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Airway epithelial cells (AEC) and airway macrophages (AM) represent the major cellular targets for infection by different respiratory viruses. These include influenza A virus (IAV), a member of the Orthomyxoviridae family, and parainfluenza virus type-3 (PIV-3), a member of the Paramyxoviridae family. Virus-infected cells rapidly induce interferons (IFNs) which in turn activate hundreds of interferon-stimulated genes (ISGs) in infected cells and in neighbouring uninfected cells. The family of interferon-induced transmembrane (IFITM) proteins are ISGs that have shown broad antiviral activity against different virus families by interfering with virus entry. In this thesis, we evaluated the antiviral activity of human IFITM1, IFITM2 and IFITM3 against IAV and PIV-3, two respiratory viruses with distinct entry requirements. Using a doxycycline (DOX)-inducible IFITM overexpression system we found that all three IFITM proteins limited IAV infection to varying degrees, with IFITM3 having the strongest effect. Our studies confirmed that IFITM proteins display antiviral activity early, but not late, in the IAV replication cycle. None of the IFITM proteins modulated PIV-3 replication at either early or late-stages in the replication cycle. Different approaches were explored for their utility to modulate IFITM1, IFITM2 or IFITM3 expression to assess potential antiviral activity against IAV and PIV-3. Herein, we highlight caveats associated with constitutive IFITM protein overexpression as well as using CRISPR/Cas9 gene editing techniques to knockout endogenous IFITM expression. Observations herein also demonstrated that entry of IAV into epithelial cells by endocytosis conferred sensitivity to restriction by endosome-localized IFITM2 and IFITM3. Indeed, key steps in the IAV replication cycle from cellular entry through to exit are relatively well defined in epithelial cells. However, comparatively less is known about all stages of the viral life cycle during IAV infection of m

HIV-1 infection leads to the acquired immunodeficiency syndrome (AIDS). Antibody-dependent cellular phagocytosis (ADCP) by macrophages was found to contribute to HIV-1 clearance. CD47 is a cell-surface marker regulating macrophage phagocytic activity in both cancer and HIV models, providing “don’t eat me” signals preventing phagocytosis. Therefore, it seems reasonable that CD47 blockade (i.e. Hu5F9 anti-CD47 antibody) will enhance macrophage phagocytosis of HIV-1 infected cells. This study investigated whether anti-CD47 antibody treatment of SIV-infected target cells led to their increased phagocytosis by monocyte-derived macrophage (MDM) as compared to SIV-negative cells, suggesting that SIV-infection causes up- regulation of CD47 for immune evasion. We applied Leica confocal microscopy, flow cytometry, as well as EVOS microscopy and imaging system to detect and evaluate the fraction of macrophages containing ingested cells (“phagocytic ratio”) under different treatment conditions. We found that SIVmac251-infected T cells treated with Hu5F9 IgG1 anti-CD47 antibody are efficiently cleared by MDMs via ADCP in vitro. Also, Hu5F9 and related antibodies (e.g., of different subclasses) were useful in supporting phagocytosis of both the latently infected and reactivated J-Lat cells. In addition, Hu5F9 IgG1 could effectively synergize with 10-1074 broadly neutralizing antibody (bNAb) to encourage engulfment of SHIV-AD8-EO infected CD4+ T cells. Similar to previous cancer studies, another cell surface marker called Calreticulin (CRT) was found to interact with CD47 in the setting of HIV infection to provide balance between pro-phagocytosis and anti-phagocytosis. CRT-related mechanisms may be a topic of future investigation, and especially how such mechanisms might be exploited when combined with anti-CD47 treatment in HIV-1 infection. In conclusion, Hu5F9 IgG1 anti-CD47 antibody promotes monocyte-derived macrophage phagocytosis of HIV/SIV infected cells at several infection

