Mycobacterium tuberculosis: Mechanisms of Protection

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1 INFECTION AND IMMUNITY, Dec. 1978, p /78/ $02.00/0 Copyright 1978 Americn Society for Microbiology Vol. 22, No. 3 Printed in U.S.A. Induction of Enhnced Resistnce Aginst Encephlomyocrditis Virus Infection of Mice by Nonvible Mycobcterium tuberculosis: Mechnisms of Protection DONALD L. LODMELL* AND LARRY C. EWALT Ntionl Institute ofallergy nd Infectious Diseses, Rocky Mountin Lbortory, Hmilton, Montn Received for publiction 19 September 1978 Nonvible Mycobcterium tuberculosis strin Jmic suspended in oil-droplet emulsions ws used to enhnce resistnce of mice ginst encephlomyocrditis virus (EMCV). The mycobcteri-injected mice were significntly resistnt to 50,000 50% lethl doses of EMCV. Similr concentrtions of virus in plsm of norml nd mycobcteri-injected mice from 1 to 120 min fter injection of EMCV showed tht resistnce ws not result of rpid elimintion of virus from the circultion. Furthermore, survivl of viremic mice indicted protective mechnisms were opertive fter EMCV hd escped primry surveillnce. Resistnce did not pper to be ssocited with the mouse mjor histocomptibility gene complex. The spleen ws intimtely ssocited with protection, nd the thymus ws nonessentil for enhnced resistnce to EMCV. Protection ws significntly diminished by cyclophosphmide injected intrperitonelly from 3 dys before to the dy of virus chllenge. Finlly, silic given intrperitonelly 24 h before virus completely brogted resistnce of mycobcteri-injected mice to EMCV. These results suggest tht mcrophges functioning independently of T-lymphocytes re importnt effector cells in resistnce to EMCV of mice injected with nonvible mycobcteri. It is well known tht mycobcteri, in prticulr the BCG strin, increse resistnce to tumors (7, 11) nd to bcteril (18, 28), virl (13, 21, 24), nd prsitic infections (3-5). In previous study, we showed tht nonvible Mycobcterium tuberculosis strin Jmic cells ssocited with oil-droplet emulsions stimulted long-lsting resistnce of mice to encephlomyocrditis virus (EMCV) (12). This protection ws shown to be systemic in tht mice dministered mycobcteri either intrvenously (i.v.) or intrperitonelly (i.p.) were resistnt to EMCV by four different routes of chllenge. Furthermore, it ws determined tht resistnce ws not dependent on interferon. Pthogenesis studies suggested tht mice might be protected by mechnisms tht inhibited erly virl repliction nd spred of virus to the centrl nervous system. The present study ws designed to determine the mechnisms) of enhnced resistnce induced by nonvible M. tuberculosis ssocited with oil-droplet emulsions by selectively removing or temporrily inctivting vrious components of the immune system. The results suggest tht mcrophges re importnt effector cells in this system nd tht they function independently of T-lymphocytes. MATERIALS AND METHODS EMCV, virus titrtions, tissue culture nd medi, C57BL/10 mice, mycobcteri, nd preprtion of nonvible M. tuberculosis strin Jmic oil-droplet emulsions hve been reported in detil previously (12). Mouse strins other thn C57BL/10. Mice of strins other thn C57BL/10 (see Tble 4) were rised t the Rocky Mountin Lbortory nd kindly provided by Bruce Chesebro. Splenectomy. Spleens were removed under nesthesi fter i.p. injection of 0.9 mg of sodium pentobrbitl (Abbott Lbortories, North Chicgo, II.) per g of body weight for norml mice, nd 1.2 mg of sodium pentobrbitl per g of body weight for mice previously injected with nonvible M. tuberculosis. The incisions were closed with wound clips. Thymectomy. Thymuses were removed by spirtion under constnt vcuum pressure from precooked (4WC) neontl mice <48 h of ge. Incisions were closed with 3/s-inch (c cm) circle tper needles ttched to sterile, silicone-treted, silk-brided 6.0 thred (Americn Cynmid Compny, Perl River, N.Y.). Silic. Min-U-Sil no. 216 (Whittker, Clrk nd Dniels, Inc., New York, N.Y.) ws gift from Crl Lrson, University of Montn, Missoul. The Min- U-Sil ws suspended in Dulbecco phosphte-buffered sline with C2" nd Mg2+ (12), soniclly disrupted, nd injected i.p. t concentrtion of 200 mg/kg. Concentrtions higher thn 200 mg/kg were toxic for mice. 740

