The dose-dependent immunoregulatory effects of the nitric oxide synthase inhibitor n(g)-nitro-L-arginine methyl ester in rats with sub-acute peritonitis

Background

Chronic inflammation accompanied by arginine deficiency, immune dysfunction, and excess nitric oxide (NO) production is a clinical condition found in patients with peritonitis. A previous study showed that the nonselective NOS inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) may facilitate the metabolism of the immune nutrient arginine without altering NO homeostasis in rats with sub-acute peritonitis. Here, we investigated the effects of L-NAME on the immunocytic subpopulation distribution and response.

Materials and Methods

Male Wistar rats with cecal puncture-induced peritonitis were administered parenteral nutrition solutions supplemented with 0 (CPP group), 5 (LNA group), 25 (MNA group) or 50 (HNA group) mg·kg⁻¹·day⁻¹ of L-NAME for 7 days. Parenteral-fed sham-operated rats (TPN group) and orally-fed healthy rats (R group) were included as controls.

Results

The TPN group had significantly increased spleen weights and levels of plasma nitrite/nitrate (NOx), circulating white blood cells (WBC), and splenocytic T cells, as well as significantly decreased levels of cytotoxic T- and B-leukocytes and B-splenocytes compared to the R group. The CPP group had significantly decreased levels of plasma NOx and concanavalin (Con) A-stimulated interferon (IFN)-γ and interleukin (IL)-2 production by leukocytes and significantly increased production of Con A-stimulated tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS)-stimulated IFN-γ in the leukocytes. In addition, the LNA and MNA groups had significantly decreased spontaneous IL-6 and Con A-stimulated TNF-α and IFN-γ production by the leukocytes while the HNA group had significantly increased LPS-stimulated TNF-α and Con A-stimulated IFN-γ and IL-2 production by the splenocytes compared to the CPP group.

Conclusions

Low-dose L-NAME infusion may suppress proinflammatory and T-helper-1 (Th1) response in leukocytes, and high-dose infusion may activate the proinflammatory response in splenic macrophages and Th1 response in T-splenocytes in rats with sub-acute peritonitis.     

Immunostimulating properties of synthetic derivatives of muramyl dipeptide and glucosaminyl muramyl dipeptide in vitro

Production of tumor necrosis factor (TNF) and interleukin-1 (IL-1) by macrophages of the spleen and peritoneal exudate of mice as well as cytotoxic factors (CFs) by murine splenocytes after in vitro activation was estimated. All the derivatives of muramyldipeptide (MDP) and glucosaminylmuramyldipeptide (GMDP) were able to induce production of TNF and CFs. In the presence of lipopolysaccharide (LPS), the effect was always higher. The response of the spleen macrophages to the effect of the preparations was higher than that of the peritoneal ones and ++non-fractionated splenocytes. GMDP and GMDP4 especially in the presence of LPS had the highest effect on induction of IL-1 by the murine peritoneal macrophages. On the contrary, MDP induced higher IL-1 synthesis by the spleen macrophages. The most active substances with respect to production of TNF, CFs and IL-1, i.e. MDP3 and GMDP4, might be recommended for immunotherapy of syngeneic tumors in animals.     

Hemoadsorption Reprograms Inflammation in Experimental Gram-negative Septic Peritonitis: Insights from In Vivo and In Silico Studies

