Charles A. Scanga

TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis

To investigate the role of Toll-like receptor (TLR)9 in the immune response to mycobacteria as well as its cooperation with TLR2, a receptor known to be triggered by several major mycobacterial ligands, we analyzed the resistance of TLR9−/− as well as TLR2/9 double knockout mice to aerosol infection with Mycobacterium tuberculosis. Infected TLR9−/− but not TLR2−/− mice displayed defective mycobacteria-induced interleukin (IL)-12p40 and interferon (IFN)-γ responses in vivo, but in common with TLR2−/− animals, the TLR9−/− mice exhibited only minor reductions in acute resistance to low dose pathogen challenge. When compared with either of the single TLR-deficient animals, TLR2/9−/− mice displayed markedly enhanced susceptibility to infection in association with combined defects in proinflammatory cytokine production in vitro, IFN-γ recall responses ex vivo, and altered pulmonary pathology. Cooperation between TLR9 and TLR2 was also evident at the level of the in vitro response to live M. tuberculosis, where dendritic cells and macrophages from TLR2/9−/− mice exhibited a greater defect in IL-12 response than the equivalent cell populations from single TLR9-deficient animals. These findings reveal a previously unappreciated role for TLR9 in the host response to M. tuberculosis and illustrate TLR collaboration in host resistance to a major human pathogen.

Effects of Tumor Necrosis Factor Alpha on Host Immune Response in Chronic Persistent Tuberculosis: Possible Role for Limiting Pathology

ABSTRACT Reactivation of latent tuberculosis contributes significantly to the incidence of disease caused by Mycobacterium tuberculosis. The mechanisms involved in the containment of latent tuberculosis are poorly understood. Using the low-dose model of persistent murine tuberculosis in conjunction with MP6-XT22, a monoclonal antibody that functionally neutralizes tumor necrosis factor alpha (TNF-α), we examined the effects of TNF-α on the immunological response of the host in both persistent and reactivated tuberculous infections. The results confirm an essential role for TNF-α in the containment of persistent tuberculosis. TNF-α neutralization resulted in fatal reactivation of persistent tuberculosis characterized by a moderately increased tissue bacillary burden and severe pulmonic histopathological deterioration that was associated with changes indicative of squamous metaplasia and fluid accumulation in the alveolar space. Analysis of pulmonic gene and protein expression of mice in the low-dose model revealed that nitric oxide synthase was attenuated during MP6-XT22-induced reactivation, but was not totally suppressed. Interleukin-12p40 and gamma interferon gene expression in TNF-α-neutralized mice was similar to that in control mice. In contrast, interleukin-10 expression was augmented in the TNF-α-neutralized mice. In summary, results of this study suggest that TNF-α plays an essential role in preventing reactivation of persistent tuberculosis, modulates the pulmonic expression of specific immunologic factors, and limits the pathological response of the host.

Cutting Edge: MyD88 Is Required for Resistance to Toxoplasma gondii Infection and Regulates Parasite-Induced IL-12 Production by Dendritic Cells

Host resistance to the intracellular protozoan Toxoplasma gondii is highly dependent on early IL-12 production by APC. We demonstrate here that both host resistance and T. gondii-induced IL-12 production are dramatically reduced in mice lacking the adaptor molecule MyD88, an important signaling element used by Toll-like receptor (TLR) family members. Infection of MyD88-deficient mice with T. gondii resulted in uncontrolled parasite replication and greatly reduced plasma IL-12 levels. Defective IL-12 responses to T. gondii Ags (soluble tachyzoite Ag (STAg)) were observed in MyD88−/− peritoneal macrophages, neutrophils, and splenic dendritic cells (DC). In contrast, DC from TLR2- or TLR4-deficient animals developed normal IL-12 responses to STAg. In vivo treatment with pertussis toxin abolished the residual IL-12 response displayed by STAg-stimulated DC from MyD88−/− mice. Taken together, these data suggest that the induction of IL-12 by T. gondii depends on a unique mechanism involving both MyD88 and G protein-coupled signaling pathways.

Mice lacking myeloid differentiation factor 88 display profound defects in host resistance and immune responses to Mycobacterium avium infection not exhibited by Toll-like receptor 2 (TLR2)- and TLR4-deficient animals.

