Temporal and Anatomic Relationship between Virus Replication and Cytokine Gene Expression after Vaginal Simian Immunodeficiency Virus Infection

(1)JOURNAL OF VIROLOGY, Oct. 2005, p. 12164–12172 0022-538X/05/$08.00⫹0 doi:10.1128/JVI.79.19.12164–12172.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved. Vol. 79, No. 19 Temporal and Anatomic Relationship between Virus Replication and Cytokine Gene Expression after Vaginal Simian Immunodeficiency Virus Infection Kristina Abel,1,2,3* David M. Rocke,2,4 Barinderpal Chohan,1,2 Linda Fritts,1,2 and Christopher J. Miller1,2,3,5 Received 16 May 2005/Accepted 5 July 2005 The current knowledge about early innate immune responses at mucosal sites of human immunodeficiency virus (HIV) entry is limited but likely to be important in the design of effective HIV vaccines against heterosexual transmission. This study examined the temporal and anatomic relationship between virus replication, lymphocyte depletion, and cytokine gene expression levels in mucosal and lymphoid tissues in a vaginal-transmission model of HIV in rhesus macaques. The results of the study show that the kinetics of cytokine gene expression levels in the acute phase of infection are positively correlated with virus replication in a tissue. Thus, cytokine responses after vaginal simian immunodeficiency virus (SIV) inoculation are earliest and strongest in mucosal tissues of the genital tract and lowest in systemic lymphoid tissues. Importantly, the early cytokine response was dominated by the induction of proinflammatory cytokines, while the induction of cytokines with antiviral activity, alpha/beta interferon, occurred too late to prevent virus replication and dissemination. Thus, the early cytokine response favors immune activation, resulting in the recruitment of potential target cells for SIV. Further, unique cytokine gene expression patterns were observed in distinct anatomic locations with a rapid and persistent inflammatory response in the gut that is consistent with the gut being the major site of early CD4 T-cell depletion in SIV infection. Women represent the majority of new human immunodeficiency virus (HIV) type 1-infected patients worldwide. Thus, an understanding of the early virus-host interactions after vaginal HIV infection is important for the design of an effective HIV vaccine and/or microbicides aimed at preventing heterosexual HIV transmission. Much of the effort in understanding anti-HIV immunity has focused on the understanding and modulation of adaptive antiviral T- and B-cell responses. Knowledge about innate immune responses to HIV at mucosal sites of virus entry is limited. However, these early innate responses are likely to be critical in limiting local virus replication and in preventing virus dissemination after vaginal HIV/simian immunodeficiency virus (SIV) transmission, as adaptive antiviral immune responses are not detectable until after systemic infection has been established (36). Innate antiviral responses may provide the host with the necessary time to activate adaptive antiviral responses important in effective control of virus replication. Cytokines play an important role in the early stages of infection, as they can have direct antiviral activity, act as danger signals that activate and recruit various immune effector cells, and direct the nature and strength of adaptive antiviral immune responses. At the same time, cytokines are key players in inflammation. Thus, the timing and duration of cytokine expression might be a critical factor determining the outcome of HIV * Corresponding author: Mailing address: UC Davis, CCM/CNPRC, County Rd. 98/Hutchison Dr., Davis, CA 95616. Phone: (530) 7545673. Fax: (530) 754-4411. E-mail: kabel@ucdavis.edu. infection, and cytokine dysregulation could potentially contribute to the immunopathology of AIDS. Alpha and beta interferon (IFN-␣/␤) are rapidly induced in viral infections. Their essential in vivo role in the control of virus infections was confirmed in IFN-␣/␤ receptor knockout mice (6, 33, 45, 46). Further, IFN-␣/␤ have evolved as key players in the bridging of innate and adaptive immune responses (5, 7, 10, 15, 30, 38). Although the role of type I interferon responses has been clearly established in mouse models of viral infections, it has not been thoroughly examined in mucosal anti-HIV immunity. Similarly, there are no studies examining the local inflammatory response after mucosal SIV transmission. However, in the case