The Cnes2 Locus on Mouse Chromosome 17 Regulates Host Defense against Cryptococcal Infection through Pleiotropic Effects on Host Immunity

The genetic basis of natural susceptibility to progressive Cryptococcus neoformans infection is not well understood. Using C57BL/6 and CBA/J inbred mice, we previously identified three chromosomal regions associated with C. neoformans susceptibility (Cnes1, Cnes2, and Cnes3). To validate and characterize the role of Cnes2 during the host response, we constructed a congenic strain on the C57BL/6 background (B6.CBA-Cnes2). Phenotypic analysis of B6.CBA-Cnes2 mice 35 days after C. neoformans infection showed a significant reduction of fungal burden in the lungs and spleen with higher pulmonary expression of gamma interferon (IFN-γ) and interleukin-12 (IL-12), lower expression of IL-4, IL-5, and IL-13, and an absence of airway epithelial mucus production compared to that in C57BL/6 mice. Multiparameter flow cytometry of infected lungs also showed a significantly higher number of neutrophils, exudate macrophages, CD11b⁺ dendritic cells, and CD4⁺ cells in B6.CBA-Cnes2 than in C57BL/6 mice. The activation state of recruited macrophages and dendritic cells was also significantly increased in B6.CBA-Cnes2 mice. Taken together, these findings demonstrate that the Cnes2 interval is a potent regulator of host defense, immune responsiveness, and differential Th1/Th2 polarization following C. neoformans infection.     

A Role for Interleukin-6 in Host Defense against Murine Chlamydia trachomatis Infection

Interleukin-6-deficient (IL-6^−/−) knockout mice had significantly increased Chlamydia trachomatis levels in lung tissue and increased mortality compared to B6129F₂/J controls early after intranasal infection. Gamma interferon production and chlamydia-specific antibody levels were consistent with a decreased but reversible Th1-like response in IL-6^−/− mice. IL-6 is needed for an optimal early host response to this infection.     

Morphine Disrupts Interleukin-23 (IL-23)/IL-17-Mediated Pulmonary Mucosal Host Defense against Streptococcus pneumoniae Infection

Streptococcus pneumoniae is a pathogen that causes serious respiratory disease and meningitis in the immunocompromised drug abuse population. However, the precise mechanisms by which drug abuse compromises the host immune defense to pulmonary S. pneumoniae infection is not fully understood. Using a well-established murine model of opiate abuse and S. pneumoniae lung infection, we explored the influence of morphine treatment on the interleukin-23 (IL-23)/IL-17 axis and related innate immunity. Impairment of early IL-23/IL-17 production caused by morphine treatment was associated with delayed neutrophil migration and decreased pneumococcal clearance. Furthermore, morphine treatment impaired MyD88-dependent IL-23 production in alveolar macrophages and dendritic cells in response to in vitro S. pneumoniae cell infection. Moreover, morphine treatment significantly inhibited the S. pneumoniae-induced phosphorylation of interferon response factor 3 (IRF3), ATF2, and NF-κBp65. T-cell receptor δ (TCRδ)-deficient mice showed a decrease in IL-17 production and a severely weakened capacity to clear lung S. pneumoniae infection. Finally, morphine treatment resulted in diminished secretion of antimicrobial proteins S100A9 and S100A8/A9 during early stages of S. pneumoniae infection. In conclusion, morphine treatment causes a dysfunction in IL-23-producing dendritic cells and macrophages and IL-17-producing γδT lymphocytes in response to S. pneumoniae lung infection. This leads to diminished release of antimicrobial S100A8/A9 proteins, compromised neutrophil recruitment, and more-severe infection.Immunocompromised individuals are at high risk for Streptococcus pneumoniae pulmonary infection (30). Previous studies have shown that opiate abuse causes immunosuppression by disrupting both innate and adaptive components of the immune system (26, 33). Opiate abuse is a critical risk factor for increasing susceptibility and severity of bacterial infection, including S. pneumoniae (31, 32). However, additional work is needed to understand the precise mechanisms by which opiate abuse increases the susceptibility to S. pneumoniae lung infection.Interleukin-23 (IL-23) has been recently identified as a cytokine closely related to IL-12 (5). The balance between IL-23 and IL-12 controls the outcome of inflammatory responses (16). IL-23 is secreted by activated macrophages and dendritic cells (DCs) and induces memory T-cell proliferation and is the critical factor required for T-cell IL-17 expression in response to bacterial challenge (3). IL-23 release leads to the production of IL-17. Furthermore, IL-17 promotes neutrophilic inflammation by upregulating CXC chemokines and hematopoietic growth factors (13). Several recent studies report the important role of IL-23 and IL-17 in the induction of neutrophil-mediated protective immune response against extracellular bacterial or fungal pathogens, such as Klebsiella pneumoniae (14), Pseudomonas aeruginosa (12), Porphyromonas gingivalis (34), Citrobacter rodentium (20), Bacteroides fragilis (9), and Escherichia coli (27).Generally, the IL-23/IL-17 axis plays an important role in the host defense against bacterial infections (11). Previous studies have demonstrated that IL-17 is critical for the recruitment of phagocytes that leads to the clearance of S. pneumoniae colonization from the mucosal surface of the nasopharynx (18, 35). However, whether the IL-23/IL-17 axis contributes to modulating the innate immunity in response to S. pneumoniae lung infection has not been addressed. Using a well-established opiate abuse and S. pneumoniae lung infection mouse model (31, 32), we demonstrate that S. pneumoniae induces IL-23 and IL-17 expression in the lungs as early as 2 h following infection. Morphine treatment causes a decrease in both IL-23 and IL-17 synthesis during the early stages of infection, leading to delayed neutrophil recruitment. This results in an increased bacterial burden within the lungs and the initiation of systemic disease.     

