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Few characteristics are more important to a bacterium than the substrates it consumes. It is hard to identify what substrates are consumed by bacteria in natural communities, however, because most bacteria have not been cultured. In this study, we developed a method that uses fluorescent substrate analogs, cell sorting, and DNA sequencing to identify substrates taken up by bacteria. We deployed this method using 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose analog, and bacteria of the bovine rumen. This method revealed over 40 different bacteria (amplicon sequence variants [ASVs]) from the rumen that take up glucose. Nearly half of these ASVs represent previously uncultured bacteria. We attempted to grow these ASVs on agar media, and we confirmed that nearly two-thirds resisted culture. In coculture experiments, the fluorescent label of 2-NBDG was not transferred to nontarget bacteria by cross-feeding. Because it is not affected by cross-feeding, our method has an advantage over stable isotope probing. Though we focus on glucose, many substrates can be labeled with the fluorophore NBD. Our method represents a new paradigm for identifying substrates used by uncultured bacteria. It will help delineate the niche of bacteria in their environment.IMPORTANCE We introduce a method for identifying what substrates are consumed by bacteria in natural communities. Our method offers significant improvement over existing methods for studying this characteristic. Our method uses a fluorescently labeled substrate which clearly labels target bacteria (glucose consumers in our case). Previous methods use isotope-labeled substrates, which are notorious for off-target labeling (due to cross-feeding of labeled metabolites). Our method can be deployed with a variety of substrates and microbial communities. It represents a major advance in connecting bacteria to the substrates they take up.

Natural killer T (NKT) -cells activated with the glycolipid ligand α-galactosylceramide (α-GalCer) stimulate a wide array of immune responses with many promising immunotherapeutic applications, including the enhancement of vaccines against infectious diseases and cancer. In the current study, we evaluated whether α-GalCer generates protective immunity against a swine influenza (SI) virus infection when applied as an intramuscular vaccine adjuvant. Immunization of newly weaned piglets with UV-killed pandemic H1N1 A/California/04/2009 (kCA04) SI virus and α-GalCer induced high titers of anti-hemagglutinin antibodies and generated virus-specific T cells that localized in intrapulmonary airways and in alveolar walls. Vaccination with α-GalCer resulted in a systemic increase in NKT-cell concentrations, including in the respiratory tract, which was associated with complete inhibition of viral replication in the upper and lower respiratory tract and much reduced viral shedding. These results indicate that NKT-cell agonists could be used to improve swine vaccine formulations in order to reduce the clinical signs of SI infection and limit the spread of influenza viruses amongst commercial pigs.

Studies in mice genetically lacking natural killer T (NKT) cells show that these lymphocytes make important contributions to both innate and adaptive immune responses. However, the usefulness of murine models to study human NKT cells is limited by the many differences between mice and humans, including that their NKT cell frequencies, subsets, and distribution are dissimilar. A more suitable model may be swine that share many metabolic, physiological, and growth characteristics with humans and are also similar for NKT cells. Thus, we analyzed genetically modified pigs made deficient for CD1d that is required for the development of Type I invariant NKT (iNKT) cells that express a semi-invariant T-cell receptor (TCR) and Type II NKT cells that use variable TCRs. Peripheral blood analyzed by flow cytometry and interferon-γ enzyme-linked immuno spot assays demonstrated that CD1d-knockout pigs completely lack iNKT cells, while other leukocyte populations remain intact. CD1d and NKT cells have been shown to be involved in shaping the composition of the commensal microbiota in mice. Therefore, we also compared the fecal microbiota profile between pigs expressing and lacking NKT cells. However, no differences were found between pigs lacking or expressing CD1d. Our results are the first to show that knocking-out CD1d prevents the development of NKT cells in a non-rodent species. CD1d-deficient pigs should offer a useful model to more accurately determine the contribution of NKT cells for human immune responses. They also have potential for understanding how NKT cells impact the health of commercial swine.

Invariant natural killer T (iNKT) cells are glycolipid-reactive T cells with potent immunoregulatory properties. iNKT cells activated with the marine-sponge-derived glycolipid, α-galactosylceramide (αGC), provide a universal source of T-cell help that has shown considerable promise for a wide array of therapeutic applications. This includes harnessing iNKT-cell-mediated immune responses to adjuvant whole inactivated influenza virus (WIV) vaccines. An important concern with WIV vaccines is that under certain circumstances, they are capable of triggering vaccine-associated enhanced respiratory disease (VAERD). This immunopathological phenomenon can arise after immunization with an oil-in-water (OIW) adjuvanted WIV vaccine, followed by infection with a hemagglutinin and neuraminidase mismatched challenge virus. This elicits antibodies (Abs) that bind immunodominant epitopes in the HA2 region of the heterologous virus, which purportedly causes enhanced virus fusion activity to the host cell and increased infection. Here, we show that αGC can induce severe VAERD in pigs. However, instead of stimulating high concentrations of HA2 Abs, αGC elicits high concentrations of interferon (IFN)-γ-secreting cells both in the lungs and systemically. Additionally, we found that VAERD mediated by iNKT cells results in distinct cytokine profiles and altered adaptation of the challenge virus following infection compared to an OIW adjuvant. Overall, these results provide a cautionary note about considering the formulation of WIV vaccines with iNKT-cell agonists as a potential strategy to modulate antigen-specific immunity. [ABSTRACT FROM AUTHOR]

