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1

2 Induction of immunostimulatory cytokine genes expression in human PBMCs

3 by a novelsemiquinone glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1

4 Raj Kumar a,⇑, Rakesh Kumar Sharma a, Deen Dayal Bansal a, Dev Dutt Patel a, Saurabh Mishra a,5 Lyuba Miteva b, Zlatka Dobreva b, Veselina Gadjeva b, Spaska Stanilova b

6 a Institute of Nuclear Medicine and Allied Sciences, Delhi, India7 b Faculty of Medicine, Trakia University, Stara Zagora, Bulgaria

8

1 0a r t i c l e i n f o

11 Article history:12 Received 21 July 201013 Accepted 15 November 201014 Available online xxxx

15 Keywords:16 Immunostimulation17 Cytokine18 PBMCs19 Semiquinone glucoside derivative20

2 1

a b s t r a c t

22In the present study, a semiquinone glucoside derivative (SQGD) isolated from a radioresistant bacterium23Bacillus sp. INM-1 was evaluated for its immunostimulatory activities. Human peripheral blood mononu-24clear cells (PBMCs) were stimulated by different doses (30–90 lg/ml) of SQGD for different time (3–12 h)25intervals at 37 �C, and IL-12p40, IL-23p19, IL-10, RelA and c-Jun gene expression analysis was carried out26by qRT-PCR method. SQGD dose dependent cytokines protein expression kinetic analysis was carried out27using Western blotting. As the results of SQGD (30 lg/ml) stimulation for 3 h at 37 �C, significant induc-28tion in IL-12p40, IL-23p19 and RelA gene expression was observed in PBMCs compared to unstimulated29control cells. However, no such induction in IL-10 and c-Jun gene expression was observed. Time depen-30dent protein expression study indicated significant increase in IL-12p40, IL-12p35, IL-23p19 and RelA31protein expression at 3–6 h, which was found decrease at 12 h upon SQGD treatment. In contrast,32IL-10 protein expression was found to enhance significantly at 12 h after SQGD treatment to the PBMCs.33SQGD dose dependent study showed approximately similar level of induction in IL-12p40, IL-12p35,34IL-23p19 and RelA proteins expression at all tested concentration (30–90 lg/ml) compared to control.35However, no significant change in the IL-10 and c-Jun protein expression was observed at any SQGD36concentration. SQGD treatment (0.25 mg/kg b wt.) was also found to enhance anti-keyhole Limpet37Hemocynin (KLH) IgM antibodies significantly in the mice immunized by KLH.38Thus, SQGD fraction stimulates cellular immunity by inducing immunostimulatory cytokines and39humoral immunity by enhancing IgM antibodies and could be a promising immunostimulant. Further40studies related to molecular mechanisms offering immunostimulation is underway, will certainly helpful41to unravel its mode of action in the biological system.42� 2010 Published by Elsevier Inc.

43

44

45

46 1. Introduction

47 Cytokines, the key modulators of immune response delivered48 diverse functions in both humoral and T cell mediated immunity49 [1–3]. Among the cytokines, IL-12, IL-23 and IL-10 have been50 reported to play a very crucial role in the regulation of pro/anti-

51inflammatory responses induced by microbial infections, oxidative52stress, septic shock and ionizing radiation [4–7]. Human IL-1253(IL-12p70) is a disulfide-linked heterodimer composed of two sub-54units p40 and p35. Earlier studies have defined that IL-12p70 is an55important factor exhibits numerous immune functions including56stimulation of T cells and natural killer (NK) cells, production of57IFN-c and regulation of several cell adhesion molecules [8–10].58Various reports demonstrated a vital role of IL-12p70 in the devel-59opment of specific immunity against number of intracellular60pathogens including Leishmania major, Mycobacterium tuberculosis,61Listeria monocytogenes, and Toxoplasma gondii [11–13]. Apart from62that IL-12 has also been shown to have adjuvant properties, and63can stimulate an effective cellular immune response against micro-64bial antigens that are otherwise not immunogenic when adminis-65tered alone. Besides immunostimulatory properties, IL-12p70 also66exhibits anti-tumor activity in several preclinical animal tumor67models [14]. Interleukin-23 (IL-23) is a heterodimeric cytokine

