Dynamics of Cytokine/Chemokine Responses in Intestinal CD4⁺ and CD8⁺ T Cells during Acute Simian Immunodeficiency Virus Infection

Abstract

Loss of intestinal CD4⁺ T cells was associated with decreased production of several T-helper 1 (T_H1) and T_H2 cytokines and increased production of interleukin 17 (IL-17), gamma interferon (IFN-γ), CCL4, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by CD8⁺ T cells 21 days after simian immunodeficiency virus (SIV) infection in rhesus macaques. Shifting of mucosal T_H1 to T_H2 or T-cytotoxic 1 (T_C1) to T_C2 cytokine profiles was not evident. Additionally, both CD4⁺ and CD8⁺ T cells showed upregulation of macrophage migration inhibition factor (MIF) and basic fibroblast growth factor (FGF-basic) cytokines that have been linked to HIV disease progression.

TEXT

Early profound loss of intestinal memory CD4⁺ T cells is a hallmark of both human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection (1–5). Our recent study showed that SIV selectively infects and eliminates dividing, non-SIV-specific “first responder” CD4⁺ T cells in acute infection (5). Our data also suggest that pathogenic SIV_MAC251 infection in rhesus macaques (RMs) induces an increased level of peripheral blood (PB) T-cell activation and proliferation and higher-level expression of several cytokines/chemokines in plasma (6). Understanding cytokine/chemokine networks during acute HIV/SIV infection is important for developing effective vaccines and therapeutics (7). The reduction in the efficacy of T-helper 1 (T_H1) cell function in PB during HIV infection has been proposed to occur due to a shift from T_H1 to T_H2 responses (8–10). However, this is controversial, as additional studies found evidence of a shift toward the T_H0 phenotype (11) or no evidence of a shift from the T_H1 to the T_H2 cytokine profile (12). A shift in CD8⁺ T-cytotoxic 1 (T_C1) to T_C2 responses in PB has also been proposed to occur during HIV infection (10, 13). However, these studies are limited to PB, with little data on how cytokine/chemokine profiles in intestinal tissues are affected during acute HIV infection, despite the fact that intestinal lamina propria lymphocytes (LPLs) are a major site of viral replication and CD4⁺ T-cell depletion (2, 14, 15). Here, we have compared the profiles of 28 different cytokines/chemokines produced by single-positive (SP) CD4⁺ and CD8⁺ T cells isolated from jejunum LPLs before and 21 days after SIV_MAC251 infection in RMs to assess the dynamics of changes in cytokine/chemokine networks in intestinal tissues.

Five adult female RMs (Macaca mulatta), negative for SIV, HIV-2, type D retrovirus, and simian T-cell leukemia virus 1 infection, were treated with depot medroxyprogesterone acetate (Depo-Provera; 30 mg intramuscularly) and 4 weeks later inoculated with 500 50% tissue culture infectious doses (TCID₅₀) of SIV_MAC251 intravaginally at the Tulane National Primate Research Center (TNPRC). Excisional biopsy specimens of jejunum (6 to 10 cm long) were collected before and 21 days after SIV infection under the supervision of TNPRC veterinarians in accordance with the standards incorporated in the Guide for the Care and Use of Laboratory Animals (44) and with the approval of the Tulane Institutional Animal Care and Use Committee. All five SIV-infected macaques had high plasma viral loads [log₁₀(6.55) to log₁₀(7.43) viral RNA copies/ml of plasma] 21 days after SIV infection. Jejunum LPLs were isolated both before and 21 days after SIV infection as previously described (3, 16–18). Jejunum LPLs were first labeled with LIVE/DEAD stain (Invitrogen), followed by surface staining with anti-CD3 (SP34.2; BD Biosciences), anti-CD4 (L200; BD Biosciences), anti-CD8 (3B5; Invitrogen), anti-programmed cell death 1 (anti-PD-1) (J105; eBioscience), and anti-CD38 (OKT10; NIH Nonhuman Primate Reagent Resource) monoclonal antibodies (MAbs). Data were acquired using a FACSAria flow cytometer (BD Biosciences, CA) within 24 h of staining as reported previously (19).

CD4⁺ T-cell depletion and increased CD38 expression in CD4⁺ T cells 21 days after SIV infection.

