Prior viral infection primes cross-reactive CD8+ T cells that respond to mouse heart allografts.

Introduction: Significant evidence suggests a connection between transplant rejection and the presence of high levels of pre-existing memory T cells. Viral infection can elicit viral-specific memory T cells that cross-react with allo-MHC capable of driving allograft rejection in mice. Despite these advances, and despite their critical role in transplant rejection, a systematic study of allo-reactive memory T cells, their specificities, and the role of cross-reactivity with viral antigens has not been performed. Methods: Here, we established a model to identify, isolate, and characterize cross-reactive T cells using Nur77 reporter mice (C57BL/6 background), which transiently express GFP exclusively upon TCR engagement. We infected Nur77 mice with lymphocytic choriomeningitis virus (LCMV-Armstrong) to generate a robust memory compartment, where quiescent LCMV-specific memory CD8+ T cells could be readily tracked with MHC tetramer staining. Then, we transplanted LCMV immune mice with allogeneic hearts and monitored expression of GFP within MHC-tetramer defined viral-specific T cells as an indicator of their ability to cross-react with alloantigens. Results: Strikingly, prior LCMV infection significantly increased the kinetics and magnitude of rejection as well as CD8+ T cell recruitment into allogeneic, but not syngeneic, transplanted hearts, relative to non-infected controls. Interestingly, as early as day 1 after allogeneic heart transplant an average of ~8% of MHC-tetramer+ CD8+ T cells expressed GFP, in contrast to syngeneic heart transplants, where the frequency of viral-specific CD8+ T cells that were GFP+ was <1%. These data show that a significant percentage of viral-specific memory CD8+ T cells expressed T cell receptors that also recognized alloantigens in vivo. Notably, the frequency of cross-reactive CD8+ T cells differed depending upon the viral epitope. Further, TCR sequences derived from cross-reactive T cells harbored distinctive motifs that may provide insight into cross-reactivity and allo-specificity. Discussion: In sum, we have established a mouse model to track viral-specific, allo-specific, and cross-reactive T cells; revealing that prior infection elicits substantial numbers of viral-specific T cells that cross-react to alloantigen, respond very early after transplant, and may promote rapid rejection.

Single-cell transcriptomic analysis of renal allograft rejection reveals insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding of how rejection occurs despite immunosuppression (IS). We performed combined single-cell RNA transcriptomic and TCR-α/ß sequencing on rBx from patients with ACR under differing IS drugs: tacrolimus, iscalimab, and belatacept. We found distinct CD8+ T cell phenotypes (e.g., effector, memory, exhausted) depending upon IS type, particularly within expanded CD8+ T cell clonotypes (CD8EXP). Gene expression of CD8EXP identified therapeutic targets that were influenced by IS type. TCR analysis revealed a highly restricted number of CD8EXP, independent of HLA mismatch or IS type. Subcloning of TCR-α/ß cDNAs from CD8EXP into Jurkat 76 cells (TCR-/-) conferred alloreactivity by mixed lymphocyte reaction. Analysis of sequential rBx samples revealed persistence of CD8EXP that decreased, but were not eliminated, after successful antirejection therapy. In contrast, CD8EXP were maintained in treatment-refractory rejection. Finally, most rBx-derived CD8EXP were also observed in matching urine samples, providing precedent for using urine-derived CD8EXP as a surrogate for those found in the rejecting allograft. Overall, our data define the clonal CD8+ T cell response to ACR, paving the next steps for improving detection, assessment, and treatment of rejection.

Single cell transcriptomic analysis of renal allograft rejection reveals novel insights into intragraft TCR clonality.

