MicroRNA 21 (miR-21) and miR-181b couple with NFI-A to generate myeloid-derived suppressor cells and promote immunosuppression in late sepsis.

The sepsis initial hyperinflammatory reaction, if not treated early, shifts to a protracted state of immunosuppression that alters both innate and adaptive immunity and is associated with elevated mortality. Myeloid-derived suppressor cells (MDSCs) are myeloid progenitors and precursors that fail to differentiate into mature innate-immunity cells and are known for their potent immunosuppressive activities. We previously reported that murine MDSCs expand dramatically in the bone marrow during late sepsis, induced by cecal ligation and puncture, and demonstrated that they contribute to late-sepsis immunosuppression. However, the molecular mechanism responsible for generating these immature Gr1(+) CD11b(+) myeloid cells during sepsis remains unknown. We show here that sepsis generates a microRNA (miRNA) signature that expands MDSCs. We found that miRNA 21 (miR-21) and miR-181b expression is upregulated in early sepsis and sustained in late sepsis. Importantly, we found that simultaneous in vivo blockade of both miRNAs via antagomiR (a chemically modified miRNA inhibitor) injection after sepsis initiation decreased the bone marrow Gr1(+) CD11b(+) myeloid progenitors, improved bacterial clearance, and reduced late-sepsis mortality by 74%. Gr1(+) CD11b(+) cells isolated from mice injected with antagomiRs were able to differentiate ex vivo into macrophages and dendritic cells and produced smaller amounts of the immunosuppressive interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß) after stimulation with bacterial lipopolysaccharide, suggesting that immature myeloid cells regained their maturation potential and have lost their immunosuppressive activity. In addition, we found that the protein level of transcription factor NFI-A, which plays a role in myeloid cell differentiation, was increased during sepsis and that antagomiR injection reduced its expression. Moreover, knockdown of NFI-A in the Gr1(+) CD11b(+) cells isolated from late-septic mice increased their maturation potential and reduced their production of the immunosuppressive mediators, similar to antagomiR injection. These data support the hypothesis that sepsis reprograms myeloid cells and thus alters the innate immunity cell repertoire to promote immunosuppression, and they demonstrate that this process can be reversed by targeting miR-21 and miR-181b to improve late-sepsis survival.

Scavenger receptor class a plays a central role in mediating mortality and the development of the pro-inflammatory phenotype in polymicrobial sepsis.

Sepsis is a frequent complication in critical illness. The mechanisms that are involved in initiation and propagation of the disease are not well understood. Scavenger receptor A (SRA) is a membrane receptor that binds multiple polyanions such as oxidized LDL and endotoxin. Recent studies suggest that SRA acts as a pattern recognition receptor in the innate immune response. The goal of the present study was to determine the role of SRA in polymicrobial sepsis. SRA deficient (SRA(-/-)) and C57BL/6JB/6J (WT) male mice were subjected to cecal ligation and puncture (CLP) to induce polymicrobial sepsis. NFκB activity, myeloperoxidase activity, and co-association of SRA with toll like receptor (TLR) 4 and TLR2 was analyzed in the lungs. Spleens were analyzed for apoptosis. Serum cytokines and chemokines were assayed. Blood and peritoneal fluid were cultured for aerobic and anaerobic bacterial burdens. Long-term survival was significantly increased in SRA(-/-) septic mice (53.6% vs. 3.6%, p < 0.05) when compared to WT mice. NFκB activity was 45.5% lower in the lungs of SRA(-/-) septic mice versus WT septic mice (p < 0.05). Serum levels of interleukin (IL)-5, IL-6, IL-10 and monocyte chemoattractant protein -1 were significantly lower in septic SRA(-/-) mice when compared to septic WT mice (p < 0.05). We found that SRA immuno-precipitated with TLR4, but not TLR2, in the lungs of WT septic mice. We also found that septic SRA(-/-) mice had lower bacterial burdens than WT septic mice. SRA deficiency had no effect on pulmonary neutrophil infiltration or splenocyte apoptosis during sepsis. We conclude that SRA plays a pivotal, and previously unknown, role in mediating the pathophysiology of sepsis/septic shock in a murine model of polymicrobial sepsis. Mechanistically, SRA interacts with TLR4 to enhance the development of the pro-inflammatory phenotype and mediate the morbidity and mortality of sepsis/septic shock.

