Alagarraju Muthukumar

Loss of CD127 expression defines an expansion of effector CD8+ T cells in HIV-infected individuals

The immunodeficiency that follows HIV infection is related to the virus-mediated killing of infected CD4+ T cells, the chronic activation of the immune system, and the impairment of T cell production. In this study we show that in HIV-infected individuals the loss of IL-7R (CD127) expression defines the expansion of a subset of CD8+ T cells, specific for HIV as well as other Ags, that show phenotypic (i.e., loss of CCR7 and CD62 ligand expression with enrichment in activated and/or proliferating cells) as well as functional (i.e., production of IFN-γ, but not IL-2, decreased ex vivo proliferative potential and increased susceptibility to apoptosis) features of effector T cells. Importantly, in HIV-infected individuals the levels of CD8+CD127− T cells are directly correlated with the main markers of disease progression (i.e., plasma viremia and CD4+ T cell depletion) as well as with the indices of overall T cell activation. In all, these results identify the expansion of CD8+CD127− effector-like T cells as a novel feature of the HIV-associated immune perturbation. Further studies are thus warranted to determine whether measurements of CD127 expression on CD8+ T cells may be useful in the clinical management of HIV-infected individuals.

Effects of Calorie Restriction on Polymicrobial Peritonitis Induced by Cecum Ligation and Puncture in Young C57BL/6 Mice

ABSTRACT Calorie restriction (CR) is known to prolong the life span and maintain an active immune function in aged mice, but it is still not known if rodents under CR can respond optimally to bacterial infection. We report here on the influence of CR on the response of peritoneal macrophages to lipopolysaccharide, splenic NF-κB and NF–interleukin-6 (IL-6) activities, and mortality in polymicrobial sepsis induced by cecal ligation and puncture (CLP). Macrophages from 6-month-old C57BL/6 mice on a calorie-restricted diet were less responsive to lipopolysaccharide, as evidenced by lower levels of IL-12 and IL-6 protein and mRNA expression. Furthermore, in vitro lipopolysaccharide-stimulated macrophages from mice under CR also expressed decreased lipopolysaccharide receptor CD14 levels as well as Toll-like receptor 2 (TLR2) and TLR4 mRNA levels. In addition, the phagocytic capacity and class II (I-Ab) expression of macrophages were also found to be significantly lower in mice under CR. Mice under CR died earlier (P < 0.005) after sepsis induced by CLP, which appeared to be a result of increased levels in serum of the proinflammatory cytokines tumor necrosis factor alpha and IL-6 and splenic NF-κB and NF–IL-6 activation 4 h after CLP. However, mice under CR survived significantly (P < 0.005) longer than mice fed ad libitum when injected with paraquat, a free radical-inducing agent. These data suggest that young mice under CR may be protected against oxidative stress but may have delayed maturation of macrophage function and increased susceptibility to bacterial infection.

Calorie restriction decreases proinflammatory cytokines and polymeric Ig receptor expression in the submandibular glands of autoimmune prone (NZB x NZW)F1 mice

Calorie restriction or fish oil (enriched in n-3 fatty acids) supplementation ameliorates glomerulonephritis and Sj¨ogrens syndrome lesions in (NZB × NZW)F1(B/W) mice. Enhanced proinflammatory cytokine expression and deposition of immune complexes are the important pathological events in the development of Sj¨ogrens syndrome. In the present study, we have examined the effect of calorie restriction and fish oil supplementation on the expression of key inflammatory cytokines [gamma interferon (INF-γ), interleukin-10 (IL-10), and IL-12] and polymeric immunoglobulin (Ig) receptor (pIgR) (receptor for IgA and IgM) and the secretion of Ig in the submandibular glands (SMG) of B/W mice. Weanling B/W mice were fed either ad libitum (AL) or calorie restricted (CR) (40% less calories than AL) diet supplemented with 5% corn oil (CO) or 5% fish oil (FO) until 4 or 9 months of age. The SMGs were removed and a portion of the tissue used for semiquantitive determinations of IFN-γ, IL-10, IL-12 (p40), and pIgR mRNA. The remaining SMG tissue was fragmented and cultured for 7 days and the culture supernatants assayed for IgA, IgM, and IgG2a levels by enzyme-linked immunosorbent assay. Results revealed a significant increase in the expression of IFN-γ, IL-10, and IL-12 mRNA with age in AL fed mice, whereas CR fed mice maintained their levels to near those seen in young animals regardless of the dietary fat. PIgR mRNA expression also remained unaltered in CR animals irrespective of age and dietary fat, while it was found significantly increased in AL fed mice. CR significantly inhibited the elevated levels of IgA and IgG2a seen in aged mice. Interestingly, CR also influenced the Ig level in young animals. In summary, these results indicate that amelioration of autoimmune disease by CR in B/W mice is possibly mediated by the lowered mRNA expression of IFN-γ, IL-10, IL-12, and pIgR and the reduced Ig secretion.

