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G protein-coupled receptor kinase 5 (GRK5) is a serine/threonine kinase previously shown to mediate polymicrobial sepsis-induced inflammation. The goal of the present study was to examine the role of GRK5 in monomicrobial pulmonary infection by using an intratracheal Escherichia coli infection model of pneumonia. We used sublethal and lethal doses of E. coli to examine the mechanistic differences between low-grade and high-grade inflammation induced by E. coli infection. With a sublethal dose of E. coli , GRK5 knockout (KO) mice exhibited higher plasma CXCL1/KC levels and enhanced lung neutrophil recruitment early after infection, and lower bacterial loads, than wild-type (WT) mice. The inflammatory response was also diminished, and resolution of inflammation advanced, in the lungs of GRK5 KO mice. In contrast to the reduced bacterial loads in GRK5 KO mice following a sublethal dose, at a lethal dose of E. coli , the bacterial burdens remained high in GRK5 KO mice relative to those in WT mice. This occurred in spite of enhanced plasma CXCL1 levels as well as neutrophil recruitment in the KO mice. But the recruited neutrophils (following high-dose infection) exhibited decreased CD11b expression and reduced reactive oxygen species production, suggesting decreased neutrophil activation or increased neutrophil exhaustion in the GRK5 KO mice. In agreement with the increased bacterial burden, KO mice showed poorer survival than WT mice following E. coli infection at a lethal dose. Overall, our data suggest that GRK5 negatively regulates CXCL1/KC levels during bacterial pneumonia but that the role of GRK5 in the clinical outcome in this model is dependent on the bacterial dose.

Recurrent urinary tract infections (UTIs), primarily caused by uropathogenic Escherichia coli (UPEC), annually affect over 13 million patients in the United States. Menopausal women are disproportionally susceptible, suggesting estrogen deficiency is a significant risk factor for chronic and recurrent UTI. How estrogen status governs susceptibility to UTIs remains unknown, and whether hormone therapy protects against UTIs remains controversial. Here, we used a mouse model of surgical menopause by ovariectomy and demonstrate a protective role for estrogen in UTI pathogenesis. We found that ovariectomized mice had significantly higher bacteriuria, a more robust inflammatory response, and increased production of the proinflammatory cytokine interleukin-6 (IL-6) upon UPEC infection compared to sham-operated controls. We further show that response of the urothelial stem cell niche to infection, normally activated to restore homeostasis after infection, was aberrant in ovariectomized mice with defective superficial urothelial cell differentiation. Finally, UPEC-infected ovariectomized mice showed a significant increase in quiescent intracellular bacterial reservoirs, which reside in the urothelium and can seed recurrent infections. Importantly, this and other ovariectomy-induced outcomes of UTI were reversible upon estrogen supplementation. Together, our findings establish ovariectomized mice as a model for UTIs in menopausal women and pinpoint specific events during course of infection that are most susceptible to estrogen deficiency. These findings have profound implications for the understanding of the role of estrogen and estrogen therapy in bladder health and pathogen defense mechanisms and open the door for prophylaxis for menopausal women with recurrent UTIs.

Bacterial pneumonia remains a significant cause of mortality in the United States. The innate immune response is the first line of defense against invading bacteria. Neutrophil recruitment to the lungs is the first step in a multistep sequence leading to bacterial clearance. Ligand interaction with pattern-recognizing receptors (PRRs) leads to chemokine production, which drives neutrophils to the site of infection. Although we demonstrated that RIP2 is important for host defense in the lungs against Escherichia coli , the individual roles of NOD1 and NOD2 in pulmonary defense have not been addressed. Here, we explored the role of NOD2 in neutrophil-mediated host defense against an extracellular pathogen, E. coli . We found enhanced bacterial burden and reduced neutrophil and cytokine/chemokine levels in the lungs of NOD2 −/− mice following E. coli infection. Furthermore, we observed reduced activation of NF-κB and mitogen-activated protein kinases (MAPKs) in the lungs of NOD2 −/− mice upon E. coli challenge. Moreover, NOD2 −/− neutrophils show impaired intracellular bacterial killing. Using NOD2/RIP2 −/− mice, we observed bacterial burden and neutrophil accumulation in the lungs similar to those seen with NOD2 −/− mice. In addition, bone marrow-derived macrophages obtained from NOD2/RIP2 −/− mice demonstrate a reduction in activation of NF-κB and MAPKs similar to that seen with NOD2 −/− mice in response to E. coli . These findings unveil a previously unrecognized role of the NOD2-RIP2 axis for host defense against extracellular Gram-negative bacteria. This pathway may represent a novel target for the treatment of lung infection/inflammation.

