Insights into gut microbiomes in stem cell transplantation by comprehensive shotgun long-read sequencing.

The gut microbiome is a diverse ecosystem, dominated by bacteria; however, fungi, phages/viruses, archaea, and protozoa are also important members of the gut microbiota. Exploration of taxonomic compositions beyond bacteria as well as an understanding of the interaction between the bacteriome with the other members is limited using 16S rDNA sequencing. Here, we developed a pipeline enabling the simultaneous interrogation of the gut microbiome (bacteriome, mycobiome, archaeome, eukaryome, DNA virome) and of antibiotic resistance genes based on optimized long-read shotgun metagenomics protocols and custom bioinformatics. Using our pipeline we investigated the longitudinal composition of the gut microbiome in an exploratory clinical study in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT; n = 31). Pre-transplantation microbiomes exhibited a 3-cluster structure, characterized by Bacteroides spp. /Phocaeicola spp., mixed composition and Enterococcus abundances. We revealed substantial inter-individual and temporal variabilities of microbial domain compositions, human DNA, and antibiotic resistance genes during the course of alloHSCT. Interestingly, viruses and fungi accounted for substantial proportions of microbiome content in individual samples. In the course of HSCT, bacterial strains were stable or newly acquired. Our results demonstrate the disruptive potential of alloHSCTon the gut microbiome and pave the way for future comprehensive microbiome studies based on long-read metagenomics.

Modular Approach for the Synthesis and Bioactivity Profiling of 8,8'-Biflavones.

In this work, we report the scalable and modular synthesis of a library of 55 monomeric and dimeric flavonoids including 14 8,8'-biflavones. The sterically demanding tetra-ortho-substituted axis of an acetophenone dimer key intermediate was constructed in a regioselective manner using Fe-mediated oxidative coupling. This step was systematically optimized and performed on up to multigram scale. The biological activities of this compound library were evaluated, including cytotoxicity against healthy and malignant human cell lines, antimicrobial activity against the apicomplexan parasite Toxoplasma gondii, and antioxidant capacity. A marked increase in activity for the 8,8'-dimeric structures compared to that of their monomeric counterparts was observed. Several biflavones were identified with high selectivity indices (low cytotoxicity and high antiprotozoal activity), showing that this class of natural products may serve as lead structures for further investigations.

Macrophages inhibit Coxiella burnetii by the ACOD1-itaconate pathway for containment of Q fever.

Infection with the intracellular bacterium Coxiella (C.) burnetii can cause chronic Q fever with severe complications and limited treatment options. Here, we identify the enzyme cis-aconitate decarboxylase 1 (ACOD1 or IRG1) and its product itaconate as protective host immune pathway in Q fever. Infection of mice with C. burnetii induced expression of several anti-microbial candidate genes, including Acod1. In macrophages, Acod1 was essential for restricting C. burnetii replication, while other antimicrobial pathways were dispensable. Intratracheal or intraperitoneal infection of Acod1-/- mice caused increased C. burnetii burden, weight loss and stronger inflammatory gene expression. Exogenously added itaconate restored pathogen control in Acod1-/- mouse macrophages and blocked replication in human macrophages. In axenic cultures, itaconate directly inhibited growth of C. burnetii. Finally, treatment of infected Acod1-/- mice with itaconate efficiently reduced the tissue pathogen load. Thus, ACOD1-derived itaconate is a key factor in the macrophage-mediated defense against C. burnetii and may be exploited for novel therapeutic approaches in chronic Q fever.

Indole Diketopiperazine Alkaloids from the Marine Sediment-Derived Fungus Aspergillus chevalieri against Pancreatic Ductal Adenocarcinoma.

