Increased Antimycobacterial Immunity in Interleukin-10-Deficient Mice

Macrophage effector functions are essential for clearing mycobacterial infections. Interleukin 10 (IL-10) negatively regulates macrophages and could be a factor inhibiting effective antimycobacterial immunity. We previously showed that transgenic mice which produce excess IL-10 from T cells are susceptible to infection, even though these mice continue to produce gamma interferon (IFN-gamma) at levels similar to those in controls. Here, we extend our genetic analysis of the functions of IL-10 in antimycobacterial immunity by testing the hypothesis that IL-10-deficient (IL-10(-/-)) mice should be more resistant to mycobacteria than control mice. Mycobacterium bovis bacillus Calmette-Guérin-infected IL-10(-/-) mice had significantly lower bacterial burdens than control mice early in the infection. Contrary to expectations, however, IL-10(-/-) mice did not have increased levels of IFN-gamma, either from T cells or in the plasma, suggesting that other mechanisms are responsible for the increased resistance. However, macrophages from IL-10(-/-) mice produced increased levels of inflammatory cytokines, including IFN-gamma, as well as nitric oxide and prostaglandins, which could account for increased antimycobacterial immunity. Our genetic analysis revealed that IL-10 is an inhibitor of early mycobacterial clearance. The data also suggest that IL-10 negatively regulates numerous macrophage functions as well as playing a role in down-regulating the general inflammatory response, especially in conditions where an infection must be controlled through macrophage activity.     

Cytolethal Distending Toxin Promotes Helicobacter cinaedi-Associated Typhlocolitis in Interleukin-10-Deficient Mice

Helicobacter cinaedi colonizes a wide host range, including rodents, and may be an emerging zoonotic agent. Colonization parameters, pathology, serology, and inflammatory responses to wild-type H. cinaedi (WT_Hc) were evaluated in B6.129P2-IL-10^tm1Cgn (IL-10^−/−) mice for 36 weeks postinfection (WPI) and in C57BL/6 (B6) mice for 12 WPI. Because cytolethal distending toxin (CDT) may be a virulence factor, IL-10^−/− mice were also infected with the cdtB_Hc and cdtB-N_Hc isogenic mutants and evaluated for 12 WPI. Consistent with other murine enterohepatic helicobacters, WT_Hc did not cause typhlocolitis in B6 mice, but mild to severe lesions developed at the cecocolic junction in IL-10^−/− mice, despite similar colonization levels of WT_Hc in the cecum and colon of both B6 and IL-10^−/− mice. WT_Hc and cdtB mutants also colonized IL-10^−/− mice to a similar extent, but infection with either cdtB mutant resulted in attenuated typhlocolitis and hyperplasia compared to infection with WT_Hc (P < 0.03), and only WT_Hc infection caused dysplasia and intramucosal carcinoma. WT_Hc and cdtB_Hc mutant infection of IL-10^−/− mice elevated mRNA expression of tumor necrosis factor alpha, inducible nitric oxide synthase, and gamma interferon in the cecum, as well as elevated Th1-associated serum immunoglobulin G2a^b compared to infection of B6 mice (P < 0.05). Although no hepatitis was noted, liver samples were PCR positive at various time points for WT_Hc or the cdtB_Hc mutant in approximately 33% of IL-10^−/− mice and in 10 to 20% of WT_Hc-infected B6 mice. These results indicate that WT_Hc can be used to model inflammatory bowel disease in IL-10^−/− mice and that CDT contributes to the virulence of H. cinaedi.The association of Helicobacter cinaedi infection with a variety of human diseases has received increasing attention in recent years. H. cinaedi was first isolated from homosexual men suffering from enteritis, proctitis, or proctocolitis (51). H. cinaedi was subsequently isolated from immunocompromised patients afflicted with meningitis, bacteremia, cellulitis, septic arthritis, and enteritis (2) and a neonate clinically ill with septicemia and meningitis (39), as well as from immunocompetent patients with metabolic disease (36). There are several reports of H. cinaedi isolation from various clinically healthy animal hosts, including dogs, cats, foxes, and hamsters (52). H. cinaedi has also been isolated from the inflamed colon, mesenteric lymph node, and liver of a rhesus monkey with chronic idiopathic colitis and hepatitis and from a baboon with hepatitis, as well as from feces of clinically normal, captive rhesus monkeys (11, 15, 18). Because H. cinaedi has been isolated from intestinal flora of normal hamsters and identified by PCR-based assays with wild rodents, rodents may be a zoonotic reservoir for humans (6, 47). Although H. cinaedi is the most commonly reported enterohepatic helicobacter isolated from humans, the pathogenic properties of this organism in humans or animals have not been thoroughly investigated (13).H. cinaedi and other enterohepatic helicobacters produce a cytolethal distending toxin (CDT), which arrests cell cycle progression and causes cell death in vitro (49). CDT activity also has been associated with a variety of other gram-negative bacterial pathogens that cause human diseases affecting mucosal surfaces, such as chancroid, periodontitis, and gastroenteritis (7, 48, 56). CDT from Escherichia coli, Campylobacter jejuni, Haemophilus ducreyi, Actinobacillus actinomycetemcomitans, and enterohepatic Helicobacter species irreversibly blocks the cell cycle at the G₂/M phase of growth in a wide range of cultured cells. CDT consists of three polypeptide subunits encoded by the closely linked cdtA, cdtB, and cdtC genes. cdtB is an enzymatically active subunit which functions as a nuclease that damages DNA and triggers cell cycle arrest. cdtA and cdtC are heterodimeric subunits required for the delivery of cdtB (5, 7, 48, 55, 56). In addition to inhibiting cell cycle progression, it has been reported that C. jejuni CDT directly mediates the release of proinflammatory interleukin-8 (IL-8) from intestinal epithelial cells, suggesting that CDT has a role in the inflammatory response to mucosal infections (24).IL-10 is an anti-inflammatory cytokine that inhibits the production of inflammatory cytokines in vitro and in vivo (38, 45). IL-10^−/− mice spontaneously develop enterocolitis when housed in conventional animal facilities but not in germfree conditions, suggesting that gut flora are critical for development of inflammation in this model (29, 46). It has been reported that Helicobacter bilis, Helicobacter hepaticus, Helicobacter typhlonius, and Helicobacter trogontum persistently colonize the lower bowel of mice and induce proliferative typhlocolitis in immune-dysregulated mice, including IL-10^−/− mice (14, 26, 30, 31, 34, 54). In this study, we used the IL-10^−/− mouse model to evaluate the pathogenic potential of H. cinaedi and the role of CDT in inducing gastrointestinal disease.     

