key: cord-0003785-r2ytccof
authors: Abushouk, Amir; Nasr, Amre; Masuadi, Emad; Allam, Gamal; Siddig, Emmanuel E.; Fahal, Ahmed H.
title: The Role of Interleukin-1 cytokine family (IL-1β, IL-37) and interleukin-12 cytokine family (IL-12, IL-35) in eumycetoma infection pathogenesis
date: 2019-04-04
journal: PLoS Negl Trop Dis
DOI: 10.1371/journal.pntd.0007098
sha: 8d0b98bb0c601d8a636e49bad7d2449b7dce32ff
doc_id: 3785
cord_uid: r2ytccof

Mycetoma is a neglected tropical disease, endemic in many tropical and subtropical regions, characterised by massive deformity and disability and can be fatal if untreated early and appropriately. Interleukins (IL) -35 and IL-37 are newly discovered cytokines that play an important role in suppressing the immune system. However, the expression of these interleukins in patients with Madurella mycetomatis (M. mycetomatis) induced eumycetoma has not yet been explored. The aim of this study is to determine the levels of IL-1 family (IL-1β, IL-37) and IL-12 family (IL-12, IL-35) in a group of these patients and the association between these cytokines levels and the patients’ demographic characteristics. The present, case-control study was conducted at the Mycetoma Research Centre, Soba University Hospital, University of Khartoum, Sudan and it included 140 individuals. They were divided into two groups; group I: healthy controls [n = 70; median age 25 years (range 12 to 70 years)]. Group II: mycetoma patients [n = 70 patients; median age 25 (range 13 to 70 years)]. Cytokines levels were measured in sera using enzyme linked immunosorbent assay (ELISA). There was a significant negative correlation between IL-1β and IL-12 levels and lesion size and disease duration, while IL-37 and IL-35 levels were significantly positively correlated with both lesion size and disease duration. The analysis of the risk factors of higher circulatory levels of IL-37 in patients of mycetoma showed a negative significant association with IL-1β cytokine, where a unit increment in IL-1β will decrease the levels of IL-37 by 35.28 pg/ml. The levels of IL-37 among the patients with a duration of mycetoma infection ≤ 1 year were significantly low by an average of 18.45 pg/ml compared to patients with a mycetoma infection’s duration of ≥ 5years (reference group). Furthermore, the risk factors of higher levels of IL-35 in mycetoma patients revealed a negative significant association with IL-12, as a unit increment in IL-12 decreases the levels of IL-35 by 8.99 pg/ml (p < 0.001). Levels of IL-35 among the patients with duration of mycetoma infection ≤ one year were significantly low on average by 41.82 pg/ml (p value = 0.002) compared to patients with a duration of mycetoma infection ≥ 5 years (reference group). In conclusion, this study indicates that both IL-35 and IL-37 are negatively associated with the levels of IL-1β and IL-12 in eumycetoma mycetoma infection; and high levels of IL-37 and IL-35 may have a negative impact on disease progression.

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Mycetoma is a chronic granulomatous subcutaneous inflammatory disease, caused by certain bacteria (actinomycetoma) or fungi (eumycetoma). This infection progresses to affect the deep structures and bones leading to massive destruction, deformities and disabilities [1] . It constitutes a major health problem in many tropical and subtropical countries and it is highly endemic in Sudan, Mexico, and India. In Sudan, more than 8500 patients were managed at the Mycetoma Research Centre in Khartoum, of whom 70% were infected with the fungus M. mycetomatis. The disease affects all age groups, but it occurs most commonly in young men at the age group 20 to 40 years [2] . The disease is usually painless and the clinical diagnosis is commonly based on the presence of subcutaneous mass, multiple sinuses and seropurulent discharge with grains. Treatment opportunities comprise of various chemotherapeutics agents and wide surgical excision of the infected tissues and may possibly end up with limb amputation [3] .

