RESUMO
Small molecules derived from symbiotic microbiota critically contribute to intestinal immune maturation and regulation1. However, little is known about the molecular mechanisms that control immune development in the host-microbiota environment. Here, using a targeted lipidomic analysis and synthetic approach, we carried out a multifaceted investigation of immunomodulatory α-galactosylceramides from the human symbiont Bacteroides fragilis (BfaGCs). The characteristic terminal branching of BfaGCs is the result of incorporation of branched-chain amino acids taken up in the host gut by B. fragilis. A B. fragilis knockout strain that cannot metabolize branched-chain amino acids showed reduced branching in BfaGCs, and mice monocolonized with this mutant strain had impaired colonic natural killer T (NKT) cell regulation, implying structure-specific immunomodulatory activity. The sphinganine chain branching of BfaGCs is a critical determinant of NKT cell activation, which induces specific immunomodulatory gene expression signatures and effector functions. Co-crystal structure and affinity analyses of CD1d-BfaGC-NKT cell receptor complexes confirmed the interaction of BfaGCs as CD1d-restricted ligands. We present a structural and molecular-level paradigm of immunomodulatory control by interactions of endobiotic metabolites with diet, microbiota and the immune system.
Assuntos
Aminoácidos de Cadeia Ramificada/imunologia , Aminoácidos de Cadeia Ramificada/metabolismo , Bacteroides fragilis/metabolismo , Galactosilceramidas/imunologia , Galactosilceramidas/metabolismo , Microbioma Gastrointestinal/imunologia , Simbiose/imunologia , Aminoácidos de Cadeia Ramificada/química , Animais , Antígenos CD1d/imunologia , Bacteroides fragilis/genética , Humanos , Camundongos , Modelos Animais , Modelos Moleculares , Células T Matadoras Naturais/citologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/imunologiaRESUMO
To broadly measure the spectrum of cellular self-antigens for natural killer T cells (NKT), we developed a sensitive lipidomics system to analyze lipids trapped between CD1d and NKT T cell receptors (TCRs). We captured diverse antigen complexes formed in cells from natural endogenous lipids, with or without inducing endoplasmic reticulum (ER) stress. After separating protein complexes with no, low, or high CD1d-TCR interaction, we eluted lipids to establish the spectrum of self-lipids that facilitate this interaction. Although this unbiased approach identified fifteen molecules, they clustered into only two related groups: previously known phospholipid antigens and unexpected neutral lipid antigens. Mass spectrometry studies identified the neutral lipids as ceramides, deoxyceramides, and diacylglycerols, which can be considered headless lipids because they lack polar headgroups that usually form the TCR epitope. The crystal structure of the TCR-ceramide-CD1d complex showed how the missing headgroup allowed the TCR to predominantly contact CD1d, supporting a model of CD1d autoreactivity. Ceramide and related headless antigens mediated physiological TCR binding affinity, weak NKT cell responses, and tetramer binding to polyclonal human and mouse NKT cells. Ceramide and sphingomyelin are oppositely regulated components of the "sphingomyelin cycle" that are altered during apoptosis, transformation, and ER stress. Thus, the unique molecular link of ceramide to NKT cell response, along with the recent identification of sphingomyelin blockers of NKT cell activation, provide two mutually reinforcing links for NKT cell response to sterile cellular stress conditions.
