Listeria Monocytogenes and Tuberculosis Mycobacterium Intracellular Pathogens Involved in Controlling Infections with the Recept

The Journal of Immunology

The Lymphotoxin ␤ Receptor Is Critically Involved in Controlling Infections with the Intracellular Pathogens Mycobacterium tuberculosis and Listeria monocytogenes1

Stefan Ehlers,2* Christoph Ho¨lscher,* Stefanie Scheu,† Christine Tertilt,† Thomas Hehlgans,‡ Johanna Suwinski,* Robert Endres,† and Klaus Pfeffer3†

Containment of intracellularly viable microorganisms requires an intricate cooperation between macrophages and T cells, the most potent mediators known to date being IFN-␥ and TNF. To identify novel mechanisms involved in combating intracellular infections, experiments were performed in mice with selective defects in the lymphotoxin (LT)/LT␤R pathway. When mice deficient in LT␣ or LT␤ were challenged intranasally with Mycobacterium tuberculosis, they showed a significant increase in bacterial loads in lungs and livers compared with wild-type mice, suggesting a role for LT␣␤ heterotrimers in resistance to ␣ ␤ ␤ infection. Indeed, mice deficient in the receptor for LT 1 2 heterotrimers (LT R-knockout (KO) mice) also had significantly higher numbers of M. tuberculosis in infected lungs and exhibited widespread pulmonary necrosis already by day 35 after intranasal infection. Furthermore, LT␤R-KO mice were dramatically more susceptible than wild-type mice to i.p. infection with Listeria monocytogenes. Compared with wild-type mice, LT␤R-KO mice had similar transcript levels of TNF and IFN-␥ and recruited similar numbers of CD3؉ T cells inside granulomatous lesions in M. tuberculosis-infected lungs. Flow cytometry revealed that the LT␤R is expressed on pulmonary macrophages obtained after digestion of M. tuberculosis-infected lungs. LT␤R-KO mice showed delayed expression of inducible NO synthase protein in granuloma macrophages, implicating deficient macrophage activation as the most likely cause for enhanced susceptibility of these mice to intracellular infections. Since LIGHT-KO mice proved ␣ ␤ to be equally resistant to M. tuberculosis infection as wild-type mice, these data demonstrate that signaling of LT 1 2 heterotrimers via the LT␤R is an essential prerequisite for containment of intracellular pathogens. The Journal of Immunology, 2003, 170: 5210–5218.

ycobacterium tuberculosis currently is the number one counter pathogens capable of surviving intracellularly need to be bacterial killer in the world. Eight to 10 million people reprogrammed by specific T cells, essentially via TNF and IFN-␥, M worldwide newly develop tuberculosis every year and to become fully competent in antibacterial functions, a process in for at least one-third of these patients the disease is lethal (1). which NO and superoxide generation act both as signaling and as These numbers are likely to rise with the emergence of multidrug- effector molecules (7). The concept of the activated macrophage resistant strains of M. tuberculosis and the increase of coinfection was originally established in the murine model of Listeria mono- with HIV. It is thus imperative to define the mechanisms that en- cytogenes infection which has since become a standard experimen- able the host to effectively contain M. tuberculosis infection to tal system for identifying the roles of newly discovered cells and enlist them in novel therapeutic strategies. mediators in response to intracellular pathogens (8, 9). Animal models have been instrumental in elucidating the mech- In addition, antibacterially inefficient host cells may need to be anisms of both protection and pathology in response to M. tuber- lysed and new effector cells, such as granulocytes and macro- culosis infection (2). In particular, the use of gene-targeted mice phages, must be recruited and activated to engulf and destroy the has provided incontrovertible evidence that an efficient coopera- released microorganisms (10Ð12). In the case of persistent myco- tion between T cells and macrophages is critical for control of bacteria, the close interaction of T cells and macrophages is facilitated infection (3Ð6). This is primarily because macrophages which en- in an organized microenvironment called granulomas, which at the same time serves as a physical barrier to dissemination of bacteria *Division of Molecular Infection Biology, Research Center Borstel, Borstel, Ger- released from incompetent macrophages (13). Therefore, granulomas many; †Institute of Medical Microbiology, Immunology and Hygiene, Technical Uni- are highly dynamic structures which are composed of effector cells versity, Munich, Germany; and ‡Institute of Pathology, University of Regensburg, that are chronically recruited and activated by stimuli such as chemo- Regensburg, Germany kines and IFN-␥- and TNF-mediated processes (14, 15). Received for publication August 8, 2002. Accepted for publication March 10, 2003. Lymphotoxin (LT)4 ␣,LT␤, and the recently identified TNF The costs of publication of this article were defrayed in part by the payment of page family member LIGHT (homologous to lymphotoxins, exhibits in- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ducible expression, and competes with HSV glycoprotein D for 1 This work was supported in part by Deutsche Forschungsgemeinschaft Grants SFB HVEM, a receptor expressed by T lymphocytes) along with the 391-B3, SFB 576-A6, and Pf 259/2-5/6 (to K.P.), Deutsche Forschungsgemeinschaft Grant SFB 367-C9 (to S.E.), and a research grant (Host Defense against Infections) from the University of Lu¬beck (to S.E.). 2 Address correspondence and reprint requests to Dr. Stefan Ehlers, Division of Mo- 4 Abbreviations used in this paper: LT, lymphotoxin; HVEM, herpesvirus entry me- lecular Infection Biology, Research Center Borstel, Parkallee 22, D-23845 Borstel, diator; LIGHT, ligand homologous to LTs, exhibits inducible expression, competes Germany. E-mail address: [email protected] with HSV glycoprotein D for HVEM, a receptor expressed by T lymphocytes; iNOS, 3 Current address: Institute of Medical Microbiology and Virology, Universita¬tsstrasse 1, inducible NO synthase; KO, knockout; BM, bone marrow; IKK, inhibitor of ␬B D-40225 Du¬sseldorf, Germany. kinase.

