Cigarette Smoke Exposure Impairs Pulmonary Bacterial Clearance and Alveolar Macrophage Complement-Mediated Phagocytosis of Streptococcus pneumoniae

Cigarette smoke exposure increases the risk of pulmonary and invasive infections caused by Streptococcus pneumoniae, the most commonly isolated organism from patients with community-acquired pneumonia. Despite this association, the mechanisms by which cigarette smoke exposure diminishes host defense against S. pneumoniae infections are poorly understood. In this study, we compared the responses of BALB/c mice following an intratracheal challenge with S. pneumoniae after 5 weeks of exposure to room air or cigarette smoke in a whole-body exposure chamber in vivo and the effects of cigarette smoke on alveolar macrophage phagocytosis of S. pneumoniae in vitro. Bacterial burdens in cigarette smoke-exposed mice were increased at 24 and 48 h postinfection, and this was accompanied by a more pronounced clinical appearance of illness, hypothermia, and increased lung homogenate cytokines interleukin-1β (IL-1β), IL-6, IL-10, and tumor necrosis factor alpha (TNF-α). We also found greater numbers of neutrophils in bronchoalveolar lavage fluid recovered from cigarette smoke-exposed mice following a challenge with heat-killed S. pneumoniae. Interestingly, overnight culture of alveolar macrophages with 1% cigarette smoke extract, a level that did not affect alveolar macrophage viability, reduced complement-mediated phagocytosis of S. pneumoniae, while the ingestion of unopsonized bacteria or IgG-coated microspheres was not affected. This murine model provides robust additional support to the hypothesis that cigarette smoke exposure increases the risk of pneumococcal pneumonia and defines a novel cellular mechanism to help explain this immunosuppressive effect.Pneumococcal pneumonia, caused by the Gram-positive pathogen Streptococcus pneumoniae, is the most common form of community-acquired pneumonia in the United States and worldwide (24, 26). This organism can disseminate from the respiratory tract and is the leading cause of death from invasive bacterial infections, with antibiotic-resistant strains becoming increasingly more common (18, 26). Cigarette smoke (CS) exposure increases the risk of serious pneumococcal infections in humans (2, 29), although the mechanisms underlying this effect are not known. Consistent with increased risks of many infectious diseases among smokers (3), animal models have been used to demonstrate impairments in host defense against viral (13, 33), fungal (8), and bacterial (11) infections in smoke-exposed animals. To our knowledge, no reports exist which demonstrate the effects of CS exposure on host defense in a murine model of pneumococcal pneumonia, despite the clinical significance of this pathogen.The alveolar macrophage (AM) is a specifically differentiated resident phagocyte in the pulmonary alveoli that acts to maintain an environment free of pathogens and debris (27). Under normal conditions, AMs constitute the majority of immune cells within the alveolar space and act as a first line of innate host defense in the lung, using an array of receptors to recognize pathogen-associated molecular patterns (PAMPs) and to facilitate phagocytic uptake (36). Normally, AM function is tightly regulated to prevent inappropriate inflammation that could result in lung damage (1), but under conditions which overwhelm their clearance capacity, AMs play additional roles in the generation and subsequent resolution of inflammation and leukocyte recruitment (28, 37). Murine models of pulmonary pneumococcal infection have shown increased mortality (22) and bacterial burden (10) following AM depletion, indicating their importance in the innate host defense against such infections. Phagocytosis of S. pneumoniae is enhanced following opsonization with complement fragments C3b and C3bi, which adhere to the surfaces of bacteria. The critical importance of C3 in this context was recently demonstrated by studies reporting defects in host defense against pneumococcal pneumonia (19, 34).The increased susceptibility of smokers to pneumococcal pneumonia is incompletely understood, and no reports to date have assessed the effects of CS exposure on AM phagocytosis of pneumococcus, although many studies have demonstrated impairments in phagocytosis of other targets (15, 16, 21, 30, 31). Therefore, we determined the effects of CS exposure on pulmonary host defense against pneumococcal pneumonia in a murine model and assessed the effects of CS on AM phagocytosis of S. pneumoniae in vitro.     

