Lupus exacerbation in ovalbumin-induced asthma in Fc gamma receptor IIb deficient mice, partly due to hyperfunction of dendritic cells.

BACKGROUND: Although allergy might be another factor that exacerbates lupus as demonstrated by several epidemiologic studies, the direct correlation between lupus activities and allergy is still in question. OBJECTIVE: To explore the correlation between allergic reaction and lupus activities. METHODS: The allergic asthma model using ovalbumin (OVA) administration in wildtype (WT) and Fc gamma receptor IIb deficient (FcgRIIb-/-) mice (a lupus-prone model) together with in vitro experiments on bone marrow-derived dendritic cells (DCs) were performed. RESULTS: At 2-weeks-post OVA, both WT and FcgRIIb-/- mice demonstrated similar allergic reaction as indicated by an elevation of IgE and IL-4 in serum with asthma-liked lung histology (lung weight, inflammatory score, and bronchial thickness) with increased spleen weight. Apoptosis in the lungs and spleens (activated caspase 3 immunohistochemistry) was detected only in OVA-administered FcgRIIb-/- mice. Surprisingly, OVA-administered FcgRIIb-/- mice, demonstrated active lupus nephritis, as indicated by anti-dsDNA, proteinuria, and renal immune complex deposition (immunohistochemistry analysis) implying an impact of allergy on lupus activities. Meanwhile, serum creatinine and gut permeability defect (FitC-dextran assay and endotoxemia) were not different between the FcgRIIb-/- mice with OVA versus with control. In parallel, FcgRIIb-/- DCs were more susceptible to activations by OVA and lipopolysaccharide (LPS) than WT DCs as demonstrated by CD80 with major histocompatibility complex II (MHC II) using flow cytometry analysis. CONCLUSION: OVA-induced allergy in FcgRIIb-/- mice exacerbated lupus activity, possibly due to hyper-responsiveness of FcgRIIb-/- DCs over WT from the loss of inhibitory FcgRIIb. The proper control of allergy might be beneficial for lupus.

Proteomic profile of naturally released extracellular vesicles secreted from Leptospira interrogans serovar Pomona in response to temperature and osmotic stresses.

Bacterial extracellular vesicles (EVs) are generally formed by pinching off outer membrane leaflets while simultaneously releasing multiple active molecules into the external environment. In this study, we aimed to identify the protein cargo of leptospiral EVs released from intact leptospires grown under three different conditions: EMJH medium at 30 °C, temperature shifted to 37 °C, and physiologic osmolarity (EMJH medium with 120 mM NaCl). The naturally released EVs observed under transmission electron microscopy were spherical in shape with an approximate diameter of 80-100 nm. Quantitative proteomics and bioinformatic analysis indicated that the EVs were formed primarily from the outer membrane and the cytoplasm. The main functional COG categories of proteins carried in leptospiral EVs might be involved in cell growth, survival and adaptation, and pathogenicity. Relative to their abundance in EVs grown in EMJH medium at 30 °C, 39 and 69 proteins exhibited significant changes in response to the temperature shift and the osmotic change, respectively. During exposure to both stresses, Leptospira secreted several multifunctional proteins via EVs, while preserving certain virulence proteins within whole cells. Therefore, leptospiral EVs may serve as a decoy structure for host responses, whereas some virulence factors necessary for direct interaction with the host environment are reserved in leptospiral cells. This knowledge will be useful for understanding the pathogenesis of leptospirosis and developing as one of vaccine platforms against leptospirosis in the future.

Less Severe Lipopolysaccharide-Induced Inflammation in Conditional mgmt-Deleted Mice with LysM-Cre System: The Loss of DNA Repair in Macrophages.

Despite the known influence of DNA methylation from lipopolysaccharide (LPS) activation, data on the O6-methylguanine-DNA methyltransferase (MGMT, a DNA suicide repair enzyme) in macrophages is still lacking. The transcriptomic profiling of epigenetic enzymes from wild-type macrophages after single and double LPS stimulation, representing acute inflammation and LPS tolerance, respectively, was performed. Small interfering RNA (siRNA) silencing of mgmt in the macrophage cell line (RAW264.7) and mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated lower secretion of TNF-α and IL-6 and lower expression of pro-inflammatory genes (iNOS and IL-1ß) compared with the control. Macrophage injury after a single LPS dose and LPS tolerance was demonstrated by reduced cell viability and increased oxidative stress (dihydroethidium) compared with the activated macrophages from littermate control mice (mgmtflox/flox; LysM-Cre-/-). Additionally, a single LPS dose and LPS tolerance also caused mitochondrial toxicity, as indicated by reduced maximal respiratory capacity (extracellular flux analysis) in the macrophages of both mgmt null and control mice. However, LPS upregulated mgmt only in LPS-tolerant macrophages but not after the single LPS stimulation. In mice, the mgmt null group demonstrated lower serum TNF-α, IL-6, and IL-10 than control mice after either single or double LPS stimulation. Suppressed cytokine production resulting from an absence of mgmt in macrophages caused less severe LPS-induced inflammation but might worsen LPS tolerance.

