Salmonella.typhi OMPs induced immunomodulation in ...

Indian Jou rnal of Experimental Biology Vol. 38, April 2000, pp. 358-362

Salmonella.typhi OMPs induced immunomodulation in peritoneal macrophages

Des Raj Kashyap, Shaloo Sood, Ram Parkash T iwari & Praveen Rishi *

Department of Microbiology, Pan jab University, Chandigarh- 1600 14, India

Received 28 July 1999; revised I December 1999

The immunomodulatory properties of outer membrane proteins (OMPs) from S.typhi Ty2 were studied in mouse model at 72 hr and 20 days post-infection. lnspite of reduction in the number of macrophages and their protein content observed in the immunized group vis-a-vis infected group, OMPs activated macrophages showed significant upregulation of NO. At 20 days post infection, the level remained almost the same suggesting tile prolonged cytotoxic and cytostatic acl!vity due to the long lasting effects of OMPs activated macrophages. Higher acti vi!y of SOD in these aged cells pointed out towards the

protective efficacy of OMPs to keep the macrophages themselves away from the noxious effects of 0 ~ . Lower level of acid

phosphatase in the macrophages from immunized mice group indicated the involvement of oxygen dependent rather than oxygen independent kil ling process. The enhanced uptake of organisms and their killing could be related to the production of oxygen and nitrogen radicals in the OMPs immunized group.

The magnitude of Salmonella infections and its socio­economic impact on the modern world are as relevant today as they were in past. Typhoid fever remains a major health problem of children and adults in developing countries like ours. Since Salmonella pathogenicity is multi-factorial , as yet, little is known about the spec ific microbial factors that determine pathogenicity and those that elicit a protective immune response in man. The new immunogens include several attenuated Salmonella typhi strains for use as oral vaccines . Various subunits vaccines have also been evaluated which include Vi polysaccharides and lipopolysaccharides alone or conj ugated to

. I proteins.

Recently, surface structures of Gram-negative organ isms have been reported to play a ro le in pathogenesis.2

-4 Owing to loca lizati on on the bacterial

surface (which comes in contact with the host first), OMPs have been considered as important anti gens in the induction of immunity. We have also demonstrated that S.typhi porins interact efficiently with the host immune system of experimentally vaccinated animals.5·

7 But the reports are st ill scanty regarding the precise mechanism of protection afforded by phagocytic cell s in response to outer membrane proteins. Since, phagocytosis is the most powerful and most important part of the host defense mechanism that can operate without any delay against the invading micro-organism, in the present study, we

*Correspondent author

tried to evaluate alterations in peritoneal macrophages in response to S.typhi OMPs with which they are likely to come in contact ini tially.

Materials and Methods

Bacterial strain -Salmonella typhi Ty2 used in this study was procured from Central Research Institute, Kasauli , India. The strain was maintained on nutrient agar stabs at 4°C and subcu ltured monthly.

Mice-Laca mice, each weighing 17-25g (8-1 0 weeks old) were used in the present study.

Isolation of Outer Membrane Proteins (OMPs)­OMPs were extracted by the method of Fernandez­Beros et a/ 8

. Briefly, 14 hr old cu lture of S.typhi Ty2 grown in nutrient broth at 37°C was centrifuged at 8000 rpm for IOmi n. The pellet was washed 'twice wi th IOmM tris-HCI buffer (pH 8.0), containing SmM MgCh and suspended in an appropri ate volume of the same buffer. This bacterial cell suspension was sonicated in 4 cycles of 30 sec each wi th an interval of I mi n in ice chest. Sonicated bacterial cell s were centrifuged at 1200 rpm for I 0 min to remove undisrupted cells. The supernatant was again centrifuged at 20,000 rpm for one hr at 4°C. Pellet was treated with Tris-buffer (oH 8.0) containing 1.5% sod ium sarkosyl. After incubation at 37°C for 30 min, centrifugation was done at I ,00,000 g for J hr. OMPs obtained as pellet were suspended in Tris-buffer (pH 8.0) containing PMSF and were stored at -20°C in small aliquotes till further use.

