Nonpathogenesis of Simian Immunodeficiency Virus Infection Is Associated with Reduced Inflammation and Recruitment of Plasmacytoid Dendritic Cells to Lymph Nodes, Not to Lack of an Interferon Type I Response, during the Acute Phase^▿

MATERIALS AND METHODS

RESULTS

By in situ hybridization, we found that cells expressing IFN-α1 (IFN-α1⁺), one of the major subtypes of type I IFN, expanded at days 7 and 11 and then declined at day 14 (Fig. 1A and 2A). Whereas the dynamics were qualitatively similar between the different groups analyzed, the magnitude of the response was clearly distinct. RMs of Indian origin (RMIs) infected by SIV produced, at the peak (day 11), higher levels of IFN-α1 (median, 16 IFN⁺ cells/2 mm²) than that observed in NC-RMs of Chinese origin (7.5 IFN⁺ cells/2 mm²; RMIs versus NC-RMs, P = 0.04), and C-RMs (2.5 IFN⁺ cells/2 mm²; RMIs versus C-RMs, P = 0.0006; NC-RMs versus C-RMs, P = 0.003) compared to uninfected monkeys (0.5 IFN⁺ cells/2 mm²). The levels at the peak were less than 2.0 IFN⁺ cells/2 mm² in SIVΔnef-infected RMs and in SIV-infected AGMs. At 2 months (Fig. 2A), the numbers of IFN-α1⁺ cells were higher in monkeys progressing faster to AIDS (RMIs [4.8 IFN⁺ cells/2 mm²] and NC-RMs of Chinese origin [3.2 IFN⁺ cells/2 mm²]; RMIs versus NC-RMs, P = 0.02) than that observed in nonprogressors (C-RMs [0.98 IFN⁺ cells/2 mm²] and AGMs [0.4 IFN⁺ cells/2 mm²]; NC-RMs versus C-RMs [P = 0.0003] and C-RMs versus AGMs [P = 0.01]). The levels of type I IFN detected in the plasma (Fig. 3) were consistent with the dynamics of IFN-α1⁺ cells in LNs and revealed at the peak higher levels in animals progressing faster to AIDS compared to nonprogressors (NC-RMs versus C-RMs [P = 0.017] and NC-RMs versus AGMs [P = 0.001]). Moreover, no significance difference was observed between C-RMs and AGMs (P = not significant). Our data also revealed a significant correlation between the numbers of IFN-α1⁺ cells at the peak and disease outcome (months of survival) (Fig. 2B). In order to analyze whether the Indian group had an impact on the correlation, we excluded this group, and found a positive correlation between the expression of type I IFN and the months of survival, including only NC- and C-RMs (r² = 0.53, P = 0.0002). Animals have been followed up for disease outcome and killed at different time points postinfection corresponding to a wasting syndrome as previously described (9, 10, 33, 35, 36). The extent of apoptosis—a predictive factor of disease outcome (20, 49)—quantified in the tissues at the peak, also correlated with the levels of IFN-α1⁺ cells (Fig. 2B).

A straightforward explanation would be that increasing viral replication in these tissues induced higher levels of IFN-α1⁺ cells. Although we found that RMIs displayed higher numbers of SIV⁺ RNA cells compared to RMs of Chinese origin at the peak (∼10-fold higher, Fig. 2A), there was no significant difference in the extent of SIV⁺ cells, as well as in plasma viral load between NC-RMs, C-RMs, and SIV-infected AGMs at the same time (Fig. 2C). Lower levels of SIV⁺ cells and lower plasma viral load compared to the other groups of macaques were detected only in SIVΔnef-infected RMs. Consistent with previous reports (9, 27), these findings suggest that the level of viral replication at the peak does not discriminate between pathogenic and nonpathogenic primate models of SIV infection (RMIs nevertheless showed higher frequencies of infected cells and faster disease progression than Chinese RMs). These results also indicate that the extent of viral replication detected in the LNs was not the only factor driving higher levels of IFN-α1⁺ cells in the tissues at the peak of viral replication between RMs and AGMs.

Thereafter, the viral load declined. Thus, at day 60, the numbers of SIV⁺ RNA cells were higher in Indian than in Chinese RMs (15.9/2 and 7.4/2 mm², P = 0.001) (Fig. 2C). However, it should be noted that at day 60, the number of SIV⁺ RNA cells in LNs was extremely low in AGMs (0.5/2 mm²), despite a detectable viral load (see Fig. 6A). Viral load in the blood also declined in the blood to reach a steady state (days 60 to 180). Higher levels in NC-RMs than in C-RMs and AGM (P = 0.001) were observed. Thus, the level of IFN-α1⁺ cells detected in the LNs at day 60 is more consistent with the notion that persistence of SIV replication in LNs was associated with higher levels of IFN-α1⁺ cells. These results are consistent with another report indicating that the expression of genes related to the IFN response in peripheral LNs was higher in progressors than in nonprogressors RMs (32).