Although most individuals in endemic areas have antibodies against the causative agent of mycetoma; only few develop the disease [4] . Both innate and adaptive immunity play a role in host resistance to causative agents and in the development of disease. Therefore, T-cell responses seem to be important in the progress of mycetoma [5, 6] . A T-helper (Th) 2-like response was reported in primary lesions and in draining lymph nodes in patients with Streptomyces somaliensis infection and after stimulation of peripheral blood mononuclear cells by M. mycetomatis antigens [5, 7] , whilst Th1 response was reported in the acute phase of infection and in healthy endemic controls [8, 9] . Macrophages stimulated with live conidia of Pseudallescheria boydii also induced a Th2 response, whereas hyphae induced a Th1 response [10] . Experimental infection of nude athymic rats and mice with Nocardia (N.) asteroids let to fatal disease dissemination [11, 12] . In addition, T lymphocytes from previously immunised animals directly killed N. asteroides [12, 13] . Moreover, Trevino-Villarreal and associates [14] reported that N. brasiliensis cell wall-associated lipids are implicated in the development of experimental actinomycetoma and act principally by inhibiting several microbicidal effects of macrophages, including the inhibition of TNF-α production, phagocytosis, production of nitric oxide (NO), and bacterial killing. In addition, they demonstrated that the N. brasiliensis wall-associated lipids suppressed the expression of major histocompatibility complex class II (MHC II), CD80, and CD40 by dendritic cells (DCs) and strongly induced the production of TGF-β by these cells. It has been suggested that pre-existing Th2 environment caused by schistosomiasis promotes the development of mycetoma as patients with mycetoma were significantly more positive for schistosoma antibodies than healthy endemic controls [9] . These findings suggested that Th2 like response and anti-inflammatory/immunosuppressive cytokines could have a negative impact on mycetoma development and disease progression.

IL-1 is a polypeptide which has two forms; IL-1α and IL-1β. It is involved in the acutephase response and is accountable for several alterations that are related to the onset of various medical disorders [15, 16] . It is demonstrated recently that higher levels of IL-1β cytokine are strongly associated with surgically treated mycetoma patients, in comparison to those treated without surgery [17] . It is known that IL-1β is a pro-inflammatory cytokine that is involved in cell death coordination [18] . IL-1β cytokine is cleaved into the mature, active form primarily by inflammasome dependent caspase activity [18] . It is possibly that mature IL-1β secretion by macrophages activates IL-1 receptor type 1 (IL-1R1) on macrophages, fibroblasts and epithelial cells, inducing production of the CXC chemokine CXCL1/KC, which binds to CXCR2 on neutrophils and mediates recruitment of neutrophils from peripheral blood to stimulate inflammation at the site of mycetoma invasion. Therefore, these higher levels of IL-1β cytokine advocate a crucial role in M. mycetomatis pathogenesis.

IL-37, which is a member of the IL-1 family, has emerged as a potent anti-inflammatory cytokine that suppresses both innate and adaptive immune responses [19] . Its role in human diseases is not completely understood yet [20] . However, the anti-inflammatory properties of IL-37 have been associated with inflammatory diseases, such as systemic lupus erythematosus (SLE) [21] , and inflammatory bowel disease [22] . It has been reported that IL-37 is negatively associated with pro-inflammatory cytokines such as IL-1β, IL-6, IL-17, TNF-α and IFN-γ in peripheral blood mononuclear cells (PBMCs) of patients with degenerative intervertebral discs [23] and Graves' disease (GD) [24] . IL-37 protein level in PBMCs and DCs is up-regulated when stimulated by Toll-like receptor (TLR) ligands or pro-inflammatory cytokines [25] . In vitro, over expression of IL-37 in macrophages or epithelial cells greatly inhibits the production of major pro-inflammatory cytokines such as TNF-α, IL-1α, IL-1β, IL-6, IFN-γ and macrophage inflammatory protein 2 [25, 26] . In vivo, IL-37 transgene protects mice from lipopolysaccharides-induced shock and chemical-induced colitis [19, 27] . IL-37 interferes with the innate protective anti-Candida host response by reducing the production of pro-inflammatory cytokines and suppressing neutrophil recruitment in response to Candida infection, resulting in an increased susceptibility to disseminated candidiasis [28] . Moreover, IL-37 markedly reduced inflammasome activation and disease severity in murine aspergillosis [29] . In addition to its role in innate immunity; IL-37 plays a pivotal role in regulating adaptive immunity by inducing T regulatory (T reg ) cells and impairing activation of effector T-cell responses [30] . To our knowledge, there has been no study to report the relationship between IL-37 and mycetoma pathogenesis so far.

