Enhancement of hematopoiesis and lymphopoiesis in diet-induced obese mice.

A rodent model of diet-induced obesity revealed that obesity significantly altered hematopoietic and lymphopoietic functions in the bone marrow and thymus. C57BL/6 mice were fed a mixed high-fat diet (HFD) of 45% fat or 10% fat diet (lean controls) for 180 d. A sustained increase in the numbers of cells found in bone marrow and thymus of HFD mice occurred from day 90 to day 180. However, with the exception of a 10-18% increase in the proportion of lymphocytes, the composition of monocytes, granulocytes, erythrocytes, and mixed progenitor lineages remained normal in the marrow. Likewise, thymuses of HFD mice increased 30-50% in size compared with controls, with analogous increases in thymocyte numbers. The overall thymus cellular composition remained normal. Although increased blood and lymphatic volume in obese mice would play a role in increased hematopoiesis, there were large and disproportionate increases in blood leukocytes of HFD mice, indicating that homeostasis was not maintained. Leptin, which promotes lymphopoiesis and myelopoiesis, reached 100 ng/mL in sera from HFD mice. Moreover, a three- to sixfold increase in adipocytes in marrow resulted in spiked leptin mRNA expression in bones of HFD mice compared with lean controls. Other cytokines and growth factors did not show any increases in obese marrow. The substantial increase in lymphopoietic and hematopoietic processes in HFD mice indicates that the primary tissues are another facet of the immune system dysregulated by obesity, which was perhaps fostered by higher amounts of leptin in marrow and serum.

Enhanced production of early lineages of monocytic and granulocytic cells in mice with colitis.

The bone marrow (BM) is a large, highly active, and responsive tissue. Interestingly, little is known about the impact of colitis on hematopoietic functions. Using dextran sodium sulfate (DSS) to induce colitis in mice, we identified significant changes in the BM. Specifically, cells of the monocytic and granulocytic lineages increased nearly 60% and 80%, respectively. This change would support and promote the large infiltration of the gut with neutrophils and monocytes that are the primary cause of inflammation and tissue damage during colitis. Conversely, the early lineages of B and T cells declined in the marrow and thymus with particularly large losses observed among pre-B and pre-T cells with heightened levels of apoptosis noted among CD4(+)CD8(+) thymocytes from DSS-treated mice. Also noteworthy was the 40% decline in cells of the erythrocytic lineages in the marrow of colitis mice, which undoubtedly contributed to the anemia observed in these mice. The peripheral blood reflected the marrow changes as demonstrated by a 2.6-fold increase in neutrophils, a 60% increase in monocytes, and a decline in the lymphocyte population. Thus, colitis changed the BM in profound ways that parallel the general outcomes of colitis including infiltration of the gut with monocytes and neutrophils, inflammation, and anemia. The data provide important understandings of the full impact of colitis that may lead to unique treatments and therapies.

Natural glucocorticoids induce expansion of all developmental stages of murine bone marrow granulocytes without inhibiting function.

Natural glucocorticoids (Gc) produced during stress have profound effects on the immune system. It is well known that Gc induce apoptosis in precursor T and B cells, markedly altering lymphopoiesis. However, it has been noted that marrow myeloid cells expanded both in proportion and absolute numbers in the mouse after Gc exposure. Mice were implanted with a corticosterone (CS) tablet that increased serum Gc and caused atrophied thymuses, both classic signs of activation of the stress axis. Blood neutrophil counts were elevated (4.8x), whereas lymphocyte counts declined. Flow cytometric analysis of the marrow revealed that the phenotypic distribution of the various major classes of cells was shifted by Gc exposure. As expected, marrow lymphocyte numbers declined >40% after 3 days of exposure to Gc. Conversely, in the myeloid compartment, both monocytes and granulocytes increased in number by >40%. Further, all granulocyte developmental stages showed large increases in both total number and percentage of cells. To investigate the functional capacity of mature granulocytes from Gc-treated mice, an improved granulocyte isolation method was developed. Gc exposure had little effect on the ability of granulocytes to produce superoxide or undergo chemotaxis or phagocytose bacteria. These results indicate that Gc treatment shifts bone marrow composition and provides evidence that granulocytes and their progenitors are selectively preserved under stressful conditions without losing function.

Susceptibility of peritoneal macrophages to infection by Theiler's virus.

Theiler's murine encephalomyelitis virus (TMEV) strains fall into two groups: high-neurovirulence GDVII virus results in rapidly fatal encephalitis, while low-neurovirulence BeAn and DA viruses produce persistent central nervous system (CNS) infection and inflammatory demyelinating disease. Because macrophages (Mphis) are key components in BeAn virus-induced demyelinating disease, we examined the susceptibility of primary peritoneal macrophages (pMphis) to BeAn infection in vitro. Freshly isolated, thioglycollate-elicited pMphis were resistant to BeAn virus infection even at high multiplicity of infection. In contrast, after incubation of thioglycollate-elicited pMphis at 37 degrees C for 4 days before infection, approximately half of the cells expressed virus antigen(s) and contained nicked DNA indicative of apoptosis. However, BeAn virus RNA replication and virus yields were highly restricted. Interestingly, about one-third of the cells were apoptotic but negative for virus RNA and antigen(s). Tumor necrosis factor-alpha (TNF-alpha) and interferon-alpha (IFN-alpha) were elevated in BeAn-infected pMphi cultures suggesting that bystander killing may be responsible for the apoptosis seen in BeAn virus antigen-negative cells. These data show for the first time that pMphis are susceptible to BeAn virus infection, although the infection is highly restricted and most of these cells undergo BeAn-induced apoptosis.

