Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival.

To study the in vivo role of alveolar macrophages (AM) in gram-negative bacterial pneumonia in mice, AM were eliminated by the intratracheal (i.t.) administration of dichloromethylene diphosphonate encapsulated liposomes. Subsequently, the AM-depleted mice were infected i.t. with 100 CFU of Klebsiella pneumoniae, and the effects of AM depletion on survival, bacterial clearance, and neutrophil (polymorphonuclear leukocyte [PMN]) recruitment were assessed. It was shown that depletion of AM decreases survival dramatically, with 100% lethality at day 3 postinfection, versus 100% long-term survival in the control group. This increased mortality was accompanied by 20- to 27- and 3- to 10-fold increases in the number of K. pneumoniae CFU in lung and plasma, respectively, compared to those in nondepleted animals. This decreased bacterial clearance was not due to an impaired PMN recruitment; on the contrary, the K. pneumoniae-induced PMN recruitment in AM-depleted lungs was sevenfold greater 48 h postinfection than that in control infected lungs. Together with an increased PMN infiltration, 3- and 10-fold increases in lung homogenate tumor necrosis factor alpha (TNF-alpha) and macrophage inflammatory protein 2 (MIP-2) levels, respectively, were measured. Neutralization of TNF-alpha or MIP-2, 2 h before infection, reduced the numbers of infiltrating PMN by 41.6 and 64.2%, respectively, indicating that these cytokines mediate PMN influx in infected lungs, rather then just being produced by the recruited PMN themselves. Our studies demonstrate, for the first time, the relative importance of the AM in the containment and clearance of bacteria in the setting of Klebsiella pneumonia.     

Immunostimulation with macrophage-activating lipopeptide-2 increased survival in murine pneumonia

Community-acquired pneumonia (CAP) is associated with high morbidity and mortality, and Streptococcus pneumoniae is the most prevalent causal pathogen identified in CAP. Impaired pulmonary host defense increases susceptibility to pneumococcal pneumonia. S. pneumoniae may up-regulate Toll-like receptor (TLR)-2 expression and activate TLR-2, contributing to pneumococcus-induced immune responses. In the current study, the course of severe murine pneumococcal pneumonia after pulmonary TLR-2-mediated immunostimulation with synthetic macrophage-activating lipopeptide-2 (MALP-2) was examined. Intratracheal MALP-2 application evoked enhanced proinflammatory cytokine and chemokine release, resulting in recruitment of polymorphonuclear neutrophils (PMN), macrophages, and lymphocytes into the alveolar space in WT, but not in TLR-2-deficient mice. In murine lungs as well as in human alveolar epithelial cells (A549), MALP-2 increased TLR-2 expression at both mRNA and protein level. Blood leukocyte numbers and populations remained unchanged. MALP-2 application 24 hours before intranasal pneumococcal infection resulted in increased levels of CCL5 associated with augmented leukocyte recruitment, and decreased levels of anti-inflammatory IL-10 in bronchoalveolar lavage fluid. Clinically, MALP-2-treated as compared with untreated mice showed increased survival, reduced hypothermia, and increased body weight. MALP-2 also reduced bacteremia and improved bacterial clearance in lung parenchyma, as examined by immunohistochemistry. In conclusion, pulmonary immunostimulation with MALP-2 before infection with S. pneumoniae improved local host defense and increased survival in murine pneumococcal pneumonia.     

PTEN limits alveolar macrophage function against Pseudomonas aeruginosa after bone marrow transplantation

