Higher Numbers Of Neutrophils Research Articles

Inflammasome-Dependent IL-1β Release from Neutrophils in Human Sickle Cell Anemia

Sickle cell anemia (SCA) is associated with a chronic inflammatory state. The inflammasome complex, responsible for IL-1β and IL-18 cytokine maturation and release, is formed by pattern recognition receptors (PRRs), such as the NLRP3 protein, which recognize damage (or pathogen) associated molecular patterns (DAMPs), in turn recruiting the ASC adapter protein, and cleaving pro-caspase-1, which processes pro-IL-1β and pro-IL-18 into their bioactive forms. While elevated plasma IL-1β and IL-18 levels have been reported in SCA, organization of the inflammasome and its components, and the cells in which this occurs, have yet to be identified in the disease. We aimed to determine whether inflammasome assembly occurs in the leukocytes of individuals with SCA, and associate this formation with levels of some circulating inflammatory DAMPs. Leukocytes (separated by ficoll or percoll gradients) and plasma were obtained from healthy control individuals (CON) and SCA patients in steady state (SCA) not on hydroxyurea therapy. Plasma cytokines and DAMPs were quantified by colorimetric/ bioluminescence assays/ ELISA. Caspase-1 activity was determined in phenotypically-characterized leukocyte populations by flow cytometry (Fam-FlicaTM kit). Both IL-1β and IL-18, hallmarks of inflammasome formation, were significantly increased in SCA plasma, compared to CON (IL-1β: 0.531±0.221, 0.056±0.026 pg/ml; IL-18: 884.0±240.7, 304.8±32.4 pg/ml, for SCA [N=13] and CON [N=11], respectively; P<0.01). Caspase-1 activity was augmented in SCA neutrophils, when compared to CON (2.16±0.36 %, 0.86±0.09 % caspase-1 positivity [N=10/13], respectively; P<0.001), the equivalent of approximately 8.47±1.78 x104 caspase-positive neutrophils/ml in the circulation of these patients. This augmented caspase-1 activity was associated with an elevated secretion of IL-1β from SCA neutrophils, compared to that of CON neutrophils (10.6±1.5 pg/106 cells, 2.1±0.5 pg/106 cells [N=8/9] respectively; P<0.001, during 2h, 37oC). In contrast, although some basal caspase-1 activity was observed in CON CD14+CD16- and CD14+CD16+ monocytes, this activity was not significantly different in SCA monocytes (P>0.05). Accordingly, IL-1β secretion from SCA monocytes was not significantly augmented (P>0.05). Interestingly, while caspase-1 inhibition (co-incubation with 40 µM YVAD-FMK) abrogated IL-1β release from SCA neutrophils (2x106 cell/ml) during 2 h (decreased from 18.9±1.9 pg/ml to 9.8±1.1 pg/ml; N=5, P<0.05), consistent with inflammasome-dependent IL-1β production, NLRP3 inhibition (5µM MCC950 co-incubation) did not affect SCA neutrophil IL-1β release (18.6±3.0 pg/ml; N=5), suggesting that an alternative PPR may participate in the SCA neutrophil inflammasome [MCC 950 inhibitor efficiency was confirmed using a NLRP3 neutrophil inflammasome control]. Furthermore, confocal microscopy and immunofluorescence indicated augmented ASC activity in unstimulated SCA neutrophils, compared to CON neutrophils (Figure 1). We also determined the concentrations of some circulating DAMPs in CON [N≥7] and SCA [N≥11] subjects. Heme, HSP70 and HMGB1 were all significantly (P<0.05) elevated in SCA plasma, compared to CON plasma (Heme: 18.9±2.7, 62.6±7.8 µM; HSP70: 5.53±0.51, 9.41±1.09; HMGB1: 1.6±0.25, 3.6±0.5; for CON and SCA, respectively), while no significant modulations in ATP or IL-33 were observed in SCA plasma (data not shown; P>0.05). However, no significant correlations were observed between DAMP concentrations and plasma IL-1β or neutrophil caspase activity (P>0.05). In conclusion, data indicate that augmented inflammasome assembly occurs in the neutrophils of individuals with SCA; in contrast, we found no evidence of augmented inflammasome activation in the monocytes of these same individuals. Given the high number of neutrophils in the circulation of SCA patients, it seems reasonable to assume that these cells may contribute to augmented IL-1β and IL-18 processing in this disease. DAMPs, associated with both red cell destruction and ischemia-reperfusion injury, were found elevated in the plasma of the SCA individuals studied and may, collectively, contribute to triggering inflammatory pathways, including inflammasome formation. In conclusion, we present data to confirm that inflammasome assembly occurs in sickle cell anemia and may represent a therapeutic target for SCA. [Display omitted] DisclosuresConran:Bayer AG: Research Funding.

