Pregnancy enables antibody protection against intracellular infection.

Adaptive immune components are thought to exert non-overlapping roles in antimicrobial host defence, with antibodies targeting pathogens in the extracellular environment and T cells eliminating infection inside cells1,2. Reliance on antibodies for vertically transferred immunity from mothers to babies may explain neonatal susceptibility to intracellular infections3,4. Here we show that pregnancy-induced post-translational antibody modification enables protection against the prototypical intracellular pathogen Listeria monocytogenes. Infection susceptibility was reversed in neonatal mice born to preconceptually primed mothers possessing L. monocytogenes-specific IgG or after passive transfer of antibodies from primed pregnant, but not virgin, mice. Although maternal B cells were essential for producing IgGs that mediate vertically transferred protection, they were dispensable for antibody acquisition of protective function, which instead required sialic acid acetyl esterase5 to deacetylate terminal sialic acid residues on IgG variable-region N-linked glycans. Deacetylated L. monocytogenes-specific IgG protected neonates through the sialic acid receptor CD226,7, which suppressed IL-10 production by B cells leading to antibody-mediated protection. Consideration of the maternal-fetal dyad as a joined immunological unit reveals protective roles for antibodies against intracellular infection and fine-tuned adaptations to enhance host defence during pregnancy and early life.

Keeping the host alive - lessons from obligate intracellular bacterial pathogens.

Mammals have evolved sophisticated host cell death signaling pathways as an important immune mechanism to recognize and eliminate cell intruders before they establish their replicative niche. However, intracellular bacterial pathogens that have co-evolved with their host have developed a multitude of tactics to counteract this defense strategy to facilitate their survival and replication. This requires manipulation of pro-death and pro-survival host signaling pathways during infection. Obligate intracellular bacterial pathogens are organisms that absolutely require an eukaryotic host to survive and replicate, and therefore they have developed virulence factors to prevent diverse forms of host cell death and conserve their replicative niche. This review encapsulates our current understanding of these host-pathogen interactions by exploring the most relevant findings of Anaplasma spp., Chlamydia spp., Rickettsia spp. and Coxiella burnetii modulating host cell death pathways. A detailed comprehension of the molecular mechanisms through which these obligate intracellular pathogens manipulate regulated host cell death will not only increase the current understanding of these difficult-to-study pathogens but also provide insights into new tools to study regulated cell death and the development of new therapeutic approaches to control infection.

Intravacuolar Pathogens Hijack Host Extracellular Vesicle Biogenesis to Secrete Virulence Factors.

Extracellular vesicles (EVs) have garnered significant interest in recent years due to their contributions to cell-to-cell communication and disease processes. EVs are composed of a complex profile of bioactive molecules, which include lipids, nucleic acids, metabolites, and proteins. Although the biogenesis of EVs released by cells under various normal and abnormal conditions has been well-studied, there is incomplete knowledge about how infection influences EV biogenesis. EVs from infected cells contain specific molecules of both host and pathogen origin that may contribute to pathogenesis and the elicitation of the host immune response. Intracellular pathogens exhibit diverse lifestyles that undoubtedly dictate the mechanisms by which their molecules enter the cell's exosome biogenesis schemes. We will discuss the current understanding of the mechanisms used during infection to traffic molecules from their vacuolar niche to host EVs by selected intravacuolar pathogens. We initially review general exosome biogenesis schemes and then discuss what is known about EV biogenesis in Mycobacterium, Plasmodium, Toxoplasma, and Leishmania infections, which are pathogens that reside within membrane delimited compartments in phagocytes at some time in their life cycle within mammalian hosts. The review includes discussion of the need for further studies into the biogenesis of EVs to better understand the contributions of these vesicles to host-pathogen interactions, and to uncover potential therapeutic targets to control these pathogens.

Intracellular and Extracellular Roles of Granzyme K.

Granzymes are a family of serine proteases stored in granules inside cytotoxic cells of the immune system. Granzyme K (GrK) has been only limitedly characterized and knowledge on its molecular functions is emerging. Traditionally GrK is described as a granule-secreted, pro-apoptotic serine protease. However, accumulating evidence is redefining the functions of GrK by the discovery of novel intracellular (e.g. cytotoxicity, inhibition of viral replication) and extracellular roles (e.g. endothelial activation and modulation of a pro-inflammatory immune cytokine response). Moreover, elevated GrK levels are associated with disease, including viral and bacterial infections, airway inflammation and thermal injury. This review aims to summarize and discuss the current knowledge of i) intracellular and extracellular GrK activity, ii) cytotoxic and non-cytotoxic GrK functioning, iii) the role of GrK in disease, and iv) GrK as a potential therapeutic target.

