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Translocator protein 18 kDa (TSPO) is a well-known outer mitochondrial membrane protein and it is widely used as a biomarker of neuroinflammation and brain injury. Although it is thought that TSPO plays key roles in a multitude of host cell functions, including steroid biosynthesis, apoptosis, generation of reactive oxygen species, and proliferation, some of these functions have recently been questioned. Here, we report the unexpected finding that circulating immune cells differentially express basal levels of TSPO on their cell surface, with a high percentage of monocytes and neutrophils expressing cell surface TSPO. In vitro stimulation of monocytes with LPS significantly increases the frequency of cells with surface TSPO expression in the absence of altered gene expression. Importantly, the LPS increase in TSPO cell surface expression in monocytes appears to be selective for LPS because two other distinct monocyte activators failed to increase the frequency of cells with surface TSPO. Finally, when we quantified immune cell TSPO surface expression in antiretroviral therapy-treated HIV+ donors, a chronic inflammatory disease, we found significant increases in the frequency of TSPO surface localization, which could be pharmacologically suppressed with ∆9-tetrahydrocannabinol. These findings suggest that cell surface TSPO in circulating leukocytes could serve as a peripheral blood-based biomarker of inflammation.

HIV infection affects an estimated 38 million people. Approximately 50% of HIV patients exhibit neurocognitive dysfunction termed HIV-Associated Neurocognitive Disorder (HAND). HAND is a consequence of chronic low-level neuroinflammation due to HIV entry into the brain. Initially, monocytes become activated in circulation and traffic to the brain. Monocytes, when activated, become susceptible to infection by HIV and can then carry the virus across the blood brain barrier. Once in the brain, activated monocytes secrete chemokines, which recruit virus-specific CD8+ T cells into the brain to further promote neuroinflammation. HAND is closely linked to systemic inflammation driven, in part, by HIV but is also due to persistent translocation of microorganisms across the GI tract. Persistent anti-viral responses in the GI tract compromise microbial barrier integrity. Indeed, HIV patients can exhibit remarkably high levels of activated (CD16+) monocytes in circulation. Recent studies, including our own, show that HIV patients using medical marijuana exhibit lower levels of circulating CD16+ monocytes than non-cannabis using HIV patients. Cannabis is a known immune modulator, including anti-inflammatory properties, mediated, in part, by ∆9-tetrahydrocannabinol (THC), as well as less characterized minor cannabinoids, such as cannabidiol (CBD), terpenes and presumably other cannabis constituents. The immune modulating activity of THC is largely mediated through cannabinoid receptors (CB) 1 and 2, with CB1 also responsible for the psychotropic properties of cannabis. Here we discuss the anti-inflammatory properties of cannabinoids in the context of HIV and propose CB2 as a putative therapeutic target for the treatment of neuroinflammation. Graphical Abstract HIV-associated neurocognitive disorder is a systemic inflammatory disease leading to activation of plasmacytoid dendritic cells, monocytes and T cells. Monocyte and CD8 T cell migration across the BBB and interaction with astrocytes promotes neurotoxic inflammatory mediators release. CB2 ligands are proposed as therapeutics capable of suppressing systemic and localized inflammation.

Dome formation can occur in cultured tubular epithelial cells originating from various tissues, including the mammary gland and the kidney. The isolation and characterization of normal kidney epithelial stem cells that give rise to dome-forming tubular cells have never been reported. We attempted to isolate and characterize canine kidney epithelial stem cells using a simple cell culture method that we have previously used to isolate other adult human stem cells. Dome-forming kidney epithelial cells were derived from dissociated adult canine kidney tissues that were cultured in a modified keratinocyte serum-free medium supplemented with N-acetyl-l-cysteine, l-ascorbic acid 2-phosphate, nicotinamide, and fetal bovine serum. These cells exhibited high self-renewal capacity in long-term culture (growth for >13 months and 30 cumulative population doublings) and exhibited characteristics of stem cells, including (1) deficiency in gap junctional intercellular communication, (2) anchorage-independent growth, (3) expression of stem cell markers octamer-binding transcription factor 4 and SRY (sex determining region Y)-box 2, (4) expression of cell surface markers CD24 and CD133, and (5) multipotent differentiation into osteoblasts, adipocytes, chondrocytes, and dome-forming tubular cells. Most of these characteristics are shared by the well-known canine renal tubule-derived immortalized Madin-Darby Canine Kidney cell line. Furthermore, the putative canine kidney stem cells developed in this study formed budding tubule-like organoids on Matrigel and required high cell density (>4,000 cells/cm2) for sustained growth and confluency for dome formation. The signal transducer and activator of transcription-3 (STAT3) phosphorylation inhibitor, AG490, inhibited colony-forming efficiency and dome formation, whereas lipopolysaccharide, an activator of STAT3, increased colony-forming efficiency in a dose-dependent manner. These results are consistent with the hypothesis that high cell density induces STAT3 expression, which promotes both stem cell self-renewal and differentiation into tubular cells. Our novel cell culture method should be useful for the future development of normal human kidney stem cells for clinical applications and for studying mechanisms of nephrotoxicity.

Hemp (Cannabis sativa L.) has recently become an important crop due to the growing market demands for products containing cannabinoids. Unintended cross-pollination of C. sativa crops is one of the most important threats to cannabinoid production and has been shown to reduce cannabinoid yield. Ploidy manipulation has been used in other crops to improve agronomic traits and reduce fertility, however, little is known about the performance of C. sativa polyploids. In this study, colchicine was applied to two proprietary, inbred diploid C. sativa inbred lines, ‘TS1-3’ and ‘P163’, to produce the tetraploids ‘TS1-3 (4x)’ and ‘P163 (4x)’. The diploid, triploid, and tetraploid F1 hybrids from ‘TS1-3’ × ‘P163’, ‘TS1-3 (4x)’ × ‘P163’, and ‘TS1-3 (4x)’ × ‘P163 (4x)’ were produced to test their fertilities, crossing compatibilities, and yields. The results indicated a reduction in fertility in the triploids and the tetraploids, relative to their diploid counterparts. When triploids were used as females, seed yields were less than 2% compared to when diploids were used as females, thus, triploids were determined to be female infertile. The triploids resulting from the crosses made herein displayed increases in biomass and inflorescence weight compared to the diploids created from the same parents in a field setting. Statistical increases in cannabinoid concentrations were not observed. Lastly, asymmetric crossing compatibility was observed between the diploids and the tetraploids of the genotypes tested. The results demonstrate the potential benefits of triploid C. sativa cultivars in commercial agriculture.

Cannabis is widely used in the United States, with an estimated prevalence of 9.5%. Certain cannabinoids in Cannabis sativa, Δ9-tetrahydrocannabinol (THC) in particular, possess immune-modulating and anti-inflammatory activity. Depending on the context, the anti-inflammatory activity of cannabinoids may be beneficial (e.g., in treating inflammatory diseases) or detrimental to normal immune defense against pathogens. The potential beneficial effect of cannabinoids on chronic neuroinflammation has gained recent attention. Monocyte migration to the brain has been implicated as a key event in chronic neuroinflammation and in the etiology of central nervous system diseases including viral infection (e.g., human immunodeficiency virus-associated neurocognitive disorder). In the brain, monocytes can contribute to neuroinflammation through interactions with astrocytes, including inducing astrocyte secretion of cytokines and chemokines. In a human coculture system, monocyte-derived interleukin (IL)-1β due to Toll-like receptor 7 (TLR7) activation has been identified to promote astrocyte production of monocyte chemoattractant protein (MCP)-1 and IL-6. THC treatment of the TLR7-stimulated coculture suppressed monocyte secretion of IL-1β, resulting in decreased astrocyte production of MCP-1 and IL-6. Furthermore, THC displayed direct inhibition of monocytes, as TLR7-stimulated monocyte monocultures treated with THC also showed suppressed IL-1β production. The cannabinoid receptor 2 (CB2) agonist, JWH-015, impaired monocyte IL-1β production similar to that of THC, suggesting that THC acts, in part, through CB2. THC also suppressed key elements of the IL-1β production pathway, including IL1B mRNA levels and caspase-1 activity. Collectively, this study demonstrates that the anti-inflammatory properties of THC suppress TLR7-induced monocyte secretion of IL-1β through CB2, which results in decreased astrocyte secretion of MCP-1 and IL-6. SIGNIFICANCE STATEMENT: Because cannabis use is highly prevalent in the United States and has putative anti-inflammatory properties, it is important to investigate the effect of cannabinoids on immune cell function. Furthermore, cannabinoids have garnered particular interest due to their potential beneficial effects on attenuating viral-induced chronic neuroinflammation. This study utilized a primary human coculture system to demonstrate that the major psychotropic cannabinoid in cannabis, Δ9-tetrahydrocannabinol, and a cannabinoid receptor-2 selective agonist suppress specific monocyte-mediated astrocyte inflammatory responses.

