CCAAT/Enhancer Binding Protein β Is a Major Mediator of Inflammation and Viral Replication in the Gastrointestinal Tract of Simian Immunodeficiency Virus-Infected Rhesus Macaques
2008; Elsevier BV; Volume: 173; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2008.080108
ISSN1525-2191
AutoresMahesh Mohan, Pyone P. Aye, Juan T. Borda, Xavier Álvarez, Andrew A. Lackner,
Tópico(s)HIV/AIDS drug development and treatment
ResumoThe gastrointestinal tract (GIT) is a major target of infection with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Chronic GIT disease and inflammation are common sequelae to HIV/SIV infection. Nonetheless, the molecular mechanisms that cause and maintain GIT dysfunction remain unclear. We investigated the contribution of CCAAT/enhancer-binding protein β (C/EBPβ) to GIT disease and viral replication in jejunum and colon collected at necropsy from 12 SIV-infected (group 1), or 10 uninfected macaques with chronic diarrhea (group 2), and 9 uninfected control macaques (group 3). All group 1 and 2 macaques had chronic diarrhea, wasting, and colitis, but group 1 animals had more severe lesions in the jejunum. C/EBPβ gene expression increased significantly in colon of groups 1 and 2 and in jejunum of only group 1 macaques compared with controls. In group 1 animals, CEBPβ expression was localized predominantly to macrophages and occasionally lymphocytes. Chromatin immunoprecipitation assays confirmed the binding of C/EBPβ and p65 to the SIV long terminal repeat region in colonic lamina propria cells, suggesting a mechanistic link between inflammation and activation of viral replication in vivo. This is the first in vivo study describing the transcriptional changes and immunophenotypic localization of C/EBPβ in the GIT of SIV-infected macaques. More importantly, these data provide a molecular mechanism for persistent inflammation and immune activation leading to increased SIV burden and GIT pathology in SIV-infected macaques and perhaps HIV-infected individuals. The gastrointestinal tract (GIT) is a major target of infection with human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). Chronic GIT disease and inflammation are common sequelae to HIV/SIV infection. Nonetheless, the molecular mechanisms that cause and maintain GIT dysfunction remain unclear. We investigated the contribution of CCAAT/enhancer-binding protein β (C/EBPβ) to GIT disease and viral replication in jejunum and colon collected at necropsy from 12 SIV-infected (group 1), or 10 uninfected macaques with chronic diarrhea (group 2), and 9 uninfected control macaques (group 3). All group 1 and 2 macaques had chronic diarrhea, wasting, and colitis, but group 1 animals had more severe lesions in the jejunum. C/EBPβ gene expression increased significantly in colon of groups 1 and 2 and in jejunum of only group 1 macaques compared with controls. In group 1 animals, CEBPβ expression was localized predominantly to macrophages and occasionally lymphocytes. Chromatin immunoprecipitation assays confirmed the binding of C/EBPβ and p65 to the SIV long terminal repeat region in colonic lamina propria cells, suggesting a mechanistic link between inflammation and activation of viral replication in vivo. This is the first in vivo study describing the transcriptional changes and immunophenotypic localization of C/EBPβ in the GIT of SIV-infected macaques. More importantly, these data provide a molecular mechanism for persistent inflammation and immune activation leading to increased SIV burden and GIT pathology in SIV-infected macaques and perhaps HIV-infected individuals. Infection with human immunodeficiency virus (HIV), the causative agent of acquired immunodeficiency syndrome (AIDS) in humans, leads to progressive immune deficiency because of massive loss of CD4+ T cells. This observation has been confirmed and extended using the macaque model of AIDS in which rhesus macaques are infected with simian immunodeficiency virus (SIV), a close relative of HIV.1Lackner AA Veazey RS Current concepts in AIDS pathogenesis: insights from the SIV/macaque model.Annu Rev Med. 2007; 58: 461-476Crossref PubMed Scopus (107) Google Scholar Based on these studies, which have been confirmed in HIV-infected humans, it is clear that the most significant and rapid depletion of CD4+ T cells occurs in the gastrointestinal tract (GIT) during the first 14 to 21 days after infection.2Veazey RS DeMaria M Chalifoux LV Shvetz DE Pauley DR Knight HL Rosenzweig M Johnson RP Desrosiers RC Lackner AA Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection.Science. 