Prenatal Cocaine Disrupts Serotonin Signaling-Dependent Behaviors: Implications for Sex Differences, Early Stress and Prenatal SSRI Exposure

Prenatal cocaine (PC) exposure negatively impacts the developing nervous system, including numerous changes in serotonergic signaling. Cocaine, a competitive antagonist of the serotonin transporter, similar to selective serotonin reuptake inhibitors (SSRIs), also blocks dopamine and norepinephrine transporters, leaving the direct mechanism through which cocaine disrupts the developing serotonin system unclear. In order to understand the role of the serotonin transporter in cocaine’s effect on the serotonergic system, we compare reports concerning PC and prenatal antidepressant exposure and conclude that PC exposure affects many facets of serotonergic signaling (serotonin levels, receptors, transporters) and that these effects differ significantly from what is observed following prenatal SSRI exposure. Alterations in serotonergic signaling are dependent on timing of exposure, test regimens, and sex. Following PC exposure, behavioral disturbances are observed in attention, emotional behavior and stress response, aggression, social behavior, communication, and like changes in serotonergic signaling, these effects depend on sex, age and developmental exposure. Vulnerability to the effects of PC exposure can be mediated by several factors, including allelic variance in serotonergic signaling genes, being male (although fewer studies have investigated female offspring), and experiencing the adverse early environments that are commonly coincident with maternal drug use. Early environmental stress results in disruptions in serotonergic signaling analogous to those observed with PC exposure and these may interact to produce greater behavioral effects observed in children of drug-abusing mothers. We conclude that based on past evidence, future studies should put a greater emphasis on including females and monitoring environmental factors when studying the impact of PC exposure

Investigating the role of NF-kappaB in the pathology of osteoarthritis

Globally the most prevalent muscoskeletal condition affecting humans, osteoarthritis is a complex, multifactorial disease characterised by deterioration of articular cartilage and varying degrees of synovial inflammation. The catabolic cytokine interleukin-1beta (IL-1beta), a potent inducer of the transcription factor NFkappaB, induces the production of cartilage destructive aggrecanases and matrix metalloproteinases (MMPs) and the inflammatory cytokine, interleukin-6 (IL-6), within the joint. The aim of the work described in this thesis was to investigate the role of NFkappaB within the osteoarthritic joint and its potential as a therapeutic target for disease intervention. NFkappaB activation was inhibited using adenoviral gene transfer or by two novel pharmacological inhibitors of IKK, R0100 and R0919. Inhibition of the NFkappaB signalling cascade in human synovial fibroblasts from osteoarthritic patients suppressed the IL-1beta induction of IL-6, MMP-1 and MMP-3 but did not affect the levels of tissue inhibitor of metalloproteinases-1 (TIMP-1). To further investigate the effects of these IKK inhibitors, cartilage degradation was investigated by culturing murine patellas with human synovial fibroblasts. Early stage cartilage deterioration, induced by IL-1beta, was prevented by NFkappaB inhibition. An animal model of OA, that reflected the early stage pathological changes, was set up as part of this study. The therapeutic efficacy of RO100 and R0919 was tested in vivo. It was observed that neither inhibitor prevented pathological changes associated with OA, for example cartilage degradation. The basis of the lack of efficacy demonstrated by RO100 and R0919 is unknown but may be due to poor bioavailability of the agents within the joint. In conclusion, the studies conducted during this thesis have shown, in various systems, that inhibiting NFkappaB can prevent changes such as cartilage degradation that occur in OA. Increasing the bioavailability of these or other inhibitors of NFkappaB may be key in the development of successful novel therapeutic modalities in the future

The role of synovial macrophages and macrophage-produced cytokines in driving aggrecanases, matrix metalloproteinases, and other destructive and inflammatory responses in osteoarthritis