Nonredundant Roles of Interleukin-17A (IL-17A) and IL-22 in Murine Host Defense against Cutaneous and Hematogenous Infection Due to Methicillin-Resistant Staphylococcus aureus

Staphylococcus aureus is the leading cause of skin and skin structure infections (SSSI) in humans. Moreover, the high frequency of recurring SSSI due to S. aureus, particularly methicillin-resistant S. aureus (MRSA) strains, suggests that infection induces suboptimal anamnestic defenses. The present study addresses the hypothesis that interleukin-17A (IL-17A) and IL-22 play distinct roles in immunity to cutaneous and invasive MRSA infection in a mouse model of SSSI. Mice were treated with specific neutralizing antibodies against IL-17A and/or IL-22 and infected with MRSA, after which the severity of infection and host immune response were determined. Neutralization of either IL-17A or IL-22 reduced T cell and neutrophil infiltration and host defense peptide elaboration in lesions. These events corresponded with increased abscess severity, MRSA viability, and CFU density in skin. Interestingly, combined inhibition of IL-17A and IL-22 did not worsen abscesses but did increase gamma interferon (IFN-γ) expression at these sites. The inhibition of IL-22 led to a reduction in IL-17A expression, but not vice versa. These results suggest that the expression of IL-17A is at least partially dependent on IL-22 in this model. Inhibition of IL-17A but not IL-22 led to hematogenous dissemination to kidneys, which correlated with decreased T cell infiltration in renal tissue. Collectively, these findings indicate that IL-17A and IL-22 have complementary but nonredundant roles in host defense against cutaneous versus hematogenous infection. These insights may support targeted immune enhancement or other novel approaches to address the challenge of MRSA infection.     

Close Association between Pulmonary Disease Manifestation in Mycoplasma pneumoniae Infection and Enhanced Local Production of Interleukin-18 in the Lung, Independent of Gamma Interferon

To investigate pathophysiologies of Mycoplasma pneumoniae infection from an immunological point of view, we measured the levels of interleukin-18 (IL-18) (originally designated gamma interferon [IFN-γ]-inducing factor) in 19 serum samples from 10 patients with pneumonia without pleural effusion (ages 1 to 16 years), 3 serum and 13 pleural fluid samples from 11 patients with pleural effusions (ages 11 months to 15 years), and 18 serum and 27 cerebrospinal fluid samples from 24 patients with central nervous system complications (ages 1 to 15 years). IL-18 was measured by a commercially available enzyme-linked immunosorbent assay kit (MBL, Nagoya, Japan). In addition, the levels of tumor necrosis factor alpha, IFN-γ, IL-6, IL-12, and KL-6 (a mucin-like glycoprotein expressed on type 2 pneumocytes) were measured in selected samples. The results concerning pleural effusions showed that elevated levels of IL-18 in pleural fluid, but not in serum, were solely associated with a sustained fibrotic change of the lung on chest roentgenography which might represent a pathological feature of intraluminal organization. All the pleural fluid samples with elevated levels of IL-18 were positive by PCR for M. pneumoniae DNA. There was no association between IL-18 and IFN-γ levels in serum or in the pleural fluid. On the other hand, elevated levels of IL-18 in serum, but not in cerebrospinal fluid samples, were observed in the cases complicated by central nervous system involvement, including profound brain dysfunction with seizures. Our study demonstrated that M. pneumoniae can induce IL-18 and that the enhanced local production of IL-18 in the lung is closely associated with pulmonary disease manifestation.     

