Dual Inhibition of Rip2 and IRAK1/4 Regulates IL-1β and IL-6 in Sarcoidosis Alveolar Macrophages and Peripheral Blood Mononuclear Cells

Abstract

Sarcoidosis is a multisystem granulomatous disease of unknown etiology that primarily affects the lungs. Our previous work indicates that activation of p38 plays a pivotal role in sarcoidosis inflammatory response. Therefore, we investigated the upstream kinase responsible for activation of p38 in sarcoidosis alveolar macrophages (AMs) and PBMCs. We identified that sustained p38 phosphorylation in sarcoidosis AMs and PBMCs is associated with active MAPK kinase 4 but not with MAPK kinase 3/6. Additionally, we found that sarcoidosis AMs exhibit a higher expression of IRAK1, IRAK-M, and receptor interacting protein 2 (Rip2). Surprisingly, ex vivo treatment of sarcoidosis AMs or PBMCs with IRAK1/4 inhibitor led to a significant increase in IL-1β mRNA expression both spontaneously and in response to TLR2 ligand. However, a combination of Rip2 and IRAK-1/4 inhibitors significantly decreased both IL-1β and IL-6 production in sarcoidosis PBMCs and moderately in AMs. Importantly, a combination of Rip2 and IRAK-1/4 inhibitors led to decreased IFN-γ and IL-6 and decreased percentage of activated CD4(+)CD25(+) cells in PBMCs. These data suggest that in sarcoidosis, both pathways, namely IRAK and Rip2, are deregulated. Targeted modulation of Rip2 and IRAK pathways may prove to be a novel treatment for sarcoidosis.

Figure 1. Increased p38 activation in sarcoidosis PBMCs and AMs as compared to healthy controls

PBMCs from patients (A) and healthy controls (B) were fixed, permeabilized and stained with monoclonal antibodies for CD14 and phospho p38 and analyzed by flow cytometry. (C) Percentage of CD14, pp38 and CD14/pp38 positive cells in PBMCs of 8 controls and 8 sarcoidosis patients presented as box plot. (D) Western blot analysis of phosphorylated p38 and total p38 from sarcoid AMs and PBMCs. Whole cell extracts were prepared from AMs or PBMCs and subjected to SDS-gel electrophoresis. Western blot analysis was performed using antibodies against the phosphop38 and total p38. Representative blot is shown for three different patients (P1–P3). (E) Densitometric values expressed as fold increase of the ratio of phosphorylated p38/total p38 (mean± SEM) from 12 patients and 10 healthy controls. Grey bars represent PBMCs and black bars represent AMs. * p-value < 0.05.

Figure 2. Increased p38 activity in sarcoidosis is associated with MKK4

AMs and PBMCs from patients were activated with TLR2 (PAM, 100 ng/mL) and TLR4 (LPS, 1 ng/mL) ligands for 30 min. Whole cell extracts of AMs and PBMCs were prepared and subjected to SDS-PAGE and Western blot analysis using specific phospho-antibodies for MKK4, MKK3/6, p38. Equal loading was confirmed using antibodies against β-actin or total p38. (A) Sarcoid AMs and (B) PBMCs showed the presence of active (phosphorylated) form of MKK4 at Ser 257/Thr 261 and pp38 both at baseline and in response to LPS and PAM. Representative blots for AMs and PBMCs are shown from one patient out of a total of 12 patients. (C) Densitometric values (mean± SEM) expressed as ratio of phosphorylated MKK4/β-actin from 12 patients. (D) Densitometry values (mean± SEM) expressed as ratio of phosphorylated p38/total p38 from 12 patients. *p-value < 0.05.

Figure 3. Increased IRAKs and Rip2 expression in sarcoidosis

AMs from sarcoid subjects and controls were treated with PAM (100 ng/mL) or LPS (1 ng/mL) or left untreated. Whole cell extracts were prepared and subjected to SDS-PAGE and Western blot analysis using specific antibodies for IRAK1, IRAK-M, Rip2, β-actin, and p38. (A) Sarcoidosis AMs exhibited higher activated form of IRAK1 as well as higher expression of IRAK-M as compared to control subjects (B) PBMCs from sarcoid subjects also exhibited higher IRAK1 but comparable IRAK-M expression (C) Densitometric values (mean±SEM) expressed as fold change of the ratio of IRAK1/β-actin in AMs of 6 control and 10 sarcoidosis subjects. (D) Densitometric values (mean+SEM) expressed as fold change of the ratio of IRAK-M/β-actin in AMs of 6 control and 10 sarcoidosis subjects. (E) Densitometric values (mean+SEM) expressed as fold change of the ratio of IRAK1/β-actin in PBMCs of 6 control and 10 sarcoidosis subjects (F) Densitometric values (mean+SEM) expressed as fold change of the ratio of IRAK-M/β-actin in PBMCs of 10 control and10 sarcoidosis subjects. (G) Rip2 expression in sarcoidosis and control AMs. Whole cell lysates of AMs of two control subjects (C1&C2) and two sarcoidosis patients (P1&P2) were immunoblotted with Rip2 and p38 antibodies. AMs from sarcoid subjects showed higher expression of Rip2 as compared to healthy controls. (H) Densitometric values (mean+ SEM) expressed as ratio of RIP2/β-actin from 6 controls and 10 sarcoidosis patients. * p- value < 0.05. (I) AMs from controls and sarcoidosis subjects were activated with PAM (100 ng/mL) for indicated time periods. Western blot analysis shows baseline expression of Rip2 and its time- dependent induction with PAM. Representative blots for AMs and PBMCs are shown out of a total of 10 patients and 6 healthy controls.