KASHYAP eta/. : S. TYPHI OMPs INDUCED IMM UNOMODULATION 359

Protein concentration was estimated by the method of Lowry as modified by Markwett et al 9

. LPS contamination in the extrac ted protein was determined by the method of Morrison and Le ive 10

. SDS-PAGE was performed by the method of Laemml i 11

.

Immunization of mice - Mice were immunized intn:iperitonea ll y w ith 0.5ml of the suspens ion

containing 50f.lg of protein followed by a second dose of same dilution and volume 15 days later as described by Kuss i et al 12

. Additional group of non­immunized mi ce was set aside at the time of immunization for testing the virulence of the challenge culture. LD50 of S.typhi Ty2 in mice was determined using 5% hog gastric muc in . LD50 and challenge dose was calcul ated by the method of Reed and Muench 13

.

Estimation of number and protein content of I M I . . 20 macrop wges - ac rop 1age count tn preparation

was determined by mixing equal amount of macrophage suspension and try pan blue (0. 1%) and counting was done in a hemocytometer. Protein content of macro-phages was estimated by the method mentioned above.

Disruption of macrophages- Macrophages were di srupted by sonicat ing the cell s for 3 cycles of 15 sec each with 30 sec interval in between the cyc les in ice chest. Cell debris was removed by centri fugati on at 1800 rpm for I 0 min .

Nitric oxide assay- The amount of nitric ox ide in the ce ll free supernatant was determined by Griess reaction as described by Green et a/ 14

. Brie fl y, 100 f.ll a liquots of supernatant mixed with equal vo lumes of Griess reagent ( I% sulfanilamide, 0 . 1% naphthylethylened iamine dihyd rochl oride, 2% H3P04)

was incubated at room te mperature for 10 min . Optical density was measured at 550nm. N02- was quantitated using NaN02 as a standard.

Superoxide dismutase (SOD) estimation - Le ve l of SOD in the supernatant was measured by method of Kono 15

• Briefl y, to 1.3 ml of Solution A (0. 1 mM EDT A containing SOmM Na2C03 (pH I 0.0), 0.5 ml of soluti on B [90 ~tM Nitroblue tetrazolium (NBT) dye] and 0.1 ml of solution C (0 .6%, T riton X-1 00 in solution A), 0.1 ml of soluti on D [20mM Hydroxylamine hydrochloride (pH 6.0)] was added and the rate of NBT reduction was recorded for one minute at 560 nm. 20f.ll of the supernatant was added to the test cuvette as well as reference cuvette which do riot contain hydroxy lamine hydrochl oride. Finally the percentage inhibition in the rate of reduction of

NBT was recorded as described above. One enzyme unit was ex pressed as inverse of the

amount of protein (mg) required to inhibit the reduction rate by 50% in one minute.

Acid phosphatase estimation- The level of acid phosphatase was measured by the method described by Linhart and Walter 16

• Briefl y, 1.0 ml of assay volume containing 0.9ml of ! .OM acetate buffer (pH 4.8), 0 .05ml of p-nitrophe nylphosphate (PNPP) and 0.05 ml of cell free supernatant , was incubated fo r 30 min at 3rc. Reacti on was terminated by additi on or 0.4 NaOH. Ye ll ow colour deve loped was read at 405 nm.

One enzy me unit was expressed as the amoun t (f.! mole) of p-nitrophenol liberated in 30 min at 7>7"C.

Uptake and in!racellu/ar killing by 1/l(tcrop/wges ­The phagocytic uptake and intracellular killing of S.typhi Ty2 by the peritonea l macrophages from control, infected and immuni zed-infected mice were studied as described by Murray and Cohn 17

•

Results A significant increase in macrophage number was

observed in S.typhi Ty2 infected mice as compared to control group. The macrophage number in immunized infected mice was comparable with control group at 72 hr post-challenge. However, a s ignificant decrease in macrophage count was observed in infec ted as well as in immunized-infected mice group a t 20 days post chall enge. Protein content of macrophages from these groups also followed the same pattern after 72 hr. Infected mice group showed a significant (P < 0.00 I) increase in the prote in content as compared to control group followed by a decrease thereafter. In the immt!:lized-infected group, prote in content was almost the same as that of control group throughout the study (Table I).

