A critical role for suppressor of cytokine signalling 3 in promoting M1 macrophage activation and function in vitro and in vivo

Abstract

Macrophages respond to their microenvironment and develop polarized functions critical for orchestrating appropriate inflammatory responses. Classical (M1) activation eliminates pathogens while alternative (M2) activation promotes regulation and repair. M1 macrophage activation is strongly associated with suppressor of cytokine signalling 3 (SOCS3) expression in vitro, but the functional consequences of this are unclear and the role of SOCS3 in M1-macrophage polarization in vivo remains controversial. To address these questions, we defined the characteristics and function of SOCS3-expressing macrophages in vivo and identified potential mechanisms of SOCS3 action. Macrophages infiltrating inflamed glomeruli in a model of acute nephritis show significant up-regulation of SOCS3 that co-localizes with the M1-activation marker, inducible nitric oxide synthase. Numbers of SOCS3(hi) -expressing, but not SOCS1(hi) -expressing, macrophages correlate strongly with the severity of renal injury, supporting their inflammatory role in vivo. Adoptive transfer of SOCS3-short interfering RNA-silenced macrophages into a peritonitis model demonstrated the importance of SOCS3 in driving production of pro-inflammatory IL-6 and nitric oxide, while curtailing expression of anti-inflammatory IL-10 and SOCS1. SOCS3-induced pro-inflammatory effects were due, at least in part, to its role in controlling activation and nuclear accumulation of nuclear factor-κB and activity of phosphatidylinositol 3-kinase. We show for the first time that SOCS3 also directs the functions of human monocyte-derived macrophages, including efficient M1-induced cytokine production (IL-1β, IL-6, IL-23, IL-12), attenuated signal transducer and activator of transcription 3 activity and ability of antigen-loaded macrophages to drive T-cell responses. Hence, M1-associated SOCS3 was a positive regulator of pro-inflammatory responses in our rodent models and up-regulated SOCS3 is essential for effective M1-macrophage activation and function in human macrophages.

Keywords: M1 macrophage; glomerulonephritis; inflammation; nuclear factor-κB; suppressor of cytokine signalling 3.

Figure 1

The majority of suppressor of cytokine signalling 3-high (SOCS3^hi) expressing macrophages strongly co-expressed the M1 marker inducible nitric oxide synthase (iNOS). Triple immunostaining of a representative glomerulus from a non-diseased (a) and diseased (c, e) animal after induction of nephrotoxic nephritis. The number of CD68-positive macrophages (green fluorescence) that were SOCS3-positive (brown chromogenic stain) and iNOS-positive (red fluorescence) (a, c), or SOCS1-positive (brown chromogenic stain) and iNOS-positive (red fluorescence) (e) are shown. Original magnification × 200. The mean ± SD percentage of macrophages expressing, SOCS1 and SOCS3 (b); iNOS and SOCS3 (d); or iNOS and SOCS1 (f) in glomeruli of rat kidney 3 days after induction of nephrotoxic nephritis. The mean number of total macrophages per nephritic glomerulus was 18·6 ± 8·2; n = 8 rats, at least five glomeruli per animal were analysed.

Figure 2

Numbers of glomerular macrophages expressing suppressor of cytokine signalling 3 (SOCS3) correlate strongly with severity of injury in experimental nephritis. Triple immunostaining identified SOCS1 and SOCS3 expressing CD68-positive macrophages. Macrophages exclusively expressing SOCS3 but not SOCS1 show a more significant correlation with levels of albuminuria as a measure of severity of disease (P = 0·001) as compared with total macrophages (P = 0·01), whereas, the number of macrophages exclusively expressing SOCS1 but not SOCS3 did not correlate with albuminuria (P = 0·120) suggesting they do not play a main role in driving disease severity.

Figure 3

Suppressor of cytokine signalling 3 (SOCS3) knockdown macrophages injected into, and conditioned within, an in vivo inflamed environment show decreased pro-inflammatory and enhanced anti-inflammatory characteristics. (a) PCR analysis showing SOCS3 knockdown macrophages (SOCS3) have enhanced mRNA for SOCS1, mannose receptor (MR), arginase I and interleukin-10 (IL-10) compared with control short interfering RNA (siRNA) transfected cells (Con) after conditioning within an inflamed rat peritoneum in vivo. (b) Gene expression was quantified using RT-PCR and normalized to the housekeeping gene. The results are shown as mean ± SEM from n = 6 rats. (c) Production of macrophage inflammatory mediators in supernatant of mock transfected, control siRNA transfected, or SOCS3 siRNA transfected macrophages isolated from an in vivo conditioning environment and cultured for 12 hr before analysis for nitrite (NO), IL-10 and IL-6. Values represent mean concentration ± SD, n = 6 animals/group (*P < 0·05). # Knockdown of SOCS3 in macrophages conditioned in an inflamed environment for 4 hr show a reciprocal up-regulation of SOCS1 protein when analysed by immunoblotting with SOCS1, SOCS3 and β-actin antibodies.

