Key Points

Abstract

The inflammatory responsiveness of phagocytes to exogenous and endogenous stimuli is tightly regulated. This regulation plays an important role in systemic inflammatory response syndromes (SIRSs). In SIRSs, phagocytes initially develop a hyperinflammatory response, followed by a secondary state of hyporesponsiveness, a so-called “tolerance.” This hyporesponsiveness can be induced by endotoxin stimulation of Toll-like receptor 4 (TLR4), resulting in an ameliorated response after subsequent restimulation. This modification of inflammatory response patterns has been described as innate immune memory. Interestingly, tolerance can also be triggered by endogenous TLR4 ligands, such as the alarmins myeloid-related protein 8 (MRP8, S100A8) and MRP14 (S100A9), under sterile conditions. However, signaling pathways that trigger hyporesponsiveness of phagocytes in clinically relevant diseases are only barely understood. Through our work, we have now identified 2 main signaling cascades that are activated during MRP-induced tolerance of phagocytes. We demonstrate that the phosphatidylinositol 3-kinase/AKT/GSK-3β pathway interferes with NF-κB–driven gene expression and that inhibition of GSK-3β mimics tolerance in vivo. Moreover, we identified interleukin-10–triggered activation of transcription factors STAT3 and BCL-3 as master regulators of MRP-induced tolerance. Accordingly, patients with dominant-negative STAT3 mutations show no tolerance development. In a clinically relevant condition of systemic sterile stress, cardiopulmonary bypass surgery, we confirmed the initial induction of MRP expression and the tolerance induction of monocytes associated with nuclear translocation of STAT3 and BCL-3 as relevant mechanisms. Our data indicate that the use of pharmacological JAK-STAT inhibitors may be promising targets for future therapeutic approaches to prevent complications associated with secondary hyporesponsiveness during SIRS.

Introduction

Systemic inflammatory response syndromes (SIRSs) are life-threatening cardiovascular conditions. SIRS is primarily driven by the innate immune system, in which phagocytes play a dominant role. SIRSs can be characterized by an early strong inflammatory phase, followed by a secondary hyporeactive state.1  During an initial inflammatory immune response, phagocytes detect conserved pathogen-associated molecular patterns through pattern recognition receptors (eg, lipopolysaccharide [LPS] by Toll-like receptor 4 [TLR4]).2 

Patients who survive this early hyperinflammatory phase often develop a prolonged counterregulatory hypoinflammatory phase. During this stage, patients are very susceptible to secondary infections resulting from an impaired reaction of phagocytes to invading pathogens.3  This hyporesponsiveness of phagocytes to a subsequent endotoxin challenge has been described as endotoxin tolerance.4 

Interestingly, hyporesponsiveness of the innate immune system can also occur under sterile conditions. Here, the immune response is initiated by endogenous alarmins.5  Myeloid-related protein 8 (MRP8; S100A8) and MRP14 (S100A9) are such endogenous ligands of TLR4 and are also 2 of the most abundant alarmins found in many inflammatory diseases.6-8 

The pathological dynamics of SIRS are not completely understood; therefore, treatment options for this immune-suppressive stage of SIRS are still inadequate. Currently, growing evidence shows that phagocytes can display adaptive characteristics after a challenge with pathogens or sterile triggers, which later modulate their subsequent inflammatory responses.9,10  This process of reprogramming has been termed “innate immune memory.”10  However, the biological relevance and signaling pathways of innate immune memory in clinically relevant diseases are only partly understood. We recently demonstrated that MRP8/14 induce a TLR4-dependent hyporesponsiveness of phagocytes under sterile conditions.9 

We now identify 2 major pathways responsible for the hyporesponsiveness of monocytes induced by MRP stimulation: the phosphatidylinositol 3-kinase (PI3K)/AKT/GSK-3β pathway and an interleukin-10 (IL-10)–dependent STAT3 activation and nuclear BCL-3 accumulation, with the latter resulting in inhibition of NF-κB transactivation. Accordingly, patients carrying a dominant-negative STAT3 mutation are not able to develop hyporesponsiveness. Lastly, we confirmed the early expression of MRP alarmins and IL-10, as well as the subsequent development of phagocyte hyporesponsiveness, in a patient cohort after cardiopulmonary bypass surgery (CBS). Our study identifies specific molecular pathways that provide a basis for innovative therapies targeting hyporesponsiveness of SIRS, because JAK/STAT3 inhibitors are already being used in clinical practice for other inflammatory diseases.

Methods

Patients

This study was approved by the institutional ethics committee (Medical Association of Westphalia-Lippe) and performed in accordance with the Helsinki Declaration. All patients provided written informed consent.

The study enrolled 12 CBS patients (Department of Cardiac Surgery, University Hospital Muenster; Table 2). Exclusion criteria included current infectious, oncologic, immunologic, or neurologic diseases and redo-surgery patients. All patients had unremarkable infection parameters at admission. Blood samples were taken on the day of admission, directly after CBS, on the first postoperative day, and at 6 days postsurgery.

The study included 3 hyperimmunoglobulin E syndrome (HIES) patients with heterozygous dominant-negative mutations in the STAT3 gene (Center for Chronic Immunodeficiency and Center for Translational Cell Research, University of Freiburg; Table 1).

Table 1.

Epidemiologic characteristics and STAT3 mutations of the HIES patients

PatientSexAge (y)Mutation in STAT3
STAT3 LOF1 Male 51 c.172C > T (p.His58Tyr) 
STAT3 LOF2 Female 21 c.172C > T (p.His58Tyr) 
STAT3 LOF3 Male 28 c.1909G > A (p.Val637Met) 
PatientSexAge (y)Mutation in STAT3
STAT3 LOF1 Male 51 c.172C > T (p.His58Tyr) 
STAT3 LOF2 Female 21 c.172C > T (p.His58Tyr) 
STAT3 LOF3 Male 28 c.1909G > A (p.Val637Met) 

Antibodies and reagents

Akt (C67E7), ERK-1/2 (9101), IκBα, p38, phosphorylated (p)-AKT (D9E), p–c-Raf, p–Erk-1/2 (Thr202/Tyr204), p–GSK-3β (D85E12), p-MEK1/2 (41G9), p-p38 (Thr180/Tyr182), p-PI3K (Tyr458/Tyr199), p-STAT3 (D3A7), and STAT3 (79D7) antibodies were purchased from Cell Signaling Technology (Danvers, MA). BCL-3 (Sc-185) was purchased from Santa Cruz Biotechnology (Dallas, TX), CD14 and IL-10R (3F9) were purchased from BioLegend (San Diego, CA), and MRP14 (S32-2) was purchased from BMA (Augst, Switzerland). Polyclonal MRP14 was purified by T.V. Secondary antibodies were purchased from Cell Signaling Technology and Dako; DRAG5 was purchased from Thermo Scientific (Waltham, MA). CHIR99021 and AKT(1/2) inhibitor were obtained from Abcam (Cambridge, UK), and LLL12 and MG132 were obtained from Merck (Darmstadt, Germany). LPS (Escherichia coli 055:B5) and D-Gal were purchased from Sigma-Aldrich (Steinheim, Germany); ONX-0941 was purchased from MedKoo Biosciences (Research Triangle Park, NC).

