Nicola J. Mason

Critical roles of c-Rel in autoimmune inflammation and helper T cell differentiation

Different members of the Rel/NF-kappaB family may play different roles in immunity and inflammation. We report here that c-Rel-deficient mice are resistant to autoimmune encephalomyelitis and are defective in Th1, but not Th2 responses. The Th1 deficiency appears to be caused by selective blockade of IL-12 production by c-Rel-deficient antigen-presenting cells, as well as by a complete abrogation of IFN-gamma expression in c-Rel-deficient T cells. Interestingly, c-Rel deficiency does not affect T-bet expression, suggesting that c-Rel may act downstream of T-bet during Th1 cell differentiation. Thus, unlike NF-kappaB1, which selectively regulates Th2 cell differentiation, c-Rel is essential for Th1 cell differentiation and Th1 cell-mediated autoimmune inflammation.

Cutting Edge: Identification of c-Rel-Dependent and -Independent Pathways of IL-12 Production During Infectious and Inflammatory Stimuli

The production of IL-12 is required for immunity to many intracellular pathogens. Recent studies have shown that c-Rel, a member of the NF-κB family of transcription factors, is essential for LPS-induced IL-12p40 production by macrophages. In this study, we demonstrate that c-Rel is also required for IL-12p40 production by macrophages in response to Corynebacterium parvum, CpG oligodeoxynucleotides, anti-CD40 and low molecular weight hyaluronic acid. However, c-Rel−/− mice infected with Toxoplasma gondii produce comparable amounts of IL-12p40 to infected wild-type mice and have an IL-12-dependent mechanism of resistance to this infection. Furthermore, c-Rel was not required for IL-12p40 production by macrophages or dendritic cells in response to soluble Toxoplasma Ag, and neutrophils from c-Rel−/− mice contain normal amounts of preformed IL-12p40. Together these studies reveal the presence of c-Rel-dependent pathways critical for IL-12p40 production in response to inflammatory stimuli and demonstrate a novel c-Rel-independent pathway of IL-12p40 production during toxoplasmosis.

Differential Requirement for NF-κB Family Members in Control of Helminth Infection and Intestinal Inflammation

The NF-κB family of transcription factors is critical in controlling the expression of a wide range of immune response genes. However, whether individual family members perform specific roles in regulating immunity and inflammation remains unclear. Here we investigated the requirement for NF-κB1, NF-κB2, and c-Rel in the expression of Th2 cytokine responses, development of host protective immunity, and regulation of intestinal inflammation following infection with the gut-dwelling helminth parasite Trichuris muris. While mice deficient in c-Rel mounted sufficient Th2 responses to expel infection, NF-κB1 knockout (KO) and NF-κB2 KO mice developed chronic infections associated with elevated production of Ag-specific IFN-γ. However, only infected NF-κB1 KO mice exhibited polarized IFN-γ responses associated with the loss of intestinal goblet cells and the development of destructive colitis-like pathology. Furthermore, blockade of IL-12 (previously shown to confer resistance in susceptible strains) recovered Ag-specific IL-13 responses and resistance to infection in NF-κB2 KO, but not NF-κB1 KO mice. Therefore, unique infection, immunological, and pathological outcomes were observed in different NF-κB KO strains. Taken together, these results provide direct evidence of nonoverlapping functions for NF-κB family members in the development of Th2 cytokine-mediated resistance to T. muris and the control of infection-induced intestinal inflammation.

T Cell-Intrinsic Expression of c-Rel Regulates Th1 Cell Responses Essential for Resistance to Toxoplasma gondii

The ability of many microbial and inflammatory stimuli to activate members of the Rel/NF-κB family of transcription factors is associated with the regulation of innate and adaptive responses required to control infection. Individual family members play distinct roles during different infectious and inflammatory responses. For example, c-Rel is essential for the production of IL-12 in response to LPS, but dispensable for IL-12 production in response to Toxoplasma Ag. To assess the role of c-Rel during immunity to the intracellular pathogen Toxoplasma gondii, wild-type (WT) and c-Rel−/− mice were infected with Toxoplasma and the immune response was analyzed. c-Rel−/− mice developed severe toxoplasmic encephalitis with increased numbers of parasites compared with WT controls and succumbed to infection within 5–8 wk. Although increased susceptibility of c-Rel−/− mice was associated with decreased T cell activation, proliferation, and production of IFN-γ, these mice were able to generate Th1 effector cells that were present in the brain during chronic infection. In vitro mixing studies using WT and c-Rel−/− dendritic cells and WT and c-Rel−/− TCR transgenic T cells indicated that c-Rel−/− dendritic cells are defective in their ability to stimulate T cell responses. However, when c-Rel−/− T cells were transferred into T cell-deficient hosts, early defects in T cell activation, proliferation, and IFN-γ production persisted, and these mice remained susceptible to infection. Together, these studies indicate that although c-Rel is an important regulator of innate immune responses, it also plays an important role in optimization and maintenance of adaptive T cell responses during infection.