All RMs showed statistically significant reductions in jejunum double-positive (DP) CD4⁺ CD8⁺ (14.2% before infection versus 0.9% at 21 days postinfection) and SP CD4⁺ (41.4% before infection versus 12.0% at 21 days postinfection) T cells 21 days after SIV infection (P < 0.001) (Fig. 1A and B). The increased expression of PD-1, a member of the CD28 family, is considered a measure of CD4⁺ and CD8⁺ T-cell exhaustion (20, 21). In this study, we observed decreased PD-1 expression in SP CD4⁺ T cells, compared to levels for SP CD8⁺ T cells during acute infection (Fig. 1C and D). However, PD-1 expression differs by subsets of CD4⁺ T cells. PD-1 is highly expressed by CCR5⁺ CD4⁺ T cells as well as in central memory (CD28⁺ CD95⁺) CD4⁺ T cells (20). We have also previously shown that the majority of the intestinal SP CD4⁺ T cells are central memory T cells and express a higher percentage of CCR5 coreceptors than PB (16). These populations of CD4⁺ T cells are rapidly depleted during acute SIV infection, and the remaining cells express lower levels of PD-1. There was a slight decrease in PD-1 expression in SP CD8⁺ T cells (31.0% preinfection versus 26.7% 21 days after SIV infection) 21 days after SIV infection. However, the differences in PD-1 expression in intestinal CD4⁺ and CD8⁺ T cells between the preinfection time point and 21 days after SIV infection are not statistically significant.

Fig 1.

Distribution of single-positive (SP) CD4⁺ CD8⁺ and double-positive (DP) CD4⁺ CD8⁺ T cells in jejunum LPLs and their expression of phenotypic markers of activation and exhaustion. (A) Representative contour plots showing SP CD4⁺, CD8⁺, and DP CD4⁺ CD8⁺ T-cell population before and 21 days after SIV infection. Plots were generated by gating CD3⁺ T cells. (B) Mean percentages (± standard errors [SE]) of SP CD4⁺, CD8⁺, and DP CD4⁺ CD8⁺ T cells of five SIV_MAC251-infected macaques before infection (pre) and 21 days after infection (post). Representative contour plots show PD-1 (C) and CD38 (E) phenotypic expression in SP CD4⁺ and SP CD8⁺ T-cell population before and after SIV infection, where cells were gated through CD3⁺ T cells. Mean percentages (± SE) of T-cell exhaustion (PD-1) (D) and activation (CD38) (F) markers are shown for SP CD4⁺ and CD8⁺ T cells of five SIV_MAC251-infected macaques before and after SIV infection. Asterisks indicate statistically significant differences in respective cell populations, compared to preinfection levels (P < 0.05). FSC, forward-angle light scatter.

T-cell activation was also indirectly assessed by CD38 expression on T-cell surfaces. Increased surface expression of CD38 on CD8⁺ T cells is considered to be an indicator of immune activation and a strong prognostic marker of disease progression and death in HIV-infected patients (22). While we did observe increased expression of CD38 on CD4⁺ T cells 21 days after SIV infection, compared to preinfection levels (Fig. 1E and F), this was not observed on CD8⁺ T cells. Using other markers (increased expression of CD25, CD69, and HLA-DR), we have previously shown that intestinal SP CD8⁺ T cells from normal macaques are highly activated (16). These data raise the question of the utility of CD38 as a marker of immune activation on intestinal CD8⁺ T cells of rhesus macaques.

Isolation, sorting, and stimulation of jejunum SP CD4⁺ and CD8⁺ T cells from jejunum lamina propria.