1998; 280: 427-431Crossref PubMed Scopus (1209) Google Scholar, 3Mehandru S Poles MA Tenner-Racz K Horowitz A Hurley A Hogan C Boden D Racz P Markowitz M Primary HIV-1 infection is associated with preferential depletion of CD4+ T lymphocytes from effector sites in the gastrointestinal tract.J Exp Med. 2004; 200: 761-770Crossref PubMed Scopus (912) Google Scholar, 4Brenchley JM Schacker TW Ruff LE Price DA Taylor JH Beilman GJ Nguyen PL Khoruts A Larson M Haase AT Douek DC CD4+ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract.J Exp Med. 2004; 200: 749-759Crossref PubMed Scopus (1435) Google Scholar Associated with the loss of GIT CD4+ T cells are a variety of symptoms of GIT dysfunction that affect most HIV-infected patients. 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In our earlier study, we observed that all SIV-infected macaques with high IL-6 and constitutive STAT3 expression had high mucosal viral loads.22Mohan M Aye PP Borda JT Alvarez X Lackner AA Gastrointestinal disease in SIV-infected rhesus macaques is characterized by proinflammatory dysregulation of the IL-6-JAK-STAT3 pathway.Am J Pathol. 2007; 171: 1952-1965Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Although constitutive STAT3 expression in the GIT was associated with inflammation,22Mohan M Aye PP Borda JT Alvarez X Lackner AA Gastrointestinal disease in SIV-infected rhesus macaques is characterized by proinflammatory dysregulation of the IL-6-JAK-STAT3 pathway.Am J Pathol. 2007; 171: 1952-1965Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar it does not have a binding site on the HIV/SIV LTR. Therefore, in the present study, using the SIV-rhesus macaque model of AIDS we attempted to identify mechanisms by which IL-6 promotes not only GIT inflammation/disease but also viral replication with a special focus on the role of C/EBPβ. The results of our study point to a significant increase in C/EBPβ mRNA and protein expression as a key event. C/EBPβ gene and protein expression was significantly increased in SIV-infected rhesus macaques with chronic diarrhea and was found to efficiently bind to the SIV LTR in infected cells isolated from the colonic lamina propria. We conclude that chronic uncontrolled intestinal inflammation, characterized by increased expression of C/EBPβ may lead to persistent localized immune activation, disruption of the intestinal architecture and function, and facilitate viral replication that ultimately leads to chronic diarrhea, wasting, and death. Tissues were collected from a total of 31 animals including 12 animals infected with pathogenic strains of SIV (group 1) that use CCR5 in vivo and 19 animals not infected with SIV. Of the uninfected animals, 10 had chronic diarrhea (group 2) and 9 did not (group 3). The animals in group 2 with chronic nonresponsive diarrhea of no known infectious etiology have been described and used as a model of inflammatory bowel disease.48Sestak K Merritt CK Borda J Saylor E Schwamberger SR Cogswell F Didier ES Didier PJ Plauche G Bohm RP Aye PP Alexa P Ward RL Lackner AA Infectious agent and immune response characteristics of chronic enterocolitis in captive rhesus macaques.Infect Immun. 2003; 71: 4079-4086Crossref PubMed Scopus (104) Google Scholar, 49Ramesh G Alvarez X Borda JT Aye PP Lackner AA Sestak K Visualizing cytokine-secreting cells in situ in the rhesus macaque model of chronic gut inflammation.Clin Diagn Lab Immunol. 2005; 12: 192-197PubMed Google Scholar It would be ideal to have a fourth group consisting of SIV-infected animals without diarrhea. Unfortunately, untreated SIV infection consistently leads to GIT dysfunction and diarrhea and hence it is not possible to include such a group. Jejunum and colon specimens were collected at necropsy from the 12 SIV-infected macaques with chronic diarrhea (group 1), 10 non-SIV-infected macaques with chronic diarrhea (group 2), and four uninfected control macaques (group 3) (Table 1). Chronic diarrhea and weight loss were major causes for euthanasia in all animals except those in group 3. In addition, pinch biopsies from jejunum and colon were collected from another five control macaques bringing the total number of control