G3BP1 Inhibition Alleviates Intracellular Nucleic Acid-Induced Autoimmune Responses.

The detection of intracellular nucleic acids is a fundamental mechanism of host defense against infections. The dysregulated nucleic acid sensing, however, is a major cause for a number of autoimmune diseases. In this study, we report that GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) is critical for both intracellular DNA- and RNA-induced immune responses. We found that in both human and mouse cells, the deletion of G3BP1 led to the dampened cGAS activation by DNA and the insufficient binding of RNA by RIG-I. We further found that resveratrol (RSVL), a natural compound found in grape skin, suppressed both intracellular DNA- and RNA-induced type I IFN production through inhibiting G3BP1. Importantly, using experimental mouse models for Aicardi-Goutières syndrome, an autoimmune disorder found in humans, we demonstrated that RSVL effectively alleviated intracellular nucleic acid-stimulated autoimmune responses. Thus, our study demonstrated a broader role of G3BP1 in sensing different kinds of intracellular nucleic acids and presented RSVL as a potential treatment for autoimmune conditions caused by dysregulated nucleic acid sensing.

Acid-electrolyzed functional water-induces Interleukin-1α release from Intracellular Storage Sites in Oral Squamous Cell Carcinoma.

The aim of this study was to examine the acid-electrolyzed functional water (FW)-mediated cytokine release in an oral squamous cell carcinoma-derived cell line (OSCC) following treatment with FW. FW is generated by the electrolysis of a sodium chloride solution and accelerate the burn wound healing. To elucidate the underlying mechanisms, the cytokine/chemokine secretion profile of HSC3 cells was examined using a cytokine array. FW treatment significantly induced interleukin (IL)-1α secretion, which was confirmed by enzyme-linked immunosorbent assay. Subsequently, the HSC3 cells were pre-treated with cycloheximide (CHX) for 1 h prior to FW stimulation to determine whether the augmented IL-1α secretion was due to enhanced protein synthesis. CHX pre-treatment did not affect IL-1α secretion suggesting that the secreted IL-1α might have been derived from intracellular storage sites. The amount of IL-1α in the cell lysate of the FW-treated HSC3 cells was significantly lower than that of the non-treated cells. Immunofluorescence staining using a polyclonal antibody against full-length IL-1α revealed a drastic reduction in IL-1α inside the FW- treated cells. IL-1α is synthesized in its precursor form (pIL-1α) and cleaved to produce pro-piece and mature IL-1α (ppIL-1α and mIL-1α) inside the cells. In the present study, only pIL-1α was detected within the HSC3 cells in its resting state. However, FW stimulation resulted in the release of the 33 kDa and two other smaller forms (about 19 kDa) of the protein. These results indicates that FW treatment induces IL-1α secretion, a typical alarmin, from the intracellular storage in OSCC cells.

Autoantibodies Targeting Intracellular and Extracellular Proteins in Autoimmunity.

Detecting autoantibodies provides foundational information for the diagnosis of most autoimmune diseases. An important pathophysiological distinction is whether autoantibodies are directed against extracellular or intracellular proteins. Autoantibodies targeting extracellular domains of proteins, such as membrane receptors, channels or secreted molecules are often directly pathogenic, whereby autoantibody binding to the autoantigen disrupts the normal function of a critical protein or pathway, and/or triggers antibody-dependent cell surface complement killing. By comparison, autoantibodies directed against intracellular proteins are recognized as useful diagnostic biomarkers of abnormal autoimmune activity, but the link between antigenicity and pathogenicity is less straightforward. Because intracellular autoantigens are generally inaccessible to autoantibody binding, for the most part, they do not directly contribute to pathogenesis. In a few diseases, autoantibodies to intracellular targets cause damage indirectly by immune complex formation, immune activation, and other processes. In this review, the general features of and differences between autoimmune diseases segregated on the basis of intracellular or extracellular autoantigens are explored using over twenty examples. Expression profiles of autoantigens in relation to the tissues targeted by autoimmune disease and the temporal appearance of autoantibodies before clinical diagnosis often correlate with whether the respective autoantibodies mostly recognize either intracellular or extracellular autoantigens. In addition, current therapeutic strategies are discussed from this vantage point. One drug, rituximab, depletes CD20+ B-cells and is highly effective for autoimmune disorders associated with autoantibodies against extracellular autoantigens. In contrast, diseases associated with autoantibodies directed predominately against intracellular autoantigens show much more complex immune cell involvement, such as T-cell mediated tissue damage, and require different strategies for optimal therapeutic benefit. Understanding the clinical ramifications of autoimmunity derived by autoantibodies against either intracellular or extracellular autoantigens, or a spectrum of both, has practical implications for guiding drug development, generating monitoring tools, stratification of patient interventions, and designing trials based on predictive autoantibody profiles for autoimmune diseases.

