Essential role for the p40 subunit of interleukin-12 in neutrophil-mediated early host defense against pulmonary infection with Streptococcus pneumoniae: involvement of interferon-gamma

Interleukin (IL)-12 is a critical cytokine in the T helper (Th)1 response and host defense against intracellular microorganisms, while its role in host resistance to extracellular bacteria remains elusive. In the present study, we elucidated the role of IL-12 in the early-phase host defense against acute pulmonary infection with Streptococcus pneumoniae, a typical extracellular bacterium, using IL-12p40 gene-disrupted (IL-12p40KO) mice. IL-12p40KO mice were highly susceptible to S. pneumoniae infection, as indicated by the shortened survival time, which was completely restored by the replacement therapy with recombinant (r) IL-12, and increased bacterial counts in the lung. In these mice, recruitment of neutrophils in the lung was significantly attenuated when compared to that in wild-type (WT) mice, which correlated well with the reduced production of macrophage inflammatory protein (MIP-2) and tumor necrosis factor (TNF)-alpha in the infected tissues at the early phase of infection. In vitro synthesis of both cytokines by S. pneumoniae-stimulated lung leukocytes was significantly lower in IL-12p40KO mice than in WT mice, and addition of rIL-12 or interferon (IFN)-gamma restored the reduced production of MIP-2 and TNF-alpha in IL-12p40KO mice. Neutralizing anti-IFN-gamma monoclonal antibody (mAb) significantly decreased the effect of rIL-12. Anti-IFN-gamma mAb shortened the survival time of infected mice and reduced the recruitment of neutrophils and production of MIP-2 and TNF-alpha in the lungs. Our results indicated that IL-12p40 plays a critical role in the early-phase host defense against S. pneumoniae infection by promoting the recruitment of neutrophils to the infected tissues.     

Pathogenesis of Sendasi virus infection in mice. On the possible role of interferon on the development of disease

Intraperitoneally (i.p.) injected interferon prolonged the survival time of mice inoculated intranasally (i.n.) with Sendai virus and reduced the mortality in mice inoculated i.n. with Haemophilus influenzae. Moderate concentrations of interferon were demonstrated in homogenized lungs of Sendai virus infected mice as long as the virus was present. Similar concentrations could be produced by i.p. injection of Sendai virus or interferon. Alveolar macrophages from mice treated i.p. with interferon or Sendai virus phagocytized more actively than control macropages. From the present and earlier data it is concluded that interferon may have a direct effect on the Sendai virus infection. The total effect of virus pneumonia is a reduction of the lung macrophage antimicrobial activity, and therefore the phagocytosis-modifying effect of interferon produced in the lungs is probably of minor importance for the outcome of the disease.     

Effect of tumor necrosis factor-alpha on intracellular Staphylococcus aureus in vascular endothelial cells.

Although tumor necrosis factor-alpha (TNF-alpha) is an important host factor against intracellular bacteria, little is known about the effect of TNF-alpha on the persistence of intracellular Staphylococcus aureus in vascular endothelial cells. It was investigated whether recombinant human TNF-alpha influences the survival of intracellular S. aureus (ATCC 29213) in human umbilical vein endothelial cells (HUVEC) under a condition with an antistaphylococcal agent, and its mechanism. The HUVECs were incubated with TNF-alpha, oxacillin, or both in 24-well plates for up to 48 h following internalization of S. aureus (10(6) CFU well(-1)) into HUVECs for 1 h. TNF-alpha (1 ng mL(-1)) significantly reduced the number of intracellular S. aureus in HUVECs, and TNF-alpha plus oxacillin eliminated more intracellular S. aureus in HUVEC than oxacillin alone. The LDH viability assay and quantification of apoptosis using photometric enzyme-immunoassay showed that TNF-alpha preferentially induced cell death and apoptosis of HUVECs infected with S. aureus compared with noninfected HUVECs. These results indicate that TNF-alpha helps antistaphylococcal antibiotics to eliminate intracellular S. aureus in vascular endothelial cells, partly because TNF-alpha preferentially induces apoptosis of endothelial cells infected by S. aureus.     

Protective effect of recombinant tumor necrosis factor-alpha in murine salmonellosis

The enhancement of resistance by i.p. administration of murine rTNF-alpha into mice against i.p. challenge with virulent Salmonella typhimurium was studied. Administration of TNF-alpha (5 x 10(4) U/mouse) into mice at 6 or 12 h before the challenge with S. typhimurium organisms enhanced the bactericidal capacity in the peritoneal cavities of the mice. The diminution of the infecting organism in the peritoneal cavities of the TNF-alpha-treated mice was not due to the systemic spread of the organism inasmuch as few organism were recovered from other areas such as the spleen and liver. The TNF-alpha treatment effected a slight increase of neutrophils in the peritoneal cavity, but did not effect an increase of macrophages, including Ia-bearing macrophages. The survival rate of mice infected with Salmonella was improved by the i.p. injection of TNF-alpha before infection. Co-administration of smaller doses of TNF-alpha (5 x 10(3) U) and murine rIFN-gamma (10(2) U) at 6 h before the challenge also effectively enhanced bactericidal activity and protectivity. The cooperative effect of TNF-alpha and IFN-gamma was only seen when these recombinant cytokines were injected together at the proper time before the challenge. Injection of rabbit anti-TNF-alpha serum abolished the effects of TNF-alpha and the cooperative effect of TNF-alpha and IFN-gamma. Furthermore, the serum could abolish the cooperative effect of IFN-gamma and LPS on bactericidal activity, suggesting participation of LPS-induced TNF-alpha in the cooperation.     

Inability of recombinant interferon-gamma to activate the antibacterial activity of mouse peritoneal macrophages against Listeria monocytogenes and Salmonella typhimurium

The effect of recombinant murine interferon-gamma (rIFN-gamma) as single stimulus for the activation of antibacterial activity of macrophages was investigated on the basis of the rate of intracellular killing of Listeria monocytogenes and Salmonella typhimurium by normal and rIFN gamma-activated peritoneal macrophages of CBA and C57BL/10 mice, which differ in natural resistance to infection by these bacteria. Eighteen hours after i.p. injection of 10 to 1 X 10(4) U rIFN-gamma, resident and exudate peritoneal macrophages which had phagocytosed L. monocytogenes or S. typhimurium in vivo, killed both species in vitro just as efficiently as did resident macrophages of normal mice. Similar results were obtained after 18 hr of in vitro incubation of resident or exudate peritoneal macrophages with 0.1 to 1 X 10(4) U/ml rIFN-gamma. Consistent with the in vitro findings, two i.v. injections of 5 X 10(4) U rIFN-gamma did not affect the rate of in vivo proliferation of L. monocytogenes or S. typhimurium in the spleens of mice during the first 2 days after i.v. injection of the bacteria. Compared with the effect on the controls, two i.p. injections of 5 X 10(2) to 5 X 10(4) U rIFN-gamma did not decrease the numbers of viable S. typhimurium in either the peritoneal cell suspension or the spleen 24 hr after i.p. injection of the bacteria. Checking the state of activation of rIFN-gamma-activated macrophages on the basis of two commonly used criteria for macrophage activation showed that rIFN-gamma-activated macrophages inhibited the intracellular replication of Toxoplasma gondii and displayed enhanced O2 consumption and H2O2 release after stimulation with phorbol myristate acetate compared with macrophages from normal CBA and C57BL/10 mice. The present findings show that as single activating stimulus, rIFN-gamma is not capable of activating the antibacterial effector functions of peritoneal macrophages against facultative intracellular pathogens such as L. monocytogenes and S. typhimurium.     