A s ignificant increase (P<O.OI ) in the producti on of NO was observed in macrophages from S.typhi T y2 infected mi ce, but the production of NO by macrophages from immunized-infected mice group was still higher. The leve l of NO decreased in both the groups after 20 days, but the decrease was not as significant in immunized-infected group as it was in infected group (Table 2) , in which the level was even lower than control group.

SOD showed a significant (P < 0.00 I) decrease in infected group. However, a significant (P < 0.00 I) increase was observed in immunized-infected mtce group as compared to control and infected mice

360 INDIA N J EXP BIOL, APR IL 2000

groups. In thi s group although the level decreased insignificantly at 20 days post-infection but it was still higher as compared to the level in control and infected mice (Table 2).

Both , infected as well as immunized-infected mice showed a decrease in levels of acid-phosphatase but the decrease was more pronou nced in immunized­infected group as compared to control and infected groups (Tab le 2). At 20 clays post-infection signi ficant decrease was observed m the level of ac id­phosphatase in infected group . Although immunized­infected group showed a slight increase in the level (20 days post challenge) but it was still lower than the control and infected groups.

Immunoprotecti ve role of OMPs against S.typhi Ty2 was assessed by monitoring phagocytic uptake and intracellular killing of Sa lmonellae by peritoneal macrophages from infected and immunized- infected mice group . Within 30 min of exposure of S.typhi Ty2 to the peritonea l macrophages enhanced uptake was observed in immunized-infected mice group as compared to control and in fec ted group, but with further exposure up to 90 min , the percentage uptake

in all the three groups was comparable. Studies at 20 days post-infection showed no net increase or decrease in the phagocyt ic uptake in immunized­infected group whereas a significant decrease was observed in uptake by infected mice group. Both, infected as well as immunized-infected mice group showed significant increase in intracellular killing. In infected group it was comparat ively less than that of immu ni zed-infected mi ce group. Macrophages taken from control and infected mice group showed killing up to two hr. of ex posure only, whereas significant killing was observed upto three hr in ide macrophages from immunized-infected mice group. Macrophages from infected group taken after 20 clays post-infection showed a significant decrease in killing, whereas no change was observed in killing of Salmonellae by macrophages from immunized-infected mice group 20 clays post-challenge (Tab les 3,4)

Discussion The increased number and hence the protein

content at 72 hr post-infection in the infected group may be attributed to the presence of heavy bacterial

Table I-Macrophage number and protei n content in three groups

Groups

Control Infected

Groups

Control Infected Immuni zed-infected

*** P<O.OO I

Macrophage count I ml Protein content (mg/ml ) 3 days 20 days 3 days 20 days

4.2 x 106

2.07 X JQ7

4.7 x l06 4.9x 106

1.57 X I 06

0. 169±0.003 1.232 ±0.008* ** 0.191 ±0.002

0.0775 ± 0.008 0. 1975±0.017

Table 2- Level s of NO, ac id phosphatase and SOD in peritoneal macrophages from three go ups Nitric ox ide cone.

(nmole) SOD (IU) Acid phosphatase (IU) 3 days 20 days 3 days 20 clays 3 days 20 days

9.687±0.2 13 9.47±0.011 688.259 ± 35.431 9.529 ± 0.243 115.0±4.933 9.624±0.29 13.0 ± 0.866** 6.242 ± 0.005 139.788 ± 2.606*** 110.278±0.37 96.5 ± 4.57* 69.87± 1.732

Immuni zed- 15.787 ±0.325*** 13.625 ± 0.072 I 001.882 ± 11.865** * 933.83±25.93 36.25 ± 6.737*** 49.75 ±4.25 infected

*?values : <0.05; **P<O.OI: ***P<O.OOI

Table 3-Phagocyti c upt ake of S.typhi Ty2 by peritoneal maerophages ( I Ox cell s/ml from three groups

