Gregory A. Gibson

Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Oral-resident natural Th17 cells and γδ T cells control opportunistic Candida albicans infections

Conti et al. show that IL-17 is produced by tongue-resident populations of γδ T cells and nTh17 cells in response to oropharyngeal candidiasis in mice.

Mitochondrial hyperfusion induced by loss of the fission protein Drp1 causes ATM-dependent G2/M arrest and aneuploidy through DNA replication stress

Summary Mitochondrial fission and fusion cycles are integrated with cell cycle progression. In this paper, we demonstrate that the inhibition of mitochondrial fission protein Drp1 causes an unexpected delay in G2/M cell cycle progression and aneuploidy. In investigating the underlying molecular mechanism, we revealed that inhibiting Drp1 triggers replication stress, which is mediated by a hyperfused mitochondrial structure and unscheduled expression of cyclin E in the G2 phase. This persistent replication stress then induces an ATM-dependent activation of the G2 to M transition cell cycle checkpoint. Knockdown of ATR, an essential kinase in preventing replication stress, significantly enhanced DNA damage and cell death of Drp1-deficienct cells. Persistent mitochondrial hyperfusion also induces centrosomal overamplification and chromosomal instability, which are causes of aneuploidy. Analysis using cells depleted of mitochondrial DNA revealed that these events are not mediated by the defects in mitochondrial ATP production and reactive oxygen species (ROS) generation. Thus dysfunctional mitochondrial fission directly induces genome instability by replication stress, which then initiates the DNA damage response. Our findings provide a novel mechanism that contributes to the cellular dysfunction and diseases associated with altered mitochondrial dynamics.

A theoretical and non-destructive experimental approach for direct inclusion of measured collagen orientation and recruitment into mechanical models of the artery wall

Gradual collagen recruitment has been hypothesized as the underlying mechanism for the mechanical stiffening with increasing stress in arteries. In this work, we investigated this hypothesis in eight rabbit carotid arteries by directly measuring the distribution of collagen recruitment stretch under increasing circumferential loading using a custom uniaxial (UA) extension device combined with a multi-photon microscope (MPM). This approach allowed simultaneous mechanical testing and imaging of collagen fibers without traditional destructive fixation methods. Fiber recruitment was quantified from 3D rendered MPM images, and fiber orientation was measured in projected stacks of images. Collagen recruitment was observed to initiate at a finite strain, corresponding to a sharp increase in the measured mechanical stiffness, confirming the previous hypothesis and motivating the development of a new constitutive model to capture this response. Previous constitutive equations for the arterial wall have modeled the collagen contribution with either abrupt recruitment at zero strain, abrupt recruitment at finite strain or as gradual recruitment beginning at infinitesimal strain. Based on our experimental data, a new combined constitutive model was presented in which fiber recruitment begins at a finite strain with activation stretch represented by a probability distribution function. By directly including this recruitment data, the collagen contribution was modeled using a simple Neo-Hookean equation. As a result, only two phenomenological material constants were required from the fit to the stress stretch data. Three other models for the arterial wall were then compared with these results. The approach taken here was successful in combining stress-strain analysis with simultaneous microstructural imaging of collagen recruitment and orientation, providing a new approach by which underlying fiber architecture may be quantified and included in constitutive equations.

Effective Immunotherapy against Murine Gliomas Using Type 1 Polarizing Dendritic Cells―Significant Roles of CXCL10

