL-13, but not IL-4, can impair epithelial barrier function by inducing epithelial apoptosis, and altering tight junctions and Interestingly, CD is usually designated as a Type 1-driven disease with an elevated activation of Th1 and Th17 cells and their related cytokines, whereas UC is more associated with Type 2 inflammation (Th2 cell activation) and subsequent cytokine production [53].

Cytokines and fungal pathogens
Recent studies have proposed tissue-resident fungi as immune modulators contributing to the pathogenesis of autoimmune, inflammatory, and neoplastic diseases [54].At the intestinal mucosal surface, C-type lectin receptors interact with fungal cell wall components and initiate a signal via spleen tyrosine kinase, a caspase recruitment domain 9, B-cell lymphoma 10, and mucosa-associated lymphoid tissue lymphoma translocation protein 1 complex, and/or Raf-1 proto-oncogene, serinethreonine kinase.These signaling pathways activate the production of IL-23, IL-6, IL-10, IL-2, IL-1, and TNF-a by phagocytes (macrophages, monocytes, dendritic cells, and neutrophils) [55].The interaction with fungi-induced phagocytes results in the development of Th1 and Th17 cells, and recruitment of neutrophils to the intestinal lamina propria [3].Commensal fungi play an important role in inflammasome activation via the spleen tyrosine kinase-caspase recruitment domain 9 pathway and its downstream IL-18, which is required for IFN-c release from T cells.This signaling pathway of commensal gut fungi could promote anti-tumorigenic T-cell responses and eventually protect against colitis and colon cancer [13].IL-1b and IL-18 generally represent similar roles in mucosal immunity against fungi after the stimulation of Dectin-1 [56, 57].Furthermore, IL-1b modulates Group 3 innate lymphoid cells to produce IL-22 [13].In response to intestinal fungi and mucosal fungal infection, IL-6 amplifies and eliminates the pathogens via recruiting neutrophils and augmenting Th1-mediated immunity [58-60].Malassezia restricta is particularly potent at inducing IL-6 expression from mouse dendritic cells [14].The detailed signaling pathway of commensal gut fungi is presented in Figure 2.
Moreover, the contribution of IL-10 to antifungal responses is associated with conflicting outcomes.IL-10 inhibits the fungicidal activity of monocytes or neutrophils [61-63], whereas it is required for the optimal development of IL-12-dependent Th1 cells and regulatory T cells [64,65].Recently a novel probiotic yeast attenuated colitis by upregulating IL-10 in mice [66].An in vitro study of murine dendritic cells showed a significant elevation of IL-10 following stimulation with Saccharomyces cerevisiae, a species decreased in IBD patients [4], suggesting an association between specific fungal strains and distinct cytokines.
IL-33 has an established role in enhancing resistance and tolerance to fungal pathogens in animal models [67,68].IL-33 administration limits fungal burden by increasing neutrophil phagocytic activity.Conversely, IL-33 stimulates Th2 cells to produce IL-13, which, in turn, drives macrophages towards the M2 subtype and subsequent immunosuppression The Th2 pathway and its mediators, including IL-4 and IL-13, are also involved in antifungal immunity (Table 1).IL-4 was found to suppress protective immune responses to Aspergillus fumigatus and Histoplasma capsulatum [77, 78]; IL-13 induced tolerance to Cryptococcus neoformans in murine models [79].Aside from the expected resistance of both IL-4 and IL-13 against fungal species [12], IL-4 takes part in the optimal development of IL-12dependent Th1 responses [80] and IL-13 increases oxidative burst and phagocytosis during fungal infections [79,81].Similarly to IBD, these cytokines have contrary effects against fungal pathogens that are generally time-and dose-dependent [82]; therefore, a regulated balance is required to maintain intestinal homeostasis during inflammation.No previous study has examined the relevance of IL-4 and IL-13 to intestinal fungi in IBD; however, Th2-related cytokines are involved in mucosal immunity against gut fungi [59].
Intriguingly, the production of TNF-a and IFN-c was strikingly increased in the inflamed mucosa of both CD and UC patients, in which intestinal mycobiome richness and diversity were positively correlated with TNF-a and IFN-c expression [16,59].Further analysis of cytokine expression demonstrated that commensal fungi such as C. albicans upregulated the production of IL-2, IL-10, IL-17, TNF-a, and IFN-c, restricted to the fungus-specific memory T cells [83].

