An agonistic anti-signal regulatory protein α antibody for chronic inflammatory diseases.

Signal regulatory protein (SIRPα) is an immune inhibitory receptor expressed by myeloid cells to inhibit immune cell phagocytosis, migration, and activation. Despite the progress of SIRPα and CD47 antagonist antibodies to promote anti-cancer immunity, it is not yet known whether SIRPα receptor agonism could restrain excessive autoimmune tissue inflammation. Here, we report that neutrophil- and monocyte-associated genes including SIRPA are increased in inflamed tissue biopsies from patients with rheumatoid arthritis and inflammatory bowel diseases, and elevated SIRPA is associated with treatment-refractory ulcerative colitis. We next identify an agonistic anti-SIRPα antibody that exhibits potent anti-inflammatory effects in reducing neutrophil and monocyte chemotaxis and tissue infiltration. In preclinical models of arthritis and colitis, anti-SIRPα agonistic antibody ameliorates autoimmune joint inflammation and inflammatory colitis by reducing neutrophils and monocytes in tissues. Our work provides a proof of concept for SIRPα receptor agonism for suppressing excessive innate immune activation and chronic inflammatory disease treatment.

A mouse knockout library for secreted and transmembrane proteins.

Large collections of knockout organisms facilitate the elucidation of gene functions. Here we used retroviral insertion or homologous recombination to disrupt 472 genes encoding secreted and membrane proteins in mice, providing a resource for studying a large fraction of this important class of drug target. The knockout mice were subjected to a systematic phenotypic screen designed to uncover alterations in embryonic development, metabolism, the immune system, the nervous system and the cardiovascular system. The majority of knockout lines exhibited altered phenotypes in at least one of these therapeutic areas. To our knowledge, a comprehensive phenotypic assessment of a large number of mouse mutants generated by a gene-specific approach has not been described previously.

Selective impairment of TLR-mediated innate immunity in human newborns: neonatal blood plasma reduces monocyte TNF-alpha induction by bacterial lipopeptides, lipopolysaccharide, and imiquimod, but preserves the response to R-848.

Newborns are at increased risk of overwhelming infection, yet the mechanisms underlying this susceptibility are incompletely defined. In this study we report a striking 1- to 3-log decrease in sensitivity of monocytes in human neonatal cord blood, compared with monocytes in adult peripheral blood, to the TNF-alpha-inducing effect of multiple TLR ligands, including bacterial lipopeptides (BLPs), LPS, and the imidazoquinoline compound, imiquimod. In marked contrast, TNF-alpha release in response to R-848, a TLR ligand that is a congener of imiquimod, was equivalent in newborn and adult blood. Differences in ligand-induced TNF-alpha release correlated with divergent ligand-induced changes in monocyte TNF-alpha mRNA levels. Newborn and adult monocytes did not differ in basal mRNA or protein expression of TLRs or mRNA expression of functionally related molecules. Newborn monocytes demonstrated diminished LPS-induced, but equivalent R-848-induced, phosphorylation of p38 mitogen-activated protein kinase and altered BLP- and LPS-induced acute modulation of cognate receptors, suggesting that the mechanism accounting for the observed differences may be localized proximal to ligand recognition by surface TLRs. Remarkably, newborn plasma conferred substantially reduced BLP-, LPS-, and imiquimod-induced TNF-alpha release on adult monocytes without any effect on R-848-induced TNF-alpha release, reflecting differences in a plasma factor(s) distinct from soluble CD14. Impaired response to multiple TLR ligands may significantly contribute to immature neonatal immunity. Conversely, relative preservation of responses to R-848 may present unique opportunities for augmenting innate and acquired immunity in the human newborn.

Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis.

