Selective macrophage suppression during sepsis

Polymicrobial sepsis induces suppression of macrophage function as determined by a reduction of pro-inflammatory cytokine production upon re-exposure to lipopolysaccharide (LPS) in vitro. We examined whether macrophages were refractory to only LPS challenge or if they were immunoparalyzed and unable to respond to other stimuli such as lipoteichoic acid (LTA) or zymosan (ZYM). This study evaluated the capacity of peritoneal macrophages to produce pro-inflammatory and anti-inflammatory cytokines as well as chemokines following mild or severe sepsis induced by cecal ligation and puncture (CLP). Peritoneal macrophages were isolated 29 h after CLP and challenged with different stimuli. LPS was a more potent stimulus for cytokine induction than LTA or ZYM in both mild and severe sepsis. In mild sepsis, the macrophage cytokine response to LPS was selective and less refractory than in severe sepsis. While production of IL-6 and KC was reduced, secretion of TNF-alpha and MIP-1alpha was enhanced in those cells isolated from mice with mild sepsis. Production of IL-10 and the IL-1 receptor antagonist , MIP-2, and MCP-1 in response to LPS stimulation was equivalent to the amount produced by naive macrophages. Our results indicate that macrophages are not immunoparalyzed during sepsis and may still be induced to secrete some inflammatory mediators.     

Role of phosphatidylinositol-3 kinase in transcriptional regulation of TLR-induced IL-12 and IL-10 by Fc gamma receptor ligation in murine macrophages

Ligation of FcgammaR concurrent with LPS stimulation of murine macrophages results in decreased IL-12 and increased IL-10 production. Because PI3K deficiency has been associated with increased IL-12, we hypothesized that PI3K was central to the anti-inflammatory effect of FcgammaR ligation on TLR-induced IL-12. FcgammaR ligation of macrophages increased pAKT, a correlate of PI3K activity, above levels induced by TLR4 or TLR2 agonists. This increase was blocked by PI3K inhibitors, wortmannin or LY294002, as was the effect of FcgammaR ligation on TLR-induced IL-12 and IL-10. LPS-induced binding of NF-kappaB to the IL-12 p40 promoter NF-kappaB-binding site was not affected by FcgammaR ligation at 1 h; however, by 4 h, NF-kappaB binding was markedly inhibited, confirmed in situ by chromatin immunoprecipitation analysis. This effect was wortmannin sensitive. Although TLR-induced IkappaBalpha degradation was not affected by FcgammaR ligation, IkappaBalpha accumulated in the nuclei of cells treated with LPS and FcgammaR ligation for 4 h, and was blocked by PI3K inhibitors. LPS-induced IFN regulatory factor-8/IFN consensus sequence-binding protein mRNA, and an IFN regulatory factor-8-dependent gene, Nos2, were inhibited by concurrent FcgammaR ligation, and this was also reversed by wortmannin. Thus, FcgammaR ligation modulates LPS-induced IL-12 via multiple PI3K-sensitive pathways that affect production, accumulation, and binding of key DNA-binding proteins required for IL-12 induction.     

Microtubule-associated serine/threonine kinase-205 kDa and Fc gamma receptor control IL-12 p40 synthesis and NF-kappa B activation

Stimulation of murine macrophages with LPS results in the coordinated activation of a set of proinflammatory cytokines and costimulatory molecules, including TNF-alpha, IL-6, IL-1, IL-8, IL-12, and CD80. Macrophage LPS-induced synthesis of IL-12 is inhibited following FcgammaR ligation; TNF-alpha secretion is unchanged. We report that microtubule-associated serine/threonine kinase-205 kDa (MAST205) is required for LPS-induced IL-12 synthesis. RNA interference-mediated suppression of MAST205 results in the inhibition of LPS-stimulated IL-12 promoter activity and IL-12 secretion, from both J774 cells and bone marrow-derived macrophages. Similarly, dominant-negative MAST205 mutants inhibit LPS-stimulated IL-12 synthesis and NF-kappaB activation, but do not affect IL-1 or TNF-alpha signaling. Finally, macrophage FcgammaR ligation regulates MAST205 by inducing the rapid ubiquitination and proteasomal degradation of the protein.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