Niche-specific MHC II and PD-L1 regulate CD4+CD8αα+ intraepithelial lymphocyte differentiation.

Conventional CD4+ T cells are differentiated into CD4+CD8αα+ intraepithelial lymphocytes (IELs) in the intestine; however, the roles of intestinal epithelial cells (IECs) are poorly understood. Here, we showed that IECs expressed MHC class II (MHC II) and programmed death-ligand 1 (PD-L1) induced by the microbiota and IFN-γ in the distal part of the small intestine, where CD4+ T cells were transformed into CD4+CD8αα+ IELs. Therefore, IEC-specific deletion of MHC II and PD-L1 hindered the development of CD4+CD8αα+ IELs. Intracellularly, PD-1 signals supported the acquisition of CD8αα by down-regulating the CD4-lineage transcription factor, T helper-inducing POZ/Krüppel-like factor (ThPOK), via the Src homology 2 domain-containing tyrosine phosphatase (SHP) pathway. Our results demonstrate that noncanonical antigen presentation with cosignals from IECs constitutes niche adaptation signals to develop tissue-resident CD4+CD8αα+ IELs.

Cytoplasmic-translocated Ku70 senses intracellular DNA and mediates interferon-lambda1 induction.

We have previously identified that human Ku70, a nuclear protein, serves as a cytosolic DNA sensor. Upon transfection with DNA or infection with DNA virus, Ku70 translocates from the nucleus into the cytoplasm and then predominately induces interferon lambda1 (IFN-λ1) rather than IFN-alpha or IFN-beta, through a STING-dependent signalling pathway. However, a detailed mechanism for Ku70 cytoplasmic translocation and its correlation with IFN-λ1 induction have not been fully elucidated. Here, we observed that cytoplasmic translocation of Ku70 only occurred in DNA-triggered IFN-λ1-inducible cells. Additionally, infection by Herpes simplex virus type-1 (HSV-1), a DNA virus, induces cytoplasmic translocation of Ku70 and IFN-λ1 induction in a strain-dependent manner: the translocation and IFN-λ1 induction were detected upon infection by HSV-1 McKrae, but not MacIntyre, strain. A kinetic analysis indicated that cytoplasmic translocation of Ku70 was initiated right after DNA transfection and was peaked at 6 hr after DNA stimulation. Furthermore, treatment with leptomycin B, a nuclear export inhibitor, inhibited both Ku70 translocation and IFN-λ1 induction, suggesting that Ku70 translocation is an essential and early event for its cytosolic DNA sensing. We further confirmed that enhancing the acetylation status of the cells promotes Ku70's cytoplasmic accumulation, and therefore increases DNA-mediated IFN-λ1 induction. These findings provide insights into the molecular mechanism by which the versatile sensor detects pathogenic DNA in a localization-dependent manner.

Transforming nanobodies into high-precision tools for protein function analysis.

Single-domain antibodies, derived from camelid heavy antibodies (nanobodies) or shark variable new antigen receptors, have attracted increasing attention in recent years due to their extremely versatile nature and the opportunities they offer for downstream modification. Discovered more than three decades ago, these 120-amino acid (∼15-kDa) antibody fragments are known to bind their target with high specificity and affinity. Key features of nanobodies that make them very attractive include their single-domain nature, small size, and affordable high-level expression in prokaryotes, and their cDNAs are routinely obtained in the process of their isolation. This facilitates and stimulates new experimental approaches. Hence, it allows researchers to formulate new answers to complex biomedical questions. Through elementary PCR-based technologies and chemical modification strategies, their primary structure can be altered almost at leisure while retaining their specificity and biological activity, transforming them into highly tailored tools that meet the increasing demands of current-day biomedical research. In this review, various aspects of camelid nanobodies are expounded, including intracellular delivery in recombinant format for manipulation of, i.e., cytoplasmic targets, their derivatization to improve nanobody orientation as a capturing device, approaches to reversibly bind their target, their potential as protein-silencing devices in cells, the development of strategies to transfer nanobodies through the blood-brain barrier and their application in CAR-T experimentation. We also discuss some of their disadvantages and conclude with future prospects.

