Impact of interleukin-32α on T helper cell-related cytokines, transcription factors, and proliferation in patients with type 2 diabetes mellitus

Abstract Objective The ability of interleukin (IL)-32α to induce T helper (Th) 1, Th17, and Treg cytokines (interferon gamma [IFN-γ], IL-17, and IL-10, respectively), and transcription factors ([signal transducer and activator of transcription (STAT) 1 and T-box (T-bet) for Th1, STAT3 and retinoid-related orphan receptor (ROR)-γt for Th17, and STAT5 and forkhead box P3 (Foxp3) for Treg]) were investigated in type 2 diabetes mellitus (T2DM). IL-32α effects on Th cell proliferation and related factors including IL-2 and NF-κB were also explored. Methods Serum levels of IL-32α in 31 patients and 31 healthy controls (HCs) were determined by ELISA assay. CD4+ T cells cultured with polyclonal activators in the presence and absence of recombinant IL-32α (rIL-32α). Gene expressions in cultured Th cells were assessed with real-time PCR. Cytokines in supernatants were measured with ELISA. Proliferation experiments were assessed by flow cytometry. Results The patients showed significant increase in IL-32α levels compared with HCs and its levels were positively correlated with fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c). rIL-32α enhanced IL-17 and IL-2 production, increased ROR-γt and NF-κB expression, and enhanced Th proliferation in both patients and HCs. In patients, IL-17, ROR-γt, NF-κB, and proliferation levels were higher than those in HCs, in cultures with and without rIL-32α (rIL-32α+ and rIL-32α-). IL-2 levels in rIL-32α+cultures of patients were significantly higher than the HCs, and it was positively correlated with proliferation rate and NF-κB expression. Conclusions Aberrant IL-32α levels are participated in T2DM pathogenesis. IL-32α potently induces Th17-related factors and amplifies the proliferative function of T cells. Highlights Enhanced serum levels of IL-32α in T2DM patients was correlated with FPG and HbA1c. IL-32α increases ROR-γt expression and IL-17 production, and induces Th17 cells. IL-32α enhances NF-κB expression and IL-2 production, and promotes Th proliferation. IL-32α is more effective for inducing Th17 cells and proliferation in the patients. IL-32α axis could be mentioned as a future therapeutic goal for the T2DM.


Introduction
Type 2 diabetes mellitus (T2DM) is a multifaceted illness diagnosed by a high concentration of glucose in the circulatory system [1]. Disruptive cytokine networks and impaired action of peripheral CD4 þ T helper (Th) cells have been reported in diabetic persons [2,3].
Interleukin (IL)-32 is a novel immune mediator that is mainly produced by immune cells and 9 different isoforms of IL-32 (a, b, c, d, e, f, g, 1, and h) has been identified in humans [4]. IL-32a is the most abundant pro-inflammatory transcript with a high biological function that has been observed to be involved in Th cell response and cytokine production [5]. IL-32a stimulates nuclear factor kappa-lightchain-enhancer of activated B cells (NF-kB), thus inducing various pro-inflammatory mediators such as tumor necrosis factor (TNF) -a and IL-1b [6,7]. High levels of IL-32a have been participated in the pathogenesis of some diseases. For example, increased plasma levels of IL-32a have been reported in myasthenia gravis and hepatocellular carcinoma [8,9]. The association of disease activity and up-regulation of IL-32a transcript has been published in the graves and psoriasis disorders [5,10]. It has been shown that IL-32a stimulates bone marrow stromal cells of multiple myeloma patients to secret IL-6 in vitro [11].
Different subsets of CD4 þ T cells play an essential role in the induction and regulation of immune functions [12]. In humans, naive CD4 þ T cells turn into pro-inflammatory (Th1 and Th17) and regulatory T (Treg) cells via unique transcription factors and cytokines [12]. Th1 cells dominantly express signal transducer and activator of transcription (STAT) 1 and T -box (T-bet) transcription factors. Th1 mainly participates in inflammatory immune responses by the secretion of interferon gamma (IFN-c) [13]. Th17 cells discriminate by the expression of STAT3 and retinoid-related orphan receptor (ROR) -ct transcription factors. Th17 triggers the immune system by producing IL-17 [13]. Treg cells express STAT5 and forkhead box P3 (Foxp3) transcription factors and produce anti-inflammatory IL-10. This subset of immune cells maintenances peripheral tolerance by the suppression of target cells [14].