Interleukin-12 (IL-12) is frequently denoted as a B cell cytokine, although it is mainly formed by innate immune cells, comprising epithelial cells, DCs, and macrophages [31] . IL-12 is a multimer that plays a fundamental role in immune regulation and is extensively involved in infections. It binds to the heterodimeric IL-12 receptor, which is principally present in T cells and on natural killer (NK) cells [31] . IL-12 induces Th1 responses [32] , which consequently increase the cytotoxic cytokines, in addition to IFN-γ by T cells [31, 32] .

IL-35 is a recently identified heterodimeric cytokine which belongs to the IL-12 cytokine family, composed of the subunits of IL-27; β chain Epstein-Barr-virus (EBV)-induced gene 3 (Ebi3) and IL-12α chain p35 [33, 34] . IL-35 is a potent immunosuppressive cytokine produced by B regulatory cells (B reg ) [35] , DCs [36] , and to a lesser extent, by endothelial cells, smooth muscle cells, and monocytes [37] . The biological effect of IL-35 is poorly understood, however IL-35 is recognised as a typical anti-inflammatory cytokine, and the predominant mechanism of suppression is associated with its ability to suppress T cell proliferation and effector functions [33, 38] . Given the direct immunosuppressive effect of IL-35, many studies have been conducted to evaluate its role in the development of several diseases. IL-35 can suppress several types of chronic inflammatory diseases such as inflammatory bowel disease [26] , and decreased the severity of collagen induced arthritis in animals via enhancement of IL-10 production [39] and suppression of Th17 cells [40] . In an asthma model, intra-tracheal instillation of IL-35 decreased disease severity by diminishing the Th2 cell counts [41] and by reducing the production of IL-17 [42] . In bacterial infections, Shen and associates [35] found that mice without IL-35 expression demonstrated an obviously improved resistance to infection with the intracellular bacterial pathogen Salmonella typhimurium. In addition, IL-35 has been increased in the serum of adults and children with sepsis, and administration of anti-IL-35 p35 antibodies diminished dissemination of the bacteria in septic animals [43] . Similarly, tuberculous patients exhibited an increase in serum IL-35 and in mRNA expression of both subunits of IL-35 (p35 and EBI3) in white blood cells and peripheral blood mononuclear cells [44] . However, the role of IL-35 in mycetoma pathogenesis has not been highlighted yet.

With this background this study was set to determine the IL-1 cytokine family (IL-1β, IL-37) and IL-12 cytokine family (IL-12, IL-35) circulating levels of in patients infected with M. mycetomatis, and to explore the association between the pro-inflammatory/anti-inflammatory cytokine levels and the patients' demographic characteristics. This is correlation may help to understand the pathogenesis of mycetoma disease.

This case-control study was conducted at the Mycetoma Research Centre, Soba University Hospital, University of Khartoum, Sudan. After a written informed consent, blood samples were taken from patients and a matched control population living in the mycetoma endemic areas of Sudan between 2015 and 2016. Samples collection was previously described in details by Nasr and associates [17] .

In this study 140 individuals were enrolled; 49 (35%) were females and 91 (65%) were males with an overall median age of 25 years (range 12-70 years). Seventy patients were infected with M. mycetomatis. The study population was divided into two groups; group I: healthy controls [n = 70; median age 25 years (range 12 to 70 years)]. Group II: mycetoma patients [n = 70 patients; median age 25 (range 13 to 70 years)]. Both groups had a similar gender distribution, 54 (80%) were males in each group.

The diagnosis of eumycetoma was established by various techniques and that included imaging, molecular and histopathological techniques, and grain culture [1, 45] . Surgical biopsies are obtained by a wide local incision under anaesthesia and appropriate surgical conditions as part of the routine patients' treatment protocol [45] .

After medical examination, healthy controls were selected from blood bank donors or healthy volunteers to match the patient's birthplace geographically. All healthy controls were questioned for acute or chronic infectious diseases, autoimmune family history and genetic disorders. Then all study participants gave their informed written consent and the study was approved by the Ethics Committee of the Faculty of Medicine, University of Khartoum, Sudan.

