Regulation of gastric B cell recruitment is dependent on IL-17 receptor A signaling in a model of chronic bacterial infection.

Th17-driven immune responses contribute to the pathogenesis of many chronic inflammatory diseases. In this study, we investigated the role of IL-17 signaling in chronic gastric inflammation induced by Helicobacter pylori, a Gram-negative bacterium that persistently colonizes the human stomach. Wild-type C57BL/6 mice and mice lacking IL-17RA (IL-17RA(-/-)) were orogastrically infected with H. pylori. Differences in bacterial colonization density and gastric inflammation were not apparent at 1 mo postinfection, but by 3 mo postinfection, H. pylori colonization density was higher and mononuclear gastric inflammation more severe in infected IL-17RA(-/-) mice than in infected wild-type mice. A striking feature was a marked increase in gastric B cells, plasma cells, and lymphoid follicles, along with enhanced H. pylori-specific serum Ab responses, in infected IL-17RA(-/-) mice. Fewer gastric neutrophils and lower levels of neutrophil-recruiting chemokines were detected in infected IL-17RA(-/-) mice than in infected wild-type mice. Gastric IL-17a and IL-21 transcript levels were significantly higher in infected IL-17RA(-/-) mice than in infected wild-type mice or uninfected mice, which suggested that a negative feedback loop was impaired in the IL-17RA(-/-) mice. These results underscore an important role of IL-17RA signaling in regulating B cell recruitment. In contrast to many chronic inflammatory diseases in which IL-17RA signaling promotes an inflammatory response, IL-17RA signaling down-regulates the chronic mononuclear inflammation elicited by H. pylori infection.

Role of the dosR-dosS two-component regulatory system in Mycobacterium tuberculosis virulence in three animal models.

The Mycobacterium tuberculosis dosR gene (Rv3133c) is part of an operon, Rv3134c-Rv3132c, and encodes a response regulator that has been shown to be upregulated by hypoxia and other in vitro stress conditions and may be important for bacterial survival within granulomatous lesions found in tuberculosis. DosR is activated in response to hypoxia and nitric oxide by DosS (Rv3132c) or DosT (Rv2027c). We compared the virulence levels of an M. tuberculosis dosR-dosS deletion mutant (DeltadosR-dosS [DeltadosR-S]), a dosR-complemented strain, and wild-type H37Rv in rabbits, guinea pigs, and mice infected by the aerosol route and in a mouse hollow-fiber model that may mimic in vivo granulomatous conditions. In the mouse and the guinea pig models, the DeltadosR-S mutant exhibited a growth defect. In the rabbit, the DeltadosR-S mutant did not replicate more than the wild type. In the hollow-fiber model, the mutant phenotype was not different from that of the wild-type strain. Our analyses reveal that the dosR and dosS genes are required for full virulence and that there may be differences in the patterns of attenuation of this mutant between the animal models studied.

Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates.

Understanding the physical characteristics of the local microenvironment in which Mycobacterium tuberculosis resides is an important goal that may allow the targeting of metabolic processes to shorten drug regimens. Pimonidazole hydrochloride (Hypoxyprobe) is an imaging agent that is bioreductively activated only under hypoxic conditions in mammalian tissue. We employed this probe to evaluate the oxygen tension in tuberculous granulomas in four animal models of disease: mouse, guinea pig, rabbit, and nonhuman primate. Following infusion of pimonidazole into animals with established infections, lung tissues from the guinea pig, rabbit, and nonhuman primate showed discrete areas of pimonidazole adduct formation surrounding necrotic and caseous regions of pulmonary granulomas by immunohistochemical staining. This labeling could be substantially reduced by housing the animal under an atmosphere of 95% O(2). Direct measurement of tissue oxygen partial pressure by surgical insertion of a fiber optic oxygen probe into granulomas in the lungs of living infected rabbits demonstrated that even small (3-mm) pulmonary lesions were severely hypoxic (1.6 +/- 0.7 mm Hg). Finally, metronidazole, which has potent bactericidal activity in vitro only under low-oxygen culture conditions, was highly effective at reducing total-lung bacterial burdens in infected rabbits. Thus, three independent lines of evidence support the hypothesis that hypoxic microenvironments are an important feature of some lesions in these animal models of tuberculosis.

