Yersinia pseudotuberculosis is a foodborne pathogen that is the ancestral strain to Yersinia pestis, the causative agent of Plague. Y. pseudotuberculosis invades a host through the intestinal epithelium. The bacteria resist mucosal innate immune defenses including antimicrobial chemokines and phagocytic cells, and replicate in local lymph nodes. They cause Tuberculosis-like symptoms, including necrosis of local tissue and granuloma formation. Like all bacteria, Y. pseudotuberculosis has a net negative charge, which contributes to its susceptibility to some cationic antimicrobial peptides. Y. pseudotuberculosis is able to reduce this negative charge by adding 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A portion of lipopolysaccharide. The production and addition of the L-Ara4N is coded for by the pmrHFIJKLM (pmrF) operon. A previous study has shown that the Y. pseudotuberculosis pmrF operon is important for resistance against polymyxin, but is not important for virulence in mice. Several previous reports have shown a strong influence of growth temperature on resistance to antimicrobial peptides and pmrF expression in pathogenic Yersinia species, but these studies also suggest significant variability between species, and even between strains of individual species. In particular, the regulation of the Y. pseudotuberculosis pmrF operon and its effect on bacterial interactions with mucosa-associated antimicrobial chemokines and neutrophils is not understood. In these studies, we investigated the environmental influences on pmrF expression in Y. pseudotuberculosis. We found that the promoter activity of the pmrHFIJKLM operon is increased at lower temperatures (21ºC) and in the presence of human serum. A ΔpmrI mutant strain of Y. pseudotuberculosis defective for addition of L-Ara4N was found to be more susceptible to killing by the antimicrobial chemokine CCL28 compared to wild-type. This suggests that this gene is important in the bacterial defense against antimicrobial chemokines. However, when the ΔpmrI mutant strain was exposed to human neutrophils, there was a decrease in phagocytosis as compared to wild-type bacteria. Our results suggest that the regulation of L-Ara4N modifications in Yersinia is more complex than previously appreciated and varies between species. Addition of L-Ara4N to Y. pseudotuberculosis appears to enhance resistance to some antimicrobial peptides like CCL28 and promote greater phagocytic engulfment by neutrophils. These opposing effects may partly explain why there is no net apparent survival defect in mutants lacking the pmrF operon during infection.

Neutrophils are important innate immune effector cells that primarily function during infection by engulfing and killing pathogens using a combination of toxic granule components and reactive oxygen species (ROS) generated by the NADPH oxidase. Francisella tularensis is a Gram-negative bacterium and the causative agent of tularemia, an infectious disease that, in the absence of treatment, results in 30-60% mortality. A closely related species, F. novicida, does not cause human disease but causes a tularemia-like illness in mice and productively infects human and murine cells in vitro; thus this organism is often employed as a model. In our previous work, we have shown that virulent and avirulent F. tularensis enters neutrophils without inducing a respiratory burst, as the NADPH oxidase fails to assemble on bacterial phagosomes. Further, this pathogen inhibits enzyme activity upon subsequent neutrophil stimulation despite successful oxidase assembly, indicating that F. tularensis employs multiple mechanisms to inhibit the NADPH oxidase. It remains unknown, however, whether F. novicida retains these mechanisms of oxidase inhibition, or whether its inability to modulate neutrophil function partially accounts for its avirulence in humans. Additional work has suggested a potential role for Francisella acid phosphatases and catalase genes in inhibited production and detoxification of neutrophil-derived ROS, respectively. In the current study, we employ subjective and objective techniques to evaluate the magnitude and location of ROS generation during infection with F. tularensis LVS, F. novicida, or F. novicida mutants acpA or katG. Our results demonstrate that serum-opsonized F. novicida, but not LVS, induced a prominent respiratory burst that coincided with oxidase assembly and intraphagosomal superoxide production in bacterial phagosomes. Furthermore, our data show for the first time that opsonized F. novicida, but not LVS, engaged Fc-gammaRIII (CD16) during phagocytosis by neutrophils suggesting that this receptor may play a role in signaling events that lead to respiratory burst induction. Despite its inability to evade burst induction, F. novicida inhibited post-assembly oxidase activity following sequential stimulation of neutrophils, similar to F. tularensis strains. Finally, we conclude that acpA and katG do not play a significant role in F. novicida-neutrophil interactions as these mutants did not induce a stronger respiratory burst during phagocytosis, and their ability to inhibit post-assembly NADPH oxidase activity and survive in neutrophils was indistinguishable from wild type organisms. Thus, these data strongly suggest that differential opsonization of F. novicida compared to F. tularensis results in engagement of specific receptors that function to activate these cells during infection. Further, the retained ability of F. novicida to inhibit post-assembly oxidase activity confirms that Francisella utilize two independent mechanisms by which they modulate NADPH oxidase function. Finally, our conclusions that acpA and katG are disposable for these interactions with neutrophils suggest that F. novicida encodes other important genes that enable them to productively infect these cells.