Hematopoietic stem cell transplant patients are susceptible to infection despite cellular reconstitution. In a murine model of syngeneic bone marrow transplantation (BMT), we previously reported that BMT mice have impaired host defense against Pseudomonas aeruginosa pneumonia due to overproduction of (PG)E(2) in lung. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is an effector in the PGE(2) signaling pathway that negatively regulates alveolar macrophage (AM) phagocytosis and bacterial killing. Therefore, examined whether overproduction of PGE(2) after BMT inhibits AM host defense by up-regulating PTEN phosphatase activity. We found that PTEN activity is elevated in BMT AMs in response to increased PGE(2) signaling and that pharmacological inhibition of PTEN activity in BMT AMs fully restores phagocytosis of serum-opsonized P. aeruginosa but only partially restores phagocytosis of nonopsonized P. aeruginosa. In wild-type mice transplanted with myeloid-specific conditional PTEN knockout (PTEN CKO) bone marrow, bacterial clearance is improved after challenge with P. aeruginosa pneumonia. Furthermore, PTEN CKO BMT AMs display improved TNF-α production and enhanced phagocytosis and killing of serum-opsonized P. aeruginosa despite overproduction of PGE(2). However, AM phagocytosis of nonopsonized P. aeruginosa is only partially restored in the absence of PTEN after BMT. This may be related to elevated AM expression of IL-1 receptor-associated kinase (IRAK)-M, a molecule previously identified in the PGE(2) signaling pathway to inhibit AM phagocytosis of nonopsonized bacteria. These data suggest that PGE(2) signaling up-regulates IRAK-M independently of PTEN and that these molecules differentially inhibit opsonized and nonopsonized phagocytosis of P. aeruginosa.     

Analysis of murine genetic predisposition to pneumococcal infection reveals a critical role of alveolar macrophages in maintaining the sterility of the lower respiratory tract

The study of pathogenic mechanisms of disease can be greatly facilitated by studying genetic differences in susceptibility to infection. In the present study, we compared the severity of pneumococcal infection in C57BL/6 (B6) and 129Sv mice. The results showed that 129Sv mice were remarkably more susceptible to pneumococcal infection than B6 mice. Bacterial clearance, proinflammatory mediators, leukocyte recruitment, and phagocyte activities were measured to examine potential immune factors associated with differences in susceptibility to pneumococcal infection. The greater susceptibility of 129Sv mice was associated only with inadequate alveolar macrophage bacterial killing, as indicated by significantly decreased initial bacterial clearance from the respiratory tract. Effective pneumococcal clearance was not dependent upon Toll-like receptor 2 (TLR2) expression, oxidative stress, or matrix metallopeptidase 12 (MMP-12) expression. Furthermore, phagocytosis analysis suggested that the deficiency found in 129Sv alveolar macrophages was not due to a lack of bacterial recognition but, rather, to reduced bacterial uptake. In conclusion, our findings indicate a crucial role of alveolar macrophage phagocytosis during innate defense against pneumococcal infection, which may explain the association of host genetic risk factors with predisposition to pneumococcal infection.     

Toll-like receptor 2 does not contribute to host response during postinfluenza pneumococcal pneumonia

Influenza A can be complicated by secondary bacterial pneumonia, which is most frequently caused by Streptococcus pneumoniae and associated with uncontrolled pulmonary inflammation. Evidence points to Toll-like receptor (TLR) 2 as a possible mediator of this exaggerated lung inflammation: (1) TLR2 is the most important "sensor" for gram-positive stimuli, (2) TLR2 contributes to S. pneumoniae-induced inflammation, and (3) influenza A enhances TLR2 expression in various cell types. Therefore, the objective of this study was to determine the role of TLR2 in the host response to postinfluenza pneumococcal pneumonia. TLR2 knockout (KO) and wild-type (WT) mice were infected intranasally with influenza A virus. Fourteen days later they were administered with S. pneumoniae intranasally. Influenza was associated with a similar transient weight loss in TLR2 KO and WT mice. Both mouse strains were fully recovered and had completely cleared the virus at Day 14. Importantly, no differences between TLR2 KO and WT mice were detected during postinfluenza pneumococcal pneumonia with respect to bacterial growth, lung inflammation, or cytokine/chemokine concentrations, with the exception of lower pulmonary levels of cytokine-induced neutrophil chemoattractant in TLR2 KO mice. Toll-like receptor 2 does not contribute to host defense during murine postinfluenza pneumococcal pneumonia.     

