Jagged-1 signaling suppresses the IL-6 and TGF-β treatment-induced Th17 cell differentiation via the reduction of RORγt/IL-17A/IL-17F/IL-23a/IL-12rb1

Jagged-1 signaling has recently been reported to be involved in the Th17 cell differentiation. However, little is known about its mechanisms. Soluble Jagged-1 was used to activate the Jagged-1–Notch signaling to interfere with the IL-6 and TGF-β-induced Th17 cell skewing. Genes relevant to the autoimmunity or inflammation were screened for the first time in this system by qPCR array for the differential expressions. The 18 genes out of 84, including Clec7a, Il12b, Il12rb1, Il12rb2, Csf3, Il15, Il17a, Il17f, Il17rc, Il17rd, Il17re, Il23a, Myd88, Socs1, Stat4, Stat5a, Sykb and Tbx21, were downregulated, but only Cxcl2, Cxcl12 and Mmp3 were upregulated. The expressions of the genes, Rorγt, Il17a, Il17f, Il12rb1 and Il23a, induced by simultaneous IL-6 and TGF-β treatment were significantly suppressed by Jagged-1, followed by the reduction of RORγt, IL-17A, and IL-17F. Consistent with the attenuation of RORγt, and the reduced production and secretion of IL-17A and IL-17F in the cell supernatant and the in situ stained cells, the number of CD4+IL-17+ cells was also diminished. It is concluded that the Jagged-1–Notch signaling can suppress the IL-6 and TGF-β treatment-induced Th17 cell skewing through the attenuation of RORγt and, hence by, the down-regulation of IL-17A, IL-17F, IL-23a, and IL-12rb1.

R ecently, the Th1/Th2 paradigm has been expanded, following the discovery of a third subset of the effector Th cells, called Th17 cells [1][2][3] and characterized by the production of IL-17A, IL-17F, and IL-22 as the signature cytokines. In the presence of TGF-b alone, naive T cells express Foxp3 that induces the regulatory T cells 4 . A relevant finding is that IL-6 is a potent inhibitor of the TGF-b-driven induction of Foxp3 1 regulatory T cells 5 . IL-6 not only inhibits the generation of these cells, but also together with TGF-b, guides the naive CD4 1 T cells to produce IL-17. Th17 cells express a unique transcription factor, RORct 6 , which induces the transcription of the Il-17 gene in naive helper T cells to promote the development of IL-17-producing cells in the presence of TGF-b and IL-6 7 . RORct activation also induces the expression of IL-23R, indicating that IL-23 acts on T cells that are already committed to the Th17 lineage 8 . It is well known that the Th17 cells play a key part in the pathogenesis of the autoimmune and inflammatory diseases as well as the tumors. Thus, it is of great significance to reveal the mechanisms that regulate the Th17 cell differentiation.
Notch signaling has been proven to decide the fates of immune cells. In mammals, there are four Notch receptors, including Notch-1, Notch-2, Notch-3 and Notch-4, and five Notch ligands, including Jagged-1, Jagged-2, delta-1 (Dll1), delta-3 (Dll3) and delta-4 (Dll4). Jagged-1 is taken as the example. While Jagged-1 interacts with the Notch-1 or Notch-2, successive cleavages are triggered in the transmembrane region of Notch receptors by the disintegrin and metalloproteinase. The endocytosis of the transmembrane fragment of the Notch contributes to the further cleavage by c-secretase, leading to the release of the Notch intracellular domain and the final activation of the Notch signaling pathway. Radtke et al. found that the deletion of Notch-1 resulted in the marked decrease in the size of thymus that lacked T cells and contained the excess of B cells 9,10 . Notch-1 inactivation causes a complete block in T lineage development, indicating that the other Notch family members cannot compensate for the loss of Notch-1 in vivo 10 . Notch signaling not only regulates the T/B cell lineage commitment, but also the differentiation and function of the peripheral T cells 11,12 . Different Notch ligands play various roles in the differentiation of the naive CD4 1 T cells.