A new prenylated indole diketopiperazine alkaloid, rubrumline P (1), was isolated along with six more analogues and characterized from the fermentation culture of a marine sediment-derived fungus, Aspergillus chevalieri, collected at a depth of 15 m near the lighthouse in Dahab, Red Sea, Egypt. In the current study, a bioassay-guided fractionation allowed for the identification of an active fraction displaying significant cytotoxic activity against the human pancreatic adenocarcinoma cell line PANC-1 from the EtOAc extract of the investigated fungus compared to the standard paclitaxel. The structures of the isolated compounds from the active fraction were established using 1D/2D NMR spectroscopy and mass spectrometry, together with comparisons with the literature. The absolute configuration of the obtained indole diketopiperazines was established based on single-crystal X-ray diffraction analyses of rubrumline I (2) and comparisons of optical rotations and NMR data, as well as on biogenetic considerations. Genome sequencing indicated the formation of prenyltransferases, which was subsequently confirmed by the isolation of mono-, di-, tri-, and tetraprenylated compounds. Compounds rubrumline P (1) and neoechinulin D (4) confirmed preferential cytotoxic activity against PANC-1 cancer cells with IC50 values of 25.8 and 23.4 µM, respectively. Although the underlying mechanism-of-action remains elusive in this study, cell cycle analysis indicated a slight increase in the sub-G1 peak after treatment with compounds 1 and 4.

Characterization of the cagA-gene in Helicobacter pylori in Mongolia and detection of two EPIYA-A enriched CagA types.

Helicobacter pylori infection is strongly associated with gastritis, gastroduodenal ulcer disease and gastric carcinoma. The virulence of H. pylori strains increases with the presence of the pathogenicity island PAI, which encodes a Type 4 Secretion System and the oncoprotein CagA. Two major CagA types can be distinguished by differences in the repetitive EPIYA region in the C-terminal sequence; the more virulent East Asian CagA type with EPIYA-A, -B, and -D motifs and the Western CagA type with EPIYA-A, -B, and C motifs, the virulence of which is associated with the multitude of EPIYA-C motifs. In this study, the cagA gene was characterized in H. pylori strains isolated from Mongolians suffering from gastritis (80%) or ulcer (20%). The EPIYA region of 53 isolates was determined by PCR-amplification of overlapping cagA regions and subsequent Sanger sequencing. Only one H. pylori isolate carried the East Asian type (ABD) and 52 isolates the Western type of CagA, thereof 30 the EPIYA type ABC, 19 the ABCC type and one each of type ABCCCC, AAABC and AAAAB. An amino acid exchange from EPIYA-B to EPIYT-B was predominantly found in CagA proteins in strains with < 2 EPIYA-C copies (n = 25/32; p = 0.015) including the two EPIYA-A enriched CagA proteins, which have not been described to date. Due to the amino acid triplet preceding the EPIYA motif and strength of predicted phosphorylation, the multiple EPIYA-A motifs A2, A3 and A4 were shown to cluster with EPIYA-B and EPIYT-B with the unique feature of amino acid E in position - 4 to Y of EPIYA. It has been described that tyrosine-phosphorylated EPIYA-A and -B motifs counteract the EPIYA-C-driven signaling towards host cell transformation and malignancy. Thus, Mongolian H. pylori strains carrying CagA proteins not only with a few EPIYA-C segments but also with multiplied EPIYA-A segments are probably less virulent; a thesis that needs further investigation at the protein level.

CD169+ macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling.

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTß) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTßR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTßR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTßR and RANK with implications for the immune response.

fuPCR as diagnostic method for the detection of rare fungal pathogens, such as Trichosporon, Cryptococcus and Fusarium.