Differential contribution of bacterial N-formyl-methionyl-leucyl- phenylalanine and host-derived CXC chemokines to neutrophil infiltration into pulmonary alveoli during murine pneumococcal pneumonia

Despite the development of new potent antibiotics, Streptococcus pneumoniae remains the leading cause of death from bacterial pneumonia. Polymorphonuclear neutrophil (PMN) recruitment into the lungs is a primordial step towards host survival. Bacterium-derived N-formyl peptides (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) and host-derived chemokines (KC and macrophage inflammatory protein 2 [MIP-2]) are likely candidates among chemoattractants to coordinate PMN infiltration into alveolar spaces. To investigate the contribution of each in the context of pneumococcal pneumonia, CD1, BALB/c, CBA/ca, C57BL/6, and formyl peptide receptor (FPR)-knockout C57BL/6 mice were infected with 10(6) or 10(7) CFU of penicillin/erythromycin-susceptible or -resistant serotype 3 or 14 S. pneumoniae strains. Antagonists to the FPR, such as cyclosporine H (CsH) and chenodeoxycholic acid, or neutralizing antibodies to KC and MIP-2 were injected either 1 h before or 30 min after infection, and then bronchoalveolar lavage fluids were obtained for quantification of bacteria, leukocytes, and chemokines. CsH was effective over a short period after infection with a high inoculum, while anti-CXC chemokine antibodies were effective after challenge with a low inoculum. CsH prevented PMN infiltration in CD1 mice infected with either serotype 3 or 14, whereas antichemokine antibodies showed better efficacy against the serotype 3 strain. When different mouse strains were challenged with serotype 3 bacteria, CsH prevented PMN migration in the CD1 mice only, whereas the antibodies were effective against CD1 and C57BL/6 mice. Our results suggest that fMLP and chemokines play important roles in pneumococcal pneumonia and that these roles vary according to bacterial and host genetic backgrounds, implying redundancy among chemoattractant molecules.     

Nitric oxide is required for effective innate immunity against Klebsiella pneumoniae.

Nitric oxide (NO) has been associated with protection against various parasitic and viral infections and may play a similar role in bacterial infections. We studied the role of NO in host defense against Klebsiella pneumoniae infection in the lung. Initial studies demonstrated a time-dependent increase in NO production of the lungs of CBA/J mice following the intratracheal administration of K. pneumoniae (7 x 10(2) CFU). To assess the role of NO in Klebsiella pneumonia, mice were treated intraperitoneally with either L-NAME (N-omega-nitro-L-arginine methylester), a competitive inhibitor of NO synthesis, or D-NAME, an inert enantiomer. The treatment of Klebsiella-infected mice with L-NAME resulted in a 10- and 46-fold increase in K. pneumoniae CFU in lungs and blood, respectively, at 48 h post-K. pneumoniae inoculation compared to treatment of mice with D-NAME. In addition, a greater-than-twofold increase in mortality was evident in L-NAME-treated mice compared to the mortality in control animals. No significant difference in bronchoalveolar lavage inflammatory cell profiles was noted between L-NAME- and D-NAME-treated mice with Klebsiella pneumonia. Interestingly, increased levels of tumor necrosis factor, gamma interferon, macrophage inflammatory protein 1alpha (MIP-1alpha), and MIP-2 mRNA and protein were noted in infected mice treated with L-NAME compared to the levels in mice treated with D-NAME. Importantly, the in vitro incubation of murine alveolar macrophages with L-NAME, but not with D-NAME, resulted in a significant impairment in both the phagocytosis and killing of K. pneumoniae. In total, these results suggest that NO plays a critical role in antibacterial host defense against K. pneumoniae, in part by regulating macrophage phagocytic and microbicidal activity.     

Galectin-3 reduces the severity of pneumococcal pneumonia by augmenting neutrophil function

The Gram-positive Streptococcus pneumoniae is the leading cause of community-acquired pneumonia worldwide, resulting in high mortality. Our in vivo studies show that galectin-3(-/-) mice develop more severe pneumonia after infection with S. pneumoniae, as demonstrated by increased bacteremia and lung damage compared to wild-type mice and that galectin-3 reduces the severity of pneumococcal pneumonia in part by augmenting neutrophil function. Specifically, we show that 1) galectin-3 directly acts as a neutrophil-activating agent and potentiates the effect of fMLP, 2) exogenous galectin-3 augments neutrophil phagocytosis of bacteria and delays neutrophil apoptosis, 3) phagocytosis of apoptotic neutrophils by galectin-3(-/-) macrophages is less efficient compared to wild type, and 4) galectin-3 demonstrates bacteriostatic properties against S. pneumoniae in vitro. Furthermore, ad-back of recombinant galectin-3 in vivo protects galectin-3-deficient mice from developing severe pneumonia. Together, these results demonstrate that galectin-3 is a key molecule in the host defense against pneumococcal infection. Therapeutic strategies designed to augment galectin-3 activity may both enhance inflammatory cell function (by directly affecting neutrophil responsiveness and prolonging neutrophil longevity) and have direct bacteriostatic activity, improving clinical outcomes after severe pneumococcal infection.     

