Donald Souza

Cellular responses to murine CD40 in a mouse B cell line may be TRAF dependent or independent

Engagement of CD40 by its ligand induces IKK and mitogen‐activated protein kinase (MAPK) phosphorylation and transcriptional activation, leading to activation and differentiation of B cells. These events are most likely transduced by adaptor molecules that are recruited to the CD40 cytoplasmic domain, called TNF receptor‐associated factors (TRAF). We have engineered a chimeric CD40 molecule using the human extracellular sequence and the murine cytoplasmic domain to assess the contribution that specific TRAF binding domains provide to the cytoplasmic signaling functions of CD40. Thedata presented here show that the shared binding site for TRAF2 and TRAF3 accounts for receptor internalization, and the majority of signaling through CD40, but is redundant with the TRAF6 binding site for activation of p38 and NFκB signaling pathways. Disruption of the TRAF2/3 binding site results in a delayed and diminished kinase pathway induction, but complete preclusion of all signals requires the disruption of more than the two known TRAF binding sites. The specific TRAF dependency of CD40‐induced growth arrest, TNF‐α production, and phosphorylation of signaling molecules are shown, while p38 MAPK activation and cell surface antigen modulation suggest TRAF independent CD40 signaling in B cells.

Nonsteroidal Dissociated Glucocorticoid Agonists Containing Azaindoles as Steroid A-Ring Mimetics

Syntheses and structure-activity relationships (SAR) of nonsteroidal glucocorticoid receptor (GR) agonists are described. These compounds contain azaindole moieties as A-ring mimetics and display various degrees of in vitro dissociation between gene transrepression and transactivation. Collagen induced arthritis studies in mouse have demonstrated that in vitro dissociated compounds (R)-16 and (R)-37 have steroid-like anti-inflammatory properties with improved metabolic side effect profiles, such as a reduced increase in body fat and serum insulin levels, compared to steroids.

Kcnn4 is a regulator of macrophage multinucleation in bone homeostasis and inflammatory disease.

Summary Macrophages can fuse to form osteoclasts in bone or multinucleate giant cells (MGCs) as part of the immune response. We use a systems genetics approach in rat macrophages to unravel their genetic determinants of multinucleation and investigate their role in both bone homeostasis and inflammatory disease. We identify a trans-regulated gene network associated with macrophage multinucleation and Kcnn4 as being the most significantly trans-regulated gene in the network and induced at the onset of fusion. Kcnn4 is required for osteoclast and MGC formation in rodents and humans. Genetic deletion of Kcnn4 reduces macrophage multinucleation through modulation of Ca2+ signaling, increases bone mass, and improves clinical outcome in arthritis. Pharmacological blockade of Kcnn4 reduces experimental glomerulonephritis. Our data implicate Kcnn4 in macrophage multinucleation, identifying it as a potential therapeutic target for inhibition of bone resorption and chronic inflammation.

A GPBAR1 (TGR5) Small Molecule Agonist Shows Specific Inhibitory Effects on Myeloid Cell Activation In Vitro and Reduces Experimental Autoimmune Encephalitis (EAE) In Vivo

GPBAR1 is a G protein-coupled receptor that is activated by certain bile acids and plays an important role in the regulation of bile acid synthesis, lipid metabolism, and energy homeostasis. Recent evidence suggests that GPBAR1 may also have important effects in reducing the inflammatory response through its expression on monocytes and macrophages. To further understand the role of GPBAR1 in inflammation, we generated a novel, selective, proprietary GPBAR1 agonist and tested its effectiveness at reducing monocyte and macrophage activation in vitro and in vivo. We have used this agonist, together with previously described agonists to study agonism of GPBAR1, and shown that they can all induce cAMP and reduce TLR activation-induced cytokine production in human monocytes and monocyte-derived macrophages in vitro. Additionally, through the usage of RNA sequencing (RNA-Seq), we identified a select set of genes that are regulated by GPBAR1 agonism during LPS activation. To further define the in vivo role of GPBAR1 in inflammation, we assessed GPBAR1 expression and found high levels on circulating mouse monocytes. Agonism of GPBAR1 reduced LPS-induced cytokine production in mouse monocytes ex vivo and serum cytokine levels in vivo. Agonism of GPBAR1 also had profound effects in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis, where monocytes play an important role. Mice treated with the GPBAR1 agonist exhibited a significant reduction in the EAE clinical score which correlated with reduced monocyte and microglial activation and reduced trafficking of monocytes and T cells into the CNS. These data confirm the importance of GPBAR1 in controlling monocyte and macrophage activation in vivo and support the rationale for selective agonists of GPBAR1 in the treatment of inflammatory diseases.