A role for inducible costimulator protein in the CD28- independent mechanism of resistance to Toxoplasma gondii.

Long-term resistance to Toxoplasma gondii is dependent on the development of parasite-specific T cells that produce IFN-γ. CD28 is a costimulatory molecule important for optimal activation of T cells, but CD28−/− mice are resistant to T. gondii, demonstrating that CD28-independent mechanisms regulate T cell responses during toxoplasmosis. The identification of the B7-related protein 1/inducible costimulator protein (ICOS) pathway and its ability to regulate the production of IFN-γ suggested that this pathway may be involved in the CD28-independent activation of T cells required for resistance to T. gondii. In support of this hypothesis, infection of wild-type or CD28−/− mice with T. gondii resulted in the increased expression of ICOS by activated CD4+ and CD8+ T cells. In addition, both costimulatory pathways contributed to the in vitro production of IFN-γ by parasite-specific T cells and when both pathways were blocked, there was an additive effect that resulted in almost complete inhibition of IFN-γ production. Although in vivo blockade of the ICOS costimulatory pathway did not result in the early mortality of wild-type mice infected with T. gondii, it did lead to increased susceptibility of CD28−/− mice to T. gondi associated with reduced serum levels of IFN-γ, increased parasite burden, and increased mortality compared with the control group. Together, these results identify a critical role for ICOS in the protective Th1-type response required for resistance to T. gondii and suggest that ICOS and CD28 are parallel costimulatory pathways, either of which is sufficient to mediate resistance to this intracellular pathogen.

TRAF6-Dependent Mitogen-Activated Protein Kinase Activation Differentially Regulates the Production of Interleukin-12 by Macrophages in Response to Toxoplasma gondii

ABSTRACT The production of interleukin-12 (IL-12) is critical to the development of innate and adaptive immune responses required for the control of intracellular pathogens. Many microbial products signal through Toll-like receptors (TLR) and activate NF-κB family members that are required for the production of IL-12. Recent studies suggest that components of the TLR pathway are required for the production of IL-12 in response to the parasite Toxoplasma gondii; however, the production of IL-12 in response to this parasite is independent of NF-κB activation. The adaptor molecule TRAF6 is involved in TLR signaling pathways and associates with serine/threonine kinases involved in the activation of both NF-κB and mitogen-activated protein kinase (MAPK). To elucidate the intracellular signaling pathways involved in the production of IL-12 in response to soluble toxoplasma antigen (STAg), wild-type and TRAF6−/− mice were inoculated with STAg, and the production of IL-12(p40) was determined. TRAF6−/− mice failed to produce IL-12(p40) in response to STAg, and TRAF6−/− macrophages stimulated with STAg also failed to produce IL-12(p40). Studies using Western blot analysis of wild-type and TRAF6−/− macrophages revealed that stimulation with STAg resulted in the rapid TRAF6-dependent phosphorylation of p38 and extracellular signal-related kinase, which differentially regulated the production of IL-12(p40). The studies presented here demonstrate for the first time that the production of IL-12(p40) in response to toxoplasma is dependent upon TRAF6 and p38 MAPK.

CD40-Activated B Cell Cancer Vaccine Improves Second Clinical Remission and Survival in Privately Owned Dogs with Non-Hodgkin's Lymphoma

Cell-based active immunotherapy for cancer is a promising novel strategy, with the first dendritic cell (DC) vaccine achieving regulatory approval for clinical use last year. Manufacturing remains arduous, especially for DC vaccines, and the prospect of using cell-based immunotherapy in the adjuvant setting or in combination with chemotherapy remains largely untested. Here, we used a comparative oncology approach to test the safety and potential efficacy of tumor RNA-loaded, CD40-activated B cells in privately owned dogs presenting with non-Hodgkins lymphoma (NHL), a clinical scenario that represents not only a major problem in veterinary medicine but also a bona fide spontaneous animal model for the human condition. When administered to NHL dogs in remission after induction chemotherapy, CD40-B cells electroporated ex vivo with autologous tumor RNA safely stimulated immunity in vivo. Although chemotherapy plus CD40-B vaccination did not improve time-to-progression or lymphoma-specific survival compared to dogs treated with chemotherapy alone, vaccination potentiated the effects of salvage therapy and improved the rate of durable second remissions as well as subsequent lymphoma-specific survival following salvage therapy. Several of these relapsed dogs are now long-term survivors and free of disease for more than a year. Overall, these clinical and immunological results suggest that cell-based CD40 cancer vaccination is safe and synergizes with chemotherapy to improve clinical outcome in canine NHL. More broadly, our findings underscore the unique value of clinical investigations in tumor-bearing companion animals.