Interleukin-4 and interleukin-13
Anrukinzumab, an anti-IL-13 antibody that impedes the binding of IL-13 and IL-4Ra, and tralokinumab (IL-13 neutralizing antibody) failed to significantly improve the clinical condition of patients with moderate-to-severe UC [84, 85].However, Dual role of fungi-derived cytokines in IBD | 3 promising effects of IL-4/IL-13 dual antagonist were observed on murine colitis models [86].Exogenous IL-4 treatment had controversial effects on clinical remission and mucosal healing in colitis models [87,88].

Interleukin-23, interleukin-17, and interleukin-22
Ustekinumab is an anti-IL-12/IL-23 agent that is approved for the treatment of both CD and UC; it binds the shared p40 subunit of cytokines IL-12 and IL-23, and prevents the cytokine from joining to its receptor [89].However, the favorable outcomes may stem from antagonizing IL-12, which is one of the primary mediators of Th1-induced immunity.The clinical efficacy of selective IL-23/ p19 inhibitors (such as mairikizumab, risankizumab, brazikumab, and guselkumab) is still an ongoing project that needs to be clarified in further clinical trials [90].
IL-17A and IL-17F functions seem to be determined by the target organ.Unlike skin, IL-17A is pivotal for intestinal barrier

Interleukin-10
Recombinant IL-10 therapies have been tested in multiple IBD clinical trials but revealed limited or no efficacy in disease remission [98, 99].

Interleukin-1 and interleukin-18
Blockade of IL-1b and IL-18 reduced intestinal inflammation in murine models of UC [100,101].However, no clinical trial has ever investigated the blockade of IL-1b and IL-18 in IBD patients.
Additionally, anakinra (IL-1R antagonist) could not reduce the need for rescue therapy or colectomy in patients with acute severe UC [102].

Interleukin-6
Different anti-IL-6/IL-6R therapeutic options have shown plausible results in IBD clinical trials [103,104].PF-04236921 is an anti-IL-6 antibody that induced clinical remission in refractory CD patients who were unresponsive to anti-TNF regimens [103].Tocilizumab and olamkicept are other prospective IL-6R inhibitors in IBD target therapies [104].

Interleukin-33
Studies have shown that both IL-33 deficiency and the administration of recombinant IL-33 ameliorated experimental colitis in mice.In fact, treatment with IL-33 exacerbates the disease Dual role of fungi-derived cytokines in IBD | 5 severity at the onset of dextran sodium sulfate-induced colitis, whereas it ameliorates the disease during the recovery phases [105].

Interleukin-35
Recombinant IL-35 reversed the inflammatory indices and mucosal damage in murine models of active colitis [106].

Interferon-gamma and tumor necrosis factor-alpha
Anti-TNF-a agents are the cornerstones of IBD biological treatments [107].Four of them are widely used in the treatment of IBD: infliximab, adalimumab, golimumab, and certolizumab pegol.They generally bind to TNF-a and cause cell lysis of macrophages and T cells [9].Intravenous administration of infliximab induces and maintains clinical remission and mucosal healing in patients with refractory or unresponsive IBD [108].In contrast to infliximab and adalimumab, etanercept was associated with the development of paradoxical IBD, suggesting that other mechanisms beyond TNF-a neutralization are involved in the therapeutic effect of anti-TNF-a in IBD [109].Additionally, AVX-470 is a new oral anti-TNF antibody that has improved the clinical, endoscopic, and inflammatory biomarkers for refractory UC with a good safety profile [110].Moreover, unlike the intestinal bacterial community, anti-TNF therapy did not alter the gut fungal composition [111].IFN-c blockade in dextran sodium sulfate-colitis mouse models had increased angiogenesis accompanied with improved vascular barrier function and reduced colonic inflammation [112].Fontolizumab, an anti-IFN-c antibody, increased the rate of clinical response in individuals with refractory CD, after receiving two doses of fontolizumab intravenously on Day 56 [113].