Current advances in combined antiretroviral therapy have rendered HIV infection a chronic, manageable disease; however, the problem of persistent immune activation still remains despite treatment. The immune cell receptor SLAMF7 has been shown to be upregulated in diseases characterized by chronic immune activation. In this study, we studied the function of the SLAMF7 receptor in immune cells of HIV patients and the impacts of SLAMF7 signaling on peripheral immune activation. We observed increased frequencies of SLAMF7+ PBMCs in HIV+ individuals in a clinical phenotype-dependent manner, with discordant and long-term nonprogressor patients showing elevated SLAMF7 levels, and elite controllers showing levels comparable to healthy controls. We also noted that SLAMF7 was sensitive to IFN-⍺ stimulation, a factor elevated during HIV infection. Further studies revealed SLAMF7 to be a potent inhibitor of the monocyte-derived proinflammatory chemokine CXCL10 (IP-10) and other CXCR3 ligands, except in a subset of HIV+ patients termed SLAMF7 silent (SF7S). Studies utilizing small molecule inhibitors revealed that the mechanism of CXCL10 inhibition is independent of known SLAMF7 binding partners. Furthermore, we determined that SLAMF7 activation on monocytes is able to decrease their susceptibility to HIV-1 infection in vitro via downregulation of CCR5 and upregulation of the CCL3L1 chemokine. Finally, we discovered that neutrophils do not express SLAMF7, are CXCL10+ at baseline, are able to secrete CXCL10 in response to IFN-⍺ and LPS, and are nonresponsive to SLAMF7 signaling. These findings implicate the SLAMF7 receptor as an important regulator of IFN-⍺–driven innate immune responses during HIV infection.

Xenobiotic-mediated activation of the aryl hydrocarbon receptor (AHR) is immunotoxic in a number of immune cell types, with the B cell being a well-established sensitive target. Recent advances have provided evidence that the B cell repertoire is a heterogeneous population, with subpopulations exhibiting vastly different cellular and functional phenotypes. Recent work from our laboratory identified the T cell specific kinase lck as being differentially regulated by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a potent activator of AHR. While LCK is primarily expressed in T cells, a subset of CD5+ B cells also express LCK. CD5 positivity describes a broad class of B lymphocytes termed innate-like B cells (ILBs) that are critical mediators of innate immunity through constitutive secretion of polyvalent natural immunoglobulin M (IgM). We hypothesized that CD5+ ILBs may be sensitive to AHR-mediated immunotoxicity. Indeed, when CD5+ B cells were isolated from the CD19+ pool and treated with TCDD, they showed increased suppression of the CD40 ligand-induced IgM response compared to CD5- B cells. Further, characterization of the CD5+ population indicated increased basal expression of AHR, AHR repressor (AHRR), and cytochrome p450 family 1 member a1 (CYP1A1). Indeed the levels of AHR-mediated suppression of the IgM response from individual donors strongly correlated with the percentage of the B cell pool that was CD5+, suggesting that CD5+ B cells are more sensitive to AHR-mediated impairment. Together these data highlight the sensitive nature of CD5+ ILBs to AHR activation and provide insight into mechanisms associated with AHR activation in human B cells.

Human cytomegalovirus (HCMV) microRNAs play essential roles in latency and reactivation in CD34+ hematopoietic progenitor cells (HPCs) via regulation of viral and cellular gene expression. In the present study, we show that HCMV miR-US25-1 targets RhoA, a small GTPase required for CD34+ HPC self-renewal, proliferation, and hematopoiesis. Expression of miR-US25-1 impairs signaling through the nonmuscle myosin II light chain, which leads to a block in cytokinesis and an inhibition of proliferation. Moreover, infection with an HCMV mutant lacking miR-US25-1 resulted in increased proliferation of CD34+ HPCs and a decrease in the proportion of genome-containing cells at the end of latency culture. These observations provide a mechanism by which HCMV limits proliferation to maintain latent viral genomes in CD34+ HPCs.IMPORTANCE Each herpesvirus family establishes latency in a unique cell type. Since herpesvirus genomes are maintained as episomes, the virus needs to devise mechanisms to retain the latent genome during cell division. Alphaherpesviruses overcome this obstacle by infecting nondividing neurons, while gammaherpesviruses tether their genome to the host chromosome in dividing B cells. The betaherpesvirus human cytomegalovirus (HCMV) establishes latency in CD34+ hematopoietic progenitor cells (HPCs), but the mechanism used to maintain the viral genome is unknown. In this report, we demonstrate that HCMV miR-US25-1 downregulates expression of RhoA, a key cell cycle regulator, which results in inhibition of CD34+ HPC proliferation by blocking mitosis. Mutation of miR-US25-1 during viral infection results in enhanced cellular proliferation and a decreased frequency of genome-containing CD34+ HPCs. These results reveal a novel mechanism through which HCMV is able to regulate cell division to prevent viral genome loss during proliferation.

Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals and is a significant problem during transplantation. The virus can establish a latent infection in CD34+ hematopoietic progenitor cells (HPCs) and periodically reactivate to cause disease in the absence of an intact immune system., Human cytomegalovirus (HCMV) infection of myeloid lineage cells, such as CD34+ hematopoietic progenitor cells (HPCs) or monocytes, results in the upregulation of antiapoptotic cellular proteins that protect the newly infected cells from programmed cell death. The mechanisms used by HCMV to regulate proapoptotic cellular proteins upon infection of CD34+ HPCs have not been fully explored. Here, we show that HCMV utilizes pUL7, a secreted protein that signals through the FLT3 receptor, and miR-US5-1 and miR-UL112-3p to reduce the abundance and activity of the proapoptotic transcription factor FOXO3a at early times after infection of CD34+ HPCs. Regulation of FOXO3a by pUL7, miR-US5-1, and miR-UL112 results in reduced expression of the proapoptotic BCL2L11 transcript and protection of CD34+ HPCs from virus-induced apoptosis. These data highlight the importance of both viral proteins and microRNAs (miRNAs) in protecting CD34+ HPCs from apoptosis at early times postinfection, allowing for the establishment of latency and maintenance of viral genome-containing cells. IMPORTANCE Human cytomegalovirus (HCMV) causes serious disease in immunocompromised individuals and is a significant problem during transplantation. The virus can establish a latent infection in CD34+ hematopoietic progenitor cells (HPCs) and periodically reactivate to cause disease in the absence of an intact immune system. What viral gene products are required for successful establishment of latency is still not fully understood. Here, we show that both a viral protein and viral miRNAs are required to prevent apoptosis after infection of CD34+ HPCs. HCMV pUL7 and miRNAs miR-US5-1 and miR-UL112-3p act to limit the expression and activation of the transcription factor FOXO3a, which in turn reduces expression of proapoptotic gene BCL2L11 and prevents virus-induced apoptosis in CD34+ HPCs.

In human cytomegalovirus (HCMV)-seropositive patients, CD34(+) hematopoietic progenitor cells (HPCs) provide an important source of latent virus that reactivates following cellular differentiation into tissue macrophages. Multiple groups have used primary CD34(+) HPCs to investigate mechanisms of viral latency. However, analyses of mechanisms of HCMV latency have been hampered by the genetic variability of CD34(+) HPCs from different donors, availability of cells, and low frequency of reactivation. In addition, multiple progenitor cell types express surface CD34, and the frequencies of these populations differ depending on the tissue source of the cells and culture conditions in vitro. In this study, we generated CD34(+) progenitor cells from two different embryonic stem cell (ESC) lines, WA01 and WA09, to circumvent limitations associated with primary CD34(+) HPCs. HCMV infection of CD34(+) HPCs derived from either WA01 or WA09 ESCs supported HCMV latency and induced myelosuppression similar to infection of primary CD34(+) HPCs. Analysis of HCMV-infected primary or ESC-derived CD34(+) HPC subpopulations indicated that HCMV was able to establish latency and reactivate in CD38(+) CD90(+) and CD38(+/low) CD90(−) HPCs but persistently infected CD38(−) CD90(+) cells to produce infectious virus. These results indicate that ESC-derived CD34(+) HPCs can be used as a model for HCMV latency and that the virus either latently or persistently infects specific subpopulations of CD34(+) cells. IMPORTANCE Human cytomegalovirus infection is associated with severe disease in transplant patients and understanding how latency and reactivation occur in stem cell populations is essential to understand disease. CD34(+) hematopoietic progenitor cells (HPCs) are a critical viral reservoir; however, these cells are a heterogeneous pool with donor-to-donor variation in functional, genetic, and phenotypic characteristics. We generated a novel system using embryonic stem cell lines to model HCMV latency and reactivation in HPCs with a consistent cellular background. Our study defined three key stem cell subsets with differentially regulated latent and replicative states, which provide cellular candidates for isolation and treatment of transplant-mediated disease. This work provides a direction toward developing strategies to control the switch between latency and reactivation.

