Mycobacterial MCE proteins as transporters that control lipid homeostasis of the cell wall.

Mammalian cell entry (mce) genes are not only present in genomes of pathogenic mycobacteria, including Mycobacterium tuberculosis (the causative agent of tuberculosis), but also in saprophytic and opportunistic mycobacterial species. MCE are conserved cell-wall proteins encoded by mce operons, which maintain an identical structure in all mycobacteria: two yrbE genes (A and B) followed by six mce genes (A, B, C, D, E and F). Although these proteins are known to participate in the virulence of pathogenic mycobacteria, the presence of the operons in nonpathogenic mycobacteria and other bacteria indicates that they play another role apart from host cell invasion. In this respect, more recent studies suggest that they are functionally similar to ABC transporters and form part of lipid transporters in Actinobacteria. To date, most reviews on mce operons in the literature discuss their role in virulence. However, according to data from transcriptional studies, mce genes, particularly the mce1 and mce4 operons, modify their expression according to the carbon source and upon hypoxia, starvation, surface stress and oxidative stress; which suggests a role of MCE proteins in the response of Mycobacteria to external stressors. In addition to these data, this review also summarizes the studies demonstrating the role of MCE proteins as lipid transporters as well as the relevance of their transport function in the interaction of pathogenic Mycobacteria with the hosts. Altogether, the evidence to date would indicate that MCE proteins participate in the response to the stress conditions that mycobacteria encounter during infection, by participating in the cell wall remodelling and possibly contributing to lipid homeostasis.

Central Nervous System-Infecting Pathogens Escherichia coli and Cryptococcus neoformans Exploit the Host Pdlim2 for Intracellular Traversal and Exocytosis in the Blood-Brain Barrier.

Microbial penetration of the blood-brain barrier, a prerequisite for the development of central nervous system (CNS) infection, involves microbial invasion, intracellular traversal, and exocytosis. Microbial invasion of the blood-brain barrier has been investigated, but the molecular basis for microbial traversal and exit from the blood-brain barrier remains unknown. We performed transcriptome analysis of human brain microvascular endothelial cells (HBMEC) infected with Escherichia coli and Cryptococcus neoformans, representative bacterial and fungal pathogens common in CNS infections. Among the targets upregulated in response to E. coli and C. neoformans infection, PDLIM2 was knocked down by small hairpin RNA (shRNA) in HBMEC for further investigation. We demonstrated that Pdlim2 specifically regulated microbial traversal and exit from HBMEC by assessing microbial invasion, transcytosis, intracellular multiplication, and egression. Additionally, the defective exocytosis of internalized E. coli cells from the PDLIM2 shRNA knockdown cells was restored by treatment with a calcium ionophore (ionomycin). Moreover, we performed proximity-dependent biotin labeling with the biotin ligase BioID2 and identified 210 potential Pdlim2 interactors. Among the nine Pdlim2 interactors enriched in response to both E. coli and C. neoformans infection, we selected MPRIP and showed that HBMEC with knockdown of MPRIP mimicked the phenotype of PDLIM2 knockdown cells. These results suggest that the CNS-infecting microbes hijack Pdlim2 and Mprip for intracellular traversal and exocytosis in the blood-brain barrier.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

Control of immune cell trafficking through inter-organ communication.

Cell migration is a cardinal feature of the immune system. Immune cell trafficking is orchestrated principally by chemokines and adhesion molecules, which guide the cells to the right place and at the right time to efficiently induce immune responses. Recent studies have demonstrated that signals from other organ systems influence the expression of and responsiveness to these guidance cues and consequentially immune cell migration. Neuronal inputs control entry and exit of immune cells to and from lymphoid and non-lymphoid tissues. The circadian clock helps establish diurnal variations in immune cell distribution among tissues. Nutritional status also alters immune cell homing to the bone marrow. In this review, we summarize the current knowledge about inter-organ control of immune cell trafficking and discuss the physiological and pathological significance of these mechanisms.

Aquaporins in Immune Cells and Inflammation: New Targets for Drug Development.

The mammalian immune system senses foreign antigens by mechanisms that involve the interplay of various kinds of immune cells, culminating in inflammation resolution and tissue clearance. The ability of the immune cells to communicate (via chemokines) and to shift shape for migration, phagocytosis or antigen uptake is mainly supported by critical proteins such as aquaporins (AQPs) that regulate water fluid homeostasis and volume changes. AQPs are protein channels that facilitate water and small uncharged molecules' (such as glycerol or hydrogen peroxide) diffusion through membranes. A number of AQP isoforms were found upregulated in inflammatory conditions and are considered essential for the migration and survival of immune cells. The present review updates information on AQPs' involvement in immunity and inflammatory processes, highlighting their role as crucial players and promising targets for drug discovery.