IL-2 mainly releases by activated CD4 þ T cells and it is identified as a growth agent for lymphocytes. This cytokine enhances the proliferation of T cells and it is an essential factor for function of Treg cells [12]. NF-jB is a pleiotropic factor that controls the expression of many genes involved in different biological activities and inflammatory response. NF-jB enhances Th cell function and cytokines production such as IL-1b, IL-12, and TNF-a [15]. This factor regulates the cell cycle by inducing anti-apoptotic factors and cytokines [15].
Previously, we and other researchers have published that the enhanced levels of pro-inflammatory cytokines and abnormal changes in Th1, Th17, and Treg subsets are involved in the formation of inflammatory condition in T2DM. For instance, increased serum levels of TNF-a, IFN-c, and IL-17 have been reported in T2DM persons [3,16]. The plasma levels of TNF-a are positively correlated with fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) in T2DM patients [16]. High frequency of Th1 (CD4 þ IFN-c þ ) and Th17 (CD4 þ IL-17 þ ) cells, and low percentage of circulating Foxp3 þ Tregs have been noticed in T2DM persons [2]. In addition, overexpression of NF-jB mRNA level has been seen in peripheral blood of patients with nephropathy and it is participates in the onset of disease [17].
Overall, IL-32a has been linked to inflammatory and autoimmune disorders but its impacts on Th-related abnormalities remains to be explored in T2DM. In this project, first, the serum concentrations of IL-32a and its relations with diabetes parameters were checked. Second, the effects of recombinant IL-32a (rIL-32a) on Th transcription factors and cytokines including Th1 (STAT1, T-bet, and IFN-c), Th17 (STAT3, ROR-ct, and IL-17), and Treg cells (STAT5, Foxp3, and IL-10) were investigated in vitro. Third, the impacts of rIL-32a on Th proliferation and related factors (IL-2 and NF-jB) were tested.

Participants
A random sample of 31 new cases with T2DM and 31 healthy controls (HCs) were enrolled from the Shahid Beheshti hospital, Department of Internal Medicine of Hamadan University of Medical Science (UMSHA). All participants were examined by physician and diagnosis of T2DM was done by the standard 2020 American Diabetes Association guideline.
Inclusion criteria for study patients were as follows: (a) FPG ! 126 mg/dl, (b) HbA1c ! 6.5%, (c) normal range of glomerular filtration rate (GFR) (>60), (d) normal range of albuminuria (<30 mg/g), and (e) normal range of creatinine in serum (<1.3 mg/dl). The available details of the subjects are presented in Supplementary Table 1. Exclusion criteria for study patients were as follows: (a) persons with neoplasia, infection diseases, autoimmune, and inflammatory diseases, (b) persons with nephropathy and neuropathy complications, (c) HbA1c > 9%, (c) use of medications including metformin, sulfonylureas, gliptins, gliflozins, insulin, and statins, (d) use of immunosuppressive or antibiotics in the past 6 months before sampling, (e) age >60 years, (f) pregnant women, (g) persons with fungal or other infections, and (h) acute complications of diabetes such as diabetic ketoacidosis, hyperketonemia, and hyperosmolar hyperglycemic state were excluded based on clinical judgment of the attending physician.
All healthy persons had normal FPG without suffering from specific diseases including T2DM confirmed by physician. All T2DM and HC persons were matched for age and gender as much as possible without any significant differences between the groups (Supplementary Table 1). The project was approved by local human ethics committee (IR.UMSHA. REC.1400. 609) and participants filled out and signed the study informed consent.

