Hepatocyte Adenosine Kinase Promotes Excessive Fat Deposition and Liver Inflammation.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease is highly associated with obesity and progresses to nonalcoholic steatohepatitis when the liver develops overt inflammatory damage. While removing adenosine in the purine salvage pathway, adenosine kinase (ADK) regulates methylation reactions. We aimed to study whether hepatocyte ADK functions as an obesogenic gene/enzyme to promote excessive fat deposition and liver inflammation. METHODS: Liver sections of human subjects were examined for ADK expression using immunohistochemistry. Mice with hepatocyte-specific ADK disruption or overexpression were examined for hepatic fat deposition and inflammation. Liver lipidomics, hepatocyte RNA sequencing (RNA-seq), and single-cell RNA-seq for liver nonparenchymal cells were performed to analyze ADK regulation of hepatocyte metabolic responses and hepatocyte-nonparenchymal cells crosstalk. RESULTS: Whereas patients with nonalcoholic fatty liver disease had increased hepatic ADK levels, mice with hepatocyte-specific ADK disruption displayed decreased hepatic fat deposition on a chow diet and were protected from diet-induced excessive hepatic fat deposition and inflammation. In contrast, mice with hepatocyte-specific ADK overexpression displayed increased body weight and adiposity and elevated degrees of hepatic steatosis and inflammation compared with control mice. RNA-seq and epigenetic analyses indicated that ADK increased hepatic DNA methylation and decreased hepatic Ppara expression and fatty acid oxidation. Lipidomic and single-cell RNA-seq analyses indicated that ADK-driven hepatocyte factors, due to mitochondrial dysfunction, enhanced macrophage proinflammatory activation in manners involving increased expression of stimulator of interferon genes. CONCLUSIONS: Hepatocyte ADK functions to promote excessive fat deposition and liver inflammation through suppressing hepatocyte fatty acid oxidation and producing hepatocyte-derived proinflammatory mediators. Therefore, hepatocyte ADK is a therapeutic target for managing obesity and nonalcoholic fatty liver disease.

Influenza C and D Viruses Demonstrated a Differential Respiratory Tissue Tropism in a Comparative Pathogenesis Study in Guinea Pigs.

Influenza C virus (ICV) is increasingly associated with community-acquired pneumonia (CAP) in children and its disease severity is worse than the influenza B virus, but similar to influenza A virus associated CAP. Despite the ubiquitous infection landscape of ICV in humans, little is known about its replication and pathobiology in animals. The goal of this study was to understand the replication kinetics, tissue tropism, and pathogenesis of human ICV (huICV) in comparison to the swine influenza D virus (swIDV) in guinea pigs. Intranasal inoculation of both viruses did not cause clinical signs, however, the infected animals shed virus in nasal washes. The huICV replicated in the nasal turbinates, soft palate, and trachea but not in the lungs while swIDV replicated in all four tissues. A comparative analysis of tropism and pathogenesis of these two related seven-segmented influenza viruses revealed that swIDV-infected animals exhibited broad tissue tropism with an increased rate of shedding on 3, 5, and 7 dpi and high viral loads in the lungs compared to huICV. Seroconversion occurred late in the huICV group at 14 dpi, while swIDV-infected animals seroconverted at 7 dpi. Guinea pigs infected with huICV exhibited mild to moderate inflammatory changes in the epithelium of the soft palate and trachea, along with mucosal damage and multifocal alveolitis in the lungs. In summary, the replication kinetics and pathobiological characteristics of ICV in guinea pigs agree with the clinical manifestation of ICV infection in humans, and hence guinea pigs could be used to study these distantly related influenza viruses. IMPORTANCE Similar to influenza A and B, ICV infections are seen associated with bacterial and viral co-infections which complicates the assessment of its real clinical significance. Further, the antivirals against influenza A and B viruses are ineffective against ICV which mandates the need to study the pathobiological aspects of this virus. Here we demonstrated that the respiratory tract of guinea pigs possesses specific viral receptors for ICV. We also compared the replication kinetics and pathogenesis of huICV and swIDV, as these viruses share 50% sequence identity. The tissue tropism and pathology associated with huICV in guinea pigs are analogous to the mild respiratory disease caused by ICV in humans, thereby demonstrating the suitability of guinea pigs to study ICV. Our comparative analysis revealed that huICV and swIDV replicated differentially in the guinea pigs suggesting that the type-specific genetic differences can result in the disparity of the viral shedding and tissue tropism.