Improper compartmentalization of the inflammatory response leads to systemic inflammation in sepsis. Hemoadsorption (HA) is an emerging approach to modulate sepsis-induced inflammation. We sought to define the effects of HA on inflammatory compartmentalization in Escherichia coli–induced fibrin peritonitis in rats. Hypothesis: HA both reprograms and recompartmentalizes inflammation in sepsis. Sprague Dawley male rats were subjected to E. coli peritonitis and, after 24 h, were randomized to HA or sham treatment (sepsis alone). Venous blood samples collected at 0, 1, 3 and 6 h (that is, 24–30 h of total experimental sepsis), and peritoneal samples collected at 0 and 6 h, were assayed for 14 cytokines along with NO₂^−/NO₃⁻. Bacterial counts were assessed in the peritoneal fluid at 0 and 6 h. Plasma tumor necrosis factor (TNF)-α, interleukin (IL)-6, CXCL-1, and CCL2 were significantly reduced in HA versus sham. Principal component analysis (PCA) suggested that inflammation in sham was driven by IL-6 and TNF-α, whereas HA-associated inflammation was driven primarily by TNF-α, CXCL-1, IL-10 and CCL2. Whereas –peritoneal bacterial counts, plasma aspartate transaminase levels and peritoneal IL-5, IL-6, IL-18, interferon (IFN)-γ and NO₂⁻/NO₃⁻ were significantly lower, both CXCL-1 and CCL2 as well as the peritoneal-to-plasma ratios of TNF-α, CXCL-1 and CCL2 were significantly higher in HA versus sham, suggesting that HA-induced inflammatory recompartmentalization leads to the different inflammatory drivers discerned in part by PCA. In conclusion, this study demonstrates the utility of combined in vivo/in silico methods and suggests that HA exerts differential effects on mediator gradients between local and systemic compartments that ultimately benefit the host.     

Role of bacterial DNA in macrophage activation by group B streptococci

Bacterial DNA (CpG DNA) induces macrophage activation and the production of inflammatory mediators, including tumor necrosis factor (TNF) and nitric oxide (NO) by these cells. However, the role of bacterial DNA in the macrophage response to whole bacteria is unknown. We used overlapping strategies to estimate the relative contribution of bacterial DNA to the upregulation of TNF and NO production in macrophages stimulated with antibiotic-treated group B streptococci (GBS). Selective inhibitors of the bacterial DNA/TLR9 pathway (chloroquine, an inhibitory oligonucleotide, and DNase I) consistently inhibited GBS-induced TNF secretion by 35-50% in RAW 264.7 macrophages and murine splenic macrophages, but had no effect on inducible nitric oxide synthase (iNOS) accumulation or NO secretion. Similarly, splenic and peritoneal macrophages from mice lacking TLR9 expression secreted 40% less TNF than macrophages from control mice after GBS challenge but accumulated comparable amounts of iNOS protein. Finally, studies in both RAW 264.7 cells and macrophages from TLR9-/- mice implicated GBS DNA in the upregulation of interleukins 6 (IL-6) and 12 (IL-12) but not interferon-beta (IFNbeta), a key intermediary in macrophage production of iNOS/NO. Our data suggest that the bacterial DNA/TLR9 pathway plays an important role in stimulating TNF rather than NO production in macrophages exposed to antibiotic-treated GBS, and that TLR9-independent upregulation of IFNbeta production by whole GBS may account for this difference.     

The role of interleukin 6 in interferon-gamma production in thermally injured mice

Following traumatic injury, patients suffer from compromised immunity increasing their susceptibility to infection. Previous studies from this laboratory demonstrated that female BALB/c mice subjected to a 15% total body surface area (TBSA) scald injury exhibit a decrease in cell-mediated immunity 10 days post-burn. Studies described herein revealed that concanavalin A (Con A; 2 microg/ml)-stimulated splenocytes from sham treated animals produced 3557+/-853 pg/ml of IFN-gamma while splenocytes from burn injured animals released two-fold more cytokine (P<0.05). To determine whether leukocyte production of IFN-gamma was under the influence of macrophages, splenic macrophage supernatants generated from burned animals were incubated with splenic lymphocytes from sham and burn animals. The amount of IFN-gamma released by lymphocytes from sham animals increased when cultured with macrophages from burned mice (P<0.05). This suggests that the increase in IFN-gamma production by unfractionated splenocytes in burned mice relative to sham treated animals is macrophage-dependent. Macrophage supernatants from burned mice released twice as much IL-6 as supernatants from sham animals (P<0.05), and when IL-6 was blocked in vivo, the amount of IFN-gamma production in burned mice decreased to sham levels (P<0.05). Thus, IL-6 mediates IFN-gamma production following burn.     