Pathogenicity of selected Toxoplasma gondii isolates in young pigs

The pathogenicity in 7-week-old pigs to five different Toxoplasma gondii strains of various host species origin was compared after i.v. inoculation of 10(4) tachyzoites. Additionally, one group of pigs was inoculated i.v. with 10(6) tachyzoites of the reference strain, SSI 119. In response to the infection a significant effect of T. gondii tachyzoite inoculation dose as well as differences among strains could be observed in several parameters. The 10(6)-dose inoculated pigs showed variable degrees of clinical illness and recurrent episodes of fever 4-17 days p.i., while pigs of four of the 10(4) tachyzoite inoculated groups experienced a short-lived rise in body temperature from day 6-8 p.i. without any apparent illness or inappetence. Control pigs and pigs infected with the least pathogenic strain had normal body temperature throughout the experiment. In all inoculated pigs, T. gondii-specific IgM and IgG antibodies appeared from day 8-10 and 10-17 p.i., respectively. Serum levels of alkaline phosphatase and the acute phase protein haptoglobin were decreased or increased, respectively, in response to the infection. Differential leukocyte count on peripheral blood revealed a significant lymphocytopenia on day 6 p.i. equal to both CD4+ and CD8+ T-cells, but shifting towards a reduced ratio of CD4+/CD8+ T-cells from day 8-14 p.i. In the 10(6)-dose inoculated pigs a considerable increase in zymosan induced and spontaneous oxidative burst capacity of peripheral blood leukocytes was observed from 6 days p.i. compared with control pigs. Oxidative burst capacity was not examined for other pigs. In conclusion, several useful parameters to identify differences in T. gondii pathogenicity other than mortality were identified. Furthermore, even at low doses, significant differences between recently collected Danish T. gondii field isolates were demonstrated after i.v. inoculation in young pigs.     

Roles of tumor necrosis factor alpha (TNF-alpha) and the p55 TNF receptor in CD1d induction and coxsackievirus B3-induced myocarditis

Giving C57BL/6 mice 10(4) PFU of coxsackievirus B3 (H3 variant) fails to induce myocarditis, but increasing the initial virus inoculum to 10(5) or 10(6) PFU causes significant cardiac disease. Virus titers in the heart were equivalent at days 3 and 7 in mice given all three virus doses, but day 3 titers in the pancreases of mice inoculated with 10(4) PFU were reduced. Tumor necrosis factor alpha (TNF-alpha) concentrations in the heart were increased in all infected mice, but cytokine levels were highest in mice given the larger virus inocula. TNF-alpha(-/-) and p55 TNF receptor-negative (TNFR(-/-)) mice developed minimal myocarditis compared to B6;129 or C57BL/6 control mice. p75 TNFR(-/-) mice were as disease susceptible as C57BL/6 animals. No significant differences in virus titers in heart or pancreas were observed between the groups, but C57BL/6 and p75 TNFR(-/-) animals showed 10-fold more inflammatory cells in the heart than p55 TNFR(-/-) mice, and the cell population was comprised of high concentrations of CD4(+) gamma interferon-positive and Vgamma4(+) cells. Cardiac endothelial cells isolated from C57BL/6 and p75 TNFR(-/-) mice upregulate CD1d, the molecule recognized by Vgamma4(+) cells, but infection of TNF(-/-) or p55 TNFR(-/-) endothelial cells failed to upregulate CD1d. Infection of C57BL/6 endothelial cells with a nonmyocarditic coxsackievirus B3 variant, H310A1, which is a poor inducer of TNF-alpha, failed to elicit CD1d expression, but TNF-alpha treatment of H310A1-infected endothelial cells increased CD1d levels to those seen in H3-infected cells. TNF-alpha treatment of uninfected endothelial cells had only a modest effect on CD1d expression, suggesting that optimal CD1d upregulation requires both infection and TNF-alpha signaling.     

Rate of increase of Autographa californica nuclear polyhedrosis virus in Trichoplusia ni larvae determined by DNA:DNA hybridization

The rate of increase and doubling time of the HOB clone of Autographa californica nuclear polyhedrosis virus (AcMNPV-HOB) in neonate Trichoplusia ni larvae was determined by measuring the increase in viral DNA through time following inoculation with average doses of 50 or 17,400 occlusion bodies per larva. Changes in total DNA and viral DNA through time were followed by fluorescence spectroscopy and quantitative slot-blot DNA:DNA hybridization, respectively. Total DNA content (i.e., larval DNA and viral DNA) of larvae infected with the intermediate dose lagged behind that of noninfected larvae 30 hr post-inoculation (p.i), reached a maximum at 51 hr p.i., and stayed constant thereafter. The total DNA content of larvae inoculated with the high dose lagged behind that of the control group from 18 hr p.i. and increased slowly until death of the larvae (ca. 48 hr p.i.). The amount of viral DNA in larvae inoculated with the intermediate dose increased exponentially between 15 and 42 hr p.i., reached a maximum at 48 hr p.i., and stayed constant until 68 hr p.i., by which time most larvae had died. The amount of viral DNA in larvae inoculated with the high dose did not increase exponentially; initially the rate of increase was the same as that for larvae inoculated with the intermediate dose but became progressively lower after 13 hr p.i. Calculations of the rate of increase for AcMNPV-HOB in neonate T. ni larvae inoculated with the intermediate dose and incubated at 29 degrees C resulted in a value of 0.264 hr-1 (doubling time: 2.63 hr).(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expansion, activation and effector functions of conventional and plasmacytoid dendritic cells in mouse tissues transiently infected with Listeria monocytogenes

