Lynne R. Prince

Selective Roles for Toll-Like Receptor (TLR)2 and TLR4 in the Regulation of Neutrophil Activation and Life Span

Neutrophil responses to commercial LPS, a dual Toll-like receptor (TLR)2 and TLR4 activator, are regulated by TLR expression, but are amplified by contaminating monocytes in routine cell preparations. Therefore, we investigated the individual roles of TLR2 and TLR4 in highly purified, monocyte-depleted neutrophil preparations, using selective ligands (TLR2, Pam3CysSerLys4 and Staphylococcus aureus peptidoglycan; TLR4, purified LPS). Activation of either TLR2 or TLR4 caused changes in adhesion molecule expression, respiratory burst (alone, and synergistically with fMLP), and IL-8 generation, which was, in part, dependent upon p38 mitogen-activated protein kinase signaling. Neutrophils also responded to Pam3CysSerLys4 and purified LPS with down-regulation of the chemokine receptor CXCR2 and, to a lesser extent, down-regulation of CXCR1. TLR4 was the principal regulator of neutrophil survival, and TLR2 signals showed relatively less efficacy in preventing constitutive apoptosis over short time courses. TLR4-mediated neutrophil survival depended upon signaling via NF-κB and mitogen-activated protein kinase cascades. Prolonged neutrophil survival required both TLR4 activation and the presence of monocytes. TLR4 activation of monocytes was associated with the release of neutrophil survival factors, which was not evident with TLR2 activation, and TLR2 activation in monocyte/neutrophil cocultures did not prevent late neutrophil apoptosis. Thus, TLRs are important regulators of neutrophil activation and survival, with distinct and separate roles for TLR2 and TLR4 in neutrophil responses. TLR4 signaling presents itself as a pharmacological target that may allow therapeutic modulation of neutrophil survival by direct and indirect mechanisms at sites of inflammation.

Alveolar Macrophage Apoptosis Contributes to Pneumococcal Clearance in a Resolving Model of Pulmonary Infection

The role of alveolar macrophages (AM) in host defense against pulmonary infection has been difficult to establish using in vivo models. This may reflect a reliance on models of fulminant infection. To establish a unique model of resolving infection, with which to address the function of AM, C57BL/6 mice received low-dose intratracheal administration of pneumococci. Administration of low doses of pneumococci produced a resolving model of pulmonary infection characterized by clearance of bacteria without features of pneumonia. AM depletion in this model significantly increased bacterial outgrowth in the lung. Interestingly, a significant increase in the number of apoptotic AM was noted with the low-dose infection as compared with mock infection. Caspase inhibition in this model decreased AM apoptosis and increased the number of bacteremic mice, indicating a novel role for caspase activation in pulmonary innate defense against pneumococci. These results suggest that AM play a key role in clearance of bacteria from the lung during subclinical infection and that induction of AM apoptosis contributes to the microbiologic host defense against pneumococci.

Translational mini-review series on toll-like receptors : Networks regulated by toll-like receptors mediate innate and adaptive immunity

The Toll‐like receptor (TLR) family provide key components of mammalian immunity and are part of the earliest surveillance mechanisms responding to infection. Their activation triggers the innate immune response, and is crucial to the successful induction of Th1/Th2‐phenotyped adaptive immunity. Innate immunity was long considered to be non‐specific and somewhat simple compared to adaptive immunity, mediated via the engulfment and lysis of microbial pathogens by phagocytic cells such as macrophages and neutrophils, and involving no complex protein–protein interactions. The emergence of the TLR field has contributed to a revision of our understanding, and innate immunity is now viewed as a highly complex process, in line with adaptive immunity. This review will give a brief overview of our current knowledge of TLR biology, and will focus on TLRs as key components in complex networks that activate, integrate and select the appropriate innate and adaptive immune responses in the face of immunological danger.