There is an increasing body of evidence that synovitis plays a role in the progression of osteoarthritis and that overproduction of cytokines and growth factors from the inflamed synovium can influence the production of degradative enzymes and the destruction of cartilage. In this study, we investigate the role of synovial macrophages and their main proinflammatory cytokines, interleukin (IL)-1 and tumour necrosis factor-alpha (TNF-α), in driving osteoarthritis synovitis and influencing the production of other pro- and anti-inflammatory cytokines, production of matrix metalloproteinases, and expression of aggrecanases in the osteoarthritis synovium. We established a model of cultures of synovial cells from digested osteoarthritis synovium derived from patients undergoing knee or hip arthroplasties. By means of anti-CD14-conjugated magnetic beads, specific depletion of osteoarthritis synovial macrophages from these cultures could be achieved. The CD14(+)-depleted cultures no longer produced significant amounts of macrophage-derived cytokines like IL-1 and TNF-α. Interestingly, there was also significant downregulation of several cytokines, such as IL-6 and IL-8 (p < 0.001) and matrix metalloproteinases 1 and 3 (p < 0.01), produced chiefly by synovial fibroblasts. To investigate the mechanisms involved, we went on to use specific downregulation of IL-1 and/or TNF-α in these osteoarthritis cultures of synovial cells. The results indicated that neutralisation of both IL-1 and TNF-α was needed to achieve a degree of cytokine (IL-6, IL-8, and monocyte chemoattractant protein-1) and matrix metalloproteinase (1, 3, 9, and 13) inhibition, as assessed by enzyme-linked immunosorbent assay and by reverse transcription-polymerase chain reaction (RT-PCR), similar to that observed in CD14(+)-depleted cultures. Another interesting observation was that in these osteoarthritis cultures of synovial cells, IL-1β production was independent of TNF-α, in contrast to the situation in rheumatoid arthritis. Using RT-PCR, we also demonstrated that whereas the ADAMTS4 (a disintegrin and metalloprotease with thrombospondin motifs 4) aggrecanase was driven mainly by TNF-α, ADAMTS5 was not affected by neutralisation of IL-1 and/or TNF-α. These results suggest that, in the osteoarthritis synovium, both inflammatory and destructive responses are dependent largely on macrophages and that these effects are cytokine-driven through a combination of IL-1 and TNF-α

Sequential targeting of PI3Kδ and LAG3 as an effective anti-cancer approach

Emerging studies have demonstrated the potential of PI3Kδ blockade as an immunotherapy for solid tumours. In pre-clinical models, we recently demonstrated that anti-LAG3 immune checkpoint blockade vastly potentiated PI3Kδ-based immunotherapy, enabling successful tumour control in all treated mice

Preferential Generation of 15-HETE-PE Induced by IL-13 Regulates Goblet Cell Differentiation in Human Airway Epithelial Cells

Type 2–associated goblet cell hyperplasia and mucus hypersecretion are well known features of asthma. 15-Lipoxygenase-1 (15LO1) is induced by the type 2 cytokine IL-13 in human airway epithelial cells (HAECs) in vitro and is increased in fresh asthmatic HAECs ex vivo. 15LO1 generates a variety of products, including 15-hydroxyeicosatetraenoic acid (15-HETE), 15-HETE-phosphatidylethanolamine (15-HETE-PE), and 13-hydroxyoctadecadienoic acid (13-HODE). In this study, we investigated the 15LO1 metabolite profile at baseline and after IL-13 treatment, as well as its influence on goblet cell differentiation in HAECs. Primary HAECs obtained from bronchial brushings of asthmatic and healthy subjects were cultured under air–liquid interface culture supplemented with arachidonic acid and linoleic acid (10 μM each) and exposed to IL-13 for 7 days. Short interfering RNA transfection and 15LO1 inhibition were applied to suppress 15LO1 expression and activity. IL-13 stimulation induced expression of 15LO1 and preferentially generated 15-HETE-PE in vitro, both of which persisted after removal of IL-13. 15LO1 inhibition (by short interfering RNA and chemical inhibitor) decreased IL-13–induced forkhead box protein A3 (FOXA3) expression and enhanced FOXA2 expression. These changes were associated with reductions in both mucin 5AC and periostin. Exogenous 15-HETE-PE stimulation (alone) recapitulated IL-13–induced FOXA3, mucin 5AC, and periostin expression. The results of this study confirm the central importance of 15LO1 and its primary product, 15-HETE-PE, for epithelial cell remodeling in HAEC