MRP purification

The human MRP8/14 heterodimer was isolated from granulocytes, as described earlier.11  The human and murine MRP8 homodimers were purified from E coli BL21(DE3), as described earlier.12  Endotoxin contaminations were excluded by an LAL Assay (BioWhittaker, Walkersville, MD).

Cells and cell culture

Buffy coat monocytes were isolated using Pancoll and Percoll gradients, as described earlier.9  Patient monocytes were isolated from EDTA blood samples by Pancoll gradients and magnetic cell separation (Monocyte Isolation Kit II; Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultivated in Teflon bags (lumox film; Sarstedt, Nümbrecht, Germany) using RPMI 1640 medium. Bone marrow–derived macrophages (BMDMs) were obtained as described earlier.9  Human monocytes were stimulated for 24 hours with low doses of LPS (25 pg/mL), MRP8/14 (10 µg/mL), or MRP8 (50 ng/mL) and subsequently restimulated with LPS (1 ng/mL) for 30 minutes to 4 hours. CHIR99021 (5 µmol/L), AKT(1/2) inhibitor (10 µmol/L), MG132 (200 nmol/L), ONX0914 (10 nmol/L), LLL12 (1 µmol/L), or IL-10R blocking antibody (50 µg/mL) was added 1 hour prior to prestimulation. Patient monocytes were stimulated with LPS (1 ng/mL) for 4 hours.

Generation of Hoxb8 cells

Hoxb8 monocytes were generated as described earlier.13  BCL-3−/− Hoxb8 cells originated from BCL-3–deficient mice. STAT3−/− Hoxb8 cells were generated using CRISPR/Cas9, as described earlier.14,15  For monocyte differentiation, Hoxb8 cells were cultivated for 3 days in RPMI 1640 medium supplemented with macrophage colony-stimulating factor or granulocyte-macrophage colony-stimulating factor.

Cell lysates

Whole-cell lysates were produced using M-PER Reagent (Thermo Scientific). For cell fractionation, a nuclear extraction kit (Epigentek, Farmingdale, NY) was used.

Phospho-kinase array

A Human Phospho-Kinase Antibody Array (R&D Systems, Minneapolis, MN) was used to determine the relative phosphorylation levels of several kinases.

Determination of MRP levels

The MRP protein concentrations in serum samples were measured by an MRP enzyme-linked immunosorbent assay (ELISA), as described earlier.16 

Determination of TNF-α and IL-10 secretion

Tumor necrosis factor α (TNF-α) secretion was quantified by commercial ELISAs (BD Bioscience, San Jose, CA). BioLegend’s Legendplex immunoassays were used to measure IL-10 release in supernatants of human monocytes or in human serum samples.

qRT-PCR

RNA was isolated using a NucleoSpin Extract II Isolation Kit (Macherey Nagel, Düren, Germany). Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was performed as described earlier.17  Sample data are presented as relative messenger RNA (mRNA) expression values compared with control cells.

ImageStream analysis

The CBS patients’ whole blood samples were stimulated with 2 ng/mL LPS for 90 minutes. Monocytes were then labeled with a CD14 antibody. Fixation of leukocytes and lysis of erythrocytes were performed with 4% paraformaldehyde and 3% diethylene glycol (DEG).

Purified buffy coat monocytes were stimulated as described above, restimulated with LPS (1 ng/mL) for 30 minutes, and fixed with 4% paraformaldehyde.

Fixed monocytes were washed and stained with antibodies in 0.1% Triton X-100 buffer. Primary antibodies were visualized by Alexa Fluor 488– or phycoerythrin-conjugated antibodies. DRAQ5 was used for nuclear imaging. Protein intensity and nuclear translocation were analyzed using an ImageStream system (Amnis, Seattle, WA) and IDEAS image analysis program.

Mice

C57BL/6 mice (10-16 weeks of age; Harlan Laboratories) were maintained under specific pathogen-free conditions. All animal experiments were approved by the local ethics committee governing animal experimentation (Landesamt für Natur, Umwelt und Verbraucherschutz NRW, Germany).

LPS/D-Gal model

Mice were treated intraperitoneally with CHIR99021 (25 mg/kg). Subsequently, endotoxin shock was induced by intraperitoneal injection of LPS (40 mg/kg) and D-Gal (680 mg/kg). The survival rate was analyzed for 24 hours. MRP8/14 serum levels were determined by ELISA, as described earlier.18  Serum cytokine concentrations were analyzed using BioLegend’s Legendplex immunoassays.

Murine ex vivo tolerance model

Hyporesponsiveness was induced in vivo by intraperitoneal injection of LPS (40 mg kg). After 24 hours, bone marrow cells (BMCs) and splenocytes were isolated and incubated on cell culture plates (1 hour) to remove lymphocytes. Macrophage-enriched cell populations were restimulated with LPS for 4 hours.

Results

Sterile and microbial TLR4 stimulation induces monocyte hyporesponsiveness by GSK-3β–dependent signaling

We first analyzed TNF-α production in response to repeated TLR4 stimulation to validate the hyporesponsiveness of cells. LPS was used as a microbial activator, and MRP8/14 hetero- or MRP8 homodimers were used as endogenous activators of TLR4 (supplemental Figure 1, available on the Blood Web site). We found that prestimulation with low doses of LPS or MRPs caused a significantly reduced TNF-α response to subsequent LPS activation in human monocytes, murine BMDMs, and Hoxb8 monocytes (Figure 1A-C). Next, we tolerized mice with LPS in vivo and detected significantly attenuated TNF-α production from their isolated BMCs following LPS activation ex vivo (Figure 1D). Subsequently we intended to identify kinases that play a role in the development of hyporesponsiveness following TLR4 stimulation. Using a Phospho-Kinase Antibody Array, we found that phosphorylation of GSK-3β contributes to the hyporesponsiveness of human monocytes (Figure 1E). GSK-3β is a constitutively active kinase that can be inactivated by phosphorylation (Ser9). After inducing hyporesponsiveness in human monocytes, inactivation of GSK-3β persisted over 24 hours and remained unaltered by subsequent restimulation (Figure 1F). To confirm the relevance in vivo, we injected low doses of LPS into mice, harvested BMCs after 24 hours, and analyzed GSK-3β phosphorylation/inhibition after restimulation ex vivo. Accordingly, BMCs of the tolerized mice showed a significantly higher GSK-3β phosphorylation in comparison with naive mice (Figure 1G). To prove our concept, we blocked GSK-3β using CHIR99021. Preincubation of human monocytes with this inhibitor mimicked hyporesponsiveness and led to significantly reduced TNF expression and secretion following LPS stimulation (Figure 1H-I).

Figure 1.

Hyporesponsive phagocytes show a prolonged inactivation of GSK-3β, protecting mice from septic shock. (A) TNF-α secretion of human monocytes 4 or 24 hours after treatment with medium (n), LPS (25 pg/mL), MRP8 (50 ng/mL), or MRP8/14 (10 µg/mL). Untreated or treated cells were restimulated with LPS (1 ng/mL) for 4 hours, and TNF-α was measured (n = 4). (B) BMDMs were stimulated with LPS (10 ng/mL) or Mrp8 (1 µg/mL) for 24 hours and subsequently restimulated with LPS (100 ng/mL) for 4 h. TNF-α production was measured in cell supernatants (n = 4). (C) WT Hoxb8 monocytes (differentiated for 3 days) were stimulated for 24 hours with LPS (10 ng/mL) or Mrp8 (5 µg/mL). After 4 hours of restimulation, TNF-α secretion was measured (n = 4). (D) Mice were tolerized in vivo with LPS (40 mg/kg) for 24 hours. Subsequently, BMCs and splenocytes were stimulated with LPS (100 ng/mL) for 4 hours ex vivo. TNF-α production was measured in cell supernatants (n = 6). (E) Whole-cell lysates of unstimulated, activated, and tolerized human monocytes were used for a human Phospho-Kinase Antibody Array (p-GSK-3β is marked in red). (F) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 24 hours and subsequently restimulated with LPS (1 ng/mL) for 30 minutes or 4 hours. GSK-3β phosphorylation was determined by western blot analysis. Results show 1 representative western blot out of 3. (G) WT mice were treated in vivo with a single LPS pulse (40 mg/kg) for 24 hours. Subsequently, GSK-3β phosphorylation in BMCs was determined by western blot analysis. One representative western blot and densitometric analysis are shown (n = 5). (H-I) Human monocytes were incubated with the GSK-3β inhibitor CHIR99021 (5 µmol/L) for 1 hour. Cells were subsequently stimulated with LPS (25 pg/mL), MRP8 (50 ng/mL), or MRP8/14 (10 µg/mL) for 24 hours and restimulated with LPS (1 ng/mL) for 4 hours. (H) qRT-PCR analysis of TNF expression is presented relative to baseline expression in unstimulated cells, normalized to ribosomal protein L (RPL) (n = 6). (I) TNF-α secretion was measured in cell supernatants via ELISA (n = 4). (J) WT mice were treated with CHIR99021 for 1 hour, and endotoxin shock was subsequently induced by LPS/D-Gal injection. The mice survival rate was analyzed for 24 hours using Kaplan-Meier analysis (n = 14). Murine serum concentrations of IL-1β (K), TNF-α (L), and MCP-1 (M) were measured 2 hours after LPS/D-Gal injection, and murine serum concentrations of IL-6 (N) and IL-10 (O) were measured 6 hours after LPS/D-Gal injection. Cytokine concentrations were analyzed using BioLegend’s Legendplex (n = 5). *P < .05, **P < .01, ***P < .001.

Figure 1.

Hyporesponsive phagocytes show a prolonged inactivation of GSK-3β, protecting mice from septic shock. (A) TNF-α secretion of human monocytes 4 or 24 hours after treatment with medium (n), LPS (25 pg/mL), MRP8 (50 ng/mL), or MRP8/14 (10 µg/mL). Untreated or treated cells were restimulated with LPS (1 ng/mL) for 4 hours, and TNF-α was measured (n = 4). (B) BMDMs were stimulated with LPS (10 ng/mL) or Mrp8 (1 µg/mL) for 24 hours and subsequently restimulated with LPS (100 ng/mL) for 4 h. TNF-α production was measured in cell supernatants (n = 4). (C) WT Hoxb8 monocytes (differentiated for 3 days) were stimulated for 24 hours with LPS (10 ng/mL) or Mrp8 (5 µg/mL). After 4 hours of restimulation, TNF-α secretion was measured (n = 4). (D) Mice were tolerized in vivo with LPS (40 mg/kg) for 24 hours. Subsequently, BMCs and splenocytes were stimulated with LPS (100 ng/mL) for 4 hours ex vivo. TNF-α production was measured in cell supernatants (n = 6). (E) Whole-cell lysates of unstimulated, activated, and tolerized human monocytes were used for a human Phospho-Kinase Antibody Array (p-GSK-3β is marked in red). (F) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 24 hours and subsequently restimulated with LPS (1 ng/mL) for 30 minutes or 4 hours. GSK-3β phosphorylation was determined by western blot analysis. Results show 1 representative western blot out of 3. (G) WT mice were treated in vivo with a single LPS pulse (40 mg/kg) for 24 hours. Subsequently, GSK-3β phosphorylation in BMCs was determined by western blot analysis. One representative western blot and densitometric analysis are shown (n = 5). (H-I) Human monocytes were incubated with the GSK-3β inhibitor CHIR99021 (5 µmol/L) for 1 hour. Cells were subsequently stimulated with LPS (25 pg/mL), MRP8 (50 ng/mL), or MRP8/14 (10 µg/mL) for 24 hours and restimulated with LPS (1 ng/mL) for 4 hours. (H) qRT-PCR analysis of TNF expression is presented relative to baseline expression in unstimulated cells, normalized to ribosomal protein L (RPL) (n = 6). (I) TNF-α secretion was measured in cell supernatants via ELISA (n = 4). (J) WT mice were treated with CHIR99021 for 1 hour, and endotoxin shock was subsequently induced by LPS/D-Gal injection. The mice survival rate was analyzed for 24 hours using Kaplan-Meier analysis (n = 14). Murine serum concentrations of IL-1β (K), TNF-α (L), and MCP-1 (M) were measured 2 hours after LPS/D-Gal injection, and murine serum concentrations of IL-6 (N) and IL-10 (O) were measured 6 hours after LPS/D-Gal injection. Cytokine concentrations were analyzed using BioLegend’s Legendplex (n = 5). *P < .05, **P < .01, ***P < .001.

To confirm the relevance of these findings in vivo, we used a murine LPS-induced model of sepsis. We artificially induced the hyporesponsiveness of phagocytes in vivo by injecting CHIR99221 1 hour prior to an LPS/D-Gal injection. Indeed, we observed significantly higher survival rates of CHIR99021-treated mice in comparison with untreated mice (Figure 1J). Furthermore, serum concentrations of IL-1β, TNF-α, IL-6, and IL-10 were markedly diminished in inhibitor-treated mice, confirming phagocyte hyporesponsiveness in vivo (Figure 1K-O).

These results demonstrate that inactivation of GSK-3β, induced by MRPs or LPS, promotes hyporesponsiveness of phagocytes.

GSK-3β inhibition is controlled by the PI3K/AKT pathway

GSK-3β is a downstream target of the PI3K/AKT pathway, and its activity has been described to be inhibited by AKT-mediated phosphorylation. We were able to find an early and persistent activation of PI3K and AKT during induction of hyporesponsiveness in monocytes (Figure 2A). Therefore, the use of an AKT inhibitor completely abolished induction of hyporesponsiveness in monocytes, which was reflected by a normal TNF response following LPS restimulation (Figure 2B-C). Further, western blot analysis demonstrated that AKT inhibition during prestimulation prevented GSK-3β phosphorylation/inactivation (Figure 2D). After induction of hyporesponsiveness in monocytes, other downstream targets of AKT, such as c-Raf, remained constantly phosphorylated and inactive (Figure 2E), thereby preventing Raf/MEK/ERK pathway reactivation during LPS restimulation. However, MAPK p38 could still be activated through LPS activation in hyporesponsive human monocytes, which further emphasizes the specificity of our findings.

Figure 2.

Involvement of the PI3K/AKT/GSK-3β pathway in phagocyte hyporesponsiveness. (A) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 4 or 24 hours and then 24-hour samples were restimulated with LPS (1 ng/mL) for 30 minutes. The phosphorylation state or total amount of different proteins of the PI3K pathway was determined by western blot analysis. Results show representative western blots. (B-C) Human monocytes were incubated with an AKT(1/2) inhibitor (10 µmol/L) for 1 hour. Cells were subsequently stimulated with LPS (25 pg/mL), MRP8 (50 ng/mL), or MRP8/14 (10 µg/mL) for 24 hours and restimulated with LPS (1 ng/mL) for 4 hours. (B) qRT-PCR analysis of TNF expression is presented relative to baseline expression in unstimulated cells, normalized to RPL. (C) TNF-α secretion was measured in cell supernatants via ELISA (n = 4). (D) Human monocytes were incubated with an AKT(1/2) inhibitor (10 µmol/L) before prestimulation. BCL-3 and IκBα protein accumulation, as well as the phosphorylation state of GSK-3β, was determined by western blot analysis. Results show 1 representative western blot of 3. (E) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 4 or 24 hours and then 24-hour samples were restimulated with LPS (1 ng/mL) for 30 minutes. The phosphorylation state or total amount of MAPK pathway proteins was determined by western blot analysis. *P < .05, **P < .01.

Figure 2.

Involvement of the PI3K/AKT/GSK-3β pathway in phagocyte hyporesponsiveness. (A) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 4 or 24 hours and then 24-hour samples were restimulated with LPS (1 ng/mL) for 30 minutes. The phosphorylation state or total amount of different proteins of the PI3K pathway was determined by western blot analysis. Results show representative western blots. (B-C) Human monocytes were incubated with an AKT(1/2) inhibitor (10 µmol/L) for 1 hour. Cells were subsequently stimulated with LPS (25 pg/mL), MRP8 (50 ng/mL), or MRP8/14 (10 µg/mL) for 24 hours and restimulated with LPS (1 ng/mL) for 4 hours. (B) qRT-PCR analysis of TNF expression is presented relative to baseline expression in unstimulated cells, normalized to RPL. (C) TNF-α secretion was measured in cell supernatants via ELISA (n = 4). (D) Human monocytes were incubated with an AKT(1/2) inhibitor (10 µmol/L) before prestimulation. BCL-3 and IκBα protein accumulation, as well as the phosphorylation state of GSK-3β, was determined by western blot analysis. Results show 1 representative western blot of 3. (E) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 4 or 24 hours and then 24-hour samples were restimulated with LPS (1 ng/mL) for 30 minutes. The phosphorylation state or total amount of MAPK pathway proteins was determined by western blot analysis. *P < .05, **P < .01.

These results indicate that the activity of GSK-3β is tightly controlled by the PI3K/AKT pathway in hyporesponsive monocytes.

GSK-3β downstream targets, IκBα and BCL-3, accumulate in hyporesponsive phagocytes

Phosphorylation of target proteins by GSK-3β frequently leads to their proteasomal degradation. Therefore, we analyzed the expression levels of 2 known GSK-3β substrates, IκBα and BCL-3, during induction of hyporesponsiveness in vitro and in vivo. In hyporesponsive monocytes, IκBα and BCL-3 showed an increase in cytosolic expression (Figure 3A), and BCL-3 also exhibited nuclear accumulation (Figure 3B). Additional imaging flow cytometry measurements confirmed a total increase in expression and nuclear localization of BCL-3 (Figure 3C). Furthermore, BMCs and splenocytes of in vivo–tolerized mice also showed a significantly higher intracellular accumulation of BCL-3 in comparison with naive cells (Figure 3D). We artificially induced hyporesponsiveness by CHIR99021 and then analyzed degradation of GSK-3β substrates. In this case, the inhibitor prevented IκBα degradation in LPS-activated cells, leading to similar protein levels compared with LPS- and MRP8-tolerized monocytes (Figure 3E). However, the amounts of BCL-3 protein remained unaffected by GSK-3β inhibition and did not accumulate in naive or in activated cells (Figure 3E). Accordingly, we used MG132 and ONX0941 to exclude proteasomal degradation of BCL-3 in these cells (Figure 3F). However, BCL-3 gene expression was significantly increased 4 and 24 hours after LPS- or MRP8-induced hyporesponsiveness (Figure 3G). This indicates regulation at the transcriptional level and a de novo synthesis of BCL-3 in a GSK-3β–independent manner. To validate the functional relevance of BCL-3 accumulation during the phagocyte hyporesponsiveness, we established BCL-3−/− Hoxb8 monocytes. First, we confirmed an increase in BCL-3 in wild-type (WT) Hoxb8 cells during induction of hyporesponsiveness (Figure 3H). Subsequently, we first stimulated WT and BCL-3−/− Hoxb8 cells and then restimulated them with LPS. Prestimulated BCL-3−/− Hoxb8 cells showed a normal TNF-α secretion in response to a secondary LPS stimulus. On the other hand, WT cells showed the characteristic attenuated TNF-α production seen in hyporesponsive phagocytes (Figure 3I).

Figure 3.

Accumulation of GSK-3β target proteins, IκBα and BCL-3, in hyporesponsive cells. (A-B) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8/14 (10 µg/mL) for 24 hours and subsequently restimulated with LPS (1 ng/mL) for 30 minutes. Cells were further fractionated into cytosolic and nuclear fractions. The protein accumulation of IκBα and BCL-3 was determined by western blot analysis. Results show 1 representative western blot of 3. (C) Accumulation of BCL-3 in hyporesponsive human monocytes was further analyzed by immunofluorescence colony staining. Representative images (upper panels), as well as quantification of total intensity (lower left panel) and nuclear intensity (lower right panel) of BCL-3, are shown (n = 3). (D) WT mice were stimulated in vivo with LPS (40 mg/kg) for 24 hours. Subsequently, BMCs and splenocytes were isolated and lysed. BCL-3 accumulation was determined by western blot analysis. One representative western blot and densitometric analysis are shown (n = 5). (E) Human monocytes were incubated with the GSK-3β inhibitor CHIR99021 (5 µmol/L) for 1 hour. Cells were subsequently stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) and then restimulated with LPS (1 ng/mL) for 30 minutes. Accumulation of BCL-3 and IκBα was analyzed by western blot. One representative western blot of 3 is shown. (F-G) BCL-3 expression was analyzed in LPS- and MRP8-tolerized human monocytes 4 and 24 hours after prestimulation. The 24-hour prestimulated samples were also restimulated with LPS for 1 hour. (F) Human monocytes were preincubated with the proteasome inhibitor MG132 (200 nmol/L) or ONX0914 (10 nmol/L). Influence of proteasome inhibition on BCL-3 protein accumulation was analyzed by western blot. One representative western blot of 3 is shown. (G) qRT-PCR analysis of BCL-3 expression in human monocytes is presented relative to baseline expression in unstimulated cells, normalized to RPL (n = 4). (H) WT Hoxb8 cells were differentiated into monocytes for 3 days and subsequently stimulated for 24 hours with LPS (10 ng/mL) or Mrp8 (5 µg/mL). Cells were restimulated with LPS (100 ng/mL) for 30 minutes and subsequently lysed for western blot analysis. Results of 1 representative western blot of 3 are shown. (I) WT and BCL-3−/− Hoxb8 cells were differentiated into monocytes for 3 days. Cells were stimulated with LPS (10 ng/mL) or Mrp8 (5 µg/mL) for 24 hours and subsequently restimulated with LPS (100 ng/m) for 4 hours. TNF-α production was measured in cell supernatants. Results are presented relative to only activated cells (n/LPS) (n = 4). **P < .01, ***P < .001.

Figure 3.

Accumulation of GSK-3β target proteins, IκBα and BCL-3, in hyporesponsive cells. (A-B) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8/14 (10 µg/mL) for 24 hours and subsequently restimulated with LPS (1 ng/mL) for 30 minutes. Cells were further fractionated into cytosolic and nuclear fractions. The protein accumulation of IκBα and BCL-3 was determined by western blot analysis. Results show 1 representative western blot of 3. (C) Accumulation of BCL-3 in hyporesponsive human monocytes was further analyzed by immunofluorescence colony staining. Representative images (upper panels), as well as quantification of total intensity (lower left panel) and nuclear intensity (lower right panel) of BCL-3, are shown (n = 3). (D) WT mice were stimulated in vivo with LPS (40 mg/kg) for 24 hours. Subsequently, BMCs and splenocytes were isolated and lysed. BCL-3 accumulation was determined by western blot analysis. One representative western blot and densitometric analysis are shown (n = 5). (E) Human monocytes were incubated with the GSK-3β inhibitor CHIR99021 (5 µmol/L) for 1 hour. Cells were subsequently stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) and then restimulated with LPS (1 ng/mL) for 30 minutes. Accumulation of BCL-3 and IκBα was analyzed by western blot. One representative western blot of 3 is shown. (F-G) BCL-3 expression was analyzed in LPS- and MRP8-tolerized human monocytes 4 and 24 hours after prestimulation. The 24-hour prestimulated samples were also restimulated with LPS for 1 hour. (F) Human monocytes were preincubated with the proteasome inhibitor MG132 (200 nmol/L) or ONX0914 (10 nmol/L). Influence of proteasome inhibition on BCL-3 protein accumulation was analyzed by western blot. One representative western blot of 3 is shown. (G) qRT-PCR analysis of BCL-3 expression in human monocytes is presented relative to baseline expression in unstimulated cells, normalized to RPL (n = 4). (H) WT Hoxb8 cells were differentiated into monocytes for 3 days and subsequently stimulated for 24 hours with LPS (10 ng/mL) or Mrp8 (5 µg/mL). Cells were restimulated with LPS (100 ng/mL) for 30 minutes and subsequently lysed for western blot analysis. Results of 1 representative western blot of 3 are shown. (I) WT and BCL-3−/− Hoxb8 cells were differentiated into monocytes for 3 days. Cells were stimulated with LPS (10 ng/mL) or Mrp8 (5 µg/mL) for 24 hours and subsequently restimulated with LPS (100 ng/m) for 4 hours. TNF-α production was measured in cell supernatants. Results are presented relative to only activated cells (n/LPS) (n = 4). **P < .01, ***P < .001.

All things considered, these results demonstrate that the GSK-3β downstream targets, IκBα and BCL-3, accumulate in hyporesponsive phagocytes. However, the accumulation of IκBα, but not that of BCL-3, seems to directly depend on the inactivity of GSK-3β.

BCL-3 accumulates in human monocytes in an IL-10–dependent manner

LPS and MRP8 stimulation of human monocytes led to an increased IL-10 secretion after 4 hours, which continued to rise over the next 12 hours (Figure 4A). Therefore, we verified the impact of IL-10 on the hyporesponsiveness of human monocytes. Pretreatment with recombinant IL-10 prevented LPS-induced TNF-α secretion that was comparable to TLR4-mediated hyporesponsiveness (Figure 4B). On the other hand, antibody-mediated blockage of the IL-10 receptor during prestimulation significantly increased the response of human monocytes to LPS restimulation (Figure 4C). Furthermore, stimulation with IL-10 alone was sufficient to induce BCL-3 accumulation after 24 hours (Figure 4D).

Figure 4.

The IL-10 pathway plays a crucial role in the hyporesponsiveness of phagocytes. (A) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 4, 6, 8, 12, or 24 hours, and IL-10 secretion was analyzed in the cell supernatants using immunoassays (Legendplex). (B) Hyporesponsiveness was induced in human monocytes by LPS (25 pg/mL) or IL-10 (10 ng/mL) for 24 hours. Subsequently, cells were restimulated with LPS (1 ng/mL) or IL-10 for 4 hours. Hyporesponsiveness was monitored by measuring TNF-α concentrations in cell supernatants using ELISA (n = 4). (C) Human monocytes were incubated with an IL-10 receptor blocking antibody (50 µg/mL); 24 hours later, cells were restimulated with LPS for 4 hours. TNF-α secretion of these cells was analyzed in the supernatants (n = 7). (D) BCL-3 protein accumulation by IL-10 was compared with LPS-induced protein accumulation. Human monocytes were stimulated with LPS or IL-10 for 4 or 24 hours. Whole-cell lysates were further analyzed by western blot. One representative western blot of 3 is shown. *P < .05, **P < .01, ***P < .001.

Figure 4.

The IL-10 pathway plays a crucial role in the hyporesponsiveness of phagocytes. (A) Human monocytes were stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 4, 6, 8, 12, or 24 hours, and IL-10 secretion was analyzed in the cell supernatants using immunoassays (Legendplex). (B) Hyporesponsiveness was induced in human monocytes by LPS (25 pg/mL) or IL-10 (10 ng/mL) for 24 hours. Subsequently, cells were restimulated with LPS (1 ng/mL) or IL-10 for 4 hours. Hyporesponsiveness was monitored by measuring TNF-α concentrations in cell supernatants using ELISA (n = 4). (C) Human monocytes were incubated with an IL-10 receptor blocking antibody (50 µg/mL); 24 hours later, cells were restimulated with LPS for 4 hours. TNF-α secretion of these cells was analyzed in the supernatants (n = 7). (D) BCL-3 protein accumulation by IL-10 was compared with LPS-induced protein accumulation. Human monocytes were stimulated with LPS or IL-10 for 4 or 24 hours. Whole-cell lysates were further analyzed by western blot. One representative western blot of 3 is shown. *P < .05, **P < .01, ***P < .001.

