Increased inflammatory response in aged mice is associated with age-related zinc deficiency and zinc transporter dysregulation

Aging is a complex process associated with physiological changes in numerous organ systems. In particular, aging of the immune system is characterized by progressive dysregulation of immune responses, resulting in increased susceptibility to infectious diseases, impaired vaccination efficacy and systemic low-grade chronic inflammation. Increasing evidence suggest that intracellular zinc homeostasis, regulated by zinc transporter expression, is critically involved in the signaling and activation of immune cells. We hypothesize that epigenetic alterations and nutritional deficits associated with aging may lead to zinc transporter dysregulation, resulting in decreases in cellular zinc levels and enhanced inflammation with age. The goal of this study was to examine the contribution of age-related zinc deficiency and zinc transporter dysregulation on the inflammatory response in immune cells. The effects of zinc deficiency and age on the induction of inflammatory responses were determined using an in vitro cell culture system and an aged mouse model. We showed that zinc deficiency, particularly the reduction in intracellular zinc in immune cells, was associated with increased inflammation with age. Furthermore, reduced Zip 6 expression enhanced proinflammatory response, and age-specific Zip 6 dysregulation correlated with an increase in Zip 6 promoter methylation. Furthermore, restoring zinc status via dietary supplementation reduced aged-associated inflammation. Our data suggested that age-related epigenetic dysregulation in zinc transporter expression may influence cellular zinc levels and contribute to increased susceptibility to inflammation with age.     

Attenuated response of aged mice to respiratory Francisella novicida is characterized by reduced cell death and absence of subsequent hypercytokinemia

Background

Pneumonia and pulmonary infections are major causes of mortality among the growing elderly population. Age associated attenuations of various immune parameters, involved with both innate and adaptive responses are collectively known as immune senescence. These changes are likely to be involved with differences in host susceptibility to disease between young and aged individuals.

Methodology/Principal Findings

The objective of this study was to assess potential age related differences in the pulmonary host response in mice to the Gram-negative respiratory pathogen, Francisella novicida. We intranasally infected mice with F. novicida and compared various immune and pathological parameters of the pulmonary host response in both young and aged mice.

Conclusions/Significance

We observed that 20% of aged mice were able to survive an intranasal challenge with F. novicida while all of their younger cohorts died consistently within 4 to 6 days post infection. Further experiments revealed that all of the aged mice tested were initially able to control bacterial replication in the lungs as well as at distal sites of replication compared with young mice. In addition, the small cohort of aged survivors did not progress to a severe sepsis syndrome with hypercytokinemia, as did all of the young adult mice. Finally, a lack of widespread cell death in potential aged survivors coupled with a difference in cell types recruited to sites of infection within the lung confirmed an altered host response to Francisella in aged mice.     

Attenuated defense response and low basal blood pressure in orexin knockout mice

The perifornical area of the hypothalamus has been known as the center for the defense response, or "fight or flight" response, which is characterized by a concomitant rise in arterial blood pressure (AP), heart rate (HR), and respiratory frequency (Rf). We examined whether orexin, a recently identified hypothalamic neuropeptide, contributes to the defense response and basal cardiovascular regulation using orexin knockout mice. Microinjection of a GABA-A receptor antagonist, bicuculline methiodide (0.1-1 mM in 20 nl), to the perifornical area in urethane-anesthetized wild-type mice elicited dose-dependent increases in AP, HR, and Rf. Although similar changes were observed in orexin knockout mice, intensities were smaller and duration was shorter than those in wild-type mice. Moreover, in an awake and freely moving condition, telemeter-indwelling orexin knockout mice showed diminished cardiovascular and behavioral responses to emotional stress in the resident-intruder test. We also found that basal AP in orexin knockout mice was significantly lower in both anesthetized (117 +/- 8 mmHg in wild type and 92 +/- 3 in knockout) and conscious (125 +/- 6 mmHg in wild type and 109 +/- 2 in knockout) conditions. alpha-Adrenergic blockade with prazosin or ganglion blockade with hexamethonium canceled the difference in basal AP. HR and cardiac contractile parameters by echocardiography did not differ between the two strains of mice. These results indicate lower sympathetic vasoconstrictor tone in knockout mice. The present study suggests that orexin-containing neurons in the perifornical area play a role as one of the efferent pathways of defense response and also operate as a regulator of AP at basal condition by activating sympathetic outflow.     