Neutrophils, essential innate immune cells, recognize danger signals through receptors on their surface. Upon receptor ligation, neutrophils may undergo priming, a process involving limited reactive oxygen species (ROS) generation and partial degranulation. Priming facilitates neutrophil migration and prepares the cell for an enhanced response to a secondary stimulus, including a spike in ROS generation by NADPH oxidase 2 (NOX2). It is well established that NOX2-derived oxidants are involved in pathogen killing and that off-target effects can cause host tissue damage; however, several lines of recent evidence also support an anti-inflammatory function for NOX2 oxidants. First, patients with chronic granulomatous disease exhibit sterile inflammatory phenomena. Second, neutrophils lacking NOX2 function (genetically or pharmacologically) have an inflammatory phenotype under resting conditions. Finally, NOX2-deficient mice exhibit enhanced localized inflammation in several disease models. The goals of this thesis were to investigate an anti-inflammatory function for NOX2 during systemic inflammation and to further elucidate mechanisms of neutrophil priming, with particular focus on priming through Toll-like receptor 2 (TLR2). Using a murine model of sterile systemic inflammatory response syndrome (SIRS), we observed that NOX2-deficient mice had dramatically increased mortality compared to WT mice. While both genotypes developed SIRS, characterized by hypothermia, hypotension, and leukopenia, the WT mice recovered within 48 h whereas the NOX2-deficient mice did not. Moreover, NOX2 function limited the extent of pulmonary pathology as significant lung injury was noted in the NOX2-deficient mice compared to the WT mice. Plasma analysis revealed that several inflammatory cytokines were persistently elevated in the NOX2-deficient mice, likely contributing to the ongoing inflammatory response. One of the complications seen in human SIRS patients is the development of multiple organ dysfunction syndrome (MODS). Thus, we next investigated the role of NOX2 in the progression from SIRS to MODS. Cellular analysis revealed continued neutrophil recruitment to the peritoneum and lungs of the NOX2-deficient mice and altered activation states of both neutrophils and macrophages. Histology showed multiple organ pathology indicative of MODS in the NOX2-deficient mice, and several inflammatory cytokines were elevated in lungs of the NOX2-deficient mice. Overall, these data suggest that NOX2 function protects against the development of MODS and is required for normal resolution of systemic inflammation. As we utilized a TLR2/6 agonist (zymosan) to induce SIRS in our in vivo model, we wanted to investigate neutrophil priming through TLR2 in an in vitro model. Notably, we determined that a TLR2/6 agonist, FSL-1, primed neutrophils from all donors to a similar extent, evidenced by direct and primed ROS generation, MAPK signaling, limited degranulation, and cytokine secretion. Surprisingly, Pam3CSK4, a TLR2/1 agonist, primed neutrophils from a subset of donors to a much greater extent than neutrophils from other donors. We demonstrated that the different neutrophil priming responses were the consequence of a common TLR1 polymorphism. In sum, the data presented here significantly advance our understanding of the roles of NOX2 and TLR2 signaling in host inflammation and neutrophil priming. This research could advance the development of therapies that target pathogenic neutrophil subsets in inflammatory conditions without compromising innate immune function

Recently, it was found that pathogens are trapped and killed by neutrophil extracellular traps (NETs). The role of Rac small GTPases is explored in the formation of NET using neutrophils lacking Rac1, Rac2 or both isoforms. NET formation was observed in both wild-type and Rac1-null neutrophils. In contrast, NET formation was markedly impaired in cells lacking either Rac2 or both Rac2 and Rac1. The defect in NET formation in Rac2-null cells was rescued in the presence of exogenous reactive oxygen species sources, suggesting that Rac2-mediated ROS generation is required. In addition, the role of nitric oxide in NET formation is assessed. Blocking NO production with the nitric oxide synthase inhibitor L-NAME significantly reduced NET formation. Moreover, Rac2-null cells produced significantly less NO than Rac1-null cells or their wild type counterparts. Our data suggest that Rac2 is essential for NET formation via pathways involving both ROS and NO.