Immunodeficient mice engrafted with human tissues provide a robust model for the in vivo investigation of human-restricted viruses such as human cytomegalovirus (HCMV). Several humanized mouse models have been developed and improved over the last 30 years. Here, we describe a protocol for the transplant of human hematopoietic stem cells with autologous fetal liver and thymic tissues into NOD.Cg-PrkdcscidIL2rγtm1Wjl mice to create a humanized bone marrow-liver-thymus model (huBLT) that can be infected with HCMV. The presence of human thymus allows the development of a functional human immune system, including HLA-restricted human T-cells and B-cells. Indeed, following infection, huBLT mice generate virus-specific CD4+ and CD8+ T-cell responses. Additionally, both HCMV-specific IgM and IgG B-cell responses can be detected. This huBLT model provides the first animal model to explore the adaptive human immune response to HCMV infection.

Herpesvirus infection of the host results in latency in unique cell-types for each sub-family of viruses. Since herpesviruses are maintained as an episome, the viruses need to devise mechanisms to retain the latent genome during cell division. α-herpesviruses overcome this obstacle by infecting nondividing neurons while γ-herpesviruses tether their genome to the host chromosome in dividing B-cells. The β-herpesvirus Human Cytomegalovirus (HCMV) establishes latency in CD34+ hematopoietic progenitor cells (HPCs) but the mechanism to maintain the viral genome during cellular proliferation is unknown. In this report we demonstrate that HCMV miR-US25-1 down-regulates expression of RhoA that results in the inhibition CD34+ HPC proliferation by blocking mitosis. Mutation of miR-US25-1 in the virus results in the loss of the HCMV genome in latently infected cells. These results reveal a novel mechanism through which HCMV is able to regulate cell division to prevent the loss of the viral genome in proliferating cells.

Patients with HIV routinely use medicinal cannabinoids to treat neuropathic pain, anxiety, and human immunodeficiency virus (HIV)–associated wasting. However, Δ9-tetrahydrocannabinol (THC), the primary psychoactive cannabinoid in cannabis, suppresses T-cell function and secretion of interferons, both critically important in the antiviral immune response. Interferon-α (IFNα), a key cytokine in T-cell activation and peripheral control of HIV infection, can potentiate responsiveness to interleukin-7 (IL-7), a crucial homeostatic cytokine for peripheral T-cell maintenance. The objective of this investigation was to compare the response of T cells to stimulation by IFNα and IL-7 in T cells from healthy and HIV+ donors in the absence and presence of THC. To compare T-cell responses between healthy and HIV+ donors signaling through IFNα receptor, IFNα-induced expression of IL-7α receptor (IL-7Rα), cognate signaling through IL-7R, and on IL-7–mediated T-cell proliferation were measured by flow cytometry and real-time quantitative polymerase chain reaction. CD8+ T cells from HIV+ donors showed a diminished response to IFNα-induced phosphorylated signal transducer and activator of transcription-1 activation compared with CD8+ T cells from healthy donors, whereas CD4+ T cells from HIV+ donors and healthy donors were comparable. Treatment with IFNα promoted IL-7R expression and potentiated IL-7–induced STAT5 phosphorylation to augment IL-7–mediated proliferation by T cells from healthy and HIV+ donors. Finally, HIV+ donors exhibited reduced sensitivity to THC-mediated suppression by IFNα- and IL-7–mediated stimulation compared with healthy donors. These results further support THC as being immune suppressive while identifying putatively beneficial aspects of cannabinoid-based therapies in HIV+ patients.

The aryl hydrocarbon receptor (AHR) is a cytosolic ligand-activated transcription factor involved in xenobiotic sensing, cell cycle regulation, and cell development. In humans, the activation of AHR by 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a high affinity AHR-ligand, impairs the secretion of immunoglobulin M (IgM) to suppress humoral immunity. However, the mechanisms bridging the activation of AHR and the impairment of IgM secretion by human primary B cells remain poorly understood. Recent transcriptomic analysis revealed upregulation of lymphocyte-specific protein tyrosine kinase (LCK) in AHR-activated human primary B cells. LCK is a well-characterized tyrosine kinase that phosphorylates critical signaling proteins involved in activation and cytokine production in T cells. Conversely, the role of LCK in human primary B cells is not well understood. In the current studies, we have verified the transcriptomic finding by detecting AHR-mediated upregulation of LCK protein in human primary B cells. We also confirmed the role of AHR in the upregulation of LCK by using a specific AHR antagonist, which abolished the AHR-mediated increase of LCK. Furthermore, we have confirmed the role of LCK in the AHR-mediated suppression of IgM by using LCK specific inhibitors, which restored the IgM secretion by human B cells in the presence of TCDD. Collectively, the current studies demonstrate a novel role of LCK in IgM response and provide new insights into the mechanism for AHR-mediated impairment of immunoglobulin secretion by human primary B cells.

OBJECTIVE: Chronic immune activation and elevated numbers of circulating activated monocytes (CD16(+)) are implicated in HIV-associated neuroinflammation. The objective was to compare the level of circulating CD16(+) monocytes and interferon-γ-inducible protein 10 (IP-10) between HIV-infected cannabis users (HIV+MJ+) and non-cannabis users (HIV+MJ−), and determine whether in vitro Δ(9)-Tetrahydrocannabinol (THC), a constituent of cannabis, affected CD16 expression as well as IP-10 production by monocytes. DESIGN: The levels of circulating CD16(+) monocytes and IP-10 from HIV+MJ− and HIV+MJ+ donors were examined. In vitro experimentation using THC was performed on primary leukocytes isolated from HIV-MJ-, HIV+MJ− and HIV+MJ+ donors to determine if THC has an impact on CD16(+) monocyte and IP-10 levels. METHODS: Flow cytometry was used to measure the number of blood CD16(+) monocytes and serum IP-10 from HIV+MJ− and HIV+MJ+ donors. Peripheral blood mononuclear cells (PBMC) were isolated from HIV-MJ− and HIV+ (MJ− and MJ+) donors for in vitro THC and IFNα treatment, and CD16(+) monocytes and supernatant IP-10 were quantified. RESULTS: HIV+MJ+ donors possessed a lower level of circulating CD16(+) monocytes and serum IP-10, compared to HIV+MJ− donors. Further, monocytes from HIV+MJ+ donors were unable to induce CD16 expression when treated with in vitro IFNα, while HIV-MJ− and HIV+MJ− donors displayed pronounced CD16 induction, suggesting anti-inflammatory effects by cannabis. Lastly, in vitro THC treatment impaired CD16(−) monocyte transition to CD16(+) and monocyte-derived IP-10. CONCLUSIONS: Components of cannabis, including THC, may decelerate peripheral monocyte processes that are implicated in HIV-associated neuroinflammation.

Segmented filamentous bacteria (SFB) and Bacteroides fragilis are known to interact with the host immune response through the aryl hydrocarbon receptor (Ahr). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), an environmental toxicant and a high-affinity Ahr ligand has the potential to modify the effect of SFB and B. fragilis. MicroRNAs (miRNA) with their role in regulating gene expression post-transcriptionally, may potentially be used to observe such interactions between SFB, B. fragilis, and TCDD. However, little is known regarding the impact of gut microbial members on miRNA expression or its modulation in the presence of an environmental toxicant. This information is important in understanding toxicant-mediated dysbiosis in gut microbiome and the resulting human health impacts. In this study, C57BL/6 germ-free (GF) mice were colonized with SFB and B. fragilis and administered 30 μg/kg TCDD every 4 d for 28 d and miRNA were measured. Compared to GF mice, colonization with SFB resulted in an increase in up- and down-regulated Ileal miRNAs. TCDD treatment of this group decreased the number of upregulated miRNA and increased the number of down-regulated miRNAs. Association with SFB and B. fragilis together had a similar but less pronounced effect in response to TCDD treatment. TCDD treatment of GF mice had no miRNA expression response. Immune and inflammatory responses and T-cell differentiation were the key functions impacted by these miRNAs. Overall, these results reveal that the host response to toxicants may also depend on the presence of specific gut microbial populations.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates biological responses to endogenous and environmental chemical cues. Increasing evidence shows that the AHR plays physiological roles in regulating development, homeostasis, and function of a variety of cell lineages in the immune system. However, the role of AHR in human B cell development has not been investigated. Toward this end, an in vitro feeder-free human B cell developmental model system was employed using human cord blood CD34+ hematopoietic stem/progenitor cells. Using this model, we found that AHR activation by the high-affinity ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin significantly suppressed the generation of early B cells and pro-B cells from hematopoietic stem/progenitor cells, indicating the impairment of B cell lineage specification and commitment. Addition of an AHR antagonist reversed 2,3,7,8-tetrachlorodibenzo-p-dioxin–elicited suppression of early B and pro-B cells, suggesting a role of AHR in regulating B lymphopoiesis. Gene expression analysis revealed a significant decrease in the messenger RNA level of early B cell factor 1 (EBF1) and paired box 5, two critical transcription factors directing B cell lineage specification and commitment. Additionally, binding of the ligand-activated AHR to the putative dioxin response elements in the EBF1 promoter was demonstrated by EMSAs and chromatin immunoprecipitation analysis, suggesting transcriptional regulation of EBF1 by AHR. Taken together, this study demonstrates a role for the AHR in regulating human B cell development, and it suggests that transcriptional alterations of EBF1 by the AHR are involved in the underlying mechanism.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which is a strong AHR agonist, causes significant suppression of human B cell activation and differentiation. The current studies describe the identification of Src homology phosphatase 1 (SHP-1) encoded by the gene PTPN6 as a putative regulator of TCDD-mediated suppression of B cell activation. Shp-1 was initially identified through a genome-wide analysis of AHR binding in mouse B cells in the presence of TCDD. The binding of AHR to the PTPN6 promoter was further confirmed using electrophoretic mobility shift assays in which, specific binding of AHR was detected at four putative DRE sites within PTPN6 promoter. Time-course measurements performed in human B cells highlighted a significant increase in SHP-1 mRNA and protein levels in the presence of TCDD. The changes in the protein levels of SHP-1 were also observed in a TCDD concentration-dependent manner. The increase in SHP-1 levels was also seen to occur due to a change in early signaling events in the presence of TCDD. We have shown that BCL-6 regulates B cell activation by repressing activation marker CD80 in the presence of TCDD. TCDD-treatment led to a significant increase in the double positive (SHP-1hi BCL-6hi) population. Interestingly, treatment of naive human B cells with SHP-1 inhibitor decreased BCL-6 protein levels suggesting possible regulation of BCL-6 by SHP-1 for the first time. Collectively, these results suggest that SHP-1 is regulated by AHR in the presence of TCDD and may, in part through BCL-6, regulate TCDD-mediated suppression of human B cell activation.