Associations between sarcopenia and circulating branched-chain amino acids: a cross-sectional study over 100,000 participants.

BACKGROUND: Emerging evidence suggests that alterations in BCAA metabolism may contribute to the pathogenesis of sarcopenia. However, the relationship between branched-chain amino acids (BCAAs) and sarcopenia is incompletely understood, and existing literature presents conflicting results. In this study, we conducted a community-based study involving > 100,000 United Kingdom adults to comprehensively explore the association between BCAAs and sarcopenia, and assess the potential role of muscle mass in mediating the relationship between BCAAs and muscle strength. METHODS: Multivariable linear regression analysis examined the relationship between circulating BCAAs and muscle mass/strength. Logistic regression analysis assessed the impact of circulating BCAAs and quartiles of BCAAs on sarcopenia risk. Subgroup analyses explored the variations in associations across age, and gender. Mediation analysis investigated the potential mediating effect of muscle mass on the BCAA-muscle strength relationship. RESULTS: Among 108,017 participants (mean age: 56.40 ± 8.09 years; 46.23% men), positive associations were observed between total BCAA, isoleucine, leucine, valine, and muscle mass (beta, 0.56-2.53; p < 0.05) and between total BCAA, leucine, valine, and muscle strength (beta, 0.91-3.44; p < 0.05). Logistic regression analysis revealed that increased circulating valine was associated with a 47% reduced sarcopenia risk (odds ratio = 0.53; 95% confidence interval = 0.3-0.94; p = 0.029). Subgroup analyses demonstrated strong associations between circulating BCAAs and muscle mass/strength in men and individuals aged ≥ 60 years. Mediation analysis suggested that muscle mass completely mediated the relationship between total BCAA, and valine levels and muscle strength, partially mediated the relationship between leucine levels and muscle strength, obscuring the true effect of isoleucine on muscle strength. CONCLUSION: This study suggested the potential benefits of BCAAs in preserving muscle mass/strength and highlighted muscle mass might be mediator of BCAA-muscle strength association. Our findings contribute new evidence for the clinical prevention and treatment of sarcopenia and related conditions involving muscle mass/strength loss.

Anti-PD-1 antibody-activated Th17 cells subvert re-invigoration of antitumor cytotoxic T-lymphocytes via myeloid cell-derived COX-2/PGE2.

Anti-PD-1 antibody-mediated activation of type 17 T-cells undermines checkpoint inhibitor therapy in the LSL-KrasG12D murine lung cancer model. Herein, we establish that the Th17 subset is the primary driver of resistance to therapy demonstrate that the ontogeny of dysplasia-associated Th17 cells is driven by microbiota-conditioned macrophages; and identify the IL-17-COX-2-PGE2 axis as the mediator of CD8+ cytotoxic T-lymphocyte de-sensitization to checkpoint inhibitor therapy. Specifically, anti-PD-1 treatment of LSL-KrasG12D mice, in which CD4+ T-cells were deficient for RORc, resulted in a 60% increase in CTL cytotoxicity and a 2.5-fold reduction in tumor burden confirming the critical role of Th17 cells in resistance to therapy. Lung-specific depletion of microbiota reduced Th17 cell prevalence and tumor burden by 5- and 2.5-fold, respectively; establishing a link between microbiota and Th17 cell-driven tumorigenesis. Importantly, lung macrophages from microbiota sufficient, but not from microbiota-deficient, mice polarized naïve CD4+ T-cells to a Th17 phenotype, highlighting their role in bridging microbiota and Th17 immunity. Further, treatment with anti-PD-1 enhanced COX-2 and PGE2 levels, whereas neutralization of IL-17 diminished this effect. In contrast, inhibition of COX-2 rescued CTL activity and restored tumor suppression in anti-PD-1-treated mice, revealing the molecular basis of IL-17-mediated resistance to checkpoint blockade. Clinical implications of these findings are discussed.