Differential IFN-α1⁺ expression in pathogenic and nonpathogenic models, despite similar levels of viral replication during the acute phase, suggests an alternative explanation such as a difference in TLR signaling. However, in contrast to the previous report on sooty mangabeys (29), we found that in vitro stimulation of PBMC with TLR7 and TLR9 ligands induced similar levels of type I IFN-α as detected by ELISA from PBMC between both RMs of Indian and Chinese origins and AGMs (Fig. 4A).

Consistent with the notion that CD123⁺ and CD11c⁻ pDCs are potent producers of type I IFNs (4), we observed, by flow cytometry, that the predominant source of IFN-α2 was pDCs both in healthy RMs of Chinese origin and in AGMs (Fig. 4B).

Therefore, we next examined the dynamics of pDCs (Fig. 5) (6, 45). The percentage of pDCs in healthy RMs exhibited a mean of 0.17% (range, 0.057 to 0.30%) in the LNs. This percentage increased early after infection by days 7 and 11 coincident with the peak of type I IFN-expressing cells. The percentage of pDCs then drastically decreased and returned to baseline levels at day 14. Interestingly, the percentages of pDCs were higher in the LNs of NC-RMs than in LNs of C-RMs both at day 11 (0.6% ± 0.11% versus 0.31% ± 0.07%, P = 0.005) and at day 60 (0.25% ± 0.07% versus 0.11% ± 0.06%, P = 0.01), a finding consistent with the dynamics of IFN-α⁺ cells. In contrast, we found no major changes in the dynamic of pDCs in LNs both in SIV-infected AGMs and SIVΔnef-infected RMs. We cannot exclude that other cells produce type I IFN in the LNs, such as macrophages which may produce 10-fold less type I IFN than pDCs. However, during the acute phase, IFN-α⁺ cells were essentially localized in the paracortical T-cell zone (Fig. 1) but not in the germinal centers, which contain a majority of B cells (37), and most of the IFN-α⁺ cells were CD123⁺ as determined by immunochemistry (data not shown), which is consistent with another report (25).

The production of chemokines and cytokines in an inflammatory environment participates in the recruitment of pDCs. Interestingly, the level of IL-8, which is a general marker of inflammation, was increased early after infection at day 7 in both RMs and AGMs (RMs of Indian origin, 300 ± 36 pg/ml; RMs of Chinese origin, 200 ± 25 pg/ml; AGMs, 220 ± 35 pg/ml) (Fig. 6B). Thereafter, IL-8 was highly expressed only in pathogenic SIV-infected RMIs (3,000 ± 232 pg/ml at day 14), as well of NC-RMs of Chinese origin (2,100 ± 270 pg/ml at day 14), compared to C-RMs (200 ± 45 pg/ml at day 14, P = 0.0003) and nonpathogenic SIV models (AGMs, 110 ± 29 pg/ml at day 14, P = 0.0006) (Fig. 6B). Other reports have also shown an increased level of IL-8 early after infection of RMs of Indian origin (5, 25). At day 60, the levels of IL-8 remained higher in monkeys progressing faster to AIDS (5,000 ± 269 pg/ml in RMIs, and 4,600 ± 320 pg/ml in NC-RMs) compared to AGMs (120 ± 14 pg/ml, P = 0.0003) and C-RMs (110 ± 23 pg/ml, P = 0.0006). However, the absence of IL-8 detection in the nonpathogenic AGM model was not related to defective TLR signaling (Fig. 6C). Thus, the inflammatory response is uncoupled from the extent of viral replication at the peak but characterizes pathogenic models of SIV infection.

DISCUSSION

This study showed an early elevation of IFN-α1 expression in peripheral LNs, as well type I IFN in the blood during the acute phase of SIV infections in both pathogenic and nonpathogenic primates. The magnitude during the acute phase and the persistent expression of IFN-α1 at the set point is associated with a more rapid progression toward AIDS. The extent of viral replication in the tissues may participate in the direct elevation of type I IFN-α1; in fact, RMs of Indian origin exhibited increased expression of type I IFN-α1 and increased levels of viral replication compared to RMs of Chinese origin and macaques infected with the Δnef SIVmac251 strain. However, despite similar levels of viral replication within LNs at the peak in AGMs and C- and NC-RMs of Chinese origin, the level of IFN-α1 expression was higher only in RMs that progress toward AIDS, compared to C-RMs and AGMs. This discrepancy was not due to a defect in TLR signaling between the different species, in contrast to an earlier report in sooty mangabey (29). Our data revealed that recruitment of pDCs, which represent the main cell type producing type I IFN (4), occurs early after SIV infection concomitantly with the peak of type I IFN. Higher recruitment of pDCs within LNs is observed in monkeys progressing more rapidly to AIDS and associated with greater inflammatory response (as measured by the presence of circulating IL-8). Recently, the examination of global gene expression changes of pathogenic (pigtail monkeys) and nonpathogenic (AGMs) SIV infections (23) has revealed in LNs that the greatest differential regulation of genes is related to (i) genes associated with cell death, which is consistent with the extent of apoptosis detected by the TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) method in the tissues of SIV-infected monkeys (9, 14, 33, 43, 49); (ii) inflammatory and chemotaxis genes, which is consistent with the detection of IL-8 and other reports (1, 25, 40, 42); (iii) higher levels of immune activation consistent with previous studies showing that in NCs the extent of CD8 activation is greater than in CS RMs of Chinese origin (34, 36); and (iv) genes associated with the IFN response (1, 21, 41). Consistent with our data, Milush et al. (32) found that expression of type I IFN expression in LNs was more highly expressed in NC-RMs than in C-RMs. However, these authors observed that genes associated with the IFN response were differently expressed in mucosal tissues and peripheral LNs. Thus, these genes were more highly expressed in the LNs of RMs that do not control infection (NC-RMs), whereas the genes were expressed less in the colon, and vice versa, in nonprogressors (C-RMs). Lederer et al. (23) also observed higher levels of IFN gene in the colon of nonpathogenic AGMs compared to pathogen SIV-infected pigtail monkeys. More recently, mRNA expression of four immune modulators—IFN-α, oligoadenylate synthetase, CXCL9, and CXCL10—was positively associated with disease progression within the LN tissues of SIV-infected RMs of Indian origin (13) and consistent with the profile of mRNAs observed in lymphatic tissue of untreated HIV-infected persons during the acute phase (26). Thus, depending on the anatomical location, the levels of expression of IFN-associated genes may distinguish pathogenic and nonpathogenic infections.