Stimulation of the Molecule 4-1BB Enhances Host Defense against Listeria monocytogenes Infection in Mice by Inducing Rapid Infiltration and Activation of Neutrophils and Monocytes

The tumor necrosis factor receptor family molecule 4-1BB (CD137) has diverse roles in adaptive and innate immune responses. However, little is known of its role in bacterial infections. Previously, we showed that 4-1BB-deficient mice have enhanced susceptibility to Listeria monocytogenes infection, and mice pretreated with agonistic anti-4-1BB antibody (3E1) were much more resistant to L. monocytogenes infection than mice treated with control antibody. In this study, we report that stimulating 4-1BB by administering 3E1 in the early phase of L. monocytogenes infection is critical for promoting the survival of mice by inducing rapid infiltration of neutrophils and monocytes into L. monocytogenes-infected livers. The levels of tumor necrosis factor alpha, interleukin 6, and monocyte chemoattractant protein 1 in the livers of 3E1-treated mice increased as early as 30 min postinfection and peaked by 1 to 2 h, while those in mice treated with control antibody started to increase only at 16 h postinfection. Monocytes and neutrophils from the 3E1-treated mice had higher levels of activation markers, phagocytic activity, and reactive oxygen species than those from control mice. In vitro stimulation of 4-1BB induced the production of the inflammatory cytokines/chemokines of neutrophils, but not those of monocytes. These results suggest that 4-1BB stimulation of neutrophils in the early phase of L. monocytogenes infection causes rapid production of inflammatory cytokines/chemokines and that the subsequent infiltration of neutrophils and monocytes is crucial for eliminating the infecting L. monocytogenes.Listeria monocytogenes is a gram-positive intracellular pathogen responsible for listeriosis, a life-threatening infection in immunocompromised patients, newborns, or elderly people. The murine model of listeriosis has been used to investigate immune responses to bacterial infection (11). L. monocytogenes infects both phagocytic and nonphagocytic cells, escapes from intracellular vacuoles into the cytosol by secreting listeriolysin, replicates, and spreads to neighboring cells by actin-based motility. Intravenous (i.v.) bacteria are rapidly cleared from the bloodstream; most of them are taken up by the liver and spleen within 10 min of infection. Although T-cell-dependent adaptive immune responses are required to clear L. monocytogenes infection, they take several days to develop. Therefore, early control of the infection is critical for the survival of mice and primarily depends on innate immunity (5); indeed, it has even been shown that lymphocytes are detrimental during the early innate immune response to L. monocytogenes (4). Neutrophils and monocytes/macrophages are thought to be the main cells responsible for killing L. monocytogenes during the innate immune response. Thus, depletion of neutrophils from mice by using anti-Gr-1 antibodies greatly enhances their susceptibility to infection with L. monocytogenes (35), and the increased number of neutrophils resulting from deficiency in LFA-1 in mice confers resistance to listeriosis (30). Recruitment of monocytes is also essential to eradicate L. monocytogenes from infected mice, as indicated by reports that blocking complement receptor 3 of monocytes exacerbates listeriosis (38), and CC chemokine receptor 2-deficient mice have defects in the emigration of monocytes from the bone marrow and are highly susceptible to L. monocytogenes infection (22, 40). Although recruitment of neutrophils/monocytes is critical for eradication of L. monocytogenes during the early phase of infection, the molecular mechanisms of bacterial killing and the receptor molecules responsible for activation of neutrophils/monocytes against the bacteria are not clearly defined.The 4-1BB (CD137) receptor, a member of the tumor necrosis factor receptor superfamily (TNFRSF 9), is expressed on activated T cells (43), and the in vivo effects of 4-1BB activation on T-cell-dependent immune responses, such as eradication of established tumors (29), antiviral responses (1), and enhancement of the memory pool of antigen-specific CD8⁺ T cells (34), have been well defined. However, recent findings indicate that 4-1BB activation also plays an important role in other immune cells. 4-1BB is constitutively expressed on innate immune cells, including neutrophils (24), dendritic cells (10), natural killer (NK) cells (28), mast cells (31), and eosinophils (9). Its activation results in proliferation, gamma interferon secretion, and tumor rejection by NK cells (28); the production of cytokines by dendritic cells and the expression of costimulatory molecules on these cells (10); proliferation, survival, and cytokine production in human monocytes (21); and abrogation of the granulocyte-macrophage colony-stimulating factor-mediated antiapoptotic functions of human neutrophils (17). Previously, we reported that 4-1BB-deficient (4-1BB^−/−) mice are very susceptible to L. monocytogenes infection because the antibacterial activity of their neutrophils is defective (24). Furthermore, pretreatment of agonistic anti-4-1BB monoclonal antibody (MAb) markedly increased the survival of L. monocytogenes-infected 4-1BB^+/+ mice. In this study, we further characterized the mechanism of 4-1BB-mediated protection of L. monocytogenes-infected mice. We found that activation of 4-1BB in the early phase of L. monocytogenes infection rapidly stimulated the induction of proinflammatory cytokines/chemokines and the subsequent recruitment and activation of neutrophils and monocytes into the bacterium-infected livers.     