GCN2-Dependent Metabolic Stress Is Essential for Endotoxemic Cytokine Induction and Pathology

Activated inflammatory macrophages can express indoleamine 2,3-dioxygenase (IDO) and thus actively deplete their own tryptophan supply; however, it is not clear how amino acid depletion influences macrophage behavior in inflammatory environments. In this report, we demonstrate that the stress response kinase GCN2 promotes macrophage inflammation and mortality in a mouse model of septicemia. In vitro, enzymatic amino acid consumption enhanced sensitivity of macrophages to the Toll-like receptor 4 (TLR4) ligand lipopolysaccharide (LPS) with significantly increased interleukin 6 (IL-6) production. Tryptophan withdrawal induced the stress response proteins ATF4 and CHOP/GADD153; however, LPS stimulation rapidly enhanced expression of both proteins. Moreover, LPS-driven cytokine production under amino acid-deficient conditions was dependent on GCN2, as GCN2 knockout (GCN2KO) macrophages had a significant reduction of cytokine gene expression after LPS stimulation. To test the in vivo relevance of these findings, monocytic-lineage-specific GCN2KO mice were challenged with a lethal dose of LPS intraperitoneally (i.p.). The GCN2KO mice showed reduced inflammatory responses, with decreased IL-6 and IL-12 expression correlating with significant reduction in animal mortality. Thus, the data show that amino acid depletion stress signals (via GCN2) synergize with proinflammatory signals to potently increase innate immune responsiveness.     

Granulocyte-macrophage colony-stimulating factor (CSF) and macrophage CSF-dependent macrophage phenotypes display differences in cytokine profiles and transcription factor activities: implications for CSF blockade in inflammation

GM-CSF and M-CSF (CSF-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. Of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. What the critical actions are of these CSFs on macrophages during inflammatory reactions are unknown. To address this issue, adherent macrophages (GM-BMM and BMM) were first derived from murine bone marrow precursors by GM-CSF and M-CSF, respectively, and stimulated in vitro with LPS to measure secreted cytokine production, as well as NF-kappaB and AP-1 activities. GM-BMM preferentially produced TNF-alpha, IL-6, IL-12p70, and IL-23 whereas, conversely, BMM generated more IL-10 and CCL2; strikingly the latter population could not produce detectable IL-12p70 and IL-23. Following LPS stimulation, GM-BMM displayed rapid IkappaBalpha degradation, RelA nuclear translocation, and NF-kappaB DNA binding relative to BMM, as well as a faster and enhanced AP-1 activation. Each macrophage population was also pretreated with the other CSF before LPS stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and NF-kappaB activity. Thus, GM-CSF and M-CSF demonstrate, at the level of macrophage cytokine production, different and even competing responses with implications for their respective roles in inflammation, including a possible dampening or suppressive role for M-CSF in certain circumstances.     

C-reactive protein mediates protection from lipopolysaccharide through interactions with Fc gamma R

C-reactive protein (CRP) is a component of the acute phase response to infection, inflammation, and trauma. A major activity of acute phase proteins is to limit the inflammatory response. It has been demonstrated that CRP protects mice from lethal doses of LPS. In the mouse, CRP binds to the regulatory receptor, FcgammaRIIb, and to the gamma-chain-associated receptor, FcgammaRI. The goal ofthis study was to determine whether FcgammaRs are necessary for the protective effect of CRP. The ability of CRP to protect mice from a lethal dose of LPS was confirmed using injections of 500 and 250 micro g of CRP at 0 and 12 h. CRP treatment of FcgammaRIIb-deficient mice increased mortality after LPS challenge and increased serum levels of TNF and IL-12 in response to LPS. CRP did not protect FcR gamma-chain-deficient mice from LPS-induced mortality. Treatment of normal mice, but not gamma-chain-deficient mice, with CRP increased IL-10 levels following LPS injection. In vitro, in the presence of LPS, CRP enhanced IL-10 synthesis and inhibited IL-12 synthesis by bone marrow macrophages from normal, but not gamma-chain-deficient mice. The protective effect of CRP appears to be mediated by binding to FcgammaRI and FcgammaRII resulting in enhanced secretion of the anti-inflammatory cytokine IL-10 and the down-regulation of IL-12. These results suggest that CRP can alter the cytokine profile of mouse macrophages by acting through FcgammaR leading to a down-regulation of the inflammatory response.     