Assuntos
Antígenos CD1d , Lipidômica , Células T Matadoras Naturais , Receptores de Antígenos de Linfócitos T , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Antígenos CD1d/imunologia , Antígenos CD1d/metabolismo , Animais , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Camundongos , Lipidômica/métodos , Humanos , Autoantígenos/imunologia , Autoantígenos/metabolismo , Ceramidas/metabolismo , Ceramidas/imunologia , Lipídeos/química , Lipídeos/imunologia , Estresse do Retículo Endoplasmático/imunologiaRESUMO
MHC class I-like CD1 molecules have evolved to present lipid-based antigens to T cells. Differences in the antigen-binding clefts of the CD1 family members determine the conformation and size of the lipids that are presented, although the factors that shape CD1 diversity remain unclear. In mice, two homologous genes, CD1D1 and CD1D2, encode the CD1d protein, which is essential to the development and function of natural killer T (NKT) cells. However, it remains unclear whether both CD1d isoforms are equivalent in their antigen presentation capacity and functions. Here, we report that CD1d2 molecules are expressed in the thymus of some mouse strains, where they select functional type I NKT cells. Intriguingly, the T cell antigen receptor repertoire and phenotype of CD1d2-selected type I NKT cells in CD1D1-/- mice differed from CD1d1-selected type I NKT cells. The structures of CD1d2 in complex with endogenous lipids and a truncated acyl-chain analog of α-galactosylceramide revealed that its A'-pocket was restricted in size compared with CD1d1. Accordingly, CD1d2 molecules could not present glycolipid antigens with long acyl chains efficiently, favoring the presentation of short acyl chain antigens. These results indicate that the two CD1d molecules present different sets of self-antigen(s) in the mouse thymus, thereby impacting the development of invariant NKT cells.
Assuntos
Apresentação de Antígeno/imunologia , Antígenos CD1d/fisiologia , Diferenciação Celular , Glicolipídeos/imunologia , Células Matadoras Naturais/imunologia , Timo/imunologia , Animais , Células Cultivadas , Cristalografia por Raios X , Células Matadoras Naturais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Conformação Proteica , Isoformas de Proteínas , Timo/citologiaRESUMO
Mucosal-associated invariant T (MAIT) cells detect microbial infection via recognition of riboflavin-based antigens presented by the major histocompatibility complex class I (MHC-I)-related protein 1 (MR1). Most MAIT cells in human peripheral blood express CD8αα or CD8αß coreceptors, and the binding site for CD8 on MHC-I molecules is relatively conserved in MR1. Yet, there is no direct evidence of CD8 interacting with MR1 or the functional consequences thereof. Similarly, the role of CD8αα in lymphocyte function remains ill-defined. Here, using newly developed MR1 tetramers, mutated at the CD8 binding site, and by determining the crystal structure of MR1-CD8αα, we show that CD8 engaged MR1, analogous to how it engages MHC-I molecules. CD8αα and CD8αß enhanced MR1 binding and cytokine production by MAIT cells. Moreover, the CD8-MR1 interaction was critical for the recognition of folate-derived antigens by other MR1-reactive T cells. Together, our findings suggest that both CD8αα and CD8αß act as functional coreceptors for MAIT and other MR1-reactive T cells.
Assuntos
Células T Invariantes Associadas à Mucosa , Receptores de Antígenos de Linfócitos T alfa-beta , Antígenos , Antígenos CD8 , Linfócitos T CD8-Positivos , Antígenos de Histocompatibilidade Classe I , Humanos , Antígenos de Histocompatibilidade MenorRESUMO
αß and γδ T cell receptors (TCRs) are highly diverse antigen receptors that define two evolutionarily conserved T cell lineages. We describe a population of γµTCRs found exclusively in non-eutherian mammals that consist of a two-domain (Vγ-Cγ) γ-chain paired to a three-domain (Vµ-Vµj-Cµ) µ-chain. γµTCRs were characterized by restricted diversity in the Vγ and Vµj domains and a highly diverse unpaired Vµ domain. Crystal structures of two distinct γµTCRs revealed the structural basis of the association of the γµTCR heterodimer. The Vµ domain shared the characteristics of a single-domain antibody within which the hypervariable CDR3µ loop suggests a major antigen recognition determinant. We define here the molecular basis underpinning the assembly of a third TCR lineage, the γµTCR.