Attenuation of Intestinal Inflammation in Interleukin-10-Deficient Mice Infected with Citrobacter rodentium

Interleukin-10 (IL-10) curtails immune responses to microbial infection and autoantigens and contributes to intestinal immune homeostasis, yet administration of IL-10 has not been effective at attenuating chronic intestinal inflammatory conditions, suggesting that its immune functions may be context dependent. To gain a broader understanding of the importance of IL-10 in controlling mucosal immune responses to infectious challenges, we employed the murine attaching and effacing pathogen Citrobacter rodentium, which colonizes primarily the surfaces of the cecum and colon and causes transient mucosal inflammation driven by Th17 and Th1 T helper cells. Infection induced macrophage and dendritic cell production of IL-10, which diminished antibacterial host defenses, because IL-10-deficient mice cleared infection faster than wild-type controls. In parallel, the mice had less acute infection-associated colitis and resolved it more rapidly than controls. Importantly, transient C. rodentium infection protected IL-10-deficient mice against the later development of spontaneous colitis that normally occurs with aging in these mice. Genome-wide expression studies revealed that IL-10 deficiency was associated with downregulation of proinflammatory pathways but increased expression of the anti-inflammatory cytokine IL-27 in response to infection. IL-27 was found to suppress in vitro Th17 and, to a lesser degree, Th1 differentiation independent of IL-10. Furthermore, neutralization of IL-27 resulted in more severe colitis in infected IL-10-deficient mice. Together, these findings indicate that IL-10 is dispensable for resolving C. rodentium-associated colitis and further suggest that IL-27 may be a critical factor for controlling intestinal inflammation and Th17 and Th1 development by IL-10-independent mechanisms.     

Early Resistance of Interleukin-10 Knockout Mice to Acute Systemic Candidiasis

In contrast to immunocompetent controls, interleukin-10 (IL-10) knockout (KO) mice eliminated an experimental intravenous inoculation with Candida albicans from their kidneys. Improved clearance of C. albicans from the kidneys of IL-10 KO mice was evident at 24 h after intravenous challenge with the fungus. Conversely, mice with a deletion of the IL-4 cytokine gene were more susceptible to systemic candidiasis than were immunocompetent controls. The hyperresistance of IL-10 KO mice to acute systemic candidiasis did not seem to correlate with nitric oxide-mediated immunity, but rather, it appeared to be associated with more efficient effector function of innate cells, possibly neutrophils. In support of the latter hypothesis, we observed that neutrophils from IL-10 KO mice were more efficient at killing C. albicans blastoconidia and hyphae than were neutrophils from immunocompetent control mice. Neither IL-10 KO nor IL-4 KO mice that were monoassociated with C. albicans for 4 weeks showed any histologic evidence of systemic candidiasis of endogenous origin. In contrast to systemic candidiasis, we observed no significant (P < 0.05) differences in susceptibility among IL-10 KO, IL-4 KO, and wild-type (immunocompetent) mice to orogastric candidiasis. Our results suggest that IL-10 exerts a negative effect on the early, innate response to acute systemic candidiasis; however, in comparison to immunocompetent control (wild-type) mice, neither IL-10 nor IL-4 deficiency enhanced susceptibility to orogastric candidiasis.     