prototype cytokine TNF may be defined as a core group of cyto- dilutions of the cultures on Middlebrook 7H10 agar plates followed by kines clustered within the growing TNF superfamily (16Ð19). The incubation at 37¡C. Before infection of experimental animals, stock solu- specific cell surface receptors for the ligands of the TNF core fam- tions of M. tuberculosis were briefly sonicated and diluted in PBS. Pul- ␤ monary infection was performed by intranasal instillation. Mice were anes- ily are TNFRp55, TNFRp75, LT R and herpesvirus entry medi- thetized by i.p. injection of 1.25% ketamine (Bayer, Leverkusen, Germany) ator (HVEM) that reveal overlapping, but distinct ligand interac- and 0.025% Rompun (WDT, Garbsen, Germany) to fully suppress swal- ␮ tions (16, 20, 21). TNF3 binds to the TNFRp55 and TNFRp75, lowing reflexes. Intranasal instillation was performed by applying 20 lof 4 whereas LT␣ engages the TNFRp55, TNFRp75, and HVEM as a suspension containing 1Ð2.5 ϫ 10 CFU of M. tuberculosis/ml to the 3 nares of anesthetized mice. Actual inoculum size was determined 24 h after homotrimer (16, 20, 22). In combination with the membrane- infection by determining the bacterial load in the lungs of infected mice ␤ ␣ ␣ ␤ ␤ bound LT ,LT binds as the LT 1 2 heterotrimer to the LT R and is indicated in the figure legends. Bacterial loads in infected organs ␣ (16, 21, 23, 24). TNF3,LT 3 in concert with the TNFRp55, were were evaluated at different time points after infection with M. tuberculosis shown to be of critical importance for the host defense against to follow the course of infection. Lungs and livers of sacrificed animals intracellularly replicating bacterial pathogens and for the forma- (four to five mice per group) were removed and weighed aseptically and homogenized in distilled water. Ten-fold serial dilutions of organ homog- tion and maintenance of fully differentiated granulomas (25Ð31). enates were plated in duplicates onto Middlebrook 7H10 agar plates con- In contrast, the detailed role of the LT␤R and its cognate ligands taining 10% OADC and incubated at 37¡C for 3 wk. Colonies on plates in antimicrobial resistance and granuloma formation has remained were enumerated and results expressed as log10 CFU per organ. unexplored. Infections with Listeria monocytogenes were performed as described ␤ previously (26). Briefly, overnight cultures of L. monocytogenes (ATCC To ascertain the functional relevance of the LT R for the im- strain 43251) were grown in brain-heart infusion (Difco), adjusted to an mune defense against intracellular microorganisms, we investi- OD of 0.75, and serially diluted in medium. Titrated numbers of L. mono- gated the course of infection in two infection models (M. tuber- cytogenes were i.p. inoculated in a volume of 350 ␮l into the mice. The culosis and L. monocytogenes) in mice deficient for the LT␤R, dose of bacteria was checked by plating 10-␮l aliquots of a serial 10-fold LT␣,LT␤, or LIGHT. Collectively, the results of this comprehen- dilution on Columbia blood agar plates and counting the CFU after over- ␣ ␤ ␤ night incubation at 37¡C. Livers and spleens were removed 2 days after sive study indicate that LT 1 2 signaling via the LT Ronmac- infection, organ homogenates prepared, and bacterial counts determined as rophages is essential for controlling the infection with both above. pathogens. Histology and immunohistology Materials and Methods One lung lobe per mouse was fixed in 4% Formalin-PBS, set in paraffin Mice blocks, and sectioned (2Ð3 ␮m). Histology was performed using standard protocols for H&E staining and for acid-fast staining. For immunohistol- LT␤R-knockout (KO) (21), TNFRp55-KO (25), and LT␣-KO (32) were ogy, tissue sections were deparaffinized, placed in 10 mM sodium citrate ␤ N5 backcross generations to C57BL/6; LT -KO (23) were N10 backcrosses buffer (pH 6.0), and pressure-cooked for exactly 1.5 min. After blocking to C57BL/6. LIGHT-KO (22) were N backcrosses to C57BL/6 and control 3 for 20 min in 1% H2O2 solution, slides were incubated with appropriately mice in experiments involving LIGHT-KO mice were N3 backcross het- diluted polyclonal rabbit anti-mouse inducible NO synthase (iNOS; Bi- erozygous mice. Wild-type mice were C57BL/6 mice purchased from omol, Hamburg, Germany) in TBS/10% FCS for 30 min in a humid cham- Charles River Breeding Laboratories (Sulzfeld, Germany). LT␣-KO and ber. Appropriately diluted goat anti-rabbit IgG (bridging Ab; Dianova, C57BL/6 mice congenic for the CD45.1 Ag were purchased from The Hamburg, Germany) and diluted rabbit anti-goat IgG-peroxidase conjugate Jackson Laboratory (Bar Harbor, ME). All other mice were bred at the GSF (tertiary Ab) (Dianova) were used in sequential incubations of 30 min each. National Research Center for Environment and Health (Martinsried, Ger- For detection of T cells, anti-CD3 mAb (clone CD3.12; Biotrend, Cologne, many) under specific pathogen-free conditions and were housed in isolator Germany) was used as the primary Ab, diluted rabbit anti-rat IgG (Di- cages under barrier conditions at the BSL 3 facility at the Research Center anova) as the secondary, and goat anti-rabbit IgG peroxidase as the tertiary Borstel (Borstel, Germany). All mice used were between 8 and 16 wk old. Ab. Development was performed using 3,3Ј-diaminobenzidine (Sigma- In any given experiment, mice were matched for age and sex. All exper- Aldrich, Deisenhofen, Germany) and urea superoxide (Sigma-Aldrich), iments performed were in accordance with the German Animal Protection and hemalum was used to counterstain the slides. Law and were approved by the Animal Research Ethics Boards of the regional Ministries of Environment, Nature and Forestation (Kiel, Ger- Flow cytometry many) and the government of Upper Bavaria (Munich, Germany). For flow cytometric determination of BM chimerism, Abs directed against Bone marrow chimeras CD3 (FITC, clone 500A2; BD Biosciences), B220 (FITC, clone RA3-6B2; BD Biosciences), Mac3 (FITC, clone M3/84; BD Biosciences), CD45.1 Bone marrow (BM) cells were harvested by flushing femurs and tibias of (biotin, clone A20; BD Biosciences), and CyChrome-conjugated strepta- donor mice with cold RPMI 1640 medium (Seromed, Biochrom KG, Ber- vidin (BD Biosciences) were used. lin, Germany) supplemented with 10% heat-inactivated FCS (Seromed), 2 For detection of LT␤R expression after infection with M. tuberculosis, ␮ mM L-glutamine (Seromed) 50 M 2-ME (Life Technologies, Eggenstein, noninfected and infected mice were anesthetized and injected i.p. with 150 ␮ Germany), 50 g/ml streptomycin (Seromed), and 100 U/ml penicillin. U heparin (Ratiopharm, Ulm, Germany). Lungs were perfused through the Recipient wild-type C57BL/6 mice congenic for the CD45.1 (Ly5.1) Ag 137 heart with warm PBS. Once lungs appeared white, they were removed and were irradiated with 10 Gy using a Cs irradiator (Buchler, Braun- sectioned. Dissected lung tissue was then incubated in collagenase A (0.7 ϫ 6 schweig, Germany). After washing and counting, 5 10 BM cells were mg/ml; Roche Diagnostics, Mannheim, Germany) and DNase (30 ␮g/ml; injected i.v. into recipients. The degree of chimerism was determined in Sigma-Aldrich) at 37¡C for 2 h. Digested lung tissue was gently disrupted PBL (B220ϩ B cells and CD3ϩ T cells) 8 wk after reconstitution and in ϩ by sequential passage through a 23-gauge cannula, a metal sieve, and a alveolar Mac3 macrophages 21 days postinfection with M. tuberculosis. 