Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Jun 12;8:691.
doi: 10.3389/fimmu.2017.00691. eCollection 2017.

Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization Through LC3-Dependent Autophagy

Affiliations
Free PMC article

Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization Through LC3-Dependent Autophagy

Yang Wang et al. Front Immunol. .
Free PMC article

Abstract

Brucellosis is caused by infection with Brucella species and exhibits diverse clinical manifestations in infected humans. Monocytes and macrophages are not only the first line of defense against Brucella infection but also a main reservoir for Brucella. In the present study, we examined the effects of Brucella infection on human peripheral monocytes and monocyte-derived polarized macrophages. We showed that Brucella infection led to an increase in the proportion of CD14++CD16- monocytes and the expression of the autophagy-related protein LC3B, and the effects of Brucella-induced monocytes are inhibited after 6 weeks of antibiotic treatment. Additionally, the production of IL-1β, IL-6, IL-10, and TNF-α from monocytes in patients with brucellosis was suppressed through the LC3-dependent autophagy pathway during Brucella infection. Moreover, Brucella infection inhibited macrophage polarization. Consistently, the addition of 3-MA, an inhibitor of LC3-related autophagy, partially restored macrophage polarization. Intriguingly, we also found that the upregulation of LC3B expression by rapamycin and heat-killed Brucella in vitro inhibits M2 macrophage polarization, which can be reversed partially by 3-MA. Taken together, these findings reveal that Brucella dysregulates monocyte and macrophage polarization through LC3-dependent autophagy. Thus, targeting this pathway may lead to the development of new therapeutics against Brucellosis.

Keywords: autophagy; brucellosis; cytokines; infection; inflammation.

Figures

Figure 1
Figure 1
Characteristics of monocyte subsets in patients with brucellosis and healthy control subjects. (A) Gating strategy for monocyte subsets. (B) The percentage of each monocyte subset in patients with Brucellia infection (BI) (n = 25) and healthy controls (HC) (n = 15). (C) The percentage of monocyte subsets in five brucellosis patients before and after a 6-week treatment with rifampicin and doxycycline. (D) The phenotypic comparisons of monocyte subsets were normalized as the ratio of brucellosis patients/HC. *P < 0.05. **P < 0.01; **P < 0.001.
Figure 2
Figure 2
LC3B expression on monocytes of patients with brucellosis. LC3B expression levels on monocytes were analyzed by flow cytometry. LC3B expression levels detected in PBMCs by flow cytometry in 25 brucellosis patients and 15 healthy volunteers were included. The results were presented as the mean fluorescence intensity. (A) LC3B expression levels on monocytes from patients with Brucellia infection (BI) (n = 25) and healthy controls (HC) (n = 15). (B) LC3B expression levels on different monocyte subsets. (C,D) LC3B expression levels in monocytes were detected by flow cytometry using PBMCs from five patients before and after a 6-week treatment with rifampicin and doxycycline. (C) LC3B expression levels of monocytes from brucellosis patients (n = 5). (D) LC3B expression levels on different monocyte subsets. **P < 0.01; **P < 0.001.
Figure 3
Figure 3
Heat-killed Brucella (HK-Br) induced LC3-dependent autophagy. (A) Purified monocytes from five healthy volunteers were pretreated with rapamycin (100 nM, 12 h) or HK-Br (MOI = 100:1) for 24 h with or without 3-MA pretreatment (3 mM, 3 h), and the LC3B levels were detected by flow cytometry. (B) Western blotting of LC3 and Beclin-1 in pretreated cells were performed, and the ratios of LC3-II/LC3-I and Beclin-1/β-actin were calculated. *P < 0.05; **P < 0.01.
Figure 4
Figure 4
Brucella infection inhibited the function of monocytes via autophagy. Purified monocytes from 10 patients with Brucellia infection (BI) and 10 healthy controls (HC) were stimulated with lipopolysaccharide (LPS) (1 µg/ml, 6 h). (A) TNF-α, (C) IL-6, (E) IL-1β, and (G) IL-10 expression levels on monocytes and their production by monocytes were examined by intracellular cytokines staining and enzyme-linked immunosorbent assay (ELISA), respectively. Purified monocytes from six brucellosis patients were pretreated with 3-MA (3 mM, 3 h), and then stimulated with LPS. (B) TNF-α, (D) IL-6, (F) IL-1β, and (H) IL-10 expression levels on monocytes and their production by monocytes were examined by intracellular cytokines staining and ELISA, respectively. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
Brucella infection inhibited macrophage polarization via autophagy. Monocytes were purified from the peripheral blood of 10 patients with Brucellia infection (BI) and 10 healthy controls (HC), and purified monocytes from 6 patients with BI were also pretreated with 3-MA. These monocytes were polarized to M1 and M2 macrophages. (A) Morphology was assessed by microscopy (scale bar 20 µm). (B) CD80, CD86, CD163, and CD206 expression of M1 and M2 macrophages from brucellosis patients and healthy volunteers were analyzed by flow cytometry. (C) CD80 and CD86 expression of M1 macrophages, CD163 and CD206 expression of M2 macrophages were determined by flow cytometry. (D) TNF-α and (E) IL-10 production were detected by enzyme-linked immunosorbent assay. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6
Figure 6
Brucella infection impaired the polarization of monocytes to M1 and M2 macrophages in vitro. Pure monocytes from five healthy volunteers were pretreated with 100 nM rapamycin or heat-killed Brucella (HK-Br) (MOI = 100:1) for 24 h in presence or absence of 3-MA-pretreatment, and then monocytes were differentiated to M1 or M2 macrophages. (A) Their morphology was observed by microscopy (scale bar 20 µm). (B) CD80 and (C) CD86 expression of M1 macrophages, (D) CD163 and (E) CD206 expression of M2 macrophages were determined by flow cytometry. (F) TNF-α production of M1 macrophages, (G) IL-10 production of M2 macrophages in the supernatants were examined by enzyme-linked immunosorbent assay. *P < 0.05, **P < 0.01, ***P < 0.001.

Similar articles

See all similar articles

Cited by 6 articles

See all "Cited by" articles

References

    1. Franco MP, Mulder M, Gilman RH, Smits HL. Human brucellosis. Lancet Infect Dis (2007) 7:775–86.10.1016/S1473-3099(07)70286-4 - DOI - PubMed
    1. Rubach MP, Halliday JE, Cleaveland S, Crump JA. Brucellosis in low-income and middle-income countries. Curr Opin Infect Dis (2013) 26(5):404–12.10.1097/QCO.0b013e3283638104 - DOI - PMC - PubMed
    1. Benard G. An overview of the immunopathology of human paracoccidioidomycosis. Mycopathologia (2008) 165:209–21.10.1007/s11046-007-9065-0 - DOI - PubMed
    1. Baldi PC, Giambartolomei GH. Immunopathology of Brucella infection. Recent Pat Antiinfect Drug Discov (2013) 8:18–26.10.2174/1574891X11308010005 - DOI - PubMed
    1. Parent MA, Goenka R, Murphy E, Levier K, Carreiro N, Golding B, et al. Brucella abortus bacA mutant induces greater pro-inflammatory cytokines than the wild-type parent strain. Microbes Infect (2007) 9:55–62.10.1016/j.micinf.2006.10.008 - DOI - PubMed
Feedback