SP CD4⁺ and CD8⁺ T cells were sorted from jejunum LPLs (2.5 × 10⁷ to 5.0 × 10⁷ cells) by use of nonhuman primate magnetic cell separation microbeads (Miltenyi Biotec) for magnetically activated cell sorting (MACS). In brief, CD20⁺ B cells were isolated first from jejunum LPLs by positive selection using anti-CD20 MAbs. The unlabeled cells were further sorted by negative selection for either SP CD4⁺ or CD8⁺ T cells by use of anti-CD8 or anti-CD4 MACS magnetic cell separation microbeads, respectively, in accordance with the manufacturer's protocols. Usage of either anti-CD8 or anti-CD4 microbeads in the first step during cell sorting would also deplete the DP CD4⁺ CD8⁺ T-cell population. Between 2.5 × 10⁶ and 10.0 × 10⁶ sorted cells for each population (SP CD4⁺ and SP CD8⁺ T cells) were obtained from the jejunum LPLs and were found to be 93 to 96% pure by flow cytometry assays using anti-CD3, -CD4, and -CD8 MAbs (data not shown). Sorted SP CD4⁺ and SP CD8⁺ T cells (5 × 10⁵ sorted cells/well) were stimulated with either anti-CD3/28 MAbs (CD3: 6G12, obtained from the NIH Nonhuman Primate Reagent Resource, used at 10 μg/ml, or CD28: CD28.2, obtained from BD Biosciences, used at 1 μg/ml) or Staphylococcus enterotoxin B (SEB; 2 μg/ml; Toxin Technologies) in RPMI 1640 medium containing 10% fetal bovine serum at 37°C in the presence of 5% CO₂ along with the appropriate medium control. For anti-CD3/28 MAb stimulation, plates were first coated overnight at 4°C with anti-CD3 MAbs diluted in phosphate-buffered saline (PBS). Purified anti-CD28 MAbs were added to the cell culture at the time point of cell stimulation. Cell culture supernatants were collected 72 h after stimulation. The concentrations of cytokines/chemokines in the cell culture supernatants were quantified using a Cytokine Monkey magnetic 28-plex panel (Invitrogen) in accordance with the manufacturer's instructions. The 28 analytes detected by this panel are as follows: interleukin 1β (IL-1β), IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-8 (CXCL8), IL-12, IL-15, IL-17, gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP-1; CCL2), macrophage inflammatory protein 1 alpha (MIP-1α) (CCL3), MIP-1β (CCL4), RANTES (CCL5), eotaxin (CCL11), macrophage-derived chemokine (MDC; CCL22), monokine induced by IFN-γ (MIG; CXCL9), IFN-γ-inducible protein 10 (IP-10; CXCL10), interferon-inducible T-cell alpha chemoattractant (I-TAC; CXCL11), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-basic), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), hepatocyte growth factor (HGF), macrophage migration inhibition factor (MIF), and vascular endothelial growth factor (VEGF). The multiplex plate was read using the Bio-Plex 200 suspension array Luminex system (Bio-Rad) as reported previously (23). Graphical presentation and statistical analysis of the data were performed using GraphPad Prism (version 5.0f; GraphPad). Eighteen out of 28 cytokines/chemokines had detectable changes in either anti-CD3/28- or SEB-stimulated CD4⁺ and CD8⁺ T cells (Fig. 2 to 4). For the majority of chemokines and cytokines, the responses detected in either anti-CD3/28- or SEB-stimulated cultures were similar; however, SEB-stimulated cultures had lower-level responses. SEB has been used as T-cell superantigen and provides useful information on T-cell signaling pathways. However, SEB stimulation is not always comparable to anti-CD3/CD28 stimulation, which is considered to be a more physiologically relevant mode of T-cell activation (24, 25). Cells stimulated with anti-CD3/28 MAbs or SEB did not induce measurable responses for 10 different cytokines/chemokines (IL-1β, IL-1RA, IL-4, IL-8, IL-12, IL-15, TNF-α, CCL22, EGF, and G-CSF).

Fig 2.

Cytokine/chemokine profile in jejunum CD3⁺ CD4⁺ T cells during acute SIV_MAC251 infection. Bar graphs show 18 different cytokine/chemokine measurements taken before and 21 days (21d) after SIV infection from culture supernatants of sorted single-positive CD4⁺ T cells stimulated with either anti-CD3/CD28 monoclonal antibodies or Staphylococcus enterotoxin B (SEB). Medium control values were subtracted from all values before the analysis (n = 5). The number and arrow in each bar for the postinfection time point show the mean fold increase (upward arrow) or decrease (downward arrow) compared to the preinfection level.

Fig 4.

Relative contribution of cytokine/chemokine expression during acute SIV_MAC251 infection. The pie charts illustrate the relative percentage contribution of each cytokine/chemokine from culture supernatants of sorted single-positive CD4⁺ and CD8⁺ T cells stimulated with anti-CD3/CD28 monoclonal antibodies before and 21 days after SIV infection. Medium control values were subtracted from all values before the analysis (n = 5). Note that cytokines having 0.01 to 0.04% overall contribution are represented as 0% in the pie chart.

Cytokine/chemokine responses in CD4⁺ T cells during acute SIV infection.

