Mutations in ace2 gene modulate cytokine levels and alter immune responses in Mycobacterium tuberculosis and SARS-CoV-2 co-infection: a Cameroonian cohort

Abstract

Introduction: SARS-CoV-2 and Mycobacterium tuberculosis (Mtb) share similarities in their modes of transmission, pathophysiological symptoms, and clinical manifestations. An imbalance in the immune response characterised by elevated levels of some inflammatory cytokines caused by tuberculosis (TB) and COVID-19 may increase the risk of developing a severe disease-like condition. It has been reported that TB increases the expression levels of Ace2 (angiotensin converting enzyme 2) and Tmprss2 (transmembrane protease serine 2) proteins, which are essential for COVID-19 pathogenesis. Single nucleotide polymorphisms (SNPs) variants of ace2 and tmprss2 genes can impact virus and host-cell interactions and alter immune responses by modulating cytokine production. This may modify the susceptibility and/or severity in COVID-19-infected people. The role of SNPs in ace2 and tmprss2 in relation to Mtb and SARS-CoV-2 co-infection is relatively underexplored.

Method: In this study, genotype frequency of 10 SNPs of ace2 and 03 SNPs of tmprss2 genes in a Cameroonian cohort consisting of COVID-19-positive (n = 31), TB-positive (n = 43), TB-COVID-19 co-infected (n = 21), and a control group (n = 24) were studied. The immune response was estimated by quantitating inflammatory cytokine levels alongside self-reported and clinically diagnosed symptoms. The relationship between specific genetic mutations in these ace2 gene SNPs and their impact on cytokine expression levels in Mtb and SARS-CoV-2 co-infected patients was investigated.

Results: We identified wild-type, heterozygous, and double-mutant genotypes in seven SNPs (rs2285666, rs6632677, rs4646116, rs4646140, rs147311723, rs2074192 and rs4646142) in ace2 gene, which showed significant variations in distribution across the study groups. Our most significant findings include the association of double mutant alleles (AA) of rs4646140 and rs2074192 in the ace2 gene with decreased IL-6 and IL-2 expression levels respectively in TB-COVID-19 participants. Also, the double mutant alleles (AA) of rs4646116 were responsible for increased expression level of IL-2 in TB-COVID-19 patients. Additionally, elevated serum levels of AST, urea, and D-dimer, as well as increased plasma concentrations of IL-10, IFN-γ, and TNF-α, have been associated with co-infections involving Mtb and SARS-CoV-2.

Conclusion: These biomarkers may reflect the complex interplay between the two pathogens and their impact on host immune responses and disease progression. This study highlights the critical role of genetic and immunological factors in shaping altered immune responses during co-infections involving Mtb and SARS-CoV-2. By elucidating these factors, the findings provide a foundation for a deeper understanding of host-pathogen interactions and their implications for disease progression and outcomes. Furthermore, this research has the potential to drive advancements in diagnostic approaches enabling more accurate detection and monitoring of co-infections.

Keywords: COVID - 19; Mycobacterium tuberculosis; SARS-CoV-2; SNPs; ace2; immune response; tmprss2; tuberculosis.

Figure 1

Study participant recruitment and classification. (A) Map showing the Centre region of Cameroon with different study sites. Samples for this study were collected at four hospitals: JH, DDH, RCH, and EBH, which are situated in Yaoundé. The sample sizes from each district were as follows: JH: 83; DDH: 102; RCH: 96; and EBH: 118. The map was created using QGIS version 3.32.3. (B) Based on COVID-19 and Tuberculosis infection status, participants were grouped into Controls: COVID-19 positive, Tuberculosis positive, and Tuberculosis and COVID-19 association. In total, our sample size consisted of 119 participants. JH, Jamot Hospital; DDH, Djoungolo District Hospital; RCH, Red Cross Hospital; EBH, Ekoumdoum Baptist Hospital.

Figure 2

Increased serum levels of AST, UREA, D-dimer and plasma levels of IL-4, IL-10, TNF-α, IFN-γ showed association with Tuberculosis and COVID-19 co-infection. Each individual’s data point is shown, with median values indicated by horizontal lines, the other lines above and below the median represent the interquartile range (IQR) and the minimum and maximum values as appropriate. (A) Biochemical parameters such as AST, ALT, D-dimer, Total-bilirubin, Direct-bilirubin, Urea and Crea were measured using spectrophotometry-based assays. (B) Plasma levels of anti-inflammatory cytokines are associated with TB/COVID-19association. Sandwich ELISA using Origene kits was employed to measure the systemic levels of anti-inflammatory cytokines (IL-10 and IL-4) and pro-inflammatory (TNF-α, IFN-γ, IL-6, IL-2 and IL-β) in plasma samples of the study population. Unpaired t-test was carried out between various combinations to determine statistically significant differences. AST, Aspartate aminotransferase; ALT, Alanine aminotransferase; CREA, Creatinine; TNF, Tumour necrosis factor; IFN, Interferon; IL, interleukin; TB, tuberculosis; COV, COVID-19; TBCOV, Tuberculosis and Covid-19 association. *p <0.05, **p <0.01, ***p <0.001.

Figure 3

Wild-type, heterozygous, and double-mutant genotypes in seven SNPs (rs2285666, rs6632677, rs4646116, rs4646140, rs147311723, rs2074192 and rs4646142) in ace2 gene showed significant variations in distribution across the four study groups: Genomic DNA isolated using Zymo Human DNA Isolation Kit was subjected to quantitative real-time PCR with TaqMan probes. TaqMan SNP Genotyping Assays were employed to identify SNPs in ace2 and tmprss2 genes. (A) specific genotype frequencies in ace2. (B) specific genotype frequencies in tmprss2. Multigroup analysis was carried out using the Chi-square test, and SNPs having a p-value of less than 0.05 were considered to have a significantly perturbed presence between the groups (Control, TB, COV, and TBCOV). TB, Tuberculosis positive; COV, COVID-19 positive and TBCOV, Tuberculosis and COVID-19 association. Asterisks represent p-values determined after multivariate analysis that was done using the Chi-square test comparing the mutation patterns in the four groups, namely, control, TB, COVID-19, and TB-COVID-19.

Figure 4

Reduced IL-6 and IL-2 levels linked to double mutant alleles of ACE2 gene polymorphisms rs4646140 and rs2074192 in TB-COVID-19 co-infected patients. The wild-type, heterozygous, and double-mutant genotypes in seven SNPs (rs2285666, rs6632677, rs4646116, rs4646140, rs147311723, rs2074192 and rs4646142) in ace2 gene were associated with expression levels of some pro-inflammatory (IL-6, IL-2, TNF-α and IFN-γ) and anti-inflammatory (IL-10) cytokines in individuals with both TB and COVID-19. Only statistically significant association are reported. The letters on the x-axis denote specific genotypes (wild-type, heterozygous mutant and double mutant). The bars represent the median values of cytokine levels, with error bars indicating the interquartile range (IQR). Horizontal lines and corresponding values above the bars indicate statistical comparisons, with p-values provided to highlight significant differences between groups.