Recent developments in in situ microscopy have enabled unparalleled resolution of the architecture of the bone marrow (BM) niche for murine hematopoietic stem and progenitor cells (HSPCs). However, the extent to which these observations can be extrapolated to human BM remains unknown. In humans, adipose tissue occupies a significant portion of the BM medullary cavity, making quantitative immunofluorescent analysis difficult due to lipid-mediated light scattering. In this study, we employed optical clearing, confocal microscopy and the nearest neighbor analysis to determine the spatial distribution of CD34+ HSPCs in the BM in a translationally relevant rhesus macaque model. Immunofluorescent analysis revealed that femoral BM adipocytes are associated with the branches of vascular sinusoids, with half of HSPCs localizing in close proximity of the nearest BM adipocyte. Immunofluorescent microscopy and flow cytometry analysis demonstrate that BM adipose tissue exists as a multicellular niche consisted of adipocytes, endothelial cells, granulocytes, and macrophages. Analysis of the BM adipose tissue conditioned media using liquid chromatography-tandem mass spectrometry revealed the presence of multiple bioactive proteins involved in regulation of hematopoiesis, inflammation and bone development with many predicted to reside inside microvesicles. Pretreatment of purified HSPCs with BM adipose tissue-conditioned media, comprising soluble and exosomal/microvesicle-derived factors, led to enhanced proliferation and an increase in granulocyte-monocyte differentiation potential ex vivo. Our work translationally extends extensive studies in murine models, indicating that BM adipose tissue is a central paracrine regulator of hematopoiesis and in nonhuman primates and possibly in humans.

Vaccines based on cytomegalovirus (CMV) demonstrate protection in animal models of infectious disease and cancer. Vaccine efficacy is associated with the ability of CMV to elicit and indefinitely maintain high frequencies of circulating effector memory T cells (TEM) providing continuous, life-long anti-pathogen immune activity. To allow for the clinical testing of human CMV (HCMV)-based vaccines we constructed and characterized as a vector backbone the recombinant molecular clone TR3 representing a wildtype genome. We demonstrate that TR3 can be stably propagated in vitro and that, despite species incompatibility, recombinant TR3 vectors elicit high frequencies of TEM to inserted antigens in rhesus macaques (RM). Live-attenuated versions of TR3 were generated by deleting viral genes required to counteract intrinsic and innate immune responses. In addition, we eliminated subunits of a viral pentameric glycoprotein complex thus limiting cell tropism. We show in a humanized mouse model that such modified vectors were able to establish persistent infection but lost their ability to reactivate from latency. Nevertheless, attenuated TR3 vectors preserved the ability to elicit and maintain TEM to inserted antigens in RM. We further demonstrate that attenuated TR3 can be grown in approved cell lines upon elimination of an anti-viral host factor using small interfering RNA, thus obviating the need for a complementing cell line. In sum, we have established a versatile platform for the clinical development of live attenuated HCMV-vectored vaccines and immunotherapies.

Reactivation of latent Human Cytomegalovirus (HCMV) in CD34+ hematopoietic progenitor cells (HPCs) is closely linked to hematopoiesis. Viral latency requires maintenance of the progenitor cell quiescence, while reactivation initiates following mobilization of HPCs to the periphery and differentiation into CD14+ macrophages. Early growth response gene 1 (EGR-1) is a transcription factor activated by Epidermal growth factor receptor (EGFR) signaling that is essential for the maintenance of CD34+ HPC self-renewal in the bone marrow niche. Down-regulation of EGR-1 results in mobilization and differentiation of CD34+ HPC from the bone marrow to the periphery. In the current study we demonstrate that the transcription factor EGR-1 is directly targeted for down-regulation by HCMV miR-US22 that results in decreased proliferation of CD34+ HPCs and a decrease in total hematopoietic colony formation. We also show that an HCMV miR-US22 mutant fails to reactivate in CD34+ HPCs, indicating that expression of EGR-1 inhibits viral reactivation. Since EGR-1 promotes CD34+ HPC self-renewal in the bone marrow niche, HCMV miR-US22 down-regulation of EGR-1 is a necessary step to block HPC self-renewal and proliferation to induce a cellular differentiation pathway necessary to promote reactivation of virus., Author summary Human cytomegalovirus (HCMV) is a widespread herpesvirus that persists in the host and remains a significant cause of morbidity and mortality in solid organ and stem cell transplant patients. HCMV latency is complex, and the molecular mechanisms for establishment, maintenance, and reactivation from latency are poorly understood. Quiescent stem cells in the bone marrow represent a critical reservoir of latent HCMV, and the mobilization and differentiation of these cells is closely linked to viral reactivation from latency. HCMV encodes small regulatory RNAs, called miRNAs that play important roles in the regulation of viral and cellular gene expression. In this study, we show that HCMV miR-US22 targets Early growth response gene 1 (EGR-1) a host transcription factor that is necessary for stem cell quiescence and self-renewal in the bone marrow. Expression of this miR-US22 down-regulates expression of EGR-1 that reduces CD34+ HPCs proliferation and total hematopoietic colony formation. An HCMV miR-US22 mutant is unable to reactivate from latency suggesting that the ability of the miRNA to disrupt CD34+ HPC renewal in the bone marrow niche to initiate a differentiation pathway is critical for viral reactivation.

The influence of interindividual variability is not typically assessed in traditional toxicological studies. Given that chemical exposures occur in heterogeneous populations, this knowledge gap has the potential to cause undue harm within the realms of public health and industrial and municipal finances. A recent report from the National Research Council (NRC) suggests that when accounting for interindividual variation in responses, traditionally assumed nonlinear dose-response relationships (DRRs) for noncancer-causing endpoints would better be explained with a linear relationship within the low-dose region. To address this knowledge gap and directly test the NRC’s assumption, this study focused on assessing the DRR between 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD) exposure and immune suppression in a cohort of unique human donors. Human B cells were isolated from 51 individual donors and treated with logarithmically increasing concentrations of TCDD (0–30 nM TCDD). Two endpoints sensitive to TCDD were assessed: (1) number of IgM-secreting B cells and (2) quantity of IgM secreted. The results show that TCDD significantly suppressed both the number of IgM-secreting B cells and the quantity of IgM secreted (P

We describe the selection of reduced chlorhexidine susceptibility during chlorhexidine use in a patient with two episodes of cutaneous USA300 methicillin-resistant Staphylococcus aureus abscess. The second clinical isolate harbors a novel plasmid that encodes the QacA efflux pump. Greater use of chlorhexidine for disease prevention warrants surveillance for resistance.

