Eric S. Sobel

Normalization of CD4+ T cell metabolism reverses lupus

Systemic lupus erythematosus is associated with enhanced CD4+ T cell metabolism and can be reversed by metabolic modulators. Normalizing immune cell metabolism treats lupus Systemic lupus erythematosus (SLE) is an autoimmune disease where the immune system attacks normal, healthy tissues. CD4+ T cells are critical to SLE pathogenesis, but it has remained unclear if metabolism in these cells contributes to disease. Now, Yin et al. report that two metabolic pathways—glycolysis and mitochondrial oxidative metabolism—are elevated in cells from SLE patients as well as in mouse models of disease. What’s more, inhibitors of these pathways currently in the clinic—2-deoxy-d-glucose (2DG) and metformin—normalized T cell metabolism and decreased markers of SLE in animal models as well as in cells from SLE patients. These data suggest that inhibiting both glycolysis and mitochondrial metabolism could be a new therapeutic strategy for treating SLE. Systemic lupus erythematosus (SLE) is an autoimmune disease in which autoreactive CD4+ T cells play an essential role. CD4+ T cells rely on glycolysis for inflammatory effector functions, but recent studies have shown that mitochondrial metabolism supports their chronic activation. How these processes contribute to lupus is unclear. We show that both glycolysis and mitochondrial oxidative metabolism are elevated in CD4+ T cells from lupus-prone B6.Sle1.Sle2.Sle3 (TC) mice as compared to non-autoimmune controls. In vitro, both the mitochondrial metabolism inhibitor metformin and the glucose metabolism inhibitor 2-deoxy-d-glucose (2DG) reduced interferon-γ (IFN-γ) production, although at different stages of activation. Metformin also restored the defective interleukin-2 (IL-2) production by TC CD4+ T cells. In vivo, treatment of TC mice and other lupus models with a combination of metformin and 2DG normalized T cell metabolism and reversed disease biomarkers. Further, CD4+ T cells from SLE patients also exhibited enhanced glycolysis and mitochondrial metabolism that correlated with their activation status, and their excessive IFN-γ production was significantly reduced by metformin in vitro. These results suggest that normalization of T cell metabolism through the dual inhibition of glycolysis and mitochondrial metabolism is a promising therapeutic venue for SLE.

Systemic Overexpression of IL-10 Induces CD4+CD25+ Cell Populations In Vivo and Ameliorates Type 1 Diabetes in Nonobese Diabetic Mice in a Dose-Dependent Fashion

Early systemic treatment of nonobese diabetic mice with high doses of recombinant adeno-associated virus (rAAV) vector expressing murine IL-10 prevents type 1 diabetes. To determine the therapeutic parameters and immunological mechanisms underlying this observation, female nonobese diabetic mice at 4, 8, and 12 wk of age were given a single i.m. injection of rAAV-murine IL-10 (104, 106, 108, and 109 infectious units (IU)), rAAV-vector expressing truncated murine IL-10 fragment (109 IU), or saline. Transduction with rAAV-IL-10 at 109 IU completely prevented diabetes in all animals injected at all time points, including, surprisingly, 12-wk-old animals. Treatment with 108 IU provided no protection in the 12-wk-old injected mice, partial prevention in 8-wk-old mice, and full protection in all animals injected at 4 wk of age. All other treatment groups developed diabetes at a similar rate. The rAAV-IL-10 therapy attenuated pancreatic insulitis, decreased MHC II expression on CD11b+ cells, increased the population of CD11b+ cells, and modulated insulin autoantibody production. Interestingly, rAAV-IL-10 therapy dramatically increased the percentage of CD4+CD25+ regulatory T cells. Adoptive transfer studies suggest that rAAV-IL-10 treatment alters the capacity of splenocytes to impart type 1 diabetes in recipient animals. This study indicates the potential for immunomodulatory gene therapy to prevent autoimmune diseases, including type 1 diabetes, and implicates IL-10 as a molecule capable of increasing the percentages of regulatory cells in vivo.