Combined effects of tumor necrosis factor-alpha, prostaglandin E2, and corticosterone on induced Ia expression on murine macrophages

Ia expression is an important marker of macrophage functional capacity. IFN-gamma induces Ia expression on perhaps all murine macrophages, whereas IL-4, granulocyte-macrophage CSF, and CSF-1 induce Ia on restricted sets of macrophages. Inhibitors of expression include PGE2, glucocorticoids, and IFN-beta. TNF has been found to augment Ia expression on several macrophage lineage cell lines but to inhibit expression on murine peritoneal macrophages. Our study shows that TNF can have opposite effects on Ia expression (induced by IFN-gamma) on thioglycollate-elicited peritoneal macrophages, depending on the length of time cells are treated and on the presence of other modulators. In particular, TNF augmented early expression induced by IFN-gamma but inhibited later expression. And although TNF synergized with PGE2 to markedly inhibit Ia induction on these cells, it partially antagonized the inhibition by corticosterone and IFN-beta. TNF and PGE2 also synergized to inhibit Ia expression induced on bone marrow-derived and splenic macrophages by either IFN-gamma or IL-4. In contrast to their effect on Ia expression, TNF and PGE2 had opposite effects on expression of gamma 2a FcR in macrophages. TNF blocked the increase in FcR expression due to any combination of PGE2, IFN-gamma, and IFN-beta. However, TNF and PGE2 both increased expression of gamma 2a FcR on WEHI-3 cells. If the different effects of TNF reflect the differentiation states of macrophages, its effects on Ia and FcR expression may vary with the progression of an immune response.     

IL-10-deficient mice demonstrate multiple organ failure and increased mortality during Escherichia coli peritonitis despite an accelerated bacterial clearance

To determine the role of endogenous IL-10 in local antibacterial host defense and in the development of a systemic inflammatory response syndrome during abdominal sepsis, IL-10 gene-deficient (IL-10(-/-)) and wild-type (IL-10(+/+)) mice received an i.p. injection with Escherichia coli. Peritonitis was associated with a bacterial dose-dependent increase in IL-10 concentrations in peritoneal fluid and plasma. The recovery of E. coli from the peritoneal fluid, blood, and lungs was diminished in IL-10(-/-) mice, indicating that endogenous IL-10 impaired bacterial clearance. Despite a lower bacterial load, IL-10(-/-) mice had higher concentrations of TNF, macrophage inflammatory protein-2 and keratinocyte in peritoneal fluid and plasma, and demonstrated more severe multiple organ damage as indicated by clinical chemistry and histopathology. Furthermore, IL-10(-/-) mice showed an increased neutrophil recruitment to the peritoneal cavity. To examine the role of elevated TNF levels in the altered host response in IL-10(-/-) mice, the effect of a neutralizing anti-TNF mAb was determined. Anti-TNF did not influence the clearance of E. coli in either IL-10(+/+) or IL-10(-/-) mice. Furthermore, anti-TNF did not affect leukocyte influx in the peritoneal fluid, multiple organ damage, or survival in IL-10(+/+) mice. In IL-10(-/-) mice, anti-TNF partially attenuated neutrophil recruitment and multiple organ damage, and prevented the increased lethality. These data suggest that although endogenous IL-10 facilitates the outgrowth and dissemination of bacteria during E. coli peritonitis, it protects mice from lethality by attenuating the development of a systemic inflammatory response syndrome by a mechanism that involves inhibition of TNF release.     

Tumor necrosis factor is markedly synergistic with interleukin 1 and interferon-γ in stimulating the production of nerve growth factor in fibroblasts

A possible interaction between tumor necrosis factor- (TNF) and other cytokines/growth factors in stimulating the production of nerve growth factor (NGF) in Swiss 3T3 cells was studied. TNF's stimulatory activity on fibroblast NGF production was synergized by interleukin-1 (IL-1), IL-1β and interferon-γ (IFN-γ), but was antagonized by transforming growth factor-β (TGF-β). The most remarkable synergistic effect was observed between TNF and IL-1/β; as little as 0.003 ng/ml of IL-1β markedly enhanced TNF's stimulatory activity on NGF production in the cells. These findings reinforce the idea that TNF, in concert with IL-1/β, plays an essential role in regulating the regeneration of peripheral nerves following injury through an indirect mechanism by which it stimulates NGF production in fibroblasts.     