Cell preparation
An aliquot of 15 ml of blood sample from each participant was drawn into EDTA tube (Becton Dickinson, BD, Franklin Lakes, NJ, USA) for isolation of immune cells and 3 ml of blood sample was collected into clot activator tube (BD, USA) for separation of serum. Isolation of peripheral blood mononuclear cells (PBMCs) was done by standard ficoll technique (Sigma, Germany, Cat no. 10771) [18]. Briefly, fresh blood samples were diluted with equal volume of sterile phosphate buffer saline (PBS) and centrifuged on ficoll at 2200 rpm for 30 min. For purification of CD4 þ T cells, PBMCs were magnetically labeled with anti-human CD4 micro beads and isolated with magnetic-activated cell separator based on the kit protocol (Miltenyi Biotec, Germany, Cat no.130045101). Trypan blue was applied as a dye to determinate cell viability. The cells (1 Â 10 6 cells/ml) stored in a complete cell culture medium containing RPMI 1640, 10% fetal bovine serum, 10,000 U/ml penicillin and 100 mg/ml streptomycin, 2 mM L-glutamine, and 1% sodium pyruvate until the next use. All medium reagents were purchased from Biosera, France.

Cell surface staining
PBMCs (2 Â 10 5 cells/100 ll) from each donor were labeled with phycoerythrin (PE)-conjugated monoclonal anti-human CD4 (clone SK3, Biolegend, San Diego, CA, USA, Cat no. 344606) and phycoerythrin-Cy5 (PE-Cy5)-conjugated monoclonal anti-human CD45RA (clone HI-100, BD, USA, Cat no. 555490) antibodies, in the standard staining buffer (0.2% BSA in PBS). Then, the cells suspension was maintained in the dark at room temperature for 40 min. After washing steps, the cells were analyzed by Attune NxT flow cytometry with version 2.5 software (Invitrogen, Carlsbad, CA, USA). Fluorescence minus one (FMO) control tubes were applied to determine the borderline between negative and positive populations in the multicolor staining experiments.

Cell culture
Isolated CD4 þ T population (1 Â 10 6 cells/ml) was cultured in flat bottom plates with purified anti-human CD3 antibody (clone OKT3, plate bound, 2 lg/ml, Cat no. 317325) and antihuman CD28 antibody (clone 28.2, soluble, 2 lg/ml, Cat no. 302934) in the absence and presence of recombinant human IL-32a (rIL-32a, soluble, 5 ng/ml, Cat no. 551004) for 96 h in a standard cell culture incubator (CO 2 at 5% and 37 C temperature). The doses of culture activators were selected based on the previous observations [18,19]. Cell supernatants were collected and frozen at À80 C for cytokine determination, whereas cells were stored in culture media for gene experiments. All culture stimulator antibodies were purchased from Biolegend.

Gene expression assay
Total RNA from cultured Th cells was isolated using the RNA purification kit (Qiagen, Germantown, MD, USA, Cat no: 74106), and the generation of cDNA was done using a PrimeScript reverse transcription kit (Takara, Kusatsu, Japan, Cat no. RR037A) according to the kit's protocol. Purity calculation of RNAs was tested by a spectrophotometer instrument and RNAs with an A260/A280 ratio above 1.8 was used for gene experiments. Ampliqon SYBR Green kit (Denmark, Cat no: A-323402) was used for performing real-time PCR and all reactions carried out in a LightCycler 96 instrument (Roche, Munich, Germany) at 95 C for 30 s, 60 C for 30 s, and 72 C for 30 s. The relative expressions of STAT1, STAT3, STAT5, T-bet, ROR-ct, Foxp3, and NF-jB were calculated by 2 -DCq method and the GAPDH as a housekeeping gene was used for normalization. The sequence of the primers is written in Supplementary Table 2.
Proliferation assay CD4 þ T cells (1 Â 10 6 cells/tube) suspended in 1 ml of PBS and stained with 2 mM of carboxy fluorescein succinimidyl ester (CFSE; Sigma, Roedermark, Germany, Cat no: 423801) at 37 C for 10 min. The cells cultured in 96-well (1 Â 10 5 cells/ well) flat bottom plates and activated with purified antihuman CD3 antibody (plate bound, 2 lg/ml) plus anti-human CD28 antibody (soluble, 2 lg/ml) in the absence and presence of soluble rIL-32a (5 lg/ml) in a 5% CO 2 incubator at 37 C for 96 h. After incubation period, the harvested cells stained with surface PE-conjugated monoclonal anti-human CD4 antibody in the dark for 40 min and proliferation rate was assayed by flow cytometry.