0008-8749/$ - see front matter � 2010 Published by Elsevier Inc.

doi:10.1016/j.cellimm.2010.11.005

Abbreviations: C3bgp, C3 binding glycoprotein; DCs, dendritic cells; DPPH, 1,1-

diphenyl 2-picrylhydrazyl; EDTA, ethlene diamine tetra acetic acid; IFN-c, inter-feron-gamma; IL, interleukin; kGy, kilo gray; LPS, lipopolysaccharide; MHC, major

histocompatibility antigen; MTCC, microbial type culture collection; NFjb, nuclearfactor kappa beta; NK, natural killer cells; PBMC, peripheral blood mononuclear

cells; PBS, phosphate buffer saline; PHA, phytoagglutinin; PWM, pok weed mitogen;

qRT-PCR, quantitative real-time polymerase chain reaction; RelA, reticuloendothe-

liosis homolog A; TLR4, toll like receptor 4; KLH, keyhole limpet hemocyanin.⇑ Corresponding author. Address: Institute of Nuclear Medicine and Allied

Sciences, Brig. S.K. Mazumdar Road, Delhi 110054, India. Fax: +91 11 23919509.

E-mail address: rajkumar790@yahoo.com (R. Kumar).

Cellular Immunology xxx (2010) xxx–xxx

Contents lists available at ScienceDirect

Cellular Immunology

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68 (p40/p19) consisting of a novel p19 subunit and the p40 subunit of69 IL-12p70. IL-23 has similar but discrete functions from IL-12p70.70 IL-23 induces activated memory T cells to proliferate and produce71 IFN-c. In addition, IL-23 induces production of a pro-inflammatory72 cytokine, IL-17, from activated T cells [15].73 In contrast to IL-12 and IL-23, IL-10 has immunosuppressive74 effect and inhibits Th1 cytokine synthesis from activated75 macrophages [16]. IL-10 is known to produce by a variety of cells,76 including activated antigen presenting cells, CD4+ Th0 and Th277 cells. Up-regulated IL-10 gene expression induced oxidative stress78 in the septic shock patients [16,17] and lead to mortality. Similarly,79 higher expression of IL-10 induced immunosupression in the80 cancer patients [18,19]. It has been reported that the up regulation81 of IL-10 gene was concomitantly down regulate the activation of82 IL-12 and IL-23 gene expression [20–22] and thus lead to83 immunosuppression.84 Therefore, to combat the IL-10 mediated immunosuppression,85 induction of IL-12 and IL-23 genes expression are highly desirable86 to initiate the Th1 cellular immune response in the cancer patients87 exposed with radiation during radiotherapy regime. Various natu-88 ral compounds like lipopolysaccharide of Gram negative bacteria,89 peptidoglycan, lipoteichoic acid, N-acetyl glucosaminyl glycan,90 b 1,4 linked N-acetylmuramic acid of gram positive bacteria,91 C3binding glycoprotein (C3bgp) isolated from Cascuta europea,

92 pokweed mitogen (PWM) a lectin isolated from Phytollaca

93 americana and phytoagglutinin (PHA) have been identified and94 evaluated for their abilities to stimulate Th1 mediated immune re-95 sponse by induction of IL-12 and IL-23 cytokine gene expression96 [4,17,23–25].97 Present study was under taken to evaluate a semiquinone glu-98 coside derivative rich fraction (SQGD) isolated from Bacillus sp.99 INM-1 for its immunomostimultory activities using human PBMCs.