Immune Cell Connection by Tunneling Nanotubes: The Impact of Intercellular Cross-Talk on the Immune Response and Its Therapeutic Applications.

Direct intercellular communication is an important prerequisite for the development of multicellular organisms, the regeneration of tissue, and the maintenance of various physiological activities. Tunnel nanotubes (TNTs), which have diameters of approximately 50-1500 nm and lengths of up to several cell diameters, can connect cells over long distances and have emerged as one of the most important recently discovered types of efficient communication between cells. Moreover, TNTs can also directly transfer organelles, vehicles, proteins, genetic material, ions, and small molecules from one cell to adjacent and even distant cells. However, the mechanism of intercellular communication between various immune cells within the complex immune system has not been fully elucidated. Studies in the past decades have confirmed the existence of TNTs in many types of cells, especially in various kinds of immune cells. TNTs display different structural and functional characteristics between and within different immunocytes, playing a major role in the transmission of signals across various kinds of immune cells. In this review, we introduce the discovery and structure of TNTs, as well as their different functional properties within different immune cells. We also discuss the roles of TNTs in potentiating the immune response and their potential therapeutic applications.

Blood-brain barrier transport using a high affinity, brain-selective VNAR antibody targeting transferrin receptor 1.

Transfer across the blood-brain barrier (BBB) remains a significant hurdle for the development of biopharmaceuticals with therapeutic effects within the central nervous system. We established a functional selection method to identify high affinity single domain antibodies to the transferrin receptor 1 (TfR1) with efficient biotherapeutic delivery across the BBB. A synthetic phage display library based on the variable domain of new antigen receptor (VNAR) was used for in vitro selection against recombinant human TfR1 ectodomain (rh-TfR1-ECD) followed by in vivo selection in mouse for brain parenchyma penetrating antibodies. TXB2 VNAR was identified as a high affinity, species cross-reactive VNAR antibody against TfR1-ECD that does not compete with transferrin or ferritin for receptor binding. IV dosing of TXB2 when fused to human Fc domain (TXB2-hFc) at 25 nmol/kg (1.875 mg/kg) in mice resulted in rapid binding to brain capillaries with subsequent transport into the brain parenchyma and specific uptake into TfR1-positive neurons. Likewise, IV dosing of TXB2-hFc fused with neurotensin (TXB2-hFc-NT) at 25 nmol/kg resulted in a rapid and reversible pharmacological response as measured by body temperature reduction. TXB2-hFc did not elicit any acute adverse reactions, bind, or deplete circulating reticulocytes or reduce BBB-expressed endogenous TfR1 in mice. There was no evidence of target-mediated clearance or accumulation in peripheral organs except lung. In conclusion, TXB2 is a high affinity, species cross-reactive, and brain-selective VNAR antibody to TfR1 that rapidly crosses the BBB and exhibits a favorable pharmacokinetic and safety profile and can be readily adapted to carry a wide variety of biotherapeutics from blood to brain.

Physicochemical Evidence that Francisella FupA and FupB Proteins Are Porins.

Responsible for tularemia, Francisella tularensis bacteria are highly infectious Gram-negative, category A bioterrorism agents. The molecular mechanisms for their virulence and resistance to antibiotics remain largely unknown. FupA (Fer Utilization Protein), a protein mediating high-affinity transport of ferrous iron across the outer membrane, is associated with both. Recent studies demonstrated that fupA deletion contributed to lower F. tularensis susceptibility towards fluoroquinolones, by increasing the production of outer membrane vesicles. Although the paralogous FupB protein lacks such activity, iron transport capacity and a role in membrane stability were reported for the FupA/B chimera, a protein found in some F. tularensis strains, including the live vaccine strain (LVS). To investigate the mode of action of these proteins, we purified recombinant FupA, FupB and FupA/B proteins expressed in Escherichia coli and incorporated them into mixed lipid bilayers. We examined the porin-forming activity of the FupA/B proteoliposomes using a fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS) probe. Using electrophysiology on tethered bilayer lipid membranes, we confirmed that the FupA/B fusion protein exhibits pore-forming activity with large ionic conductance, a property shared with both FupA and FupB. This demonstration opens up new avenues for identifying functional genes, and novel therapeutic strategies against F. tularensis infections.

