Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize ... more Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells....
Interferon-b induction occurs during acute simian immunodeficiency virus (SIV) infection in the b... more Interferon-b induction occurs during acute simian immunodeficiency virus (SIV) infection in the brain. We have examined expression and function of cytosolic RNA sensors, retinoic acid inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5), in vivo in the brain of our consistent, accelerated SIVmacaque model and in vitro in SIV-infected macaque macrophages to identify the pathway of type I interferon (IFN) induction. MDA5 messenger RNA (mRNA) and protein were expressed at higher levels in the brain than RIG-I, with protein expression correlating with the severity of disease from 42 until 84 days post-inoculation. The siRNA experiments reveal that mRNA expression of IFN-inducible gene MxA is dependent on MDA5, but not RIG-I. Finally, we demonstrate that SIV infection leads to the production of double-stranded RNA in vivo, which may act as the MDA5 ligand. We have shown for the first time to our knowledge the functional role of MDA5 in the innate immune resp...
Purpose of Review: In addition to preventive protocols and antiretroviral therapy, HIV-1 eradicat... more Purpose of Review: In addition to preventive protocols and antiretroviral therapy, HIV-1 eradication has been considered as additional strategy to help fight the AIDS epidemic. With the support of multiple funding agencies, research groups worldwide have been developing protocols to achieve either a sterilizing or a functional cure for HIV-infection. Recent Findings: Most of the studies focus on the elimination or suppression of circulating CD4+ T cells, the best characterized HIV-1 latent reservoir. The role of the central nervous system (CNS) as a latent reservoir is still controversial. Although brain macrophages and astrocytes are susceptible to HIV-1 infection, it has not been ascertained whether the CNS carries latent HIV-1 during cART and, if so, whether the virus can be reactivated and spread to other compartments after ART interruption. Summary: Here we examine the implications of HIV-1 eradication strategies on the CNS, regardless of whether it is a true latent reservoir and, if so, whether it is present in all patients.
Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known... more Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt clinical hepatitis, with rise in ALT and serum cytokines, and increased intrahepatic F4/80 expression. Despite ongoing viral replication, whole liver transcriptome showed baseline expression levels of inflammatory cytokines, interferons, and interferon induced genes during the chronic infection phase. Transcriptome analyses of sorted KC and IMs using NanoString technology revealed two unique phenotypes with only minimal overlap. At the chronic viral infection phase, KC showed no increased transcription of activation markers Cd80 and Cd86, but an increased expression of genes related to antigen presentation, whereas monocytes were more activated and expressed higher levels of Tnf transcripts. Although both KCs and intrahepatic IM share the surface markers F4/80 and CD11b, their transcriptomes point towards distinctive roles during virusinduced chronic hepatitis.
Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expre... more Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expression. Classical monocytes express the CD14CD16 CCR2 phenotype and migrate to inflammatory sites by quickly responding to CCL2 signaling. Here, we identified and characterized the expansion of a novel monocyte subset during HIV and SIV infection, which were undistinguishable from classical monocytes, based on CD14 and CD16 expression, but expressed significantly lower surface CCR2. Transcriptome analysis of sorted cells demonstrated that the CCR2 low/neg cells are a distinct subpopulation and express lower levels of inflammatory cytokines and activation markers than their CCR2 high counterparts. They exhibited impaired phagocytosis and greatly diminished chemotaxis in response to CCL2 and CCL7. In addition, these monocytes are refractory to SIV infection and suppress CD8 T cell proliferation in vitro. These cells express higher levels of STAT3 and NOS2, suggesting a phenotype similar to ...