Functional capacity of neutrophils from class III obese patients.

Class III obesity is associated with chronic inflammation and a variety of changes in immune function. Yet surprisingly little was known about the status of neutrophils that represent the first line of immune defense. The aim of this study was to assess key functions of neutrophils from class III obese patients, namely phagocytosis, superoxide production, chemotaxis, and response to endotoxin challenge, and compare their responses with lean controls. Thirty obese patients (BMI 48.8 ± 6.6 kg/m(2)) with comorbidities such as diabetes, hyperlipidemia, high blood pressure, etc. and nine lean (BMI between 20 and 25) subjects were enrolled in the study. Neutrophils from class III obese patients phagocytosed Escherichia coli (E. coli) at similar rates and with adequate numbers of bacteria taken up per cell compared with cells from lean subjects. Neutrophil production of superoxide, which is key to rapid killing of pathogens, showed modest diminution in the class III obese, which increased among patients with BMI >50. Chemotactic activity of neutrophils from class III obese patients was not altered. However, neutrophils from obese subjects showed an increased response to low-dose endotoxin, with concomitant reduced apoptosis and extension of their half-life compared with lean subjects, which suggests possible hyperresponsiveness of these neutrophils. Overall, neutrophil activity was not significantly altered by age, gender, diabetic status, or hyperlipidemia. Collectively, these results suggest that class III obese patients, even with comorbidities, have normal or nearly normal phagocytic, chemotactic, and superoxide generating capacity.

Peripheral, but not central nervous system, type I interferon expression in mice in response to intranasal vesicular stomatitis virus infection.

Type I interferon (IFN) is critical for resistance of mice to infection with vesicular stomatitis virus (VSV). Wild type (wt) VSV infection did not induce type I IFN production in vitro or in the central nervous system (CNS) of mice; however IFN-beta was detected in lungs, spleen, and serum within 24 h. The M protein mutant VSV, T1026R1 (also referred to as M51R), induced type I IFN production in vitro and in the CNS, with poor expression in spleens. In addition, VSV T1026R1 was not pathogenic to mice after intranasal infection, illustrating the importance of IFN in controlling VSV replication in the CNS. Experiments with chemical sympathectomy, sRAGE, and neutralizing antibody to VSV were performed to investigate the mechanism(s) utilized for induction of peripheral IFN; neither sRAGE infusion nor chemical sympathectomy had an effect on peripheral IFN production. In contrast, administration of neutralizing antibody (Ab) readily blocked the response. Infectious VSV was transiently present in lungs and spleens at 24 h post infection. The results are consistent with VSV traffic from the olfactory neuroepithelium to peripheral lymphoid organs hematogenously or via lymphatic circulation. These results suggest that VSV replicates to high titers in the brains of mice because of the lack of IFN production in the CNS after intranasal VSV infection. In contrast, replication of VSV in peripheral organs is controlled by the production of large amounts of IFN.

VSV replication in neurons is inhibited by type I IFN at multiple stages of infection.

Vesicular stomatitis virus (VSV) is a rhabdovirus which causes acute encephalitis in mice after intranasal infection. Because type I interferon (IFN) has been shown to be a potent inhibitor of VSV, we investigated the role of type I IFN in viral replication in neurons in culture. Pre-treatment of NB41A3 neuroblastoma cells or primary neuron cultures with IFN-beta or IFN-alpha strongly inhibits virus replication, with 1000-fold inhibition of infectious virus release occurring at 7 h post-infection, and maximum inhibition of 14,000-fold occurring at 14 h. Type I IFN inhibited both viral protein and RNA synthesis, but not enough to account for the inhibition of infectious virus yield. The influenza virus protein NS1 binds dsRNA and antagonizes induction of PKR activity, an IFN-inducible antiviral protein which phosphorylates and inactivates the elongation factor eIF-2alpha, resulting in cessation of translation. In NS1-expressing neuroblastoma cells, VSV replication was inhibited by IFN-beta as well as in control NB41A3 cells, and eIF-2alpha phosphorylation was blocked, suggesting that PKR activity was not involved in inhibition of viral protein synthesis. Similarly, inhibition of VSV by IFN-beta was not affected by addition of inhibitors of nitric oxide synthase, indicating that IFN-beta activity is not mediated by nitric oxide or superoxide. This contrasts with the essential role of NOS-1 in inhibition of VSV replication when neurons are treated with IFN-gamma. Analysis of cell culture supernatants revealed suppression of release of VSV particles from both NB41A3 cells and primary neurons treated with IFN. The inhibition of virion release closely matched the overall suppression of infectious VSV particle release, suggesting that type I IFN plays a role in inhibition of VSV assembly.