Th1 and Th17 cells regulate innate immune responses and bacterial clearance during central nervous system infection

Brain abscesses arise following parenchymal infection with pyogenic bacteria and are typified by inflammation and edema, which frequently results in a multitude of long-term health problems. The impact of adaptive immunity in shaping continued innate responses during late-stage brain abscess formation is not known but is important, because robust innate immunity is required for effective bacterial clearance. To address this issue, brain abscesses were induced in TCR αβ knockout (KO) mice, because CD4(+) and NKT cells represented the most numerous T cell infiltrates. TCR αβ KO mice exhibited impaired bacterial clearance during later stages of infection, which was associated with alterations in neutrophil and macrophage recruitment, as well as perturbations in cytokine/chemokine expression. Adoptive transfer of either Th1 or Th17 cells into TCR αβ KO mice restored bacterial burdens and innate immune cell infiltrates to levels detected in wild-type animals. Interestingly, adoptively transferred Th17 cells demonstrated plasticity within the CNS compartment and induced distinct cytokine secretion profiles in abscess-associated microglia and macrophages compared with Th1 transfer. Collectively, these studies identified an amplification loop for Th1 and Th17 cells in shaping established innate responses during CNS infection to maximize bacterial clearance and differentially regulate microglial and macrophage secretory profiles.     

Antigen-specific responses accelerate bacterial clearance in the bladder

Urinary tract infections (UTIs) cause patient morbidity and have a substantial economic impact. Half of all women will suffer a UTI at least once, and 25% of these women will have recurrent infections. That 75% of previously infected women do not become reinfected strongly suggests a role for an adaptive immune response. The goal of this study was to characterize the adaptive immune responses to uropathogenic Escherichia coli (UPEC), the predominant uropathogen. A novel murine model of UTI reinfection was developed using the prototypic cystitis UPEC isolate NU14 harboring a plasmid encoding OVA as a unique antigenic marker. Bacterial colonization of the bladder was quantified following one or more infections with NU14-OVA. Animals developed anti-OVA serum IgG and IgM titers after the initial infection and marked up-regulation of activation markers on splenic T cells. We observed a 95% reduction in bacterial colonization upon reinfection, and splenic leukocytes showed Ag-specific proliferation in vitro. Adoptive transfer of splenic T cells or passive transfer of serum from previously infected mice protected naive syngeneic mice from UPEC colonization. These findings support our hypothesis that adaptive immune responses to UPEC protect the bladder from reinfection and form the basis of understanding susceptibility to recurrent UTI in women.     

Enhancement of the innate immune response of bladder epithelial cells by Astragalus polysaccharides through upregulation of TLR4 expression

The innate host defenses at mucosal surfaces are critical in the early stages of urinary tract bacterial infection. Recent studies have shown that uroepithelial cells aid innate immune cells in fighting off infection, although the exact mechanism by which the uroepithilium participates in immunity remains unclear. TLR4 has been implicated to possess antimicrobial activities specific for bladder epithelial cells (BECs). TLR4 promotes secretion of IL-6 and IL-8, mediates inhibition of bladder epithelial cell (BEC) bacterial invasion, and mediates expulsion of uropathogenic Escherichia coli from BECs. In this study, cultured 5637 cells and Balb/C mice were treated with Astragalus polysaccharides (APS) against invading E. coli. To determine the contribution of TLR4 upregulation to immune response, TLR4 expression and bacterial colony numbers were monitored. After 24 h incubation, only 5637 cells treated with 500 μg/ml APS expressed higher levels of TLR4 compared with the untreated group. However, after 48 h, all 5637 cells treated by APS showed higher levels of TLR4 expression than the control cells. The TLR4 expression in the bladder and macrophages mice that received APS was higher than that in the controls. Bacterial colonization in 5637 cells and the bladders of mice treated with APS was significantly reduced compared with the controls. These results demonstrate that at certain concentrations, APS can induce increased TLR4 expression in vivo and in vitro. Further, TLR4 expression upregulation can enhance innate immunity during mucosal bacterial infection. The findings establish the use of APS to modulate the innate immune response of the urinary tract through TLR4 expression regulation as an alternative option for UTI treatment.     