The CsHSFA-CsJAZ6 module-mediated high temperature regulates flavonoid metabolism in Camellia sinensis.

High temperatures (HTs) seriously affect the yield and quality of tea. Catechins, derived from the flavonoid pathway, are characteristic compounds that contribute to the flavour of tea leaves. In this study, we first showed that the flavonoid content of tea leaves was significantly reduced under HT conditions via metabolic profiles; and then demonstrated that two transcription factors, CsHSFA1b and CsHSFA2 were activated by HT and negatively regulate flavonoid biosynthesis during HT treatment. Jasmonate (JA), a defensive hormone, plays a key role in plant adaption to environmental stress. However, little has been reported on its involvement in HT response in tea. Herein, we demonstrated that CsHSFA1b and CsHSFA2 activate CsJAZ6 expression through directly binding to heat shock elements in its promoter, and thereby repress the JA pathway. Most secondary metabolites are regulated by JA, including catechin in tea. Our study reported that CsJAZ6 directly interacts with CsEGL3 and CsTTG1 and thereby reduces catechin accumulation. From this, we proposed a CsHSFA-CsJAZ6-mediated HT regulation model of catechin biosynthesis. We also determined that negative regulation of the JA pathway by CsHSFAs and its homologues is conserved in Arabidopsis. These findings broaden the applicability of the regulation of JAZ by HSF transcription factors and further suggest the JA pathway as a valuable candidate for HT-resistant breeding and cultivation.

Macular edema after siponimod treatment for multiple sclerosis: a case report and literature review.

BACKGROUND: As a modulator of the sphingosine 1-phosphate receptor, siponimod is administered as a therapeutic intervention for multiple sclerosis. A previous phase 3 study first reported siponimod-associated macular edema. Since that report, there were only few relevant reports in clinical settings. Here, we report a case of secondary progressive multiple sclerosis developed macular edema after siponimod treatment. We also review the progress of sphingosine 1-phosphate receptor modulators, elaborate on accepted mechanisms in treating multiple sclerosis, and discuss the causation of siponimod-associated macular edema. CASE PRESENTATION: A 38-year-old Chinese female patient with secondary progressive multiple sclerosis, who had recurrent numbness of the limbs and right leg fatigue, developed mild macular edema following 4 months of siponimod treatment. The macular edema resolved after discontinuing the medication, and did not recur after resuming siponimod. CONCLUSION: Although siponimod-associated macular edema may be rare, mild, transitory, and manageable, it cannot be ignored and requires ongoing vigilance.

Shading-Dependent Greening Process of the Leaves in the Light-Sensitive Albino Tea Plant 'Huangjinya': Possible Involvement of the Light-Harvesting Complex II Subunit of Photosystem II in the Phenotypic Characteristic.

The light-sensitive albino tea plant can produce pale-yellow shoots with high levels of amino acids which are suitable to process high-quality tea. In order to understand the mechanism of the albino phenotype formation, the changes in the physio-chemical characteristics, chloroplast ultrastructure, chlorophyll-binding proteins, and the relevant gene expression were comprehensively investigated in the leaves of the light-sensitive albino cultivar 'Huangjinya' ('HJY') during short-term shading treatment. In the content of photosynthetic pigments, the ultrastructure of the chloroplast, and parameters of the photosynthesis in the leaves of 'HJY' could be gradually normalized along with the extension of the shading time, resulting in the leaf color transformed from pale yellow to green. BN-PAGE and SDS-PAGE revealed that function restoration of the photosynthetic apparatus was attributed to the proper formation of the pigment-protein complexes on the thylakoid membrane that benefited from the increased levels of the LHCII subunits in the shaded leaves of 'HJY', indicating the low level of LHCII subunits, especially the lack of the Lhcb1 might be responsible for the albino phenotype of the 'HJY' under natural light condition. The deficiency of the Lhcb1 was mainly subject to the strongly suppressed expression of the Lhcb1.x which might be modulated by the chloroplast retrograde signaling pathway GUN1 (GENOMES UNCOUPLED 1)-PTM (PHD type transcription factor with transmembrane domains)-ABI4 (ABSCISIC ACID INSENSITIVE 4).