An Inside Job: Applications of Intracellular Single Domain Antibodies.

Sera of camelid species contain a special kind of antibody that consists only of heavy chains. The variable antigen binding domain of these heavy chain antibodies can be expressed as a separate entity, called a single domain antibody that is characterized by its small size, high solubility and oftentimes exceptional stability. Because of this, most single domain antibodies fold correctly when expressed in the reducing environment of the cytoplasm, and thereby retain their antigen binding specificity. Single domain antibodies can thus be used to target a broad range of intracellular proteins. Such intracellular single domain antibodies are also known as intrabodies, and have proven to be highly useful tools for basic research by allowing visualization, disruption and even targeted degradation of intracellular proteins. Furthermore, intrabodies can be used to uncover prospective new therapeutic targets and have the potential to be applied in therapeutic settings in the future. In this review we provide a brief overview of recent advances in the field of intracellular single domain antibodies, focusing on their use as research tools and potential therapeutic applications. Special attention is given to the available methods that allow delivery of single domain antibodies into cells.

Covering All the Bases: Complementary MR1 Antigen Presentation Pathways Sample Diverse Antigens and Intracellular Compartments.

The ubiquitously expressed, monomorphic MHC class Ib molecule MHC class I-related protein 1 (MR1) presents microbial metabolites to mucosal-associated invariant T (MAIT) cells. However, recent work demonstrates that both the ligands bound by MR1 and the T cells restricted by it are more diverse than originally thought. It is becoming increasingly clear that MR1 is capable of presenting a remarkable variety of both microbial and non-microbial small molecule antigens to a diverse group of MR1-restricted T cells (MR1Ts) and that the antigen presentation pathway differs between exogenously delivered antigen and intracellular microbial infection. These distinct antigen presentation pathways suggest that MR1 shares features of both MHC class I and MHC class II antigen presentation, enabling it to sample diverse intracellular compartments and capture antigen of both intracellular and extracellular origin. Here, we review recent developments and new insights into the cellular mechanisms of MR1-dependent antigen presentation with a focus on microbial MR1T cell antigens.

Circulating versus lipopolysaccharide-induced inflammatory markers as correlates of subthreshold depressive symptoms in older adults.

Objectives: Circulating cytokines have been associated with depression, but their detection has limitations, which may be overcome by direct detection of intracellular cytokines (ICCs) after lipopolysaccharide (LPS) stimulation in vitro. This study compared circulating versus LPS-induced inflammatory markers as correlates of subthreshold depressive symptoms.Methods: Secondary data analysis of a cross-sectional insomnia study in healthy community-dwelling older adults was conducted. In 117 participants (≥55 years), plasma tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), C-reactive protein (CRP) and in vitro LPS-induced monocyte production of IL-6 and TNF-α were assayed. Depressive symptoms were assessed using the clinician-rated Inventory of Depressive Symptomatology (IDS-C). Multivariate linear regression was conducted to test the associations between inflammatory markers and subthreshold depressive symptoms in the entire sample as well as in subgroups stratified into higher and lower inflammation levels.Results: LPS-induced TNF-α (adjusted ß = 0.28, p = .04), IL-6 (0.29, p = .03) and TNF-α + IL-6 (0.43, p = .001) significantly positively correlated with subthreshold depressive symptoms only in higher inflammation subgroups. No circulating biomarkers positively correlated in any subgroups. In the entire sample, no biomarkers were significantly associated with subthreshold depressive symptoms.Conclusions: LPS-induced cytokines may be more sensitive correlates of subthreshold depressive symptoms than circulating cytokines, particularly in older adults with higher systemic inflammation.Clinical Trials Registry: ClinicalTrials.gov NCT00280020.

HIF-1α as a central mediator of cellular resistance to intracellular pathogens.