2 VOL. 22, 1978 Cyclophosphmide. Cytoxn (Med Johnson Lbortories, Evnsville, Ind.) ws suspended in Dulbecco phosphte-buffered sline nd injected i.p. t concentrtion of 300 mg/kg. Concentrtions higher thn 300 mg/kg were toxic for mice. RESULTS Effect of nonvible M. tuberculosis on resistnce of mice to high concentrtions of EMCV. Previous experiments showed tht 10 50% lethl doses (LD50) of EMCV killed 100% of norml mice but less thn 20% of mice previously injected with nonvible M. tuberculosis suspended in n oil emulsion (12). The mgnitude of this protection ws prticulrly obvious with very high concentrtions of virus; ll mycobcteri-injected mice survived intrmusculr chllenge with 5,000 LD50 of EMCV, nd sttisticlly significnt protection ws detected fter i.v. or intrmusculr chllenge with 50,000 LD5o of EMCV (Tble 1). Clernce of high concentrtions of EMCV from blood of norml mice nd mice injected with nonvible M. tuberculosis A possible explntion for resistnce of M. tuberculosis-injected mice to EMCV might involve the rpid removl of virus from the circultion. The dt in Tble 2 indicte this ws not the cse becuse virus titers in plsm of norml nd mycobcteri-injected mice did not differ 1, 3, nd 5 min fter the i.v. injection of 10,000 LD5o of EMCV. Furthermore, dditionl experiments showed tht virus titers in plsm were similr in norml nd mycobcteri-injected nimls 10, 60, nd 120 min fter the i.v. injection of similr mounts of virus (dt not shown). A difference ws detected, however, fter sufficient time hd elpsed for EMCV repliction to occur; 24 h fter injection of 10 LD5o of EMCV, concentrtions of virus rnging from 102 to 104 plque-forming units per ml of plsm were present in norml nimls, nd miniml, if ny, virus ws present in plsm of mice previously injected with M. tuberculosis (12). Viremi nd survivl of mice injected with nonvible M. tuberculosis. We hve observed nd it recently hs been reported tht, once circulting virus is present in norml EMCV-injected mice, they uniformly die (23). We utilized this observtion s mens to study enhnced resistnce becuse survivl of viremic mice would indicte tht protective mechnisms were opertive fter virus hd replicted. The dt in Tble 3 show tht 10 of 20 mice injected i.v. with 100 LD50 of EMCV survived; 7 of these 10 survivors were viremic t 72 h, time t which the initil virus inoculum would hve been undetectble unless there hd been virl repliction (12). Additionl evidence for virl repliction ws shown in the five survivors who ENHANCED RESISTANCE AGAINST EMCV INFECTION 741 TABLE 1. Effect of nonvible M. tuberculosis on resistnce of mice to high concentrtions of EMCV LD2 of Survivors/totl (%) EMCV injected i.v.b P vlues i.m./ P vlue 50 10/10 (100) /10 (100) /10 (100) /10 (100) ,000 7/10 (70) /10 (100) ,000 5/10 (50) /10 (50) Control" 0/10 (0) 0/10 (0) Mice inoculted i.v. with 500,ug of nonvible M. tuberculosis in oil-droplet emulsions were chllenged 5 weeks lter with different concentrtions of EMCV. broute of c EMCV injection. i.m., intrmusculr. Treted versus untreted controls. According to Minlnd (14) the levels of significnce re <0.025 significnt nd - < = highly significnt. d Norml mice were injected with