Neutrophil Subsets Research Articles

Interaction between CD177 and platelet endothelial cell adhesion molecule-1 downregulates membrane-bound proteinase-3 (PR3) expression on neutrophils and attenuates neutrophil activation induced by PR3-ANCA

BackgroundA recent study found that CD177 served as a receptor of membrane-bound proteinase-3 (mPR3) in a subset of neutrophils. Furthermore, CD177 has been identified as a high-affinity heterophilic binding partner for the endothelial cell platelet endothelial cell adhesion molecule-1 (PECAM-1). The current study aimed to investigate whether the interaction between PECAM-1 and CD177 could influence mPR3 expression as well as PR3-antineutrophil cytoplasmic antibody (ANCA)-induced neutrophil activation and glomerular endothelial cell (GEnC) injury.MethodsThe effect of interaction between CD177 and PECAM-1 on mPR3 expression was explored by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The effect of PECAM-1 on neutrophil activation and GEnC injury induced by PR3-ANCA-positive immunoglobulin (Ig)Gs was evaluated by dihydrorhodamine (DHR) assay and ELISA. CD177-negative neutrophils were selected by magnetic cell sorting (MACS), and the inhibitory effect of PECAM-1 on CD177-negative and mixed neutrophils was explored by measuring neutrophil degranulation.ResultsThe level of specific interaction between CD177 and PECAM-1 was elevated with increasing CD177 concentration. The expression of mPR3 significantly decreased in neutrophils preincubated with PECAM-1 in a dose-dependent manner. Consistently, the levels of respiratory burst and degranulation induced by PR3-ANCA-positive IgGs in recombinant human tumor necrosis factor-alpha (TNF-α)-primed neutrophils was significantly reduced by preincubation with PECAM-1 (440.6 ± 123.0 vs. 511.4 ± 95.5, p < 0.05; and 3155.0 ± 1733.0 ng/ml vs. 5903.0 ± 717.5 ng/ml, p < 0.05, respectively). In CD177-negative neutrophils incubated with PR3-ANCA-positive IgGs, the level of degranulation was not significantly changed by preincubation with PECAM-1. However, in mixed neutrophils, PECAM-1 significantly decreased the level of degranulation induced by PR3-ANCA-positive IgGs (1015.9 ± 229.2% vs. 1725.2 ± 412.4%, p < 0.01). Furthermore, with preincubation of TNF-α-primed neutrophils with PECAM-1, the level of soluble intercellular cell adhesion molecule-1 (sICAM-1), a marker of endothelial cell activation and injury, in the supernatant of GEnCs treated with primed neutrophils plus PR3-ANCA-positive IgGs was significantly attenuated (112.7 ± 24.2 pg/ml vs. 167.5 ± 27.7 pg/ml, p < 0.05).ConclusionsPECAM-1 can decrease the level of mPR3 expression on neutrophils, resulting in attenuation of neutrophil activation and subsequent GEnC injury induced by PR3-ANCA-positive IgGs.

Dysregulated neutrophil responses and neutrophil extracellular trap formation and degradation in PAPA syndrome

ObjectivesPyogenic arthritis, pyoderma gangrenosum and acne (PAPA) syndrome is characterised by flares of sterile arthritis with neutrophil infiltrate and the overproduction of interleukin (IL)-1β. The purpose of this study was...

Interactions among myeloid regulatory cells in cancer.

Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.