The antigenic stimulation of the naive CD4 1 T cells in the context of antigen-presenting cells engineered to express Dll1 leads to the secretion of Th1 cytokines, such as IFN-c 13 , whereas the Jagged-1 promotes the Th2 cytokine production, such as IL-4 14 . Mukherjee et al. found that the dendritic cells (DCs) were activated by TLR-specific signals, and Dll4 up-regulated the RORct expression in T cells; both rorct and il17 gene promoters were the direct transcriptional notch targets that further enhance the differentiation of Th17 cell populations, while anti-Dll4Ab significantly inhibited the differentiation 15 . Ito et al. showed that the TLR9-deficient mice challenged with a mycobacterium antigen displayed an altered Th17 cytokine profile, the decreased accumulation of granuloma-associated myeloid DCs, and profoundly reduced Dll4 expression, suggesting that the Dll4 plays an important role in promoting the Th17 activity during mycobacterium challenge 16 . Notch-3 antibody could down-regulate the expression of IL-17 in experimental autoimmune encephalomyelitis. These data suggest that the Dll-Notch signaling may be involved in the differentiation of Th17 cells 17 . Our previous study showed that the Jagged-1-Hes-1 signaling could reduce the production of IL-17 in CD4 1 T cells, which was reversed by Hes-1-targeting siRNA 18 . However, it is still unclear how genes relevant to autoimmunity or inflammation are altered in the Jagged-1-treated cells. Therefore, in the current study, qPCR array was used for the first time to explore the genes that may play an important part in the Jagged-1-mediated Th17 cell differentiation from different angles.

Results
Jagged-1-Notch signaling inhibits the differentiation of the CD4 1 T cells into Th17 cells. Flow cytometry was performed to analyze the phenotypes of CD4 1 T cells treated with Jagged-1. As shown in Fig. 1, compared with the anti-CD3/CD28 group, the group, treated simultaneously by IL-6 and TGF-b, showed the increased percentage of CD4 1 IL-17 1 T cells. This augment can be abolished by Jagged-1. However, the percentage of IFN-c 1 IL-17 1 T cells was unchanged in each group. The results indicate that the activation of the Jagged-1 signaling can inhibit the polarization of Th17 cells without the enhancement of Th1-like cytokine.
The reduction in the expressions of il-17a, il-17f, il-12rb1 and il-23a by Jagged-1-Notch signaling. The Jagged-1-Notch-1 signaling induced changes in il-17a, il-17f, il-12rb1 and il-23a were further confirmed by RT-PCR ( Fig. 3A-D) and qPCR ( Fig. 3E-H). As shown in Fig. 3, compared with the group treated with IL-6/TGFb/IL-23a in the presence of anti-CD3/CD28, the activation of Jagged-1-Notch signaling indeed led to the decrease of il-17a, il-17f, il-12rb1 and il-23a levels in the CD4 1 T cells, consistent with the alterations observed by qPCR array. However, there was no difference between IL-17A and IL-17F, different from the qPCR array data. These results show that the Jagged-1 can inhibit the Th17 cell differentiation, maturation and stability.
Jagged-1-Notch-1 signaling inhibits the specific cytokine production in Th17 cells via RORct. IL-17A and IL-17F were considered as functional executants of Th17 cells. qPCR and Western Blot were first used to evaluate the relationship among RORct, IL-17A and IL-17F. Compared with the anti-CD3/CD28 group, both IL-6 and TGF-b could obviously increase the expressions of RORct, IL-17A and IL-17F mRNAs, but Jagged-1 could decrease their expressions in the CD4 1 T cells induced by simultaneous IL-6 and TGF-b treatment ( Fig. 4A-C). Following the alterations in RORct, IL-17A and IL-17F mRNAs, RORct, IL-17A and IL-17F protein levels were also decreased. Both IL-6 and TGF-b upregulated the expressions of RORct, IL-17A and IL-17F, whereas this augment could be abrogated by Jagged-1 ( Fig. 4D-F). Furthermore, the secretion of IL-17A and IL-17F in the treated cells was tested by ELISA. Compared with the anti-CD3/CD28 group, simultaneous IL-6 and TGF-b treatment boosted the cells to secrete IL-17A and IL-17F, which could be reversed by Jagged-1 ( Fig. 4G-H). These findings further support that the Jagged-1-Notch-1 signaling inhibits the production of IL-17A and IL-17F in the CD4 1 T cells via RORct. The qPCR array data showed that the level of IL-17F in the treated cells was lower than that of IL-17A. It seems that IL-17F might play a more important role in Th17 cell polarization, but other results demonstrate that there is no significant difference between the both.