the stndrd 10-LD50 chllenge dose of EMCV. TABLE 2. Presence ofemcv in plsm of norml mice nd mice injected with nonvible M. tuberculosis" Time Plque-forming units per 0.2 ml of plsm (log0o) (min) in: post-injection of M. tuberculosis-in- Norml mice EMCV jected mice 1 5.7, 5.7, 5.4 (5.6)b 5.7, 5.9, 6.0 (5.9) 3 5.9, 5.4, 5.4 (5.6) 5.7, 5.7, 6.0 (5.8) 5 5.4, 5.3, 5.7 (5.5) 5.4, 5.7, 5.6 (5.6) Three mice were injected i.v. 4 weeks previously with nonvible M. tuberculosis in oil-droplet emulsions, nd three norml mice were injected i.v. with 10,000 LD5o of EMCV. At vrious intervls therefter, blood ws collected from the infrorbitl sinus of ech mouse, nd the plsm ws titrted for EMCV. b Averge titer. TABLE 3. Viremi nd survivl of mice injected with nonvible M. tuberculosis" Survivors (mouse Viremi t: no.) 24h 72h 'Twenty mice inoculted i.v. with 500 ug of nonvible M. tuberculosis in oil-droplet emulsions were injected i.v. 4 weeks lter with 100 LD50 of EMCV. At 24 nd 72 h post-emcv injection, 0.1 ml of blood ws removed from the infrorbitl plexus, nd the plsm ws checked for virus on ML cells. Virus in positive cultures ws confirmed s EMCV by neutrliztion tests.

3 742 LODMELL AND EWALT were not viremic 24 h fter virus injection, but were viremic 48 h lter (72 h postinjection). The decresed survivl rte (50%) of mycobcteri-injected mice ws probbly due to the stress of the bleeding schedule. Protection ginst EMCV in different mouse strins injected with nonvible M. tuberculosis. To determine whether resistnce to EMCV ws influenced by the mouse mjor histocomptibility gene complex, severl strins of mice were injected with oil emulsions of nonvible M. tuberculosis. It ws found tht ll mycobcteri-injected strins, regrdless of H-2 genotype, were resistnt to EMCV (Tble 4). Effect of splenectomy before or fter injection of nonvible M. tuberculosis on resistnce of mice to EMCV. Mice injected with nonvible M. tuberculosis in oil-droplet suspensions hve prominent heptosplenomegly (12). To determine the importnce of the spleen in resistnce to EMCV, spleens were removed from mice either before or fter injection of the oil emulsion. Splenectomy performed before injection of M. tuberculosis hd no effect on resistnce of nimls to EMCV (Tble 5). In contrst, splenectomy performed fter injection of M. tuberculosis mrkedly reduced resistnce of mice to virus chllenge. This reduced resistnce ws especilly pprent if EMCV ws dministered i.v.; 20% of the mice survived, s compred with 85% of the mycobcteri-injected mice with spleens. TABLE 4. Protection ginst EMCV in different mouse strins injected with nonvible M. tuberculosis Survivors/totl (%) Mouse strins H-2 genotype M. tuberculosis Norml injected C57BL/10 b/b 7/8 (87) 2/8 (25) BALB.B b/b 6/8 (75) 0/8) (0) (C57BL/10 x b/b 8/8 (100) 5/8 (62) BALB.B)F1 (C57BL/10 x b/b 8/8 (100) 2/8 (25) A.BY)F1 A.BY b/b 4/8 (50) 0/8 (0) (B1O.D2 x d/d 7/8 (87) 2/8 (25) BALB/c)F1 (B1O.A x b/ 7/8 (87) 2/8 (25) A.BY)F1 (B1O.A x / 8/8 (100) 0/8 (0) A)F1 Eight mice of ech group were inoculted i.v. with 500 jig of nonvible M. tuberculosis in oil-droplet emulsions. Four weeks lter the mice injected with M. tuberculosis nd 8 norml mice of ech group were chllenged i.v. with 10 LD5o of EMCV. Thirty dys fter injection of virus the experiment ws terminted. INFECT. IMMUN. TABLE 5. Effect of splenectomy before or fter injection of nonvible M. tuberculosis on resistnce of mice to EMCV Survivors/totl (%) Route of Splenecto- Splenecto- Mice with spleens EMCV mized be- mized fter injec- fore M. tu- M. tubercu- M. tubercution berculosis losis injec- losi Norml injection tion iv. 18/20 (90) 4/20 (20)b 17/20 (85) 0/20 (0) i.m. 19/20 (95) 13/20 (65) 18/20 (90) 0/20 (0) Mice were splenectomized either 1 week before or 3 weeks fter the i.v. injection of 500,ug of nonvible M. tuberculosis in oil-droplet emulsions. Four weeks fter injection of the M. tuberculosis, mice were chllenged either i.v. or i.m. with 10 LD50 of EMCV. Shm splenectomy before or fter injection of M. tuberculosis did not ffect the resistnce of mice to EMCV. Norml mice with or without spleens were eqully susceptible to EMCV. 