Role of Gamma Interferon in the Pathogenesis of Severe Schistosomiasis in Interleukin-4-Deficient Mice

In the absence of interleukin-4 (IL-4), infection with Schistosoma mansoni leads to a severe fatal disease rather than the chronic survivable condition that occurs in wild-type (WT) mice. Because the sustained production of NO most closely correlates to weight loss and fatality in infected IL-4(-/-) mice and because gamma interferon (IFN-gamma) is an important inducer of inducible NO synthase, infected IL-4(-/-) mice were treated with anti-IFN-gamma antibodies to determine the role of IFN-gamma during schistosomiasis in WT and IL-4(-/-) animals. When IFN-gamma was neutralized, Th2 responses were enhanced and NO production was reduced in both WT and IL-4(-/-) mice. The decreased NO production correlated with a rescue of proliferation in splenocytes from infected IL-4(-/-) mice. Furthermore, the neutralization of IFN-gamma in vivo improved the gross appearance of the liver and led to a reduction in granuloma size in infected IL-4(-/-) but not WT mice. However, the neutralization of IFN-gamma in vivo did not affect the development of severe disease in infected IL-4(-/-) mice. These results suggest that while the increased production of IFN-gamma does lead to some of the pathology observed in infected IL-4(-/-) mice, it is not ultimately responsible for cachexia and death.     

Interleukin-15 Complex Treatment Protects Mice from Cerebral Malaria by Inducing Interleukin-10-Producing Natural Killer Cells

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Increased Resistance to Taenia crassiceps Murine Cysticercosis in Qa-2 Transgenic Mice

We previously reported important differences in resistance to Taenia crassiceps murine cysticercosis between BALB/c substrains. It was suggested that resistance might correlate with expression of the nonclassic class I major histocompatibility complex (MHC) Qa-2 antigen; BALB/cAnN is Qa-2 negative and highly susceptible to T. crassiceps, whereas BALB/cJ expresses Qa-2 and is highly resistant. In this study, we investigated the role of Qa-2 in mediating resistance to cysticercosis by linkage analysis and infection of Qa-2 transgenic mice. In BALB/cAnN × (C57BL/6J × BALB/cAnN)F₁ and BALB/cAnN × (BALB/cJ × BALB/cAnN)F₁ backcrosses, the expression of Qa-2 antigen correlated with resistance to cysticercosis. Significantly fewer parasites were recovered from infected Qa-2 transgenic male and female mice than from nontransgenic mice of similar genetic background. These results clearly demonstrate that the Qa-2 MHC antigen is involved in resistance to T. crassiceps cysticercosis.

