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Summary Toll-like receptor 4 (TLR4) plays a central role in host responses to bacterial infection, but the precise mechanism(s) by which its downstream signaling components coordinate the bone marrow response to sepsis is poorly understood. Using mice deficient in TLR4 downstream adapters MYD88 or TRIF, we demonstrate that both cell-autonomous and non-cell-autonomous MYD88 activation are major causes of myelosuppression during sepsis, while having a modest impact on hematopoietic stem cell (HSC) functions. In contrast, cell-intrinsic TRIF activation severely compromises HSC self-renewal without directly affecting myeloid cells. Lipopolysaccharide-induced activation of MYD88 or TRIF contributes to cell-cycle activation of HSC and induces rapid and permanent changes in transcriptional programs, as indicated by persistent downregulation of Spi1 and CebpA expression after transplantation. Thus, distinct mechanisms downstream of TLR4 signaling mediate myelosuppression and HSC exhaustion during sepsis through unique effects of MyD88 and TRIF., Graphical Abstract, Highlights • Activation of TLR4 by LPS causes HSC injury, myelosuppression, and neutropenia • LPS-induced MYD88 activation leads to apoptosis and myelosuppression • LPS causes HSC damage and exhaustion by TRIF activation • HSC retain long-term memory of LPS injury, In this article, Carlesso and colleagues investigate the role of the TLR4 downstream adapters, MyD88 and TRIF, in the regulation of bone marrow response to sepsis. Their work shows that MyD88 activation is a major cause of myelosuppression during sepsis while having a modest impact on HSC, whereas cell-intrinsic TRIF activation compromises HSC self-renewal without directly affecting myeloid cells. Cell-intrinsic activation in HSC results in HSC exhaustion and transcriptional changes persisting over the long term even when the endotoxic environment is removed.

Sepsis-induced organ damage is caused by systemic inflammatory response syndrome (SIRS), which results in substantial comorbidities. Therefore, it is of medical importance to identify molecular brakes that can be exploited to dampen inflammation and prevent the development of SIRS. We investigated the role of phosphatase and tensin homolog (PTEN) in suppressing SIRS, increasing microbial clearance, and preventing lung damage. Septic patients and mice with sepsis exhibited increased PTEN expression in leukocytes. Myeloid-specific Pten deletion in an animal model of sepsis increased bacterial loads and cytokine production, which depended on enhanced myeloid differentiation primary response gene 88 (MyD88) abundance and resulted in mortality. PTEN-mediated induction of the microRNAs (miRNAs) miR125b and miR203b reduced the abundance of MyD88. Loss- and gain-of-function assays demonstrated that PTEN induced miRNA production by associating with and facilitating the nuclear localization of Drosha-Dgcr8, part of the miRNA-processing complex. Reconstitution of PTEN-deficient mouse embryonic fibroblasts with a mutant form of PTEN that does not localize to the nucleus resulted in retention of Drosha-Dgcr8 in the cytoplasm and impaired production of mature miRNAs. Thus, we identified a regulatory pathway involving nuclear PTEN-mediated miRNA generation that limits the production of MyD88 and thereby limits sepsis-associated mortality.

Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell’s DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B., Author summary Hepadnavirus cccDNA is the persistent form of viral genome, and in terms of human hepatitis B virus (HBV), cccDNA is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure with current medications. Therefore, the elucidation of molecular mechanism of cccDNA formation will aid HBV research at both basic and medical levels. In this study, we screened a total of 107 cellular DNA repair genes and identified DNA ligase 1 and 3 as key factors for cccDNA formation from viral relaxed (open) circular DNA. In addition, we found that the cellular DNA ligase 4 is responsible for converting viral double-stranded linear DNA into cccDNA. Our study further confirmed the involvement of host DNA repair machinery in cccDNA formation, and may reveal new antiviral targets for treatment of hepatitis B in future.

Sepsis can induce an overwhelming systemic inflammatory response, resulting in organ damage and death. Suppressor of cytokine signaling 1 (SOCS1) negatively regulates signaling by cytokine receptors and Toll-like receptors (TLRs). However, the cellular targets and molecular mechanisms for SOCS1 activity during polymicrobial sepsis are unknown. To address this, we utilized a cecal ligation and puncture (CLP) model for sepsis; C57BL/6 mice subjected to CLP were then treated with a peptide (iKIR) that binds the SOCS1 kinase inhibitory region (KIR) and blocks its activity. Treatment with iKIR increased CLP-induced mortality, bacterial burden, and inflammatory cytokine production. Myeloid cell-specific SOCS1 deletion (Socs1Δmyel) mice were also more susceptible to sepsis, demonstrating increased mortality, higher bacterial loads, and elevated inflammatory cytokines, compared with Socs1fl littermate controls. These effects were accompanied by macrophage metabolic reprograming, as evidenced by increased lactic acid production and elevated expression of the glycolytic enzymes hexokinase, lactate dehydrogenase A, and glucose transporter 1 in septic Socs1Δmyel mice. Upregulation was dependent on the STAT3/HIF-1α/glycolysis axis, and blocking glycolysis ameliorated increased susceptibility to sepsis in iKIR-treated CLP mice. These results reveal a role of SOCS1 as a regulator of metabolic reprograming that prevents overwhelming inflammatory response and organ damage during sepsis.

Made available in DSpace on 2015-10-21T20:56:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2015-02-23. Added 1 bitstream(s) on 2015-10-22T09:52:44Z : No. of bitstreams: 1 WOS000350662100020.pdf: 1366601 bytes, checksum: dc2295060a1ced093a5a667e81900462 (MD5) National Institutes of Health Ralph W. and Grace M. Showalter Research Trust Fund Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Macrophages dictate both initiation and resolution of inflammation. During acute inflammation classically activated macrophages (M1) predominate, and during the resolution phase alternative macrophages (M2) are dominant. The molecular mechanisms involved in macrophage polarization are understudied. MicroRNAs are differentially expressed in M1 and M2 macrophages that influence macrophage polarization. We identified a role of miR-21 in macrophage polarization, and found that cross-talk between miR-21 and the lipid mediator prostaglandin E-2 (PGE(2)) is a determining factor in macrophage polarization. miR-21 inhibition impairs expression of M2 signature genes but not M1 genes. PGE(2) and its downstream effectors PKA and Epac inhibit miR-21 expression and enhance expression of M2 genes, and this effect is more pronounced in miR-21-/-cells. Among potential targets involved in macrophage polarization, we found that STAT3 and SOCS1 were enhanced in miR-21-/-cells and further enhanced by PGE2. We found that STAT3 was a direct target of miR-21 in macrophages. Silencing the STAT3 gene abolished PGE(2)-mediated expression of M2 genes in miR-21-/-macrophages. These data shed light on the molecular brakes involved in homeostatic macrophage polarization and suggest new therapeutic strategies to prevent inflammatory responses. Indiana Univ Sch Med, Dept Microbiol &Immunol, Indianapolis, IN 46202 USA Univ Estadual Paulista, Fac Ciencias Farmaceut, Dept Ciencias Biol, BR-14801902 Araraquara, SP, Brazil Univ Estadual Paulista, Fac Ciencias Farmaceut, Dept Ciencias Biol, BR-14801902 Araraquara, SP, Brazil National Institutes of Health: 1R21AI079349-01 National Institutes of Health: HL-103777-01 National Institutes of Health: HL-124159-01 National Institutes of Health: T32AI060519

MicroRNAs are known to control TLR activation in phagocytes. We have shown that leukotriene (LT) B4 (LTB4) positively regulates macrophage MyD88 expression by decreasing suppressor of cytokine signaling-1 (SOCS-1) mRNA stability. In this study, we investigated the possibility that LTB4 control of MyD88 expression involves the generation of microRNAs. Our data show that LTB4, via its receptor B leukotriene receptor 1 (BLT1) and Gαi signaling, increased macrophage expression of inflammatory microRNAs, including miR-155, miR-146b, and miR-125b. LTB4-mediated miR-155 generation was attributable to activating protein-1 activation. Furthermore, macrophage transfection with antagomirs against miR-155 and miR-146b prevented both the LTB4-mediated decrease in SOCS-1 and increase in MyD88. Transfection with miR-155 and miR-146b mimics decreased SOCS-1 levels, increased MyD88 expression, and restored TLR4 responsiveness in both wild type and LT-deficient macrophages. To our knowledge, our data unveil a heretofore unrecognized role for the GPCR BLT1 in controlling expression of microRNAs that regulate MyD88-dependent activation of macrophages.

Induction of the urokinase type plasminogen activator receptor (uPAR) promotes cell adhesion through its interaction with vitronectin (VN) in the extracellular matrix, and facilitates cell migration and invasion by localizing uPA to the cell surface. We provide evidence that this balance between cell adhesion and cell detachment is governed by PA inhibitor-1 (PAI-1). First, we demonstrate that uPAR and PAI-1 bind to the same site in VN (i.e., the amino-terminal somatomedin B domain; SMB), and that PAI-1 competes with uPAR for binding to SMB. Domain swapping and mutagenesis studies indicate that the uPAR-binding sequence is located within the central region of the SMB domain, a region previously shown to contain the PAI-1-binding motif. Second, we show that PAI-1 dissociates bound VN from uPAR and detaches U937 cells from their VN substratum. This PAI-1 mediated release of cells from VN appears to occur independently of its ability to function as a protease inhibitor, and may help to explain why high PAI-1 levels indicate a poor prognosis for many cancers. Finally, we show that uPA can rapidly reverse this effect of PAI-1. Taken together, these results suggest a dynamic regulatory role for PAI-1 and uPA in uPAR-mediated cell adhesion and release.