Assuntos
Monodelphis/imunologia , Receptores de Antígenos de Linfócitos T/química , Subpopulações de Linfócitos T/imunologia , Animais , Linhagem da Célula , Regiões Determinantes de Complementaridade/química , Cristalografia por Raios X , Modelos Moleculares , Monodelphis/genética , Conformação Proteica , Domínios Proteicos , Multimerização Proteica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Antígenos de Linfócitos T gama-deltaRESUMO
The function of a protein in a cell often involves coordinated interactions with one or several regulatory partners. It is thus imperative to characterize a protein both in isolation as well as in the context of its complex with an interacting partner. High resolution structural information determined by X-ray crystallography and Nuclear Magnetic Resonance offer the best route to characterize protein complexes. These techniques, however, require highly purified and homogenous protein samples at high concentration. This requirement often presents a major hurdle for structural studies. Here we present a strategy based on co-expression and co-purification to obtain recombinant multi-protein complexes in the quantity and concentration range that can enable hitherto intractable structural projects. The feasibility of this strategy was examined using the σ factor/anti-σ factor protein complexes from Mycobacterium tuberculosis. The approach was successful across a wide range of σ factors and their cognate interacting partners. It thus appears likely that the analysis of these complexes based on variations in expression constructs and procedures for the purification and characterization of these recombinant protein samples would be widely applicable for other multi-protein systems.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Bioquímica/métodos , Mycobacterium tuberculosis/química , Fator sigma/isolamento & purificação , Complexos Multiproteicos/isolamento & purificaçãoRESUMO
Type I and type II natural killer T (NKT) cells are restricted to the lipid antigen-presenting molecule CD1d. While we have an understanding of the antigen reactivity and function of type I NKT cells, our knowledge of type II NKT cells in health and disease remains unclear. Here we describe a population of type II NKT cells that recognise and respond to the microbial antigen, α-glucuronosyl-diacylglycerol (α-GlcADAG) presented by CD1d, but not the prototypical type I NKT cell agonist, α-galactosylceramide. Surprisingly, the crystal structure of a type II NKT TCR-CD1d-α-GlcADAG complex reveals a CD1d F'-pocket-docking mode that contrasts sharply with the previously determined A'-roof positioning of a sulfatide-reactive type II NKT TCR. Our data also suggest that diverse type II NKT TCRs directed against distinct microbial or mammalian lipid antigens adopt multiple recognition strategies on CD1d, thereby maximising the potential for type II NKT cells to detect different lipid antigens.
Assuntos
Antígenos CD1d/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Apresentação de Antígeno , Antígenos CD1d/metabolismo , Cristalografia por Raios X , Citometria de Fluxo , Galactosilceramidas/imunologia , Glicolipídeos/imunologia , Camundongos , Camundongos Knockout , Simulação de Acoplamento Molecular , Células T Matadoras Naturais/metabolismo , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Subpopulações de Linfócitos T/metabolismoRESUMO
T cell receptors (TCRs) recognize antigens presented by major histocompatibility complex (MHC) and MHC class I-like molecules. We describe a diverse population of human γδ T cells isolated from peripheral blood and tissues that exhibit autoreactivity to the monomorphic MHC-related protein 1 (MR1). The crystal structure of a γδTCR-MR1-antigen complex starkly contrasts with all other TCR-MHC and TCR-MHC-I-like complex structures. Namely, the γδTCR binds underneath the MR1 antigen-binding cleft, where contacts are dominated by the MR1 α3 domain. A similar pattern of reactivity was observed for diverse MR1-restricted γδTCRs from multiple individuals. Accordingly, we simultaneously report MR1 as a ligand for human γδ T cells and redefine the parameters for TCR recognition.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Menor/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Cristalografia por Raios X , Células HEK293 , Antígenos de Histocompatibilidade Classe I/química , Humanos , Antígenos de Histocompatibilidade Menor/química , Domínios Proteicos , Receptores de Antígenos de Linfócitos T gama-delta/químicaRESUMO
Crucial to Natural Killer T (NKT) cell function is the interaction between their T-cell receptor (TCR) and CD1d-antigen complex. However, the diversity of the NKT cell repertoire and the ensuing interactions with CD1d-antigen remain unclear. We describe an atypical population of CD1d-α-galactosylceramide (α-GalCer)-reactive human NKT cells that differ markedly from the prototypical TRAV10-TRAJ18-TRBV25-1(+) type I NKT cell repertoire. These cells express a range of TCR α- and ß-chains that show differential recognition of glycolipid antigens. Two atypical NKT TCRs (TRAV21-TRAJ8-TRBV7-8 and TRAV12-3-TRAJ27-TRBV6-5) bind orthogonally over the A'-pocket of CD1d, adopting distinct docking modes that contrast with the docking mode of all type I NKT TCR-CD1d-antigen complexes. Moreover, the interactions with α-GalCer differ between the type I and these atypical NKT TCRs. Accordingly, diverse NKT TCR repertoire usage manifests in varied docking strategies and specificities towards CD1d-α-GalCer and related antigens, thus providing far greater scope for diverse glycolipid antigen recognition.