Discussion
Here, we reviewed the dual-faced cytokines involved in IBD pathogenesis as well as intestinal and mucosal antifungal responses.Despite a few controversial results, the cytokine network of both IBD and fungal-induced mucosal immunity had similar functions.Interestingly, distinct fungal species induced pro-or anti-inflammatory reactions during IBD, as M. restricta upregulated IL-6 [14] and S. cerevisiae induced IL-10 expression [4].Dectin-1 played the vital role of the gatekeeper in initiating the immune responses against fungal flora.In general, the intestinal fungal composition seems to determine the type and the dose of cytokine expression and their role in the intestinal environment (Figure 2).Thus, an increase in opportunistic fungi such as C. albicans induced inflammation, whereas commensal fungi richness resulted in epithelial regeneration.However, no cytokine is specific for a distinct type of microorganism including bacteria, fungi, and viruses.It should be considered that, during intestinal inflammation, some bacteria and fungi may act via their interkingdom network rather than directly via the host [5].Therefore, missing data regarding the probable confounding effect of other intestinal residents on fungal load need to be determined in future studies.
The biological therapies targeting the dual-faced cytokines have mainly failed to achieve remission in IBD clinical trials.In the reviewed literature, IFN-c and IL-6 were the only cytokines with inflammatory effects against fungal species despite their dual function in IBD pathogenesis.On the other hand, anti-IL-6 and anti-IFN-c antibodies such as olamkicept and fontolizumab significantly improved the clinical condition of IBD-affected patients.Thus, it could be speculated that treatments attenuating inflammatory responses against intestinal fungi are probably more effective.However, the scarce information regarding therapeutic options in the current study should be considered.
Most of our data were obtained from in vitro or animal models.Besides, considering the novelty of fungi to be regarded as the driving agent of cytokines and other limitations of the current study including lack of attention to the individual-specific composition of human intestinal fungi and insufficient sample size in a substantial number of reviewed studies, these conclusions should be regarded with caution.Of note, although the cytokines with a predictable role also work in the interaction of fungi with IBD pathogenesis, we reviewed the cytokines with dual functions to highlight the probable role of this association in IBD development.

Conclusions
This review suggests that the intestinal fungal flora could probably contribute more to IBD genesis and drug resistance than we expected.We propose that the type of immune responses and cytokine expression in IBD might be influenced by the composition of intestinal fungal residents.Thus, altering the fungal flora in IBD patients may improve their clinical condition.However, further investigation should be followed by more clinical data and a larger sample size.
https://doi.org/10.1093/gastro/goad068Review Article drive intestinal inflammation[19, 20].IL-6 signaling also prevents apoptosis and promotes the survival of Th1, Th2, or Th17 CD4þ T cells through activating signal transducers and the activator of the transcription 3 (STAT3) signaling pathway, which explains the contrasting effects of IL-6[21].Thus, the pro-inflammatory effects arise from increasing T-cell survival and the antiinflammatory effects from maintaining intestinal epithelial cells[22].Additionally, IL-23 is involved in the expansion and survival of Th17, natural killer cells, and innate lymphoid cells[23].Figure1provides a better understanding of the cytokine signaling pathways in IBD.Upon stimulation, IL-10 is produced primarily by dendritic cells and lymphocytes, especially regulatory T cells [24].After binding to its heterotetrameric receptor, IL-10 initiates an immune cascade to phosphorylate STAT3 and enhances immune tolerance through balancing polymorphonuclear leukocyteregulatory T-cell interaction [25].IL-10/IL-10R signaling defects are a known underlying etiology for early-onset IBD in pediatric patients [26].Despite the well-known immunomodulatory effects of IL-10, its overexpression could lead to tissue fibrosis by activating fibroblasts [15, 27].IL-33 and IL-35 are other innate immune-associated cytokines, primarily derived from intestinal epithelial cells, sub-epithelial myofibroblasts, and dendritic cells.IL-33 and Toll-like receptors commonly share the MyD88-dependent pathway, which leads to nuclear factor kappa B (NF-jB) and mitogen-activated protein kinase activation.Subsequently, a set of pro-inflammatory cytokines is expressed, disrupting the tolerance against intestinal pathogens [28].Additionally, IL-33 promotes Type 2 immune responses and could act as an alarm in response to cellular stress, to induce intestinal epithelial cell proliferation and repair [28].IL-35 is a member of the IL-12 cytokine family and consists of IL-12p35 and IL-27b subunits that exert function through activating Janus tyrosine kinase/STAT3 signaling.While the IL-12p35 subunit demonstrates a suppressive role in inflammatory/autoimmune conditions [29], IL-27b is highly expressed on regulatory T cells and Th17 cells, contributing to immunomodulation [30]; IL-35 is associated with colitis exacerbation [31].Following the activation of Th17, cytokines such as IL-17 (commonly IL-17A and IL-17F) and IL-22 are released [32].In addition to Th17, innate lymphoid cells, Th1, and natural killer T cells are the other sources of IL-22 expression [6].IL-17A, IL-17F, and IL-22 act on Th1 and intestinal fibroblasts to secrete pro-inflammatory mediators and matrix metalloproteinases, respectively, leading to either intestinal inflammation or fibrosis [33-36].Furthermore, IL-17 and IL-22 along with IL-6 enhance anti-microbial peptide secretion and intestinal epithelial cell permeability and proliferation via STAT3 phosphorylation [37-39].In turn, STAT3 downregulates inflammatory mediators and maintains intestinal barrier integrity [24, 37].

