Characterization of spontaneous air space enlargement in mice lacking microfibrillar-associated protein 4.

Microfibrillar-associated protein 4 (MFAP4) is localized to elastic fibers in blood vessels and the interalveolar septa of the lungs and is further present in bronchoalveolar lavage. Mfap4 has been previously suggested to be involved in elastogenesis in the lung. We tested this prediction and aimed to characterize the pulmonary function changes and emphysematous changes that occur in Mfap4-deficient (Mfap4(-/-)) mice. Significant changes included increases in total lung capacity and compliance, which were evident in Mfap4(-/-) mice at 6 and 8 mo but not at 3 mo of age. Using in vivo breath-hold gated microcomputed tomography (micro-CT) in 8-mo-old Mfap4(-/-) mice, we found that the mean density of the lung parenchyma was decreased, and the low-attenuation area (LAA) was significantly increased by 14% compared with Mfap4(+/+) mice. Transmission electron microscopy (TEM) did not reveal differences in the organization of elastic fibers, and there was no difference in elastin content, but a borderline significant increase in elastin mRNA expression in 3-mo-old mice. Stereological analysis showed that alveolar surface density in relation to the lung parenchyma and total alveolar surface area inside of the lung were both significantly decreased in Mfap4(-/-) mice by 25 and 15%, respectively. The data did not support an essential role of MFAP4 in pulmonary elastic fiber organization or content but indicated increased turnover in young Mfap4(-/-) mice. However, Mfap4(-/-) mice developed a spontaneous loss of lung function, which was evident at 6 mo of age, and moderate air space enlargement, with emphysema-like changes.

Rho kinase inhibition modulates microglia activation and improves survival in a model of amyotrophic lateral sclerosis.

Disease progression in amyotrophic lateral sclerosis (ALS) is characterized by degeneration of motoneurons (MN) and their axons, but is also influenced by neighboring cells such as astrocytes and microglial cells. The role of microglia in ALS is complex as it switches from an anti-inflammatory and neuroprotective phenotype in early disease to a proinflammatory and neurotoxic phenotype in later stages. Our previous studies in models of neurodegeneration identified rho kinase (ROCK) as a target, which can be manipulated to beneficially influence disease progression. Here, we examined the neuroprotective potential of the ROCK inhibitor Fasudil to target the central pathogenic features of ALS. Application of Fasudil to kainic acid-lesioned primary MN in vitro resulted in a strong prosurvival effect. In vivo, SOD1(G93A) mice benefited from oral treatment with Fasudil showing prolonged survival and improved motor function. These findings were correlated to an improved survival of motor neurons and a pronounced alteration of astroglial and microglial cell infiltration of the spinal cord under Fasudil treatment. Modeling a proinflammatory microglial phenotype by stimulation with LPS in vitro, Fasudil decreased the release of proinflammatory cytokines and chemokines TNFα, Il6, CCL2, CCL3, and CCL5 while CXCL1 release was only transiently suppressed. In sciatic nerve motor axons, neuromuscular junction remodeling processes were increased. In conclusion, we provide preclinical and neurobiological evidence that inhibition of ROCK by the clinically approved small molecule inhibitor Fasudil may be a novel therapeutic approach in ALS combining both neuroprotection and immunomodulation for the cure of this devastating disease.

Standardized, systemic phenotypic analysis of Umod(C93F) and Umod(A227T) mutant mice.

Uromodulin-associated kidney disease (UAKD) summarizes different clinical features of an autosomal dominant heritable disease syndrome in humans with a proven uromodulin (UMOD) mutation involved. It is often characterized by hyperuricemia, gout, alteration of urine concentrating ability, as well as a variable rate of disease progression inconstantly leading to renal failure and histological alterations of the kidneys. We recently established the two Umod mutant mouse lines Umod(C93F) and Umod(A227T) on the C3H inbred genetic background both showing kidney defects analogous to those found in human UAKD patients. In addition, disease symptoms were revealed that were not yet described in other published mouse models of UAKD. To examine if further organ systems and/or metabolic pathways are affected by Umod mutations as primary or secondary effects, we describe a standardized, systemic phenotypic analysis of the two mutant mouse lines Umod(A227T) and Umod(C93F) in the German Mouse Clinic. Different genotypes as well as different ages were tested. Beside the already published changes in body weight, body composition and bone metabolism, the influence of the Umod mutation on energy metabolism was confirmed. Hematological analysis revealed a moderate microcytic and erythropenic anemia in older Umod mutant mice. Data of the other analyses in 7-10 month-old mutant mice showed single small additional effects.