Dendritic cells (DC) are crucial in generating immunity to infection. Here we characterize changes in DC in terms of number, activation and effector functions, focusing on conventional DC (cDC) and plasmacytoid DC (pDC), in Listeria-infected mice. Kinetic studies showed a subset- and tissue-specific expansion of cDC and upregulation of CD80 and CD86 on splenic and mesenteric lymph node (MLN) cDC after intragastric infection. Expansion of pDC was more prolonged than cDC, and pDC upregulated CD86 and MHC-II, but not CD80, in both the spleen and MLN. cDC were an important source of IL-12 but not TNF-alpha during infection, while pDC made neither of these cytokines. Instead other CD11c(int) cells produced these cytokines. Using five-colour flow cytometry and double intracellular cytokine staining, we detected phenotypically similar CD11c(int)CD11b(+)Gr1(+) cells with distinct capacities to produce TNF-alpha/IL-12 or TNF-alpha/iNOS (inducible nitric oxide synthase) in Listeria-infected tissues. IL-12p70 was also produced by sorted CD11c(hi) and CD11c(int)CD11b(+)Gr1(+) cells. Furthermore, production of TNF-alpha, iNOS and IL-12 was differentially dependent on cellular localization of the bacteria. Cytosol-restricted bacteria induced TNF-alpha and iNOS-producing cells, albeit at lower frequency than wild-type bacteria. In contrast, IL-12 was induced only with wild-type bacteria. These data provide new insight into the relative abundance and function of distinct CD11c-expressing populations during the early stage of Listeria infection.     

Construction and properties of pseudorabies virus recombinants with altered control of immediate-early gene expression.

To investigate how altered control of expression of the essential immediate-early (IE) gene of pseudorabies virus influences virus replication and virulence, we replaced the IE promoter with the tissue-specific promoters of the bovine cytokeratin IV gene (CKIV), the bovine cytokeratin VIb gene (CKVIb), or the inducible promoter of Drosophila heat shock gene HSP70. We compared expression of the IE gene of the wild-type virus and recombinant viruses in different cell types and at different temperatures and found that IE expression had become cell type or temperature dependent. When a recombinant virus was titrated on nonpermissive cells or was titrated at nonpermissive temperatures in vitro, the plating efficiency was reduced by more than 99%. Mice were inoculated subcutaneously (s.c.), intraperitoneally (i.p.), or intranasally (i.n.) with a dose equal to 100 times the 50% lethal dose of the wild-type virus. After inoculation with temperature-sensitive recombinant N-HSP, two (s.c.), two (i.p.), and four (i.n.) of five mice died. However, at this dose, recombinant N-CKIV, which contains a promoter specific for stratified epithelial tissue of the tongue mucosa, was not lethal when inoculated s.c. or i.p. but killed four mice when inoculated i.n. Recombinant N-CKVIb, which contains a promoter specific for the suprabasal layers of the epidermis, was not lethal after inoculation by any of the three routes. In explant cultures of nasal mucosa of pigs, replication of N-CKIV and N-CKVIb was not markedly reduced in the epithelium. However, in contrast to results obtained with wild-type virus, infection of the stroma was not observed. We conclude that the replicative ability and virulence of pseudorabies virus can be influenced by altering control of expression of the IE gene.     