Magnitude and complexity of rectal mucosa HIV-1-specific CD8+ T-cell responses during chronic infection reflect clinical status

Background

The intestinal mucosa displays robust virus replication and pronounced CD4+ T-cell loss during acute human immunodeficiency virus type 1 (HIV-1) infection. The ability of HIV-specific CD8+ T-cells to modulate disease course has prompted intensive study, yet the significance of virus-specific CD8+ T-cells in mucosal sites remains unclear.

Methods and Findings

We evaluated five distinct effector functions of HIVgag-specific CD8+ T-cells in rectal mucosa and blood, individually and in combination, in relationship to clinical status and antiretroviral therapy (ART). In subjects not on ART, the percentage of rectal Gag-specific CD8+ T-cells capable of 3, 4 or 5 simultaneous effector functions was significantly related to blood CD4 count and inversely related to plasma viral load (PVL) (p<0.05). Polyfunctional rectal CD8+ T-cells expressed higher levels of MIP-1β and CD107a on a per cell basis than mono- or bifunctional cells. The production of TNFα, IFN-γ, and CD107a by Gag-specific rectal CD8+ T-cells each correlated inversely (p<0.05) with PVL, and MIP-1β expression revealed a similar trend. CD107a and IFN-γ production were positively related to blood CD4 count (p<0.05), with MIP-1β showing a similar trend. IL-2 production by rectal CD8+ T-cells was highly variable and generally low, and showed no relationship to viral load or blood CD4 count.

Conclusions

The polyfunctionality of rectal Gag-specific CD8+ T-cells appears to be related to blood CD4 count and inversely related to PVL. The extent to which these associations reflect causality remains to be determined; nevertheless, our data suggest a potentially important role for mucosal T-cells in limiting virus replication during chronic infection.     

Macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce IL-10 in response to MyD88- and TRIF-dependent TLR signals, and TLR-independent signals

We have previously reported that mouse plasmacytoid dendritic cells (DC) produce high levels of IL-12p70, whereas bone marrow-derived myeloid DC and splenic DC produce substantially lower levels of this cytokine when activated with the TLR-9 ligand CpG. We now show that in response to CpG stimulation, high levels of IL-10 are secreted by macrophages, intermediate levels by myeloid DC, but no detectable IL-10 is secreted by plasmacytoid DC. MyD88-dependent TLR signals (TLR4, 7, 9 ligation), Toll/IL-1 receptor domain-containing adaptor-dependent TLR signals (TLR3, 4 ligation) as well as non-TLR signals (CD40 ligation) induced macrophages and myeloid DC to produce IL-10 in addition to proinflammatory cytokines. IL-12p70 expression in response to CpG was suppressed by endogenous IL-10 in macrophages, in myeloid DC, and to an even greater extent in splenic CD8alpha(-) and CD8alpha(+) DC. Although plasmacytoid DC did not produce IL-10 upon stimulation, addition of this cytokine exogenously suppressed their production of IL-12, TNF, and IFN-alpha, showing trans but not autocrine regulation of these cytokines by IL-10 in plasmacytoid DC.     

Immune dysfunction following trauma-haemorrhage: influence of gender and age

Recent studies indicate that young female proestrus mice show an enhanced immune response following trauma-haemorrhage, as opposed to the immunodepression observed in males of comparable age. Testosterone is suggested as the cause of immunodepression in males, whereas oestradiol seems to be responsible for the enhanced immune response in females, however, sex hormone levels decrease with age. To determine if the sexual dimorphism in immune responses observed in young mice following trauma-haemorrhage changes with age, young (2-3 months) and aged (18-19 months) male and female CBA/J NIA mice were subjected to soft-tissue trauma (laparatomy) and haemorrhage (35+5 mmHg for 90 min and fluid resuscitation) or sham operation. Mice were killed 24 h later, and whole blood, as well as splenic and peritoneal macrophages (Mstraight phi) obtained. Plasma 17beta-oestradiol and free testosterone decreased in aged females and males, respectively. Mstraight phi from young females had enhanced IL-1beta and suppressed IL-10 production following trauma-haemorrhage, while aged females had unchanged production IL-1beta and IL-6 production and enhanced IL-10 release. In contrast, IL-1beta and IL-6 production by Mbeta from young males was suppressed and IL-10 production enhanced following trauma-haemorrhage, whereas Mstraight phi from aged males produced elevated levels of IL-1beta and IL-6 and suppressed levels of IL-10 following trauma-haemorrhage. Thus, the gender-related changes in the immune response to trauma-haemorrhage were reversed in aged mice.     