Carbon-nanoparticle-triggered acute lung inflammation and its resolution are not altered in PPARγ-defective (P465L) mice.

BACKGROUND: The alveolar macrophage (AM) - first line of innate immune defence against pathogens and environmental irritants - constitutively expresses peroxisome-proliferator activated receptor γ (PPARγ). PPARγ ligand-induced activation keeps the AM quiescent, and thereby contributes to combat invaders and resolve inflammation by augmenting the phagocytosis of apoptotic neutrophils and inhibiting an excessive expression of inflammatory genes. Because of these presumed anti-inflammatory functions of PPARγ we tested the hypothesis, whether reduced functional receptor availability in mutant mice resulted in increased cellular and molecular inflammatory response during acute inflammation and/or in an impairment of its resolution. METHODS: To address this hypothesis we examined the effects of a carbon-nanoparticle (CNP) lung challenge, as surrogate for non-infectious environmental irritants, in a murine model carrying a dominant-negative point mutation in the ligand-binding domain of PPARγ (P465L/wt). Animals were instilled intratracheally with Printex 90 CNPs and bronchoalveolar lavage (BAL) was gained 24 h or 72 h after instillation to investigate its cellular and protein composition. RESULTS: Higher BAL cell numbers - due to higher macrophage counts - were found in mutants irrespective of treatment. Neutrophil numbers in contrast were slightly lower in mutants. Intratracheal CNP instillation resulted in a profound recruitment of inflammatory neutrophils into the alveolus, but genotype related differences at acute inflammation (24 h) and resolution (72 h) were not observed. There were no signs for increased alveolar-capillary membrane damage or necrotic cell death in mutants as determined by BAL protein and lactate-dehydrogenase content. Pro-inflammatory macrophage-derived cytokine osteopontin was higher, but galectin-3 lower in female mutants. CXCL5 and lipocalin-2 markers, attributed to epithelial cell stimulation did not differ. CONCLUSIONS: Despite general genotype-related differences, we had to reject our hypothesis of an increased CNP induced lung inflammation and an impairment of its resolution in PPARγ defective mice. Although earlier studies showed ligand-induced activation of nuclear receptor PPARγ to promote resolution of lung inflammation, its reduced activity did not provide signs of resolution impairment in the settings investigated here.

Comparison of particle-exposure triggered pulmonary and systemic inflammation in mice fed with three different diets.

BACKGROUND: Obesity can be linked to disease risks such as diabetes and cardiovascular disorders, but recently, the adipose tissue (AT) macrophage also emerges as actively participating in inflammation and immune function, producing pro- and anti-inflammatory factors. Connections between the AT and chronic lung diseases, like emphysema and asthma and a protective role of adipocyte-derived proteins against acute lung injury were suggested.In this study we addressed the question, whether a diet challenge increases the inflammatory response in the alveolar and the blood compartment in response to carbon nanoparticles (CNP), as a surrogate for ambient/urban particulate air pollutants. METHODS: Mice were fed a high caloric carbohydrate-rich (CA) or a fat-rich (HF) diet for six weeks and were compared to mice kept on a purified low fat (LF) diet, respectively. Bronchoalveolar lavage (BAL) and blood samples were taken 24 h after intratracheal CNP instillation and checked for cellular and molecular markers of inflammation. RESULTS AND DISCUSSION: The high caloric diets resulted in distinct effects when compared with LF mice, respectively: CA resulted in increased body and fat mass without affecting blood cellular immunity. Conversely, HF activated the blood system, increasing lymphocyte and neutrophil counts, and resulted in slightly increased body fat content. In contrast to higher pro-inflammatory BAL Leptin in CA and HF mice, on a cellular level, both diets did not lead to an increased pro-inflammatory basal status in the alveolar compartment per se, nor did result in differences in the particle-triggered response. However both diets resulted in a disturbance of the alveolar capillary barrier as indicated by enhanced BAL protein and lactate-dehydrogenase concentrations. Systemically, reduced serum Adiponectin in HF mice might be related to the observed white blood cell increase. CONCLUSION: The increase in BAL pro-inflammatory factors in high caloric groups and reductions in serum concentrations of anti-inflammatory factors in HF mice, clearly show diet-specific effects, pointing towards augmented systemic inflammatory conditions. Our data suggest that extended feeding periods, leading to manifest obesity, are necessary to generate an increased susceptibility to particle-induced lung inflammation; although the diet-challenge already was efficient in driving pro-inflammatory systemic events.

