NDRG1 is induced by antigen-receptor signaling but dispensable for B and T cell self-tolerance.

Peripheral tolerance prevents the initiation of damaging immune responses by autoreactive lymphocytes. While tolerogenic mechanisms are tightly regulated by antigen-dependent and independent signals, downstream pathways are incompletely understood. N-myc downstream-regulated gene 1 (NDRG1), an anti-cancer therapeutic target, has previously been implicated as a CD4+ T cell clonal anergy factor. By RNA-sequencing, we identified Ndrg1 as the third most upregulated gene in anergic, compared to naïve follicular, B cells. Ndrg1 is upregulated by B cell receptor activation (signal one) and suppressed by co-stimulation (signal two), suggesting that NDRG1 may be important in B cell tolerance. However, though Ndrg1-/- mice have a neurological defect mimicking NDRG1-associated Charcot-Marie-Tooth (CMT4d) disease, primary and secondary immune responses were normal. We find that B cell tolerance is maintained, and NDRG1 does not play a role in downstream responses during re-stimulation of in vivo antigen-experienced CD4+ T cells, demonstrating that NDGR1 is functionally redundant for lymphocyte anergy

Regulation of Chemokines, CCL3 and CCL4, by interferon gamma and Nitric oxide synthase 2 in mouse macrophages and during Salmonella enterica Serovar Typhimurium infection

Background. Interferon gamma (IFN-gamma) increases the expression of multiple genes and responses; however, the mechanisms by which IFN-gamma downmodulates cellular responses is not well understood. In this study, the repression of CCL3 and CCL4 by IFN-gamma and nitric oxide synthase 2 (NOS2) in macrophages and upon Salmonella typhimurium infection of mice was investigated. Methods. Small molecule regulators and adherent peritoneal exudates cells (A-PECs) from Nos2(-/-)mice were used to identify the contribution of signaling molecules during IFN-gamma-mediated in vitro regulation of CCL3, CCL4, and CXCL10. In addition, infection of bone marrow-derived macrophages (BMDMs) and mice (C57BL/6, Ifn-gamma(-/), and Nos2(-/-)) with S. typhimurium were used to gain an understanding of the in vivo regulation of these chemokines. Results. IFN-gamma repressed CCL3 and CCL4 in a signal transducer and activator of transcription 1 (STAT1)-NOS2-p38 mitogen activated protein kinase (p38MAPK)-activating transcription factor 3 (ATF3) dependent pathway in A-PECs. Also, during intracellular replication of S. typhimurium in BMDMs, IFN-gamma and NOS2 repressed CCL3 and CCL4 production. The physiological roles of these observations were revealed during oral infection of mice with S. typhimurium, wherein endogenous IFN-gamma and NOS2 enhanced serum amounts of tumor necrosis factor alpha and CXCL10 but repressed CCL3 and CCL4. Conclusions. This study sheds novel mechanistic insight on the regulation of CCL3 and CCL4 in mouse macrophages and during S. typhimurium oral infection

Interferon-gamma- and glucocorticoid-mediated pathways synergize to enhance death of CD4(+) CD8(+) thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy is known to occur during infections; however, there is limited understanding of its causes and of the cross-talk between different pathways. This study investigates mechanisms involved in thymic atrophy during a model of oral infection by Salmonella enterica serovar Typhimurium (S.typhimurium). Significant death of CD4+CD8+ thymocytes, but not of single-positive thymocytes or peripheral lymphocytes, is observed at later stages during infection with live, but not heat-killed, bacteria. The death of CD4+CD8+ thymocytes is Fas-independent as shown by infection studies with lpr mice. However, apoptosis occurs with lowering of mitochondrial potential and higher caspase-3 activity. The amounts of cortisol, a glucocorticoid, and interferon- (IFN-), an inflammatory cytokine, increase upon infection. To investigate the functional roles of these molecules, studies were performed using Ifn/ mice together with RU486, a glucocorticoid receptor antagonist. Treatment of C57BL/6 mice with RU486 does not affect colony-forming units (CFU), amounts of IFN- and mouse survival; however, there is partial rescue in thymocyte death. Upon infection, Ifn/ mice display higher CFU and lower survival but more surviving thymocytes are recovered. However, there is no difference in cortisol amounts in C57BL/6 and Ifn/ mice. Importantly, the number of CD4+CD8+ thymocytes is significantly higher in Ifn/ mice treated with RU486 along with lower caspase-3 activity and mitochondrial damage. Hence, endogenous glucocorticoid and IFN--mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S.typhimurium infection. The implications of this study for host responses during infection are discussed

