Indiana University-Purdue University Indianapolis (IUPUI) / Acute kidney injury (AKI) affects millions of patients worldwide yet has few treatment options. There is a critical need to identify novel interventions for AKI, especially approaches targeting cell types that are central to the disease, such as neutrophils. Neutrophils are professional phagocytic cells that respond early to tissue injury. In rodent models of severe ischemic-reperfusion-injury AKI, neutrophils transiently infiltrate the injured kidney, appearing within 6 hours, and are gone by 72 hours. These infiltrating neutrophils are considered proinflammatory and harmful to tissue repair and recovery of kidney function. However, neutrophils can exhibit atypical activity such as antigen presentation and have a central role in recovery from myocardial ischemic injury. Furthermore, little is known of neutrophil polarization, atypical activity, or neutrophil diversity in AKI. Lastly, the kidney generated and renal-protective immunomodulatory protein uromodulin (Tamm-Horsfall Protein, THP) regulates granulopoiesis. In the absence of uromodulin, there is a systemic increase in neutrophils and mouse kidneys are sensitive to injury in AKI. To elucidate neutrophil diversity in AKI and their sensitivity to uromodulin, I performed a series of single-cell sequencing experiments to generate transcriptional profiles of neutrophils from the blood and kidneys of wild-type and THPknockout mice after renal ischemic-reperfusion-injury (IRI). Neutrophil diversity was detected following IRI of the mouse kidney in the blood and kidney. The distribution of subpopulations was sensitive to the kidney milieu. Within the kidney, this diversity and the transcriptional programs of neutrophil subpopulations was sensitive to the severity of ischemic injury. Lastly, Cxcl3 was uniquely upregulated in specific neutrophils after severe ischemic injury. Using single-cell sequencing of uromodulin knock-out mice, I detected the upregulation of toll-like receptor pathways and complement cascades across neutrophil subpopulations in a THP sensitive manner. Furthermore, CXCR2 ligand expression was a combination of moderate and severe injury in wild-type mice. This confirmed previously reported cytokine dysregulation in the uromodulin knock-out mouse after IRI and uncovers a novel role for Cxcl3. Thus, upon revisiting the well-studied neutrophil, I have uncovered novel neutrophil diversity that correlates with recovery of kidney function in AKI and suggests new roles for an old player.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24798号 / 医博第4990号 / 新制||医||1066(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 妹尾 浩, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The neutrophil is the first haemopoetic cell to arrive at the site of infection. In acute respiratory distress syndrome (ARDS), dense neutrophilic infiltrates are found in the lung in response to bacterial infection as well as generalised inflammatory stimuli, such as pancreatitis. At sites of infection, phagocytosis of bacteria by neutrophils enhances their subsequent apoptosis and clearance by macrophages however at inflammatory sites, the lifespan of the neutrophil is influenced by both pro- and antiapoptotic factors in the inflammatory milieu. Furthermore subsequent macrophage phagocytosis of apoptotic neutrophils induces the macrophage to switch to an antiinflammatory phenotype thereby hastening resolution of inflammation. The Fas death receptor pathway is important in T lymphocyte apoptosis but its role in neutrophil apoptosis is controversial. We have shown that neutrophils express the Fas receptor (CD95) on their surface but there is no evidence of expression of its natural ligand (FasL). An agonistic anti-Fas monoclonal antibody (CH-11) accelerated neutrophil apoptosis under certain culture conditions. Lipopolysaccharide (LPS) originating from Gram-negative bacteria is often found at sites of inflammation. We have shown that LPS attenuated CH-11 - induced neutrophil apoptosis unless the Fas/FasL death receptor pathway was activated prior to the LPS signalling