Type I Interferon Modulates Monocyte Recruitment and Maturation in Chronic Inflammation

Chronic inflammation is characterized by continuous recruitment and activation of immune cells such as monocytes in response to a persistent stimulus. Production of proinflammatory mediators by monocytes leads to tissue damage and perpetuates the inflammatory response. However, the mechanism(s) responsible for the sustained influx of monocytes in chronic inflammation are not well defined. In chronic peritonitis induced by pristane, the persistent recruitment of Ly6C(hi) inflammatory monocytes into the peritoneum was abolished in type I interferon (IFN-I) receptor-deficient mice but was unaffected by the absence of IFN-gamma, tumor necrosis factor-alpha, interleukin-6, or interleukin-1. IFN-I signaling stimulated the production of chemokines (CCL2, CCL7, and CCL12) that recruited Ly6C(hi) monocytes via interactions with the chemokine receptor CCR2. Interestingly, after 2,6,10,14-tetramethylpentadecane treatment, the rapid turnover of inflammatory monocytes in the inflamed peritoneum was associated with a lack of differentiation into Ly6C(lo) monocytes/macrophages, a more mature subset with enhanced phagocytic capacity. In contrast, Ly6C(hi) monocytes differentiated normally into Ly6C(lo) cells in IFN-I receptor-deficient mice. The effects of IFN-I were specific for monocytes as granulocyte migration was unaffected in the absence of IFN-I signaling. Taken together, our findings reveal a novel role of IFN-I in promoting the recruitment of inflammatory monocytes via the chemokine receptor CCR2. Continuous monocyte recruitment and the lack of terminal differentiation induced by IFN-I may help sustain the chronic inflammatory response.

High sensitivity C-reactive protein in systemic lupus erythematosus: relation to disease activity, clinical presentation and implications for cardiovascular risk

Measurement of high sensitivity C-reactive protein (hs-CRP), has been used in the assessment of disease activity in numerous rheumatic conditions including systemic lupus erythematosus (SLE). However, the utility of hs-CRP measurement in patients with lupus is uncertain. This study examined if hs-CRP can be used to assess disease activity, severity and cardiovascular risk in SLE. Serum samples from 601 visits of 213 SLE patients and 134 controls were analysed for hs-CRP by nephelometry. Detailed demographic data were obtained from all subjects and medication history and key laboratory parameters were collected. Disease activity was assessed using the SLEDAI. High sensitivity CRP was not associated with disease activity (SLEDAI), number of ACR SLE criteria or presence of any particular organ involvement. hs-CRP levels were significantly correlated with standard cardiovascular risk factors including body weight (P = 0.0002), hypertension (P = 0.001), and apolipoprotein A-I (P < 0.0001). Interestingly an inverse correlation was seen between hs-CRP levels and antimalarial use (P = 0.0018). Our results suggest that measurement of hs-CRP, though not valuable as marker of disease activity in SLE may be of some use in the assessment of cardiovascular risk. We speculate that antimalarials may help to reduce cardiovascular risk in patients with SLE.

The Major Murine Systemic Lupus Erythematosus Susceptibility Locus Sle1 Results in Abnormal Functions of Both B and T Cells

Sle1 is a major susceptibility locus in the NZM2410 murine model of systemic lupus erythematosus. When isolated on a C57BL/6 background in the B6.Sle1 congenic strain, Sle1 results in the production of high levels of anti-chromatin IgG Abs, histone-specific T cells, and increased B and T cell activation. We have shown by mixed bone marrow chimeras with allotypic markers that Sle1 is expressed in B cells. Using the same technique, we now show that it is also expressed in T cells. To assess whether Sle1 results in intrinsic defects in B or T cells, we have bred the μMT and Tcrα−/− mutations onto B6.Sle1 resulting in the absence of circulating B cells and αβ T cells in B6.Sle1.μMT and B6.Sle1.Tcrα−/−, respectively. The immune phenotypes in these two strains were compared with that of B6.Sle1 and B6.μMT or B6.Tcrα−/−. Sle1-expressing B cells broke tolerance to chromatin in the absence of T cells, as shown by high levels of anti-ssDNA IgM Abs in B6.Sle1.Tcrα−/− mice, and had an increased expression of activation markers. Conversely, increased expression of activation markers and increased cytokine production were observed in Sle1-expressing T cells in the absence of B cells in B6.Sle1.μMT mice. However, the production of IgG antinuclear Abs required the presence of both T and B cells. These experiments showed that Sle1 expression results in both B and T cells intrinsic defects and demonstrate that the documented involvement of each cell compartment in the production of anti-chromatin Abs corresponds to genetic defects rather than bystander effects.

