Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment.

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

Toll-like receptor 4 signaling by follicular dendritic cells is pivotal for germinal center onset and affinity maturation.

Germinal centers (GCs) are specialized microenvironments where antigen-activated B cells undergo proliferation, immunoglobulin (Ig) class switch recombination, somatic hypermutation (SHM), and affinity maturation. Within GCs, follicular dendritic cells (FDCs) are key players in driving these events via direct interaction with GC B cells. Here, we provide in vivo evidence that FDCs express and upregulate Toll-like-receptor (TLR) 4 in situ during germinal center reactions, confirm that their maturation is driven by TLR4, and associate the role of FDC-expressed TLR4 with quantitative and qualitative affects of GC biology. In iterative cycles of predictions by in silico modeling subsequently verified by in vivo experiments, we demonstrated that TLR4 signaling modulates FDC activation, strongly impacting SHM and generation of Ig class-switched high-affinity plasma and memory B cells. Thus, our data place TLR4 in the heart of adaptive humoral immunity, providing further insight into mechanisms driving GCs arising in both health and disease.

Selective antibody intervention of Toll-like receptor 4 activation through Fc γ receptor tethering.

Inflammation is mediated mainly by leukocytes that express both Toll-like receptor 4 (TLR4) and Fc γ receptors (FcγR). Dysregulated activation of leukocytes via exogenous and endogenous ligands of TLR4 results in a large number of inflammatory disorders that underlie a variety of human diseases. Thus, differentially blocking inflammatory cells while sparing structural cells, which are FcγR-negative, represents an elegant strategy when targeting the underlying causes of human diseases. Here, we report a novel tethering mechanism of the Fv and Fc portions of anti-TLR4 blocking antibodies that achieves increased potency on inflammatory cells. In the presence of ligand (e.g. lipopolysaccharide (LPS)), TLR4 traffics into glycolipoprotein microdomains, forming concentrated protein platforms that include FcγRs. This clustering produces a microenvironment allowing anti-TLR4 antibodies to co-engage TLR4 and FcγRs, increasing their avidity and thus substantially increasing their inhibitory potency. Tethering of antibodies to both TLR4 and FcγRs proves valuable in ameliorating inflammation in vivo. This novel mechanism of action therefore has the potential to enable selective intervention of relevant cell types in TLR4-driven diseases.

A polyglutamic acid motif confers IL-27 hydroxyapatite and bone-binding properties.

The p28 subunit of the composite cytokine IL-27 comprises a polyglutamic acid domain, which is unique among type I cytokines. This domain is very similar to the acidic domain known to confer hydroxyapatite (HA)-binding properties and bone tropism to bone sialoprotein. We observed IL-27 binding to HA, in accordance with previous studies reporting successful p28 HA chromatography. The IL-27 polyglutamic acid domain is located in a flexible inter-α helix loop, and HA-bound IL-27 retained biological activity. Using IL-27 alanine mutants, we observed that the p28 polyglutamic acid domain confers HA- and bone-binding properties to IL-27 in vitro and bone tropism in vivo. Because IL-27 is a potent regulator of cells residing in endosteal bone marrow niches such as osteoclasts, T regulatory, memory T, plasma, and stem cells, this specific property could be beneficial for therapeutic applications. IL-27 has potent antitumoral and antiosteoclastogenic activities. It could therefore also be useful for therapies targeting hematologic cancer or solid tumors metastasis with bone tropism. Furthermore, these observations suggest that polyglutamic motifs could be grafted onto other type I cytokine inter-α helix loops to modify their pharmacological properties.

The composite cytokine p28/cytokine-like factor 1 sustains B cell proliferation and promotes plasma cell differentiation.

IL-27 is an APC-derived IL-6/IL-12 family composite cytokine with multiple functions such as regulation of Th1, Th17, and regulatory T cell differentiation, B cell proliferation, and Ig class switching. The IL-27 complex is formed by the association of the cytokine p28 with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27 cytokine and soluble receptor subunits p28 and EBI3 can be secreted independently. The p28 subunit has been shown to have IL-27-independent biological activities. We previously demonstrated that p28 can form an alternative composite cytokine with the EBI3 homolog cytokine-like factor 1 (CLF; CRLF1). p28/CLF modulates NK cell activity and CD4 T cell cytokine production in vitro. In this study we used IL-6-dependent plasmacytoma cell line B9 and CD4 T cells from IL-27Rα-deficient mice to demonstrate that p28/CLF activates IL-27-unresponsive cells, indicating that p28/CLF and IL-27 signal through different receptors. The observation that p28/CLF, unlike IL-27, sustains B9 plasmacytoma cell proliferation prompted us to investigate the effects of p28/CLF on mouse B cells. We observed that p28/CLF induces IgM, IgG2c, and IgG1 production and plasma cell differentiation. p28/CLF therefore has the potential to contribute to B and plasma cell function, differentiation, and proliferation in normal and pathological conditions such as Castelman's disease and multiple myeloma.

