Huiqing Fang

Lipopolysaccharide-Induced Macrophage Inflammatory Response Is Regulated by SHIP

LPS stimulates monocytes/macrophages through TLR4, resulting in the activation of a series of signaling events that potentiate the production of inflammatory mediators. Recent reports indicated that the inflammatory response to LPS is diminished by PI3K, through the activation of the serine/threonine kinase Akt. SHIP is an inositol phosphatase that can reverse the activation events initiated by PI3K, including the activation of Akt. However, it is not known whether SHIP is involved in TLR4 signaling. In this study, we demonstrate that LPS stimulation of Raw 264.7 mouse macrophage cells induces the association of SHIP with lipid rafts, along with IL-1R-associated kinase. In addition, SHIP is tyrosine phosphorylated upon LPS stimulation. Transient transfection experiments analyzing the function of SHIP indicated that overexpression of a wild-type SHIP, but not the SHIP Src homology 2 domain-lacking catalytic activity, up-regulates NF-κB-dependent gene transcription in response to LPS stimulation. These results suggest that SHIP positively regulates LPS-induced activation of Raw 264.7 cells. To test the validity of these observations in primary macrophages, LPS-induced events were compared in bone marrow macrophages derived from SHIP+/+ and SHIP−/− mice. Results indicated that LPS-induced MAPK phosphorylation is enhanced in SHIP+/+ cells, whereas Akt phosphorylation is enhanced in SHIP−/− cells compared with SHIP+/+ cells. Finally, LPS-induced TNF-α and IL-6 production was significantly lower in SHIP−/− bone marrow-derived macrophages. These results are the first to demonstrate a role for SHIP in TLR4 signaling, and propose that SHIP is a positive regulator of LPS-induced inflammation.

The Inositol 3-Phosphatase PTEN Negatively Regulates Fcγ Receptor Signaling, but Supports Toll-Like Receptor 4 Signaling in Murine Peritoneal Macrophages

FcγR clustering in macrophages activates signaling events that result in phagocytosis. Phagocytosis is accompanied by the generation harmful byproducts such as reactive oxygen radicals and production of inflammatory cytokines, which mandate that the phagocytic process be subject to a tight regulation. The molecular mechanisms involved in this regulation are not fully understood. In this study, we have examined the role of the inositol 3-phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in FcγR-induced macrophage function. We demonstrate that in ex vivo murine peritoneal macrophages that are deficient in PTEN expression, FcγR-induced Akt and extracellular signal-regulated kinase phosphorylation are enhanced. Notably, PTEN−/− macrophages showed constitutively high phosphorylation of Akt. However, PTEN did not seem to influence tyrosine phosphorylation events induced by FcγR clustering. Furthermore, PTEN−/− macrophages displayed enhanced phagocytic ability. Likewise, FcγR-induced production of TNF-α, IL-6, and IL-10 was significantly elevated in PTEN−/− macrophages. Surprisingly, LPS-induced TNF-α production was down-regulated in PTEN−/− macrophages. Analyzing the molecular events leading to PTEN influence on LPS/Toll-like receptor 4 (TLR4) signaling, we found that LPS-induced activation of mitogen-activated protein kinases is suppressed in PTEN−/− cells. Previous reports indicated that LPS-induced mitogen-activated protein kinase activation is down-regulated by phosphatidylinositol 3-kinase through the activation of Akt. Our observation that Akt activation is basally enhanced in PTEN−/− cells suggests that PTEN supports TLR4-induced inflammatory responses by suppressing the activation of Akt. Thus, we conclude that PTEN is a negative regulator of FcγR signaling, but a positive regulator of TLR4 signaling. These findings are the first to demonstrate a role for PTEN in FcγR- and TLR4-mediated macrophage inflammatory response.

Analysis of the Effects of the Bruton's tyrosine kinase (Btk) Inhibitor Ibrutinib on Monocyte Fcγ Receptor (FcγR) Function.

The irreversible Brutons tyrosine kinase (Btk) inhibitor ibrutinib has shown efficacy against B-cell tumors such as chronic lymphocytic leukemia and B-cell non-Hodgkin lymphoma. Fcγ receptors (FcγR) on immune cells such as macrophages play an important role in tumor-specific antibody-mediated immune responses, but many such responses involve Btk. Here we tested the effects of ibrutinib on FcγR-mediated activities in monocytes. We found that ibrutinib did not affect monocyte FcγR-mediated phagocytosis, even at concentrations higher than those achieved physiologically, but suppressed FcγR-mediated cytokine production. We confirmed these findings in macrophages from Xid mice in which Btk signaling is defective. Because calcium flux is a major event downstream of Btk, we tested whether it was involved in phagocytosis. The results showed that blocking intracellular calcium flux decreased FcγR-mediated cytokine production but not phagocytosis. To verify this, we measured activation of the GTPase Rac, which is responsible for actin polymerization. Results showed that ibrutinib did not inhibit Rac activation, nor did the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). We next asked whether the effect of ibrutinib on monocyte FcγR-mediated cytokine production could be rescued by IFNγ priming because NK cells produce IFNγ in response to antibody therapy. Pretreatment of monocytes with IFNγ abrogated the effects of ibrutinib on FcγR-mediated cytokine production, suggesting that IFNγ priming could overcome this Btk inhibition. Furthermore, in monocyte-natural killer cell co-cultures, ibrutinib did not inhibit FcγR-mediated cytokine production despite doing so in single cultures. These results suggest that combining ibrutinib with monoclonal antibody therapy could enhance chronic lymphocytic leukemia cell killing without affecting macrophage effector function.

