Optimized Detection of Acute MHV68 Infection With a Reporter System Identifies Large Peritoneal Macrophages as a Dominant Target of Primary Infection.

Investigating the dynamics of virus-host interactions in vivo remains an important challenge, often limited by the ability to directly identify virally infected cells. Here, we utilize a beta-lactamase activated fluorescent substrate to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response. MHV68 infection leads to a pronounced increase in immune cells, with CD8+ T cells increasing by 3 days, and total infiltrate peaking around 8 days post-infection. MHV68 infection results in near elimination of large peritoneal macrophages (LPMs) by 8 days post-infection, and a concordant increase in small peritoneal macrophages (SPMs) and monocytes. Infection is associated with prolonged changes to myeloid cells, with a distinct population of MHC IIhigh LPMs emerging by 14 days. Targets of MHV68 infection could be readily detected. Between 1 and 3 days post-infection, MHV68 infects ∼5-10% of peritoneal cells, with >75% being LPMs. By 8 days post-infection, the frequency of MHV68 infection is reduced at least 10-fold, with infection primarily in SPMs, with few infected dendritic cells and B cells. Importantly, limiting dilution analysis indicates that at 3 days post-infection, the majority of MHV68-infected cells harbor latent rather than lytic virus at frequencies consistent with those identified based on reporter gene expression. Our findings demonstrate the utility of the beta-lactamase MHV68 reporter system for high throughput single-cell analysis and identify dynamic changes during primary gammaherpesvirus infection.

Therapeutic potential of FLANC, a novel primate-specific long non-coding RNA in colorectal cancer.

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

Full Spectrum Flow Cytometry as a Powerful Technology for Cancer Immunotherapy Research.

The Nobel Prize-deserving concept of blocking inhibitory pathways in T cells, to unleash their anti-tumoral capacity, became one of the pillars of cancer treatment in the last decade and has resulted in durable clinical responses for multiple cancer types. Currently, two of the most important goals in cancer immunotherapy are to understand the mechanisms resulting in failure to checkpoint blockade and to identify predictive immunological biomarkers that correlate to treatment response, disease progression or adverse effects. The identification and validation of biomarkers for routine clinical use is not only critical to monitor disease or treatment progression, but also to personalize and develop new therapies. To achieve these goals, powerful research tools are needed. Flow cytometry stands as one of the most successful single-cell analytical tools used to characterize immune cell phenotypes to monitor solid tumors, hematological malignancies, minimal residual disease or metastatic progression. This technology has been fundamental in diagnosis, treatment and translational research in cancer clinical trials. Most recently, the need to evaluate simultaneously more features in each cell has pushed the field to implement more powerful adaptations beyond conventional flow cytometry, including Full Spectrum Flow Cytometry (FSFC). FSFC captures the full emission spectrum of fluorescent molecules using arrays of highly sensitive light detectors, and to date has enabled characterization of 40 parameters in a single sample. We will summarize the contributions of this technology to the advancement of research in immunotherapy studies and discuss best practices to obtain reliable, robust and reproducible FSFC results.

A secondary wave of neutrophil infiltration causes necrosis and ulceration in lesions of experimental American cutaneous leishmaniasis.

We evaluated the importance of neutrophils in the development of chronic lesions caused by L. Viannia spp. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R2 = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p<0.001 each). In contrast, reduced neutrophil infiltration via cyclophosphamide-mediated depletion led to more benign lesions and lower parasite loads compared to controls (p<0.001 each). Neutrophils of the second wave expressed significantly lower GM-CSF, reactive oxygen species and nitric oxide than those of the first wave, suggesting that they had less efficient anti-leishmania activity. However, there was increased inflammatory cytokines and expression of neutrophil proteases (myeloperoxidase, cathepsin G and elastase) in lesions during the second wave of neutrophil infiltration compared with the levels reached during the first wave (6h p.i.). This suggests that augmented neutrophil proteases and inflammatory cytokines during the secondary wave of neutrophils could contribute to skin inflammation, ulceration and necrosis in ACL. The overall results indicate that neutrophils were unable to clear the infection in this model, and that the second wave of neutrophils played an important role in the severity of ACL.

Adult cancer-related hemophagocytic lymphohistiocytosis - a challenging diagnosis: a case report.