A human immunodeficiency virus (HIV) infection cure requires an understanding of the cellular and... more A human immunodeficiency virus (HIV) infection cure requires an understanding of the cellular and anatomical sites harboring virus that contribute to viral rebound upon treatment interruption. Despite antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are reported in HIV-infected individuals on ART. Biomarkers for macrophage activation and neuronal damage in cerebrospinal fluid (CSF) of HIV-infected individuals demonstrate continued effects of HIV in brain and suggest that the central nervous system (CNS) may serve as a viral reservoir. Using a simian immunodeficiency virus (SIV)/macaque model for HIV encephalitis and AIDS, we evaluated whether infected cells persist in brain despite ART. Eight SIV-infected pig-tailed macaques were virally suppressed with ART, and plasma and CSF viremia levels were analyzed longitudinally. To assess whether virus persisted in brain macrophages (BrMΦ) in these macaques, we used a macrophage quantitative viral outgrowth assay...
Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal an... more Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, ...
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Understanding the cellular and anatomical sites of latent virus that contribute to HIV rebound is... more Understanding the cellular and anatomical sites of latent virus that contribute to HIV rebound is essential for eradication. In HIV+ patients, CD4+ T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4+ T-cells in compartments other than blood and lymph nodes is unclear. Macrophages (MΦ) are infected by HIV/SIV, and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4+ T-cell and MΦ functional latent reservoirs were measured in various tissues using cell specific QVOAs. Our results show that blood, spleen, and lung in the majority of suppressed animals contained l...
Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, Jan 6, 2018
HIV-associated neurocognitive disorders (HAND) occur in ~50% of HIV infected individuals despite ... more HIV-associated neurocognitive disorders (HAND) occur in ~50% of HIV infected individuals despite combined antiretroviral therapy. Transmigration into the CNS of CD14CD16 monocytes, particularly those that are HIV infected and express increased surface chemokine receptor CCR2, contributes to neuroinflammation and HAND. To examine whether in HIV infected individuals CCR2 on CD14CD16 monocytes serves as a potential peripheral blood biomarker of HAND, we examined a cohort of 45 HIV infected people. We correlated CCR2 on CD14CD16 monocytes with cognitive status, proton magnetic resonance spectroscopy (H-MRS) measured neurometabolite levels, and peripheral blood mononuclear cell (PBMC) HIV DNA copies. We determined that CCR2 was increased specifically on CD14CD16 monocytes from people with HAND (median [interquartile range (IQR)]) (63.3 [51.6, 79.0]), compared to those who were not cognitively impaired (38.8 [26.7, 56.4]) or those with neuropsychological impairment due to causes other tha...
Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative an... more Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative and well-characterized animal model for HIV neuropathogenesis studies that provides an excellent opportunity to study and develop prognostic markers of HIV-associated neurocognitive disorders (HAND) for HIV-infected individuals. SIV studies can be performed in a controlled setting that enhances reproducibility and offers high-translational value. Similar to observations in HIV-infected patients receiving antiretroviral therapy (ART), ongoing neurodegeneration and inflammation are present in SIV-infected pigtailed macaques treated with suppressive ART. By developing quantitative viral outgrowth assays that measure both CD4+ T cells and macrophages harboring replication competent SIV as well as a highly sensitive mouse-based viral outgrowth assay, we have positioned the SIV/pigtailed macaque model to advance our understanding of latent cellular reservoirs, including potential CNS reservoirs,...
HIV reservoirs persist despite antiretroviral therapy (ART) and are established within a few days... more HIV reservoirs persist despite antiretroviral therapy (ART) and are established within a few days after infection. Infected myeloid cells in the central nervous system (CNS) may contribute to the establishment of a CNS viral reservoir. The mature CD14(+) CD16(+) monocyte subset enters the CNS in response to chemokines, including CCL2. Entry of infected CD14(+) CD16(+) monocytes may lead to infection of other CNS cells, including macrophages or microglia and astrocytes, and to release of neurotoxic early viral proteins and additional cytokines. This contributes to neuroinflammation and neuronal damage leading to HIV-associated neurocognitive disorders (HAND) in ~50% of HIV-infected individuals despite ART. We examined the mechanisms of monocyte entry in the context of HIV infection and report for the first time that HIV(+) CD14(+) CD16(+) monocytes preferentially transmigrate across the blood-brain barrier (BBB). The junctional proteins JAM-A and ALCAM and the chemokine receptor CCR2...
Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immuno... more Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transc...
Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize ... more Fluorescence-activated cell sorting (FACS) is a sensitive and valuable technique to characterize cellular subpopulations and great advances have been made using this approach. Cells are often fixed with formaldehyde prior to the sorting process to preserve cell morphology and maintain the expression of surface molecules, as well as to ensure safety in the sorting of infected cells. It is widely recognized that formaldehyde fixation alters RNA and DNA structure and integrity, thus analyzing gene expression in these cells has been difficult. We therefore examined the effects of formaldehyde fixation on the stability and quantitation of nucleic acids in cell lines, primary leukocytes and also cells isolated from SIV-infected pigtailed macaques. We developed a method to extract RNA from fixed cells that yielded the same amount of RNA as our common method of RNA isolation from fresh cells. Quantitation of RNA by RT-qPCR in fixed cells was not always comparable with that in unfixed cells....
Interferon-b induction occurs during acute simian immunodeficiency virus (SIV) infection in the b... more Interferon-b induction occurs during acute simian immunodeficiency virus (SIV) infection in the brain. We have examined expression and function of cytosolic RNA sensors, retinoic acid inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5), in vivo in the brain of our consistent, accelerated SIVmacaque model and in vitro in SIV-infected macaque macrophages to identify the pathway of type I interferon (IFN) induction. MDA5 messenger RNA (mRNA) and protein were expressed at higher levels in the brain than RIG-I, with protein expression correlating with the severity of disease from 42 until 84 days post-inoculation. The siRNA experiments reveal that mRNA expression of IFN-inducible gene MxA is dependent on MDA5, but not RIG-I. Finally, we demonstrate that SIV infection leads to the production of double-stranded RNA in vivo, which may act as the MDA5 ligand. We have shown for the first time to our knowledge the functional role of MDA5 in the innate immune resp...
Purpose of Review: In addition to preventive protocols and antiretroviral therapy, HIV-1 eradicat... more Purpose of Review: In addition to preventive protocols and antiretroviral therapy, HIV-1 eradication has been considered as additional strategy to help fight the AIDS epidemic. With the support of multiple funding agencies, research groups worldwide have been developing protocols to achieve either a sterilizing or a functional cure for HIV-infection. Recent Findings: Most of the studies focus on the elimination or suppression of circulating CD4+ T cells, the best characterized HIV-1 latent reservoir. The role of the central nervous system (CNS) as a latent reservoir is still controversial. Although brain macrophages and astrocytes are susceptible to HIV-1 infection, it has not been ascertained whether the CNS carries latent HIV-1 during cART and, if so, whether the virus can be reactivated and spread to other compartments after ART interruption. Summary: Here we examine the implications of HIV-1 eradication strategies on the CNS, regardless of whether it is a true latent reservoir and, if so, whether it is present in all patients.
Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known... more Due to the scarcity of immunocompetent animal models for chronic viral hepatitis, little is known about the role of the innate intrahepatic immune system during viral replication in the liver. These insights are however fundamental for the understanding of the inappropriate adaptive immune responses during the chronic phase of the infection. We apply the Lymphocytic Choriomenigitis Virus (LCMV) clone 13 mouse model to examine chronic virus-host interactions of Kupffer cells (KC) and infiltrating monocytes (IM) in an infected liver. LCMV infection induced overt clinical hepatitis, with rise in ALT and serum cytokines, and increased intrahepatic F4/80 expression. Despite ongoing viral replication, whole liver transcriptome showed baseline expression levels of inflammatory cytokines, interferons, and interferon induced genes during the chronic infection phase. Transcriptome analyses of sorted KC and IMs using NanoString technology revealed two unique phenotypes with only minimal overlap. At the chronic viral infection phase, KC showed no increased transcription of activation markers Cd80 and Cd86, but an increased expression of genes related to antigen presentation, whereas monocytes were more activated and expressed higher levels of Tnf transcripts. Although both KCs and intrahepatic IM share the surface markers F4/80 and CD11b, their transcriptomes point towards distinctive roles during virusinduced chronic hepatitis.
Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expre... more Monocytes have been categorized in three main subpopulations based on CD14 and CD16 surface expression. Classical monocytes express the CD14CD16 CCR2 phenotype and migrate to inflammatory sites by quickly responding to CCL2 signaling. Here, we identified and characterized the expansion of a novel monocyte subset during HIV and SIV infection, which were undistinguishable from classical monocytes, based on CD14 and CD16 expression, but expressed significantly lower surface CCR2. Transcriptome analysis of sorted cells demonstrated that the CCR2 low/neg cells are a distinct subpopulation and express lower levels of inflammatory cytokines and activation markers than their CCR2 high counterparts. They exhibited impaired phagocytosis and greatly diminished chemotaxis in response to CCL2 and CCL7. In addition, these monocytes are refractory to SIV infection and suppress CD8 T cell proliferation in vitro. These cells express higher levels of STAT3 and NOS2, suggesting a phenotype similar to ...
A human immunodeficiency virus (HIV) infection cure requires an understanding of the cellular and... more A human immunodeficiency virus (HIV) infection cure requires an understanding of the cellular and anatomical sites harboring virus that contribute to viral rebound upon treatment interruption. Despite antiretroviral therapy (ART), HIV-associated neurocognitive disorders (HAND) are reported in HIV-infected individuals on ART. Biomarkers for macrophage activation and neuronal damage in cerebrospinal fluid (CSF) of HIV-infected individuals demonstrate continued effects of HIV in brain and suggest that the central nervous system (CNS) may serve as a viral reservoir. Using a simian immunodeficiency virus (SIV)/macaque model for HIV encephalitis and AIDS, we evaluated whether infected cells persist in brain despite ART. Eight SIV-infected pig-tailed macaques were virally suppressed with ART, and plasma and CSF viremia levels were analyzed longitudinally. To assess whether virus persisted in brain macrophages (BrMΦ) in these macaques, we used a macrophage quantitative viral outgrowth assay...
Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal an... more Treatment of HIV infection with either antiretroviral (ARV) therapy or neutralizing monoclonal antibodies (NAbs) leads to a reduction in HIV plasma virus. Both ARVs and NAbs prevent new rounds of viral infection, but NAbs may have the additional capacity to accelerate the loss of virus-infected cells through Fc gamma receptor (FcγR)-mediated effector functions, which should affect the kinetics of plasma-virus decline. Here, we formally test the role of effector function in vivo by comparing the rate and timing of plasma-virus clearance in response to a single-dose treatment with either unmodified NAb or those with either reduced or augmented Fc function. When infused into viremic simian HIV (SHIV)-infected rhesus macaques, there was a 21% difference in slope of plasma-virus decline between NAb and NAb with reduced Fc function. NAb engineered to increase FcγRIII binding and improve antibody-dependent cellular cytotoxicity (ADCC) in vitro resulted in arming of effector cells in vivo, ...
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Understanding the cellular and anatomical sites of latent virus that contribute to HIV rebound is... more Understanding the cellular and anatomical sites of latent virus that contribute to HIV rebound is essential for eradication. In HIV+ patients, CD4+ T lymphocytes comprise a well-defined functional latent reservoir, defined as cells containing transcriptionally silent genomes able to produce infectious virus once reactivated. However, the persistence of infectious latent virus in CD4+ T-cells in compartments other than blood and lymph nodes is unclear. Macrophages (MΦ) are infected by HIV/SIV, and are likely to carry latent viral genomes during antiretroviral therapy (ART), contributing to the reservoir. Currently, the gold standard assay used to measure reservoirs containing replication competent virus is the quantitative viral outgrowth assay (QVOA). Using an SIV-macaque model, the CD4+ T-cell and MΦ functional latent reservoirs were measured in various tissues using cell specific QVOAs. Our results show that blood, spleen, and lung in the majority of suppressed animals contained l...