The Alveolar Microenvironment of Patients Infected with Human Immunodeficiency Virus Does Not Modify Alveolar Macrophage Interactions with Streptococcus pneumoniae

We tested the hypothesis that HIV infection results in activation of alveolar macrophages and that this might be associated with impaired defense against pneumococcus. We compared alveolar macrophages and lymphocytes in 131 bronchoalveolar lavage samples from HIV-infected and healthy controls using inflammatory gene microarrays, flow cytometry, real-time PCR, and enzyme-linked immunosorbent assay (ELISA) to determine the pattern of macrophage activation associated with HIV infection and the effect of this activation on defense against pneumococcus. We used gamma interferon (IFN-γ) priming to mimic the cellular milieu in HIV-infected lungs. InnateDB and BioLayout 3D were used to analyze the interactions of the upregulated genes. Alveolar macrophages from HIV-infected adults showed increased gene expression and cytokine production in a classical pattern. Bronchoalveolar lavage from HIV-infected subjects showed excess CD8⁺ lymphocytes with activated phenotype. Toll-like receptor 4 (TLR4) expression was increased in macrophages from HIV-infected subjects, but function was similar between the groups; lung lavage fluid did not inhibit TLR function in transfected HeLa cells. Alveolar macrophages from HIV-infected subjects showed normal binding and internalization of opsonized pneumococci, with or without IFN-γ priming. Alveolar macrophages from HIV-infected subjects showed classical activation compared to that of healthy controls, but this does not alter macrophage interactions with pneumococci.     

Porous Implants Modulate Healing and Induce Shifts in Local Macrophage Polarization in the Foreign Body Reaction

The foreign body reaction (FBR) to implanted materials is of critical importance when medical devices require biological integration and vascularization to support their proper function (e.g., transcutaneous devices, implanted drug delivery systems, tissue replacements, and sensors). One class of materials that improves FBR outcomes is made by sphere-templating, resulting in porous structures with uniform, interconnected 34 μm pores. With these materials we observe reduced fibrosis and increased vascularization. We hypothesized that improved healing is a result of a shift in macrophage polarization, often measured as the ratio of M1 pro-inflammatory cells to M2 pro-healing cells. In this study, macrophage polarity of 34 μm porous implants was compared to non-porous and 160 μm porous implants in subcutaneous mouse tissue. Immunohistochemistry revealed that macrophages in implant pores displayed a shift towards an M1 phenotype compared to externalized cells. Macrophages in 34 μm porous implants had up to 63% greater expression of M1 markers and up to 85% reduction in M2 marker expression (p < 0.05). Macrophages immediately outside the porous structure, in contrast, showed a significant enrichment in M2 phenotypic cells. This study supports a role for macrophage polarization in driving the FBR to implanted materials.     

Effect of Trace Metals on Phagocytosis by Alveolar Macrophages

Experiments were performed to measure the effect of trace metals on a vital function of the alveolar macrophage (AM), phagocytosis. Since certain trace metals were found to reduce the viability of AMs, a technique was developed to permit examination of live cells only for phagocytosis. Evidence is presented that Ni(2+) selectively altered the phagocytic activity of AMs at concentrations lower than those which caused cell death. It is further shown that a level of VO(3) (-) that caused extensive lysis and death did not reduce phagocytosis in surviving cells. The effects of Cd(2+), Cr(3+), and Mn(2+) on AMs were also examined.     

Phagocytosis and Killing of Streptococcus pyogenes by Human Alveolar Macrophages

In contrast to human polymorphonuclear leukocytes and monocytes, alveolar macrophages were able to readily phagocytose and kill an M protein-positive Streptococcus pyogenes strain after opsonization in normal human serum.     