Methionine- and Choline-Deficient Diet-Induced Nonalcoholic Steatohepatitis Is Associated with Increased Intestinal Inflammation.

Inflammation drives the pathogenesis of nonalcoholic steatohepatitis (NASH). The current study examined changes in intestinal inflammation during NASH. In male C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in severe hepatic steatosis and inflammation relative to feeding a chow diet (CD). MCD-fed mice exhibited characteristics of mucosal and submucosal inflammatory responses compared with CD-fed mice. Moreover, intestinal phosphorylation states of c-Jun N-terminal protein kinase p46 and mRNA levels of IL-1B, IL-6, tumor necrosis factor alpha, and monocyte chemoattractant protein-1 were significantly higher and intestinal mRNA levels of IL-4 and IL-13 were significantly lower in MCD-fed mice compared with those in CD mice. Surprisingly, upon treatment with MCD-mimicking media, the proinflammatory responses in cultured intestinal epithelial CMT-93 cells did not differ significantly from those in CMT-93 cells treated with control media. In contrast, in RAW264.7 macrophages, MCD-mimicking media significantly increased the phosphorylation states of c-Jun N-terminal protein kinase p46 and mitogen-activated protein kinases p38 and mRNA levels of IL-1B, IL-6, IL-10, and tumor necrosis factor alpha under either basal or lipopolysaccharide-stimulated conditions. Collectively, these results suggest that increased intestinal inflammation is associated with NASH phenotype. Thus, elevated proinflammatory responses in macrophages likely contribute to, in large part, increased intestinal inflammation in NASH.

Anti-PD-1 antibody-mediated activation of type 17 T-cells undermines checkpoint blockade therapy.

Tumors that develop in the genetic LSL-K-rasG12D murine lung cancer model are resistant to anti-PD-1 antibody treatment. Analysis of tumor-bearing lungs from anti-PD-1-treated mice revealed an up to 2.5-fold increase in IL-17-producing T-cells, with minimal change in CD8+ T-cell activity. Neutralization of IL-17 concurrent with anti-PD-1 treatment on the other hand, resulted in robust CD8+ T-cell activation and a threefold reduction in tumor burden. Loss-of-function studies demonstrated that anti-PD-1 driven activation of CD4+ and γδTCR+ T-cells contributed to IL-17-mediated de-sensitization of CD8+ cytotoxic T-cells (CTL) to therapy; and that CTL activation was critical to tumor eradication. Importantly, post-therapy lung Th17 cell prevalence and activity prognosticated treatment efficacy. Consistent with the murine data, analysis of tumor biopsy samples from non-small cell lung cancer (NSCLC) patients revealed that pre-therapy intratumoral CD8+/RORc+ cell ratio correlated with response to immune checkpoint blockade (ICB). These findings provide the initial evidence for a new mechanism of ICB resistance in lung cancer.

Indole Alleviates Diet-Induced Hepatic Steatosis and Inflammation in a Manner Involving Myeloid Cell 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3.