TMIC-30. ROLE OF MATRIX METALLOPROTEINASE 9 (MMP9) IN RESISTANCE TO ANTI-ANGIOGENIC THERAPY

While bevacizumab initially showed great promise as an anti-angiogenic therapy for glioblastoma, randomized clinical trials showed limited duration of efficacy that fails to impact overall survival. Preclinical studies from our group and others found tumors resistant to anti-angiogenic therapy to be more invasive with an increased metastatic potential. This enhanced level of malignant cell invasion is thought to occur through hijacking of natural epithelial-to-mesenchymal transition (EMT) pathways activated during development, a set of complex spatial and temporal interactions for migration involving multiple cellular pathways and transcription factors. To better understand the mechanisms of glioblastoma resistance to anti-angiogenic therapy, we developed a generational xenograft model of resistance and profiled transcriptional changes and cells in the microenvironment contributing to resistance. Genome-wide expression profile of selected generations discovered a shift over time towards a mesenchymal molecular profile, with a similar shift seen in pre- and post-bevacizumab treated patient samples. Detailed analysis of the protein interactions between these mesenchymal genes elucidated MMP9, a member of the matrix metalloproteinase family, as the strongest potential driver of the shift towards the mesenchymal phenotype in our xenograft model. Previous work has found that neutrophils, drawn to inflamed tumors sites, are unique producers of MMP9 without its regulator TIMP1. Fluorescent imaging of intracranial xenograft tumors treated with PBS or bevacizumab discovered increased levels of MMP9, which occurred within the higher number of neutrophils seen in the invading edge of the tumor mass in bevacizumab-treated samples, confirming these tumors are better able to recruit neutrophils and hijack their innate immune response machinery for increased tumor survival. These findings challenge the ideas that gelatinases are solely downstream targets of higher level regulators and that neutrophils are passive inflammatory bystanders, instead defining neutrophil-secreted MMP9 as an early activator responsible for downstream effects driving resistance to anti-angiogenic therapy.

IL-4R signaling in B cells mediates control of IL-17A dependent emphysema triggered by N. brasiliensis infection.

Abstract The intestinal nematode parasite, Nippostrongylus brasiliensis (Nb), transiently resides in the lung and causes lung damage, which is then rapidly repaired. However, the repaired lung eventually develops emphysema and the mechanisms contributing to this chronic lung pathology remain poorly defined. Here we show that emphysema, as measured by mean linear intercept measurements (Lm) of alveolar spaces, develops as early as 7 days after Nb inoculation and is associated with increased gene expression of Il17A, neutrophil elastase (ELA), and macrophage matrix metalloproteinase 12 (Mmp12) in the lung. Transfer of Nb-primed neutrophils or macrophages to naïve recipients results in emphysema, and these cells expressed high level of ELA and Mmp12, respectively. Neutralization of IL-17A using anti-IL-17A antibody curbed development of emphysema, and decreased influx of neutrophils in the lung and gene expression of Il17A. Parasite infection in B cell deficient Jh−/− mice results in a more severe emphysema when compared to wild-type mice or FcRγ−/− mice, and is associated with higher numbers of neutrophils and macrophages, as well as increased expression of Il17a, ELA2 and Mmp12 in the lung. Transfer of B lymphocytes from wild-type mice or IL-10−/− mice, but not from IL4Rα−/− mice, to recipient Jh−/− mice can restore control of emphysema and downregulate IL-17A. B cells from lung tissue of Nb-primed wild type but not IL4Rα−/− mice expressed high level of Relmα. Taken together, these data suggest that IL-17A triggers infiltration of neutrophils and macrophages that contribute to chronic emphysema, which is controlled by IL-4R dependent B regulatory cells expressing high levels of Relmα.