Management of KPC-producing Klebsiella pneumoniae infections

BackgroundKlebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) has become one of the most important contemporary pathogens, especially in endemic areas.AimsTo provide practical suggestion for physicians dealing with the management of KPC-KP infections in critically ill patients, based on expert opinions.SourcesPubMed search for relevant publications related to the management of KPC-KP infections.ContentsA panel of experts developed a list of 12 questions to be addressed. In view of the current lack of high-level evidence, they were asked to provide answers on the bases of their knowledge and experience in the field. The panel identified several key aspects to be addressed when dealing with KPC-KP in critically ill patients (preventing colonization in the patient, preventing infection in the colonized patient and colonization of his or her contacts, reducing mortality in the infected patient by rapidly diagnosing the causative agent and promptly adopting the best therapeutic strategy) and provided related suggestions that were based on the available observational literature and the experience of panel members.ImplicationsDiagnostic technologies could speed up the diagnosis of KPC-KP infections. Combination treatment should be preferred to monotherapy in cases of severe infections. For non–critically ill patients without severe infections, results from randomized clinical trials are needed for ultimately weighing benefits and costs of using combinations rather than monotherapy. Multifaceted infection control interventions are needed to decrease the rates of colonization and cross-transmission of KPC-KP.     

自制复方中药汤剂灌胃对小鼠淋巴细胞转化率和巨噬细胞吞噬功能的影响

目的:观察自制复方中药汤剂对实验小鼠淋巴细胞转化率和巨噬细胞吞噬功能的影响。方法:取昆明小鼠40只,分为对照组、复方中药低剂量组(2.5mg/kg/天)、中剂量组(2.5mg/kg/天)和高剂量组(50mg/kg/天),连续灌胃给药15天后,采尾静脉血瑞氏染色观测淋巴细胞转化率,取腹腔液瑞氏染色观测巨噬细胞吞噬功能。结果:三种剂量复方中药均可明显提高淋巴细胞的转化率(与对照组比较,P<0.01)。各组巨噬细胞吞噬功能与对照组比较显著增强(P<0.01),且中剂量组明显高于低、高剂量组(P<0.05)。结论:复方中药汤剂灌胃对小鼠免疫功能有明显的调节和增强作用。     

Effect of Azelastine Hydrochloride on Macrophage Chemotaxis and Phagocytosis in Vitro

Azelastine, a newly synthesized anti-allergic agent, was tested for its effects on guinea pig macrophage chemotaxis and phagocytosis. As specific macrophage chemo-attractants, we used macrophage chemotactic factors a and c; separated and highly purified from inflamed skin sites. Macrophage chemotaxis induced by skin extract or chemotactic factors was significantly suppressed by a low concentration of the agent (1 microgram/ml); the effect was dose-dependent. The inhibition of chemotaxis was reversible, because chemotactic activity was restored when the agents was removed by washing cells before chemotactic assay. Inactivation of chemotactic factors was not detected by mixing azelastine and factors a and c. Azelastine may directly interact with macrophages to decrease their chemotactic responsiveness. beta-Glucuronidase activity in the medium and macrophages after phagocytosis of polystyrene latex particles was not affected by this agent at concentrations ranging from 1 to 10 micrograms/ml. The phagocytosis of latex particles or sheep red blood cells opsonized with IgG antibodies (EA) and anchoring of macrophages to substrate were not inhibited and azelastine did not damage the macrophages as determined by lactate dehydrogenase (LDH) release assay.     

Leptin modulates neutrophil phagocytosis of Klebsiella pneumoniae

Leptin is a pleiotropic hormone-cytokine known to regulate energy homeostasis and immune function. Neutrophils from leptin-deficient mice exhibited impaired phagocytosis of Klebsiella pneumoniae opsonized with serum containing complement and reduced CD11b expression that could be restored with exogenous leptin. These results suggest that leptin is required for normal neutrophil complement-mediated phagocytosis of bacteria.     

异形矿物粉尘巨噬细胞毒性研究

为研究粉尘样品对兔肺泡巨噬细胞（AM）产生的不同毒性。探讨巨噬细胞受损的机制，采用体外细胞培养技术，观测兔肺泡巨噬细胞死亡率，丙二醛（MDA）、乳酸脱氢酶（LDH）及超氧化物歧化酶（SOD）的活性及变化。结果表明．沸石、硅灰石无细胞毒性，而其它的纤维状及颗粒状矿物粉尘则表现出不同程度的细胞毒性。纤维状矿物尘对AM毒性大于颗粒状矿物，毒性程度与粉尘中的活性OH^-含量正相关，但并不一定与SiO2含量相关。粉尘所形成的高pH值不利于细胞的生存，低生物持久性的粉尘对人体是安全的。粉尘中变价元素的含量可能影响其毒性。表面电位是粉尘毒性的非稳定因素。矿物尘的微形态是影响其毒性的因素之一．而矿物尘的毒性主要依赖于其特性。     

