Ameliorated ConA-Induced Hepatitis in the Absence of PKC-theta

Severe liver injury that occurs when immune cells mistakenly attack an individual's own liver cells leads to autoimmune hepatitis. In mice, acute hepatitis can be induced by concanavalin A (ConA) treatment, which causes rapid activation of CD1d-positive natural killer (NK) T cells. These activated NKT cells produce large amounts of cytokines, which induce strong inflammation that damages liver tissues. Here we show that PKC-θ−/− mice were resistant to ConA-induced hepatitis due to essential function of PKC-θ in NKT cell development and activation. A dosage of ConA (25 mg/kg) that was lethal to wild-type (WT) mice failed to induce death resulting from liver injury in PKC-θ−/− mice. Correspondingly, ConA-induced production of cytokines such as IFNγ, IL-6, and TNFα, which mediate the inflammation responsible for liver injury, were significantly lower in PKC-θ−/− mice. Peripheral NKT cells had developmental defects at early stages in the thymus in PKC-θ−/− mice, and as a result their frequency and number were greatly reduced. Furthermore, PKC-θ−/− bone marrow adoptively transferred to WT mice displayed similar defects in NKT cell development, suggesting an intrinsic requirement for PKC-θ in NKT cell development. In addition, upon stimulation with NKT cell-specific lipid ligand, peripheral PKC-θ−/− NKT cells produced lower levels of inflammatory cytokines than that of WT NKT cells, suggesting that activation of NKT cells also requires PKC-θ. Our results suggest PKC-θ is an essential molecule required for activation of NKT cell to induce hepatitis, and thus, is a potential drug target for prevention of autoimmune hepatitis

Enhanced oxidative stress by alcohol use in HIV+ patients: possible involvement of cytochrome P450 2E1 and antioxidant enzymes

BACKGROUND: Alcohol consumption is prevalent amongst HIV positive population. Importantly, chronic alcohol use is reported to exacerbate HIV pathogenesis. Although alcohol is known to increase oxidative stress, especially in the liver, there is no clinical evidence that alcohol increases oxidative stress in HIV positive patients. The mechanism by which alcohol increases oxidative stress in HIV positive patients is also unknown. METHODS: To examine the effects of alcohol use on oxidative stress we recruited HIV+ patients who reported mild-to-moderate alcohol use. Strict inclusion and exclusion criteria were applied to reduce the effect of other therapeutic drugs metabolized via the hepatic system as well as the effect of co-morbidities such as active tuberculosis on the interaction between alcohol and HIV infection, respectively. Blood samples were collected from HIV-negative alcohol-users and HIV positive alcohol-users followed by collection of plasma and isolation and fractionation of monocytes from peripheral blood. We then determined oxidative DNA damage, glutathione level, alcohol level, transcriptional level of cytochrome P450 2E1 (CYP2E1) and several antioxidant enzymes, and plasma level of cytokines. RESULTS: Compared to HIV-negative alcohol users, HIV-positive alcohol users demonstrated an increase in oxidative DNA damage in both plasma and CD14+ monocytes, as well as, a relative increase in oxidized/reduced glutathione (GSSG/GSH) in plasma samples. These results suggest an increase in oxidative stress in HIV-positive alcohol users compared with HIV-negative alcohol users. We also examined whether alcohol metabolism, perhaps by CYP2E1, and antioxidant enzymes are involved in alcohol-mediated increased oxidative stress in HIV-positive patients. The results showed a lower plasma alcohol level, which was associated with an increased level of CYP2E1 mRNA in monocytes, in HIV-positive alcohol users compared with HIV-negative alcohol users. Furthermore, the transcription of major antioxidants enzymes (catalase, SOD1, SOD2, GSTK1), and their transcription factor, Nrf2, were reduced in monocytes obtained from HIV positive alcohol users compared to the HIV-negative alcohol user group. However, no significant change in levels of five major cytokines/chemokines were observed between the two groups. CONCLUSIONS: The data suggests that alcohol increases oxidative stress in HIV+ patients, perhaps through CYP2E1- and antioxidant enzymes-mediated pathways. The enhanced oxidative stress is accompanied by a failure of cellular antioxidant mechanisms to maintain redox homeostasis. Overall, the enhanced oxidative stress in monocytes may exacerbate HIV pathogenesis in HIV positive alcohol users

HIV Protein Sequence Hotspots for Crosstalk with Host Hub Proteins

HIV proteins target host hub proteins for transient binding interactions. The presence of viral proteins in the infected cell results in out-competition of host proteins in their interaction with hub proteins, drastically affecting cell physiology. Functional genomics and interactome datasets can be used to quantify the sequence hotspots on the HIV proteome mediating interactions with host hub proteins. In this study, we used the HIV and human interactome databases to identify HIV targeted host hub proteins and their host binding partners (H2). We developed a high throughput computational procedure utilizing motif discovery algorithms on sets of protein sequences, including sequences of HIV and H2 proteins. We identified as HIV sequence hotspots those linear motifs that are highly conserved on HIV sequences and at the same time have a statistically enriched presence on the sequences of H2 proteins. The HIV protein motifs discovered in this study are expressed by subsets of H2 host proteins potentially outcompeted by HIV proteins. A large subset of these motifs is involved in cleavage, nuclear localization, phosphorylation, and transcription factor binding events. Many such motifs are clustered on an HIV sequence in the form of hotspots. The sequential positions of these hotspots are consistent with the curated literature on phenotype altering residue mutations, as well as with existing binding site data. The hotspot map produced in this study is the first global portrayal of HIV motifs involved in altering the host protein network at highly connected hub nodes

PKN1 Exerts Neurodegenerative Effects in an In Vitro Model of Cerebellar Hypoxic–Ischemic Encephalopathy via Inhibition of AKT/GSK3β Signaling

We recently identified protein kinase N1 (PKN1) as a negative gatekeeper of neuronal AKT protein kinase activity during postnatal cerebellar development. The developing cerebellum is specifically vulnerable to hypoxia-ischemia (HI), as it occurs during hypoxic-ischemic encephalopathy, a condition typically caused by oxygen deprivation during or shortly after birth. In that context, activation of the AKT cell survival pathway has emerged as a promising new target for neuroprotective interventions. Here, we investigated the role of PKN1 in an in vitro model of HI, using postnatal cerebellar granule cells (Cgc) derived from Pkn1 wildtype and Pkn1−/− mice. Pkn1−/− Cgc showed significantly higher AKT phosphorylation, resulting in reduced caspase-3 activation and improved survival after HI. Pkn1−/− Cgc also showed enhanced axonal outgrowth on growth-inhibitory glial scar substrates, further pointing towards a protective phenotype of Pkn1 knockout after HI. The specific PKN1 phosphorylation site S374 was functionally relevant for the enhanced axonal outgrowth and AKT interaction. Additionally, PKN1pS374 shows a steep decrease during cerebellar development. In summary, we demonstrate the pathological relevance of the PKN1-AKT interaction in an in vitro HI model and establish the relevant PKN1 phosphorylation sites, contributing important information towards the development of specific PKN1 inhibitors