The majority of cytokines/chemokines expressed by intestinal SP CD4⁺ T cells prior to infection were downregulated during acute SIV infection. IL-2 (growth factor), IL-5, IL-6 (proinflammatory cytokine), IL-17, IFN-γ, CCL2, CCL3, CCL4, CCL5, CCL11, CXCL9, CXCL10, CXCL11, GM-CSF, HGF, and VEGF showed at-least-1.5-fold reductions after infection, suggesting an overall dysfunction of intestinal CD4⁺ T cells (Fig. 2). There was no evidence of a shift from T_H1 (primarily IFN-γ) to T_H2 (primarily IL-5) cytokine responses or vice versa in intestinal SP CD4⁺ T cells (Fig. 2).

Chemokines (CCL2, CCL3, CCL4, CCL5, CCL11, CXCL9, CXCL10, and CXCL11) that play major roles in immune activation and recruitment of immune cells, including T cells, granulocytes, and monocytes, were downregulated, which is suggestive of a failing immune response. For example, CXCL9 is associated with immune activation and CD8⁺ T-cell recruitment (26, 27), and CXCL11 induces recruitment of CD4⁺ T cells and is an important regulator of HIV pathogenesis (28).

Growth factors, including IL-2, GM-CSF, HGF, and VEGF, were downregulated. IL-2 and GM-CSF were found to play important roles in differentiation and recruitment of immune cells (29). Similarly, HGF and VEGF stimulate vasculogenesis and angiogenesis, besides being associated with HIV neuropathogenesis (30).

However, MIF and FGF-basic cytokines were upregulated by CD4⁺ T cells during acute infection. The growth factor FGF-basic and proinflammatory cytokine MIF were elevated more than 2-fold and 7-fold, respectively, after stimulation with anti-CD3/28 MAb 21 days after SIV infection, compared to preinfection levels (Fig. 2). FGF-basic modulates normal processes of angiogenesis, wound healing, and tissue repair and has been shown to be elevated in HIV-infected patients and patients with neurological symptoms (31–33). Increased MIF in plasma during acute HIV infection is thought to play an important role in promoting HIV transcription and viral replication (34, 35). In summary, significant loss of LPL CD4⁺ T cells, substantially decreased production of several T_H1 and T_H2 cytokines, and an increased production of FGF-basic and MIF proteins are all suggestive of poor prognosis of the intestinal effector function of CD4⁺ T cells during acute SIV infection (Fig. 1 and 2). However, none of these cytokine responses between the preinfection period and 21 days after SIV infection were found to be statistically significant (P > 0.05) by a 2-tailed paired t test.

Cytokine/chemokine responses in CD8⁺ T cells during acute SIV infection.

CD8⁺ T-lymphocyte cytokine responses were either maintained (IL-2, CCL2, CCL3, CCL5, CXCL9, CXCL11, and VEGF; less-than-1.5-fold change in anti-CD3/28 MAb-stimulated cultures, compared to preinfection levels), decreased (IL-6, CCL11, and CXCL10; more-than-1.5-fold reduction in anti-CD3/28 MAb-stimulated cultures, compared to preinfection levels), or increased (IL-5, IL-17, IFN-γ, CCL4, FGF-basic, GM-CSF, HGF, and MIF; more-than-1.5-fold increase in anti-CD3/28 MAb-stimulated cultures, compared to preinfection levels) during acute SIV infection (Fig. 3). These data do not support a shift from T_C1 to T_C2 in jejunum SP CD8⁺ T cells, as both IL-5 and IFN-γ cytokine levels increased more than 1.5-fold during acute infection, compared to preinfection levels (Fig. 3). Upregulation of different cytokines/chemokines by CD8⁺ T cells is indicative of immune activation in CD8⁺ T cells, despite the absence of surface CD38 expression in this population during acute infection. These data further bring into question the utility of CD38 as a marker of immune activation in intestinal CD8⁺ T cells in rhesus macaques and warrant further investigation. The increased expression of IL-17 produced by CD8⁺ T cells also suggests an influx of T_C17 cells during acute SIV infection that might play a role in regulating functional immune responses and intestinal homeostasis. IL-17 also plays a vital role in pathogen clearance and mediates proinflammatory responses by inducing the production of several cytokines, including GM-CSF, which was upregulated at least 4-fold from preinfection levels. Additionally, increased IFN-γ expression produced by CD8⁺ T cells may play an important role in inhibiting HIV pathogenesis (36, 37). However, as with CD4⁺ T cells, upregulation of MIF is likely to be detrimental to progression of disease (35).

