Yersinia Enterocolitica Encodes a Suppressor for Tumor Necrosis Factor Alpha Expression
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INFECTION AND IMMUNITY, Apr. 1995, p. 1270–1277 Vol. 63, No. 4 0019-9567/95/$04.0010 Copyright q 1995, American Society for Microbiology Bacterial Evasion of Host Immune Defense: Yersinia enterocolitica Encodes a Suppressor for Tumor Necrosis Factor Alpha Expression 1 1 2 1 HORST ULRICH BEUSCHER, * FRANZ RO¨ DEL, ÅKE FORSBERG, AND MARTIN RO¨ LLINGHOFF Institute for Clinical Microbiology and Immunology, University of Erlangen/Nu¨rnberg, 91054 Erlangen, Germany,1 and Department of Cell Biology and Microbiology, National Defence Research Institute, S-901 Umeå, Sweden2 Received 11 August 1994/Returned for modification 4 October 1994/Accepted 28 December 1994 The ability of the enteropathogenic Yersinia enterocolitica to survive and proliferate in host tissue depends on a 70-kb plasmid known to encode a number of released Yersinia outer proteins that act as virulence factors by inducing cytotoxicity and inhibiting phagocytosis. This study demonstrates that one of the Yersinia outer proteins, the 41-kDa YopB, suppresses the production of tumor necrosis factor alpha (TNF-a), a macrophage- derived cytokine with central roles in the regulation of immune and inflammatory responses to infection. This conclusion is based on several lines of evidence. First, in macrophage cultures, suppression of TNF-a mRNA expression was induced by culture supernatant (CS1) of plasmid-bearing yersiniae, the effect which was blocked by anti-YopB antiserum. Second, suppression of TNF-a production, but not of interleukin-1 (IL-1) and IL-6, was induced by purified YopB. Third, in Yersinia-infected mice, no increase in TNF-a mRNA expression was observed in Peyer’s patches, the primary site of bacterial invasion, compared with IL-1 (a and b) mRNA. Finally, administration of anti-YopB antiserum to mice prior to infection with Y. enterocolitica increased TNF activity levels in Peyer’s patches and coincided with a reduction in bacterial growth. The results thus provide direct evidence for a secreted eubacterial virulence factor that mediates selective suppression of TNF-a production. Although suppression of this single cytokine response is probably not sufficient to facilitate survival of the infecting organisms, the results suggest that suppression of TNF-a production by YopB significantly contributes to the evasion of Y. enterocolitica from antibacterial host defense. Infection with the facultative intracellular bacterium Yersinia Consequently, it has been proposed that the V antigen or other enterocolitica is a frequently recognized cause of food-borne plasmid-derived virulence factors of pathogenic yersiniae may diseases that range from gastroenteritis to invasive colitis and be able to suppress the endogenous production of either cyto- lymphadenitis. After oral infection of a mammalian host, the kine. Apart from bacterial infections, a number of viruses have bacteria cross the epithelium overlying the Peyer’s patches been reported to encode several cytokine response modifiers (PP) of the gut and multiply within the lymphoid follicles that inhibit synthesis, functional maturation, and bioactivity of, before spreading to the adjacent lamina propria (7, 8, 20). e.g., IL-1 or TNF-a, thereby contributing to the pathology Similar to other intestinal pathogens, e.g., Salmonella, Shigella, associated with viral infections (13, 37). In view of these ob- and Campylobacter spp., the persistence of yersiniae in mucosal servations, we initiated our studies to identify plasmid-encoded tissue is considered to be an important factor for the develop- proteins of Y. enterocolitica that were able to suppress the ment of polyarticular, nonsuppurative arthritis (14). Although endogenous elaboration of proinflammatory cytokines, includ- a number of virulence properties, including resistance to ing IL-1, IL-6, and TNF-a. phagocytosis, cytotoxicity, and host protein-dephosphorylation Previous data from our laboratory demonstrated that inva- (5, 10, 35) have been characterized and linked to the expres- sion of Y. enterocolitica into PP caused an infiltration of mono- sion of a set of released, plasmid-encoded Yersinia outer pro- cytes and inflammatory macrophages, which rapidly produced teins (Yops), escape of yersiniae from the ensuing immune increasing amounts of the cytokines IL-1a and IL-1b (3, 33). response is not fully understood. Here we report a lack of TNF-a induction in infected PP. Recent studies have demonstrated central roles for the cy- Results are presented to show that the 41-kDa plasmid-en- tokines gamma interferon (IFN-g), interleukin-1 (IL-1), and coded YopB can act as a potent suppressor of TNF-a mRNA tumor necrosis factor alpha (TNF-a) in inflammatory reactions expression in macrophages and infected tissue. associated with the development of cellular immunity and granulomas, leading to eradication of obligate and facultative MATERIALS AND METHODS intracellular pathogens (19, 22, 27, 30). In particular, treatment of mice with antibodies to IFN-g and TNF-a has been shown Bacteria. The