Bulk analysis of renal allograft biopsies (rBx) identified RNA transcripts associated with acute cellular rejection (ACR); however, these lacked cellular context critical to mechanistic understanding. We performed combined single cell RNA transcriptomic and TCRα/ß sequencing on rBx from patients with ACR under differing immunosuppression (IS): tacrolimus, iscalimab, and belatacept. TCR analysis revealed a highly restricted CD8 + T cell clonal expansion (CD8 EXP ), independent of HLA mismatch or IS type. Subcloning of TCRα/ß cDNAs from CD8 EXP into Jurkat76 cells (TCR -/- ) conferred alloreactivity by mixed lymphocyte reaction. scRNAseq analysis of CD8 EXP revealed effector, memory, and exhausted phenotypes that were influenced by IS type. Successful anti-rejection treatment decreased, but did not eliminate, CD8 EXP , while CD8 EXP were maintained during treatment-refractory rejection. Finally, most rBx-derived CD8 EXP were also observed in matching urine samples. Overall, our data define the clonal CD8 + T cell response to ACR, providing novel insights to improve detection, assessment, and treatment of rejection.

MDA5-dependent responses contribute to autoimmune diabetes progression and hindrance.

Type 1 diabetes (T1D) is an autoimmune disease resulting in pancreatic ß cell destruction. Coxsackievirus B3 (CVB3) infection and melanoma differentiation-associated protein 5-dependent (MDA5-dependent) antiviral responses are linked with T1D development. Mutations within IFIH1, coding for MDA5, are correlated with T1D susceptibility, but how these mutations contribute to T1D remains unclear. Utilizing nonobese diabetic (NOD) mice lacking Ifih1 expression (KO) or containing an in-frame deletion within the ATPase site of the helicase 1 domain of MDA5 (ΔHel1), we tested the hypothesis that partial or complete loss-of-function mutations in MDA5 would delay T1D by impairing proinflammatory pancreatic macrophage and T cell responses. Spontaneous T1D developed in female NOD and KO mice similarly, but was significantly delayed in ΔHel1 mice, which may be partly due to a concomitant increase in myeloid-derived suppressor cells. Interestingly, KO male mice had increased spontaneous T1D compared with NOD mice. Whereas NOD and KO mice developed CVB3-accelerated T1D, ΔHel1 mice were protected partly due to decreased type I IFNs, pancreatic infiltrating TNF+ macrophages, IFN-γ+CD4+ T cells, and perforin+CD8+ T cells. Furthermore, ΔHel1 MDA5 protein had reduced ATP hydrolysis compared with wild-type MDA5. Our results suggest that dampened MDA5 function delays T1D, yet loss of MDA5 promotes T1D.

Maternal-fetal conflict averted by progesterone- induced FOXP3+ regulatory T cells.

Pregnancy stimulates an intricately coordinated assortment of physiological changes to accommodate growth of the developing fetus, while simultaneously averting rejection of genetically foreign fetal cells and tissues. Despite increasing evidence that expansion of immune-suppressive maternal regulatory T cells enforces fetal tolerance and protects against pregnancy complications, the pregnancy-associated signals driving this essential adaptation remain poorly understood. Here we show that the female reproductive hormone, progesterone, coordinates immune tolerance by stimulating expansion of FOXP3+ regulatory T cells. Conditional loss of the canonical nuclear progesterone receptor in maternal FOXP3+ regulatory T cells blunts their proliferation and accumulation, which is associated with fetal wastage and decidual infiltration of activated CD8+ T cells. Reciprocally, the synthetic progestin 17α-hydroxyprogesterone caproate (17-OHPC) administered to pregnant mice reinforces fetal tolerance and protects against fetal wastage. These immune modulatory effects of progesterone that promote fetal tolerance establish a molecular link between immunological and other physiological adaptions during pregnancy.

Auranofin-Mediated NRF2 Induction Attenuates Interleukin 1 Beta Expression in Alveolar Macrophages.