Fungal respiratory tract colonization is a common finding in patients with hematologic neoplasms due to immunosuppression inherent in the diseases and exacerbated by therapy. This greatly increases the risk of fungal infections of the lungs, which is associated with significant mortality. Therefore, reliable diagnostic methods with rapidly available results are needed to administer adequate antifungal therapy. We have established an improved method for fungal DNA extraction and amplification that allows simultaneous detection of fungal families based on a set of multiplexed real-time PCR reactions (fuPCR). We analyzed respiratory rinses and blood of 94 patients with hematological systemic diseases by fuPCR and compared it with the results of culture and serological diagnostic methods. 40 healthy subjects served as controls. Regarding Candida species, the highest prevalence resulted from microbiological culture of respiratory rinses and from detection of antibodies in blood serum in patients (61 and 47%, respectively) and in the control group (29 and 51%, respectively). Detection of other pathogenic yeasts, such as Cryptococcus and Trichosporon, and molds, such as Fusarium, was only possible in patients by fuPCR from both respiratory rinses and whole blood and serum. These fungal species were found statistically significantly more frequent in respiratory rinses collected from patients after myeloablative therapy for stem cell transplantation compared to samples collected before treatment (P < 0.05i). The results show that fuPCR is a valuable complement to culturing and its inclusion in routine mycological diagnostics might be helpful for early detection of pathophysiologically relevant respiratory colonization for patients with hematologic neoplasms.

We validated a set of PCR reactions (fuPCR) for use in routine diagnostic. In contrast to culture and serological methods, only by fuPCR pathogenic yeasts (Cryptococcus and Trichosporon) and molds (Aspergillus and Fusarium) were detected in respiratory rinses and blood of hematological patients.

Lymphotoxin-ß-receptor (LTßR) signaling on hepatocytes is required for liver regeneration after partial hepatectomy.

Lymphotoxin-ß-receptor deficient (LTßR-/-) and Tumor Necrosis Factor Receptor p55 deficient (TNFRp55-/-) mice show defects in liver regeneration (LR) after partial hepatectomy (PHx) with significantly increased mortality. LTßR and TNFRp55 belong to the core members of the TNF/TNFR superfamily. Interestingly, combined failure of LTßR and TNFRp55 signaling after PHx leads to a complete defect in LR. Here, we first addressed the question which liver cell population crucially requires LTßR signaling for efficient LR. To this end, mice with a conditionally targeted LTßR allele (LTßRfl/fl) were crossed to AlbuminCre and LysozymeMCre mouse lines to unravel the function of the LTßR on hepatocytes and monocytes/macrophages/Kupffer cells, respectively. Analysis of these mouse lines clearly reveals that LTßR is required on hepatocytes for efficient LR while no deficit in LR was found in LTßRfl/fl × LysMCre mice. Second, the molecular basis for the cooperating role of LTßR and TNFRp55 signaling pathways in LR was investigated by transcriptome analysis of etanercept treated LTßR-/- (LTßR-/-/ET) mice. Bioinformatic analysis and subsequent verification by qRT-PCR identified novel target genes (Cyclin-L2, Fas-Binding factor 1, interferon-related developmental regulator 1, Leucyl-tRNA Synthetase 2, and galectin-4) that are upregulated by LTßR/TNFRp55 signaling after PHx and fail to be upregulated after PHx in LTßR-/-/ET mice.

Lymphotoxin ß Receptor: a Crucial Role in Innate and Adaptive Immune Responses against Toxoplasma gondii.

The lymphotoxin ß receptor (LTßR) plays an essential role in the initiation of immune responses to intracellular pathogens. In mice, the LTßR is crucial for surviving acute toxoplasmosis; however, until now, a functional analysis was largely incomplete. Here, we demonstrate that the LTßR is a key regulator required for the intricate balance of adaptive immune responses. Toxoplasma gondii-infected LTßR-deficient (LTßR-/-) mice show globally altered interferon-γ (IFN-γ) regulation, reduced IFN-γ-controlled host effector molecule expression, impaired T cell functionality, and an absent anti-parasite-specific IgG response, resulting in a severe loss of immune control of the parasites. Reconstitution of LTßR-/- mice with toxoplasma immune serum significantly prolongs survival following T. gondii infection. Notably, analysis of RNA-seq data clearly indicates a specific effect of T. gondii infection on the B cell response and isotype switching. This study uncovers the decisive role of the LTßR in cytokine regulation and adaptive immune responses to control T. gondii.

An aberrant STAT pathway is central to COVID-19.