Effect of treatment with free and liposomized albendazole on selected immunological parameters and cyst growth in mice infected with Echinococcus multilocularis

Selected immunological parameters in healthy mice and mice infected with Echinococcus multilocularis and the effect of free and liposomized albendazole (lip.ABZ) upon these parameters in relation to the reduction of parasite growth were investigated over 26 weeks. Proliferative response of splenic T and B lymphocytes, number of CD4+ and CD8+ spleen T cell subpopulations, serum concentration of IFN-γ and IL-5, and generation of superoxide anion (O₂⁻) by peritoneal macrophages were the chosen parameters. Both drug forms were given to mice at a dose of 10 mg kg⁻¹ twice a week from week 4 to week 10 post infection (p.i.) (6 weeks in total). The reduction of cyst growth after treatment with ABZ and lip.ABZ was similar up to week 4 after last dose, but the parasitostatic effect of lip.ABZ lasted 4 weeks longer than the effect of free drug. After administration of both drug forms, the proliferative responses of T and B cells were restored, and also the number of CD4+ and CD8+ increased markedly. In lip.ABZ-treated mice, stimulation of mentioned lymphocyte parameters, except that of CD8+ numbers, persisted for longer period than after ABZ therapy, where values peaked at week 12 p.i., then declined more rapidly. A very strong stimulatory effect was seen on B lymphocytes during the period of lip.ABZ administration, although interestingly, numbers of CD8+ cells were higher in free ABZ-treated group. Low concentrations of IFN-γ (Th1 response) were present in infected, untreated mouse serum. Only moderate IFN-γ elevation was observed after treatment with free ABZ. A profound increase of its concentration was seen shortly after administration of lip.ABZ, and persisted until the experiment ended. In infected untreated mice, concentration of IL-5 (Th2 response) was highest on week 2 p.i. Significantly more IL-5 was recorded in serum of mice treated with free ABZ treatment than with lip.ABZ from week 12 to 18 p.i. (weeks 2–8 after the last dose). After the initial increase of superoxide anions (weeks 4–11 p.i.), generation of O₂⁻ by peritoneal macrophages was gradually inhibited by E. multilcoularis infection. In general, treatment abolished this suppression and macrophages from lip.ABZ-treated mice produced elevated amounts of O₂⁻ over a longer period than macrophages from ABZ-treated mice. Our data indicate that anthelmintic potency of ABZ could be increased after incorporation into liposomes, not only because of improved pharmacokinetics and consequent bioavailability, but also because of significant stimulation of Th1-type cytokine IFN-γ response and effector macrophage functions.     

β-Arrestin 2 Negatively Regulates Toll-like Receptor 4 (TLR4)-triggered Inflammatory Signaling via Targeting p38 MAPK and Interleukin 10

The control of IL-10 production in Toll-like receptor (TLR) signals remains to be elucidated. Here, we report that β-arrestin 2 positively regulates TLR-triggered IL-10 production in a p38 mitogen-activated protein kinase (MAPK)-dependent mechanism. In vitro studies with cells including peritoneal macrophages and HEK293/TLR4 cells have demonstrated that β-arrestin 2 forms complexes with p38 and facilitates p38 activation after lipopolysaccharide (LPS) stimulation. Deficiency of β-arrestin 2 and inhibition of p38 MAPK activity both ameliorate TLR4-stimulated IL-10 response. Additionally, in vivo experiments show that mice lacking β-arrestin 2 produce less amount of IL-10, and are more susceptible to LPS-induced septic shock which is further enhanced by blocking IL-10 signal. These results reveal a novel mechanism by which β-arrestin 2 negatively regulates TLR4-mediated inflammatory reactions.     