Recognition of microbial patterns by host receptors is the first step in a multistep sequence leading to neutrophil-dependent host resistance. Although the role of membrane-bound sensors in bacterial recognition has been examined in detail, the importance of cytosolic sensors in the lungs is largely unexplored. In this context, there is a major lack of understanding related to the downstream signaling mediators, such as cells and/or molecules, during acute extracellular Gram-negative bacterial pneumonia. In order to determine the role of NOD-like receptors (NLRs), we used an experimental Escherichia coli infection model using mice deficient in the gene coding for the NLR adaptor, receptor-interacting protein 2 (RIP2). RIP2 −/− mice with E. coli infection displayed higher bacterial burden and reduced neutrophil recruitment and tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP-2), and CXCL5/LIX expression, along with attenuated histopathological changes in the lungs. Decreased IL-17A levels were observed, along with lower numbers of IL-17A-producing T cells, in RIP2 −/− mice after infection. RIP2 −/− mice also show reduced IL-6 and IL-23 levels in the lungs, along with decreased activation of STAT3 after infection. Furthermore, activation of NF-κB and mitogen-activated protein kinases (MAPKs) and expression of intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) in the lungs of infected RIP2 −/− mice were attenuated following infection. Although neutrophil mobilization to the blood was impaired in RIP2 −/− mice following infection, the expression of CD62P, CD11a/18, CD11b, and CXCR2 on blood and lung neutrophils was not altered between infected wild-type (WT) and RIP2 −/− mice. Thus, RIP2 contributes to neutrophil-dependent host defense against an extracellular Gram-negative pathogen via (i) IL-17A regulation and (ii) neutrophil mobilization to the blood.

The interaction among gram-negative bacteria, the innate immune system, and soluble CD14 (sCD14) has not been well documented. The effect of recombinant bovine sCD14 (rbosCD14) on milk somatic cell count (SCC), bacterial clearance, and cytokine production was investigated by using a bovine intramammaryEscherichia coliinfection model. We first determined whether rbosCD14 would increase the SCC during a lipopolysaccharide (LPS) challenge. Three quarters of each of six healthy lactating cows were injected with either 0.3 μg of LPS, 0.3 μg of LPS plus 100 μg of rbosCD14, or saline. In comparison with quarters injected with LPS alone, the SCC was twofold higher (P< 0.05) in quarters injected with LPS plus rbosCD14 after the challenge. We therefore hypothesized that whenE.colibacteria invade the mammary gland, sCD14 in milk would interact with LPS and rapidly recruit neutrophils from the blood to eliminate the bacteria before establishment of infection. To test this hypothesis, two quarters of each of nine healthy cows were challenged with either 50 CFU ofE.coliplus saline or 50 CFU ofE.coliplus 100 μg of rbosCD14. Quarters challenged withE.coliplus rbosCD14 had a more rapid recruitment of neutrophils, which was accompanied by a faster clearance of bacteria, lower concentrations of tumor necrosis factor alpha and interleukin-8 in milk, and milder clinical symptoms, than challenged quarters injected with saline. Results indicate that increasing the concentration of sCD14 in milk may be a potential strategy with which to prevent or reduce the severity of infection by coliform bacteria.