Time of Control Group In fec ted Group Immunized ln fecled Group

Exposure s. typhi s. typhi s. typhi (min) ( I 000 x I 06 cfu/ml) ( I 000 x I Oli cfu/ml) % uptake ( I OOOx I 06 cfu/ml ) % uptake

% uptake 3 days 20 days 3 days 20 days 3 days 20 days 3 days 20 days

0 1.4 x l07' 1.37 x l07

' 3.3 X ]Q6' 6.9x l06

' 3.8x lOr.•

30 1.9 x !Or, 86.43 I X l Or, 2.7 x 106 88.32 18. 18 3.0x 105 1. 2 X I 0(, 95 .65 68.4 60 7.0 x 105 96.0 4.0x 105 1.2 X I 06 97.08 63.63 2.83 X 105 5.0x 105 95.89 86.84 90 I.O x 105 99 .28 I.O x 105 1.6x 106 99.28 5 1.51 1.06 x 105 8.4x 104 98 .46 97.79

*Initial value count of S. typhi Ty2 before exposure to maerophages .

KAS HYAP el a/. : S. TYPHI OMPs INDUCED IMMUNOMODULATION 36 1

Table 4 - Intracellul ar kill ing of S.trph i Ty2 by peritoneal macro phages ( I Oxcells/ml) from three groups

Time of Control Graue Infected Graue Immunized Infected Graue

Exposure s. t\'! lhi % ki ll ing S. 1yphi % kill ing s. lyphi % kill ing (hr) ( I OOO x IOr.cfu/ml) ( I 000 x I O" cfu/ml) ( I OOOx 10" cfu/ml)

3 days 20 days 3 days 20 days 3 days 20 days 3 days 20 days 0 8.2 x 106

• 8.9 x 10"· 5.2 x lOr.• 9.7 x l Or'· 4.7 x l or.•

I 3.7 x l Or. 54.88 3.2 x 106 3.8 x lOr. 64.04 26.92 2.5 X 106 2.9 x l06 79.23 38.297

2 2.2 x l 05 73 .1 7 1.3 x l Or. 2.0 x 106 85.39 6 1.54 7.0 x 105 1.6 x 105 92.78 96.59

3 4.9 x l 05 40.24 2.9 x 105 3.6 x 106 67.42 30.77 1.05x 105 8.5 X 104 98.92 98.19

* Initial viable count of S.l)'phi Ty2 inside eeritoneal macroehages before intracellular killing.

peptides which are known potent chemoattractants for phagocytes. The reduced number of macrophages and their lower protein content in the immuni zed group vis-a-v is infected grou p at 72 hr, may be because of specific fu ncti ons attributed to macrophage popul ati on i.e . some being di verted towards immune response expression as anti gen presenting cells and others serving as phagocytic cells (macrophages) which inturn may be due to the immunomodulatory properties of OMPsl4. Reduced number of macrophages in thi s group may also be attributed to the · macrophages undergoing apoptosis, which is likely to pl ay an essent ial physiological role in the course of disease process . No net change in the number and hence protein content at 20 days post­in fec tion in the immuni zed group may be due to the long lasting effects of OMPs acti vated macrophages .

Macrophages fro m immunized-in fec ted group also showed a signifi cant upregul ati on of NO as compared to that of control and infected groups. These results are consistent with the observations reported by Alder et al. 18

. NO produced, in particular by the acti vated macrophages has been shown to functi on as a cytotox ic and cytostatic molecule and inhibit the growth of a di verse array of pathogens 19

. In infected group, the NO leve l declined significantly at 20 days post-infec tion but in immunized group the level remained almost the same which again suggested the prolonged cytotox ic and cytostatic acti vity due to the long lasting effects of OMPs m acti vating macrophages .