Jagged-1-Notch-1 signaling inhibits the in situ expressions of RORct, IL-17A and IL-17F induced by simultaneous IL-6 and TGF-b treatment. As shown in Fig. 5-6, CD4 1 T cells were treated with IL-6, TGF-b and Jagged-1 for 72 h to find out the effects of Jagged-1-Notch-1 signaling on the in situ expressions of RORct, IL-17A and IL-17F in the CD4 1 T cells. Compared with the anti-CD3/CD28 group, the group, treated simultaneously by IL-6 and TGF-b, showed the enhanced in situ expressions of RORct, IL-17A, and IL-17F. The in situ expressions of RORct, IL-17A and IL-17F in the CD4 1 T cells induced by simultaneous IL-6 and TGF-b treatment were obviously decreased after the treatment by Jagged-1. Moreover, the changes in IL-17A and IL-17F levels were consistent with the RORct expression. The data further support that the activation of Jagged-1-Notch-1 signaling pathway may inhibit the differentiation of CD4 1 T cells towards Th17 cells induced by simultaneous IL-6 and TGF-b treatment via the reduction of RORct/IL-17A/IL-17F/IL-23a/IL-12rb1.

Discussion
Naive CD4 1 T cells express Notch-1 and Notch-2 receptor mRNAs, while they do not express Notch-3 and Notch-4 mRNAs 14 . T cells express Notch ligands Jagged-1 24 , Jagged-2 25 , and Delta-1 26  and Jagged-2 as well as Delta-4 14,24,25 . Jagged-1 was originally isolated as a mammalian ligand that activates the Notch-1 signaling. The activated domain has been mapped to a N-terminal extracellular region of Jagged-1, as a specific peptide from the Delta/Serrate/ Lag2 (DSL)-domain of Jagged-1 that can mediate the activation of Notch signaling 27 .
There are debates about the suppression of T cell activation or the enhancement of T cell function by Notch signaling in murine and human systems 28 . Notch ligands have been shown to differently influence the T cell differentiation [29][30][31] . Yasutomo et al. first demon-strated that Dll1 could promote the skewing of naive CD4 1 T cells toward Th1 29 . Subsequently, Amsen et al. found that Jagged-1 could induce naïve CD4 1 T cells to differentiate into Th2 lineage by increasing the expression of IL-4 in naïve CD4 1 T cells 14 . Other investigators showed that Jagged-1 directly activated a Jagged-1-Notch signaling pathway, inducing naïve peripheral T cells to differentiate into regulatory T cells 30,31 . Furthermore, Ito et al. found that Dll4 would improve the secretion of Th17 cytokines, such as IL-17A and IL-17F in vitro and in vivo. Dll4 influences the generation of IL-17-producing T cells in the presence of both IL-6 and TGF-b 16 . Our previous data indicate that the Jagged-1-Hes-1 signaling can suppress the skewing of CD4 1 T cells toward Th17 cells by means of siRNA to knockdown hes-1 gene, but the genes that function in the Th17 cell skewing remain poorly understood 18 . In this study, we observed the alterations in 84 genes possibly related to the differentiation of Th17 cells for the first time to find out the genes that play a prominent part in the differentiation induced by Jagged-1. The novel findings demonstrate that cxcl12, cxcl2 and mmp3 gene expressions are increased, but clec7a, csf3, il12b, il12rb1, il12rb2, il15, il17a, il17d, il17f, il17rc, il17rd, il17re, il23a, myd88, socs1, stat4, stat5a, sykb and tbx21 expressions are decreased. Some of the downregulated genes, such as il12b, il12rb1, il12rb2, stat4 and tbx21, might be related to Th1 cell polarization, but others, mainly including il17a, il17d, il17f, il17rc, il17rd, il17re and il23a, might be related to Th17 cell skewing. Furthermore, il17a, il17f, il12rb1 and il23a were selected for further confirming their relationship to Jagged-1 signaling. They were indeed reduced in the treated cells, which is consistent with the attenuation of CD4 1 IL17 1 T cells. The downregulation of IL17a, IL17f, IL17rc, IL17rd and IL17re contributes to the inhibition of Th17 cell skewing by Jagged-1. Are they correlated to RORct change? The reductions in both IL17A and IL17F are attributed to the decreased expression of RORct by Jagged-1, and that Jagged-1 signaling is responsible for the reduction in IL12rb1, which may avail the inhibition of naïve CD4 1 T cell skewing toward Th1. In addition, the reduction in IL23a by Jagged-1 may preclude the maturation of Th17, contributing to the decreased number of CD4 1 IL17 1 T cells by Jagged-1 signaling, too. Undoubtedly, these findings provide a novel insight into the mechanisms of Th17 cell differentiation. Of course, it merits to exploring further the roles that Cxcl12, Cxcl2 and Mmp3 play in the Jagged-1-inhibited Th17 cell skewing.
