Interleukin-18 Is Critical for Mucosa-Associated Invariant T Cell Gamma Interferon Responses to Francisella Species In Vitro but Not In Vivo.

Mucosa-associated invariant T (MAIT) cells are a subset of innate T cells that express a semi-invariant Vα chain paired with limited Vß chains. MAIT cells are activated by riboflavin metabolite derivatives presented by the nonpolymorphic major histocompatibility complex class I (MHC-I)-like molecule MR1. The precise mechanisms required to activate MAIT cells are an area of intense interest. Here we used two closely related intracellular pathogens with distinct inflammasome activation phenotypes to probe the role of innate cytokines in MAIT cell activation. Using an in vitro assay containing transgenic murine MAIT cells, we show that macrophages infected with Francisella novicida, a strong inflammasome activator, released high levels of interleukin-18 (IL-18) and stimulated high levels of MAIT cell gamma interferon (IFN-γ) through a partially MR1-independent pathway. In contrast, macrophages infected with Francisella tularensis live vaccine strain (LVS), a weak inflammasome activator, generated little IL-18 and stimulated low MAIT cell IFN-γ through an MR1-dependent pathway. By manipulating the quantities of IL-18 in these cultures, we show that the IL-18 concentration is sufficient to influence the magnitude of MAIT cell IFN-γ production. Correspondingly, infected IL-18-deficient macrophages failed to induce substantial MAIT cell IFN-γ. In contrast, we found that MAIT cell IFN-γ production in the lungs of IL-18-deficient mice was not significantly different from that in WT mice during F. tularensis LVS pulmonary infection. Overall, we demonstrate that while IL-18 is essential for the MAIT cell IFN-γ response in vitro, it is not essential for MAIT cell IFN-γ production during in vivo LVS pulmonary infection, suggesting that additional signals can drive MAIT cell IFN-γ production in vivo.

MAIT cells are critical for optimal mucosal immune responses during in vivo pulmonary bacterial infection.

Mucosa-associated invariant T (MAIT) cells are "innate" T cells that express an invariant T-cell receptor α-chain restricted by the nonclassical MHC class I molecule MHC-related protein 1 (MR1). A recent discovery that MR1 presents vitamin B metabolites, presumably from pathogenic and/or commensal bacteria, distinguishes MAIT cells from peptide- or lipid-recognizing αß T cells in the immune system. MAIT cells are activated by a wide variety of bacterial strains in vitro, but their role in defense against infectious assaults in vivo remains largely unknown. To investigate how MAIT cells contribute to mucosal immunity in vivo, we used a murine model of pulmonary infection by using the live vaccine strain (LVS) of Francisella tularensis. In the early acute phase of infection, MAIT cells expanded robustly in the lungs, where they preferentially accumulated after reaching their peak expansion in the late phase of infection. Throughout the course of infection, MAIT cells produced the critical cytokines IFN-γ, TNF-α, and IL-17A. Mechanistic studies showed that MAIT cells required both MR1 and IL-12 40 kDa subunit (IL-12p40) signals from infected antigen presenting cells to control F. tularensis LVS intracellular growth. Importantly, pulmonary F. tularensis LVS infection of MR1-deficient (MR1(-/-)) mice, which lack MAIT cells, revealed defects in early mucosal cytokine production, timely recruitment of IFN-γ-producing CD4(+) and CD8(+) T cells to the infected lungs, and control of pulmonary F. tularensis LVS growth. This study provides in vivo evidence demonstrating that MAIT cells are an important T-cell subset with activities that influence the innate and adaptive phases of mucosal immunity.

MAIT cells and pathogen defense.

Mucosa-associated invariant T (MAIT) cells are a unique population of innate T cells that are abundant in humans. These cells possess an evolutionarily conserved invariant T cell receptor α chain restricted by the nonpolymorphic class Ib major histocompatibility (MHC) molecule, MHC class I-related protein (MR1). The recent discovery that MAIT cells are activated by MR1-bound riboflavin metabolite derivatives distinguishes MAIT cells from all other αß T cells in the immune system. Since mammals lack the capacity to synthesize riboflavin, intermediates from the riboflavin biosynthetic pathway are distinct microbial molecular patterns that provide a unique signal to the immune system. Multiple lines of evidence suggest that MAIT cells, which produce important cytokines such as IFN-γ, TNF, and IL-17A, have the potential to influence immune responses to a broad range of pathogens. Here we will discuss our current understanding of MAIT cell biology and their role in pathogen defense.