Frontline Science: Splenic progenitors aid in maintaining high neutrophil numbers at sites of sterile chronic inflammation.

Neutrophils are constantly generated from hematopoietic stem and progenitor cells in the bone marrow to maintain high numbers in circulation. A considerable number of neutrophils and their progenitors have been shown to be present in the spleen too; however, their exact role in this organ remains unclear. Herein, we sought to study the function of splenic neutrophils and their progenitors using a mouse model for sterile, peritoneal inflammation. In this microcapsule device implantation model, we show chronic neutrophil presence at implant sites, with recruitment from circulation as the primary mechanism for their prevalence in the peritoneal exudate. Furthermore, we demonstrate that progenitor populations in the spleen play a key role in maintaining elevated neutrophil numbers. Our results provide new insight into the role for splenic neutrophils and their progenitors and establish a model to study neutrophil function during sterile inflammation.

CML Mouse Model Generated from Leukemia Stem Cells.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder with a high number of well-differentiated neutrophils in peripheral blood and myeloid cells in bone marrow (BM). CML is derived from the hematopoietic stem cells (HSCs) with the Philadelphia chromosome (Ph(+), t(9;22)-(q34;q11)), resulting in generating a fusion oncogene, BCR/ABL1. HSCs with Ph(+) are defined as leukemia stem cells (LSCs), a subpopulation cell at the apex of hierarchies in leukemia cells and responsible for the disease continuous propagation. Several kinds of CML models have been developed to reveal the mechanism of CML pathogenesis and evaluate therapeutic drugs in the past three decades. Here, we describe the procedures to generate a CML mouse model by introducing BCR/ABL1 into Lin(-)Sca1(+) cKit(+) population cells purified from mouse bone marrow. In CML retroviral transduction/transplantation mouse models, this modified model can mimic CML pathogenesis on high fidelity.

Comparative Efficiency and Impact on the Activity of Blood Neutrophils Isolated by Percoll, Ficoll and Spontaneous Sedimentation Methods

ABSTRACTStudies on the role of cells in physiological and pathological processes generally require isolation of some populations, such as neutrophils. In the literature, several methods used for isolating neutrophils are described; however, there is no consensus on the best technique to be used in cell functional studies. The present study compares the efficiency and impact on the chemotactic and phagocytic activity of neutrophils isolated from blood by three different methods: Percoll and Ficoll density centrifugation gradients and spontaneous sedimentation technique. The neutrophil chemotaxis, stimulated with lipopolysaccharide (LPS), autologous serum or homologous serum, was determined by using Boyden chambers. The phagocytic capacity was assessed by ingestion of zimosan particles, and digestion phase was analyzed by nitroblue tetrazolium test (NBT). The results obtained from neutrophil isolation by Percoll and Ficoll density gradients, as compared to spontaneous sedimentation technique, showed similar degrees of cell yields and higher purity; however, these methods affected neutrophil responsiveness, accompanied by elevated chemotaxis and reduced chemotactic capacity to respond to subsequent stimulation. Neutrophil isolation by spontaneous sedimentation, in contrast, did not affect cellular activity and resulted in cell preparation with high number of neutrophils. Although neutrophil phagocytosis results were similar between the different methods, digestion phase of phagocytosis was significantly enhanced after LPS-stimulation, only in the neutrophils isolated by spontaneous sedimentation technique. In conclusion, the present study shows that isolation of blood neutrophils by the spontaneous sedimentation technique is appropriate for the assessment of cellular activity, since it neither primes or activates the neutrophils nor does it affect their functional responsiveness.