Taenia solium cysticercosis is a parasitic disease that seriously affects human health (24) and causes important economic losses in pig farming of developing countries (1) where conditions that favor parasite transmission persist. The essential role of pigs as an obligatory intermediate host in the parasite life cycle offers the opportunity to interfere with transmission by inducing acquired immunity through vaccination (10, 13, 18), by decreasing susceptibility through genetic manipulation (14), or both. Systematic exploration of the role of genetic factors in cysticercosis and the identification of protective immunogens are hampered by the high costs and slow data retrieval involved in studies with pigs. However, another cestode, Taenia crassiceps, that naturally infects rodents (3) is highly suitable for experimentation. It shows extensive antigenic cross-reactivity and cross-protective immunity with T. solium (7, 21); the antigenic similarity is such that T. crassiceps antigens can be used for immunodiagnosis of human cysticercosis (9). Furthermore, T. crassiceps and T. solium both have a typical two-host taeniid life cycle and morphologically and structurally related larval stages. Since T. crassiceps can reproduce asexually, experimental infection is readily attained by injecting the cysticerci in the peritoneal cavity of the mouse (3). Thus, T. crassiceps murine cysticercosis has been shown to be a useful experimental model of metacestode infection in the study of genetic factors involved in host resistance (2, 20) and underlying immunological mechanisms (19, 23, 25).Initial findings showed that genes linked to H-2 affect T. crassiceps growth in mice (20). Thus, significant differences in the extent of the parasitosis were found between mice carrying the H-2^d (BALB/cAnN and DBA/2) haplotype, which were the most susceptible, and mice with H-2^b (BALB/B, C57BL/6J, and C57BL/10J) or H-2^k (BALB/K, C3H/HeJ, and C3H/FeJ) haplotype, which were comparatively resistant. Further studies (2) showed low susceptibility of congenic and recombinant B10 mice, regardless of H-2 haplotype, indicating that genes in C57BL background confer resistance to the parasitosis such that they override the effect of H-2. The effect of genes outside H-2 on the control of parasite growth was also revealed by the differential susceptibility of three H-2^d BALB/c substrains, of which BALB/cAnN was highly susceptible, whereas BALB/cJ was highly resistant and BALB/cByJ displayed and intermediate degree of susceptibility (2). BALB/cAnN and BALB/cJ, which are genetically quite similar strains, differ in several phenotypes, including the expression of the Qa-2 antigen (11, 16). This antigen is a nonclassical class I major histocompatibility complex (MHC) molecule encoded by four genes (Q6 to Q9) located telomeric to the H-2D loci (11, 22). BALB/cJ (Qa-2^low), a Qa-2 expressor substrain, has only active Q6 and Q7 genes because Q8 and Q9 have fused, resulting in an inactive Q8/Q9^d gene (11, 15). In BALB/cAnN (Qa-2^null), an additional deletion of genomic DNA has occurred between the Q6 and Q7 genes, leading to their inactivation and accounting for the Qa-2 null expression (11). We proposed previously that differences in susceptibility to T. crassiceps observed between BALB/cJ and BALB/cAnN might be related to Qa-2 antigen expression (2). Here we describe that results of genetic linkage studies are entirely consistent with this hypothesis. Furthermore, a role of Qa-2 in mediating resistance to T. crassiceps was directly established by the diminution of parasite loads in infected Qa-2 transgenic mice.     

Lack of Interleukin-12 in p40-Deficient Mice Leads to Poor CD8+ T-Cell Immunity against Encephalitozoon cuniculi Infection

A CD8⁺ T-cell response is critical for protection against Encephalitozoon cuniculi infection. However, the factors responsible for the generation of CD8⁺ T-cell immunity during E. cuniculi infection and the cytokines involved in this process have not been identified. In the present study, we demonstrated that p40-deficient animals, which are unable to produce interleukin-12 (IL-12), have a serious defect in expansion of the CD8⁺ T-cell response which compromises the survival of an infected host. Adoptive transfer of CD8⁺ T cells from immunocompetent donors protected SCID mice infected with E. cuniculi, whereas administration of CD8⁺ T cells from p40^−/− mice failed to protect infected SCID mice. In vitro dendritic cell (DC) cultures from knockout mice pulsed with E. cuniculi spores were unable to develop a robust CD8⁺ T-cell immune response. Addition of exogenous IL-12 or transfer of CD8⁺ T cells that were initially primed with DC from p40^−/− animals to DC cultures from immunocompetent mice (directly or via transwells) led to optimal expansion of these cells. This IL-12-mediated reinstatement of CD8⁺ T-effector immunity was independent of gamma interferon (IFN-γ) as addition of antibody to the cultures failed to have an effect. These studies demonstrated that IL-12 plays a predominant role in the expansion of effector CD8⁺ T-cell immunity against E. cuniculi, which is critical for host survival. These findings are very important for understanding the protective immune mechanisms needed to protect an immunocompromised host against an opportunistic infection and can be extended to other microsporidial pathogens.Encephalitozoon cuniculi is an opportunistic pathogen recently associated with HIV infection, organ transplants, travelers, and the elderly (8, 37). Cell-mediated immunity has been shown to be critical for host resistance against E. cuniculi infection (32), and previous studies in our laboratory have suggested that CD8⁺ T cells play a predominant role during this type of infection (16). Mice lacking CD8⁺ T cells are unable to survive E. cuniculi challenge, and a CD8⁺ T-cell cytotoxic response is essential for protective immunity against this parasite.Priming of the CD8⁺ T-cell response against several intracellular pathogens has been shown to be largely dependent on interleukin-12 (IL-12) (25, 40), a heterodimeric proinflammatory cytokine that has been postulated to serve as a bridge between the innate and adaptive immune responses (35). Formed by a light-chain p35 and a heavy-chain p40, this cytokine has multiple functions. It stimulates the development of Th1 lymphocytes (18), induces the production of gamma interferon (IFN-γ) by mouse CD4⁺ Th1 clones (12), and induces the production of cytotoxic CD8⁺ T lymphocytes (34). In the present study, we demonstrate that the absence of IL-12 causes a severe defect in the expansion of the effector CD8⁺ T-cell response against E. cuniculi infection. In vitro studies demonstrate that addition of exogenous IL-12 even 48 to 72 h postculture can reverse the defect in a CD8⁺ T-cell population primed with p40^−/− dendritic cells (DC).     

