Lyn M. Hillyer

Elevated Bioactivity of the Tolerogenic Cytokines, Interleukin-10 and Transforming Growth Factor-β, in the Blood of Acutely Malnourished Weanling Mice

The main objective of this investigation was to determine the influence of acute deficits of protein and energy on the blood levels of interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), physiologically the main anti-inflammatory and tolerogenic cytokines. In four 14-day experiments, male and female C57BL/6J mice, initially 19 days old, consumed a complete purified diet either ad libitum or in restricted daily quantities, or had free access to an isocaloric purified low-protein diet. A zero-time control group (19 days old) was included. In the first two experiments, serum IL-10 levels were assessed by sandwich enzyme-linked immunosorbent assay (ELISA) and bioassay. The mean serum IL-10 bioactivities were higher (P ≤ 0.05) in both malnourished groups (low-protein and restricted intake: 15.8 and 12.2 ng/ml, respectively) than in the zero-time and age-matched control groups (6.3 and 7.3 ng/ml, respectively), whereas serum IL-10 immunoactivity was high only in the restricted intake group (e.g., second experiment: 17.0 pg/ml vs. 5.4, 3.7, and 3.1 pg/ml in the zero-time control, age-matched control and low-protein group, respectively). The third and fourth experiments centered on plasma TGF-β immunoactivity (sandwich ELISA) and bioactivity, respectively. The ELISA revealed a high mean plasma TGF-β1 level (P < 0.05) in the low-protein group only, but TGF-β bioactivity (β1 isoform, although 15% β2 in the restricted intake group) was high in both malnourished groups (8.7 and 9.3 ng/ml in the low-protein and restricted groups, respectively) relative to the age-matched control group (0.5 ng/ml). Thus, metabolically distinct weanling systems mimicking marasmus and incipient kwashiorkor both exhibit a blood cytokine profile that points to a tolerogenic microenvironment within immune response compartments. A model emerges in which malnutrition-associated immune competence, at least in advanced weight loss, centers on cytokine-mediated peripheral tolerance that reduces the risk of catabolically induced autoimmune disease, but this is at the cost of attenuated responsiveness to infectious agents.

Lifelong exposure to n-3 PUFA affects pubertal mammary gland development.

There is growing evidence that early developmental periods may importantly influence future breast cancer risk. Also, there is great interest in the role of dietary fat in breast cancer risk, but the role of dietary fat during pubertal mammary gland development remains poorly understood. This study investigated the effect of n-3 polyunsaturated fatty acids (PUFA) using complementary dietary and genetic approaches to examine the effect of lifelong exposure of n-3 PUFA or n-6 PUFA (control) on mammary gland development and fatty acid composition. n-3 PUFA from both diet and genetics were enriched in mammary glands as early as 3 weeks of age. Parameters related to mammary gland development, including number of terminal end buds (TEB), percent coverage of ductal tree, and infiltration of TEB, were influenced by n-3 PUFA at 3 and 4 weeks of age. Overall, findings suggest that n-3 PUFA incorporation into the mammary gland early in life plays a role in the morphological development of the mammary gland during puberty.

The delta 6 desaturase knock out mouse reveals that immunomodulatory effects of essential n-6 and n-3 polyunsaturated fatty acids are both independent of and dependent upon conversion

Typically fatty acids (FA) exert differential immunomodulatory effects with n-3 [α-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)] and n-6 [linoleic acid (LA) and arachidonic acid (AA)] exerting anti- and pro-inflammatory effects, respectively. This over-simplified interpretation is confounded by a failure to account for conversion of the parent FA (LA and ALA) to longer-chain bioactive products (AA and EPA/DHA, respectively), thereby precluding discernment of the immunomodulatory potential of specific FA. Therefore, we utilized the Δ6-desaturase model, wherein knockout mice (D6KO) lack the Fads2 gene encoding for the rate-limiting enzyme that initiates FA metabolism, thereby providing a model to determine specific FA immunomodulatory effects. Wild-type (WT) and D6KO mice were fed one of four isocaloric diets differing in FA source (9weeks): corn oil (LA-enriched), arachidonic acid single cell oil (AA-enriched), flaxseed oil (ALA-enriched) or menhaden fish oil (EPA/DHA-enriched). Splenic mononuclear cell cytokine production in response to lipopolysaccharide (LPS), T-cell receptor (TCR) and anti-CD40 stimulation was determined. Following LPS stimulation, AA was more bioactive compared to LA, by increasing inflammatory cytokine production of IL-6 (1.2-fold) and TNFα (1.3-fold). Further, LPS-stimulated IFNγ production in LA-fed D6KO mice was reduced 5-fold compared to LA-fed WT mice, indicating that conversion of LA to AA was necessary for cytokine production. Conversely, ALA exerted an independent immunomodulatory effect from EPA/DHA and all n-3 FA increased LPS-stimulated IL-10 production versus LA and AA. These data definitively identify specific immunomodulatory effects of individual FA and challenge the simplified view of the immunomodulatory effects of n-3 and n-6 FA.

