Evaluation of 34 Cytokines and Vitamin D Status Reveal A Sexually-Dimorphic Active Immune Response to SARS-CoV-2

Background: Several observational studies have inconsistently demonstrated that vitamin D deficiency is a risk factor for coronavirus disease-19 (COVID-19) infection and severity. Discrepancies in results may partially be explained by the individuals’ immune profiles, which are modulated, in varying degrees, by vitamin D status and sex hormones. Methods: In this study we evaluated the differences and associations of serum levels of 25(OH)D with 34 cytokines in 220 adults (82 controls (41 males; 41 females) and 138 SARS-CoV-2 patients (79 males and 59 females)) with and without COVID-19. Results: Serum 25(OH)D levels were significantly lower in the SARS-CoV-2 group than in the controls. Serum IP-10, MCP-1, CRP, IFNγ, IL-10, IL-13, IL-17α, IL-23, and IL-6 were significantly higher in COVID-19 patients compared to controls. Serum levels of VEGF, IFNγ, IL-13, and IL-5 were significantly higher in male patients than in females. 25(OH)D was significantly correlated with EFG (R = 0.39, p < 0.05) and IL-15 (R = 0.39, p < 0.05) in male patients, while it was inversely correlated with CRP (R = −0.51, p < 0.05) in female patients. Conclusions: Altered levels of cytokines, chemokines, and vitamin D were observed in SARS-CoV-2 adult patients. These expressions were sexually dimorphic and thus highlight the sex-specific nature of the active immune response following SARS-CoV-2 infection.


Introduction
The ongoing coronavirus disease 2019 (COVID- 19) pandemic, now in its third year, has been responsible for an estimated 18 million human deaths, reported as excess mortality by the University of Washington [1]. The pathogen responsible for this global tragedy is the severe acute respiratory syndrome novel coronavirus 2 (SARS-CoV-2), the latest from a family of highly pathogenic human coronaviruses also including SARS-CoV and the Middle East respiratory syndrome coronavirus (MERS-CoV) [2]. COVID-19 can cause a wide range of clinical presentations, ranging from asymptomatic to severe viral pneumonia with respiratory failure and even death [3][4][5]. While there is abundant evidence on the clinical sequelae of COVID-19 infections, the pathogenesis of COVID-19 has not been well defined [6,7].
All study participants provided hand-written informed consent prior to inclusion. A general questionnaire was administered, which included medical history (hypertension (HPN), diabetes mellitus (DM), congestive heart failure (CHF), cardiovascular disease (CVD), chronic kidney disease (CKD), and stroke, along with smoking history), as well as a list of medications (angiotensin receptor blockers (ARB), β-blockers, angiotensin converting enzyme (ACE) inhibitors, anti-coagulants, anti-platelet drugs, statins, calcium channel blockers (CCB), oral hypoglycemic agents, and insulin) prior to recruitment. Ethical approval was obtained from the Institutional Review Board (IRB) of the College of Medicine in KSUMC-KKUH in Riyadh, Saudi Arabia (E-20-4803, 11 June 2020).

Anthropometry and Blood Collection
Fasting and non-fasting blood samples were collected following nasal and oral swabs. Anthropometric measurements taken included height (cm), weight (kg), body mass index (BMI, kg/m2), and waist (cm) and hip (cm) circumferences. Trained nurses were assigned to take blood pressure (systolic and diastolic in mmHg) (mean of two readings) using standard procedures (Table S1).

Data Analysis and Sample Size Determination
Data were analyzed using SPSS version 21. Results are presented as mean ± standard deviation for continuous normal variables and median (1st quartile-3rd quartile) for nonnormal continuous variables. Independent sample T-test and Mann-Whitney U-test were used to examine SARS-CoV-2 and gender difference in continuous normal and non-normal biochemical variables, respectively. Analysis of covariance (ANCOVA) was used to obtain age and BMI adjustment. Logistic regression was used with forward stepwise selection with selected cytokines to identify the most significant associations with SARS-CoV-2. Pearson and Spearman correlation coefficients were used to test for association between vitamin D and other normal and non-normal biochemical parameters, respectively. Significance was set at p < 0.05.
A post-hoc power analysis revealed that this sample size achieves a power higher than 80% with an effect size of 0.196 (two-sided α of 0.05), as calculated using the G*Power.

Associations of Vitamin D Status, Cytokines, and Chemokines
No significant associations were observed between 25(OH)D and measured cytokines and chemokines regardless of SARS-CoV-2 status (Table S2). However, when stratified according to sex (Table 4)   Logistic regression was used with forward stepwise selection to identify the cytokines with the highest association with SARS-CoV-2 (Table 5). All cytokines that were significantly elevated in SARS-CoV-2 patients were entered into the regression along with age and BMI. Results showed that only IP-10, IFNγ and IL-23 were independently associated with SAR-CoV-2. IFNγ was the cytokine showing the highest association with COVID-19 infection, and was therefore considered a significant risk factor for COVID-19 in our cohort (OR 169.2 (95% CI 5.5-5200.4); p = 0.003)).