Figure 1 .
Figure 1.The cytokine network in pathogenesis of inflammatory bowel disease.These signaling pathways could be driven by intestinal fungal residents, leading to the maintenance of barrier integrity through increased IEC survival or mucosal injury and tissue fibrosis.Dectin-1, as the main receptor of fungi, induces cytokine expression via the SYK-CARD9 pathway.Red arrows and green arrows represent pro-inflammatory and antiinflammatory pathways, respectively.CARD9 ¼ caspase recruitment domain 9, IECs ¼ intestinal epithelial cells, IFN-c ¼ interferon-gamma, IL ¼ interleukin, ILC ¼ innate lymphoid cells, JAK/STAT ¼ Janus kinase/signal transducers and activators of transcription, MMP ¼ matrix metalloproteinases, NF-jB ¼ nuclear factor kappa B, SYK ¼ spleen tyrosine kinase, Th ¼ T helper, TNF-a ¼ tumor necrosis factor-alpha.
[69].Moreover, IL-33 mediated regulatory T-cell differentiation and restricted the IL-23 signaling pathway in a bacteria-driven colitis model [70].Additionally, IL-35 exerts a p35-dependent antifungal response downstream of IL-22 [71].Furthermore, IL12p35 -/-mice had a lower fungal burden during oral candidiasis, indicating the participation of IL-35 in reducing tissue damage [72], while Candida albicans raised the IL-27b level and suppressed inflammation [73].In a similar way, the IL-23/Th17 signaling pathway has a controversial role in tolerance to fungal flora by acting on both regulatory T-and Th1-cell responses [73].Dectin-1 is the main fungal receptor that initiates a cascade leading to Th17 activation, followed by IL-17 and IL-22 production [74].IL-17 is involved in both response and resistance to fungal species.It promotes neutrophil recruitment and natural killer cell trafficking in murine models [75, 76].Additionally, a former study reported increased levels of mucosal IL-23 and IL-17A in UC patients and their correlation with specific fungal genera in the intestine [60].IL-22 has a more crucial role than IL-17 in the mucosal host defense against C. albicans.In animal models, IL-22-deficient mice were highly susceptible to intestinal candidiasis [71].

Figure 2 .
Figure 2. Negative effects of mucosal immunity and cytokine induction in response to opportunistic fungi (A) and positive effects of cytokines in response to commensal fungi (B).In addition to the type of expressed cytokines, the dose of induction determines the type of immune responses to different fungal flora.Opportunistic fungi upregulate IL-1b, IL-17, IL-22, IL-23, and TNF-a production, leading to mucosal inflammation, whereas these cytokines are downregulated in response to commensal fungi, leading to regeneration.The level of expression of various cytokines is displayed with a dot in the same color.BCL-10 ¼ B-cell lymphoma 10, CARD9 ¼ caspase recruitment domain 9, IFN-c ¼ interferon-gamma, Ig ¼ immunoglobulin, IL ¼ interleukin, ILC ¼ innate lymphoid cells, MALT-1 ¼ mucosa-associated lymphoid tissue lymphoma translocation protein 1, NFAT ¼ nuclear factor of activated T cells, NF-jB ¼ nuclear factor kappa B, SYK ¼ spleen tyrosine kinase, Th ¼ T helper, TNF-a ¼ tumor necrosis factor-alpha.

Table 2 .
The summary of IBD clinical trials targeting the selected cytokines Ustekinumab is the only anti-IL-12/IL-23 agent approved for the treatment of both CD and UC TNF-a Anti-TNF-a agents (infliximab, adalimumab, golimumab, AVX-470, and certolizumab pegol) are the main biologic therapies in IBD IFN-c Fontolizumab (anti-IFN-c antibody) increased the rate of clinical response in individuals with refractory CD IL ¼ interleukin, TNF-a ¼ tumor necrosis factor-alpha, IBD ¼ inflammatory bowel disease, UC ¼ ulcerative colitis, CD ¼ Crohn's disease, R ¼ receptor.