These data clearly illustrate that the IL-10 pathway has a crucial role in the mechanism of BCL-3–dependent hyporesponsiveness of phagocytes.

STAT3 is a central regulator of phagocyte hyporesponsiveness

To characterize the observed transcriptional regulation and the de novo synthesis of BCL-3 in more detail, we analyzed transcription factor binding sites within the BCL-3 gene by performing in silico analyses. We identified 2 binding sites of STAT3 inside the BCL-3 gene (Figure 5A). Because STAT3 is a known signaling molecule of the IL-10 pathway, we next analyzed the activation of STAT3 in hyporesponsive phagocytes. At 4 hours after the induction of hyporesponsiveness, we observed STAT3 activation by phosphorylation. This active state was maintained over 24 hours (Figure 5B). In addition, the amounts of total STAT3 protein were markedly enhanced after prestimulation, whereas STAT3 mRNA expression remained unaltered (Figure 5B-C). Furthermore, BMCs from in vivo–tolerized mice showed a significantly higher accumulation of STAT3 in comparison with naive mice (Figure 5D). Inhibition of STAT3 (LLL12) completely reversed the hyporesponsiveness in human monocytes, as indicated by a normal TNF-α response to LPS restimulation (Figure 5E-F). Furthermore, preincubation with LLL12 reduced the upregulation of BCL-3 mRNA and protein in prestimulated monocytes (Figure 5G), whereas the amount of STAT3 protein remained unaffected (Figure 5H). Finally, we confirmed the functional relevance of our findings in STAT3−/− Hoxb8 monocytes, in which BCL-3 protein accumulation was not seen after tolerization (Figure 5I). Previously, we reported that the hyporesponsiveness of phagocytes persists for >24 hours.9  We now demonstrate that the PI3K/AKT/GSK-3β and IL-10/STAT3 pathways remain active as long as the hyporesponsiveness exists (Figure 5J).

Figure 5.

STAT3-dependent mechanisms during hyporesponsiveness of phagocytes. (A) In silico analysis of STAT3 transcription factor binding sites inside the intron region between exons 2 and 3 of the BCL-3 gene. For analysis, the program Patch 1.0 was used, which is based on the manually curated database of eukaryotic transcription factors TRANSFAC. (B) The phosphorylation state and total protein amount of STAT3 were analyzed in LPS- and MRP8- stimulated human monocytes in comparison with unstimulated and activated cells. One representative western blot of 3 is shown. (C) mRNA expression of STAT3 was analyzed in LPS- and MRP8-stimulated human monocytes after 4 and 24 hours. The samples stimulated for 24 hours were restimulated with LPS for 1 hour. qRT-PCR analysis of STAT3 expression in these cells is presented relative to baseline expression in unstimulated cells, normalized to RPL (n = 4). (D) WT mice were tolerized in vivo with LPS (40 mg/kg) for 24 hours. Subsequently, BMCs were isolated and lysed. Protein accumulation of STAT3 was determined by western blot analysis. One representative western blot and densitometric analysis are shown (n = 3). (E-G) Human monocytes were incubated with the STAT3 inhibitor LLL12 (1 µmol/L) for 1 hour, stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 24 hours, and restimulated with LPS (1 ng/mL) for 4 hours. (E-G) qRT-PCR analysis of TNF and BCL-3 in these cells is presented relative to baseline expression in unstimulated cells, normalized to RPL. (F) TNF-α secretion was measured in cell supernatants via ELISA (n = 4). (H) Human monocytes were incubated with the STAT3 inhibitor LLL12 before stimulation with LPS, MRP8, or IL-10 for 4 or 24 hours. Influence on BCL-3 and STAT3 accumulation, as well as STAT3 phosphorylation, was determined by western blot analysis. Representative western blots are shown (1 of 3). (I) WT and STAT3−/− Hoxb8 cells were differentiated into monocytes for 3 days. Cells were stimulated with LPS for 24 hours and compared with unstimulated and activated cells. The protein amount of BCL-3 and STAT3, as well as STAT3 phosphorylation, was compared in these 2 cell types by western blot analysis. Results show 1 representative western blot of 3. (J) The phosphorylation state of GSK-3 and total protein amount of STAT3 and BCL-3 were analyzed in LPS- and MRP8-stimulated human monocytes 24 and 48 h after removing the tolerizing stimulus. (K) qRT-PCR analysis of TNF and BCL-3, presented relative to baseline expression, in unstimulated cells of healthy donors (HD) or immune-deficient phenotype of HIES patients, normalized to GAPDH (n = 2). *P < .05, **P < .05, ***P < .001.

Figure 5.

STAT3-dependent mechanisms during hyporesponsiveness of phagocytes. (A) In silico analysis of STAT3 transcription factor binding sites inside the intron region between exons 2 and 3 of the BCL-3 gene. For analysis, the program Patch 1.0 was used, which is based on the manually curated database of eukaryotic transcription factors TRANSFAC. (B) The phosphorylation state and total protein amount of STAT3 were analyzed in LPS- and MRP8- stimulated human monocytes in comparison with unstimulated and activated cells. One representative western blot of 3 is shown. (C) mRNA expression of STAT3 was analyzed in LPS- and MRP8-stimulated human monocytes after 4 and 24 hours. The samples stimulated for 24 hours were restimulated with LPS for 1 hour. qRT-PCR analysis of STAT3 expression in these cells is presented relative to baseline expression in unstimulated cells, normalized to RPL (n = 4). (D) WT mice were tolerized in vivo with LPS (40 mg/kg) for 24 hours. Subsequently, BMCs were isolated and lysed. Protein accumulation of STAT3 was determined by western blot analysis. One representative western blot and densitometric analysis are shown (n = 3). (E-G) Human monocytes were incubated with the STAT3 inhibitor LLL12 (1 µmol/L) for 1 hour, stimulated with LPS (25 pg/mL) or MRP8 (50 ng/mL) for 24 hours, and restimulated with LPS (1 ng/mL) for 4 hours. (E-G) qRT-PCR analysis of TNF and BCL-3 in these cells is presented relative to baseline expression in unstimulated cells, normalized to RPL. (F) TNF-α secretion was measured in cell supernatants via ELISA (n = 4). (H) Human monocytes were incubated with the STAT3 inhibitor LLL12 before stimulation with LPS, MRP8, or IL-10 for 4 or 24 hours. Influence on BCL-3 and STAT3 accumulation, as well as STAT3 phosphorylation, was determined by western blot analysis. Representative western blots are shown (1 of 3). (I) WT and STAT3−/− Hoxb8 cells were differentiated into monocytes for 3 days. Cells were stimulated with LPS for 24 hours and compared with unstimulated and activated cells. The protein amount of BCL-3 and STAT3, as well as STAT3 phosphorylation, was compared in these 2 cell types by western blot analysis. Results show 1 representative western blot of 3. (J) The phosphorylation state of GSK-3 and total protein amount of STAT3 and BCL-3 were analyzed in LPS- and MRP8-stimulated human monocytes 24 and 48 h after removing the tolerizing stimulus. (K) qRT-PCR analysis of TNF and BCL-3, presented relative to baseline expression, in unstimulated cells of healthy donors (HD) or immune-deficient phenotype of HIES patients, normalized to GAPDH (n = 2). *P < .05, **P < .05, ***P < .001.

