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. 2022 Dec;10(12):e740.
doi: 10.1002/iid3.740.

Overexpression of CD86 enhances the ability of THP-1 macrophages to defend against Talaromyces marneffei

Rifeng Chen  1 , Di Yang  1 , Linxia Shen  1 , Jinling Fang  1 , Raqib Khan  1 , Donghua Liu  1
Affiliations

Overexpression of CD86 enhances the ability of THP-1 macrophages to defend against Talaromyces marneffei

Rifeng Chen et al. Immun Inflamm Dis. 2022 Dec.

Abstract

Background: Macrophages are the first line of defense against Talaromyces marneffei. CD86 is a surface molecule expressed on antigen-presenting cells, such as macrophages, that provide costimulatory signals necessary for T cell activation and survival. In a prior study, it was shown that as infection progressed, CD86 expression levels in macrophages considerably declined while CD86 concentrations in the supernatant significantly increased. Additionally, M1 macrophage polarization was insufficient and switched to M2 macrophage polarization. Besides costimulation, however, additional roles of CD86 are not known or well-studies. Therefore, we hypothesized that upregulating CD86 on macrophages might promote T. marneffei defense.

Methods: A lentivirus vector, called Lenti-CD86, was used to infect THP-1 cells to overexpress secretory CD86. Through killing assay, nitric oxide detection, and cytokine detection, the capacity of THP-1 macrophages to phagocytose and kill T. marneffei was examined.

Results: In the current study, Lenti-CD86 transfection of THP-1 cells resulted in a signifant expression of CD86. Additionally, the THP-1 macrophages stably transfected with Lenti-CD86 showed higher nitric oxide and IL-1β production, faster polarization, and stronger phagocytosis and killing capabilities than the non-transfected or control virus transfected cells.

Conclusion: Our study shows that lentivirus-mediated CD86 overexpression improves THP-1 macrophages' capacity to phagocytose and eliminate T. marneffei.

Keywords: CD86; THP-1 macrophages; Talaromyces marneffei; lentivirus; overexpression.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Efficiency of lentivirus‐mediated CD86 overexpression cells. (A) GFP expression was observed in the infected THP‐1 cells by a fluorescence microscopy at 72 h after infection (×200). (B) CD86 expression was clearly increased at the RNA level by LV‐mediated overexpression in THP‐1 cells. (C) Results of western blot revealed the increased CD86 expression in OE group. Data are means ± SD. **p < .01, ***p < .001, ns p > .05. SD, standard deviation.
Figure 2
Figure 2
The morphological changes of macrophages during Talaromyces marneffei infection were observed under an optical microscope. As early as 24 h OE group had obvious irregular macrophages with long pseudopodia (C), while CON and NC group showed round and oval macrophages (A, B). After 48 h and 72 h T. marnaffei infection, the macrophages of OE group still had obvious irregular morphology with long pseudopodia (F, I) in comparison with CON group (D, G) and NC group (E, H). Black arrows indicate irregular macrophages with long pseudopodia. Light microscopy ×400.
Figure 3
Figure 3
The phagocytosis of macrophages (red) after infection with Talaromyces marneffei (bright blue) was observed under fluorescence microscope. (A−F) At 24 and 48 h, there was no significant difference in the number of T. marneffei conidia phagocytized by macrophages in CON, NC, and OE group. (G−I) At 72 h, the number of T. marneffei conidia phagocytized by macrophages in OE group was significantly higher than that CON and NC group. White arrows indicate macrophages that have engulfed large amounts of T. marneffei conidia. Light microscopy ×400.
Figure 4
Figure 4
Macrophage killing assay. Colony count of each group = colony count of culture dish × dilution ratio (100 times), compared with CON and NC group, the colony count in OE group decreased significantly. There was no significant difference in colony count between CON and NC group. Data are means ± SD. **p < .01, ns p > .05. SD, standard deviation.
Figure 5
Figure 5
The relative expression of IL‐1β (A), TNF‐α (B), and γ‐IFN (C) mRNA in macrophages. Data are means ± SD. *p < .05, **p < .01, ***p < .001, ns p > .05. SD, standard deviation.
Figure 6
Figure 6
The protein concentration of IL‐1β (A), TNF‐α (B), and γ‐IFN (C) in the supernatant. Data are means ± SD. *p < .05, **p < .01, ***p < .001, ns p > .05. SD, standard deviation.
Figure 7
Figure 7
(A−C) NO concentration in the supernatant. Data are means ± SD. *p < .05, **p < .01, ns p > .05. NO, nitric oxide; SD, standard deviation.
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