Characterization of the interaction of African swine fever virus with monocytes and derived macrophage subsets.

Abstract African swine fever (ASF) is a devastating disease for which there is no vaccine available. The ASF virus (ASFV) primarily infects cells of the myeloid lineage and this tropism is thought to be crucial for disease pathogenesis. A detailed in vitro characterization of the interactions of a virulent Sardinian isolate (22653/14) and a tissue culture adapted avirulent strain (BA71V) of ASFV with porcine monocytes, un-activated (moMΦ), classically (moM1) and alternatively (moM2) activated monocyte-derived macrophages was conducted in an attempt to better understand this relationship. Using a multiplicity-of-infection (MOI) of 1, both viruses were able to infect monocytes and macrophage subsets, but BA71V presented a reduced ability to infect moM1 compared to 22653/14, with higher expression of early compared to late proteins. Using an MOI of 0.01, only 22653/14 was able to replicate in all the macrophage subsets, with initially lowest in moM1 and moM2. No differences were observed in the expression of CD163 between ASFV infected and uninfected bystander cells. ASFV down-regulated CD16 expression but did not modulate MHC class II levels in monocytes and macrophage subsets. BA71V-infected but not 22653/14-infected moMΦ and moM2 presented with a reduced expression of MHC class I compared to the mock-infected controls. Higher levels of IL-18, IL1-β and IL-1α were released from moM1 after infection with BA71V compared to 22653/14 or mock-infected control. These results revealed differences between these ASFV strains, suggesting that virulent isolates have evolved mechanisms to counteract activated macrophages responses, promoting their survival, dissemination in the host and so ASF pathogenesis

Interferon regulatory factor 8-deficiency determines massive neutrophil recruitment but T cell defect in fast growing granulomas during tuberculosis

Following Mycobacterium tuberculosis (Mtb) infection, immune cell recruitment in lungs is pivotal in establishing protective immunity through granuloma formation and neogenesis of lymphoid structures (LS). Interferon regulatory factor-8 (IRF-8) plays an important role in host defense against Mtb, although the mechanisms driving anti-mycobacterial immunity remain unclear. In this study, IRF-8 deficient mice (IRF-8−/−) were aerogenously infected with a low-dose Mtb Erdman virulent strain and the course of infection was compared with that induced in wild-type (WT-B6) counterparts. Tuberculosis (TB) progression was examined in both groups using pathological, microbiological and immunological parameters. Following Mtb exposure, the bacterial load in lungs and spleens progressed comparably in the two groups for two weeks, after which IRF-8−/− mice developed a fatal acute TB whereas in WT-B6 the disease reached a chronic stage. In lungs of IRF-8−/−, uncontrolled growth of pulmonary granulomas and impaired development of LS were observed, associated with unbalanced homeostatic chemokines, progressive loss of infiltrating T lymphocytes and massive prevalence of neutrophils at late infection stages. Our data define IRF-8 as an essential factor for the maintenance of proper immune cell recruitment in granulomas and LS required to restrain Mtb infection. Moreover, IRF-8−/− mice, relying on a common human and mouse genetic mutation linked to susceptibility/severity of mycobacterial diseases, represent a valuable model of acute TB for comparative studies with chronically-infected congenic WT-B6 for dissecting protective and pathological immune reactions

Goat milk extracellular vesicles: immuno-modulation effects on porcine monocyte-derived macrophages in vitro

IntroductionExtracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages.MethodsIn this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays.ResultsThese particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected.DiscussionOverall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors

A study of multiple Felis catus papillomavirus types (1, 2, 3, 4) in cat skin lesions in Italy by quantitative PCR

Objectives The aim of the study was to investigate, by quantitative PCR (qPCR) the presence of papillomavirus in feline viral plaques (VPs), Bowenoid in situ carcinoma (BISC), squamous cell carcinoma (SCC) and actinic keratosis (AK). Methods Twenty-nine cases with previously established diagnoses of feline VPs, BISC, invasive SCC and AK were selected from a dermatopathological database. A critical re-evaluation of diagnosis was performed by defining clear criteria toward carcinomatous vs non-carcinomatous, in situ vs invasive (if carcinomatous) and viral vs actinic. Cases were evaluated for p16 immunolocalisation. The presence of the target viral genes for Felis catus papillomavirus (FcaPV)-1, FcaPV-2, FcaPV-3 and FcaPV-4 was determined by qPCR. The data generated ΔΔCq values, which represent a normalised measure of DNA viral quantity. Samples with a positive ΔΔCq value were submitted to sequence analysis. Results Four VPs, 19 BISCs, four SCCs and one AK were included. By ΔΔCq analysis we found that all VPs were positive for FcaPV-1 or FcaPV-2; eight BISCs were positive for FcaPV-1, FcaPV-2 and FcaPV-4. FcaPV-2 was the most prevalent among the group of VPs and BISCs. Conclusions and relevance Using the ΔΔCq method we report the first evidence of FcaPV-1, FcaPV-2 and FcaPV-4 in Italy; FcaPV-2 was the most frequently detected. To a lesser extent, FcaPV-1 and FcaPV-4 were detected in the examined samples. FcaPV-3 was never associated with viral-induced lesions by ΔΔCq investigation. Compared with conventional PCR the ΔΔCq method has the advantage of establishing a possible role of the virus in the outcome of infection

Assessment of the Impact of a Toll-like Receptor 2 Agonist Synthetic Lipopeptide on Macrophage Susceptibility and Responses to African Swine Fever Virus Infection

Toll-like receptor 2 (TLR2) ligands are attracting attention as prophylactic and immunopotentiator agents against pathogens, including viruses. We previously reported that a synthetic diacylated lipopeptide (Mag-Pam2Cys_P48) polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. Here, we investigated its role in modulating monocyte-derived macrophage (moMΦ) responses against African swine fever virus (ASFV), the etiological agent of one of the greatest threats to the global pig industry. Two ASFV isolates were compared: the attenuated NH/P68 and the virulent 26544/OG10. No effect on virus infection nor the modulation of surface markers’ expression (MHC I, MHC II DR, CD14, CD16, and CD163) were observed when Mag-Pam2Cys_P48 treated moMΦ were infected using a multiplicity of infection (MOI) of 1. Mag-Pam2Cys_P48 treated moMΦ released higher levels of IL-1α, IL-1β, IL-1Ra, and IL-18 in response to infection with NH/P68 ASFV compared to 26544/OG10-infected and mock-infected controls. Surprisingly, when infected using a MOI of 0.01, the virulent ASFV 26544/OG10 isolate replicated even slightly more efficiently in Mag-Pam2Cys_P48 treated moMΦ. These effects also extended to the treatment of moMΦ with two other lipopeptides: Mag-Pam2Cys_P80 and Mag-Pam2Cys_Mag1000. Our data suggested limited applicability of TLR2 agonists as prophylactic or immunopotentiator agents against virulent ASFV but highlighted the ability of the virulent 26544/OG10 to impair macrophage defenses

Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells.

Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction