How Neutrophils Shape the Immune Response: Reassessing Their Multifaceted Role in Health and Disease.

Neutrophils are the most abundant of the circulating immune cells and are the first to be recruited to sites of inflammation. Neutrophils are a heterogeneous group of immune cells from which are derived extracellular traps (NETs), reactive oxygen species, cytokines, chemokines, immunomodulatory factors, and alarmins that regulate the recruitment and phenotypes of neutrophils, macrophages, dendritic cells, T cells, and B cells. In addition, cytokine-stimulated neutrophils can express class II major histocompatibility complex and the internal machinery necessary for successful antigen presentation to memory CD4+ T cells. This may be relevant in the context of vaccine memory. Neutrophils thus emerge as orchestrators of immune responses that play a key role in determining the outcome of infections, vaccine efficacy, and chronic diseases like autoimmunity and cancer. This review aims to provide a synthesis of current evidence as regards the role of these functions of neutrophils in homeostasis and disease.

Host defense against viral infection involves interferon mediated down-regulation of sterol biosynthesis.

Little is known about the protective role of inflammatory processes in modulating lipid metabolism in infection. Here we report an intimate link between the innate immune response to infection and regulation of the sterol metabolic network characterized by down-regulation of sterol biosynthesis by an interferon regulatory loop mechanism. In time-series experiments profiling genome-wide lipid-associated gene expression of macrophages, we show a selective and coordinated negative regulation of the complete sterol pathway upon viral infection or cytokine treatment with IFNγ or ß but not TNF, IL1ß, or IL6. Quantitative analysis at the protein level of selected sterol metabolic enzymes upon infection shows a similar level of suppression. Experimental testing of sterol metabolite levels using lipidomic-based measurements shows a reduction in metabolic output. On the basis of pharmacologic and RNAi inhibition of the sterol pathway we show augmented protection against viral infection, and in combination with metabolite rescue experiments, we identify the requirement of the mevalonate-isoprenoid branch of the sterol metabolic network in the protective response upon statin or IFNß treatment. Conditioned media experiments from infected cells support an involvement of secreted type 1 interferon(s) to be sufficient for reducing the sterol pathway upon infection. Moreover, we show that infection of primary macrophages containing a genetic knockout of the major type I interferon, IFNß, leads to only a partial suppression of the sterol pathway, while genetic knockout of the receptor for all type I interferon family members, ifnar1, or associated signaling component, tyk2, completely abolishes the reduction of the sterol biosynthetic activity upon infection. Levels of the proteolytically cleaved nuclear forms of SREBP2, a key transcriptional regulator of sterol biosynthesis, are reduced upon infection and IFNß treatment at both the protein and de novo transcription level. The reduction in srebf2 gene transcription upon infection and IFN treatment is also found to be strictly dependent on ifnar1. Altogether these results show that type 1 IFN signaling is both necessary and sufficient for reducing the sterol metabolic network activity upon infection, thereby linking the regulation of the sterol pathway with interferon anti-viral defense responses. These findings bring a new link between sterol metabolism and interferon antiviral response and support the idea of using host metabolic modifiers of innate immunity as a potential antiviral strategy.

Reversible inhibition of murine cytomegalovirus replication by gamma interferon (IFN-γ) in primary macrophages involves a primed type I IFN-signaling subnetwork for full establishment of an immediate-early antiviral state.

Activated macrophages play a central role in controlling inflammatory responses to infection and are tightly regulated to rapidly mount responses to infectious challenge. Type I interferon (alpha/beta interferon [IFN-α/ß]) and type II interferon (IFN-γ) play a crucial role in activating macrophages and subsequently restricting viral infections. Both types of IFNs signal through related but distinct signaling pathways, inducing a vast number of interferon-stimulated genes that are overlapping but distinguishable. The exact mechanism by which IFNs, particularly IFN-γ, inhibit DNA viruses such as cytomegalovirus (CMV) is still not fully understood. Here, we investigate the antiviral state developed in macrophages upon reversible inhibition of murine CMV by IFN-γ. On the basis of molecular profiling of the reversible inhibition, we identify a significant contribution of a restricted type I IFN subnetwork linked with IFN-γ activation. Genetic knockout of the type I-signaling pathway, in the context of IFN-γ stimulation, revealed an essential requirement for a primed type I-signaling process in developing a full refractory state in macrophages. A minimal transient induction of IFN-ß upon macrophage activation with IFN-γ is also detectable. In dose and kinetic viral replication inhibition experiments with IFN-γ, the establishment of an antiviral effect is demonstrated to occur within the first hours of infection. We show that the inhibitory mechanisms at these very early times involve a blockade of the viral major immediate-early promoter activity. Altogether our results show that a primed type I IFN subnetwork contributes to an immediate-early antiviral state induced by type II IFN activation of macrophages, with a potential further amplification loop contributed by transient induction of IFN-ß.