In an attempt to develop effective vaccines against central nervous system (CNS) tumors, we evaluated the ability of vaccines with standard dendritic cells (DC) versus type 1 polarizing DCs (DC1) to induce glioma-specific type 1 CTLs with CNS tumor-relevant homing properties and the mechanism of their action. C57BL/6 mouse-derived bone marrow cells were cultured with mouse granulocyte/macrophage colony-stimulating factor (GM-CSF) for 6 days, and CD11c(+) cells were subsequently cultured with GM-CSF, rmIFN-gamma, rmIFN-alpha, rmIL-4, and polyinosinic-polycytidylic acid stabilized by lysine and carboxymethylcellulose for 24 hours to generate DC1s. In analogy to their human counterparts, mouse DC1s exhibited surface marker profiles of mature DCs and produced high levels of IL-12 and CXCL10. Importantly for their application as cancer vaccines, such DC1s stably retained their type 1 phenotype even when exposed to type 2-promoting or regulatory T cell (Treg)-promoting environments. Consistently, mouse DC1s induced antigen-specific type 1 CTLs more efficiently than nonpolarized DCs in vitro. DC1s given s.c. migrated into draining lymph nodes, induced antigen-specific CTLs, and suppressed Treg accumulation. In addition, s.c. immunization with DC1s loaded with glioma-associated antigen (GAA)-derived CTL epitope peptides prolonged the survival of CNS GL261 glioma-bearing mice, which was associated with efficient CNS glioma homing of antigen-specific CTLs. Intratumoral injections of GAA peptide-loaded DC1s further enhanced the anti-CNS glioma effects of DC1-based s.c. immunization. Interestingly, the antitumor functions were abrogated with CXCL10(-/-) mouse-derived DC1s. Collectively, these findings show the anti-CNS glioma effects of DC1-based therapy and a novel role of CXCL10 in the immunologic and therapeutic activity of DC-based cancer vaccines.

Lipopolysaccharide Clearance, Bacterial Clearance, and Systemic Inflammatory Responses Are Regulated by Cell Type–Specific Functions of TLR4 during Sepsis

The morbidity associated with bacterial sepsis is the result of host immune responses to pathogens, which are dependent on pathogen recognition by pattern recognition receptors, such as TLR4. TLR4 is expressed on a range of cell types, yet the mechanisms by which cell-specific functions of TLR4 lead to an integrated sepsis response are poorly understood. To address this, we generated mice in which TLR4 was specifically deleted from myeloid cells (LysMTLR4KO) or hepatocytes (HCTLR4KO) and then determined survival, bacterial counts, host inflammatory responses, and organ injury in a model of cecal ligation and puncture (CLP), with or without antibiotics. LysM-TLR4 was required for phagocytosis and efficient bacterial clearance in the absence of antibiotics. Survival, the magnitude of the systemic and local inflammatory responses, and liver damage were associated with bacterial levels. HCTLR4 was required for efficient LPS clearance from the circulation, and deletion of HCTLR4 was associated with enhanced macrophage phagocytosis, lower bacterial levels, and improved survival in CLP without antibiotics. Antibiotic administration during CLP revealed an important role for hepatocyte LPS clearance in limiting sepsis-induced inflammation and organ injury. Our work defines cell type–selective roles for TLR4 in coordinating complex immune responses to bacterial sepsis and suggests that future strategies for modulating microbial molecule recognition should account for varying roles of pattern recognition receptors in multiple cell populations.

Rapid Host Defense against Aspergillus fumigatus Involves Alveolar Macrophages with a Predominance of Alternatively Activated Phenotype

The ubiquitous fungus Aspergillus fumigatus is associated with chronic diseases such as invasive pulmonary aspergillosis in immunosuppressed patients and allergic bronchopulmonary aspergillosis (ABPA) in patients with cystic fibrosis or severe asthma. Because of constant exposure to this fungus, it is critical for the host to exercise an immediate and decisive immune response to clear fungal spores to ward off disease. In this study, we observed that rapidly after infection by A. fumigatus, alveolar macrophages predominantly express Arginase 1 (Arg1), a key marker of alternatively activated macrophages (AAMs). The macrophages were also found to express Ym1 and CD206 that are also expressed by AAMs but not NOS2, which is expressed by classically activated macrophages. The expression of Arg1 was reduced in the absence of the known signaling axis, IL-4Rα/STAT6, for AAM development. While both Dectin-1 and TLR expressed on the cell surface have been shown to sense A. fumigatus, fungus-induced Arg1 expression in CD11c+ alveolar macrophages was not dependent on either Dectin-1 or the adaptor MyD88 that mediates intracellular signaling by most TLRs. Alveolar macrophages from WT mice efficiently phagocytosed fungal conidia, but those from mice deficient in Dectin-1 showed impaired fungal uptake. Depletion of macrophages with clodronate-filled liposomes increased fungal burden in infected mice. Collectively, our studies suggest that alveolar macrophages, which predominantly acquire an AAM phenotype following A. fumigatus infection, have a protective role in defense against this fungus.