Figure 4. IRAK-1/4 inhibitor does not inhibit IL-1β, IL-6 production and p38 phosphorylation in sarcoidosis AMs and PBMCs

AMs or PBMCs of sarcoid subjects were pretreated with IRAK-1/4 inhibitor (20μM) for 1h and stimulated with PAM (100 ng/mL) or LPS (1 ng/mL) for 24h. The conditioned medium was assessed for IL-1β and IL-6 using ELISA. IRAK1/4 inhibitor did not significantly inhibit either IL-1β or IL-6 production both in sarcoid AMs (A & C) and IL-1β in PBMCs (B). Data represent mean± SEM from at least six different patients. * Represents p value < 0.05 was considered significant. (D) Sarcoidosis AMs were treated with TLR2, TLR4 ligands in the presence or absence of IRAK1/4 inhibitor. Cell lysates were subjected to immunoblotting using phospho p38 and total p38 (equal loading) antibodies. IRAK1/4 inhibitor did not decrease p38 phosphorylation in AMs. (E) Densitometric values (mean+ SEM) expressed as ratio of pp38/p38 from 6 different sarcoidosis patients.

Figure 5. Effect of gefitinib and IRAK1/4 inhibitor on IL-1β and IL-6 synthesis in sarcoid AMs and PBMCs

AMs (A&B) or PBMCs (C&D) were pretreated with combination of IRAK1/4 inhibitor (20μM) and gefitinib (1μM) for 30min and then activated with PAM (100 ng/mL) for 24h. Conditioned media were analyzed for IL-1β and IL-6 via ELISA. Data represent mean± SEM from at least ten different patients. * represents p value < 0.05 were considered significant.

Figure 6. Effect of gefitinib and IRAK inhibitor on the relative gene expression of IL-1β, Rip2 and p38 phosphorylation in sarcoid AMs and PBMCs

PBMCs and AMs from sarcoidosis subjects were activated with PAM (100 ng/mL) in the presence or absence of IRAK1/4 (20μM) inhibitor or gefitinib (1μM) or combination of IRAK1/4 inhibitor and gefitinb for 1h. RNA was isolated from the cells and subjected to RT-PCR for IL-1β gene expression (A & B) and (C) RIP2 gene expression. Values were normalized to β-actin and are shown as fold change. Data represent mean± SEM from at least five different patients. * represents p value< 0.05. (D) AMs and PBMCs were activated with PAM (100 ng/mL) in presence or absence of inhibitor alone or a combination of both inhibitors for 30 min. Whole cell lysates were subjected to immunoblotting to assess the phosphorylation of p38 and total p38 (equal loading). Data presented is a representative blot out of 10 different patients. (E) Densitometric values (mean± SEM) expressed as ratio of pp38/ p38 in AMs and PBMCs samples from 10 patients in presence and absence of both inhibitors at baseline and in response to PAM. * p- value < 0.05.

Figure 7. Effect of gefitinib and IRAK1/4 inhibitor on CD4⁺CD25⁺ cells and IFN-γ and IL-6 production in PBMCs

PBMCs were cultured at the density of 2×10⁶ cells per well in the presence of rhIL-2 (10 ng/mL) and activated with anti-CD3 (1 μg/mL) in the presence or absence of IRAK1/4 inhibitor (20μM) and gefitinib (1μM). Inhibitors were added 30 min prior to activation. Cells were harvested after 96h of culture and immunostained with fluorescein conjugated antibodies CD4 and CD25 and analyzed by flow cytometry using Flow-jo software. (A–D) Representative scatter plots show FACS analysis of CD4 and CD25 expression of sarcoidosis PBMCs. (A) In unstimulated PBMCs the CD4 and CD25 double positive cells were about 10% in sarcoidosis subjects. (B) Sarcoidosis PBMCs cultured in the presence of gefitinib and IRAK1/4 inhibitor for 96h. The percentage of CD4 and CD25 double positive cells decreased from 10% to 5%. (C) Sarcoidosis PBMCs stimulated with anti-CD3. The percentage of CD4 and CD25 double positive T-cells increased to 62%. (D) Percentage of activated T-cells decreased from 62% after anti-CD3 challenged to 18% in the presence of gefitinib and IRAK1/4inhibitor. Data presented is a representative plot of at least 5 different patients. (E) Percentage of CD4⁺CD25⁺ cells (mean±SEM) of five patients at baseline, in response to anti-CD3 treatment and in presence and absence of both inhibitors. (F&G) The conditioned medium was quantified for IFN-γ (F) and IL-6 (G) production. Data represents mean± SEM from at least five different patients. * represents p value < 0.05 and was considered significant. There is a significant increase of IFN- γ and IL-6 after anti-CD3 stimulation which is significantly decreased by pretreatment with a combination of gefitinib and IRAK 1/4 inhibitor.