Macrophage-Derived Factors with the Potential to Contribute to Pathogenicity of HIV-1 and HIV-2: Role of CCL-2/MCP-1.

Human immunodeficiency virus type 2 (HIV-2) is known to be less pathogenic than HIV-1. However, the mechanism(s) underlying the decreased HIV-2 pathogenicity is not fully understood. Herein, we report that ß-chemokine CCL2 expression was increased in HIV-1-infected human monocyte-derived macrophages (MDM) but decreased in HIV-2-infected MDM when compared to uninfected MDM. Inhibition of CCL2 expression following HIV-2 infection occurred at both protein and mRNA levels. By microarray analysis, quantitative PCR, and Western blotting, we identified that Signal Transducer and Activator of Transcription 1 (STAT1), a critical transcription factor for inducing CCL2 gene expression, was also reduced in HIV-2-infected MDM. Blockade of STAT1 in HIV-infected MDM using a STAT1 inhibitor significantly reduced the production of CCL2. In contrast, transduction of STAT1-expressing pseudo-retrovirus restored CCL2 production in HIV-2-infected MDM. These findings support the concept that CCL2 inhibition in HIV-2-infected MDM is meditated by reduction of STAT1. Furthermore, we showed that STAT1 reduction in HIV-2-infected MDM was regulated by the CUL2/RBX1 ubiquitin E3 ligase complex-dependent proteasome pathway. Knockdown of CUL2 or RBX1 restored the expression of STAT1 and CCL2 in HIV-2-infected MDM. Taken together, our findings suggest that differential regulation of the STAT1-CCL2 axis may be one of the mechanisms underlying the different pathogenicity observed for HIV-1 and HIV-2.

Development of a New Cell-Based AP-1 Gene Reporter Potency Assay for Anti-Anthrax Toxin Therapeutics.

Anthrax toxin is a critical virulence factor of Bacillus anthracis. The toxin comprises protective antigen (PA) and two enzymatic moieties, edema factor (EF) and lethal factor (LF), forming bipartite lethal toxin (LT) and edema toxin (ET). PA binds cellular surface receptors and is required for intracellular translocation of the enzymatic moieties. For this reason, anti-PA antibodies have been developed as therapeutics for prophylaxis and treatment of human anthrax infection. Assays described publicly for the control of anti-PA antibody potency quantify inhibition of LT-mediated cell death or the ET-induced increase in c-AMP levels. These assays do not fully reflect and/or capture the pathological functions of anthrax toxin in humans. Herein, we report the development of a cell-based gene reporter potency assay for anti-PA antibodies based on the rapid LT-induced degradation of c-Jun protein, a pathogenic effect that occurs in human cells. This new assay was developed by transducing Hepa1c1c7 cells with an AP-1 reporter lentiviral construct and has been qualified for specificity, accuracy, repeatability, intermediate precision, and linearity. This assay not only serves as a bioassay for LT activity, but has applications for characterization and quality control of anti-PA therapeutic antibodies or other products that target the AP-1 signaling pathway.

Variable Induction of Pro-Inflammatory Cytokines by Commercial SARS CoV-2 Spike Protein Reagents: Potential Impacts of LPS on In Vitro Modeling and Pathogenic Mechanisms In Vivo.

Proinflammatory cytokine production following infection with severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) is associated with poor clinical outcomes. Like SARS CoV-1, SARS CoV-2 enters host cells via its spike protein, which attaches to angiotensin-converting enzyme 2 (ACE2). As SARS CoV-1 spike protein is reported to induce cytokine production, we hypothesized that this pathway could be a shared mechanism underlying pathogenic immune responses. We herein compared the capabilities of Middle East Respiratory Syndrome (MERS), SARS CoV-1 and SARS CoV-2 spike proteins to induce cytokine expression in human peripheral blood mononuclear cells (PBMC). We observed that only specific commercial lots of SARS CoV-2 induce cytokine production. Surprisingly, recombinant SARS CoV-2 spike proteins from different vendors and batches exhibited different patterns of cytokine induction, and these activities were not inhibited by blockade of spike protein-ACE2 binding using either soluble ACE2 or neutralizing anti-S1 antibody. Moreover, commercial spike protein reagents contained varying levels of lipopolysaccharide (LPS), which correlated directly with their abilities to induce cytokine production. The LPS inhibitor, polymyxin B, blocked this cytokine induction activity. In addition, SARS CoV-2 spike protein avidly bound soluble LPS in vitro, rendering it a cytokine inducer. These results not only suggest caution in monitoring the purity of SARS CoV-2 spike protein reagents, but they indicate the possibility that interactions of SARS CoV-2 spike protein with LPS from commensal bacteria in virally infected mucosal tissues could promote pathogenic inflammatory cytokine production.