Myelodysplastic Syndrome Caused By Activating Kras Mutation in the Non-Hematopoietic Bone Marrow Microenvironment

Oncogenic Ras mutations occur frequently in myelodysplastic and myeloproliferative syndromes as juvenile myelomonocytic leukemia (JMML) and the myeloproliferative variant of chronic myelomonocytic leukemia (MP-CMML) as well as in acute myeloid leukemia. However in these reports the mutations were in the hematopoietic cells. Here, we show that an activating mutation of Kras in the non-hematopoietic system leads to hematologic disorder resembling human myelodysplastic syndrome (MDS).Rosa26CreERT2;LSL-KrasG12D mice (CD45.2) were lethally irradiated and transplanted with wild-type bone marrow (CD45.1). After control of engraftment efficiency (above 99.6%), the mice were treated with Tamoxifen to induce the expression of KrasG12D in non-hematopoietic cells.6-8 weeks after Tamoxifen treatment, the mice developed anemia, leukocytopenia and thrombocytopenia and had a highly increased percentage of myeloid cells in peripheral blood, spleen and bone marrow. FACS-analysis confirmed that these cells were donor-derived and therefore of wild-type origin. The frequency of immature myeloid progenitors (CD11b+ c-kit+) was increased in bone marrow of Rosa26CreERT2;LSL-KrasG12D mice compared to littermate controls suggesting a disturbed differentiation. Morphological analysis of blood smears and bone marrow revealed a high number of dysplastic hypersegmented neutrophils as well as the occurrence of myeloid blasts.Additionally, a significant decrease of B-lymphocytes was observed in the bone marrow of KrasG12D recipient mice which has also been described in human MDS. Osteoblasts have been shown to contribute to B-cell lymphopoiesis which implicates that decreased B-cell lymphopoiesis in this study may be a result of oncogenic Kras expression in osteoblasts.All these data indicate that a single mutation in the hematopoietic microenvironment can initiate a severe hematologic disorder. The expression of oncogenic Kras in bone marrow stroma cells leads to a shift to myeloid differentiation, severe anemia and thrombocytopenia as well as reduced B-cell counts recapitulating main signs of human myelodysplastic syndrome. DisclosuresNo relevant conflicts of interest to declare.

Low Vitamin D Status Is Associated with Systemic and Gastrointestinal Inflammation in Dogs with a Chronic Enteropathy

IntroductionVitamin D deficiency, as assessed by serum concentrations of 25 hydroxyvitamin D (25(OH)D), has been linked to the development of over-zealous and inappropriate inflammation in humans. However, the relationship between vitamin D status and inflammation in dogs is ill-defined. Chronic enteropathies (CE) are frequently diagnosed in client owned dogs, have a wide range of serum 25(OH)D concentrations, and represent a spontaneous model in which to probe the relationship between vitamin D and inflammation. The hypothesis of this study was that vitamin D status would be negatively associated with systemic and gastrointestinal inflammation in dogs with a CE. The aim of this study was to examine the relationship between serum 25(OH)D concentrations and markers of systemic and gastrointestinal inflammation in a cohort of dogs with CE.Methods and MaterialsSerum 25(OH)D concentrations, together with neutrophil, monocyte, eosinophil and lymphocyte counts, duodenal histopathology scores, serum IL-2, IL-6, IL-8 and TNFα concentrations and were measured in 39 dogs with histologically confirmed CE. A linear regression model examined the relationship between serum 25(OH)D status and measures of inflammation.ResultsSerum 25(OH)D concentrations were negatively associated with neutrophil and monocyte counts, duodenal histopathology scores and serum IL-2 and IL-8 concentrations. Dogs with low serum 25(OH)D concentrations typically had an inflammatory signature characterised by high monocyte and neutrophil numbers together with low lymphocyte numbers. There is a need to establish whether low vitamin D status is a cause or consequence of inflammation.

Possible role of interferon tau on the bovine corpus luteum and neutrophils during the early pregnancy.