70-␮m pore size nylon cell strainer. Recovered lung cells were washed and For bronchoalveolar lavage, M. tuberculosis-infected mice were anesthe- incubated with FcR-blocking Ab (clone 2.4G2; BD Biosciences). Cells tized. Cells were collected by performing five intratracheal lavages with were then incubated for 30 min with either anti-LT␤R-mAb (clone 5G11b, 0.8 ml of cold PBS containing 0.5 mM EDTA. Flow cytometric analysis of rat IgG2a; T. Hehlgans et al., manuscript in preparation) or isotype control the differential CD45 Ag expression on donor and recipient BM-derived (rat IgG2a, clone A110-2; BD Biosciences). Cells were washed again and cells was performed. Eighty-nine percent of peripheral blood T cells and incubated with biotinylated isotype-specific anti-rat IgG2a mAb (clone Ͼ ϩ 95% peripheral blood B cells and alveolar Mac3 macrophages were RG7/1.30; BD Biosciences). After subsequent washing, cells were stained found to be donor derived. with streptavidin-CyChrome (BD Biosciences) and FITC-coupled anti- Bacterial infections Mac3 (clone M3/84; BD Biosciences). CyChrome fluorescence intensity was analyzed after gating on macrophages identified by side scatter profile M. tuberculosis (H37Rv) was grown in Middlebrook 7H9 broth (Difco, and high expression of Mac3. Detroit, MI) supplemented with Middlebrook OADC enrichment medium For detection of LT␤R expression on peritoneal macrophages, thiogly- (BD Biosciences, Heidelberg, Germany), 0.002% glycerol, and 0.05% colate-induced peritoneal exudate cells were recovered 4 days after i.p. Tween 80. Midlog phase cultures were harvested, aliquoted, and frozen at injection of 1 ml of thioglycolate by peritoneal lavage, washed, and incu- Ϫ80¡C. After thawing, viable cell counts were determined by plating serial bated with FcR-blocking Ab (clone 2.4G2; BD Biosciences). Cells were 5212 LT␤R AND INTRACELLULAR BACTERIAL INFECTIONS then incubated for 30 min with either anti-LT␤R-mAb (clone 5G11b, rat IgG2a) or buffer alone. Cells were washed again and stained with allophy- cocyanin-coupled anti-Mac1␣ (clone M1/70; BD Biosciences) and FITC- coupled isotype-specific anti-rat IgG1/2a mAb (clone G28-5; BD Bio- sciences). FITC fluorescence intensity was analyzed after gating on macrophages identified by side scatter profile and high expression of Mac1␣ (CD11b). RT-PCR RT-PCR was performed essentially as described previously (33). In brief, weighed lung tissue samples were homogenized in 5 ml of 4 M guani- dinium-isothiocynanate buffer and diluted to obtain equalized amounts of milligrams of lung per milliliter of buffer. Total RNA was purified using an RNA isolation kit (High Pure RNA Tissue kit; Roche Diagnostics), and 1 ␮g RNA was transcribed into cDNA using Moloney murine leukemia virus-RT (Life Technologies) and oligo(dT) (12Ð18 mer; Sigma-Aldrich) as a primer. PCR was performed on a Light Cycler (Roche Diagnostics) using the proprietary Light-Cycler-DNA Master SYBR Green I kit (Roche Di- ␤ Ј agnostics) and the following primer sets: 2-microglobulin: sense, 5 - TGACCGGCTTGTATGCTATC-3Ј; antisense, 5Ј-CAGTGTGAGCCAG GATATAG-3Ј; IFN-␥: sense, 5Ј-AACGCTACACACTGCATCTTGG-3Ј; antisense, 5Ј-GACTTCAAAGAGTCTGAGG-3Ј; TNF: sense, 5Ј-GATCT CAAAGACAACCAACTAGTG-3Ј; antisense, 5Ј-CTCCAGCTGGAAG ACTCCTCCCAG-3Ј. After amplification (denaturation at 94¡C for 1 s, annealing at 60¡C for FIGURE 1. Bacterial load in the lungs and livers of LT␣-KO, LT␤-KO, 5 s, and extension at 72¡C for 5 s), melting curve analysis was performed to exclude the presence of confounding primer-dimers. Semiquantitative and wild-type mice 33 days after intranasal challenge with M. tuberculosis ␣ ␤ f comparisons of amplified products were made based on the crossing points H37Rv. LT -KO (A), LT -KO (B), and C57BL/6 ( in A and B) mice obtained for each sample compared with a serially diluted, arbitrarily se- were intranasally infected with ϳ500 CFU of M. tuberculosis H37Rv. lected standard cDNA run in parallel. In this way, arbitrary units could be Mice were sacrificed on day 33 after infection and lungs and livers were assigned to mRNA levels present in each sample. Units were normalized removed to determine CFU counts. Data shown are the means of five mice ␤ by calculating the mRNA ratios of cytokine/ 2-microglobulin for each Ϯ SD and are from one representative experiment of three performed, p Ͻ 0.05 ,ء .sample. giving nearly identical results Quantification of TNF by ELISA TNF levels in lung homogenates prepared with a proteinase inhibitor mix- ture (Roche Diagnostics) were analyzed in 3-fold serial dilutions by a sand- First, LT␤R-KO and congenic C57BL/6 mice were compared wich ELISA (OptEia; BD Biosciences) using a modified protocol. Before for their capacity to resist an intrapulmonary infection with M. adding biotinylated Abs, endogenous biotin was blocked by incubating samples with an avidin/biotin block reagent (Vector Laboratories, Peter- tuberculosis H37Rv. In three independent experiments, all borough, U.K.). After incubation with HRP coupled to avidin and devel- LT␤R-KO mice intranasally infected with ϳ500 CFU of H37Rv oping with tetramethylbenzidine substrate reagent, the absorbance was read succumbed to infection by day 45, whereas all C57BL/6 mice sur- on a microplate reader (Sunrise; Tecan, Ma¬nnedorf, Switzerland). Using a vived this infecting dose and showed no signs of illness until ter- test wavelength of 450 nm and a reference wavelength of 630 nm, samples mination of the experiment on day 60 (Fig. 2). were compared with appropriate recombinant cytokine standards. The detection limit for TNF was 5 pg/ml. To investigate the course of infection with M. tuberculosis in LT␤R-KO mice, mycobacterial replication in the lungs, livers, and Statistics spleens of mice infected intranasally with ϳ500 CFU of M. tu- Quantifiable data are expressed as the means of individual determinations berculosis was monitored over time. Although C57BL/6 mice and SD. Statistical analysis was performed using the Student’s t test. were capable of containing the growth of M. tuberculosis organ- Results isms in the lungs after day 14 postinfection, bacterial multiplication progressed almost uncurtailed in LT␤R-KO mice in this organ Pulmonary infection with M. tuberculosis is exacerbated in mice deficient for LT␣ or LT␤ LT␣ is biologically active both in its homotrimeric form and in ␤ ␣ ␤ ␣ ␤ heterotrimers along with LT (LT 1 2 or LT 2 1) (16, 21, 23, 24). To elucidate whether the homotrimeric and the heterotrimeric forms are involved in early resistance against pulmonary M. tuberculosis infection, bacterial multiplication was assessed in LT␣- and LT␤-KO mice 5 wk following intranasal challenge with ϳ500 CFU of strain H37Rv (Fig. 1). Both LT␣- and LT␤-KO mice had significantly higher CFU counts in their lungs and livers than infected congenic C57BL/6 mice at this time point, suggesting that not only LT␣ homotrimers, but also LT␣␤ heterotrimers participate in the early immune response to M. tuberculosis infection. LT␤R-KO mice are markedly more susceptible to pulmonary M. FIGURE 2. Survival of LT␤R-KO and wild-type mice following intra- tuberculosis infection nasal challenge with 500 CFU of M. tuberculosis H37Rv. LT␤R-KO (E) and C57BL/6 mice (-F-; six mice per group) were intranasally infected ␣ ␤ ␤ LT 1 2 heterotrimers bind to and signal via the LT R (16, 21). To with ϳ500 CFU of M. tuberculosis H37Rv. Mice were euthanized when directly test the hypothesis whether the LT␤R pathway is involved moribund. Data are from one experiment of three performed, with a total in early defense against M. tuberculosis, the outcome of infection of 12 of 12 infected LT␤R-KO mice dead by day 45 postinfection and 12 was examined in mice deficient for the LT␤R. of 12 infected C57BL/6 mice alive by day 60 postinfection. The Journal of Immunology 5213