Acceleration of Autoimmunity by Organochlorine Pesticides in (NZB × NZW)F1 Mice

Systemic lupus erythematosus (SLE) is an autoimmune disorder that affects women more frequently than men. In the (NZB × NZW)F1 mouse, a murine SLE model, the presence or absence of estrogen markedly influences the rate of progression of disease. Three organochlorine pesticides with estrogenic effects were administered chronically to ovariectomized female (NZB × NZW)F1 mice, and we measured the time to development of renal disease, the principal clinical manifestation of lupus in this model. Treatment with chlordecone, methoxychlor, or o,p′-dichlorodiphenyl-trichloroethane (o,p′-DDT) significantly decreased the time to onset of renal impairment, as did treatment with 17β-estradiol used as a positive control. In an expanded study of chlordecone, we found a dose-related early appearance of elevated anti–double-strand DNA autoantibody titers that corresponded with subsequent development of glomerulonephritis. Immunohistofluorescence confirmed early deposition of immune complexes in kidneys of mice treated with chlordecone. These observations are consistent with an effect of these organochlorine pesticides to accelerate the natural course of SLE in the (NZB × NZW)F1 mouse. Although we originally hypothesized that the effect on progression of autoimmunity was due to estrogenic properties of the pesticides, autoimmune effects and estrogenicity, assessed through measurement of uterine hypertrophy, were not well correlated. This may indicate that uterine hypertrophy is a poor indicator of comparative estrogenic effects of organochlorine pesticides on the immune system, or that the pesticides are influencing autoimmunity through a mode of action unrelated to their estrogenicity.

Genetic Dissection of Systemic Lupus Erythematosus Pathogenesis: Evidence for Functional Expression of Sle3/5 by Non-T Cells

On the non-autoimmune C57BL/6 (B6) background, the chromosome 7-derived lupus susceptibility loci Sle3 and Sle5 have been shown to mediate an elevated CD4:CD8 ratio with an increase in activated CD4+ T cells, decreased susceptibility to apoptosis, and a break in humoral tolerance. Development of subcongenic strains has subsequently shown that the elevated CD4:CD8 ratio is due to Sle3 but that both loci contribute to the development of autoantibodies. To elucidate the functional expression patterns of these loci, adoptive transfer experiments were conducted. All possible combinations of bone marrow reconstitution, including syngenic, were conducted between the congenic B6 and B6.Sle3/5 strains. It was found that the Sle3/5 locus was functionally expressed by bone marrow-derived cells, but not by host cells, and that the elevated CD4:CD8 phenotype could be reconstituted in radiation chimeras. Using Ly5-marked congenic strains and B6 host mice, additional experiments surprisingly demonstrated that the elevated CD4:CD8 ratio was neither an intrinsic property of the T cells nor of single positive thymocytes. Allotype-marked chimeras indicated that autoantibody production by B cells was also an extrinsic property, as shown by the fact that B cells without the Sle3/5 interval contributed to autoantibody production. These experiments strongly suggest that a gene within the B6.Sle3/5 interval was expressed by a bone marrow-derived, nonlymphocyte population in the thymus and periphery and was affecting T cell selection and/or survival.