100 Modulation of IL-12p40 and IL-23p19 genes expression was stud-101 ied and compared with immunosuppressive cytokine IL-10 gene102 expression using qRT-PCR methods. The effect of SQGD on Th1 im-103 mune response, regulatory transcription factor RelA and c-Jun gene104 expression was evaluated and compared with LPS and C3bgp med-105 iated gene expression. To further validate the qRT-PCR based gene106 expression profile of IL-12p40, IL-12p35, IL-23p19, RelA, IL-10 and107 c-Jun, time dependent protein expression studies also carried out.108 Effect of SQGD on keyhole limpet hemocynin (KLH) stimulated hu-109 moral response was also evaluated in vivo using Swiss Albino male110 mice.

111 2. Material and methods

112 2.1. Reagent

113 Lipopolysaccharides (LPS) from Escherichia coli serotype 026:B6,114 Antibodies, Histopaque-1077, RPMI-1640 medium, PHA, PWM etc.115 were procured from Sigma, USA and all cell culture plastic ware116 were purchased from Nunc, Denmark. C3 binding glycoprotein117 (C3bgp) was isolated at Trakia University, Stara Zagora, Bulgaria118 from a water extract of the seeds of the parasitic plant Cuscuta

119 europea by affinity chromatography as described [26].120 SQGD, an endotoxin free semiquinone glucoside derivative121 (SQGD) rich fractions was isolated from Bacillus sp. INM-1 (MTCC122 No 1026) at our laboratory at INMAS, Delhi, India.

123 2.2. PBMC isolation

124 The blood samples were collected from five healthy volunteers125 after obtaining consent form each of them and diluted (1:1 ratio)126 with sterile phosphate buffer saline (PBS). The diluted blood sam-127 ples were carefully transferred to histopaque-1077 followed by

128centrifugation at 1800 rpm at 4 �C for 20 min. The interface of129histopaque-1077 and diluted blood samples containing peripheral130blood mononuclear cells (PBMC) was gently harvested and washed131twice with sterile cold serum-free RPMI-1640 medium. After132washing, cell pellet of PBMC was resuspended in RPMI-1640133medium and cells viability was tested with Trypan blue exclusion134method.

1352.3. Cell culture and stimulation

136PBMCs (1 � 106) were cultured in sterile 24-well polystyrene137plates containing 2.0 ml RPMI-1640 media supplemented with138100 U/ml penicillin, 100 lg/ml gentamicin and 0.3 mg/ml L-gluta-139mine. The cultures were stimulated with 30 lg/ml SQGD, 30 lg/ml140C3 binding glycoprotein (C3bgp), and 30 lg/ml LPS. Both stimulated141and unstimulated cultures were incubated at 37 �C for 3 h.

1422.4. RNA isolation

143PBMCs (1 � 106 cells) stimulated with SQGD, C3bgp, or LPS for1443 h, were used for RNA isolation. Unstimulated cells were used as145calibrators. After incubation, cells were washed and resuspended146in lysis solution RL supplied with a column-based In-Prep blood147RNA isolation kit (AJ Roboscreen, Leipzig; Germany). Total RNA iso-148lation was performed according to the manufacturer’s instructions.149To remove traces of genomic DNA, total RNAs (1 lg) were treated150with RNase-free DNase I (Fermentas, USA) as per the manufacture’s151instruction.

1522.5. Reverse transcription

153Synthesis of cDNA was performed manually as per the instruc-154tions supplied by manufacturers with high-capacity cDNA Archive155kit (Applied Biosystems, USA) that uses random primers and156MultiScribeTM MuLV reverse transcriptase enzyme. Incubate for15710 min at 25 �C followed by 2 h at 37 �C.