Psychosocial maternal stress during pregnancy: effects on reproduction for F0 and F1 generation laboratory rats.

The impact of daily social maternal stress on reproduction parameters was studied in F0 and F1 generation female Long-Evans rats. Chronic maternal stress was induced in pregnant females (F0 females) by 2 h social confrontation with a dominant female throughout pregnancy. Social stress of F0 females was associated with lower maternal body mass investment in litters. While maternal stress did not cause a decline in the F0 female mass, it resulted in reduced litter sizes and lower litter masses. The individual body mass of offspring from stressed (= prenatally stressed offspring, PS) and control F0 generation mothers (= prenatal control offspring, PC) did not differ at birth. However, PS offspring grew faster during lactation and were therefore heavier than PC offspring at weaning. Reproduction parameters of F1 generation females were determined until an age of 180 days. Investigation revealed that PS females did not differ from PC females in any reproduction parameter assessed, except for higher PS offspring body mass at birth. It was also observed that a higher percentage of PS females gave birth outside the core breeding period during the light (= inactive) period. This study shows that exposure to a relatively mild social stressor during pregnancy alters female reproduction in rats. However, there was no indication of higher infant mortality which is often associated with severe laboratory stress. We argue that the reduced litter sizes in stressed F0 mothers represent an adaptive strategy from an evolutionary point of view.

Maternal social stress during pregnancy alters immune function and immune cell numbers in adult male Long-Evans rat offspring during stressful life-events.

The impact of social confrontations on distribution and function of blood immune cells in adult male rat offspring from stressed and non-stressed pregnancies was studied. Repeated 2 h resident-intruder confrontations were performed on ten consecutive days using a protective cage. Prenatally stressed intruder males (PSI) had a generally lower number of neutrophiles, monocytes, T and NK cells and reduced lymphocyte proliferation in whole blood cultures than prenatally non-stressed control intruders (PCI). Differences also existed in the temporal dynamics of immunological changes. On confrontation day 1, stress-induced reductions in lymphocyte and monocyte numbers but increased granulocyte counts were observed in both groups. However, only PCI showed a partial recovery of T cell and monocyte numbers on confrontation day 10 and a full restoration in all immune cell numbers 5 days post-confrontation. Thus, the immunological response to a psychosocial stressor in adult rats can be modified by the mothers' exposure to stress during pregnancy.

Psychosocial maternal stress during pregnancy affects serum corticosterone, blood immune parameters and anxiety behaviour in adult male rat offspring.

Exposure to prenatal stress can impair the behavioural and hormonal development in mammals. However, the consequences for the immune system are rarely investigated and there is only limited evidence that naturalistic prenatal stressors do also have the potential to affect the offspring. Thus, by using a social conflict model in female Long-Evans rats, we investigated the effects of prenatal social stress on several behavioural, hormonal and immunological parameters. Offspring from stressed and non-stressed pregnant females were housed in pairs after weaning, and tested at an age of 4-6 months. Prenatally stressed (PS) males were more active in the elevated plus-maze test as indicated by significantly more frequent entries into the open arms compared to prenatal control males (PC). In addition, PS males had significantly lower serum corticosterone concentrations under basal conditions as well as after ACTH-challenge. The basal number of total leukocytes was significantly lower in the PS group due to significantly lower lymphocyte counts. In particular, the CD4+ T-helper cell subset was affected. The lymphocyte proliferation to pokeweed mitogen was lower in PS males. Because some of the present findings do not correspond to previous studies using conventional stressors, we assume that the nature of the stressor plays an important role for pregnancy outcome and behaviour and physiology of the offspring in later life.