Protection against Intestinal Amebiasis by a Recombinant Vaccine Is Transferable by T Cells and Mediated by Gamma Interferon

We have previously shown that vaccination with purified Entamoeba histolytica Gal/GalNAc lectin or recombinant subunits can protect mice from intestinal amebiasis upon intracecal challenge. In this study, we demonstrated with adoptive-transfer experiments that this lectin vaccine protection is mediated by T cells but not serum. The cell-mediated immune (CMI) response was characterized by significant gamma interferon (IFN-γ), interleukin 12 (IL-12), IL-2, IL-10, and IL-17 production. To move toward a human vaccine, we switched to a recombinant protein and tested a range of adjuvants and routes appropriate for humans. We found that subcutaneous delivery of LecA with IDRI''s adjuvant system EM014 elicited a potent Th1-type CMI profile and provided significant protection, as measured by culture negativity (79% efficacy); intranasal immunization with cholera toxin provided 56% efficacy; and alum induced a Th2-type response that protected 62 to 68% of mice. Several antibody and CMI cytokine responses were examined for correlates of protection, and prechallenge IFN-γ⁺ or IFN-γ-, IL-2-, and tumor necrosis factor alpha-triple-positive CD4 cells in blood were statistically associated with protection. To test the role of IFN-γ in LecA-mediated protection, we neutralized IFN-γ in LecA-immunized mice and found that it abrogated the protection conferred by vaccination. These data demonstrate that CMI is sufficient for vaccine protection from intestinal amebiasis and reveal an important role for IFN-γ, even in the setting of alum.The enteric protozoan parasite Entamoeba histolytica is the causative pathogen of amebic dysentery and liver abscess that affects millions of people worldwide. Bangladeshi children experience a 40% annual incidence of E. histolytica infection (24), and evidence of prior E. histolytica infection can be detected in 8.4% of the general population in Mexico (6). Despite the availability of effective antibiotics, the World Health Organization estimates that up to 100,000 deaths occur annually, highlighting the need for alternate approaches to control amebiasis. One approach is to develop a vaccine to prevent intestinal infection (26).Several vaccine candidates for amebiasis have been proposed (48), including the serine-rich E. histolytica protein, peroxiredoxin, the EhCP112 molecule, and the galactose/N-acetyl-d-galactosamine-inhibitable lectin (Gal/GalNAc lectin). A significant body of work has focused on the latter: vaccination with either parasite-purified Gal/GalNAc lectin (10, 29, 32, 38, 40) or recombinant lectin subunits has provided protection in rodent models against amebic liver abscess and amebic colitis (29, 37, 46, 47, 53). Although these results are encouraging, two limitations remain. First, in most of these vaccine studies, the adjuvants and delivery routes are not compatible with eventual use in humans. Second, the mechanisms of amebiasis vaccine-mediated protection are still not fully understood. For instance, in the intestinal model, there was an association between the presence of an antiparasite lectin fecal immunoglobulin A (IgA) response and subsequent protection, but the association did not extend to the recombinant antigen, and fecal IgA-negative mice remained statistically protected, suggesting that other immune mechanisms exist (29). Indeed, there is increasing evidence for a role of cell-mediated immunity (CMI) in protection from intestinal amebiasis (14, 20, 28). We have found that gamma interferon (IFN-γ), the canonical Th1 cytokine, can clear intestinal amebic infection in CBA mice (21). In this study, we demonstrate for the first time that CMI plays a critical role in lectin-elicited protective immunity to intestinal amebic infection. A variety of vaccine adjuvants and delivery routes were tested for their effectiveness in protection and in eliciting CMI, and the tests demonstrated a clear role for CMI and IFN-γ in lectin-based vaccine protection.     

Protection against Mycobacterium tuberculosis Infection by CD8+ T Cells Requires the Production of Gamma Interferon

The role of CD8 T cells in controlling Mycobacterium tuberculosis infections in mice was confirmed by comparing the levels of growth of the organism in control, major histocompatibility complex class II knockout, and athymic mice and by transferring T-cell populations into athymic mice. By using donor mice which were incapable of making gamma interferon (IFN-γ), it was shown that IFN-γ production was essential for CD8 cell mediation of protective immunity against M. tuberculosis.