COVID-19 is caused by SARS-CoV-2 infection and characterized by diverse clinical symptoms. Type I interferon (IFN-I) production is impaired and severe cases lead to ARDS and widespread coagulopathy. We propose that COVID-19 pathophysiology is initiated by SARS-CoV-2 gene products, the NSP1 and ORF6 proteins, leading to a catastrophic cascade of failures. These viral components induce signal transducer and activator of transcription 1 (STAT1) dysfunction and compensatory hyperactivation of STAT3. In SARS-CoV-2-infected cells, a positive feedback loop established between STAT3 and plasminogen activator inhibitor-1 (PAI-1) may lead to an escalating cycle of activation in common with the interdependent signaling networks affected in COVID-19. Specifically, PAI-1 upregulation leads to coagulopathy characterized by intravascular thrombi. Overproduced PAI-1 binds to TLR4 on macrophages, inducing the secretion of proinflammatory cytokines and chemokines. The recruitment and subsequent activation of innate immune cells within an infected lung drives the destruction of lung architecture, which leads to the infection of regional endothelial cells and produces a hypoxic environment that further stimulates PAI-1 production. Acute lung injury also activates EGFR and leads to the phosphorylation of STAT3. COVID-19 patients' autopsies frequently exhibit diffuse alveolar damage (DAD) and increased hyaluronan (HA) production which also leads to higher levels of PAI-1. COVID-19 risk factors are consistent with this scenario, as PAI-1 levels are increased in hypertension, obesity, diabetes, cardiovascular diseases, and old age. We discuss the possibility of using various approved drugs, or drugs currently in clinical development, to treat COVID-19. This perspective suggests to enhance STAT1 activity and/or inhibit STAT3 functions for COVID-19 treatment. This might derail the escalating STAT3/PAI-1 cycle central to COVID-19.

CTX-M-9 group ESBL-producing Raoultella planticola nosocomial infection: first report from sub-Saharan Africa.

BACKGROUND: Raoultella are Gram-negative rod-shaped aerobic bacteria which grow in water and soil. They mostly cause nosocomial infections associated with surgical procedures. This case study is the first report of a Raoultella infection in Africa. Case presentation We report a case of a surgical site infection (SSI) caused by Raoultella planticola which developed after caesarean section (CS) and surgery for secondary small bowel obstruction. The patient became febrile with neutrophilia (19,157/µL) 4 days after laparotomy and started to develop clinical signs of a SSI on the 8th day after laparotomy. The patient continued to be febrile and became critically ill despite empirical treatment with ceftriaxone and vancomycin. Raoultella species with extended antimicrobial resistance (AMR) carrying the CTX-M-9 ß-lactamase was isolated from the wound discharge. Considering the antimicrobial susceptibility test, ceftriaxone was replaced by ceftazidime. The patient recovered and could be discharged on day 29 after CS. CONCLUSIONS: Raoultella planticola was isolated from an infected surgical site after repeated abdominal surgery. Due to the infection the patient's stay in the hospital was prolonged for a total of 4 weeks. It is noted that patients undergoing surgical and prolonged inpatient treatment are at risk for infections caused by Raoultella. The development of a SSI caused by Raoultella planticola with extended AMR has to be assumed to be a consequence of ineffective antibiotic utilization. The presented case advices that rare bacteria as Raoultella should be considered as potential cause of nosocomial SSI with challenging treatment due to high levels of AMR.

Cutting Edge: TNF Is Essential for Mycobacteria-Induced MINCLE Expression, Macrophage Activation, and Th17 Adjuvanticity.

TNF blockade is a successful treatment for human autoimmune disorders like rheumatoid arthritis and inflammatory bowel disease yet increases susceptibility to tuberculosis and other infections. The C-type lectin receptors (CLR) MINCLE, MCL, and DECTIN-2 are expressed on myeloid cells and sense mycobacterial cell wall glycolipids. In this study, we show that TNF is sufficient to upregulate MINCLE, MCL, and DECTIN-2 in macrophages. TNF signaling through TNFR1 p55 was required for upregulation of these CLR and for cytokine secretion in macrophages stimulated with the MINCLE ligand trehalose-6,6-dibehenate or infected with Mycobacterium bovis bacillus Calmette-Guérin. The Th17 response to immunization with the MINCLE-dependent adjuvant trehalose-6,6-dibehenate was specifically abrogated in TNF-deficient mice and strongly attenuated by TNF blockade with etanercept. Together, interference with production or signaling of TNF antagonized the expression of DECTIN-2 family CLR, thwarting vaccine responses and possibly increasing infection risk.