Statistical analysis
Normality assumption of data was examined by Shapiro-Wilk test. Independent-samples t-test (for checking between the patients and controls) and paired-samples t-test (for checking between the parameters in one group) were used. Pearson test was applied for correlations. All analyses were carried out with the SPSS version 22.0 (SPSS Inc., Chicago, IL, USA) and the presented graphs were drawn with the GraphPad Prism version 6.07 GraphPad Software, La Jolla, CA, USA). p Value less than 0.05 was considered statistically significant. Data are summarized as mean ± standard deviation (SD) in the text and mean ± standard error of the mean (SEM) was used for graphs.

Serum levels of IL-32a in patients and healthy controls
In the first step of this study, serum levels of IL-32a were measured in T2DM patients and HCs. Increased concentrations of IL-32a were observed in the patients compared to HCs (p ¼ 0.001, Figure 1 positively correlated with FPG and HbA1C in the patients (p ¼ 0.017, r ¼ 0.52, and p ¼ 0.018, r ¼ 0.61, Figure 1(B,C)). There were no conspicuous correlations between IL-32a and other study parameters including BMI, GFR, creatinine, and albuminuria (data not shown).

T cells in patients and HCs
To analyze the number of total CD4 þ T cells as well as naïve and memory subgroups, isolated PBMCs from each participant stained with fluorescent-conjugated antibodies and tested with a flow cytometer. Principle of gating strategy is presented in Supplementary Figure 1(A). The results showed that the percentages of total CD4 þ T cells, CD4 þ CD45RA þ T cells, and CD4 þ CD45RA -T cells were similar between the groups (Supplementary Figure 1(B-D)).
Effect of rIL-32a on cytokine production of CD4 1 T cells To realize the impact of rIL-32a on Th cytokines, purified CD4 þ T cells cultured with polyclonal activators in the absence and presence of rIL-32a, and supernatant cytokines were assessed. In both patients and HCs, the addition of rIL-32a (rIL-32a þ ) to the culture cause an increase in IL-2 levels, compared to cultures without rIL-32a (rIL-32a À ) (p ¼ 0.006, p < 0.001, Figure 2(A), left graph). The production of IL-2 in rIL-32a þ cultures of patients was higher than the HCs (p ¼ 0.002, Figure 2(A), left graph). Fold change analysis indicated that rIL-32a had a higher potential to increase IL-2 from CD4 þ T cells in patients versus HCs (p < 0.001, Figure  2(A), right graph). We observed no changes in IFN-c levels between the rIL-32a þ and rIL-32a À cultures, and also between the patients and HCs. Fold change in IFN-c levels of patients was similar to that in HCs (Figure 2(B), left and right graphs).
No considerable changes in IL-10 levels (and its fold change) were seen between the study groups or between the rIL-32a þ and rIL-32acultures ( Figure 2(D), left and right graphs).
Effect of rIL-32a on gene expression profile of CD4 1 T cells To determine whether rIL-32a can affect the gene expression of T cell transcription factors, Th cells were activated by polyclonal activators with and without rIL-32a, and the expressions of the Th-related genes were investigated.
The data demonstrated that rIL-32a did not change the expression of STAT1, STAT3, STAT5, and T-bet in the cell cultures. Additionally, the expression and fold change of them did not show apparent changes between the patients and HCs (Figure 3(A-D) left and right graphs).
rIL-32a treatment causes a significant increase in ROR-ct expression in both patient and HC groups (p< 0.001 for both, Figure 3(E), left graph). In patients, ROR-ct expression was higher in both rIL-32a þ and rIL-32acultures compared to those in HCs (p ¼ 0.02, p¼ 0.001, Figure 3E, left graph). Fold change analysis showed that ROR-ct expressions in the patients were higher than that of HCs (p ¼ 0.042, Figure 3(E), right graph).