Hypoxia-inducible transcription factor-1α (HIF-1α) was originally identified as a master regulator of cellular responses to hypoxia. More recently, HIF-1α has emerged as a critical regulator of immune cell function that couples shifts in cellular metabolism to cell type-specific transcriptional outputs. Activation of macrophages with inflammatory stimuli leads to induction of the metabolic program aerobic glycolysis and to HIF-1α stabilization, which reinforce one another in a positive feedback loop that helps drive macrophage activation. This activation of aerobic glycolysis and HIF-1α is important both for production of inflammatory cytokines, such as IL-1ß, and for cell intrinsic control of infection. Here, we review the importance of HIF-1α for control of bacterial, fungal, and protozoan intracellular pathogens, highlighting recent findings that reveal mechanisms by which HIF-1α is activated during infection and how HIF-1α coordinates antimicrobial responses of macrophages.

Metabolic reprogramming: an innate cellular defence mechanism against intracellular bacteria?

The limited metabolic resources of a cell represent an intriguing 'conflict of interest' during host-pathogen interactions, as the battle for nutrients might determine the outcome of an infection. To adapt their metabolic needs, innate immune cells such as monocytes, macrophages or dendritic cells reprogram their metabolism upon activation by microbial compounds. In turn, infection by intracellular bacteria provokes metabolic alterations of the host cell that benefit the pathogen. Here, we discuss the state-of-the-art knowledge on metabolic reprogramming of host cells upon activation or infection with intracellular bacteria. The study of the host-driven and pathogen-driven metabolic alterations that seem to co-exist during infection is an emerging field that will define the metabolic pathways that might be targeted to combat infection.

Apoptosis inhibition by intracellular bacteria and its consequence on host immunity.

Regulated cell death via apoptosis not only is important for organismal homeostasis but also serves as an innate defense mechanism. The engulfment of apoptotic infected cells, a process known as efferocytosis, is a common pathway for the destruction of many intracellular bacteria. Some pathogens take advantage of efferocytosis to prevent activation of macrophages and thereby facilitate their dissemination. Conversely, many obligate intracellular bacterial pathogens and some facultative-intracellular bacteria inhibit apoptosis, preventing efferocytosis, and evading innate host defenses. The molecular mechanism of bacterial effectors includes secreted proteins that bind to and inhibit apoptosis cell signaling pathways. We provide an overview of the known bacterial effectors, their host cell targets and their importance for the virulence of human pathogens.

Autophagy and Its Interaction With Intracellular Bacterial Pathogens.

Cellular responses to stress can be defined by the overwhelming number of changes that cells go through upon contact with and stressful conditions such as infection and modifications in nutritional status. One of the main cellular responses to stress is autophagy. Much progress has been made in the understanding of the mechanisms involved in the induction of autophagy during infection by intracellular bacteria. This review aims to discuss recent findings on the role of autophagy as a cellular response to intracellular bacterial pathogens such as, Streptococcus pyogenes, Mycobacterium tuberculosis, Shigella flexneri, Salmonella typhimurium, Listeria monocytogenes, and Legionella pneumophila, how the autophagic machinery senses these bacteria directly or indirectly (through the detection of bacteria-induced nutritional stress), and how some of these bacterial pathogens manage to escape from autophagy.

Intracellular complement activation-An alarm raising mechanism?

It has become increasingly apparent that the complement system, being an ancient defense mechanism, is not operative only in the extracellular milieu but also intracellularly. In addition to the known synthetic machinery in the liver and by macrophages, many other cell types, including lymphocytes, adipocytes and epithelial cells produce selected complement components. Activation of e.g. C3 and C5 inside cells may have multiple effects ranging from direct antimicrobial defense to cell differentiation and possible influence on metabolism. Intracellular activation of C3 and C5 in T cells is involved in the maintenance of immunological tolerance and promotes differentiation of T helper cells into Th1-type cells that activate cell-mediated immune responses. Adipocytes are unique in producing many complement sensor proteins (like C1q) and Factor D (adipsin), the key enzyme in promoting alternative pathway amplification. The effects of complement activation products are mediated by intracellular and cell membrane receptors, like C3aR, C5aR1, C5aR2 and the complement regulator MCP/CD46, often jointly with other receptors like the T cell receptor, Toll-like receptors and those of the inflammasomes. These recent observations link complement activation to cellular metabolic processes, intracellular defense reactions and to diverse adaptive immune responses. The complement components may thus be viewed as intracellular alarm molecules involved in the cellular danger response.