Regina Maus

Basophils enhance immunological memory responses

The cellular basis of immunological memory remains a controversial issue. Here we show that basophils bound large amounts of intact antigens on their surface and were the main source of interleukins 6 and 4 in the spleen and bone marrow after restimulation with a soluble antigen. Depletion of basophils resulted in a much lower humoral memory response and greater susceptibility of immunized mice to sepsis induced by Streptococcus pneumoniae. Adoptive transfer of antigen-reactive basophils significantly increased specific antibody production, and activated basophils, together with CD4+ T cells, profoundly enhanced B cell proliferation and immunoglobulin production. These basophil-dependent effects on B cells required interleukins 6 and 4 and increased the capacity of CD4+ T cells to provide B cell help. Thus, basophils are important contributors to humoral memory immune responses.

Lung-Specific Overexpression of CC Chemokine Ligand (CCL) 2 Enhances the Host Defense to Streptococcus pneumoniae Infection in Mice: Role of the CCL2-CCR2 Axis

Mononuclear phagocytes are critical components of the innate host defense of the lung to inhaled bacterial pathogens. The monocyte chemotactic protein CCL2 plays a pivotal role in inflammatory mononuclear phagocyte recruitment. In this study, we tested the hypothesis that increased CCL2-dependent mononuclear phagocyte recruitment would improve lung innate host defense to infection with Streptococcus pneumoniae. CCL2 transgenic mice that overexpress human CCL2 protein in type II alveolar epithelial cells and secrete it into the alveolar air space showed a similar proinflammatory mediator response and neutrophilic alveolitis to challenge with S. pneumoniae as wild-type mice. However, CCL2 overexpressing mice showed an improved pneumococcal clearance and survival compared with wild-type mice that was associated with substantially increased lung mononuclear phagocyte subset accumulations upon pneumococcal challenge. Surprisingly, CCL2 overexpressing mice developed bronchiolitis obliterans upon pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block the CCL2-CCR2 axis in CCL2 overexpressing mice, though completely abrogating bronchiolitis obliterans, led to progressive pneumococcal pneumonia. Collectively, these findings demonstrate the importance of the CCL2-CCR2 axis in the regulation of both the resolution/repair and remodelling processes after bacterial challenge and suggest that overwhelming innate immune responses may trigger bronchiolitis obliterans formation in bacterial lung infections.

Importance of CXC Chemokine Receptor 2 in Alveolar Neutrophil and Exudate Macrophage Recruitment in Response to Pneumococcal Lung Infection

ABSTRACT Sustained neutrophilic infiltration is known to contribute to organ damage, such as acute lung injury. CXC chemokine receptor 2 (CXCR2) is the major receptor regulating inflammatory neutrophil recruitment in acute and chronic inflamed tissues. Whether or not the abundant neutrophil recruitment observed in severe pneumonia is essential for protective immunity against Streptococcus pneumoniae infections is incompletely defined. Here we show that CXCR2 deficiency severely perturbs the recruitment of both neutrophils and exudate macrophages associated with a massive bacterial outgrowth in distal airspaces after infection with S. pneumoniae, resulting in 100% mortality in knockout (KO) mice within 3 days. Moreover, irradiated wild-type mice reconstituted with increasing amounts of CXCR2 KO bone marrow (10, 25, 50, and 75% KO) have correspondingly decreased numbers of both neutrophils and exudate macrophages, which is associated with a stepwise increase in bacterial burden and a reciprocal stepwise decrease in survival in S. pneumoniae-induced pulmonary infection. Finally, application of the CXCR2 antagonist SB-225002 resulted in decreased alveolar neutrophil and exudate macrophage recruitment in mice along with increased lung bacterial loads after infection with S. pneumoniae. Together, these data show that CXC chemokine receptor 2 serves a previously unrecognized nonredundant role in the regulation of both neutrophil and exudate macrophage recruitment to the lung in response to S. pneumoniae infection. In addition, we demonstrate that a threshold level of 10 to 25% of reduced neutrophil recruitment is sufficient to cause increased mortality in mice infected with S. pneumoniae.

TNF-related apoptosis-inducing ligand (TRAIL) exerts therapeutic efficacy for the treatment of pneumococcal pneumonia in mice

Neutrophil-derived TRAIL induces apoptosis of alveolar macrophages, limiting the spread of S. pneumoniae infection.

