CCR2-dependent CX3CR1+ colonic macrophages promote Enterococcus faecalis dissemination.

Enterococci are common commensal bacteria that colonize the gastrointestinal tracts of most mammals, including humans. Importantly, these bacteria are one of the leading causes of nosocomial infections. This study examined the role of colonic macrophages in facilitating Enterococcus faecalis infections in mice. We determined that depletion of colonic phagocytes resulted in the reduction of E. faecalis dissemination to the gut-draining mesenteric lymph nodes. Furthermore, we established that trafficking of monocyte-derived CX3CR1-expressing macrophages contributed to E. faecalis dissemination in a manner that was not reliant on CCR7, the conventional receptor involved in lymphatic migration. Finally, we showed that E. faecalis mutants with impaired intracellular survival exhibited reduced dissemination, suggesting that E. faecalis can exploit host immune cell migration to disseminate systemically and cause disease. Our findings indicate that modulation of macrophage trafficking in the context of antibiotic therapy could serve as a novel approach for preventing or treating opportunistic infections by disseminating enteric pathobionts like E. faecalis.

Method for the Determination of Oxygen in FLiBe via Inert Gas Fusion.

In nuclear reactors that use molten fluoride salts, either as coolants or as the medium for the fuel, the purity of the salts is critical for controlling salt chemistry and mitigating corrosion. Water is a particularly important contaminant in this regard, as it participates in a number of important corrosion reactions, so the careful measurement of oxygen, which is principally present in the salts due to water contamination, is a critical step in salt characterization. Here, we present an analytical method for quantifying oxygen contamination in Li2BeF4 (FLiBe), a technologically important and suitably representative fluoride salt, with a detection limit of 22 µg of oxygen, or 110 ppm in a 200 mg sample. To test the method, four FLiBe samples from different batches were tested. Two of these showed oxygen concentrations below the method detection limit, while two showed concentrations above it. In particular, the difference in the oxygen concentration between purified and un-purified batches of material from Kairos Power showed the efficacy of this method in characterizing the degree of oxygen removal obtained from purification methods.

Clonal lineage tracing reveals mechanisms skewing CD8+ T cell fate decisions in chronic infection.

Although recent evidence demonstrates heterogeneity among CD8+ T cells during chronic infection, developmental relationships and mechanisms underlying their fate decisions remain incompletely understood. Using single-cell RNA and TCR sequencing, we traced the clonal expansion and differentiation of CD8+ T cells during chronic LCMV infection. We identified immense clonal and phenotypic diversity, including a subset termed intermediate cells. Trajectory analyses and infection models showed intermediate cells arise from progenitor cells before bifurcating into terminal effector and exhausted subsets. Genetic ablation experiments identified that type I IFN drives exhaustion through an IRF7-dependent mechanism, possibly through an IFN-stimulated subset bridging progenitor and exhausted cells. Conversely, Zeb2 was critical for generating effector cells. Intriguingly, some T cell clones exhibited lineage bias. Mechanistically, we identified that TCR avidity correlates with an exhausted fate, whereas SHP-1 selectively restricts low-avidity effector cell accumulation. Thus, our work elucidates novel mechanisms underlying CD8+ T cell fate determination during persistent infection and suggests two potential pathways leading to exhaustion.

MyD88 is an essential regulator of NK cell-mediated clearance of MCMV infection.

The signaling adapter MyD88 is critical for immune cell activation in response to viral or bacterial pathogens via several TLRs, IL-1ßR and IL-18R. However, the essential role of MyD88 during activations mediated by germline-encoded NK cell receptors (NKRs), such as Ly49H or NKG2D, has yet to be investigated. To define the NK cell-intrinsic function of MyD88, we generated a novel NK cell conditional knockout mouse for MyD88 (Myd88fl/flNcr1Cre/+). Phenotypic characterization of these mice demonstrated that MyD88 is dispensable for NK cell development and maturation. However, the MyD88-deficient NK cells exhibited significantly reduced cytotoxic potentials in vivo. In addition, the lack of MyD88 significantly reduced the NKG2D-mediated inflammatory cytokine production in vitro. Consistent with this, mice lacking MyD88 were unable to respond and clear MCMV infection. Transcriptomic analyses of splenic NK cells following MCMV infection revealed that inflammatory gene signatures were upregulated in Ly49H+. In contrast, Ly49H- NK cells have significant enrichment in G2M checkpoint genes, revealing distinct transcriptomic profiles of these subsets. Our results identify a central role for MyD88 in Ly49H-dependent gene signatures, including alterations in genes regulating proliferation in Ly49H+ NK cells. In summary, our study reveals a previously unknown function of MyD88 in Ly49H-dependent signaling and in vivo functions of NK cells.