Interferon-γ inhibits interleukin-1β-induced matrix metalloproteinase production by synovial fibroblasts and protects articular cartilage in early arthritis

Introduction: The first few months after symptom onset represents a pathologically distinct phase in rheumatoid arthritis (RA). We used relevant experimental models to define the pathological role of interferon-γ (IFN-γ) during early inflammatory arthritis. Methods: We studied IFN-γ's capacity to modulate interleukin-1β (IL-1β) induced degenerative responses using RA fibroblast-like synoviocytes (FLS), a bovine articular cartilage explant (BACE)/RA-FLS co-culture model and an experimental inflammatory arthritis model (murine antigen-induced arthritis (AIA)). Results: IFN-γ modulated IL-1β driven matrix metalloproteinases (MMP) synthesis resulting in the down-regulation of MMP-1 and MMP-3 production in vitro. IFN-γ did not affect IL-1β induced tissue inhibitor of metalloproteinase-1 (TIMP-1) production by RA FLS but skewed the MMP/TIMP-1 balance sufficiently to attenuate glycosaminoglycan-depletion in our BACE model. IFN-γ reduced IL-1β expression in the arthritic joint and prevented cartilage degeneration on Day 3 of AIA. Conclusions: Early therapeutic intervention with IFN-γ may be critical to orchestrate tissue-protective responses during inflammatory arthritis

Paracetamol reduces influenza-induced immunopathology in a mouse model of infection without compromising virus clearance or the generation of protective immunity

Background: Seasonal influenza A infection affects a significant cohort of the global population annually, resulting in considerable morbidity and mortality. Therapeutic strategies are of limited efficacy, and during a pandemic outbreak would only be available to a minority of the global population. Over-the-counter medicines are routinely taken by individuals suffering from influenza, but few studies have been conducted to determine their effectiveness in reducing pulmonary immunopathology or the influence they exert upon the generation of protective immunity. Methods: A mouse model of influenza infection was utilised to assess the efficacy of paracetamol (acetaminophen) in reducing influenza-induced pathology and to examine whether paracetamol affects generation of protective immunity. Results: Administration (intraperitoneal) of paracetamol significantly decreased the infiltration of inflammatory cells into the airway spaces, reduced pulmonary immunopathology associated with acute infection and improved the overall lung function of mice, without adversely affecting the induction of virus-specific adaptive responses. Mice treated with paracetamol exhibited an ability to resist a second infection with heterologous virus comparable with that of untreated mice. Conclusions: Our results demonstrate that paracetamol dramatically reduces the morbidity associated with influenza but does not compromise the development of adaptive immune responses. Overall, these data support the utility of paracetamol for reducing the clinical symptoms associated with influenza virus infection

