Divergent Cytokine and Chemokine Responses at Early Acute Simian Immunodeficiency Virus Infection Correlated with Virus Replication and CD4 T Cell Loss in a Rhesus Macaque Model

Cytokine and chemokine levels remain one of the significant predictive factors of HIV pathogenesis and disease outcome. Understanding the impact of cytokines and chemokines during early acute infection will help to recognize critical changes during HIV pathogenesis and might assist in establishing improved HIV treatment and prevention methods. Sixty-one cytokines and chemokines were evaluated in the plasma of an SIV-infected rhesus macaque model. A substantial change in 11 cytokines/growth factors and 9 chemokines were observed during acute infection. Almost all the cytokines/chemokines were below the baseline values for an initial couple of days of infection. We detected six important cytokines/chemokines, such as IL-18, IP-10, FLT3L, MCP-1, MCP-2, and MIP-3β, that can be used as biomarkers to predict the peripheral CD4+ T cell loss and increased viral replication during the acute SIV/HIV infection. Hence, regulating IL-18, IP-10, FLT3L, MCP-1, MCP-2, and MIP-3β expression might provide an antiviral response to combat acute SIV/HIV infection.


Introduction
HIV, the causative agent of AIDS, is one of the world's most serious public healthrelated sexually transmissible diseases, with 38.4 million people living with HIV infection as of 2021 (UNAIDS 2022 epidemiological estimates; aidsinfo.unaids.org). However, until today, more than 10 million HIV-infected people have no access to antiretroviral therapy (ART) (aidsinfo.unaids.org) despite its first reported case in June of 1981 [1]. ART has reduced AIDS mortality significantly. However, the treatment is not curative and the latent virus rebounds if the ART treatment is discontinued. Cytokine dysregulation and chronic inflammation are major clinical manifestations of HIV or simian immunodeficiency virus (SIV) infection [2][3][4][5][6][7]. HIV/SIV causes the early depletion of CD4+ T cells and induces dysregulation of protective immune responses, including the lack of antiviral mechanism and delay in the generation of effective neutralizing antibodies. The early depletion of intestinal CD4+ T cells during HIV/SIV infection was also associated with changes in both T-helper 1 (T h 1) and T h 2 cytokines that were also indicative of the failure of functional adaptive immune responses and the dysregulation of protective immunity [3,4]. In our earlier studies, a lack of T h 1 to T h 2 shift, as well as T cytotoxic 1(T C 1) to T C 2 cytokine shift in CD4+ and CD8+ T cells, respectively, in cells isolated from the intestine, peripheral blood, bone marrow, and axillary lymph node (LN) at 21 days post-SIV infection were detected when compared to the pre-infection phase [3,4]. However, limited data are available on the dynamics of different cytokines, chemokines, and growth factors at the early acute period of HIV infection and how those are related to the viral dynamics and peripheral CD4+ T cell loss. Since the cytokine and chemokine statuses remain one of the significant predictive factors of the viral load, disease progression, and the depletion of CD4+ T cells, understanding the significance of cytokines and chemokines during early acute infection will help to recognize critical changes during HIV pathogenesis. The knowledge will also help manage infected individuals' clinical outcomes and assist in establishing improved HIV treatment and prevention methods.
In the present study, we have assessed 61 different cytokines and chemokines in plasma at the early acute SIV infection starting from 0, 0.25 (6 h.), 1, 2, 3, 4, 5, 7, 14, and 21 days of an SIV-infected rhesus macaque (RhM) model. RhMs remain an invaluable animal model for understanding HIV pathogenesis, which will mimic the cytokine/chemokine changes that happen during acute HIV infection in humans. Study related to early acute HIV infection in humans is likely impossible to implement. This study will provide insight into the cytokine status at the early stage of disease and has the potential to identify novel biomarkers that can predict virus replication and peripheral CD4+ T cell levels during early infection.

Ethics Statement
All Indian Rhesus macaques (RhMs, Macaca mulatta) were housed at the Tulane National Primate Research Center (TNPRC) biosafety level 2 facility by the standards incorporated in the guide for the cure and use of laboratory animals. The study was reviewed and approved by Tulane University IACUC protocol number 3818. TNPRC is fully accredited by AALAC, Animal Welfare Assurance No. A4499-01.
Macaques were singly housed indoors in climate-controlled buildings with a 12/12light/dark cycle and were fed a commercially prepared nonhuman primate diet twice daily, supplemented by different feeding enrichments. Water was accessible ad libitum in each cage. All the subjects were monitored twice daily for pain, distress, and disease signs. In addition, the subjects were anesthetized intramuscularly with ketamine hydrochloride (10 mg/kg of BW) or tiletamine hydrochloride/zolazepam (Telazol, Zoetis, Parsippany, NJ, USA) (5-8 mg/kg of BW) for blood collection, physical exams, and virus inoculation [8].