Less Severe Polymicrobial Sepsis in Conditional mgmt-Deleted Mice Using LysM-Cre System, Impacts of DNA Methylation and MGMT Inhibitor in Sepsis.

The O6-methylguanine-DNA methyltransferase (MGMT) is a DNA suicide repair enzyme that might be important during sepsis but has never been explored. Then, the proteomic analysis of lipopolysaccharide (LPS)-stimulated wild-type (WT) macrophages increased proteasome proteins and reduced oxidative phosphorylation proteins compared with control, possibly related to cell injury. With LPS stimulation, mgmt null (mgmtflox/flox; LysM-Crecre/-) macrophages demonstrated less profound inflammation; supernatant cytokines (TNF-α, IL-6, and IL-10) and pro-inflammatory genes (iNOS and IL-1ß), with higher DNA break (phosphohistone H2AX) and cell-free DNA, but not malondialdehyde (the oxidative stress), compared with the littermate control (mgmtflox/flox; LysM-Cre-/-). In parallel, mgmt null mice (MGMT loss only in the myeloid cells) demonstrated less severe sepsis in the cecal ligation and puncture (CLP) model (with antibiotics), as indicated by survival and other parameters compared with sepsis in the littermate control. The mgmt null protective effect was lost in CLP mice without antibiotics, highlighting the importance of microbial control during sepsis immune modulation. However, an MGMT inhibitor in CLP with antibiotics in WT mice attenuated serum cytokines but not mortality, requiring further studies. In conclusion, an absence of mgmt in macrophages resulted in less severe CLP sepsis, implying a possible influence of guanine DNA methylation and repair in macrophages during sepsis.

Less Severe Sepsis in Cecal Ligation and Puncture Models with and without Lipopolysaccharide in Mice with Conditional Ezh2-Deleted Macrophages (LysM-Cre System).

Despite a previous report on less inflammatory responses in mice with an absence of the enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase of epigenetic regulation, using a lipopolysaccharide (LPS) injection model, proteomic analysis and cecal ligation and puncture (CLP), a sepsis model that more resembles human conditions was devised. As such, analysis of cellular and secreted protein (proteome and secretome) after a single LPS activation and LPS tolerance in macrophages from Ezh2 null (Ezh2flox/flox; LysM-Crecre/-) mice (Ezh2 null) and the littermate control mice (Ezh2fl/fl; LysM-Cre-/-) (Ezh2 control) compared with the unstimulated cells from each group indicated fewer activities in Ezh2 null macrophages, especially by the volcano plot analysis. Indeed, supernatant IL-1ß and expression of genes in pro-inflammatory M1 macrophage polarization (IL-1ß and iNOS), TNF-α, and NF-κB (a transcription factor) were lower in Ezh2 null macrophages compared with the control. In LPS tolerance, downregulated NF-κB compared with the control was also demonstrated in Ezh2 null cells. In CLP sepsis mice, those with CLP alone and CLP at 2 days after twice receiving LPS injection, representing sepsis and sepsis after endotoxemia, respectively, symptoms were less severe in Ezh2 null mice, as indicated by survival analysis and other biomarkers. However, the Ezh2 inhibitor improved survival only in CLP, but not LPS with CLP. In conclusion, an absence of Ezh2 in macrophages resulted in less severe sepsis, and the use of an Ezh2 inhibitor might be beneficial in sepsis.

Lipopolysaccharide Tolerance Enhances Murine Norovirus Reactivation: An Impact of Macrophages Mainly Evaluated by Proteomic Analysis.