Macrophages from immunized infected mice showed a significant increase in the level of SOD as compared to other two groups. The hi gher ac tivity of SOD in these aged cells, since they are acti vated, points towards the protecti ve efficacy of OMPs to keep the macrophages themselves away from the nox ious effects of 0 2-. Khanna and Sri vastava 20 also quoted that, as the monocytes mature into

macrophages, their capacity to produce o2- dec I ine precipitously and at the same time, spec ific acti vity of SOD increases . SOD acts as oxygen scavenging system, hence it may be possible that hi gher level of o2- generated by the macrophages require hi gher amount of SOD. The elevated level of 0 2- thus produced, although short li ved, is hi ghl y tox ic to pathogens21

. Low levels of SOD in the infected group may be because of the lesser amount of SOD required fo r lesser dismutati on of lesser re lease of o2- than normal macrophages, because of lower efficacy and lesser acti vati on of di seased macro phages to produce 0 2-. Enhanced generat ion of 0 2- in conjunction with hi gher production of NO in response to OMPs may pl ay a role in causing inacti vati on of organism by phagocytes because o2- has been repot1ed to interact wi th NO and their reacti on product perox ini trite (ONOO) not onl y cytotox ic to pathogens but is also

. fl d" 19 an 111 ammatory me tator .

The level of ac id phosphatase in the macrophages from immunized mi ce group was lower than the control and S.typhi Ty2 infected mice groups. This may be due to altered physiological activ ity of macrophages in immunized mice group lead ing to generati on of additi onal amount of ROS and RNJs, indicating thereby oxygen dependent kill ing process.

In order to find out the effect of ox idati ve path way in providing protection against Salmonellosis, the phagocytic uptake as well as intracellular killing of Sa lmonellae by macrophages in these groups was also studied. The enhanced uptake of the organism and their killing could be correlated to the enhanced production of oxygen and nitrogen radicals in the immunized group. Moreover, the organi sms were cleared not only fas ter in this group but the clearance was also effec ti ve upto 3 hr as compared to the 2 hr. in the infected group . These results are consistent with the reports given by Gonzalez et al. 22

, showing substantial killing of S.typhi by macrophages taken

362 INDI AN J EXP BIOL, APRIL 2000

from mice immun ized with OMPs than macrophages from na·ive mice. OMPs elic it T-cell response 22

' 6

,

which may confer protection by increasing the macrophage intracellular killing capacity, through the

release of lymphokines such as IFN-)P. IFN-y enhances release of TNF-a24

'25

, which in turn enhances macrophage capacity to release ROJs and RNis . Although, the enhanced microbial activity of macrophages from mice immuni zed with OMPs seems to be due to the enhanced production of reactive oxygen and reactive nitrogen intermediates, a more detailed study using inhibitors of ROS and ROI and on the cytokines is being carried out in our laboratory.

References I Acharya V L, Lowe C V, Thapa R, Guraba Charya V L,

Shreshta M B, Cadoz M & Schul z D. Prevention of typhoid fever in Nepal with the Vi capsu lar polysaccharide of Salmonella ryphi. N. Engl. J. Med., 317 ( 1987) I I 0 I.

2 lsibasi A, Navarrete V 0, f'aniajua J, Pelayo R, Gonzalez C R, Gorcia J A & Kumata J. Active protection of mice against Salmonella ryphi by immunization with strain-specific porins. Vaccine. 10(12) (1992), 811.

3 Matsui K & Arai T. Protective immunity induced by porins in experimental mouse Salmonellosis. Microbial. lmmunol. , 33(4) (1989) 699.

4 Galdiero M, De Lero D C, Donnarumma G, Marcatili A & Galdiero F. IL-l and IL-6 gene expression to human monocytes stimulated with Salmonella typhimurium po rin s. Jmmunol., 86 (1995) 612.

5 Rishi- Sharma P, Ganguli N K, Sharma B K, Sharma S & Sehgal R. Specific immunoglobuline response in mice immunized with porins and challenged with Salmonella typhi. Micro. lmmunol., 33(7) ( 1989) 519.

6 Rishi- Sharma P, Sharma B K, Sharma S & Ganguli N K. · Humoral and cell mediated immune response to porins of

Salmonella typhi. l ap. J. Exp. Med., 59 ( 1989) 73. 7 Tabaraie B, Sharma B K, Rishi P & Ganguli · K. Evaluation

of Salmonella porins as a broad-spectrum vaccine candidate. Micro. lmmunol. 38 (7) ( 1994) 553.