Enhanced efficacy of BCG vaccine formulated in adjuvant is dependent on IL-17A expression.

A new, more effective vaccine against tuberculosis (TB) is urgently needed to curtail the current TB problem. The only licensed vaccine, BCG, has been shown to have highly variable protective efficacy in several clinical trials ranging from zero to 80 % against TB disease. We have previously reported that BCG formulated in dimethyl dioctadecyl-ammonium bromide (DDA) with D-(+)-Trehalose 6,6'-Dibehenate (TDB) adjuvant (BCG + Adj) is significantly more protective than BCG alone following murine aerosol Mycobacterium tuberculosis infection. Here we investigate the immunological basis for this improved efficacy by examining expression of different immune markers and cytokines in the lungs of vaccinated mice after M. tuberculosis aerosol challenge. We found significantly greater numbers of pulmonary IL-17A-expressing CD4+ T cells in mice immunized with BCG+Adj as compared to nonvaccinated and BCG-immunized mice at one-month post-challenge and that the enhanced protection was abrogated in IL-17A-deficient mice. Furthermore, we found significantly higher levels of IL-17A, IL-12p40 and IL-33 expression in the lungs of BCG + Adj immunized animals relative to nonvaccinated mice after M. tuberculosis challenge. These results demonstrate that the DDA/TDB adjuvant increases expression of IL-17A in response to the BCG vaccine and that these augmented IL-17A levels enhance control of M. tuberculosis infection.

Lung CD4-CD8- double-negative T cells are prominent producers of IL-17A and IFN-gamma during primary respiratory murine infection with Francisella tularensis live vaccine strain.

For several intracellular infections, pulmonary vaccination provides measurably better protection against pulmonary challenge. The unique factors that contribute to pulmonary immune responses are not well characterized. In this study, we show that CD4(-)CD8(-) double negative (DN) T cells are a major responding T cell subset in the lungs of mice during pulmonary Francisella tularensis live vaccine strain (LVS) infection. DN T cells were a minor (<2%) subset in spleens and lungs of mice during sublethal intradermal infection with LVS. In contrast, they were a major responding T cell subset in lungs during pulmonary LVS infection, producing large quantities of IFN-gamma and IL-17A. The numbers of IL-17A(+) DN T cells in the lungs exceeded that of CD4(+) and CD8(+) T cells on day 7 postinfection; by day 14 postinfection, all three IL-17A-producing T cell subsets were present in equivalent numbers. CD4(+), CD8(+), and DN T cell production of IL-17A was not observed in the spleens of pulmonary-infected mice or the lungs and spleens of intradermally infected mice. Correspondingly, IL-17A knockout mice were more susceptible to respiratory than intradermal LVS infection, with delayed clearance 1-3 wk postinfection. Finally, in vitro treatment of LVS-infected macrophages and alveolar type II epithelial cells with IFN-gamma and IL-17A affected significantly greater LVS growth control than treatment with either cytokine alone. The data presented in this study demonstrate that DN cells contribute to production of IL-17A and IFN-gamma in the lungs during inhalational Francisella infection and that these cytokines additively activate host cells to control LVS intracellular growth.

CD4-CD8- T cells control intracellular bacterial infections both in vitro and in vivo.