产超广谱β-内酰胺酶肺炎克雷伯菌耐药基因分析

目的 了解邢台地区产超广谱β-内酰胺酶(ESBLs)肺炎克雷伯菌的分子流行病学特点,为临床感染预防和治疗提供依据.方法 采用最小抑菌浓度(MIC)法检测产ESBLs肺炎克雷伯菌的耐药性,选取13种特异性引物采用聚合酶链反应(PCR)的方法检测产ESBLs肺炎克雷伯菌的基因类型.结果 2013年1月至2014年3月从邢台人民医院分离出的125株非重复的产ESBLs肺炎克雷伯菌菌株,耐药基因以blaCTX-M的阳性率最高,为92.0％,blaSHV和blaTEM次之,为82.9％和73.2％.所有菌株均携带1种或1种以上的耐药基因.结论 产ESBLs肺炎克雷伯菌多重耐药现象严重,耐药基因以blaCTX-M、blaSHV和blaTEM为主.医院应加强对ESBLs的监测,降低ESBLs的感染率以及耐药基因的突变,提高治疗感染的效率,防止医院感染的发生.     

Epidemiology of Quinolone Resistance of Klebsiella pneumoniae and Klebsiella oxytoca in Europe

 The epidemiology of quinolone resistance and the concomitant resistance to other antibiotic classes was investigated in 445 Klebsiella pneumoniae and 238 Klebsiella oxytoca isolates. Decreased susceptibility to ciprofloxacin was found in 7.2% and 3.4% of these two species, respectively. Ciprofloxacin resistance was significantly linked to ceftazidime resistance, the hallmark of extended-spectrum β-lactamase production, as well as to resistance to all antibiotic classes tested. Using automated ribotyping, seven intrahospital- and interhospital-transmitted clones of ciprofloxacin-resistant isolates were found. The newer fluoroquinolones sitafloxacin and clinafloxacin may become increasingly valuable, since they proved to be active also against ciprofloxacin-resistant isolates.     

Glucose Stimulates Phagocytosis of Unopsonized Pseudomonas aeruginosa by Cultivated Human Alveolar Macrophages

Glucose has previously been shown to increase the in vitro phagocytosis of unopsonized Pseudomonas aeruginosa by freshly explanted murine peritoneal macrophages (PM) and cultivated alveolar macrophages (AM). This study examined the effect of glucose on the same phagocytosis process in human AM in order to determine whether this phenomenon is conserved among species. Freshly explanted human AM phagocytosed unopsonized P. aeruginosa at a low level (2 bacteria/macrophage/30 min), whereas mouse AM ingested a negligible number of P. aeruginosa (0.01 bacterium/macrophage/30 min). Glucose had no effect on this or other phagocytic processes in freshly explanted mouse or human AM. However, following in vitro cultivation for 72 h, human AM phagocytosed three to four times more unopsonized P. aeruginosa than did freshly explanted cells, but only in the presence of glucose. This glucose-inducible phagocytic response had also been observed in cultivated murine AM. Although similar increases were also detected for the phagocytosis of latex particles and complement-coated sheep erythrocytes by cultivated human AM, these processes were not glucose dependent. The lack of response to glucose in freshly explanted mouse AM was attributed to insufficient glucose transport; however, freshly explanted human AM exhibited significant facilitative glucose transport activity that was inhibitable by cytochalasin B and phloretin. Taken together, these results suggest that the process of glucose-inducible phagocytosis of unopsonized P. aeruginosa is conserved among macrophages from different species, including humans, and that AM, but not PM, required cultivation for this glucose effect to occur. Glucose transport by AM appears to be necessary but not sufficient for phagocytosis of unopsonized P. aeruginosa.     