The role of rodlet cells in the inflammatory response in Phoxinus phoxinus brains infected with Diplostomum

European minnows, Phoxinus phoxinus L., are commonly infected with Diplostomum phoxini Faust, 1918 metacercariae. A sub-sample of 34 minnows collected from the River Endrick, Stirlingshire, Scotland revealed that 50% of the population were infected (n = 17), with the cerebellum, medulla oblongata and the optic lobe regions of the minnow brain bearing the heaviest infections (13.7 +/- 2.6 mean +/- S.E.; 1-38 range). Serial histological sections through the brains of both uninfected and infected minnows revealed the presence of rodlet cells in the latter, which were occasionally observed in close proximity to the tegument of a metacercaria. Rodlet cells were the only type of host inflammatory cells found in this study and their role in the host's immune response to parasitic infection is commented upon.     

Impaired resolution of inflammatory response in the lungs of JF1/Msf mice following carbon nanoparticle instillation

Background

Declined lung function is a risk factor for particulate matter associated respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD). Carbon nanoparticles (CNP) are a prominent component of outdoor air pollution that causes pulmonary toxicity mainly through inflammation. Recently we demonstrated that mice (C3H/HeJ) with higher than normal pulmonary function resolved the elicited pulmonary inflammation following CNP exposure through activation of defense and homeostasis maintenance pathways. To test whether CNP-induced inflammation is affected by declined lung function, we exposed JF1/Msf (JF1) mice with lower than normal pulmonary function to CNP and studied the pulmonary inflammation and its resolution.

Methods

5 μg, 20 μg and 50 μg CNP (Printex 90) were intratracheally instilled in JF1 mice to determine the dose response and the time course of inflammation over 7 days (20 μg dosage). Inflammation was assessed using histology, bronchoalveolar lavage (BAL) analysis and by a panel of 62 protein markers.

Results

24 h after instillation, 20 μg and 50 μg CNP caused a 25 fold and 19 fold increased polymorphonuclear leucocytes (PMN) respectively while the 5 μg represented the ''no observable adverse effect level'' as reflected by PMN influx (9.7 × 10E3 vs 8.9 × 10E3), and BAL/lung concentrations of pro-inflammatory cytokines. Time course assessment of the inflammatory response revealed that compared to day1 the elevated BAL PMN counts (246.4 × 10E3) were significantly decreased at day 3 (72.9 × 10E3) and day 7 (48.5 × 10E3) but did not reach baseline levels indicating slow PMN resolution kinetics. Strikingly on day 7 the number of macrophages doubled (455.0 × 10E3 vs 204.7 × 10E3) and lymphocytes were 7-fold induced (80.6 × 10E3 vs 11.2 × 10E3) compared to day1. At day 7 elevated levels of IL1B, TNF, IL4, MDC/CCL22, FVII, and vWF were detected in JF1 lungs which can be associated to macrophage and lymphocyte activation.

Conclusion

This explorative study indicates that JF1 mice with impaired pulmonary function also exhibits delayed resolution of particle mediated lung inflammation as evident from elevated PMN and accumulation of macrophages and lymphocytes on day7. It is plausible that elevated levels of IL1B, IL4, TNF, CCL22/MDC, FVII and vWF counteract defense and homeostatic pathways thereby driving this phenomenon.     

Rescue of the tumor-specific immune response of aged mice in vitro

In contrast to young mice, old mice fail to reject a transplanted challenge of the highly immunogenic, ultraviolet light-induced tumor 1591-RE. Old mice also fail to mount a cytolytic tumor-specific immune response in vivo, and spleen cells of old mice are defective in their ability to generate tumor-specific T cells in vitro. In the present study we report the results of cell culture mixing experiments that show that this deficiency is due to a decreased responsiveness of the Lyt-2+ tumor-specific cytolytic T cell precursors of the old animals. We also demonstrate with limiting dilution analysis that the defective responsiveness is not due to a clonal exhaustion of the precursors. In fact, the responsiveness could be restored in vitro by culturing the spleen cells of old animals at high density or by the addition of excess Lyt-1-/Lyt-2-/2000-rad-resistant spleen cells from young or old mice. Our results suggest that the rescue of tumor immunity in old individuals may be possible, perhaps by educating effector cells in vitro for adoptive immunotherapy.     

