Donald A. Cohen

The Phytoestrogen Genistein Reduces Bone Loss in Short-Term Ovariectomized Rats

Abstract: The incidence of fractures and of osteoporosis differs between Oriental and Western Caucasian women. This may depend, at least in part, on nutritional factors, including dissimilarities in dietary intake of phytoestrogens. To investigate this possibility, 2-month-old female rats were ovariectomized (OVX) or sham-operated (SHAM), fed a casein-based diet, injected daily with subcutaneous genistein (GEN), the most abundant and best characterized phytoestrogen, or vehicle (Veh) and killed 21 days after surgery. As expected, ovariectomy resulted in loss of bone mineral density (BMD) and in uterine atrophy. However, administration of 5 mg GEN per gram body weight (b.w.) ameliorated the ovariectomy-induced loss of BMD (189 ± 2 mg/cm2 in OVX and 192 ± 2 in OVX with 5 mg GEN/g b.w. per day; p<0.05). One microgram GEN per gram body weight did not affect the BMD loss and the effect of the 5 mg and 25 mg GEN per gram body weight were statistically not different. A trend toward reduced uterine atrophy (21% reduction) was noted with the 25 mg GEN dose, but not with the 1 mg and 5 mg doses. A separate experiment with 2 x 2 factorial design was conducted to elucidate the mechanism by which GEN ameliorates ovariectomy-induced bone loss. In this experiment, histomorphometry demonstrated a dramatic reduction in trabecular bone volume after ovariectomy (7.6 ± 0.7% of total bone volume in SHAM-Veh vs 3.3 ± 0.2% in OVX-Veh; p<0.01) and less bone loss in OVX rats injected with 5 mg GEN per gram per day (3.3 ± 0.2% of total bone volume in OVX-Veh vs 5.2 ± 0.4% in OVX-GEN; p<0.01). Administration of GEN was associated with higher bone formation rate per tissue volume and with a trend toward a higher number of osteoblasts per bone perimeter. The parameters of bone resorption were not affected by GEN. The concentration of serum osteocalcin and the urinary excretion of deoxypyridinoline provided corroborating results. Since production of proinflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis, the effect of GEN on lipopolysaccharide-induced in vitro production of Tumor necrosis factor-alpha (TNFa) was tested in monocytic cells from the same four rat groups. Production of TNFa was markedly elevated in OVX-Veh as compared with the SHAM-Veh rats, but this was blocked by GEN in the OVX rats. This study shows that GEN reduces both trabecular and compact bone loss after ovariectomy and that this protective effect differs from that of estrogen, since it depends on stimulation of bone formation rather than on suppression of bone resorption. Lack of action of GEN on uterine atrophy supports the possibility that this GEN dose affects target tissues via non-estrogenic mechanisms. Modulation of cytokine production may be involved in the effect of GEN on bone.

Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene

Transgenic mice expressing an inducible suicide gene, which allows systemic and reversible elimination of macrophages, were developed. A macrophage‐specific c‐fms promoter was used to express enhanced green fluorescent protein and a drug‐inducible suicide gene that leads to Fas‐mediated apoptosis in resting and cycling cells of the macrophage lineage. Transgenic mice were fertile, of normal weight, and showed no abnormal phenotype before drug exposure. The transgene was expressed constitutively in macrophages and dendritic cells (DC) but not significantly in T cells or B cells. Induction of the suicide gene led to depletion of 70–95% of macrophages and DC in nearly all tissues examined. Depletion reduced the ability to clear bacteria from the blood and led to increased bacterial growth in the liver. Depleted mice displayed several abnormalities, including splenomegaly, lymphadenopathy, thymic atrophy, extramedullary hematopoiesis, and development of peritoneal adhesions. This new, transgenic line will be useful in investigating the role of macrophages and DC.