OBJECTIVE: CD16 (IgG Fcgamma receptor type IIIA [FcgammaRIIIA])-expressing CD14+ monocytes express high levels of Toll-like receptor 2 (TLR-2) and are able to efficiently produce proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha). To understand the role of CD16 and TLR-2 in monocyte and macrophage activation in rheumatoid arthritis (RA), we investigated the expression of TLR-2 on CD16+ blood monocytes and synovial tissue macrophages and the effect of CD16 and TLR-2 activation on cytokine production. METHODS: The expression of CD14, CD16, TLR-2, and TLR-4 on blood monocytes was measured by flow cytometric analysis. CD16 and TLR-2 expression in RA synovial tissue was detected by 2-color immunofluorescence labeling. CD16+ mature monocytes were prepared by incubating blood monocytes in plastic plates for 24 hours. These adhered monocytes were stimulated with lipoteichoic acid (LTA), anti-FcgammaRIII antibody, and Hsp60 for 5 hours, and culture supernatants were measured for various cytokines by immunoassay. The activation of NF-kappaB was detected by electrophoretic mobility shift assay. RESULTS: The frequency of CD16+ cells in all blood monocytes was significantly increased in patients with RA compared with healthy controls. TLR-2 was expressed at higher levels on CD16+ monocytes than on CD16- monocytes, while TLR-4 was expressed similarly on both monocytes. In RA synovial tissue, CD16+/TLR-2+ cells were distributed mainly in the lining layer. TLR-2 expression on monocytes was enhanced by macrophage colony-stimulating factor (M-CSF) and interleukin-10 (IL-10), but was reduced by transforming growth factor beta1, while CD16 expression was inducible by these cytokines. Adhered monocytes ( approximately 50% CD16+) produced TNFalpha, IL-1beta, IL-6, IL-8, IL-12 p40, IL-1 receptor antagonist, and IL-10 after LTA stimulation. This cytokine response was inhibited significantly by anti-TLR-2 antibody and partly by anti-TLR-4 antibody. Anti-FcgammaRIII antibody stimulation markedly enhanced the LTA-induced TNFalpha response. Hsp60 could stimulate TNFalpha production by adhered monocytes, which was inhibited similarly by anti-TLR-2 antibody and anti-TLR-4 antibody. NF-kappaB activation in adhered monocytes was induced by LTA, but this NF-kappaB activity was not augmented by anti-FcgammaRIII antibody stimulation. CONCLUSION: These results suggest that CD16+ monocytes and synovial tissue macrophages with high TLR-2 expression may be induced by M-CSF and IL-10, and their production of TNFalpha could be simulated by endogenous TLR ligands such as Hsp60 and FcgammaRIIIA ligation by small immune complexes in RA joints.

APC-independent activation of NK cells by the Toll-like receptor 3 agonist double-stranded RNA.

Toll-like receptors (TLRs) play a fundamental role in the recognition of bacteria and viruses. TLR3 is activated by viral dsRNA and polyinosinic-polycytidylic acid (poly(I:C)), a synthetic mimetic of viral RNA. We show that NK cells, known for their capacity to eliminate virally infected cells, express TLR3 and up-regulate TLR3 mRNA upon poly(I:C) stimulation. Treatment of highly purified NK cells with poly(I:C) significantly augments NK cell-mediated cytotoxicity. Poly(I:C) stimulation also leads to up-regulation of activation marker CD69 on NK cells. Furthermore, NK cells respond to poly(I:C) by producing proinflammatory cytokines like IL-6 and IL-8, as well as the antiviral cytokine IFN-gamma. The induction of cytokine production by NK cells was preceded by activation of NF-kappaB. We conclude that the ability of NK cells to directly recognize and respond to viral products is important in mounting effective antiviral responses.

Activation of Toll-like receptor 2 on human tracheobronchial epithelial cells induces the antimicrobial peptide human beta defensin-2.

As pattern recognition receptors capable of eliciting responses to a diverse array of microbial products, Toll-like receptors (TLRs) participate in the activation of host defense mechanisms that protect against infectious pathogens. Given that epithelial cells lie at the interface between the host and its environment, we designed experiments to determine whether human airway epithelial cells express TLRs and respond to TLR agonists. Immunohistochemical labeling of TLR2 in normal human airways revealed TLR2 expression throughout the epithelium, with an apparently higher level of expression on noncolumnar basal epithelial cells. Two-color immunofluorescent labeling of TLR2 and cytokeratins 8 and 15 revealed that TLR2 is coexpressed with the epithelial cell markers. In addition, airway epithelial cells grown at air-liquid interface responded to bacterial lipopeptide in a TLR2-dependent manner with induction of mRNA and protein of the antimicrobial peptide human beta defensin-2. Stimulation of epithelial cell cultures with lipopeptide resulted in a small and variable reduction of bacteria on the apical surface. Together, these data suggest that TLRs monitor epithelial surfaces to enhance host defense by inducing the production of an antimicrobial peptide.