IL-17F co-expression improves cell growth characteristics and enhances recombinant protein production during CHO cell line engineering.

The generation of a high productivity cell line is a critical step in the production of a therapeutic protein. Many innovative engineering strategies have been devised in order to maximize the expression rate of production cells for increased process efficiency. Less effort has focused on improvements to the cell line generation process, which is typically long and laborious when using mammalian cells. Based on unexpected findings when generating stable CHO cell lines expressing human IL-17F, we studied the benefit of expressing this protein during the establishment of production cell lines. We demonstrate that IL-17F expression enhances the rate of selection and overall number of selected cell lines as well as their transgene expression levels. We also show that this benefit is observed with different parental CHO cell lines and selection systems. Furthermore, IL-17F expression improves the efficiency of cell line subcloning processes. IL-17F can therefore be exploited in a standard manufacturing process to obtain higher productivity clones in a reduced time frame.

IL-6 activates STAT5 in T cells.

BACKGROUND: IL-6 is a pleiotropic cytokine which emerged recently as a key regulator of CD4 T cell function. IL-6 alone or in combination with other cytokines promotes T helper 1, T helper 17 and T follicular helper cell differentiation whilst inhibiting the induction of regulatory T cell generation. IL-6 activates multiple pathways among which JAK/STAT3 is the most clearly validated in the control of CD4 T helper differentiation. Activation of STAT5 by cytokines such as IL-2 can counteract IL-6-induced T helper 17 and T follicular helper cell differentiation and promote the induction of regulatory T cell generation. STAT5 and STAT3 are known to compete for promoter binding sites in CD4 T cells and the two transcription factors are believed to have opposite functions in the control of CD4 T cell differentiation. METHODS: We analyzed IL-6-induced STAT1, 3 and 5 activation by flow cytometry (phosflow) in mouse mononuclear cells and its effect on the level of the mRNA coding for cytokine-inducible SH2-containing protein (CIS). RESULTS: The results show that IL-6 also induces STAT5 activation in both CD4 and CD8 T as well as NK cells. Analysis of STAT5 phosphorylation in CD4 T cells indicates that it is transient and requires higher cytokine concentrations than that of STAT3. CD4 T cell stimulation with IL-6 induces the synthesis of CIS, which is encoded by a gene known to be regulated by STAT5. CONCLUSIONS: Thus, IL-6 at concentrations corresponding to levels observed in the serum during inflammation may activate, in CD4 T cells, a STAT5-negative feedback loop which alters the balance between STAT3-dependent pro-inflammatory helper T cells and STAT5-induced T regulatory cells. STAT5 activation may modulate the differentiation of T helper cells through attenuation of TGF-ß stability and production. Since STAT5 is directly activated by Janus kinases, therapeutic approaches designed to inhibit STAT3 activation or to recruit STAT3 phosphatases may be useful in altering the balance of activated STAT3 and STAT5 in favor a profile that would be beneficial in pathologies involving IL-6.

The cytokines cardiotrophin-like cytokine/cytokine-like factor-1 (CLC/CLF) and ciliary neurotrophic factor (CNTF) differ in their receptor specificities.