IL-23 is a member of IL-12 family of heterodimeric cytokines. It is composed of two subunits: IL-12p40, which is common to IL-12, and IL-23-specific p19 subunit 32 . The reported relationships of IL-12 and Th1 cells with autoimmunity can be explained by the requirement for IL-23 33 . Many of the proinflammatory functions of IL-23 seem to be related to Th17 cell subset 34 . The stimulation of IL-23 maintains the IL-17 production by Th17 cells 35 , that enhances the Th17 cell differentiation 32 , or promotes the survival of Th17 cells 36 . In the presence of proinflammatory cytokines particularly, TGF-b and IL-6 can trigger the Th17 differentiation through Th17-specific transcription factor RORct, which leads to the production of IL-17A and IL-17F 3,5,37 . IL-23R is up-regulated on the membrane of the naïve CD4 1 T cells after activation in the presence of IL-6 6 . IL-23R is induced in the developing Th17 cells, and IL-12Rb2 is produced in the developing Th1 cells to pair with the constitutively expressed IL-12Rb1 chain to combine with IL-23 and IL-12, respectively. IL-12Rb1 chain binds a common subunit of IL-23 and IL-12 heterodimers (IL-12p40 or IL-12b) to pair with IL-23p19 (IL-23a) or IL-12p35 (IL-12a), respectively 32,38,39 . In our study, the expressions of Il17a, Il17f, Il12rb1 and Il23a were also significantly inhibited by Jagged-1, which was directly related to the observed Th17 phenotype, suggesting that the IL-23 signaling pathway plays a role in the development of Th17 cells. Some reports suggest that the activation of the IL-23 or IL-12 receptor induces late events in the downstream of early Th17 or Th1 lineage commitment 1,37 . Whereas the IL-12 receptor potently activates Stat4 in Th1 cells, the IL-23 receptor predominantly activates Stat3, but also recruits some Stat4 32,40,41 . Stat3 is required for programming TGF-b1 and IL-6, and the IL-23-stimulated IL-17-secreting phenotype. Moreover, the retroviral transduction of the constitutively active Stat3 into the differentiating T cells enhances IL-17 production, suggesting that the Stat3 may directly regulate the forming of Th17 cells 40 . Moreover, the IFN-c-and IL-17-producing T cells were typically found in association with the IFN-c-producing T cells in the context of both autoimmune inflammation and infection. These data suggest that there might be a parallel between Th1 and Th17 developmental programs. Our results show that the Jagged-1 does not influence the percentage of IFN-c-producing CD4 1 T cells, but significantly decreases the number of IL-17-producing CD4 1 T cells. The critical cytokines and their receptors to promote Th1 cell skewing, including IL12b, IL12rb1 and IL12rb2, are reduced, and the crucial transcriptional factors to facilitate Th1 cell skewing, including Stat4 and T-box 21 are also attenuated, further supporting our findings.
Although several up to date reports implicate the impact of Notch signaling on the Th17 differentiation, there still exist the opposite effects. Elyaman et al. reported that Jagged-1 could protect from the Experimental Autoimmune Encephalomyelitis (EAE), but Dll1 exacerbated its development. On the contrary, the administration of anti-Jagged-1 Ab exacerbated the EAE, while anti-Dll1 Ab reduced its severity. These data suggest that Jagged-1 may inhibit Th17 cell deviation or function 42 . In our study, Jagged-1 could reduce the number of CD4 1 IL-17 1 T cells with the reduction of IL-17A and IL-17F, and did not influence the expression of IFN-c, indicating that it may suppress the skewing of CD4 1 T cells toward Th17 nor Th1 cells. Keerthivasan et al. obtained the opposite evidence that if Notch signaling was blocked; the production of Th17-associated cytokines was diminished, suggesting that the Notch signaling is required for the Th17 differentiation 43 . Of note, Bailis' team found that Notch did not direct the Th cell differentiation, but instead concurrently regu-lated the Th1, Th2, and Th17 cell genetic programs mainly by the addition of a gamma secretase inhibitor 44 . Through comparison and analysis for the associated reports, we consider that this contradiction or difference might be the result from the differentially polarizing conditions, dissimilarly inducing protocols, different host cells (or models) and evaluating standards.