BACKGROUND: Alveolar macrophages (AMs) are resident inflammatory cells in the lung that serve as early sentinels of infection or injury. We have identified thioredoxin reductase 1 inhibition by gold compounds increases activation of nuclear factor erythroid 2-related factor 2 (NRF2)-dependent pathways to attenuate inflammatory responses. The present studies utilized murine alveolar macrophages (MH-S) to test the hypothesis that the gold compound, auranofin (AFN), decreases interleukin (IL)-1ß expression through NRF2-mediated interactions with nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway genes and/or increases in glutathione synthesis. METHODS: MH-S cells were treated with AFN and lipopolysaccharide (LPS) and analyzed at 6 and 24 h. The Il1b promoter was analyzed by chromatin immunoprecipitation for direct interaction with NRF2. RESULTS: Expression of IL-1ß, p-IκBα, p-p65 NF-kB, and NOD-, LRR-, and pyrin domain-containing protein 3 were elevated by LPS exposure, but only IL-1ß expression was suppressed by AFN treatment. Both AFN and LPS treatments increased cellular glutathione levels, but attenuation of glutathione synthesis by buthionine sulfoximine (BSO) did not alter expression of Il-1ß. Analysis revealed direct NRF2 binding to the Il1b promoter which was enhanced by AFN and inhibited the transcriptional activity of DNA polymerase II. CONCLUSIONS: Our data demonstrate that AFN-induced NRF2 activation directly suppresses IL-1ß synthesis independent of NFκB and glutathione-mediated antioxidant mechanisms. NRF2 binding to the promoter region of IL1ß directly inhibits transcription of the IL1ß gene. Collectively, our research suggests that gold compounds elicit NRF2-dependent pulmonary protection by suppressing macrophage-mediated inflammation.

Persistent Zika Virus Clinical Susceptibility despite Reduced Viral Burden in Mice with Expanded Virus-Specific CD8+ T Cells Primed by Recombinant Listeria monocytogenes.

Vaccines against Zika virus (ZIKV) infection that target CD8+ T cells are of considerable interest because Abs may enhance infection susceptibility. However, whether CD8+ T cells are protective or promote susceptibility to clinical infection symptoms remains uncertain. To more precisely investigate ZIKV-specific CD8+ T cells in isolation, we engineered a Listeria monocytogenes-based vector to express a single MHC class I-restricted immune dominant peptide, E294-302, from ZIKV envelope protein. We show accumulation of activated ZIKV-specific CD8+ T cells primed by recombinant L. monocytogenes is associated with reductions in circulating virus levels after ZIKV challenge in type I IFN receptor-deficient mice and wildtype mice administered neutralizing Abs against type I IFN receptor. Interestingly, susceptibility to ZIKV clinical infection including weight loss and mortality each persists and is neither significantly improved nor worsened compared with isogenic L. monocytogenes-primed control mice. These data demonstrating persistent ZIKV clinical susceptibility despite reduced viral burden in mice with expanded virus-specific CD8+ T cells highlights the need for targeting other adaptive immune components in developing vaccines against ZIKV infection.

Commensal Candida albicans Positively Calibrates Systemic Th17 Immunological Responses.

Mucosal barriers are densely colonized by pathobiont microbes such as Candida albicans, capable of invasive disseminated infection. However, systemic infections occur infrequently in healthy individuals, suggesting that pathobiont commensalism may elicit host benefits. We show that intestinal colonization with C. albicans drives systemic expansion of fungal-specific Th17 CD4+ T cells and IL-17 responsiveness by circulating neutrophils, which synergistically protect against C. albicans invasive infection. Protection conferred by commensal C. albicans requires persistent fungal colonization and extends to other extracellular invasive pathogens such as Staphylococcus aureus. However, commensal C. albicans does not protect against intracellular influenza virus infection and exacerbates allergic airway inflammation susceptibility, indicating that positively calibrating systemic Th17 responses is not uniformly beneficial. Thus, systemic Th17 inflammation driven by CD4+ T cells responsive to tonic stimulation by commensal C. albicans improves host defense against extracellular pathogens, but with potentially harmful immunological consequences.