Gammaherpesviruses and B Cells: A Relationship That Lasts a Lifetime.

Gammaherpesviruses are highly prevalent pathogens that establish life-long infection and are associated with diverse malignancies, including lymphoproliferative diseases and B cell lymphomas. Unlike other viruses that either do not infect B cells or infect B cells transiently, gammaherpesviruses manipulate physiological B cell differentiation to establish life-long infection in memory B cells. Disruption of such viral manipulation by genetic or environmental causes is likely to seed viral lymphomagenesis. In this review, we discuss physiological and unique host and viral mechanisms usurped by gammaherpesviruses to fine tune host B cell biology for optimal infection establishment and maintenance.

γ-herpesvirus latency attenuates Mycobacterium tuberculosis infection in mice.

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a bacterial pathogen which is transmitted via aerosol and establishes a chronic lung infection. In naïve hosts, Mtb grows for several weeks without being restricted by IFNγ-producing T cells, which eventually accumulate and limit Mtb dissemination. In this study, we used a mouse model of Mtb/γ-herpesvirus (γHV) coinfection to test the hypothesis that latent γHV infection alters host resistance to Mtb. γHVs are DNA viruses which elicit a polyclonal T cell response and attenuate some acute bacterial pathogens in mice; whether γHVs modulate infection with Mtb is unknown. Here, mice harboring latent mouse gammaherpesvirus 68 (MHV68)-a γHV genetically and biologically related to human Epstein Barr virus (EBV)-were infected via aerosol with a low dose of virulent Mtb. Mtb burdens and IFNγ+ T cell frequencies in mice with latent MHV68 (MHV68POS mice) were subsequently measured and compared to control mice that did not harbor latent MHV68 (MHV68NEG mice). Relative to MHV68NEG controls, MHV68POS mice more effectively limited Mtb growth and dissemination, and had higher frequencies of CD4+IFNγ+ cells in lung-draining lymph nodes. Collectively, our results support a model wherein latent γHV confers moderate protection against subsequent Mtb infection.

Conserved Gammaherpesvirus Protein Kinase Selectively Promotes Irrelevant B Cell Responses.

Gammaherpesviruses are ubiquitous pathogens that are associated with B cell lymphomas. In the early stages of chronic infection, these viruses infect naive B cells and subsequently usurp the B cell differentiation process through the germinal center response to ensure latent infection of long-lived memory B cells. A unique feature of early gammaherpesvirus chronic infection is a robust differentiation of irrelevant, virus-nonspecific B cells with reactivities against self-antigens and antigens of other species. In contrast, protective, virus-specific humoral responses do not reach peak levels until a much later time. While several host factors are known to either promote or selectively restrict gammaherpesvirus-driven germinal center response, viral mechanisms that contribute to the irrelevant B cell response have not been defined. In this report we show that the expression and the enzymatic activity of the gammaherpesvirus-encoded conserved protein kinase selectively facilitates the irrelevant, but not virus-specific, B cell responses. Further, we show that lack of interleukin-1 (IL-1) receptor attenuates gammaherpesvirus-driven B cell differentiation and viral reactivation. Because germinal center B cells are thought to be the target of malignant transformation during gammaherpesvirus-driven lymphomagenesis, identification of host and viral factors that promote germinal center responses during gammaherpesvirus infection may offer an insight into the mechanism of gammaherpesvirus pathogenesis.IMPORTANCE Gammaherpesviruses are ubiquitous cancer-associated pathogens that usurp the B cell differentiation process to establish life-long latent infection in memory B cells. A unique feature of early gammaherpesvirus infection is the robust increase in differentiation of B cells that are not specific for viral antigens and instead encode antibodies that react with self-antigens and antigens of other species. Viral mechanisms that are involved in driving such irrelevant B cell differentiation are not known. Here, we show that gammaherpesvirus-encoded conserved protein kinase and host IL-1 signaling promote irrelevant B cell responses and gammaherpesvirus-driven germinal center responses, with the latter thought to be the target of viral transformation.