(Fig. 3A). Simultaneously infected TNFRp55-KO mice were sim- were found to develop the typical circumscript granulomatous le- ilarly unable to restrict the growth of M. tuberculosis and suc- sions in their lungs (Fig. 4, A and C). Early during infection (day cumbed to infection around day 30, consistent with previously 21), LT␤R-KO mice showed less inflammatory infiltration in both published data (Fig. 3A). A reduction in the rate of M. tuberculosis interstitial and alveolar spaces, compared with infected wild-type growth was evident in the livers and spleens of immunocompetent mice. By day 35 after infection, widespread pulmonary necrosis mice at 3 wk after infection. In contrast, LT␤R-KO mice were developed in LT␤R-KO mice, eventually leading to destruction of permissive for M. tuberculosis growth in these organs up to the almost the entire lung (Fig. 4, B and D). No signs of necrosis were fifth week of infection when bacterial replication plateaued at a observed in the livers or spleens of infected LT␤R-KO mice (data comparatively high level of ϳ5 ϫ 106 CFU (Fig. 3, B and C). not shown). Whereas in C57BL/6 mice epithelioid cell differenti- However, most LT␤R-KO mice sacrificed on day 35 for analysis ation was well advanced on day 35 of infection and acid-fast ba- of bacterial loads already showed severe signs of infection (de- cilli were only sparsely detectable (Fig. 4, C and E), LT␤R-KO creased body weight and scruffy hair). Mice infected with lower mice showed extensive sheets of dead or degenerating cells sur- inocula (ϳ250 and 100 CFU of M. tuberculosis) also had signif- rounding necrotic tissue with fewer signs of epithelioid cell dif- icantly increased bacterial burdens in their lungs 5 wk after infec- ferentiation at this time point (Fig. 4D). Instead, acid-fast bacilli tion (data not shown). Thus, LT␤R-KO mice were invariably more were abundant within areas of tissue necrosis and numerous within susceptible than wild-type mice to intrapulmonary challenge with individual macrophages (Fig. 4F). M. tuberculosis. LT␤R-KO mice are highly susceptible to L. monocytogenes LT␤R-KO mice develop necrotic granulomatous lesions infection harboring abundant acid-fast bacilli It appeared possible that the observed essential role for LT␤Rin When tissue sections of mice infected intranasally with ϳ500 CFU resistance was due to the specific pathogen chosen (M. tuberculo- of M. tuberculosis were examined microscopically, C57BL/6 mice sis) or the specific route of infection (intranasal challenge). We therefore infected LT␤R-KO mice and wild-type mice with a fac- ultative intracellular pathogen, L. monocytogenes (strain ATCC 43251), and performed infection via the i.p. route (Fig. 5). Wild- ϳ ϫ 5 type mice had an LD50 of 3 10 CFU of L. monocytogenes.At this inoculum dose, LT␤R-KO mice all succumbed to infection. ␤ ϳ ϫ 3 The LD50 for LT R-KO mice was determined to be 3 10 CFU. Thus, LT␤R-KO mice were 100-fold more susceptible to infection. In additional experiments, Listeria CFU were determined in the livers and spleens of infected mice on days 2 and 4 after infection