Attenuation of the pulmonary inflammatory response following butylated hydroxytoluene treatment of cytosolic phospholipase A2 null mice

Administration of butylated hydroxytoluene (BHT) to mice causes lung damage characterized by the death of alveolar type I pneumocytes and the proliferation and subsequent differentiation of type II cells to replace them. Herein, we demonstrate this injury elicits an inflammatory response marked by chemokine secretion, alveolar macrophage recruitment, and elevated expression of enzymes in the eicosanoid pathway. Cytosolic phospholipase A(2) (cPLA(2)) catalyzes release of arachidonic acid from membrane phospholipids to initiate the synthesis of prostaglandins and other inflammatory mediators. A role for cPLA(2) in this response was examined by determining cPLA(2) expression and enzymatic activity in distal respiratory epithelia and macrophages and by assessing the consequences of cPLA(2) genetic ablation. BHT-induced lung inflammation, particularly monocyte infiltration, was depressed in cPLA(2) null mice. Monocyte chemotactic protein-1 (MCP-1) content in bronchoalveolar lavage fluid increases after BHT treatment but before monocyte influx, suggesting a causative role. Bronchiolar Clara cells isolated from cPLA(2) null mice secrete less MCP-1 than Clara cells from wild-type mice, consistent with the hypothesis that cPLA(2) is required to secrete sufficient MCP-1 to induce an inflammatory monocytic response.     

Pathogenic natural antibodies propagate cerebral injury following ischemic stroke in mice

Self-reactive natural Abs initiate injury following ischemia and reperfusion of certain tissues, but their role in ischemic stroke is unknown. We investigated neoepitope expression in the postischemic brain and the role of natural Abs in recognizing these epitopes and mediating complement-dependent injury. A novel IgM mAb recognizing a subset of phospholipids (C2) and a previously characterized anti-annexin IV mAb (B4) were used to reconstitute and characterize injury in Ab-deficient Rag1(-/-) mice after 60 min of middle cerebral artery occlusion and reperfusion. Reconstitution with C2 or B4 mAb in otherwise protected Rag1(-/-) mice restored injury to that seen in wild-type (wt) mice, as demonstrated by infarct volume, demyelination, and neurologic scoring. IgM deposition was demonstrated in both wt mice and reconstituted Rag1(-/-) mice, and IgM colocalized with the complement activation fragment C3d following B4 mAb reconstitution. Further, recombinant annexin IV significantly reduced infarct volumes in wt mice and in Rag1(-/-) mice administered normal mouse serum, demonstrating that a single Ab reactivity is sufficient to develop cerebral ischemia reperfusion injury in the context of an entire natural Ab repertoire. Finally, C2 and B4 mAbs bound to hypoxic, but not normoxic, human endothelial cells in vitro. Thus, the binding of pathogenic natural IgM to postischemic neoepitopes initiates complement-dependent injury following murine cerebral ischemia and reperfusion, and, based also on previous data investigating IgM reactivity in human serum, there appears to be a similar recognition system in both mouse and man.     

Impaired immune responses in the lungs of aged mice following influenza infection

Background

Each year, influenza virus infection causes severe morbidity and mortality, particularly in the most susceptible groups including children, the elderly (>65 years-old) and people with chronic respiratory diseases. Among the several factors that contribute to the increased susceptibility in elderly populations are the higher prevalence of chronic diseases (e.g. diabetes) and the senescence of the immune system.

Methods

In this study, aged and adult mice were infected with sublethal doses of influenza virus (A/Puerto Rico/8/1934). Differences in weight loss, morbidity, virus titer and the kinetics of lung infiltration with cells of the innate and adaptive immune responses were analyzed. Additionally, the main cytokines and chemokines produced by these cells were also assayed.