Selective reconstitution of IFN­Î³ gene function in Ncr1+ NK cells is sufficient to control systemic vaccinia virus infection.

IFN-γ is an enigmatic cytokine that shows direct anti-viral effects, confers upregulation of MHC-II and other components relevant for antigen presentation, and that adjusts the composition and balance of complex cytokine responses. It is produced during immune responses by innate as well as adaptive immune cells and can critically affect the course and outcome of infectious diseases, autoimmunity, and cancer. To selectively analyze the function of innate immune cell-derived IFN-γ, we generated conditional IFN-γOFF mice, in which endogenous IFN-γ expression is disrupted by a loxP flanked gene trap cassette inserted into the first intron of the IFN-γ gene. IFN-γOFF mice were intercrossed with Ncr1-Cre or CD4-Cre mice that express Cre mainly in NK cells (IFN-γNcr1-ON mice) or T cells (IFN-γCD4-ON mice), respectively. Rosa26RFP reporter mice intercrossed with Ncr1-Cre mice showed selective RFP expression in more than 80% of the NK cells, while upon intercrossing with CD4-Cre mice abundant RFP expression was detected in T cells, but also to a minor extent in other immune cell subsets. Previous studies showed that IFN-γ expression is needed to promote survival of vaccinia virus (VACV) infection. Interestingly, during VACV infection of wild type and IFN-γCD4-ON mice two waves of serum IFN-γ were induced that peaked on day 1 and day 3/4 after infection. Similarly, VACV infected IFN-γNcr1-ON mice mounted two waves of IFN-γ responses, of which the first one was moderately and the second one profoundly reduced when compared with WT mice. Furthermore, IFN-γNcr1-ON as well as IFN-γCD4-ON mice survived VACV infection, whereas IFN-γOFF mice did not. As expected, ex vivo analysis of splenocytes derived from VACV infected IFN-γNcr1-ON mice showed IFN-γ expression in NK cells, but not T cells, whereas IFN-γOFF mice showed IFN-γ expression neither in NK cells nor T cells. VACV infected IFN-γNcr1-ON mice mounted normal cytokine responses, restored neutrophil accumulation, and showed normal myeloid cell distribution in blood and spleen. Additionally, in these mice normal MHC-II expression was detected on peripheral macrophages, whereas IFN-γOFF mice did not show MHC-II expression on such cells. In conclusion, upon VACV infection Ncr1 positive cells including NK cells mount two waves of early IFN-γ responses that are sufficient to promote the induction of protective anti-viral immunity.

Fragile X mental retardation protein protects against tumour necrosis factor-mediated cell death and liver injury.

OBJECTIVE: The Fragile X mental retardation (FMR) syndrome is a frequently inherited intellectual disability caused by decreased or absent expression of the FMR protein (FMRP). Lack of FMRP is associated with neuronal degradation and cognitive dysfunction but its role outside the central nervous system is insufficiently studied. Here, we identify a role of FMRP in liver disease. DESIGN: Mice lacking Fmr1 gene expression were used to study the role of FMRP during tumour necrosis factor (TNF)-induced liver damage in disease model systems. Liver damage and mechanistic studies were performed using real-time PCR, Western Blot, staining of tissue sections and clinical chemistry. RESULTS: Fmr1null mice exhibited increased liver damage during virus-mediated hepatitis following infection with the lymphocytic choriomeningitis virus. Exposure to TNF resulted in severe liver damage due to increased hepatocyte cell death. Consistently, we found increased caspase-8 and caspase-3 activation following TNF stimulation. Furthermore, we demonstrate FMRP to be critically important for regulating key molecules in TNF receptor 1 (TNFR1)-dependent apoptosis and necroptosis including CYLD, c-FLIPS and JNK, which contribute to prolonged RIPK1 expression. Accordingly, the RIPK1 inhibitor Necrostatin-1s could reduce liver cell death and alleviate liver damage in Fmr1null mice following TNF exposure. Consistently, FMRP-deficient mice developed increased pathology during acute cholestasis following bile duct ligation, which coincided with increased hepatic expression of RIPK1, RIPK3 and phosphorylation of MLKL. CONCLUSIONS: We show that FMRP plays a central role in the inhibition of TNF-mediated cell death during infection and liver disease.