Lung Bacterial Clearance in Murine Pneumococcal Pneumonia

We studied the bactericidal capacity of the rat lung during the development of pneumococcal pneumonia. Pneumonia was produced in a lower lobe by the intrabronchial instillation of 10(4)Streptococcus pneumoniae cells in buffer. Lung bacterial counts progressively increased, reaching 10(7) per lung within 48 h, and the increase was associated with localized atelectasis and consolidation. Bacterial multiplication was inhibited with tetracycline at various intervals after infection, and the subsequent clearance of pneumococci was determined. Viable pneumococci were rapidly killed by lung defenses if bacterial multiplication was inhibited within 12 h of the onset of infection. No change occurred in the bacterial populationif tetracycline was delayed until 24 h after infection, indicating that pneumococcal killing by lung defenses had ceased. This effect could be reproduced with the addition of pneumococcal capsular polysaccharide to the inoculum, which produced a dose-related inhibition of pneumococcal clearance. The clearance of S. epidermidis was not impaired in the presence of pneumococcal pneumonia or by administration of exogenous capsular polysaccharide. These data indicate that pneumococcal pneumonia causes a marked impairment in lung antipneumococcal defenses within 24 h of the onset of infection. This acquired defect in antibacterial defenses may be due to the accumulation of pneumococcal capsular material in the lungs of infected animals.     

The role of CD44 in the acute and resolution phase of the host response during pneumococcal pneumonia

Streptococcus pneumoniae is the most prevalent pathogen causing community-acquired pneumonia. CD44 is a transmembrane adhesion molecule, expressed by a wide variety of cell types, that has several functions in innate and adaptive immune responses. In this study, we tested the hypothesis that CD44 is involved in the host response during pneumococcal pneumonia. On intranasal infection with a lethal dose of S. pneumoniae CD44-knockout (KO) mice showed a prolonged survival when compared with wild-type mice, which was accompanied by a diminished pulmonary bacterial growth and reduced dissemination to distant body sites. Whereas, proinflammatory cytokine responses and lung pathology were not affected, CD44 deficiency resulted in increased early neutrophil influx into the lung. In separate experiments, we confirmed a detrimental role of CD44 in host defense against pneumococci during sublethal pneumonia, as demonstrated by an improved capacity of CD44 KO mice to clear a low infectious dose. In addition, CD44 appeared important for the resolution of lung inflammation during sublethal pneumonia, as shown by histopathology of lung tissue slides. In conclusion, we show here that CD44 facilitates bacterial outgrowth and dissemination during pneumococcal pneumonia, which in lethal infection results in a prolonged survival of CD44 KO mice. Moreover, during sublethal pneumonia CD44 contributes to the resolution of the inflammatory response.     

Fas/Fas ligand system mediates epithelial injury, but not pulmonary host defenses, in response to inhaled bacteria

The Fas/Fas ligand (FasL) system has been implicated in alveolar epithelial cell apoptosis during pulmonary fibrosis and acute respiratory distress syndrome. However, Fas ligation can also lead to cell activation and cytokine production. The goal of this study was to determine the role of the Fas/FasL system in host defenses against Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. We administered bacteria by aerosolization into the lungs of Fas-deficient (lpr) mice and wild-type (C57BL/6) mice and measured bacterial clearance at 6 and 12 h. One hour prior to euthanasia, the mice received an intraperitoneal injection of human serum albumin (HSA) for alveolar permeability determinations. At all times after bacterial challenges, the lungs of the lpr mice contained similar or lower numbers of bacteria than those of the C57BL/6 mice. Alveolar permeability changes, as determined by bronchoalveolar lavage fluid HSA concentrations, were less severe in the lpr mice 6 h after the challenges. In response to E. coli, the lpr mice had significantly more polymorphonuclear leukocytes (PMN) and macrophage inflammatory protein 2 in the lungs, whereas histopathologic changes were less severe. In contrast, in response to the gram-positive cocci, the lpr animals had similar or lower numbers of PMN. We conclude that the Fas/FasL system contributes to the development of permeability changes and tissue injury during-gram negative bacterial pneumonia. The Fas/FasL system did not have a major role in the clearance of aerosolized bacteria from the lungs at the bacterial doses tested.     