plasmidless strain (NCTC 10598), here named O82, and the to exacerbate primary infection with Y. enterocolitica (2), indi- isogenic plasmid-bearing strain (NCTC 10938), here named O81,ofY. entero- cating that either cytokine is important for antibacterial host colitica O8 were obtained from the National Collection of Type Cultures (Cen- tral Public Health Laboratory, London, England). The Yersinia pseudotubercu- defense. Moreover, in infection with Yersinia pestis, treatment losis serotype II was taken from the strain collection of the Institute for Clinical of mice with antibodies to the plasmid-encoded V antigen Microbiology (Erlangen, Federal Republic of Germany). This strain of Y. increased the levels of both circulating IFN-g and TNF-a (29). pseudotuberculosis exhibited a marked defect in the expression of three Yops, namely, YopH, YopD, and YopB. The bacteria were routinely grown in defined TMH medium at 268C for up to 18 h (35). Experimental infection of mice. Female BALB/c mice (age, 6 to 8 weeks; * Corresponding author. Mailing address: Institute for Clinical Mi- weight, about 20 g) were orally infected with 0.1 ml of bacterial suspension crobiology and Immunology, Wasserturmstrasse 3, 91054 Erlangen, containing 107 cells. At different times (0 to 10 days) after infection, mice were Germany. Phone (49) 9131/852650. Fax: (49) 9131/852573. bled and killed by cervical dislocation. Inflamed PP and spleens were then 1270 VOL. 63, 1995 YopB-INDUCED SUPPRESSION OF TNF-a 1271 removed and processed for immunohistochemical staining and RNA analysis (1, phoretically transferred onto nitrocellulose membranes (Bio-Rad) by using the 3, 33). Pharmacia semidry blotting apparatus. To inhibit nonspecific binding, mem- Determination of bacterial colonization of PP. At various times after infection, branes were blocked with 3% (wt/vol) gelatin in buffered saline. Thereafter, mice either untreated or treated with antiserum to YopB, preimmune serum immunoblots were incubated (2 h, 258C) with saturating amounts (dilution (pIS), or phosphate-buffered saline (PBS), were killed, and the distal part of the 1:1,000) of the various rabbit anti-Yop antisera. Following incubation (1 h, 258C) small intestine was removed and gently washed with 5 ml of saline. The PP were with a POX-conjugated goat anti-rabbit antiserum (Sigma), the POX reaction excised, counted, suspended in saline, and homogenized in a Potter homoge- was performed with 4-chloro-1-naphthol and H2O2 as substrates (Merck, Darm- nizer. Appropriate dilutions of the homogenates were placed onto Yersinia se- stadt, Federal Republic of Germany). lective agar containing the selective supplement SR 109 (Oxoid, Basingstoke, Induction of anti-YopB antiserum. New Zealand White rabbits (Charles England), and the number of CFU were counted. River, Sulzfeld, Federal Republic of Germany), were immunized with YopB Bacterial CS. Cultures of yersiniae grown overnight were diluted 1/20 and purified as described above. YopB (100 mg/250 ml) was mixed with an equal incubated at 378C for 4 h (6) to induce secretion of Yops. Culture supernatants volume of complete Freund’s adjuvant (Sigma, Mu¨nchen, Federal Republic of (Cs) of plasmid-bearing (CS1) and plasmidless (CS2) bacteria were then col- Germany), and injected subcutaneously into two sites in the back, and animals lected by centrifugation (5 min, 3,000 3 g), sterile filtered, concentrated 100 were given booster injections of 10 mg of YopB in incomplete Freund’s adjuvant each week for a month. Blood samples taken 7 days after the last booster times by using an Amicon ultrafiltration cell equipped with a YM10 membrane, 1 and dialyzed against tissue culture medium RPMI 1640 (GIBCO/BRL, Eggen- injection were routinely assayed by Western blot analysis with concentrated CS . stein, Federal Republic of Germany). For depletion of individual Yops, aliquots Antisera (dilution 1:1,000) that identified a single band in size similar to the (500 ml) of dialyzed CS1 were incubated (18 h at 48C) with 100 ml of protein A 41-kDa YopB were chosen. Sepharose beads (Pharmacia, Freiburg, Federal Republic of Germany) adsorbed Bioassays. Cytokine activities were assayed by using three different cell lines: with 50 ml of rabbit antiserum (10) to either YopH-M, YopB, YopD-N, YopE, the WEHI 164 fibrosarcoma cell line which is sensitive to lysis by TNF-a and the or normal rabbit serum. After centrifugation, CS1 was removed, sterile filtered, D10G4.1 T helper cell line and the B9 hybridoma cell line (9, 25, 32) which and used for incubation with murine macrophages. proliferate in response to IL-1 or IL-6, respectively. After incubation with serial Cells and tissue culture. Primary macrophages were prepared from the peri- dilutions of either macrophage culture fluids or tissue homogenates that had toneal cavities of BALB/c mice 3 days after intraperitoneal injection of 1 ml of been adjusted to 0.1 mg of protein per ml, cytokine activities were analyzed by 10% Proteose Peptone (Difco), plated at 106 per well in RPMI 1640, supple- measuring the number of viable cells (28) with 3-(4,5-dimethythioazol-2-yl-2,5- diphenyltetrazolium bromide.