Human CD4+ T cell subsets differ in their abilities to cross endothelial and epithelial brain barriers in vitro.

BACKGROUND: The brain barriers establish compartments in the central nervous system (CNS) that significantly differ in their communication with the peripheral immune system. In this function they strictly control T-cell entry into the CNS. T cells can reach the CNS by either crossing the endothelial blood-brain barrier (BBB) or the epithelial blood-cerebrospinal fluid barrier (BCSFB) of the choroid plexus (ChP). OBJECTIVE: Analysis of the cellular and molecular mechanisms involved in the migration of different human CD4+ T-cell subsets across the BBB versus the BCSFB. METHODS: Human in vitro models of the BBB and BCSFB were employed to study the migration of circulating and CNS-entry experienced CD4+ T helper cell subsets (Th1, Th1*, Th2, Th17) across the BBB and BCSFB under inflammatory and non-inflammatory conditions in vitro. RESULTS: While under non-inflammatory conditions Th1* and Th1 cells preferentially crossed the BBB, under inflammatory conditions the migration rate of all Th subsets across the BBB was comparable. The migration of all Th subsets across the BCSFB from the same donor was 10- to 20-fold lower when compared to their migration across the BBB. Interestingly, Th17 cells preferentially crossed the BCSFB under both, non-inflamed and inflamed conditions. Barrier-crossing experienced Th cells sorted from CSF of MS patients showed migratory characteristics indistinguishable from those of circulating Th cells of healthy donors. All Th cell subsets could additionally cross the BCSFB from the CSF to ChP stroma side. T-cell migration across the BCSFB involved epithelial ICAM-1 irrespective of the direction of migration. CONCLUSIONS: Our observations underscore that different Th subsets may use different anatomical routes to enter the CNS during immune surveillance versus neuroinflammation with the BCSFB establishing a tighter barrier for T-cell entry into the CNS compared to the BBB. In addition, CNS-entry experienced Th cell subsets isolated from the CSF of MS patients do not show an increased ability to cross the brain barriers when compared to circulating Th cell subsets from healthy donors underscoring the active role of the brain barriers in controlling T-cell entry into the CNS. Also we identify ICAM-1 to mediate T cell migration across the BCSFB.

Accumulation and trafficking of zinc oxide nanoparticles in an invertebrate model, Bombyx mori, with insights on their effects on immuno-competent cells.

Zinc oxide nanoparticles (ZnO NPs) are used in many applications; however, their interactions with cells, immune cells in particular, and potential health risk(s) are not fully known. In this manuscript, we have demonstrated the potential of ZnO NPs to cross the gut barrier in an invertebrate model, Bombyx mori, and that they can reach the hemolymph where they interact with and/or are taken up by immune-competent cells resulting in various toxic responses like decline in hemocyte viability, ROS generation, morphological alterations, apoptotic cell death, etc. Exposure to these NPs also resulted in alteration of hemocyte dynamics including an immediate increase in THC, possibly due to the release of these hemocytes either from enhanced rate of cell divisions or from attached hemocyte populations, and decline in percentage of prohemocytes and increase in percentage of two professional phagocytes, i.e., granulocytes and plasmatocytes, possibly due to the differentiation of prohemocytes into phagocytes in response to a perceived immune challenge posed by these NPs. Taken together, our data suggest that ZnO NPs have the potential to cross gut barrier and cause various toxic effects that could reverse and the insects could return to normal physiological states as there is restoration and repair of various systems and their affected pathways following the clearance of these NPs from the insect body. Our study also indicates that B. mori has the potential to serve as an effective alternate animal model for biosafety, environmental monitoring and screening of NPs, particularly to evaluate their interactions with invertebrate immune system.

Extracellular Vesicles in Mycobacterial Infections: Their Potential as Molecule Transfer Vectors.

Extracellular vesicles are membrane-bound structures released by living cells and present in body fluids. Their composition includes proteins, lipids, carbohydrates, and nucleic acids and are involved in transfers between cells. Extracellular vesicles can deliver molecules to cells and tissues even if distant. As a consequence, they have a role in information transmission and in the modulation of the biological function of recipient cells. Among other things, they are involved in antigen presentation and the induction of secretion events by immune cells. Thus, extracellular vesicles participate in the regulation of immune responses during infections. We will discuss their potential as effectors and disease biomarkers concerning only mycobacterial infections.