Modulation of the lung inflammatory response to serotype 8 pneumococcal infection by a human immunoglobulin m monoclonal antibody to serotype 8 capsular polysaccharide

The human monoclonal antibody to serotype 8 pneumococcal capsular polysaccharide D11 [immunoglobulin M(kappa)] protects wild-type and complement component 4 knockout (C4 KO) mice against lethal intratracheal challenge with serotype 8 pneumococcus, but it does not promote polymorphonuclear leukocyte (PMN)-mediated pneumococcal killing in vitro. In this study, we investigated the effect of D11 on the blood and lung bacterial burdens and the serum and lung expression of inflammatory chemokines and cytokines in an intratracheal challenge model with serotype 8 pneumococcus in C4 KO mice. Pneumococcus was not detected in the blood of D11-treated mice, whereas control mice had high-grade bacteremia with >10(7) CFU. Control mice had a >5-log increase in lung CFU and D11-treated mice manifested a nearly 3-log increase in lung CFU compared to the original inoculum 24 h after infection. Serum and lung levels of soluble macrophage inflammatory protein 2 (MIP-2) and interleulin-6 (IL-6) as measured by an enzyme-linked immunosorbent assay were lower in D11-treated mice than in control mice 24 h after infection. Real-time PCR was performed to examine lung mRNA chemokine and cytokine expression. The results showed that D11-treated mice had significantly less gamma interferon, MIP-2, IL-12, monocyte chemoattractant protein 1/JE, and tumor necrosis factor alpha expression than control mice 24 h after infection. Histopathology and immunohistochemical staining of lung tissues revealed that D11-treated mice had less inflammation, fewer PMNs, and less myeloperoxidase staining than control mice 24 h after infection. These findings suggest that the efficacy of certain serotype-specific antibodies against pneumococcal pneumonia could be associated with modulation of the lung inflammatory response and a reduction in host damage.     

Cathepsin G and neutrophil elastase play critical and nonredundant roles in lung-protective immunity against Streptococcus pneumoniae in mice

Neutrophil serine proteases cathepsin G (CG), neutrophil elastase (NE), and proteinase 3 (PR3) have recently been shown to contribute to killing of Streptococcus pneumoniae in vitro. However, their relevance in lung-protective immunity against different serotypes of S. pneumoniae in vivo has not been determined so far. Here, we examined the effect of CG and CG/NE deficiency on the lung host defense against S. pneumoniae in mice. Despite similar neutrophil recruitment, both CG knockout (KO) mice and CG/NE double-KO mice infected with focal pneumonia-inducing serotype 19 S. pneumoniae demonstrated a severely impaired bacterial clearance, which was accompanied by lack of CG and NE but not PR3 proteolytic activity in recruited neutrophils, as determined using fluorescence resonance energy transfer (FRET) substrates. Moreover, both CG and CG/NE KO mice but not wild-type mice responded with increased lung permeability to infection with S. pneumoniae, resulting in severe respiratory distress and progressive mortality. Both neutrophil depletion and ablation of hematopoietic CG/NE in bone marrow chimeras abolished intra-alveolar CG and NE immunoreactivity and led to bacterial outgrowth in the lungs of mice, thereby identifying recruited neutrophils as the primary cellular source of intra-alveolar CG and NE. This is the first study showing a contribution of neutrophil-derived neutral serine proteases CG and NE to lung-protective immunity against focal pneumonia-inducing serotype 19 S. pneumoniae in mice. These data may be important for the development of novel intervention strategies to improve lung-protective immune mechanisms in critically ill patients suffering from severe pneumococcal pneumonia.     