When pregnancy is established, interferon tau (IFNT), a well-known pregnancy recognition signal in ruminants, is secreted by embryonic trophoblast cells and acts within the uterus to prepare for pregnancy. IFNT acts as an endocrine factor on the corpus luteum (CL) to induce refractory ability against the luteolytic action of PGF2 α. Hypothesising that IFNT may influence not only the uterine environment but also the CL in cows via local or peripheral circulation, we investigated qualitative changes in the CL of pregnant cows during the maternal recognition period (day 16) and the CL of non-pregnant cows. The CL of pregnant animals had a higher number of neutrophils, and the expression of interleukin 8 (IL8) mRNA and its protein was higher as well as compared with the CL of non-pregnant animals. Although IFNT did not affect progesterone (P4) secretion and neutrophil migration directly, it stimulated IL8 mRNA expression on luteal cells (LCs), influencing the neutrophils, resulting in the increased migration of IFNT-activated neutrophils. Moreover, both IFNT-activated neutrophils and IL8 increased P4 secretion from LCs in vitro. Our novel finding was the increase in neutrophils and IL8 within the CL of pregnant cows, suggesting the involvement of IFNT function within the CL toward establishment of pregnancy in cows. The present results suggest that IFNT upregulates neutrophil numbers and function via IL8 on LCs in the CL of early pregnant cows and that both neutrophils and IL8, stimulated by IFNT, are associated with an increase in P4 concentrations during the maternal recognition period in cows.

B lymphocytes limit the severity of emphysema caused by N. brasiliensis infection in mice (INC4P.332)

Abstract The intestinal nematode parasite, Nippostrongylus brasiliensis (Nb), transiently resides in the lung causing lung damage, resulting in emphysema. However, the mechanisms contributing to this chronic lung pathology remain not well-defined. Here we show that emphysema develops as early as 7 days after Nb inoculation, as measured by alveolar mean linear chord length and is associated with increased neutrophil elastase (NE) and macrophage matrix metalloproteinase 12 (Mmp12) gene expression in the lung. Transfer of sorted Nb-primed neutrophils or macrophages to naïve recipients results in emphysema, indicating that either of these populations can trigger lung emphysema in naïve mice. Parasite infection also results in more severe emphysema in B cell deficient Jh-/- mice compared to wild-type mice or FcRγ-/-mice and is associated with higher numbers of neutrophils and macrophages, as well as increased expression of NE and Mmp12 in the lung. Transfer of B lymphocytes from wild-type mice to the Jh-/- mice can restore control of emphysema. These data suggest that Nb-primed neutrophils and macrophages contribute to lung emphysema. Furthermore, parasite-induced B cells exert regulatory effects that can markedly reduce the severity of emphysema.

Angels and demons: Th17 cells represent a beneficial response, while neutrophil IL-17 is associated with poor prognosis in squamous cervical cancer

The role of interleukin (IL)-17 in cancer remains controversial. In view of the growing interest in the targeting of IL-17, knowing its cellular sources and clinical implications is crucial. In the present study, we unraveled the phenotype of IL-17 expressing cells in cervical cancer using immunohistochemical double and immunofluorescent triple stainings. In the tumor stroma, IL-17 was found to be predominantly expressed by neutrophils (66%), mast cells (23%), and innate lymphoid cells (8%). Remarkably, T-helper 17 (Th17) cells were a minor IL-17 expressing population (4%). A similar distribution was observed in the tumor epithelium. The Th17 and granulocyte fractions were confirmed in head and neck, ovarian, endometrial, prostate, breast, lung, and colon carcinoma. An above median number of total IL-17 expressing cells was an independent prognostic factor for poor disease-specific survival in early stage disease (p = 0.016). While a high number of neutrophils showed at trend toward poor survival, the lowest quartile of mast cells correlated with poor survival (p = 0.011). IL-17 expressing cells and neutrophils were also correlated with the absence of vaso-invasion (p < 0.01). IL-17 was found to increase cell growth or tightness of cervical cancer cell lines, which may be a mechanism for tumorigenesis in early stage disease. These data suggest that IL-17, primarily expressed by neutrophils, predominantly promotes tumor growth, correlated with poor prognosis in early stage disease. Strikingly, a high number of Th17 cells was an independent prognostic factor for improved survival (p = 0.026), suggesting Th17 cells are part of a tumor suppressing immune response.