Because of endotoxemia during sepsis (a severe life-threatening infection), lipopolysaccharide (LPS) tolerance (the reduced responses to the repeated LPS stimulation) might be one of the causes of sepsis-induced immune exhaustion (the increased susceptibility to secondary infection and/or viral reactivation). In LPS tolerance macrophage (twice-stimulated LPS, LPS/LPS) compared with a single LPS stimulation (N/LPS), there was (i) reduced energy of the cell in both glycolysis and mitochondrial activities (extracellular flux analysis), (ii) decreased abundance of the following proteins (proteomic analysis): (a) complex I and II of the mitochondrial electron transport chain, (b) most of the glycolysis enzymes, (c) anti-viral responses with Myxovirus resistance protein 1 (Mx1) and Ubiquitin-like protein ISG15 (Isg15), (d) antigen presentation pathways, and (iii) the down-regulated anti-viral genes, such as Mx1 and Isg15 (polymerase chain reaction). To test the correlation between LPS tolerance and viral reactivation, asymptomatic mice with and without murine norovirus (MNV) infection as determined in feces were tested. In MNV-positive mice, MNV abundance in the cecum, but not in feces, of LPS/LPS mice was higher than that in N/LPS and control groups, while MNV abundance of N/LPS and control were similar. Additionally, the down-regulated Mx1 and Isg15 were also demonstrated in the cecum, liver, and spleen in LPS/LPS-activated mice, regardless of MNV infection, while N/LPS more prominently upregulated these genes in the cecum of MNV-positive mice compared with the MNV-negative group. In conclusion, defects in anti-viral responses after LPS tolerance, perhaps through the reduced energy status of macrophages, might partly be responsible for the viral reactivation. More studies on patients are of interest.

Chlorhexidine Promotes Psl Expression in Pseudomonas aeruginosa That Enhances Cell Aggregation with Preserved Pathogenicity Demonstrates an Adaptation against Antiseptic.

Because Pseudomonas aeruginosa is frequently in contact with Chlorhexidine (a regular antiseptic), bacterial adaptations are possible. In comparison with the parent strain, the Chlorhexidine-adapted strain formed smaller colonies with metabolic downregulation (proteomic analysis) with the cross-resistance against colistin (an antibiotic for several antibiotic-resistant bacteria), partly through the modification of L-Ara4N in the lipopolysaccharide at the outer membrane. Chlorhexidine-adapted strain formed dense liquid-solid interface biofilms with enhanced cell aggregation partly due to the Chlorhexidine-induced overexpression of psl (exopolysaccharide-encoded gene) through the LadS/GacSA pathway (c-di-GMP-independence) in 12 h biofilms and maintained the aggregation with SiaD-mediated c-di-GMP dependence in 24 h biofilms as evaluated by polymerase chain reaction (PCR). The addition of Ca2+ in the Chlorhexidine-adapted strain facilitated several Psl-associated genes, indicating an impact of Ca2+ in Psl production. The activation by Chlorhexidine-treated sessile bacteria demonstrated a lower expression of IL-6 and IL-8 on fibroblasts and macrophages than the activation by the parent strain, indicating the less inflammatory reactions from Chlorhexidine-exposed bacteria. However, the 14-day severity of the wounds in mouse caused by Chlorhexidine-treated bacteria versus the parent strain was similar, as indicated by wound diameters and bacterial burdens. In conclusion, Chlorhexidine induced psl over-expression and colistin cross-resistance that might be clinically important.

The Acquired Vulnerability Caused by CDK4/6 Inhibition Promotes Drug Synergism Between Oxaliplatin and Palbociclib in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult to treat cancers, and its nature of being largely refractory to most, if not all, current treatments results in generally poor prognosis and high mortality. Efficacious alternative therapies that can be used ubiquitously are urgently needed. Using acquired vulnerability screening, we observed that CCA cells that reprofile and proliferate under CDK4/6 inhibition became vulnerable to ribosomal biogenesis stress and hypersensitive to the anti-ribosome chemotherapy oxaliplatin. CCA cells overexpress the oncogenic ribosomal protein RPL29 under CDK4/6 inhibition in a manner that correlated with CDK4/6 inhibitor resistance. Depletion of RPL29 by small interfering RNAs (siRNAs) restored the sensitivity of CCA cells to CDK4/6 inhibition. Oxaliplatin treatment suppressed the RPL29 expression in the CDK4/6 inhibitor treated CCA cells and triggered RPL5/11-MDM2-dependent p53 activation and cancer apoptosis. In addition, we found that combination treatment with oxaliplatin and the CDK4/6 inhibitor palbociclib synergistically inhibited both parental and CDK4/6 inhibitor-resistant CCA, and prevented the emergence of CDK4/6 and oxaliplatin-resistant CCA. This drug combination also exerted suppressive and apoptosis effects on CCA in the in vitro 3-dimensional culture, patient-derived organoid, and in vivo xenograft CCA models. These results suggest the combination of the CDK4/6 inhibitor palbociclib and the anti-ribosome drug oxaliplatin as a potentially promising treatment for cholangiocarcinoma.

