Julie A. Preston

The Identification of Markers of Macrophage Differentiation in PMA-Stimulated THP-1 Cells and Monocyte-Derived Macrophages

Differentiated macrophages are the resident tissue phagocytes and sentinel cells of the innate immune response. The phenotype of mature tissue macrophages represents the composite of environmental and differentiation-dependent imprinting. Phorbol-12-myristate-13-acetate (PMA) and 1,25-dihydroxyvitamin D3 (VD3) are stimuli commonly used to induce macrophage differentiation in monocytic cell lines but the extent of differentiation in comparison to primary tissue macrophages is unclear. We have compared the phenotype of the promonocytic THP-1 cell line after various protocols of differentiation utilising VD3 and PMA in comparison to primary human monocytes or monocyte-derived macrophages (MDM). Both stimuli induced changes in cell morphology indicative of differentiation but neither showed differentiation comparable to MDM. In contrast, PMA treatment followed by 5 days resting in culture without PMA (PMAr) increased cytoplasmic to nuclear ratio, increased mitochondrial and lysosomal numbers and altered differentiation-dependent cell surface markers in a pattern similar to MDM. Moreover, PMAr cells showed relative resistance to apoptotic stimuli and maintained levels of the differentiation-dependent anti-apoptotic protein Mcl-1 similar to MDM. PMAr cells retained a high phagocytic capacity for latex beads, and expressed a cytokine profile that resembled MDM in response to TLR ligands, in particular with marked TLR2 responses. Moreover, both MDM and PMAr retained marked plasticity to stimulus-directed polarization. These findings suggest a modified PMA differentiation protocol can enhance macrophage differentiation of THP-1 cells and identify increased numbers of mitochondria and lysosomes, resistance to apoptosis and the potency of TLR2 responses as important discriminators of the level of macrophage differentiation for transformed cells.

Haemophilus influenzae infection drives IL-17-mediated neutrophilic allergic airways disease.

A subset of patients with stable asthma has prominent neutrophilic and reduced eosinophilic inflammation, which is associated with attenuated airways hyper-responsiveness (AHR). Haemophilus influenzae has been isolated from the airways of neutrophilic asthmatics; however, the nature of the association between infection and the development of neutrophilic asthma is not understood. Our aim was to investigate the effects of H. influenzae respiratory infection on the development of hallmark features of asthma in a mouse model of allergic airways disease (AAD). BALB/c mice were intraperitoneally sensitized to ovalbumin (OVA) and intranasally challenged with OVA 12–15 days later to induce AAD. Mice were infected with non-typeable H. influenzae during or 10 days after sensitization, and the effects of infection on the development of key features of AAD were assessed on day 16. T-helper 17 cells were enumerated by fluorescent-activated cell sorting and depleted with anti-IL-17 neutralizing antibody. We show that infection in AAD significantly reduced eosinophilic inflammation, OVA-induced IL-5, IL-13 and IFN-γ responses and AHR; however, infection increased airway neutrophil influx in response to OVA challenge. Augmented neutrophilic inflammation correlated with increased IL-17 responses and IL-17 expressing macrophages and neutrophils (early, innate) and T lymphocytes (late, adaptive) in the lung. Significantly, depletion of IL-17 completely abrogated infection-induced neutrophilic inflammation during AAD. In conclusion, H. influenzae infection synergizes with AAD to induce Th17 immune responses that drive the development of neutrophilic and suppress eosinophilic inflammation during AAD. This results in a phenotype that is similar to neutrophilic asthma. Infection-induced neutrophilic inflammation in AAD is mediated by IL-17 responses.

Chlamydial respiratory infection during allergen sensitization drives neutrophilic allergic airways disease.