New lessons from old pathogens: what parasitic infections have taught us about the role of nuclear factor‐κB in the regulation of immunity

Summary:  The nuclear factor‐κB (NF‐κB) family of transcription factors is activated by many infectious and inflammatory stimuli. This family regulates the expression of multiple genes, whose products include cytokines, chemokines, adhesion molecules, and antiapoptotic factors that are important components of the innate and adaptive immune response. A prominent role of NF‐κB transcription factors in resistance to a variety of infectious diseases was revealed by studies with mice that lack individual family members. However, little is known about the basis for these effects or about the role of individual family members during a coordinated immune response to infection. Diverse parasites such as Toxoplasma gondii, Leishmania major, and Trichuris muris provide a unique opportunity to understand the role of the NF‐κB system in the development of innate and adaptive immunity to these infections. The basis for resistance and susceptibility to these parasites is well understood, and studies using these experimental systems have provided unique insights into the role of NF‐κB in the regulation of T‐helper 1 cell (Th1) and Th2 type responses. It has become clear that NF‐κB family members have cell lineage‐specific functions and that their relative importance varies with type of infection as well as route of pathogen entry. Thus, studies with models of parasitic infection have revealed that individual NF‐κB family members perform distinct, nonoverlapping, and biologically significant functions in the regulation of immunity and inflammation.

NEMO Binding Domain peptide inhibits constitutive NF-κB activity and reduces tumor burden in a canine model of relapsed, refractory Diffuse Large B-Cell Lymphoma

Purpose: Activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) is an aggressive, poorly chemoresponsive lymphoid malignancy characterized by constitutive canonical NF-κB activity that promotes lymphomagenesis and chemotherapy resistance via overexpression of antiapoptotic NF-κB target genes. Inhibition of the canonical NF-κB pathway may therefore have therapeutic relevance in ABC-DLBCL. Here, we set out to determine whether dogs with spontaneous DLBCL have comparative aberrant constitutive NF-κB activity and to determine the therapeutic relevance of NF-κB inhibition in dogs with relapsed, resistant DLBCL. Experimental Design: Canonical NF-κB activity was evaluated by electrophoretic mobility shift assays and immunoblot analyses, and NF-κB target gene expression was measured by quantitative real time PCR. Primary malignant canine B lymphocytes were treated with the selective IKK complex inhibitor NF-κB essential modulator-binding domain (NBD) peptide and evaluated for NF-κB activity and apoptosis. NBD peptide was administered intranodally to dogs with relapsed B-cell lymphoma and NF-κB target gene expression and tumor burden were evaluated pre- and post-treatment. Results: Constitutive canonical NF-κB activity and increased NF-κB target gene expression were detected in primary DLBCL tissue. NBD peptide inhibited this activity and induced apoptosis of primary malignant B cells in vitro. Intratumoral injections of NBD peptide to dogs with relapsed DLBCL inhibited NF-κB target gene expression and reduced tumor burden. Conclusions: This work shows that dogs with spontaneous DLBCL represent a clinically relevant, spontaneous, large animal model for human ABC-DLBCL and shows the therapeutic relevance of NF-κB inhibition in the treatment of ABC-DLBCL. These results have important translational relevance for ABC-DLBCL treatment in human patients. Clin Cancer Res; 17(14); 4661–71. ©2011 AACR.

Evaluation of equine peripheral blood apheresis product, bone marrow, and adipose tissue as sources of mesenchymal stem cells and their differentation potential

OBJECTIVE To evaluate effects of apheresis on mesenchymal stem cells (MSCs) and compare those MSCs with MSCs obtained from adipose tissue or bone marrow (BM). SAMPLE POPULATION Samples obtained from 6 adult horses. PROCEDURES Samples of blood from a peripheral vein, adipose tissue, and BM aspirate were obtained from each horse. Samples were processed via apheresis of blood and techniques reported elsewhere for adipose tissue and BM. Cultures were maintained until adherence and subsequently were subjected to differentiation protocols to evaluate adipogenic, osteoblastogenic, and chondrogenic potential. RESULTS Apheresis product had a significantly higher mononuclear percentage, higher platelet count, and lower RBC count, compared with values for peripheral blood. No cell adherence to the tissue culture plates was detected for the apheresis product. Adherence was detected for 6 of 6 adipose-derived and 4 of 6 BM-derived samples. Variations in efficiency were detected for differentiation of adipose- and BM-derived cells into adipocytes, chondrocytes, and osteoblasts. CONCLUSIONS AND CLINICAL RELEVANCE Apheresis was able to concentrate mononuclear cells and reduce RBC contamination. However, the apheresis product was unable to adhere to the tissue culture plates. In matched horses, adipose- and BM-derived MSCs were capable of producing lipids, glycosaminoglycan, and mineral. The BM was vastly superior to adipose tissue as a source of MSCs with osteoblastogenic potential in matched horses. Additional studies will be necessary to optimize apheresis techniques for horses before peripheral blood can be considered a suitable source for multipotential cells for use in cell-based treatments.