The functional response of human monocyte-derived macrophages to serum amyloid A and Mycobacterium tuberculosis infection

Abstract

Introduction: In the course of tuberculosis (TB), the level of major acute phase protein, namely serum amyloid A (hSAA-1), increases up to a hundredfold in the pleural fluids of infected individuals. Tubercle bacilli infecting the human host can be opsonized by hSAA-1, which affects bacterial entry into human macrophages and their intracellular multiplication.

Methods: We applied global RNA sequencing to evaluate the functional response of human monocyte-derived macrophages (MDMs), isolated from healthy blood donors, under elevated hSAA-1 conditions and during infection with nonopsonized and hSAA-1-opsonized Mycobacterium tuberculosis (Mtb). In the same infection model, we also examined the functional response of mycobacteria to the intracellular environment of macrophages in the presence and absence of hSAA-1. The RNASeq analysis was validated using qPCR. The functional response of MDMs to hSAA-1 and/or tubercle bacilli was also evaluated for selected cytokines at the protein level by applying the Milliplex system.

Findings: Transcriptomes of MDMs cultured in the presence of hSAA-1 or infected with Mtb showed a high degree of similarity for both upregulated and downregulated genes involved mainly in processes related to cell division and immune response, respectively. Among the most induced genes, across both hSAA-1 and Mtb infection conditions, CXCL8, CCL15, CCL5, IL-1β, and receptors for IL-7 and IL-2 were identified. We also observed the same pattern of upregulated pro-inflammatory cytokines (TNFα, IL-6, IL-12, IL-18, IL-23, and IL-1) and downregulated anti-inflammatory cytokines (IL-10, TGFβ, and antimicrobial peptide cathelicidin) in the hSAA-1 treated-MDMs or the phagocytes infected with tubercle bacilli. At this early stage of infection, Mtb genes affected by the inside microenvironment of MDMs are strictly involved in iron scavenging, adaptation to hypoxia, low pH, and increasing levels of CO₂. The genes for the synthesis and transport of virulence lipids, but not cholesterol/fatty acid degradation, were also upregulated.

Conclusion: Elevated serum hSAA-1 levels in tuberculosis enhance the response of host phagocytes to infection, including macrophages that have not yet been in contact with mycobacteria. SAA induces antigen processing and presentation processes by professional phagocytes reversing the inhibition caused by Mtb infection.

Keywords: host-pathogen transcriptomics; human serum amyloid A; immunological response; monocyte-derived macrophages; tuberculosis.

Figure 1

GO biological process enrichment analysis and dotplot charts. MQ represents control macrophages, MQ+Mtb, macrophages infected with Mtb; MQ+SAA, macrophages treated with SAA. The diagram was generated by ShinyGO 0.77.

Figure 2

RTPCR (TaqMan) analysis of transcripts for CCL19, CSF2, and CXCL8 genes of MDMs. MQ represents control macrophages, MQ+Mtb, macrophages infected with Mtb; MQ+Mtb/SAA, macrophages infected with Mtb opsonized with SAA; MQ+SAA, macrophages treated with SAA. The number of tested transcripts was normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeping gene and relative fold changes in gene expression in comparison to the control strain were calculated using the delta method (2-ΔΔCT). The data distribution was evaluated by the Shapiro-Wilk normality test. Statistical analysis was performed by one-way ANOVA with a post-hoc Tukey test. *represents p<0.05, **p<0.0021, ***p<0.0002, ****p<0.0001.

Figure 3

Phagosome and phagocytosis promoting receptors of MDMs. The expression level of genes compared to the control is presented as a heatmap. MDMs infected with Mtb are represented by A, hSAA-1 treated MDMs by B, MDMs infected with hSAA-1 opsonized Mtb compared to MDMs infected with nonopsonized Mtb by C. The analysis was completed based on total RNA sequencing isolated from MDMs of three blood donors (control MDMs and hSAA-1 treated MDMs) or four blood donors (MDMs infected with hSAA-1 opsonized or nonopsonized Mtb) in three biological repeats each, completed separately for each comparison using the iDEP.96 platform and presented at each gene as A, B, and C.

Figure 4

MDM gene expression of selected cytokines. CPM (counts per million) analysis of transcripts for selected cytokines in control MDMs (MQ), MDMs infected with nonopsonized Mtb (MQ+Mtb), hSAA-1 opsonized Mtb (MQ+Mtb/SAA), and MDMs treated with hSAA-1 (MQ+SAA). The assay was performed for three (MQ, MQ+SAA) or four (MQ+Mtb, MQ+Mtb/SAA) independent healthy blood donors in three biological repeats each. Statistical analysis was performed by nonparametric data distribution, which was evaluated by the Shapiro-Wilk normality test. Furthermore, statistical analysis was performed by Kruskal-Wallis one-way ANOVA with post-hoc Dunn’s test or one-way ANOVA with post-hoc Tukey’s test (IL12B). *represents p<0.05, **p<0.0021, ***p<0.0002, ****p<0.0001.

Figure 5

The concentrations of selected cytokines were determined using the Milliplex system. The protein level was assessed in the cell supernatant of control MDMs (MQ), MDMs infected with nonopsonized Mtb (MQ+Mtb), hSAA-1 opsonized Mtb (MQ+Mtb/SAA), and MDMs treated with hSAA-1 (MQ+SAA). The assay was performed for three independent healthy blood donors and the samples of collected culture supernatants were run in triplicate. The data distribution was evaluated by the Shapiro-Wilk normality test. Furthermore, statistical analysis was performed by Kruskal-Wallis one-way ANOVA with post-hoc Dunn’s test. *represents p<0.05, **p<0.0021, ***p<0.0002, ****p<0.0001.

Figure 6

GO biological process enrichment analysis of Mtb intracellular environment-induced changes. Mtb represents control bacilli, MtbMQ represents Mtb isolated from MDMs. The diagram was generated by ShinyGO 0.77.

Figure 7

The functional response of tubercle bacilli to the intracellular environment of MDMs. Red arrows, upregulation; blue arrows, downregulation; IM, inner membrane; OM, outer membrane; SL, sulfolipids; PAT, polyacylated trehalose; PGL, phenolic glycolipids.