A Zebrafish Compound Screen Reveals Modulation of Neutrophil Reverse Migration as an Anti-Inflammatory Mechanism

The proresolution therapeutic tanshinone IIA drives inflammation resolution by reverse migration. An Anti-Inflammatory Fish Story Inflammation is one way the body tries to protect itself from injury and begin the healing process. However, as with any good thing, too much inflammation can be harmful, causing bystander injuries to healthy tissue. Hence, there is an active mechanism to resolve inflammation; failed resolution contributes to diseases of chronic inflammation such as atherosclerosis and rheumatoid arthritis. Now, Robertson et al. use a zebrafish screening platform to identify new means of resolving inflammation. The authors used a transgenic zebrafish model of sterile tissue injury to screen potential factors involved in inflammation resolution. They found that tanshinone IIA, which is derived from a Chinese medicinal herb, had proresolving activity by both inducing neutrophil apoptosis and promoting reverse migration of neutrophils. What’s more, these effects were not limited to their zebrafish model but held true in human neutrophils. Although efficacy remains to be tested in actual patients, these data support “fishing” for new drug candidates for resolving inflammation. Diseases of failed inflammation resolution are common and largely incurable. Therapeutic induction of inflammation resolution is an attractive strategy to bring about healing without increasing susceptibility to infection. However, therapeutic targeting of inflammation resolution has been hampered by a lack of understanding of the underlying molecular controls. To address this drug development challenge, we developed an in vivo screen for proresolution therapeutics in a transgenic zebrafish model. Inflammation induced by sterile tissue injury was assessed for accelerated resolution in the presence of a library of known compounds. Of the molecules with proresolution activity, tanshinone IIA, derived from a Chinese medicinal herb, potently induced inflammation resolution in vivo both by induction of neutrophil apoptosis and by promoting reverse migration of neutrophils. Tanshinone IIA blocked proinflammatory signals in vivo, and its effects are conserved in human neutrophils, supporting a potential role in treating human inflammation and providing compelling evidence of the translational potential of this screening strategy.

The role of TLRs in neutrophil activation.

Neutrophils are key innate immune effector cells that are rapidly recruited to sites of infection and inflammation to provide early defence against invading microorganisms. This function is facilitated by the expression of Toll-like receptor (TLR) family members by neutrophils, allowing the recognition of an extensive repertoire of pathogen-associated molecular patterns (PAMPs) and thus triggering the response to invading pathogens. TLR activation leads to important cellular processes including reactive oxygen species (ROS) generation, cytokine production and increased survival, all of which can contribute to the pathogenesis of chronic inflammation when signalling becomes dysregulated. In turn, inflammation and tissue injury results in the release of endogenous TLR ligands, known as damage-associated molecular patterns (DAMPs), which are a rapidly growing class of potent inflammatory stimuli. DAMPs act in an autocrine manner, alerting the host of damage, but can also amplify inflammation leading to further tissue damage. This review highlights recent literature on neutrophil TLR function and regulation during disease, and provides an overview of the recently emerging area of neutrophil responses to DAMPs.

Inhibition of Neutrophil Apoptosis by ATP Is Mediated by the P2Y11 Receptor

Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen- and host-derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found that physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, annexin V/To-Pro3 staining, and mitochondrial membrane permeabilization). We found that even brief exposure to ATP (10 min) was sufficient to cause a long-lasting delay of apoptosis and showed that the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the antiapoptotic actions of ATP was identified using a combination of more selective ATP analogs, receptor expression studies, and study of downstream signaling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signaling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type I cAMP-dependent protein kinases activated downstream of P2Y11, without effects on constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation.

Subversion of a Lysosomal Pathway Regulating Neutrophil Apoptosis by a Major Bacterial Toxin, Pyocyanin

Neutrophils undergo rapid constitutive apoptosis that is accelerated following bacterial ingestion as part of effective immunity, but is also accelerated by bacterial exotoxins as a mechanism of immune evasion. The paradigm of pathogen-driven neutrophil apoptosis is exemplified by the Pseudomonas aeruginosa toxic metabolite, pyocyanin. We previously showed pyocyanin dramatically accelerates neutrophil apoptosis both in vitro and in vivo, impairs host defenses, and favors bacterial persistence. In this study, we investigated the mechanisms of pyocyanin-induced neutrophil apoptosis. Pyocyanin induced early lysosomal dysfunction, shown by altered lysosomal pH, within 15 min of exposure. Lysosomal disruption was followed by mitochondrial membrane permeabilization, caspase activation, and destabilization of Mcl-1. Pharmacological inhibitors of a lysosomal protease, cathepsin D (CTSD), abrogated pyocyanin-induced apoptosis, and translocation of CTSD to the cytosol followed pyocyanin treatment and lysosomal disruption. A stable analog of cAMP (dibutyryl cAMP) impeded the translocation of CTSD and prevented the destabilization of Mcl-1 by pyocyanin. Thus, pyocyanin activated a coordinated series of events dependent upon lysosomal dysfunction and protease release, the first description of a bacterial toxin using a lysosomal cell death pathway. This may be a pathological pathway of cell death to which neutrophils are particularly susceptible, and could be therapeutically targeted to limit neutrophil death and preserve host responses.