Assuntos
Antígenos CD1d/imunologia , Galactosilceramidas/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Cristalografia por Raios X , Gangliosídeos/imunologia , Glucosilceramidas/imunologia , Humanos , Lipídeos/imunologia , Simulação de Acoplamento Molecular , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Ressonância de Plasmônio de SuperfícieAssuntos
Proteínas de Bactérias/química , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Corismato Mutase/química , Cristalografia por Raios X , Proteínas Mitocondriais/química , Modelos Moleculares , Dados de Sequência Molecular , Mycobacterium tuberculosis/genética , Proteínas de Saccharomyces cerevisiae/química , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Background: With a steep fall in maternal mortality and morbidity and with much more liberalization of indications, the incidence of cesarean section rate has greatly increased over the last thirty years and almost doubled in the current decade. The objective of this study was to study incidence of maternal morbidity in emergency lower segment cesarean section.Methods: Hospital based prospective study was carried out among 200 women undergoing emergency lower segment cesarean section. Data relating to demographic characteristics, clinical characteristics, maternal and fetal indications, incidence of morbidity, and types of morbidities, Intra operative complications, and Post operative complications was noted down. Chi square and odds ratio was used for statistical analysis.Results: The incidence of LSCS was 24.21%. Incidence of emergency LSCS was 96.74%. 71% were having primary LSCS and 20.5% were booked cases. The most common maternal indication for emergency LSCS was pregnancy induced hypertension and eclampsia in 32.5% of the cases. The most common fetal indication for emergency LSCS was fetal distress in 60%. Incidence of morbidity was 35% and it was associated with booking status, parity and social class. Incidence of intraoperative complications was 23.5%. Majority (18.5%) developed febrile morbidity followed by wound sepsis in 12.5%, urinary tract infection in 8%, mastitis in 7.5%, respiratory tract infection in 7%, wound gaping in 4%, paralytic ileus in 3%, endometritis in 2.5%, postpartum hemorrhage in 1.5%, 2 cases of burst abdomen and one case of small bowel obstruction.Conclusions: Emergency LSCS was more common than elective LSCS and it was associated with booking status, parity and social class.
RESUMO
An effective transcriptional response to redox stimuli is of particular importance for Mycobacterium tuberculosis, as it adapts to the environment of host alveoli and macrophages. The M. tuberculosis sigma factor sigma(L) regulates the expression of genes involved in cell-wall and polyketide syntheses. sigma(L) interacts with the cytosolic anti-sigma domain of a membrane-associated protein, RslA. Here we demonstrate that RslA binds Zn(2+) and can sequester sigma(L) in a reducing environment. In response to an oxidative stimulus, proximal cysteines in the CXXC motif of RslA form a disulfide bond, releasing bound Zn(2+). This results in a substantial rearrangement of the sigma(L)/RslA complex, leading to an 8-fold decrease in the affinity of RslA for sigma(L). The crystal structure of the -35-element recognition domain of sigma(L), sigma(4)(L), bound to RslA reveals that RslA inactivates sigma(L) by sterically occluding promoter DNA and RNA polymerase binding sites. The crystal structure further reveals that the cysteine residues that coordinate Zn(2+) in RslA are solvent exposed in the complex, thus providing a structural basis for the redox sensitivity of RslA. The biophysical parameters of sigma(L)/RslA interactions provide a template for understanding how variations in the rate of Zn(2+) release and associated conformational changes could regulate the activity of a Zn(2+)-associated anti-sigma factor.