The phosphocholine-binding pocket on C-reactive protein is necessary for initial protection of mice against pneumococcal infection.

Human C-reactive protein (CRP) protects mice from lethal Streptococcus pneumoniae infection when injected into mice within the range of 6 h before to 2 h after the administration of pneumococci. Because CRP binds to phosphocholine-containing substances and subsequently activates the complement system, it has been proposed that the antipneumococcal function of CRP requires the binding of CRP to phosphocholine moieties present in pneumococcal cell wall C-polysaccharide. To test this proposal experimentally, in this study, we utilized a new CRP mutant incapable of binding to phosphocholine. Based on the structure of CRP-phosphocholine complexes, which showed that Phe(66), Thr(76), and Glu(81) formed the phosphocholine-binding pocket, we constructed a CRP mutant F66A/T76Y/E81A in which the pocket was blocked by substituting Tyr for Thr(76). When compared with wild-type CRP, mutant CRP bound more avidly to phosphoethanolamine and could be purified by affinity chromatography using phosphoethanolamine-conjugated Sepharose. Mutant CRP did not bind to phosphocholine, C-polysaccharide, or pneumococci. Mutant CRP was free in the mouse serum, and its rate of clearance in vivo was not faster than that of wild-type CRP. When either 25 µg or 150 µg of CRP was administered into mice, unlike wild-type CRP, mutant CRP did not protect mice from lethal pneumococcal infection. Mice injected with mutant CRP had higher mortality rates than mice that received wild-type CRP. Decreased survival was due to the increased bacteremia in mice treated with mutant CRP. We conclude that the phosphocholine-binding pocket on CRP is necessary for CRP-mediated initial protection of mice against lethal pneumococcal infection.

MicroRNA-146a and RBM4 form a negative feed-forward loop that disrupts cytokine mRNA translation following TLR4 responses in human THP-1 monocytes.

Within hours after its initiation, the severe systemic inflammatory response of sepsis shifts to an adaptive anti-inflammatory state with coincident immunosuppression. This anti-inflammatory phenotype is characterized by diminished proinflammatory cytokine gene expression in response to toll-like receptor (TLR) stimulation with bacterial endotoxin/lipopolysaccharide (LPS), also known as endotoxin tolerance/adaptation. Our and other studies have established that gene-specific reprogramming following TLR4 responses independently represses transcription and translation of proinflammatory genes such as tumor necrosis factor alpha (TNFα). We also previously demonstrated that TNFα and interleukin (IL)-6 mRNA translation is repressed in endotoxin-adapted THP-1 human monocytes by an miRNA-based mechanism involving the argonaute family protein argonaute 2 (Ago2). Here, we further define the molecular nature of reprogramming translation by showing that TLR4-induced microRNA-146 promotes a feed-forward loop that modifies the subcellular localization of the RNA-binding protein RBM4 (RNA-binding motif protein 4) and promotes its interaction with Ago2. This interaction results in the assembly of a translation-repressor complex that disrupts TNFα and IL-6 cytokine synthesis in endotoxin-adapted THP-1 monocytes. This novel molecular path prevents the phosphorylation of RBM4 on serine-309 by p38 MAPK (mitogen-activated protein kinase), which leads to RBM4 accumulation in the cytosol and interaction with Ago2. We further find that microRNA-146a knockdown by antagomirs or protein phosphatase inhibition by okadaic acid increases p38 MAPK phosphorylation and results in RBM4 serine-309 phosphorylation and nuclear relocalization, which disrupts RBM4 and Ago2 interactions and restores TLR4-dependent synthesis of TNFα and IL-6. We conclude that miR-146a has a diverse and critical role in limiting an excessive acute inflammatory reaction.