Patients With Pulmonary Tuberculosis Are Frequently Positive for Anti–Cyclic Citrullinated Peptide Antibodies, but Their Sera Also React With Unmodified Arginine-Containing Peptide

OBJECTIVE The anti-cyclic citrullinated peptide (anti-CCP) enzyme-linked immunosorbent assay (ELISA) has high sensitivity and specificity for rheumatoid arthritis (RA). However, detection of anti-CCP in patients with active pulmonary tuberculosis (TB) has recently been reported. To determine whether this activity was specific for the citrullinated residue, the specificity of anti-CCP-positive sera for CCP versus that for unmodified arginine-containing peptide (CAP) was examined in patients with TB and compared with that in patients with RA. METHODS Anti-CCP and anti-CAP in sera from patients with pulmonary TB (n = 49), RA patients (n = 36), and controls (n = 18) were tested by ELISA. Sera were available at diagnosis from most TB patients. All TB patients were treated with a combination of 2-4 antibiotics for at least 6 months, and sera were collected over time. RESULTS Anti-CCP was found in 37% of TB patients and in 43% of RA patients. CAP reactivity was more common in TB than in RA. High anti-CCP:anti-CAP ratios (>2.0) were seen far more commonly in anti-CCP-positive RA patients than in anti-CCP-positive TB patients (94% versus 22%). Anti-CCP was inhibited by CCP peptide in sera from RA patients, but not in sera from TB patients. A slight increase in anti-CCP was common after initiating treatment for TB, although the anti-CCP level decreased after 1-2 months. CONCLUSION Anti-CCP is frequently present in patients with active TB. However, many anti-CCP-positive TB sera also reacted with CAP, and anti-CCP:anti-CAP ratios in TB sera were low. Anti-CCP:anti-CAP ratios should be useful clinically for distinguishing CCP-specific reactivity seen in RA from reactivity with both CCP and CAP frequently seen in pulmonary TB.

Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus

IntroductionMore than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy.MethodsMonocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR.ResultsCirculating CD14+ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy.ConclusionsExpression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients.

Citrulline Dependence of Anti-Cyclic Citrullinated Peptide Antibodies in Systemic Lupus Erythematosus as a Marker of Deforming/Erosive Arthritis

Objective. Anti-cyclic citrullinated peptide (CCP) antibodies are a serological marker for rheumatoid arthritis (RA); up to 10%–15% of patients with systemic lupus erythematosus (SLE) are also positive. While anti-CCP in RA is citrulline-dependent, anti-CCP in some other diseases is citrulline-independent and reacts with both CCP and the unmodified (arginine-containing) cyclic arginine peptide (CAP). We investigated the citrulline dependence of anti-CCP and its significance in the arthritis of SLE. Methods. IgG anti-CCP was compared by ELISA to anti-CAP in sera from patients with SLE (n = 335) and RA (n = 47) and healthy controls (n = 35). SLE patients were divided into 5 groups based on their joint involvement: subset I: deforming/erosive arthritis (n = 20); II: arthritis fulfilling (or likely fulfilling) American College of Rheumatology criteria for RA but without erosions (n = 18); III: joint swelling but not fulfilling RA criteria (n = 39); IV: arthritis without documented joint swelling (n = 194); and V: no arthritis (n = 58). Results. Anti-CCP (> 1.7 units) was found in 68% (32/47) of patients with RA and 17% (55/329) of those with SLE. It was more common in SLE patients with deforming/erosive arthritis (38%). High anti-CCP (> 10 units) was found in RA (26%) and deforming/erosive SLE (12%). High anti-CCP/CAP ratios (> 2, indicating a selectivity to CCP) were found in 91% of anti-CCP-positive RA and 50% of anti-CCP-positive SLE patients with deforming/erosive arthritis. Patients from subset II did not have high anti-CCP/CAP. Conclusion. Citrulline dependence or high levels (> 10) of anti-CCP were common in SLE patients with deforming/erosive arthritis, while most anti-CCP in SLE patients was citrulline-independent. This may be useful in identifying a subset of SLE patients with high risk for development of deforming/erosive arthritis.