Memory T cells, including the well-known CD4(+) and CD8(+) T cells, are central components of the acquired immune system and are the basis for successful vaccination. After infection, CD4(+) and CD8(+) T cells expand into effector cells, and then differentiate into long-lived memory cells. We show that a rare population of CD4(-)CD8(-)CD3(+)alphabeta(+)gammadelta(-)NK1.1(-) T cells has similar functions. These cells potently and specifically inhibit the growth of the intracellular bacteria Mycobacterium tuberculosis (M. tb.) or Francisella tularensis Live Vaccine Strain (LVS) in macrophages in vitro, promote survival of mice infected with these organisms in vivo, and adoptively transfer immunity to F. tularensis LVS. Furthermore, these cells expand in the spleens of mice infected with M. tb. or F. tularensis LVS, and then acquire a memory cell phenotype. Thus, CD4(-)CD8(-) T cells have a role in the control of intracellular infection and may contribute to successful vaccination.

Multiple T cell subsets control Francisella tularensis LVS intracellular growth without stimulation through macrophage interferon gamma receptors.

A variety of data suggest that in vivo production of interferon (IFN)-gamma is necessary, but not sufficient, for expression of secondary protective immunity against intracellular pathogens. To discover specific IFN-gamma-independent T cell mediated mechanisms, we took advantage of an in vitro culture system that models in vivo immune responses to the intracellular bacterium Francisella tularensis live vaccine strain (LVS). LVS-immune lymphocytes specifically controlled 99% of the growth of LVS in wild-type murine bone marrow-derived macrophages. Surprisingly, LVS-immune lymphocytes also inhibited LVS intracellular growth by as much as 95% in macrophages derived from IFN-gamma receptor knockout (IFNgammaR KO) mice. CD8+ T cells, and to a lesser degree CD4+ T cells, controlled LVS intracellular growth in both wild-type and IFNgammaR KO macrophages. Further, a unique population of Thy1+alphabeta+CD4-CD8- cells that was previously suggested to operate during secondary immunity to LVS in vivo strongly controlled LVS intracellular growth in vitro. A large proportion of the inhibition of LVS intracellular growth in IFNgammaR KO macrophages by all three T cell subsets could be attributed to tumor necrosis factor (TNF) alpha. Thus, T cell mechanisms exist that control LVS intracellular growth without acting through the IFN-gamma receptor; such control is due in large part to TNF-alpha, and is partially mediated by a unique double negative T cell subpopulation.

CXCL16 Stimulates Antigen-Induced MAIT Cell Accumulation but Trafficking During Lung Infection Is CXCR6-Independent.

Mucosa-associated invariant T (MAIT) cells are a unique T cell subset that contributes to protective immunity against microbial pathogens, but little is known about the role of chemokines in recruiting MAIT cells to the site of infection. Pulmonary infection with Francisella tularensis live vaccine strain (LVS) stimulates the accrual of large numbers of MAIT cells in the lungs of mice. Using this infection model, we find that MAIT cells are predominantly CXCR6+ but do not require CXCR6 for accumulation in the lungs. However, CXCR6 does contribute to long-term retention of MAIT cells in the airway lumen after clearance of the infection. We also find that MAIT cells are not recruited from secondary lymphoid organs and largely proliferate in situ in the lungs after infection. Nevertheless, the only known ligand for CXCR6, CXCL16, is sufficient to drive MAIT cell accumulation in the lungs in the absence of infection when administered in combination with the MAIT cell antigen 5-OP-RU. Overall, this new data advances the understanding of mechanisms that facilitate MAIT cell accumulation and retention in the lungs.

Artificially induced MAIT cells inhibit M. bovis BCG but not M. tuberculosis during in vivo pulmonary infection.

There is significant interest in targeting MAIT cells with immunostimulatory agents to enhance immune responses. Mycobacterium tuberculosis (M. tb.) is a pervasive respiratory disease that could benefit from treatments that augment immunity. Here we investigate the role of MAIT cells in M. tb. infection and the potential for MAIT cell-targeted immunotherapy to control bacterial burdens. We find that MAIT cells fail to substantially accumulate in the lungs during murine pulmonary M. bovis BCG and M. tb. infections but this defect is overcome by intranasal installation of a TLR2/6 agonist and a MAIT cell antigen. Although artificially induced MAIT cells produce important cytokines in both infections, they control BCG but not M. tb. growth in the lungs. Correspondingly, M. tb.-infected mouse macrophages are relatively resistant to MAIT cell antimicrobial activities in vitro. Thus, MAIT cell antigen-mediated immunotherapy for M. tb. presents a complex challenge.

