Study of Inflammatory Biomarkers in Treatment-Naive HIV Patients and Their Correlation With Clusters of Differentiation 4 (CD4) Count

Introduction The human immunodeficiency virus (HIV) primarily targets clusters of differentiation 4 (CD4)+ T cells and other immune cells, leading to immune dysfunction. Cytokines such as interleukin (IL)-23 and IL-27 have complex roles in HIV-associated disease progression, affecting viral replication and immune responses. This study aimed to explore the correlation between HIV-related CD4 lymphopenia and the inflammatory cytokines IL-23 and IL-27 in treatment-naive HIV patients. Materials and methods This is a single-center, prospective, observational study conducted at the Antiretroviral Treatment (ART) Center of Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, Uttar Pradesh, India. Sixty-five treatment-naive HIV seropositive patients were recruited in this study. Quantitative estimation of inflammatory biomarkers (IL-23 and IL-27) was performed using enzyme-linked immunosorbent assay (ELISA). The fluorescent-activated cell sorter count (FACSCount) technology was used to determine the CD4+ T-cell count. Results Our study revealed that HIV-infected individuals had significantly higher levels of IL-23 (868.9±246.7 pg/mL vs 98.3±86.6 pg/mL, p < 0.01) and IL-27 (1629.5±518.5 pg/mL vs 291.3±225.2 pg/mL, p < 0.01) compared to healthy controls. Additionally, we found a strong positive correlation between CD4 count and IL-23 titers (r = 0.93, p < 0.01), as well as between CD4 count and IL-27 titers (r = 0.92, p < 0.01) in HIV-positive individuals. Conclusion The findings suggest that these cytokines respond to HIV infection and may potentially play a crucial role in restraining HIV replication and slowing down the progression of the disease.


Introduction
In 1981, the emergence of acquired immune deficiency syndrome (AIDS) marked a watershed moment in medical history, initially impacting young homosexual men and posing perplexing questions for the scientific community [1].This pursuit led to the identification of human immunodeficiency virus type 1 (HIV-1) as the causative agent, ushering in one of the most devastating infectious diseases in history [2].Its most profound impact has been in developing nations, particularly sub-Saharan Africa, where young adults bear a disproportionate burden of infection.As of 2022, an estimated 39 million individuals were living with HIV/AIDS, with approximately 0.63 million deaths attributed to AIDS-related illnesses worldwide.In India, approximately 2.5 million individuals were living with HIV, with around 40 thousand deaths due to AIDSrelated diseases [3].
Significant advancements in treating HIV largely emerged with the introduction of highly active antiretroviral therapy (HAART), which effectively reduces viral loads in the peripheral blood to undetectable levels.However, despite the availability of HAART, the virus persists within reservoirs in the body, primarily within the genomes of non-activated T lymphocytes and other latent cells [4].Therefore, there is a pressing need to explore immunology-based therapies that empower the host to maintain control over the infection.In this context, cytokines emerge as promising targets for immunomodulatory interventions.

Data collection
Enrolled patients were interviewed in detail regarding sociodemographic information such as age, gender, residence, education, sexual orientation, marital status, and mode of HIV transmission.Patients were given information about the known routes of HIV transmission and asked to select the most likely route for HIV acquisition in their case to record the mode of transmission of HIV.If a patient was unsure of their possible route of HIV acquisition or refused to disclose sexual exposure history, declined to disclose prior intravenous (IV) drug use, or refused to disclose or could not recall past medical, surgical, or blood transfusion history, that patient was labeled as "unknown" in the possible mode of transmission (MOT).The WHO standards for clinical staging of HIV/AIDS in adults were used to establish the clinical stage [8].

Measurement of plasma cytokines
Blood samples were collected from each patient in anticoagulant tubes containing ethylenediaminetetraacetic acid (EDTA).The blood samples were centrifuged for 15 minutes at 2000 rpm to separate plasma, following which plasma samples were immediately stored at -80º C until use.Plasma levels of IL-23 and IL-27 were measured by the enzyme-linked immunoassay (ELISA) method using a commercial kit (Diaclone Research SAS, Besançon, France) by employing appropriate and specific biotinylated antibody pairs according to the manufacturer's protocol.
The sensitivity of kits for IL-23 and IL-27 were 20 pg/ml and 12.8 pg/ml, respectively.Briefly, 100 μl of the purified capture antibodies were adsorbed overnight on polystyrene microtitre plates at 4⁰C in the carbonate-bicarbonate buffer of pH 9.5.Plates were washed five times with phosphate-buffered saline with Tween 20 (PBST) and blocked with 5% skimmed milk.After the usual washing steps, 100 μl of the patient's plasma was dispensed in each well to determine its cytokine content.After the stipulated incubation time, the plates were thoroughly washed and incubated with a biotinylated mouse anti-human cytokine detection antibody.Afterward, the plate was washed three times with PBST.Subsequently, 100 μl of the streptavidinhorseradish peroxidase (HRP) conjugate was added to each well, and the plate was incubated for 30 minutes at room temperature.The plate was again washed three times with PBST, and finally, a colored complex was developed with tetramethylbenzidine (TMB).The reaction was then stopped with 2N H 2 SO 4 , and the absorbance was read at 450 nm with a microtitre ELISA plate reader (Bio-Rad Laboratories, Inc., Hercules, California, United States) within 15 minutes after stopping the reaction.A known specific recombinant cytokine was used as a standard for calculating the level of the given cytokine in the samples tested, and the concentration was expressed as pg/mL relative to the concentration of the standard as determined by plotting the standard curve.