Identification of Early Biomarkers during Acetaminophen-Induced Hepatotoxicity by Fourier Transform Infrared Microspectroscopy

<div><p>Acetaminophen is a widely prescribed drug used to relieve pain and fever; however, it is a leading cause of drug-induced liver injury and a burden on public healthcare. In this study, hepatotoxicity in mice post oral dosing of acetaminophen was investigated using liver and sera samples with Fourier Transform Infrared microspectroscopy. The infrared spectra of acetaminophen treated livers in BALB/c mice show decrease in glycogen, increase in amounts of cholesteryl esters and DNA respectively. Rescue experiments using L-methionine demonstrate that depletion in glycogen and increase in DNA are abrogated with pre-treatment, but not post-treatment, with L-methionine. This indicates that changes in glycogen and DNA are more sensitive to the rapid depletion of glutathione. Importantly, analysis of sera identified lowering of glycogen and increase in DNA and chlolesteryl esters earlier than increase in alanine aminotransferase, which is routinely used to diagnose liver damage. In addition, these changes are also observed in C57BL/6 and <em>Nos2</em>^−/− mice. There is no difference in the kinetics of expression of these three molecules in both strains of mice, the extent of damage is similar and corroborated with ALT and histological analysis. Quantification of cytokines in sera showed increase upon APAP treatment. Although the levels of Tnfα and Ifnγ in sera are not significantly affected, <em>Nos2</em>^−/− mice display lower Il6 but higher Il10 levels during this acute model of hepatotoxicity. Overall, this study reinforces the growing potential of Fourier Transform Infrared microspectroscopy as a fast, highly sensitive and label-free technique for non-invasive diagnosis of liver damage. The combination of Fourier Transform Infrared microspectroscopy and cytokine analysis is a powerful tool to identify multiple biomarkers, understand differential host responses and evaluate therapeutic regimens during liver damage and, possibly, other diseases.</p> </div

Biochemical and histological analysis of APAP induced liver damage in BALB/c mice.

<p>Total GSH amounts (A), malonaldehyde amounts (B) and hematoxylin and eosin staining (C) of livers upon APAP treatment in BALB/c mice. Arrows indicate necrotic lesions (C). Each panel represents data from experiments with n = 3 mice.</p

Cytokine analysis in sera of BALB/c mice during APAP-induced liver damage.

<p>Fold changes in the levels of Ifnγ (A), Tnfα (B), Il6 (C) and Il10 (D) with time in APAP treated with respect to untreated mice sera. All the data are shown as mean ± S.E. with n = 5 mice or more.</p

Kinetic analysis of spleen post oral dosing with APAP in mice using FTIR.

<p>Changes in glycogen (1030 cm<b>⁻</b>¹/1080 cm<b>⁻</b>¹; A), cholesteryl ester (1171 cm<b>⁻</b>¹/1152 cm<b>⁻</b>¹; B) and DNA (966 cm<b>⁻</b>¹/996 cm<b>⁻</b>¹; C) in sera from APAP treated BALB/c mice. All the data are represented as mean ± S.E. with n = 3 mice.</p

Band assignments for the IR spectra [26], [27].

<p>Band assignments for the IR spectra <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045521#pone.0045521-Toyran1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045521#pone.0045521-Movasaghi1" target="_blank">[27]</a>.</p