Myeloid Takl Acts as a Negative Regulator of the LPS Response and Mediates Resistance to Endotoxemia

TGFβ-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, is considered a key intermediate in a multitude of innate immune signaling pathways. Yet, the specific role of TAK1 in the myeloid compartment during inflammatory challenges has not been revealed. To address this question, we generated myeloid-specific kinase-dead TAK1 mutant mice. TAK1 deficiency in macrophages results in impaired NF-κB and JNK activation upon stimulation with lipopolysaccharide (LPS). Moreover, TAK1-deficient macrophages and neutrophils show an enhanced inflammatory cytokine profile in response to LPS stimulation. Myeloid-specific TAK1 deficiency in mice leads to increased levels of circulating IL-1β, TNF and reduced IL-10 after LPS challenge and sensitizes them to LPS-induced endotoxemia. These results highlight an antiinflammatory role for myeloid TAK1, which is essential for balanced innate immune responses and host survival during endotoxemia.     

Myeloid TAKI [corrected] acts as a negative regulator of the LPS response and mediates resistance to endotoxemia

TGFβ-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAP3K) family, is considered a key intermediate in a multitude of innate immune signaling pathways. Yet, the specific role of TAK1 in the myeloid compartment during inflammatory challenges has not been revealed. To address this question, we generated myeloid-specific kinase-dead TAK1 mutant mice. TAK1 deficiency in macrophages results in impaired NF-κB and JNK activation upon stimulation with lipopolysaccharide (LPS). Moreover, TAK1-deficient macrophages and neutrophils show an enhanced inflammatory cytokine profile in response to LPS stimulation. Myeloid-specific TAK1 deficiency in mice leads to increased levels of circulating IL-1β, TNF and reduced IL-10 after LPS challenge and sensitizes them to LPS-induced endotoxemia. These results highlight an antiinflammatory role for myeloid TAK1, which is essential for balanced innate immune responses and host survival during endotoxemia.     

Control of infection with Leishmania major in susceptible BALB/c mice lacking the common gamma-chain for FcR is associated with reduced production of IL-10 and TGF-beta by parasitized cells

Previous studies have shown that the in vitro ligation of FcgammaRs with IgG-opsonized Leishmania amastigotes promotes IL-10 production by macrophages. In addition, infection of either BALB/c mice lacking the common gamma-chain of Fc receptors (FcgammaR(-/-)) or mice genetically altered to lack circulating Ab (J(H)D) with Leishmania pifanoi results in reduced and delayed lesion development and a deficit in the recruitment of inflammatory cells into infected lesions. We show in this study that FcgammaR(-/-) mice can control infection with Leishmania major and totally resolve cutaneous lesions. The ability to eventually control infection is not associated with a reduction in lesion inflammation or a reduction in the ability of Leishmania to parasitize cells through week 6 of infection. The immune response in healing FcgammaR(-/-) mice is associated with a reduction in numbers of cells producing Th2-type cytokines, including IL-4 and IL-10, but not an increase in numbers of IFN-gamma-producing cells characteristic of a dominant Th1-type response. Instead, we observe a reduction in levels of IL-10 and TGF-beta within infected lesions, including reduced levels of these cytokines within parasitized macrophages. Together, these results suggest that uptake of opsonized parasites via FcgammaRs may be a strong in vivo stimulus for the production of anti-inflammatory cytokines that play a role in susceptibility to infection.     

Adenosine inhibits IL-12 and TNF-[alpha] production via adenosine A2a receptor-dependent and independent mechanisms.