Abstract 5069: Inflammatory cytokine localization in the prostate tumor microenvironment and their association with prostate cancer racial disparities

Abstract Prostate cancer (PCa) is the 3rd leading cause of cancer-related deaths in American men. The major risk factors for PCa development include advanced age, family history and African ancestry. Chronic inflammation has been implicated as a major contributor to PCa and genes involved in inflammatory pathways are reported as more prevalent in tumors from African American (AA) versus Caucasian American (CA) men. Specifically, interleukin-1β (IL1β), IL6, IL8, and IL10 have been identified as over-expressed in the tumor microenvironment of AA men. These interleukins are in involved in the attraction, activation or maintenance of innate immune cells such as neutrophils and macrophages. However, it is not well understood where these interleukins are expressed in the prostate and what cell types produce them. We aim to address these specific questions in this study. The expression pattern and cellular localization of IL1β, IL6, IL8 and IL10 were evaluated using a highly specific RNA in situ hybridization (RISH) assay to analyze formalin-fixed paraffin-embedded tissues of low grade (Gleason ≤ 3+4) and higher grade (Gleason ≥ 4+3) PCa from AA and CA men. IL8 expression was additionally assessed by RISH in two unique tissue microarray (TMA) sets; i) primary prostate tumors obtained by radical prostatectomy from AA and CA men, matched for patient age, tumor grade and stage; ii) distant metastatic tissues obtained by autopsy. Immunohistochemical analysis of neutrophils (CD66ce) and RISH for macrophages (CD68) was used to assess the expression pattern of these immune cells within the same cohorts. Limited IL1β and IL10 expression was observed across all cases. IL6 expression was largely confined to the stromal compartment in endothelial cells and areas of acute inflammation. Modest IL6 expression was observed in atrophic epithelial cells. IL6 was never seen in tumor cells. IL8 was the most abundantly detected cytokine within our cohort and was significantly increased in higher grade cases. In benign regions, IL8 expression was predominantly observed in epithelial cells in regions of prostatic atrophy and in areas surrounding corpora amylacea. We observed marked IL8 expression in urothelial cells in most cases. IL8 expression was also apparent in multiple tumors, both in tumor cells and infiltrating immune cells. Tumor-infiltrating macrophages were markedly increased within a subset of tumors, but did not appear to express IL8. Conversely, a subset of neutrophils expressed IL8. Neutrophils were significantly increased in benign tissues from men with higher grade PCa, particularly in AA men. IL8 expression was also observed in a subset of distant metastases, particularly in liver metastases. There is differential expression of cytokines among men with PCa, and IL8 is the most abundant among the ones we analyzed. Our future studies aim to determine a role for IL8 in prostate carcinogenesis. Citation Format: Janielle P. Maynard, Onur Ertunc, Angelo M. De Marzo, Karen S. Sfanos. Inflammatory cytokine localization in the prostate tumor microenvironment and their association with prostate cancer racial disparities [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5069.

Cancer-related circulating and tumor-associated neutrophils - subtypes, sources and function.

In recent years, the role of neutrophils in cancer biology has been a matter of increasing interest. Many patients with advanced cancer show high levels of neutrophilia, tumor neutrophils are connected to dismal prognosis, and the neutrophil-to-lymphocyte ratio has been introduced as a significant prognostic factor for survival in many types of cancer. Neutrophils constitute an important portion of the infiltrating immune cells in the tumor microenvironment, but controversy has long surrounded the function of these cells in the context of cancer. Multiple evidences have shown that neutrophils recruited to the tumor can acquire either protumor or antitumor function. These findings have led to the identification of multiple and heterogeneous neutrophil subsets in the tumor and circulation. In addition, tumor-associated neutrophils (TANs) were shown to demonstrate functional plasticity, driven by multiple factors present in the tumor microenvironment. In this review, we examine the current knowledge on cancer-related circulating neutrophils, their source and the function of the different subtypes, both mature and immature. We then discuss the pro vs antitumor nature of TANs in cancer, their functional plasticity and the mechanisms that regulate neutrophil recruitment and polarization. Although the vast majority of the knowledge on neutrophils in cancer comes from murine studies, recent work has been done on human cancer-related neutrophils. In the final paragraphs, we expand on the current knowledge regarding the role of neutrophils in human cancer and examine the question whether cancer-related neutrophils (circulating or intratumoral) could be a new possible target for cancer immunotherapy.