Antiretroviral therapy potentiates high-fat diet induced obesity and glucose intolerance.

OBJECTIVE: Breakthroughs in HIV treatment, especially combination antiretroviral therapy (ART), have massively reduced AIDS-associated mortality. However, ART administration amplifies the risk of non-AIDS defining illnesses including obesity, diabetes, and cardiovascular disease, collectively known as metabolic syndrome. Initial reports suggest that ART-associated risk of metabolic syndrome correlates with socioeconomic status, a multifaceted finding that encompasses income, race, education, and diet. Therefore, determination of causal relationships is extremely challenging due to the complex interplay between viral infection, ART, and the many environmental factors. METHODS: In the current study, we employed a mouse model to specifically examine interactions between ART and diet that impacts energy balance and glucose metabolism. Previous studies have shown that high-fat feeding induces persistent low-grade systemic and adipose tissue inflammation contributing to insulin resistance and metabolic dysregulation via adipose-infiltrating macrophages. Studies herein test the hypothesis that ART potentiates the inflammatory effects of a high-fat diet (HFD). C57Bl/6J mice on a HFD or standard chow containing ART or vehicle, were subjected to functional metabolic testing, RNA-sequencing of epididymal white adipose tissue (eWAT), and array-based kinomic analysis of eWAT-infiltrating macrophages. RESULTS: ART-treated mice on a HFD displayed increased fat mass accumulation, impaired glucose tolerance, and potentiated insulin resistance. Gene set enrichment and kinomic array analyses revealed a pro-inflammatory transcriptional signature depicting granulocyte migration and activation. CONCLUSION: The current study reveals a HFD-ART interaction that increases inflammatory transcriptional pathways and impairs glucose metabolism, energy balance, and metabolic dysfunction.

Superoxide Production by NADPH Oxidase Intensifies Macrophage Antiviral Responses during Diabetogenic Coxsackievirus Infection.

Coxsackievirus B infections are suspected environmental triggers of type 1 diabetes (T1D) and macrophage antiviral responses may provide a link to virus-induced T1D. We previously demonstrated an important role for NADPH oxidase (NOX)-derived superoxide production during T1D pathogenesis, as NOX-deficient NOD mice (NOD.Ncf1m1J ) were protected against T1D due, in part, to impaired proinflammatory TLR signaling in NOD.Ncf1m1J macrophages. Therefore, we hypothesized that loss of NOX-derived superoxide would dampen diabetogenic antiviral macrophage responses and protect from virus-induced diabetes. Upon infection with a suspected diabetogenic virus, Coxsackievirus B3 (CB3), NOD.Ncf1m1J mice remained resistant to virus-induced autoimmune diabetes. A concomitant decrease in circulating inflammatory chemokines, blunted antiviral gene signature within the pancreas, and reduced proinflammatory M1 macrophage responses were observed. Importantly, exogenous superoxide addition to CB3-infected NOD.Ncf1m1J bone marrow-derived macrophages rescued the inflammatory antiviral M1 macrophage response, revealing reduction-oxidation-dependent mechanisms of signal transducer and activator of transcription 1 signaling and dsRNA viral sensors in macrophages. We report that superoxide production following CB3 infection may exacerbate pancreatic ß cell destruction in T1D by influencing proinflammatory M1 macrophage responses, and mechanistically linking oxidative stress, inflammation, and diabetogenic virus infections.

Redox-Sensitive Innate Immune Pathways During Macrophage Activation in Type 1 Diabetes.