The number of γδ T cells in the peritoneal cavity was increased after an intraperitoneal (i.p.) infection with Escherichia coli in lipopolysaccharide (LPS)-responsive C3H/HeN mice but not in LPS-hyporesponsive C3H/HeJ mice. The γδ T cells preferentially expressed invariant Vγ6 and Vδ1 chains and proliferated to produce a large amount of gamma interferon in the presence of LPS. Mice depleted of γδ T cells by T-cell receptor δ gene mutation showed impaired resistance against E. coli as assessed by bacterial growth. Macrophages from C3H/HeN mice infected with E. coli expressed higher levels of interleukin-15 (IL-15) mRNA than those from the infected C3H/HeJ mice. Administration of anti-IL-15 monoclonal antibody inhibited, albeit partially, the appearance of γδ T cells in C3H/HeN mice after E. coli infection and diminished the host defense against the infection. These results suggest that LPS-stimulated γδ T cells play an important role in the host defense against E. coli infection and that IL-15 may be partly involved in the protection via an increase in the γδ T cells.

Interleukin-6 (IL-6) is a multipotential cytokine detected in the serum of patients or experimental animals undergoing bacterial sepsis. To date, the role of IL-6 in gram-negative sepsis models has been controversial. We have used IL-6-deficient mice to investigate the role of IL-6 during virulent Escherichia coli infection and in lipopolysaccharide (LPS)-induced mortality. In this report we describe an increased susceptibility of IL-6-deficient mice to E. coli infection in terms of mortality and accumulation of viable bacteria in tissues, indicating a protective role for IL-6 during the immune response against E. coli. In contrast, mortality rates of IL-6-deficient mice and control animals undergoing LPS-induced shock did not differ, indicating that IL-6 was inconsequential for survival in this model. Furthermore, we have shown that neutrophils were crucial for resistance to E. coli in normal mice. IL-6-deficient mice were unable to efficiently induce neutrophilia in the bloodstream immediately following challenge with E. coli, in contrast to a characteristic neutrophilia induced in control animals. Prophylactic treatment of the mutant animals with recombinant IL-6 protein reverted both the deficit of neutrophilia and the accumulation of bacteria in tissues. These data clarify the role of IL-6 as protective in virulent E. coli infection and suggest that the protective effect may be at least partially mediated through neutrophils.

Serum obtained after immunization with an O18 polysaccharide-toxin A conjugate vaccine was evaluated for the estimation of protective levels of anti-O-specific lipopolysaccharide (LPS) immunoglobulin G (IgG) antibody against bacteremia and death caused by a homologous serotype of Escherichia coli K1 strains. Passive transfer of rabbit serum conferred significant protection from a lethal E. coli infection in a neonatal rat model. The overall incidence of bacteremia and mortality was 4% in rat pups receiving undiluted postvaccination serum, while that in control animals was 100% (P < 0.001). The overall incidences of bacteremia were 5 and 72% for animals with serum anti-O18 LPS IgG concentrations of > 1.0 and < 1.0 microgram/ml, respectively, while the overall incidences of mortality for animals with serum anti-O18 LPS IgG levels of > 1.0 and < 1.0 microgram/ml were 0 and 72%, respectively (P < 0.001). Protection against E. coli infection was also demonstrated with human anti-O18 polysaccharide IgG. None of the animals with human anti-O18 LPS IgG levels of > 1 microgram/ml had bacteremia after bacterial challenge, whereas all animals with bacteremia at 18 h had levels of < 1 microgram/ml. These findings suggest that serum anti-O18 LPS IgG concentrations of > 1.0 microgram/ml may provide protection against bacteremia and death caused by a homologous E. coli K1 infection.

Acyloxyacyl hydrolase (AOAH), an enzyme that removes the secondary acyl chains of gram-negative bacterial lipid A (endotoxin), has been identified previously in human neutrophils and mouse macrophages. We report here that bovine leukocytes also contain AOAH activity. Although bovine AOAH deacylates bacterial lipopolysaccharide in a manner similar to human AOAH, it is active in vitro over a broader pH range, from 4.0 to 7.0. By using Escherichia coli infection of the bovine mammary gland as a model of localized gram-negative bacterial disease and associated tissue inflammation, AOAH activity per leukocyte increased. In addition, AOAH activity increased in the cell-free portion of infected mammary secretions. These data indicate that AOAH activity increases in leukocytes associated with inflammation induced by gram-negative bacteria and provide additional evidence of its potential involvement in the defense against the effects of bacterial endotoxin.