These data demonstrate that the expression of BCL-3 is controlled by activation of the IL-10 downstream transcription factor STAT3; therefore, STAT3 is a pivotal factor in LPS- or MRP-induced hyporesponsiveness of phagocytes.

To strengthen this conclusion, we subsequently analyzed the expression of BCL-3, as well as of proinflammatory mediators, in monocytes from patients with dominant-negative mutations in the STAT3 gene (Table 1). Monocytes obtained from these patients showed a significant lack of hyporesponsiveness after prestimulation. This is reflected by TNF mRNA induction after restimulation and the lack of BCL-3 accumulation (Figure 5K). These data demonstrate the central role that STAT3 plays as a master regulator during induction of monocyte hyporesponsiveness.

Phagocytes from patients with CBS show hyporesponsiveness

The inflammatory response induced during CBS has been implicated in many of the postoperative clinical problems faced by these patients.19  We now demonstrate that LPS-stimulated monocytes from CBS patients (Table 2) exhibit a state of hyporesponsiveness that is characterized by a remarkably reduced TNF-α expression and secretion (Figure 6A-B). As a potential mediator of hyporesponsiveness, the MRP8/14 levels in serum were measured. These levels were already significantly increased at the time of cardiopulmonary bypass removal and remained elevated until 6 days after surgery (Figure 6C). In accordance with our findings that IL-10 upregulation is an important step in the STAT3/BCL-3 pathway in vitro, we detected an early accumulation of IL-10 in the postoperative samples, with a clear peak directly after the end of surgery (Figure 6D). On day 1 after CBS, this peak preceded the peak of tolerance. Imaging flow cytometry analysis also demonstrated a significant increase in BCL-3 and STAT3 accumulation and nuclear translocation in monocytes from CBS patients 1 day after surgery. This increase persisted until day 6 (Figure 6E-F).

Table 2.

Epidemiologic and clinical characteristics of the CBS patients (n = 12)

Age, mean ± SD (y)Male (%)Diagnosis (n)Surgery (%)CR (%)
71.4 ± 9.2 41.7 CHD 7 ACB 33.3 8.3 
  V 7 CV 41.7  
  AA 2 A 8.3  
  ASD 0 Combination 25  
  T 0 MIS 50  
Age, mean ± SD (y)Male (%)Diagnosis (n)Surgery (%)CR (%)
71.4 ± 9.2 41.7 CHD 7 ACB 33.3 8.3 
  V 7 CV 41.7  
  AA 2 A 8.3  
  ASD 0 Combination 25  
  T 0 MIS 50  

A, aorta; AA, aortic aneurysm; ACB, aortocoronary bypass; ASD, atrial septal defect; CHD, coronary heart disease; CR, cardiopulmonary resuscitation; CV, cardiac valve; MIS, minimally invasive surgery; T, tumor; V, valvular.

Figure 6.

Phagocytes from CBS patients show hyporesponsiveness. Isolated CBS patient monocytes were stimulated with LPS (1 ng/mL) for 4 hours, and TNF-α secretion (A) and gene expression of TNF (B) were analyzed on the day of admission (d-1), directly after CBS (d0), on the first postoperative day (d1), and 6 days postsurgery (d6). qRT-PCR analysis of TNF expression is presented relative to baseline expression in control cells at time point d-1, set as 1 normalized to GAPDH. (C) MRP8/14 secretion was measured in human serum samples using ELISA (n = 19). (D) IL-10 secretion was measured in human serum samples using Legendplex bead-based immunoassays (n = 12). *P < .05, **P < .01, ***P < .001. Accumulation of STAT3 (E) and BCL-3 (F) in unstimulated human whole blood samples was analyzed by immunofluorescence colony staining. Representative images and total intensity and nuclear intensity of STAT3 and BCL-3 are shown (n = 12). (A-D), *P < .05, **P < .01, ***P < .001. (E-F), *P < .1, **P < .01.

Figure 6.

Phagocytes from CBS patients show hyporesponsiveness. Isolated CBS patient monocytes were stimulated with LPS (1 ng/mL) for 4 hours, and TNF-α secretion (A) and gene expression of TNF (B) were analyzed on the day of admission (d-1), directly after CBS (d0), on the first postoperative day (d1), and 6 days postsurgery (d6). qRT-PCR analysis of TNF expression is presented relative to baseline expression in control cells at time point d-1, set as 1 normalized to GAPDH. (C) MRP8/14 secretion was measured in human serum samples using ELISA (n = 19). (D) IL-10 secretion was measured in human serum samples using Legendplex bead-based immunoassays (n = 12). *P < .05, **P < .01, ***P < .001. Accumulation of STAT3 (E) and BCL-3 (F) in unstimulated human whole blood samples was analyzed by immunofluorescence colony staining. Representative images and total intensity and nuclear intensity of STAT3 and BCL-3 are shown (n = 12). (A-D), *P < .05, **P < .01, ***P < .001. (E-F), *P < .1, **P < .01.

These results clearly indicate that the hyporesponsiveness of monocytes induced during CBS is preceded by the upregulation of MRP alarmins and involves the induction of IL-10 and the activation of the transcriptional regulators BCL-3 and STAT3.

Discussion

Although most SIRS patients survive the initial hyperinflammatory phase, a substantial number develop a secondary hyporesponsive state that can be even more life-threatening due to secondary microbial infections. A treatment option for this phagocyte tolerance does not exist.

Several signaling pathways have already been described in the context of pathogen triggered tolerance.4  It has been reported that prolonged activation of TLR4 can lead to prevention in the activation of downstream kinases, such as IRAK and MAPKs. However, existing results vary significantly between model systems, and the blocking of the hitherto described mechanisms does not abrogate tolerance induction.20  Furthermore, it should be noted that the majority of published data in vivo were obtained using mouse models. Therefore the relevance of these specific pathways in relation to tolerance induction as seen in humans during clinically relevant diseases, such as in sterile SIRS, is only partially understood.

We have now identified 2 distinct signaling pathways, the PI3K/AKT/GSK-3β and the IL-10/STAT3/BCL-3 pathway, that play a dominant role in hyporesponsive monocytes. We were able to confirm the relevance of these 2 pathways through a specific inhibition and a targeted gene knock-out in monocytes in vitro, as well as through additional in vivo experimental models and in clinically relevant disorders, ie, CBS and STAT3 deficiency.

We specifically chose CBS as a clinical model on the account of the major sterile stress created during surgery and CBS circulation. Additionally, the time course of the surgery, in the absence of an infectious agent, is well defined. In our clinically relevant experiments, we were able to show an initial upregulation of MRP8/14 during CBS and demonstrate that this MRP8/14 trigger induces an early, although persistent, PI3K/AKT-dependent inactivation of GSK-3β resulting in hyporesponsive monocytes.