1582.6. Quantitative real-time polymerase chain reaction (qRT-PCR)

159Quantitative real-timepolymerase chain reaction (qRT-PCR)was160performed on a 7500 Real-Time PCR System (Applied Biosystems,161CA, USA). The following validate PCR primers and TaqMan162MGB probes (6FAM-labeled) were used: IL-12p40 (assay ID:163Hs00233688_m1), IL-23p19 (Hs00372324_m1), IL-10 (Hs00116474086_m1), RelA (Hs00153294_m1) and c-Jun (Hs99999141_s1).165Eukaryotic 18S ribosomal RNA (Hs99999903_m1) was used as166endogenous control. An aliquot of 5 ll of the RT reactionwas ampli-167fied in duplicate in final volume of 20 ll of TaqMan Universal PCR168master mix and gene expression assay mix, containing specific for-169ward and reverse primers and labeled probes for target genes and170endogenous control (Applied Biosystems, USA).The thermocycling171conditions were applied as followed: initial 10 min incubation at17295 �C followed by 40 cycles of denaturation for 15 s at 95 �C and173extension for 1 min at 60 �C. PCR datawere collectedwith Sequence174Detection Systemsoftware, version 1.3.1. Relative quantitative eval-175uation of cytokinemRNAswas performed by the comparativeDDCt176method. The mean DCt obtained in unstimulated PBMC for each177cytokine mRNA was used as calibrator, after normalization to178endogenous control-18S rRNA. The results are presents as an n-fold179difference relative to calibrator (RQ = 2�DDCt).

1802.7. Kinetics of cytokines protein expression at different SQGD

181concentrations

182PBMCs were stimulated with different doses (30–90 lg/ml) of183SQGD at 37 �C temperature for 3, 6 and 12 h. After completion of

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glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1, Cell. Immunol. (2010), doi:10.1016/j.cellimm.2010.11.005

184 the incubation, cells were harvested by centrifugation at 5000 rpm185 for 10 min. Cells were then sonicated (two cycles of 50 Hz) in PBS186 and total soluble proteins were extracted by centrifugation187 (5000 rpm, 20 min) at 4 �C.

188 2.7.1. Protein estimation

189 Total soluble protein in the cell lysate was estimated by190 Bradford method (28) and the amount (lg) of protein was quanti-191 fied using BSA standard curve.

192 2.7.2. SDS–PAGE and western blot analysis

193 For SDS–PAGE and western blot analysis of IL-12p40, IL-12p35,194 IL-23p19, RelA, IL-10 and c-Jun protein expression was carried out195 using the protocols standardized in our laboratory (29). IL-12p40,196 IL-12p35, IL-23p19, RelA, IL-10 and c-Jun expression levels were197 analyzed by probing with respective mouse monoclonal antibodies198 (1:1000 dilution) having cross reactivity with human proteins. The199 protein bands obtained were further subjected to densitometric

200analysis. The quantification of individual protein bands was done201using Alpha Ease FC 4.0.0 software (Alpha-Innotech, India).

2022.8. Experimental animals and immunization protocol

203Swiss Albino male mice (aged 8–10 week and weight approxi-204mately 25 gm) were used to evaluate the stimulatory effect of205SQGD on KLH induced humoral response. All animals provided206standard food and water ad libitum with 12:12 h light:dark cycle.207The rules and regulations of Institutional Animal Ethics Committee208were strictly followed during experimentation.

2092.8.1. Primary humoral response protocol

210To find out the effect of SQGD on induction of humoral response211initiated by KLH, all animals were divided into following three212groups.213Group 1. Control mice administered with sterile PBS (0.5 ml).

Fig. 1. Effect of SQGD treatment on transcriptional induction of IL-12p40 (A), IL-23p19 (B) and RelA (C). Human PBMCs were stimulated for 3 h at 37 �C either with SQGD

(30 lg/ml), C3bgp (30 lg/ml) and bacterial LPS (30 lg/ml) and expressions of IL-12p40, IL-23p19 and RelA gene transcript was analyzed. Relative quantitative analysis of

the genes expression levels was carried out using real time PCR method. Genes expression in control group of cells which were not stimulated by SQGD was used as

calibrator. ±S.D.