One hundred μl of blood were collected on Whatman qualitative filter paper, Grade 1, circles, diam. 42.5 mm (Sigma-Aldrich Chemical Co., St. Louis, MO, USA) for determination of cytokines. The use of filter paper dried whole blood spots (DBS) for specimen collection was preferred to facilitate collection, storage and transportation of specimens and it is in line with the World Health Organization recommendations and also used in several previous studies [46] [47] [48] . Sera were extracted from filter-paper samples as described previously in details [17] .

IL-1β, IL-37, and IL-12 were measured in the sera using commercially available enzyme linked immunosorbent assay (ELISA) kits (Abcam, Cambridge, UK). Serum levels of IL-35 were estimated using a sandwich ELISA commercial kit (Colorful Gene Biological Technology, Wuhan, China). Cytokine assays were performed in duplicates according to the manufacturers' protocols. The sensitivity of Human ELISA kits for IL-1β, IL-37, IL-12 and IL-35 cytokines was 0.5 pg/ml.

The data were managed by SPSS version 24.0 statistical software for Windows (IBM SPSS statistics) and appropriate statistical tests were used. The results are expressed as mean ± standard deviation (SD) or median with interquartile range (IQR). Spearman correlation test was used to evaluate the associations between serum IL-37 and IL-35 levels and laboratory values as well as serum cytokine levels. For non-parametric data, comparisons between the groups were performed using the Kruskal-Wallis test. One-way ANOVA was used for parametric data. General linear models were used to assess the risk factors for circulating IL-37 and IL-35 among mycetoma patients with different disease duration and lesions size of mycetoma infection adjusted with other variables. A test with a p value <0.05 was considered statistically significant.

This study was approved by the Ethics Committee of Soba University Hospital, Khartoum, Sudan (SUH, EC-17-029). Written informed consent was taken from all the participants before enrolment in the study. The work described here was performed in accordance with the Declaration of Helsinki [49] .

This study included 70 confirmed mycetoma patients and 70 healthy controls; gender and age matched. Fifty-six patients (80%) were males and 14 (20%) were females. Their ages ranged between 12 and 77 years and the median age was 25.5 years. The common age group [22 (31.4%)] was 19-24 years, 17 (22.9%) in the age group (25) (26) (27) (28) (29) years, and nine (12.9%) were 40 years old or more ( 

The levels of cytokines (IL-1β, IL-37, IL-12 and IL-35) were constitutively correlated among mycetoma patients with different lesions diameters. The levels of IL-1β were constitutively positively correlated with IL-12 and lesions diameter ( Table 2) . On the other hand, the levels of cytokine IL-1β were constitutively negatively correlated with IL-37 and IL-35 (Table 2) . Furthermore, the levels of cytokine IL-37 were constitutively positively correlated with IL-35 (Table 2) . However, the levels of cytokine IL-37 and IL-35 were constitutively negatively correlated with IL-12 (Table 2) .

In the patients group, the levels of cytokines (IL-1β, IL-37, IL-12 and IL-35) were constitutively correlated with the duration of mycetoma infection. Levels of IL-1β showed a consistent positive correlation with IL-12 and negative correlation with IL-37 and IL-35 (Table 3) . Whereas, levels of IL-37 were constitutively positively correlated with IL-35 (Table 3) . However, the levels of IL-37 and IL-35 were constitutively negatively correlated with IL-12 ( Table 3) .

Circulating serum cytokine levels were determined in all mycetoma patients and were compared between the different lesion diameters among mycetoma patients and healthy controls. Overall, there was a significant difference in the levels of all studied cytokines between the four groups (Three levels of lesion diameter and the healthy controls), ( Table 4 ). Distribution of IL-1β levels has decreased dramatically with lesion diameter [for lesion diameter � 5 cm: the mean ± SD (3.39 ± 1.07); for 5-10 cm: (2.32 ± 0.05); for � 10 cm: (2.08 ± 0.11), p value < 0.001]. However, the circulating serum levels of IL-37 were significantly increased with lesion diameter [for lesion diameter � 5 cm: the mean ± SD (107.92 ± 5.96); for 5-10 cm: (141.45 ± 12.96) and for � 10 cm: (193.20 ± 15.01), p value < 0.001], (Table 4) .