with 1/10 LD50. At least 10- to 100-fold higher bacterial burdens were found in LT␤R-KO mice compared with wild-type mice (data not shown), indicating that in the absence of the LT␤R, the replication of L. monocytogenes cannot be sufficiently controlled. Taken together, these data indicate that the LT␤R initiates antibacterial protection against prototypic intracellular bacteria inde- pendently of the route of infection. Pulmonary infection with M. tuberculosis is exacerbated in lethally irradiated wild-type recipients of LT␤R-deficient BM LT␣-, LT␤-, and LT␤R-KO mice all exhibit anomalies in the development of lymph nodes, albeit to a varying extent (21, 23, 24, 34, 35). To exclude that a defunct lymphoid anatomy was the sole factor responsible for the loss of early resistance to M. tuberculosis infection in these mice, BM chimeras were constructed in lethally irradiated C57BL/6 mice infused with either BM obtained from LT␤R-KO mice or from wild-type mice. Reconstitution resulted in ϳ90% of donor-derived T cells and Ͼ95% donor-derived B cells in the peripheral blood and Ͼ95% donor-derived Mac3ϩ macrophages in bronchoalveolar lavage cells (data not shown). Recipi- ents of LT␤R-KO BM were highly impaired in resisting a M. tuberculosis intrapulmonary challenge, showing significantly increased bacterial CFU counts in their lungs and livers when directly compared with recipients of wild-type BM at 6 wk postinfection (Fig. 6). FIGURE 3. Course of mycobacterial replication in the lungs, livers, and Thus, decreased resistance of the LT␤R-KO mice used in this spleens of TNFRp55-KO, LT␤R-KO, and wild-type mice intranasally in- F Ⅺ study cannot be attributed solely to lymph node abnormalities, but fected with 250 CFU of M. tuberculosis. C57BL/6 ( ), TNFRp55-KO ( ), ␤ and LT␤R-KO (E) mice were intranasally infected with 500 CFU of M. is directly caused by the lack of signaling via the LT R expressed tuberculosis H37Rv and sacrificed at indicated time points. Organs were on hemopoietic cells during pulmonary M. tuberculosis infection. removed and bacterial CFU counts were determined. Data represent the In the experiments detailed below, we therefore addressed possible means of four to five mice Ϯ SD and are from one experiment of three defects in LT␤R-KO mice which might account for the observed performed, showing almost identical results. impairment of host defense mechanisms. 5214 LT␤R AND INTRACELLULAR BACTERIAL INFECTIONS

