Gastrointestinal Disease in Simian Immunodeficiency Virus-Infected Rhesus Macaques Is Characterized by Proinflammatory Dysregulation of the Interleukin-6-Janus Kinase/Signal Transducer and Activator of Transcription3 Pathway
2007; Elsevier BV; Volume: 171; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2007.070017
ISSN1525-2191
AutoresMahesh Mohan, Pyone P. Aye, Juan T. Borda, Xavier Álvarez, Andrew A. Lackner,
Tópico(s)Immunodeficiency and Autoimmune Disorders
ResumoGastrointestinal disease and inflammation are common sequelae of human and simian immunodeficiency virus (SIV) infection. Nevertheless, the molecular mechanisms that lead to gastrointestinal dysfunction remain unclear. We investigated regulation of the interleukin (IL)-6-JAK-STAT3 pathway in jejunum and colon, collected at necropsy, from 10 SIV-infected macaques with diarrhea (group 1), 10 non-SIV-infected macaques with diarrhea (group 2), and 7 control uninfected macaques (group 3). All group 1 and 2 macaques had chronic diarrhea, wasting, and colitis, but group 1 animals had more frequent and severe lesions in the jejunum. A significant increase in IL-6 and SOCS-3 gene expression along with constitutive STAT3 activation was observed in the colon of all group 1 and 2 macaques and in the jejunum of only group 1 macaques compared to controls. Further, in colon, histopathology severity scores correlated significantly with IL-6 (groups 1 and 2) and SOCS-3 (group 2) gene expression. In jejunum, a similar correlation was observed only in group 1 animals. Phosphorylated STAT3 (p-STAT3) was localized to lymphocytes (CD3+) and macrophages (CD68+), with fewer CD3+ lymphocytes expressing p-STAT3 in group 1 macaques. Despite high SOCS-3 expression, STAT3 remained constitutively active, providing a possible explanation for persistent intestinal inflammation and immune activation that may favor viral replication and disease pro-gression. Gastrointestinal disease and inflammation are common sequelae of human and simian immunodeficiency virus (SIV) infection. Nevertheless, the molecular mechanisms that lead to gastrointestinal dysfunction remain unclear. We investigated regulation of the interleukin (IL)-6-JAK-STAT3 pathway in jejunum and colon, collected at necropsy, from 10 SIV-infected macaques with diarrhea (group 1), 10 non-SIV-infected macaques with diarrhea (group 2), and 7 control uninfected macaques (group 3). All group 1 and 2 macaques had chronic diarrhea, wasting, and colitis, but group 1 animals had more frequent and severe lesions in the jejunum. A significant increase in IL-6 and SOCS-3 gene expression along with constitutive STAT3 activation was observed in the colon of all group 1 and 2 macaques and in the jejunum of only group 1 macaques compared to controls. Further, in colon, histopathology severity scores correlated significantly with IL-6 (groups 1 and 2) and SOCS-3 (group 2) gene expression. In jejunum, a similar correlation was observed only in group 1 animals. Phosphorylated STAT3 (p-STAT3) was localized to lymphocytes (CD3+) and macrophages (CD68+), with fewer CD3+ lymphocytes expressing p-STAT3 in group 1 macaques. Despite high SOCS-3 expression, STAT3 remained constitutively active, providing a possible explanation for persistent intestinal inflammation and immune activation that may favor viral replication and disease pro-gression. Since its initial description in 1981, human immunodeficiency virus (HIV), the causative agent of acquired immune deficiency syndrome (AIDS), has been known to cause a wide variety of illnesses by specifically targeting CD4+ T cells. 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In the present study, using SIV-infected rhesus macaques with chronic diarrhea, we have observed constitutive activation of p-STAT3 and dysregulation of the IL-6-STAT3 signal transduction pathway in the GI tract providing a link between SIV infection and GI inflammation that could also play a role in enhancing viral replication. Tissues were collected from a total of 27 animals including 10 animals infected with pathogenic strains of SIV (group 1) that use CCR5 in vivo and 17 animals not infected with SIV. Of the uninfected animals, 10 had chronic diarrhea (group 2) and 7 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.43Sestak 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 (102) Google Scholar, 44Ramesh 