'Treted versus untreted injected with M. tuberculosis. P = < highly significnt (14). Protection ginst EMCV in neontlly thymectomized mice injected with nonvible M. tuberculosis. Becuse T-lymphocytes re known to be importnt for protection ginst vrious virus infections, we questioned whether thymectomized mice injected with M. tuberculosis would be protected ginst EMCV. The dt in Tble 6 show tht neontl thymectomy did not limit the protective effect of M. tuberculosis; regrdless of the route ofvirus chllenge, 93% of the thymectomized mice survived. Mcroscopic exmintion of survivors showed >80% without thymus; histologicl exmintion of thymic res from four mice without evidence of thymus indicted no evidence of thymus remnnts. Furthermore, in comprison with norml nimls, low or bsent hemolysin titers were detected in thymectomized mice 7 dys fter the i.p. injection of 10% suspension of sheep erythrocytes. Preliminry dt in thymic nude mice corroborte the thymectomy results in tht nude mice lso were protected ginst EMCV by nonvible M. tuberculosis. Effect of silic on resistnce to EMCV of mice injected with nonvible M. tuberculosis. The possibility tht mcrophges medite M. tuberculosis-enhnced ctivity ws investigted by the i.p. injection of silic, known mcrophge inctivtor. The results in Tble 7 show tht silic completely brogted the protective effect of M. tuberculosis. The totl number of peritonel exudte cells ws similr in silic-treted nd untreted mycobcteri-injected mice. However, differentil cell counts of silic-treted mice indicted ninefold decrese in mcrophges nd lymphoid cells nd twofold increse in neutrophils.

4 VOL. 22, 1978 TABLE 6. Protection ginst EMCV in neontlly thymectomized mice injected with nonvible M. tuberculosis Survivors/totl (%) Group i.p. injection im. injection M. tuberculosis + 34/38 (90) 24/26 (93) thymus M. tuberculosis - 26/28 (93) 26/28 (93) thymus Norml + thymus 0/10 (0) 0/10 (0) Norml - thymus 1/10 (10) 0/10 (0) Neontl thymectomies of precooled (40C) mice <48 h old were done by spirtion. The treted nimls were wened t 30 dys of ge, fed utoclved food, nd mintined on wter contining 10 mg of neomycin nd 100,000 U of polymyxin B sulfte per liter. Approximtely 8 weeks fter wening, mice were injected i.v. with 500 itg of nonvible M. tuberculosis in oil-droplet emulsions nd chllenged with 10 LD50 of EMCV 4 weeks lter. Nonthymectomized littermtes were held seprtely so tht pproprite gemted controls would be vilble for ll experiments. All thymectomized survivors were mcroscopiclly exmined for thymus (>80% were free of thymus tissue), nd thymic res of four survivors without mcroscopic evidence of thymus were exmined histologiclly by W. J. Hdlow for evidence of thymic remnnts-none ws detected. TABLE 7. Effect of silic on resistnce to EMCV of mice injected with nonvible M. tuberculosis Survivors/totl Group i.p. i.v. i.m. None M. tuberculosis + 0/20 0/20 0/20 20/20 silic M. tuberculosis - 18/20 20/20 20/20 silic Norml + silic 0/20 0/20 0/20 20/20 Norml - silic 0/20 0/20 0/20 Mice were injected i.v. with 500 yg of nonvible M. tuberculosis in oil-droplet emulsions. Four weeks lter they were injected i.p. with 200 mg of silic nd then chllenged with 10 LD50 of EMCV 24 h lter. i.m., intrmusculr. Effect of cyclophosphmide