Suppression of experimental colitis by intestinal mononuclear phagocytes

The contribution of innate immunity to inflammatory bowel disease (IBD) remains an area of intense interest. Macrophages (MØ) and dendritic cells (DC) are considered important factors in regulating the onset of IBD. The goal of this study was to determine if intestinal mononuclear phagocytes (iMNP) serve a pathological or protective role in dextran sulfate sodium (DSS)‐induced colitis in mice. Using a conditional MØ/DC depletion transgenic mouse line—MØ Fas‐induced apoptosis—to systemically deplete iMNP, DSS colitis histopathology was shown to be more severe in MØ/DC‐depleted compared with MØ/DC‐intact mice. Similarly, localized iMNP depletion by clodronate‐encapsulated liposomes into C57BL/6, BALB/c, and CB.17/SCID mice also increased DSS colitis severity, as indicated by increased histopathology, weight loss, rectal bleeding, decreased stool consistency, and colon length compared with MØ/DC‐intact, DSS‐treated mice. Histology revealed that iMNP depletion during DSS treatment led to increased neutrophilic inflammation, increased epithelial injury, and enhanced mucin depletion from Goblet cells. iMNP depletion did not further elevate DSS‐induced expression of TNF‐α and IFN‐γ mRNA but significantly increased expression of CXCL1 chemokine mRNA. Myeloperoxidase activity was increased in colons of MØ/DC‐depleted, DSS‐treated mice, compared with DSS alone, coincident with increased neutrophil infiltration in diseased colons. Neutrophil depletion combined with MØ/DC depletion prevented the increase in DSS colitis severity compared with MØ/DC depletion alone. This study demonstrates that iMNP can serve a protective role during development of acute colitis and that protection is associated with MØ/DC‐mediated down‐regulation of neutrophil infiltration.

Secretory antibodies in breast milk promote long-term intestinal homeostasis by regulating the gut microbiota and host gene expression

Significance An experimental system was developed in mice to study the long-term benefits of early exposure to secretory antibodies of the IgA class (SIgA) in breast milk. We found that breast milk-derived SIgA promoted intestinal epithelial barrier function in suckling neonates, preventing systemic infection by potential pathogens. Long-term benefits of early exposure to SIgA included maintenance of a healthy gut microbiota and regulation of gene expression in intestinal epithelial cells. These findings suggest that maternal antibodies provide benefits to the intestinal immune system of the breast-fed infant, which persist into adulthood. Maintenance of intestinal homeostasis requires a healthy relationship between the commensal gut microbiota and the host immune system. Breast milk supplies the first source of antigen-specific immune protection in the gastrointestinal tract of suckling mammals, in the form of secretory IgA (SIgA). SIgA is transported across glandular and mucosal epithelial cells into external secretions by the polymeric Ig receptor (pIgR). Here, a breeding scheme with polymeric Ig receptor-sufficient and -deficient mice was used to study the effects of breast milk-derived SIgA on development of the gut microbiota and host intestinal immunity. Early exposure to maternal SIgA prevented the translocation of aerobic bacteria from the neonatal gut into draining lymph nodes, including the opportunistic pathogen Ochrobactrum anthropi. By the age of weaning, mice that received maternal SIgA in breast milk had a significantly different gut microbiota from mice that did not receive SIgA, and these differences were magnified when the mice reached adulthood. Early exposure to SIgA in breast milk resulted in a pattern of intestinal epithelial cell gene expression in adult mice that differed from that of mice that were not exposed to passive SIgA, including genes associated with intestinal inflammatory diseases in humans. Maternal SIgA was also found to ameliorate colonic damage caused by the epithelial-disrupting agent dextran sulfate sodium. These findings reveal unique mechanisms through which SIgA in breast milk may promote lifelong intestinal homeostasis, and provide additional evidence for the benefits of breastfeeding.

The Plague Virulence Protein YopM Targets the Innate Immune Response by Causing a Global Depletion of NK Cells