SIV Infection and Sample Collection
This study used 14 RhMs of both sexes (six females and eight males) between 6.3 and 8.3 years of age. The macaques were seronegative for SIV, simian T-cell leukemia virus type 1, and type-D retroviruses antibodies at the initiation of the study. The subjects were randomly assigned into two groups (infected and uninfected control), with ten and four RhMs per group. The blood collected from all the SIV-infected RhMs was studied longitudinally for 21 days post-SIV infection for hematology, virological, and immunological assays ( Figure 1A). The macaques from the infected group were inoculated with 100 TCID 50 pathogenic SIV MAC 251 using an intravenous route to mimic one of the primary routes of HIV transmission in humans. The blood was also collected from all the uninfected controls for all those longitudinal time points to perform hematology and immunological assays ( Figure 1A). For analysis, plasma and serum samples were collected at various time points, including 0, 0.25, 1, 2, 3, 4, 5, 7, 14, and 21 days post-infection (dpi) or study periods ( Figure 1A).  of SIV infection, as determined using RT-PCR (n = 10). (C) Absolute counts of peripheral CD4+ T cells at pre-and post-SIV infection. Each point of the scattered plot represents data from individual macaques. Asterisks indicate statistical differences between time points as calculated using Dunnett's multiple comparison analysis (**, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001). Absolute counts of lymphocytes (D), monocytes (E), neutrophils (F), and RBCs (G) in peripheral blood of SIV infected (n = 10) and uninfected control (n = 4) macaques are shown. Data presented as mean ± SE of different cell populations for the entire 21 days study period. Mann-Whitney t-test analysis was used to determine statistically significant differences between SIV-infected and uninfected-control groups in all those cell populations for that specific time (*, p ≤ 0.05; **, p ≤ 0.01).

Hematology
Hematology was performed on EDTA-anticoagulated blood using a Sysmex XT2000i analyzer (Sysmex Corporation, Kobe, Japan). Fresh blood was used for all the hematology analyses.

Plasma Isolation
The plasma was isolated from EDTA-anticoagulated blood after centrifugation. The frozen plasma samples were used for plasma viral load and cytokine/chemokine analysis.

Quantitative SIV Plasma Virus Load (PVL)
Quantitative reverse transcription-PCR (qRT-PCR) was used to measure plasma viral RNA copies at the Wisconsin National Primate Research Center with a lower detection limit of 60 SIV RNA copies/mL of plasma [9].

Quantification of Cytokines and Chemokines in Plasma
Sixty-one cytokines/chemokines in plasma were quantified using the U-plex biomarker , VEGF-α (vascular endothelial growth factor-α), and YKL-40 (chitinase-3-like protein 1) following the manufacturer instruction with minor modification [8]. U-plex plates were coated with respective biotinylated capture antibodies and incubated overnight on a shaker at 4 • C. Calibrator standards and diluted plasma samples were added to the individual wells after washing with wash buffer. The plate was incubated overnight on a shaker at 4 • C. The next day, the plate was washed with wash buffer and the detection antibody was added to the well and incubated on a shaker at room temperature for 1 h. The plate was finally washed and a read buffer was added. The plate was read immediately on an MSD microplate reader (MSD). The concentration of each cytokine and chemokine was determined based on the calibration standard curve and its respective signals.

Flow Cytometry of Peripheral Blood Mononuclear Cells (PBMCs)
The PBMCs were isolated from whole heparinized blood using density gradient centrifugation [10,11]. The CD4 and CD8 T cell frequencies were quantified in the PBMCs using directly conjugated monoclonal antibodies. Anti-CD45 (clone D058-1283), anti-CD3 (clone SP34.2), anti-CD4 (clone L200), and anti-CD8 (clone SK1) monoclonal antibodies obtained from BD Biosciences were used for staining. Live/Dead stain (Thermo Fisher Scientific, Waltham, MA, USA) was used to exclude dead cells from the flow analysis. After surface staining and fixation, the cells were acquired on a Becton Dickinson LSRII instrument. At least 50,000 events were acquired from each sample using lymphocyte gating and analyzed using FlowJo software (v10.8, BD Biosciences, Franklin Lakes, NJ, USA). The absolute CD4 count was calculated using complete blood count (CBC) data from hematology and flow cytometry CD4+ T cells percentage analysis.