In rIL-32acultures of patients, Foxp3 had a lower expression than controls (p ¼ 0.02, Figure 3(F), left graph). In both patient and HC groups, no considerable changes in Foxp3 expression were found between the rIL-32aand rIL-32a þ cultures. Fold change in Foxp3 expression had no changes between the groups (Figure 3(F), right graphs). rIL-32a treatment increased baseline expression of NF-jB in both study groups (p ¼ 0.001, p < 0.001, Figure 3(G), left graph). mRNA expression of NF-jB in patients was higher in both rIL-32aand rIL-32a þ cultures compared to those in HCs (p ¼ 0.02, p ¼ 0.001, Figure 3(G), left graph). Fold change analysis illustrated that NF-jB expressions in patients were higher than in HCs (p ¼ 0.04, Figure 3(G), right graph).
Effect of rIL-32a on proliferation capacity of CD4 1 T cells To test the effect of rIL-32a on proliferation rate, purified Th cells were activated by polyclonal activators and the proliferation rate was determined in vitro. Flow cytometry gating strategy for the assays is shown in Figure 4(A). The addition of rIL-32a to the cultures elevated the proliferation ability of CD4 þ T cells in both patients and HCs (p < 0.001 for both, Figure 4(B), left graph). Both rIL-32aand rIL-32a þ cultures of T2DM patients had a higher proliferation rate compared to those in HCs (p ¼ 0.04, p < 0.001, Figure 4 had a higher proliferation ability to response to rIL-32a compared with HCs (p ¼ 0.018, Figure 4(B), right graph).
In rIL-32a þ cultures of patients, the concentration of IL-2 was positively correlated with NF-jB expression and proliferation rate (p ¼ 0.019, p ¼ 0.022, Figure 4(C,D)). In both patient and HC groups, no significant changes were seen in all studied parameters between the genders (data not shown).

Discussion
Aggressive action of pro-inflammatory Th1 and Th17 subsets, but defective function of Treg cells is involved in the pathogenesis of T2DM [20]. IL-32a is mainly produced by CD4 þ T cells and it has a vital role in controlling inflammatory immune response [4]. In this study, we attempted to clarify the roles of IL-32a in T2DM pathogenesis. The impact of IL-32a on Th subsets was tested in vitro. The effects of IL-32a on proliferation rate of CD4 þ T cells and related factors (IL-2 and NF-jB) were also investigated.
The first finding of this study showed the serum concentrations of IL-32a were increased in untreated T2DM patients compared to HCs, and its levels were correlated with HbA1C and FPG. Previous reports have demonstrated that the plasma levels of IL-32a increased in myasthenia gravis, hepatocellular carcinoma, and periodontitis diseases [8,9,21]. In addition, overexpression of IL-32a transcript in PBMCs of patients with graves and psoriasis has been reported to be associated with disease activity [5,10]. The associations between pro-inflammatory cytokines and the glucose parameters in the patients have been shown by other studies. For example, the high levels of TNF-a and IL-18 have been shown to be correlated with the levels of insulin resistance and HbA1c [16,22]. IL-32 transgenic animal models showed impaired glucose tolerance and IL-32 transcript was upregulated in T1DM [23]. According to the present data, it seems that IL-32a has an important effect on glucose metabolism parameters. However further investigations are required to clarify the effects of this cytokine on other diabetes factors including insulin resistance, and the evolution of IL-32a during the treatment should be explored.
Abnormal changes in proportion and function of Th cells have been reported in T2DM patients [2,3,20]. Few studies have been reported that IL-32 induces the function of immune cells [19,23]. For instance, it has been demonstrated that IL-32 is a potent inducer of TNF-a and IL-32c-stimulated dendritic cells could induce a T helper response [19,24]. In this study, we explored the impact of rIL-32a on Th1, Th17, and Tregs mediators and transcription factors in the patients compared to HCs.