Myeloid-derived suppressor cells evolve during sepsis and can enhance or attenuate the systemic inflammatory response.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous Gr1(+) CD11b(+) population of immature cells containing granulocytic and monocytic progenitors, which expand under nearly all inflammatory conditions and are potent repressors of T-cell responses. Studies of MDSCs during inflammatory responses, including sepsis, suggest they can protect or injure. Here, we investigated MDSCs during early and late sepsis. To do this, we used our published murine model of cecal ligation and puncture (CLP)-induced polymicrobial sepsis, which transitions from an early proinflammatory phase to a late anti-inflammatory and immunosuppressive phase. We confirmed that Gr1(+) CD11b(+) MDSCs gradually increase after CLP, reaching ∼88% of the bone marrow myeloid series in late sepsis. Adoptive transfer of early (day 3) MDSCs from septic mice into naive mice after CLP increased proinflammatory cytokine production, decreased peritoneal bacterial growth, and increased early mortality. Conversely, transfer of late (day 12) MDSCs from septic mice had the opposite effects. Early and late MDSCs studied ex vivo also differed in their inflammatory phenotypes. Early MDSCs expressed nitric oxide and proinflammatory cytokines, whereas late MDSCs expressed arginase activity and anti-inflammatory interleukin 10 (IL-10) and transforming growth factor ß (TGF-ß). Late MDSCs had more immature CD31(+) myeloid progenitors and, when treated ex vivo with granulocyte-macrophage colony-stimulating factor (GM-CSF), generated fewer macrophages and dendritic cells than early MDSCs. We conclude that as the sepsis inflammatory process progresses, the heterogeneous MDSCs shift to a more immature state and from being proinflammatory to anti-inflammatory.

Hematopoietic stem-progenitor cells restore immunoreactivity and improve survival in late sepsis.

Sepsis progresses from an early/acute hyperinflammatory to a late/chronic hypoinflammatory phase with immunosuppression. As a result of this phenotypic switch, mortality in late sepsis from persistent primary infection or opportunistic new infection often exceeds that in acute sepsis. Emerging data support that persistence of the hypoinflammatory (hyporesponsive) effector immune cells during late sepsis might involve alterations in myeloid differentiation/maturation that generate circulating repressor macrophages that do not readily clear active infection. Here, we used a cecal ligation and puncture (CLP) murine model of prolonged sepsis to show that adoptive transfer of CD34(+) hematopoietic stem-progenitor cells after CLP improves long-term survival by 65%. CD34(+) cell transfer corrected the immunosuppression of late sepsis by (i) producing significantly higher levels of proinflammatory mediators upon ex vivo stimulation with the Toll-like receptor 4 (TLR4) agonist lipopolysaccharide, (ii) enhancing phagocytic activity of peritoneal macrophages, and (iii) clearing bacterial peritonitis. Improved immunity by CD34(+) cell transfer decreased inflammatory peritoneal exudate of surviving late-sepsis mice. Cell tracking experiments showed that the transferred CD34(+) cells first appeared in the bone marrow and then homed to the spleen and peritoneum. Because CD34(+) cells did not affect the early-phase hyperinflammatory response, it is likely that the newly incorporated pluripotent CD34(+) cells differentiated into competent immune cells in blood and tissue, thereby reversing or replacing the hyporesponsive endotoxin-tolerant cells that occur and persist after the initiation of early sepsis.

Adoptive transfer of CD34(+) cells during murine sepsis rebalances macrophage lipopolysaccharide responses.

Effective treatment of the acute systemic inflammatory response associated with sepsis is lacking, but likely will require new ways to rebalance dysregulated immune responses. One challenge is that human sepsis often is diagnosed too late to reduce the hyperinflammation of early sepsis. Another is that the sequential response to sepsis inflammation rapidly generates an adaptive and immunosuppressive state, which by epigenetic imprint may last for months or years. Emerging data support that the immunosuppressive phase of sepsis can both directly reprogram gene expression of circulating and tissue cells, and disrupt development and differentiation of myeloid precursor cells into competent immunocytes. We recently reported that adoptive transfer of bone marrow CD34(+) cells into mice after sepsis induction by cecal ligation and puncture significantly improves late-sepsis survival by enhancing bacterial clearance through improved neutrophil and macrophage phagocytosis. That study, however, did not examine whether CD34(+) transfer can modify noninfectious acute systemic inflammatory responses. Here, we report that CD34(+) cell transfer mice that have survived late sepsis also resist lethal lipopolysaccharide (LPS)-induced inflammatory shock (88% lived vs 0% of naive mice). The CD34(+) cell-recipient survivor mice administered LPS had globally reduced levels of circulating inflammatory mediators compared with naive mice, but their peritoneal and bone marrow-derived macrophages (BMDMs), unlike those from naïve mice, remained LPS responsive ex vivo. We further found that CD34(+) cell transfer into LPS-challenged naïve mice had diminished immunosuppression, as assessed by ex vivo responses of peritoneal and BMDMs to LPS challenge. We conclude that CD34(+) cell adoptive transfer rebalances dysregulated immune responses associated with sepsis and endotoxin shock.