macaques (group 3) used in this study to nine. Colon specimens were collected for all 31 macaques. Jejunum specimens were available for 9 of 12 group 1, 6 of 10 group 2, and 9 of 9 group 3 control macaques. All animals in groups 1 and 2 were euthanized when they became unresponsive to treatment (eg, subcutaneous or intravenous fluids and antibiotics as appropriate based on a culture and sensitivity) or lost greater than 20% of their body weight. After euthanasia with an intravenous overdose of pentobarbital all animals received a complete necropsy and histopathological examination. All tissues were collected in RNAlater (Ambion, Austin, TX) for RNA quantification and confocal microscopy. According to the manufacturer, RNAlater protects both RNA and protein (by reversible inhibition of nucleases and proteases) in addition to preserving tissue architecture. Tissues were also collected in cryovials and snap-frozen by immersion in a 2-methylbutane/dry-ice mixture for protein extraction.Table 1Animals, Inoculum, Viral Load, CD4+ T-Cell Count in Group 1 MacaquesAnimal group and no.Duration of infection (days)InoculumCD4 count cells/μl*The lower end of the normal range for CD4+ T cells/μl of blood in the rhesus macaques is 800 cells/μl of blood.Plasma viral copies/ml × 106Viral copies/mg of total RNA × 106, colonViral copies/mg of total RNA × 106, jejunumSIV-infected with diarrhea (group 1) AJ82232SIVmac251NANA1.950.078 DD88388SIVmac239501.226.893.35 CI65377SIVmac2394501.54.592.5 L441170SIVmac251 and 2396321.2615.533.2 H405232SIVmac23952371.417,20015,480 V205973SIVmac2392960.38.03.45 AT81171SIVsmG9326400.060.50.057 T561460SIVmac251 and 23956360213,00016,380 DT56265SIVmac2399311.25.9NA DI2881SIVmac2519540.018411.27 DE6870SIVmac239275159NA EB17111SIVmac2519353.62.7NANA, not applicable.* The lower end of the normal range for CD4+ T cells/μl of blood in the rhesus macaques is 800 cells/μl of blood. Open table in a new tab NA, not applicable. GIT tissues were collected immediately after euthanasia and fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 6 μm, and stained with hematoxylin and eosin (H&E) for analysis. Sections of jejunum and colon were examined in a blinded manner and inflammation was scored semiquantitatively on a scale of 0 to 3 as follows: 0, within normal limits; 1, mild; 2, moderate; 3, severe. In addition, the presence of crypt dilatation, villous blunting, diverticulosis, and amyloidosis were recorded (Table 2).Table 2Intestinal Histopathology in Groups 1, 2, and 3 MacaquesIntestinal histopathology*Sections of jejunum and colon were examined in a blinded fashion and inflammation was scored semiquantitatively on a scale of 0 to 3 as follows: 0, within normal limits; 1, mild; 2, moderate; 3 severe. In addition the presence of crypt dilatation (CD), villous blunting (VB), diverticulosis (DV), and amyloidosis (AMD) were recorded.Animal group and no.ColonJejunumSIV-infected with diarrhea (group 1) AJ8223 DD881 and AMD1 and AMD CI651 and AMD1 and AMD L44111 H4053 and CD2 V20531 and AMD, VB AT8133 and VB AT563 and AMD, CD3 DT563 and CD0 DI2811 DE68†Macaques DE68 and EB17 were only used for the ChIP assay and their histopathology severity scores were not included for the statistical analysis.3 and CDNA EB17†Macaques DE68 and EB17 were only used for the ChIP assay and their histopathology severity scores were not included for the statistical analysis.1NANon-SIV-infected with diarrhea (group 2) EI903 and CDNA EL453 and CDNA EC493 and CDNA EB123 and CDNA EM4111 EL713 and CD, DV0 EB273 and CD, DV1 DJ153 and CD1 CT7721 EJ5431Uninfected controls (group 3) BV5200 EH7000 CB9800 CF3300 M30200 CC9600 R84200 EL6600 EH8000NA, not applicable.* Sections of jejunum and colon were examined in a blinded fashion and inflammation was scored semiquantitatively on a scale of 0 to 3 as follows: 0, within normal limits; 1, mild; 2, moderate; 3 severe. In addition the presence of crypt dilatation (CD), villous blunting (VB), diverticulosis (DV), and amyloidosis (AMD) were recorded.† Macaques DE68 and EB17 were only used for the ChIP assay and their histopathology severity scores were not included for the statistical analysis. Open table in a new tab NA, not applicable. Gene expression for C/EBPβ in the jejunum and colon tissues was evaluated by the Quantitative Real-Time SYBR Gr
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