Th1 cells express STAT1 and T-bet transcription factors and produce large quantities of IFN-c [3,13]. High frequency of circulating CD3 þ CD8 -IFN-c þ Th1 cells in diabetes patients have been reported previously [2]. We have recently published that the serum concentrations of IFN-c and its production from PHA-stimulated PBMCs enhanced in T2DM patients treated with metformin and gliclazide compared to HCs [3,25]. However, in this study, no significant changes in IFN-c, STAT1, and T-bet levels were seen in isolated CD4 þ T cells activated with anti-CD3 and anti-CD28 between the patients and HCs. This disconfirmation could be as a result of therapy or various types of cultured cells and stimulators. Moreover, we found that in vitro administration of IL-32a did not change Th1-related factors, and no correlations were seen between the Th1 cells and glucose metabolism parameters such as FPG and HbA1c. However, the effects of other isoforms of IL-32 on cytokine production have been reported in other diseases. For example, IL-32c induces the secretion of IFN-c1 from PBMCs of HBV-infected healthy volunteers and low mRNA expression of total IL-32 cause to decrease in IFN-c levels in HIV-infected PBMCs of healthy volunteers [26,27]. Th17 is another pro-inflammatory subset of Th cells that recognized by STAT3 and ROR-ct transcription factors as well as IL-17 secretion [12,13]. Th17 is strictly embroiled in the pathogenesis of T2DM. In this study, we showed that the expression of ROR-ct and production of IL-17 from Th cells increased in untreated T2DM patients. In consonance with this study, we have previously revealed that the gene expression of ROR-ct and the serum levels of IL-17 have enhanced in the patients treated with metformin and gliclazide [3,28]. In addition, high frequency of circulating and adipose tissue resident CD4 þ IL-17 þ Th17 cells has been reported in the patients [2,29]. We also observed that the addition of rIL-32a to the Th cultures enhanced the expression of ROR-ct and production of IL-17, and rIL-32a had a higher potential to increase them in the patients versus HCs. In line with our results, Moon and collogues have reported that total IL-32 promotes IL-17 production and it can induce the differentiation of CD4 þ T cells to Th17 cells in both RA model mice and RA patients in vitro [19]. Other study has indicated that IL-32 was able to induce IL-6 production by the interaction of IL-32a with protein kinase C (PKC) in human promyelomonocytic cell line [30].
This study suggests that IL-32a may play a pathologic role in induction of Th17 cells by enhancement of ROR-ct expression and IL-17 production in the patients, and the relationships between IL-32a and IL-17 exists in T2DM, contributing to accelerated Th17-related inflammation. In the immune system, ROR-ct expressions can help Th17 cells to produce IL-17 and vice versa. It is possible that IL-32a causes to produce IL-17 by the induction of ROR-ct. In this study, we did not observe significant effect of IL-32a on STAT3. It seems that other intracellular signaling molecules expect STAT3 contribute to ROR-ct expression. IL32 production is induced by Tcell activation [31], and one can hypothesize that IL-32a may modulates the function of naïve T cells by promoting Th17 responses, and both IL-32 and Th17 cells have been linked to T2DM pathogenesis. Further studies are needed to examine these possibilities.
Diabetic patients are usually susceptible to fungal infections and Th17 cells are essential for host defense against fungal infections [32,33]. In this study, none of the subjects were infected by fungal infections and the role or function of isolated Th17 cells on fungal infections was not assessed in the patients. Further investigations are required to explore this possibility.
Treg subset has a critical role in T2DM pathogenesis. Treg cells are identified by STAT5 and Foxp3 transcription factors, and IL-10 secretion [18]. Recent findings have been shown that the number and suppressive function of circulating CD4 þ Treg cells (by phenotype: CD25 þ Foxp3 þ and CD25 þ CD45RA þ ) decreased in diabetes persons, and the impairment of Treg cells is correlated with diabetes parameters including insulin resistance [14,20]. Alteration in ratio of Th1 and Th17 to Treg cells was also reported in T2DM [2,20]. We have recently reported that the mRNA levels of Foxp3 in PBMCs have decreased in the patients treated with anti-diabetic agents compared to HCs [28]. In this study, we confirmed that the mRNA expression of Foxp3 was significantly reduced in isolated CD4 þ T cells of the patients, but no significant effects of IL-32a on Treg-related factors (STAT5, Foxp3, and IL-10) were observed in both patients and HCs in vitro.