FIGURE 4. Lung histopathology of M. tuberculosis-infected LT␤R-KO and wild-type mice. C57BL/6 (A, C, and E) and LT␤R-KO (B, D, and F) mice were intranasally infected with 500 CFU of M. tuberculosis H37Rv and sacrificed on day 35 after infection. Lungs were removed and processed for histology. A and B, H&E stain; magnification, ϫ40; C and D, H&E stain; magnification, ϫ200; E and F, Ziehl-Neelsen stain; magnification, ϫ400. White arrows indicate epithelioid macrophages, black arrows indicate dead or dying cells and nuclei, and red arrow indicates macrophages filled with acid-fast rods. X denotes area of necrosis. Histologies shown are representative of four to five mice per group in three independent experiments.

The defective host response in LT␤R-KO mice is not caused by tein in lung homogenates taken at the same time points, but TNF deficient TNF or IFN-␥ expression protein levels could be determined and, again, were similar in ␤ To exclude the possibility that the lack of LT␤R signaling was wild-type and LT R-KO mice (day 21 after infection: 312 pg/g ␤ associated with decreased TNF or IFN-␥ production known to be lung in wild-type and 455 pg/g lung in LT R-KO mice; day 30 critical for controlling infection with intracellular bacteria, the ex- after infection: 1733 pg/g lung in wild-type and 3442 pg/g lung in ␤ pression of mRNAs for TNF and IFN-␥ was determined in the LT R-KO mice). These results clearly demonstrate that the defi- ␤ lungs of LT␤R-KO and wild-type mice 21 and 30 days after in- cient antibacterial response in LT R-KO mice is not caused by a ␥ tranasal infection with M. tuberculosis. As shown in Fig. 7, mRNA flawed TNF or IFN- regulation. levels for both cytokines were identical or higher in the lungs of Activated pulmonary macrophages express LT␤R infected LT␤R-KO mice. We were unable to measure IFN-␥ pro- It is generally assumed that the LT␤R is expressed solely on stromal (nonhemopoietic) cell types (16), although there is some evidence that it may also be detectable on macrophage-like cells (36). Indeed, the insufficient control of infections with intracellular bacteria by chimeric mice after transfer of LT␤R-KO BM into lethally irradiated wild-type mice suggests that the LT␤R is expressed in

FIGURE 5. Course of infection in LT␤R-KO and wild-type mice in- FIGURE 6. Bacterial load in the lungs and livers of BM-chimeric mice fected with L. monocytogenes. C57BL/6 (f, F, Œ) and LT␤R-KO (Ⅺ, E, intranasally infected with M. tuberculosis. Lethally irradiated C57BL/6 ‚) mice were i.p. infected with the following doses of L. monocytogenes: mice were infused with BM derived from either C57BL/6 (f, five mice per 300,000 CFU (Œ, ‚), 30,000 CFU (F, E), 3,000 CFU (f, Ⅺ). Mice were group) or LT␤R-KO (Ⅺ, seven mice per group) mice and infected intra- ␤ euthanized when moribund. LD50 values for wild-type and LT R-KO mice nasally with 250 CFU of M. tuberculosis. Mice were sacrificed on day 42 p Ͻ ,ء .differed significantly (p Ͻ 0.05) in three independent experiments postinfection and CFU counts were determined in lungs and livers performed. 0.05 Data shown are means Ϯ SD. The Journal of Immunology 5215

FIGURE 8. LT␤R expression on pulmonary macrophages. Cells recovered after digestion of lung tissue were labeled with anti-Mac3 and anti- LT␤R mAb. Macrophages were identified and gated as side scatter-positive Mac3high cells (upper left panel). LT␤R expression is depicted as a single parameter histogram for macrophages obtained before infection (upper right panel), day (d) 28 postinfection (lower left panel), and day 42 postinfection (lower right panel). Isotype-matched control IgG staining is shown in gray and staining for LT␤R is indicated as a black line. On day 28 postinfection, controls included both matched isotype IgG-labeled macrophages from infected wild-type mice and macrophages from infected LT␤R-KO mice (dotted line; identical to staining with isotype-matched ␥ FIGURE 7. TNF and IFN- mRNA expression in the lungs of M. tu- Ab). Data shown are representative of four to five mice per group. berculosis- infected LT␤R-KO and wild-type mice. C57BL/6 (f) and LT␤R-KO (Ⅺ) mice were intranasally infected with 500 CFU of M. tuberculosis H37Rv. Mice were sacrificed at the indicated time points and lung homogenates were prepared for RNA extraction and semiquantitative contained highly activated macrophages as evidenced by marked RT-PCR analysis of TNF (A) and IFN-␥ (B) mRNA production normalized staining for iNOS protein (Fig. 9A). In LT␤R-KO mice, however, ␤ Ϯ to 2-microglobulin mRNA expression. Data represent the means SD of pulmonary lesions present on day 21 postinfection showed little or four mice per group and are from one experiment of three performed, no material reactive with the Ab (Fig. 9B), whereas just before Ͻ ء giving similar results. , p 0.05 death, when large necrotic lesions had developed, some positive staining for iNOS was detected, particularly within smaller lesions or at the circumference of necrotic lesions (data not shown). Thus, the hemopoietic system. To re-evaluate the expression of LT␤Ron expression of this important antibacterial effector protein was sig- macrophages, macrophages were obtained by tryptic lung diges- nificantly delayed in M. tuberculosis-infected LT␤R-KO mice and tion from wild-type and, as a control, LT␤R-KO mice, and were could not be compensated for by TNF or IFN-␥ alone. labeled with mAbs directed against the LT␤R and Mac3. As de- Recently, it was suggested that defective recruitment of T cells picted in Fig. 8, the LT␤R was not detected on quiescent, resident into granulomatous lesions was responsible for the ineffective de- macrophages, but became detectable following M. tuberculosis in- fense observed in M. tuberculosis-infected LT␣-deficient mice fection on activated wild-type macrophages (days 28 and 42 (31). However, in the lungs of LT␤R-KO mice infected with M. postinfection), whereas on LT␤R-deficient macrophages (day 28 tuberculosis, T cells were readily found to be colocalized with postinfection), no labeling was observed. Thioglycolate-elicited macrophages inside granulomas as evidenced by staining with a peritoneal macrophages from wild-type, but not LT␤R-KO mice, monoclonal anti-CD3 Ab (small arrows in Fig. 9, C and D). also expressed the LT␤R (data not shown). Thus, it appears possible that the LT␤R directly induces antibacterial processes in LIGHT-KO mice effectively control intranasal infection with M. macrophages. tuberculosis The LT␤R recognizes not only LT␣␤ heterotrimers, but also the ␤ M. tuberculosis-infected LT R-KO mice show impaired iNOS recently identified ligand LIGHT (20). To define the role of protein expression in lung granulomas LIGHT in antimycobacterial protection, the bacterial load was de- Activation of macrophages for production of NO is a known cor- termined in the lungs of LIGHT-KO mice (22) infected intrana- ollary of effective antimycobacterial immunity in mice. Immuno- sally with M. tuberculosis. In contrast to mice deficient in LT␣, histological staining with a polyclonal Ab specific for iNOS re- LT␤,orLT␤R, LIGHT-KO mice effectively controlled bacterial vealed that LT␤R-KO were deficient in timely expression of iNOS replication in their lungs (Fig. 10) and exhibited similar inflam- protein within developing granulomatous lesions. Twenty-one and matory responses as wild-type mice (data not shown). To examine 30 days after infection, granulomatous lesions in C57BL/6 mice the possibility that LIGHT may influence the course of infection at 5216 LT␤R AND INTRACELLULAR BACTERIAL INFECTIONS

