Endosomal MR1 Trafficking Plays a Key Role in Presentation of Mycobacterium tuberculosis Ligands to MAIT Cells

Mucosal-Associated Invariant T (MAIT) cells, present in high frequency in airway and other mucosal tissues, have Th1 effector capacity positioning them to play a critical role in the early immune response to intracellular pathogens, including Mycobacterium tuberculosis (Mtb). MR1 is a highly conserved Class I-like molecule that presents vitamin B metabolites to MAIT cells. The mechanisms for loading these ubiquitous small molecules are likely to be tightly regulated to prevent inappropriate MAIT cell activation. To define the intracellular localization of MR1, we analyzed the distribution of an MR1-GFP fusion protein in antigen presenting cells. We found that MR1 localized to endosomes and was translocated to the cell surface upon addition of 6-formyl pterin (6-FP). To understand the mechanisms by which MR1 antigens are presented, we used a lentiviral shRNA screen to identify trafficking molecules that are required for the presentation of Mtb antigen to HLA-diverse T cells. We identified Stx18, VAMP4, and Rab6 as trafficking molecules regulating MR1-dependent MAIT cell recognition of Mtb-infected cells. Stx18 but not VAMP4 or Rab6 knockdown also resulted in decreased 6-FP-dependent surface translocation of MR1 suggesting distinct pathways for loading of exogenous ligands and intracellular mycobacterially-derived ligands. We postulate that endosome-mediated trafficking of MR1 allows for selective sampling of the intracellular environment.Career Development Award: (#IK2 CX000538); U.S. Department of Veterans Affairs Clinical Sciences Research and Development Program (MJH); U.S.Department of Veterans Affairs Biomedical Laboratory Research and Development Program (DML) Merit Award: (#I01 BX000533); American Lung Association: (RT-350058)

Role of innate T cells in anti-bacterial immunity

Innate T cells are a heterogeneous group of αβ and γδ T cells that respond rapidly (<2 h) upon activation. These innate T cells also share a non MHC class I or II restriction requirement for antigen recognition. Three major populations within the innate T cell group are recognized, namely, invariant NKT cells, mucosal associated invariant T cells, and gamma delta T cells. These cells recognize foreign/self-lipid presented by non-classical MHC molecules, such as CD1d, MR1, and CD1a. They are activated during the early stages of bacterial infection and act as a bridge between the innate and adaptive immune systems. In this review, we focus on the functional properties of these three innate T cell populations and how they are purposed for antimicrobial defense. Furthermore, we address the mechanisms through which their effector functions are targeted for bacterial control and compare this in human and murine systems. Lastly, we speculate on future roles of these cell types in therapeutic settings such as vaccination

Cellular immune function in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS)

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition with unknown aetiology, Myalgic encephalomyelitis unclear pathophysiology and with no diagnostic test or biomarker available. Many patients report their ME/CFS began after an acute infection, and subsequent increased frequency of infections, such as colds or influenza, is common. These factors imply an altered immunological status exists in ME/CFS, in at least a proportion of patients, yet previous studies of peripheral immunity have been discrepant and inconclusive. The UK ME/CFS Biobank, which has collected blood samples from nearly 300 clinically-confirmed ME/CFS patients, enables large-scale studies of immunological function in phenotypically well-characterised participants. In this study, herpes virus serological status and T cell, B cell, NK cell and monocyte populations were investigated in 251 ME/CFS patients, including 54 who were severely affected, and compared with those from 107 healthy participants and with 46 patients with Multiple Sclerosis. There were no differences in seroprevalence for six human herpes viruses between ME/CFS and healthy controls, although seroprevalence for the Epstein-Barr virus was higher in multiple sclerosis patients. Contrary to previous reports, no significant differences were observed in NK cell numbers, subtype proportions or in vitro responsiveness between ME/CFS patients and healthy control participants. In contrast, the T cell compartment was altered in ME/CFS, with increased proportions of effector memory CD8+ T cells and decreased proportions of terminally differentiated effector CD8+ T cells. Conversely, there was a significantly increased proportion of mucosal associated invariant T cells (MAIT) cells, especially in severely affected ME/CFS patients. These abnormalities demonstrate that an altered immunological state does exist in ME/CFS, particularly in severely affected people. This may simply reflect ongoing or recent infection, or may indicate future increased susceptibility to infection. Longitudinal studies of ME/CFS patients are needed to help to determine cause and effect and thus any potential benefits of immuno-modulatory treatments for ME/CFS

Deficiency in CCR2 increases susceptibility of mice to infection with an intracellular pathogen, Francisella tularensis LVS, but does not impair development of protective immunity.

CCR2 is the major chemokine receptor that regulates appropriate trafficking of inflammatory monocytes, but the role of this chemokine receptor and its ligands during primary and secondary infection with intracellular infections remains incompletely understood. Here we used murine infection with the Live Vaccine Strain (LVS) of Francisella tularensis to evaluate the role of CCR2 during primary and secondary parenteral responses to this prototype intracellular bacterium. We find that mice deficient in CCR2 are highly compromised in their ability to survive intradermal infection with LVS, indicating the importance of this receptor during primary parenteral responses. Interestingly, this defect could not be readily attributed to the activities of the known murine CCR2 ligands MCP-1/CCL2, MCP-3/CCL7, or MCP-5/CCL12. Nonetheless, CCR2 knockout mice vaccinated by infection with low doses of LVS generated optimal T cell responses that controlled the intramacrophage replication of Francisella, and LVS-immune CCR2 knockout mice survived maximal lethal Francisella challenge. Thus, fully protective adaptive immune memory responses to this intracellular bacterium can be readily generated in the absence of CCR2

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-\u3b3, 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-\u3b3, 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.Peer reviewed: YesNRC publication: Ye

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

<i>F</i>. <i>tularensis</i> subspecies <i>tularensis</i> Schu S4-infected pulmonary epithelial cells are relatively resistant to the antimicrobial effects of cytokine treatment.

<p>(A) TC-1 lung epithelial cells were infected with Schu S4, and intracellular bacteria were enumerated after 72 h. Cultures were treated with recombinant cytokines immediately after infection as indicated, or untreated (‘Schu S4 alone’). (B) Changes in NOS2 gene expression in TC-1 cell cultures infected with Schu S4 and treated with recombinant cytokines. Fold induction values are expressed relative to ‘Schu S4 alone’ cultures. * = significantly different from cultures treated only with individual cytokines (P<0.05). Data are expressed as mean ± SD of triplicate cultures, and plots are representative of two experiments of similar design.</p