Differential Responses by Human Macrophages to Infection With Mycobacterium tuberculosis and Non-tuberculous Mycobacteria.

Xiuhong Nie,Xiyuan Bai,Zhihong Feng,An Bai,Tao Wang,Edward D. Chan,Cindy Garcia,Li Li,Jennifer R. Honda

doi:10.3389/fmicb.2020.00116

Abstract

Mycobacterium tuberculosis (MTB) and non-tuberculous mycobacteria (NTM) are formidable causes of lung diseases throughout the world. While MTB is considered to be more virulent than NTM, host factors also play a key role in disease development. To elucidate whether there are differential immune responses to various mycobacteria, THP-1 macrophages were temporally infected with MTB H37Rv or with four different NTM species. We found that cells infected with MTB had greater bacterial burden and p65 nuclear factor-kappa B (NF-κB) activation than cells infected with NTM. There was also differential expression of mRNA for interleukin-1-β (IL-1β), IL-8, IL-10, and tumor necrosis factor-alpha (TNF-α) with no distinct pattern of mRNA expression among the different mycobacteria. In contrast, at the protein level, some generalizations can be made of the cytokines and chemokines expressed. Compared to uninfected cells, the rapid-growing Mycobacterium smegmatis but not Mycobacterium abscessus induced significantly greater pro-inflammatory cytokines and IL-10, whereas both NTM individually induced greater levels of chemokines. Compared to uninfected control cells, the two slow-growing NTM and MTB differentially induced cytokine expression with Mycobacterium avium inducing more pro-inflammatory cytokines and IL-10, whereas M. avium, Mycobacterium intracellulare, and MTB inducing greater but similar levels of chemokines. MTB-infected THP-1 cells also demonstrated lower level of phagosome–lysosome fusion and apoptosis than NTM-infected cells while there were differences in these macrophage functions among the NTM species. Interestingly, M. intracellulare, M. avium, and MTB have similar levels of autophagosome formation, but the levels displayed by all three were lower than for M. smegmatis and M. abscessus. This study demonstrates the differences in bacterial burden and macrophage effector functions among several clinically relevant mycobacterial species. Such disparities may, in part, account for differences in clinical outcomes among patients infected with various species of NTM as has been seen for different strains of MTB.

Highlights

MATERIALS AND METHODSIn the past two decades, the prevalence of non-tuberculous mycobacterial lung disease (NTM-LD) has been increasing in the United States and several parts of the world (DanielWayman et al, 2019; Wagner et al, 2019)
With the slow-growing mycobacteria (SGM) and Mycobacterium tuberculosis (MTB), the cell-associated burden of M. avium was significantly more than M. intracellulare at 2 days after infection, while the bacterial burden of MTB H37Rv was significantly more than M. intracellulare at 4 days after infection (Figure 1B)
We previously reported that MTB infection of human macrophages induced nuclear factor-kappa B (NF-κB) activation, which inhibited autophagosome formation

Summary

MATERIALS AND METHODS

In the past two decades, the prevalence of non-tuberculous mycobacterial lung disease (NTM-LD) has been increasing in the United States and several parts of the world (DanielWayman et al, 2019; Wagner et al, 2019). Penicillin/streptomycin, LysoTracker Red DND-99, Cy3-goat anti-rabbit IgG (H + L), Lab-Tek II Chamber Slide System, NE-PER Nuclear and Cytoplasmic Extraction Reagent, and Annexin-V Human ELISA Kit were purchased from Thermo Fisher Scientific/Life Technologies (Carlsbad, CA, United States). After 1 h, 2 and 4 days of infection, the cells were washed, lysed, and the lysates serially diluted and plated on 7H10 solid medium to quantify cell-associated M. smegmatis, M. abscessus, M. intracellulare, M. avium, and MTB H37Rv. GFP-labeled Mycobacterium species were generated as previously described (Bai et al, 2013). Supernatants of differentiated THP-1 macrophages infected with different mycobacterial species for 1 h, 2, and 4 days were quantified for lL-1β, IL-6, lL-8, lL-10, lL-12p40, IL12p70, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1-alpha (MIP1α), and TNF-α using HCYTOMAG-60K/MILLIPLEX R MAP Human Cytokine/Chemokine Magnetic Bead Panel-Immunology Multiplex Assay (EMDMILLIPORE Inc., Temecula, CA, United States) with the Luminex MAGPIX instrument (Luminex Inc.). Group means were compared by repeated-measures ANOVA using Fisher’s least significant test or by two-way ANOVA with Bonferroni’s post hoc test

RESULTS

DISCUSSION

CONCLUSION

DATA AVAILABILITY STATEMENT