A further observation was the persistence of IFN-α expression and pDC recruitment in the peripheral LNs of animals progressing to AIDS. These findings provide evidence that productive infection persists despite a prolonged IFN-α response within peripheral LNs. This was consistent with gene expression (23), since most of the significant changes between days 10 and 45 postinfection in the LNs and the colon involved genes associated with IFN responses and were significantly attenuated during nonpathogenic SIV infection (AGMs) compared to pathogenic SIV infection (pigtail monkeys). The decreased expression of these genes and the lower levels of IFN-α1⁺ cells detected in the LNs of AGMs may in part be due to decreased viral replication in these LNs, despite similar levels of viral load in the blood. However, we do not know whether this also occurs in the colon. Thus, it has been assumed that the strong and early IFN signaling in the colons of SIV-infected AGMs should be a mechanism for limiting SIV pathogenesis, and such early attenuation of the inflammatory response in AGMs may help to reduce damage (23). Similar observations have been reported in macaques that progress slowly (32). Thus, depending on the anatomical location, the production of type I IFN should be beneficial or detrimental, controlling or favoring an inflammatory response.

Activities and therapeutic uses of type I IFNs have clearly established a wide breadth of biological activities. In particular, it has been reported in the past that low-dose IFN-α treatment exerts an anti-inflammatory control action associated with a significant reduction in tissue infiltration of neutrophils, macrophages, and lymphocytes. In contrast, high doses can directly induce proinflammatory and pyretic responses in humans, and a common adverse reaction is lymphopenia associated with flulike symptoms (for reviews, see references 2 and 38).

The notion that type I IFN could be more detrimental than beneficial during HIV infection was originally proposed a decade ago; in fact, it has been demonstrated that type I IFN in the sera of chronically HIV-infected patients was associated with disease progression, instead of resolution (17, 22), and the loss of uninfected T cells in vitro due to HIV-1 is mediated by IFN-α (50). It has also been reported that IFN-α derived from pDCs, following in vitro exposure to HIV, was implicated in the killing of T cells (19). Thus, type I IFNs may lead to immune deviation by altering CD4 Th-cell differentiation through the promotion of CD4 T-cell apoptosis during the acute phase (9, 14, 24, 33, 43, 49). Indeed, the events occurring early after infection play a key role in conditioning further disease evolution in particular the loss of memory CD4 T cells (24, 30, 35, 39, 44, 48). However, it remains to be determined whether pDCs from SIV-infected monkeys are capable of inducing CD4 T-cell death.

Moreover, models of viral infection in mice, such as infection with lymphocytic choriomeningitis virus or measles virus, have established that there is induction of a generalized immune suppression in their natural hosts. This occurs early after infection concomitantly with a change in the balance of type I IFN and IL-12 expression (3, 11, 18) in which type I IFN antagonizes IL-12 production (8, 31). IL-12 is a cytokine essential in the development of cell-mediated immunity (47) and cell survival (7, 15, 16). The peak of type I IFN observed in our study early after infection may lead to decreased IL-12 expression in LNs during the acute phase. Thus, the recruitment of pDCs in the peripheral LNs during the acute phase of SIV infection could have a detrimental indirect effect on CD4 T-cell function. It remains to be determined whether the balance of IL-12 expression is affected during the acute phase in both pathogenic and nonpathogenic primate models.

In conclusion, our data demonstrate that the increased number of IFN-α1⁺ cells in the peripheral LNs of RMs of Chinese origin pathogenically infected with SIV is at least related to an early elevation in the level of recruitment of pDCs and associated with an increased inflammatory environment during the acute phase. Conversely, reduced recruitment of pDCs and reduced expression of IFN-α1 in the peripheral LNs is correlated with nonpathogenic disease outcome, despite an intense peak of viral replication.