BACKGROUND: Adult hemophagocytic lymphohistiocytosis is a secondary immunopathologic phenomenon, mainly secondary to malignancy, infection, or autoimmune disorders. The performance of diagnostic criteria, studied in the pediatric population, is yet to be validated in the adult population. Some of the criteria include cytopenias and organomegaly that are inherent features to malignant processes, thus making the diagnosis of hemophagocytic lymphohistiocytosis a challenge in patients with cancer. CASE PRESENTATION: We describe the case of a 54-year-old white man with history of metastatic maxillary sinus adenoid cystic carcinoma who had severe liver injury and cytopenias with progressive clinical deterioration. We performed an evaluation, by flow cytometry, of the expression of surface markers in his natural killer cells that revealed remarkable abnormalities. His syndrome eventually fulfilled criteria for hemophagocytic lymphohistiocytosis and he received therapy with steroids with interval clinical improvement. Unfortunately, he refused further cytotoxic treatment and died 2 weeks later. CONCLUSIONS: The conventional criteria for the diagnosis of hemophagocytic lymphohistiocytosis are suboptimal for adult patients with cancer resulting in delays in diagnosis and timely initiation of treatment. The diagnostic criteria have to be re-evaluated in patients with cancer; novel, easily available, and accurate diagnostic methods are needed.

Autophagy Is Required for Neutrophil-Mediated Inflammation.

Autophagy, an intracellular degradation and energy recycling mechanism, is emerging as an important regulator of immune responses. However, the role of autophagy in regulating neutrophil functions is not known. We investigated neutrophil biology using myeloid-specific autophagy-deficient mice and found that autophagy deficiency reduced neutrophil degranulation in vitro and in vivo. Mice with autophagy deficiency showed reduced severity of several neutrophil-mediated inflammatory and autoimmune disease models, including PMA-induced ear inflammation, LPS-induced breakdown of blood-brain barrier, and experimental autoimmune encephalomyelitis. NADPH oxidase-mediated reactive oxygen species generation was also reduced in autophagy-deficient neutrophils, and inhibition of NADPH oxidase reduced neutrophil degranulation, suggesting NADPH oxidase to be a player at the intersection of autophagy and degranulation. Overall, this study establishes autophagy as an important regulator of neutrophil functions and neutrophil-mediated inflammation in vivo.

Mutually exclusive recurrent somatic mutations in MAP2K1 and BRAF support a central role for ERK activation in LCH pathogenesis.

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder characterized by lesions composed of pathological CD207(+) dendritic cells with an inflammatory infiltrate. BRAFV600E remains the only recurrent mutation reported in LCH. In order to evaluate the spectrum of somatic mutations in LCH, whole exome sequencing was performed on matched LCH and normal tissue samples obtained from 41 patients. Lesions from other histiocytic disorders, juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease were also evaluated. All of the lesions from histiocytic disorders were characterized by an extremely low overall rate of somatic mutations. Notably, 33% (7/21) of LCH cases with wild-type BRAF and none (0/20) with BRAFV600E harbored somatic mutations in MAP2K1 (6 in-frame deletions and 1 missense mutation) that induced extracellular signal-regulated kinase (ERK) phosphorylation in vitro. Single cases of somatic mutations of the mitogen-activated protein kinase (MAPK) pathway genes ARAF and ERBB3 were also detected. The ability of MAPK pathway inhibitors to suppress MAPK kinase and ERK phosphorylation in cell culture and primary tumor models was dependent on the specific LCH mutation. The findings of this study support a model in which ERK activation is a universal end point in LCH arising from pathological activation of upstream signaling proteins.

Autophagy regulates phagocytosis by modulating the expression of scavenger receptors.

Autophagy and phagocytosis are conserved cellular functions involved in innate immunity. However, the nature of their interactions remains unclear. We evaluated the role of autophagy in regulating phagocytosis in macrophages from myeloid-specific autophagy-related gene 7-deficient (Atg7⁻/⁻) mice. Atg7⁻/⁻ macrophages exhibited higher bacterial uptake when infected with Mycobacterium tuberculosis (Mtb) or with M. tuberculosis var. bovis BCG (BCG). In addition, BCG-infected Atg7⁻/⁻ mice showed increased bacterial loads and exacerbated lung inflammatory responses. Atg7⁻/⁻ macrophages had increased expression of two class A scavenger receptors: macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor 1 (MSR1). The increase in scavenger receptors was caused by increased activity of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) transcription factor resulting from accumulated sequestosome 1 (SQSTM1 or p62) in Atg7⁻/⁻ macrophages. These insights increase our understanding of the host-pathogen relationship and suggest that therapeutic strategies should be designed to include modulation of both phagocytosis and autophagy.

Congenital transmission of experimental leishmaniasis in a hamster model.