IL-10 and TGF-beta redundantly protect against severe liver injury and mortality during acute schistosomiasis

The cytokines IL-10 and TGF-beta regulate immunity and inflammation. IL-10 is known to suppress the extent of hepatic damage caused by parasite ova during natural infection with Schistosoma mansoni, but the role of TGF-beta is less clear. Cytokine blockade studies in mice revealed that anti-IL-10R mAb treatment during acute infection modestly increased cytokine production and liver damage, whereas selective anti-TGF-beta mAb treatment had marginal effects. In contrast, mice administered both mAbs developed severe hepatic inflammation, with enlarged, necrotic liver granulomas, cachexia, and >80% mortality by 8 wk postinfection, despite increased numbers of CD4(+)CD25(+)Foxp3(+) T regulatory cells. Blocking both IL-10 and TGF-beta at the onset of egg production also significantly increased IL-4, IL-6, TNF, IFN-gamma, and IL-17 production and markedly increased hepatic, peritoneal, and splenic neutrophilia. In contrast, coadministration of anti-IL-10R and TGF-beta mAbs had little effect upon parasite ova-induced intestinal pathology or development of alternatively activated macrophages, which are required to suppress intestinal pathology. This suggests that inflammation is controlled during acute S. mansoni infection by two distinct, organ-specific mechanisms: TGF-beta and IL-10 redundantly suppress hepatic inflammation while intestinal inflammation is regulated by alternatively activated macrophages.     

Soluble immune complexes shift the TLR-induced cytokine production of distinct polarized human macrophage subsets towards IL-10

Background

Costimulation of murine macrophages with immune complexes (ICs) and TLR ligands leads to alternative activation. Studies on human myeloid cells, however, indicate that ICs induce an increased pro-inflammatory cytokine production. This study aimed to clarify the effect of ICs on the pro- versus anti-inflammatory profile of human polarized macrophages.

Materials and Methods

Monocytes isolated from peripheral blood of healthy donors were polarized for four days with IFN-γ, IL-4, IL-10, GM-CSF, M-CSF, or LPS, in the presence or absence of heat aggregated gamma-globulins (HAGGs). Phenotypic polarization markers were measured by flow cytometry. Polarized macrophages were stimulated with HAGGs or immobilized IgG alone or in combination with TLR ligands. TNF, IL-6, IL-10, IL-12, and IL-23 were measured by Luminex and/or RT-qPCR.

Results

HAGGs did not modulate the phenotypic polarization and the cytokine production of macrophages. However, HAGGs significantly altered the TLR-induced cytokine production of all polarized macrophage subsets, with the exception of MΦ_IL-4. In particular, HAGGs consistently enhanced the TLR-induced IL-10 production in both classically and alternatively polarized macrophages (M1 and M2). The effect of HAGGs on TNF and IL-6 production was less pronounced and depended on the polarization status, while IL-23p19 and IL-12p35 expression was not affected. In contrast with HAGGs, immobilized IgG induced a strong upregulation of not only IL-10, but also TNF and IL-6.

Conclusion

HAGGs alone do not alter the phenotype and cytokine production of in vitro polarized human macrophages. In combination with TLR-ligands, however, HAGGs but not immobilized IgG shift the cytokine production of distinct macrophage subsets toward IL-10.     

Regulation of macrophage inflammatory protein-1 alpha expression and function by endogenous interleukin-10 in a model of acute inflammation

In this study we have determined the role of endogenous interleukin (IL)-10 on leucocyte recruitment and production of the CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) in a murine model of acute inflammation. Intraperitoneal injection of zymosan produced a dose-dependent cellular infiltration which was concomitant with MIP-1alpha release in the lavage fluids. Release of this chemokine had a functional role since treatment of mice with a specific anti-MIP-1alpha antibody reduced both neutrophil and monocyte accumulation into the peritoneal cavity. An unexpected increase in cell influx and MIP-1alpha production was measured following depletion of resident peritoneal macrophages, as achieved by a 3-day liposome treatment. A similar result was obtained when the zymosan peritonitis response was elicited in IL-10 knock-out mice. In summary we propose a functional cross talk between endogenous IL-10 and this CC chemokine during the host inflammatory response.     