Enhanced Bacteremia in Dextran Sulfate-Induced Colitis in Splenectomy Mice Correlates with Gut Dysbiosis and LPS Tolerance.

Because both endotoxemia and gut dysbiosis post-splenectomy might be associated with systemic infection, the susceptibility against infection was tested by dextran sulfate solution (DSS)-induced colitis and lipopolysaccharide (LPS) injection models in splenectomy mice with macrophage experiments. Here, splenectomy induced a gut barrier defect (FITC-dextran assay, endotoxemia, bacteria in mesenteric lymph nodes, and the loss of enterocyte tight junction) and gut dysbiosis (increased Proteobacteria by fecal microbiome analysis) without systemic inflammation (serum IL-6). In parallel, DSS induced more severe mucositis in splenectomy mice than sham-DSS mice, as indicated by mortality, stool consistency, gut barrier defect, serum cytokines, and blood bacterial burdens. The presence of green fluorescent-producing (GFP) E. coli in the spleen of sham-DSS mice after an oral gavage supported a crucial role of the spleen in the control of bacteria from gut translocation. Additionally, LPS administration in splenectomy mice induced lower serum cytokines (TNF-α and IL-6) than LPS-administered sham mice, perhaps due to LPS tolerance from pre-existing post-splenectomy endotoxemia. In macrophages, LPS tolerance (sequential LPS stimulation) demonstrated lower cell activities than the single LPS stimulation, as indicated by the reduction in supernatant cytokines, pro-inflammatory genes (iNOS and IL-1ß), cell energy status (extracellular flux analysis), and enzymes of the glycolysis pathway (proteomic analysis). In conclusion, a gut barrier defect after splenectomy was vulnerable to enterocyte injury (such as DSS), which caused severe bacteremia due to defects in microbial control (asplenia) and endotoxemia-induced LPS tolerance. Hence, gut dysbiosis and gut bacterial translocation in patients with a splenectomy might be associated with systemic infection, and gut-barrier monitoring or intestinal tight-junction strengthening may be useful.

Surface proteomics and label-free quantification of Leptospira interrogans serovar Pomona.

Leptospirosis is a re-emerging zoonosis with a global distribution. Surface-exposed outer membrane proteins (SE-OMPs) are crucial for bacterial-host interactions. SE-OMPs locate and expose their epitope on cell surface where is easily accessed by host molecules. This study aimed to screen for surface-exposed proteins and their abundance profile of pathogenic Leptospira interrogans serovar Pomona. Two complementary approaches, surface biotinylation and surface proteolytic shaving, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) were employed to identify SE-OMPs of intact leptospires. For quantitative comparison, in-depth label-free analysis of SE-OMPs obtained from each method was performed using MaxQuant. The total number of proteins identified was 1,001 and 238 for surface biotinylation and proteinase K shaving, respectively. Among these, 39 were previously known SE-OMPs and 68 were predicted to be localized on the leptospiral surface. Based on MaxQuant analysis for relative quantification, six known SE-OMPs including EF- Tu, LipL21, LipL41, LipL46, Loa22, and OmpL36, and one predicted SE-OMPs, LipL71 were found in the 20 most abundant proteins, in which LipL41 was the highest abundant SE-OMP. Moreover, uncharacterized LIC14011 protein (LIP3228 ortholog in serovar Pomona) was identified as a novel predicted surface ßb-OMP. High-abundance leptospiral SE-OMPs identified in this study may play roles in virulence and infection and are potential targets for development of vaccine or diagnostic tests for leptospirosis.

A Comparison Between 1 Day versus 7 Days of Sepsis in Mice with the Experiments on LPS-Activated Macrophages Support the Use of Intravenous Immunoglobulin for Sepsis Attenuation.