of HIV/SIV infection, a robust inflammatory response, necessary to recruit immune effector cells to the site of virus replication, may be detrimental to the host. In HIV type 1-infected patients, HIV-infected chimpanzees, and SIV-infected rhesus macaques, disease progression is associated with increased T-cell activation (9, 18, 23, 26, 28, 34, 39, 41, 42), while in African green monkeys and sooty mangabeys, the natural hosts of SIV, the virus replicates at high levels without causing disease and excessive immune activation (11, 16, 24, 35, 41). Thus, knowledge and understanding of the local cytokine milieu induced early after vaginal transmission may provide significant insight into new therapeutic interventions for virus transmission. We have recently determined the pattern of virus dissemination in the acute phase after vaginal inoculation of rhesus macaques with pathogenic SIV (32). In the first 5 days after vaginal SIV inoculation, virus replication is confined to genital 12164 Downloaded from http://jvi.asm.org/ on November 8, 2019 by guest Center for Comparative Medicine,1 California National Primate Research Center,2 Department of Internal Medicine, Division of Infectious Diseases, School of Medicine,3 Division of Biostatistics,4 and Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine,5 University of California at Davis, Davis, California

(2) VOL. 79, 2005 TISSUE CYTOKINE AND vRNA LEVELS IN ACUTE SIV INFECTION MATERIALS AND METHODS Animals and SIV infection. Adult female rhesus macaques were inoculated intravaginally with pathogenic SIVmac239 or SIVmac251 and necropsied in the acute phase of infection as described previously (32). Six animals were necropsied between days 3 and 5 p.i., six animals were necropsied between days 6 and 10 p.i., and three animals each were necropsied on days 14, 21, and 28 p.i. Genital tract tissues (vaginal mucosa, cervix, and genital lymph nodes), gut tissues (colon, jejunum, and mesenteric lymph nodes), and distal lymphoid tissues (spleen and axillary lymph nodes) were collected and analyzed for viral RNA (vRNA) and for cytokine mRNA expression levels. Virological analysis. Plasma and tissue vRNA levels were determined using a branched DNA (bDNA) assay as described previously (2, 14, 32). The bDNA assay, developed by Bayer Diagnostics, was originally designed to quantify vRNA levels in the plasma of SIV-infected animals. The detection limit of the assay is 125 vRNA copies per ml of plasma. There are no data regarding the detection limit of the assay for vRNA in total RNA isolated from tissues of SIV-infected animals. Samples from 13 different tissues of the genital tract, the gut, and lymphoid tissues were collected from three animals that had not been exposed to SIV to determine the specificity of the tissue assay (32). To be able to compare vRNA levels across various tissue samples, vRNA copies are reported per microgram of total tissue RNA. Excluding one spurious result, average values for the bDNA assay were 113 copies/␮g tissue RNA in the uninfected animals. Thus, the value of 113 copies would have been considered negative in the standard vRNA assay for plasma. To avoid any false-positive results, we set the cutoff for the assay at 200 copies/␮g tissue RNA, the average copy number per uninfected tissue plus 2 standard deviations (SD). Copy numbers of less than 200 copies in animals exposed to live virus were reported as negative. Average vRNA data for each tissue of all animals collected on days 3 to 5 p.i., days 6 to 10 p.i., and day 14, day 21, and day 28 p.i. are reported. RNA isolation and cDNA preparation. Prior to RNA isolation, tissue samples were homogenized using a power homogenizer (PowerGen 700; 7 mm by 195 mm; Fisher Scientific, Santa Clara, CA). In addition, tissues of the vaginal mucosa and cervix were treated with proteinase K (QIAGEN, Valencia, CA) at 200 ␮g/ml Trizol for 10 min at 55°C. Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions from tissue samples stored in RNAlater (Ambion, Austin, TX). RNA samples were DNase treated with DNA-free (Ambion) for 1 h at 37°C. cDNA was prepared using random hexamer primers (Amersham-Pharmacia Biotech, Inc., Piscataway, NJ) and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Relative quantification of cytokine mRNA expression levels. Cytokine mRNA levels were determined by real-time PCR as described previously (1, 2). Briefly, samples were tested in duplicate, and the PCR for the GAPDH (glyceraldehyde3-phosphate dehydrogenase) housekeeping gene and the target gene from each sample were run in parallel on the same plate. The reaction was carried out on a 96-well optical plate (Applied Biosystems, Foster City, CA) in a 25-␮l reaction volume containing 5 ␮l cDNA plus 20 ␮l Mastermix (Applied Biosystems). All sequences were amplified using the 7900 default amplification program: 2 min at 50°C, 10 min at 95°C, followed by 40 to 45 cycles of 15 s at 95°C and 1 min at 60°C. The results were analyzed with the SDS 7900 system software, version 2.1 (Applied Biosystems). Cytokine mRNA expression levels were calculated from normalized ⌬CT values. CT values correspond to the cycle number at which the fluorescence due to enrichment of the PCR product reaches significant levels above the background fluorescence (threshold). In this analysis, the CT value for the housekeeping gene (GAPDH) is subtracted from the CT value of the target (cytokine) gene (⌬CT). In general, the ⌬CT value for the SIV-infected tissue sample is then subtracted from the mean ⌬CT value of the corresponding control samples (⌬⌬CT). Assuming that the target gene (cytokine) and the reference gene (GAPDH) are amplified with the same efficiency (data not shown), the increase in cytokine mRNA levels in tissue samples of SIV-infected monkeys compared to tissue samples of uninfected animals is then calculated as follows: increase ⫽ 2⫺⌬⌬CT (user bulletin no. 2, ABI Prism 7700 Sequence Detection System; Applied Biosystems). In the present study, a minimum of six normal tissues were analyzed to determine baseline mRNA levels for each cytokine in each tissue of uninfected animals. Cytokine mRNA levels (⌬CT) in each tissue of each individual uninfected monkey were determined, and the mean value for each cytokine mRNA level of all animals in each tissue (mean ⌬CT) was calculated. The ⌬CT value for each cytokine of each individual tissue sample was then subtracted from the mean ⌬CT value [⌬⌬CT (individual control monkey) ⫽ ⌬CT (individual monkey) ⫺ mean⌬CT (control monkeys)]. Next, the increase in mRNA levels for each individual sample compared to the mean mRNA level of all uninfected animals was determined to control for variation in cytokine mRNA levels among individual control animals: increase ⫽ 2⫺⌬⌬CT. The increases in all the samples from the uninfected monkeys were averaged and are reported as average increase in cytokine mRNA levels ⫾ SD of the uninfected control animal population to show the variation of cytokine mRNA levels in uninfected control animals, likely a result of the outbred nature of the animals. Similarly, the increases in cytokine mRNA levels in distinct issues of individual SIV-infected monkeys are calculated by subtracting the ⌬CT value for each cytokine of each individual tissue sample from the mean ⌬CT value for the same cytokine in the same tissue of the uninfected control animals [⌬⌬CT (individual SIV monkey) ⫽ ⌬CT ⫺ mean ⌬CT (control monkeys)]. The increase in cytokine mRNA levels of a tissue sample from a SIV-infected animal was then calculated as follows: increase ⫽ 2⫺⌬⌬CT共SIV兲. This reflects changes in cytokine mRNA levels of SIV-infected monkeys compared to normal, SIV-naı¨ve animals as a result of SIV infection. Changes in relative tissue cytokine mRNA levels at distinct time points after SIV infection (days 3 to 5 p.i., days 6 to 10 p.i., and day 14, day 21, and day 28 p.i.) are reported as average increase in cytokine mRNA levels ⫾ SD of all SIVinfected animals. Statistical analysis. The main analysis of cytokine levels over time since infection and in different tissues (anatomic location) was done using two-way analysis of variance on log mRNA levels. To determine whether specific pairs of tissues differed in cytokine mRNA expression levels, we used Tukey’s studentized range test, as implemented in the Tukey HSD function in the R statistical package, which produces confidence intervals for all pairs of levels at the chosen family-wise confidence level, in this case 95% (see Fig. 1, 3, and 4). A similar analysis was done to distinguish pairs of time points. The influence of vRNA levels on cytokine expression was examined using the analysis of covariance with log mRNA cytokine expression as the response and tissue, time, and log vRNA concentration as predictors. Note that the comparative analysis of cytokine mRNA levels between various tissues considered changes in cytokine mRNA levels observed in each tissue throughout the