Microbiota of MR1 deficient mice confer resistance against Clostridium difficile infection.

Clostridium difficile (Cd) infection (CDI) typically occurs after antibiotic usage perturbs the gut microbiota. Mucosa-associated invariant T cells (MAIT) are found in the gut and their development is dependent on Major histocompatibility complex-related protein 1 (MR1) and the host microbiome. Here we were interested in determining whether the absence of MR1 impacts resistance to CDI. To this end, wild-type (WT) and MR1-/- mice were treated with antibiotics and then infected with Cd spores. Surprisingly, MR1-/- mice exhibited resistance to Cd colonization. 16S rRNA gene sequencing of feces revealed inherent differences in microbial composition. This colonization resistance was transferred from MR1-/- to WT mice via fecal microbiota transplantation, suggesting that MR1-dependent factors influence the microbiota, leading to CDI susceptibility.

Innate and adaptive immunity to Francisella.

Studies of immune responses to Francisella have been conducted for well over 50 years. Here, the basic parameters of innate and adaptive immune responses to Francisella are reviewed, with an emphasis on those that may contribute directly to protection against infection. Although older literature provides a wealth of information on human immune responses to infection and vaccination, most recent information has been derived largely from studies in animals and using animal cells, particularly mice. In experimental animals, activation of macrophages, a major and probably preferred host cell for Francisella, appears central to control of infection. Thus, in animal models and in vitro studies using mouse macrophages, cytokines such as IFN-gamma and TNF-alpha, derived first from both nonspecific cells such as natural killer cells and later from Francisella-specific T cells, collaborate to effect intracellular killing. In mice, these intracellular killing mechanisms include reactive nitrogen and oxygen species, but killing mechanisms remain to be identified in humans. Ultimately both CD4(+) and CD8(+) T cells develop into Francisella-specific memory cells and are important for control of primary Francisella infection or vaccination-induced protection. The effector mechanisms invoked by either CD4(+) or CD8(+) T cells, beyond production of IFN-gamma and TNF-alpha, are the subject of ongoing studies. Both specific antibodies and B cells may contribute to control of primary infection or vaccination-induced protection in some circumstances, particularly against lower virulence Francisella strains. Thus a number of known proinflammatory and Th-1 T cell related components of the immune system combat this virulent bacterium; no doubt others remain to be discovered.

MAIT cells promote inflammatory monocyte differentiation into dendritic cells during pulmonary intracellular infection.

Mucosa-associated invariant T (MAIT) cells are a unique innate T cell subset that is necessary for rapid recruitment of activated CD4+ T cells to the lungs after pulmonary F. tularensis LVS infection. Here, we investigated the mechanisms behind this effect. We provide evidence to show that MAIT cells promote early differentiation of CCR2-dependent monocytes into monocyte-derived DCs (Mo-DCs) in the lungs after F. tularensis LVS pulmonary infection. Adoptive transfer of Mo-DCs to MAIT cell-deficient mice (MR1-/- mice) rescued their defect in the recruitment of activated CD4+ T cells to the lungs. We further demonstrate that MAIT cell-dependent GM-CSF production stimulated monocyte differentiation in vitro, and that in vivo production of GM-CSF was delayed in the lungs of MR1-/- mice. Finally, GM-CSF-deficient mice exhibited a defect in monocyte differentiation into Mo-DCs that was phenotypically similar to MR1-/- mice. Overall, our data demonstrate that MAIT cells promote early pulmonary GM-CSF production, which drives the differentiation of inflammatory monocytes into Mo-DCs. Further, this delayed differentiation of Mo-DCs in MR1-/- mice was responsible for the delayed recruitment of activated CD4+ T cells to the lungs. These findings establish a novel mechanism by which MAIT cells function to promote both innate and adaptive immune responses.