In both humans and NOD mice, type 1 diabetes (T1D) develops from the autoimmune destruction of pancreatic beta cells by T cells. Interactions between both helper CD4+ and cytotoxic CD8+ T cells are essential for T1D development in NOD mice. Previous work has indicated that pathogenic T cells arise from deleterious interactions between relatively common genes which regulate aspects of T cell activation/effector function (Ctla4, Tnfrsf9, Il2/Il21), peptide presentation (H2-A g7, B2m), and T cell receptor (TCR) signaling (Ptpn22). Here, we used a combination of subcongenic mapping and a CRISPR/Cas9 screen to identify the NOD-encoded mammary tumor virus (Mtv)3 provirus as a genetic element affecting CD4+/CD8+ T cell interactions through an additional mechanism, altering the TCR repertoire. Mtv3 encodes a superantigen (SAg) that deletes the majority of Vß3+ thymocytes in NOD mice. Ablating Mtv3 and restoring Vß3+ T cells has no effect on spontaneous T1D development in NOD mice. However, transferring Mtv3 to C57BL/6 (B6) mice congenic for the NOD H2 g7 MHC haplotype (B6.H2 g7) completely blocks their normal susceptibility to T1D mediated by transferred CD8+ T cells transgenically expressing AI4 or NY8.3 TCRs. The entire genetic effect is manifested by Vß3+CD4+ T cells, which unless deleted by Mtv3, accumulate in insulitic lesions triggering in B6 background mice the pathogenic activation of diabetogenic CD8+ T cells. Our findings provide evidence that endogenous Mtv SAgs can influence autoimmune responses. Furthermore, since most common mouse strains have gaps in their TCR Vß repertoire due to Mtvs, it raises questions about the role of Mtvs in other mouse models designed to reflect human immune disorders. [ABSTRACT FROM AUTHOR]

Type 1 diabetes development in the NOD mouse model is widely reported to be dependent on high-level production by autoreactive CD4+ and CD8+ T cells of interferon-γ (IFN-γ), generally considered a proinflammatory cytokine. However, IFN-γ can also participate in tolerance-induction pathways, indicating it is not solely proinflammatory. This study addresses how IFN-γ can suppress activation of diabetogenic CD8+ T cells. CD8+ T cells transgenically expressing the diabetogenic AI4 T-cell receptor adoptively transferred disease to otherwise unmanipulated NOD.IFN-γnull , but not standard NOD, mice. AI4 T cells only underwent vigorous intrasplenic proliferation in NOD.IFN-γnull recipients. Disease-protective IFN-γ could be derived from any lymphocyte source and suppressed diabetogenic CD8+ T-cell responses both directly and through an intermediary nonlymphoid cell population. Suppression was not dependent on regulatory T cells, but was associated with increased inhibitory STAT1 to STAT4 expression levels in pathogenic AI4 T cells. Importantly, IFN-γ exposure during activation reduced the cytotoxicity of human-origin type 1 diabetes-relevant autoreactive CD8+ T cells. Collectively, these results indicate that rather than marking the most proinflammatory lymphocytes in diabetes development, IFN-γ production could represent an attempted limitation of pathogenic CD8+ T-cell activation. Thus, great care should be taken when designing possible diabetic intervention approaches modulating IFN-γ production. [ABSTRACT FROM AUTHOR]

Bats are natural reservoirs of coronaviruses and other viruses with zoonotic potential. Florida has indigenous non-migratory populations of Brazilian free-tailed bats (Tadarida brasiliensis) that mostly roost in colonies in artificial structures. Unlike their counterparts in Brazil and Mexico, the viruses harbored by the Florida bats have been underexplored. We report the detection of an alphacoronavirus RNA-dependent RNA polymerase (RdRp) gene sequence in the feces of two of 19 different T. brasiliensis that were capture/release bats that had been evaluated for overall health. The RdRp sequence is similar but not identical to previously detected sequences in the feces of two different species of bats (T. brasiliensis and Molossus molossus) in Brazil. In common with the experience of others doing similar work, attempts to isolate the virus in cell cultures were unsuccessful. We surmise that this and highly related alphacoronavirus are carried by Brazilian free-tailed bats living in a wide eco-spatial region. As various coronaviruses (CoVs) that affect humans emerged from bats, our study raises the question whether CoVs such as the one detected in our work are yet-to-be-detected pathogens of humans and animals other than bats. [ABSTRACT FROM AUTHOR]

Suppressors of cytokine signaling (SOCS) are a family of eight proteins that negatively regulate Janus kinase and signal transducers and activators of transcription signaling in cells that utilize this pathway to respond to extracellular stimuli. SOCS are best known for attenuating cytokine signaling in the immune system. However, they are also expressed in many other cell types, including pancreatic β-cells, where there is considerable interest in harnessing SOCS molecules to prevent cytokine-mediated apoptosis during diabetes and allogeneic transplantation. Apart from their potential as therapeutic targets, SOCS molecules play a central role for regulating important functions in β-cells, including growth, glucose sensing, and insulin secretion. This review will discuss SOCS proteins as central regulators for diverse cellular processes important for normal β-cell function as well as their protective anti-apoptotic effects during β-cell stress. [ABSTRACT FROM AUTHOR]