To assess the role of Toll-like receptor (TLR) signaling in host resistance to Mycobacterium avium infection, mice deficient in the TLR adaptor molecule myeloid differentiation factor 88 (MyD88), as well as TLR2−/− and TLR4−/− animals, were infected with a virulent strain of M. avium, and bacterial burdens and immune responses were compared with those in wild-type (WT) animals. MyD88−/− mice failed to control acute and chronic M. avium growth and succumbed 9–14 wk postinfection. Infected TLR2−/− mice also showed increased susceptibility, but displayed longer survival and lower bacterial burdens than MyD88−/− animals, while TLR4−/− mice were indistinguishable from their WT counterparts. Histopathological examination of MyD88−/− mice revealed massive destruction of lung tissue not present in WT, TLR2−/−, or TLR4−/− mice. In addition, MyD88−/− and TLR2−/−, but not TLR4−/−, mice displayed marked reductions in hepatic neutrophil infiltration during the first 2 h of infection. Although both MyD88−/− and TLR2−/− macrophages showed profound defects in IL-6, TNF, and IL-12p40 responses to M. avium stimulation in vitro, in vivo TNF and IL-12p40 mRNA induction was impaired only in infected MyD88−/− mice. Similarly, MyD88−/− mice displayed a profound defect in IFN-γ response that was not evident in TLR2−/− or TLR4−/− mice or in animals deficient in IL-18. These findings indicate that resistance to mycobacterial infection is regulated by multiple MyD88-dependent signals in addition to those previously attributed to TLR2 or TLR4, and that these undefined elements play a major role in determining bacterial induced proinflammatory as well as IFN-γ responses.

MyD88-deficient mice display a profound loss in resistance to Mycobacterium tuberculosis associated with partially impaired Th1 cytokine and nitric oxide synthase 2 expression.

ABSTRACT Mycobacterium tuberculosis possesses agonists for several Toll-like receptors (TLRs), yet mice with single TLR deletions are resistant to acute tuberculosis. MyD88−/− mice were used to examine whether TLRs play any role in protection against aerogenic M. tuberculosis H37Rv infection. MyD88−/− mice failed to control mycobacterial replication and rapidly succumbed. Moreover, expressions of interleukin 12, tumor necrosis factor alpha, gamma interferon, and nitric oxide synthase 2 were markedly decreased in the knockout animals. These results argue that resistance to M. tuberculosis must depend on MyD88-dependent signals mediated by an as-yet-undetermined TLR or a combination of TLRs.

Host control of Mycobacterium tuberculosis is regulated by 5-lipoxygenase–dependent lipoxin production

Th1 type cytokine responses are critical in the control of Mycobacterium tuberculosis infection. Recent findings indicate that 5-lipoxygenase-dependent (5-LO-dependent) lipoxins regulate host IL-12 production in vivo. Here, we establish lipoxins as key chemical mediators in resistance to M. tuberculosis infection. High levels of lipoxin A4 (LXA4) were detected in sera from infected WT but not infected 5-LO-deficient mice. Moreover, lungs from M. tuberculosis-infected 5-lo-/- animals showed increased IL-12, IFN-gamma, and NO synthase 2 (NOS2) mRNA levels compared with the same tissues in WT mice. Similarly, splenocyte recall responses were enhanced in mycobacteria-infected 5-lo-/- versus WT mice. Importantly, bacterial burdens in 5-lo-/- lungs were significantly lower than those from WT mice, and this enhancement in the resistance of the 5-lo-/- animals to M. tuberculosis was completely prevented by administration of a stable LXA4 analog. Together our results demonstrate that lipoxins negatively regulate protective Th1 responses against mycobacterial infection in vivo and suggest that the inhibition of lipoxin biosynthesis could serve as a strategy for enhancing host resistance to M. tuberculosis.

CD8 + CTL from Lungs of Mycobacterium tuberculosis -Infected Mice Express Perforin In Vivo and Lyse Infected Macrophages

CD8+ T lymphocytes have been implicated in the protective immune response against human and murine tuberculosis. However, the functional role that this cell subset plays during the resolution of infection remains controversial. In this study, we demonstrate the presence of Mycobacterium tuberculosis-specific CD8+ CTL in the lungs and lung-draining lymph nodes of mice infected with M. tuberculosis via the aerosol or i.v. route. These cells expressed perforin in vivo and specifically recognized and lysed M. tuberculosis-infected macrophages in a perforin-dependent manner after a short period of in vitro restimulation. The efficiency of lysis of infected macrophages was dependent upon the time allowed for interaction between macrophage and M. tuberculosis bacilli. Recognition of infected targets by CD8+ CTL was β2-microglobulin and MHC class I dependent and was not CD1d restricted. The presented data indicate that CD8+ T cells contribute to the protective immune response during M. tuberculosis infection by exerting cytotoxic function and lysing infected macrophages.