We were also able to mimic this hyporesponsiveness in vitro and in vivo using the GSK-3β inhibitor CHIR99021. The use of CHIR99021 significantly increased the survival rate of mice during endotoxin shock. Conversely, the inhibition of the GSK-3β opponent AKT led to a complete attenuation of hyporesponsiveness in human monocytes. Furthermore, we identified a prolonged inactivation of the Raf/MEK/ERK pathway that is important for the induction of TNF expression downstream of AKT.21  In contrast, MAPK p38 could still be activated in hyporesponsive cells, indicating very specific effects seen during the induction of hyporesponsiveness of monocytes rather than a complete inactivation of the MAPK pathway, contradicting some earlier publications.4,22  During normal signal transduction, activation/deactivation processes of these kinases can last anywhere from a few minutes to 1 hour.23,24  We found that the phosphorylation of GSK-3β persisted for several days. These long-lasting posttranslational protein modifications reflect a novel principle of innate immune memory.25  Additionally, we demonstrated that there was an accumulation of GSK-3β target proteins, IκBα and BCL-3, in hyporesponsive monocytes. The inactivation of GSK-3β and the resulting accumulation of IκBα interfere with the translocation of active NF-κB to the nucleus and, in turn, prevent the induction of proinflammatory cytokines.26  BCL-3 is also a member of the IκB family and functions as a transcriptional repressor by promoting the binding of inactive NF-κB dimers to the TNF promoter.27  Moreover, NF-κB and BCL-3 are known to interact with histone deacetylases HDAC1, HDAC3, and HDAC6 and, thereby, influence the transcription of genes through the regulation of histone acetylation and promoter accessibility.28,29  Our results show that GSK-3β does not directly act as a modifier of BCL-3. These findings suggest the existence of a new and unknown regulation of BCL-3 during hyporesponsiveness. We now identified an IL-10–dependent feedback loop that resulted in the activation of STAT3, which, in turn, initiated BCL-3 gene expression. Blocking or deletion of STAT3 in monocytes almost completely abolished hyporesponsiveness and BCL-3 accumulation. We found no self-regulation of STAT3, as described in an earlier study.30  The functional relevance of these findings in the human system was further confirmed in monocytes obtained from HIES patients suffering from negative STAT3 gene mutations.31  Monocytes from HIES patients are unable to develop hyporesponsiveness after prolonged TLR4 stimulation. Accordingly, they show a reduction in BCL-3 gene expression. Therefore, we provide strong evidence that STAT3 deficiency prohibits the induction of an anti-inflammatory counterregulation, which thereby enhances the inflammatory pathogenesis of HIES. However, it must be taken into account that STAT3 regulates the signaling of multiple cytokines that might be implicated in the complex pathophysiology of HIES.32 

The recent concept of “innate immune memory” describes several mechanisms and pathways that lead to reprogramming of innate immune cells and result in the adaptation of response patterns to subsequent or repetitive stimulation.10  These mechanisms can be divided into tolerance, classified by the downregulation of proinflammatory genes, and trained immunity, resulting in an increased production of proinflammatory cytokines.10  However, our studies revealed that tolerance involves the up- and downregulation of genes in parallel, which is in line with studies describing up- and downregulated gene expression in LPS-induced tolerance and β-glycan–induced trained immunity.2,33  Hyper- and hyporesponsive mechanisms in SIRS are also not strictly separated during the clinical course of disease; “tolerance,” as well as “trained immunity,” induced by endogenous and exogenous triggers of TLRs may drive, in parallel, the complex pathogenesis of SIRS and sepsis.

It is well known that epigenetic chromatin modifications are 1 essential factor for the hyporesponsiveness seen in the innate immune cells. These modifications eventually result in major functional and metabolic cell changes.34  However, the relevance of initial triggers and signaling pathways resulting in innate immune memory in specific clinically relevant pathologic conditions is less clear. We now demonstrate that, in monocytes from CBS patients, the initial induction of MRP8/14 secretion is indeed followed by a hyporesponsive state of diminished TLR4-dependent cytokine production. This state reaches a peak on the first day after surgery and lasts ≥6 days. Using imaging flow cytometry, we could show an elevated expression and increased nuclear localization of BCL-3 and STAT3 in monocytes from CBS patients during the critical phase of hyporesponsiveness. Furthermore, STAT3 translocation was preceded by a high induction of IL-10, indicating that the mechanisms and pathways described above are also relevant in a clinical setting of sterile systemic stress induction.

In summary, we could identify the AKT/GSK-3β signaling cascade and activation of the IL-10/STAT3/BCL-3 pathway as 2 distinct and functionally relevant mechanisms for the development of monocyte hyporesponsiveness during alarmin-driven sterile stress reactions. Whether these pathways are relevant for reprogramming of other phagocytes (eg, granulocytes or dendritic cells) during SIRS remains to be investigated. Our cellular studies using blocking and knockout approaches in vitro and experimental models in mice demonstrated the functional role of these specific pathways for tolerance induction, whereas the clinical relevance was confirmed in parallel by our ex vivo analysis of patient-derived monocytes and serum samples during CBS. STAT3 seems to be an especially promising candidate for new treatment options for this hyporesponsive state. Several specific JAK/STAT3 inhibitors have already been tested in clinical studies and have shown a good safety profile.35  Because MRP8/14 are the most abundant alarmins in many acute and chronic inflammatory processes,36  MRP-mediated hyporesponsiveness of phagocytes might be a mechanism that is relevant in the development of complications in several inflammatory disease profiles.

For original data, please contact Johannes Roth (rothj@uni-muenster.de).

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

The authors thank U. Nordhues, H. Berheide, and H. Harter for excellent technical support and Ingo Schmitz (Centre for Infection Research, Brunswick) for providing BCL-3−/− mice.

This work was supported by grants from Innovative Medical Research of the University of Muenster (AU121327 and AU211603) (J.A.), the Interdisciplinary Center of Clinical Research at the University of Muenster (Vo2/014/09, Ro2/003/15, Ro2/023/19) (T.V. and J. Roth), the German Research Foundation CRC 1009 B8, B9, and Z2 (T.V. and J. Roth), ERARE2 networks Treat-AID and Cure-AID (J. Roth), and the Federal Ministry of Education and Research project AID-NET (J. Roth and T.V.).

The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authorship

Contribution: N.F., A.B., A.I.C., J.H., J. Roth, and J.A. conceived and designed the experiments; N.F., A.B., T.O., N.D., M.S., J.H., J. Rojas, S.-L.J., O.F. and J.A. performed experiments; A.B., J.H., M.S., J. Rojas, B.G., A.H., and S.M. collected and provided CBS and HIES patient samples; N.F., A.B., A.I.C., T.V., B.G., S.M., J. Roth, and J.A. analyzed the data; and N.F., A.B., J. Roth, and J.A. wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Johannes Roth, Institute of Immunology, University of Muenster, Roentgenstraße 21, D-48149 Muenster, Germany; e-mail: rothj@uni-muenster.de.

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Author notes

*

N.F. and A.B. contributed equally to this work.

Supplemental data