Differential antibacterial control by neutrophil subsets

AbstractNeutrophils comprise a heterogeneous population of cells essential for bacterial eradication, and defects in neutrophil function are associated with increased susceptibility to infection. In this study, neutrophils from healthy controls were shown to prevent bacterial proliferation for at least 48 hours when cocultured with methicillin-resistant Staphylococcus aureus (MRSA) in tissue-like scaffolds by establishing a bacteriostatic environment inside their phagolysosome. This intracellular bacterial containment is independent of reactive oxygen species because neutrophils that lack a functional nicotinamide adenine dinucleotide phosphate–oxidase complex displayed no defect in intracellular bacterial containment, whereas killing of the pathogen was impaired. During acute inflammation, a subset of CD16bright/CD62Ldim hypersegmented neutrophils displayed normal phagocytosis associated with a remarkably poor capacity to contain bacteria intracellularly. Conversely, CD16dim-banded neutrophils were the only neutrophil subset that adequately contained MRSA. These findings demonstrate a clear neutrophil heterogeneity in their antimicrobial capacity and the appearance of neutrophil subsets with a clear differentiation in functionality during acute inflammation. Furthermore, this study provides an evolutionary basis for the rapid release of banded neutrophils into the circulation during acute inflammation.

An unappreciated role for neutrophil-DC hybrids in immunity to invasive fungal infections.

Neutrophils are classically defined as terminally differentiated, short-lived cells; however, neutrophils can be long-lived with phenotypic plasticity. During inflammation, a subset of neutrophils transdifferentiate into a population called neutrophil-DC hybrids (PMN-DCs) having properties of both neutrophils and dendritic cells. While these cells ubiquitously appear during inflammation, the role of PMN-DCs in disease remains poorly understood. We observed the differentiation of PMN-DCs in pre-clinical murine models of fungal infection: blastomycosis, aspergillosis and candidiasis. Using reporter strains of fungal viability, we found that PMN-DCs associate with fungal cells and kill them more efficiently than undifferentiated canonical neutrophils. During pulmonary blastomycosis, PMN-DCs comprised less than 1% of leukocytes yet contributed up to 15% of the fungal killing. PMN-DCs displayed higher expression of pattern recognition receptors, greater phagocytosis, and heightened production of reactive oxygen species compared to canonical neutrophils. PMN-DCs also displayed prominent NETosis. To further study PMN-DC function, we exploited a granulocyte/macrophage progenitor (GMP) cell line, generated PMN-DCs to over 90% purity, and used them for adoptive transfer and antigen presentation studies. Adoptively transferred PMN-DCs from the GMP line enhanced protection against systemic infection in vivo. PMN-DCs pulsed with antigen activated fungal calnexin-specific transgenic T cells in vitro and in vivo, promoting the production of interferon-γ and interleukin-17 in these CD4+ T cells. Through direct fungal killing and induction of adaptive immunity, PMN-DCs are potent effectors of antifungal immunity and thereby represent innovative cell therapeutic targets in treating life-threatening fungal infections.

Augmentation of the expression of the eotaxin receptor on duodenal neutrophils by IL-21

Inflammation can occur via different mechanisms, such as via acute and chronic responses, on numerous occasions and function accordingly through various roles. There are more than five subsets of neutrophils; neutrophilic heterogeneity is modulated by the inflammatory condition. To understand the characteristics of inflammation, identification of atypical neutrophils is important. In this study, we found that the expression of eotaxin receptor (CD193) on atypical neutrophils in the duodenum is augmented in IL-21 isoform transgenic (Tg) mice. In a series of studies, we have established a Tg mouse strain to further investigate the functions of IL-21 in vivo. Interestingly, Tg mice immunized with ovalbumin (OVA) were more sensitive to OVA-induced systemic anaphylaxis as compared with wild type mice with duodenal and splenic gross congestion. Further analysis conducted in the duodenum of Tg mice revealed that only the number of neutrophils migrating into the duodenum was significantly increased prior to immunization. Previous studies have shown that the gastrointestinal compartment and the spleen constantly produce eotaxin, which regulates basal levels of tissue eosinophils. Therefore, we analyzed CD193 expression on neutrophils and eosinophils. As expected, its expression by duodenal neutrophils was upregulated in Tg mice. Furthermore, the addition of IL-21 into bone marrow cell culture increased the number of CD193+ neutrophils, which easily migrated into the duodenum. These observations suggested that CD193+ neutrophils increase in number under inflammatory conditions due to chronic IL-21 production.