A major cause of severe skin infections is methicillin-resistant Staphylococcus aureus (MRSA). Immunotherapeutic strategies are needed to treat infections. Neutrophils and macrophages control MRSA skin infection but undue inflammation causes injury. The bioactive eicosanoid leukotriene B4 is a potent chemoattractant and enhances phagocyte effector functions in naïve mice. However we have shown that diabetic mice produce chronic LTB4 which promotes susceptibility to sepsis but the role of LTB4 in localized infections is unknown. We hypothesize that aberrant LTB4 drive excessive inflammation during MRSA skin infection in diabetics. Diabetic mice at day 1 post infection had larger infected areas, bacterial and apoptotic cell burdens than nondiabetic controls. By intravital microscopy we observed more neutrophil recruitment in diabetic mice. These effects correlated with high levels of LTB4 and its receptor BLT1. At day 9 post infection nondiabetic mice were nearly healed but diabetic mice still had cell infiltration, high bacterial and apoptotic burdens, which associated with high levels of inflammatory mediators. To test whether chronic LTB4 causes uncontrolled infection, diabetic mice were treated daily with a topical ointment containing BLT1 antagonist. The treatment reduced infection area, bacterial burden and neutrophil recruitment. We also detected fewer apoptotic cells and lower expression of inflammatory mediators. These findings reveal that LTB4 is detrimental for diabetics by chronically inducing a damaging inflammatory response. We observe that topical BLT1 antagonist treatment improves host defense in diabetic mice and promises to be an effective therapeutic to treat MRSA infections in diabetic patients.

With the recent completion of the human genome sequencing project, scientists now face the daunting challenge of deciphering the function of these newly found genes quickly and efficiently. For biotechnology, it is equally important to identify the therapeutically relevant genes as quickly as possible. Mammalian expression systems provide many advantages to aid in this task. Mammalian cell lines have the capacity for proper post-translational modifications, including proper protein folding and glycosylation. In response to these needs, a CHO-K1 cell line that grows in suspension and in serum-free media was initially established and designated CHO-K1-S. An antibody gene of interest was chosen as the target for optimization rather than a reporter gene system. A comparison of various lipid transfection reagents was made using recombinant protein expression as the endpoint readout. Various other parameters including lipid:DNA ratios, cell density, and transfections in shaker versus spinner flasks were tested using the CHO-K1-S cell line. As a result, a rapid and reliable transient transfection protocol was developed. Using this procedure, we have produced milligram/per liter quantities of bioactive recombinant proteins from several genes of interest.

Type I diabetes (T1DM) is characterized by hyperglycemia and insulin deficiency. T1DM is associated with constitutive production of pro-inflammatory cytokine such as the members of IL-1 family. IL-1β binding to its MyD88-associated receptor IL-1R is a major cause of tissue injury in T1DM. We hypothesize that hyperglycemia (HG) enhances MyD88 in macrophages (MA) amplifying IL-1βR responsiveness. We found that both in vitro and in vivo HG increases the expression of MyD88 and its transcription factor STAT-1. Speculating that enhanced MyD88/STAT-1 levels could be due to epigenetic changes, we analyzed the expression of enzymes involved in DNA methylation (DNMT) and Histone deacetylation (HDAC) in MA from T1DM mice. We observed that diabetic mice expressed at least 2 times more HDAC 8, 10 and 11, and DNMT 3b than nondiabetic mice. In vitro inhibition of HDAC (TSA) or DNMT (5-azacyd) in MA impaired HG-enhanced MyD88 and STAT-1 expression. These data suggest a heretofore unrecognized epigenetic changes associated with hyperinflammatory pattern of MA from T1DM, leading to high MyD88 expression and possibly allowing overly IL-1β receptor and other MyD88-dependent receptor activation

Plasminogen activator inhibitor 1 (PAI-1) binds to the somatomedin B (SMB) domain of vitronectin (VN), a domain present in at least seven other proteins. In this study, we investigate the PAI-1 binding activity of these SMB homologs and attempt to more specifically localize the PAI-1 binding site within this domain. SMBVN and several of its homologs were expressed in Escherichia coli, purified, and tested for PAI-1 binding activity in a competitive ligand binding assay. Although recombinant SMBVN was fully active in this assay, none of the homologs bound to PAI-1 or competed with VN for PAI-1 binding. These inactive homologs are structurally related to SMBVN, having 33-45% sequence identity and containing all 8 cysteines at conserved positions. Thus, homolog-scanning experiments were conducted by exchanging progressively larger portions of the NH2- or COOH-terminal regions of active SMBVN with the corresponding regions of the inactive homologs. These experiments revealed that the minimum PAI-1-binding sequence was present in the central region (residues 12-30) of SMBVN. Alanine scanning mutagenesis further demonstrated that each of the 8 cysteines as well as Gly12, Asp22, Leu24, Try27, Tyr28, and Asp34 were critical for PAI-1 binding and were required to stabilize PAI-1 activity. These results indicate that the PAI-1 binding motif is localized to residues 12-30 of SMBVN and suggest that this motif is anchored in the active conformation by disulfide bonds.