Induction of Unconventional T Cells by a Mutant Mycobacterium bovis BCG Strain Formulated in Cationic Liposomes Correlates with Protection against Mycobacterium tuberculosis Infections of Immunocompromised Mice.

Earlier studies aimed at defining protective immunity induced by Mycobacterium bovis BCG immunization have largely focused on the induction of antituberculosis CD4(+) and CD8(+) T cell responses. Here we describe a vaccine consisting of a BCGΔmmaA4 deletion mutant formulated in dimethyl dioctadecyl-ammonium bromide (DDA) with d-(+)-trehalose 6,6'-dibehenate (TDB) (DDA/TDB) adjuvant (A4/Adj) that protected TCRδ(-/-) mice depleted of CD4(+), CD8(+), and NK1.1(+) T cells against an aerosol challenge with M. tuberculosis These mice were significantly protected relative to mice immunized with a nonadjuvanted BCGΔmmaA4 (BCG-A4) mutant and nonvaccinated controls at 2 months and 9 months postvaccination. In the absence of all T cells following treatment with anti-Thy1.2 antibody, the immunized mice lost the ability to control the infection. These results indicate that an unconventional T cell population was mediating protection in the absence of CD4(+), CD8(+), NK1.1(+), and TCRγδ T cells and could exhibit memory. Focusing on CD4(-) CD8(-) double-negative (DN) T cells, we found that these cells accumulated in the lungs postchallenge significantly more in A4/Adj-immunized mice and induced significantly greater frequencies of pulmonary gamma interferon (IFN-γ)-producing cells than were seen in the nonvaccinated or nonadjuvanted BCG control groups. Moreover, pulmonary DN T cells from the A4/Adj group exhibited significantly higher IFN-γ integrated median fluorescence intensity (iMFI) values than were seen in the control groups. We also showed that enriched DN T cells from mice immunized with A4/Adj could control mycobacterial growth in vitro significantly better than naive whole-spleen cells. These results suggest that formulating BCG in DDA/TDB adjuvant confers superior protection in immunocompromised mice and likely involves the induction of long-lived memory DN T cells.

Control of Francisella tularensis Intracellular Growth by Pulmonary Epithelial Cells.

The virulence of F. tularensis is often associated with its ability to grow in macrophages, although recent studies show that Francisella proliferates in multiple host cell types, including pulmonary epithelial cells. Thus far little is known about the requirements for killing of F. tularensis in the non-macrophage host cell types that support replication of this organism. Here we sought to address this question through the use of a murine lung epithelial cell line (TC-1 cells). Our data show that combinations of the cytokines IFN-γ, TNF, and IL-17A activated murine pulmonary epithelial cells to inhibit the intracellular growth of the F. tularensis Live Vaccine Strain (LVS) and the highly virulent F. tularensis Schu S4 strain. Although paired combinations of IFN-γ, TNF, and IL-17A all significantly controlled LVS growth, simultaneous treatment with all three cytokines had the greatest effect on LVS growth inhibition. In contrast, Schu S4 was more resistant to cytokine-induced growth effects, exhibiting significant growth inhibition only in response to all three cytokines. Since one of the main antimicrobial mechanisms of activated macrophages is the release of reactive nitrogen intermediates (RNI) via the activity of iNOS, we investigated the role of RNI and iNOS in Francisella growth control by pulmonary epithelial cells. NOS2 gene expression was significantly up-regulated in infected, cytokine-treated pulmonary epithelial cells in a manner that correlated with LVS and Schu S4 growth control. Treatment of LVS-infected cells with an iNOS inhibitor significantly reversed LVS killing in cytokine-treated cultures. Further, we found that mouse pulmonary epithelial cells produced iNOS during in vivo respiratory LVS infection. Overall, these data demonstrate that lung epithelial cells produce iNOS both in vitro and in vivo, and can inhibit Francisella intracellular growth via reactive nitrogen intermediates.

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS, and contributes to F. novicida murine pathogenesis.

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.

Innate and adaptive immune responses to an intracellular bacterium, Francisella tularensis live vaccine strain.