CD93 is a cell surface lectin receptor involved in the control of the inflammatory response stimulated by exogenous DNA.

Bacterial DNA contains CpG oligonucleotide (ODN) motifs to trigger innate immune responses through the endosomal receptor Toll-like receptor 9 (TLR9). One of the cell surface receptors to capture and deliver microbial DNA to intracellular TLR9 is the C-type lectin molecule DEC-205 through its N-terminal C-type lectin-like domain (CTLD). CD93 is a cell surface protein and member of the lectin group XIV with a CTLD. We hypothesized that CD93 could interact with CpG motifs, and possibly serve as a novel receptor to deliver bacterial DNA to endosomal TLR9. Using ELISA and tryptophan fluorescence binding studies we observed that the soluble histidine-tagged CD93-CTLD was specifically binding to CpG ODN and bacterial DNA. Moreover, we found that CpG ODN could bind to CD93-expressing IMR32 neuroblastoma cells and induced more robust interleukin-6 secretion when compared with mock-transfected IMR32 control cells. Our data argue for a possible contribution of CD93 to control cell responsiveness to bacterial DNA in a manner reminiscent of DEC-205. We postulate that CD93 may act as a receptor at plasma membrane for DNA or CpG ODN and to grant delivery to endosomal TLR9.

Concise Review: Intercellular Communication Via Organelle Transfer in the Biology and Therapeutic Applications of Stem Cells.

The therapeutic potential of stem cell-based therapies may be largely dependent on the ability of stem cells to modulate host cells rather than on their differentiation into host tissues. Within the last decade, there has been considerable interest in the intercellular communication mediated by the transfer of cytoplasmic material and organelles between cells. Numerous studies have shown that mitochondria and lysosomes are transported between cells by various mechanisms, such as tunneling nanotubes, microvesicles, and cellular fusion. This review will focus on the known instances of organelle transfer between stem cells and differentiated cells, what effects it has on recipient cells and how organelle transfer is regulated. Stem Cells 2019;37:14-25.

Biological function of SPNS2: From zebrafish to human.

Sphingosine-1-phosphate (S1P), a bioactive metabolite of sphingolipid, has an important role in lymphocyte trafficking, immune responses, vascular and embryonic development, cancer, bone homeostasis, etc. S1P is produced intracellularly and then secreted into the circulation to engage in the above physiological or pathological processes by regulating the proliferation, differentiation and survival of target cells; however, the underlying mechanisms of S1P secretion and function remain poorly understood. Recently, Spinster 2 (SPNS2), a newly identified transporter of S1P, was shown to act as a mediator of intracellular S1P release and play an important role in the regulation of S1P. In this review, we focus on the primary biological characteristics and functions of SPNS2 and provide novel insights into the development of therapies for S1P-related disorders.

A minimal physiologically based pharmacokinetic model to investigate FcRn-mediated monoclonal antibody salvage: Effects of Kon, Koff, endosome trafficking, and animal species.

Manipulation of binding affinity between monoclonal antibodies (mAbs) and the neonatal Fc receptor (FcRn) has been leveraged to extend mAb half-life; however, the steps required for success remain ambiguous and experimental observations are inconsistent. Recent models have considered the time course of endosomal transit a major contributor to the relationship between FcRn affinity and antibody half-life. Our objective was to develop a minimal physiologically based pharmacokinetic model to explain how changes in IgG-FcRn association rate constant (Kon), dissociation rate constant (Koff), and endosomal transit time [T(w)] translate to improved IgG clearance across mice, monkeys and humans. By simulating mAb clearance across physiological values of Kon, Koff, and T(w), we found that lowering Koff improves clearance only until the dissociation half-life reaches endosomal transit time. In contrast, Kon influenced clearance independently of T(w).The model was then applied to fit 66 mAb plasma profiles across species digitized from the literature, and clearance of mAb (CLIgG) and vascular fluid-phase endocytosis rate (CLup) were estimated. We found that CLIgG scaled well with body weight (allometric exponent of 0.90). After accounting for mAbs with significant FcRn binding at physiological pH, CLup was allometrically scalable (exponent 0.72). For the antibodies surveyed, Kon was more highly correlated with CLIgG across all species. The relationship between Koff and KD with CLIgG was largely inconsistent. Taken together, this model provides a parsimonious approach to evaluate endosomal transit kinetics using only mAb plasma concentrations. These findings reinforce the idea that endosomal transit kinetics should be considered when modeling FcRn salvage.