Fig 3.

Cytokine/chemokine profile in jejunum CD3⁺ CD8⁺ T cells during acute SIV_MAC251 infection. Bar graphs show 18 different cytokine/chemokine measurements taken before and 21 days (21d) after SIV infection from culture supernatants of sorted single-positive CD8⁺ T cells stimulated with either anti-CD3/CD28 monoclonal antibodies or Staphylococcus enterotoxin B (SEB). Medium control values were subtracted from all values before the analysis (n = 5). The number and arrow in each bar for the postinfection time point show the mean fold increase (upward arrow) or decrease (downward arrow) compared to the preinfection level.

Increased CXCL10 RNA levels in plasma and lymph nodes has been associated with rapid HIV disease progression and viral replication (38–40). CXCL10 also acts as a chemoattractant for monocytes/macrophages, T cells, natural killer cells, and dendritic cells. However, in this study, intestinal CXCL10 was decreased 2.9- to 3.2-fold in both jejunum CD4⁺ and CD8⁺ T cells 21 days after infection, compared to preinfection levels. This occurred despite the increased levels of intestinal IFN-γ and IL-17 produced by CD8⁺ T cells, which have a role in upregulating CXCL10. This suggests either that the regulation of CXCL10 expression in jejunum differs from that in PB and lymph nodes during SIV/HIV infection or that the major sources of CXCL10 in the intestine are cells other than SP CD4⁺ and CD8⁺ T cells.

CCL11, which was shown to inhibit HIV replication in vitro (41) and is important in recruitment of eosinophils, was also downregulated during SIV infection in both CD4⁺ and CD8⁺ T cells. However, the chemokine CCL4 was upregulated, and expression of CD8⁺-specific CCL4 may play an important role in immune response against SIV/HIV by inhibiting SIV/HIV replication and killing of virus-infected cells (36, 37). Three growth factors, namely, FGF-basic, GM-CSF, and HGF, were also upregulated in CD8⁺ T cells, and as with CD4⁺ cells, the increase in FGF-basic is indicative of a poor prognosis. Increased levels of HGF during HIV infection are often associated with secondary infection complications such as Kaposi's sarcoma (42). Conversely, GM-CSF is thought to be involved in inducing immune responses by recruiting and stimulating antigen-presenting cells (29) and has been used as a promising adjuvant in HIV vaccine studies (43). In summary, CD8⁺ showed evidence of upregulation of cytokines, indicating a functional immune response; however, none of these described differences in cytokine responses were found to be statistically significant (P > 0.05) by a 2-tailed paired t test. When the data are displayed by SIV infection time point and T-cell phenotype, the relationship between SIV infection and overall contribution of cytokines/chemokines expression becomes more apparent (Fig. 4). Intestinal CD4⁺ T cells demonstrated marked reduction of IL-2, IL-17, IFN-γ, CCL4, and CCL5 and increased production of FGF-basic and MIF cytokines/chemokines 21 days after SIV infection, compared to their preinfection levels (Fig. 4). In contrast, marked increases in IL-17, IFN-γ, GM-CSF, and MIF cytokines/chemokines in CD8⁺ T cells were observed 21 days after SIV infection (Fig. 4).

In conclusion, loss of intestinal CD4⁺ T cells was associated with downregulation of several T_H1 and T_H2 cytokines in CD4⁺ T cells during acute SIV infection. In isolated populations of CD4 and CD8⁺ T cells, no evidence of a T_H1-to-T_H2 shift for CD4⁺ T cells or a T_C1-to-T_C2 shift for CD8⁺ T cells (or vice versa) was found in gut-associated lymphoid tissue during acute SIV infection. The experimental design does not address cytokine/chemokine changes for other populations or at time points beyond the acute infection period. Decreased CD4⁺-specific and increased CD8⁺ T-cell-specific cytokine/chemokine responses suggest that CD8⁺ T cells are actively mounting functional immune responses and potentially mitigating some of the impact of the loss of functional T_H cells. The absence of surface PD-1 expression upregulation suggests that intestinal CD8⁺ T cells are not exhausted. Despite upregulation of several essential effector cytokines/chemokines (IL-17, IFN-γ, and GM-CSF) by CD8⁺ T cells, upregulation of FGF-basic, HGF, and MIF chemokines may provide poor prognosis in SIV/HIV infection either by favoring HIV/SIV replication or by other, unknown mechanisms.