Important Role for CC Chemokine Ligand 2-Dependent Lung Mononuclear Phagocyte Recruitment to Inhibit Sepsis in Mice Infected with Streptococcus pneumoniae

The monocyte chemoattractant CCL2 is of major importance in inflammatory monocyte recruitment to the lungs in response to bacterial infection. Streptococcus pneumoniae is the most prevalent pathogen in community-acquired pneumonia causing significant morbidity and mortality worldwide. In the current study, we examined the role of CCL2 in lung-protective immunity against two strains of S. pneumoniae exhibiting different virulence profiles. Both wild-type mice and CCL2 knockout (KO) mice became septic within 24 h of infection with serotype 3 S. pneumoniae and died of infection by day 4 after challenge. In contrast, wild-type mice challenged with serotype 19 S. pneumoniae did not become septic or succumb to pneumococcal pneumonia, whereas CCL2 KO mice showed an early bacterial outgrowth in their lungs and sepsis starting by day 2 after infection, finally resulting in ∼50% decreased survival compared with wild-type mice. This phenotype was not due to impaired lung neutrophil recruitment in the KO mice, but was characterized by a significantly reduced recruitment of lung exudate macrophages and conventional lung dendritic cells, suggesting that these two phagocyte subsets critically regulate protection against septic disease progression in mice. In conclusion, we show here a differential role for CCL2-dependent lung exudate macrophage and conventional dendritic cell recruitment that critically contributes to lung protective immunity against S. pneumoniae.

Role of Mincle in Alveolar Macrophage-Dependent Innate Immunity against Mycobacterial Infections in Mice

The role of macrophage-inducible C-type lectin Mincle in lung innate immunity against mycobacterial infection is incompletely defined. In this study, we show that wild-type (WT) mice responded with a delayed Mincle induction on resident alveolar macrophages and newly immigrating exudate macrophages to infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG), peaking by days 14–21 posttreatment. As compared with WT mice, Mincle knockout (KO) mice exhibited decreased proinflammatory mediator responses and leukocyte recruitment upon M. bovis BCG challenge, and they demonstrated increased mycobacterial loads in pulmonary and extrapulmonary organ systems. Secondary mycobacterial infection on day 14 after primary BCG challenge led to increased cytokine gene expression in sorted alveolar macrophages of WT mice, but not Mincle KO mice, resulting in substantially reduced alveolar neutrophil recruitment and increased mycobacterial loads in the lungs of Mincle KO mice. Collectively, these data show that WT mice respond with a relatively late Mincle expression on lung sentinel cells to M. bovis BCG infection. Moreover, M. bovis BCG-induced upregulation of C-type lectin Mincle on professional phagocytes critically shapes antimycobacterial responses in both pulmonary and extrapulmonary organ systems of mice, which may be important for elucidating the role of Mincle in the control of mycobacterial dissemination in mice.

Local Delivery of GM-CSF Protects Mice from Lethal Pneumococcal Pneumonia

The growth factor GM-CSF has an important role in pulmonary surfactant metabolism and the regulation of antibacterial activities of lung sentinel cells. However, the potential of intra-alveolar GM-CSF to augment lung protective immunity against inhaled bacterial pathogens has not been defined in preclinical infection models. We hypothesized that transient overexpression of GM-CSF in the lungs of mice by adenoviral gene transfer (Ad-GM-CSF) would protect mice from subsequent lethal pneumococcal pneumonia. Our data show that intra-alveolar delivery of Ad-GM-CSF led to sustained increased pSTAT5 expression and PU.1 protein expression in alveolar macrophages during a 28-d observation period. Pulmonary Ad-GM-CSF delivery 2–4 wk prior to infection of mice with Streptococcus pneumoniae significantly reduced mortality rates relative to control vector-treated mice. This increased survival was accompanied by increased inducible NO synthase expression, antibacterial activity, and a significant reduction in caspase-3–dependent apoptosis and secondary necrosis of lung sentinel cells. Importantly, therapeutic treatment of mice with rGM-CSF improved lung protective immunity and accelerated bacterial clearance after pneumococcal challenge. We conclude that prophylactic delivery of GM-CSF triggers long-lasting immunostimulatory effects in the lung in vivo and rescues mice from lethal pneumococcal pneumonia by improving antibacterial immunity. These data support use of novel antibiotic-independent immunostimulatory therapies to protect patients against bacterial pneumonias.

Mediator responses of alveolar macrophages and kinetics of mononuclear phagocyte subset recruitment during acute primary and secondary mycobacterial infections in the lungs of mice