BACKGROUND: Because survival and death after sepsis are partly due to a proper immune adaptation and immune dysregulation, respectively, survivors and moribund mice after cecal ligation and puncture (CLP) sepsis surgery and in vitro macrophage experiments were explored. METHODS: Characteristics of mice at 1-day and 7-days post-CLP, the representative of moribund mice (an innate immune hyper-responsiveness) and survivors (a successful control on innate immunity), respectively. In parallel, soluble heat aggregated immunoglobulin (sHA-Ig), a representative of immune complex, was tested in lipopolysaccharide (LPS)-activated macrophages together with a test of intravenous immunoglobulin (IVIG), a molecule of adaptive immunity, on CLP sepsis mice. RESULTS: Except for a slight increase in alanine transaminase (liver injury), IL-10, endotoxemia, and gut leakage (FITC-dextran assay), most of the parameters in survivors (7-days post-CLP) were normalized, with enhanced adaptive immunity, including serum immunoglobulin (using serum protein electrophoresis) and activated immune cells in spleens (flow cytometry analysis). The addition of sHA-Ig in LPS-activated macrophages reduced supernatant cytokines, cell energy (extracellular flux analysis), reactive oxygen species (ROS), several cell activities (proteomic analysis), and Fc gamma receptors (FcgRs) expression. The loss of anti-inflammatory effect of sHA-Ig in LPS-activated macrophages from mice with a deficiency on Fc gamma receptor IIb (FcgRIIb-/-), the only inhibitory signaling of FcgRs family, when compared with wild-type macrophages, implying the FcgRIIb-dependent mechanism. Moreover, IVIG attenuated sepsis severity in CLP mice as evaluated by serum creatinine, liver enzyme (alanine transaminase), serum cytokines, spleen apoptosis, and abundance of dendritic cells in the spleen (24-h post-CLP) and survival analysis. CONCLUSION: Immunoglobulin attenuated LPS-activated macrophages, partly, through the reduced cell energy of macrophages and might play a role in sepsis immune hyper-responsiveness. Despite the debate over IVIG's use in sepsis, IVIG might be beneficial in sepsis with certain conditions.

Erratum: STING Mediates Lupus via the Activation of Conventional Dendritic Cell Maturation and Plasmacytoid Dendritic Cell Differentiation.

[This corrects the article DOI: 10.1016/j.isci.2020.101530.].

Systems biology analysis of osteogenic differentiation behavior by canine mesenchymal stem cells derived from bone marrow and dental pulp.

Utilization of canine mesenchymal stem cells (cMSCs) for regenerating incorrigible bone diseases has been introduced. However, cMSCs harvested from different sources showed distinct osteogenicity. To clarify this, comparative proteomics-based systems biology analysis was used to analyze osteogenic differentiation behavior by cMSCs harvested from bone marrow and dental pulp. The results illustrated that canine dental pulp stem cells (cDPSCs) contained superior osteogenicity comparing with canine bone marrow-derived MSCs (cBM-MSCs) regarding alkaline phosphatase activity, matrix mineralization, and osteogenic marker expression. Global analyses by proteomics platform showed distinct protein clustering and expression pattern upon an in vitro osteogenic induction between them. Database annotation using Reactome and DAVID revealed contrast and unique expression profile of osteogenesis-related proteins, particularly on signaling pathways, cellular components and processes, and cellular metabolisms. Functional assay and hierarchical clustering for tracking protein dynamic change confirmed that cBM-MSCs required the presences of Wnt, transforming growth factor (TGF)-beta, and bone-morphogenetic protein (BMP) signaling, while cDPSCs mainly relied on BMP signaling presentation during osteogenic differentiation in vitro. Therefore, these findings illustrated the comprehensive data regarding an in vitro osteogenic differentiation behavior by cBM-MSCs and cDPSCs which is crucial for further mechanism study and the establishment of cMSC-based bone tissue engineering (BTE) for veterinary practice.

STING Mediates Lupus via the Activation of Conventional Dendritic Cell Maturation and Plasmacytoid Dendritic Cell Differentiation.