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214 Group 2. The first injection of KLH antigen (50 lg) was given at215 day 0 and booster dose (50 lg) of the KLH antigen in saline was216 administered (i.p.) to the mice on days 7.217 Group 3. Mice administered with primary (at day 0) and sec-218 ondary (at day 7) doses of KLH antigen (50 lg) were subsequently219 administered with different (250, 500 and 750 lg) concentrations220 of SQGD.221 Seven days after final treatment, all the mice were sacrificed.222 Blood was collected and anti-KLH IgM and IgG antibody titer in223 the serum were estimated.

224 2.8.2. ELISA for serum anti-KLH antibodies estimation

225 The surface of 96-well Maxisorb immunoplates (Nunc,226 Danmark) were coated with 10 lg KLH per ml of 0.5 M sodium227 carbonate buffer (pH 9.6) and incubated overnight at 4 �C. Then,228 plates were washed, kept at room temperature and blocked for229 1 h with PBS containing 1% BSA and subjected for two washes with230 PBS. The plates were then incubated with a 1:100 dilution of

231immunized mice sera or pre immune sera. After three washes,232plates were incubated with peroxidase-conjugated mouse mono-233clonal anti-mice antibodies, specific to mice IgM and IgG (Clone234No. RG-16; Sigma). After three washes, 0.1 ml of substrate solution235(0.005% O-phenylenediamine in 0.1 M citric buffer, pH 5.0, with23610 ll H2O2) was added to each well and incubated for 10 min at237room temperature. The developing color reaction was stopped by238addition of 10% H2SO4 and the optical density was measured at239492 nm using an ELISA plate reader (Rosys Antos-2010, Austria).

2402.9. Statistical analysis

241The data was expressed as means ± standard deviation of three242independent repeated experiments. The statistical analysis of var-243iance (One-Way ANOVA) was performed for multiple comparisons244followed by a post hoc test. All the statistical tests were performed245using statistical software SPSS-Version-11. Differences were con-246sidered significant when the p-value was less than 0.05.

Fig. 2. Influence of SQGD treatment on IL-10 (A) and cJun (B) gene expression. Human PBMCs were treated by SQGD (30 lg/ml), C3bgp (30 lg/ml) and bacterial LPS (30 lg/ml)

and expressions of IL-10 and cJun gene transcript was analyze by real time relative quantitative analysis. Gene expression in control group of cells which were not treated by

SQGD was used as calibrator. ±S.D.

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247 3. Results

248 3.1. Induction of IL-12p40 cytokine gene expression by SQGD

249 The quantitative real time gene expression analysis of IL-12p40250 was carried out using human PBMCs. Cells were stimulated with251 SQGD for 3 h, and level of IL-12p40 gene transcription analyzed252 and compared with the untreated control cells as well as with253 the cells stimulated with known immunostimulatory agents (i.e.,254 LPS and C3bgp). The results were calculated and presented as the255 fold increase compared to unstimulated controls. As the results256 of SQGD treatment, maximum (p = 0.016) induction in the tran-257 scription of IL-12p40 (5.74-fold increase) gene was observed in hu-258 man PBMCs as compared to unstimulated control (Fig. 1A).259 Similarly, significant induction in IL-12p40 gene expression was260 observed in the group of cells stimulated with SQGD as compared261 to the cells stimulated with C3bgp (decreased by 2.7-fold;262 p = 0.021) and LPS (decreased by 1.7-fold; p = 0.032), respectively263 (Fig. 1A).

2643.2. Effect of SQGD on IL-23p19 gene expression

265qRT-PCR gene expression analysis was indicated that SQGD266treatment significantly (p = 0.02) induced IL-23p19 gene expres-267sion as compared to unstimulated control (Fig. 1B). Almost similar268level of IL-23p19 gene expression was observed in the cells stimu-269lated with C3bgp as compared to unstimulated control. Expression270of IL-23p19 though increased in the cells stimulated by LPS as com-271pared to unstimulated control, however, the level of the expression272was remained low significantly (p = 0.04) as compared to the cells273stimulated with SQGD (Fig. 1B).

