Donald A. Ferguson

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

ABSTRACT 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 β (TGF-β). 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.

Early Activation of Hepatic Nfκb and Nf-il6 in Polymicrobial Sepsis Correlates With Bacteremia, Cytokine Expression, and Mortality

BACKGROUND The role of transcription factor activation in the pathophysiology of sepsis syndrome has not been established. This study investigated the relation between tissue nuclear factor kappaB (NFkappaB) and nuclear factor interleukin 6 (NF-IL6 or C/EBP) activation and bacteremia, inflammatory cytokine expression, and mortality in a murine model of cecal ligation and puncture (CLP). METHODS Transcription factor activation was assessed by the electrophoretic mobility shift assay. Cytokine mRNA levels were established by reverse transcription-polymerase chain reaction and quantified by scanning densitometry. Bacteremia was evaluated by standard aerobic and anaerobic microbiologic methods. RESULTS CLP stimulated hepatic NFkappaB activation at 2, 3, 4, 5, 6, and 8 hours compared with control and sham-operated mice. Hepatic NFkappaB activation during CLP peaked at 4 hours (1114% vs. no surgery, 609% vs. sham). Hepatic NF-IL6 activation was observed at 3, 4, and 6 hours after CLP. Hepatic and splenic levels of tumor necrosis factor-alpha and IL-6 mRNA were also elevated after CLP. Bacteremia in CLP mice consisted of Bacteroides species and to a lesser extent facultative gram-negative bacilli and group D Enterococcus. CONCLUSIONS Early activation of hepatic and splenic NFkappaB and NF-IL6 positively correlates with tissue cytokine mRNA expression and mortality in a surgical model of polymicrobial sepsis. The data suggest that transcription factor activation is an early event in the pathophysiology of sepsis.

MicroRNA 21 (miR-21) and miR-181b Couple with NFI-A To Generate Myeloid-Derived Suppressor Cells and Promote Immunosuppression in Late Sepsis

ABSTRACT 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 β (TGF-β) 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.

Early Activation of Pulmonary Nuclear Factor kappa B and Nuclear Factor Interleukin-6 in Polymicrobial Sepsis

BACKGROUND Transcription factor activation may be a pivotal step in the pathophysiology of sepsis syndrome and adult respiratory distress syndrome. This study investigated the activation of lung nuclear factor kappaB (NFkappaB) and nuclear factor interleukin-6 (NF-IL6) and how they correlate to proinflammatory cytokine expression and mortality in a murine model of cecal ligation and puncture (CLP). METHODS Polymicrobial sepsis was induced by CLP. Transcription factor activation was assessed at 0, 1, 2, 3, 4, 5, 6, 8, and 24 hours after CLP by the electrophoretic mobility-shift assay. Lung cytokine mRNA levels were established by reverse transcriptase-polymerase chain reaction. RESULTS CLP induced pulmonary NFkappaB activation at 3, 4, and 8 hours (p < 0.05). Lung NFkappaB activation peaked at 3 hours (533% vs. no surgery, 2,900% vs. sham treatment) after CLP. Supershift analysis revealed a predominance of p50 subunits in the lung nuclear extracts of septic mice 3 hours after CLP, indicating the presence of p50 homodimer. In contrast, liver nuclear extracts from septic mice indicated the presence of both p65 and p50 subunits at 3 hours. Lung NF-IL6 activation (p < 0.05) was observed at 4 hours (649% vs. no surgery, 296% vs. sham treatment) and 6 hours after CLP. Lung tumor necrosis factor-alpha mRNA levels were increased (p < 0.05) at all time intervals after CLP. Lung IL-6 mRNA levels were increased at 3, 6, and 8 hours after CLP. CONCLUSION Early activation of lung NFkappaB and NF-IL6 and lung cytokine mRNA expression correlated with mortality in polymicrobial sepsis. Although IL-6 mRNA levels correlated with NFkappaB and NF-IL6 activation, tumor necrosis factor-alpha mRNA levels did not, in that they preceded transcription factor activation. These data suggest a potential role for NFkappaB and NF-IL6 activation in the initiation and propagation of acute lung injury.

Hematopoietic Stem-Progenitor Cells Restore Immunoreactivity and Improve Survival in Late Sepsis

ABSTRACT 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.

Identification of H. pylori in saliva by a nested PCR assay derived from a newly cloned DNA probe

A novel probe was developed from genomic DNA ofHelicobacter pylori ATCC type strain 43629. Ithybridized with all 73 H. pylori clinical isolatestested but not with any of 183 non-H. pylori DNAs in dot blot hybridization. Typing tests revealed 41different HaeIII-digestion patterns from 57 H. pyloristrains tested. Based on the sequence of the probe, anested PCR was developed that detected as little as 2 fg of H. pylori DNA or approximatelyequivalent to one cell. No PCR products were amplifiedfrom any of 21 non-H. pylori strains tested. Using thisnested PCR, H. pylori DNA was detected in 33 of 45 (73%) saliva samples collected from patientswith gastric H. pylori infection. These data suggestthat the probe is useful for typing H. pylori and thatthe nested PCR is a valuable tool for detecting H. pylori DNA in saliva.

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.

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 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.

Evaluation of two string tests for obtaining gastric juice for culture, nested-PCR detection, and combined single- and double-stranded conformational polymorphism discrimination of Helicobacter pylori.

We have compared two gastric string tests forobtaining gastric juice for culture of Helicobacterpylori and for nested-PCR detection and PCR-basedcombined single- and double-stranded conformationalpolymorphism (SDSCP) discrimination of infecting strains.String test specimens were obtained from oneseropositive volunteer for 13 consecutive weeks. Thedistal 10 cm of each string was suspended in 1 ml salineand quantitatively cultured. An additional ninevolunteers with histories of upper-gastrointestinalcomplaints were given a string test for culture andnested-PCR assay. H. pylori isolates and/or gastricjuice from each volunteer were extracted for DNA andanalyzed by PCR-based SDSCP. Quantitative culture showedthat the Entero-test was four times as sensitive as theGastro-test but was more prone to contamination by oral flora. However, the two string testsare equally sensitive by PCR assays. Thus, theGastro-test is more suitable for culture detection of H.pylori, since it is less prone to oral contamination and its shorter length is better tolerated.SDSCP analysis of H. pylori DNA from four PCR-positivevolunteers without requiring culture showed fourdistinct profiles, indicating different infectingstrains. SDSCP analysis of strains isolated before andafter treatment of one volunteer had the same SDSCPprofile, suggesting endogenous reinfection by the samestrain.