Direct Visualization of HIV-1 with Correlative Live-Cell Microscopy and Cryo-Electron Tomography

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-native state and therefore has the potential to help elucidate early events of HIV-1 infection in host cells. However, structural details of infecting HIV-1 have not been observed, due to technological challenges in working with rare and dynamic HIV-1 particles in human cells. Here, we report structural analysis of HIV-1 and host-cell interactions by means of a correlative high-speed 3D live-cell-imaging and cryoET method. Using this method, we showed under near-native conditions that intact hyperstable mutant HIV-1 cores are released into the cytoplasm of host cells. We further obtained direct evidence to suggest that a hyperstable mutant capsid, E45A, showed delayed capsid disassembly compared to the wild-type capsid. Together, these results demonstrate the advantages of our correlative live-cell and cryoET approach for imaging dynamic processes, such as viral infection.

Donor dendritic cell–derived exosomes promote allograft-targeting immune response

The immune response against transplanted allografts is one of the most potent reactions mounted by the immune system. The acute rejection response has been attributed to donor dendritic cells (DCs), which migrate to recipient lymphoid tissues and directly activate alloreactive T cells against donor MHC molecules. Here, using a murine heart transplant model, we determined that only a small number of donor DCs reach lymphoid tissues and investigated how this limited population of donor DCs efficiently initiates the alloreactive T cell response that causes acute rejection. In our mouse model, efficient passage of donor MHC molecules to recipient conventional DCs (cDCs) was dependent on the transfer of extracellular vesicles (EVs) from donor DCs that migrated from the graft to lymphoid tissues. These EVs shared characteristics with exosomes and were internalized or remained attached to the recipient cDCs. Recipient cDCs that acquired exosomes became activated and triggered full activation of alloreactive T cells. Depletion of recipient cDCs after cardiac transplantation drastically decreased presentation of donor MHC molecules to directly alloreactive T cells and delayed graft rejection in mice. These findings support a key role for transfer of donor EVs in the generation of allograft-targeting immune responses and suggest that interrupting this process has potential to dampen the immune response to allografts.

Selective Perturbation of Early Endosome and/ortrans-Golgi Network pH but Not Lysosome pH by Dose-dependent Expression of Influenza M2 Protein

Many sorting stations along the biosynthetic and endocytic pathways are acidified, suggesting a role for pH regulation in protein traffic. However, the function of acidification in individual compartments has been difficult to examine because global pH perturbants affect all acidified organelles in the cell and also have numerous side effects. To circumvent this problem, we have developed a method to selectively perturb the pH of a subset of acidified compartments. We infected HeLa cells with a recombinant adenovirus encoding influenza virus M2 protein (an acid-activated ion channel that dissipates proton gradients across membranes) and measured the effects on various steps in protein transport. At low multiplicity of infection (m.o.i.), delivery of influenza hemagglutinin from thetrans-Golgi network to the cell surface was blocked, but there was almost no effect on the rate of recycling of internalized transferrin. At higher m.o.i., transferrin recycling was inhibited, suggesting increased accumulation of M2 in endosomes. Interestingly, even at the higher m.o.i., M2 expression had no effect on lysosome morphology or on EGF degradation, suggesting that lysosomal pH was not compromised by M2 expression. However, delivery of newly synthesized cathepsin D to lysosomes was slowed in cells expressing active M2, suggesting that acidification of the TGN and endosomes is important for efficient delivery of lysosomal hydrolases. Fluorescence labeling using a pH-sensitive dye confirmed the reversible effect of M2 on the pH of a subset of acidified compartments in the cell. The ability to dissect the role of acidification in individual steps of a complex pathway should be useful for numerous other studies on protein processing and transport.