Abstract 028: Neutrophils Aged Under Oxidative Stress Impede Vascular Repair

Objectives: The link between chronic inflammation and poor cardiovascular outcomes has long been appreciated, but the regulation of immune cellular targets to improve cardiovascular health remains a challenge. Mounting evidence suggests that neutrophils, previously considered acute first responder cells, play critical role in chronic inflammation. Following systemic surveillance of tissues, patrolling neutrophils age in the circulation and traffic to the bone marrow, where they undergo macrophage-mediated clearance. Here, we assessed how chronic inflammation and oxidative stress modify neutrophil trafficking and function during ischemia. Methods: Neutrophils from the Townes humanized sickle cell (SS) mice - surrogate for chronic inflammation and oxidative stress - were compared to neutrophils from MerTK deficient mice (MerKO), which age in the circulation owing to defective efferocytosis. Hind limb ischemia (HLI) was used as model of vascular injury. LASER Doppler perfusion imaging (LDPI) measured perfusion recovery after HLI. Results: The proportion of CXCR4 Hi CD62L lo aged neutrophils in circulation was markedly elevated in the SS (34 ± 4%) and MerKO mice (38 ± 4%) when compared to wild-type mice (7 ± 1.2%). However, in contrast with MerKO neutrophils, aged neutrophils from SS showed impaired phagocytosis, increased capacity to produce reactive oxygen species, and pronounced release of extracellular traps (NETs). In response to HLI, neutrophils accumulated in the MerKO and SS mice, but perfusion recovery on LDPI was only impaired in the SS mice, suggesting differential role of neutrophils aged by oxidative stress. Importantly, the phenotype of SS mice was reversed by treatment with the anti-oxidant N-acetyl cysteine (NAC), which had no impact on perfusion recovery in MerKO mice. Conclusion: Although SS and MerKO mice demonstrated elevated numbers of aged neutrophils, our data suggest that chronic inflammation and oxidative stress modifies the function of aged CXCR4 Hi CD62L lo neutrophils to impede vascular repair after ischemia. This specialized subset of neutrophils may be targeted to improve vascular health without compromising overall anti-microbial host defenses