丛枝菌根化翅果油树幼苗根际土壤微环境

以我国二级濒危保护植物翅果油(Elaeagnus mollis)为供试植物, 通过温室盆栽试验, 研究接种丛枝菌根真菌对翅果油树幼苗根际土壤微生态环境的影响。试验设计分4个组: 摩西球囊霉(Glomus mosseae)单独接种组(GM)、脆无梗囊霉(Acaulospora delicata)单独接种组(AD)、混合接种组(GM + AD)、不接种的对照组(CK)。测定了菌根侵染率、生物量、根际微生物数量、土壤pH值、土壤酶活性及其对N、P营养的影响等指标。结果显示: 菌根真菌对3个接种组均有侵染, 其中, GM + AD的侵染率最大(90.5%), 生态学效应最好; 与对照组相比, 接种组的生物量均明显提高(p < 0.05), 其中GM + AD组生物量显著增加, 是CK组的2.2倍; AM菌根对根部微生物种群数量产生一定的影响, 主要是使根面上的细菌、放线菌、固氮菌的数量显著增加(p < 0.05); AM菌根使根际pH值降低, 与菌根侵染率呈显著负相关关系(p < 0.05); 接种组根际土壤磷酸酶、脲酶、蛋白酶的活性增加, 根际土壤的磷酸酶、蛋白酶的活性增加量与菌根侵染率呈极显著相关关系(p < 0.01); 接种组的根际土壤中, 可直接被植物吸收利用的N、P元素出现富集现象, 与菌根侵染率呈显著相关关系(p < 0.05)。研究表明: 丛枝菌根的形成改善了翅果油树幼苗的微生态环境, 提高了根际土壤肥力。     

Protection of OK-432, a Streptococcus pyogenes preparation, against lethal infection of mice with herpes simplex virus

We have studied the protective effect of OK-432, a biological response modifier (BRM) of Streptococcus pyogenes origin, on the lethal infection of mice with herpes simplex virus (HSV)-1. A single intraperitoneal (i.p.) injection of more than 10 micrograms of OK-432, when given at least two days before the infection, gave a marked effect yielding nearly 100% protection against ordinarily lethal infection. The protection was independent of the amount of infected virus inoculated. When given after the infection, the agent even at the maximal dose (100 micrograms), produced only a marginal effect. A single i.p. administration of OK-432 augmented the natural killer (NK) activity of peritoneal exudate cells and spleen mononuclear cells in mice 2 to 3 days after injection of OK-432, coinciding with the times when it induced a survival effect on HSV-infection. Treating OK-432-treated mice with a combination of an anti-macrophage agent, silica, and an anti-NK cell agent, anti-asialo GM1 serum, before infection diminished the antiviral effect of OK-432. The OK-432 protection against HSV infection was also markedly diminished in athymic nude mice. Thus, the protective effect of OK-432 on lethal HSV infection seems to be based on the activation of NK cells, macrophages, and T lymphocytes.     

Adoptive transfer of BALb/c mouse splenocytes reduces lesion severity and induces intestinal pathophysiologic changes in the Mycobacterium avium Subspecies paratuberculosis beige/scid mouse model

Successful immune reconstitution would enhance resistance of beige/scid mice to chronic infection with Mycobacterium avium subspecies paratuberculosis, but may cause damage to intestinal tissue. Therefore, we investigated the effect of adoptive transfer of BALB/c mouse splenocytes on lesion severity and intestinal physiology in beige/scid mice infected with M. paratuberculosis. Mice were inoculated intraperitoneally (i.p.) with M. paratuberculosis, and two weeks later were inoculated i.p. with viable spleen cells from immune-competent BALB/c mice. Mice were necropsied 12 weeks after infection when engraftment of lymphocytes, clinical disease, pathologic lesions, and intestinal electrophysiologic parameters were evaluated. Lymphocytes were rare in control beige/scid mice not inoculated with spleen cells. In contrast, high numbers of CD4+, CD8+, and B220+ lymphocytes were detected in the spleen of all beige/scid mice (n = 24) inoculated with spleen cells, indicating that adoptive transfer resulted in successful engraftment of donor lymphocytes (immune reconstitution). Immune reconstitution of M. paratuberculosis-infected beige/ scid mice significantly reduced the severity of clinical disease and pathologic lesions, and numbers of bacteria in the liver. However, intestinal electrophysiologic parameters studied in vitro indicated that intestinal tissues from reconstituted beige/scid mice had reduced short-circuit current responses (due to reduced ion secretion) following electrical, glucose, and forskolin stimulation. These abnormal responses suggested that neural or epithelial cells in the intestine were damaged. We conclude that successful immune reconstitution of beige/scid mice enhance their resistance to M. paratuberculosis infection, but may cause pathophysiologic changes associated with intestinal inflammation.     