whole first 4 weeks postinfection. Downloaded from http://jvi.asm.org/ on November 8, 2019 by guest tract tissues (32). While SIV can disseminate to lymphoid tissues in the first 5 days, SIV replication in distal lymphoid tissues is not detected until day 6 postinoculation (p.i.) (32). Despite a similar delay in replication in the colon, profound CD4⫹ T-cell depletion occurs by day 10 (29). The signal responsible for the onset of SIV replication in distal lymphoid tissues and the mechanism(s) responsible for rapid CD4⫹ Tcell depletion in the gut are not known. The goal of the current study was to determine the temporal and anatomic relationship between virus replication, lymphocyte depletion, and cytokine gene expression levels in mucosal and lymphoid tissues of the same animals. To this end, gene expression levels of cytokines with predominantly antiviral activity (type I interferons) and cytokines important in the early inflammatory response for the activation and recruitment of multiple innate and adaptive effector cells to the site of virus entry (IFN-␥, tumor necrosis factor alpha [TNF-␣], macrophage inflammatory protein 1␣ [MIP-1␣], interleukin 6 [IL-6], IL-12, and IL-8) were determined in tissues close to the portal of entry (cervicovaginal mucosa), in gut tissues, and in distal lymphoid tissues of the same animals. Our results show that increases in cytokine mRNA expression in mucosal and systemic lymphoid tissues occur at the same time that SIV replication becomes detectable in the same tissues. Thus, cytokine responses are earliest and strongest in mucosal tissues at the portal of entry and in the draining genital lymph nodes and lowest in systemic lymphoid tissues. In all tissues, the early cytokine response is dominated by the induction of proinflammatory cytokines, while the induction of antiviral type I interferons occurs too late to prevent virus replication and dissemination. Further, there are distinct gene expression patterns in different anatomic locations, with a rapid and pronounced inflammatory response that may contribute to the rapid loss of CD4⫹ T cells from the colon (29). 12165

(3) 12166 ABEL ET AL. J. VIROL. Correlations between levels of virus replication and cytokine gene expression levels in the same tissue (see Fig. 2,) were determined using the GraphPad Prism software (GraphPad Software, Inc., San Diego, CA), version 4.0, by graphing tissue vRNA levels of all time points versus cytokine mRNA levels in the same tissue at the same time points. Correlations were considered significant if the P value was ⬍0.05. RESULTS Temporal relationship between virus replication and tissue cytokine mRNA levels. We have previously shown that, while the infection rapidly disseminates, virus replication in the first 5 days after vaginal inoculation is generally confined to the portal of entry (32). However, by day 6 p.i., virus replication simultaneously begins in all distal mucosal and systemic lymphoid tissues (Fig. 1A) (32). Thus, to determine the relationship between cytokine gene expression patterns in mucosal and lymphoid tissues and virus dissemination and replication, cytokine gene expression levels were measured in tissues collected between days 3 and 5 p.i. (when virus replication was Downloaded from http://jvi.asm.org/ on November 8, 2019 by guest FIG. 1. Viral RNA levels and IFN-␣/␤ responses in tissues after vaginal SIV infection. (A) Average vRNA levels at various time points after vaginal SIV inoculation for each tissue are shown. Viral RNA levels are reported as log10 vRNA copies per ␮g tissue RNA. The different tissues are grouped by anatomic location, with tissues of the genital tract shown in red (vaginal mucosa, solid bars; cervix, striped bars; genital lymph nodes [LN], open bars), tissues of the gut in black (colon, solid bars; jejunum, striped bars; mesenteric LN, open bars), and distal lymphoid tissues (spleen, solid bars; axillary LN, open bars) in blue. The numbers underneath each bar on the x axis indicate the days after SIV inoculation when tissues were collected. (B and C) Increases in IFN-␤ and IFN-␣ mRNA levels compared to IFN-␤ and IFN-␣ mRNA levels in the same tissues of SIV-naı¨ve animals. The lines above the bars show statistically significant differences in cytokine mRNA levels between tissues connected by the line, with the tissue with statistically higher cytokine mRNA levels compared to the other tissues indicated by an asterisk. The error bars represent SD.

