Down-Regulated NOD2 by Immunosuppressants in Peripheral Blood Cells in Patients with SLE Reduces the Muramyl Dipeptide-Induced IL-10 Production

Pattern recognition receptors (PRRs) such as Toll-like receptors are aberrantly expressed of peripheral blood mononuclear cells (PBMCs) in systemic lupus erythematosus (SLE) patients, for playing immunopathological roles. basal productions of cytokines (IL-6, IL-8 and IL-10) were significantly increased in immunosuppressant naïve patients and patients with active disease despite immunosuppressants compared with HCs. Upon MDP stimulaiton, relative induction (%) of cytokines (IL-1β) from PBMC was significantly increased in immunosuppressant naïve patients with inactive disease, and patients with active disease despite immunosuppressant treatment compared with HCs. Immunosuppressant usage was associated with a decreased basal production and MDP induced relative induction (%) of IL-10 in patients with inactive disease compared with immunosuppressant naïve patients and HCs.Bacterial exposure may increase the NOD2 expression in monocytes in immunosuppressant naïve SLE patients which can subsequently lead to aberrant activation of PBMCs to produce proinflammatory cytokines, implicating the innate immune response for extracellular pathogens in the immunopathological mechanisms in SLE. Immunosuppressant therapy may downregulate NOD2 expression in CD8+ T lymphocytes, monocytes, and DCs in SLE patients which subsequently IL-10 reduction, contributing towards the regulation of immunopathological mechanisms of SLE, at the expense of increasing risk of bacterial infection

<i>Ex vivo</i> basal production and relative induction of cytokines from NOD2 ligand activated PBMC in various groups.

<p>Culture supernatant was obtained from PBMCs cultured with medium or NOD2 ligand (Muramyl dipeptide, MDP) for 24 hours. The basal production (pg/ml) and relative induction (%) of cytokines (IL-1β, TNF-α, IL-6, IL-8 and IL-10) by PBMCs were analyzed by flow cytometry. Numerical data are expressed as median (interquartile range, IQR). Group 1) Patients with inactive disease (SLEDAI <4) who were never treated with immunosuppressants since the diagnosis or within the past 10 years, whichever longer; Group 2) Patients with inactive disease (SLEDAI <4) who had received or are currently on immunosuppressants; Group 3) Patients with active disease (SLEDAI >4) who had received or are currently on immunosuppressants.</p>&<p><i>p</i><0.05, comparing between Group 1 and Group 2;</p>Δ<p><i>p</i><0.05,</p>ΔΔΔ<p><i>p</i><0.001, comparing between Group 1 with healthy control subjects;</p>Φ<p><i>p</i><0.05,</p>ΦΦ<p><i>p</i><0.01, comparing between Group 2 with healthy control subjects;</p>§<p><i>p</i><0.05,</p>§§<p><i>p</i><0.01 comparing between Group 3 with healthy control subjects.</p

Demographic and clinical characteristics of patients with systemic lupus erythematosus.

<p>Values are median (interquartile range, IQR) unless stated otherwise; s.d., standard deviation; n.a., not applicable. SLE, systemic lupus erythematosus; Group 1) Patients with inactive disease (SLEDAI <4) who were never treated with immunosuppressants since the diagnosis or within the past 10 years, whichever longer; Group 2) Patients with inactive disease (SLEDAI <4) who had received or are currently on immunosuppressants; Group 3) Patients with active disease (SLEDAI >4) who had received or are currently on immunosuppressants. SLEDAI, systemic lupus erythematosus disease activity index; SLICC, Systemic Lupus International Collaborating Clinics Score;</p>┼<p>“Never” refers to SLE patients never be given treatment of immunosuppressants [prednisolone, hydroxychloroquine, azathioprine, cyclophosphamide (oral or IV), cyclosporin A and mycophenolate mofetil] since the diagnosis of SLE or within recent 10 years;</p>┼┼<p>“Ever” refer to use of immunosuppressants since the diagnosis of SLE.</p>┼┼┼<p>“<i>Flare</i>” is defined as increase in the SLEDAI score by 3 or more;</p>&<p><i>p</i><0.05,</p>&&<p><i>p</i><0.01,</p>&&&<p><i>p</i><0.001, comparing between Group 1 and Group 2;</p><p>*<i>p</i><0.05,</p><p>**<i>p</i><0.01,</p><p>***<i>p</i><0.001, comparing between Group 1 and Group 3;</p>#<p><i>p</i><0.05,</p>##<p><i>p</i><0.01,</p>###<p>p<0.001, comparing between Group 2 and Group 3.</p

Univariate analysis: the relationship between clinical characteristics, the use of drugs ever and the expression of NOD2 in SLE patients (categorical variables).

┼<p>“<i>Flare</i>” is defined as increase in the SLEDAI score by 3 or more;</p>┼┼<p>“Ever” refers to use of immunosuppressants [prednisolone, hydroxychloroquine, azathioprine, cyclophosphamide (oral or IV), cyclosporin A and mycophenolate mofetil] since the diagnosis of SLE;</p><p>*<i>p</i><0.05.</p

Expression of intracellular NOD2 in CD4⁺ T, CD8⁺ T, CD19⁺ B lymphocytes, monocytes, myeloid dendritic cells and plasmacytoid dendritic cells using flow cytometry.

<p>Group 1) Patients with inactive disease (SLEDAI <4) who were never treated with immunosuppressants since the diagnosis or within the past 10 years, whichever longer; Group 2) Patients with inactive disease (SLEDAI <4) who had received or are currently on immunosuppressants; Group 3) Patients with active disease (SLEDAI >4) who had received or are currently on immunosuppressants. The differential protein expression of intracellular pathogen recognition receptor NOD2 in (A) CD4+ T lymphocytes, (B) CD8+ T lymphocytes, (C) CD19+ B lymphocytes, (D) Monocytes, (E) myeloid dendritic cells (F) plasmacytoid dendritic cells of SLE patients and healthy controls by flow cytometry were shown as median (IQR) of mean fluorescence intensity (MFI) subtracting corresponding isotypic controls in scatter plots.</p