HIV-1 infection leads to the acquired immunodeficiency syndrome (AIDS). Antibody-dependent cellular phagocytosis (ADCP) by macrophages was found to contribute to HIV-1 clearance. CD47 is a cell-surface marker regulating macrophage phagocytic activity in both cancer and HIV models, providing “don’t eat me” signals preventing phagocytosis. Therefore, it seems reasonable that CD47 blockade (i.e. Hu5F9 anti-CD47 antibody) will enhance macrophage phagocytosis of HIV-1 infected cells. This study investigated whether anti-CD47 antibody treatment of SIV-infected target cells led to their increased phagocytosis by monocyte-derived macrophage (MDM) as compared to SIV-negative cells, suggesting that SIV-infection causes up- regulation of CD47 for immune evasion. We applied Leica confocal microscopy, flow cytometry, as well as EVOS microscopy and imaging system to detect and evaluate the fraction of macrophages containing ingested cells (“phagocytic ratio”) under different treatment conditions. We found that SIVmac251-infected T cells treated with Hu5F9 IgG1 anti-CD47 antibody are efficiently cleared by MDMs via ADCP in vitro. Also, Hu5F9 and related antibodies (e.g., of different subclasses) were useful in supporting phagocytosis of both the latently infected and reactivated J-Lat cells. In addition, Hu5F9 IgG1 could effectively synergize with 10-1074 broadly neutralizing antibody (bNAb) to encourage engulfment of SHIV-AD8-EO infected CD4+ T cells. Similar to previous cancer studies, another cell surface marker called Calreticulin (CRT) was found to interact with CD47 in the setting of HIV infection to provide balance between pro-phagocytosis and anti-phagocytosis. CRT-related mechanisms may be a topic of future investigation, and especially how such mechanisms might be exploited when combined with anti-CD47 treatment in HIV-1 infection. In conclusion, Hu5F9 IgG1 anti-CD47 antibody promotes monocyte-derived macrophage phagocytosis of HIV/SIV infected cells at several infection

Background: Macrophages play a key role in coronary plaque destabilization. In-vitro human monocyte-derived macrophages (MDMs) are used to study macrophages infiltrating tissue. Optical coherence tomography (OCT) provides an in-vivo insight of the coronary arteries. We compared the MDMs morpho-phenotype and culprit plaque features at OCT in acute coronary syndrome (ACS) patients according to the underlying plaque pathobiology. Methods: Sixty-six patients undergoing coronary angiography and pre-angioplasty OCT of the culprit vessel were allocated to three groups according to mechanism of ACS at OCT and C-reactive protein levels (cut-off: 2 mg/Ll): 1) plaque rupture with systemic inflammation; 2) plaque rupture without systemic inflammation, 3) plaque with intact fibrous cap. A blood sample was collected to obtain MDMs, categorized as having “round” or “spindle” morphology. Results: Thirty-two patients (48.5%) were assigned to Group 1, 10 (15.2%) to Group 2 and 24 (36.4%) to Group 3. The “round” MDMs were significantly more frequent in Group 1 (39.25 ± 4.98%) than in Group 2 (23.89 ± 3.10%) and Group 3 (23.02 ± 7.89%), p = 0.008. MDMs in Group 1 as compared to Groups 2 and 3 showed lower efferocytosis (8.74 ± 1.38 vs 9.74 ± 2.15 vs 11.41 ± 2.41; p = 0.012), higher tissue factor levels (369.84 ± 101.13 vs 301.89 ± 59.78 vs 231.74 ± 111.47; p = 0.001) and higher heme oxygenase-1 expression (678.78 ± 145.43 vs 419.12 ± 74.44 vs 409.78 ± 64.33; p = 0.008). Conclusions: MDMs of ACS patients show morpho-phenotypic heterogeneity with prevalence of pro-thrombotic and pro-oxidative properties in case of plaque rupture and systemic inflammation. Such MDMs subpopulation may take part to the cellular pathways leading to fibrous cap rupture with the subsequent thrombus formation.

Netrin-1 is a laminin-like protein that plays a pivotal role in cell migration and, according to the site of its release, exerts both pro and anti-atherosclerotic functions. Macrophages, key cells in atherosclerosis, are heterogeneous in morphology and function and different subpopulations may support plaque progression, stabilization, and/or regression. Netrin-1 was evaluated in plasma and, together with its receptor UNC5b, in both spindle and round monocyte-derived macrophages (MDMs) morphotypes from coronary artery disease (CAD) patients and control subjects. In CAD patients, plaque features were detected in vivo by optical coherence tomography. CAD patients had lower plasma Netrin-1 levels and a higher MDMs expression of both protein and its receptor compared to controls. Specifically, a progressive increase in Netrin-1 and UNC5b was evidenced going from controls to stable angina (SA) and acute myocardial infarction (AMI) patients. Of note, spindle MDMs of AMI showed a marked increase of both Netrin-1 and its receptor compared to spindle MDMs of controls. UNC5b expression is always higher in spindle compared to round MDMs, regardless of the subgroup. Finally, CAD patients with higher intracellular Netrin-1 levels showed greater intraplaque macrophage accumulation in vivo. Our findings support the role of Netrin-1 and UNC5b in the atherosclerotic process.