Leukocyte Dectin-1 expression is differentially regulated in fungal versus polymicrobial sepsis.

OBJECTIVE: To examine peripheral leukocyte Dectin-1 regulation in clinically relevant models of fungal and polymicrobial sepsis. DESIGN: Prospective animal study. SETTING: University medical school research laboratory. SUBJECTS: Age, weight, and sex matched ICR/HSD mice. INTERVENTIONS: Mice were infected with Candida albicans (1 x 10, intravenously) or were subjected to cecal ligation and puncture to induce polymicrobial sepsis. MEASUREMENTS: Blood, spleen, and peritoneal exudate were harvested and leukocytes were isolated. Leukocytes were evaluated for membrane-associated Dectin-1 expression and cell phenotype by flow cytometry. MAIN RESULTS: In C. albicans infection, Dectin-1-positive blood and splenic leukocytes were increased from 23.5% to 58.9% over the course of infection. The increased percentage of Dectin-1-expressing cells was primarily attributable to neutrophilia. However, the amount of Dectin-1 expressed by blood and splenic neutrophils in C. albicans-infected mice was decreased by a range of 49.0% to 53.3%. C. albicans infection also resulted in an infiltration of Dectin-1-positive macrophages and neutrophils into the kidney. In contrast, polymicrobial sepsis decreased blood leukocyte Dectin-1-expressing cells by up to 51.4%. This reduction was due to a decrease in Dectin-1-positive neutrophils in the periphery. However, the percentage of Dectin-1-expressing cells in the peritoneal cavity increased by 774% with cecal ligation and puncture. Treatment of isolated neutrophils with three soluble glucans, mannan, lipopolysaccharide, or a variety of cytokines revealed that glucans, alone or in combination, were the only treatment that resulted in a decrease in Dectin-1-positive neutrophils. CONCLUSIONS: We conclude that peripheral leukocyte Dectin-1 expression is differentially regulated in fungal vs. polymicrobial sepsis. These data demonstrate that leukocyte Dectin-1 levels are modulated in response to infections of fungal and nonfungal origin.

Corynebacterium striatum: an underappreciated community and nosocomial pathogen.

Corynebacterium striatum (CS) is an underappreciated human pathogen that has been associated with serious infections in both immunocompetent and immunocompromised hosts. CS infections tend to be more frequent in males and major infection sites have included blood stream, lung, and central nervous system. Most are nosocomially acquired and there is a significant association with medical devices ranging from intravascular catheters to central nervous system drainage devices. Empiric therapy with vancomycin is advisable as susceptibility to other agents is variable. Treatment may also include removal of foreign material such as an intravascular catheter. The present review describes the wide spectrum of infections associated with CS and we add a unique case of CS pancreatic abscess where treatment included linezolid.

Structural characterization of (1-->3)-beta-D-glucans isolated from blastospore and hyphal forms of Candida albicans.

Glucans are (1-->3)-beta-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.

Mitogen-activated protein kinase phosphatase 1 disrupts proinflammatory protein synthesis in endotoxin-adapted monocytes.

Autotoxic production of proinflammatory mediators during early sepsis induces excessive inflammation, and their later suppression may limit the immune response. We previously reported that sepsis differentially represses transcription and translation of tumor necrosis factor alpha (TNF-α) and interleukin 1ß (IL-1ß) to reprogram sepsis inflammation. This switch is gene specific and plays a crucial role in the clinically relevant syndrome of endotoxin adaptation/tolerance, multiorgan failure, and poor sepsis outcome. To further define the mechanisms responsible for translation disruption that follows inflammation induction, we used THP-1 human promonocytes as a model of Toll-like receptor 4 (TLR4) responses found in sepsis. We showed that phosphorylation-dependent activation of p38 mitogen-activated protein kinase (MAPK) and translation disruption of TNF-α and IL-6 follow increased MAPK phosphatase 1 (MKP-1) expression and that MKP-1 knockdown rephosphorylates p38 and restores the capacity to translate TNF-α and IL-6 mRNAs. We also observed that the RNA-binding protein motif 4 (RBM4), a p38 MAPK target, accumulates in an unphosphorylated form in the cytosol in endotoxin-adapted cells, suggesting that dephosphorylated RBM4 may function as a translational repressor. Moreover, MKP-1 knockdown promotes RBM4 phosphorylation, blocks its transfer from the nucleus to the cytosol, and reverses translation repression. We also found that microRNA 146a (miR-146a) knockdown prevents and miR-146a transfection induces MKP-1 expression, which lead to increases or decreases in TNF-α and IL-6 translation, respectively. We conclude that a TLR4-, miR-146a-, p38 MAPK-, and MKP-1-dependent autoregulatory pathway regulates the translation of proinflammatory genes during the acute inflammatory response by spatially and temporally modifying the phosphorylation state of RBM4 translational repressor protein.