Myocardial collagen deposition and inflammatory cell infiltration in cats with pre-clinical hypertrophic cardiomyopathy

The histological features of feline hypertrophic cardiomyopathy (HCM) have been well documented, but there are no reports describing the histological features in mild pre-clinical disease, since cats are rarely screened for the disease in the early stages before clinical signs are apparent. Histological changes at the early stage of the disease in pre-clinical cats could contribute to an improved understanding of disease aetiology or progression. The aim of this study was to evaluate the histological features of HCM in the left ventricular (LV) myocardium of cats diagnosed with pre-clinical HCM. Clinically healthy cats with normal (n = 11) and pre-clinical HCM (n = 6) were identified on the basis of echocardiography; LV free wall dimensions (LVFWd) and/or interventricular septal wall (IVSd) dimensions during diastole of 6–7 mm were defined as HCM, while equivalent dimensions <5.5 mm were defined as normal. LV myocardial sections were assessed and collagen content and inflammatory cell infiltrates were quantified objectively. Multifocal areas of inflammatory cell infiltration, predominantly lymphocytes, were observed frequently in the left myocardium of cats with pre-clinical HCM. Tissue from cats with pre-clinical HCM also had a higher number of neutrophils and a greater collagen content than the myocardium of normal cats. The myocardium variably demonstrated other features characteristic of HCM, including arteriolar mural hypertrophy and interstitial fibrosis and, to a lesser extent, myocardial fibre disarray and cardiomyocyte hypertrophy. These results suggest that an inflammatory process could contribute to increased collagen content and the myocardial fibrosis known to be associated with HCM.

Neutrophil extracellular traps regulate IL-1β-mediated inflammation in familial Mediterranean fever

ObjectiveInflammatory attacks of familial Mediterranean fever (FMF) are characterised by circulation and influx of high number of polymorphonuclear neutrophils (PMN) in the affected sites and profound therapeutic effect of IL-1β...

PTU-130 Regulatory T Cells In Acute Liver Failure Are Functionally Intact And May Contribute To Retention Of Neutrophils In Areas Of Hepatic Necrosis

IntroductionAcute liver failure (ALF) is a sterile inflammation with a high mortality. The immunological response to acute liver injury is initiated by the infiltration of innate neutrophils and is followed...

Kinetics of pulmonary immune cells, antibody responses and their correlations with the viral clearance of influenza A fatal infection in mice

Fatal influenza A virus infection is a major threat to public health throughout the world. Lung macrophages and neutrophils have critical roles for both the pathogenesis and viral clearance of fatal viral infections. These are complicated by the interaction of innate immunity and adaptive immunity against viral infection. In this study, we investigated the overall kinetics of lung macrophages, neutrophils, CD4+T cells, CD8+T cells, CD38+ cells, and CD138+ cells, the levels of antibody and cytokine responses, both in the early and late phases of fatal infection with A/PR/8/34 (H1N1) virus in mice. The changes in lung viral load were also evaluated. We found that pulmonary macrophages and neutrophils both accumulated in the early and late phases of fatal infections and they positively correlated with the lung and serum antibody titers, and negatively correlated with the viral load locally. The secretion of IL-6 might relate to high numbers of macrophages and neutrophils in the early infection. The work implies that pulmonary macrophages, neutrophils and the antibody response all have an essential role in virus elimination of fatal influenza A viral infection. These findings may have implications for the development of prophylactic and therapeutic strategies in fatal influenza A viral infection. Further evaluation of the cooperation among macrophages, neutrophils and antibody responses in eliminating the virus with fatal infection is needed.

Neutrophils mediate edema formation but not mechanical allodynia during zymosan-induced inflammation

Inflammatory pain is based on stimulation and sensitization of peripheral endings of sensory neurons (nociceptors) by pronociceptive mediators. These mediators can be released by resident cells, as well as invading immune cells. Although neutrophils are known to release various mediators, which can stimulate or sensitize nociceptors, the extent of their contribution to nociceptive responses is unclear. Here, we studied the contribution of neutrophils to zymosan-induced inflammatory pain, which is characterized by an early recruitment of high numbers of neutrophils. Surprisingly, antibody-mediated neutrophil depletion caused a complete loss of edema formation but had no effect on mechanical pain thresholds. Blockage of the interaction between neutrophils and platelets or endothelial cells using antibodies directed against CD11b and CD162 reduced neutrophil recruitment to the site of inflammation. Again, the treatment decreased zymosan-induced edemas without altering mechanical pain thresholds. Also, HLB-219 mice, which have five to 10 times less platelets than WT mice, showed reduced neutrophil recruitment to the site of inflammation and decreased edema sizes, whereas, again, mechanical thresholds were unaltered. The effects observed in HLB-219 mice were relatively small and not reproduced in vWF-deficient mice or after antibody-mediated blockage of GPIbα. Flow chamber and transmigration assays showed that platelets were not necessary for neutrophil adhesion to endothelial cells but increased their transmigration. Taken together, zymosan-induced mechanical allodynia is, in contrast to edema formation, independent of neutrophils, and recruitment of neutrophils is only partly influenced by interactions with platelets.