IL-6 Trans-signaling Controls Liver Regeneration After Partial Hepatectomy.

Interleukin-6 (IL-6) is critically involved in liver regeneration after partial hepatectomy (PHX). Previous reports suggest that IL-6 trans-signaling through the soluble IL-6/IL-6R complex is involved in this process. However, the long-term contribution of IL-6 trans-signaling for liver regeneration after PHX is unknown. PHX-induced generation of the soluble IL-6R by ADAM (a disintegrin and metallo) proteases enables IL-6 trans-signaling, in which IL-6 forms an agonistic complex with the soluble IL-6 receptor (sIL-6R) to activate all cells expressing the signal-transducing receptor chain glycoprotein 130 (gp130). In contrast, without activation of ADAM proteases, IL-6 in complex with membrane-bound IL-6R and gp130 activates classic signaling. Here, we describe the generation of IL-6 trans-signaling mice, which exhibit boosted IL-6 trans-signaling and abrogated classic signaling by genetic conversion of all membrane-bound IL-6R into sIL-6R proteins phenocopying hyperactivation of ADAM-mediated shedding of IL-6R as single substrate. Importantly, although IL-6R deficient mice were strongly affected by PHX, survival and regeneration of IL-6 trans-signaling mice was indistinguishable from control mice, demonstrating that IL-6 trans-signaling fully compensates for disabled classic signaling in liver regeneration after PHX. Moreover, we monitored the long-term consequences of global IL-6 signaling inhibition versus IL-6 trans-signaling selective blockade after PHX by IL-6 monoclonal antibodies and soluble glycoprotein 130 as fragment crystallizable fusion, respectively. Both global IL-6 blockade and selective inhibition of IL-6 trans-signaling results in a strong decrease of overall survival after PHX, accompanied by decreased signal transducer and activator of transcription 3 phosphorylation and proliferation of hepatocytes. Mechanistically, IL-6 trans-signaling induces hepatocyte growth factor production by hepatic stellate cells. Conclusion: IL-6 trans-signaling, but not classic signaling, controls liver regeneration following PHX.

Fetal origin confers radioresistance on liver macrophages via p21cip1/WAF1.

BACKGROUND & AIMS: Cells of hematopoietic origin, including macrophages, are generally radiation sensitive, but a subset of Kupffer cells (KCs) is relatively radioresistant. Here, we focused on the identity of the radioresistant KCs in unmanipulated mice and the mechanism of radioresistance. METHODS: We employed Emr1- and inducible CX3Cr1-based fate-mapping strategies combined with the RiboTag reporter to identify the total KCs and the embryo-derived KCs, respectively. The KC compartment was reconstituted with adult bone-marrow-derived KCs (bm-KCs) using clodronate depletion. Mice were lethally irradiated and transplanted with donor bone marrow, and the radioresistance of bone-marrow- or embryo-derived KCs was studied. Gene expression was analyzed using in situ mRNA isolation via RiboTag reporter mice, and the translatomes were compared among subsets. RESULTS: Here, we identified the radioresistant KCs as the long-lived subset that is derived from CX3CR1-expressing progenitor cells in fetal life, while adult bm-KCs do not resist irradiation. While both subsets upregulated the Cdkn1a gene, encoding p21-cip1/WAF1 protein, radioresistant embryo-derived KCs showed a greater increase in response to irradiation. In the absence of this molecule, the radioresistance of KCs was compromised. Replacement KCs, derived from adult hematopoietic stem cells, differed from radioresistant KCs in their expression of genes related to immunity and phagocytosis. CONCLUSIONS: Here, we show that, in the murine liver, a subset of KCs of embryonic origin resists lethal irradiation through Cdkn1a upregulation and is maintained for a long period, while bm-KCs do not survive lethal irradiation. LAY SUMMARY: Kupffer cells (KCs) are the tissue-resident macrophages of the liver. KCs can be originated from fetal precursors and from monocytes during the fetal stage and post-birth, respectively. Most immune cells in mice are sensitive to lethal-irradiation-induced death, while a subset of KCs resists radiation-induced death. These radioresistant KCs continue to live in the irradiated mice. We discovered that this relatively radioresistant KC subset are the fetal-derived KCs, and they achieve this through cell-cycle arrest. Understanding the radiobiology of KCs will provide valuable insights into the mechanisms that elicit radiation-induced liver disease.