on resistnce to EMCV of mice injected with nonvible M. tuberculosis The effect of cyclophosphmide on resistnce of M. tuberculosis-injected nimls to EMCV ws evluted by vrying the time t which the immunosuppressnt ws injected in reltion to EMCV (Tble 8). Cyclophosphmide mrkedly decresed, but did not completely brogte resistnce of mice if it ws given from 3 dys before to the dy of EMCV injection. Administrtion of the drug 2 dys before EMCV resulted in optiml brogtion of resistnce (27% survivors); t this time there ws ENHANCED RESISTANCE AGAINST EMCV INFECTION 743 TABLE 8. Effect of cyclophosphmide on resistnce to EMCV of mice injected with nonvible M. tuberculosis Survivors/totl Mouse group (%) p Cytoxn injected -3 dysc 20/40 (50) dys 11/40 (27) dy 19/40 (48) /40 (60) dy 33/40 (82) +3 dys 32/40 (80) Controls M. tuberculosis + cy- 40/40 (100) toxn (no EMCV) M. tuberculosis + EMCV 38/40 (95) (no cytoxn) Norml mice + EMCV 1/40 (2.5) (no cytoxn) Mice were inoculted i.p. with 500 Ig of nonvible M. tuberculosis in oil-droplet emulsions. During week 5 post-m. tuberculosis injection, mice were inoculted i.p. with 300 mg of cyclophosphmide per kg nd 10 LD50 of EMCV. b Treted versus M. tuberculosis mice injected only with EMCV. According to Minlnd (14) the levels of significnce re <0.025 = significnt, <0.005 = highly significnt. Except s noted, ll other probbility vlues re > ' Time of cytoxn injection in reltion to EMCV injection. threefold decrese in the totl number of peritonel exudte cells fter the i.p. injection of cyclophosphmide in treted s compred with untreted mycobcteri-injected mice. Differentil cell counts, however, indicted no significnt difference in the percentge of mcrophges, lymphoid cells, nd neutrophils. DISCUSSION The present study ws initited to define the in vivo mechnism(s) responsible for enhnced resistnce to EMCV of mice injected with nonvible M. tuberculosis. It ws determined tht mycobcteri-injected mice were significntly resistnt to 50,000 LD50 of EMCV. Furthermore, similr concentrtions of virus in plsm of norml nd mycobcteri-injected mice from 1 to 120 min fter i.v. injection of EMCV indicted tht possible mechnism of resistnce ws not due to rpid elimintion of virus from the circultion. In corrobortion of these results, it ws shown tht protective mechnism ws opertive fter EMCV hd escped primry surveillnce; 7 of 10 mice tht were viremic 72 h fter virus injection survived until termintion of their respective experiments. Supportive evidence tht viremi hd indeed developed in these mice ws shown by the 5 of 10 survivors

5 744 LODMELL AND EWALT who were not viremic 24 h fter injection of EMCV, but were viremic 48 h lter (72 h postinjection). Severl strins of mice with different H-2 genotypes were protected eqully well ginst EMCV by the oil emulsion preprtion of nonvible M. tuberculosis. This result indicted tht protection pprently ws not ssocited with the mouse mjor histocomptibility gene complex. These results re in contrst to those of Civil nd Mhmoud who showed tht vible BCG induced nonspecific resistnce to the multicellulr helminth prsite Schistosom mnsoni in only certin strins of inbred mice (3); BALB/c, C3H/He, nd CBA mice developed no or miniml nonspecific protection. Studies re in progress to determine whether nonresponder strins of mice to BCG similr to those used by Civil nd Mhmoud, nd other strins we hve not previously tested, differ in their resistnce to EMCV nd other viruses fter injection with oil emulsion preprtions of nonvible M. tuberculosis. Becuse of our previous detection of heptosplenomegly in mycobcteri-injected mice (12), the importnce of the spleen in enhnced resistnce to EMCV ws evluted. Interestingly, if