Resident Enteric Bacteria Are Necessary for Development of Spontaneous Colitis and Immune System Activation in Interleukin-10-Deficient Mice

Mice with targeted deletion of the gene for interleukin-10 (IL-10) spontaneously develop enterocolitis when maintained in conventional conditions but develop only colitis when kept in specific-pathogen-free (SPF) environments. This study tested the hypothesis that enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice. IL-10-deficient mice were maintained in either SPF conditions or germfree conditions or were populated with bacteria known to cause colitis in other rodent models. IL-10-deficient mice kept in SPF conditions developed colitis in all segments of the colon (cecum and proximal and distal colon). These mice exhibited immune system activation as evidenced by increased expression of CD44 on CD4⁺ T cells; increased mesenteric lymph node cell numbers; and increased production of immunoglobulin A (IgA), IgG1, and IL-12 p40 from colon fragment cultures. Mice populated with bacterial strains, including Bacteroides vulgatus, known to induce colitis in other rodent models had minimal colitis. Germfree IL-10-deficient mice had no evidence of colitis or immune system activation. We conclude therefore that resident enteric bacteria are necessary for the development of spontaneous colitis and immune system activation in IL-10-deficient mice.     

Increased Activity and Altered Subcellular Distribution of Lysosomal Enzymes Determine Neuronal Vulnerability in Niemann-Pick Type C1-Deficient Mice

Niemann-Pick disease type C (NPC), caused by mutations in the Npc1 or Npc2 genes, is a progressive neurodegenerative disorder characterized by intracellular accumulation/redistribution of cholesterol in a number of tissues including the brain. This is accompanied by a severe loss of neurons in selected brain regions. In this study, we evaluated the role of lysosomal enzymes, cathepsins B and D, in determining neuronal vulnerability in NPC1-deficient (Npc1^−/−) mouse brains. Our results showed that Npc1^−/− mice exhibit an age-dependent degeneration of neurons in the cerebellum but not in the hippocampus. The cellular level/expression and activity of cathepsins B and D are increased more predominantly in the cerebellum than in the hippocampus of Npc1^−/− mice. In addition, the cytosolic levels of cathepsins, cytochrome c, and Bax2 are higher in the cerebellum than in the hippocampus of Npc1^−/− mice, suggesting a role for these enzymes in the degeneration of neurons. This suggestion is supported by our observation that degeneration of cultured cortical neurons treated with U18666A, which induces an NPC1-like phenotype at the cellular level, can be attenuated by inhibition of cathepsin B or D enzyme activity. These results suggest that the increased level/activity and altered subcellular distribution of cathepsins may be associated with the underlying cause of neuronal vulnerability in Npc1^−/− brains. Therefore, their inhibitors may have therapeutic potential in attenuating NPC pathology.Niemann-Pick disease type C (NPC) is an autosomal recessive neurovisceral disorder caused by mutations in the Npc1 or Npc2 gene. NPC1 is a membrane protein that contains a sterol-sensing domain and resides primarily in late endosomes/lysosomes, whereas NPC2 is a soluble protein that resides primarily in lysosomes.^1,2,3,4 The loss of function of either protein results in intracellular accumulation of unesterified cholesterol and glycosphingolipids within the endosomal-lysosomal (EL) system in a number of tissues including the brain. In addition, there is evidence that homeostatic responses to exogenously supplied cholesterol and activation of cholesterol esterification are severely impaired in cells lacking functional NPC1. These defects in cholesterol accumulation/homeostasis trigger abnormal liver and spleen function as well as widespread neurological deficits including ataxia, dystonia, seizures, and dementia that eventually lead to premature death.^5,6,7,8,9 Interestingly, BALB/cNctr-Npc^N/N mice, which do not express NPC1 protein because of a spontaneous deletion/insertion mutation in the Npc1 gene, have been shown to recapitulate pathological features associated with NPC disease. These Npc1^−/− mice are asymptomatic at birth but gradually develop tremor and ataxia, dying prematurely at ∼3 months.^10,11,12,13 As in the human disease, Npc1^−/− mice show accumulation of unesterified cholesterol in the EL system and exhibit activation of microglia and astrocytes as well as degradation of the myelin sheath throughout the central nervous system. Progressive loss of neurons is particularly evident in the prefrontal cortex, thalamus, brainstem, and cerebellum but not in the hippocampal formation.^{13,14,15,16,17,18} However, at present, very little is known about the underlying mechanisms associated with the vulnerability of select populations of neurons in Npc1^−/− mice.A number of earlier studies have shown that the EL system, the major site of cholesterol accumulation in NPC pathology, consists of two dynamic interrelated cellular pathways: the endocytic pathway and the lysosomal system. Under normal conditions, the EL system serves as an important site for intracellular protein turnover and proteolytic processing of certain proteins mediated by lysosomal hydrolases termed cathepsins.^19,20,21 After their synthesis in the endoplasmic reticulum, cathepsins bind to the insulin-like growth factor-II (IGF-II)/mannose 6-phosphate (M6P) receptor on the trans face of the Golgi complex and are transported in vesicles to the EL system.^22,23,24 The importance of lysosomal enzymes in the proper functioning of the EL system is underscored by the fact that altered synthesis, sorting, or targeting of lysosomal enzymes is the molecular basis of more than 40 inherited disorders associated with extensive neurodegeneration, mental retardation and often progressive cognitive decline.^19,25,26,27There is evidence that increased endosome volumes and/or levels of cathepsins, such as cathepsins B and D, can mediate cell death by inducing lysosomal destabilization and enzyme leakage into cell cytosol, as is observed during oxidative stress²⁸ and experimental brain ischemia in primates.²⁹ Conversely, a number of recent studies have shown that lysosomal enzyme expression/levels can be up-regulated in the absence of cell death as a compensatory mechanism to repair damage/injury.^30,31,32,33 Thus, it seems that lysosomal enzymes are not only involved in the degeneration of neurons but also in the protection of neurons against toxicity in a variety of experimental as well as pathological paradigms. Although the EL system, the major site of cholesterol accumulation in NPC1-deficient cells, has been suggested to play a critical role in the development of NPC pathology,^6,7,8 very little is known about the significance of lysosomal cathepsins in determining neuronal vulnerability associated with the disease. To address this issue, we measured age-related changes in the levels, distribution, and activity of cathepsins B and D in the hippocampus and cerebellum of Npc1^−/− and age-matched control mice. In parallel, we evaluated the levels and distribution of the IGF-II/M6P receptor in Npc1^−/− and control mice to establish whether factors regulating cathepsin bioavailability can also influence the development of pathological changes. In addition, using cultured mouse cortical neurons we determined the significance of cathepsins B and D in the degeneration of neurons after accumulation of cholesterol. Our results reveal that alterations in the levels/activity as well as subcellular distribution of the lysosomal enzymes may be one of the underlying mechanisms associated with the selective neuronal vulnerability observed in NPC pathology.     