Summary Infection with human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality following hematopoietic stem cell transplant (HSCT) because of various hematologic problems, including myelosuppression. Here, we demonstrate that latently expressed HCMV miR-US5-2 downregulates the transcriptional repressor NGFI-A binding protein (NAB1) to induce myelosuppression of uninfected CD34+ hematopoietic progenitor cells (HPCs) through an increase in TGF-β production. Infection of HPCs with an HCMVΔmiR-US5-2 mutant resulted in decreased TGF-β expression and restoration of myelopoiesis. In contrast, we show that infected HPCs are refractory to TGF-β signaling as another HCMV miRNA, miR-UL22A, downregulates SMAD3, which is required for maintenance of latency. Our data suggest that latently expressed viral miRNAs manipulate stem cell homeostasis by inducing secretion of TGF-β while protecting infected HPCs from TGF-β-mediated effects on viral latency and reactivation. These observations provide a mechanism through which HCMV induces global myelosuppression following HSCT while maintaining lifelong infection in myeloid lineage cells.

2,3,7,8-Tetrachlordibenzo-p-dioxin (TCDD) is an environmental pollutant that can cause various toxic effects, including chloracne, metabolic syndrome, and immune suppression. Most of the toxicity associated with TCDD is mediated through activation of the aryl hydrocarbon receptor (AHR). Recent research has suggested the presence of a wide-range of interindividual variability in TCDD-mediated suppression of the Immunoglobulin-M (IgM) response across the human population. In an attempt to identify putative modifiers of AHR-mediated immunosuppression beyond the AHR, B cells were isolated from a panel of genetically diverse mouse strain to scan for modulators that drive interstrain differences in TCDD-mediated suppression of the IgM response. Results implicated a region of mouse Chromosome 1 near a gene encoding serine peptidase inhibitor, clade B, member 2 (Serpinb2) whose human ortholog is plasminogen activator inhibitor 2 (PAI2). Further downstream analyses indicated that Serpinb2 is dysregulated by TCDD and, furthermore, that B cells from Serpinb2(−/−) mice are significantly more sensitive to TCDD-mediated suppression as compared to littermate controls. This study suggests a protective role of Serpinb2 within TCDD-mediated immunosuppression and, furthermore, a novel function of Serpinb2-related activity in the IgM response.

The ability of human cytomegalovirus (HCMV) to reactivate from latent infection of hematopoietic progenitor cells (HPCs) is intimately linked to cellular differentiation. HCMV encodes UL7 that our group has shown is secreted from infected cells and induces angiogenesis. In this study, we show that UL7 is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R), a well-known critical factor in HPC differentiation. We observed that UL7 directly binds Flt-3R and induces downstream signaling cascades, including phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathways. Importantly, we show that UL7 protein induces differentiation of both CD34+ HPCs and CD14+ monocytes. Last, we show that an HCMV mutant lacking UL7 fails to reactivate in CD34+ HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients., IMPORTANCE Human cytomegalovirus (HCMV) remains a significant cause of morbidity and mortality in allogeneic hematopoietic stem cell transplant recipients. CD34+ hematopoietic progenitor cells (HPCs) represent a critical reservoir of latent HCMV in the transplant population, thereby providing a source of virus for dissemination to visceral organs. HCMV reactivation has been linked to HPC/myeloid cellular differentiation; however, the mechanisms involved in these events are poorly understood at the molecular level. In this study, we show that a viral protein is a ligand for Fms-like tyrosine kinase 3 receptor (Flt-3R) and that the binding of HCMV UL7 to the Flt-3R triggers HPC and monocyte differentiation. Moreover, the loss of UL7 prevents viral reactivation in HPCs in vitro as well as in humanized mice. These observations define the first virally encoded differentiation factor with significant implications not only for HCMV reactivation but also for alteration of the hematopoietic compartment in transplant patients.

Bisphenol A (BPA) is commonly used in the manufacturing of a wide range of consumer products, including polycarbonate plastics, epoxy resin that lines beverage and food cans, and some dental sealants. Consumption of food and beverages containing BPA represents the primary route of human BPA exposure, which is virtually ubiquitous. An increasing number of studies have evaluated the effects of BPA on immune responses in laboratory animals that have reported a variety of effects some of which have been contradictory. To address the divergent findings surrounding BPA exposure, a comprehensive chronic treatment study of BPA was conducted in Sprague-Dawley rats, termed the Consortium Linking Academic and Regulatory Insights on Toxicity of BPA (CLARITY-BPA). As a participant in the CLARITY-BPA project, our studies evaluated the effects of BPA on a broad range of immune function endpoints using spleen cells isolated from BPA or vehicle treated rats. This comprehensive assessment included measurements of lymphoproliferation in response to mitogenic stimuli, immunoglobulin production by B cells, and cellular activation of T cells, NK cells, monocytes, granulocytes, macrophages and dendritic cells. In total, 630 different measurements in BPA treated rats were performed of which 35 measurements were statistically different from vehicle controls. The most substantive alteration associated with BPA treatment was the augmentation of lymphoproliferation in response to pokeweed mitogen stimulations in 1 year old male rats, which was also observed in the reference estrogen ethinyl estradiol treated groups. With the exception of the aforementioned, the statistically significant changes associated with BPA treatment were mostly sporadic and not dose-dependent with only one out of five BPA dose groups showing a statistical difference. In addition, the observed BPA-associated alterations were mostly moderate in magnitude and showed no persistent trend over the one-year time period. Based on these findings, we conclude that the observed BPA-mediated changes observed in this study are unlikely to alter immune competence in adult rats.

Bisphenol A (BPA) is extensively used in manufacturing of a broad range of consumer products worldwide. Due to its widespread use, human exposure to BPA is virtually ubiquitous. Broad human exposure coupled with a large scientific literature describing estrogenic activity of BPA in animals has raised public health concerns. To comprehensively evaluate the health effects of BPA exposure, a chronic toxicity study using a wide-range of BPA doses (2.5–25000 μg/kg bw/day) was conducted jointly by the NTP, thirteen NIEHS-supported grantees, and the FDA, which is called the Consortium Linking Academic and Regulatory Insights on Toxicity of BPA (CLARITY-BPA). As a participant in the CLARITY-BPA project, the objective of the current study was to evaluate the effects of chronic BPA exposure in Sprague-Dawley rats on the relative number and proportion of defined leukocyte populations in the spleen and the thymus. Toward this end, lymphoid tissues from a total of 641 rats were assayed after being continuously dosed with BPA or controls for up to one year. To comprehensively evaluate the effects of BPA on leukocyte compositions, extensive endpoints that cover major populations of leukocytes were assessed, including B cells, T cells, NK cells, granulocytes, monocytes, macrophages and dendritic cells. In total, of the 530 measurements in BPA-treated rats, 10 measurements were statistically different from vehicle controls and were mainly associated with either the macrophage or dendritic cell populations. Most, if not all, of these alterations were found to be transient with no persistent trend over the one-year time period. In addition, the observed BPA-associated alterations were mostly moderate in magnitude and not dose-dependent. Due to the aforementioned, it is unlikely that the observed BPA-mediated changes alone would adversely affect immune competence.

The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes., Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes. To overcome these limitations, we adapted synthetic biology tools to clone CMV genomes in Saccharomyces cerevisiae. Using an early passage of the human CMV isolate Toledo, we first applied transformation-associated recombination (TAR) to clone 16 overlapping fragments covering the entire Toledo genome in Saccharomyces cerevisiae. Then, we assembled these fragments by TAR in a stepwise process until the entire genome was reconstituted in yeast. Since next-generation sequence analysis revealed that the low-passage-number isolate represented a mixture of parental and fibroblast-adapted genomes, we selectively modified individual DNA fragments of fibroblast-adapted Toledo (Toledo-F) and again used TAR assembly to recreate parental Toledo (Toledo-P). Linear, full-length HCMV genomes were transfected into human fibroblasts to recover virus. Unlike Toledo-F, Toledo-P displayed characteristics of primary isolates, including broad cellular tropism in vitro and the ability to establish latency and reactivation in humanized mice. Our novel strategy thus enables de novo cloning of CMV genomes, more-efficient genome-wide engineering, and the generation of viral genomes that are partially or completely derived from synthetic DNA. IMPORTANCE The genomes of large DNA viruses, such as human cytomegalovirus (HCMV), are difficult to manipulate using current genetic tools, and at this time, it is not possible to obtain, molecular clones of CMV without extensive tissue culture. To overcome these limitations, we used synthetic biology tools to capture genomic fragments from viral DNA and assemble full-length genomes in yeast. Using an early passage of the HCMV isolate Toledo containing a mixture of wild-type and tissue culture-adapted virus. we directly cloned the majority sequence and recreated the minority sequence by simultaneous modification of multiple genomic regions. Thus, our novel approach provides a paradigm to not only efficiently engineer HCMV and other large DNA viruses on a genome-wide scale but also facilitates the cloning and genetic manipulation of primary isolates and provides a pathway to generating entirely synthetic genomes.