Altered inflammatory responses following transforming growth factor-beta neutralization in experimental guinea pig tuberculous pleurisy.

The predominant extrapulmonary form of tuberculosis, which develops in 10% of diseased individuals, is pleurisy. The immune response mounted against Mycobacterium tuberculosis in the pleural cavity is one that is sufficient for clearing the organism without therapeutic intervention. Thus, examining the role of immune constituents in this context will provide understanding of the vital role they play in controlling tuberculosis. In this study, experimental tuberculous pleurisy was induced in guinea pigs, and anti-TGF-beta was administered intrapleurally to the guinea pigs daily throughout the study (8 days). Neutralizing TGF-beta resulted in a significant reduction in the percentage of lymphocytes and CD8+ cells present in the pleural exudate, decreased proliferative responses of pleural cells to ConA and PPD, and decreased mRNA expression of IFN-gamma and CCL5 in pleural effusion cells. Conversely, the percentage of neutrophils was significantly increased in anti-TGF-beta-treated guinea pigs, along with upregulated mRNA expression of CXCL8. The percentage of macrophages in the pleural exudate, TNF-alpha and IL-12p40 mRNA expression, and the histopathological response were not significantly altered. While TGF-beta is generally thought of as an immunosuppressive cytokine, the results of this study demonstrate its importance in promoting an inflammatory response, and highlight its bipolar nature.

Recombinant guinea pig tumor necrosis factor alpha stimulates the expression of interleukin-12 and the inhibition of Mycobacterium tuberculosis growth in macrophages.

Tumor necrosis factor alpha (TNF-alpha) plays an important role in the host immune response to infection with the intracellular pathogen Mycobacterium tuberculosis. It is essential for the formation of protective tuberculous granulomas and regulates the expression of other cytokines which contribute to a protective immune response. Interleukin-12 (IL-12) is known to promote a Th1 response, which is essential for antimycobacterial resistance. Recombinant guinea pig TNF-alpha (rgpTNF-alpha) protein (17 kDa) was purified, and its bioactivity was confirmed by its cytotoxicity for L929 fibroblasts. High titers of polyclonal anti-gpTNF-alpha antibody were obtained by immunization of rabbits. Resident alveolar and peritoneal macrophages were isolated from guinea pigs and infected with either the H37Ra or H37Rv strain of M. tuberculosis. The mRNA levels for TNF-alpha and IL-12 p40 were measured using real-time PCR. IL-12 p40 mRNA was up-regulated in a dose-dependent manner by rgpTNF-alpha alone. In infected macrophages, a lower dose of rgpTNF-alpha intensified the mRNA levels of TNF-alpha and IL-12 p40. However, higher doses of rgpTNF-alpha suppressed TNF-alpha and IL-12 p40 mRNA. The antimycobacterial activity of macrophages was assessed by metabolic labeling of M. tuberculosis with [3H]uracil. Resident alveolar and peritoneal macrophages treated with anti-gpTNF-alpha antibody to block endogenous TNF-alpha exhibited increased intracellular mycobacterial growth. These data suggest that the dose of TNF-alpha is crucial to the stimulation of optimal expression of protective cytokines and that TNF-alpha contributes to the control of mycobacterial replication to promote host resistance against M. tuberculosis.

Coordinate cytokine gene expression in vivo following induction of tuberculous pleurisy in guinea pigs.