Herpes Simplex Virus 1 Latency and the Kinetics of Reactivation Are Regulated by a Complex Network of Interactions between the Herpesvirus Entry Mediator, Its Ligands (gD, BTLA, LIGHT, and CD160), and the Latency-Associated Transcript.

Recently, we reported that the herpesvirus entry mediator (HVEM; also called TNFRSF14 or CD270) is upregulated by the latency-associated transcript (LAT) of herpes simplex virus 1 (HSV-1) and that the absence of HVEM affects latency reactivation but not primary infection in ocularly infected mice. gD has been shown to bind to HVEM. LIGHT (TNFSF14), CD160, and BTLA (B- and T-lymphocyte attenuator) also interact with HVEM and can interfere with HSV gD binding. It was not known if LIGHT, CD160, or BTLA affected the level of latency reactivation in the trigeminal ganglia (TG) of latently infected mice. To address this issue, we ocularly infected LIGHT-/-, CD160-/-, and BTLA-/- mice with LAT(+) and LAT(-) viruses, using similarly infected wild-type (WT) and HVEM-/- mice as controls. The amount of latency, as determined by the levels of gB DNA in the TG of the LIGHT-/-, CD160-/-, and BTLA-/- mice infected with either LAT(+) or LAT(-) viruses, was lower than that in WT mice infected with LAT(+) virus and was similar in WT mice infected with LAT(-) virus. The levels of LAT RNA in HVEM-/-, LIGHT-/-, CD160-/-, and BTLA-/- mice infected with LAT(+) virus were similar and were lower than the levels of LAT RNA in WT mice. However, LIGHT-/-, CD160-/-, and BTLA-/- mice, independent of the presence of LAT, had levels of reactivation similar to those of WT mice infected with LAT(+) virus. Faster reactivation correlated with the upregulation of HVEM transcript. The LIGHT-/-, CD160-/-, and BTLA-/- mice had higher levels of HVEM expression, and this, along with the absence of BTLA, LIGHT, or CD160, may contribute to faster reactivation, while the absence of each molecule, independent of LAT, may have contributed to lower latency. This study suggests that, in the absence of competition with gD for binding to HVEM, LAT RNA is important for WT levels of latency but not for WT levels of reactivation.IMPORTANCE The effects of BTLA, LIGHT, and CD160 on latency reactivation are not known. We show here that in BTLA, LIGHT, or CD160 null mice, latency is reduced; however, HVEM expression is upregulated compared to that of WT mice, and this upregulation is associated with higher reactivation that is independent of LAT but dependent on gD expression. Thus, one of the mechanisms by which BTLA, LIGHT, and CD160 null mice enhance reactivation appears to be the increased expression of HVEM in the presence of gD. Thus, our results suggest that blockade of HVEM-LIGHT-BTLA-CD160 contributes to reduced HSV-1 latency and reactivation.

B Cell-Mediated Maintenance of Cluster of Differentiation 169-Positive Cells Is Critical for Liver Regeneration.

