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Chronic viral infections are often established by the exploitation of immune-regulatory mechanisms that result in nonfunctional T cell responses. Viruses that establish persistent infections remain a serious threat to human health. Sphingosine kinase 2 (SphK2) generates sphingosine 1-phosphate, which is a molecule known to regulate multiple cellular processes. However, little is known about SphK2’s role during the host immune responses to viral infection. Here, we demonstrate that SphK2 functions during lymphocytic choriomeningitis virus Cl 13 (LCMV Cl 13) infection to limit T cell immune pathology, which subsequently aids in the establishment of virus-induced immunosuppression and the resultant viral persistence. The infection of Sphk2-deficient (Sphk2(–/–)) mice with LCMV Cl 13 led to the development of nephropathy and mortality via T cell–mediated immunopathology. Following LCMV infection, Sphk2(–/–) CD4(+) T cells displayed increased activity and proliferation, and these cells promoted overactive LCMV Cl 13–specific CD8(+) T cell responses. Notably, oral instillation of an SphK2-selective inhibitor promoted protective T cell responses and accelerated the termination of LCMV Cl 13 persistence in mice. Thus, SphK2 is indicated as an immunotherapeutic target for the control of persistent viral infections.

The influenza virus is one of the major public health threats. However, the development of efficient vaccines and therapeutic drugs to combat this virus is greatly limited by its frequent genetic mutations. Because of this, targeting the host factors required for influenza virus replication may be a more effective strategy for inhibiting a broader spectrum of variants. Here, we demonstrated that inhibition of a motor protein kinesin family member 18A (KIF18A) suppresses the replication of the influenza A virus (IAV). The expression of KIF18A in host cells was increased following IAV infection. Intriguingly, treatment with the selective and ATP‐competitive mitotic kinesin KIF18A inhibitor BTB‐1 substantially decreased the expression of viral RNAs and proteins, and the production of infectious viral particles, while overexpression of KIF18A enhanced the replication of IAV. Importantly, BTB‐1 treatment attenuated the activation of AKT, p38 MAPK, SAPK and Ran‐binding protein 3 (RanBP3), which led to the prevention of the nuclear export of viral ribonucleoprotein complexes. Notably, administration of BTB‐1 greatly improved the viability of IAV‐infected mice. Collectively, our results unveiled a beneficial role of KIF18A in IAV replication, and thus, KIF18A could be a potential therapeutic target for the control of IAV infection.

Influenza A virus (IAV) continues to threaten human health. To date, two classes of antiviral drugs have been approved to treat IAV infection, but the continuous emergence of the drug-resistant IAV mutant reinforces the need to develop new antiviral drugs. In this study, we aimed to investigate the anti-IAV activity of an aqueous mixture of Agrimonia pilosa and Galla rhois extracts (APRG64). We demonstrated that APRG64 significantly reduced the IAV-induced cytopathic effect, the transcription/expression of viral proteins, and the production of infectious viral particles. Among nine major components of APRG64, apigenin was identified as the main ingredient responsible for the anti-IAV activity. Interestingly, APRG64 and apigenin inhibited the cell attachment and entry of virus and polymerase activity. Importantly, intranasal administration of APRG64 or apigenin strongly reduced viral loads in the lungs of IAV-infected mice. Furthermore, oral administration of APRG64 significantly reduced the level of viral RNAs and the expression level of pro-inflammatory cytokines in the lungs, which protected mice from IAV-induced mortality. In conclusion, APRG64 could be an attractive antiviral drug to treat IAV infection.

KIF18A is a kinesin family member that is involved in various cellular processes including cell division, cell transformation, and carcinogenesis. However, its possible role in the regulation of host immunity has not been examined. The aim of this study is to investigate the functional role of KIF18A in the differentiation and activation of dendritic cells (DCs) that are the most efficient antigen-presenting cells. A bioinformatic analysis of the KIF18A gene family was performed to understand its sequence variability and evolutionary history. To inhibit KIF18A activity, a highly specific small molecule inhibitor for KIF18A, BTB-1 was used. DCs were differentiated from mouse bone marrow (BM) cells from 6 to 7 week old C57BL/6 mice with recombinant granulocyte–macrophage colony-stimulating factor (GM-CSF). Expression of KIF18A was measured by Western blotting. The surface expression of differentiation and activation markers on DCs were analyzed by flow cytometry. The phylogenetic analysis revealed that the KIF18A gene family is remarkably conserved across vertebrates. Interestingly, the expression of KIF18A was increased as BM precursor cells differentiated into DCs. BTB-1 treatment strongly inhibited the differentiation of BM cells into DCs in a dose-dependent manner. Furthermore, treatment of immature DCs with BTB-1 significantly impaired the expression of activation markers on DCs including MHC class I, CD80, and CD86 upon TLR4 or TLR7 treatment. Our results reveal that KIF18A is a critical DC differentiation and activation regulator. Therefore, KIF18A could be a potential therapeutic target for immune-mediated disorders.