Estimation of CD4 count
The anticoagulated whole blood sample was incubated with fluorescent antibodies.The processed sample was then analyzed, and the CD4+ T-cell count was determined by flow cytometry using the FACSCount system (Becton, Dickinson and Company, Franklin Lakes, New Jersey, United States) as per the manufacturer's instructions.

Statistical analysis
Continuous quantitative variables were presented as mean ± standard deviation, while qualitative variables were expressed as percentages.Independent samples t-test was used to compare plasma cytokines between patients with HIV and healthy controls.Pearson's correlation coefficient was used to evaluate the association between plasma cytokine titers and CD4 cell counts.Statistical analyses were performed using the IBM SPSS Statistics for Windows, Version 25.0 (Released 2017; IBM Corp., Armonk, New York, United States).A p-value of <0.05 was considered statistically significant.

Results
In this prospective, observational study, 65 HIV seropositive treatment-naive patients were enrolled.The sociodemographic profiles of the patients are shown in Table 1.

TABLE 1: Sociodemographic profile of the HIV patients
Age is expressed as mean±SD, and qualitative variables are expressed as frequency (percentages).

Association between cytokines and CD4+ count
Additionally, within the patient group, there was a strong positive correlation between CD4 count and IL-23 titers (r = 0.93, p < 0.01), as well as between CD4 counts and IL-27 titers (r = 0.92, p < 0.01) in treatmentnaive HIV patients (Figure 3).

Discussion
This prospective, observational study examined the correlation between HIV-related CD4 lymphopenia and inflammatory cytokines, namely IL-23 and IL-27.In our study, 65 newly diagnosed HIV-positive patients were included, with a mean age of 33±8.52,predominantly male.The majority of the patients belonged to the middle-aged group (76.9%).The demographic profile was similar to previous studies [9].Most of our patients had no or low literacy levels (n=34; 52.30%), consistent with findings of earlier studies [10].Marital status analysis revealed that 73.84% of patients were married, aligning with the survey by Bishnu et al. in 2014 (72.50%).The most common mode of transmission in our study was unprotected heterosexual contact (81.5%), in line with national figures and previous studies [9,11].However, the transmission through blood and blood products was alarmingly high (7.7%),primarily attributed to blood transfusions performed in private hospitals using blood from private blood banks.This issue highlights the importance of stringent regulations and screening procedures [12].Intravenous (IV) drug abuse, which was previously less common in our region [13], showed an increasing trend (4.6%).This behavior has played a critical role in the HIV epidemic in various regions, particularly Asia and southern Europe.Effective prevention strategies employed in developed countries, such as outreach to IV drug abusers, peer education programs, and social network interventions, are now being adopted in developing countries [12,14].
In the present study, the levels of IL-23 were considerably increased in persons who tested positive for HIV compared to a group of healthy individuals serving as controls (p < 0.01).IL-23 is crucial in the activation of the Th17 pathway.A heterodimeric receptor for IL-23 that transmits signals is made up of both a distinct IL-23 receptor (IL-23R) chain and the IL-12 receptor beta 1 (IL-12Rβ1) chain [15].Furthermore, the study discovered a strong positive correlation between CD4+ T cell counts and IL-23 levels (r = 0.93, p < 0.01) among individuals with HIV, indicating that IL-23 may contribute to supporting CD4+ T cell populations in HIV-infected individuals.The elevated IL-23 levels could potentially help mitigate the adverse effects of HIV replication on the immune system.Notably, this research represents the first report of a positive relationship between IL-23 and CD4+ T cell counts in treatment-naive HIV seropositive individuals.
The study additionally examined IL-27 levels in HIV-infected individuals and observed a significant elevation in IL-27 levels compared to healthy controls (p < 0.001).This finding is consistent with previous reports describing the inhibition of HIV-1 replication by IL-27, where the increased cytokine levels are attributed to the early-stage immune response [16,17].IL-27 is a member of the IL-6/IL-12 family produced by macrophages, endothelial cells, and dendritic cells.IL-27 stimulates Th1 responses and enhances the survival of naive T and B lymphocytes [7].IL-27 inhibits HIV-1 replication through various mechanisms.We also observed a significant positive association (r = 0.92, < 0.01) between IL-27 and CD4+ T cell counts.
Our findings suggest that IL-27 promotes the expansion of naive CD4+ T cells in vivo.He et al., in their study of 120 HIV-infected treatment-naive Chinese individuals, also found a positive correlation between IL-27 levels and CD4+ T cell counts [17].
The results of our study strongly indicate that both IL-23 and IL-27 may play a pivotal role in directly inhibiting HIV replication.Additionally, these cytokines may facilitate the rapid expansion of naive CD4+ T cells, potentially mitigating the adverse consequences of HIV replication.Conversely, individuals with low titers of IL-23 and IL-27 may face challenges in effectively controlling the virus, as these cytokines are integral to the immune response against HIV infection.

FIGURE 1 :
FIGURE 1: Flowchart depicting patient enrollment in the study.HIV: human immunodeficiency virus; HAART: highly active antiretroviral therapy; ART: antiretroviral therapy; HCV: hepatitis C virus; HBsAg: hepatitis B surface antigen