pathway. This LPS-mediated attenuation did not involve the p42/44 ERK, protein kinase C or phosphatidylinositol 3-kinase signalling pathway however the p38 MAPK and NF-κB pathway appeared to be partially involved. We have shown that neutrophils express the protein cFLIPs and that CH-11 and inflammatory mediators altered its expression. Although macrophages are principally phagocytes, they are also important in determining the composition of the milieu at an inflammatory site. Macrophages have been shown to express FasL which can be shed and may contribute to the pools of sFasL found in the bronchoalveolar lavage fluid (BALF) in ARDS patients. We have shown that the conditioned supernatants from LPS-activated macrophages induced neutrophil apoptosis at early time points. The pro-apoptotic activity was mediated by TNF-α and was found in the fraction containing proteins with molecular weights greater than 50kD. Macrophage phagocytosis of apoptotic neutrophils suppressed TNF-α production by LPS-activated macrophages and this was associated with loss of the pro-apoptotic activity. In summary, our data suggest that Fas/FasL fratricide does not appear to be involved in spontaneous neutrophil apoptosis. However LPS attenuates Fas-induced apoptosis unless the Fas/FasL death receptor pathway is activated prior to LPS signalling pathways. The signalling pathways involved in this attenuation are not clear but may involve cellular FLIP. Furthermore, activated macrophages secrete inflammatory mediators and at early time points, TNF-α appears to be the most important in inducing neutrophil apoptosis.

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Galectins-1, -3 and -9, are endowed with many immune-regulatory properties, with galectins-1 and -9 largely regarded as anti-inflammatory and galectin-3 as pro-inflammatory. Expression levels increase in activated adaptive immune cells, with peak expression often correlating with peak inflammation. Galectin actions are not only determined by their expression levels but also target tissue permissibility to galectin binding, which is in turn determined by the profile of specific carbohydrate residues, namely N-acetyllactosamine, recognised by these lectins. How expression levels and actions are modulated in innate immune cells during inflammation has not been systematically characterised. This study therefore set out to delineate the effects of inflammation on neutrophil glycophenotype, as well as elucidate the temporal and spatial modulation of galectins during resolving inflammation. The neutrophil glycophenotype was modulated during trafficking with decreased levels of all terminal glycan residues assessed. However, this did not correlate with galectin binding permissibility suggesting this is not a useful indicator in this model. The overall change in glycosylation may theoretically be a consequence of rapid modulation of cell surface glycoproteins by activated neutrophils (i.e. CD62L shedding) rather than the actions of specific glycosylation enzymes as demonstrated in T- and endothelial cells. Assessment of galectin levels in leukocytes over a 96h zymosan-induced resolving peritonitis demonstrated alterations both spatially and temporally with increased galectin-3 expression in neutrophils at the inflammatory site compared to the periphery and a peak expression at 24h adding supporting evidence that modulation of galectin expression allows delineation of galectin responses by neutrophils. This study also demonstrated a novel pro-resolution effect of galectin-3 with defective resolution observed in galectin-3 null mice. In conclusion this work demonstrated that neutrophil permissibility for galectins-1, -3 and -9 binding is more likely a consequence of the exposure to galectins at specific time points in the resolving inflammatory response rather than due to a modulation of the glycophenotype upon activation. This study also 3 demonstrated that as well as an important role in the induction of an inflammatory response galectin-3 is involved in resolution, a novel finding which may lead to a better understanding of the resolution process.

Neutrophils are a central component of the innate immune system, whose major role is to defend the host against invading microorganisms. As such they are integral players in the process of inflammation, the response of vascular tissues to injury. They are frequently the first immune cells recruited from the systemic circulation into a site of tissue injury or infection where they themselves play a key antimicrobial role. Direct killing of microbes can be accomplished by phagocytosis, degranulation, production of reactive oxygen species (ROS) or the release of DNA and antimicrobial peptides into the extracellular milieu (NETosis). In addition neutrophils orchestrate the recruitment and activation of other leucocytes, further contributing to host defence. The central importance of neutrophils in immunity is revealed by defects in either number or function leading to recurrent life threatening infection. However, as the toxic arsenal of neutrophil constituents lack specificity they can also be damaging to surrounding host tissues causing exacerbated inflammation. It is therefore essential that neutrophil function is tightly controlled to allow an appropriate response to be mounted against invading pathogens while simultaneously minimising host tissue injury. Therefore, once the inciting inflammatory insult has been successfully cleared or controlled it is imperative that these non-tissue resident specialised immune cells are rapidly ‘switched off’ or cleared to allow the return to homeostasis. This resolution phase of the inflammatory cascade is now recognised as an energy dependent, finely controlled endogenous process, the beginnings of which are activated at the onset of inflammation. One of the main aims of resolution is to ensure efficient clearance of leucocytes that are no longer necessary. It is likely that a major clearance route is by the highly regulated and energy dependent processes of neutrophil programmed cell death (apoptosis) with subsequent uptake and disposal of apoptotic neutrophils by tissue macrophages. This process of neutrophil apoptosis renders the neutrophils nonfunctional and preserves cell membrane integrity, thus preventing further release of histotoxic neutrophil-derived inflammatory mediators into the extracellular environment. Furthermore, the recognition, uptake and disposal of apoptotic neutrophils cause a dynamic change in the phagocytosing macrophage phenotype with alterations in inflammatory mediator production. The fundamental importance of neutrophil apoptosis and subsequent efferocytosis in inflammation resolution is highlighted by the pathological consequences of neutrophil necrosis or failed apoptotic cell clearance, which leads to enhanced tissue injury and autoimmunity. Acute lung infection (pneumonia) is a common and serious condition affecting both developed and developing countries; globally, childhood pneumonia is the leading cause of death in children aged less than 5 years and pneumonia is the most common fatal infection in the developed world. In over half of patients with community acquired pneumonia no causative organism is ever isolated suggesting that although the immune response has successfully controlled infection, continued uncontrolled neutrophilic inflammation in the lung continues to cause morbidity and mortality. Indeed, pneumonia frequently progresses to acute respiratory distress syndrome (ARDS), a devastating acute inflammatory condition of the lungs characterized by inflammatory cell recruitment and accumulation of protein rich oedema fluid leading to impaired lung function. ARDS affects 200,000 critically ill patients in the USA per year, and has a substantial mortality rate of up to 40%. Despite advances in intensive care treatment and antimicrobial therapy mortality from pneumonia has not fallen since the 1950s, and at present there are no specific therapies for infection-related lung inflammation or ARDS. Understanding the mechanism behind such uncontrolled, persisting inflammation, and the need for novel approaches to target infection related lung injury are therefore both urgent and essential. This thesis examines the potential of neutrophil apoptosis-inducing pharmacological agents as potential treatments for infection-associated lung inflammation. The primary agents used include a cyclin-dependent kinase inhibitor as well as plant-derived polyphenolic flavones. The ability of these compounds to induce human neutrophil apoptosis in vitro, the key importance of the intracellular neutrophil survival protein Mcl-1 in mediating this process, and the effect of targeting Mcl-1 in human macrophages is investigated. In addition, neutrophilic inflammation is modelled in zebrafish and mice with both sterile and bacterial-driven models of inflammation. A key role for Mcl-1 is delineated in vivo, with it acting as an endogenous controller of the innate immune response by influencing neutrophil apoptosis, but without effects on macrophage apoptosis or ability to phagocytose apoptotic cells. Driving neutrophil apoptosis by down-regulation of Mcl-1 accelerates resolution of inflammation in vivo. This therapeutic approach is also found to have indirect anti-bacterial effects in a model of E. Coli induced pneumonia, in stark contrast to established anti-inflammatory approaches which routinely cause immune paresis and life threatening infection. As such, targeting inflammatory cell apoptosis by changes in Mcl-1 offers a potential new therapeutic approach for the treatment of infection-associated inflammation.