Erk1/2 Inactivation-Induced c-Jun Degradation Is Regulated by Protein Phosphatases, UBE2d3, and the C-Terminus of c-Jun.

Constitutive photomorphogenic 1 (COP1) is the ubiquitin E3 ligase that mediates degradation of c-Jun protein upon Erk1/2 inactivation. It remains unknown how this protein degradation pathway is regulated. In this study, we investigated the roles of protein phosphatases, ubiquitin-conjugating E2 enzymes (UBE2), and an intrinsic motif of c-Jun in regulating this degradation pathway. By using pharmacological inhibitors and/or gene knockdown techniques, we identified protein phosphatase 1 (PP1) and PP2A as the phosphatases and UBE23d as the UBE2 promoting c-Jun degradation, triggered by Erk1/2 inactivation. In addition, we report that the C-terminus of c-Jun protein facilitates its degradation. The addition of a C-terminal tag or deletion of the last four amino acid residues from the C-terminus of c-Jun protects it from degradation under Erk1/2-inactivating conditions. Taken together, this study reveals that the Erk1/2 inactivation-triggered and COP1-mediated c-Jun degradation is extrinsically and intrinsically regulated, providing a new understanding of the mechanisms underlying this protein degradation pathway.

Identification of SRY-box 30 as an age-related essential gatekeeper for male germ-cell meiosis and differentiation.

Although important factors governing the meiosis have been reported in the embryonic ovary, meiosis in postnatal testis remains poorly understood. Herein, we first report that SRY-box 30 (Sox30) is an age-related and essential regulator of meiosis in the postnatal testis. Sox30-null mice exhibited uniquely impaired testis, presenting the abnormal arrest of germ-cell differentiation and irregular Leydig cell proliferation. In aged Sox30-null mice, the observed testicular impairments were more severe. Furthermore, the germ-cell arrest occurred at the stage of meiotic zygotene spermatocytes, which is strongly associated with critical regulators of meiosis (such as Cyp26b1, Stra8 and Rec8) and sex differentiation (such as Rspo1, Foxl2, Sox9, Wnt4 and Ctnnb1). Mechanistically, Sox30 can activate Stra8 and Rec8, and inhibit Cyp26b1 and Ctnnb1 by direct binding to their promoters. A different Sox30 domain required for regulating the activity of these gene promoters, providing a "fail-safe" mechanism for Sox30 to facilitate germ-cell differentiation. Indeed, retinoic acid levels were reduced owing to increased degradation following the elevation of Cyp26b1 in Sox30-null testes. Re-expression of Sox30 in Sox30-null mice successfully restored germ-cell meiosis, differentiation and Leydig cell proliferation. Moreover, the restoration of actual fertility appeared to improve over time. Consistently, Rec8 and Stra8 were reactivated, and Cyp26b1 and Ctnnb1 were reinhibited in the restored testes. In summary, Sox30 is necessary, sufficient and age-associated for germ-cell meiosis and differentiation in testes by direct regulating critical regulators. This study advances our understanding of the regulation of germ-cell meiosis and differentiation in the postnatal testis.

Erk1/2 inactivation promotes a rapid redistribution of COP1 and degradation of COP1 substrates.