Combined genome-wide expression profiling and targeted RNA interference in primary mouse macrophages reveals perturbation of transcriptional networks associated with interferon signalling.

BACKGROUND: Interferons (IFNs) are potent antiviral cytokines capable of reprogramming the macrophage phenotype through the induction of interferon-stimulated genes (ISGs). Here we have used targeted RNA interference to suppress the expression of a number of key genes associated with IFN signalling in murine macrophages prior to stimulation with interferon-gamma. Genome-wide changes in transcript abundance caused by siRNA activity were measured using exon-level microarrays in the presence or absence of IFNgamma. RESULTS: Transfection of murine bone-marrow derived macrophages (BMDMs) with a non-targeting (control) siRNA and 11 sequence-specific siRNAs was performed using a cationic lipid transfection reagent (Lipofectamine2000) prior to stimulation with IFNgamma. Total RNA was harvested from cells and gene expression measured on Affymetrix GeneChip Mouse Exon 1.0 ST Arrays. Network-based analysis of these data revealed six siRNAs to cause a marked shift in the macrophage transcriptome in the presence or absence IFNgamma. These six siRNAs targeted the Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2 transcripts. The perturbation of the transcriptome by the six siRNAs was highly similar in each case and affected the expression of over 600 downstream transcripts. Regulated transcripts were clustered based on co-expression into five major groups corresponding to transcriptional networks associated with the type I and II IFN response, cell cycle regulation, and NF-KB signalling. In addition we have observed a significant non-specific immune stimulation of cells transfected with siRNA using Lipofectamine2000, suggesting use of this reagent in BMDMs, even at low concentrations, is enough to induce a type I IFN response. CONCLUSION: Our results provide evidence that the type I IFN response in murine BMDMs is dependent on Ifnb1, Irf3, Irf5, Stat1, Stat2 and Nfkb2, and that siRNAs targeted to these genes results in perturbation of key transcriptional networks associated with type I and type II IFN signalling and a suppression of macrophage M1 polarization.

Emergence of novel methicillin-resistant Staphylococcus aureus strains in a tertiary care facility in Riyadh, Saudi Arabia.

PURPOSE: There is a need for continuous surveillance of methicillin-resistant Staphylococcus aureus (MRSA) to identify emergence of new strains. We hypothesize that MRSA strains are evolving with ongoing acquisition of SCCmec elements. This study was carried out to evaluate the evolution of MRSA at a tertiary care facility in Saudi Arabia. METHODS: MRSA isolates associated with invasive clinical infection, which were identified in 2017 at the microbiology laboratory, King Khalid University Hospital (KKUH) in Riyadh, Saudi Arabia, were studied. The molecular characterization of isolates was carried out using StaphyType DNA microarray (Alere Technologies GmbH/Abbott, Jena, Germany). RESULTS: The 125 MRSA isolates studied belonged to 18 clonal complexes (CC) which were distributed into 32 strain assignments. The predominant CC were CC5 (n=30), CC6 (n=17), CC80 (n=13), CC22 (n=12), CC361 (n=12). The findings demonstrated the first identification of CC152, CC361 and CC1153 MRSA as well as ST5-MRSA-[I+fus], "Geraldine Clone", CC6-MRSA-IV (PVL+) and CC88-MRSA-V (PVL+), WA MRSA-117 in Saudi Arabia. Four novel variants were identified: CC5-MRSA-[VI+fus+tirS], CC22-MRSA-[V/VT+fus](PVL+), CC152-MRSA-[V+fus](PVL+) and CC361-MRSA-[VT+fus]. Fifty-four isolates (n/N=54/125; 43.2%) including the novel strains carried the Q6GD50 SCCfusC gene while the Panton-Valentine leukocidin genes were present in 30.4% (n/N=38/125). CONCLUSION: The findings demonstrate an expanding MRSA repertoire in our setting including emergence of previously unreported clonal complexes and novel strains. The high carriage of fusC gene suggests a role for fusidic acid misuse in driving the evolution of the MRSA genome and underscores the need for increased monitoring of antibiotic use.