Little is known about the activation programme induced in alveolar macrophages upon phagocytosis of mycobacteria and the concomitant mononuclear phagocyte migratory responses that shape the acute phase of mycobacterial infection. Using high‐speed cell sorting in conjunction with real‐time RT‐PCR analysis, we show that sorted alveolar macrophages of transgenic CX3CR1+/GFP mice infected with red fluorescent‐labelled Mycobacterium bovis BCG exhibited weak transcriptional changes of lysosomal cathepsins B, L, D, K and S, whereas antimicrobial cathepsin G was strongly induced upon infection. Moreover, mRNA levels of pattern recognition receptors TLR2, TLR4 and NOD2 were downregulated as were neutrophil chemoattractants KC, MIP‐2 and IP‐10, whereas highly upregulated mRNA levels of the monocyte chemoattractant CCL2 were observed. M. bovis BCG infection of the mice elicited the alveolar accumulation of highly CX3CR1‐positive alveolar dendritic cells but not neutrophils within the alveolar compartment, whereas no increased accumulation of CX3CR1high lung parenchymal dendritic cells (lung DC) or CX3CR1neg lung macrophages compared with controls was noted. In contrast, CX3CR1+/GFP mice previously immunized with M. bovis BCG rapidly responded with increased accumulations of both CX3CR1high alveolar and lung parenchymal DC and CX3CR1neg lung macrophages upon intratracheal M. bovis BCG challenge. Moreover, alveolar and lung macrophages and lung DC were found to contribute to early uptake of M. bovis BCG. Together, acute mycobacterial infection triggers both rapid changes in gene expression profiles in infected alveolar macrophages and a compartment‐specific accumulation of mononuclear phagocyte subsets contributing to M. bovis BCG uptake in vivo.

Cathepsin G and Neutrophil Elastase Contribute to Lung-Protective Immunity against Mycobacterial Infections in Mice

The neutrophil serine proteases cathepsin G (CG) and neutrophil elastase (NE) are involved in immune-regulatory processes and exert antibacterial activity against various pathogens. To date, their role and their therapeutic potential in pulmonary host defense against mycobacterial infections are poorly defined. In this work, we studied the roles of CG and NE in the pulmonary resistance against Mycobacterium bovis bacillus Calmette-Guérin (BCG). CG-deficient mice and even more pronounced CG/NE-deficient mice showed significantly impaired pathogen elimination to infection with M. bovis BCG in comparison to wild-type mice. Moreover, granuloma formation was more pronounced in M. bovis BCG-infected CG/NE-deficient mice in comparison to CG-deficient and wild-type mice. A close examination of professional phagocyte subsets revealed that exclusively neutrophils shuttled CG and NE into the bronchoalveolar space of M. bovis BCG-infected mice. Accordingly, chimeric wild-type mice with a CG/NE-deficient hematopoietic system displayed significantly increased lung bacterial loads in response to M. bovis BCG infection. Therapeutically applied human CG/NE encapsulated in liposomes colocalized with mycobacteria in alveolar macrophages, as assessed by laser scanning and electron microscopy. Importantly, therapy with CG/NE-loaded liposomes significantly reduced mycobacterial loads in the lungs of mice. Together, neutrophil-derived CG and NE critically contribute to deceleration of pathogen replication during the early phase of antimycobacterial responses. In addition, to our knowledge, we show for the first time that liposomal encapsulated CG/NE exhibit therapeutic potential against pulmonary mycobacterial infections. These findings may be relevant for novel adjuvant approaches in the treatment of tuberculosis in humans.

FMS-Like Tyrosine Kinase 3 Ligand Aggravates the Lung Inflammatory Response to Streptococcus pneumoniae Infection in Mice: Role of Dendritic Cells

Pretreatment of mice with the hemopoietic growth factor, FMS-like tyrosine kinase 3 ligand (Flt3L), has been shown to increase monocyte-derived myeloid dendritic cells (DC) in lung parenchymal tissue, with possible implications for protective immunity to lung bacterial infections. However, whether Flt3L treatment improves lung innate immunity of mice to challenge with Streptococcus pneumoniae has not been investigated previously. Mice pretreated with Flt3L exhibited a peripheral monocytosis and a strongly expanded lung myeloid DC pool, but responded with a similar proinflammatory cytokine release (TNF-α, IL-6, keratinocyte derived cytokine, MIP-2, CCL2) and neutrophilic alveolitis upon infection with S. pneumoniae as did control mice with a normal lung DC pool. Unexpectedly, however, Flt3L-pretreated mice, but not control mice, infected with S. pneumoniae developed vasculitis and increased lung permeability by days 2–3 postinfection, and florid pneumonia accompanied by sustained increased bacterial loads by days 3–4 postinfection. This was associated with an overall increased mortality of ∼35% by day 4 after pneumococcal challenge. Application of anti-CCR2 Ab MC21 to block inflammatory monocyte-dependent lung mononuclear phagocyte mobilization significantly reduced the lung leakage, but not vasculitis in Flt3L-pretreated mice infected with S. pneumoniae, without affecting the intra-alveolar cytokine liberation or the concomitantly developing neutrophilic alveolitis. Together, the data demonstrate that previous Flt3L-induced lung DC accumulation is not protective in lung innate immunity to challenge with S. pneumoniae, and support the concept that CCR2-dependent mononuclear phagocyte as opposed to neutrophil recruitment contributes to increased lung leakage in Flt3L-pretreated mice challenged with S. pneumoniae.