Recently, it was found that pathogens are trapped and killed by neutrophil extracellular traps (NETs). The role of Rac small GTPases is explored in the formation of NET using neutrophils lacking Rac1, Rac2 or both isoforms. NET formation was observed in both wild-type and Rac1-null neutrophils. In contrast, NET formation was markedly impaired in cells lacking either Rac2 or both Rac2 and Rac1. The defect in NET formation in Rac2-null cells was rescued in the presence of exogenous reactive oxygen species sources, suggesting that Rac2-mediated ROS generation is required. In addition, the role of nitric oxide in NET formation is assessed. Blocking NO production with the nitric oxide synthase inhibitor L-NAME significantly reduced NET formation. Moreover, Rac2-null cells produced significantly less NO than Rac1-null cells or their wild type counterparts. Our data suggest that Rac2 is essential for NET formation via pathways involving both ROS and NO.

Neutrophil (PMN) inflammatory functions, including cell adhesion, diapedesis, and phagocyto-sis, are dependent on the mobilization and release of various intracellular granules/vesicles. In this study, I found that treating PMN with damnacanthal, a Ras family GTPase inhibitor, resulted in a specific release of secondary granules, but not primary or tertiary granules, and caused dy-sregulation of PMN chemotactic transmigration and cell surface protein interactions. Analysis of the activities of Ras members identified Ral GTPase as a key regulator during PMN activation and degranulation. In particular, Ral was active in freshly isolated PMN, while chemoattractant stimulation induced a quick deactivation of Ral that correlated with PMN degranulation. Over-expression of a constitutively active Ral (Ral23V) in PMN inhibited chemoattractant-induced secondary granule release. By subcellular fractionation, I found that Ral, which was associatedwith the plasma membrane under the resting condition, was redistributed to secondary granules after chemoattractant stimulation. Blockage of cell endocytosis appeared to inhibit Ral transloca-tion intracellularly. In conclusion, these results demonstrate that Ral is a critical regulator in PMN that specifically controls secondary granule release during PMN response to chemoattrac-tant stimulation.

<p>Normally, neutrophil pools are maintained by "steady-state" granulopoiesis. Infections and inflammation, however, trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as "emergency" granulopoiesis. Steady-state and emergency granulopoiesis are thought to depend on distinct members of the CCAAT enhancer binding protein (C/EBP) family of transcription factors, yet the extracellular cues that determine these developmental pathways are unclear. I hypothesize that inflammation elicits IL-1 which acts directly on hematopoietic progenitor cells for the induction of emergency granulopoiesis. Indeed, IL-1RI<super>-/-</super> mice fail to mount reactive neutrophilias in response to adjuvant-induced inflammation. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by granulocyte/macrophage progenitors (GMP) and, surprisingly, more primitive multipotent progenitors (MPP) and hematopoietic stem cells (HSC). Whereas IL-1 drives HSC proliferation directly <italic>in vitro</italic>, inflammation induces comparable rates of proliferation in IL-1RI deficient and -sufficient HSC, MPP, and GMP in mixed chimeric mice. Thus, IL-1RI signals play a necessary, but indirect role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis.</p><p>The lack of alum-induced neutrophilia in IL-1RI<super>-/-</super> mice is due to defective mobilization of bone marrow (BM) neutrophils and impaired proliferation of hematopoietic stem and progenitor cells (HSPC). Coincident defects in neutrophil mobilization and HSPC proliferation suggest that the trigger for emergency granulopoiesis might be the exhaustion of neutrophil compartments rather than inflammatory inductions of growth factors. Consistent with this hypothesis, non-inflammatory reductions in BM neutrophil numbers elicit granulopoietic responses similar to those induced by adjuvant. Alum mobilizes BM neutrophils via G-CSF, but increased HSPC proliferation results from a density-dependent mechanism that is only partially dependent on G-CSF. Notably, C/EBP&beta;, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC, but plays a role in the terminal differentiation of neutrophils. These observations indicate that the draining of BM neutrophil pools is sufficient to activate a latent, homeostatic mechanism of accelerated granulopoiesis. I propose a common model for the regulation of neutrophil production that explains both steady-state and emergency granulopoiesis through negative feedback.</p> / Dissertation