Immunological responses induced by <Emphasis Type="Italic">asd</Emphasis> and <Emphasis Type="Italic">wzy/asd</Emphasis> mutant strains of <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium in BALB/c mice

Attenuated bacteria have long been developed as vaccine candidates but can have some disadvantages, such as the potential for damage to immune organs due to insufficient clearance. To minimize these disadvantages, we generated Salmonella enterica serovar Typhimurium mutants SHJ2104 (asd::cm) and HTSaYA (wzy::km, asd::cm). The wzy gene codes for the O-antigen polymerase, which is involved in lipopolysaccharide (LPS) biosynthesis, and asd codes for aspartate β-semialdehyde dehydrogenase, which participates in cell wall formation. The strains synthesized LPS with a short-chain length, and showed lower cytotoxicity and reduced intracellular proliferation in animal cells compared to wild-type bacteria. After oral infection, the mutants were cleared in immune tissues, including the Peyer’s patch, mesenteric lymph node, and spleen, within 5 days. The LD50 of the mutants in Balb/c mice was estimated to be 10⁶ higher than wild-type bacteria when administered either via an oral or i.p. route, indicating that the two strains are highly attenuated. To compare the immune response to and protective effects of the mutants against wild-type bacterial infection, we inoculated the mutants into mice via an oral (1×10¹⁰CFU) or i.p. (1×10⁷ CFU) route once or twice at a two week interval. All immune responses, such as serum IgG and secretory IgA levels, cytokine production, and delayed hypersensitivity, were highly induced by two rounds of immunization. HTSaYA and SHJ2104 induced similar immune responses, and mice immunized with HTSaYA or SHJ2104 via an i.p. route were protected against wild-type Salmonella infection even at 100-fold of the LD₅₀ (5×10⁶ CFU). Taken together, these data indicate that HTSaYA and SHJ2104 could be developed as live attenuated Salmonella vaccine candidates.     

Protective effect of beta-glucan against systemic Streptococcus pneumoniae infection in mice

The antimicrobial effect of soluble beta-1,3-D-glucan from Sclerotinia sclerotiorum (SSG) was examined in mice experimentally infected intraperitoneally (i.p.) with Streptococcus pneumoniae serotypes 4 and 6B. SSG was administered i.p. either 3 days before challenge or 3-48 h after challenge. The number of bacteria in blood samples and the mouse survival rates were recorded. Pre-challenge SSG administration protected dose-dependently against both S. pneumoniae type 4 and 6B infections. SSG injected 24 h post-challenge had a curative effect against type 6B but not type 4 pneumococcal infection. The data demonstrate that SSG administered systemically protects against pneumococcal infection in mice.     

Fatal recall responses mediated by CD8 T cells during intracellular bacterial challenge infection

The roles(s) of CD8 T cells during infections by intracellular bacteria that reside in host cell endocytic compartments are not well understood. Our previous studies in a mouse model of human monocytotropic ehrlichiosis indicated that CD8 T cells are not essential for immunity. However, we have observed an unexpected role for these cells during challenge infection. Although immunocompetent mice cleared a primary low-dose (nonfatal) Ixodes ovatus ehrlichia infection, a secondary low-dose challenge infection resulted in fatal disease and loss of control of infection. The outcome was CD8-dependent, because CD8-deficient mice survived secondary low-dose challenge infection. Moreover, effector and/or memory phenotype CD8 T cells were responsible, because adoptive transfer of purified CD44(high) CD8 T cells to naive mice induced fatal responses following a primary low-dose infection. The fatal responses were perforin- and Fas ligand-independent, and were associated with high serum concentrations of TNF-alpha and CCL2, and low levels of IL-10. Accordingly, blockade of either TNF-alpha or CCL2 ameliorated fatal recall responses, and in vitro coculture of memory CD8 T cells and Ixodes ovatus ehrlichia-infected peritoneal exudate cells resulted in substantial increases in TNF-alpha and CCL2. Thus, during monocytotropic ehrlichiosis, inflammatory cytokine production, by CD8 T cells and/or other host cells, can trigger chemokine-dependent disease. These findings highlight a novel role for CD8 T cells, and reveal that live vaccines for intracellular bacteria can, under some conditions, induce undesirable consequences.     

The intestinal microflora regulates cytokine production positively in spleen-derived macrophages but negatively in bone marrow-derived macrophages.