Biofilm Formation Avoids Complement Immunity and Phagocytosis of Streptococcus pneumoniae

Streptococcus pneumoniae is a frequent member of the microbiota of the human nasopharynx. Colonization of the nasopharyngeal tract is a first and necessary step in the infectious process and often involves the formation of sessile microbial communities by this human pathogen. The ability to grow and persist as biofilms is an advantage for many microorganisms, because biofilm-grown bacteria show reduced susceptibility to antimicrobial agents and hinder recognition by the immune system. The extent of host protection against biofilm-related pneumococcal disease has not been determined yet. Using pneumococcal strains growing as planktonic cultures or as biofilms, we have investigated the recognition of S. pneumoniae by the complement system and its interactions with human neutrophils. Deposition of C3b, the key complement component, was impaired on S. pneumoniae biofilms. In addition, binding of C-reactive protein and the complement component C1q to the pneumococcal surface was reduced in biofilm bacteria, demonstrating that pneumococcal biofilms avoid the activation of the classical complement pathway. In addition, recruitment of factor H, the downregulator of the alternative pathway, was enhanced by S. pneumoniae growing as biofilms. Our results also show that biofilm formation diverts the alternative complement pathway activation by a PspC-mediated mechanism. Furthermore, phagocytosis of pneumococcal biofilms was also impaired. The present study confirms that biofilm formation in S. pneumoniae is an efficient means of evading both the classical and the PspC-dependent alternative complement pathways the host immune system.     

Impact of Interleukin-17 on Macrophage Phagocytosis of Apoptotic Neutrophils and Particles

There is now substantial evidence that the cytokine interleukin-17 orchestrates the accumulation of neutrophils in mammals and thereby contributes to host defense. However, the role of IL-17 in controlling neutrophil turnover is not fully understood. Here, we demonstrate that IL-17 stimulates the apoptosis of mouse neutrophils and, simultaneously, the release of the microbicidal compound, myeloperoxidase. IL-17 also stimulates mouse macrophages to phagocytose aged neutrophils and latex beads, and it induces an increase in a soluble form of the phagocytic receptor, lectin-like oxidized low-density lipoprotein receptor-1 as well. In contrast, IL-17 does not markedly increase the release of the archetype neutrophil-recruiting cytokine, macrophage inflammatory protein-2 in mouse macrophages. Importantly, IL-17 also stimulates the phagocytosis of latex beads in human monocyte-derived macrophages. Thus, IL-17 bears the potential to control both phagocytosis and neutrophil turnover during activation of host defense.     

Streptococcus pneumoniae Autolysis Prevents Phagocytosis and Production of Phagocyte-Activating Cytokines