Discussion
The present study correlated differences in serum levels of 25(OH)D with 34 cytokines and chemokines in a cohort of 220 adult Arab males and females (138 COVID-19 patients and 82 controls) in Riyadh, Saudi Arabia. Studies from previous epidemics revealed that inflammatory responses and abnormal cytokine profiles were correlated with acute lung injury and pulmonary inflammation in SARS- [10] and MERS-CoV [11] infections. In the present study, we detected significantly higher serum levels of measured cytokines, chemokines, and inflammatory proteins (IP-10, CRP, IFNγ, IL-10, IL-13, IL-17α, IL-23, and IL-6) in COVID-19 patients than in controls. SARS-CoV-2 infection induces over-production of pro-inflammatory cytokines and chemokines as a defense mechanism against the viral infection. On the other hand, a dysregulation, i.e., hyper-inflammation, results in cytokine release syndrome (CRS) leading to apoptosis of immune cells, impaired cytotoxic function, and tissue damage [13]. More importantly, our results showed that male patients had significantly higher serum concentrations of VEGF, IFNγ, IL-13, and IL-5 than female patients. This is in line with previous studies demonstrating a stronger response in cytokines and chemokines in SARS-CoV-2-infected male than in female patients [6,13]. Differences in the expression of cytokines and chemokines secondary to infection are partially yet substantially controlled by sex hormones, which modulate the innate immune function at various stages of human life concurrent with hormonal changes. This can explain why males and females may present different clinical symptoms and time-to-recovery given the same infection [6,[34][35][36].
Our data indicated an elevation in IP-10/CXCL-10, CRP, IL-10, and IL-6. A study by Notz et al. on 39 patients admitted to the intensive care unit with confirmed COVID-19, reported that IL-6, CRP, and IL-10 levels were substantially increased in patients in comparison to controls [37]. IP-10 is produced by monocytes and macrophages and has been suggested as a biomarker for severity in COVID-19 [38]. Previous studies reported that anti-retrovirus treatment reduced plasma IP-10 indicating an important role of IP-10 in the pathogenesis of the diseases [38]. Huang et al. [7] showed a significant increase in IFN-γ, IP-10, and MCP-1 levels in COVID-19 patients.
Remarkably, our results showed decreased serum levels of TNFα, IL-4, IL-2, IL-1β, IL-2, IL-7, and GM-CSF in COVID-19 patients compared to controls, which indicates that the cytokine profile in mild cases of COVID-19 does not match a classical cytokine storm where TNFα is considered the leading cytokine and a therapeutic target. Recently, Liu et al. detected that levels of TNFα and IFNγ did not increase in mild COVID-19 [39]. However, to maintain balance between pro-and anti-inflammatory mechanisms is a significant factor in a robust immune response. Cytokines and chemokines are vital to initiate and augment the response of the innate and adaptive immune system [40]; concomitant hyper-inflammation has been linked to severe pulmonary dysfunction though [41]. 25(OH)D serum levels were significantly lower in COVID-19 patients than in controls after adjusting for the main confounding factors. In addition, 25(OH)D was positively correlated with EFG and IL-15 in male patients, and inversely correlated with CRP in female patients. In a retrospective study on 107 individuals screened for COVID-19 (27 RT-PCR positive and 80 negative), the authors reported that COVID-19 patients had lower median 25(OH)D levels (11.1 ng/mL) compared to their RT-PCR-negative counterparts (24.6 ng/mL) [42]. Recently, Ilie et al. detected that serum 25(OH)D levels were significantly associated with COVID-19 number of cases and mortality in some European countries [43]. Additionally, experimental studies showed that 25(OH)D can inhibit the inflammatory cytokines IL-6 and TNFα by attenuating the activation of p38 MAP kinase in human macrophages/monocytes. Moreover, 25(OH)D promotes the stimulation of T regulatory cells, thus inhibiting production of pro-inflammatory cytokines, including IL-21, IFNγ, and IL-17 [44]. However, recently, Hastie et al. reported no relationship between 25(OH)D serum levels and COVID-19 risk in a cohort of more than 340,000 participants from the UK Biobank [45]. Despite the inconsistencies in associations, the use of vitamin D as an adjuvant therapy for COVID-19 has merits, as it has been observed to resolve COVID-19 symptoms faster even in mild-moderate cases known to have vitamin D deficiency [46,47].
The authors acknowledge several limitations that should be considered in interpreting the findings. Corrections for multiple comparisons were not performed given the small sample size. However, we believe this approach only increases the likelihood of type 2 errors and truly significant differences become non-significant [48]. Another limitation is that the actual virus loads were not obtained since all SARS-Cov-2 testing was centralized by the Ministry of Health, hence correlations with viral infection/replication were not performed and analysis was limited to only presence and absence of SARS-CoV-2. Nevertheless, the present study is one of the few to demonstrate differences in the circulating cytokine and chemokine profiles of male and female Arab adult patients with and without COVID-19 and their associations with serum 25(OH)D levels. A prospective design with a larger cohort could yield temporal changes in the immune response after infection with COVID-19.

Conclusions
Our results indicated a sexual dimorphism in the expression of circulating 25(OH)D, cytokines, and chemokines among COVID-19 patients as compared to controls, suggesting a sex-bias in the active immune response. Serum levels of IP-10, CRP, IL-10, IFNγ, IL-13, IL-17α, IL-23, IL-6 were particularly upregulated in COVID-19 patients compared to controls. Further studies are needed to prospectively investigate and consider the role of sexual dimorphism in the strength of innate and adaptive immunity as it may play a pivotal role in vaccine efficiency and overall immune response.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/healthcare10122571/s1, Figure S1: Serum cytokine and chemokine levels of SARS-CoV-2 patients (positive) and healthy controls (negative); Table S1: General anthropometric and clinical characteristics of study participants; Table S2: Correlation between vitamin D and selected study parameters according to COVID-19 status. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author. The data are not publicly available due to privacy protection.