Mohammad B. Nickdel

IL-33 receptor (T1/ST2) signalling is necessary to prevent the development of encephalitis in mice infected with Toxoplasma gondii

T1/ST2 is an immunoregulatory protein of the IL‐1 receptor family that has recently been reported as being a component of the IL‐33 receptor. IL‐33 is a newly described cytokine known to amplify the Th2 response and reduce production of Th1 cytokines. The function of T1/ST2 during Toxoplasma gondii infection is as yet undescribed. Given the requirement of a balanced type 1/type 2 response for effective control of parasite number and immunopathology, it is likely that T1/ST2 may play a part in aiding this process. Accordingly, we have shown that T1/ST2 mRNA transcripts are upregulated in the brains of mice infected with T. gondii and that mice deficient in T1/ST2 demonstrated increased susceptibility to infection with T. gondii that correlated with increased pathology and greater parasite burden in the brains. Real‐time PCR analysis of cerebral cytokine levels revealed increased mRNA levels of iNOS, IFN‐γ and TNF‐α in infected T1/ST2−/− mice. These effects were independent of changes in IL‐10 production. This study provides the first evidence of a specific role for IL‐33 receptor signalling in the brain as well as highlighting the requirement of this mechanism in limiting infection with an intracellular parasite.

Toll-like receptor-4-mediated macrophage activation is differentially regulated by progesterone via the glucocorticoid and progesterone receptors.

Macrophage function has been demonstrated to be subject to modulation by progesterone. However, as this steroid hormone can act through the glucocorticoid receptor as well as the progesterone receptor, the mechanism of action has not been precisely characterized. To determine the mode of action, we compared the ability of progesterone, norgestrel (a synthetic progesterone‐receptor‐specific agonist) and dexamethasone (a synthetic glucocorticoid receptor agonist) to modulate macrophage function following stimulation of the Toll‐like receptor‐4 (TLR‐4) ligand lipopolysaccharide (LPS). The results demonstrate that following stimulation of TLR‐4 with LPS and cotreatment with either progesterone or dexamethasone, but not norgestrel, there is a significant reduction in nitric oxide (NO) production, indicating that this progesterone‐mediated effect is through ligation of the glucocorticoid receptor. In contrast, LPS‐induced interleukin‐12 (IL‐12) production could be downregulated by all three steroids, indicating that ligation by progesterone of either the glucocorticoid or the progesterone receptors or both could mediate this effect. While progesterone downmodulated NO‐mediated killing of Leishmania donovani by activated macrophages in vitro, most probably via the glucocorticoid receptor, it had little effect on Toxoplasma gondii growth in these cells. This would suggest that progesterone‐mediated increased susceptibility to T. gondii during pregnancy is more likely to be related to the ability of the hormone to downregulate IL‐12 production and a type‐1 response utilizing the progesterone as well as the glucocorticoid receptors.

Counter-Protective Role for Interleukin-5 during Acute Toxoplasma gondii Infection

ABSTRACT The role of interleukin-5 (IL-5) during Toxoplasma gondii infection was investigated by comparing disease progression in IL-5 gene deficient (IL-5−/−) mice and their wild-type (WT) counterparts on a C57BL/6 background. IL-5−/− mice infected orally with T. gondii were less susceptible to infection than WT mice as demonstrated by reduced mortality rates. Consistent with this data, orally infected IL-5−/− mice had less severe pathological changes in their small intestines than WT mice at 8 days postinfection. At this time, splenocytes and mesenteric lymph node cells derived from IL-5−/− mice produced levels of IL-12, interferon-γ (IFN-γ), IL-4, IL-10, and nitric oxide (measured as nitrite) similar to those derived from WT mice when stimulated withToxoplasma lysate antigen. However, peak serum IL-12 and IFN-γ levels (at days 6 and 8, respectively) were significantly higher in IL-5−/− mice than in WT mice. In addition, WT mice but not IL-5−/− mice had raised levels of eosinophils in their peripheral blood between days 5 and 8 following infection. Oral administration ofNω-nitro-l-arginine methyl (from day 4 postinfection) increased mortality rates in both IL-5−/− and WT mice, indicating a protective role for nitric oxide during the early stages of oral T. gondii infection. In comparison with oral infection, no difference in mortality was observed between IL-5−/− and WT mice following intraperitoneal infection with T. gondii, with all mice surviving until 35 days postinfection. Similarly, no significant differences were observed in the severity of the meningitis, perivascular cuffing, or number of microglial nodules or parasites in the brains of intraperitoneally infected mice. Together, these results demonstrate a detrimental role for IL-5 during the early stage of oral infection with T. gondii which is associated with increased small-intestine pathology, eosinophilia, and reduced plasma IL-12 and IFN-γ levels.