Signaling through stimulator of interferon genes (STING) leads to the production of type I interferons (IFN-Is) and inflammatory cytokines. A gain-of-function mutation in STING was identified in an autoinflammatory disease (STING-associated vasculopathy with onset in infancy; SAVI). The expression of cyclic GMP-AMP, DNA-activated cGAS-STING pathway, increased in a proportion of patients with SLE. The STING signaling pathway may be a candidate for targeted therapy in SLE. Here, we demonstrated that disruption of STING signaling ameliorated lupus development in Fcgr2b-deficient mice. Activation of STING promoted maturation of conventional dendritic cells and differentiation of plasmacytoid dendritic cells via LYN interaction and phosphorylation. The inhibition of LYN decreased the differentiation of STING-activated dendritic cells. Adoptive transfer of STING-activated bone marrow-derived dendritic cells into the FCGR2B and STING double-deficiency mice restored lupus phenotypes. These findings provide evidence that the inhibition of STING signaling may be a candidate targeted treatment for a subset of patients with SLE.

Deep Proteomic Deconvolution of Interferons and HBV Transfection Effects on a Hepatoblastoma Cell Line.

Interferons are commonly utilized in the treatment of chronic hepatitis B virus (HBV) infection but are not effective for all patients. A deep understanding of the limitations of interferon treatment requires delineation of its activity at multiple "omic" levels. While myriad studies have characterized the transcriptomic effects of interferon treatment, surprisingly, few have examined interferon-induced effects at the proteomic level. To remedy this paucity, we stimulated HepG2 cells with both IFN-α and IFN-λ and performed proteomic analysis versus unstimulated cells. Alongside, we examined the effects of HBV transfection in the same cell line, reasoning that parallel IFN and HBV analysis might allow determination of cases where HBV transfection counters the effects of interferons. More than 6000 proteins were identified, with multiple replicates allowing for differential expression analysis at high confidence. Drawing on a compendium of transcriptomic data, as well as proteomic half-life data, we suggest means by which transcriptomic results diverge from our proteomic results. We also invoke a recent multiomic study of HBV-related hepatocarcinoma (HCC), showing that despite HBV's role in initiating HCC, the regulated proteomic landscapes of HBV transfection and HCC do not strongly align. Special focus is applied to the proteasome, with numerous components divergently altered under IFN and HBV-transfection conditions. We also examine alterations of other protein groups relevant to HLA complex peptide display, unveiling intriguing alterations in a number of ubiquitin ligases. Finally, we invoke genome-scale metabolic modeling to predict relevant alterations to the metabolic landscape under experimental conditions. Our data should be useful as a resource for interferon and HBV researchers.

Deficiency of STING Promotes Collagen-Specific Antibody Production and B Cell Survival in Collagen-Induced Arthritis.

The levels of interferon-alpha are high in the serum and synovial fluid of rheumatoid arthritis (RA) patients. Activation of the stimulator of type I interferon genes (STING) mediates the productions of type I interferon and promotes chronic inflammation. STING plays a significant role in autoimmune lupus mice. However, the function of STING in collagen-induced arthritis (CIA) model has never been described. This study aimed to test the function of STING in CIA. The Sting-deficient mice developed arthritis comparable to WT mice. The levels of anti-collagen antibody from Sting-deficient mice were significantly higher than the WT mice. The B cells derived from Sting-deficient mice showed better survival than WT mice in response to the B cell receptor (BCR) stimulation. Activation of STING also induced B cell death, especially in activated B cells. This study demonstrated that the inhibition of STING promotes anti-collagen antibodies and B cell survival, which suggested that STING acts as a negative regulator of B cell function in the CIA model.

Increased susceptibility against Cryptococcus neoformans of lupus mouse models (pristane-induction and FcGRIIb deficiency) is associated with activated macrophage, regardless of genetic background.

The severity of cryptococcosis in lupus from varying genetic-backgrounds might be different due to the heterogeneity of lupus-pathogenesis. This study explored cryptococcosis in lupus mouse models of pristane-induction (normal genetic-background) and FcGRIIb deficiency (genetic defect). Because the severity of lupus nephritis, as determined by proteinuria and serum creatinine, between pristane and FcGRIIb-/- mice were similar at 6-month-old, Cryptococcus neoformans was intravenously administered in 6-month-old mice and were age-matched with wild-type. Indeed, the cryptococcosis disease severity, as evaluated by mortality rate, internal-organ fungal burdens and serum cytokines, between pristane and FcGRIIb-/- mice was not different. However, the severity of cryptococcosis in wild-type was less severe than the lupus mice. On the other hand, phagocytosis activity of peritoneal macrophages from lupus mice (pristane and FcGRIIb-/-) was more predominant than the wild-type without the difference in macrophage killing-activity among these groups. In addition, the number of active T helper cells (Th-cell) in the spleen, including Th-cells with intracellular IFN-γ, from lupus mice (pristane and FcGRIIb-/-) was higher than wildtype. Moreover, these active Th-cells were even higher after 2 weeks of cryptococcal infection. These data support enhanced macrophage activation through prominent Th-cells in both lupus models. In conclusion, an increased susceptibility of cryptococcosis in both lupus models was independent to genetic background. This might due to Th-cell enhanced macrophage phagocytosis with the interference of macrophage killing activity from Cryptococcal immune-evasion properties.

Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells.

Interferon lambda (IFN-λ) is a relatively unexplored, yet promising antiviral agent. IFN-λ has recently been tested in clinical trials of chronic hepatitis B virus infection (CHB), with the advantage that side effects may be limited compared with IFN-α, as IFN-λ receptors are found only in epithelial cells. To date, IFN-λ's downstream signaling pathway remains largely unelucidated, particularly via proteomics methods. Here, we report that IFN-λ3 inhibits HBV replication in HepG2.2.15 cells, reducing levels of both HBV transcripts and intracellular HBV DNA. Quantitative proteomic analysis of HBV-transfected cells was performed following 24-hour IFN-λ3 treatment, with parallel IFN-α2a and PBS treatments for comparison using a dimethyl labeling method. The depth of the study allowed us to map the induction of antiviral proteins to multiple points of the viral life cycle, as well as facilitating the identification of antiviral proteins not previously known to be elicited upon HBV infection (e.g. IFITM3, XRN2, and NT5C3A). This study also shows up-regulation of many effectors involved in antigen processing/presentation indicating that this cytokine exerted immunomodulatory effects through several essential molecules for these processes. Interestingly, the 2 subunits of the immunoproteasome cap (PSME1 and PSME2) were up-regulated whereas cap components of the constitutive proteasome were down-regulated upon both IFN treatments, suggesting coordinated modulation toward the antigen processing/presentation mode. Furthermore, in addition to confirming canonical activation of interferon-stimulated gene (ISG) transcription through the JAK-STAT pathway, we reveal that IFN-λ3 restored levels of RIG-I and RIG-G, proteins known to be suppressed by HBV. Enrichment analysis demonstrated that several biological processes including RNA metabolism, translation, and ER-targeting were differentially regulated upon treatment with IFN-λ3 versus IFN-α2a. Our proteomic data suggests that IFN-λ3 regulates an array of cellular processes to control HBV replication.

Helicobacter pylori Infection Increased Anti-dsDNA and Enhanced Lupus Severity in Symptomatic FcγRIIb-Deficient Lupus Mice.

The defect on Fc gamma receptor IIb (FcγRIIb), the only inhibitory FcγR, has been identified as one of the genetic factors increasing susceptibility to lupus. The prevalence of Helicobacter pylori (HP) and FcγRIIb dysfunction-polymorphisms are high among Asians, and their co-existence is possible. Unfortunately, the influence of HP against lupus progression in patients with lupus is still controversial. In this study, the interactions between these conditions were tested with HP infection in 24-week-old FcγRIIb-/- mice (symptomatic lupus). HP induced failure to thrive, increased stomach bacterial burdens and stomach injury (histology and cytokines) in both wild-type and FcγRIIb-/- mice. While the severity of HP infection, as determined by these parameters, was not different between both strains, antibodies production (anti-HP, anti-dsDNA and serum gammaglobulin) were higher in FcγRIIb-/- mice compared to wild-type. Accordingly, HP infection also accelerated the severity of lupus as determined by proteinuria, serum creatinine, serum cytokines, renal histology, and renal immune complex deposition. Although HP increased serum cytokines in both wild-type and FcγRIIb-/- mice, the levels were higher in FcγRIIb-/- mice. As such, HP also increased spleen weight and induced several splenic immune cells responsible for antibody productions (activated B cell, plasma cell and follicular helper T cell) in FcγRIIb-/- mice, but not in wild-type. These data describe the different systemic responses against localized HP infection from diverse host genetic background. In conclusion, the mutual interactions between HP and lupus manifestations of FcγRIIb-/-mice were demonstrated in this study. With the prominent immune responses from the loss of inhibitory signaling in FcγRIIb-/- mice, HP infection in these mice induced intense chronic inflammation, increased antibody production, and enhanced lupus severity. Thus, the increased systemic inflammatory responses due to localized HP inducing gastritis in some patients with lupus may enhance lupus progression. More studies are needed.