Role of granulocyte macrophage colony-stimulating factor during gram-negative lung infection with Pseudomonas aeruginosa

Granulocyte macrophage colony-stimulating factor (GM-CSF) stimulates survival, proliferation, differentiation, and function of myeloid cells. Recently, GM-CSF has been shown to be important for normal pulmonary homeostasis. We report that GM-CSF is induced in lung leukocytes during infection with Gram-negative bacteria. Therefore, we postulated that deficiencies in GM-CSF would increase susceptibility to Gram-negative infection in vivo. After an intratracheal inoculum with Pseudomonas aeruginosa, GM-CSF-/- mice show decreased survival compared with wild-type mice. GM-CSF-/- mice show increased lung, spleen, and blood bacterial CFU. GM-CSF-/- mice are defective in the production of cysteinyl leukotrienes, prostaglandin E2, macrophage inflammatory protein, and keratinocyte-derived chemokine in lung leukocytes postinfection. Despite these defects, inflammatory cell recruitment is not diminished at 6 or 24 h postinfection, and the functional activity of polymorphonuclear leukocytes from the lung and peritoneum against P. aeruginosa is enhanced in GM-CSF-/- mice. In contrast, alveolar macrophage (AM) phagocytosis, killing, and H2O2 production are defective in GM-CSF-/- mice. Although the absence of GM-CSF has profound effects on AMs, peritoneal macrophages seem to have normal bactericidal activities in GM-CSF-/- mice. Defects in AM function may be related to diminished levels of IFN-gamma and TNF-alpha postinfection. Thus, GM-CSF-/- mice are more susceptible to lung infection with P. aeruginosa as a result of impaired AM function.     

Molecular analysis of the contribution of the capsular polysaccharide and the lipopolysaccharide O side chain to the virulence of Klebsiella pneumoniae in a murine model of pneumonia

Klebsiella pneumoniae is a common cause of gram-negative bacterial nosocomial pneumonia. Two surface polysaccharides, lipopolysaccharide (LPS) O side chain and capsular polysaccharide (CPS), are critical for the microorganism in causing sepsis, but little is known about their role in pneumonia. To investigate their contribution in the pathogenesis of K. pneumoniae pneumonia, we characterized the host response to bacterial challenge with a highly virulent clinical isolate or with isogenic insertion-duplication mutants deficient in CPS or LPS O side chain in a murine model of pneumonia. Animals challenged intratracheally with the wild-type or LPS O side chain-deficient strain developed pneumonia and became bacteremic before death. Extensive lung lesions as well as pleuritis, vasculitis, and edema were observed by histopathological examination, and polymorphonuclear infiltration was also demonstrated. In contrast, none of the animals challenged with the unencapsulated strain developed pneumonia or bacteremia. Examination of tissue from this group did not identify lung lesions, and none of the infected animals died. Analysis of the early host defense mechanisms that contributed to the clearance of the unencapsulated mutant showed that the levels of C3 deposited on the unencapsulated mutant surface were threefold higher than those for the wild-type and LPS O side chain-deficient strains. Furthermore, phagocytosis of the unencapsulated mutant by human alveolar macrophages (AM) was more efficient than that of the wild-type and LPS O side chain-deficient strains. We conclude that CPS, but not LPS O side chain, is essential for Klebsiella pneumonia because it modulates the deposition of C3 and protects the microorganisms against human AM phagocytosis.     

Reduced cholesterol accumulation by leptin deficient (ob/ob) mouse macrophages

Objective Although presenting many aspects of the metabolic syndrome, leptin deficient (ob/ob) mice do not spontaneously develop atherosclerosis. To examine the role of leptin in foam cell formation we analyzed ob/ob leukocyte inflammation markers and macrophage cholesterol accumulation. Methods Resident and thioglycollate (TG) elicited peritoneal cells of ob/ob and wildtype mice were studied. Activation markers, scavenger receptors (SR) and cholesterol accumulation were analyzed using flow cytometry and Taqman analysis. Cytokines, haptoglobin, adiponectin and amyloid A levels were analyzed with ELISA. Results Macrophages of ob/ob mice had reduced expression of MHC class II, CD11b, CD40, SR-A and CD36 and reduced cholesterol accumulation in vitro. Plasma haptoglobin was increased and T-cell IFNγ was reduced in ob/ob mice. Peritoneal TG instillation induced an unexpectedly weak inflammatory response in ob/ob mice. Conclusions The ob/ob mice had a reduced inflammatory response and reduced macrophage cholesterol accumulation in vitro. The data suggest decreased foam cell formation and atherosclerosis development in ob/ob mice. Received 27 January 2006; returned for revision 27 February 2006; accepted by A. Falus 27 March 2006     