Neutrophilic asthma is a prevalent, yet recently described phenotype of asthma. It is characterized by neutrophilic rather than eosinophilic airway inflammation and airways hyperresponsiveness (AHR) and may have an infectious origin. Chlamydial respiratory infections are associated with asthma, but how these Th1-inducing bacteria influence Th2-mediated asthma remains unknown. The effects of chlamydial infection on the development of asthma were investigated using a BALB/c mouse model of OVA-induced allergic airways disease (AAD). The effects of current and resolved Chlamydia muridarum infection during OVA sensitization on AAD were assessed and compared with uninfected and nonsensitized controls. Current, but not resolved, infection attenuated hallmark features of AAD: pulmonary eosinophil influx, T cell production of IL-5, mucus-secreting cell hyperplasia, and AHR. Current infection also induced robust OVA-driven neutrophilic inflammation and IFN-γ release from T cells. The phenotype of suppressed but persistent Th2 responses in association with enhanced neutrophilia is reminiscent of neutrophilic asthma. This phenotype was also characterized by increased pulmonary IL-12 and IL-17 expression and activation of APCs, as well as by reduced thymus- and activation-regulated chemokine. Inhibition of pulmonary neutrophil influx during infection blocked OVA-induced neutrophilic inflammation and T cell IFN-γ production and reversed the suppressive effects on mucus-secreting cell hyperplasia and AHR during AAD. These changes correlated with decreased IL-12 and IL-17 expression, increased thymus- and activation-regulated chemokine and altered APC activation. Blocking IFN-γ and IL-17 during OVA challenge had no effect. Thus, active chlamydial respiratory infection during sensitization enhances subsequent neutrophilic inflammation and Th1/Th17 responses during allergen exposure and may have a role in the pathogenesis of neutrophilic asthma.

Streptococcus pneumoniae infection suppresses allergic airways disease by inducing regulatory T-cells

An inverse association exists between some bacterial infections and the prevalence of asthma. We investigated whether Streptococcus pneumoniae infection protects against asthma using mouse models of ovalbumin (OVA)-induced allergic airway disease (AAD). Mice were intratracheally infected or treated with killed S. pneumoniae before, during or after OVA sensitisation and subsequent challenge. The effects of S. pneumoniae on AAD were assessed. Infection or treatment with killed S. pneumoniae suppressed hallmark features of AAD, including antigen-specific T-helper cell (Th) type 2 cytokine and antibody responses, peripheral and pulmonary eosinophil accumulation, goblet cell hyperplasia, and airway hyperresponsiveness. The effect of infection on the development of specific features of AAD depended on the timing of infection relative to allergic sensitisation and challenge. Infection induced significant increases in regulatory T-cell (Treg) numbers in lymph nodes, which correlated with the degree of suppression of AAD. Tregs reduced T-cell proliferation and Th2 cytokine release. The suppressive effects of infection were reversed by anti-CD25 treatment. Respiratory infection or treatment with S. pneumoniae attenuates allergic immune responses and suppresses AAD. These effects may be mediated by S. pneumoniae-induced Tregs. This identifies the potential for the development of therapeutic agents for asthma from S. pneumoniae.

Distinct Cell Death Programs in Monocytes Regulate Innate Responses Following Challenge with Common Causes of Invasive Bacterial Disease

Peripheral blood monocytes represent the rapid response component of mononuclear phagocyte host defense, generating vigorous but finite antibacterial responses. We investigated the fate of highly purified primary human monocytes following phagocytosis of different bacteria. Exposure to high bacterial loads resulted in rapid loss of cell viability and decreased functional competence. Cell death typically involved classical apoptosis. Exposure to high numbers of Escherichia coli and Klebsiella pneumoniae induced nonapoptotic death with loss of cell membrane integrity, marked disruption of phagolysosomes, and caspase-1 activation, while a subset of cells also released caspase-1–regulated extracellular traps. Classical apoptosis increased if extracellular bacterial replication was reduced and decreased if intracellular ATP levels were reduced during these infections. Both classical apoptosis and the alternative forms of cell death allowed monocytes, whose functional competence was exhausted, to downregulate reactive oxygen species and proinflammatory cytokine responses. In contrast, sustained stimulation of glycolytic metabolism and mitochondrial oxidative phosphorylation, with associated hypoxia inducible factor-1α upregulation, maintained intracellular ATP levels and prolonged monocyte functional longevity, as assessed by maintenance of phagocytosis, reactive oxygen species production, and proinflammatory cytokine generation. Monocyte innate responses to bacteria are short-lived and are limited by an intrinsic program of apoptosis, a response that is subverted by overwhelming infection with E. coli and K. pneumoniae or bacterial stimulation of cell metabolism. In this regard, the fate of monocytes following bacterial challenge more closely resembles neutrophils than macrophages.