Fatal Mycobacterium bovis BCG Infection in TNF-LT-α-Deficient Mice

Neutralization of TNF or disruption of TNF-R1 leads to fatal Mycobacterium bovis BCG infection. Here we used TNF-LT-α-deficient mice to test whether a complete disruption of TNF and LT-α reduces further host resistance to BCG infection. The bacterial burden especially in the lungs of TNF-LT-α-deficient mice was significantly increased and the mice succumbed to infection between 8 and 10 weeks. In the absence of TNF-LT-α the granulomatous response was severely impaired and delayed. The cells in the granulomas of TNF-LT-α-deficient mice expressed low levels of MHC class II and ICAM-1. They contained a few T cells and F4/80-positive macrophages expressing little iNOS and acid phosphatase activity. By contrast, the lethal action of endotoxin was dramatically reduced in BCG-infected TNF-LT-α-deficient mice. In summary, in the absence of TNF-LT-α the recruitment and activation of mononuclear cells in response to BCG infection were significantly delayed and reduced resulting in immature granulomas allowing uncontrolled fatal infection.     

Deficient Humoral Responses Underlie Susceptibility to Toxoplasma gondii in CD4-Deficient Mice

Resistance to infection with Toxoplasma gondii was studied in mice lacking CD4 expression. Such mice developed more brain cysts and survived for a shorter time than did wild-type controls after peroral infection with ME49 cysts. After immunization with the ts-4 strain of T. gondii, CD4-deficient mice exhibited impaired resistance to a challenge infection with virulent RH tachyzoites. Thus, deficient CD4 expression increases the susceptibility of mice to a primary peroral T. gondii infection with cysts and impairs their ability to be successfully vaccinated. CD8(+) T cells from blood or spleens of Toxoplasma-infected, CD4-deficient mice expressed markers of activation at frequencies similar to those of infected wild-type mice. Production of IFN-gamma in vitro was moderately depressed, and levels of Toxoplasma-specific immunoglobulin G2a in serum were substantially lower than in wild-type mice. Administration of Toxoplasma-immune serum to ts-4-vaccinated CD4-deficient mice significantly improved their resistance to RH challenge. Also, the survival of CD4-deficient mice chronically infected with ME49 was significantly prolonged by administration of immune serum. These results demonstrate that in addition to CD8(+) T cells and IFN-gamma, which are known to be critical for resistance, CD4(+) cells also contribute significantly to protection against chronic T. gondii infections and against challenge infections with highly virulent tachyzoites in immunized mice via their role as helper cells for production of isotype-switched antibodies.     