FIGURE 9. Lung immunohistology of M. tuberculosis-infected LT␤R-KO and wild-type mice. C57BL/6 (A and C) and LT␤R-KO (B and D) mice were intranasally infected with ϳ500 CFU of M. tuberculosis H37Rv and sacrificed on day 21 after infection. Lungs were removed and processed for histology. A and B, Anti-iNOS-peroxidase stain; magnification, ϫ200; C and D, anti- CD3-peroxidase stain; magnification, ϫ200. Small arrows indicate individual CD3ϩ cells, black arrow indicates lymphocytic cuff around vessels typical of even uninfected LT␤R-KO mice (21). Histologies shown are representative of four to five mice per group in three independent experiments.

a later stage, mice were monitored until day 118 after infection. but correlated with the absence or grossly delayed expression of Again CFU counts in the lungs were similar in control heterozy- iNOS in mycobacteria-infected macrophages. Ϯ gous (6.4 0.1 log10 CFU) and homozygous LIGHT-KO mice It is well known that the core members of the TNF superfamily Ϯ ␣␤ (6.63 0.2 log10 CFU). In conclusion, LT heterotrimers, but exhibit intrinsic functions during the organogenesis of the second- not LIGHT, are the critical ligands initiating the protective host ary lymphoid organs (21, 23, 24, 34, 35, 37). For example, LT␤R- immune response against M. tuberculosis. deficient animals lack lymph nodes and Peyer’s patches (21). LT␣-KO mice principally share this phenotype; however, within Discussion the mesenterium the presence of a lymph node-like structure was The data presented here demonstrate that activation of macro- described (34, 35). In most LT␤-deficient mice, mesenteric and phages for antibacterial effector function in the course of infections cervical lymph nodes can be observed, but other lymph nodes and with intracellular bacteria critically requires intact LT␤R signal- Peyer’s patches are absent (23, 24). Taken together, the LT␤R ing. Since LIGHT was found not to be essential, the cognate ligand controls the formation of secondary lymphoid organs with the ␤ ␣ ␤ ␣ ␤ for LT R interaction is LT 1 2. This conclusion was reached us- LT 1 2 heterotrimer playing a major role. In fact, when the other ing a number of gene-targeted mouse strains in two different ex- known ligand of the LT␤R, LIGHT (20), was inactivated in the perimental models of intracellular infection. Specifically, LT␣-, germline of the mouse, no gross anomalies within lymphoid tissues LT␤-, LT␤R-, but not LIGHT-KO mice had significantly in- were observed (22). creased bacterial loads leading to necrotic lesions following pul- To investigate whether the increased host susceptibility evident monary infection with M. tuberculosis, and LT␤R-KO mice in mice lacking LT␣,LT␤, or the LT␤R was merely caused by the proved dramatically more susceptible to L. monocytogenes infec- absence of lymph nodes, a BM transfer approach was chosen. tion. Furthermore, the observed defective antibacterial response in Wild-type hosts were lethally irradiated and LT␤R-KO BM or, as LT␤R-KO mice could not be attributed to defunct anatomy of a control, wild-type BM was infused. Leukocytes derived from secondary lymphoid organs, impaired TNF production, or dys- LT␤R-deficient and control BM stem cells readily colonized the ϩ regulated recruitment of CD3 cells into granulomatous lesions, secondary lymphoid organs and other tissues of the recipient (Ref. 38 and data not shown). In concordance with results obtained in LT␤R-KO mice, chimeras reconstituted with LT␤R-KO BM were unable to restrain mycobacterial growth, indicating that this defect is intrinsic to the hemopoietic compartment and cannot solely be attributed to anatomical defects in secondary lymphoid organs. However, it cannot be excluded that the absence of lymph nodes may also have contributed to exacerbation of M. tuberculosis infection in the KO mice, particularly in view of the fact that CFU counts in LT␤R-KO mice consistently reached higher levels than in irradiated wild-type mice reconstituted with BM derived from LT␤R-KO mice. In this regard, it is noteworthy that the induction of experimental autoimmune encephalomyelitis is severely re- FIGURE 10. Bacterial loads in the lungs and livers of LIGHT-KO and duced in LT␣-KO mice lacking lymph nodes, whereas in chimeric wild-type mice infected with M. tuberculosis. Control (heterozygous ϩ Ϫ mice reconstituted with LT␣-deficient BM, the development of LIGHT / , f) or LIGHT-KO (Ⅺ) mice were intranasally infected with 250 CFU of M. tuberculosis and sacrificed on day 42 postinfection. CFU experimental autoimmune encephalomyelitis is similar to that in counts were determined in the lungs and livers and are given as means of wild-type mice (39, 40). four mice ϩ SD. There was no significant difference between groups. Data Previous studies documented important functions for TNF and ␣ are from one experiment of two performed, giving identical results. for LT 3 in the defense against intracellular infections (25, 30, 31, The Journal of Immunology 5217