Besides its role as a barrier against potential pathogens, intestinal flora is presumed to protect the host by priming the immunological defense mechanisms. In this respect, the influence of intestinal flora on macrophage precursors was examined, and its modulating effect was compared on LPS-induced cytokine production by macrophages derived from bone marrow and spleen precursors (BMDM and SDM respectively). The regulation of IL-1, IL-6, TNF-alpha and IL-12 production in macrophages from germ-free and from three groups of flora-associated mice, conventional, conventionalized and E. coli-mono-associated mice, was investigated. The whole flora inhibited IL-1, TNF-alpha and IL-12 secretion by BMDM, whereas it had a stimulatory effect on IL-12 secretion by SDM. Implantation of E. coli alone enhanced cytokine secretion by BMDM but had a more limited effect than whole flora on SDM, enhancing only TNF-alpha and IL-12 secretion. Study of expression of mRNA showed a correlation with protein secretion for IL-6 but not for TNF-alpha and IL-1. IL-12 enhancement in BMDM seemed to be dependent on regulation of p35 mRNA expression while it was correlated to increased p40 mRNA expression in SDM. The results demonstrated that intestinal flora modulated bone marrow and spleen macrophage cytokine production in a differential manner and suggested a role for bacteria other than E. coli among the whole flora. The contrasting effects exerted by the intestinal flora on bone marrow and spleen precursors are an interesting observation in view of the different functions of these organs in immunity. The finding that intestinal flora enhanced IL-12 production in spleen is also potentially important since this cytokine is implicated in the determination of the relative levels of Th1 and Th2 responses and plays a pivotal role in host defense against intracellular microorganisms.     

Identification of antimicrobial compounds active against intracellular Staphylococcus aureus

Small-colony variants (SCVs) of Staphylococcus aureus exhibit characteristics of bacteria that can penetrate mammalian cells and remain intracellular and innocuous for indefinite periods. These properties make SCVs a convenient tool that can be used to identify new antibacterial agents having activity against intracellular, quiescent bacteria. Agents active against SCVs could be useful in the treatment of chronic staphylococcal infections such as bovine mastitis. An hemB deletion mutant of S. aureus Newbould, a bovine mastitis isolate, having a stable, genetically defined SCV phenotype, was used in a screening program to identify compounds active against intracellular, gram-positive bacteria. Out of more than 260,000 compounds screened, nine compounds having the desired properties were identified. The range of MICs against gram-positive bacteria was < or = 0.12-32 microg ml-1. One of the compounds (no. 8) showed excellent activity against gram-positive (MICs < or = 0.12 microg ml-1) and gram-negative (MICs < or = 0.12-4 microg ml-1) bacteria. Each of the nine compounds demonstrated efficacy in a neutropenic mouse thigh infection model. Two compounds, including compound no. 8, reduced numbers of bacteria in a mouse mastitis model of infection. Application of a stepwise screening process has identified lead compounds that may be useful for treating persistent, intracellular infections.     

Role of interferon-gamma in Valpha14+ natural killer T cell-mediated host defense against Streptococcus pneumoniae infection in murine lungs

Previously, we demonstrated that Valpha14+ NKT cells and IFN-gamma are important upstream components in neutrophil-mediated host defense against infection with Streptococcus pneumoniae. In the present study, we extended these findings by elucidating the role of IFN-gamma in this Valpha14+ NKT cell-promoted process. Administration of recombinant IFN-gamma to Jalpha18KO mice prolonged the shortened survival, promoted the attenuated clearance of bacteria and improved the reduced accumulation of neutrophils and synthesis of MIP-2 and TNF-alpha in the lungs, in comparison to wild-type (WT) mice. In addition, intravenous transfer of liver mononuclear cells (LMNC) from WT mice into Jalpha18KO mice resulted in complete recovery of the depleted responses listed above, whereas such effects were not detected when LMNC were obtained from IFN-gammaKO or Jalpha18KO mice. Activation of Valpha14+ NKT cells by alpha-galactosylceramide (alpha-GalCer) significantly enhanced the clearance of bacteria, accumulation of neutrophils and synthesis of MIP-2 and TNF-alpha in the infected lungs; this effect was significantly inhibited by a neutralizing anti-IFN-gamma antibody. Finally, in a flow cytometric analysis, TNF-alpha synthesis was detected largely by CD11b(bright+) cells in the infected lungs. Our results demonstrated that IFN-gamma plays an important role in the neutrophil-mediated host protective responses against pneumococcal infection promoted by Valpha14+ NKT cells.