Taken together, 84 genes relevant to the autoimmunity and inflammation were screened in this study for the first time to explore the potential roles of these genes in the Jagged-1-inhibited Th17 cell differentiation. The novel findings indicate that the activation of Jagged-1 signaling leads to the downregulation of clec7a, il12b, il12rb1, il12rb2, csf3, il15, il17a, il17f, il17rc, il17rd, il17re, il23a, myd88, socs1, stat4, stat5a, sykb and tbx21, and the upregulation of cxcl12, cxcl2 and mmp3 under Th17 cell polarization, and that the Jagged-1 signaling suppresses the simultaneous IL-6 and TGF-b treatment-induced Th17 cell differentiation via the reduction of RORct/IL-17A/IL-17F/IL-23a/IL-12rb1. The results provide a new insight into Th17 cell differentiation and a potential strategy to inhibit its deviation. Additionally, our results also suggest that the correlation between the screened Il12rb1/Clec7a/Socs1 and RORct deserves to be further explored in the Jagged-1-inhibited Th17 polarization.

Methods
Mice. BALB/c mice were purchased from the Guangzhou Medical Animal Center (Guangzhou, China). Eight week-old animals were used. All animals were bred and maintained under the specific pathogen-free condition. All animal handling and experiment procedures were approved by the Animal Care and Use Committee of Guangzhou Medical Animal Center. All experiments below were performed in accordance with relevant guidelines and regulations.
Immunomagnetic bead isolation. Peripheral lymph nodes in mice were triturated mechanically and filtered by using the 200 meshes of stainless wire net for the isolation of lymphocytes. Then, the cells were harvested, washed twice with PBS, and re-suspended in Roswell Park Memorial Institute 1640 complete culture medium containing 10% (v/v) fetal bovine serum (Gibco BRL, Gaithersburg, MD, USA) at the concentration of 1 3 10 6 cells/ml. CD4 1 T cells were segregated from the above separated lymphocytes using the mouse naïve CD4 1 T cell isolation kit (EasySep Negative Selection Kit, StemCell, Vancouver, BC, CA). The lymphocytes were suspended at a concentration of 1 3 10 8 cells/ml with medium plus 5% normal rat serum in a polystyrene tube. According to the manufacturer's instructions, CD4 1 T Cell Enrichment Cocktail, Biotin Selection Cocktail and Magnetic Nano particles were added into the tube, respectively. The CD4 1 T cells were enriched in suspension and the unwanted cells were discarded through magnetic labeling. Then, the purity of the separated CD4 1 T cell population was tested under a flow cytometer, reaching 98%. The collected CD4 1 T cells were cultured at 37uC in 5% CO 2 in the complete medium. Quantitative PCR array. The isolated CD4 1 T cells were treated with or without 1 mg/ml Jagged-1 for 72 h. Total RNA was extracted using the Trizol RNA Extraction Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. RNA samples were tested for the concentration and purity by Nanodrop 2000 (ThermoFisher Scientific). One microgram of the total RNA was reversetranscribed to obtain cDNA with a RT 2 First Strand Kit (QIAGEN GmbH, Hildden, Germany), and applied to PCR array plates. Then, the 84 genes examined using RT 2 Profiler PCR Array Mouse Th17 for Autoimmunity and Inflammation (PAMM-073A, Qiagen, Germany) are listed in Table 1 beside 12 genes as genomic DNA contamination control, reverse transcription control and internal control, respectively. 2700 ml of qPCR mixture contained 102 ml of cDNA, 1350 ml of 2 3 RT 2 SYBR Green Mastermix, and 1248 ml of RNase free water. qPCR mixture was added to the array at 25 ml/well. The qPCR reaction conditions were as follows: 95uC for 10 min, 95uC for 15 s, 55uC for 40 s, 72uC for 30 s, and 40 cycles in total. The qPCR was performed on a Bio-Rad/MJ Research Chromo4 (BIO-RAD, Berkeley, CA, USA), and each sample with and without Jagged-1 treatment was repeatedly tested using the array for average of CT values. The 2 2DDCT method was performed for the relative quantification of the mRNA expression using the web-based software for the cataloged and custom arrays (http://pcrdataanalysis.sabiosciences.com/pcr/ arrayanalysis.php) (Qiagen, Germany). The gene expressions of the Jagged-1-treated group with changes greater than 2.0 fold were obtained, compared with the control group.