Peripheral blood from healthy sheep (n = 3) and goats (n = 3) were employed to establish an efficient method for simultaneous isolation of peripheral blood mononuclear cells (PBMCs) and neutrophils and to standardize protocols for monocyte purification and generation of monocyte-derived macrophages (MDMs). In both species, a significantly enriched population of PBMCs, with higher purity and number of cells determined by flow cytometry, was achieved when processing through a density gradient a mixture of buffy-coat and red blood cell layer (RBC) in comparison to the use of just the buffy-coat (p < 0.05). Neutrophils could be subsequently isolated from the layer, located underneath PBMCs fraction with significant higher purity rates, higher than 85 % determined by flow cytometry, than those obtained with protocols without density gradients (< 60 %) (p < 0.05). This technique would allow the isolation of both cell populations from the same sample of blood. A pure cell population of monocytes, CD14+ cells, was purified from PBMCs when using immunomagnetic columns, which allow for 17 % (nº monocytes/nº PBMCs) of yield and high percentages of expression of CD14+ (88 %), MHC-II+ (91.5 %) and CD11b+ (94 %) established by flow cytometry. On the other hand, the classical and nonexpensive purification of monocytes from PBMCs based on the adherence capacity of the former, allowed significantly lower yield of monocytes (4.6 %), with percentages of surface markers expression that dropped to 35 %, 65 % and 55 %, respectively (p < 0.001), suggesting the isolation of a mixed population of cells. The addition of GM-CSF to the culture, at concentration from 25 to 125 ng/mL, enhanced proportionally the number of MDMs generated compared to the absence of supplementation or the use of autologous serum from 5% to 20 %. However, purification of monocytes through the adherence method achieved higher yields of MDMs than those isolated through immunomagnetic columns in both species (p < 0.001

Paratuberculosis is chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Whole RNA-sequencing (RNA-Seq) is a promising source of novel biomarkers for early MAP infection and disease progression in cattle. Since the blood transcriptome is widely used as a source of biomarkers, we analyzed whether it recapitulates, at least in part, the transcriptome of the ileocecal valve (ICV), the primary site of MAP colonization. Total RNA was prepared from peripheral blood (PB) and ICV samples, and RNA-Seq was used to compare gene expression between animals with focal or diffuse histopathological lesions in gut tissues versus control animals with no detectable signs of infection. Our results demonstrated both shared, and PB and ICV-specific gene expression in response to a natural MAP infection. As expected, the number of differentially expressed (DE) genes was larger in the ICV than in the PB samples. Among the DE genes in the PB and ICV samples, there were some common genes irrespective of the type of lesion including the C-X-C motif chemokine ligand 8 (CXCL8/IL8), apolipoprotein L (APOLD1), and the interferon inducible protein 27 (IF127). The biological processes (BP) enriched in the PB gene expression profiles from the cows with diffuse lesions included the killing of cells of other organism, defense response, immune response and the regulation of neutrophil chemotaxis. Two of these BP, the defense and immune response, were also enriched in the ICV from the cows with diffuse lesions. Metabolic analysis of the DE genes revealed that the N-glycan biosynthesis, bile secretion, one-carbon pool by folate and purine metabolism were significantly enriched in the ICV from the cows with focal lesions. In the ICV from cows with diffuse lesions; the valine, leucine and isoleucine degradation route, purine metabolism, vitamin digestion and absorption and the cholesterol routes were enriched. Some of the identified DE genes, BP and meta

Paratuberculosis is chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Whole RNA-sequencing (RNA-Seq) is a promising source of novel biomarkers for early MAP infection and disease progression in cattle. Since the blood transcriptome is widely used as a source of biomarkers, we analyzed whether it recapitulates, at least in part, the transcriptome of the ileocecal valve (ICV), the primary site of MAP colonization. Total RNA was prepared from peripheral blood (PB) and ICV samples, and RNA-Seq was used to compare gene expression between animals with focal or diffuse histopathological lesions in gut tissues versus control animals with no detectable signs of infection. Our results demonstrated both shared, and PB and ICV-specific gene expression in response to a natural MAP infection. As expected, the number of differentially expressed (DE) genes was larger in the ICV than in the PB samples. Among the DE genes in the PB and ICV samples, there were some common genes irrespective of the type of lesion including the C-X-C motif chemokine ligand 8 (CXCL8/IL8), apolipoprotein L (APOLD1), and the interferon inducible protein 27 (IF127). The biological processes (BP) enriched in the PB gene expression profiles from the cows with diffuse lesions included the killing of cells of other organism, defense response, immune response and the regulation of neutrophil chemotaxis. Two of these BP, the defense and immune response, were also enriched in the ICV from the cows with diffuse lesions. Metabolic analysis of the DE genes revealed that the N-glycan biosynthesis, bile secretion, one-carbon pool by folate and purine metabolism were significantly enriched in the ICV from the cows with focal lesions. In the ICV from cows with diffuse lesions; the valine, leucine and isoleucine degradation route, purine metabolism, vitamin digestion and absorption and the cholesterol routes were enriched. Some of the identified DE genes, BP and meta