Development and Characterization of Titanium Dioxide Gel with Encapsulated Bacteriorhodopsin for Hydrogen Production.

We study bacteriorhodopsin (BR) in its native purple membrane encapsulated within amorphous titanium dioxide, or titania, gels and in the presence of titania sol particles to explore this system for hydrogen production. Förster resonance energy transfer between BR and titanium dioxide sol particles was used to conclude that there is nanometer-scale proximity of bacteriorhodopsin to the titanium dioxide. The detection of BR-titania sol aggregates by fluorescence anisotropy and particle sizing indicated the affinity amorphous titania has for BR without the use of additional cross-linkers. UV-vis spectroscopy of BR-titania gels shows that methanol addition did not denature BR at a 25 mM concentration presence as a sacrificial electron donor. Additionally, confinement of BR in the gels significantly limited protein denaturation at higher concentration of added methanol or ethanol. Subsequently, titania gels fabricated through the sol-gel process using a titanium ethoxide precursor, water, and the addition of 25 mM methanol were used to encapsulate BR and a platinum reduction catalyst for the production of hydrogen gas under white light irradiation. The inclusion of 5 µM bacteriorhodopsin resulted in a hydrogen production rate of about 3.8 µmol hydrogen mL-1 h-1, an increase of 52% compared to gels containing no protein. Electron transfer and proton pumping by BR in close proximity to the titania gel surface are feasible explanations for the enhanced production of hydrogen without the need to cross-link BR to the titania gel. This work sets the stage for further developments of amorphous, rather than crystalline, titania-encapsulated bacteriorhodopsin for solar-driven hydrogen production through water splitting.

Biomembrane-Compatible Sol-Gel-Derived Photocatalytic Titanium Dioxide.

Titanium dioxide gel monoliths were synthesized using an organic precursor and 0-30 vol % ethanol in water. The visible-light-activated proton pump, bacteriorhodopsin, in its native purple membrane form, was successfully encapsulated within the titanium dioxide gels. Absorption spectra showed that the folded functional state of the protein remained intact within gels made with 0 and 15 vol % ethanol and retained the ability to make reversible conformational changes associated with the photocycle within the gel made with 0 vol % ethanol. The photocatalytic activity of gels made with no ethanol was significantly detectable and gels made with 0-30 vol % ethanol were comparable to commercial crystalline nanoparticles in similar solution conditions when irradiated with UV light. Our results show that sol-gel-derived photocatalytic titanium dioxide can be made biocompatible for a membrane-associated protein by minimizing the amount of ethanol and maximizing the amount of water in the synthesis procedure. The entrapment of the membrane protein, bacteriorhodopsin, in sol-gel-derived titanium dioxide provides the first step in future explorations of this bionanocomposite for visible light photocatalysis, including hydrogen production.

Dynamics of Crowding-Induced Mixing in Phase Separated Lipid Bilayers.

We use fluorescence microscopy to examine the dynamics of the crowding-induced mixing transition of liquid ordered (Lo)-liquid disordered (Ld) phase separated lipid bilayers when the following particles of increasing size bind to either the Lo or Ld phase: Ubiquitin, green fluorescent protein (GFP), and nanolipoprotein particles (NLPs) of two diameters. These proteinaceous particles contained histidine-tags, which were phase targeted by binding to iminodiacetic acid (IDA) head groups, via a Cu2+ chelating mechanism, of lipids that specifically partition into either the Lo phase or Ld phase. The degree of steric pressure was controlled by varying the size of the bound particle (10-240 kDa) and the amount of binding sites present (i.e., DPIDA concentrations of 9 and 12 mol%) in the supported lipid multibilayer platform used here. We develop a mass transfer-based diffusional model to analyze the observed Lo phase domain dissolution that, along with visual observations and activation energy calculations, provides insight into the sequence of events in crowding-induced mixing. Our results suggest that the degree of steric pressure and target phase influence not only the efficacy of steric-pressure induced mixing, but the rate and controlling mechanism for which it occurs.