Role of glutathione on renal mitochondrial status in hyperoxaluria

Role of glutathione on kidney mitochondrial integrity and function during stone forming process in hyperoxaluric state was investigated in male albino rats of Wistar strain. Hyperoxaluria was induced by feeding ethylene glycol (EG) in drinking water. Glutathione was depleted by administering buthionine sulfoximine (BSO), a specific inhibitor of glutathione biosynthesis. Glutathione monoester (GME) was administered for supplementing glutathione. BSO treatment alone or along with EG, depleted mitochondrial GSH by 40% and 51% respectively. Concomitantly, there was remarkable elevation in lipid peroxidation and oxidation of protein thiols. Mitochondrial oxalate binding was enhanced by 74% and 129% in BSO and BSO + EG treatment. Comparatively, EG treatment produced only a 33% increase in mitochondrial oxalate binding. Significant alteration in calcium homeostasis was seen following BSO and BSO + EG treatment. This may be due to altered mitochondrial integrity and function as evidenced from decreased activities of mitochondrial inner membrane marker enzymes, succinate dehydrogenase and cytochrome-c-oxidase and respiratory control ratio and enhanced NADH oxidation by mitochondria in these two groups. NADH oxidation (r = -0.74) and oxalate deposition in the kidney (r = -0.70) correlated negatively with mitochondrial glutathione depletion. GME supplementation restored normal level of GSH and maintained mitochondrial integrity and function, as a result of which oxalate deposition was prevented despite hyperoxaluria. These results suggest that mitochondrial dysfunction resulting from GSH depletion could be a contributing factor in the development of calcium oxalate stones.

Bone marrow–based homeostatic proliferation of mature T cells in nonhuman primates: implications for AIDS pathogenesis

Bone marrow (BM) is the key hematopoietic organ in mammals and is involved in the homeostatic proliferation of memory CD8(+) T cells. Here we expanded on our previous observation that BM is a preferential site for T-cell proliferation in simian immunodeficiency virus (SIV)-infected sooty mangabeys (SMs) that do not progress to AIDS despite high viremia. We found high levels of mature T-cell proliferation, involving both naive and memory cells, in healthy SMs and rhesus macaques (RMs). In addition, we observed in both species that lineage-specific, BM-based T-cell proliferation follows antibody-mediated in vivo CD4(+) or CD8(+) T-cell depletion, thus indicating a role for the BM in maintaining T-cell homeostasis under depleting circumstances. We also observed that, in SIV-infected SMs, but not RMs, the level of proliferation of BM-based CD4(+) T cells is higher than that of circulating CD4(+) T cells. Interestingly, limited BM-based CD4(+) T-cell proliferation was found in SIV-infected SMs with low CD4(+) T-cell counts, suggesting a regenerative failure in these animals. Collectively, these results indicate that BM is involved in maintaining T-cell homeostasis in primates and suggest a role for BM-based CD4(+) T-cell proliferation in determining the benign nature of natural SIV infection of SMs.

Effect of depletion of reduced glutathione and its supplementation by glutathione monoester on renal oxalate retention in hyperoxaluria

Abstract The effect of glutathione (GSH) depletion followed by administration of glutathione monoester (GME) on the metabolism of oxalate in hyperoxaluric condition was investigated. Renal GSH was depleted by intraperitoneal administration of buthionine sulfoximine (BSO, 4 mmol/kg b.w) twice a day for 20 days to rats with or without hyperoxaluria induced by adding 0.75% ethylene glycol (EG) in drinking water. GME was administered intraperitoneally (5 m mol in water/kg body weight) simultaneously. Tissue GSH was depleted by 47% and 58% by treatment with BSO and BSO + EG, respectively. Oxalate content was enhanced maximally (125% of control) only in BSO + EG treated group. A polarized light microscopic examination showed prominent deposition of calcium oxalate crystals only in the kidney of BSO + EG-treated rats. GME treatment brought down kidney oxalate and calcium content dramatically and reduced calcium oxalate retention. However, GME did not have any effect on urinary oxalate level. The observed decreased creatinine clearance, elevated urinary excretion of lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (γ-GT), and decreased tissue nonenzymatic and enzymatic antioxidants, and thiol status in BSO + EG treated rats were all restored to normal values on GME supplementation. GSH depletion increases the retention of calcium oxalate in renal cells and normalization of GSH by administration of glutathione monoester prevents it.