Environmental toxicants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR), are known to induce host toxicity and structural shifts in the gut microbiota. Key bacterial populations with similar or opposing functional responses to AhR ligand exposure may potentially help regulate expression of genes associated with immune dysfunction. To examine this question and the mechanisms for AhR ligand-induced bacterial shifts, C57BL/6 gnotobiotic mice were colonized with and without segmented filamentous bacteria (SFB)- an immune activator. Mice were also colonized with polysaccharide A producing Bacteroides fragilis-an immune suppressor to serve as a commensal background. Following colonization, mice were administered TCDD (30 µg/kg) every 4 days for 28 days by oral gavage. Quantified with the nCounter® mouse immunology panel, opposing responses in ileal gene expression (e.g., genes associated with T-cell differentiation via the class II major histocompatibility complex-MHC II) as a result of TCDD dosing and SFB colonization were observed. Genes that responded to TCDD in the presence of SFB did not show a significant response in the absence of SFB, and vice-versa. Regulatory T-cells, examined in the mesenteric lymph-nodes, spleen, and blood were also less impacted by TCDD in mice colonized with SFB. TCDD-induced shifts in abundance of SFB and B. fragilis compared with previous studies in mice with a traditional gut microbiome. With regard to the mouse model colonized with individual populations, results indicate that TCDD-induced host response was significantly modulated by the presence of SFB in the gut microbiome, providing insight into therapeutic potential between AhR ligands and key commensals.

Plasmacytoid dendritic cells (pDCs) play a crucial role in host antiviral immune response through secretion of type I interferon. Interferon alpha (IFNα), a type I IFN, is critical for mounting the initial response to viral pathogens. A consequence of Human Immunodeficiency Virus-1 (HIV) infection is a decrease in both pDC number and function, but prolonged pDC activity has been linked with progression from HIV infection to the development of AIDS. Patients with HIV in the United States routinely use cannabinoid-based therapies to combat the side effects of HIV infection and antiretroviral therapy. However, cannabinoids, including Δ-tetrahydrocannabinol (THC), are well-characterized immunosuppressants. Here, we report that THC suppressed secretion of IFNα by pDC from both healthy and HIV+ donors through a mechanism involving impaired phosphorylation of interferon regulatory factor 7. These results suggest that THC can suppress pDC function during the early host antiviral response by dampening pDC activation.

The strict species specificity of Human Cytomegalovirus (HCMV) has impeded our understanding of antiviral adaptive immune responses in the context of a human immune system. We have previously shown that HCMV infection of human hematopoietic progenitor cells engrafted in immune deficient mice (huNSG) results in viral latency that can be reactivated following G-CSF treatment. In this study, we characterized the functional human adaptive immune responses in HCMV latently-infected huBLT (humanized Bone marrow-Liver-Thymus) mice. Following infection, huBLT mice generate human effector and central memory CD4+ and CD8+ T-cell responses reactive to peptides corresponding to both IE and pp65 proteins. Additionally, both HCMV specific IgM and IgG B-cell responses with the ability to neutralize virus were detected. These results indicate that the HCMV huBLT mouse model may provide a valuable tool to study viral latency and reactivation as well as evaluate HCMV vaccines and immune responses in the context of a functional human immune system.

Activated carbon (AC) is an increasingly attractive remediation alternative for the sequestration of dioxins at contaminated sites globally. However, the potential for AC to reduce the bioavailability of dioxins in mammals and the residing gut microbiota has received less attention. This question was partially answered in a recent study examining 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced hallmark toxic responses in mice administered with TCDD sequestered by AC or freely available in corn oil by oral gavage. Results from that study support the use of AC to significantly reduce the bioavailability of TCDD to the host. Herein, we examined the bioavailability of TCDD sequestered to AC on a key murine gut commensal and the influence of AC on the community structure of the gut microbiota. The analysis included qPCR to quantify the expression of segmented filamentous bacteria (SFB) in the mouse ileum, which has responded to TCDD-induced host toxicity in previous studies and community structure via sequencing the 16S ribosomal RNA (rRNA) gene. The expression of SFB 16S rRNA gene and functional genes significantly increased with TCDD administered with corn oil vehicle. Such a response was absent when TCDD was sequestered by AC. In addition, AC appeared to have a minimal influence on murine gut community structure and diversity, affecting only the relative abundance of Lactobacillaceae and two other groups. Results of this study further support the remedial use of AC for eliminating bioavailability of TCDD to host and subsequent influence on the gut microbiome.

The homeostasis of peripheral B cell compartment requires lifelong B lymphopoiesis from hematopoietic stem cells (HSC). As a result, the B cell repertoire is susceptible to disruptions of hematopoiesis. Increasing evidence, primarily from rodent models, shows that the aryl hydrocarbon receptor (AHR) regulates hematopoiesis. To study the effects of persistent AHR activation on human B cell development, a potent AHR agonist and known environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was utilized. An in vitro B cell development model system was established by co-culturing human cord blood-derived HSCs with irradiated human primary bone marrow stromal cells. Using this in vitro model, we found that TCDD significantly suppressed the total number of hematopoietic stem and progenitor cells (HSPC) in a concentration-dependent manner. Cell death analysis demonstrated that the decrease in cell number was not due to cytotoxicity by TCDD. In addition, TCDD markedly decreased CD34 expression on HSPCs. Structure-activity relationship studies using dioxin congeners demonstrated a correlation between the relative AHR binding affinity and the magnitude of decrease in the number of HSPCs and CD34 expression, suggesting that AHR mediates the observed TCDD-elicited changes in HSPCs. Moreover, a significant reduction in lineage committed B cell-derived from HSCs was observed in the presence of TCDD, indicating impairment of human B cell development. Similar effects of TCDD were observed regardless of the use of stromal cells in cultures indicating a direct effect of TCDD on HSCs. Collectively, we demonstrate that AHR activation by TCDD on human HSCs impairs early stages of human B lymphopoiesis.

While it is evident that nasal colonization with S. aureus increases the likelihood of SSTI, there is a significant lack of information regarding the contribution of extranasal colonization to the overall risk of a subsequent SSTI. Furthermore, the impact of S. aureus colonization on bacterial community composition outside the nasal microbiota is unclear. Thus, this report represents the first investigation that utilized both culture and high-throughput sequencing techniques to analyze microbial dysbiosis at multiple body sites of healthy and diseased/colonized individuals. The results described here may be useful in the design of future methodologies to treat and prevent SSTIs., Skin and soft tissue infections (SSTIs) are common in the general population, with increased prevalence among military trainees. Previous research has revealed numerous nasal microbial signatures that correlate with SSTI development and Staphylococcus aureus colonization. Thus, we hypothesized that the ecology of the inguinal, oropharynx, and perianal regions may also be altered in response to SSTI and/or S. aureus colonization. We collected body site samples from 46 military trainees with purulent abscess (SSTI group) as well as from 66 asymptomatic controls (non-SSTI group). We also collected abscess cavity samples to assess the microbial composition of these infections. Samples were analyzed by culture, and the microbial communities were characterized by high-throughput sequencing. We found that the nasal, inguinal, and perianal regions were similar in microbial composition and significantly differed from the oropharynx. We also observed differences in Anaerococcus and Streptococcus abundance between the SSTI and non-SSTI groups for the nasal and oropharyngeal regions, respectively. Furthermore, we detected community membership differences between the SSTI and non-SSTI groups for the nasal and inguinal sites. Compared to that of the other regions, the microbial compositions of the nares of S. aureus carriers and noncarriers were dramatically different; we noted an inverse correlation between the presence of Corynebacterium and the presence of Staphylococcus in the nares. This correlation was also observed for the inguinal region. Culture analysis revealed elevated methicillin-resistant S. aureus (MRSA) colonization levels for the SSTI group in the nasal and inguinal body sites. Together, these data suggest significant microbial variability in patients with SSTI as well as between S. aureus carriers and noncarriers. IMPORTANCE While it is evident that nasal colonization with S. aureus increases the likelihood of SSTI, there is a significant lack of information regarding the contribution of extranasal colonization to the overall risk of a subsequent SSTI. Furthermore, the impact of S. aureus colonization on bacterial community composition outside the nasal microbiota is unclear. Thus, this report represents the first investigation that utilized both culture and high-throughput sequencing techniques to analyze microbial dysbiosis at multiple body sites of healthy and diseased/colonized individuals. The results described here may be useful in the design of future methodologies to treat and prevent SSTIs.