Vaccenic acid in serum triglycerides is associated with markers of insulin resistance in men.

Serum triglyceride levels are associated with metabolic disorders; however, it remains unclear whether the fatty acid (FA) composition of triglycerides is also changed. Although there were no differences in circulating triglyceride levels between normoglycaemic-normoinsulinaemic and hyperglycaemic-hyperinsulinaemic men, inspection of individual FA revealed that vaccenic acid was enriched with hyperglycaemia-hyperinsulinaemia. Moreover, vaccenic acid levels were positively correlated with insulin and HOMA-IR. This reinforces that examination of individual FA in the context of insulin resistance is warranted.

Splenic dendritic cell populations of the weanling mouse involute proportionately with total nucleated cell numbers throughout acute protein and energy deficiencies except in the most advanced stages of nitrogen-to-energy imbalance

Abstract Male and female C57BL/6J mice, initially 19 d old, had free access to a complete purified diet, were fed this diet in restricted daily quantities to produce energy deficit, or had free access to a low-protein diet to produce combined protein and energy deficit. Two experiments were conducted, each with feeding trials of 3, 9, and 14 d. Respectively, the two experiments addressed the proportion of nucleated splenocytes represented by the full population of dendritic cells (CD11c + F4/80 -/low ) and by the DEC-205 + dendritic cell subset. These indices were affected only by acute nitrogen-to-energy deficit (at the most advanced stage examined) in which the proportion of nucleated spleen cells exhibiting CD11c + F4/80 -/low and DEC-205 + phenotypes decreased and increased, respectively. In this form and stage of acute malnutrition, primary thymus-dependent responses in the spleen may be limited both by dendritic cell numbers and by an overabundance of tolerogenic DEC-205 + dendritic cells.

Constitutive, but Not Challenge-Induced, Interleukin-10 Production Is Robust in Acute Pre-Pubescent Protein and Energy Deficits: New Support for the Tolerance Hypothesis of Malnutrition-Associated Immune Depression Based on Cytokine Production in vivo

The tolerance model of acute (i.e., wasting) pre-pubescent protein and energy deficits proposes that the immune depression characteristic of these pathologies reflects an intact anti-inflammatory form of immune competence that reduces the risk of autoimmune reactions to catabolically released self antigens. A cornerstone of this proposition is the finding that constitutive (first-tier) interleukin(IL)-10 production is sustained even into the advanced stages of acute malnutrition. The IL-10 response to inflammatory challenge constitutes a second tier of anti-inflammatory regulation and was the focus of this investigation. Weanling mice consumed a complete diet ad libitum, a low-protein diet ad libitum (mimicking incipient kwashiorkor), or the complete diet in restricted daily quantities (mimicking marasmus), and their second-tier IL-10 production was determined both in vitro and in vivo using lipopolysaccharide (LPS) and anti-CD3 as stimulants of innate and adaptive defences, respectively. Both early (3 days) and advanced (14 days) stages of wasting pathology were examined and three main outcomes emerged. First, classic in vitro systems are unreliable for discerning cytokine production in vivo. Secondly, in diverse forms of acute malnutrition declining challenge-induced IL-10 production may provide an early sign that anti-inflammatory control over immune competence is failing. Thirdly, and most fundamentally, the investigation provides new support for the tolerance model of malnutrition-associated inflammatory immune depression.

Adoptively Transferred Dendritic Cells Restore Primary Cell-Mediated Inflammatory Competence to Acutely Malnourished Weanling Mice

Immune depression associated with prepubescent malnutrition underlies a staggering burden of infection-related morbidity. This investigation centered on dendritic cells as potentially decisive in this phenomenon. C57BL/6J mice, initially 19 days old, had free access for 14 days to a complete diet or to a low-protein formulation that induced wasting deficits of protein and energy. Mice were sensitized by i.p. injection of sheep red blood cells on day 9, at which time one-half of the animals in each dietary group received a simultaneous injection of 10(6) syngeneic dendritic cells (JAWS II). All mice were challenged with the immunizing antigen in the right hind footpad on day 13, and the 24-hour delayed hypersensitivity response was assessed as percentage increase in footpad thickness. The low-protein diet reduced the inflammatory immune response, but JAWS cells, which exhibited immature phenotypic and functional characteristics, increased the response of both the malnourished group and the controls. By contrast, i.p. injection of 10(6) syngeneic T cells did not influence the inflammatory immune response of mice subjected to the low-protein protocol. Antigen-presenting cell numbers limited primary inflammatory cell-mediated competence in this model of wasting malnutrition, an outcome that challenges the prevailing multifactorial model of malnutrition-associated immune depression. Thus, a new dendritic cell-centered perspective emerges regarding the cellular mechanism underlying immune depression in acute pediatric protein and energy deficit.