Figure 4

Nuclear factor-кB (NF-кB)/p65 activity is decreased in suppressor of cytokine signalling 3 (SOCS3) knocked down M1 activated macrophages. Bone marrow-derived macrophages (BMDM) were transfected with control short interfering RNA (siRNA) or SOCS3 siRNA. Forty-eight hours following transfection, cells were stimulated without or with lipopolysaccharide (LPS). (a) Nuclear protein extracts were prepared and assessed for NF-кB/p65 activity by a DNA binding ELISA as described in the Materials and methods. Values show mean percentage ±SD of NF-κB/p65 activity in cells transfected with SOCS3 siRNA relative to LPS-stimulated cells transfected with control siRNA; n = 6. (b) Immunoblot analysis of SOCS3, IκB and phospho-p65 in lysates of BMDM transfected with control siRNA or SOCS3 siRNA treated with interferon-γ (IFN-γ)/LPS for 0, 15, 30 and 60 min with β-actin as a loading control. SOCS3 knockdown decreased SOCS3 protein expression, decreased the rate of IκB degradation and increased phosphorylation of p65. Figure shows expression of proteins as determined by an immunoblot, representative of three individual experiments (b) and normalized densitometric analysis of scanned bands in arbitary units (a.u.) from three separate immunoblots (c). *P < 0·05.

Figure 5

Knocking down suppressor of cytokine signalling 1 (SOCS1) and SOCS3 simultaneously does not restore the SOCS3-mediated decrease in nuclear factor-кB (NF-кB)/p65 activity in M1 activated macrophages. Bone marrow-derived macrophage (BMDM) were transfected with control short interfering RNA (siRNA), SOCS3 siRNA or SOCS1 siRNA (Invitrogen) in a single or combined manner. (a) Forty-eight hours following transfection, cells were incubated with interferon-γ (IFN-γ)/lipopolysaccharide (LPS) for 30 min. SOCS1, SOCS3 and β-actin protein expression was detected by Western blotting showing successful knockdown at the protein level. The intensity of SOCS1 and SOCS3 bands on the blots were determined by densitometry and normalised to β-actin; n = 3. (b) Nuclear NF-κB/p65 DNA binding activity was measured using a TransAM NF-κB kit and demonstrated simultaneous SOCS1&3 knockdown does not restore nuclear NF-κB activity. Data shows the NF-κB/p65 activity in cells transfected with SOCS1 and/or SOCS3 siRNA relative to cells transfected with control siRNA, *P < 0·02, n = 5.

Figure 6

Suppressor of cytokine signalling 3 (SOCS3) knockdown results in an increase in phosphatidylinositol 3-kinase (PI3K) (pAKT) activity on M1 activation and inhibition of PI3K activity reverses the SOCS3 knockdown inhibitory effects on nuclear factor-κB (NF-κB). (a) Bone marrow-derived macrophages (BMDM) were transfected with control short interfering RNA (siRNA) or SOCS3 siRNA. Forty-eight hours following transfection, cells were stimulated without or with interferon-γ (IFN-γ)/lipopolysaccharide (LPS) and PI3K activity assessed by Western blotting for pAKT with loading control β-actin. Figure shows expression of proteins as determined by an immunoblot, representative of three individual experiments. (b) Normalized densitometric analysis of scanned bands in arbitary units (A. U.); n = 3; *P < 0·05. (c) Incubation of SOCS3 knockdown macrophages for 1 hr with the PI3K inhibitor LY294002 (20 μm) before M1 activation for 30 min abrogated the significant decrease in NF-κB/p65 activity in SOCS3 siRNA-transfected macrophages; DMSO (0·15%) served as a vehicle control. The inhibition of nitrite (d) and IL-6 (e) production in the culture supernatant of M1 activated, SOCS3 silenced cells compared with control siRNA-transfected cells was also prevented after pre-incubation with LY294002 for 40 min before M1 activation for an additional period of 10 hr. As LY294002 can interfere with basal NF-κB activity, values for control siRNA transfected cells with or without LY294002 were set to 100% for (c) to (e). n = 4; *P < 0·05.

Figure 7

Suppressor of cytokine signalling 3 (SOCS3) knockdown macrophages show distinct cytokine secretion profile following differential activation. (a) Macrophages were transfected with control or SOCS3-specific short interfering RNA (siRNA). Macrophages were differentially activated with 100 ng/ml lipopolysaccharide (LPS) alone or in combination with 20 ng/ml IFN-γ and cells were loaded with nominal antigen (keyhole limpet haemocyanin). Supernatants of macrophage cultures were collected 24 hr after activation and analysed by cytometric bead array [interleukin-6 (IL-6), IL-12, tumour necrosis factor-α (TNF-α), IL-10] and ELISA (IL-1β, IL-23); n = 6. (b) Autologous T cells were added to macrophages treated as above and cells were co-cultured for 8 days after which T-cell proliferation ([³H]thymidine incorporation) and T-cell cytokine secretion levels (ELISA) were measured. The plots show the mean ± SEM. Statistical significance was based on Wilcoxon matched-pairs signed rank test. n = 8; * P < 0·05.