The role of neutrophil apoptosis in the resolution of acute lung injury in newborn infants

Background: The persistent airway neutrophilia observed in chronic lung disease of prematurity (CLD) may reflect inappropriate suppression of neutrophil apoptosis. Methods: 134 bronchoalveolar lavage (BAL) samples were obtained from 32 infants requiring mechanical ventilation for respiratory distress syndrome (RDS): 13 infants (median gestation 26 weeks, range 23 to 28) subsequently developed CLD (CLD group), and 19 infants (gestation 31 weeks, range 25 to 39) recovered fully (RDS group). A further 73 BAL samples were obtained from 20 infants (median age 2 days, range 1 to 402) receiving extracorporeal membrane oxygenation (ECMO) for severe respiratory failure. Results: Neutrophil apoptosis was increased in the RDS group (mean (SEM) neutrophil apoptosis on day 7 BAL: RDS 17.0 (8.6)% v CLD 0.7 (0.2)% (p<0.05)). BAL fluid obtained from RDS but not CLD patients was proapoptotic to neutrophils (apoptosis ratio BAL fluid/saline control: day 1, RDS 9.8 (5.5) v CLD 1.2 (0.1) (p<0.05); day 2, RDS 4.32 (2.8) v CLD 0.5 (0.4) (p<0.05)). There were similar findings in the ECMO group: survivors had proapoptotic BAL fluid compared with non-survivors (apoptosis ratio day 1, survivors 7.9 (2.1) v non-survivors 2.1 (0.7) (p<0.05)). Conclusions: Inappropriate suppression of neutrophil apoptosis may be associated with a poor outcome in newborn infants with respiratory failure.

The rise and rise of Staphylococcus aureus: laughing in the face of granulocytes

Recent developments in the study of host–pathogen interactions have fundamentally altered our understanding of the nature of Staphylococcus aureus infection, and previously held tenets regarding the role of the granulocyte are being cast aside. Novel mechanisms of pathogenesis are becoming evident, revealing the extent to which S. aureus can evade neutrophil responses successfully by resisting microbicides, surviving intracellularly and subverting cell death pathways. Developing a detailed understanding of these complex strategies is especially relevant in light of increasing staphylococcal virulence and antibiotic resistance, and the knowledge that dysfunctional neutrophil responses contribute materially to poor host outcomes. Unravelling the biology of these interactions is a challenging task, but one which may yield new strategies to address this, as yet, defiant organism.

The Role of Interleukin-1β in Direct and Toll-Like Receptor 4-Mediated Neutrophil Activation and Survival

The regulation of systemic and local neutrophil activation is crucial to the clearance of infections and the successful resolution of inflammation without progress to tissue damage or disseminated inflammatory reactions. Using purified lipopolysaccharide (pLPS) and highly purified neutrophils, we have previously shown that Toll-like receptor 4 signaling is a potent neutrophil activator, but a poor stimulator of survival. In the presence of peripheral blood mononuclear cells (PBMCs), however, pLPS becomes a potent neutrophil survival factor. Interleukin (IL)-1beta has been identified as an important neutrophil activator and prosurvival cytokine, and is produced in abundance by LPS-stimulated PBMCs. We now show that IL-1beta fails to activate highly purified neutrophils or enhance their survival, but in the presence of PBMCs, IL-1beta induces neutrophil survival. We hypothesized that LPS-primed neutrophils might become responsive to IL-1beta, but were unable to demonstrate this. Moreover, IL-1ra failed to prevent pLPS + PBMC-dependent neutrophil survival. In studies of IL-1R1(-/-) mice, we found that LPS was still able to mediate neutrophil survival, and neutrophil survival was enhanced by the addition of monocytic cells. Thus an important paradigm of neutrophil regulation needs to be viewed in the context of a cellular network in which actions of IL-1beta on neutrophils are indirect and mediated by other cells.