Ciliary neurotrophic factor (CNTF) and cardiotrophin-like cytokine (CLC) are two cytokines with neurotrophic and immunomodulatory activities. CNTF is a cytoplasmic factor believed to be released upon cellular damage, while CLC requires interaction with a soluble cytokine receptor, cytokine-like factor 1 (CLF), to be efficiently secreted. Both cytokines activate a receptor complex comprising the cytokine binding CNTF receptor α (CNTFRα) and two signaling chains namely, leukemia inhibitory factor receptor ß (LIFRß) and gp130. Human CNTF can recruit and activate an alternative receptor in which CNTFRα is substituted by IL-6Rα. As both CNTF and CLC have immune-regulatory activities in mice, we compared their ability to recruit mouse receptors comprising both gp130 and LIFRß signaling chains and either IL-6Rα or IL-11Rα which, unlike CNTFRα, are expressed by immune cells. Our results indicate that 1) mouse CNTF, like its human homologue, can activate cells expressing gp130/LIFRß with either CNTFRα or IL-6Rα and, 2) CLC/CLF is more restricted in its specificity in that it activates only the tripartite CNTFR. Several gp130 signaling cytokines influence T helper cell differentiation. We therefore investigated the effect of CNTF on CD4 T cell cytokine production. We observed that CNTF increased the number of IFN-γ producing CD4 T cells. As IFN-γ is considered a mediator of the therapeutic effect of IFN-ß in multiple sclerosis, induction of IFN-γ by CNTF may contribute to the beneficial immunomodulatory effect of CNTF in mouse multiple sclerosis models. Together, our results indicate that CNTF activates the same tripartite receptors in mouse and human cells and further validate rodent models for pre-clinical investigation of CNTF and CNTF derivatives. Furthermore, CNTF and CLC/CLF differ in their receptor specificities. The receptor α chain involved in the immunomodulatory effects of CLC/CLF remains to be identified.

A monoclonal antibody targeting nonjunctional claudin-1 inhibits fibrosis in patient-derived models by modulating cell plasticity.

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.

The IL-27 p28 subunit binds cytokine-like factor 1 to form a cytokine regulating NK and T cell activities requiring IL-6R for signaling.

IL-27 is formed by the association of a cytokine subunit, p28, with the soluble cytokine receptor EBV-induced gene 3 (EBI3). The IL-27R comprises gp130 and WSX-1. The marked difference between EBI3(-/-) and WSX-1(-/-) mice suggests that p28 has functions independent of EBI3. We have identified an alternative secreted complex formed by p28 and the soluble cytokine receptor cytokine-like factor 1 (CLF). Like IL-27, p28/CLF is produced by dendritic cells and is biologically active on human NK cells, increasing IL-12- and IL-2-induced IFN-gamma production and activation marker expression. Experiments with Ba/F3 transfectants indicate that p28/CLF activates cells expressing IL-6Ralpha in addition to the IL-27R subunits. When tested on CD4 and CD8 T cells, p28/CLF induces IL-6Ralpha-dependent STAT1 and STAT3 phosphorylation. Furthermore, p28/CLF inhibits CD4 T cell proliferation and induces IL-17 and IL-10 secretion. These results indicate that p28/CLF may participate in the regulation of NK and T cell functions by dendritic cells. The p28/CLF complex engages IL-6R and may therefore be useful for therapeutic applications targeting cells expressing this receptor. Blocking IL-6R using humanized mAbs such as tocilizumab has been shown to be beneficial in pathologies like rheumatoid arthritis and juvenile idiopathic arthritis. The identification of a new IL-6R ligand is therefore important for a complete understanding of the mechanism of action of this emerging class of immunosuppressors.

IL-10 enhances MD-2 and CD14 expression in monocytes and the proteins are increased and correlated in HIV-infected patients.

Soluble proteins that bind LPS, like myeloid differentiation-2 (MD-2) and CD14, have essential roles in regulating LPS signaling through TLR4. During a gram-negative bacterial infection, the host may control the response by adjusting the levels of soluble MD-2 and CD14. To address the surface expression of MD-2 on human leukocytes, we developed a mAb, IIC1, that recognized MD-2 both free and when bound to TLR4. MD-2 was found on the surface of freshly isolated monocytes, on a subpopulation of CD19(+) B-cells and on CD15(+) neutrophils. LPS transiently reduced the MD-2 levels on monocytes, which is most likely due to endocytosis of the LPS receptor complex since MD-2 colocalized with TLR4 in early endosomes after LPS stimulation. In the absence of LPS, MD-2 partly colocalized with TLR4 in Golgi trans and medial compartments. Cultivating monocytes for 18-20 h resulted in loss of MD-2 expression on the surface, which was reversed either by LPS or IL-10. Furthermore, addition of IL-10, but not LPS, resulted in a considerable increase in mRNA for both MD-2 and CD14. Using ELISA, we demonstrated that IL-10 had a profound dose- and time-related effect on the release of soluble MD-2 and soluble CD14 from monocytes. In HIV-infected patients, the amounts of MD-2, CD14, and IL-10 increased significantly in the patient group with AIDS. Of interest, we found that IL-10, CD14, and MD-2 levels were positively correlated, suggesting that IL-10 may be a driving force for increased release of MD-2 and CD14 during systemic inflammation.