2743.3. Effect of SQGD on RelA gene expression

275RelA is a component of NFjb transcription factor which play a276very crucial role in the regulation of prosurvivalmolecular signaling277under various stresses. A significant (p = 0.033) induction (0.7-fold)278in the RelA gene expression was observed in the PBMCs stimulated279with SQGD as compared with unstimulated controls (Fig. 1C).280Similar level of induction in RelA expression was observed in the

Fig. 3. SQGD dose dependent analysis of IL-12p40, IL-12p35, IL-23p19, RelA, IL-10, and cJun protein expression. PBMCs were stimulated with different concentrations

(30–90 lg/ml) of SQGD and incubated for 3 h at 37 �C. Total soluble proteins were extracted and quantified. To target the individual protein antigen, specific anti-mouse

monoclonal antibodies having cross reactivity with human was used during western blot analysis. Relative quantitative analysis of the protein expression was carried using

Alpha Ease FC 4.0.0 software (Alpha-Innotech, India). ±S.D. $IL-12p40, IL-12p35, IL-23p19, RelA: SQGD (30, 60 and 90 lg/ml) Vs Control p < 0.05. #IL-10 and cJun: SQGD (30, 60

and 90 lg/ml) Vs Control, p > 0.05.

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281 cells stimulated with LPS. However, significant (p = 0.034) decrease282 in the RelA expression was noticed in the cells stimulated with283 C3bgp as compared to SQGD (p = 0.034). Therefore, results of the284 present study indicated that SQGDwas equally induce the transcrip-285 tion of RelA gene as LPS and thus may share the similar mechanism286 of RelA or NFjb activation as adopted by LPS.

287 3.4. Influence of SQGD on IL-10 and c-Jun genes expression

288 No significant (p > 0.05) change in the level of IL-10 and c-Jun289 gene expression was observed in the cells after 3 h, stimulation290 with SQGD as compared to unstimulated controls (Fig. 2A, B).291 Though expression of IL-10 was found to be increased (�1.7-fold)292 significantly (p = 0.03) in the cells stimulated with C3bgp as com-293 pared to SQGD stimulated cells. However, the expression of c-Jun294 gene was observed to be higher in the LPS stimulated cells as com-295 pared to the cells treated with C3bgp and SQGD (Fig. 2B).

296 3.5. Kinetics of cytokines expression after stimulation of PBMCs by SQGD

297 To evaluate the effect of different concentrations (30–90 lg/ml)298 of SQGD on cytokines protein expression, western blot analysis

299was carried out. Results of the study was clearly indicated a sharp300induction (�2.0-fold) in the proteins expression of IL-12p40,301IL-12p35, IL-23p19 and RelA in the cells pretreated by SQGD at30230 lg/ml concentration as compared to untreated control cells.303Further increase in the SQGD concentrations (60–90 lg/ml) did304not affect IL-12p40, IL-12p35, IL-23p19 and RelA expression signif-305icantly as compared to the cells stimulated by SQGD at 30 lg/ml306(Fig. 3). However, no significant change was observed in IL-10307and c-Jun protein expression upon SQGD treatment at any tested308concentration (30–90 lg/ml).309Time dependent protein expression studies demonstrated that,310as the results of SQGD (30 lg/ml) treatment, IL-12p40, IL-12p35,311IL-23p19 and RelA protein expression was increased significantly312at 3 and 6 h, however, it was found decrease at 12 h post SQGD313treatment as compared to corresponding untreated control. In con-314trast, IL-10 protein expression was found decreased significantly at3153–6 h post SQGD treatment whereas, it was found increased signif-316icantly at 12 h post SQGD treatment as compared to control.317Though, no significant change in c-Jun protein expression was318observed at 3 h after SQGD treatment to the PBMCs. However, a319mild decrease in the expression was observed at 6 h post SQGD320treatment (Fig. 4).