Anthrax lethal toxin (LT) is a protease virulence factor produced by Bacillus anthracis that is required for its pathogenicity. LT treatment causes a rapid degradation of c-Jun protein that follows inactivation of the MEK1/2-Erk1/2 signaling pathway. Here we identify COP1 as the ubiquitin E3 ligase that is essential for LT-induced c-Jun degradation. COP1 knockdown using siRNA prevents degradation of c-Jun, ETV4, and ETV5 in cells treated with either LT or the MEK1/2 inhibitor, U0126. Immunofluorescence staining reveals that COP1 preferentially localizes to the nuclear envelope, but it is released from the nuclear envelope into the nucleoplasm following Erk1/2 inactivation. At baseline, COP1 attaches to the nuclear envelope via interaction with translocated promoter region (TPR), a component of the nuclear pore complex. Disruption of this COP1-TPR interaction, through Erk1/2 inactivation or TPR knockdown, leads to rapid COP1 release from the nuclear envelope into the nucleoplasm where it degrades COP1 substrates. COP1-mediated degradation of c-Jun protein, combined with LT-mediated blockade of the JNK1/2 signaling pathway, inhibits cellular proliferation. This effect on proliferation is reversed by COP1 knockdown and ectopic expression of an LT-resistant MKK7-4 fusion protein. Taken together, this study reveals that the nuclear envelope acts as a reservoir, maintaining COP1 poised for action. Upon Erk1/2 inactivation, COP1 is rapidly released from the nuclear envelope, promoting the degradation of its nuclear substrates, including c-Jun, a critical transcription factor that promotes cellular proliferation. This regulation allows mammalian cells to respond rapidly to changes in extracellular cues and mediates pathogenic mechanisms in disease states.

The ELISA Detectability and Potency of Pegfilgrastim Decrease in Physiological Conditions: Key Roles for Aggregation and Individual Variability.

PEGylated recombinant human granulocyte colony stimulating factor (pegfilgrastim) is used clinically to accelerate immune reconstitution following chemotherapy and is being pursued for biosimilar development. One challenge to overcome in pegfilgrastim biosimilar development is establishing pharmacokinetic (PK) similarity, which is partly due to the degree of PK variability. We herein report that commercially available G-CSF and PEG ELISA detection kits have different capacities to detect pegfilgrastim aggregates that rapidly form in vitro in physiological conditions. These aggregates can be observed using SDS-PAGE, size-exclusion chromatography, dynamic light scattering, and real-time NMR analysis and are associated with decreased bioactivity as reflected by reduced drug-induced cellular proliferation and STAT3 phosphorylation. Furthermore, individual variability in the stability and detectability of pegfilgrastim in human sera is also observed. Pegfilgrastim levels display marked subject variability in sera from healthy donors incubated at 37 °C. The stability patterns of pegfilgrastim closely match the stability patterns of filgrastim, consistent with a key role for pegfilgrastim's G-CSF moiety in driving formation of inactive aggregates. Taken together, our results indicate that individual variability and ELISA specificity for inactive aggregates are key factors to consider when designing and interpreting studies involving the measurement of serum pegfilgrastim concentrations.

Epigenetic Inactivation of SOX30 Is Associated with Male Infertility and Offers a Therapy Target for Non-obstructive Azoospermia.

Non-obstructive azoospermia (NOA) is the most severe form of male infertility. However, the etiology of NOA is largely unknown, resulting in a lack of clinical treatments. Here, we performed a comparative genome-wide profiling of DNA methylation and identified SOX30 as the most notably hyper-methylated gene at promoter in testicular tissues from NOA patients. This hyper-methylation at promoter of SOX30 directly causes its silencing of expression in NOA. The reduced levels of SOX30 expression are correlated with severity of NOA disease. Deletion of Sox30 in mice uniquely impairs testis development and spermatogenesis with complete absence of spermatozoa in testes leading to male infertility, but does not influence ovary development and female fertility. The pathology and testicular size of Sox30 null mice highly simulate those of NOA patients. Re-expression of Sox30 in Sox30 null mice at adult age reverses the pathological damage of testis and restores the spermatogenesis. The re-presented spermatozoa after re-expression of Sox30 in Sox30 null mice have the ability to start a pregnancy. Moreover, the male offspring of Sox30 re-expression Sox30 null mice still can father children, and these male offspring and their children can live normally more than 1 year without significant difference of physical appearance compared with wild-type mice. In summary, methylated inactivation of SOX30 uniquely impairs spermatogenesis, probably causing NOA disease, and re-expression of SOX30 can successfully restore the spermatogenesis and actual fertility. This study advances our understanding of the pathogenesis of NOA, offering a promising therapy target for NOA disease.

