Alternatively activated and immunoregulatory monocytes in human filarial infections

Abstract

Background: Monocytes/macrophages from filaria-infected animals exhibit an alternatively activated phenotype; however, very little is known about the alternative activation phenotype of monocytes in human filarial infection.

Methods: To elucidate the activation and cytokine profile of monocytes in human filarial infection, we examined the expression patterns of genes encoding arginase, nitric oxide synthase 2, alternative activation markers, and cytokines in monocytes from individuals with asymptomatic filarial infection and individuals without filarial infection, ex vivo and in response to filarial antigen (Brugia malayi antigen [BmA]).

Results: Monocytes from patients with asymptomatic filarial infection exhibited significantly diminished expression of NOS2 and significantly enhanced expression of ARG1. These changes were associated with significantly increased expression of the genes encoding resistin, mannose receptor C type 1 (MRC1), macrophage galactose type C lectin (MGL), and chemokine ligand 18 (CCL18). In response to BmA, purified monocytes from infected individuals also expressed significantly lower levels of interleukin (IL)-12 and IL-18 but, in contrast, expressed significantly higher levels of transforming growth factor beta, IL-10, and suppressor of cytokine signaling 1 mRNA. Inhibition of arginase-1 resulted in significantly diminished expression of the genes encoding resistin, MRC1, MGL, and CCL18, as well as significantly enhanced expression of NOS2 and the genes encoding IL-12 and IL-18.

Conclusion: Patent human filarial infection is associated with the presence of monocytes characterized by an alternatively activated immunoregulatory phenotype.

Figure 1

Representative dot plot showing the purity of CD14⁺ monocytes after positive selection. FITC, fluorescein isothiocyanate; SSC, side scatter.

Figure 2

Filarial infection is associated with elevated ARG1 and decreased NOS2 expression. Peripheral blood mononuclear cells from 10 patients with asymptomatic filarial infection (AFI) and 10 uninfected patients (UN) were examined at baseline (A) or were stimulated with Brugia malayi antigen (BmA; 10 μg/mL; B ) or purified protein derivative (PPD; 10 μg/mL; C ) for 24 h, after which monocytes were isolated. NOS2 and ARG1 messenger RNA (mRNA) levels were measured by real-time reverse transcription–polymerase chain reaction and normalized to the levels of the 18S ribosomal RNA control gene in the isolated monocytes. Results are shown as fold change versus media control. D, Correlation between baseline levels of ARG1 and NOS2 mRNA is shown as an x-y scatterplot. P values were calculated using the Mann-Whitney U test or the Spearman rank correlation test. 1/dCT, 1/[change in cycle threshold for the target gene minus that for the control gene].

Figure 3

Filarial infection is associated with enhanced expression of alternative activation genes. Peripheral blood mononuclear cells from 10 patients with asymptomatic filarial infection (AFI) and 10 uninfected patients (UN) were examined at baseline (A) or were stimulated with Brugia malayi antigen (BmA; 10 μg/mL) or purified protein derivative (PPD; 10 μg/mL) for 24 h, after which monocytes were isolated. Resistin (B), mannose receptor C type 1 (MRC1; C ), chemokine ligand 18 (CCL18; D ), and macrophage galactose type C lectin (MGL; E ) messenger RNA levels were measured by real-time reverse transcription–polymerase chain reaction and normalized to the levels of the 18S ribosomal RNA control gene in the isolated monocytes. Results are shown as relative transcript levels or fold change versus media control. P values were calculated using the Mann-Whitney U test. 1/dCT, 1/[change in cycle threshold for the target gene minus that for the control gene].

Figure 4

Baseline levels of ARG1 correlate with alternative activation genes. Baseline correlation between messenger RNA levels of ARG1 and alternative activation genes encoding resistin, mannose receptor C type 1 (MRC1), chemokine ligand 18 (CCL18), and macrophage galactose type C lectin (MGL), as measured by real-time reverse transcription–polymerase chain reaction. Results are shown as x-y scatterplots for the correlation studies and as fold change versus media control for the inhibition studies. P values were calculated using the Spearman rank correlation test. 1/dCT, 1/[change in cycle threshold for the target gene minus that for the control gene].

Figure 5

Filarial infection is associated with decreased expression of proinflammatory cytokines and increased expression of down-regulatory cytokines. Peripheral blood mononuclear cells from 6 patients with asymptomatic filarial infection (AFI) and 6 uninfected patients (UN) were stimulated with Brugia malayi antigen (BmA; 10 μg/mL) or purified protein derivative (PPD; 10 μg/mL) for 24 h, after which monocytes were isolated. Interleukin (IL)–12, IL-18, tumor necrosis factor–α (TNF-α), IL-8 messenger RNA levels (A) and transforming growth factor β (TGFβ) and IL-10 messenger RNA levels (B) were measured by real-time reverse transcription–polymerase chain reaction and normalized to the levels of the 18S ribosomal RNA control gene in the isolated monocytes. Results are shown as fold change versus media control. P values were calculated using the Mann-Whitney U test.

Figure 6

Filarial infection is associated with increased expression of SOCS-1. Peripheral blood mononuclear cells from 6 patients with asymptomatic filarial infection (AFI) and 6 uninfected patients (UN) were stimulated with Brugia malayi antigen (BmA; 10 μg/mL) or purified protein derivative (PPD; 10 μg/mL) for 24 h, after which monocytes were isolated. Cytokine inducible SH2– containing protein (CIS), suppressor of cytokine signaling (SOCS)–1, SOCS-2, SOCS-3, SOCS-4, SOCS-5, and SOCS-7 messenger RNA levels were measured by real-time reverse transcription–polymerase chain reaction and normalized to the levels of the 18S ribosomal RNA control gene in the isolated monocytes. Results are shown as fold change over media control. P values were calculated using the Mann-Whitney U test.

Figure 7

Inhibition of ARG1 leads to down-regulation of alternative activation genes and up-regulation of NOS2 and the genes encoding interleukin (IL)–12 and IL-18. Peripheral blood mononuclear cells from 6 patients with asymptomatic filarial infection were stimulated with Brugia malayi antigen (10 μg/mL) for 24 h in the presence or absence of ARG1 inhibitor N^ω-hydroxy-nor-L-arginine, after which monocytes were isolated. Resistin, mannose receptor C type 1 (MRC1), chemokine ligand 18 (CCL18), and macrophage galactose type C lectin (MGL) messenger RNA levels were measured by real-time reverse transcription–polymerase chain reaction and normalized to the levels of the 18S ribosomal RNA control gene in the isolated monocytes. Results are shown as fold change versus media control in the presence or absence of inhibitor. P values were calculated using the Wilcoxon signed rank test.

Figure 8

Inhibition of ARG1 leads to up-regulation of NOS2 and the genes encoding interleukin (IL)–12 and IL-18. Monocytes from 6 patients with asymptomatic filarial infection were stimulated with Brugia malayi antigen (10 μg/mL) for 24 h in the presence or absence of ARG1 inhibitor N^ω-hydroxy-nor-L-arginine. NOS2, interleukin (IL)–12, IL-18, and suppressor of cytokine signaling–1 (SOCS-1) messenger RNA levels were measured by real-time reverse transcription–polymerase chain reaction and normalized to the levels of the 18S ribosomal RNA control gene. Results are shown as fold change versus media control in the presence or absence of inhibitor. P values were calculated using the Wilcoxon signed rank test.