Journal of neuroimmune pharmacology : the official journal of the Society on NeuroImmune Pharmacology, Jan 6, 2018
HIV-associated neurocognitive disorders (HAND) occur in ~50% of HIV infected individuals despite ... more HIV-associated neurocognitive disorders (HAND) occur in ~50% of HIV infected individuals despite combined antiretroviral therapy. Transmigration into the CNS of CD14CD16 monocytes, particularly those that are HIV infected and express increased surface chemokine receptor CCR2, contributes to neuroinflammation and HAND. To examine whether in HIV infected individuals CCR2 on CD14CD16 monocytes serves as a potential peripheral blood biomarker of HAND, we examined a cohort of 45 HIV infected people. We correlated CCR2 on CD14CD16 monocytes with cognitive status, proton magnetic resonance spectroscopy (H-MRS) measured neurometabolite levels, and peripheral blood mononuclear cell (PBMC) HIV DNA copies. We determined that CCR2 was increased specifically on CD14CD16 monocytes from people with HAND (median [interquartile range (IQR)]) (63.3 [51.6, 79.0]), compared to those who were not cognitively impaired (38.8 [26.7, 56.4]) or those with neuropsychological impairment due to causes other tha...
Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative an... more Simian immunodeficiency virus (SIV) infection of pigtailed macaques is a highly representative and well-characterized animal model for HIV neuropathogenesis studies that provides an excellent opportunity to study and develop prognostic markers of HIV-associated neurocognitive disorders (HAND) for HIV-infected individuals. SIV studies can be performed in a controlled setting that enhances reproducibility and offers high-translational value. Similar to observations in HIV-infected patients receiving antiretroviral therapy (ART), ongoing neurodegeneration and inflammation are present in SIV-infected pigtailed macaques treated with suppressive ART. By developing quantitative viral outgrowth assays that measure both CD4+ T cells and macrophages harboring replication competent SIV as well as a highly sensitive mouse-based viral outgrowth assay, we have positioned the SIV/pigtailed macaque model to advance our understanding of latent cellular reservoirs, including potential CNS reservoirs,...
HIV reservoirs persist despite antiretroviral therapy (ART) and are established within a few days... more HIV reservoirs persist despite antiretroviral therapy (ART) and are established within a few days after infection. Infected myeloid cells in the central nervous system (CNS) may contribute to the establishment of a CNS viral reservoir. The mature CD14(+) CD16(+) monocyte subset enters the CNS in response to chemokines, including CCL2. Entry of infected CD14(+) CD16(+) monocytes may lead to infection of other CNS cells, including macrophages or microglia and astrocytes, and to release of neurotoxic early viral proteins and additional cytokines. This contributes to neuroinflammation and neuronal damage leading to HIV-associated neurocognitive disorders (HAND) in ~50% of HIV-infected individuals despite ART. We examined the mechanisms of monocyte entry in the context of HIV infection and report for the first time that HIV(+) CD14(+) CD16(+) monocytes preferentially transmigrate across the blood-brain barrier (BBB). The junctional proteins JAM-A and ALCAM and the chemokine receptor CCR2...
Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immuno... more Antiviral treatment options for chronic Hepatitis E Virus (HEV) infections are limited and immunological determinants of viral persistence remain largely unexplored. We studied the antiviral potency of pegylated interferon-α (pegIFNα) against HEV infections in humanized mice and modelled intrahepatic interferon stimulated gene (ISG) responses. Human gene expression levels in humanized mouse livers were analyzed by qPCR and Nanostring. Human CXCL10 was measured in mouse serum. HEV genotype 3 (gt3) infections were cleared from liver and feces within 8 pegIFNα doses in all mice and relapsed after a single pegIFNα injection in only half of treated animals. Rapid viral clearance by pegIFNα was confirmed in HEV gt1, but not in Hepatitis B Virus infected animals. No ISG induction was observed in untreated HEV gt3 and gt1 infected humanized livers compared to control chimeric mice, irrespective of the human hepatocyte donor, viral isolate or HEV infection duration. Human specific ISG transc...
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