Cell-mediated immunity is crucial for the control of mycobacterial infections. Athymic mice (4) and mice whose T cells have been depleted (22, 23) are much more susceptible to infection with mycobacteria than euthymic or unmanipulated mice. However, the contributions of the different components of the T-cell response are unclear. CD4 T cells are thought to play a major role in controlling infections with the primary human tubercle bacillus, Mycobacterium tuberculosis; individuals with reduced CD4 counts, from infection with human immunodeficiency virus, for example, are known to be more susceptible to M. tuberculosis infections (12). Activation of CD4 cells by antigen in association with major histocompatibility complex (MHC) class II molecules results in clonal expansion and the production of cytokines, most notably gamma interferon (IFN-γ), which activate macrophages so that they become mycobactericidal. Mice with deletions of the IFN-γ gene are much more susceptible to M. tuberculosis infection than wild-type mice (5, 9). However, in addition to CD4 cells, other components of the cell-mediated response are thought to play roles in controlling infection with M. tuberculosis. For example, CD8 T cells have been shown to be involved (20, 24): β2 microglobulin-deficient knockout mice, which lack an effective CD8 response, show increased susceptibility to M. tuberculosis infection (10). Other cell types, such as T cells bearing the γ/δ T-cell receptor (19) and NK cells (1), are also thought to have roles in protection against intracellular bacteria, while a number of T cells with novel phenotypes and unknown functions have been shown to recognize mycobacterial antigens (2, 28).CD8 T cells are known to contribute to the protective response against M. tuberculosis, but the mechanism(s) by which they exert this protective effect is unknown. CD8 T cells produce a range of cytokines, including IFN-γ (11, 17, 25, 26), but their primary role is thought to be cytotoxic. However, it has recently been shown that mice with a targeted disruption in either the perforin gene or the granzyme gene and mice which are Fas receptor defective are no more susceptible to infection with M. tuberculosis than are wild-type mice (6, 16). Since perforin (13, 18) and Fas-Fas ligand interactions (21, 27, 31) are thought to be the primary mechanisms of cytotoxicity mediated by CD8 T cells, such cells may contribute their antimycobacterial activity through noncytotoxic pathways.In this study, we have used MHC class II-deficient mice and athymic mice to confirm the role of non-CD4 T-cell-mediated mechanisms in protection against M. tuberculosis infection. Using transfer of purified CD4 and CD8 cells into athymic mice, we have demonstrated that these cells contribute equally to protective immunity in this system. However, by using mice with deletions of the IFN-γ gene as T-cell donors, we have shown that production of IFN-γ is required in order for CD8 T cells to exert their antimycobacterial effect.In preliminary experiments, the levels of growth of M. tuberculosis in MHC class II knockout, athymic, and normal mice were compared. MHC class II knockout (Aβ^−/−) mice were obtained as a breeding nucleus (kindly provided by D. Gray, Hammersmith Hospital, London, United Kingdom, with permission from D. Mathis, Institut National de la Santé et de la Recherche Médicale). These mice were bred from heterozygous (Aβ^+/−) parents and genotyped as described previously (7). Heterozygous littermates were used as controls. Stock cultures of M. tuberculosis H₃₇Rv were grown in Dubos 7H9 broth for 14 days, and then they were aliquoted and stored in liquid nitrogen. For infection, aliquots were thawed, diluted in phosphate-buffered saline, and inoculated intraperitoneally into mice. The infection was monitored by removing the lungs and spleens of infected mice at various intervals; the baseline level of infection of each tissue was estimated by harvesting organs from the mice 18 h after infection and determining viable counts. The tissues were weighed and homogenized by shaking with 2-mm-diameter glass beads in chilled saline with a Mini-Bead Beater (Biospec Products, Bartlesville, Okla.), and 10-fold dilutions of the suspension were plated onto Dubos 7H11 agar with Dubos oleic albumic complex supplement (Difco Laboratories, Surrey, United Kingdom). Numbers of CFU were determined after the plates had been incubated at 37°C for approximately 20 days. The results are shown in Fig. ​Fig.1A1A and B. In control mice, there was a transient increase in bacterial counts in the spleen, followed by a steady decline over 60 days and then by a levelling out of the infection at approximately 10⁴ CFU per g of tissue. In MHC class II knockout mice, there was an initial growth of the infection over the first 60 days, followed by a plateau phase during which the infection appeared to be controlled but was significantly more severe than in wild-type mice (Fig. ​(Fig.1A).1A). In lung tissue (Fig. ​(Fig.1B),1B), a similar pattern emerged, except that in the MHC class II knockout mice, control of the infection broke down in some of the mice after about 60 days, when there was a sudden increase in bacterial counts. By day 80, counts had reached approximately 10⁷ CFU per g of tissue, a 10,000-fold increase over the counts seen in wild-type mice. Open in a separate windowFIG. 1Growth of M. tuberculosis in the tissues of MHC class II knockout, control, and athymic mice. (A and B) Growth in spleens and lungs, respectively, of MHC class II knockout mice (•) and their wild-type littermates (▪). (C and D) Growth in spleens and lungs, respectively, of MHC class II knockout (•) and athymic (▴) mice. Data are the geometric means ± the standard errors of the means for three to five mice. An asterisk indicates a significant difference between values for MHC class II knockout and control mice (P < 0.05 by Students’ t test). A double asterisk denotes that at the indicated time, all remaining mice in the group were killed because of the widely disseminated nature of the infection.These results emphasize the importance of the MHC class II-CD4 T-cell pathway in controlling M. tuberculosis infection. However, in spite of the fact that after the first few days of infection there was always a highly significant difference between the level of viable M. tuberculosis organisms in MHC class II knockout mice and the level in control mice, some control of bacterial multiplication did appear to occur in the MHC class II knockout mice. In order to demonstrate that this apparent partial control of the infection in MHC class II knockout mice was mediated by T cells, we compared growth in these mice with growth in athymic mice. Athymic (nude) BALB/c mice were obtained from a breeding colony at the National Institute for Medical Research. Athymic and MHC class II knockout mice were infected intraperitoneally, and the infections were monitored as described above. Whereas the MHC class II knockout mice were again able to control the infection to some degree, growth in athymic mice was unchecked and the mice had to be killed at 40 days because of overwhelming infection (Fig. ​(Fig.1C1C and D).These results confirm the importance of CD4 cells in controlling M. tuberculosis infections but also suggest that a contribution is made by non-CD4-mediated mechanisms. It has previously been shown that depletion of CD8 cell populations in mice with anti-CD8 antibodies (20) or abolition of a CD8 response by disruption of the β2 microglobulin gene (10) renders mice highly susceptible to infection with M. tuberculosis. CD8 T cells have also been implicated in human tuberculosis; CD8⁺ T cells with specificity for mycobacterium-pulsed target cells have been described (14, 32), and an individual with recurrent tuberculosis was found to have a specific reduction in CD8 T cells (3).In order to investigate the contribution of CD8 T cells to the control of M. tuberculosis infections in mice, total spleen cells, CD4 T cells, and CD8 T cells were transferred from control BALB/c mice into infected athymic BALB/c mice. Splenocytes were incubated in hypotonic medium to lyse erythrocytes and washed twice. To obtain highly purified populations of CD4 and CD8 cells, cell suspensions were enriched by negative selection with T-cell-subset columns (R & D Systems Inc., Minneapolis, Minn.) according to the manufacturer’s instructions. The resulting populations were >90% CD4 or CD8 T cells, as determined by flow cytometric analysis. The cells were washed, resuspended in sterile saline, and injected intravenously such that recipient mice received 5 × 10⁶ cells. The mice were then infected with M. tuberculosis, and organs were harvested 21 days later for CFU counts. The results of a typical experiment are shown in Fig. ​Fig.2.2. In athymic mice which had not received any transferred cells, the infection reached approximately 10⁷ CFU per g in the lung (Fig. ​(Fig.2A)2A) and 10⁸ CFU per g in the spleen (Fig. ​(Fig.2B).2B). Transfer of total spleen cells from naive BALB/c mice reduced the number of CFU 100- to 1,000-fold in both tissues. It appeared that CD4 and CD8 T cells contributed approximately equally to the observed protection. Open in a separate windowFIG. 2Infection of athymic mice with M. tuberculosis following transfer of splenocytes from euthymic mice. (A and B) Results for the lungs and spleen, respectively, of mice infected intravenously with approximately 10⁶ CFU 21 days prior to harvest. Transfer of cells was carried out 24 h before infection. Data are the means ± the standard errors of the means for three to five mice. Mice received either no cells, total spleen cells, CD4 cells, or CD8 cells. All three groups of mice which received cells showed significantly reduced CFU counts compared to controls (P < 0.05 by Student’s t test).The mechanism by which CD8 T cells exert this antimycobacterial response is not understood. It has been suggested that the cytotoxicity of mycobacterium-laden target cells could be involved, perhaps through the release of M. tuberculosis bacilli from ineffective macrophages to cells with greater antimycobacterial potential (15). However, perforin or granzyme knockout mice and Fas receptor-defective mice, when infected, did not display any increased susceptibility to infection, compared to wild-type controls (6, 16). Interestingly, both the perforin knockout mice and the Fas receptor-defective mice had elevated levels of cytokines, including IFN-γ, in the absence of infection, and levels in infected mice were similar to those seen in wild-type mice (16). Thus, neither perforin-, granzyme-, nor Fas-mediated cytotoxicity appeared to be involved in the control of these experimental infections (6, 16). Conversely, however, Silva and colleagues (29) produced CD8⁺ T-cell clones which were capable of conferring protection against M. tuberculosis in recipient mice, and the level of protection correlated with the level of cytotoxic activity rather than with the level of IFN-γ secretion.In a recent study of human cytotoxic cells with mycobacterial specificity, it was found that CD4⁻ CD8⁻ T cells lysed macrophages through a Fas-Fas ligand interaction but the lysis was not associated with mycobacterial killing, whereas CD8⁺ T-cells lysed macrophages by a Fas-independent pathway and the lysis resulted in the killing of mycobacteria (30). The human T-cell lines used for these experiments were unusual in that they were CD1 restricted.Since CD8 T cells were clearly able to confer significant levels of protection against M. tuberculosis in our cell transfer model, we next investigated the role of IFN-γ in this protection. Again athymic mice were recipients of either total spleen cells or CD8 cells. This time, however, donor mice were either normal BALB/c mice or IFN-γ knockout mice (8) and recipient mice received 3 × 10⁶ cells. The results (Fig. ​(Fig.3)3) clearly demonstrate the requirement for IFN-γ. Transfer of total spleen cells or CD8 T cells from normal mice gave protection, although the level of protection was slightly lower than that seen in the previous experiment (Fig. ​(Fig.2).2). This was probably because the number of cells transferred was lower (3 × 10⁶ rather than 5 × 10⁶). However, the protection seen in both organs was significant (P < 0.05). Importantly, transfer of cells from IFN-γ knockout mice gave no protection. Open in a separate windowFIG. 3Infection of athymic mice with M. tuberculosis following transfer of splenocytes from control BALB/c and IFN-γ knockout (IFN-γ −VE) BALB/c mice. (A and B) Results for the lungs and spleen, respectively. The experimental design was identical to that for Fig. ​Fig.2.2. Mice received either no cells, total spleen cells from wild-type BALB/c mice, CD8 cells from BALB/c mice, total spleen cells from IFN-γ knockout mice, or CD8 cells from IFN-γ knockout mice. Mice which received cells from control BALB/c mice (total spleen or CD8 cells) showed significantly reduced CFU counts compared to naive athymic mice (P < 0.05); there were no significant differences between values for naive athymic mice and mice which received either total spleen cells or CD8 cells from IFN-γ knockout BALB/c mice.Thus, the results reported in this study confirm the role of CD8 T cells in the control of M. tuberculosis infections in mice. We have also demonstrated that this control requires the ability of the CD8 cells to produce IFN-γ, suggesting that such cells may exert their effects through classical cytokine-mediated macrophage activation rather than through a cytotoxic mechanism. The recent demonstration that human CD1-restricted CD8 T cells were able to kill mycobacteria in vitro through a cytotoxicity-mediated pathway (30) suggests that different subpopulations of CD8 cells may have different effector mechanisms; since no murine equivalent of the CD1-restricted CD8 T cell has been described, this mechanism may be absent in mice. Alternatively, the results reported earlier for murine CD8 T-cell lines (29) or human CD8, CD1-restricted T-cell lines (30) may reflect the activity of primed or memory T cells, whereas the results reported in the present study reflect the activity of unprimed cells. Primed CD8 T cells have been shown to be hyperreactive to antigenic challenge in vitro and may employ different effector mechanisms. That production of IFN-γ by CD8 T cells is required in order to control infection has also been reported for viral infections (11, 26), where cytotoxicity has long been thought to be the major mechanism of CD8-mediated antiviral activity. IFN-γ and other cytokines have been shown to be major components of the mechanism by which hepatitis B virus is controlled in mice by CD8 cells without the killing of hepatocytes (11). The results reported in this study demonstrate that IFN-γ is essential for CD8-mediated protection against M. tuberculosis infection in mice.     