Our results showed a significant reduction of circulating IL-12 levels versus lesion diameter [for lesion diameter � 5 cm: the mean ± SD (25.22±3.34); for 5-10 cm: (14.45± 3.32); for � 10 cm: (9.65 ± 0.36), p value < 0.001], (Table 4 ). Circulating levels of IL-35 were significantly increased with increasing lesions' diameter [for lesion diameter � 5 cm: the mean ± SD (255.15 ± 1.72); for 5-10 cm: (263.23 ± 3.26); � 10 cm: (449.71 ± 22.2), p value < 0.001], ( Table 4 ).

Circulating levels of IL-1β had significantly decreased with increasing disease duration [(� 1 year; median = 2.3 pg/ml), (2-4 years; median = 2.2 pg/ml) and (� 5 years; median = 2.2 pg/ ml)], p value = 0.017 (Table 5 ). Serum levels of IL-12 dramatically decreased with the increase in disease duration [(� 1 year; median = 12.5 pg/ml), (2-4 years; median = 10.2 pg/ml) and (� 5 years; median = 9.8 pg/ml)] and p value < 0.001 (Table 5) (Table 5) . Similarly, serum levels of IL-35 were also significantly increased 

The analysis of the risk factors of higher levels of IL-37 in patients of mycetoma showed a significant negative association with IL-1β, where a unit increment in IL-1β decreases the levels of IL-37 by 9.1 pg/ml, p value = 0.008 (Table 6 ). Serum levels of IL-37 among the patients with lesion diameter � 5 cm and 5-10 cm have significantly lower on average by 75.4% and 52.6%, respectively, compared to patients with lesion diameter � 10 cm (reference group). Serum levels of IL-37 among the patients of mycetoma showed no significant difference between males and females, p value = 0.176 (Table 6 ). Circulating levels of IL-37 significantly decreased with 

The analysis of the risk factors of higher serum levels of IL-35 in mycetoma patients showed no significant association with IL-12, p value = 0.182 (Table 7) . Circulating levels of IL-35 among the patients with lesions' diameter � 5 cm and 5-10 cm were significantly decreased by 174.4% and 176.5, respectively; compared to patients with lesion diameter � 10 cm (reference group), p value < 0.001 (Table 7) . Serum levels of IL-35 among mycetoma patients showed no significant difference between males and female, p value = 0.575 (Table 7) . Circulating levels of IL-35 showed no significant association with the different age groups and different types of antifungal medication given (Table 7) .

The analysis of the risk factors of higher circulatory levels of IL-37 in patients of mycetoma showed a negative significant association with IL-1β, where a unit increment in IL-1β decreases the levels of IL-37 by 35.28 pg/ml, p < 0.001 (Table 8 ). Levels of IL-37 among patients with a disease duration � 1 year had significantly decreased on average by 18.45 pg/ ml compared to patients with a disease duration � 5years (reference group). However, there was no significant difference in levels of IL-37 between patients with infection duration 2-4 years and � 5 years, p value = 0.793 (Table 8 ). In addition, serum levels of IL-37 among the mycetoma patients showed no significant difference between males and females, p value = 0.627 (Table 8) . Furthermore, the circulating levels of IL-37 had significantly decreased with increasing age groups [(19-24 years; p value = 0.022), 25-29 years; p value = 0.030 and (30-39) years; p value = 0.022], (Table 8) . Interestingly, levels of IL-37 among mycetoma patients showed a statistically significant difference between itraconazole compared to ketoconazole, p < 0.001 (Table 8 ).

The analysis of the risk factors of higher levels of IL-35 in patients of mycetoma revealed a negative significant association with IL-12; as a unit increment in IL-12 decreases the levels of IL-35 by 8.99 pg/ml, p value < 0.001 (Table 9 ). Levels of IL-35 among the patients with a mycetoma with a disease duration of � 1 year had significantly decreased, p value = 0.002, on average by 41.82 pg/ml compared to patients with a disease duration � 5years (reference group), (Table 9 ). However, patients with an infection duration of 2-4 years and � 5 years showed no significant difference in IL-35 levels, p value = 0.391. Furthermore, there was no significant difference (p value = 0.49) in IL-35 levels between male and female mycetoma sufferers (Table 9) . Additionally, the circulating levels of IL-35 showed no significant association with the different age groups (Table 9) . Interestingly, mycetoma patients who were treated with itraconazole showed significant increased levels of IL-35 compared to the patients treated with ketoconazole, p value <0.001 (Table 9 ).