Critical role of the complement system in group B streptococcus-induced tumor necrosis factor alpha release.

Group B Streptococcus (GBS) is a major cause of newborn sepsis and meningitis and induces systemic release of tumor necrosis factor alpha (TNF-alpha), believed to play a role in morbidity and mortality. While previous studies have shown that GBS can induce TNF-alpha release from monocytes and macrophages, little is known about the potential modulating effect of plasma or serum on GBS-induced TNF-alpha release, and there are conflicting reports as to the host receptors involved. In a human whole-blood assay system, GBS type III COH-1 potently induced substantial monocyte TNF-alpha release in adult peripheral blood and, due to a higher concentration of monocytes, 10-fold-greater TNF-alpha release in newborn cord blood. Remarkably, GBS-induced TNF-alpha release from human monocytes was enhanced approximately 1000-fold by heat-labile serum components. Experiments employing C2-, C3-, or C7-depleted serum demonstrated that C3 activation via the alternative pathway is crucial for potent GBS-induced TNF-alpha release. Accordingly, whole blood from C3-deficient mice demonstrated significantly reduced GBS-induced TNF-alpha release. Preincubation with human serum enhanced the TNF-alpha-inducing activity of GBS in a C3- and factor B-dependent manner, implying deposition of complement components via the alternative pathway. GBS-induced TNF-alpha release was inhibited by monoclonal antibodies directed against each of the components of CR3 and CR4: the common integrin beta subunit CD18 and the alpha subunits CD11b (of CR3) and CD11c (of CR4). Blood derived from CR3 (CD11b/CD18)-deficient mice demonstrated a markedly diminished TNF-alpha response to GBS. We conclude that the ability of plasma and serum to greatly amplify GBS-induced TNF-alpha release reflects the activity of the alternative complement pathway that deposits fragments on GBS and thereby enhances CR3- and CR4-mediated monocyte activation.

Activation and regulation of Toll-like receptors 2 and 1 in human leprosy.

The expression and activation of Toll-like receptors (TLRs) was investigated in leprosy, a spectral disease in which clinical manifestations correlate with the type of immune response mounted toward Mycobacterium leprae. TLR2-TLR1 heterodimers mediated cell activation by killed M. leprae, indicating the presence of triacylated lipoproteins. A genome-wide scan of M. leprae detected 31 putative lipoproteins. Synthetic lipopeptides representing the 19-kD and 33-kD lipoproteins activated both monocytes and dendritic cells. Activation was enhanced by type-1 cytokines and inhibited by type-2 cytokines. In addition, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced TLR1 expression in monocytes and dendritic cells, respectively, whereas IL-4 downregulated TLR2 expression. TLR2 and TLR1 were more strongly expressed in lesions from the localized tuberculoid form (T-lep) as compared with the disseminated lepromatous form (L-lep) of the disease. These data provide evidence that regulated expression and activation of TLRs at the site of disease contribute to the host defense against microbial pathogens.

Distribution of Toll-like receptor 1 and Toll-like receptor 2 in human lymphoid tissue.

To determine how distinct receptors of the immune system can contribute to innate immunity, we investigated the pattern of Toll-like receptor 1 (TLR1) and TLR2 expression in human lymphoid tissue. We found that TLR1 and TLR2 were co-expressed on cells of the innate immune system, including macrophages and dendritic cells. In addition, TLR1 and TLR2 were expressed in mucosa-associated lymphoid tissue on tonsillar crypt epithelium. Of the lymphoid tissue examined, spleen expressed the highest levels of TLR2. Although TLR1- and TLR2-positive cells were in close proximity to T lymphocytes in vivo, lymphocytes themselves were devoid of TLR1 and TLR2 expression. The co-expression of TLR1 and TLR2 on myeloid cells in lymphoid tissue provides the host with the ability to respond to a variety of microbial ligands at sites conducive to the generation of an immune response.

Toll-like receptor 2 ligands as adjuvants for human Th1 responses.