Statistical Analyses
GraphPad Prism was used for all the statistical analysis and generating graphs (v9.4.1., GraphPad Software, San Diego, CA, USA). One-way repeated ANOVA measured significant differences between post-SIV time points and baseline data. Dunnett's multiple comparison tests were applied to examine any statistically significant changes in the cytokine/chemokine level at different post-infection time points compared to the baseline 0 day or pre-infection. A Mann-Whitney t-test as a nonparametric method was performed to determine the statistical differences between the various blood cell counts of the SIV-infected and the uninfected control group. A correlation analysis between PVL and the significantly upregulated or downregulated cytokine/chemokine expression during acute SIV infection and correlation analysis between the CD4+ T cell count and cytokine/chemokine level expressions were performed using the two-tailed Pearson correlation method. A p-value of < 0.05 was considered statistically significant throughout the analysis.

Dynamics of PVL, CD4, Lymphocytes, Monocytes, Neutrophils, and Red Blood Cells Count in SIV-Infected Macaques
Ten SIV-infected RhMs were used to evaluate the cytokine and chemokine profile during the early 21 days of the infection. Since the blood samples were drawn very often during the initial stage of this study, we have also studied four healthy, SIV-uninfected RhMs as a control to determine whether the changes detected in the SIV-infected RhMs are not due to the results of repetitive blood collection. All 10 SIV-infected RhMs remained SIV-infected throughout this study. The mean plasma viral load significantly increased at day 7 (log 10 6.1 copies/mL of plasma, p ≤ 0.0001) and peaked at day 14 post-SIV infection (10 7.4 , p ≤ 0.0001) ( Figure 1B). Similar to PVL, the peripheral CD4 count also significantly decreased at day 14 (mean ± SE, 605 ± 96 cells/µL of blood, p ≤ 0.001) compared to the baseline (1022 ± 112 cells/µL of blood) and the count remained lower at 21 days post-SIV infection ( Figure 1C). We were unable to detect any significant difference in the PVL and cell counts between the male and female SIV-infected RhMs. The CD4+ T cell count in uninfected control RhMs remained normal throughout the study.
The lymphocyte population in both the SIV-infected and control RhMs remained within the range of 1.97−3.80 × 10 3 /µL of blood. The lymphocyte counts remained stable primarily between groups except on day 2 (p = 0.048, Figure 1D), when the lymphocyte count was significantly elevated in the SIV-infected RhMs compared to the controls. Similarly, the monocyte count ranged between 0.09−0.82 × 10 3 /µL of blood. The monocyte count increased dramatically at the day 14 time point (p = 0.019, Figure 1E) in the SIVinfected RhMs compared to the controls. The neutrophil absolute counts decreased in the SIV-infected RhMs at 3-(p = 0.002), 5-(p = 0.011), 7-(p = 0.008), and 14-(p = 0.002, Figure 1F) day study periods when compared to the control group. No significant changes were detected in the red blood cell (RBC) counts between those two groups ( Figure 1G).
Several other chemokines, including CTACK, eotaxin-2, eotaxin-3, I-TAC, MIP-1β, GRO-α, MIP-3α, and MIP-5, were also evaluated during the early acute time points. However, no significant changes were detected at any time compared to the baseline (Table S1). We also did not see any substantial differences in chemokine levels from SIV-uninfected control macaques for any time points compared to day 0 ( Figure S1 and Table S1).