Dissecting the contribution of innate and antigen-specific pathways to the breach of self-tolerance observed in a murine model of arthritis

Background: The relative roles of innate immunity and antigen-specific T cells in rheumatoid arthritis remain controversial. Previous studies demonstrated that T-helper type 1 cells of irrelevant antigen specificity (ovalbumin) induced a transient arthritis in BALB/c mice, which recapitulates many of the pre-articular and articular features of human disease and is associated with the emergence of autoreactive T and B-cell responses to joint-specific antigens. However, the mechanisms underlying this phenomenon were unclear. Objectives: The aim of this study was to dissect the relative contribution of innate and heterologous antigen-specific pathways to the breach of self-tolerance and pathology observed in this model and how this may result from modified T and B-cell interactions. Methods: To address this issue, experimental arthritis was elicited either by a non-specific inflammatory stimulus alone, by activation of T cells of an irrelevant specificity or a combination of both. Results: The non-specific inflammatory response generated by lipopolysaccharide led to articular inflammation and cartilage erosion, but did not break tolerance to joint-specific antigens. In contrast, local activation of T cells of an irrelevant specificity produced a similar pathological picture but, in addition, induced T-cell responses to unrelated joint-specific antigens with associated activation of autoreactive B cells. These effects could be further potentiated by the addition of lipopolysaccharide. Conclusion: These data demonstrate that non-specific inflammation alone is insufficient to breach self-tolerance. In contrast, T cells of an irrelevant specificity, when triggered locally in an antigen-specific manner, can breach self-tolerance leading to arthritis and autoantibody production, which can then be amplified in a non-specific manner.

Intestinal pathology during acute toxoplasmosis is IL-4 dependent and unrelated to parasite burden

The role of interleukin‐4 (IL‐4) during the course of Toxoplasma gondii infection was studied using IL‐4–/– mice and their wild‐type (WT) counterparts on a C57BL/6 background. Following oral infection with T. gondii tissue cysts an exacerbative role for IL‐4 was demonstrated and IL‐4–/– mice were found to be more resistant to infection than WT mice as measured by significantly reduced mortality. Furthermore pathology in the small intestine was less severe in IL‐4–/– mice although conversely liver pathology was greater than in wild‐type mice. Significantly, plasma IL‐12 and IFN‐γ levels, which peaked at days 6 and 8, respectively, were higher in IL‐4–/– mice. The exacerbatory role of IL‐4 in the intestine was found by competitive RT‐PCR not to be associated with increased parasite burdens but was related to comparative expression of IL‐10.

Immunomodulatory role of proteinase-activated receptor-2

Objective Proteinase-activated receptor-2 (PAR2) has been implicated in inflammatory articular pathology. Using the collagen-induced arthritis model (CIA) the authors have explored the capacity of PAR2 to regulate adaptive immune pathways that could promote autoimmune mediated articular damage. Methods Using PAR2 gene deletion and other approaches to inhibit or prevent PAR2 activation, the development and progression of CIA were assessed via clinical and histological scores together with ex vivo immune analyses. Results The progression of CIA, assessed by arthritic score and histological assessment of joint damage, was significantly (p<0.0001) abrogated in PAR2 deficient mice or in wild-type mice administered either a PAR2 antagonist (ENMD-1068) or a PAR2 neutralising antibody (SAM11). Lymph node derived cell suspensions from PAR2 deficient mice were found to produce significantly less interleukin (IL)-17 and IFNγ in ex vivo recall collagen stimulation assays compared with wild-type littermates. In addition, substantial inhibition of TNFα, IL-6, IL-1β and IL-12 along with GM-CSF and MIP-1α was observed. However, spleen and lymph node histology did not differ between groups nor was any difference detected in draining lymph node cell subsets. Anticollagen antibody titres were significantly lower in PAR2 deficient mice. Conclusion These data support an important role for PAR2 in the pathogenesis of CIA and suggest an immunomodulatory role for this receptor in an adaptive model of inflammatory arthritis. PAR2 antagonism may offer future potential for the management of inflammatory arthritides in which a proteinase rich environment prevails.