Human cytomegalovirus (HCMV), a betaherpesvirus, persists indefinitely in the human host through poorly understood mechanisms. The UL136 gene is carried within a genetic locus important to HCMV latency termed the UL133/8 locus, which also carries UL133, UL135, and UL138. Previously, we demonstrated that UL136 is expressed as five protein isoforms ranging from 33-kDa to 19-kDa, arising from alternative transcription and, likely, translation initiation mechanisms. We previously showed that the UL136 isoforms are largely dispensable for virus infection in fibroblasts, a model for productive virus replication. In our current work, UL136 has emerged as a complex regulator of HCMV infection in multiple contexts of infection relevant to HCMV persistence: in an endothelial cell (EC) model of chronic infection, in a CD34+ hematopoietic progenitor cell (HPC) model of latency, and in an in vivo NOD-scid IL2Rγcnull humanized (huNSG) mouse model for latency. The 33- and 26-kDa isoforms promote replication, while the 23- and 19-kDa isoforms suppress replication in ECs, in CD34+ HPCs, and in huNSG mice. The role of the 25-kDa isoform is context dependent and influences the activity of the other isoforms. These isoforms localize throughout the secretory pathway, and loss of the 33- and 26-kDa UL136 isoforms results in virus maturation defects in ECs. This work reveals an intriguing functional interplay between protein isoforms that impacts virus replication, latency, and dissemination, contributing to the overall role of the UL133/8 locus in HCMV infection., IMPORTANCE The persistence of DNA viruses, and particularly of herpesviruses, remains an enigma because we have not completely defined the viral and host factors important to persistence. Human cytomegalovirus, a herpesvirus, persists in the absence of disease in immunocompetent individuals but poses a serious disease threat to transplant patients and the developing fetus. There is no vaccine, and current therapies do not target latent reservoirs. In an effort to define the viral factors important to persistence, we have studied viral genes with no known viral replication function in contexts important to HCMV persistence. Using models relevant to viral persistence, we demonstrate opposing roles of protein isoforms encoded by the UL136 gene in regulating latent and replicative states of infection. Our findings reveal an intriguing interplay between UL136 protein isoforms and define UL136 as an important regulator of HCMV persistence.

The aryl hydrocarbon receptor (AHR) has been extensively characterized for the essential role it plays in mediating the toxic responses elicited by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Despite similarities across animal species, species-specific differences exist in the profile of toxicity and sensitivity to TCDD owing, in part, to differences in the AHR. Newer reports have implicated the importance of AHR in the development and regulation of the immune system. Our present studies seek to further explore the essential role of AHR in lymphoid tissue composition, B cell function and the immunological responses after TCDD administration using the recently established AHR KO rats. Comprehensive immune cell phenotyping showed a decrease in the CD8+ T cell, CD11c+ populations and an increase in NKT cells in 3-week-old AHR KO rats compared to the WT controls. The lipopolysaccharide-induced IgM response and proliferation was markedly suppressed in the WT but not in the AHR KO B cells in the presence of TCDD. However, the percentage of LPS-activated IgM+ B cells was significantly higher in the AHR KO B cells as compared to that of WT suggesting the role of AHR in regulating the IgM response. The use of an AHR antagonist further alluded to the endogenous role of AHR in regulating B cell responses in the rat. Overall, the studies report for the first time, comprehensive immune cell phenotyping of the AHR KO rat and the endogenous role of AHR in the regulation of B cell function in the rat.

Suppression of the primary antibody response is particularly sensitive to suppression by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in mice; however, surprisingly little is known concerning the effects of TCDD on humoral immunity or B cell function in humans. Results from a limited number of previous studies, primarily employing in vitro activation models, suggested that human B cell effector function is suppressed by TCDD. The present study sought to extend these findings by investigating, in primary human B cells, the effects of TCDD on several critical stages leading to antibody secretion including activation and plasmacytic differentiation using an in vitro CD40 ligand activation model. These studies revealed important differences in the response of human and mouse B cells to TCDD, the most striking being altered expression of plasmacytic differentiation regulators, B lymphocyte-induced maturation protein 1 and paired box protein 5, in mouse but not human B cells. The activation of human B cells was profoundly impaired by TCDD, as evidenced by decreased expression of activation markers CD80, CD86, and CD69. The impaired activation correlated with decreased cell viability, which prevented the progression of human B cells toward plasmacytic differentiation. TCDD treatment also attenuated the early activation of mitogen-activated protein kinases (MAPK) and Akt signaling in human B cells. Collectively, the present study provided experimental evidence for novel mechanisms by which TCDD impairs the effector function of primary human B cells.

We and others have reported that simultaneous targeted deletion of CB1 and CB2 resulted in exacerbation of immune reactivity, suggesting a role of endocannabinoids in down-regulating immune function. In this study, we demonstrate that APC function is enhanced specifically in the absence of CB1 and CB2 signaling, resulting in an exacerbated immune response phenotype. After influenza infection, CB1−/−CB2−/− mice showed more pronounced pulmonary damage, increased inflammatory cell infiltrate, inflammation, and a greater cellular immune responses compared with WT mice, as evidenced by transcriptome analysis, more robust T cell activation, and effector cell cytokine production. After direct activation in vitro, there were no differences in the percentages of cytokine-producing CD4+ T cells between CB1−/−CB2−/− and WT mice. However, untreated CB1−/−CB2−/− mice routinely had fewer naïve T cells compared with WT, suggesting dysregulation of APC immune homeostasis. Moreover, bmDCs and AM isolated from CB1−/−CB2−/− mice exhibited a more mature phenotype, with and without TLR stimulation, and bmDCs elicited T cells more robustly than WT mice. Collectively, these findings implicate a role for CB1 and CB2 on APCs in regulating immune responses and immune homeostasis.

Obesity is a risk factor for the development of hormone receptor–positive breast cancer in postmenopausal women and has been associated with an increased risk of recurrence and reduced survival. In humans, obesity causes subclinical inflammation in visceral and subcutaneous adipose tissue, characterized by necrotic adipocytes surrounded by macrophages forming crown-like structures (CLS). Recently, we found increased numbers of CLS, activation of the NF-κB transcription factor, and elevated aromatase levels and activity in the mammary glands of obese mice. These preclinical findings raised the possibility that the obesity → inflammation axis is important for the development and progression of breast cancer. Here, our main objective was to determine if the findings in mouse models of obesity translated to women. Breast tissue was obtained from 30 women who underwent breast surgery. CLS of the breast (CLS-B) was found in nearly 50% (14 of 30) of patient samples. The severity of breast inflammation, defined as the CLS-B index, correlated with both body mass index (P < 0.001) and adipocyte size (P = 0.01). Increased NF-κB binding activity and elevated aromatase expression and activity were found in the inflamed breast tissue of overweight and obese women. Collectively, our results suggest that the obesity → inflammation → aromatase axis is present in the breast tissue of most overweight and obese women. The presence of CLS-B may be a biomarker of increased breast cancer risk or poor prognosis. Cancer Prev Res; 4(7); 1021–9. ©2011 AACR.

This study evaluated a calorie labeling intervention in Kaiser Permanente Hospital cafeterias. Three cafeteria conditions were no labeling; calorie and nutrient labeling on posters only; posters plus point-of-purchase menu board calorie labeling. More respondents noticed calorie information at poster plus menu board sites than at poster-only sites (P < 0.05). Over 80% of patrons supported provision of calorie information. There was a significant increase in purchases of lower calorie side dishes (P < 0.0001) and snacks (P = 0.006) at the menu board site, compared with the no-labeling site. These findings support the premise that menu labeling can be useful in worksite wellness initiatives.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the prototypical aryl hydrocarbon receptor (AhR) ligand, exhibits immune suppression in vivo and in vitro. Suppression of primary humoral immune responses in particular has been well characterized as one of the most sensitive functional immune endpoints in animals treated with TCDD. Previous studies have used purified TCDD to elucidate the mechanisms by which TCDD and dioxin-like compounds (DLC) impair IgM production by B cells, but did not represent the route by which animals and humans are likely to be exposed environmentally. In the studies reported here, mice were treated with TCDD adsorbed onto a well-defined synthetic silica phase of known purity and physical properties, followed by sensitization with sheep erythrocytes to initiate a humoral immune. We found that surfactant-templated mesoporous forms of amorphous silica provided an ideal combination of purity, dispersibility and textural properties for immobilizing TCDD. TCDD-adsorbed silica distributed to the spleen and liver after oral administration as assessed by induction of cyp1a1 gene expression. Most notably, TCDD delivered in the adsorbed state on amorphous silica and as a solute in corn oil (CO) produced similar suppression of the anti-sheep red blood cell immunoglobulin M antibody forming cell response (sRBC IgM AFC) response at equivalent doses of TCDD. These results suggest that TCDD immobilized on silicate particles found in soils distributes to the spleen and suppresses humoral immunity.

We describe a cutaneous abscess caused by catalase-negative methicillin-susceptible Staphylococcus aureus subsp. aureus in a patient who was concomitantly colonized with virulent USA300 methicillin-resistant S. aureus (MRSA). Sequencing of the katA gene demonstrated a thymine insertion leading to a frameshift mutation and premature truncation of catalase to 21 amino acids.