(4) VOL. 79, 2005 TISSUE CYTOKINE AND vRNA LEVELS IN ACUTE SIV INFECTION 12167 Downloaded from http://jvi.asm.org/ on November 8, 2019 by guest FIG. 2. Correlation between tissue vRNA levels and IFN-␣ mRNA levels. Each individual graph shows the log10 increase of IFN-␣ mRNA levels (y axis) over log10 vRNA copies/␮g tissue RNA (x axis) in the same tissue. Data are shown for all animals collected for each tissue throughout the first 28 days p.i. Each symbol represents an individual animal. If significant correlations were determined (P ⬍ 0.05; NS ⫽ nonsignificant), the correlation efficient (r2 value) is also shown. limited to the site of virus inoculation), between days 6 and 10 p.i. (when peak vRNA levels were reached), and on days 14, 21, and 28 (when virus levels started to decline and reached set point). Overall, there was a statistically significant positive correlation between vRNA levels and increased cytokine mRNA expression levels (P ⬍ 0.01) in a tissue. Thus, coincident with peak vRNA levels, cytokine mRNA levels were reached between days 6 and 10 p.i. in all tissues (see Fig. 1, 3, and 4), while from day 14 to 28, vRNA levels and cytokine mRNA levels declined in all tissues (see Fig. 1, 3, and 4). Thus, there was a consistent temporal relationship between SIV replication and the level of cytokine induction in a tissue. While the kinetics of cytokine induction were similar in all tissues, the relative

(5) 12168 ABEL ET AL. J. VIROL. strength of the cytokine expression differed significantly among tissues (P ⬍ 0.01) (see Fig. 1, 3, and 4), and distinct cytokine gene expression patterns were observed for different tissues (see Fig. 1, 3, and 4). Cytokine mRNA levels were generally highest in mucosal tissues of the genital tract and lowest in systemic lymphoid tissues. The latter might be related to the fact that when the tissues were collected, the period of SIV replication in each of the various tissues was different, with the longest period of SIV replication having occurred in tissues of the genital tract and the shortest time of SIV replication in distal lymphoid tissues. Type I interferon induction in mucosal and lymphoid tissues occurs after SIV infection is established. Vaginal SIV infection did not result in a rapid induction of type I interferon mRNA in cervicovaginal mucosa. IFN-␤ mRNA levels were only minimally (two- to fivefold) increased in the first 3 to 5 days p.i. (Fig. 1B). Coinciding with peak vRNA levels, IFN-␤ mRNA levels in the vaginal mucosa and cervix peaked at days 6 to 10 p.i. Increases in spleen and lymph node IFN-␤ mRNA levels were also noted during this period but were transient, and by day 28, IFN-␤ mRNA levels were indistinguishable from IFN-␤ mRNA levels in the same tissues of uninfected control animals (Fig. 1B). The apparent sustained increase in IFN-␤ mRNA levels in the vaginal mucosa on days 13 and 21 p.i. was due to increased IFN-␤ mRNA levels in one out of three animals. Increased IFN-␣ mRNA levels were not detectable in any tissue before days 6 to 10 p.i. (Fig. 1C). IFN-␣ mRNA levels in Downloaded from http://jvi.asm.org/ on November 8, 2019 by guest FIG. 3. Comparative analysis of proinflammatory cytokine gene expression levels in the genital tract, gut tissues, and distal lymphoid tissues. (A) Increases in TNF-␣ mRNA levels compared to cytokine mRNA levels in the same tissues of SIV-naı¨ve animals. (B) Increase in IL-6 mRNA levels compared to cytokine mRNA levels in the same tissues of SIV-naı¨ve animals. (C) Increase in MIP-1␣ mRNA levels compared to cytokine mRNA levels in the same tissues of SIV-naı¨ve animals. See the legend to Fig. 1 for details.