Although mycetoma represents a major health problem in many tropical and subtropical areas, there is no prevention or control measures for this neglected disease [22, 23] . In mycetoma endemic areas, most individuals have antibodies against the causative agents, however only few develop disease [4] . Few researchers believed that patients who develop mycetoma seem to be deficient in their cell-mediated immunity [6] . Hence, we aimed to investigate the profiles of the pro-inflammatory (IL-1β and IL-12) and the anti-inflammatory immunosuppressive (IL-37 and IL-35) cytokines among mycetoma patients and their association with disease characteristics. As far as we know, this is the first study addressing the relation between immunosuppressive cytokines (IL-37 and IL-35) and mycetoma infection. Our data showed that eumycetoma patients presented higher circulating levels of IL-1β, IL-12, IL-37 and IL-35 compared to controls. In addition, serum levels of IL-1β and IL-12 were significantly decreased with increasing lesions' diameter and disease duration, whereas levels of IL-37 and IL-35 were significantly higher with increasing lesions' diameter and disease duration. These findings indicate that immunosuppressive cytokines like IL-37 and IL-35, which could suppress cellmediated immune responses, may exacerbate the disease progression. Data of the current study clearly showed the serum levels of IL-1β and IL-12 in eumycetoma patients with different lesion size and disease duration were positively correlated with each other, and negatively correlated with IL-35 and IL-37. It has been demonstrated that the first line of innate immune response against mycetoma infection is by phagocytes, from which macrophages represent the major phagocytic cells [50] . In general, protective immunity to fungal infections [51] involves activation of TLRs generating inflammatory cytokines through pattern-recognition receptors and pathogen associated molecular patterns [52, 53] . IL-1β and other pro-inflammatory cytokines are produced early in response to fungal infections and promote phagocytosis and other means of innate immune response [54, 55] . Following inflammatory stimuli, several cell types including immune and non-immune cells produce IL-37, as a protective mechanism to prevent runaway inflammation and excessive tissue damage [56] . IL-37 directly inhibits generation of pro-inflammatory cytokines and down-regulates macrophage cytokine release, and therefore innate immunity [57, 58] . Moreover, IL-37 induces macrophages towards an M2-like phenotype [59] . M1 macrophages are the most critical effector cells in the innate immune defense system and are characterised by high expression levels of iNOS, subsequent NO production and secretion of pro-inflammatory cytokines, such as IL-1β and IL-12 [60] . However, M2 macrophages secrete antiinflammatory cytokines, such as IL-10 [61] and express arginase 1, which inhibits NO production, thus rendering these cells ineffective in killing infectious agents including fungal agents [61, 62] . Furthermore, DCs expressing IL-37 secreted higher levels of IL-10 and reduced levels of IL-1β and IL-12. Therefore, the presence of IL-37 in DCs impairs their function in prime T cells and promotes their ability to induce Treg cells that produce IL-10, which is also a potent anti-inflammatory cytokine [30] . Our results have consistently shown higher circulatory levels of IL-37 in patients of mycetoma which is negatively associated with IL-1β, as a unit increment in IL-1β decreases the levels of IL-37 by 35.28 pg/ml. Based on the aforementioned data, we can speculate that IL-37 could play a role in damping inflammatory response in mycetoma infection which leads to disease progression and this is not in the patient's favor.