The systemic immune state of super-shedder mice is characterized by a unique neutrophil-dependent blunting of TH1 responses.

Host-to-host transmission of a pathogen ensures its successful propagation and maintenance within a host population. A striking feature of disease transmission is the heterogeneity in host infectiousness. It has been proposed that within a host population, 20% of the infected hosts, termed super-shedders, are responsible for 80% of disease transmission. However, very little is known about the immune state of these super-shedders. In this study, we used the model organism Salmonella enterica serovar Typhimurium, an important cause of disease in humans and animal hosts, to study the immune state of super-shedders. Compared to moderate shedders, super-shedder mice had an active inflammatory response in both the gastrointestinal tract and the spleen but a dampened TH1 response specific to the secondary lymphoid organs. Spleens from super-shedder mice had higher numbers of neutrophils, and a dampened T cell response, characterized by higher levels of regulatory T cells (Tregs), fewer T-bet+ (TH1) T cells as well as blunted cytokine responsiveness. Administration of the cytokine granulocyte colony stimulating factor (G-CSF) and subsequent neutrophilia was sufficient to induce the super-shedder immune phenotype in moderate-shedder mice. Similar to super-shedders, these G-CSF-treated moderate-shedders had a dampened TH1 response with fewer T-bet+ T cells and a loss of cytokine responsiveness. Additionally, G-CSF treatment inhibited IL-2-mediated TH1 expansion. Finally, depletion of neutrophils led to an increase in the number of T-bet+ TH1 cells and restored their ability to respond to IL-2. Taken together, we demonstrate a novel role for neutrophils in blunting IL-2-mediated proliferation of the TH1 immune response in the spleens of mice that are colonized by high levels of S. Typhimurium in the gastrointestinal tract.

AB1195 Our experience with anakinra treatment in a tertiary centre

BackgroundAnakinra (ANK) is a recombinant interleukin-1 receptor antagonist currently used for treating autoinflammatory syndromes.ObjectivesTo describe patient characteristics and their response to ANK in a tertiary centre between 2004 and 2011.MethodsRetrospective...

Neutrophils, neutrophil extracellular traps and interleukin-17 associate with the organisation of thrombi in acute myocardial infarction

Neutrophils are important cellular sources of interleukin (IL) 17A and -F. Moreover, upon activation neutrophils are able to excrete chromatin embedded with components from their cytoplasmic granules to form 'neutrophil extracellular traps' (NETs). Recent studies suggested that NETs contribute to thrombosis by promoting fibrin deposition and platelet aggregation. IL17A may also promote thrombosis by enhancing platelet aggregation. In the present study we investigated the presence of neutrophils, NETs and IL17A and -F in coronary thrombosuction specimens obtained from patients after acute myocardial infarction. Neutrophils and NETs were identified using histochemical (HE, Feulgen procedure) and immunohistochemical stainings (Histone H1, myeloperoxidase, neutrophil elastase) in 15 fresh, 15 lytic and 15 organised thrombi. The presence and distribution of IL17A and -F was studied using (immuno)histochemical double staining and spectral image analysis, rtPCR and Western blot. High numbers of neutrophils are present (10-30% of the thrombus mass) in fresh and lytic, but not in organized thrombus. NETs were frequently observed in fresh (4/15) and lytic (12/15), but never in organised thrombus specimens. Double staining combining the Feulgen reaction with Histone-H1, MPO or neutrophil elastase confirmed colocalisation with DNA. Cytoplasmatic IL17A/F staining was found in the majority of the neutrophils, extracellularly and in NETs. Western blotting confirmed the presence of IL17A and IL17F in thrombus specimens. In conclusion, a large burden of neutrophils, neutrophil extracellular traps and IL17A and -F are important constituents of fresh and lytic thrombus after acute myocardial infarction. The specific colocalisation of these indicates a role during thrombus stabilisation and growth.