Fig. 4. Time dependent kinetics of IL-12p40, IL-12p35, IL-23p19, RelA, IL-10, and cJun protein expression. PBMCs were treated by SQGD (30 lg/ml) concentration at 37 �C for

different time (3, 6 and 12 h) intervals. Cells were then separated by centrifugation (5000 rpm; 10 min) at 4 �C and total soluble proteins were extracted with PBS and

quantified. Protein’s expression level was evaluated using monoclonal antibodies specific to individual cytokine by western blot analysis. Relative quantitative analysis of the

protein expression was carried using Alpha Ease FC 4.0.0 software (Alpha-Innotech, India). ±S.D. $IL-12p40, IL-12p35, IL-23p19, RelA: SQGD (3–6 h) Vs Control p < 0.05. #IL-10:

SQGD (3–6 h) Vs Control p < 0.05. *IL-10: SQGD (9 h) Vs Control p < 0.05.

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321 3.6. Dynamics of anti-KLH antibodies formation and their modulation

322 by SQGD treatment

323 Significant (p < 0.05) induction in the anti-KLH antibodies (IgM)324 in the group of mice immunized with KLH antigen followed by325 SQGD (0.25 mg/kg b wt. i.p.) administration was observed as com-326 pared to the mice immunized with KLH antigen only. Further327 increment (0.5–0.75 mg/kg b wt. i.p) in the SQGD concentration328 was found to decrease anti-KLH antibodies titer significantly329 (Fig. 5A). However, no significant change in IgG antibodies titer330 was observed with any SQGD concentration (Fig. 5B). Therefore,331 SQGD treatment at 0.25 mg/kg b wt. i.p. able to stimulates overall332 induction of anti-KLH-IgM antibodies and thus induces humoral333 immune response in the mice.

334 4. Discussion

335 Present study was carried out to evaluate immunostimulatory336 properties as the function of immunostimulatory cytokine induc-337 tion in human PBMCs by semiquinone glucoside derivative (SQGD)338 rich fractions isolated from Bacillus sp. INM-1. Gene and protein

339expression analysis indicated higher induction of IL-12p40 gene340in human PBMCs stimulated by SQGD as compared to LPS and341C3bgp suggested that SQGD is more potent inducer of IL-12p40342then LPS and C3bgp. However, mechanism associated with IL-34312p40 induction by SQGD is not known completely. Bacterial344LPS, an agonist of TLR4 receptor and C3bgp known to induce345IL-12p40 expression [17,23] was used as positive control in the346present study. Positive effect of IL-12p40 in the reactivation of sup-347pressed immune system and protection of hematopoietic system348against gamma radiation [27,30] providing the gain support to349the present findings. IL-12p40 induction upon SQGD treatment350may facilitate recovery of endogenous hematopoiesis and engraft-351ment of stem cells after ionizing radiation [7] and thus may protect352the immune system effectively from oxidative stress.353Expression of IL-23p19 gene was also found to be increased354significantly in the cells stimulated with SQGD as compared to355unstimulated control or LPS stimulated cells. IL-23 does effectively356mediate proliferation and IFN-c production from activated357memory/effector T cells and thus contribute to host immunity and358anti-tumor activity [31]. Therefore, present evidences strongly sug-359gested a role of SQGD in activation of IL-23p19 gene which may

Fig. 5. Effect of SQGD administration on anti-KLH IgM (A) and anti-KLH IgG (B) antibodies production in the mice pre-immunized by Keyhole Limpet Hemocynin (KLH)

antigen. Swiss albino male mice were immunized by KLH antigen (50 lg) followed by SQGD (0.25, 0.5, 0.75 mg/kg b wt) administration. After seven days, secondary

immunization was done with the same dose of KLH and SQGD. Seven days after second immunization blood and then serum of the animals was collected and concentration of

anti-KLH IgM and IgG antibodies was estimated and compared with the SQGD untreated but KLH treated control mice using ELISA assay. ±S.D.