First Report of blaCTX-M-28 in Enterobacteriaceae Isolates in the United Arab Emirates.

BACKGROUND: The CTX-M family of extended-spectrum beta lactamase (ESBL) enzymes is comprised of over 60 blaCTX-M gene variants with the predominance of blaCTX-M-15 in many regions. In this report, we present the first description of blaCTX-M-28 in the United Arab Emirates. METHODS: Forty-five non-duplicate ESBL producing isolates identified in a secondary care facility in the United Arab Emirates from June to July 2016 were studied. Gene sequencing was performed and DNA sequences were annotated using the BLAST program to identify the gene subtypes. RESULTS: The majority of the ESBL positive isolates were E. coli (n/N = 39/45; 86.6%) followed by K. pneumoniae (n = 5) and K. oxytoca (n = 1). All isolates harboured blaCTX-M and blaTEM genes, 18 had blaSHV, and 2 were blaVIM positive. Thirty-seven isolates (82.2%) were positive for blaCTX-M-28. Other blaCTX-M genes identified include blaCTX-M-167 (n = 2; isolates #1 and 26) and one each for blaCTX-M-38, blaCTX-M-163, and blaCTX-M-198. No blaCTX-M-15 was identified. The predominant blaTEM subtype was blaTEM-171 (n = 8) followed by one of each of blaTEM-120, blaTEM-163, and blaTEM-206. The blaSHV subtypes were blaSHV-148 and blaSHV-187. CONCLUSION: The findings indicate the first description of blaCTX-M-28 in a setting where blaCTX-M-15 was previously predominant.

GPX-Macrophage Expression Atlas: a database for expression profiles of macrophages challenged with a variety of pro-inflammatory, anti-inflammatory, benign and pathogen insults.

BACKGROUND: Macrophages play an integral role in the host immune system, bridging innate and adaptive immunity. As such, they are finely attuned to extracellular and intracellular stimuli and respond by rapidly initiating multiple signalling cascades with diverse effector functions. The macrophage cell is therefore an experimentally and clinically amenable biological system for the mapping of biological pathways. The goal of the macrophage expression atlas is to systematically investigate the pathway biology and interaction network of macrophages challenged with a variety of insults, in particular via infection and activation with key inflammatory mediators. As an important first step towards this we present a single searchable database resource containing high-throughput macrophage gene expression studies. DESCRIPTION: The GPX Macrophage Expression Atlas (GPX-MEA) is an online resource for gene expression based studies of a range of macrophage cell types following treatment with pathogens and immune modulators. GPX-MEA follows the MIAME standard and includes an objective quality score with each experiment. It places special emphasis on rigorously capturing the experimental design and enables the searching of expression data from different microarray experiments. Studies may be queried on the basis of experimental parameters, sample information and quality assessment score. The ability to compare the expression values of individual genes across multiple experiments is provided. In addition, the database offers access to experimental annotation and analysis files and includes experiments and raw data previously unavailable to the research community. CONCLUSION: GPX-MEA is the first example of a quality scored gene expression database focussed on a macrophage cellular system that allows efficient identification of transcriptional patterns. The resource will provide novel insights into the phenotypic response of macrophages to a variety of benign, inflammatory, and pathogen insults. GPX-MEA is available through the GPX website at http://www.gti.ed.ac.uk/GPX.

Genetic relatedness of clinical and environmental Acinetobacter baumanii isolates from an intensive care unit outbreak.

