Nalini Agrawal

Increased Ribonuclease Expression Reduces Inflammation and Prolongs Survival in TLR7 Transgenic Mice

TLR7 activation is implicated in the pathogenesis of systemic lupus erythematosus. Mice that overexpress TLR7 develop a lupus-like disease with autoantibodies and glomerulonephritis and early death. To determine whether degradation of the TLR7 ligand RNA would alter the course of disease, we created RNase A transgenic (Tg) mice. We then crossed the RNase Tg to TLR7 Tg mice to create TLR7 × RNase double Tg (DTg) mice. DTg mice had a significantly increased survival associated with reduced activation of T and B lymphocytes and reduced kidney deposition of IgG and C3. We observed massive hepatic inflammation and cell death in TLR7 Tg mice. In contrast, hepatic inflammation and necrosis were strikingly reduced in DTg mice. These findings indicate that high concentrations of serum RNase protect against immune activation and inflammation associated with TLR7 stimulation and that RNase may be a useful therapeutic strategy in the prevention or treatment of inflammation in systemic lupus erythematosus and, possibly, liver diseases.

Mice Lacking Hepatic Lipase Are Lean and Protected against Diet-Induced Obesity and Hepatic Steatosis

Hepatic lipase (HL)-mediated lipoprotein hydrolysis provides free fatty acids for energy, storage, and nutrient signaling and may play a role in energy homeostasis. Because HL-activity increases with increased visceral fat, we hypothesized that increased HL-activity favors weight gain and obesity and consequently, that HL deficiency would reduce body fat stores and protect against diet-induced obesity. To test this hypothesis, we compared wild-type mice (with endogenous HL) and mice genetically deficient in HL with respect to daily body weight and food intake, body composition, and adipocyte size on both chow and high-fat (HF) diets. Key determinants of energy expenditure, including rate of oxygen consumption, heat production, and locomotor activity, were measured by indirect calorimetry. HL-deficient mice exhibited reduced weight gain on both diets (by 32%, chow; by 50%, HF; both P < 0.0001, n = 6-7 per genotype), effects that were associated with reduced average daily food intake (by 22-30% on both diets, P < 0.0001) and a modest increase in the rate of oxygen consumption (by 25%, P < 0.003) during the light cycle. Moreover, in mice fed the HF diet, HL deficiency reduced both body fat (by 30%, P < 0.0001) and adipocyte size (by 53%, P < 0.01) and fully prevented the development of hepatic steatosis. Also, HL deficiency reduced adipose tissue macrophage content, consistent with reduced inflammation and a lean phenotype. Our results demonstrate that in mice, HL deficiency protects against diet-induced obesity and its hepatic sequelae. Inhibition of HL-activity may therefore have value in the prevention and/or treatment of obesity.

Divergent Effects of the Catalytic and Bridging Functions of Hepatic Lipase on Atherosclerosis

Objective—Increased expression of human hepatic lipase (HL) or a catalytically inactive (ci) HL clears plasma cholesterol in mice deficient in low-density lipoprotein receptors (LDLr) and murine HL. We hypothesized that increased expression of both HL and ciHL reduces atherosclerosis in these mice. Methods and Results—Mice deficient in both LDLr and murine HL, alone or transgenically expressing similar levels of either human HL or ciHL, were fed a high-fat, cholesterol-enriched “Western” diet for 3 months to accelerate the development of atherosclerosis. Levels of plasma lipids, insulin, glucose, and liver enzymes were measured monthly, and aortic atherosclerosis was quantitated after 3 months. Plasma insulin, glucose, and liver enzyme levels did not differ significantly from controls. After 3 months, expression of HL reduced plasma cholesterol by 55% to 65% and reduced atherosclerosis by 40%. Surprisingly, expression of ciHL did not reduce plasma cholesterol or atherosclerosis. Conclusions—High levels of HL, but not ciHL, delay the development of atherosclerosis in mice deficient in LDLr and mHL.

Altered IgG autoantibody levels and CD4+ T cell subsets in lupus-prone Nba2 mice lacking the nuclear progesterone receptor