Abstract DL-1: Leveraging Preclinical Models of Breast Cancer

Abstract Jeffrey M. Rosen1, Kevin Roarty1, Se Jin Kim1, Charles M. Perou2, Sendurai Mani3, Paul Ik Sun Kim4 and Xiang Zhang1,4 1Department of Molecular &amp; Cellular Biology and 1,4Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, 2University of North Carolina, Chapel Hill, NC and 3MD Anderson Cancer Center, Houston, TX Breast cancer is a disease that displays both inter- and intra-tumoral heterogeneity. More than a decade ago we asked if a subpopulation of breast cancer stem cells might be resistant to chemotherapy and responsible for relapse in breast cancer patients given neoadjuvant therapy. These clinical studies provided evidence for population of chemotherapy-resistant breast cancer-stem cells. The gene expression signature derived from these comparisons overlapped with an identified “claudin-low” molecular subtype characterized by the low to absent expression of luminal differentiation markers, high enrichment for many EMT-associated genes, and immune-response genes. The claudin-low subtype also most closely resembled a subset mammary epithelial stem cells. The “claudin-low” signature was enriched in residual tumors remaining after either endocrine therapy or chemotherapy treatment. Double positive cells expressing mesenchymal and epithelial markers were enriched in resistant tumors. This suggests the presence of an intermediate or “partial EMT”, which has been proposed to be a hybrid E/M phenotype between the epithelial to mesenchymal transition. Recently we have developed a set of EMT/MET sensors to identify cells undergoing this transition, and we have used these to screen an FDA approved compound library. Genetically engineered mouse (GEM) syngeneic p53-null mammary tumor models that closely mimic several of the subtypes in human breast cancer are being used as preclinical models to study the response to both novel targeted therapies and chemotherapy. We have developed, extensively characterized and “credentialed” a bank of these tumors, which represent the different subtypes of human breast cancer including a subset of these tumors with a similar gene expression signature as the human claudin-low tumors. These murine claudin-low tumors showed high expression of EMT inducers, low expression of members of the miR-200 family and resistance to most standard-of-care therapies. Re-expression of miR-200 family members reversed EMT, decreased the CSC population and sensitized cells to chemotherapy. The ZEB1/miR-200 axis has additional, non-cell autonomous roles in cancer pathogenesis, and has recently been shown to affect immune recognition of cancer cells whereby ZEB1 suppression of the miR-200 family leads to upregulation of PD-L1, a direct miR-200 family target. Therefore, the EMT transition also may play a role in immunosuppression exacerbating treatment response. Accordingly, immunoprofiling has identified unique subsets of immunosuppressive neutrophils and macrophages in mice with the different subtypes of p53 null tumors. Hierarchical clustering revealed "extrinsic" subtypes of breast cancers in terms of their innate immune cell profiles. Finally, we have employed specific Wnt and Stat3 pathway reporters to better understand the signaling pathways involved in intratumoral heterogeneity and metastasis. Citation Format: Rosen JM. Leveraging Preclinical Models of Breast Cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr DL-1.

A heterogeneous Ly-6B2+ leukocyte population consists of yet undescribed iNOS-expressing cell types in murine skin wounds

The gaseous mediator nitric oxide (NO) is a central regulatory molecule during the inflammatory phase of cutaneous tissue repair. The inducible NO-synthase (iNOS) represents the main isoform of the three NO producing enzymes at the wound site. In particular, keratinocytes and macrophages are described as main sources of iNOS-derived NO in skin wounds. Here we provide experimental evidence that Ly-6B2+ leukocytes are an additional cellular source of iNOS-derived NO in wounds. As wound iNOS protein expression temporally coincides with both macrophage and neutrophil infiltration, we used immunohistochemistry (IHC) and fluorescence-activated cell sorting (FACS) to address iNOS expression in both macrophages and neutrophil subsets. IHC analyses excluded F4/80+ macrophages as iNOS producers, but indicated Ly-6G/C (Gr-1)+ neutrophils to express iNOS in wound granulation tissue. A subsequent FACS-based analysis from cellular wound tissue preparations revealed an iNOS-expressing fraction of Ly-6B2-determined leukocytes that consisted of Ly-6G+ and Ly-6G− cells, meaning that mainly mature neutrophils (Ly-6B2+/Ly-6G+) as well as inflammatory monocytes (Ly-6B2+/Ly-6G−) are dominant iNOS-expressing cell types in the developing granulation tissue of acute wounds.

Developmental Analysis of Bone Marrow Neutrophils Reveals Populations Specialized in Expansion, Trafficking, and Effector Functions

Neutrophils are specialized innate cells that require constant replenishment from proliferative bone marrow (BM) precursors as a result of their short half-life. Although it is established that neutrophils are derived from the granulocyte-macrophage progenitor (GMP), the differentiation pathways from GMP to functional mature neutrophils are poorly defined. Using mass cytometry (CyTOF) and cell-cycle-based analysis, we identified three neutrophil subsets within the BM: a committed proliferative neutrophil precursor (preNeu) which differentiates into non-proliferating immature neutrophils andmature neutrophils. Transcriptomic profiling and functional analysis revealed that preNeu require the C/EBPε transcription factor for their generation from the GMP, and their proliferative program is substituted by a gain of migratory and effector function as they mature. preNeus expand under microbial and tumoral stress, and immature neutrophils are recruited to the periphery of tumor-bearing mice. Insummary, our study identifies specialized BM granulocytic populations that ensure supply under homeostasis and stress responses.