Dysregulation of Akt-FOXO1 Pathway Leads to Dysfunction of Regulatory T Cells in Patients with Psoriasis.

Psoriasis is a T lymphocyte-driven systemic inflammatory disease. Regulatory T cells (Tregs) are essential for establishing and maintaining immune tolerance. In this study, we found that patients with psoriasis and healthy controls had comparable percentages of circulating CD4+CD25+FOXP3+ Tregs, but psoriatic Tregs had reduced suppressive function. Thereafter, mRNA arrays were performed to study the gene expression profile of psoriatic Tregs. Psoriatic Tregs expressed high levels of a T helper type 1-like transcription factor and cytokines such as T-bet and IFN-γ. Furthermore, we found that FOXO1 can bind to the promoter of TBX21 to inhibit its expression, thus keeping the suppressive function of Tregs. However, an increase in protein kinase B-mediated phosphorylation of FOXO1 was observed in psoriatic Tregs, which subsequently caused FOXO1 inactivation by nuclear exclusion. In addition, incubation of healthy Tregs with psoriatic serum led to the activation of protein kinase B, nuclear exclusion of FOXO1, and the loss of FOXO1 transcription activity. The role of FOXO1 in regulating the function of Tregs was corroborated using a psoriasis-like mouse model in which Foxo1-deficient Tregs failed to protect mice from developing psoriasis. In conclusion, our findings reveal that the dysregulation of the protein kinase B-FOXO1 pathway may be a critical cause of Treg dysfunction in psoriasis.

Anthrax lethal toxin rapidly reduces c-Jun levels by inhibiting c-Jun gene transcription and promoting c-Jun protein degradation.

Anthrax is a life-threatening disease caused by infection with Bacillus anthracis, which expresses lethal factor and the receptor-binding protective antigen. These two proteins combine to form anthrax lethal toxin (LT), whose proximal targets are mitogen-activated kinase kinases (MKKs). However, the downstream mediators of LT toxicity remain elusive. Here we report that LT exposure rapidly reduces the levels of c-Jun, a key regulator of cell proliferation and survival. Blockade of proteasome-dependent protein degradation with the 26S proteasome inhibitor MG132 largely restored c-Jun protein levels, suggesting that LT promotes degradation of c-Jun protein. Using the MKK1/2 inhibitor U0126, we further show that MKK1/2-Erk1/2 pathway inactivation similarly reduces c-Jun protein, which was also restored by MG132 pre-exposure. Interestingly, c-Jun protein rebounded to normal levels 4 h following U0126 exposure but not after LT exposure. The restoration of c-Jun in U0126-exposed cells was associated with increased c-Jun mRNA levels and was blocked by inactivation of the JNK1/2 signaling pathway. These results indicate that LT reduces c-Jun both by promoting c-Jun protein degradation via inactivation of MKK1/2-Erk1/2 signaling and by blocking c-Jun gene transcription via inactivation of MKK4-JNK1/2 signaling. In line with the known functions of c-Jun, LT also inhibited cell proliferation. Ectopic expression of LT-resistant MKK2 and MKK4 variants partially restored Erk1/2 and JNK1/2 signaling in LT-exposed cells, enabling the cells to maintain relatively normal c-Jun protein levels and cell proliferation. Taken together, these findings indicate that LT reduces c-Jun protein levels via two distinct mechanisms, thereby inhibiting critical cell functions, including cellular proliferation.

Genome Wide Mapping of Foxo1 Binding-sites in Murine T Lymphocytes.