Abstract B86: Immunomodulatory effects of LL-37/CRAMP in prostate cancer progression.

Abstract Prostate cancer (PCa) is the most common type of cancer among males in the United States. However, even with advanced diagnostic capacity and conventional therapies such as surgery and chemotherapy, the 5-year survival rate for disseminated disease is approximately 36 months suggesting the need for finding alterate targets and therapies to increase patient survival. We have recently demonstrated that LL-37, an antimicrobial peptide that plays an important role in innate immune response against microbial pathogens, and its mouse orthologue CRAMP (Cathelicidin-related antimicrobial peptide) plays an important role in the progression of PCa both in humans and in a spontaneously developing mouse PCa model, respectively. Over-expression of CRAMP has also been recently reported in a few human epithelial cancers including carcinomas of the breast and lung. Results of our recent studies also demonstrated that targeted knock-down of CRAMP expression in a mouse PCa cell line, derived from the transgenic adenocarcinoma mouse prostate (TRAMP) model, TRAMP C1, resulted in decreased level of myeloid-derived suppressor cells (MDSC) in the spleen upon syngeneic transplantation. Based on these findings, the present study determined the role of LL-37/CRAMP in immune modulation during PCa progression. C57BL/6 mice were subcutaneously implanted with 3 different PCa cell lines; TRAMP C1 (CRAMPhigh), TRAMP C1scrambled (CRAMPhigh) and TRAMP C1CRAMPshRNA (CRAMPlow). Mice in TRAMP C1 and TRAMP C1scrambled groups developed measurable tumors starting from 45 days post-implantation, whereas mice implanted with TRAMP C1CRAMPshRNA showed measurable tumors from 120 days post-implantation suggesting that high levels of CRAMP expression in TRAMP C1 and TRAMP C1scrambled groups promote prostate tumor growth in vivo. Immune cell phenotyping was performed on cells collected from spleen and tumor at day 50 and 55 post-implantation to identify modulation in infiltrating immune cells as a surrogate for decrease in tumor growth due to abrogation of CRAMP expression. The result indicated a higher number of neutrophils infiltrating to the tumor site from the spleen during the initial stages of tumor growth in TRAMP C1 and TRAMP C1scrambled groups, while high numbers of neutrophils remained in the spleen of TRAMP C1CRAMPshRNA group during that time point. Elevated numbers of MDSC were observed both in spleen and in tumor microenvironment in TRAMP C1 and TRAMP C1scrambled groups suggesting that MDSC may further promote PCa growth whereas mice in the TRAMP C1CRAMPshRNA group showed significantly low level of MDSC in spleen, even when compared to control mice, at earlier time point. The number of plasmacytoid dendritic cells (pDC) both in spleen and tumor in TRAMP C1 and TRAMP C1scrambled groups was low while significantly high numbers of pDC were observed in spleen in TRAMP C1CRAMPshRNA group suggesting that high numbers of pDC may suppress the tumor growth by exerting pro-inflammatory response through cytokines such as IL-12. Altogether, the data suggest that CRAMP promotes PCa growth and plays an important role in immunomodulation during PCa progression. The CRAMP induces infiltration of neutrophils to tumor site and increased number of MDSC in both spleen and tumor, which may induce immunosuppression. Since CRAMP triggers protumorigenic stimuli including angiogenesis, invasion and key immune modulation to promote the tumor growth, LL-37/CRAMP may be used as a possible therapeutic target for PCa treatment. Ongoing tumor challenge studies in CRAMP knockout mouse model will shed more light to confirm these observations and to possibly extend the findings in human patients as a potential therapeutic target. Citation Format: Ha-Ram Cha, Anandi Sawant, Carnella Lee, Jonathan Hensel, George Tsuladze, Selvarangan Ponnazhagan. Immunomodulatory effects of LL-37/CRAMP in prostate cancer progression. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B86.