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.     

Protective Immunity against Recurrent Staphylococcus aureus Skin Infection Requires Antibody and Interleukin-17A

Although many microbial infections elicit an adaptive immune response that can protect against reinfection, it is generally thought that Staphylococcus aureus infections fail to generate protective immunity despite detectable T and B cell responses. No vaccine is yet proven to prevent S. aureus infections in humans, and efforts to develop one have been hampered by a lack of animal models in which protective immunity occurs. Our results describe a novel mouse model of protective immunity against recurrent infection, in which S. aureus skin and soft tissue infection (SSTI) strongly protected against secondary SSTI in BALB/c mice but much less so in C57BL/6 mice. This protection was dependent on antibody, because adoptive transfer of immune BALB/c serum or purified antibody into either BALB/c or C57BL/6 mice resulted in smaller skin lesions. We also identified an antibody-independent mechanism, because B cell-deficient mice were partially protected against secondary S. aureus SSTI and adoptive transfer of T cells from immune BALB/c mice resulted in smaller lesions upon primary infection. Furthermore, neutralization of interleukin-17A (IL-17A) abolished T cell-mediated protection in BALB/c mice, whereas neutralization of gamma interferon (IFN-γ) enhanced protection in C57BL/6 mice. Therefore, protective immunity against recurrent S. aureus SSTI was advanced by antibody and the Th17/IL-17A pathway and prevented by the Th1/IFN-γ pathway, suggesting that targeting both cell-mediated and humoral immunity might optimally protect against secondary S. aureus SSTI. These findings also highlight the importance of the mouse genetic background in the development of protective immunity against S. aureus SSTI.     

CD8+ T Cell Exhaustion,Suppressed Gamma Interferon Production,and Delayed Memory Response Induced by Chronic Brucella melitensis Infection

Brucella melitensis is a well-adapted zoonotic pathogen considered a scourge of mankind since recorded history. In some cases, initial infection leads to chronic and reactivating brucellosis, incurring significant morbidity and economic loss. The mechanism by which B. melitensis subverts adaptive immunological memory is poorly understood. Previous work has shown that Brucella-specific CD8⁺ T cells express gamma interferon (IFN-γ) and can transition to long-lived memory cells but are not polyfunctional. In this study, chronic infection of mice with B. melitensis led to CD8⁺ T cell exhaustion, manifested by programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) expression and a lack of IFN-γ production. The B. melitensis-specific CD8⁺ T cells that produced IFN-γ expressed less IFN-γ per cell than did CD8⁺ cells from uninfected mice. Both memory precursor (CD8⁺ LFA1^HI CD127^HI KLRG1^LO) and long-lived memory (CD8⁺ CD27^HI CD127^HI KLRG1^LO) cells were identified during chronic infection. Interestingly, after adoptive transfer, mice receiving cells from chronically infected animals were able to contain infection more rapidly than recipients of cells from acutely infected or uninfected donors, although the proportions of exhausted CD8⁺ T cells increased after adoptive transfer in both challenged and unchallenged recipients. CD8⁺ T cells of challenged recipients initially retained the stunted IFN-γ production found prior to transfer, and cells from acutely infected mice were never seen to transition to either memory subset at all time points tested, up to 30 days post-primary infection, suggesting a delay in the generation of memory. Here we have identified defects in Brucella-responsive CD8⁺ T cells that allow chronic persistence of infection.     