Trypanosoma musculi Infections in Normocomplementemic, C5-Deficient, and C3-Depleted Mice

The role of complement in host resistance to infection with Trypanosoma musculi was studied in normal, C5-deficient, and C3-depleted mice. Infections in normocomplementemic strains (CBA and B10.D2/n) were generally similar to those in strains genetically deficient in C5 (A and B10.D2/o). There were no differences in inhibition of reproduction, duration of infection, persistence of parasites in the kidneys, or resistance to reinfection. However, peak parasitemias in B10.D2/o mice were slightly greater than in B10.D2/n mice. In addition, B10.D2/o mice had slightly decreased serum levels of C1 early in the course of infection and of C3 early during the elimination of adult forms. These components were unchanged or increased in infections of B10.D2/n. Depletion of C3 and late-acting components in B10.D2/n mice by treatment with cobra venom factor during the reproductive stage of infection resulted in an increase of reproductive forms before the apparent development of ablastic immunity as well as slightly greater peak parasitemias when compared with those of untreated controls. Cobra venom factor treatment of B10.D2/o mice during the reproductive stage did not alter the course of infection. Cobra venom factor treatment of C3H mice during the adult stage prolonged infections by interfering with parasite elimination. It is concluded that complement-mediated lysis is not involved in control of T. musculi. It is not clear whether a C3-dependent function such as phagocytosis may facilitate elimination of the parasites. The major difference in degree of parasitemias among the various strains of mice studied is due to genetic factors rather than the levels of C3, C5, or late-acting complement components.     

Genes Influencing Resistance to Coccidioides immitis and the Interleukin-10 Response Map to Chromosomes 4 and 6 in Mice

Coccidioidomycosis is a fungal infection that is endemic in the southwestern United States. Infection is more severe in blacks and Filipinos, which suggests that there is a genetic basis for susceptibility to this infection in humans. We found that there is also a difference in resistance to Coccidioides immitis infection among inbred mouse strains: B6 mice are susceptible, while DBA/2 mice are resistant (T. N. Kirkland and J. Fierer, Infect. Immun. 40:912-916, 1983). In this paper we report the results of our efforts to map the genes responsible for resistance to this infection in mice. Mice were infected by intraperitoneal inoculation, and 15 days later the numbers of viable fungi in their lungs and spleens were enumerated. We also determined the amounts of interleukin-10 mRNA made in the infected lungs. These three phenotypes were mapped as quantitative traits by using the 26 available lines of recombinant inbred mice derived from a cross between B6 and DBA/2 mice. The best associations were those between the regions near the Lv locus on chromosome 4 and the Tnfr1 locus on chromosome 6. We then infected backcross mice [(B6 x DBA/2) x B6] and confirmed these associations; 14 of 16 (87%) mice that were heterozygous at both Lv and Tnfr1 were resistant to infection, whereas only 4 of 16 (25%) mice that were homozygous B6 at both loci were resistant. These are the first genetic loci to be associated with susceptibility to C. immitis, but there may be additional genes involved in murine resistance to this infection.     

Characteristics of Invasive Candidiasis in Gamma Interferon- and Interleukin-4-Deficient Mice: Role of Macrophages in Host Defense against Candida albicans