Chios mastic gum (CMG), a resin of the mastic tree (Pistacia lentiscus var. chia), has been used to treat multiple disorders caused by gastrointestinal malfunctions and bacterial infections for more than 2500 years. However, little is known about CMG’s antiviral activity. CMG is known to influence multiple cellular processes such as cell proliferation, differentiation and apoptosis. As virus replication is largely dependent on the host cellular metabolism, it is conceivable that CMG regulates virus infectivity. Therefore, in this study, we evaluated CMG’s potential as an antiviral drug to treat influenza A virus (IAV) infection. CMG treatment dramatically reduced the cytopathogenic effect and production of RNAs, proteins and infectious particles of IAV. Interestingly, CMG interfered with the early stage of the virus life cycle after viral attachment. Importantly, the administration of CMG greatly ameliorated morbidity and mortality in IAV-infected mice. The results suggest that CMG displays a potent anti-IAV activity by blocking the early stage of viral replication. Thus, mastic gum could be exploited as a novel therapeutic agent against IAV infection.

Viral infectious diseases are a significant burden on public health and the global economy, and new viral threats emerge continuously. Since CD4+ and CD8+ T cell responses are essential to eliminating viruses, it is important to understand the underlying mechanisms of anti-viral T cell-mediated immunopathogenesis during viral infections. Remarkable progress in transgenic (Tg) techniques has enabled scientists to more readily understand the mechanisms of viral pathogenesis. T cell receptor (TCR) Tg mice are extremely useful in studying T cell-mediated immune responses because the majority of T cells in these mice express specific TCRs for partner antigens. In this review, we discuss the important studies utilizing TCR Tg mice to unveil underlying mechanisms of T cell-mediated immunopathogenesis during viral infections.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) have been known to exert anti-inflammatory effects on various disease states. However, its effect on CD8+ T cell-mediated immunopathology upon viral infection has not been well elucidated yet. In this study, we investigated the possible implication of n-3 PUFAs in CD8+ T cell responses against an acute viral infection. Infection of FAT-1 transgenic mice that are capable of synthesizing n-3 PUFAs from n-6 PUFAs with lymphocytic choriomeningitis virus (LCMV) resulted in significant reduction of anti-viral CD8+ T cell responses. Interestingly, expansion of adoptively transferred wild-type (WT) LCMV-specific T cell receptor (TCR) transgenic CD8+ (P14) T cells into FAT-1 mice was significantly decreased. Also, activation of anti-viral CD4+ helper T cells was reduced in FAT-1 mice. Importantly, P14 cells carrying the fat-1 gene that were adoptively transferred into WT mice exhibited a substantially decreased ability to proliferate and produce cytokines against LCMV infection. Together, n-3 PUFAs attenuated anti-viral CD8+ T cell responses against an acute viral infection and thus could be used to alleviate immunopathology mediated by the viral infection.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs) have been known to exert anti-inflammatory effects on various inflammatory diseases. However, its role in CD8+ T cell responses against an acute viral infection has not been well elucidated yet. To determine the role of n-3 PUFAs in anti-viral CD8+ T cell responses, we used FAT-1 transgenic mice that are able to convert n-6 PUFAs to n-3 PUFAs. The FAT-1 mice or mice orally administrated with n-3 PFUAs exhibited a significant reduction of anti-viral CD8+ T cell responses against an acute strain of lymphocytic choriomeningitis virus (LCMV). When LCMV-specific P14 CD8+ T cells carrying fat-1 gene (P14/FAT-1) were adoptively transferred into mice that were subsequently infected with LCMV, expansion of the cells was substantially suppressed. Similarly, when P14/FAT-1 cells were stimulated with LCMV gp33 peptide in vitro, their expansion was significantly reduced as compared to P14 cells. Collectively, our results indicate that n-3 PUFAs attenuate anti-viral CD8+ T cell responses against acute viral infection.

Digital holographic microscopy (DHM) is an emerging imaging technique that permits instantaneous capture of a relatively large sample volume. However, large volumes usually come at the expense of lower spatial resolution, and the technique has rarely been used with prokaryotic cells due to their small size and low contrast. In this paper we demonstrate the use of a Mach-Zehnder dual-beam instrument for imaging of labeled and unlabeled bacteria and microalgae. Spatial resolution of 0.3 μm is achieved, providing a sampling of several pixels across a typical prokaryotic cell. Both cellular motility and morphology are readily recorded. The use of dyes provides both amplitude and phase contrast improvement and is of use to identify cells in dense samples., Frontiers in Chemistry, 4, ISSN:2296-2646

Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed.

Holographic microscopy is an emerging biological technique that provides amplitude and quantitative phase imaging, though the contrast provided by many cell types and organelles is low, and until now no dyes were known that increased contrast. Here we show that the metallocorrole Ga(tpfc)(SO_3H)_2, which has a strong Soret band absorption, increases contrast in both amplitude and phase and facilitates tracking of Escherichia coli with minimal toxicity. The change in phase contrast may be calculated from the dye-absorbance spectrum using the Kramers–Kronig relations, and represents a general principle that may be applied to any dye or cell type. This enables the use of holographic microscopy for all applications in which specific labeling is desired.