Volume 80, no. 7, p. [2558–2569][1], 2012. The authors hereby retract this article. After publication, this article was found to have evidence of data duplication as follows: (i) in Fig. 5F, the GAPDH (glyceraldehyde-3-phosphate dehydrogenase) control bands for NOD2/RIP2−/− mice at 6 h and

Neutrophil accumulation is a critical event to clear bacteria. Since uncontrolled neutrophil recruitment can cause severe lung damage, understanding neutrophil trafficking mechanisms is important to attenuate neutrophil-mediated damage. While monocyte chemoattractant protein 1 (MCP-1) is known to be a monocyte chemoattractant, its role in pulmonary neutrophil-mediated host defense against Gram-negative bacterial infection is not understood. We hypothesized that MCP-1/chemokine (C-C motif) ligand 2 is important for neutrophil-mediated host defense. Reduced bacterial clearance in the lungs was observed in MCP-1 −/− mice following Escherichia coli infection. Neutrophil influx, along with cytokines/chemokines, leukotriene B 4 (LTB 4 ), and vascular cell adhesion molecule 1 levels in the lungs, was reduced in MCP-1 −/− mice after infection. E. coli -induced activation of NF-κB and mitogen-activated protein kinases in the lung was also reduced in MCP-1 −/− mice. Administration of intratracheal recombinant MCP-1 (rMCP-1) to MCP-1 −/− mice induced pulmonary neutrophil influx and cytokine/chemokine responses in the presence or absence of E. coli infection. Our in vitro migration experiment demonstrates MCP-1-mediated neutrophil chemotaxis. Notably, chemokine receptor 2 is expressed on lung and blood neutrophils, which are increased upon E. coli infection. Furthermore, our findings show that neutrophil depletion impairs E. coli clearance and that exogenous rMCP-1 after infection improves bacterial clearance in the lungs. Overall, these new findings demonstrate that E. coli -induced MCP-1 causes neutrophil recruitment directly via chemotaxis as well as indirectly via modulation of keratinocyte cell-derived chemokine, macrophage inflammatory protein 2, and LTB 4 .

Mouse hybridoma antibody of immunoglobulin class M prepared with live group B meningococci was evaluated for its ability to protect against and treat Escherichia coli infections in a newborn-rat model. In these studies, antibody was administered intraperitoneally and bacteria were administered subcutaneously to avoid introducing the antibody and bacteria to the same site. The activity of this hybridoma antibody was specific; the antibody provided protection against the K-1 strain, but not against the K-92 strain. In addition, the amount of the antibody required for protection was dependent upon the size of bacterial challenge. With an increase of the bacterial inocula from the 100% lethal dose to 10 times the 100% lethal dose there was a threefold increase in the amount of the antibody required for 50% protection. Similarly, therapeutic efficacy of the antibody was also dependent upon the magnitude of bacteremia before therapy. The antibody successfully cleared the bacteremia only when the pretherapy bacterial counts in blood were less than 10(4) CFU/ml. These findings suggest that the monoclonal immunoglobulin M antibody against the capsular polysaccharide of the group B meningococcus may be useful in the prevention and treatment of K-1 E. coli infections.

To determine whether expression of type 1 pili varies during the course of Escherichia coli infection in vivo, mice were injected intraperitoneally with 5 X 10(7) CFU of piliated or nonpiliated phase variants per ml, and the degree of piliation was measured in peritoneal exudate by an enzyme-linked immunosorbent assay inhibition method. In the animals challenged with the piliated bacteria, the numbers of organisms increased a log over 9 h and the amount of pilus antigen decreased from 3 to 0.075 micrograms/10 bacteria. After a 4-h delay, nonpiliated bacteria also increased by one log over 9 h; however, the amount of piliation remained virtually undetectable. Piliated E. coli were more virulent than nonpiliated variants in this model (50% lethal dose of 7.5 X 10(6) versus 3 X 10(7), respectively). The difference was significantly reduced by prior passive immunization with rabbit serum containing high titers of antipili antibody. Piliated bacteria adhered in significantly greater numbers to isolated mouse peritoneal membranes than did nonpiliated variants (15,400 +/- 2,700 versus 1,300 +/- 700 bacteria/mm2, respectively; P = 0.05). Adherence was inhibited by the presence of 0.1 M alpha methyl mannose (1,500 +/- 1,800 bacteria/mm2, P = 0.01). These results confirm the results of previous qualitative studies showing that phase variation of type 1 pili occurs in vivo and suggest that these pili may confer an initial advantage for growth of E. coli in the peritoneal cavity, presumably by fostering colonization of the peritoneal serosal surface.