RT-PCR and real-time quantitative PCR. To further confirm the changes of the related genes in the CD4 1 T cells treated with Jagged-1, the cells were treated with or without 1 mg/ml Jagged-1 in the presence of anti-CD3/CD28, IL-6, TGF-b and IL-23 for 72 h, and the total RNA was extracted using a Trizol RNA Extraction Reagent (Invitrogen, Carlsbad, CA, USA). One microgram of the total RNA was used in each reaction primed with oligo-dT to obtain cDNA. Then, 1 ml of the synthesized cDNA was used as a template for RT-PCR or real-time quantitative PCR. The primers were as follows: Il-17a 19   www.nature.com/scientificreports AGC AC-39, b-actin antisense 59-CGT TGA CAT CCG TAA AGA CC-39. The amplified products were separated by electrophoresis on a 1.5% agarose gel, analyzed using the FluorChem 8000 system(Alpha Innotech, Santa Clara, CA, USA) and the Jagged-1/b-actin ratio was calculated.
Real-time qPCR was performed on an ABI7300 sequence detection system (Applied Biosystems). The real-time qPCR reaction was amplified with the following procedure: 95uC for 10 min, and 35 repeats at 95uC for 30 s, 60uC for 30 s and 72uC for 30 s. The 2 2DDCt method was performed for the relative quantification of mRNA expression.
Western Blot. The cells were treated as described in the ''Flow cytometry'' section. Six hours before the end of the treatment, the cells were stimulated with 50 ng/ml phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) plus 1 mg/ml of ionomycin (Alexis, Lausen, Switzerland) for the below experiments. Meanwhile, 10 mg/ml of GolgiStop (Becton Dickinson, Franklin Lakes, NJ, USA) was added to the cells. The treated cells were lysed with radio-immunoprecipitation assay (RIPA) lysis buffer (BioColors, Shanghai, China) containing phenylmethanesulfonyl fluoride. The proteins were analyzed on SDS-PAGE gels and electrophoretically transferred onto the nitrocullose membranes. The membranes were incubated with rabbit anti-RORct (Santa Cruz biotechnology, Santa Cruz, CA, USA), rat anti-IL-17A (Cell Signaling, Danvers, MA, USA) or goat anti-IL-17F (R&D Systems, Minneapolis, MN) primary antibody respectively overnight. The membranes were washed three times with TBST, followed by the incubation with the appropriate HRP-conjugated secondary antibody for 1 hour at the room temperature. The specific bands were identified with an Enhanced Chemiluminescence Detection Kit (Amersham Biosciences, Piscataway, NJ, USA). This test was repeated for at least three times.

Socs1
Suppressor of cytokine signaling 1 Socs3 Suppressor of cytokine signaling 3 Stat3 Signal transducer and activator of transcription 3 Stat4 Signal transducer and activator of transcription4 Stat5a Signal transducer and activator of transcription 5a Stat6 Signal transducer and activator of transcription 6  Sykb  Spleen tyrosine kinase  Tbx21  T-box 21  Tgfb1 Transforming growth factor, beta 1 Tirap Toll-interleukin 1 receptor (TIR) domain-containing adaptor protein Tlr4 Toll-like receptor 4 Tnf Tumor necrosis factor Traf6 Tnf receptor-associated factor 6 Yy1 YY1 transcription factor www.nature.com/scientificreports SCIENTIFIC REPORTS | 5 : 8234 | DOI: 10.1038/srep08234 under 10 fields of view selected randomly for each group were counted for average percentages.
Statistical analysis. Student's t-test was performed for the paired data comparison and one way ANOVA was followed for comparing the sets of more than two groups. P value ,0.05 was considered statistically significant.