Pneumolysin Activates Macrophage Lysosomal Membrane Permeabilization and Executes Apoptosis by Distinct Mechanisms without Membrane Pore Formation

ABSTRACT Intracellular killing of Streptococcus pneumoniae is complemented by induction of macrophage apoptosis. Here, we show that the toxin pneumolysin (PLY) contributes both to lysosomal/phagolysosomal membrane permeabilization (LMP), an upstream event programing susceptibility to apoptosis, and to apoptosis execution via a mitochondrial pathway, through distinct mechanisms. PLY is necessary but not sufficient for the maximal induction of LMP and apoptosis. PLY’s ability to induce both LMP and apoptosis is independent of its ability to form cytolytic pores and requires only the first three domains of PLY. LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β). LMP involves progressive and selective permeability to 40-kDa but not to 250-kDa fluorescein isothiocyanate (FITC)-labeled dextran, as PLY accumulates in the cytoplasm. In contrast, the PLY-dependent execution of apoptosis requires phagocytosis and is part of a host response to intracellular bacteria that also includes NO generation. In cells challenged with PLY-deficient bacteria, reconstitution of LMP using the lysomotrophic detergent LeuLeuOMe favored cell necrosis whereas PLY reconstituted apoptosis. The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP. Following bacterial phagocytosis, PLY triggers apoptosis and prevents macrophage necrosis as a component of a broad-based antimicrobial strategy. This illustrates how a key virulence factor can become the focus of a multilayered and coordinated innate response by macrophages, optimizing pathogen clearance and limiting inflammation. IMPORTANCE Streptococcus pneumoniae, the commonest cause of bacterial pneumonia, expresses the toxin pneumolysin, which can make holes in cell surfaces, causing tissue damage. Macrophages, resident immune cells essential for responses to bacteria in tissues, activate a program of cell suicide called apoptosis, maximizing bacterial clearance and limiting harmful inflammation. We examined pneumolysin’s role in activating this response. We demonstrate that pneumolysin did not directly form holes in cells to trigger apoptosis and show that pneumolysin has two distinct roles which require only part of the molecule. Pneumolysin and other bacterial factors released by bacteria that have not been eaten by macrophages activate macrophages to release inflammatory factors but also make the cell compartment containing ingested bacteria leaky. Once inside the cell, pneumolysin ensures that the bacteria activate macrophage apoptosis, rather than necrosis, enhancing bacterial killing and limiting inflammation. This dual response to pneumolysin is critical for an effective immune response to S. pneumoniae. Streptococcus pneumoniae, the commonest cause of bacterial pneumonia, expresses the toxin pneumolysin, which can make holes in cell surfaces, causing tissue damage. Macrophages, resident immune cells essential for responses to bacteria in tissues, activate a program of cell suicide called apoptosis, maximizing bacterial clearance and limiting harmful inflammation. We examined pneumolysin’s role in activating this response. We demonstrate that pneumolysin did not directly form holes in cells to trigger apoptosis and show that pneumolysin has two distinct roles which require only part of the molecule. Pneumolysin and other bacterial factors released by bacteria that have not been eaten by macrophages activate macrophages to release inflammatory factors but also make the cell compartment containing ingested bacteria leaky. Once inside the cell, pneumolysin ensures that the bacteria activate macrophage apoptosis, rather than necrosis, enhancing bacterial killing and limiting inflammation. This dual response to pneumolysin is critical for an effective immune response to S. pneumoniae.

Virulence factors in pneumococcal respiratory pathogenesis

Streptococcus pneumoniae (the pneumococcus) is a major global cause of human disease. Since the publication of the entire sequence of TIGR4 in 2001, our understanding of this human pathogen has increased significantly. Genetic studies, and the use of mutant strains have refined our understanding of the pathogenic mechanisms of classic pneumococcal virulence factors, including the polysaccharide capsule, pneumolysin and surface-expressed proteins. Genetic screens are identifying novel virulence factors. Characterization of pili and bacteriocins, as well as genes associated with competence, metabolism and resistance to oxidative stress has provided new insights into the genetic diversity of the pneumococcus. Further appreciation of the molecular basis of pneumococcal pathogenesis will lead to more effective strategies for the prevention and management of pneumococcal disease.