Increased release of sMD-2 during human endotoxemia and sepsis: a role for endothelial cells.

MD-2 is the crucial cofactor of TLR4 in the detection of LPS. Here, we show that soluble MD-2 (sMD-2) circulates in plasma of healthy individuals as a polymeric protein. The total amount of sMD-2 in septic plasma was strongly elevated and contained both sMD-2 polymers and monomers, the latter representing the putative biologically active form of MD-2. Moreover, during experimental human endotoxemia, the monomeric and total sMD-2 content in plasma increased with the kinetics of an acute phase protein. The increase in sMD-2 monomers was paralleled by enhanced TLR4 costimulatory activity. The presence of functional sMD-2 during endotoxemia and sepsis was confirmed by immunodepletion. Immunohistochemistry revealed that MD-2 expression in septic patients was strongly enhanced on endothelium and multiple inflammatory cells in lung and liver. In vitro studies showed that sMD-2 release appears to be restricted to endothelial cells and dendritic cells. Release of sMD-2 by endothelial cells was strongly enhanced by LPS and TNF-alpha stimulation. Taken together, this study demonstrates the increase of both circulating polymeric and functional monomeric sMD-2 during endotoxemia and sepsis, and evidence is provided that the endothelium is involved in this process.

Effect of Toll-like receptor 4 blockade on pulmonary inflammation caused by mechanical ventilation and bacterial endotoxin.

Mechanical ventilation (MV) and lipopolysaccharide (LPS) synergistically increase inflammation and lung injury. The goal of this study was to determine whether blockade of CD14 or Toll-like receptor 4 (TLR4) would reduce inflammation caused by LPS and MV. Rabbits were pretreated with anti-TLR4 or anti-CD14 monoclonal antibodies, followed by endobronchial LPS and MV. Blockade of TLR4 reduced the number of neutrophils and the amount of CXCL8 in bronchoalveolar lavage fluid. In contrast, blockade of CD14 did not significantly decrease the number of neutrophils or the amount of CXCL8. These data show that TLR4 blockade reduces pulmonary inflammation caused by the combination of LPS and Mechanical ventilation.

Short-term treatment with anti-CD3 antibody reduces the development and progression of atherosclerosis in mice.

BACKGROUND: Atherosclerosis is a chronic inflammatory disease of the large arteries that is the primary cause of heart disease and stroke. Anti-CD3-specific antibodies suppress immune responses by antigenic modulation of the CD3 antibody/T-cell receptor complex. Their unique capacity to restore self-tolerance in a mouse model of diabetes and, importantly, in patients with recent-onset type 1 diabetes involves transforming growth factor-beta-dependent mechanisms via expansion and/or activation of regulatory T cells. We hypothesized that treatment with anti-CD3-specific antibodies might inhibit atherosclerosis development and progression in mice. METHODS AND RESULTS: Low-density lipoprotein receptor-deficient mice were fed a high-cholesterol diet for 13 or 24 weeks. Anti-CD3 antibody was administered on 5 consecutive days beginning 1 week before or 13 weeks after the high-cholesterol diet was initiated, respectively. Control mice were injected in parallel with phosphate-buffered saline. Anti-CD3 antibody therapy reduced plaque development when administered before a high-cholesterol diet and markedly decreased lesion progression in mice with already established atherosclerosis. We found increased production of the antiinflammatory cytokine transforming growth factor-beta in concanavalin A-stimulated lymph node cells and enhanced expression of the regulatory T-cell marker Foxp3 in spleens of anti-CD3 antibody-treated mice. A higher percentage of apoptotic cells within the plaques of anti-CD3 antibody-treated mice was also observed. CONCLUSIONS: Altered disease progression, combined with the emergence of this particular cytokine pattern, indicates that short-term treatment with an anti-CD3 antibody induces a regulatory T-cell phenotype that restores self-tolerance in a mouse model of atherosclerosis.

Interleukin-6 biology is coordinated by membrane-bound and soluble receptors: role in inflammation and cancer.