Pulmonary Mycobacterium tuberculosis infection in leptin-deficient ob/ob mice

The development of active tuberculosis after infection with Mycobacterium tuberculosis is almost invariably caused by a persistent or transient state of relative immunodeficiency. Leptin, the product of the obese (ob) gene, is a pleiotropic protein produced mainly by adipocytes and is down-regulated during malnutrition and starvation, conditions closely connected with active tuberculosis. To investigate the role of leptin in tuberculosis, we intranasally infected wild-type (Wt) and leptin-deficient ob/ob mice with live virulent M. tuberculosis. Ob/ob mice displayed higher mycobacterial loads in the lungs after 5 and 10 weeks of infection, although the difference with Wt mice remained 1 log of M. tuberculosis colony forming unit. Nevertheless, ob/ob mice were less able to form well-shaped granuloma and lung lymphocyte numbers were reduced compared with Wt mice early during infection. In addition, ob/ob mice had a reduced capacity to produce the protective cytokine IFNgamma at the site of the infection early during infection and upon antigen-specific recall stimulation, and showed reduced delayed-type hypersensitivity reaction to intra-dermal tuberculin purified protein derivative. Leptin replacement restored the reduced IFNgamma response observed in ob/ob mice. Mortality did not differ between ob/ob and Wt mice. These data suggest that leptin plays a role in the early immune response to pulmonary tuberculosis.     

Leptin regulates olfactory-mediated behavior in ob/ob mice

We have investigated olfactory-mediated pre-ingestive behavior in leptin (ob/ob) and leptin receptor (db/db) mutant mice compared to age- and gender-matched wild-type (wt) mice. Olfactory-mediated behavior was tested using a buried food paradigm 5 times/day at 2-h intervals for 6 days. Mean food-finding times of ob/ob and db/db mice were approximately 10 times shorter than those of wt mice. To test the effect of leptin replacement in ob/ob mice, leptin (1 or 5 microg/g body weight in sterile saline) or carrier was injected i.p. once daily prior to testing. Mean food finding times in ob/ob mice injected with carrier or with 1 microg/g leptin were similar and were 2-3 times faster than in wt mice. Mean food finding times in ob/ob mice injected with 5 microg/g leptin tripled compared to carrier-injected ob/ob mice and were of the same order of magnitude as those of wt mice, suggesting functional leptin replacement. A 3-factor repeated measures ANOVA demonstrated significant differences between the 6 cohorts (P = 0.0001), food finding times (P< or = 0.0001), and cohort by day interaction (P< or = 0.0001). Post hoc tests suggested that the ob/ob+5 mug/g leptin cohort performed more like the wt cohort in the food-finding test than like the ob/ob or ob/ob+carrier cohort. Potential local sites of leptin production and action were identified with immunohistochemistry and in situ hybridization in epithelial and gland cells of the olfactory and nasal mucosae. Our results strongly suggest that leptin acting through leptin receptors modulates olfactory-mediated pre-ingestive behavior.     

Induction of necrosis and apoptosis of neutrophil granulocytes by Streptococcus pneumoniae

Apoptosis followed by macrophage phagocytosis is the principal mechanism by which neutrophil granulocytes (PMN) are removed from the site of inflammation. To investigate whether Streptococcus pneumoniae causes apoptosis of PMN, we exposed PMN to viable and heat-killed pneumococci and purified pneumococcal cell walls (PCW). The occurrence of PMN cell death was quantified by flow cytometry using annexin V/propidium iodide labelling of the cells. Intracellular histone-associated DNA fragments were quantified by ELISA. The presence of apoptosis was confirmed by in situ tailing. Exposure of PMN to viable pneumococci caused necrosis of the cells. The pneumococcal cytotoxin pneumolysin, the bacterial production of hydrogen peroxide, and PCW contributed to necrosis. Heat-killed pneumococci accelerated the process of apoptosis observed in cultivated non-stimulated PMN in vitro. These results demonstrated that pneumococci induce PMN cell death. Depending on the intensity of the stimulus, PMN necrosis and apoptosis were observed.