The Forkhead box O (Foxo) family of transcription factors has a critical role in controlling the development, differentiation, and function of T cells. However, the direct target genes of Foxo transcription factors in T cells have not been well characterized. In this study, we focused on mapping the genome wide Foxo1-binding sites in naïve CD4+ T cells, CD8+ T cells, and Foxp3+ regulatory T (Treg) cells. By using chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq), we identified Foxo1 binding sites that were shared among or specific to the three T cell populations. Here we describe the experiments, quality controls, as well as the deep sequencing data. Part of the data analysis has been published by Ouyang W et al. in Nature 2012[1] and Kim MV et al. in Immunity 2013[2], and the associated data set were uploaded to NCBI Gene Expression Omnibus.

Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an inhibitory receptor found on immune cells. The consequences of mutations in CTLA4 in humans are unknown. We identified germline heterozygous mutations in CTLA4 in subjects with severe immune dysregulation from four unrelated families. Whereas Ctla4 heterozygous mice have no obvious phenotype, human CTLA4 haploinsufficiency caused dysregulation of FoxP3(+) regulatory T (Treg) cells, hyperactivation of effector T cells, and lymphocytic infiltration of target organs. Patients also exhibited progressive loss of circulating B cells, associated with an increase of predominantly autoreactive CD21(lo) B cells and accumulation of B cells in nonlymphoid organs. Inherited human CTLA4 haploinsufficiency demonstrates a critical quantitative role for CTLA-4 in governing T and B lymphocyte homeostasis.

Anthrax lethal toxin inhibits translation of hypoxia-inducible factor 1α and causes decreased tolerance to hypoxic stress.

Hypoxia is considered to be a contributor to the pathology associated with administration of anthrax lethal toxin (LT). However, we report here that serum lactate levels in LT-treated mice are reduced, a finding inconsistent with the anaerobic metabolism expected to occur during hypoxia. Reduced lactate levels are also observed in the culture supernatants of LT-treated cells. LT inhibits the accumulation of hypoxia-inducible factor (HIF)-1α, a subunit of HIF-1, the master regulator directing cellular responses to hypoxia. The toxin has no effect on the transcription or protein turnover of HIF-1α, but instead it acts to inhibit HIF-1α translation. LT treatment diminishes phosphorylation of eIF4B, eIF4E, and rpS6, critical components of the intracellular machinery required for HIF-1α translation. Moreover, blockade of MKK1/2-ERK1/2, but not p38 or JNK signaling, lowers HIF-1α protein levels in both normoxic and hypoxic conditions, consistent with a role for MKK1 and MKK2 as the major targets of LT responsible for the inhibition of HIF-1α translation. The physiological importance of the LT-induced translation blockade is demonstrated by the finding that LT treatment decreases the survival of hepatocyte cell lines grown in hypoxic conditions, an effect that is overcome by preinduction of HIF-1α. Taken together, these data support a role for LT in dysregulating HIF-1α and thereby disrupting homeostatic responses to hypoxia, an environmental characteristic of certain tissues at baseline and/or during disseminated infection with Bacillus anthracis.

Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency.

The p110δ subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report fourteen patients from seven families who were heterozygous for three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ). These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and viremia due to cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV). Strikingly, they had a substantial deficiency in naive T cells but an over-representation of senescent effector T cells. In vitro, T cells from patients exhibited increased phosphorylation of the kinase Akt and hyperactivation of the metabolic checkpoint kinase mTOR, enhanced glucose uptake and terminal effector differentiation. Notably, treatment with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely 'rescued' the in vitro T cell defects and improved the clinical course.

Murine in vivo CD8+ T Cell Killing Assay.

Antigen-specific killing ability of effector CD8+ T cells is critical for protective immunity against infection. Here, we describe in vivo cytotoxic T cell assay to examine effector function of antigen-specific CD8+ T cells. Mice infected with Listeria monocytogenes (L. monocytogenes) expressing chicken ovalbumin as a model antigen mount ovalbumin-specific CD8+ T cell responses. Effector CD8+ T cell function in vivo is determined by mixed transfer of OVA peptide-pulsed target cells with control target cells into the previously immunized mice. Difference in CFSE expression levels clearly marks two distinct populations: Antigen-pulsed target cells-CFSElow vs. unpulsed target cells-CFSEhi. The frequencies between antigen-pulsed target cells and control target cells are used as readouts of antigen-specific killing.

