c 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 ϳ88% 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 ␤ (TGF-␤). 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.

Aims-To investigate the prevalence of Helicobacterpylori in the saliva ofpatients infected with this bacterium. Methods-A novel polymerase chain reaction (PCR) assay was developed to detect Hpylori in saliva and gastric biopsy specimens from patients undergoing endoscopy. Results-Our PCR assay amplified a 417 base pair fragment of DNA from all 21DNAs derived from H pylon clinical isolates but did not amplify DNA from 23 non-H pylon strains. Sixty three frozen gastric biopsy and 56 saliva specimens were tested. H pylon specific DNA was detected by PCR in all 39 culture positive biopsy specimens and was also identified from another seven biopsy specimens which were negative by culture but positive by histology. H pylon specific DNA was identified by PCR in saliva specimens from 30 (75%) of 40 patients with H pylori infection demonstrated by culture or histological examination, or both, and in three patients without H pylon infection in the stomach. Conclusion-The results indicate that the oral cavity harbours H pylon and may be the source of infection and transmission. (J Clin Pathol 1995;48:662-666)

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 ␤ (TGF-␤) 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 miR181b to improve late-sepsis survival.

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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 endotoxintolerant cells that occur and persist after the initiation of early sepsis. Sepsis is a major clinical problem (9, 52), with more than a 40% mortality rate, and is the leading cause of death in intensive care units (5,17). Evidence supports that the pathophysiology of sepsis varies as it moves from an initiating early/acute hyperinflammatory phase to a late/chronic hypoinflammatory and immunosuppressive phase (31,47,51,67). The early phase of sepsis is typified by a systemic inflammatory response syndrome (SIRS) characterized by excessive production of proinflammatory mediators by neutrophils and macrophages (53), increased generation of reactive oxygen species, and leukocyte-induced microvascular injury and organ failure (35). These destructive inflammatory responses occur in human (28) and animal (46, 51) sepsis, producing multiorgan dysfunction.While the early systemic inflammatory reaction of sepsis often spans several days (47, 61) and is considered a normal defense, the transition to a compensatory anti-inflammatory response syndrome (sometimes called CARS) to limit damage generates immunosuppression and promotes chronic infection (6, 12). CARS is characterized by downregulation in the ability of leukocytes to express proinflammatory mediators, impaired phagocytic capacity of neutrophils and macrophages (33,40,50), and significant apoptosis of lymphocytes and dendritic cells (16,29). Previous studies ha...

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.

Background: CRP exerts antipneumococcal function in mice if administered during the early stages of pneumococcal infection. Results: CRP loses such antipneumococcal function when its phosphocholine-binding pocket is blocked. Conclusion:The phosphocholine-binding pocket on CRP participates in antipneumococcal function of CRP during the early stages of infection. Significance: Remodeling of CRP may be required for successful treatment of mice during the late stages of infection.

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 Fcγ 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.