Correlation of Cytokines/Chemokines Concentration with PVL and Absolute CD4+ T Cell Count
To determine if the cytokine/chemokine responses detected early during SIV infection correlated with increased PVL and loss of peripheral CD4+ T cell count, we have performed correlation coefficient analysis between PVL and each of the significantly upregulated or downregulated cytokines/chemokines during acute SIV infection. A significant positive correlation was observed between IL-18 and PVL (r = 0.46, p = 0.003, Figure 5A), IP-10 and PVL (r = 0.51, p = 0.0007, Figure 5B), FLT3L and PVL (r = 0.48, p = 0.002, Figure  5C), MCP-1 and PVL (r = 0.38, p = 0.017, Figure 5D), and MIP-3β and PVL (r = 0.51, p = 0.0008, Figure 5E) suggesting that either increased PVL has an impact on upregulating the

Discussion
Tightly regulated cytokine and chemokine production are ideal for an effective antiviral immune response. The virus-induced increased cytokine/chemokine responses are associated with diseases such as influenza, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), SARS-CoV, and Middle East respiratory syndrome coronavirus [17]. The association of CD4+ T cell loss, increased viral load, and viral set-point with cytokine storm during HIV/SIV infection was well documented earlier [3][4][5][6]18]. The dysregulation of cytokines after HIV/SIV infection was also reported to be associated with various clinical events such as comorbidities, disease progression, and mortality [19,20].
Multiple HIV infection studies have shown changes in systemic plasma cytokine/ chemokine levels, including IFN-α, TNF-α, IFN-γ, IL-2, IL-4, IL-10, IP-10, and IL-1RA during the acute phase [2,[21][22][23][24][25]. However, a significant limitation of all these human studies was the shortage of information about the exact time of initial HIV infection. Similarly, studies with acute SIV infection have been performed in non-human primates by measuring cytokine mRNA expressions in tissues [26][27][28][29][30][31][32][33][34]. The changes in MIP-1α, TNF-α, IL-6, and IFN-α were detected in different mucosal and LN tissues at 3-10 days post-mucosally infected macaques [32]. Increased MIP-3α mRNA expression has also been reported in endocervical epithelium 24 h after mucosal SIV infection [33]. Conversely, the relationship between the abundance of mRNA expression and the manifestation of cellular protein levels is complex. The mRNA transcript levels are insufficient to determine the predicted protein levels as it depends on the cells' steady or highly dynamic state and the post-transcriptional and post-translational regulation [35][36][37]. Immunohistochemistry analysis showed increased TGF-β in LN tissue expression at day 7 from a cohort of female RhMs infected intravenously with 1 MID 50 SIV MAC 239 [38]. Our earlier study using flow cytometry and immunohistochemistry assays showed a significant increase in TGF-β expression in intestinal mucosal tissue at 21 days post-SIV infection [39]. Nevertheless, a clear understanding of the regulation of different cytokines/chemokines during acute SIV infection is needed when the most sensitive and robust MSD technology [40] allows us to measure multiple cytokines/chemokines in circulation. The SIV-RhM model strongly recapitulated the HIV infection in humans and was used here to understand the dynamics of 61 different cytokines and chemokines involved in controlling immune cell trafficking and regulating the nature of immune responses during acute infection. We observed a significant change in 11 cytokines/growth factors and 9 chemokines during acute SIV infection. The deviations in the amounts were detected as early as 6 h post-infection and were below the baseline values in all the instances for at least four days post-infection.
IL-15 upregulates early during SIV/HIV infection, which concurs with our findings and impacts increasing CD4 expression on memory CD4+ T cells [21,41]. However, we did not find any correlation between IL-15 and PVL or CD4 counts. IL-1RA plays an essential role in immune regulation by inhibiting proinflammatory cytokines such as IL-1α and IL-1β [42]. Even though nearly the same stimuli induce IL-1RA and IL-1, increased expression of IL-1RA and no change in the IL-1β production was observed. IL-1RA production was also described to be correlated with the markers of HIV progression [43]. Increased circulatory IL-18 is thought to contribute to HIV pathogenesis by enhancing the death of NK cells, as observed in in vitro studies [44]. A significant correlation between increased circulatory IL-18 and decreased CD4 count suggests that IL-18 can be considered a potential biomarker of disease progression at the acute stage of infection. IL-18 was also reported to promote HIV replication by upregulating the CXCR4 expression [12]. Our data confirm that IP-10 can also be a potential biomarker to predict PVL and CD4 levels during acute infection. An earlier in vitro study with PBMCs and monocyte-derived macrophages demonstrated that IP-10 induces increased HIV-1 replication [45] by dysregulating T-and NK-cell functions [46,47]. Increased IP-10 after HIV infection and its contribution to disease progression have been reported elsewhere [21,[48][49][50]. FLT3L has been reported as a protective cytokine in reducing viremia by expanding and mobilizing the plasmacytoid dendritic cells (pDCs) during acute HIV infection in a humanized mice model [51]. However, the significant correlation between FLT3L and PVL or CD4 levels suggests that increased FLT3L is fueling more viral replication by enhancing lymphopoiesis. It is also possible that protective FLT3L is trying to improve the pDC population, but those cells are not functional enough to control the CD4+ T cell loss and viral replication.
MCP-1 upregulates CXCR4 coreceptor expression in resting CD4+ T cells, facilitated CXCR4-tropic HIV to infect the CD4+ T cells [52], and was associated with CNS disorder [53]. Here, we have also shown a significant correlation between MCP-1 and PVL or CD4 levels suggesting that MCP-1 can be used as a biomarker to demonstrate acute SIV pathogenesis and disease progression, which agrees with previous studies [54,55]. MCP-2/MCP-4/MIP-1α and Eotaxin-1 bind to CCR5 and CCR3, respectively, and regulate viral entry to the target cells [56,57]. MCP-2 is also involved in monocyte migration and inflammatory responses and has been recently shown to be involved in acute respiratory distress syndrome [58][59][60]. Increased MCP-2 expression during acute infection might have a significant role in inflammation and the inhibition of MCP-2 expression might be beneficial in controlling early inflammation, which needs future study.
MIP-3β facilitates HIV entry and the establishment of latency by allowing the integration of viral genomes in resting CD4+ T cells from in vitro studies [61,62]. Our study also suggests that MIP-3β can be used as an additional biomarker to determine the viral replication and the loss of CD4+ T cells during acute infection. Some dysregulated chemokines regulate the HIV infection or replication by targeting virus co-receptors directly. These chemokines include SDF-1α, MCP-2, MCP-4, MIP-1α, and Eotaxin-1, which bind either to CXCR4, CXCR5, CCR5, or CCR3 and regulate viral infection [63]. The downregulation of SDF-1α in our study might indicate that SIV tends to infect CD4+ T cells more efficiently due to the availability of CXCR4 receptors at the early stage of infection.
Neutrophils are the first cells responsible for primary defense against pathogens and regulating inflammation and immune functions [64]. Neutropenia develops in HIVinfected individuals at the chronic stage of the disease. However, neutropenia has also been reported during acute infection [65,66], which is in agreement with our data suggesting that neutropenia might have a significant effect in inducing primary defense mechanisms as well as downregulations of several major cytokines/chemokines during the early few days after infection, which leads to increased viral replication and CD4 T cell loss.