Enhanced antitumor immunity through sequential targeting of PI3Kδ and LAG3

Background Despite striking successes, immunotherapies aimed at increasing cancer-specific T cell responses are unsuccessful in most patients with cancer. Inactivating regulatory T cells (Treg) by inhibiting the PI3Kδ signaling enzyme has shown promise in preclinical models of tumor immunity and is currently being tested in early phase clinical trials in solid tumors. Methods Mice bearing 4T1 mammary tumors were orally administered a PI3Kδ inhibitor (PI-3065) daily and tumor growth, survival and T cell infiltrate were analyzed in the tumor microenvironment. A second treatment schedule comprised PI3Kδ inhibitor with anti-LAG3 antibodies administered sequentially 10 days later. Results As observed in human immunotherapy trials with other agents, immunomodulation by PI3Kδ-blockade led to 4T1 tumor regressor and non-regressor mice. Tumor infiltrating T cells in regressors were metabolically fitter than those in non-regressors, with significant enrichments of antigen-specific CD8+ T cells, T cell factor 1 (TCF1)+ T cells and CD69− T cells, compatible with induction of a sustained tumor-specific T cell response. Treg numbers were significantly reduced in both regressor and non-regressor tumors compared with untreated tumors. The remaining Treg in non-regressor tumors were however significantly enriched with cells expressing the coinhibitory receptor LAG3, compared with Treg in regressor and untreated tumors. This striking difference prompted us to sequentially block PI3Kδ and LAG3. This combination enabled successful therapy of all mice, demonstrating the functional importance of LAG3 in non-regression of tumors on PI3Kδ inhibition therapy. Follow-up studies, performed using additional cancer cell lines, namely MC38 and CT26, indicated that a partial initial response to PI3Kδ inhibition is an essential prerequisite to a sequential therapeutic benefit of anti-LAG3 antibodies. Conclusions These data indicate that LAG3 is a key bottleneck to successful PI3Kδ-targeted immunotherapy and provide a rationale for combining PI3Kδ/LAG3 blockade in future clinical studies

Treg-driven tumour control by PI3Kδ inhibition limits myeloid-derived suppressor cell expansion

Background Recent studies have demonstrated that blocking the PI3Kδ signalling enzyme (by administering a small molecule inhibitor, PI-3065) can potently improve the anti-tumour T-cell response through direct inhibition of Tregs. This treatment also has a negative impact on MDSC numbers but the primary mechanism driving this effect has remained unclear. Methods The 4T1 breast cancer mouse model was used in combination with PI-3065 to gain insights into the effect of PI3Kδ inhibition on MDSCs. Results PI-3065 treatment resulted in a concomitant reduction in MDSC expansion and tumour size. However, targeting Tregs independent of PI-3065 was also associated with reduced tumour volume and MDSC numbers. Surgical removal of tumours resulted in a rapid and significant decline in MDSC numbers, whilst ex vivo studies using cells from PI-3065-treated mice demonstrated no direct effect of the inhibitor on MDSC activity. Conclusions Our data suggest that MDSCs are not inhibited directly by PI-3065 treatment but that their reduced recruitment and immunosuppression within the tumour microenvironment is an indirect consequence of PI3Kδ-inhibition-driven tumour control. This indicates that PI3Kδ inhibition drives tumour immunity by breaking down multiple immunosuppressive pathways through both direct mechanisms (on Treg) and indirect mechanisms, secondary to tumour control (on MDSCs)

Myeloid 12/15-LOX regulates B cell numbers and innate immune antibody levels in vivo

Background. The myeloid enzyme 12/15-lipoxygenase (LOX), which generates bioactive oxidized lipids, has been implicated in numerous inflammatory diseases, with several studies demonstrating an improvement in pathology in mice lacking the enzyme. However, the ability of 12/15-LOX to directly regulate B cell function has not been studied. Methods. The influence of 12/15-LOX on B cell phenotype and function, and IgM generation, was compared using wildtype (WT) and 12/15-LOX (Alox15-/-) deficient mice. The proliferative and functional capacity of splenic CD19+ B cells was measured in vitro in response to various toll-like receptor agonists. Results. WT and Alox15-/- displayed comparable responses. However in vivo, splenic B cell numbers were significantly elevated in Alox15-/- mice with a corresponding elevation in titres of total IgM in lung, gut and serum, and lower serum IgM directed against the 12/15-LOX product, 12-hydroxyeicosatetraenoic acid-phosphatidylethanolamine (HETE-PE). Discussion. Myeloid 12/15-LOX can regulate B cell numbers and innate immune antibody levels in vivo, potentially contributing to its ability to regulate inflammatory disease. Furthermore, the alterations seen in 12/15-LOX deficiency likely result from changes in the equilibrium of the immune system that develop from birth. Further studies in disease models are warranted to elucidate the contribution of 12/15-LOX mediated alterations in B cell numbers and innate immune antibody generation to driving inflammation in vivo