Neutrophil hyperactivation correlates with Alzheimer's disease progression.

Recent studies have underlined the effect of systemic inflammation on the pathophysiology of Alzheimer's disease (AD). Neutrophils are key components of early innate immunity and contribute to uncontrolled systemic inflammation if not tightly regulated. The aim of our study was to fully characterize human circulating neutrophils at different disease stages in AD. We analyzed neutrophil phenotypes and functions in 42 patients with AD (16 with mild cognitive impairment and 26 with dementia), and compared them to 22 age-matched healthy subjects. This study was performed directly in whole blood to avoid issues with data interpretation related to cell isolation procedures. Blood samples from AD patients with dementia revealed neutrophil hyperactivation associated with increased reactive oxygen species production and increased levels of intravascular neutrophil extravascular traps. The homeostasis of circulating neutrophils in these patients also changed: The ratio between the harmful hyperreactive CXCR4high /CD62Llow senescent and the CD16bright /CD62Ldim immunosuppressive neutrophil subsets rose in the later stage of the disease. These abnormalities were greater in fast-decliner than in slow-decliner patients. Our results indicate that the inflammatory properties of circulating neutrophils shift as the percentage of aged neutrophils expands in patients with AD-changes that may play an instrumental role in establishing systemic chronic inflammation. Most important, our data strongly suggest that the neutrophil phenotype may be associated with the rate of cognitive decline and may thus constitute an innovative and prognostic blood biomarker in patients with AD. Ann Neurol 2018;83:387-405.

CIRP increases ICAM-1+ phenotype of neutrophils exhibiting elevated iNOS and NETs in sepsis.

Sepsis represents uncontrolled inflammation due to an infection. Cold-inducible RNA-binding protein (CIRP) is a stress-induced damage-associated molecular pattern (DAMP). A subset of neutrophils expressing ICAM-1+ neutrophils was previously shown to produce high levels of reactive oxygen species. The role of CIRP for the development and function of ICAM-1+ neutrophils during sepsis is unknown. We hypothesize that CIRP induces ICAM-1 expression in neutrophils causing injury to the lungs during sepsis. Using a mouse model of cecal ligation and puncture (CLP)-induced sepsis, we found increased expression of CIRP and higher frequencies and numbers of ICAM-1+ neutrophils in the lungs. Conversely, the CIRP-/- mice showed significant inhibition in the frequencies and numbers of ICAM-1+ neutrophils in the lungs compared to wild-type (WT) mice in sepsis. In vitro treatment of bone marrow-derived neutrophils (BMDN) with recombinant murine CIRP (rmCIRP) significantly increased ICAM-1+ phenotype in a time- and dose-dependent manner. The effect of rmCIRP on increasing frequencies of ICAM-1+ neutrophils was significantly attenuated in BMDN treated with anti-TLR4 Ab or NF-κB inhibitor compared, respectively, with BMDN treated with isotype IgG or DMSO. The frequencies of iNOS producing and neutrophil extracellular traps (NETs) forming phenotypes in rmCIRP-treated ICAM-1+ BMDN were significantly higher than those in ICAM-1- BMDN. Following sepsis the ICAM-1+ neutrophils in the lungs showed significantly higher levels of iNOS and NETs compared to ICAM-1- neutrophils. We further revealed that ICAM-1 and NETs were co-localized in the neutrophils treated with rmCIRP. CIRP-/- mice showed significant improvement in their survival outcome (78% survival) over that of WT mice (48% survival) in sepsis. Thus, CIRP could be a novel therapeutic target for regulating iNOS producing and NETs forming ICAM-1+ neutrophils in the lungs during sepsis.