SIGNIFICANCE: Type 1 diabetes (T1D) is an autoimmune disease resulting in ß-cell destruction mediated by islet-infiltrating leukocytes. The role of oxidative stress in human and murine models of T1D is highly significant as these noxious molecules contribute to diabetic complications and ß-cell lysis, but their direct impact on dysregulated autoimmune responses is highly understudied. Pro-inflammatory macrophages play a vital role in the initiation and effector phases of T1D by producing free radicals and pro-inflammatory cytokines to facilitate ß-cell destruction and to present antigen to autoreactive T cells. Recent Advances: Redox modulation of macrophage functions may play critical roles in autoimmunity. These include enhancing pro-inflammatory innate immune signaling pathways in response to environmental triggers, enforcing an M1 macrophage differentiation program, controlling antigen processing, and altering peptide recognition by oxidative post-translational modification. Therefore, an oxidative environment may act on multiple macrophage functions to orchestrate T1D pathogenesis. CRITICAL ISSUES: Mechanisms involved in the initiation of T1D remain unclear, making preventive and early therapeutics difficult to develop. Although many of these advances in the redox regulation of macrophages are in their infancy, they provide insight into how oxidative stress aids in the precipitating event of autoimmune activation. FUTURE DIRECTIONS: Future studies should be aimed at mechanistically determining which redox-regulated macrophage functions are pertinent in T1D pathogenesis, as well as at investigating potential targetable therapeutics to halt and/or dampen innate immune activation in T1D.

Orally administered ß-glucan attenuates the Th2 response in a model of airway hypersensitivity.

ß-Glucan is a polysaccharide that can be extracted from fungal cell walls. Wellmune WGP(®), a preparation of ß-1,3/1,6-glucans, is a dietary supplement that has immunomodulating properties. Here we investigated the effect WGP had on a mouse model of asthma. OVA-induced asthma in mice is characterized by infiltration of eosinophils into the lung, production of Th2 cytokines and IgE. Daily oral administration of WGP (400 µg) significantly reduced the influx of eosinophils into the lungs of OVA-challenged mice compared to control mice. In addition, WGP inhibited pulmonary production of Th2 cytokines (IL-4, IL-5, IL-13), however serum IgE levels were unaffected by WGP treatment. These data indicate that WGP could potentially be useful as an oral supplement for some asthma patients, however, it would need to be combined with therapies that target other aspects of the disease such as IgE levels. As such, further studies that examine the potential of WGP in combination with other therapies should be explored.

Loss of NADPH oxidase-derived superoxide skews macrophage phenotypes to delay type 1 diabetes.

Macrophages are early islet-infiltrating cells seen in type 1 diabetes (T1D). While proinflammatory M1 macrophages induce T1D, M2 macrophages have been shown to delay this autoimmune disease in nonobese diabetic (NOD) mice, but the environmental cues that govern macrophage polarization and differentiation remain unresolved. We previously demonstrated the importance of reactive oxygen species (ROS) in T1D, as NOD mice deficient in NADPH oxidase (NOX)-derived superoxide (Ncf1(m1J)) were protected against T1D partly because of blunted Toll-like receptor-dependent macrophage responses. We provide evidence that NOX-derived ROS contribute to macrophage differentiation in T1D. During spontaneous diabetes progression, T1D-resistant NOD.Ncf1(m1J) islet-resident macrophages displayed a dampened M1 and increased M2 phenotype. The transfer of diabetogenic T cells into NOX-deficient NOD.Rag.Ncf1(m1J) recipients resulted in decreased TNF-α(+) and IL-1ß(+) islet-infiltrating M1 macrophages and a concomitant enhancement in arginase-1(+) M2 macrophages. Mechanistic analysis of superoxide-deficient bone marrow-derived macrophages revealed a marked diminution in a proinflammatory M1 phenotype due to decreased P-STAT1 (Y701) and interferon regulatory factor 5 compared with NOD mice. We have therefore defined a novel mechanistic link between NOX-derived ROS and macrophage phenotypes, and implicated superoxide as an important factor in macrophage differentiation. Thus, targeting macrophage redox status may represent a promising therapy in halting human T1D.

Eosinophil deficiency compromises lung defense against Aspergillus fumigatus.