Little information is available on transplacental transmission of Leishmania spp. We determined the frequency and impact of congenital infection caused by Leishmania panamensis or L. donovani in experimentally infected hamsters. A polymerase chain reaction showed that congenital transmission occurred in 25.8% (24 of 93) of offspring born to L. panamensis-infected hamsters and 14.6% (11 of 75) offspring born to L. donovani-infected hamsters. Mortality during lactation was higher in offspring born to L. panamensis-infected hamsters and offspring born to L. donovani-infected hamsters than controls, and lymphoproliferation to Leishmania was more frequent in offspring born to L. panamensis-infected hamsters (17.4%, 11 of 63) than in offspring born to L. donovani-infected hamsters (8.5%, 3 of 35). After weaning, only offspring born to L. donovani-infected hamsters had lower weight gain (P < 0.001) and hematocrit levels (P = 0.0045) than controls. Challenge of offspring born to L. panamensis-infected hamsters with L. panamensis showed no differences in lesion evolution, and offspring born to L. donovani-infected hamsters were more susceptible to L. donovani challenge than controls. Consequently, prenatal exposure of hamsters to L. donovani significantly increased the mortality risk and susceptibility to secondary homologous infection.

n-3 Fatty acids uniquely affect anti-microbial resistance and immune cell plasma membrane organization.

It is now well established that dietary lipids are incorporated into macrophage and T-cell membrane microdomains, altering their structure and function. Within cell membranes, there are specific detergent-resistant domains in which key signal transduction proteins are localized. These regions are classified as "lipid rafts". Rafts are composed mostly of cholesterol and sphingolipids and therefore do not integrate well into the fluid phospholipid bilayers causing them to form microdomains. Upon cell activation, rafts compartmentalize signal-transducing molecules, thus providing an environment conducive to signal transduction. In this review, we discuss recent novel data describing the effects of n-3 PUFA on alterations in the activation and functions of macrophages and T-cells. We believe that the modifications in these two disparate immune cell types are linked by fundamentally similar changes in membrane lipid composition and transmembrane signaling functions. We conclude that the outcomes of n-3 PUFA-mediated immune cell alterations may be beneficial (e.g., anti-inflammatory) or detrimental (e.g., loss of microbial immunity) depending upon the cell type interrogated.

Incorporation of a dietary omega 3 fatty acid impairs murine macrophage responses to Mycobacterium tuberculosis.

BACKGROUND: Beside their health benefits, dietary omega 3 polyunsaturated fatty acids (n-3 PUFA) might impair host resistance to Mycobacterium tuberculosis (Mtb) by creating an immunosuppressive environment. We hypothesized that incorporation of n-3 PUFA suppresses activation of macrophage antimycobacterial responses and favors bacterial growth, in part, by modulating the IFNgamma-mediated signaling pathway. METHODOLOGY/PRINCIPAL FINDINGS: Murine macrophage-like J774A.1 cells were incubated with bovine serum albumin (BSA)-conjugated docosahexaenoic acid (DHA; 22:6n-3) or BSA alone, activated with recombinant IFNgamma, and infected with a virulent strain (H37Rv) of M. tuberculosis. The fatty acid composition of macrophage membranes was modified significantly by DHA treatment. DHA-treated macrophages were less effective in controlling intracellular mycobacteria and showed impaired oxidative metabolism and reduced phagolysosome maturation. Incorporation of DHA resulted in defective macrophage activation, as characterized by reduced production of pro-inflammatory cytokines (TNFalpha, IL-6 and MCP-1), and lower expression of co-stimulatory molecules (CD40 and CD86). DHA treatment impaired STAT1 phosphorylation and colocalization of the IFNgamma receptor with lipid rafts, without affecting surface expression of IFNgamma receptor. CONCLUSIONS/SIGNIFICANCE: We conclude that DHA reduces the ability of J774A.1 cells to control M. tuberculosis in response to activation by IFNgamma, by modulation of IFNgamma receptor signaling and function, suggesting that n-3 PUFA-enriched diets may have a detrimental effect on host immunity to tuberculosis.

Transgenic mice enriched in omega-3 fatty acids are more susceptible to pulmonary tuberculosis: impaired resistance to tuberculosis in fat-1 mice.