8 Fernandez-Beros M E, Gonzalez C & McLntosh M F C. Immune response to the iron deprivation induced proteins of Salmonella typhi in typhoid fever. Infect. lniiiiWI. , 57(4) ( 1989) 1271.

9 Markwell M A K, Suzanne M, Haas L L & Bi lber Tal bet N E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Analyt. Biochem., 87 ( 1978) 206.

10 Morrison DC & Leive L Fractions of LPS from E.coli 0111: B4 prepared by two extraction proced ures. J.Bioi.Chem., 250 (1975)2911.

II Laemmli U K Cleavage of structural properties during the assembly of the head of bacteriophage T4. Nature., 227 (1970) 68.

12 Kussi N, Nurminen M, Saxen H, Vottomen M & Makela PH. Immunisation with major OMPs in experimental Salmonellosis of mice. lnfec!.lmmun ., 25 (1979) 857.

13 Reed I J & Muench H A. A simple method of estimating fifty percent points. Am. J. Hyg., 27 ( 1938) 493.

14 Green L C, Wagner D A, Glogowski J, Skipper PI , Wishnok J S & Tannenbaum S R. Analysis of nitrate, nitrite and ( 15N) nitrate in biological fluid s. Annai.Biochem., 126 (1982) 13 1.

15 Kono Y. Generation of superoxide rad ical during auto­oxidation of hydroxylamine and an assay for superoxide dismutase. Arch. Biochem. Biophys., 186( I) ( 1978) 189.

16 Linhart K & Walter K. Phosphatase (phosphomono-esterases). In : Methods in Enzymology, edi ted by H C Bergmeyer, (Academic Press Inc. , New York and London) 1965 ,944.

17 Murray H W & Cohn Z A. Macrophage oxygen dependent antimicrobial activity. Ill. Enh<mced oxidative metabolism as an expression of macrophage activation. 1. Exp. Mecl., 152 ( 1980) 1596.

18 Adler H, Adler B, Peveri P, Werner E R, Wachter H, Peterhans E & Jungi T W. Differenti al regulat ion of inducible nitric oxide synthase produclion in bovine and caprinc macrophages. }.Infect Dis., 173 (1996) 971.

19 Urnezawa K, Akaike T, Jujii S, Suga M, Setoguchi K, Ozawa A & Maeda H. Induction of nitric ox ide synthesis and xanthin oxidase and their role in the antimicrobial mechan ism against Salmonella ryphimurium infection in mice. Infect. lmmun., 65 ( 1997) 2932.

20 Khanna M & Srivastava L M. Release of superoxide anion from activated mouse peritoneal macrophages. Indian . 1. txp. Med., 34 (1996) 468.

21 Fields PI , Swanson R V, Haidari s C J & Heffron F. Mutants of Salmonella typh imurium that can not survive within macrophages are avi rulent. Proc.Nati.Acad.Sci. U.S.A., 83 ( 1986) 5 189.

22 Gonzolez C R, lsi basi A, Ortiz-Navarrete V, Pan iagua J, Gorcia J A, Blanco F & Kumata J. Lymphocyte proliferative re ponse to OMPs isolated from Salmonella. Microbial. lmmwwl. , 37(10) (1993) 793.

23 Kauffmann S H E, Munk ME, Koga T, Stei nhoff V, Wand­Wurllenberger A, Gatrill A J, Flesch I & Schoel B. Effector T-cells in bacterial infections. In : Progress in Imm unology (VII ), edited by F Melchers et al (Springer-Verlag, Germany) 1989,963.

24 Denis M. Modulation of Mycobacterium lepramurium grown in murine macrophages: Beneticial effects of tumor necrosis factor alpha and granulocyte-macrophage colony stimulati ng factor. Infect. lmm1111. , 59 ( 1991) 705.

25 Bermudez L E M & Yong L S. Tumor necrosis factor, alone or in combination with IL-2 but not with IF -y, is associated with macrophage killing of Mycobacterium avium. 1. lmmunol., 140 (1988) 3006.