Inhibitory Effects of Histamine on Interleukin 2 and Gamma Interferon Production by Different Human T Helper Cell Subsets

We have previously demonstrated that histamine can inhibit human helper T cells by direct interaction with these cells. It has now been investigated whether histamine inhibits lymphokine production by various subsets of CD4+ human T cells separated with the Leu-8 (p80) and Leu-18 (anti-CD45R;p220) monoclonal antibodies (MoAb). Histamine was shown to suppress to a similar extent the production of interleukin 2 (IL-2) and gamma interferon (IFN-gamma) by Leu 3+, Leu 3+8+, and Leu 3+8- cell subsets. Mitogen-activated, unseparated Leu 3+ and purified Leu 3+8- cells produced maximal amounts of IL-2 after 24 h and IFN-gamma after 72 h of culture. In contrast, the Leu 3+18+ subset produced no IL-2 after 24 h, and maximal amounts of IL-2 no sooner than 48 h of culture, and only small amounts of IFN-gamma during the entire culture period of 96 h. Histamine suppressed the production of IL-2 by both subsets, both when produced early (after 24 h), as in the case of the Leu 3+18- subset, and late (after 48 h of culture), as for the Leu 3+18+ subset. The IFN-gamma production by the Leu 3+ and Leu 3+18- cells and the marginal production by Leu 3+18+ cells were significantly suppressed by histamine. Dual staining with Leu 8 and Leu 18 MoAb demonstrated that the Leu 18- cell compartment included both Leu 8+ and Leu 8- cells. It was shown that the inhibitory effect of histamine on the early production of IL-2 and the major production of IFN-gamma by T helper cells is mediated via action on both the Leu 3+18-8- and the Leu 3+18-8+ cells. The inhibitory effect of histamine on the late production of IL-2 is mediated mainly via action on Leu-18+ cells.     

Lactobacilli and Streptococci Induce Interleukin-12 (IL-12), IL-18, and Gamma Interferon Production in Human Peripheral Blood Mononuclear Cells

Human peripheral blood mononuclear cells (PBMC) were stimulated with three nonpathogenic Lactobacillus strains and with one pathogenic Streptococcus pyogenes strain, and cytokine gene expression and protein production were analyzed. All bacteria strongly induced interleukin-1β (IL-1β), IL-6, and tumor necrosis factor alpha mRNA expression and protein production. S. pyogenes was the most potent inducer of secretion of IL-12 and gamma interferon (IFN-γ), and two of three Lactobacillus strains induced IL-12 and IFN-γ production. All strains induced IL-18 protein production. IL-10 and IL-4 production was induced weakly and not at all, respectively. Our data show that nonpathogenic lactobacilli and pathogenic streptococci can induce Th1 type cytokines IL-12, IL-18, and IFN-γ in human PBMC.     

Clearance of Staphylococcus aureus Nasal Carriage Is T Cell Dependent and Mediated through Interleukin-17A Expression and Neutrophil Influx

The anterior nares of humans are the major reservoir for Staphylococcus aureus colonization. Approximately 20% of the healthy human population is persistently and 80% is intermittently colonized with S. aureus in the nasal cavity. Previous studies have shown a strong causal connection between S. aureus nasal carriage and increased risk of nosocomial infection, as well as increased carriage due to immune dysfunction. However, the immune responses that permit persistence or mediate clearance of S. aureus on the nasal mucosa are fundamentally undefined. In this study, we developed a carriage model in C57BL/6J mice and showed that clearance begins 14 days postinoculation. In contrast, SCID mice that have a deficient adaptive immune response are unable to eliminate S. aureus even after 28 days postinoculation. Furthermore, decolonization was found to be T cell mediated but B cell independent by evaluating carriage clearance in T-cell receptor β/δ (TCR-β/δ) knockout (KO) and IgH-μ KO mice, respectively. Upregulation of the cytokines interleukin 1β (IL-1β), KC (also termed CXC ligand 1 [CXCL1]), and IL-17A occurred following inoculation with intranasal S. aureus. IL-17A production was crucial for clearance, since IL-17A-deficient mice were unable to effectively eliminate S. aureus carriage. Subsequently, cell differential counts were evaluated from nasal lavage fluid obtained from wild-type and IL-17A-deficient colonized mice. These counts displayed IL-17A-dependent neutrophil migration. Antibody-mediated depletion of neutrophils in colonized mice caused reduced clearance compared to that in isotype-treated controls. Our data suggest that the Th17-associated immune response is required for nasal decolonization. This response is T cell dependent and mediated via IL-17A production and neutrophil influx. Th17-associated immune responses may be targeted for strategies to mitigate distal infections originating from persistent S. aureus carriage in humans.