Interleukin 12 (IL-12) is a crucial cytokine in the regulation of T helper 1 vs. T helper 2 immune responses. In the present study, we investigated the effect of the endogenous purine nucleoside adenosine on the production of IL-12. In mouse macrophages, adenosine suppressed IL-12 production. Although the order of potency of adenosine receptor agonists suggested the involvement of A2a receptors, data obtained with A2a receptor-deficient mice showed that the adenosine suppression of IL-12 and even TNF-alpha production is only partly mediated by A2a receptor ligation. Studies with adenosine receptor antagonists or the adenosine uptake blocker dipyridamole showed that adenosine released endogenously also decreases IL-12. Although adenosine increases IL-10 production, the inhibition of IL-12 production is independent of the increased IL-10. The mechanism of action of adenosine was not associated with alterations of the activation of the p38 and p42/p44 mitogen-activated protein kinases or the phosphorylation of the c-Jun terminal kinase. Adenosine failed to affect steady-state levels of either IL-12 p35 or p40 mRNA, but augmented IL-10 mRNA levels. In summary, adenosine inhibits IL-12 production via various adenosine receptors. These results support the notion that adenosine-based therapies might be useful in certain autoimmune and/or inflammatory diseases.     

IFN-gamma-independent autocrine cytokine regulatory mechanism in reprogramming of macrophage responses to bacterial lipopolysaccharide.

Macrophages are now well recognized to have a critical role in both innate and acquired immunity. The sentinel macrophage function is highly regulated and serves to allow for intrinsic plasticity of the innate immune responses to potential environmental signals. However, the mechanisms underlying the dynamic properties of the cellular arm of innate immunity are poorly understood. Therefore, we have conducted a series of in vitro studies to evaluate the contribution of immunoregulatory cytokines, such as IFN-gamma, IL-10, and IL-12, in modulation of macrophage responses. We found that macrophages from IFN-gamma knockout (IFN-gamma(-/-)) mice exhibit only marginal LPS-induced TNF-alpha, IL-12, and NO responses, all of which can be fully restored in the presence of rIFN-gamma. Pretreatment with substimulatory LPS concentrations led to reprogramming of IFN-gamma(-/-) macrophage responses in a dose-dependent manner that manifested by an increased TNF-alpha and IL-12, but not NO, production upon the subsequent LPS challenge. These reprogramming effects were substantially attenuated and profoundly enhanced in macrophages from IL-12(-/-) and IL-10(-/-) mice, respectively, as compared with those modulated in macrophages from the congenic wild-type mice. LPS-dependent reprogramming was also fully reproduced in macrophages isolated from SCID mice after immunodepletion of NK cells. Our data strongly imply that cytokine (TNF-alpha and IL-12), but not NO, responses in macrophages may, at least in part, be governed by an autocrine IFN-gamma-independent regulatory mechanism reciprocally controlled by IL-10 and IL-12. This mechanism may serve as an alternative/coherent pathway to the canonical IFN-gamma-dependent induction of antimicrobial and tumoricidal activity in macrophages.     

Bruton's tyrosine kinase is required for TLR-induced IL-10 production

Bruton's tyrosine kinase (Btk) is a critical signaling mediator downstream of the B cell Ag receptor. X-linked agammaglobulinemia is caused by mutations in Btk resulting in multiple defects in B cell development and function, and recurrent bacterial infections. Recent evidence has also supported a role for Btk in TLR signaling. We demonstrate that Btk is activated by TLR4 in primary macrophages and is required for normal TLR-induced IL-10 production in multiple macrophage populations. Btk-deficient bone marrow-derived macrophages secrete decreased levels of IL-10 in response to multiple TLR ligands, compared with wild-type (WT) cells. Similarly, Btk-deficient peritoneal and splenic macrophages secrete decreased IL-10 levels compared with WT cultures. This phenotype correlates with Btk-dependent induction of NF-kappaB and AP-1 DNA binding activity, and altered commensal bacteria populations. Decreased IL-10 production may be responsible for increased IL-6 because blocking IL-10 in WT cultures increased IL-6 production, and supplementation of IL-10 to Btk-deficient cultures decreased IL-6 production. Similarly, injection of IL-10 in vivo with LPS decreases the elevated IL-6 serum levels during endotoxemia in Btk-deficient mice. These data further support a role for Btk in regulating TLR-induced cytokine production from APCs and provide downstream targets for analysis of Btk function.