BACKGROUND AND AIMS: Indole is a microbiota metabolite that exerts anti-inflammatory responses. However, the relevance of indole to human non-alcoholic fatty liver disease (NAFLD) is not clear. It also remains largely unknown whether and how indole acts to protect against NAFLD. The present study sought to examine the association between the circulating levels of indole and liver fat content in human subjects and explore the mechanisms underlying indole actions in mice with diet-induced NAFLD. APPROACH AND RESULTS: In a cohort of 137 subjects, the circulating levels of indole were reversely correlated with body mass index. In addition, the circulating levels of indole in obese subjects were significantly lower than those in lean subjects and were accompanied with increased liver fat content. At the whole-animal level, treatment of high-fat diet (HFD)-fed C57BL/6J mice with indole caused significant decreases in the severity of hepatic steatosis and inflammation. In cultured cells, indole treatment stimulated the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a master regulatory gene of glycolysis, and suppressed macrophage proinflammatory activation in a PFKFB3-dependent manner. Moreover, myeloid cell-specific PFKFB3 disruption exacerbated the severity of HFD-induced hepatic steatosis and inflammation and blunted the effect of indole on alleviating diet-induced NAFLD phenotype. CONCLUSIONS: Taken together, our results demonstrate that indole is relevant to human NAFLD and capable of alleviating diet-induced NAFLD phenotypes in mice in a myeloid cell PFKFB3-dependent manner. Therefore, indole mimetic and/or macrophage-specific PFKFB3 activation may be the viable preventive and/or therapeutic approaches for inflammation-associated diseases including NAFLD.

Transcription factor Foxo1 represses T-bet-mediated effector functions and promotes memory CD8(+) T cell differentiation.

The evolutionary conserved Foxo transcription factors are important regulators of quiescence and longevity. Although, Foxo1 is known to be important in regulating CD8(+) T cell trafficking and homeostasis, its role in functional differentiation of antigen-stimulated CD8(+) T cells is unclear. Herein, we demonstrate that inactivation of Foxo1 was essential for instructing T-bet transcription factor-mediated effector differentiation of CD8(+) T cells. The Foxo1 inactivation was dependent on mTORC1 kinase, given that blockade of mTORC1 abrogated mTORC2-mediated Akt (Ser473) kinase phosphorylation, resulting in Foxo1-dependent switch from T-bet to Eomesodermin transcription factor activation and increase in memory precursors. Silencing Foxo1 ablated interleukin-12- and rapamycin-enhanced CD8(+) T cell memory responses and restored T-bet-mediated effector functions. These results demonstrate an essential role of Foxo1 in actively repressing effector or terminal differentiation processes to promote memory CD8(+) T cell development and identify the functionally diverse mechanisms utilized by Foxo1 to promote quiescence and longevity.

Temporospatial shifts within commercial laboratory mouse gut microbiota impact experimental reproducibility.

BACKGROUND: Experimental reproducibility in mouse models is impacted by both genetics and environment. The generation of reproducible data is critical for the biomedical enterprise and has become a major concern for the scientific community and funding agencies alike. Among the factors that impact reproducibility in experimental mouse models is the variable composition of the microbiota in mice supplied by different commercial vendors. Less attention has been paid to how the microbiota of mice supplied by a particular vendor might change over time. RESULTS: In the course of conducting a series of experiments in a mouse model of malaria, we observed a profound and lasting change in the severity of malaria in mice infected with Plasmodium yoelii; while for several years mice obtained from a specific production suite of a specific commercial vendor were able to clear the parasites effectively in a relatively short time, mice subsequently shipped from the same unit suffered much more severe disease. Gut microbiota analysis of frozen cecal samples identified a distinct and lasting shift in bacteria populations that coincided with the altered response of the later shipments of mice to infection with malaria parasites. Germ-free mice colonized with cecal microbiota from mice within the same production suite before and after this change followed by Plasmodium infection provided a direct demonstration that the change in gut microbiota profoundly impacted the severity of malaria. Moreover, spatial changes in gut microbiota composition were also shown to alter the acute bacterial burden following Salmonella infection, and tumor burden in a lung tumorigenesis model. CONCLUSION: These changes in gut bacteria may have impacted the experimental reproducibility of diverse research groups and highlight the need for both laboratory animal providers and researchers to collaborate in determining the methods and criteria needed to stabilize the gut microbiota of animal breeding colonies and research cohorts, and to develop a microbiota solution to increase experimental rigor and reproducibility.

Antiviral Effects of Green Tea EGCG and Its Potential Application against COVID-19.