Murine models of invasive candidiasis were used to study the in vivo importance of gamma interferon (IFN-γ) and interleukin-4 (IL-4) in host defense against Candida albicans and to characterize the tissue inflammatory reactions, with special reference to macrophages (Mφ). Knockout (KO) IFN-γ-deficient (GKO) and IL-4-deficient (IL-4 KO) and C57BL/6 parental mouse strains were challenged intraperitoneally with 10⁸ C. albicans blastoconidia. Survival of GKO mice was significantly lower (16.7%) than that of C57BL/6 control (55.5%) and IL-4 KO (61.1%) animals, but was not correlated with the extent of organ colonization. Immunohistological analysis with a panel of myeloid and lymphoid markers revealed multiple renal abscesses, myocarditis, hepatitis, meningoencephalitis, and pneumonia in each strain, with a dominant presence of Mφ. In the absence of IFN-γ, C. albicans induced striking changes in the phenotype of alveolar Mφ and extensive perivascular lymphoid infiltrates in the lung. Impairment in nitric oxide production by peritoneal Mφ was shown only in GKO mice, and they produced Candida-specific immunoglobulin G (IgG), IgM, IgA, and IgG subclasses in lower titers. Our in vivo studies with KO mice elucidate a critical role for IFN-γ, but not IL-4, in host defense against C. albicans.Candida albicans is a common commensal organism in humans, and its importance as an opportunistic pathogen, particularly in immunocompromised patients, has continued to increase over the last two decades. According to the National Nosocomial Infections Surveillance System, the ratio of C. albicans isolates among nosocomial fungal infections increased from 52% to 63% in the 1980s (4). Phagocytic cell defects generally predispose to disseminated candidiasis; candidemia was calculated to result in 38% excess mortality and extend hospitalization by approximately 30 days (40). Besides the efforts to develop more effective and safer antifungal agents, a new therapeutic approach to augment the antifungal capacity of the host’s immune system should be investigated.The mechanisms of host defense and pathogenesis of candidiasis are not completely understood. Optimal phagocytosis of C. albicans requires opsonization; however, unopsonized yeast can be internalized by macrophages (Mφ) through the mannose receptor (21). Efficient killing of C. albicans by mononuclear phagocytes requires respiratory burst-associated toxic compounds (22), and recent data suggest that nitric oxide (NO) may also be involved in anticandidal functions of Mφ (5). Experimental evidence suggests that mononuclear phagocytes could play an important role in eradication of this pathogen, and their anticandidal activity can be augmented in vitro with granulocyte-Mφ and Mφ colony-stimulating factors and cytokines (no significant change could be measured in the level of specific immunoglobulin A [IgA] in serum or among the levels of interleukin-3 [IL-3] and gamma interferon [IFN-γ]) in both human and murine systems (23, 25, 28, 39).The in vivo benefit of cytokine treatment in disseminated candidiasis has not been established, and data from different murine models are controversial. Administration of IFN-γ has been reported to be associated with improved survival of mice after lethal challenge with C. albicans, which correlated with the anticandidal activity of peritoneal Mφ (28); another study showed a reduction in tissue fungal burden in IFN-γ-treated mice (19). However, in a different murine model, in vivo administration of IFN-γ resulted in increased susceptibility and organ colonization of four infected inbred strains (13). In vivo administration of IL-12, which has been reported to prime naive T cells for high IFN-γ expression and skew cytokine production toward a Th1-type response (38), did not modify the course of systemic candidiasis (32). In contrast, Th2-type cytokines IL-4 and IL-10 have been reported to exacerbate infection, and neutralization of IL-4 by specific antibody or soluble IL-4 receptor resulted in an enhanced production of Th1 cytokines, associated with increased resistance to systemic murine candidiasis (26, 30, 37). The controversial results of in vivo cytokine treatment may be the consequence of genetic differences among the infected strains and also the variation in protocols; the kinetics of cytokine production are influenced by several host and pathogen factors, and the effect of exogenous cytokine might depend on the condition of the infected host and stage of infection.Cytokine and receptor gene disruption strategies make it possible to examine the role of cytokines in host response to different pathogens directly. Recent studies showed an increased susceptibility of IFN-γ–receptor knockout (KO) mice to Mycobacterium bovis or Mycobacterium tuberculosis, but not to Schistosoma mansoni (1, 7, 8). Another study reported that disruption of the IFN-γ receptor gene was associated with higher susceptibility to Leishmania major and that IL-4 deficiency resulted in increased resistance, but only in certain inbred strains (17).Our study was undertaken to investigate the in vivo role of IFN-γ and IL-4 in disseminated C. albicans infection and characterize the tissue inflammatory cells by immunohistochemistry and by functional assays ex vivo. We demonstrate that IFN-γ, but not IL-4, is essential for survival in invasive candidiasis and show the dominant participation of Mφ in the inflammatory lesions of different tissues in KO as well as wild-type mice. In the absence of IFN-γ, a striking local immune regulatory alteration was observed in the lungs.     

Increased Capacity for Interleukin-2 Synthesis Parallels Disease Progression in Mice Infected with Leishmania major

Lymph node cells of BALB/c mice with progressive leishmaniasis produced sixfold more interleukin-2 (IL-2) in culture than those of healing C57BL/6 mice. IL-2 synthesis also increased in C57BL/6 mice made susceptible by IL-12 or gamma interferon deficiency. However, IL-2 mRNA levels in vivo did not reflect IL-2 production in vitro. Because IL-2 contributes to the pathogenesis of progressive leishmaniasis, the functional significance of these findings should be further explored.