BACKGROUND. Besides their health benefits, dietary omega-3 fatty acids (n-3 PUFAs) can impair host resistance to intracellular pathogens. Previously, we and others have showed that n-3 PUFA-treated macrophages poorly control Mycobacterium tuberculosis infection in vitro. METHODS. Wild-type and fat-1 transgenic mice were infected with virulent H37Rv M. tuberculosis via the aerosol route. We evaluated bacteriological and histopathological changes in lungs, as well as differences in activation and antimycobacterial capacity in primary macrophages ex vivo. RESULTS. fat-1 mice were more susceptible to tuberculosis, as demonstrated by higher bacterial loads and less robust inflammatory responses in lungs. Macrophages obtained from fat-1 mice were more readily infected with M. tuberculosis in vitro, compared with wild-type macrophages. This impaired bacterial control in cells from fat-1 mice correlated with reduced proinflammatory cytokine secretion, impaired oxidative metabolism, and diminished M. tuberculosis-lysotracker colocalization within phagosomes. CONCLUSIONS. We showed that endogenous production of n-3 PUFAs in fat-1 mice increases their susceptibility to tuberculosis, which could be explained in part by diminished activation and antimycobacterial responses in cells from fat-1 mice. These data suggest that n-3 PUFA-supplemented diets might have a detrimental effect on immunity to M. tuberculosis and raise concerns regarding the safety of omega-3 dietary supplementation in humans.

Pregnancy enhances the innate immune response in experimental cutaneous leishmaniasis through hormone-modulated nitric oxide production.

The maintenance of host defense during pregnancy may depend on heightened innate immunity. We evaluated the immune response of pregnant hamsters during early infection with Leishmania (Viannia) panamensis, a cause of American cutaneous leishmaniasis. At 7 days post-infection, pregnant animals showed a lower parasite burden compared with nonpregnant controls at the cutaneous infection site (P=0.0098) and draining lymph node (P=0.02). Resident peritoneal macrophages and neutrophils from pregnant animals had enhanced Leishmania killing capacity compared with nonpregnant controls (P=0.018 each). This enhanced resistance during pregnancy was associated with increased expression of inducible NO synthase (iNOS) mRNA in lymph node cells (P=0.02) and higher NO production by neutrophils (P=0.0001). Macrophages from nonpregnant hamsters infected with L. panamensis released high amounts of NO upon estrogen exposure (P=0.05), and addition of the iNOS inhibitor L-N6-(1-iminoethyl) lysine blocked the induction of NO production (P=0.02). Infected, nonpregnant females treated with estrogen showed a higher percentage of cells producing NO at the infection site than controls (P=0.001), which correlated with lower parasite burdens (P=0.036). Cultured macrophages or neutrophils from estrogen-treated hamsters showed significantly increased NO production and Leishmania killing compared with untreated controls. iNOS was identified as the likely source of estrogen-induced NO in primed and naïve macrophages, as increased transcription was evident by real-time PCR. Thus, the innate defense against Leishmania infection is heightened during pregnancy, at least in part as a result of estrogen-mediated up-regulation of iNOS expression and NO production.

Retrieval of Gunther Tulip optional vena cava filters 30 days after implantation: a prospective clinical study.

PURPOSE: To report on the feasibility and safety of retrieval of the Günther Tulip optional vena cava filter 30 days after initial implantation. MATERIALS AND METHODS: From March 2004 to September 2005, a single-center prospective study was undertaken in 35 patients who required inferior vena cava (IVC) filtration. All the Günther Tulip filters (GTFs) were implanted with the intention to be removed 30 days after initial implantation. A modified commercial dynamometer was used to measure the force required to remove the device. The degree of difficulty to remove the GTF was classified into four levels: N (no difficulty, force of 0-4.41 N), M (medium difficulty, force of 4.41-5.88 N), G (great difficulty, force of 5.88-9.8 N), and U (unable to remove). Clinical follow-up was performed 1, 3, 6, and 12 months after filter retrieval by review of medical records and imaging. RESULTS: Two of the 35 patients experienced extensive thrombosis in the IVC as revealed by abdominal computed tomography, and their filters were left in place on a permanent basis. One patient died of respiratory and cardiac failure during follow-up within the first 30 days after GTF insertion. Filter retrieval was attempted in the remaining 32 patients, and 31 of these attempts were successful (98%). The force necessary to disengage the GTF from the caval wall was less than 9.8 N (N, 79%; M, 13%; G, 6%). Attempts to remove the GTF failed in only one patient (2%). On follow-up times ranging between 14 and 640 days (mean, 342.5 d), no complications or cases of recurrent pulmonary embolism were observed in this patient population. CONCLUSION: The Günther Tulip optional IVC filter can be safely placed and retrieved percutaneously 30 days after initial implantation.