Cysteinyl leukotriene receptor 1 expression identifies a subset of neutrophils during the antiviral response that contributes to postviral atopic airway disease

Viral respiratory tract infections increase the risk of development and exacerbation of atopic disease. Previously, we demonstrated the requirement for a neutrophil (PMN) subset expressing CD49d to drive development of postviral atopic airway disease in mice. We sought to determine whether human CD49d+ PMNs are present in the nasal mucosa during acute viral respiratory tract infections and further characterize this PMN subset in human subjects and mice. Sixty subjects (5-50years old) were enrolled within 4days of acute onset of upper respiratory symptoms. Nasal lavage for flow cytometry and nasal swabs for viral PCR were performed at enrollment and during convalescence. The Sendai virus mouse model was used to investigate the phenotype and functional relevance of CD49d+ PMNs. CD49d+ PMN frequency was significantly higher in nasal lavage fluid during acute respiratory symptoms in all subjects (2.9% vs 1.0%, n=42, P<.001). In mice CD49d+ PMNs represented a "proatopic" neutrophil subset that expressed cysteinyl leukotriene receptor 1 (CysLTR1) and produced TNF, CCL2, and CCL5. Inhibition of CysLTR1 signaling in the first days of a viral respiratory tract infection was sufficient to reduce accumulation of CD49d+ PMNs in the lungs and development of postviral atopic airway disease. Similar to the mouse, human CD49d+ PMNs isolated from nasal lavage fluid during a viral respiratory tract infection expressed CysLTR1. CD49d and CysLTR1-coexpressing PMNs are present during symptoms of an acute viral respiratory tract infection in human subjects. Further study is needed to examine selective targeting of proatopic neutrophils as a potential therapeutic strategy to prevent development of postviral atopic airway disease.

Interleukin 10 modulation of neutrophil subsets infiltrating lungs during Streptococcus pneumoniae infection

Interleukin-10 production and lung neutrophil infiltration are two essential components of the balanced immune response to pneumonia caused by Streptococcus pneumoniae. Here we describe the existence of two neutrophil subsets in lungs during experimental S. pneumoniae infection in mice, which have different size, granularity and expression of activation markers. During infection, both neutrophils subsets were increased in the lungs of IL-10 producing mice, however this increment was significantly higher in the absence of this cytokine. These results suggest that IL-10 is a key cytokine that regulates lung inflammation during bacterial infection caused by specific neutrophil subsets infiltrating the lungs.

Neutrophil-mediated Suppression of Influenza-induced Pathology Requires CD11b/CD18 (MAC-1).

Severe influenza virus infection can lead to life-threatening pathology through immune-mediated tissue damage. In various experimental models, this damage is dependent on T cells. There is conflicting evidence regarding the role of neutrophils in influenza-mediated pathology. Neutrophils are often regarded as cells causing tissue damage, but, in recent years, it has become clear that a subset of human neutrophils is capable of suppressing T cells, which is dependent on macrophage-1 antigen (CD11b/CD18). Therefore, we tested the hypothesis that immune suppression by neutrophils can reduce T cell-mediated pathology after influenza infection. Wild-type (WT) and CD11b-/- mice were infected with A/HK/2/68 (H3N2) influenza virus. Disease severity was monitored by weight loss, leukocyte infiltration, and immunohistochemistry. We demonstrated that CD11b-/- mice suffered increased weight loss compared with WT animals upon infection with influenza virus. This was accompanied by increased pulmonary leukocyte infiltration and lung damage. The exaggerated pathology in CD11b-/- mice was dependent on T cells, as it was reduced by T cell depletion. In addition, pathology in CD11b-/- mice was accompanied by higher numbers of T cells in the lungs early during infection compared with WT mice. Importantly, these differences in pathology were not associated with an increased viral load, suggesting that pathology was immune-mediated rather than caused by virus-induced damage. In contrast to adoptive transfer of CD11b-/- neutrophils, a single adoptive transfer of WT neutrophils partly restored protection against influenza-induced pathology, demonstrating the importance of neutrophil CD11b/CD18. Our data show that neutrophil CD11b/CD18 limits pathology in influenza-induced, T cell-mediated disease.