Cytokine receptors, which exist in membrane-bound and soluble forms, bind their ligands with comparable affinity. Although most soluble receptors are antagonists and compete with their membrane-associated counterparts for the ligands, certain soluble receptors are agonists. In these cases, complexes of ligand and soluble receptor bind on target cells to second receptor subunits and initiate intracellular signaling. The soluble receptors of the interleukin (IL)-6 family of cytokines (sIL-6R, sIL-11R, soluble ciliary neurotrophic factor receptor) are agonists capable of transmitting signals through interaction with the universal signal-transducing receptor for all IL-6 family cytokines, gp130. In vivo, the IL-6/sIL-6R complex stimulates several types of cells, which are unresponsive to IL-6 alone, as they do not express the membrane IL-6R. We have named this process trans-signaling. The generation of soluble cytokine receptors occurs via two distinct mechanisms-limited proteolysis and translation-from differentially spliced mRNA. We have demonstrated that a soluble form of the IL-6 family signaling receptor subunit gp130, which is generated by differential splicing, is the natural inhibitor of IL-6 trans-signaling responses. We have shown that in many chronic inflammatory diseases, including chronic inflammatory bowel disease, peritonitis, rheumatoid arthritis, asthma, as well as colon cancer, IL-6 trans-signaling is critically involved in the maintenance of a disease state, by promoting transition from acute to chronic inflammation. Moreover, in all these models, the course of the disease can be disrupted by specifically interfering with IL-6 trans-signaling using the soluble gp130 protein. The pathophysiological mechanisms by which the IL-6/sIL-6R complex regulates the inflammatory state are discussed.

Toll-Like Receptor 4 on both Myeloid Cells and Dendritic Cells Is Required for Systemic Inflammation and Organ Damage after Hemorrhagic Shock with Tissue Trauma in Mice.

Trauma combined with hemorrhagic shock (HS/T) leads to systemic inflammation, which results in organ injury. Toll-like Receptor 4 (TLR4)-signaling activation contributes to the initiation of inflammatory pathways following HS/T but its cell-specific roles in this setting are not known. We assessed the importance of TLR4 on leukocytes of myeloid lineage and dendritic cells (DCs) to the early systemic inflammatory response following HS/T. Mice were subjected to HS/T and 20 inflammatory mediators were measured in plasma followed by Dynamic Bayesian Network (DBN) Analysis. Organ damage was assessed by histology and plasma ALT levels. The role of TLR4 was determined using TLR4-/-, MyD88-/-, and Trif-/- C57BL/6 (B6) mice, and by in vivo administration of a TLR4-specific neutralizing monoclonal antibody (mAb). The contribution of TLR4 expressed by myeloid leukocytes and DC was determined by generating cell-specific TLR4-/- B6 mice, including Lyz-Cre × TLR4loxP/loxP, and CD11c-Cre × TLR4loxP/loxP B6 mice. Adoptive transfer of bone marrow-derived TLR4+/+ or TLR4-/- DC into TLR4-/- mice confirmed the contribution of TLR4 on DC to the systemic inflammatory response after HS/T. Using both global knockout mice and the TLR4-blocking mAb 1A6 we established a central role for TLR4 in driving systemic inflammation. Using cell-selective TLR4-/- B6 mice, we found that TLR4 expression on both myeloid cells and CD11chigh DC is required for increases in systemic cytokine levels and organ damage after HS/T. We confirmed the capacity of TLR4 on CD11chigh DC to promote inflammation and liver damage using adoptive transfer of TLR4+/+ conventional (CD11chigh) DC into TLR4-/- mice. DBN inference identified CXC chemokines as proximal drivers of dynamic changes in the circulating levels of cytokines/chemokines after HS/T. TLR4 on DC was found to contribute selectively to the elevations in these proximal drivers. TLR4 on both myeloid cells and conventional DC is required for the initial systemic inflammation and organ damage in a mouse model of HS/T. This includes a role for TLR4 on DC in promoting increases in the early inflammatory networks identified in HS/T. These data establish DC along with macrophages as essential to the recognition of tissue damage and stress following tissue trauma with HS.

Detection of epsilon class switching and IgE synthesis in human B cells.

We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.

Tlr2 on Bone Marrow and Non-Bone Marrow Derived Cells Regulates Inflammation and Organ Injury in Cooperation with Tlr4 During Resuscitated Hemorrhagic Shock.