In the current work, the circulating levels of IL-1β and IL-12 in eumycetoma patients with different lesion size and disease duration were negatively correlated with IL-35; whereas serum levels of IL-35 were increased with increasing lesion size and disease duration, and levels of IL-1β and IL-12 simultaneously decreased. This may probably be an attempt to dampen ongoing inflammation. Both IL-1β and IL-12 have a pivotal role in inflammatory and cell-mediated immune responses. Macrophages, Th1 and cytotoxic T-cells (CTLs), which constitute the main component of cell-mediated immunity, play an important role in the protective immunity against mycetoma infection [2] . As fatal dissemination of N. asteroids infection occurs in nude athymic rats and mice [11, 12] , T cells from previously immunized animals are able to kill N. asteroids in new infections [12, 13] . IL-35 could suppress Th1 and macrophage responses [63] , whereas deficiency in IL-35 increases macrophage's activation and induces Th1 responses [35, 63] . The increased immunity found in mice lacking IL-35 is associated with a higher activation of macrophages and inflammatory T cells, as well as enhancing function of antigen-presenting cells [35] . In another infection model, Cao and his co-workers reported higher serum levels of IL-35 in septic patients compared to controls, and IL-35 gradually increased with increased sepsis severity. Moreover, administration of anti-IL-35 antibodies diminished dissemination of the bacteria in septic animals and enhanced local neutrophil recruitment with increasing in inflammatory cytokines and chemokines production [43] . Furthermore, IL-35 suppressed the proliferation of antigen-specific CTLs and IFN-γ production [64] . Our data revealed that higher levels of IL-35 in patients with mycetoma is negatively associated with IL-12, where a unit increment in IL-12 decreases the levels of IL-35 by 8.99 pg/ml. This finding indicates that IL-35 may be a risk factor for mycetoma infection and have a negative role in the clinical presentation of the disease.

Prevalence of mycetoma infection may vary with age. Data from this study showed variation of mycetoma prevalence with age; 74.3% of the patients' age were 19-39 years. This finding is consistent with previous studies which reported that mycetoma mostly affects ages between 20 and 40 years-old, In addition, our data demonstrated that mycetoma infection was predominant in males, as the male to female ratio in patient's group was 4:1. This finding is running parallel with the results of a previous study which demonstrated that in a tertiary facility in Khartoum, Sudan, the male to female ratio is 4:1, whereas at the primary care level in White Nile State, Sudan, the reported male to female ratio was 1.6:1. Another studies reported that male to female ratios in mycetoma infections were in the range of 1.6-6.6:1 [2] . The predominance of mycetoma in males may be attributed to increased exposure in men who engage in different manual labors including agricultural work. Moreover, influence of sex hormones might have a role in susceptibility to mycetoma infections and disease progression [4, 65] .

Our data showed that about, 50% of the patients have lesion diameter more than 10 cm. This result reflected that most mycetoma patients tend to present late with massive lesions. This finding could be attributed to the nature of mycetoma which is usually painless and slowly progressive. In addition, the lack of health facilities in endemic areas, the low socio-economic status of the affected patients and their poor health education [1, 4, 66] are amongst the reasons why the current treatment of mycetoma is suboptimal, characterised by low cure rates and frequent recurrence often leading to amputation [67, 68] . However, clinical experience shows that early and small mycetoma lesions are associated with good outcome and prevent severe complications of the disease.

One of the remarkable findings of the current study is the significant increase of IL-37 and IL-35 levels with itraconazole treatment compared to the ketoconazole. A previous study by Friccius and colleagues suggested that the dose of 10 μg/ml itraconazole leads to strong inhibition of the cytokines IL-2, IL-4, IL-9 and IFN-γ and slight inhibition of TNF-α cytokine production in PBMC after 6 and 24 hours of incubation. These results demonstrate that IL-35 and IL-37 can be one of the underline factors associated with inhibition of the cytokines related to Itraconazole [69] .