Conclusions
The loss of peripheral CD4 T cells and increased PVL detected at day 14 and day 7 post-SIV infection, respectively, as well as neutropenia seen from day 3 to day 14 in SIV-infected RhMs, suggest that the early loss of neutrophil-mediated primary defense has a significant impact on initial virus replication and the loss of peripheral CD4+ T cells. A substantial change in 11 cytokines/growth factors and 9 chemokines out of 61 cytokines/chemokines were observed during acute SIV infection. Almost all the cytokines/chemokines were below the baseline values for an initial couple of days of infection. Our experimental design does not address any responses detected beyond the 21 days of acute infection. Since we have only quantified cytokines and chemokines and performed the correlation analysis, the role of each cytokine/chemokine in modulating disease progression requires future study. We detected six important cytokines/chemokines (IL-18, IP-10, FLT3L, MCP-1, MCP-2, and MIP-3β) that can be used as biomarkers to predict the peripheral CD4+ T cell loss and increased viral replication during the acute phase of SIV/HIV infection. Hence, vaccines or drugs modulating IL-18, IP-10, FLT3L, MCP-1, MCP-2, and MIP-3β expression might be promising novel tools to induce early antiviral responses to combat acute SIV/HIV infection.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/vaccines11020264/s1, Figure S1: Twenty cytokine/chemokine profiles from plasma for a 21-day study period in uninfected control macaques are shown (n = 4). Each red circle of the scattered plot (including mean ± SE) represents cytokine/chemokine concentration for an individual macaque. There was no significant difference in the cytokine/chemokine values at any time point compared to day 0.

Funding:
The study was supported by National Institutes of Health grant funding R01DK109883 (BP).

Institutional Review Board Statement:
The study was reviewed and approved by Tulane University IACUC protocol number 3818. TNPRC is fully accredited by AALAC, Animal Welfare Assurance No. A4499-01.

Informed Consent Statement: Not applicable.
Data Availability Statement: All relevant data are included within the manuscript. The raw data are available on request from the corresponding author.