PAR2 expression in peripheral blood monocytes of patients with rheumatoid arthritis

Objectives Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteinases with proinflammatory activity. A study was undertaken to investigate the presence and functio©nal significance of PAR2 expression on rheumatoid arthritis (RA)-derived leucocyte subsets. Methods Venous blood was obtained from patients with RA and osteoarthritis (OA) as well as healthy control subjects. Surface expression of PAR2 on peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry and interleukin 6 (IL-6) generation by ELISA. Results Patients with RA had elevated but variable surface expression of PAR2 on CD14+ monocytes compared with control subjects (median (1st to 3rd quartiles) 1.76% (0.86–4.10%) vs 0.06% (0.03–0.81%), p<0.0001). CD3+ T cells showed a similar pattern with significantly higher PAR2 expression in patients with RA compared with controls (3.05% (0.36–11.82%) vs 0.08% (0.02–0.28%), p<0.0001). For both subsets, PAR2 expression was significantly higher (p<0.00001) in patients with high levels of disease activity: PAR2 expression for both CD14+ and CD3+ cells correlated to C reactive protein and erythrocyte sedimentation rate. Furthermore, in a cohort of patients with newly diagnosed RA, elevated PAR2 expression in both CD14+ and CD3+ cells was significantly reduced 3 months after methotrexate or sulfasalazine treatment and this reduction correlated significantly with the reduction in the 28-joint Disease Activity Scale score (p<0.05). PAR2 expression on cells from patients with OA was low, similar to levels seen in control subjects. Generation of IL-6 by monocytes in response to a selective PAR2 agonist was significantly greater in patients with RA than in patients with OA and control subjects (p<0.05). Conclusions These findings are consistent with a pathogenic role for PAR2 in RA.

PAR 2 expression in peripheral blood monocytes of patients with rheumatoid arthritis

Objectives Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteinases with proinflammatory activity. A study was undertaken to investigate the presence and functio©nal significance of PAR2 expression on rheumatoid arthritis (RA)-derived leucocyte subsets. Methods Venous blood was obtained from patients with RA and osteoarthritis (OA) as well as healthy control subjects. Surface expression of PAR2 on peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry and interleukin 6 (IL-6) generation by ELISA. Results Patients with RA had elevated but variable surface expression of PAR2 on CD14+ monocytes compared with control subjects (median (1st to 3rd quartiles) 1.76% (0.86–4.10%) vs 0.06% (0.03–0.81%), p<0.0001). CD3+ T cells showed a similar pattern with significantly higher PAR2 expression in patients with RA compared with controls (3.05% (0.36–11.82%) vs 0.08% (0.02–0.28%), p<0.0001). For both subsets, PAR2 expression was significantly higher (p<0.00001) in patients with high levels of disease activity: PAR2 expression for both CD14+ and CD3+ cells correlated to C reactive protein and erythrocyte sedimentation rate. Furthermore, in a cohort of patients with newly diagnosed RA, elevated PAR2 expression in both CD14+ and CD3+ cells was significantly reduced 3 months after methotrexate or sulfasalazine treatment and this reduction correlated significantly with the reduction in the 28-joint Disease Activity Scale score (p<0.05). PAR2 expression on cells from patients with OA was low, similar to levels seen in control subjects. Generation of IL-6 by monocytes in response to a selective PAR2 agonist was significantly greater in patients with RA than in patients with OA and control subjects (p<0.05). Conclusions These findings are consistent with a pathogenic role for PAR2 in RA.

PAR2expression in peripheral blood monocytes of patients with rheumatoid arthritis

Objectives Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteinases with proinflammatory activity. A study was undertaken to investigate the presence and functio©nal significance of PAR2 expression on rheumatoid arthritis (RA)-derived leucocyte subsets. Methods Venous blood was obtained from patients with RA and osteoarthritis (OA) as well as healthy control subjects. Surface expression of PAR2 on peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry and interleukin 6 (IL-6) generation by ELISA. Results Patients with RA had elevated but variable surface expression of PAR2 on CD14+ monocytes compared with control subjects (median (1st to 3rd quartiles) 1.76% (0.86–4.10%) vs 0.06% (0.03–0.81%), p<0.0001). CD3+ T cells showed a similar pattern with significantly higher PAR2 expression in patients with RA compared with controls (3.05% (0.36–11.82%) vs 0.08% (0.02–0.28%), p<0.0001). For both subsets, PAR2 expression was significantly higher (p<0.00001) in patients with high levels of disease activity: PAR2 expression for both CD14+ and CD3+ cells correlated to C reactive protein and erythrocyte sedimentation rate. Furthermore, in a cohort of patients with newly diagnosed RA, elevated PAR2 expression in both CD14+ and CD3+ cells was significantly reduced 3 months after methotrexate or sulfasalazine treatment and this reduction correlated significantly with the reduction in the 28-joint Disease Activity Scale score (p<0.05). PAR2 expression on cells from patients with OA was low, similar to levels seen in control subjects. Generation of IL-6 by monocytes in response to a selective PAR2 agonist was significantly greater in patients with RA than in patients with OA and control subjects (p<0.05). Conclusions These findings are consistent with a pathogenic role for PAR2 in RA.