Neutrophil killing of Staphylococcus aureus in diabetes, obesity and metabolic syndrome: a prospective cellular surveillance study

BackgroundObesity, metabolic syndrome (MetS), and diabetes are frequent in surgical populations and can enhance susceptibility to postoperative surgical site infections. Reduced neutrophil function has been linked with diabetes and risk of Staphylococcus aureus infection. Therefore, neutrophil function in diabetic and obese subjects (± MetS) was assessed in this prospective serological and cellular surveillance study to determine whether vaccines administered to protect against infections after surgery could be effective in these populations.MethodsNeutrophil function (chemotaxis, phagocytosis, and opsonophagocytic killing of S. aureus) was assessed in subjects classified according to diabetes status, body mass index, and presence/absence of MetS. Neutrophils were characterized within functional subsets by flow cytometry. A serologic assay was used to measure baseline antibody presence to each antigen in SA4Ag: capsular polysaccharide (CP) type 5, CP8, recombinant mutant Clumping factor A (rmClfA), and recombinant Manganese transport protein C (rMntC).ResultsNeutrophil function was similar for comorbid and healthy cohorts, with no significant between-group differences in cell counts, migration, phagocytosis ability, neutrophil subset proportions, and S. aureus killing ability when neutrophils were isolated 3–6 months apart (Visit 1 [n = 90] and Visit 2 [n = 70]) and assessed. Median pre-existing antibody titers to CP5, CP8, and rmClfA were comparable for all cohorts (insufficient subjects with rMntC titers for determination).ConclusionsMetS, diabetes, and obesity do not impact in vitro neutrophil function with regard to S. aureus killing, suggesting that if an effective S. aureus vaccine is developed it may be effective in individuals with these comorbidities.

Neutrophil programming dynamics and its disease relevance.

Neutrophils are traditionally considered as first responders to infection and provide antimicrobial host defense. However, recent advances indicate that neutrophils are also critically involved in the modulation of host immune environments by dynamically adopting distinct functional states. Functionally diverse neutrophil subsets are increasingly recognized as critical components mediating host pathophysiology. Despite its emerging significance, molecular mechanisms as well as functional relevance of dynamically programmed neutrophils remain to be better defined. The increasing complexity of neutrophil functions may require integrative studies that address programming dynamics of neutrophils and their pathophysiological relevance. This review aims to provide an update on the emerging topics of neutrophil programming dynamics as well as their functional relevance in diseases.

Pandemic 2009 H1N1 Influenza Venus reporter virus reveals broad diversity of MHC class II-positive antigen-bearing cells following infection in vivo

Although it is well established that Influenza A virus infection is initiated in the respiratory tract, the sequence of events and the cell types that become infected or access viral antigens remains incompletely understood. In this report, we used a novel Influenza A/California/04/09 (H1N1) reporter virus that stably expresses the Venus fluorescent protein to identify antigen-bearing cells over time in a mouse model of infection using flow cytometry. These studies revealed that many hematopoietic cells, including subsets of monocytes, macrophages, dendritic cells, neutrophils and eosinophils acquire influenza antigen in the lungs early post-infection. Surface staining of the viral HA revealed that most cell populations become infected, most prominently CD45neg cells, alveolar macrophages and neutrophils. Finally, differences in infection status, cell lineage and MHC class II expression by antigen-bearing cells correlated with differences in their ability to re-stimulate influenza-specific CD4 T cells ex vivo. Collectively, these studies have revealed the cellular heterogeneity and complexity of antigen-bearing cells within the lung and their potential as targets of antigen recognition by CD4 T cells.