Bacterial lipopeptides (bLPs) are increasingly used as adjuvants to activate cell-mediated immune responses to foreign Ags. To explore mechanisms whereby bLPs adjuvant T cell responses, we stimulated human PBMCs with bLPs. We found that bLPs stimulate T cells to proliferate and produce IFN-gamma in an accessory cell-dependent manner and in the absence of exogenous protein Ags. The ability of bLPs to stimulate T cell proliferation was Toll-like receptor 2 dependent and required IL-12, interaction with costimulatory molecules, and MHC proteins. Our data suggest that bLPs adjuvant adaptive Th1 responses by enhancing Ag presentation of endogenous peptides.

Activation and regulation of toll-like receptors 2 and 1 in human leprosy

The expression and activation of Toll-like receptors (TLRs) was investigated in leprosy, a spectral disease in which clinical manifestations correlate with the type of immune response mounted toward Mycobacterium leprae. TLR2-TLR1 heterodimers mediated cell activation by killed M. leprae, indicating the presence of triacylated lipoproteins. A genome-wide scan of M. leprae detected 31 putative lipoproteins. Synthetic lipopeptides representing the 19-kD and 33-kD lipoproteins activated both monocytes and dendritic cells. Activation was enhanced by type-1 cytokines and inhibited by type-2 cytokines. In addition, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced TLR1 expression in monocytes and dendritic cells, respectively, whereas IL-4 downregulated TLR2 expression. TLR2 and TLR1 were more strongly expressed in lesions from the localized tuberculoid form (T-lep) as compared with the disseminated lepromatous form (L-lep) of the disease. These data provide evidence that regulated expression and activation of TLRs at the site of disease contribute to the host defense against microbial pathogens.

Activation of toll-like receptor 2 in acne triggers inflammatory cytokine responses.

One of the factors that contributes to the pathogenesis of acne is Propionibacterium acnes; yet, the molecular mechanism by which P. acnes induces inflammation is not known. Recent studies have demonstrated that microbial agents trigger cytokine responses via Toll-like receptors (TLRs). We investigated whether TLR2 mediates P. acnes-induced cytokine production in acne. Transfection of TLR2 into a nonresponsive cell line was sufficient for NF-kappa B activation in response to P. acnes. In addition, peritoneal macrophages from wild-type, TLR6 knockout, and TLR1 knockout mice, but not TLR2 knockout mice, produced IL-6 in response to P. acnes. P. acnes also induced activation of IL-12 p40 promoter activity via TLR2. Furthermore, P. acnes induced IL-12 and IL-8 protein production by primary human monocytes and this cytokine production was inhibited by anti-TLR2 blocking Ab. Finally, in acne lesions, TLR2 was expressed on the cell surface of macrophages surrounding pilosebaceous follicles. These data suggest that P. acnes triggers inflammatory cytokine responses in acne by activation of TLR2. As such, TLR2 may provide a novel target for treatment of this common skin disease.

Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines.

Members of the Toll-like receptor (TLR) family mediate dorsoventral patterning and cellular adhesion in insects as well as immune responses to microbial products in both insects and mammals. TLRs are characterized by extracellular leucine-rich repeat domains and an intracellular signaling domain that shares homology with cytoplasmic sequences of the mammalian IL-1 receptor and plant disease resistance genes. Ten human TLRs have been cloned as well as RP105, a protein similar to TLR4 but lacking the intracellular signaling domain. However, only five TLRs have described functions as receptors for bacterial products (e.g., LPS, lipoproteins). To identify potential sites of action, we used quantitative real-time RT-PCR to examine systematically the expression of mRNAs encoding all known human TLRs, RP105, and several other proteins important in TLR functions (e.g., MD-1, MD-2, CD14, MyD88). Most tissues tested expressed at least one TLR, and several expressed all (spleen, peripheral blood leukocytes). Analysis of TLR expression in fractionated primary human leukocytes (CD4(+), CD8(+), CD19(+), monocytes, and granulocytes) indicates that professional phagocytes express the greatest variety of TLR mRNAs although several TLRs appear more restricted to B cells, suggesting additional roles for TLRs in adaptive immunity. Monocyte-like THP-1 cells regulate TLR mRNA levels in response to a variety of stimuli including phorbol esters, LPS, bacterial lipoproteins, live bacteria, and cytokines. Furthermore, addition of Escherichia coli to human blood ex vivo caused distinct changes in TLR expression, suggesting that important roles exist for these receptors in the establishment and resolution of infections and inflammation.