(-)-Epigallocatechin-3-O-gallate (EGCG), the most abundant component of catechins in tea (Camellia sinensis (L.) O. Kuntze), plays a role against viruses through inhibiting virus invasiveness, restraining gene expression and replication. In this paper, the antiviral effects of EGCG on various viruses, including DNA virus, RNA virus, coronavirus, enterovirus and arbovirus, were reviewed. Meanwhile, the antiviral effects of the EGCG epi-isomer counterpart (+)-gallocatechin-3-O-gallate (GCG) were also discussed.

Screening the Key Region of Sunlight Regulating the Flavonoid Profiles of Young Shoots in Tea Plants (Camellia sinensis L.) Based on a Field Experiment.

Both UV and blue light have been reported to regulate the biosynthesis of flavonoids in tea plants; however, the respective contributions of the corresponding regions of sunlight are unclear. Additionally, different tea cultivars may respond differently to altered light conditions. We investigated the responses of different cultivars ('Longjing 43', 'Zhongming 192', 'Wanghai 1', 'Jingning 1' and 'Zhonghuang 2') to the shade treatments (black and colored nets) regarding the biosynthesis of flavonoids. For all cultivars, flavonol glycosides showed higher sensitivity to light conditions compared with catechins. The levels of total flavonol glycosides in the young shoots of different tea cultivars decreased with the shade percentages of polyethylene nets increasing from 70% to 95%. Myricetin glycosides and quercetin glycosides were more sensitive to light conditions than kaempferol glycosides. The principal component analysis (PCA) result indicated that shade treatment greatly impacted the profiles of flavonoids in different tea samples based on the cultivar characteristics. UV is the crucial region of sunlight enhancing flavonol glycoside biosynthesis in tea shoots, which is also slight impacted by light quality according to the results of the weighted correlation network analysis (WGCNA). This study clarified the contributions of different wavelength regions of sunlight in a field experiment, providing a potential direction for slightly bitter and astringent tea cultivar breeding and instructive guidance for practical field production of premium teas based on light regimes.

Interleukin-21 enhances the antibody avidity elicited by DNA prime and MVA boost vaccine.

Enhancement of the magnitude or affinity of protective antibodies (Abs) induced by vaccine adjuvant is highly desirable to prevent challenging pathogens such as HIV-1. IL-21 plays a crucial role in germinal center reactions during humoral immune responses. However, the effect of IL-21 as a vaccine adjuvant on the quantity and quality of antigen-specific Abs elicited by DNA prime and MVA boost vaccine, a commonly used vaccine strategy, remains unknown. To close this knowledge gap, female adult B6N mice were primed with DNA vaccine twice (days 0, 14, 100 µg, I.M.) and boosted with MVA vaccine (day 28, 2 × 107 pfu, I.M.) with or without an IL-21 DNA adjuvant (days 3, 17, 31, 40 µg, I.M.), in which HIV-1 gag was expressed as a model antigen. With the addition of an IL-21 adjuvant, we found significantly increased avidity of antigen-specific Abs at multiple time points in a longitudinal follow up. Collectively, our results suggest that an IL-21 immune adjuvant can significantly increase Ab quality induced by heterologous DNA-MVA prime-boost vaccine strategy.

A central role for mTOR kinase in homeostatic proliferation induced CD8+ T cell memory and tumor immunity.

The cell-intrinsic mechanisms guiding naive CD8+ T cells for clonal expansion and memory generation via homeostatic proliferation (HP) are unclear. Here, we have shown that HP of naive CD8+ T cells requires IL-7-, but not IL-15-induced mTOR kinase activation. HP-induced mTOR enhances transcription factor T-bet for functional maturation and CD122 expression, which sensitizes for an IL-15-dependent memory transition by favoring transcription factor Eomesodermin over T-bet. Inhibition of mTOR blocks T-bet and CD122 expression but preserves memory in an IL-15-independent manner by promoting Eomesodermin expression. The ability of rapamycin to augment HP-induced memory was cell-intrinsic given that silencing mTOR in CD8+ T cells generated identical outcomes. Strikingly, HP-induced CD8+ T cell memory generated by IL-15-dependent or -independent mechanisms demonstrated identical tumor efficacy. These results indicate a central role for mTOR in HP-induced CD8+ T cell responses and demonstrate the importance for CD8+ memory in HP-induced tumor efficacy.