BACKGROUND: Although the role of TLR4 in driving inflammation and organ injury after hemorrhagic shock and resuscitation (H/R) is well established, the role of TLR2-another receptor for damage-associated molecular pattern (DAMP) molecules-is not. In this study, we used a combination of TLR2 and wild type (WT) mice treated with anti-TLR2 and anti-TLR4 neutralizing monoclonal antibodies (mAb) to discern the contribution of TLR2 relative to TLR4 to the systemic inflammatory response in murine H/R. MATERIAL AND METHODS: WT mice, TLR2, and WT mice receiving an anti-TLR2 or an anti-TLR4 mAB (given as a pretreatment) were sacrificed at 6 or 20 h post-H/R. Bone marrow TLR2/WT chimeric mice were created to assess the importance of immune and nonimmune cell-associated TLR2. RESULTS: TLR2 mice subjected to H/R exhibited significantly less liver damage and lower markers of systemic inflammation only at 20 h. Bone marrow chimeric mice using combinations of TLR2 mice and WT mice demonstrated that TLR2 on non-bone marrow derived cells played a dominant role in the differences at 20 h. Interestingly, WT mice treated with anti-TLR2 mAB demonstrated a reduction in organ damage and systemic inflammation at both 6 and 20 h following H/R. A combination of anti-TLR2 mAB and anti-TLR4 mAB showed that both receptors drive IP-10 and KC levels and that there is cooperation for increases in IL-6, MIG, and MCP-1 levels between TLR2 and TLR4. CONCLUSION: These data also support the conclusion that TLR2 and TLR4 act in concert as important receptors in the host immune response to H/R.

A specific anti-citrullinated protein antibody profile identifies a group of rheumatoid arthritis patients with a toll-like receptor 4-mediated disease.

BACKGROUND: Increased expression of toll-like receptor 4 (TLR4) and its endogenous ligands, is characteristic of rheumatoid arthritis (RA) synovitis. In this study, we evaluated how these TLR4 ligands may drive pathogenic processes and whether the fine profiling of anti-citrullinated protein antibodies (ACPA) based on their target specificity might provide a simple means to predict therapeutic benefit when neutralizing TLR4 in this disease. METHODS: The capacity of RA synovial fluids (RASF) to stimulate cytokine production in monocytes from patients with RA was analyzed by ELISA. The presence of TLR4 activators in RASF was determined by measuring the levels of ACPA, ACPA subtypes with reactivity to specific citrullinated peptides and other TLR4 ligands. Neutralization of TLR4 signaling was investigated using NI-0101, a therapeutic antibody that targets TLR4. RESULTS: RASF exhibited a heterogeneous capacity to induce production of proinflammatory cytokines by monocytes isolated from patients with RA. Such cytokine responses were significantly modified by TLR4 blockade achieved using NI-0101. The analysis of the content of RASF and matched sera demonstrated that ACPA fine specificities in patient samples predict cellular response to anti-TLR4 exposure in vitro. CONCLUSION: TLR4 represents a possible therapeutic target in RA. Our study demonstrates that TLR4 inhibition in an ex vivo model of RA pathogenesis can significantly modulate cytokine release and does so in specific subgroups of RA patient-derived samples. It also suggests that ACPA fine profiling has the potential to identify RA patients with a predominantly TLR4-driven pathotype that could be used to predict preferential response to TLR4 antagonism.

Gene expression of Toll-like receptor-2, Toll-like receptor-4, and MD2 is differentially regulated in rabbits with Escherichia coli pneumonia.

Sepsis, a common sequela to Gram-negative pneumonia, results in considerable morbidity and mortality in hospitalized patients. The goal of this study was to determine whether Gram-negative pneumonia alters the expression TLR2, TLR4, and MD2 in lungs or in organs distant to the site of the primary infection. The cDNA sequence coding open reading frames for rabbit TLR2, TLR4, and MD2 were cloned and expressed in Escherichia coli, and specific polyclonal antibodies and polymerase chain reaction (PCR) probes were produced to identify changes in these receptors in rabbits with Gram-negative pneumonia. Using tissues from lungs and distant organs, we show that TLR2, TLR4, and MD2 gene expression is differentially regulated in rabbits with E. coli pneumonia. The increased expression of TLR2 and TLR4 could play an important role in the innate immune response to bacterial infection in the lungs, and improve pathogen recognition and bacterial clearance. In contrast, the increased gene expression of TLR2, TLR4, and MD2 in organs distant to the primary site of infection may contribute to the deleterious systemic inflammatory response observed in patients with sepsis.