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.

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.

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.

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.

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.

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.

Infection of mice with Francisella as an immunological model.

This unit describes the utility of various mouse models of infection for studying pathogenesis and adaptive immune responses to the facultative intracellular bacteria pathogen Francisella tularensis. By judicious use of different combinations of mouse and bacterial strains, as well as different routes of infection, murine tularemia models may be used to explore a complete picture of F. tularensis infection and immunity. Moreover, studies using Francisella, particularly the Live Vaccine Strain (LVS), serve as a convenient and tractable model system that appears to be representative of mammalian host responses to intracellular pathogens in general.

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.

Survival of secondary lethal systemic Francisella LVS challenge depends largely on interferon gamma.

Although survival of primary infection with the live vaccine strain (LVS) of Francisella tularensis depends on interferon gamma (IFN-gamma), the relative importance of IFN-gamma to secondary protective immunity in vivo has not been clearly established. Here we examine the role of IFN-gamma in T cell priming and expression of vaccine-induced protection against lethal intraperitoneal challenge of mice. Large amounts of IFN-gamma were detected between days 3 and 7 in the sera of LVS-immunized mice, while relatively small amounts were found transiently after secondary LVS challenge. Consistent with the production of this cytokine, mice lacking IFN-gamma (gamma interferon knockout, GKO, mice) could not be successfully vaccinated with LVS or an attenuated mglA mutant of F. novicida to withstand secondary Francisella LVS challenge. Further, splenocytes from such primed mice did not adoptively transfer protection to naive GKO recipient mice in vivo, nor control the intramacrophage growth of LVS in vitro. Finally, LVS-immune WT mice depleted of IFN-gamma prior to intraperitoneal challenge survived only the lowest doses of challenge. Thus successful priming of protective LVS-immune T cells, as well as complete expression of protection against Francisella during secondary challenge, depends heavily on IFN-gamma.

The membrane form of tumor necrosis factor is sufficient to mediate partial innate immunity to Francisella tularensis live vaccine strain.

Here we characterize Francisella tularensis live vaccine strain (LVS) infection in total tumor necrosis factor (TNF) knockout (KO) mice and in transgenic mice expressing only the membrane form of TNF (memTNF). MemTNF mice, but not TNF KO mice, survived low-dose, sublethal LVS infections. Splenic nitric oxide production was impaired in infected memTNF mice and was absent in infected TNF KO mice. Spleen cell production of interferon-gamma, RANTES, and monocyte chemotactic protein-1 was elevated in TNF KO mice, compared with that in WT mice, by days 4-5 after infection, along with transiently increased numbers of CCR2(+) cells, whereas memTNF mice had an intermediate phenotype. By day 6 after infection, TNF KO mice, but not memTNF mice, exhibited massive apoptosis in spleens and livers, which shortly preceded their death. Thus, memTNF partially functions to regulate chemokine expression, cell recruitment, and nitric oxide production during primary LVS infection and protects against the induction of apoptosis observed in TNF KO mice.

Differential requirements by CD4+ and CD8+ T cells for soluble and membrane TNF in control of Francisella tularensis live vaccine strain intramacrophage growth.

During primary infection with intracellular bacteria, the membrane-associated form of TNF provides some TNF functions, but the relative contributions during memory responses are not well-characterized. In this study, we determined the role of T cell-derived secreted and membrane-bound TNF (memTNF) during adaptive immunity to Francisella tularensis live vaccine strain (LVS). Although transgenic mice expressing only the memTNF were more susceptible to primary LVS infection than wild-type (WT) mice, LVS-immune WT and memTNF mice both survived maximal lethal secondary Francisella challenge. Generation of CD44(high) memory T cells and clearance of bacteria were similar, although more IFN-gamma and IL-12(p40) were produced by memTNF mice. To examine T cell function, we used an in vitro tissue coculture system that measures control of LVS intramacrophage growth by LVS-immune WT and memTNF-T cells. LVS-immune CD4(+) and CD8(+) T cells isolated from WT and memTNF mice exhibited comparable control of LVS growth in either normal or TNF-alpha knockout macrophages. Although the magnitude of CD4(+) T cell-induced macrophage NO production clearly depended on TNF, control of LVS growth by both CD4(+) and CD8(+) T cells did not correlate with levels of nitrite. Importantly, intramacrophage LVS growth control by CD8(+) T cells, but not CD4(+) T cells, was almost entirely dependent on T cell-expressed TNF, and required stimulation through macrophage TNFRs. Collectively, these data demonstrate that T cell-expressed memTNF is necessary and sufficient for memory T cell responses to this intracellular pathogen, and is particularly important for intramacrophage control of bacterial growth by CD8(+) T cells.

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.

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.

A Francisella tularensis pathogenicity island required for intramacrophage growth.

Francisella tularensis is a gram-negative, facultative intracellular pathogen that causes the highly infectious zoonotic disease tularemia. We have discovered a ca. 30-kb pathogenicity island of F. tularensis (FPI) that includes four large open reading frames (ORFs) of 2.5 to 3.9 kb and 13 ORFs of 1.5 kb or smaller. Previously, two small genes located near the center of the FPI were shown to be needed for intramacrophage growth. In this work we show that two of the large ORFs, located toward the ends of the FPI, are needed for virulence. Although most genes in the FPI encode proteins with amino acid sequences that are highly conserved between high- and low-virulence strains, one of the FPI genes is present in highly virulent type A F. tularensis, absent in moderately virulent type B F. tularensis, and altered in F. tularensis subsp. novicida, which is highly virulent for mice but avirulent for humans. The G+C content of a 17.7-kb stretch of the FPI is 26.6%, which is 6.6% below the average G+C content of the F. tularensis genome. This extremely low G+C content suggests that the DNA was imported from a microbe with a very low G+C-containing chromosome.

CD4+ T cells mediate IFN-gamma-independent control of Mycobacterium tuberculosis infection both in vitro and in vivo.

Although IFN-gamma is necessary for survival of Mycobacterium tuberculosis infection in people and animal models, it may not be sufficient to clear the infection, and IFN-gamma is not a reliable correlate of protection. To determine whether IFN-gamma-independent mechanisms of immunity exist, we developed a murine ex vivo culture system that directly evaluates the ability of splenic or lung lymphocytes to control the growth of M. tuberculosis within infected macrophages, and that models in vivo immunity to tuberculosis. Surprisingly, CD4(+) T cells controlled >90% of intracellular M. tuberculosis growth in the complete absence of IFN-gamma stimulation of macrophages, via a NO-dependent mechanism. Furthermore, bacillus Calmette-Guerin-vaccinated IFN-gamma-deficient mice exhibited significant protection against M. tuberculosis challenge that was lost upon depletion of CD4(+) T cells. These findings demonstrate that CD4(+) T cells possess IFN-gamma-independent mechanisms that can limit the growth of an intracellular pathogen and are dominant in secondary responses to M. tuberculosis.

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.

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.