Exposure to the mold Aspergillus fumigatus may result in allergic bronchopulmonary aspergillosis, chronic necrotizing pulmonary aspergillosis, or invasive aspergillosis (IA), depending on the host's immune status. Neutrophil deficiency is the predominant risk factor for the development of IA, the most life-threatening condition associated with A. fumigatus exposure. Here we demonstrate that in addition to neutrophils, eosinophils are an important contributor to the clearance of A. fumigatus from the lung. Acute A. fumigatus challenge in normal mice induced the recruitment of CD11b+ Siglec F+ Ly-6G(lo) Ly-6C(neg) CCR3+ eosinophils to the lungs, which was accompanied by an increase in lung Epx (eosinophil peroxidase) mRNA levels. Mice deficient in the transcription factor dblGATA1, which exhibit a selective deficiency in eosinophils, demonstrated impaired A. fumigatus clearance and evidence of germinating organisms in the lung. Higher burden correlated with lower mRNA expression of Epx (eosinophil peroxidase) and Prg2 (major basic protein) as well as lower interleukin 1ß (IL-1ß), IL-6, IL-17A, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL1 levels. However, examination of lung inflammatory cell populations failed to demonstrate defects in monocyte/macrophage, dendritic cell, or neutrophil recruitment in dblGATA1-deficient mice, suggesting that the absence of eosinophils in dlbGATA1-deficient mice was the sole cause of impaired lung clearance. We show that eosinophils generated from bone marrow have potent killing activity against A. fumigtaus in vitro, which does not require cell contact and can be recapitulated by eosinophil whole-cell lysates. Collectively, our data support a role for eosinophils in the lung response after A. fumigatus exposure.

LAB/NTAL facilitates fungal/PAMP-induced IL-12 and IFN-γ production by repressing ß-catenin activation in dendritic cells.

Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2-/- mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear ß-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2-/- DCs. Accordingly, Lat2-/- DCs directed reduced Th1 polarization in vitro and Lat2-/- mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and ß-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

Dysregulated TLR3-dependent signaling and innate immune activation in superoxide-deficient macrophages from nonobese diabetic mice.

In type 1 diabetes (T1D), reactive oxygen species (ROS) and proinflammatory cytokines produced by macrophages and other innate immune cells destroy pancreatic ß cells while promoting autoreactive T cell maturation. Superoxide-deficient nonobese diabetic mice (NOD.Ncf1(m1J)) are resistant to spontaneous diabetes, revealing the integral role of ROS signaling in T1D. Here, we evaluate the innate immune activation state of bone marrow-derived macrophages (BM-Mϕ) from NOD and NOD.Ncf1(m1J) mice after poly(I:C)-induced Toll-like receptor 3 (TLR3) signaling. We show that ROS synthesis is required for efficient activation of the NF-κB signaling pathway and concomitant expression of TLR3 and the cognate adaptor molecule, TRIF. Poly(I:C)-stimulated NOD.Ncf1(m1J) BM-Mϕ exhibited a 2- and 10-fold decrease in TNF-α and IFN-ß proinflammatory cytokine synthesis, respectively, in contrast to NOD BM-Mϕ. Optimal expression of IFN-α/ß is not solely dependent on superoxide synthesis, but requires p47(phox) to function in a NOX-independent manner to mediate type I interferon synthesis. Interestingly, MHC-II I-A(g7) expression necessary for CD4 T cell activation is increased 2-fold relative to NOD, implicating a role for superoxide in I-A(g7) downregulation. These findings suggest that defective innate immune-pattern-recognition receptor activation and subsequent decrease in TNF-α and IFN-ß proinflammatory cytokine synthesis necessary for autoreactive T cell maturation may contribute to the T1D protection observed in NOD.Ncf1(m1J) mice.

Automated dose-response analysis and comparative toxicogenomic evaluation of the hepatic effects elicited by TCDD, TCDF, and PCB126 in C57BL/6 mice.