In conclusion, our study revealed that the levels of IL-37 and IL-35 were consistently positively correlated with different diameters of mycetoma lesions as well as its duration. However, the levels of IL-1β and IL-12 were consistently negatively correlated with different diameters of lesions and the duration of mycetoma infection. The analysis of the risk factors of higher circulatory levels of IL-37 in patients of mycetoma showed a negative significant association with IL-1β cytokine, where a unit increment in IL-1β will decrease the levels of IL-37 by 35.28 pg/ ml. Levels of IL-37 among the patients with a mycetoma infection duration � 1 year had significantly decreased on average by 18.45 pg/ml compared to patients with a mycetoma infection duration � 5years (reference group). Furthermore, the risk factors of higher levels of IL-35 in patients of mycetoma revealed a negative significant association with IL-12, as a unit increment in IL-12 decreases the levels of IL-35 decrease by 8.99 pg/ml, p < 0.001. Levels of IL-35 among the patients with a mycetoma infection duration � 1 year had significantly decreased (p value = 0.002) on average by 41.82 pg/ml compared to patients with a mycetoma infection duration � 5years (reference group). More investigations are needed to explore the mechanism by which IL-35 and IL-37 contribute in the mycetoma infection outcomes. This will help in understanding the role of these cytokines IL-35 and IL-37 in the pathogenesis of mycetoma, and may exploit it as a potential therapeutic target to prevent mycetoma diseases recurrence.

Supporting information S1 Checklist. STROBE checklist. (DOC) 

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IL-12 family cytokines: immunological playmakers

IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases

Human tolerogenic dendritic cells produce IL-35 in the absence of other IL-12 family members

IL-35 and autoimmunity: a comprehensive perspective

Epstein-Barr virus-induced gene 3 negatively regulates neuroinflammation and T cell activation following coronavirus-induced encephalomyelitis

IL-35 stimulation of CD39+ regulatory T cells confers protection against collagen II-induced arthritis via the production of IL-10

IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells

Airway inflammation and IgE production induced by dust mite allergen-specific memory/effector Th2 cell line can be effectively attenuated by IL-35

IL-35 production by inducible costimulator (ICOS)-positive regulatory T cells reverses established IL-17-dependent allergic airways disease

IL-35 is elevated in clinical and experimental sepsis and mediates inflammation

Elevated serum IL-35 and increased expression of IL-35-p35 or-EBI3 in CD4+ CD25+ T cells in patients with active tuberculosis

Mycetoma laboratory diagnosis: Review article

Use of filter paper for the collection and analysis of human whole blood specimens

Comparison of available methods to elute serum from dried blood spot samples for measles serology

The use of the dried blood spot sample in epidemiological studies

Helsinki Declaration revisited over concerns about human subjects

Differential activity of innate defense antimicrobial peptides against Nocardia species

Immunity to fungi. Current opinion in immunology

MyD88-deficient Hydra reveal an ancient function of TLR signaling in sensing bacterial colonizers

Role of pathogen-associated molecular patterns (PAMPS) in immune responses to fungal infections

Alexithymia and its relationships with inflammatory response mediated by IL-1 family members

Comparison of the interleukin-1β-inducing potency of allergenic spores from higher fungi (basidiomycetes) in a cryopreserved human whole blood system. International archives of allergy and immunology

Suppression of inflammation and acquired immunity by IL-37. Immunological reviews

Impact of Fungi on Immune Responses

IL-37 suppresses MyD88-mediated inflammatory responses in human aortic valve interstitial cells

IL-37 Expression is Upregulated in Patients with Tuberculosis and Induces Macrophages Towards an M2-like Phenotype

Obstacles and opportunities for understanding macrophage polarization

Autocrine IL-10 induces hallmarks of alternative activation in macrophages and suppresses antituberculosis effector mechanisms without compromising T cell immunity

Toll-like receptor-induced arginase 1 in macrophages thwarts effective immunity against intracellular pathogens

Interleukin 35: A novel candidate biomarker to diagnose early onset sepsis in neonates

IL-35 inhibits HBV antigen-specific IFN-γ-producing CTLs in vitro

Nocardia brasiliensis: in vitro and in vivo growth response to steroid sex hormones

A new model for management of mycetoma in the Sudan

The Surgical Treatment of Mycetoma

Predictors of Post-operative Mycetoma Recurrence Using Machine-Learning Algorithms: The Mycetoma Research Center Experience

The effects of the antifungal azoles itraconazole, fluconazole, ketoconazole and miconazole on cytokine gene expression in human lymphoid cells

The authors are grateful to the patients, controls and their families and the staff at the Mycetoma Research Centre at Soba University Hospital, University of Khartoum-Sudan, for their continued collaboration and generous hospitality during a decade of samples collection. The authors are thankful to Dr. Hamza A., Dundee University, UK for improving the English language and proof reading the Manuscript.