Discovery of a potent and dissociated non-steroidal glucocorticoid receptor agonist containing an alkyl carbinol pharmacophore.

Synthesis and structure-activity relationship (SAR) of a series of alkyl and cycloalkyl containing non-steroidal dissociated glucocorticoid receptor (GR) agonists is reported. This series of compounds was identified as part of an effort to replace the CF3 group in a scaffold represented by 1a. The study culminated in the identification of compound 14, a t-butyl containing derivative, which has shown potent activity for GR, selectivity against the progesterone receptor (PR) and the mineralocorticoid receptor (MR), in vitro anti-inflammatory activity in an IL-6 transrepression assay, and dissociation in a MMTV transactivation counter-screen. In a collagen-induced arthritis mouse model, 14 displayed prednisolone-like efficacy, and lower impact on body fat and free fatty acids than prednisolone at an equivalent anti-inflammatory dose.

The role of leukocyte function-associated antigen-1 in animal models of inflammation.

Both preclinical and clinical data have identified leukocyte function-associated antigen-1 (LFA-1) as an important component of inflammatory disease states. We evaluated small molecule inhibitors of this glycoprotein in several animal models in which the inflammatory process is dependent on human or non-human primate LFA-1. (R)-5(4-bromobenzyl)-3(3,5-dichlorophenyl)-1,5-dimethylimidazolidine-2,4-dione, BIRT 377, effectively suppressed the production of human immunoglobulin (IgG) following reconstitution of severe combined immunodeficient (SCID) mice with human peripheral blood mononuclear cells. The BIRT 377 analog, BIX 642, inhibited the cellular infiltrate and increase in skin thickness associated with the delayed-type hypersensitivity reaction in previously immunized squirrel monkeys challenged with antigen. BIX 642 also inhibited the trans-vivo delayed-type hypersensitivity response in the footpads of SCID mice injected with human peripheral blood mononuclear cells and donor-sensitive antigen. These results demonstrate the efficacy of small molecule inhibitors of LFA-1 in preclinical models of inflammation dependent on human or non-human primate LFA-1.

Function-regulating pharmacophores in a sulfonamide class of glucocorticoid receptor agonists

A class of α-methyltryptamine sulfonamide glucocorticoid receptor (GR) modulators was optimized for agonist activity. The design of ligands was aided by molecular modeling, and key function-regulating pharmacophoric points were identified that are critical in achieving the desired agonist effect in cell based assays. Compound 27 was profiled in vitro and in vivo in models of inflammation. Analogs could be rapidly prepared in a parallel approach from aziridine building blocks.

CCR1 Plays a Critical Role in Modulating Pain through Hematopoietic and Non-Hematopoietic Cells

Inflammation is associated with immune cells infiltrating into the inflammatory site and pain. CC chemokine receptor 1 (CCR1) mediates trafficking of leukocytes to sites of inflammation. However, the contribution of CCR1 to pain is incompletely understood. Here we report an unexpected discovery that CCR1-mediated trafficking of neutrophils and CCR1 activity on non-hematopoietic cells both modulate pain. Using a genetic approach (CCR1−/− animals) and pharmacological inhibition of CCR1 with selective inhibitors, we show significant reductions in pain responses using the acetic acid-induced writhing and complete Freunds adjuvant-induced mechanical hyperalgesia models. Reductions in writhing correlated with reduced trafficking of myeloid cells into the peritoneal cavity. We show that CCR1 is highly expressed on circulating neutrophils and their depletion decreases acetic acid-induced writhing. However, administration of neutrophils into the peritoneal cavity did not enhance acetic acid-induced writhing in wild-type (WT) or CCR1−/− mice. Additionally, selective knockout of CCR1 in either the hematopoietic or non-hematopoietic compartments also reduced writhing. Together these data suggest that CCR1 functions to significantly modulate pain by controlling neutrophil trafficking to the inflammatory site and having an unexpected role on non-hematopoietic cells. As inflammatory diseases are often accompanied with infiltrating immune cells at the inflammatory site and pain, CCR1 antagonism may provide a dual benefit by restricting leukocyte trafficking and reducing pain.