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glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1, Cell. Immunol. (2010), doi:10.1016/j.cellimm.2010.11.005

360 delivered diverse functions to enhance the cellular immunity and361 cell proliferation in the surrounding normal tissues damaged362 during radiotherapy of the cancer patients without effecting tumor363 regression rate [32,33].364 RelA (p65) is a subunit of NFjb, form a heterodimer with sister365 peptide p50 and act as transcription factor. Expression of RelA gene366 and corresponding protein was found to be increased significantly367 in the cells expressing higher level of IL-12p40 and IL-23p19 gene368 after SQGD stimulation (Figs. 1A–C, 3 and 4). This observation was369 suggested an involvement of SQGD in activation of prosurvival370 NFjb signal transduction pathway. NFjb has already been known371 for its selective up regulation of pro-inflammatory cytokine genes372 [34,35] further provided a gain support to the present investiga-373 tion. Though, expression of IL-12p40 and IL-23p19 was increased374 (Fig.1A, B) significantly, while, no significant change in IL-10 gene375 expression was noticed in the PBMCs stimulated by SQGD at 3 h376 (Fig. 2A). However, significant decrease in IL-10 protein expression377 at 6 h after SQGD treatment (Fig. 4) suggested immunostimulatory378 potential of SQGD. Down regulation of IL-10 synthesis accelerates379 IL-12 secretion by induction of IL-12 secreting cell population380 [36]. Hoffmann and co-workers [37] reported that in the absence381 of IL-10, mice exposed with radiation attenuated larvae developed382 Th1 type cytokine (IL-12) profile and high level of T cells mediated383 immunity against Schistosoma mansoni infection. Similar results384 were obtained in our previous study conducted on the septic shock385 survivor’s, which, revealed significant reduction in IL-10 expres-386 sion along with higher level of IL-12p40 expression in human387 PBMCs stimulated by C3bgp compared to non survivors [17] pro-388 vides a gain support to the present investigation. Apart from cell389 mediated primary immune response, SQGD was also evaluated390 for its secondary immune response stimulatory properties. Results391 of the study showed significant induction in the level of anti-KLH392 antibodies in the serum of the mice treated with KLH antigen393 followed by SQGD administration as compared to only KLH immu-394 nized mice (Fig. 5A, B) suggested that SQGD effectively potentiate395 the secondary immune response in the mice immunized with KLH396 antigen. Similar results were also reported by Stanilova [38] with397 C3bgp plant derive antigen in the rabbit provided a gain support398 to the present investigation.399 In conclusion, present study provided strong evidence that400 semiquinone glucoside derivative (SQGD) rich fraction stimulates401 primary immune response by inducing IL-12p40, IL-12p35,402 IL-23p19 and RelA cytokine genes and corresponding protein403 without significantly effecting IL-10 cytokine gene expression404 and protein in the human PBMCs. SQGD was also found to acceler-405 ate secondary immune response by inducing anti-KLH IgM406 antibodies in the mice. Thus in future, this novel biologically active407 semiquinone glucoside derivative may qualify as a potential408 immunostimulatory agent of therapeutic significance.

409 5. Uncited references

410 Refs. [28,29].

411 Acknowledgments

412 The work was carried out under the aegis of Indo–Bulgaria413 collaborative project (B-57) financially supported by the Depart-414 ment of Science and Technology (DST), Government of India Delhi,415 India and Ministry of Education, Science and Technology, Govern-416 ment of Bulgaria. Author is highly thankful to the Defence Research417 and Development Organization (DRDO), Ministry of Defence, Govt.418 of India and Trakia University, Stara Zagora, Bulgaria to provide419 financial support and the basic facilities to undertake the present420 work.

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glucoside derivative (SQGD) isolated from a Bacillus sp. INM-1, Cell. Immunol. (2010), doi:10.1016/j.cellimm.2010.11.005