Novel recombinant BCG expressing perfringolysin O and the over-expression of key immunodominant antigens; pre-clinical characterization, safety and protection against challenge with Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), has infected approximately two billion individuals worldwide with approximately 9.2 million new cases and 1.6 million deaths annually. Current efforts are focused on making better BCG priming vaccines designed to induce a comprehensive and balanced immunity followed by booster(s) targeting a specific set of relevant antigens in common with the BCG prime. We describe the generation and immunological characterization of recombinant BCG strains with properties associated with lysis of the endosome compartment and over-expression of key Mtb antigens. The endosome lysis strain, a derivative of BCG SSI-1331 (BCG(1331)) expresses a mutant form of perfringolysin O (PfoA(G137Q)), a cytolysin normally secreted by Clostridium perfringens. Integration of the PfoA(G137Q) gene into the BCG genome was accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q) under the control of the Ag85B promoter. The resultant BCG construct, designated AERAS-401 (BCG(1331) DeltaureC::OmegapfoA(G137Q)) secreted biologically active Pfo, was well tolerated with a good safety profile in immunocompromised SCID mice. A second rBCG strain, designated AFRO-1, was generated by incorporating an expression plasmid encoding three mycobacterial antigens, Ag85A, Ag85B and TB10.4, into AERAS-401. Compared to the parental BCG strain, vaccination of mice and guinea pigs with AFRO-1 resulted in enhanced immune responses. Mice vaccinated with AFRO-1 and challenged with the hypervirulent Mtb strain HN878 also survived longer than mice vaccinated with the parental BCG. Thus, we have generated improved rBCG vaccine candidates that address many of the shortcomings of the currently licensed BCG vaccine strains.

CD40, but Not CD40L, Is Required for the Optimal Priming of T Cells and Control of Aerosol M. tuberculosis Infection

CD40(-/-) mice succumbed to low-dose aerosol infection with M. tuberculosis due to deficient IL-12 production leading to impaired priming of IFN-gamma T cell responses. In contrast, CD40L(-/-) mice were resistant to M. tuberculosis. This asymmetry in outcome of infection between the two knockout strains is likely due to the existence of an alternative ligand for CD40. Both in vitro M. tuberculosis infection and recombinant M. tuberculosis Hsp70 elicited IL-12 production from WT dendritic cells. This response was absent in both CD40(-/-) dendritic cells and CD40(-/-) mice, suggesting that M. tuberculosis Hsp70 serves as an alternative ligand for CD40 in vivo.

Maintenance of Pulmonary Th1 Effector Function in Chronic Tuberculosis Requires Persistent IL-12 Production

The mechanisms that prevent reactivation of latent Mycobacterium tuberculosis infection in asymptomatic individuals are poorly understood. Although IL-12 is critical for the induction of IFN-γ-dependent host control of M. tuberculosis, the requirement for the cytokine in the maintenance of host resistance and pulmonary Th1 effector function has not yet been formally examined. In this study, we reconstituted IL-12p40-deficient mice with IL-12 during the first 4 wk of infection and then assessed the effects of cytokine withdrawal. Although IL-12 administration initially resulted in restricted mycobacterial growth and prolonged survival, the reconstituted animals eventually succumbed to infection. This breakdown in bacterial control was accompanied by a marked reduction in the numbers of IFN-γ-producing CD4+ T cells in lungs. Moreover, whereas CD4+ T cells isolated from chronically infected wild-type mice expanded and transferred long-term protection to M. tuberculosis-challenged RAG−/− mice, they failed to do so in IL-12p40-deficient RAG−/− recipients and were clearly reduced in frequency within pulmonary granulomas in the latter animals. These studies establish that continuous IL-12 production is necessary for maintenance of the pulmonary Th1 cells required for host control of persistent M. tuberculosis infection and suggest that breakdown of this mechanism could be a contributing factor in reactivated disease.