(6) VOL. 79, 2005 TISSUE CYTOKINE AND vRNA LEVELS IN ACUTE SIV INFECTION 12169 the genital tract increased several hundred- to 1,000-fold (Fig. 1C). There was also a marked increase in IFN-␣ mRNA levels in all the lymph nodes by day 6 p.i., coincident with the onset of SIV replication in systemic lymphoid tissues. However, spleen IFN-␣ mRNA levels were only minimally (1- to 11-fold) and transiently increased between days 6 and 10 p.i. (Fig. 1C). In all the lymph nodes, increased IFN-␣ mRNA levels were still detectable at day 28 p.i., albeit at much lower levels, while IFN-␣ expression was no longer detectable in mucosal tissues (Fig. 1C). This was consistent with slightly higher vRNA levels in lymphoid than in mucosal tissues at day 28 p.i. (Fig. 1A). It should be noted that type I interferon induction in the gut-associated tissues was less pronounced than in lymph nodes. In the jejunum, IFN-␤ mRNA levels did not change at any time after virus infection, while IFN-␣ mRNA levels increased only slightly (⬍5-fold) at days 6 to 10 p.i. (Fig. 1B and C). A marked increase (840-fold) in colon IFN-␣ mRNA levels was seen in a single animal, while colon IFN-␣ mRNA levels in the remaining four animals were only marginally increased (1- Downloaded from http://jvi.asm.org/ on November 8, 2019 by guest FIG. 4. Tissue mRNA levels for IFN-␥, IL-8, and IL-12 after vaginal SIV inoculation. (A) Increase in IFN-␥ mRNA levels compared to cytokine mRNA levels in the same tissues of SIV-naı¨ve animals. (B) Increase in IL-8 mRNA levels compared to cytokine mRNA levels in the same tissues of SIV-naı¨ve animals. (C) Increase in IL-12 mRNA levels compared to cytokine mRNA levels in the same tissues of SIV-naı¨ve animals. See the legend to Fig. 1 for details.

(7) 12170 ABEL ET AL. infection was observed in the colon, and elevated IL-8 mRNA levels also persisted throughout the acute infection (Fig. 4B). It was noteworthy, though, that the inflammatory response observed in the colon was not detectable in the jejunum (Fig. 4A and B) or ileum (data not shown). The finding that IFN-␥ and IL-8 mRNA levels were most rapidly and persistently induced in the colon may be critical for AIDS pathogenesis in light of studies demonstrating that the gut is the major site of CD4 depletion and of general immune activation after SIV infection (29, 31, 44). Importantly, there was no correlation between vRNA levels and IFN-␥ or IL-8 mRNA levels in the colon (data not shown). Thus, while initially and rapidly induced by SIV, the inflammatory response initiated in the colon was sustained as the level of virus replication declined. In contrast, mRNA levels for IL-12, a cytokine important in the induction of T helper responses, were lowest in the colon and jejunum and higher in the lymph nodes (Fig. 4C). In fact, IL-12 mRNA levels were significantly higher in mesenteric and axillary lymph nodes than in the colon and jejunum. Of note, IL-12 was the only cytokine that was more rapidly induced in systemic lymphoid tissues than in mucosal tissues, and elevated IL-12 mRNA levels persisted in lymph nodes throughout the first 28 days p.i. (Fig. 4C). In mucosal tissues, there was also a trend toward increasing IL-12 mRNA levels over time (Fig. 4C), but there was no statistically significant difference in the kinetics of IL-12 mRNA induction in mucosal and systemic lymphoid tissues. DISCUSSION Antiviral immune responses need to be elicited rapidly in local tissues at the site of virus entry in order to reduce virus transmission and to prevent virus dissemination. After intravaginal SIV inoculation, the early cytokine response in the genital tract is dominated by the induction of proinflammatory cytokines, while cytokines with antiviral activity, the type I interferons, are barely detectable in the first few days postinfection. This result is in sharp contrast to many animal models of viral infections (3, 12, 20, 22, 37) and acute human viral diseases (27), in which IFN-␣ is induced within hours of virus exposure. However, by days 6 to 10 p.i., IFN-␣ mRNA levels increase in the genital tract as SIV replication levels become very high. Thus, the lack of a more immediate response to SIV transmission is not due to an inherent inability of the genital tract to produce IFN-␣ but may rather reflect the scattered and multifocal nature of infection in the first few days after intravaginal SIV inoculation (32). As the infection