Food restriction and fish oil suppress atherogenic risk factors in lupus-prone (NZB x NZW) F1 mice

Atherosclerosis-mediated coronary artery disease is a significant cause of mortality in lupus patients. Both an activated immune system and hyperlipidemia are implicated in the pathogenesis of the atherosclerotic lesions of lupus. In this study, the increases in anticardiolipin antibodies, total cholesterol, and LDL cholesterol with age were significantly lowered by fish oil and food restriction, either alone or in combination. Food restriction also significantly decreased the elevation in anti-dsDNA antibody production seen with age in ad libitum groups. Interestingly, effects of food restriction and fish oil on both lipid profile and autoantibody production were seen from a young age. Accumulation of leukocytes in the blood vessels and deposition of IgG in the glomerular mesangium also were suppressed by food restriction. Thus, beneficial effects of fish oil and food restriction on lupus nephritis and survival could be, at least in part, due to their selective effect on atherogenic risk factors.

Calorie restriction increases Fas/Fas-ligand expression and apoptosis in murine splenic lymphocytes

One‐month‐old male ICR mice were fed a nutritionally adequate, semipurified diet, either ad libitum (AL) or calorie restricted (CR) (40% less food) for 6 months and were killed to obtain spleens. Flow cytometric analysis revealed increased proportions of both CD4+ and CD8+ T cells in CR‐fed mice compared to AL‐fed mice. The T cell subsets of CR‐fed mice were also found to have higher levels of plasma membrane Fas receptor expression. Similarly, Fas‐ligand (Fas‐L) expression was higher in anti‐CD3‐stimulated CD4+ and CD8+ T cells. CR‐fed mice also had increased numbers of annexin V‐positive CD4+ and CD8+ T cells in stimulated splenic lymphocytes suggesting an increased potential for apoptosis. Fas and Fas‐L gene expression in splenic lymphocytes, which correlated closely with the observed increased rate of apoptosis, was significantly increased in CR‐fed mice compared to AL‐fed mice. In conclusion, these results indicate that CR increases the expression of Fas and Fas‐L which may contribute to the known beneficial effects of CR such as prolongation of life span by activating chronic physiologically mediated apoptosis.

Renal Injury Mediated Calcium Oxalate Nephrolithiasis: Role of Lipid Peroxidation

The role of lipid peroxidation (LPO) in renal tubular damage mediated calcium oxalate retention was investigated in a rat model. Hyperoxaluria, without deposition of oxalate in kidney, was induced by administration of ethylene glycol (EG), a precursor of oxalate. Oxidative stress condition was produced by administration of buthionine sulfoximine (BSO), an inhibitor of glutathione biosynthesis. BSO-treated rats showed a significant (p < 0.001) increase in LPO over EG-treated rats and it was almost doubled in BSO + EG treated rats. LPO was accompanied by significant urinary excretion of renal damage marker enzymes such as gamma-glutamyl transpeptidase (gamma-GT), alkaline phosphatase (ALP) and cathepsin D, mucoproteins, and glycosaminoglycans (GAGs) in the BSO and BSO + EG groups but not in the EG group. Urinary excretion of gamma-GT (r = +0.90) (p < 0.001) and deposition of oxalate (r = +0.78) (p < 0.001) in kidney positively correlated with LPO. These results suggest that LPO initiates renal damage, thereby leading to calcium oxalate retention and stone formation.

Calorie restriction modulates Th-1 and Th-2 cytokine-induced immunoglobulin secretion in young and old C57BL/6 cultured submandibular glands

Immunoglobulin production by the salivary gland plays an important role in oral and upper respiratory tract immunity. Age and/or disease may compromise salivary gland function. In order to gain insight into the role of calorie restriction (CR) on immunoglobulin (Ig) production, we determined the effect of ad libitum (AL) feeding and CR in young (3 months) and old (18–24 months) C57BL/6 mouse submandibular glands (SM). The SM tissues were fragmented and cultured in the absence (control) or presence of either Th-1 cytokines, such as interleukin-2 (IL-2) and interferon-γ (IFN-γ), or Th-2 cytokines, e.g. IL-4 and IL-5, for seven days. Culture supernatants were then analyzed for immunoglobulin A (IgA), IgM, and IgG2a levels by ELISA. Aging increased basal (control) IgA and IgM production by 3.1- and 3.7-fold, respectively, in AL mice. CR prevented the age-dependent rise of both IgA and IgM, maintaining levels equal to those of young AL mice. Interestingly, age resulted in a decrease of Th-1 cytokine-induced IgA and IgM, and increased IgG2a secretion in AL mice, while Th-2 cytokines did not appear to have an age effect. In general, CR suppressed Ig production induced by both Th-1 and Th-2 cytokines in young mice. In contrast, CR in old mice resulted in enhanced IgA and IgM production to levels similar to those in their young counterparts, while IgG2a was predominantly suppressed by Th-1 and not Th-2 cytokines. The data presented herein show, for the first time, the ability of CR to offset age-induced changes in submandibular gland Ig production, which may play a role in maintaining mucosal immune function, including proper oral health.