Murine In vitro Memory T Cell Differentiation.

Upon pathogen encounter, naïve CD8+ T cells are primed and undergo massive clonal expansion. A fraction of effector CD8+ T cells remains during the contraction phase and differentiate into memory T cells critical for mounting robust recall responses in response to secondary infection. Low frequency of memory T cells in vivo is a major obstacle to investigate their functional aspects including migration capacity and genetic regulation. Here, we describe detailed protocol for memory T cell differentiation developed by von Andrian's group to generate large number of CD44hiCD62Lhi antigen-specific memory T cells in vitro.

The transcription factor Foxo1 controls central-memory CD8+ T cell responses to infection.

Memory T cells protect hosts from pathogen reinfection, but how these cells emerge from a pool of antigen-experienced T cells is unclear. Here, we show that mice lacking the transcription factor Foxo1 in activated CD8+ T cells have defective secondary, but not primary, responses to Listeria monocytogenes infection. Compared to short-lived effector T cells, memory-precursor T cells expressed higher amounts of Foxo1, which promoted their generation and maintenance. Chromatin immunoprecipitation sequencing revealed the transcription factor Tcf7 and the chemokine receptor Ccr7 as Foxo1-bound target genes, which have critical functions in central-memory T cell differentiation and trafficking. These findings demonstrate that Foxo1 is selectively incorporated into the genetic program that regulates memory CD8+ T cell responses to infection.

TGF-ß cytokine signaling promotes CD8+ T cell development and low-affinity CD4+ T cell homeostasis by regulation of interleukin-7 receptor α expression.

Interleukin-7 receptor α chain (IL-7Rα) is induced upon T cell positive selection and controls thymic CD8-lineage specification and peripheral naive T cell homeostasis. How IL-7Rα expression is regulated in developing thymocytes is unclear. Here, we show that transforming growth factor ß (TGF-ß) signaling promoted IL-7Rα expression and CD8+ T cell differentiation. In addition, TGF-ß signaling was required for high IL-7Rα expression in CD4+ T cells bearing low-affinity T cell receptors, and the abrogation of TGF-ß receptor expression led to failed maintenance of peripheral CD4+ T cells. Compromised IL-7Rα expression in TGF-ß-receptor-deficient T cells was associated with increased expression of the Il7ra transcriptional repressor, Gfi-1. IL-7Rα transgenesis or T-cell-specific ablation of Gfi-1 restored IL-7Rα expression and largely ameliorated the development and homeostasis defects of TGF-ß-receptor-deficient T cells. These findings reveal functions for TGF-ß signaling in controlling IL-7Rα expression and in promoting T cell repertoire diversification.

A New Murine Model for Gastrointestinal Anthrax Infection.

The scientific community has been restricted by the lack of a practical and informative animal model of gastrointestinal infection with vegetative Bacillus anthracis. We herein report the development of a murine model of gastrointestinal anthrax infection by gavage of vegetative Sterne strain of Bacillus anthracis into the complement-deficient A/J mouse strain. Mice infected in this manner developed lethal infections in a dose-dependent manner and died 30 h-5 d following gavage. Histological findings were consistent with penetration and growth of the bacilli within the intestinal villi, with subsequent dissemination into major organs including the spleen, liver, kidney and lung. Blood cultures confirmed anthrax bacteremia in all moribund animals, with approximately 1/3 showing co-infection with commensal enteric organisms. However, no evidence of immune activation was observed during infection. Time-course experiments revealed early compromise of the intestinal epithelium, characterized by villus blunting and ulceration in the ileum and jejunum. A decrease in body temperature was most predictive of near-term lethality. Antibiotic treatment of infected animals 24 h following high-dose bacterial gavage protected all animals, demonstrating the utility of this animal model in evaluating potential therapeutics.