ABSTRACT Yersinia pestis, the etiologic agent of plague, delivers six Yersinia outer proteins (Yops) into host cells upon direct bacterial contact. One of these, YopM, is necessary for virulence in a mouse model of septicemic plague, but its pathogenic function is unknown. We report here the immune processes affected by YopM during infection. To test whether the innate or adaptive immune system is targeted by YopM, C57BL/6 (B6) and B6 SCID mice were infected with either the conditionally virulent Y. pestis KIM5 or a yopM deletion mutant and evaluated for bacterial growth in spleen and liver. Both B6 and SCID mice succumbed to infection with Y. pestis KIM5, whereas both mouse strains survived infection by the YopM− mutant. These data showed that YopM counteracts innate defenses present in SCID mice. The YopM− strain grew more slowly than the parent Y. pestis during the first 4 days of infection in both mouse strains, indicating an early pathogenic role for YopM. In B6 mice, populations of cells of the immune system were not differentially affected by the two Y. pestis strains, with one major exception: the parent Y. pestis KIM5 but not the YopM− mutant caused a significant global decrease in NK cell numbers (blood, spleen, and liver), beginning early in infection. NK cells and macrophages isolated early (day 2) from livers and spleens of mice infected with either Y. pestis strain contained comparable levels of cytokine mRNA: interleukin (IL)-1β, IL-12, IL-15, IL-18, and tumor necrosis factor alpha in macrophages and gamma interferon in NK cells. However, by day 4 postinfection, cells from mice infected with the parent Y. pestis expressed lower levels of these messages, while those from mice infected with the mutant retained strong expression. Significantly, mRNA for the IL-15 receptor α chain was not expressed in NK cells from Y. pestis KIM5-infected mice as early as day 2 postinfection. These findings suggest that YopM interferes with innate immunity by causing depletion of NK cells, possibly by affecting the expression of IL-15 receptor α and IL-15.

Targeted deletion of MyD88 in intestinal epithelial cells results in compromised antibacterial immunity associated with downregulation of polymeric immunoglobulin receptor, mucin-2, and antibacterial peptides.

Intestinal epithelial cells (IECs) form a physical and immunological barrier that separates the vast gut microbiota from host tissues. MyD88-dependent Toll-like receptor signaling is a key mediator of microbial–host cross-talk. We examined the role of epithelial MyD88 expression by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88ΔIEC). Loss of epithelial MyD88 signaling resulted in increased numbers of mucus-associated bacteria; translocation of bacteria, including the opportunistic pathogen Klebsiella pneumoniae, to mesenteric lymph nodes; reduced transmucosal electrical resistance; impaired mucus-associated antimicrobial activity; and downregulated expression of polymeric immunoglobulin receptor (the epithelial IgA transporter), mucin-2 (the major protein of intestinal mucus), and the antimicrobial peptides RegIIIγ and Defa-rs1. We further observed significant differences in the composition of the gut microbiota between MyD88ΔIEC mice and wild-type littermates. These physical, immunological, and microbial defects resulted in increased susceptibility of MyD88ΔIEC mice to experimental colitis. We conclude that MyD88 signaling in IECs is crucial for maintenance of gut homeostasis.

T CELL INDEPENDENCE OF BLEOMYCIN-INDUCED PULMONARY FIBROSIS

The role of T cells and cytokines in bleomycin (BLM)‐induced fibrosis was evaluated in susceptible and resistant strains of normal and SCID mice. Histology and hydroxyproline analysis showed that BLM induced pulmonary fibrosis in C57BL/6 and (C57BL/6 × BALB/c)F1 mice, whereas BALB/c mice were resistant to the disease. To test whether lymphocytes were required for the induction of BLM‐induced pulmonary fibrosis, SCID mice were injected intratracheally with BLM and evaluated for the development of pulmonary inflammation and fibrosis. Similar morphological changes and increases in hydroxyproline were observed in both C57BL/6 SCID and (C57BL/6 × CB.17)F1 SCID animals compared to those seen in wild‐type C57BL/6 and (C57BL/6 × BALB/c)F1 mice. In contrast, CB.17 SCID mice, which are genetically similar to BALB/c mice, were resistant to disease induction. Analysis of the cellular infiltrate in BLM‐treated C57Bl/6 SCID mice confirmed a lack of T cells in the lungs of SCID mice and demonstrated a pronounced accumulation of eosinophils in areas of developing pulmonary fibrosis. NK cells were significantly elevated in untreated SCID mice and did not increase further after BLM treatment. Analysis of selected cytokines 1 day after initiation of BLM‐induced pulmonary fibrosis indicated that the levels of TNF‐α and IFN‐γ appeared to segregate with fibrosis in both the SCID and wild‐type mice. The data demonstrate that T cells are not required for the induction of fibrosis by BLM and suggest that responses by non‐lymphoid cells may be sufficient for the induction of fibrosis. J. Leukoc. Biol. 65: 187–195; 1999.