BTK inhibition ameliorates kidney disease in spontaneous lupus nephritis

Lupus nephritis is a common disease manifestation of SLE, in which immune complex deposition and macrophage activation are important contributors to disease pathogenesis. Brutons tyrosine kinase (BTK) plays an important role in both B cell and FcgammaR mediated myeloid cell activation. In the current study, we examined the efficacy of BI-BTK-1, a recently described irreversible BTK inhibitor, in the classical NZB × NZW F1 (NZB/W) and MRL/lpr spontaneous mouse models of SLE. NZB/W mice were randomly assigned to a treatment (0.3 mg/kg, 1 mg/kg, 3 mg/kg and 10 mg/kg) or control group and began treatment at 22 weeks of age. The experimental setup was similar in MRL/lpr mice, but with a single treated (10 mg/kg, beginning at 8-9 weeks of age) and control group. A separate experiment was performed in the MRL/lpr strain to assess the ability of BI-BTK-1 to reverse established kidney disease. Early treatment with BI-BTK-1 significantly protected NZB/W and MRL/lpr mice from the development of proteinuria, correlating with significant renal histological protection, decreased anti-DNA titers, and increased survival in both strains. BI-BTK-1 treated mice displayed a significant decrease in nephritis-associated inflammatory mediators (e.g. LCN2 and IL-6) in the kidney, combined with a significant inhibition of immune cell infiltration and accumulation. Importantly, BI-BTK-1 treatment resulted in the reversal of established kidney disease. BTK inhibition significantly reduced total B cell numbers and all B cell subsets (immature, transitional, follicular, marginal zone, and class switched) in the spleen of NZB/W mice. Overall, the significant efficacy of BI-BTK-1 in ameliorating multiple pathological endpoints associated with kidney disease in two distinct murine models of spontaneous lupus nephritis provides a strong rationale for BTK inhibition as a promising treatment approach for lupus nephritis.

02.50 Intestinal overexpression of human tnf triggers sacro-iliitis

An intriguing link exists between gut and joint inflammation in spondyloarthritis (SpA). About 50% of patients has subclinical (eg, microscopic) gut inflammation, which represents a risk factor for development of Crohn’s disease, sacroiliac inflammation and evolution in to ankylosing spondylitis. However, the underlying mechanisms are still relatively poorly understood. Our goal was to examine the relationship between TNF, microscopic gut inflammation and axial inflammation. Therefore, we examined in situ expression of TNF, TNFR1 and TNFR2 using triple in situ hybridisation in gut biopsies of human SpA patients and found marked upregulation of TNF in inflamed versus non-inflamed biopsies. We also noted a predominant upregulation of TNFR1 on intestinal epithelium and TNFR2 in lamina propria respectively. Of interest, IL-17 and IL-23 were also markedly increased while IL-22 was most abundant in chronically inflamed samples. In line with this, we found that patients with gut inflammation had a higher need for anti-TNF therapy and their degree of clinical response after anti-TNF was also markedly higher. We speculated that TNF in the gut represents an important risk factor for disease severity and progression in SpA. To investigate this further we generated intestinal specific human TNF transgenic mice, in which hTNF is under control of a rat iFABP (fatty acid binding protein) promoter, generating a mouse-model over-expressing human TNF in the ileum. These mice, together with wild type littermates, were evaluated for the development of arthritis up until the age of 13 weeks after which they were euthanized and ankle and sacroiliac joints as well as ileum were processed for histology. Transgenic mice exhibit a runt phenotype and hallmarks of inflammatory bowel disease, including increased intestinal permeability and inflammation compared to their wild-type littermates. While in peripheral joints no clear signs of arthritis were observed, the sacroiliac joints in transgenic mice, by contrast, showed marked signs of inflammation as well as bone erosion and destruction. These data propose a new paradigm that gut-derived TNF is sufficient to trigger sacroiliitis and provide an alternate explanation on the relationship between gut inflammation, evolution to inflammatory bowel disease and axial inflammation in SpA.