Paratuberculosis is chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Whole RNA-sequencing (RNA-Seq) is a promising source of novel biomarkers for early MAP infection and disease progression in cattle. Since the blood transcriptome is widely used as a source of biomarkers, we analyzed whether it recapitulates, at least in part, the transcriptome of the ileocecal valve (ICV), the primary site of MAP colonization. Total RNA was prepared from peripheral blood (PB) and ICV samples, and RNA-Seq was used to compare gene expression between animals with focal or diffuse histopathological lesions in gut tissues versus control animals with no detectable signs of infection. Our results demonstrated both shared, and PB and ICV-specific gene expression in response to a natural MAP infection. As expected, the number of differentially expressed (DE) genes was larger in the ICV than in the PB samples. Among the DE genes in the PB and ICV samples, there were some common genes irrespective of the type of lesion including the C-X-C motif chemokine ligand 8 (CXCL8/IL8), apolipoprotein L (APOLD1), and the interferon inducible protein 27 (IF127). The biological processes (BP) enriched in the PB gene expression profiles from the cows with diffuse lesions included the killing of cells of other organism, defense response, immune response and the regulation of neutrophil chemotaxis. Two of these BP, the defense and immune response, were also enriched in the ICV from the cows with diffuse lesions. Metabolic analysis of the DE genes revealed that the N-glycan biosynthesis, bile secretion, one-carbon pool by folate and purine metabolism were significantly enriched in the ICV from the cows with focal lesions. In the ICV from cows with diffuse lesions; the valine, leucine and isoleucine degradation route, purine metabolism, vitamin digestion and absorption and the cholesterol routes were enriched. Some of the identified DE genes, BP and meta

Paratuberculosis is chronic granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Whole RNA-sequencing (RNA-Seq) is a promising source of novel biomarkers for early MAP infection and disease progression in cattle. Since the blood transcriptome is widely used as a source of biomarkers, we analyzed whether it recapitulates, at least in part, the transcriptome of the ileocecal valve (ICV), the primary site of MAP colonization. Total RNA was prepared from peripheral blood (PB) and ICV samples, and RNA-Seq was used to compare gene expression between animals with focal or diffuse histopathological lesions in gut tissues versus control animals with no detectable signs of infection. Our results demonstrated both shared, and PB and ICV-specific gene expression in response to a natural MAP infection. As expected, the number of differentially expressed (DE) genes was larger in the ICV than in the PB samples. Among the DE genes in the PB and ICV samples, there were some common genes irrespective of the type of lesion including the C-X-C motif chemokine ligand 8 (CXCL8/IL8), apolipoprotein L (APOLD1), and the interferon inducible protein 27 (IF127). The biological processes (BP) enriched in the PB gene expression profiles from the cows with diffuse lesions included the killing of cells of other organism, defense response, immune response and the regulation of neutrophil chemotaxis. Two of these BP, the defense and immune response, were also enriched in the ICV from the cows with diffuse lesions. Metabolic analysis of the DE genes revealed that the N-glycan biosynthesis, bile secretion, one-carbon pool by folate and purine metabolism were significantly enriched in the ICV from the cows with focal lesions. In the ICV from cows with diffuse lesions; the valine, leucine and isoleucine degradation route, purine metabolism, vitamin digestion and absorption and the cholesterol routes were enriched. Some of the identified DE genes, BP and meta

IVIg is an approved therapy for immunodeficiency and for several autoimmune and inflammatory diseases. However, the molecular basis for the IVIg anti-inflammatory activity remains to be fully explained and cannot be extrapolated from studies on animal models of disease. We now report that IVIg impairs the generation of human monocyte-derived anti-inflammatory macrophages by inducing JNK activation and activin A production and limits proinflammatory macrophage differentiation by inhibiting GM-CSF-driven STAT5 activation. In vivo, IVIg provokes a rapid increase in peripheral blood activin A, CCL2, and IL-6 levels, an effect that can be recapitulated in vitro on human monocytes. On differentiating monocytes, IVIg promotes the acquisition of altered transcriptional and cytokine profiles, reduces TLR expression and signaling, and upregulates negative regulators of TLR-initiated intracellular signaling. In line with these effects, in vivo IVIg infusion induces a state tolerant toward subsequent stimuli that results in reduced inflammatory cytokine production after LPS challenge in human peripheral blood and significant protection from LPS-induced death in mice. Therefore, IVIg conditions human macrophages toward the acquisition of a state of cross-tolerance against inflammatory stimuli, an effect that correlates with the net anti-inflammatory action of IVIg in vivo.