Alcohol Abstinence Does Not Fully Reverse Abnormalities of Mucosal-Associated Invariant T Cells in the Blood of Patients With Alcoholic Hepatitis

OBJECTIVES: Alcoholic hepatitis (AH) develops in approximately 30% of chronic heavy drinkers. The immune system of patients with AH is hyperactivated, yet ineffective against infectious diseases. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that are highly enriched in liver, mucosa, and peripheral blood and contribute to antimicrobial immunity. We aimed to determine whether MAIT cells were dysregulated in heavy drinkers with and without AH and the effects of alcohol abstinence on MAIT cell recovery. METHODS: MR1 tetramers loaded with a potent MAIT cell ligand 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil were used in multiparameter flow cytometry to analyze peripheral blood MAIT cells in 59 healthy controls (HC), 56 patients with AH, and 45 heavy drinkers without overt liver disease (HDC) at baseline and 6- and 12-month follow-ups. Multiplex immunoassays were used to quantify plasma levels of cytokines related to MAIT cell activation. Kinetic Turbidimetric Limulus Amebocyte Lysate Assay and ELISA were performed to measure circulating levels of 2 surrogate markers for bacterial translocation (lipopolysaccharide and CD14), respectively. RESULTS: At baseline, patients with AH had a significantly lower frequency of MAIT cells than HDC and HC. HDC also had less MAIT cells than HC (median 0.16% in AH, 0.56% in HDC, and 1.25% in HC). Further, the residual MAIT cells in patients with AH expressed higher levels of activation markers (CD69, CD38, and human leukocyte antigen [HLA]-DR), the effector molecule granzyme B, and the immune exhaustion molecule PD-1. Plasma levels of lipopolysaccharide and CD14 and several cytokines related to MAIT cell activation were elevated in patients with AH (interferon [IFN]-α, interleukin [IL]-7, IL-15, IL-17, IL-18, IL-23, IFN-γ, and tumor necrosis factor α). Decreased MAIT cell frequency and upregulated CD38, CD69, and HLA-DR correlated negatively and positively, respectively, with aspartate aminotransferase level. MAIT cell frequency negatively correlated with IL-18. HLA-DR and CD38 levels correlated with several cytokines. At follow-ups, abstinent patients with AH had increased MAIT cell frequency and decreased MAIT cell activation. However, MAIT cell frequency was not fully normalized in patients with AH (median 0.31%). DISCUSSION: We showed that HDC had a reduction of blood MAIT cells despite showing little evidence of immune activation, whereas patients with AH had a severe depletion of blood MAIT cells and the residual cells were highly activated. Alcohol abstinence partially reversed those abnormalities

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)

Polyclonal mucosa-associated invariant T cells have unique innate functions in bacterial infection

Mucosa-associated invariant T (MAIT) cells are a unique population of αβ T cells in mammals that reside preferentially in mucosal tissues and express an invariant Vα paired with limited Vβ T-cell receptor (TCR) chains. Furthermore, MAIT cell development is dependent upon the expression of the evolutionarily conserved major histocompatibility complex (MHC) class Ib molecule MR1. Using in vitro assays, recent studies have shown that mouse and human MAIT cells are activated by antigen-presenting cells (APCs) infected with diverse microbes, including numerous bacterial strains and yeasts, but not viral pathogens. However, whether MAIT cells play an important, and perhaps unique, role in controlling microbial infection has remained unclear. To probe MAIT cell function, we show here that purified polyclonal MAIT cells potently inhibit intracellular bacterial growth of Mycobacterium bovis BCG in macrophages (MΦ) in coculture assays, and this inhibitory activity was dependent upon MAIT cell selection by MR1, secretion of gamma interferon (IFN-γ), and an innate interleukin 12 (IL-12) signal from infected MΦ. Surprisingly, however, the cognate recognition of MR1 by MAIT cells on the infected MΦ was found to play only a minor role in MAIT cell effector function. We also report that MAIT cell-deficient mice had higher bacterial loads at early times after infection compared to wild-type (WT) mice, demonstrating that MAIT cells play a unique role among innate lymphocytes in protective immunity against bacterial infection

Drugs and drug-like molecules can modulate the function of mucosal-associated invariant T cells

The major-histocompatibility-complex-(MHC)-class-I-related molecule MR1 can present activating and non-activating vitamin-B-based ligands to mucosal-associated invariant T cells (MAIT cells). Whether MR1 binds other ligands is unknown. Here we identified a range of small organic molecules, drugs, drug metabolites and drug-like molecules, including salicylates and diclofenac, as MR1-binding ligands. Some of these ligands inhibited MAIT cells ex vivo and in vivo, while others, including diclofenac metabolites, were agonists. Crystal structures of a T cell antigen receptor (TCR) from a MAIT cell in complex with MR1 bound to the non-stimulatory and stimulatory compounds showed distinct ligand orientations and contacts within MR1, which highlighted the versatility of the MR1 binding pocket. The findings demonstrated that MR1 was able to capture chemically diverse structures, spanning mono- and bicyclic compounds, that either inhibited or activated MAIT cells. This indicated that drugs and drug-like molecules can modulate MAIT cell function in mammals

MAIT cells launch a rapid, robust and distinct hyperinflammatory response to bacterial superantigens and quickly acquire an anergic phenotype that impedes their cognate antimicrobial function: Defining a novel mechanism of superantigen-induced immunopathology and immunosuppression

Superantigens (SAgs) are potent exotoxins secreted by Staphylococcus aureus and Streptococcus pyogenes. They target a large fraction of T cell pools to set in motion a "cytokine storm" with severe and sometimes life-threatening consequences typically encountered in toxic shock syndrome (TSS). Given the rapidity with which TSS develops, designing timely and truly targeted therapies for this syndrome requires identification of key mediators of the cytokine storm's initial wave. Equally important, early host responses to SAgs can be accompanied or followed by a state of immunosuppression, which in turn jeopardizes the host's ability to combat and clear infections. Unlike in mouse models, the mechanisms underlying SAg-associated immunosuppression in humans are ill-defined. In this work, we have identified a population of innate-like T cells, called mucosa-associated invariant T (MAIT) cells, as the most powerful source of pro-inflammatory cytokines after exposure to SAgs. We have utilized primary human peripheral blood and hepatic mononuclear cells, mouse MAIT hybridoma lines, HLA-DR4-transgenic mice, MAIThighHLA-DR4+ bone marrow chimeras, and humanized NOD-scid IL-2Rγnull mice to demonstrate for the first time that: i) mouse and human MAIT cells are hyperresponsive to SAgs, typified by staphylococcal enterotoxin B (SEB); ii) the human MAIT cell response to SEB is rapid and far greater in magnitude than that launched by unfractionated conventional T, invariant natural killer T (iNKT) or γδ T cells, and is characterized by production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2, but not IL-17A; iii) high-affinity MHC class II interaction with SAgs, but not MHC-related protein 1 (MR1) participation, is required for MAIT cell activation; iv) MAIT cell responses to SEB can occur in a T cell receptor (TCR) Vβ-specific manner but are largely contributed by IL-12 and IL-18; v) as MAIT cells are primed by SAgs, they also begin to develop a molecular signature consistent with exhaustion and failure to participate in antimicrobial defense. Accordingly, they upregulate lymphocyte-activation gene 3 (LAG-3), T cell immunoglobulin and mucin-3 (TIM-3), and/or programmed cell death-1 (PD-1), and acquire an anergic phenotype that interferes with their cognate function against Klebsiella pneumoniae and Escherichia coli; vi) MAIT cell hyperactivation and anergy co-utilize a signaling pathway that is governed by p38 and MEK1/2. Collectively, our findings demonstrate a pathogenic, rather than protective, role for MAIT cells during infection. Furthermore, we propose a novel mechanism of SAg-associated immunosuppression in humans. MAIT cells may therefore provide an attractive therapeutic target for the management of both early and late phases of severe SAg-mediated illnesses

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

IL-17 in the immunopathogenesis of spondyloarthritis

pondyloarthritis (SpA) is a term that refers to a group of inflammatory diseases that includes psoriatic arthritis, axial SpA and nonradiographic axial SpA, reactive arthritis, enteropathic arthritis and undifferentiated SpA. The disease subtypes share clinical and immunological features, including joint inflammation (peripheral and axial skeleton); skin, gut and eye manifestations; and the absence of diagnostic autoantibodies (seronegative). The diseases also share genetic factors. The aetiology of SpA is still the subject of research by many groups worldwide. Evidence from genetic, experimental and clinical studies has accumulated to indicate a clear role for the IL-17 pathway in the pathogenesis of SpA. The IL-17 family consists of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, of which IL-17A is the best studied. IL-17A is a pro-inflammatory cytokine that also has the capacity to promote angiogenesis and osteoclastogenesis. Of the six family members, IL-17A has the strongest homology with IL-17F. In this Review, we discuss how IL-17A and IL-17F and their cellular sources might contribute to the immunopathology of SpA

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Результаты внедрения новой формы управляемой самостоятельной работы студентов в учебный процесс на кафедре патофизиологии

МЕДИЦИНСКИЕ ИНСТИТУТЫОБУЧЕНИ