Tuberculous pleurisy is a severe inflammatory response induced by Mycobacterium tuberculosis organisms that have escaped from lung granulomata into the pleural space during pulmonary infection. We have used the guinea pig model of tuberculous pleurisy to examine several aspects of the immune response to this antigen-specific inflammatory event. Pleurisy was induced by injection of heat-killed M. tuberculosis H37Rv directly into the pleural space of guinea pigs previously vaccinated with M. bovis BCG. Four animals were euthanized each day over a period of 9 days. Fluid in the pleural cavity was analyzed for transforming growth factor beta 1 (TGF-beta 1) and total interferon (IFN) protein levels. In addition, RNA was obtained from pleural cells and examined for TGF-beta 1, tumor necrosis factor alpha (TNF-alpha), IFN-gamma, and interleukin-8 (IL-8) expression by real-time PCR. Finally, pleural cells were examined for the ability to proliferate in response to concanavalin A and purified protein derivative (PPD) in vitro. In the pleural fluid, TGF-beta 1 protein concentrations increased over the course of the inflammatory response while IFN protein levels were not significantly altered. Expression of TGF-beta 1 mRNA peaked on days 3 and 4, and IFN-gamma mRNA expression peaked on day 3 and then returned to background levels. TNF-alpha mRNA expression was highest on days 2 to 4, and IL-8 mRNA levels remained elevated between days 2 and 5, peaking on day 3 before returning to background levels. PPD-induced proliferative responses were evident by day 3 and remained present throughout the study. Analysis of cytokine expression during tuberculous pleurisy may lead to a better understanding of the self-healing nature of this manifestation of tuberculosis.

Effect of neutralizing transforming growth factor beta1 on the immune response against Mycobacterium tuberculosis in guinea pigs.

Transforming growth factor beta (TGF-beta) is a cytokine which has been shown to suppress the antimycobacterial immune responses of humans and experimental animals. In this study, the contributions of TGF-beta to cytokine production in vivo were investigated by using the established guinea pig model of tuberculous pleurisy. Mycobacterium bovis BCG-vaccinated guinea pigs were injected intrapleurally with heat-killed virulent Mycobacterium tuberculosis. Eight days following induction of an antigen-specific pleural effusion, guinea pigs were injected intrapleurally with anti-TGF-beta1 or isotype control antibody. The following day, pleural exudates were removed, and the fluid volume and characteristics of the infiltrating cells were determined. Pleural fluid was analyzed for total interferon (IFN) and tumor necrosis factor (TNF) protein levels by using appropriate bioassays. RNA from pleural effusion cells was examined to determine TGF-beta1, TNF-alpha, IFN-gamma, and interleukin-8 mRNA levels by using real-time PCR. Proliferative responses of pleural effusion lymphocytes were examined in response to concanavalin A and purified protein derivative (PPD) in vitro. Treatment with anti-TGF-beta1 resulted in decreased pleural fluid volume and decreased cell numbers in the pleural space along with an increased percentage of lymphocytes and a decreased percentage of neutrophils. The bioactive TNF protein levels in pleural fluid were increased in guinea pigs treated with anti-TGF-beta1, while the bioactive IFN protein concentrations were not altered. Expression of TGF-beta1 and TNF-alpha mRNA was significantly increased following TGF-beta1 neutralization. Finally, PPD-induced proliferative responses of pleural cells from anti-TGF-beta1-treated animals were significantly enhanced. Thus, TGF-beta1 may be involved in the resolution of this local, mycobacterial antigen-specific inflammatory response.

Effect of Mycobacterium bovis BCG vaccination on Mycobacterium-specific cellular proliferation and tumor necrosis factor alpha production from distinct guinea pig leukocyte populations.

In this study, we focused on three leukocyte-rich guinea pig cell populations, bronchoalveolar lavage (BAL) cells, resident peritoneal cells (PC), and splenocytes (SPC). BAL cells, SPC, and PC were stimulated either with live attenuated Mycobacterium tuberculosis H37Ra or with live or heat-killed virulent M. tuberculosis H37Rv (multiplicity of infection of 1:100). Each cell population was determined to proliferate in response to heat-killed virulent H37Rv, whereas no measurable proliferative response could be detected upon stimulation with live mycobacteria. Additionally, this proliferative capacity (in SPC and PC populations) was significantly enhanced upon prior vaccination with Mycobacterium bovis BCG. Accordingly, in a parallel set of experiments we found a strong positive correlation between production of antigen-specific bioactive tumor necrosis factor alpha (TNF-alpha) and prior vaccination with BCG. A nonspecific stimulus, lipopolysaccharide, failed to induce this effect on BAL cells, SPC, and PC. These results showed that production of bioactive TNF-alpha from mycobacterium-stimulated guinea pig cell cultures positively correlates with the vaccination status of the host and with the virulence of the mycobacterial strain.