41Ð43). In particular, TNFRp55-KO mice succumbed to chal- of the macrophage in the absence of the LT␤R. Significantly, we lenges with L. monocytogenes, Mycobacterium avium, or M. tu- and others found this receptor to be expressed on macrophages berculosis, showing unaltered or only slightly delayed recruitment (Ref. 36 and this study). From our data, it is evident that some form of inflammatory cells into infectious lesions (26, 27, 29). To dis- of macrophage activation is necessary for LT␤R expression to be- sect the ligands involved, studies were also performed in TNF-KO come detectable, and this would explain why enhanced mycobac- mice and in LT␣-KO mice (31, 44). TNF-KO and TNF/LT␣-dou- terial growth in LT␤R-KO mice is not evident before day 21 ble-KO mice rapidly died of infection with L. monocytogenes, postinfection when T cell-dependent macrophage activation showing at least 10,000-fold higher bacterial titers in infected or- emerges. Only at this stage would activated macrophages then be gans in comparison to wild-type mice (45). Unfortunately, the his- poised to receive costimulatory signals from T cells (which we topathology of infected tissues derived from these mice was not demonstrated to be present within granulomatous lesions at this ␣ ␤ reported. Collectively, these studies support a model where TNF point, Fig. 9B) expressing membrane-bound LT 1 2. It would also ␣ ␤ and possibly LT 3, via TNFRp55, effectively prevent intercellular appear that a deficiency in LT R signaling cannot be fully com- spreading of L. monocytogenes, although the ultimate effector pensated by TNF and IFN-␥ nor direct T cell-macrophage contact- mechanisms remain ill-defined. dependent events. However, a more indirect effect involving a Experimental infections with M. tuberculosis in these KO mice defect in the maturation of APC or flawed priming of the immune have yielded a slightly different picture. TNF-KO mice infected response is still possible. In this respect, the LT␤R may be re- with M. tuberculosis showed structural deficiencies in granuloma quired also on other hemopoietic cells to provide coactivating formation which were interpreted to be the crucial malfunction stimuli for full macrophage antibacterial activity. underlying their heightened susceptibility to infection (30, 46). Ligation of the LT␤R leads to activation of NF-␬B engaging the Likewise, chimeric mice created by infusing LT␣-deficient BM “classical” pathway via inhibitor of ␬B kinase (IKK) ␤/IKK␥, re- into irradiated wild-type recipients appeared to have no major de- sulting in RelA containing NF-␬B species and an “alternative” fect in macrophage activation when compared with wild-type pathway where NF-␬B complexes composed of NF-␬B2/RelB are mice, as evidenced by similar amounts of NO reaction products in activated via NF␬B-inducing kinase and IKK␣ (48Ð52). Ulti- the serum in the course of M. tuberculosis infection (31). However, mately, the signaling of LT␤R appears to be mainly required for these mice showed a delay in chemokine induction and appeared to the generation of sufficient amounts of type I IFNs which in turn be unable to recruit T cells into inflammatory lesions, suggesting are potent activators of iNOS in vitro and in vivo (53Ð55). Im- that a defective organization of the granulomatous response was the pairment of iNOS expression in LT␤R-deficient mice may thus be critical factor leading to unrestrained mycobacterial growth (30, 46). attributed to deficient signaling via type I IFN receptors (54Ð56), The results from experiments detailed in this report indicate that, a hypothesis that will be addressed in future studies. Alternatively, with respect to initiating antibacterial responses to intracellular additional, as yet unidentified, mediators of the host defense may ␤ ␣ ␤ ␤ infections, the role of the LT R and its ligand LT 1 2 may be be under the control of the LT R. distinct from that served by the TNFRp55 and its ligands TNF and Our studies add another receptor-ligand system to the already ␣ LT 3. A prominent and consistent feature of the inflammatory intricate network orchestrating the immune response to intracellu- response in LT␤R-KO mice was the grossly delayed expression of lar infections. Since LTs and the LT␤R clearly are essential for iNOS protein within granuloma macrophages. This could not be antibacterial protection, it appears possible that specificdeficien- attributed to a decreased presence of CD3ϩ cells within granulo- cies in these molecules may account for the heightened suscepti- matous lesions nor to defective regulation of TNF or IFN-␥ mRNA bility to intracellular infections of some patients. A similar sce- expression. Thus, inefficient macrophage activation, rather than nario was described for patients suffering from disseminated abnormal granuloma formation, was the principal factor leading to mycobacterial infections who had deficiencies in the IL-12/IFN-␥ excessive M. tuberculosis growth in the lungs of LT␤R-KO mice. pathway of macrophage activation (57). The elucidation of the It is, however, extremely difficult to quantitatively assess the ki- LT␤R-initiated processes that contribute to arming macrophages netics of granulomatous inflammation in the lung, and it remains with antibacterial effector functions may eventually enable the de- possible that a slight delay in granuloma formation may also have velopment of novel immunotherapeutic tools for adjuvant treat- contributed to decreased resistance in these mice. ment of patients with intracellular infections, e.g., those with mul- To our knowledge, this is the first report implicating LT␣␤ het- tidrug-resistant M. tuberculosis. erotrimers in the generation of macrophage antibacterial activity. These findings are in contrast to a recent report in which chimeric mice reconstituted with LT␤-deficient BM efficiently controlled Acknowledgments infection with M. tuberculosis (31). 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