Blood serum interferon-γ bioactivity is low in weanling mice subjected to acute deficits of energy or both protein and energy

The main objective of the present study was to determine the influence of acute deficits of protein and energy on the blood serum level of interferon-gamma, a signature type 1 polarising inflammatory cytokine. In two 14 d experiments, male and female C57BL/6J mice, initial age 19 d, consumed a complete purified diet ad libitum or in restricted daily quantities, or had free access to an isoenergetic purified low-protein diet. A zero-time control group (age 19 d) was included in the second experiment. Serum interferon-gamma was assessed in both experiments by sandwich ELISA and, in the second experiment, also by a bioassay based on inhibition of proliferation by WEHI-279 B lymphoma cells. The immunoassay detected interferon-gamma inconsistently in all groups (range 0-14 pg/ml; detection limits 1 x 5 and 0 x 7 pg/ml in experiments 1 and 2, respectively). By contrast, interferon-gamma bioactivity was found in all animals of each group (means 339, 499, 124 and 200 pg/ml in zero-time controls, age-matched controls, low-protein and restricted intake groups, respectively; detection limit, 12 pg/ml), and the mean serum bioactivity of each malnourished group was low compared with the age-matched control (P <or=0 x 05). The present study defines the physiological serum interferon-gamma bioactivity of the adolescent mouse. Moreover, to the extent achievable by way of the blood, the results reflect the influence of metabolically diverse forms of acute malnutrition on the polarising type 1 cytokine profile within lymphoid microenvironments wherein immune responses arise. Therefore, the results suggest a mechanism underlying the cell-mediated inflammatory incompetence that characterises acute, prepubescent malnutrition.

Effector/memory T cells of the weanling mouse exhibit Type 2 cytokine polarization in vitro and in vivo in the advanced stages of acute energy deficit.

Our objective was to determine whether the polarizing cytokine profile of the effector/memory T-cell compartment reflects the profound decline of cell-mediated inflammatory competence that characterizes acute prepubescent malnutrition. Weanling C57BL/6J mice were permitted free access to a complete purified diet, free access to an isocaloric low-protein diet or restricted intake of the complete diet for 14 days. First, interleukin (IL)-4 and interferon (IFN)-gamma concentrations generated in vitro by splenic and nodal effector/memory T cells were assessed following exposure to plate-bound anti-CD3. Second, net systemic production of IFN-gamma and IL-4 by the effector/memory T-cell compartment was assessed by the in vivo cytokine capture assay following anti-CD3 stimulation. In vitro stimulation generated less IFN-gamma (P=.002) but more IL-4 (P=.05) by T cells from the restricted-intake group relative to the age-matched control group. Similarly, in vivo stimulation generated low serum levels of antibody-captured IFN-gamma in the restricted-intake group vis-à-vis the age-matched control group (P=.01), while the IL-4 response was sustained (P=.39). By contrast, the 14-day low-protein model exhibited no change in T-cell cytokine signature either in vitro or in vivo. However, following extended consumption of the low-protein diet (26 days), carcass energy losses exceeded those of the 14-day protocol and serum levels of in vivo antibody-captured IFN-gamma were low after anti-CD3 challenge relative to the age-matched control group (P=.02), while levels of captured IL-4 remained unaffected (P=.07). Acute weanling malnutrition elicits a Type 2 polarizing cytokine character on the part of the effector/memory T-cell compartment, but only in the most advanced stages of energy decrement.

Omega-3/omega-6 fatty acid ratios in different phospholipid classes and depressive symptoms in coronary artery disease patients.

Depressive symptoms are highly incident among coronary artery disease (CAD) patients and increase mortality. Reduced ratios of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (omega-3 fatty acids) to arachidonic acid (AA, omega-6 fatty acid) concentrations have been linked with depressive symptoms in CAD. It remains unclear whether depressive symptoms are differentially associated with that ratio in different phospholipid classes, and this may have mechanistic implications. This study investigated associations between depressive symptoms in CAD patients and the EPA+DHA to AA ratio in the major phospholipid classes. This was a cross-sectional study of stable CAD patients. Sociodemographic, medical, medication, and cardiopulmonary fitness data were collected from each patient. Each patient was assessed for depressive symptoms using the 17-item Hamilton Depression Rating Scale (HAM-D). The percentage of EPA, DHA, and AA in each erythrocyte phospholipid class was determined using gas chromatography from fasting blood. Relationships between EPA+DHA to AA ratios and depressive symptoms were assessed using linear regression and were corrected for multiple comparisons. Seventy-six CAD patients were included (age=61.9 ± 8.5, 74% male, HAM-D=7.2 ± 5.9). In a backward elimination linear regression model, lower EPA+DHA to AA in erythrocyte phosphatidylinositol (B=-12.71, β=-0.33, p<.01) and sphingomyelin (B=-2.52, β=-0.37, p<.01) was associated with greater depressive symptom severity, independently of other known predictors. Other phospholipid classes were not associated with depressive symptoms. In conclusion, the relationship between EPA+DHA to AA ratios and depressive symptoms in CAD may not be consistent across phospholipid classes. Continued investigation of these potentially differential relationships may clarify underlying disease mechanisms.