the spleen ws removed fter injection of mycobcteri, resistnce of mice to EMCV ws mrkedly decresed, indicting tht resistnce ws intimtely ssocited with splenic function. If the spleen ws removed before injection of mycobcteri, however, ntivirl resistnce ws not ffected. It would pper, therefore, tht bsence of the spleen led to n ltertion in the norml distribution of M. tuberculosis fter i.v. injection which resulted in locliztion of mycobcteri in res tht were entirely sufficient to enhnce resistnce to lethl chllenge of EMCV. The spleen, therefore, ws not the only orgn which hrbored effector cells in this system. The thymus ws shown to be nonessentil in estblishment of enhnced ntivirl resistnce INFECT. IMMUN. by nonvible M. tuberculosis, s it hs previously been shown to be unimportnt in thymectomized mice injected with the immunopotentitor pyrn nd then chllenged with herpes simplex virus type 2 (17). In our studies, neontl mice thymectomized t <48 h of ge were s resistnt to EMCV s their littermtes tht possessed thymus or tht hd been shm thymectomized. Supportive evidence tht the T-lymphocyte ws not importnt ws shown by our preliminry dt in which resistnce to EMCV ws estblished in thymic nude mice injected with nonvible M. tuberculosis-oil-droplet emulsions. Thus, t lest one mechnism of resistnce to EMCV fter injection of mycobcteri ppers to involve component of the immunologicl system other thn the T-lymphocyte. The most likely cndidte ppers to be the mcrophge. The importnce of the mcrophge in our studies ws shown by the complete brogtion of resistnce to EMCV fter the i.p. injection of silic, which is known to be toxic for mcrophges (1, 6, 15). Supportive evidence implicting the mcrophge ws shown by the ninefold decrese in the percentge of mcrophges nd lymphoid cells fter silic tretment. Furthermore, threefold decrese in the totl number of lymphoid cells nd mcrophges in the peritonel cvity ws ssocited with decresed resistnce to EMCV fter i.p. injection of cyclophosphmide, known T-lymphocyte (16, 19, 22), B-lymphocyte (22, 25), nd monocyte-mcrophge (2, 8, 26) immunosuppressnt. Becuse our previous dt hve shown tht enhnced resistnce of mycobcteri-injected mice to EMCV is not ssocited with nti-emcv ntibody (12), nd therefore B-lymphocytes by ssocition, nd experiments reported here hve indicted nonessentil role for T-lymphocytes in resistnce, it ppers tht the reduction in mcrophge numbers fter tretment with silic nd cyclophosphmide ws ssocited with decresed resistnce to EMCV. Becuse nonvible M. tuberculosis ws effective in thymectomized nd nude mice, it would pper tht mcrophges cted independently of T-lymphocytes in enhncing resistnce to EMCV. This finding of mcrophge ctivtion in the bsence of T-lymphocytes is not novel in tht others hve shown stimultion of phgocytic ctivity in T-lymphocyte-deprived mice (27), n increse in resistnce to Listeri infection in nude mice (20), nd genertion of cytotoxic ctivity to tumor cells in T-lymphocytedeprived nd nude mice (9). Tht ctivted mcrophges were present in our mice ws highly probble becuse Kelly hs identified these cells in nimls injected with oil-droplet emulsions of M. bovis strin BCG cell wlls (10). Studies re in progress to identify the peritonel exudte cells from mice injected with nonvible mycobcteri tht inhibit EMCV repliction in mouse embryo cells (dt not shown) nd to determine the mens by which this inhibition occurs. ACKNOWLEDGMENTS We thnk Shirley Houston for typing the mnuscript, John Portis for the differentil cell counts, nd Ry Holt for ssistnce with the experimentl nimls. LITERATURE CITED 1. Allison, A. C., J. S. Hrington, nd M. Birbeck

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