INTRODUCTION: Determination of microbial genetic relatedness is important for improving efficiency of infection control measures during hospital outbreaks. This study aimed to analyze the clonal relationships of clinical and environmental Acinetobacter baumannii strains isolated during an outbreak in the intensive care unit (ICU) of a secondary care hospital in Saudi Arabia. METHODOLOGY: Twelve clinical and fourteen environmental A. baumannii isolates identified during an outbreak in February 2013 in the 14-bed adult intensive care unit of a tertiary care hospital in Riyadh, Saudi Arabia, were studied. Bacterial identification and antimicrobial susceptibility testing were carried out using Microscan Walkaway 96 automated system. Determination of clonal diversity was investigated by repetitive-sequence-based polymerase chain reaction (rep-PCR) with the semi-automated DiversiLab system. RESULTS: The majority of the clinical isolates were from endotracheal tube aspirates (n = 9), one from a wound swab and two were from urine and sputum, respectively. Environmental isolates were from bed railings (n = 10) and with one each from curtain, stethoscope, computer mouse and telephone. Isolates were categorized into 6 clusters (Groups 1-6). Most of the isolates were associated with two clusters namely Groups 2 (n = 11) and 3 (n = 9). All isolates were multidrug resistant showing resistance to three or more classes of antibiotics. One clinical strain from Cluster 3 was resistant to colistin (MIC > 4ug/ml). CONCLUSION: This outbreak was caused by two clonal groups of multidrug resistant A. baumannii. The presence of multiple environmental clones was suggestive of environmental source dissemination via healthcare workers within the ICU.

Time-lapse ultrashort pulse microscopy of infection in three-dimensional versus two-dimensional culture environments reveals enhanced extra-chromosomal virus replication compartment formation.

The mechanisms that enable viruses to harness cellular machinery for their own survival are primarily studied in cell lines cultured in two-dimensional (2-D) environments. However, there are increasing reports of biological differences between cells cultured in 2-D versus three-dimensional (3-D) environments. Here we report differences in host-virus interactions based on differences in culture environment. Using ultrashort pulse microscopy (UPM), a form of two-photon microscopy that utilizes sub-10-fs pulses to efficiently excite fluorophores, we have shown that de novo development of extra-chromosomal virus replication compartments (VRCs) upon murine cytomegalovirus (mCMV) infection is markedly enhanced when host cells are cultured in 3-D collagen gels versus 2-D monolayers. In addition, time-lapse imaging revealed that mCMV-induced VRCs have the capacity to grow by coalescence. This work supports the future potential of 3-D culture as a useful bridge between traditional monolayer cultures and animal models to study host-virus interactions in a more physiologically relevant environment for the development of effective anti-viral therapeutics. These advances will require broader adoption of modalities, such as UPM, to image deep within scattering tissues.

Transcriptional responses of murine macrophages to the adenylate cyclase toxin of Bordetella pertussis.

Three different recombinant forms of CyaA were used to investigate transcriptional responses of murine bone marrow-derived macrophages (BMMs) using Affymetrix Mouse Genome GeneChips. These forms were enzymically active, invasive CyaA, non-enzymically active, invasive CyaA (CyaA*) and non-enzymically active, non-invasive CyaA (proCyaA*). BMMs, treated with 20 ng/ml of CyaA for 24h, showed over 1000 significant changes in gene transcription compared with control cells. CyaA caused an increase in transcription of many inflammatory genes and genes associated with various signalling cascades such as those involved in cyclic AMP-dependent protein kinase A signalling. Most strikingly, CyaA caused down-regulation of numerous genes involved in cell proliferation. CyaA* at 20 ng/ml significantly up-regulated the transcription of only twelve genes after 24h whereas proCyaA* at this concentration significantly increased the transcription of only two genes.

Treatment of non-resectable hepatocellular carcinoma with autologous tumor-pulsed dendritic cells.

BACKGROUND: The response of hepatocellular carcinoma (HCC) to therapy is often disappointing and new modalities of treatment are clearly needed. Active immunotherapy based on the injection of autologous dendritic cells (DC) co-cultured ex vivo with tumor antigens has been used in pilot studies in various malignancies such as melanoma and lymphoma with encouraging results. METHODS: In the present paper, the preparation and exposure of patient DC to autologous HCC antigens and re-injection in an attempt to elicit antitumor immune responses are described. RESULTS: Therapy was given to two patients, one with hepatitis C and one with hepatitis B, who had large, multiple HCC and for whom no other therapy was available. No significant side-effects were observed. The clinical course was unchanged in one patient, who died a few months later. The other patient, whose initial prognosis was considered poor, is still alive and well more than 3 years later with evidence of slowing of tumor growth based on organ imaging. CONCLUSIONS: It is concluded that HCC may be a malignancy worthy of DC trials and sufficient details in the present paper are given for the protocol to be copied or modified.