Previous work from our laboratories has demonstrated that purified, recombinant human astrovirus coat protein (HAstV CP) binds C1q and mannose-binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. Analysis of the 787 amino acid CP molecule revealed that residues 79-139 share limited sequence homology with human neutrophil defensin-1 (HNP-1), a molecule previously demonstrated to bind C1q and MBL, inhibiting activation of the classical and lectin pathways of complement, respectively. A 30 amino acid peptide derived from this region of the CP molecule competitively inhibited the binding of wild-type CP to C1q. The parent peptide and various derivatives were subsequently assayed for C1q binding, inhibition of C1 and C4 activation as well as suppression of complement activation in hemolytic assays. The parent peptide and several derivatives inhibited complement activation in these functional assays to varying degrees. One peptide derivative in particular (E23A) displayed superior inhibition of complement activation in multiple assays of classical complement pathway activation. Further analysis revealed homology to a plant defensin allowing development of a proposed structural model for E23A. Based upon these findings, we hypothesize that further rationale optimization of E23A may result in a promising therapeutic inhibitor for the treatment of inflammatory and autoimmune diseases in which dysregulated activation of the classical and lectin pathways of complement contribute to pathogenesis.

Summary MCL-1 is a BCL-2 family protein implicated in the development and chemoresistance of human cancer. Unlike its anti-apoptotic homologs, Mcl-1 deletion has profound physiologic consequences, indicative of a broader role in homeostasis. We report that the BCL-2 homology 3 (BH3) α helix of MCL-1 can directly engage very long-chain acyl-CoA dehydrogenase (VLCAD), a key enzyme of the mitochondrial fatty acid β-oxidation (FAO) pathway. Proteomic analysis confirmed that the mitochondrial matrix isoform of MCL-1 (MCL-1 Matrix ) interacts with VLCAD. Mcl-1 deletion, or eliminating MCL-1 Matrix alone, selectively deregulated long-chain FAO, causing increased flux through the pathway in response to nutrient deprivation. Transient elevation in MCL-1 upon serum withdrawal, a striking increase in MCL-1 BH3/VLCAD interaction upon palmitic acid titration, and direct modulation of enzymatic activity by the MCL-1 BH3 α helix are consistent with dynamic regulation. Thus, the MCL-1 BH3 interaction with VLCAD revealed a separable, gain-of-function role for MCL-1 in the regulation of lipid metabolism.

Human astroviruses (HAstVs) constitute a family of non-enveloped, RNA viruses which cause infantile gastroenteritis. We have previously demonstrated that purified HAstV coat protein (CP), multiple copies of which compose the viral capsid, bind C1q resulting in inhibition of classical complement pathway activity. The objective of this study was to further analyze the mechanism by which CP inhibits C1 activation. CP inhibited C1 activation, preventing cleavage of C1s to its active form in the presence of heat-aggregated IgG, a potent classical pathway activator. CP also inhibited generation of the potent anaphylatoxin C5a. CP dose-dependently bound to C1q, the isolated globular heads and the collagen-like regions of the C1q molecule. When CP was added to C1, C1s dissociated from C1q suggesting that CP functionally displaces the protease tetramer (C1s-C1r-C1r-C1s). Given the structural and functional relatedness of C1q and MBL, we subsequently investigated the interactions between CP and MBL. CP bound to purified MBL and was able to inhibit mannan-mediated activation of the lectin pathway. Interestingly, CP did not bind to a variant of MBL that replaces a lysine residue (Lys55) critical for binding to MASP-2, a functional homolog of C1s. Finally, CP was shown to cross the species barrier to inhibit C3 activation and MAC formation in rat serum. These findings suggest CP inhibits C1 and MBL activation via a novel mechanism of interference with the normal interaction of the recognition molecule with its cognate serine proteases.

Rodent models have been extensively utilized to identify putative human immunotoxicants; however, even when immunotoxicity is established, uncertainty remains whether the effects are predictive of human risk. Therefore, the objective of this study was to establish a polyclonal immunoglobulin M (IgM) antibody-forming cell (AFC) response model to directly characterize immunotoxicity in primary mouse or human B cells. CD40 ligand (CD40L) was selected to activate B cells because it effectively drives both primary human and mouse B cells in vitro to AFC in a physiologically relevant manner to mimic T-cell-dependent antibody responses in vivo. In this model, the IgM AFC response is induced by cell surface–expressed CD40L and promoted by recombinant cytokines. Reported here are the conditions required to induce IgM AFC responses using mouse splenic B cells or human peripheral blood B cells, allowing for species comparisons. Moreover, less than one order of magnitude difference was observed in the CD40L-induced B-cell AFC responses based on data from multiple donors. In addition to antibody production, proliferation and phenotypic changes characteristic of B-cell activation as well as the plasma cell phenotype were also significantly induced. Finally, two well-characterized immunotoxicants, arsenic and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide, using the CD40L-induced IgM AFC response were compared in both mouse and human B cells. Collectively, an IgM AFC response model is described that can be applied to assess the sensitivity of antibody responses to modulation by xenobiotics using mouse as well as human primary B cells.

Plasma C-reactive protein (CRP) is an inflammatory biomarker that predicts cardiovascular disease. Lowering elevated CRP with statins has reduced the incidence of cardiovascular disease. We investigated whether vitamin C or E could reduce CRP. Healthy nonsmokers (N=396) were randomized to three groups, 1000 mg/day vitamin C, 800 IU/day vitamin E, or placebo, for 2 months. Median baseline CRP was low, 0.85 mg/L. No treatment effect was seen when all participants were included. However, a significant interaction was found, indicating that treatment effect depends on baseline CRP concentration. Among participants with CRP indicative of elevated cardiovascular risk (or =1.0 mg/L), vitamin C reduced the median CRP by 25.3% vs placebo (p=0.02) (median reduction in the vitamin C group, 0.25 mg/L, 16.7%). These effects are similar to those of statins. The vitamin E effect was not significant. In summary, treatment with vitamin C but not vitamin E significantly reduced CRP among individuals with CRPor =1.0 mg/L. Among the obese, 75% had CRPor =1.0 mg/L. Research is needed to determine whether reducing this inflammatory biomarker with vitamin C could reduce diseases associated with obesity. But research on clinical benefits of antioxidants should limit participants to persons with elevations in the target biomarkers.

Suppression of humoral immune responses by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been well established to require the aryl hydrocarbon receptor; however, the downstream mechanisms for this immunotoxic response remain poorly understood. Based on evidence demonstrating that primary hepatocytes pretreated with interferon-gamma (IFN-gamma) exhibited decreased induction of cytochrome P450 1A1 (CYP1A1) by TCDD, and that serum factors alter the sensitivity of the in vitro T-cell-dependent IgM antibody forming cell (AFC) response, it was hypothesized that IFN-gamma attenuates suppression of humoral immune responses by TCDD. In fact, concomitant addition of IFN-gamma (100 U/ml) produced a concentration-related attenuation of TCDD-mediated suppression of the anti-sheep erythrocyte (anti-sRBC) IgM AFC response. Time-of-addition studies performed by adding 100 U/ml IFN-gamma at 0, 1, 2, 4, 12, 24, 48, and 72 h post-TCDD showed that suppression of the AFC response was prevented only when IFN-gamma was added within 2 h of TCDD treatment. mRNA levels of the IgM components, immunoglobulin kappa light chain, immunoglobulin mu heavy chain, and immunoglobulin J-chain were significantly decreased by TCDD treatment, an effect that was completely reversed by IFN-gamma (100 U/ml) cotreatment. Further studies showed that IFN-alpha, IFN-beta, and IFN-gamma significantly attenuate TCDD-induced increases in CYP1A1 mRNA levels to varying degrees, but concentrations as high as 1000 U/ml of type I IFNs did not reverse the effect of TCDD on the anti-sRBC IgM AFC response. In summary, IFN-gamma prevents TCDD-mediated suppression of the IgM AFC response in a concentration- and time-related manner by altering transcriptional effects associated with TCDD treatment.

Sheep-associated malignant catarrhal fever (MCF) due to infection with ovine herpesvirus 2 (OvHV-2) is common in commercial herds of American bison ( Bison bison). Inability to propagate OvHV-2 in vitro has been a constraint on experimental studies of the disease. We sought to establish whether nasal secretions from sheep that shed OvHV-2 might induce the disease in bison and to define a minimum challenge dose. Fourteen bison were nebulized with sheep nasal sections containing 103–107 OvHV-2 deoxyribonucleic acid (DNA) copies. Most challenged bison (11/14, 78.6%) developed clinical signs at 29–52 days postnebulization (DPN). The mean incubation time was 42.18 (±7.33 SD) DPN. Using real-time polymerase chain reaction, we detected OvHV-2 DNA in peripheral blood leukocytes at 21–31 DPN. All bison that developed MCF had antibodies against the MCF group viruses. Gross and histologic lesions were typical of the acute disease. There was no morphologic evidence of a dose-related difference in the severity or distribution of lesions. This is the first successful reproduction of MCF in bison using a nasal route of exposure. Experimentally challenged bison are more susceptible to MCF, compared with experimentally challenged domestic cattle in a previous experiment. Bison are a pertinent ruminant species in which the pathogenesis of the disease can be investigated.