Granzyme B Expression Is Enhanced in Human Monocytes by TLR8 Agonists and Contributes to Antibody-Dependent Cellular Cytotoxicity

FcγRs are critical mediators of mAb cancer therapies, because they drive cytotoxic processes upon binding of effector cells to opsonized targets. Along with NK cells, monocytes are also known to destroy Ab-coated targets via Ab-dependent cellular cytotoxicity (ADCC). However, the precise mechanisms by which monocytes carry out this function have remained elusive. In this article, we show that human monocytes produce the protease granzyme B upon both FcγR and TLR8 activation. Treatment with TLR8 agonists elicited granzyme B and also enhanced FcγR-mediated granzyme B production in an additive fashion. Furthermore, monocyte-mediated ADCC against cetuximab-coated tumor targets was enhanced by TLR8 agonist treatment, and this enhancement of ADCC required granzyme B. Hence we have identified granzyme B as an important mediator of FcγR function in human monocytes and have uncovered another mechanism by which TLR8 agonists may enhance FcγR-based therapies.

Toll-like Receptor 2 Ligands Regulate Monocyte Fcγ Receptor Expression and Function

Background: Toll-like receptors recognize bacterial components, leading to immune activation. Results: TLR2 ligands alter monocyte/macrophage FcγR and phosphatase expression to improve function both in vitro and in vivo. Conclusion: TLR2 ligands enhance monocyte/macrophage function. Significance: FcγR function is critical for antibody therapy. Deciphering the effect of TLR2 ligands provides a potential means to enhance therapy. Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy.

Toll-like Receptor 4 Ligands Down-regulate Fcγ Receptor IIb (FcγRIIb) via MARCH3 Protein-mediated Ubiquitination

Monocytes and macrophages are critical for the effectiveness of monoclonal antibody therapy. Responses to antibody-coated tumor cells are largely mediated by Fcγ receptors (FcγRs), which become activated upon binding to immune complexes. FcγRIIb is an inhibitory FcγR that negatively regulates these responses, and it is expressed on monocytes and macrophages. Therefore, deletion or down-regulation of this receptor may substantially enhance therapeutic outcomes. Here we screened a panel of Toll-like receptor (TLR) agonists and found that those selective for TLR4 and TLR8 could significantly down-regulate the expression of FcγRIIb. Upon further examination, we found that treatment of monocytes with TLR4 agonists could lead to the ubiquitination of FcγRIIb protein. A search of our earlier microarray database of monocytes activated with the TLR7/8 agonist R-848 (in which FcγRIIb was down-regulated) revealed an up-regulation of membrane-associated ring finger (C3HC4) 3 (MARCH3), an E3 ubiquitin ligase. Therefore, we tested whether LPS treatment could up-regulate MARCH3 in monocytes and whether this E3 ligase was involved with LPS-mediated FcγRIIb down-regulation. The results showed that LPS activation of TLR4 significantly increased MARCH3 expression and that siRNA against MARCH3 prevented the decrease in FcγRIIb following LPS treatment. These data suggest that activation of TLR4 on monocytes can induce a rapid down-regulation of FcγRIIb protein and that this involves ubiquitination.

Interferon-γ promotes antibody-mediated fratricide of Acute Myeloid Leukemia cells

Acute myeloid leukemia (AML) is characterized by the proliferation of immature myeloid lineage blasts. Due to its heterogeneity and to the high rate of acquired drug resistance and relapse, new treatment strategies are needed. Here, we demonstrate that IFNγ promotes AML blasts to act as effector cells within the context of antibody therapy. Treatment with IFNγ drove AML blasts toward a more differentiated state, wherein they showed increased expression of the M1-related markers HLA-DR and CD86, as well as of FcγRI, which mediates effector responses to therapeutic antibodies. Importantly, IFNγ was able to up-regulate CD38, the target of the therapeutic antibody daratumumab. Because the antigen (CD38) and effector receptor (FcγRI) were both simultaneously up-regulated on the AML blasts, we tested whether IFNγ treatment of the AML cell lines THP-1 and MV4-11 could stimulate them to target one another after the addition of daratumumab. Results showed that IFNγ significantly increased daratumumab-mediated cytotoxicity, as measured both by 51Cr release and lactate dehydrogenase release assays. We also found that the combination of IFNγ and activation of FcγR led to the release of granzyme B by AML cells. Finally, using a murine NSG model of subcutaneous AML, we found that treatment with IFNγ plus daratumumab significantly attenuated tumor growth. Taken together, these studies show a novel mechanism of daratumumab-mediated killing and a possible new therapeutic strategy for AML.

Anti-leukemic effects of all-trans retinoic acid in combination with Daratumumab in acute myeloid leukemia

Acute myeloid leukemia (AML) remains a significant health problem, with poor outcomes despite chemotherapy and bone marrow transplants. Although one form of AML, acute promyelocytic leukemia (APL), is successfully treated with all-trans retinoic acid (ATRA), this drug is seemingly ineffective against all other forms of AML. Here, we show that ATRA up-regulates CD38 expression on AML blasts to sufficient levels that promote antibody-mediated fratricide following the addition of anti-CD38 daratumumab (DARA). The combination of ATRA plus DARA induced Fc-dependent conjugate formation and cytotoxicity among AML blasts in vitro. Combination treatment also led to reduction in tumor volume and resulted in increased overall survival in murine engraftment models of AML. These results suggest that, although ATRA does not induce differentiation of non-APL, it may be effective as a therapy in conjunction with DARA.