spreads and coalesces within the genital tract, strong type I interferon responses become detectable. In intravenously inoculated monkeys, systemic SIV replication begins by day 3 p.i. (30), and IFN-␣ mRNA levels are also increased by day 3 in lymphoid tissues of these same monkeys (authors’ unpublished observation). Thus, IFN-␣ production is tightly linked to the onset of SIV replication, and the delay in the IFN-␣ response in intravaginally versus intravenously inoculated monkeys is consistent with a delay of virus replication (19). This conclusion is also consistent with data demonstrating that increased IFN-␣ levels are rapidly detectable in the plasma and lymph nodes of monkeys infected intravenously with a high-replicating pathogenic Downloaded from http://jvi.asm.org/ on November 8, 2019 by guest to 26-fold) compared to the cervicovaginal mucosa and lymph nodes (Fig. 1A). In fact, while IFN-␣ mRNA levels in the vaginal mucosa and in the lymph nodes positively correlated with vRNA levels, there was no correlation between virus replication and IFN-␣ mRNA levels in the colon (Fig. 2). Thus, the delayed induction of type I interferons at the portal of entry (vaginal mucosa) and the relatively low induction of type I interferons in the colon, a major site of virus replication, indicate that there was little recognition of the virus by antiviral host immune defense mechanisms in these tissues in the first few days after SIV infection. This may explain why virus can rapidly disseminate undetected from the genital tract to systemic lymphoid tissues. Proinflammatory cytokine genes are preferentially expressed in mucosal compared to systemic lymphoid tissues. Several proinflammatory cytokines (TNF-␣, MIP-1␣, and IL-6) were consistently induced in the vaginal mucosa at days 3 to 5 p.i. (1- to 14-fold, 3- to 38-fold, and 1- to 9-fold, respectively) (Fig. 3A to C). This finding stands in contrast to the limited induction of type I interferons from day 3 to 5 p.i. in the vaginal mucosa. Peak mRNA levels for these cytokines in the genital tract were reached by days 6 to 10 p.i., coinciding with peak vRNA levels (Fig. 3). Importantly, despite similar vRNA levels in mucosal and lymphoid tissues by days 6 to 10 p.i., proinflammatory cytokine mRNA levels were significantly higher in the tissues closest to the portal of virus entry (Fig. 3). Thus, TNF-␣ mRNA levels were slightly increased in the colon at day 6 to 10 p.i., but not in the jejunum or any of the systemic lymphoid tissues (Fig. 3A). Similarly, IL-6 mRNA levels in systemic lymphoid tissues were only minimally (average increase, ⬍5-fold) increased by days 6 to 10 p.i. compared to IL-6 mRNA levels in the same tissues of uninfected control animals (Fig. 3B). In fact, among TNF-␣, IL-6, and MIP-1␣, only MIP-1␣ mRNA levels were consistently increased in all lymphoid tissues after SIV inoculation. Increased MIP-1␣ mRNA levels became detectable in all lymphoid tissues between days 6 and 10 p.i., at the same time viral replication became detectable in these tissues (32), and increased MIP-1␣ mRNA levels persisted at low levels in some of the animals (Fig. 3C). Of the proinflammatory cytokines assessed, IFN-␥ mRNA levels were the most markedly increased after SIV inoculation. It is noteworthy that the greatest increase in IFN-␥ mRNA levels in the first 3 to 5 days p.i. occurred in the colon (average increase, 74-fold; range, 3- to 215-fold). Unlike the other proinflammatory cytokines, elevated colon IFN-␥ mRNA levels persisted throughout the first 28 days of SIV infection (Fig. 4A). During days 3 to 5 p.i., the increase in IFN-␥ mRNA levels in the vaginal mucosa and cervix was only minimal (average IFN-␥ mRNA levels, 3.88 and 8.26, respectively). However, IFN-␥ mRNA levels increased up to several hundredfold by days 6 to 10 p.i., when peak vRNA levels were reached in the genital tract. Once systemic SIV replication was established (days 6 to 10 p.i.), increased IFN-␥ mRNA levels were also detectable in lymphoid tissues (Fig. 4A). However, IFN-␥ mRNA levels in the cervicovaginal mucosal tissues and in the colon were 10- to 100-fold higher than in lymphoid tissues at days 6 to 10 p.i., and this difference was statistically significant (Fig. 4A). As vRNA levels in all tissues decreased from day 14 to 28 p.i., IFN-␥ mRNA levels also decreased. Similar to IFN-␥, the highest increase in IL-8 mRNA levels after SIV J. VIROL.