The immune response to intracellular bacterium, Francisella tularensis, which causes tularemia and is proposed to be a potential bioterrorism pathogen, has been studied in mice using the attenuated live vaccine strain (LVS). Here we review this infection model, which provides a convenient means of studying protective immune mechanisms not only for Francisella, but also for the large and important class of intracellular pathogens.

Expression and localization of the Mycobacterium tuberculosis protein tyrosine phosphatase PtpA.

The Mycobacterium tuberculosis open reading frame Rv2234 encodes a low molecular weight tyrosine phosphatase named PtpA. Kinetic analyses of PtpA activity reveal that it is capable of dephosphorylation of p-nitrophenyl phosphate, as well as a variety of phosphotyrosine-containing substrates. In contrast, PtpA showed no detectable activity towards substrates containing phosphoserine or -threonine residues. Transcriptional analysis reveals that the M. tuberculosis ptpA promoter is expressed in the slow-growing Mycobacterium species M. bovis BCG, but not in the fast-growing species M. smegmatis. Furthermore, ptpA expression is upregulated upon entry of BCG cultures into stationary phase and increases upon infection of human monocytes. We also show that, despite the lack of a general export pathway signal sequence, the M. tuberculosis PtpA protein can be released from both M. tuberculosis and M. smegmatis during growth.

The identification of five genetic loci of Francisella novicida associated with intracellular growth.

Five transposon mutants of Francisella novicida were isolated that are compromised in their ability to grow in mouse macrophages in vitro. Sequence analysis of the DNA flanking the transposon insertions identified the genes that were interrupted in these mutants. One of the inactivated loci corresponds to the Francisella tularensis gene that encodes a 23-kDa protein that is the most prominently induced protein following macrophage infection. Another insertion was localised to approximately 2 kb upstream of the gene encoding the 23-kDa protein. By analysis of the incomplete Francisella genome sequence it was surmised that these two insertions disrupt different portions of a putative operon that encodes four proteins, none of which have discernible functions. Three other interrupted loci associated with poor intramacrophage growth showed similarity at the deduced amino acid level to alanine racemase, the ClpB heat-shock protease, and the purine biosynthetic enzyme, glutamine phosphoribosylpyrophosphate amidotransferases.

Immunity to Francisella.

In recent years, studies on the intracellular pathogen Francisella tularensis have greatly intensified, generating a wealth of new information on the interaction of this organism with the immune system. Here we review the basic elements of the innate and adaptive immune responses that contribute to protective immunity against Francisella species, with special emphasis on new data that has emerged in the last 5 years. Most studies have utilized the mouse model of infection, although there has been an expansion of work on human cells and other new animal models. In mice, basic immune parameters that operate in defense against other intracellular pathogen infections, such as interferon gamma, TNF-α, and reactive nitrogen intermediates, are central for control of Francisella infection. However, new important immune mediators have been revealed, including IL-17A, Toll-like receptor 2, and the inflammasome. Further, a variety of cell types in addition to macrophages are now recognized to support Francisella growth, including epithelial cells and dendritic cells. CD4(+) and CD8(+) T cells are clearly important for control of primary infection and vaccine-induced protection, but new T cell subpopulations and the mechanisms employed by T cells are only beginning to be defined. A significant role for B cells and specific antibodies has been established, although their contribution varies greatly between bacterial strains of lower and higher virulence. Overall, recent data profile a pathogen that is adept at subverting host immune responses, but susceptible to many elements of the immune system's antimicrobial arsenal.

Measurement of macrophage-mediated killing of intracellular bacteria, including Francisella and mycobacteria.

Macrophages activated by T cell cytokines are a critical defense mechanism against intracellular bacterial pathogens. This unit presents two general methods for assessing the capacity of mouse macrophages, activated with either soluble cytokines or whole immune T lymphocytes, to control or reduce numbers of intracellular bacteria residing within them. "Measurement of killing" is inferred from a reduction in the number of colony-forming units (cfu) of bacteria at the end of a culture period, compared to the input numbers of cfu at initiation of culture, to the peak numbers of cfu measured during culture, or to a control group in which killing is expected to be poor.