Critical Role of Regulator G-Protein Signaling 10 (RGS10) in Modulating Macrophage M1/M2 Activation

Regulator of G protein signaling 10 (RGS10), a GTPase accelerating protein (GAP) for G alpha subunits, is a negative regulator of NF-κB in microglia. Here, we investigated the role of RGS10 in macrophages, a closely related myeloid-derived cell type. Features of classical versus alternative activation were assessed in Rgs10-/- peritoneal and bone marrow-derived macrophages upon LPS or IL-4 treatments, respectively. Our results showed that Rgs10-/- macrophages produced higher levels of pro-inflammatory cytokines including TNF, IL-1β and IL-12p70 in response to LPS treatment and exerted higher cytotoxicity on dopaminergic MN9D neuroblastoma cells. We also found that Rgs10-/- macrophages displayed a blunted M2 phenotype upon IL-4 priming. Specifically, Rgs10-/- macrophages displayed lower YM1 and Fizz1 mRNA levels as measured by QPCR compared to wild type macrophages upon IL-4 treatment and this response was not attributable to differences in IL-4 receptor expression. Importantly, phagocytic activities of Rgs10-/- macrophages were blunted in response to IL-4 priming and/or LPS treatments. However, there was no difference in chemotaxis between Rgs10-/- and WT macrophages. Our data indicate that Rgs10-/- macrophages displayed dysregulated M1 responses along with blunted M2 alternative activation responses, suggesting that RGS10 plays an important role in determining macrophage activation responses.     

Both lymphotoxin-alpha and TNF are crucial for control of Toxoplasma gondii in the central nervous system

Immunity to Toxoplasma gondii critically depends on TNFR type I-mediated immune reactions, but the precise role of the individual ligands of TNFR1, TNF and lymphotoxin-alpha (LTalpha), is still unknown. Upon oral infection with T. gondii, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice failed to control intracerebral T. gondii and succumbed to an acute necrotizing Toxoplasma encephalitis, whereas wild-type (WT) mice survived. Intracerebral inducible NO synthase expression and-early after infection-splenic NO levels were reduced. Additionally, peritoneal macrophages produced reduced levels of NO upon infection with T. gondii and had significantly reduced toxoplasmastatic activity in TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-) mice as compared with WT animals. Frequencies of parasite-specific IFN-gamma-producing T cells, intracerebral and splenic IFN-gamma production, and T. gondii-specific IgM and IgG titers in LTalpha(-/-) and TNF/LTalpha(-/-) mice were reduced only early after infection. In contrast, intracerebral IL-10 and IL-12p40 mRNA expression and splenic IL-2, IL-4, and IL-12 production were identical in all genotypes. In addition, TNF(-/-), LTalpha(-/-), and TNF/LTalpha(-/-), but not WT, mice succumbed to infection with the highly attenuated ts-4 strain of T. gondii or to a subsequent challenge infection with virulent RH toxoplasms, although they had identical frequencies of IFN-gamma-producing T cells as compared with WT mice. Generation and infection of bone marrow reconstitution chimeras demonstrated an exclusive role of hematogeneously produced TNF and LTalpha for survival of toxoplasmosis. These findings demonstrate the crucial role of both LTalpha and TNF for control of intracerebral toxoplasms.     

Influence of aging on murine neutrophil and macrophage function against Candida albicans

Previous work by our group showed that aged C57BL/6 mice develop an altered innate and adaptive immune response to Candida albicans and are more susceptible to systemic primary candidiasis. In this work, we used young (2-3 months old) and aged (18-20 months old) C57BL/6 mice to study in vitro the influence of aging on (1) the fungicidal activity of neutrophils and macrophages, (2) the production of cytokines by resident peritoneal macrophages in response to C. albicans, and (3) cell surface Toll-like receptor (TLR) 2 expression on resident peritoneal macrophages. Our results indicate that murine phagocytes have a fungicidal activity well preserved with aging. In vitro production of proinflammatory cytokines (IL-6, IL-1beta, and tumor necrosis factor-alpha and chemokines (MIP-2) by purified (CD11b(+)) peritoneal macrophages in response to yeasts and hyphae of C. albicans was significantly lower in aged mice as compared with young mice. However, the production of IL-10 by macrophages, in response to C. albicans, was similar in both young and aged animals. Moreover, baseline TLR2 surface expression level was lower on aged macrophages than on control macrophages. Taken together, these data indicate that the increased susceptibility to C. albicans disseminated infections in aged mice is correlated with defects in TLR2 expression and in cytokine production, but not with an impaired fungicidal activity.