(8) VOL. 79, 2005 TISSUE CYTOKINE AND vRNA LEVELS IN ACUTE SIV INFECTION ACKNOWLEDGMENTS We thank A. T. Haase for helpful discussions about the manuscript. This work was supported by NIH grant DE016541-01 to K.A.; NIH grants AI44480, RR14555, RR0169, AI055793, and AI57264 to C.J.M.; and the CNPRC base grant U51RR00169. REFERENCES 1. Abel, K., L. Compton, T. Rourke, D. Montefiori, D. Lu, K. Rothaeusler, L. Fritts, K. Bost, and C. J. Miller. 2003. Simian-human immunodeficiency virus SHIV89.6-induced protection against intravaginal challenge with pathogenic SIVmac239 is independent of the route of immunization and is associated with a combination of cytotoxic T-lymphocyte and alpha interferon responses. J. Virol. 77:3099–3118. 2. Abel, K., L. La Franco-Scheuch, T. Rourke, Z. M. Ma, V. De Silva, B. Fallert, L. Beckett, T. A. Reinhart, and C. J. Miller. 2004. 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Route of simian immunodeficiency virus inoculation determines the complexity but not the identity of viral variant populations that infect rhesus macaques. J. Virol. 75:3753–3765. 20. Guha-Thakurta, N., and J. A. Majde. 1997. Early induction of proinflammatory cytokine and type I interferon mRNAs following Newcastle disease virus, poly [rI:rC], or low-dose LPS challenge of the mouse. J. Interferon Cytokine Res. 17:197–204. 21. Guimbaud, R., V. Bertrand, L. Chauvelot-Moachon, G. Quartier, N. Vidon, J. P. Giroud, D. Couturier, and S. Chaussade. 1998. Network of inflammatory cytokines and correlation with disease activity in ulcerative colitis. Am. J. Gastroenterol. 93:2397–2404. 22. Heremans, H., A. Billiau, and P. De Somer. 1980. Interferon in experimental viral infections in mice: tissue interferon levels resulting from the virus Downloaded from http://jvi.asm.org/ on November 8, 2019 by guest SIV, but not in animals inoculated with a replication-attenuated SIV (25). While the focal nature of SIV replication in the first 5 days after intravaginal inoculation is initially not sufficient to elicit a type I interferon response, the proinflammatory cytokine cascade is immediately initiated in the genital tract. Thus, the early cytokine milieu induced after vaginal SIV inoculation favors immune activation and results in the recruitment and activation of target cells to support viral replication and does not promote an antiviral response in a timely manner. Defining the danger signals necessary and sufficient to elicit these distinct innate responses by SIV and determining if the observed inflammation is the result of an abnormal effector cell response and/or the result of impaired regulatory mechanisms might be critical in designing therapies aimed at limiting the inflammatory response and thereby reducing virus replication. Furthermore, the results of the current study show that there are unique patterns of cytokine gene expression in different anatomic locations. The colon is the earliest and most important site of CD4⫹ T-cell depletion after intravaginal SIV inoculation (29), and thus, it is important to understand the immunopathology of SIV infection in this tissue. We found that there was a distinct cytokine profile in the colon compared to the genital tract and systemic lymphoid tissues. Most striking was the observation that the colon has high and persistent levels of proinflammatory cytokines but is a relatively inefficient site for the induction of type I interferons. The cytokine profile in the colon is reminiscent of chronic inflammatory diseases of the gut. Persistently increased levels of TNF-␣, IFN-␥, IL-6, and IL-8 are characteristic of Crohn’s disease and ulcerative colitis (4, 8, 13, 17, 21, 43). Consequently, there is a constant recruitment of neutrophils, monocytes, and activated effector T cells that results in an uncontrolled, sustained inflammatory response of the gut. In the case of SIV/HIV infection, the persistent recruitment of activated T cells provides a potential source of target cells for SIV/HIV. In fact, it has been demonstrated that the gut is a major site of CD4 depletion (29, 31, 44, 47, 48). The mechanisms of CD4 depletion in the gut remain controversial, with some investigators proposing a pure viral cytopathic effect (31) and others suggesting a role for apoptosis-mediated killing of CD4 T cells (29). The cytokine milieu of the colon is consistent with the viral lysis and apoptotic models of T-cell depletion, as both IFN-␥ and IL-8 have been shown to upregulate Fas and Fas ligand (40, 49). In summary, the data are consistent with the conclusion that the virus in the first few days can replicate and spread without any major interference from the host immune system, as the only responses induced are of proinflammatory nature and favor virus replication by attracting more activated target cells and are less likely to have direct antiviral function. Thus, an effective HIV vaccine must be able to elicit an effective antiviral response within the first days after infection and at the same time prevent the inflammatory environment that is driving virus replication. 12171

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