Abstract Important interactions between female reproduction and autoimmunity are suggested by the female-predominance of systemic lupus erythematosus (SLE) and other autoimmune diseases and the amelioration of certain autoimmune diseases during pregnancy. Sexually dimorphic risk of developing SLE involves modulation of genetic risk by environmental factors, sex hormones and non-hormonal factors encoded on the sex chromosomes. In some lupus models, estrogen, via estrogen receptor alpha (ER-α), enhances production of highly pathogenic IgG2a/c autoantibodies (autoAbs). Some studies indicate that treatment with progesterone, a chief female reproductive steroid, can suppress IgG2a/2c autoAb production. Little is known about how endogenous progesterone impacts lupus autoimmunity. To investigate this, we introduced a disruptive progesterone receptor (PR) gene mutation into lupus-prone mice and tracked the development of spontaneous IgG autoAbs. Here, we present evidence that PR can suppress the emergence of class-switched IgG2c autoAbs, suggesting that PR and ER-α counter-regulate a critical step in lupus autoimmunity. PRs control of IgG2c autoAb production correlates with alterations in the relative abundance of splenic T follicular helper (TFH) cells and non-TFH CD4+ T cells, especially regulatory T cells (TREGS). Surprisingly, PR also appears to help to maintain sexually dimorphic abundance of splenic leukocytes, a feature common to many mouse models of SLE. Together our results identify a novel molecular link between female reproduction and lupus autoimmunity. Further investigation into the immunomodulatory functions of PR promises to inform reproductive health care in women and offers mechanistic insight into important immunologic phenomena of pregnancy.

In vivo structure-function studies of human hepatic lipase: the catalytic function rescues the lean phenotype of HL-deficient (hl / ) mice

The lean body weight phenotype of hepatic lipase (HL)–deficient mice (hl−/−) suggests that HL is required for normal weight gain, but the underlying mechanisms are unknown. HL plays a unique role in lipoprotein metabolism performing bridging as well as catalytic functions, either of which could participate in energy homeostasis. To determine if both the catalytic and bridging functions or the catalytic function alone are required for the effect of HL on body weight, we studied (hl−/−) mice that transgenically express physiologic levels of human (h)HL (with catalytic and bridging functions) or a catalytically‐inactive (ci)HL variant (with bridging function only) in which the catalytic Serine 145 was mutated to Alanine. As expected, HL activity in postheparin plasma was restored to physiologic levels only in hHL‐transgenic mice (hl−/−hHL). During high‐fat diet feeding, hHL‐transgenic mice exhibited increased body weight gain and body adiposity relative to hl−/−ciHL mice. A similar, albeit less robust effect was observed in female hHL‐transgenic relative to hl−/−ciHL mice. To delineate the basis for this effect, we determined cumulative food intake and measured energy expenditure using calorimetry. Interestingly, in both genders, food intake was 5–10% higher in hl−/−hHL mice relative to hl−/−ciHL controls. Similarly, energy expenditure was ~10% lower in HL‐transgenic mice after adjusting for differences in total body weight. Our results demonstrate that (1) the catalytic function of HL is required to rescue the lean body weight phenotype of hl−/− mice; (2) this effect involves complementary changes in both sides of the energy balance equation; and (3) the bridging function alone is insufficient to rescue the lean phenotype of hl−/−ciHL mice.

Genetic and functional investigation of a Mendelian form of systemic lupus erythematosus

Abstract We have shown that bi-allelic mutations in ACP5 result in a deficiency of the encoded protein, tartrate-resistant acid phosphatase (TRAP), which causes the immuno-osseous disease spondyloenchondrodysplasia. In addition to having a bone and neurological phenotype, patients with spondyloenchondrodysplasia exhibit a wide spectrum of autoimmune manifestations, including autoantibody production, systemic lupus erythematosus, increased interferon α (IFNα), and an interferon signature. In bone, TRAP produced by osteoclasts regulates cell migration by dephosphorylation of osteopontin. However, TRAP is also produced by immune cells, particularly macrophages and dendritic cells (DCs). In murine plasmacytoid dendritic cells (pDC), phosphorylated osteopontin is integral to IFN-α production. Whereas osteopontin has been shown to be a TRAP substrate in murine bone cells, nothing is known about whether TRAP regulates osteopontin in immune cells. We hypothesise that TRAP dephosphorylates osteopontin in human pDCs and negatively regulates IFN-α production. We studied the interaction between TRAP and osteopontin. By confocal microscopy, we showed that TRAP co-localised with osteopontin in the Golgi in primary human macrophages, DCs, and pDCs. In a TRAP and osteopontin overexpression system in human embryonic kidney 293 cells, we co-immunoprecipitated TRAP and osteopontin, suggesting that they interact with each other. We also showed in an in-vitro assay that recombinant human TRAP could dephosphorylate osteopontin, demonstrating that osteoponin is a substrate for TRAP. These findings suggest that osteopontin is a target for TRAP in immune cells. To test whether TRAP deficiency leads to hyperphosphorylation of osteopontin and increased IFNα, we are generating TRAP knockdowns in a pDC cell line. Understanding of the mechanism by which TRAP regulates IFNα, potentially via osteopontin, may eventually allow for directed therapeutic approaches in spondyloenchondrodysplasia and other autoimmune diseases associated with an interferon signature, particularly systemic lupus erythematosus. Funding Wellcome Trust.