Diabetic nephropathy is a common diabetic microvascular disease with a prevalence of about 10% to 42%. Research has shown that neutrophil gelatinase-associated lipocalin (NGAL) levels would increase rapidly in the urine and blood of patients with acute kidney failure. NGAL may represent an early and predictive kidney injury biomarker due to the increase of NGAL occurs earlier than that of molecules (creatinine, cystatin C and £]2-microglobulin) for traditional assessment of renal injury in renal disease samples. To evaluate the association of plasma level of NGAL and £]2-microglobulin with diabetic nephropathy, this study was performed on 21 diabetic patients without nephropathy as the control group and 21 patients with diabetic nephropathy stage 2, 26 patients with stage 3, 9 patients with stage 4 and 16 patients stage 5 as the study group. Collection of blood and measurements of all cases were approved by the ethical committee. The results indicate that the levels of blood urea nitrogen (BUN), creatinine, NGAL, and £]2-microglobulin of study group were significantly higher than control group (P<0.001), while the glomerular filtration rate (GFR) was significantly lower than the control group (P<0.001). Linear regression analysis show that NGAL was positively correlated with white blood cells, BUN, creatinine, £]2-micrglobulin and negatively correlated with GFR; and £]2-micrglobulin was positively correlated with BUN, creatinine, NGAL and negatively correlated with GFR. All results indicate that plasma NGAL levels in diabetic nephropathy were positively correlated with renal function parameters, and closely correlated with kidney injury, suggesting that NGAL may play an important role in the progression of diabetic nephropathy.

Abstract Neutrophils are the major cellular component of acute inflammatory response. The mechanism by which fMLP or PAF activates neutrophils is not fully elucidated. Stimulation of MAPKs and activation of NF-kappa B in neutrophils regulate various cell functions, including superoxide production. Neutrophils isolated from blood taken from healthy donors, were incubated with specific inhibitors, GF109203X (PKC inhibitor), calphostin C (PKC-gamma isoform inhibitor), wortmannin (PI3K inhibitor), U73122 (PLC inhibitor), aristolochic acid (PLA2 inhibitor), SKF96365 (SOC channel inhibitor), EGTA (extracellular calcium chelator), SB203580 (p38 MAPK inhibitor), and PD98059 (MEK inhibitor), followed by fMLP or PAF treatment. MAPK activation by fMLP or PAF is based on immunoblot analysis. NF-kappa B activation is detected by EMSA, and superoxide production is measured by flow cytometry. The data indicate that neutrophil MAPK signaling pathways mediated by fMLP and PAF are different. PAF-induced ERK MAPK phosphorylation was involved PI3K, PKC, PLA2, PLC, and extracellular calcium, wheres fMLP-induced phosphorylation doesn¡¦t involve PKC

Systemic lupus erythematosus (SLE) is a chronic multiorgan autoimmune disorder characterized by abundant immune complex (IC) deposition, with nephritis being a major cause of morbidity and mortality. Yet, IC deposition alone is not sufficient for disease development suggesting that additional factors dictate the propensity for developing target organ injury. Genome-wide association studies have identified polymorphisms in the leukocyte integrin Mac-1 (CD11b/CD18, ITGAM) that associate with lupus nephritis. Although Mac-1 promotes inflammation by triggering leukocyte recruitment and cytotoxic functions, there is emerging evidence that it may also serve protective roles under certain conditions. We demonstrate that Mac-1 deficiency in the context of the uniquely human FcgRIIA a receptor that binds IgG-IC, promotes susceptibility to lupus nephritis in two independent animal models. Analysis of renal tissue and intravital microscopy revealed that Mac-1 modulates neutrophil recruitment by FcgRIIA. The SLE-associated variant of Mac-1 rs1143679 (R77H), results in reduced Mac-1 functions, but the underlying mechanism remains undefined. CD18 integrin mediated adhesion is a multistep process that begins with affinity changes for ligand via transmission of allosteric signals. Moreover, mechanical forces (e.g. shear flow) paradoxically increase the lifetime of integrin-ligand bonds, referred to as "catch-bonds". Here, we show that expression of Mac-1 R77H on neutrophils, and blocking antibodies to the extracellular b-propeller domain in which it resides, markedly impairs Mac-1 adhesion to ligand under shear flow. R77H expressing cells exhibit a shift in equilibrium towards a bent conformation, a lower affinity and on- and off- rate for ligand and an inability to form catch-bonds. Additional mutants and activating antibodies reveal that R77H prevents allosteric signal transmission to the aI-domain required for productive ligand binding.