Inhaled IL-10 Suppresses Lung Tumorigenesis via Abrogation of Inflammatory Macrophage-Th17 Cell Axis.

Intratracheal administration of a novel IL-10 formulation suppressed IL-17-driven, CD4+ T cell-dependent tumorigenesis in the LSL-K-rasG12D murine lung cancer model. Analysis of lung lymphocyte populations demonstrated that antitumor activity of IL-10 was associated with a 5-fold decline in Th17 cell prevalence and a concurrent suppression of inflammatory M1-like macrophage activity. Further phenotypic characterization revealed that macrophages and dendritic cells, but not Th17 cells, expressed IL-10RA on the cell surface with the CD11b+F4/80+CX3CR1+ interstitial macrophages representing the dominant IL-10RA+ subset. Consistent with these observations, in vitro stimulation of sorted CD4+ T cells with IL-10 did not affect their ability to produce IL-17, whereas similar treatment of purified interstitial macrophages resulted in a dramatic M1 to M2 phenotypic switch. Importantly, preconditioning of macrophages (but not of CD4+ T cells) with IL-10 led to potent suppression of CD4+ T cell IL-17 production in an in vitro coculture assay, suggesting that IL-10 suppressed Th17 cell activity primarily via its upstream effects on macrophages. In support of this notion, in vivo macrophage depletion resulted in a 5-fold decline in Th17 cell numbers and a concurrent 6-fold reduction in tumor burden. Collectively, these data demonstrate that in the LSL-K-rasG12D murine lung cancer model, inflammatory macrophage-Th17 cell axis is critical to tumorigenesis and that IL-10 blocks this process primarily via a direct effect on the former. Inhaled IL-10 formulations may be of use in prophylaxis against lung cancer in high-risk patients.

Altered Ratio of T Follicular Helper Cells to T Follicular Regulatory Cells Correlates with Autoreactive Antibody Response in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

Individuals with chronic HIV-1 infection have an increased prevalence of autoreactive Abs. Many of the isolated HIV broadly neutralizing Abs from these individuals are also autoreactive. However, the underlying mechanism(s) that produce these autoreactive broadly neutralizing Abs remains largely unknown. The highly regulated coordination among B cells, T follicular helper (TFH) cells, and T follicular regulatory (TFR) cells in germinal centers (GCs) of peripheral lymphatic tissues (LTs) is essential for defense against pathogens while also restricting autoreactive responses. We hypothesized that an altered ratio of TFH/TFR cells in the GC contributes to the increased prevalence of autoreactive Abs in chronic HIV infection. We tested this hypothesis using a rhesus macaque (RM) SIV model. We measured the frequency of TFH cells, TFR cells, and GC B cells in LTs and anti-dsDNA and anti-phospholipid Abs from Indian RMs, with and without SIV infection. We found that the frequency of anti-dsDNA and anti-phospholipid Abs was much higher in chronically infected RMs (83.3% [5/6] and 66.7% [4/6]) than in acutely infected RMs (33.3% [2/6] and 18.6% [1/6]) and uninfected RMs (0% [0/6] and 18.6% [1/6]). The increased ratio of TFH/TFR cells in SIV infection correlated with anti-dsDNA and anti-phospholipid autoreactive Ab levels, whereas the frequency of TFR cells alone did not correlate with the levels of autoreactive Abs. Our results provide direct evidence that the ratio of TFH/TFR cells in LTs is critical for regulating autoreactive Ab production in chronic SIV infection and possibly, by extension, in chronic HIV-1 infection.

Associations of Simian Immunodeficiency Virus (SIV)-Specific Follicular CD8+ T Cells with Other Follicular T Cells Suggest Complex Contributions to SIV Viremia Control.