Results

Compared to adult mice, aged mice had higher morbidity, lost weight more rapidly, and recovered more slowly from infection. There was a delay in the accumulation of granulocytic cells and conventional dendritic cells (cDCs), but not macrophages in the lungs of aged mice compared to adult animals. The delayed infiltration kinetics of APCs in aged animals correlated with alteration in their activation (CD40 expression), which also correlated with a delayed detection of cytokines and chemokines in lung homogenates. This was associated with retarded lung infiltration by natural killer (NK), CD4⁺ and CD8⁺ T-cells. Furthermore, the percentage of activated (CD69+) influenza-specific and IL-2 producer CD8+ T-cells was higher in adult mice compared to aged ones. Additionally, activation (CD69+) of adult B-cells was earlier and correlated with a quicker development of neutralizing antibodies in adult animals.

Conclusion

Overall, alterations in APC priming and activation lead to delayed production of cytokines and chemokines in the lungs that ultimately affected the infiltration of immune cells following influenza infection. This resulted in delayed activation of the adaptive immune response and subsequent delay in clearance of virus and prolonged illness in aged animals. Since the elderly are the fastest growing segment of the population in developed countries, a better understanding of the changes that occur in the immune system during the aging process is a priority for the development of new vaccines and adjuvants to improve the immune responses in this population.     

Attenuated response to liver injury in moesin-deficient mice: impaired stellate cell migration and decreased fibrosis

Hepatic stellate cells (HSCs) respond to injury with a coordinated set of events (termed activation), which includes migration and upregulation of matrix protein production. Cell migration requires an intact actin cytoskeleton that is linked to the plasma membrane by ezrin-radixin-moesin (ERM) proteins. We have previously found that the linker protein in HSCs is exclusively moesin. Here, we describe HSC migration and fibrogenesis in moesin-deficient mice. We developed an acute liver injury model that involved focal thermal denaturation and common bile duct ligation. HSC migration and collagen deposition were assessed by immunohistology and quantitative real-time PCR. Activated HSCs were isolated from wild-type or moesin-deficient mice for direct examination of migration. Activated HSCs from wild-type mice were positive for moesin. Migration of moesin-deficient HSCs was significantly reduced. In a culture assay, 22.1% of normal HSCs migrated across a filter in 36h. In contrast, only 1.3% of activated moesin-deficient HSCs migrated. Collagen deposition around the injury area similarly was reduced in moesin-deficient liver. The linker protein moesin is essential for HSC activation and migration in response to injury. Fibrogenesis is coupled to migration and reduced in moesin-deficient mice. Agents that target moesin may be beneficial for chronic progressive fibrosis.     

Attenuated neuropathic pain in Cav3.1 null mice

To assess the role of alpha(1G) T-type Ca2+ channels in neuropathic pain after L5 spinal nerve ligation, we examined behavioral pain susceptibility in mice lacking CaV3.1 (alpha1G(-/-)), the gene encoding the pore-forming units of these channels. Reduced spontaneous pain responses and an increased threshold for paw withdrawal in response to mechanical stimulation were observed in these mice. The alpha1G(-/-) mice also showed attenuated thermal hyperalgesia in response to both low-(IR30) and high-intensity (IR60) infrared stimulation. Our results reveal the importance of alpha(1G) T-type Ca2+ channels in the development of neuropathic pain, and suggest that selective modulation of alpha1G subtype channels may provide a novel approach to the treatment of allodynia and hyperalgesia.     

Attenuated cold sensitivity in TRPM8 null mice

Thermosensation is an essential sensory function that is subserved by a variety of transducer molecules, including those from the Transient Receptor Potential (TRP) ion channel superfamily. One of its members, TRPM8 (CMR1), a ligand-gated, nonselective cation channel, is activated by both cold and chemical stimuli in vitro. However, its roles in cold thermosensation and pain in vivo have not been fully elucidated. Here, we show that sensory neurons derived from TRPM8 null mice lack detectable levels of TRPM8 mRNA and protein and that the number of these neurons responding to cold (18 degrees C) and menthol (100 microM) is greatly decreased. Furthermore, compared with WT mice, TRPM8 null mice display deficiencies in certain behaviors, including icilin-induced jumping and cold sensation, as well as a significant reduction in injury-induced responsiveness to acetone cooling. These results suggest that TRPM8 may play an important role in certain types of cold-induced pain in humans.     