Enhancing IgG distribution to lung mucosal tissue improves protective effect of anti-Pseudomonas aeruginosa antibodies.

IgG antibodies are abundantly present in the vasculature but to a much lesser extent in mucosal tissues. This contrasts with antibodies of the IgA and IgM isotype that are present at high concentration in mucosal secretions due to active delivery by the polymeric Ig receptor (pIgR). IgG is the preferred isotype for therapeutic mAb development due to its long serum half-life and robust Fc-mediated effector function, and it is utilized to treat a diverse array of diseases with antigen targets located in the vasculature, serosa, and mucosa. As therapeutic IgG antibodies targeting the luminal side of mucosal tissue lack an active transport delivery mechanism, we sought to generate IgG antibodies that could be transported via pIgR, similarly to dimeric IgA and pentameric IgM. We show that an anti-Pseudomonas aeruginosa IgG fused with pIgR-binding peptides gained the ability to transcytose and be secreted via pIgR. Consistent with these results, pIgR-binding IgG antibodies exhibit enhanced localization to the bronchoalveolar space when compared with the parental IgG antibody. Furthermore, pIgR-binding mAbs maintained Fc-mediated functional activity and promoted enhanced survival compared with the parental mAb in a P. aeruginosa acute pneumonia model. Our results suggest that increasing IgG accumulation at mucosal surfaces by pIgR-mediated active transport can improve the efficacy of therapeutic mAbs that act at these sites.

Effect of short-term liver X receptor activation on epidermal barrier features in mild to moderate atopic dermatitis: A randomized controlled trial.

BACKGROUND: Liver X receptors (LXRs) are involved in maintaining epidermal barrier and suppressing inflammatory responses in model systems. The LXR agonist VTP-38543 showed promising results in improving barrier function and inflammatory responses in model systems. OBJECTIVE: To assess the safety, tolerability, cellular and molecular changes, and clinical efficacy of the topical VTP-38543 in adults with mild to moderate atopic dermatitis (AD). METHODS: A total of 104 ambulatory patients with mild to moderate AD were enrolled in this randomized, double-blind, vehicle-controlled trial between December 2015 and September 2016. VTP-38543 cream in 3 concentrations (0.05%, 0.15%, and 1.0%) or placebo was applied twice daily for 28 days. Pretreatment and posttreatment skin biopsy specimens were obtained from a subset of 33 patients. Changes in SCORing of Atopic Dermatitis, Eczema Area and Severity Index, Investigator's Global Assessment, and tissue biomarkers (by real-time polymerase chain reaction and immunostaining) were evaluated. RESULTS: Topical VTP-38543 was safe and well tolerated. VTP-38543 significantly increased messenger RNA (mRNA) expression of epidermal barrier differentiation (loricrin and filaggrin, P = .02) and lipid (adenosine triphosphate-binding cassette subfamily G member 1 and sterol regulatory element binding protein 1c, P < .01) measures and reduced epidermal hyperplasia markers (thickness, keratin 16 mRNA). VTP-38543 nonsignificantly suppressed cellular infiltrates and down-regulated mRNA expression of several TH17/TH22-related (phosphatidylinositol 3, S100 calcium-binding protein A12) and innate immunity (interleukin 6) markers. CONCLUSION: Topical VTP-38543 is safe and well tolerated. Its application led to improvement in barrier differentiation and lipids. Longer-term studies are needed to clarify whether a barrier-based approach can induce meaningful suppression of immune abnormalities. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02655679.

Transcriptomic changes in the pre-implantation uterus highlight histotrophic nutrition of the developing marsupial embryo.

Early pregnancy is a critical time for successful reproduction; up to half of human pregnancies fail before the development of the definitive chorioallantoic placenta. Unlike the situation in eutherian mammals, marsupial pregnancy is characterised by a long pre-implantation period prior to the development of the short-lived placenta, making them ideal models for study of the uterine environment promoting embryonic survival pre-implantation. Here we present a transcriptomic study of pre-implantation marsupial pregnancy, and identify differentially expressed genes in the Sminthopsis crassicaudata uterus involved in metabolism and biosynthesis, transport, immunity, tissue remodelling, and uterine receptivity. Interestingly, almost one quarter of the top 50 genes that are differentially upregulated in early pregnancy are putatively involved in histotrophy, highlighting the importance of nutrient transport to the conceptus prior to the development of the placenta. This work furthers our understanding of the mechanisms underlying survival of pre-implantation embryos in the earliest live bearing ancestors of mammals.