We have also tested the impact of IL-32a on proliferation ability of T helper cells and related factors. We observed that in vitro proliferation rate of activated CD4 þ T cells and NF-jB expression levels were higher in IL-32acultures of untreated patients, in agreement with our previous reports [14]. Increased proliferation of T cells was shown in the inflammatory and autoimmune diseases including lupus nephritis and inflammatory bowel diseases [34,35]. Other study has also demonstrated that the gene expression of NF-jB was increased in PBMCs of diabetes patients [17].
We also observed that the addition of rIL-32a to the cultures enhanced the proliferation of Th cells, NF-jB expression, and IL-2 production, and rIL-32a was more efficient to increase them in the patients. It has been shown that IL-32 enhances the proliferation of cutaneous T-Cell lymphoma cell lines by activating MAPK and NF-jB signaling pathway [36]. Lin et al. have reported that IL-32a induces the proliferation of bone marrow stromal cells line in vitro by activating NF-jB and STAT3 [11]. They have suggested that IL-32a induces the production of IL-6 in this cells line which leads to increasing proliferation rate.
IL-2 is a growth mediator for Th cells and the NF-jB is a multi-roll transcription factor that promotes T cells function [12,15]. The relationship between NF-jB and IL-2 is explained by other studies and both NF-jB and IL-2 have an additive effect on each other [12,15]. In this study, we found positive correlations between the IL-2 with both NF-jB transcript and proliferation rate of CD4 þ T cell in the IL-32a þ cultures of patients. However, we did not find any correlations between the proliferation assays (or NF-jB expression) with other proinflammatory cytokines such as IL-17 and IFN-c, and also Th transcription factors. It seems that IL-32a enhances the proliferation of T cells by promoting NF-jB and IL-2 in T2DM.
Some limitations could be addressed in future works. We evaluated the impact of IL-32a on Th cytokines and transcription factors, however, the effect of this cytokine on the percentage of T cell subsets and Th surface markers (flow cytometric assays) could be explored. The proliferative potential of IL-32a was only explored in total CD4 þ T cells. The effect of IL-32a on proliferation of individual Th subpopulations such as Th1 and Th17 could be determined in future works. The evaluation of other isoforms of IL-32 including IL-32b and IL-32c in T2DM patients is suggested for future studies. The effect of IL-32a on other Th subsets including Th9 and Th22 could be analyzed in future studies. It has been reported that patients with an HbA1c higher than 9% were associated with an enhanced risk of heart failure and hospitalization [37]. This study was performed on pure and moderate form of untreated T2DM patients (with FPG between 126 and 250 mg/dl and/and HbA1c 6.5-9%) without other clinical complications. It is suggested that the impact of IL-32a on prediabetes patients (with FPG between 100 and 125 mg/dl and/or HbA1c 5.7-6.4%) and severe forms of diabetes (HbA1c >9%) are investigated in future studies. The effects of anti-diabetic agents on IL-32a and the roles of IL-32a in diabetes patients with severe complications such as nephropathy, retinopathy, diabetic ketoacidosis, and hyperosmolar hyperglycemic state could also be examined in future research.

Conclusion
It has been previously published that aggressive Th function and enhanced proliferative response of Th cells are involved in the pathogenesis of T2DM. The molecular mechanisms underlying the immune abnormalities are unknown. This project showed that recombinant IL-32a activates pro-inflammatory Th17 cells by inducing the ROR-ct gene expression and IL-17 production, and IL-32a may accelerate the Th17 -related inflammation. Moreover, IL-32a enhances NF-jB expression and IL-2 secretion, and promotes CD4 þ T cell proliferation. IL-32a was more effective to enhance the abovementioned factors in the patients compared to healthy individuals. Moreover, enhanced serum levels of IL-32a in the patients were correlated with FPG and HbA1c. The present findings provide the in vitro functional role of IL-32a in T2DM, and suggest possible mechanisms underlying the aggressive actions of Th cells in diabetic persons. However, in vivo biological roles of IL-32a in the T2DM should be tested in the next projects and blocking IL-32a signaling pathway could be mentioned as a therapeutic approach in the future.