Inhibition of IL-10 Receptor Function in Alveolar Macrophages by Toll-Like Receptor Agonists

Despite an immunosuppressive lung environment, alveolar macrophages (AM) retain the capacity to respond to microorganisms. This report demonstrates that IL-10, constitutively produced by normal alveolar epithelium, stimulates signal transduction through the IL-10R on AM and that IL-10R function can be inhibited by stimulation of Toll-like receptor (TLR) on AM. IL-10 mRNA and protein were constitutively expressed in normal alveolar epithelium of mice, and IL-10R were constitutively expressed on normal murine AM. Stimulation of AM through TLR2, TLR4, or TLR9 was sufficient to inhibit IL-10R signal transduction, including phosphorylation and nuclear translocation of STAT3 transcription factor. Inhibition of IL-10R function by TLRs was not associated with a decrease in IL-10R expression, but did require expression of the myeloid differentiation factor 88 adaptor protein. Continuous exposure of macrophages to IL-10 caused sustained expression of the chemokine receptors CCR1 and CCR5. However, the addition of TLR ligands inhibited IL-10-induced expression of CCR1 and CCR5. Finally, exposure of macrophages to TLR ligands blocked the ability of IL-10 to inhibit the induction of TNF-α by C2-ceramide. These findings demonstrate a novel regulatory mechanism that may allow AM to overcome inhibitory effects of constitutive IL-10 in the lungs that may permit a more effective response to pulmonary infections.

Interleukin-1 (IL-1) depresses cytochrome P450 levels and activities in mice.

Endotoxin depresses cytochrome P450 levels when injected into animals. The purpose of this study was to determine whether endotoxin itself, or monokine(s) released in response to endotoxin administration are responsible for this effect. Cytochrome P450 levels and drug metabolizing activities were measured in endotoxin resistant C3H/HeJ mice 24h after single intraperitoneal injections of either lipopolysaccharide (LPS), a semipurified murine monokine preparation containing interleukin-1 (IL-1), or murine recombinant IL-1. In endotoxin sensitive C3H/HeN mice, LPS (0.5 mg/Kg) decreased total cytochrome P450 levels, benzphetamine demethylase activities, and ethoxyresorufin-0-deethylase activities. This dose of LPS did not alter cytochrome P450 levels or activities in the C3H/HeJ mice. However, after injection of the semipurified monokine preparation or the recombinant IL-1, there were significant decreases in cytochrome P450 levels and activities similar to the decreases observed with LPS in the C3H/HeN mice. These findings suggest that the alterations in hepatic cytochrome P450 seen with endotoxin injection are mediated, at least in part, by IL-1.

Zinc attenuates tumor necrosis factor-mediated activation of transcription factors in endothelial cells

OBJECTIVE The objective of the study was to test the hypothesis that zinc can protect against endothelial dysfunction by interfering with oxidative stress-mediated cellular signaling and subsequent inhibition of an endothelial cell inflammatory response. Our approach was to compare alterations on molecular and biochemical levels with changes in endothelial barrier function that occur in zinc deficient conditions. METHODS To investigate our hypothesis, endothelial cells were exposed to zinc deficient media for 2 to 10 days to deplete cellular zinc stores. Following this, half of the groups received zinc supplementation (9.2 microM) for 48 hours. The other half served as zinc deficient controls. These cells were then challenged with tumor necrosis factor-alpha (TNF) for varying time periods. Nuclear extracts were prepared from cells and analyzed for nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) binding. Media from cells were analyzed for interleukin 8 (IL-8) production, and cellular proteins were determined. RESULTS Zinc supplementation resulted in a 74% increase in cellular zinc content. It was also shown that a 1.5 hour exposure to TNF (100 U/mL medium) significantly increased NF-kappa B and AP-1 binding, which was lowered considerably when cells were supplemented with physiological levels of zinc. Zinc supplementation also caused a marked attenuation in IL-8 expression by endothelial cells in response to TNF-mediated cell activation. DISCUSSION Our previous data clearly show that zinc is a protective and critical nutrient for maintenance of endothelial integrity. The present data suggest that zinc may protect against cytokine-mediated activation of oxidative stress sensitive transcription factors, upregulation of inflammatory cytokines and endothelial cell dysfunction. This may have implications in understanding mechanisms of atherosclerosis.