The effect of a chemically synthesized polyprenol derivative, dihydroheptaprenol (DHP), on the nonspecific resistance of mice to infection with Escherichia coli was investigated. Mice that had been injected intramuscularly with 100 mg of DHP per kg of body weight, prepared as a microemulsion with lecithin, 1 to 4 days before infection showed enhanced resistance to subcutaneous (s.c.) infection with E. coli. When DHP-injected mice were inoculated s.c. with 3 X 10(8) E. coli, which induces fatal acute systemic infection in normal mice, propagation of bacteria in the blood, liver, and spleen was significantly inhibited. Enhanced resistance of athymic (nude) mice to E. coli infection was also induced by DHP. DHP markedly stimulated the generation of peripheral blood neutrophils, significantly enhanced clearance of E. coli from the bloodstream, and activated neutrophils and peritoneal macrophages for H2O2 generation. DHP restored the resistance to E. coli infection in cyclophosphamide-treated mice over the normal level. Furthermore, DHP shortened the period of the recovery of neutrophils and also enhanced clearance of E. coli from the bloodstream in cyclophosphamide-treated mice. DHP was nontoxic for mice and rats (400 mg/kg intramuscularly and 800 mg/kg s.c.) and nonpyrogenic at a dose of 30 mg/kg when administered intravenously to rabbits. These results suggest that the mechanism of action of DHP for enhancing resistance in mice may be, at least in part, its ability to stimulate the generation of potent neutrophils and to activate macrophages in the reticuloendothelial system.

Protection against Escherichia coli inoculated intraperitoneally into mice was enhanced by intraperitoneal, intravenous, or subcutaneous administration of a water-soluble, high-molecular-weight fraction extracted from a dialyzed hot-water extract from a strain of Chlorella vulgaris (CVE-A). The enhancing effect was detected with doses over 2.0 mg/kg and when doses were administered 1, 4, or 7 days before the infection. The elimination of bacteria from the spleen of CVE-A-treated mice was increased, and this enhanced elimination may have been related to the acceleration of superoxide generation and chemokinesis in polymorphonuclear leucocytes by CVE-A treatment. A cyclophosphamide-induced decrease in protection against E. coli could be prevented by subcutaneous administration of CVE-A.

Lipid X, the major monosaccharide precursor of lipid A, is nontoxic and has previously been shown to protect mice and sheep from the harmful effects of endotoxin. To test the hypothesis that lipid X could be therapeutic against infections with gram-negative organisms, neutropenic ICR mice were infected by intramuscular inoculation of Escherichia coli and subsequently treated with lipid X alone or in combination with the antibiotic ticarcillin. Lipid X slightly prolonged survival; treated mice had a significantly improved rate of survival 18 h after intramuscular inoculation as compared with controls (P less than 0.025). By 24 h, however, this difference disappeared. When lipid X was combined with ticarcillin, survival differences were both significant and prolonged. Treatment of mice with one to two doses of lipid X for a total dose of 1 mg intravenously and with 1,200 mg of ticarcillin per kg every 6 h improved survival over a 48-h treatment period from 5 to 23% (P less than 0.0025). Treatment with lipid X and ticarcillin over a broad range of antibiotic dosages in 362 mice demonstrated improved survival of two- to fourfold (P less than 0.0001 at 24 h after inoculation, P less than or equal to 0.0005 at 48 h, and P less than or equal to 0.0001 at 5 days). Lipid X enabled the dose of ticarcillin necessary to protect 50% of mice from death to be reduced by two- to fivefold. Pretreatment with lipid X was not necessary to improve survival: 16 of 17 (94%) infected and visibly ill animals that received lipid X and ticarcillin 6 h after thigh inoculation survived versus 30 of 44 (68%) control animals treated with ticarcillin alone (P less than 0.0001). Lipid X had no antimicrobial activity in vitro. Lipid X is a novel agent that enhances survival in an animal model of severe infection with gram-negative organisms.