Follicular CD8+ T (fCD8) cells reside within B cell follicles and are thought to be immune-privileged sites of HIV/SIV infection. We have observed comparable levels of fCD8 cells between chronically SIV-infected rhesus macaques with low viral loads (LVL) and high viral loads (HVL), raising the question concerning their contribution to viremia control. In this study, we sought to clarify the role of SIV-specific fCD8 cells in lymph nodes during the course of SIV infection in rhesus macaques. We observed that fCD8 cells, T follicular helper (Tfh) cells, and T follicular regulatory cells (Tfreg) were all elevated in chronic SIV infection. fCD8 cells of LVL animals tended to express more Gag-specific granzyme B and exhibited significantly greater killing than did HVL animals, and their cell frequencies were negatively correlated with viremia, suggesting a role in viremia control. Env- and Gag-specific IL-21+ Tfh of LVL but not HVL macaques negatively correlated with viral load, suggesting better provision of T cell help to fCD8 cells. Tfreg positively correlated with fCD8 cells in LVL animals and negatively correlated with viremia, suggesting a potential benefit of Tfreg via suppression of chronic inflammation. In contrast, in HVL macaques, Tfreg and fCD8 cell frequencies tended to be negatively correlated, and a positive correlation was seen between Tfreg number and viremia, suggesting possible dysfunction and suppression of an effective fCD8 cell immune response. Our data suggest that control of virus-infected cells in B cell follicles not only depends on fCD8 cell cytotoxicity but also on complex fCD8 cell associations with Tfh cells and Tfreg.

A DNA Vaccine Expressing Consensus Hemagglutinin-Esterase Fusion Protein Protected Guinea Pigs from Infection by Two Lineages of Influenza D Virus.

Two lineages of influenza D virus (IDV) have been found to infect cattle and promote bovine respiratory disease complex, one of the most commonly diagnosed causes of morbidity and mortality within the cattle industry. Furthermore, IDV can infect other economically important domestic livestock, including pigs, and has the potential to infect humans, which necessitates the need for an efficacious vaccine. In this study, we designed a DNA vaccine expressing consensus hemagglutinin-esterase fusion (HEF) protein (FluD-Vax) and tested its protective efficacy against two lineages of IDV (D/OK and D/660) in guinea pigs. Animals that received FluD-Vax (n = 12) developed appreciable titers of neutralizing antibodies against IDV lineage representatives, D/OK and D/660. Importantly, vaccinated animals were protected against intranasal challenge with IDV [3 × 105 50% tissue culture infective dose(s) (TCID50)] D/OK (n = 6) or D/600 (n = 6), based on the absence of viral RNA in necropsied tissues (5 and 7 days postchallenge) using quantitative reverse transcription-PCR and in situ hybridization. In contrast, animals that received a sham DNA vaccine (n = 12) had no detectable neutralizing antibodies against IDV, and viral RNA was readily detectable in respiratory tract tissues after intranasal challenge (3 × 105 TCID50) with IDV D/OK (n = 6) or D/660 (n = 6). Using a TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) assay, we found that IDV D/OK and D/600 infections induced apoptosis in epithelial cells lining alveoli and bronchioles, as well as nonepithelial cells in lung tissues. Our results demonstrate for the first time that the consensus IDV HEF DNA vaccine can elicit complete protection against infection from two lineages of IDV in the guinea pig model.IMPORTANCE Influenza D virus (IDV) infection has been associated with bovine respiratory disease complex, one of the most devastating diseases of the cattle population. Moreover, with broad host range and high environmental stability, IDV has the potential to further gain virulence or even infect humans. An efficacious vaccine is needed to prevent infection and stop potential cross-species transmission. In this study, we designed a DNA vaccine encoding the consensus hemagglutinin-esterase fusion (HEF) protein of two lineages of IDV (D/OK and D/660) and tested its efficacy in a guinea pig model. Our results showed that the consensus DNA vaccine elicited high-titer neutralizing antibodies and achieved sterilizing protection against two lineage-representative IDV intranasal infections. To our knowledge, this is the first study showing that a DNA vaccine expressing consensus HEF is efficacious in preventing different lineages of IDV infections.