Genetic background affects susceptibility in nonfatal pneumococcal bronchopneumonia

A nonfatal pneumococcal lung infection model was required to investigate immune responses during recovery, and the interaction of other diseases subsequent to infection. A murine model of nonfatal pneumococcal lung infection was developed and the effect of genetic background on susceptibility was determined in BALB/c and C57BL/6 mice. Bacteria colonised the lungs and mice developed mild clinical illness with pathophysiology similar to human bronchopneumonia. Recovery was associated with immune cell influx, which cleared bacteria but induced tissue damage characteristic of pneumococcal bronchopneumonia. After clearance, immune cell populations returned to normal and tissues appeared less inflamed. Although bacterial exposure and clearance were similar, the extent of immune cell influx and tissue damage differed significantly. Larger numbers of neutrophils and lymphocytes entered lung tissue and the affected area was greater in BALB/c compared with C57BL/6 mice. An inflammatory basis for differences was determined with greater levels of phagocytosis and oxidative burst observed in BALB/c mice. C57BL/6 mice cleared the low inoculum with a reduced immune response; however, C57BL/6 mice are more susceptible to larger inocula, which overwhelms the immune system. These different susceptibilities result from a greater inflammatory response in BALB/c compared with C57BL/6 mice.

Monocytes Regulate the Mechanism of T-cell Death by Inducing Fas-Mediated Apoptosis during Bacterial Infection

Monocytes and T-cells are critical to the host response to acute bacterial infection but monocytes are primarily viewed as amplifying the inflammatory signal. The mechanisms of cell death regulating T-cell numbers at sites of infection are incompletely characterized. T-cell death in cultures of peripheral blood mononuclear cells (PBMC) showed ‘classic’ features of apoptosis following exposure to pneumococci. Conversely, purified CD3+ T-cells cultured with pneumococci demonstrated necrosis with membrane permeabilization. The death of purified CD3+ T-cells was not inhibited by necrostatin, but required the bacterial toxin pneumolysin. Apoptosis of CD3+ T-cells in PBMC cultures required ‘classical’ CD14+ monocytes, which enhanced T-cell activation. CD3+ T-cell death was enhanced in HIV-seropositive individuals. Monocyte-mediated CD3+ T-cell apoptotic death was Fas-dependent both in vitro and in vivo. In the early stages of the T-cell dependent host response to pneumococci reduced Fas ligand mediated T-cell apoptosis was associated with decreased bacterial clearance in the lung and increased bacteremia. In summary monocytes converted pathogen-associated necrosis into Fas-dependent apoptosis and regulated levels of activated T-cells at sites of acute bacterial infection. These changes were associated with enhanced bacterial clearance in the lung and reduced levels of invasive pneumococcal disease.

Impaired Mitochondrial Microbicidal Responses in Chronic Obstructive Pulmonary Disease Macrophages

Rationale: Chronic obstructive pulmonary disease (COPD) is characterized by impaired clearance of pulmonary bacteria. Objectives: The effect of COPD on alveolar macrophage (AM) microbicidal responses was investigated. Methods: AMs were obtained from bronchoalveolar lavage from healthy donors or patients with COPD and challenged with opsonized serotype 14 Streptococcus pneumoniae. Cells were assessed for apoptosis, bactericidal activity, and mitochondrial reactive oxygen species (mROS) production. A transgenic mouse line in which the CD68 promoter ensures macrophage‐specific expression of human induced myeloid leukemia cell differentiation protein Mcl‐1 (CD68.hMcl‐1) was used to model the molecular aspects of COPD. Measurements and Main Results: COPD AMs had elevated levels of Mcl‐1, an antiapoptotic B‐cell lymphoma 2 family member, with selective reduction of delayed intracellular bacterial killing. CD68.hMcl‐1 AMs phenocopied the microbicidal defect because transgenic mice demonstrated impaired clearance of pulmonary bacteria and increased neutrophilic inflammation. Murine bone marrow‐derived macrophages and human monocyte‐derived macrophages generated mROS in response to pneumococci, which colocalized with bacteria and phagolysosomes to enhance bacterial killing. The Mcl‐1 transgene increased oxygen consumption rates and mROS expression in mock‐infected bone marrow‐derived macrophages but reduced caspase‐dependent mROS production after pneumococcal challenge. COPD AMs also increased basal mROS expression, but they failed to increase production after pneumococcal challenge, in keeping with reduced intracellular bacterial killing. The defect in COPD AM intracellular killing was associated with a reduced ratio of mROS/superoxide dismutase 2. Conclusions: Up‐regulation of Mcl‐1 and chronic adaption to oxidative stress alter mitochondrial metabolism and microbicidal function, reducing the delayed phase of intracellular bacterial clearance in COPD.