The protective function of human C-reactive protein in mouse models of Streptococcus pneumoniae infection.

Human C-reactive protein (CRP), injected intravenously into mice or produced inside mice by a human transgene, protects mice from death following administration of lethal numbers of Streptococcus pneumoniae. The protective effect of CRP is due to reduction in the concentration of bacteria in the blood. The exact mechanism of CRP-dependent killing of pneumococci and the partners of CRP in this process are yet to be defined. The current efforts to determine the mechanism of action of CRP in mice are directed by four known in vitro functions of CRP: 1. the ability of pneumococcal C-polysaccharide-complexed CRP to activate complement pathways, 2. the ability of CRP to bind to Fcgamma receptors on phagocytic cells, 3. the ability of CRP to bind to immobilized complement regulator protein factor H which can also be present on pneumococci, and, 4. the ability of CRP to interact with dendritic cells. CRP-treated dendritic cells may well be as host-defensive as CRP alone. An interesting condition for the protective function of CRP is that CRP must be given to mice within a few hours of the administration of pneumococci. CRP does not protect mice if given later, suggesting that CRP works prophylactically but not as a treatment for infection. However, full knowledge of CRP may lead to the development of CRP-based treatment strategies to control pneumococcal infection. Also, because CRP deficiency in humans has not yet been reported, it becomes important to investigate the deficiency of the mechanism of action of CRP in CRP-positive individuals.

Human C-reactive protein protects mice from Streptococcus pneumoniae infection without binding to pneumococcal C-polysaccharide.

Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection of mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcgamma receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.

Role of the property of C-reactive protein to activate the classical pathway of complement in protecting mice from pneumococcal infection.

C-reactive protein (CRP) is not an acute-phase protein in mice, and therefore, mice are widely used to investigate the functions of human CRP. It has been shown that CRP protects mice from pneumococcal infection, and an active complement system is required for full protection. In this study, we assessed the contribution of CRP's ability of activating the classical pathway of complement in the protection of mice from lethal infection with virulent Streptococcus pneumoniae type 3. We used two CRP mutants, Y175A and K114A. The Y175A CRP does not bind C1q and does not activate complement in human serum. The K114A CRP binds C1q and activates complement more efficiently than wild-type CRP. Passively administered, both CRP mutants and the wild-type CRP protected mice from infection equally. Infected mice injected with wild-type or mutant CRP had reduced bacteremia, resulting in lower mortality and increased longevity compared with mice that did not receive CRP. Thus, the protection of mice was independent of CRP-mediated activation of the classical pathway of complement. To confirm that human CRP does not differentiate between human and mouse complement, we analyzed the binding of human CRP to mouse C1q. Surprisingly, CRP did not react with mouse C1q, although both mutant and wild-type CRP activated mouse C3, indicating species specificity of CRP-C1q interaction. We conclude that the mouse is an unfit animal for exploring CRP-mediated activation of the classical complement pathway, and that the characteristic of CRP to activate the classical complement pathway has no role in protecting mice from infection.

Comparison of cytotoxin genotypes of Helicobacter pylori in stomach and saliva.

We have previously reported a high prevalence of H. pylori DNA in saliva. In this study, the cytotoxin genotypes of H. pylori strains from both stomach and saliva were compared in 31 patients with gastritis and peptic ulcer. The cagA, vacA m1, vacA m2, and vacA s1 genotypes were analyzed by PCR. The 417 bp PCR products from three patients were also subjected to DNA sequencing analysis. There was 95% agreement between stomach H. pylori isolates and their corresponding saliva DNA in at least one cytotoxin genotype; 86% agreement with two cytotoxin genotypes; 59% agreement with three cytotoxin genotypes; and 27% agreement with all four cytotoxin genotypes studied. DNA sequencing from three patients showed 78.0%, 64.0%, and 66.9% homology of H. pylori from both sources, respectively. The data suggest that more than one H. pylori strain may exist in the stomach and saliva in the same patient.