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 GI dysfunction and diarrhea, and hence it is not possible to include such a group. Jejunum and colon specimens were collected at necropsy from the 10 SIV-infected macaques with chronic diarrhea (group 1), 10 non-SIV-infected macaques with chronic diarrhea (group 2), and 2 uninfected control macaques (group 3) (Table 1, Table 2). In addition, pinch biopsies from jejunum and colon were collected from another five control macaques bringing the total of control macaques (group 3) used in this study to seven. Colon specimens were collected for all 27 macaques. Jejunum specimens were available for 9 of 10 group 1, 6 of 10 group 2, and 7 of 7 group 3 control macaques. All animals in groups 1 and 2 were euthanized when they became unresponsive to treatment (subcutaneous or intravenous fluids and antibiotics as appropriate based on 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 AT561460SIVmac251 and 23956360213,00016,380 DT56265SIVmac2399311.25.9NA DI2881SIVmac2519540.018411.27NA, 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 Table 2Intestinal Histopathology in Group 1, 2, and 3 MacaquesIntestinal histopathology*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 (CD), villous blunting (VB), diverticulosis (DV), and amyloidosis (AMD) were recordedAnimal 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 DI2811Non-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 R84200NA, not applicable.* 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 (CD), villous blunting (VB), diverticulosis (DV), and amyloidosis (AMD) were recorded Open table in a new tab NA, not applicable. NA, not applicable. GI 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). Gene expression for IL-6 and SOCS-3 in the jejunum and colon was evaluated by quantitative real-time SYBR green one-step RT-PCR assay (qRT-PCR) (Qiagen Inc., Valencia, CA). Total RNA was extracted from both jejunum and colon samples using the SV total RNA isolation kit (Promega Corporation, Madison, WI), and an RNA sample representing the colon and jejunum from each macaque was assayed in triplicate wells. Each qRT-PCR reaction (25 μl) contained the following: 2× Master mix without uracil-N-glycosylase (12.5 μl), reverse transcriptase (0.25 μl), target forward and reverse primer, and total RNA (200 ng) quantified spectrophotometrically based on A260:A280 ratios. Forward and reverse primer sequence, concentration, and product size for both targets including β-actin are shown in Table 3. The PCR amplification was performed in the ABI Prism 7700 sequence detection system (PE Applied Biosystems, Foster City, CA). Thermal cycling conditions were 50°C for 30 minutes, 95°C for 15 minutes, followed by 40 repetitive cycles of 95°C for 15 seconds, 54°C for 30 seconds, 72°C for 30 seconds. As a normalization control for RNA loading, parallel reactions in the same multiwell plate were performed using β-actin mRNA.Table 3Primer Sequences Used for Real Time SYBR Green One-Step RT-PCRName of genePrimer sequenceProduct size (bp)Primer concentrationIL-6Forward: 5′-CCAGTACTCCCAGGAGAAGATTCCAA-3′103500 nmol/LReverse: 5′-CGTCGAGGATGTACCGAATGTGTT-3′SOCS-3Forward: 5′-TCTTCAGCATCTCTGTCGGAAGACC-3′106500 nmol/LReverse: 5′-GGCATCGTACTGGTCCAGGAACT-3′β-ActinForward: 5′-AGGCTCTCTTCCAACCTTCCTT-3′108300 nmol/LReverse: 5′-CGTACAGGTCTTTACGGATGTCCA-3′ Open table in a new tab Quantification of gene amplification after RT-PCR was made by determining the threshold cycle (CT) number for SYBR Green fluorescence within the geometric region of the semilog plot generated during PCR. Within this region of the amplification curve, each difference of one cycle is equivalent to a doubling of the amplified product of the PCR. The relative quantification of target gene expression across treatments was evaluated using the comparative CT method. The ΔCT value was determined by subtracting the β-actin CT value for each sample from the target CT value of that sample. Calculation of ΔΔCT involved using the highest sample ΔCT value (ie, sample with the lowest target expression) as an arbitrary constant to subtract from all other ΔCT sample values. Fold changes in the relative gene expression of target was determined by evaluating the expression, 2−ΔΔ CT. Small 2.5-cm2 pieces of colon and jejunum were crushed using a disposable polypropylen
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