Attenuated response to cold of brown adipose tissue of mice made obese with gold thioglucose

In a first study, mice made obese with gold thioglucose became hypothermic when exposed to 4 degrees C. In a second study, lean mice and mice made obese with gold thioglucose (dynamic phase) were acclimated to 14 degrees C for up to 2 weeks and their brown adipose tissue was studied. The cold-induced increase in thyroxine 5'-deiodinase activity was initially slightly smaller in obese mice, but by 24 h and 2 weeks in the cold the activity of thyroxine 5'-deiodinase was the same in lean and obese mice. Unexpectedly, the elevated activity of 5'-deiodinase returned to the low level seen in warm-acclimated mice in both lean and obese mice after 2 weeks of cold acclimation. In gold thioglucose obese mice, a progressive cold-induced increase in the binding of guanosine diphosphate to isolated mitochondria, an index of both acute thermogenic activation and a long-term increase in uncoupling protein concentration, paralleled that seen in normal lean mice and remained at a high level after 2 weeks in the cold, although still remaining slightly lower than normal. It is not clear how a high level of mitochondrial GDP binding is maintained in cold-acclimated mice at the same time as a low level of thyroxine 5'-deiodinase activity when both are believed to be controlled by the sympathetic nervous system. We conclude that the gold thioglucose obese mouse can activate its brown adipose tissue fairly normally when it is exposed to cold, but that some attenuation of this process may contribute to the impaired survival of this mouse at low temperatures.     

Madecassoside attenuates inflammatory response on collagen-induced arthritis in DBA/1 mice

Madecassoside (MA), a triterpenoid product isolated from Centella asiatica, has been described to exhibit antioxidant and anti-inflammatory activities. The present study was undertaken to determine whether madecassoside (MA) is efficacious against collagen-induced arthritis (CIA) in mice and its possible mechanisms. DBA/1J mice were immunized with bovine type II collagen and treated with MA (3, 10 and 30 mg/kg d, i.g.) from days 21 to 42 after immunization. Arthritis was evaluated by hind paw swelling, polyarthritis index, and histological examination. In vitro proliferation of spleen cells was examined using 3-[4,5-dimethylthylthiazol-2-yl]-2, 5-diphenyltetrazoliumbromide (MTT) assay. Plasma levels of cytokines tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10) and the expression of prostaglandin E₂ (PGE₂), cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) in synovial tissues were also determined. The results showed that comparing with untreated CIA mice, treated with MA dose-dependently suppressed the clinical arthritis score and joints tissues pathological damage, reduced the proliferation of spleen cells, plasma levels of TNF-α and IL-6, synovial tissues PGE₂ production and COX-2 protein expression, however, the expression of COX-1 in symovial tissues did not change and the plasma levels of IL-10 were increased. These results suggest that MA can effectively alleviate inflammatory response on CIA, and anti-inflammatory effects of MA can be attributed, at least partially, to the inhibition of pro-inflammatory mediators, including COX-2 expression, PGE₂ production, TNF-α and IL-6 levels and the up-regulation anti-inflammatory molecule IL-10.     

Nramp1 drives an accelerated inflammatory response during Salmonella-induced colitis in mice

A recently developed model for enterocolitis in mice involves pre-treatment with the antibiotic streptomycin prior to infection with Salmonella enterica serovar Typhimurium ( S.  Typhimurium). The contribution of Nramp1/Slc11a1 protein, a critical host defence mechanism against S.  Typhimurium, to the development of inflammation in this model has not been studied. Here, we analysed the impact of Nramp1 expression on the early development of colitis using isogenic Nramp1^+/+ and Nramp1^−/− mice. We hypothesized that Nramp1 acts by rapidly inducing an inflammatory response in the gut mucosa creating an antibacterial environment and limiting spread of S.  Typhimurium to systemic sites. We observed that Nramp1^+/+ mice showed lower numbers of S.  Typhimurium in the caecum compared with Nramp1^−/− mice at all times analysed. Acute inflammation was much more pronounced in Nramp1^+/+ mice 1 day after infection. The effect of Nramp1 on development of colitis was characterized by higher secretion of the pro-inflammatory cytokines IFN-γ, TNF-α and MIP-1α and a massive infiltration of neutrophils and macrophages, compared with Nramp1^−/− animals. These data show that an early and rapid inflammatory response results in protection against pathological effects of S.  Typhimurium infection in Nramp1^+/+ mice.