The liver has an extraordinary capacity to regenerate through activation of key molecular pathways. However, central regulators controlling liver regeneration remain insufficiently studied. Here, we show that B cell-deficient animals failed to induce sufficient liver regeneration after partial hepatectomy (PHx). Consistently, adoptive transfer of B cells could rescue defective liver regeneration. B cell-mediated lymphotoxin beta production promoted recovery from PHx. Absence of B cells coincided with loss of splenic cluster of differentiation 169-positive (CD169+ ) macrophages. Moreover, depletion of CD169+ cells resulted in defective liver regeneration and decreased survival, which was associated with reduced hepatocyte proliferation. Mechanistically, CD169+ cells contributed to liver regeneration by inducing hepatic interleukin-6 (IL-6) production and signal transducer and activator of transcription 3 activation. Accordingly, treatment of CD169+ cell-depleted animals with IL-6/IL-6 receptor rescued liver regeneration and severe pathology following PHx. Conclusion: We identified CD169+ cells to be a central trigger for liver regeneration, by inducing key signaling pathways important for liver regeneration.

Tumor Necrosis Factor-Mediated Survival of CD169+ Cells Promotes Immune Activation during Vesicular Stomatitis Virus Infection.

Innate immune activation is essential to mount an effective antiviral response and to prime adaptive immunity. Although a crucial role of CD169+ cells during vesicular stomatitis virus (VSV) infections is increasingly recognized, factors regulating CD169+ cells during viral infections remain unclear. Here, we show that tumor necrosis factor is produced by CD11b+ Ly6C+ Ly6G+ cells following infection with VSV. The absence of TNF or TNF receptor 1 (TNFR1) resulted in reduced numbers of CD169+ cells and in reduced type I interferon (IFN-I) production during VSV infection, with a severe disease outcome. Specifically, TNF triggered RelA translocation into the nuclei of CD169+ cells; this translocation was inhibited when the paracaspase MALT-1 was absent. Consequently, MALT1 deficiency resulted in reduced VSV replication, defective innate immune activation, and development of severe disease. These findings indicate that TNF mediates the maintenance of CD169+ cells and innate and adaptive immune activation during VSV infection.IMPORTANCE Over the last decade, strategically placed CD169+ metallophilic macrophages in the marginal zone of the murine spleen and lymph nodes (LN) have been shown to play a very important role in host defense against viral pathogens. CD169+ macrophages have been shown to activate innate and adaptive immunity via "enforced virus replication," a controlled amplification of virus particles. However, the factors regulating the CD169+ macrophages remain to be studied. In this paper, we show that after vesicular stomatitis virus infection, phagocytes produce tumor necrosis factor (TNF), which signals via TNFR1, and promote enforced virus replication in CD169+ macrophages. Consequently, lack of TNF or TNFR1 resulted in defective immune activation and VSV clearance.

TNFα induced up-regulation of Na+,K+,2Cl- cotransporter NKCC1 in hepatic ammonia clearance and cerebral ammonia toxicity.

The devastating consequences of hepatic failure include hepatic encephalopathy, a severe, life threatening impairment of neuronal function. Hepatic encephalopathy is caused by impaired hepatic clearance of NH4+. Cellular NH4+ uptake is accomplished mainly by the Na+,K+,2Cl- cotransporter. Here we show that hepatic clearance of NH4+ is impaired in TNFα deficient as well as TNFR1&TNFR2 double knockout mice, which both develop hyperammonemia. Despite impaired hepatic clearance of NH4+, TNFα deficient mice and TNFR1 deficient mice were protected against acute ammonia intoxication. While 54% of the wild-type mice and 60% of TNFR2 deficient mice survived an NH4+ load, virtually all TNFα deficient mice and TNFR1 deficient mice survived the treatment. Conversely, TNFα treatment of wild type mice sensitized the animals to the toxic effects of an NH4+ load. The protection of TNFα-deficient mice against an NH4+ load was paralleled by decreased cerebral expression of NKCC1. According to the present observations, inhibition of TNFα formation and/or NKCC1 may be strategies to favorably influence the clinical course of hepatic encephalopathy.