The toxic equivalency factor (TEF) approach recommended by the World Health Organization is used to quantify dioxin-like exposure concentrations for mixtures of polychlorinated dibenzo-dioxins, -furans, and polychlorinated biphenyls (PCBs), including 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Whole-genome microarrays were used to evaluate the hepatic gene expression potency of TCDF and PCB126 relative to TCDD with complementary histopathology, tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay results. Immature ovariectomized C57BL/6 mice were gavaged with 0.001, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, and 300 µg/kg TCDD and TEF-adjusted doses (TEF for TCDF and PCB126 is 0.1) of TCDF or PCB126 (1, 3, 10, 30, 100, 300, 1000, and 3000 µg/kg of TCDF or PCB126) or sesame oil vehicle and sacrificed 24 h post dose. In general, TCDD, TCDF, and PCB126 tissue levels, as well as histopathological effects, were comparable when comparing TEF-adjusted doses. Automated dose-response modeling (ToxResponse Modeler) of the microarray data identified 210 TCDF and 40 PCB126 genes that exhibited sigmoidal dose-response curves with comparable slopes when compared with TCDD. These similar responses were used to calculate a median TCDF gene expression relative potency (REP) of 0.06 and a median PCB126 gene expression REP of 0.02. REPs of 0.02 were also calculated for EROD induction for both compounds. Collectively, these data suggest that differences in the ability of the liganded aryl hydrocarbon receptor:AhR nuclear translocator complex to elicit differential hepatic gene expression, in addition to pharmacokinetic differences between ligands, influence their potency in immature ovariectomized C57BL/6 mice.

Automated dose-response analysis of the relative hepatic gene expression potency of TCDF in C57BL/6 mice.

Toxic equivalency factors (TEFs) are assigned to dioxin-like chemicals based on relative potency (REP) values of individual adaptive and toxic responses compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Agilent 4x44K oligonucleotide microarrays were used to examine the hepatic gene expression potency of 2,3,7,8-tetrachlorodibenzofuran (TCDF), relative to TCDD with complementary histopathology, TCDD and TCDF tissue level analysis, and ethoxyresorufin-O-deethylase (EROD) assay data. Immature ovariectomized C57BL/6 mice were gavaged with 0.03, 0.1, 0.3, 1, 3, 10, 30, or 100 microg/kg TCDD, the World Health Organization TEF-adjusted doses (10 x TCDD dose) of TCDF (0.3, 1, 3, 10, 30, 100, or 300 microg/kg), or sesame oil vehicle and killed at 72 h. Two thousand two hundred eighty-eight and 1347 genes were differentially expressed (P1(t) > 0.90) at one or more doses by TCDD and TCDF, respectively. Automated dose-response modeling (ToxResponse Modeler) identified a total of 1027 and 837 genes with either a sigmoidal, exponential, linear, Gaussian, or quadratic dose-response relationship 72 h after treatment in TCDD and TCDF, respectively. Two hundred seventy genes exhibited a sigmoidal TCDD-induced dose-response (ED(50s) from 0.08 to 42.2 microg/kg) compared to only 179 sigmoidal responsive genes (ED(50s) from 0.74 to 299.9 microg/kg) elicited by TCDF. Of the 1027 TCDD dose-responsive genes, 654 were not examined further due to the lack of a dose response elicited by TCDF. Of the 373 genes that exhibited a TCDD and TCDF dose response, REPs were calculated for the 83 genes that exhibited comparable sigmoidal curve shapes and slopes. The median REP for these 83 genes was 0.10, with a maximum REP of 0.56 and a minimum of 0.01. REPs of 0.04 were also calculated for EROD and increase in relative liver weight (RLW) at 72 h. Collectively, the lower number of TCDF-induced genes compared to TCDD and the 0.04 REPs for EROD activity and increased RLW are not consistent with the TEF of 0.10 for the hepatotoxicity of TCDF in C57BL/6 mice at 72 h.