Streptococcus pneumoniae is a major pathogen in humans. The pathogenicity of this organism is related to its many virulence factors, the most important of which is the thick pneumococcal capsule that minimizes phagocytosis. Another virulence-associated trait is the tendency of this bacterium to undergo autolysis in stationary phase through activation of the cell wall-bound amidase LytA, which breaks down peptidoglycan. The exact function of autolysis in pneumococcal pathogenesis is, however, unclear. Here, we show the selective and specific inefficiency of wild-type S. pneumoniae for inducing production of phagocyte-activating cytokines in human peripheral blood mononuclear cells (PBMC). Indeed, clinical pneumococcal strains induced production of 30-fold less tumor necrosis factor (TNF), 15-fold less gamma interferon (IFN-γ), and only negligible amounts of interleukin-12 (IL-12) compared with other closely related Streptococcus species, whereas the levels of induction of IL-6, IL-8, and IL-10 production were similar. If pneumococcal LytA was inactivated by mutation or by culture in a medium containing excess choline, the pneumococci induced production of significantly more TNF, IFN-γ, and IL-12 in PBMC, whereas the production of IL-6, IL-8, and IL-10 was unaffected. Further, adding autolyzed pneumococci to intact bacteria inhibited production of TNF, IFN-γ, and IL-12 in a dose-dependent manner but did not inhibit production of IL-6, IL-8, and IL-10 in response to the intact bacteria. Fragments from autolyzed bacteria inhibited phagocytosis of intact bacteria and reduced the in vitro elimination of pneumococci from human blood. Our results suggest that fragments generated by autolysis of bacteria with reduced viability interfere with phagocyte-mediated elimination of live pneumococci.The pneumococcus Streptococcus pneumoniae is a leading cause of community-acquired pneumonia, meningitis, otitis media, and sinusitis and is a common cause of infection-related mortality in children and elderly people (28, 37).There is a large number of streptococcal species whose taxonomic classification is debated (14, 31). A number of streptococci, including alpha-hemolytic and nonhemolytic variants, constitute the viridans group, which can be further subdivided into the mitis, sanguinis, anginosus, salivarius, and mutans groups based on biochemical tests (14). Phenotypic and genetic tests consistently show that S. pneumoniae is closely related to and may be placed in the mitis subgroup (14, 30). Although the other members of the mitis group can cause sepsis and endocarditis (53), they are considerably less virulent than S. pneumoniae.Pneumococci are considered strictly extracellular pathogens, whose elimination depends on ingestion and killing by phagocytes (i.e., alveolar and tissue-resident macrophages and neutrophils recruited during the inflammatory process). Accordingly, an important determinant of pneumococcal pathogenicity is the thick, hydrophilic polysaccharide capsule, which impedes elimination by phagocytes in the absence of capsule-specific antibodies.The ability of phagocytes to kill microbes is augmented by the phagocyte-activating cytokines gamma interferon (IFN-γ) and tumor necrosis factor (TNF), which boost the bactericidal machinery and enhance killing and digestion of bacteria present within the phagosome (4, 39, 47). TNF is produced by monocytes/macrophages and activated T cells, while IFN-γ is produced by NK cells and T cells in response to interleukin-12 (IL-12) from macrophages. Thus, production of TNF, IFN-γ, and IL-12 is necessary for host defense against intracellular bacteria (8, 11, 21, 34, 48). More recently, these phagocyte-activating cytokines have also been shown to be essential for controlling extracellular gram-positive bacteria, including S. pneumoniae (36, 42, 50, 52, 54). Thus, a patient with an IL-12 deficiency was shown to suffer from recurrent episodes of pneumococcal infection (20). Phagocyte activation by TNF and/or IFN-γ might be required for decomposition of the thick, sturdy peptidoglycan (PG) layer of gram-positive bacteria after phagocytosis, while gram-negative bacteria may be more easily digested. Thus, human leukocytes produce more TNF, IFN-γ, and IL-12 when they are stimulated with gram-positive bacteria than when they are stimulated with gram-negative bacteria (23, 24).A peculiar property of S. pneumoniae is its tendency to undergo autolysis when it reaches the stationary phase of growth. This process is mediated by enzymes called autolysins (ALs), which, when activated, degrade cell wall PG. The major AL is an N-acetyl-muramyl-l-alanine amidase called LytA (27). Other pneumococcal ALs include LytB and LytC, which are believed to be involved mainly in modification of the cell wall during growth and division (16, 17). ALs are anchored to the cell wall via interactions with choline moieties on teichoic acid and lipoteichoic acid (LTA). Choline is necessary for pneumococcal growth, but culture in the presence of high concentrations of choline renders the bacteria incapable of undergoing autolysis (6, 19).Studies with mice have shown that S. pneumoniae with mutated LytA is less virulent than wild-type pneumococci (2, 7, 25). The reason for this is not clear, but two main hypotheses have been put forward. First, autolysis promotes the release of the intracellular toxin pneumolysin (Ply) (5, 33). Ply is an important determinant of virulence (3, 41) and interferes with several defense systems, including inhibition of ciliary beating (15), complement activation (38), and induction of intracellular oxygen radical production (33). Second, cell wall degradation products, such as soluble PG fragments and LTA released upon autolysis, have been suggested to augment the inflammatory response (9, 10, 44, 49).Here we examine a third possibility, that autolysis interferes with the generation of phagocyte-activating cytokines. We have previously shown that intact gram-positive bacteria provide a very efficient stimulus for IL-12 production by human monocytes, regardless of whether they are dead or alive (1, 23, 24), but that decomposed bacteria are inactive in this process and soluble components of the gram-positive cell wall, such as PG and LTA, even downregulate the production of IL-12 in response to intact bacteria in a dose-dependent manner (1). These observations led us to speculate that autolysis may promote virulence by generating bacterial cell wall fragments that block IL-12 production and thereby reduce IFN-γ production and phagocyte activation. Indeed, our data demonstrate that AL-mediated disintegration of pneumococci inhibits production of IFN-γ and also TNF in response to intact bacteria. Further, the presence of autolyzed bacteria reduced elimination of live pneumococci by blood cells in vitro.