NOD2-deficient mice have impaired resistance to Mycobacterium tuberculosis infection through defective innate and adaptive immunity

NOD2/CARD15 mediates innate immune responses to mycobacterial infection. However, its role in the regulation of adaptive immunity has remained unknown. In this study, we examined host defense, T cell responses, and tissue pathology in two models of pulmonary mycobacterial infection, using wild-type and Nod2-deficient mice. During the early phase of aerosol infection with Mycobacterium tuberculosis, Nod2(-/-) mice had similar bacterial counts but reduced inflammatory response on histopathology at 4 and 8 wk postchallenge compared with wild-type animals. These findings were confirmed upon intratracheal infection of mice with attenuated Mycobacterium bovis bacillus Calmette-Guérin. Analysis of the lungs 4 wk after bacillus Calmette-Guérin infection demonstrated that Nod2(-/-) mice had decreased production of type 1 cytokines and reduced recruitment of CD8(+) and CD4(+) T cells. Ag-specific T cell responses in both the spleens and thoracic lymph nodes were diminished in Nod2(-/-) mice, indicating impaired adaptive antimycobacterial immunity. The immune regulatory role of NOD2 was not restricted to the lung since Nod2 disruption also led to reduced type 1 T cell activation following i.m. bacillus Calmette-Guérin infection. To determine the importance of diminished innate and adaptive immunity, we measured bacterial burden 6 mo after aerosol infection with M. tuberculosis and followed a second infected group for assessment of survival. Nod2(-/-) mice had a higher bacterial burden in the lungs 6 mo after infection and succumbed sooner than did wild-type controls. Taken together, these data indicate that NOD2 mediates resistance to mycobacterial infection via both innate and adaptive immunity.     

C-reactive protein enhances immunity to Streptococcus pneumoniae by targeting uptake to Fc gamma R on dendritic cells

C-reactive protein (CRP) is an acute phase reactant with roles in innate host defense, clearance of damaged cells, and regulation of the inflammatory response. These activities of CRP depend on ligand recognition, complement activation, and binding to FcgammaR. CRP binds to phosphocholine in the Streptococcus pneumoniae cell wall and provides innate defense against pneumococcal infection. These studies examine the effect of this early innate defense molecule on the development of Abs and protective immunity to S. pneumoniae. Dendritic cells (DC) initiate and direct the adaptive immune response by integrating innate stimuli with cytokine synthesis and Ag presentation. We hypothesized that CRP would direct uptake of S. pneumoniae to FcgammaR on DC and enhance Ag presentation. CRP opsonization of the R36a strain of S. pneumoniae increased the uptake of bacteria by DC. DC pulsed with untreated or CRP-opsonized R36a were transferred into recipient mice, and Ab responses were measured. In mice challenged with free R36a, CRP opsonization resulted in higher secondary and memory IgG responses to both phosphocholine and pneumococcal surface protein A. Furthermore, mice immunized with DC that had been pulsed with CRP-opsonized R36a showed increased resistance to intranasal infection with virulent S. pneumoniae. The effects of CRP on Ag uptake, Ab responses, and protection from infection all required FcR gamma-chain expression on DC. The results indicate that innate recognition by CRP enhances effective uptake and presentation of bacterial Ags through FcgammaR on DC and stimulates protective adaptive immunity.     

Mast cells initiate early anti-Listeria host defences

The Gram-positive bacterium Listeria monocytogenes ( L. m. ) is the aetiological agent of listeriosis. The early phase listeriosis is characterized by strong innate host responses that play a major role in bacterial clearance. This is emphasized by the fact that mice deficient in T and B cells have a remarkable ability to control infection. Mast cells, among the principal effectors of innate immunity, have largely been studied in the context of hyper-reactive conditions such as allergy and autoimmune diseases. In the present study, we evaluated the significance of mast cells during the early phase of listeriosis. Compared with controls, mice depleted of mast cells showed hundred-fold higher bacterial burden in spleen and liver and were significantly impaired in neutrophil mobilization. Although L. m. interacts with and triggers mast cell degranulation, bacteria were hardly found within such cells. Mainly neutrophils and macrophages phagozytosed L. m . Thus, mast cells control infection not via direct bacterial uptake, but by initiating neutrophils influx to the site of infection. We show that this is initiated by pre-synthesized TNF-α, rapidly secreted by mast cell upon activation by L. m . We also show that upon recruitment, neutrophils also become activated and additionally secrete TNF-α thus amplifying the anti- L. m. inflammatory response.     

Aging enhances the production of reactive oxygen species and bactericidal activity in peritoneal macrophages by upregulating classical activation pathways

Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3-4 months) and aged (14-15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LPS). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many proteins involved in immune cell function in aged Balb/c mice. Collectively, these results indicate that macrophages isolated from old mice are in a preactivated state that enhances their sensitivities to LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection by general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the level of formation of reactive oxygen species are likely to contribute to observed age-dependent increases in the level of oxidative damage that underlie many diseases of the elderly.     

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

Cutting edge: protective cell-mediated immunity to Listeria monocytogenes in the absence of myeloid differentiation factor 88

In addition to their role in triggering innate immune responses, Toll-like receptors are proposed to play a key role in linking the innate and adaptive arms of the immune response. The majority of cellular responses downstream of Toll-like receptors are mediated through the adapter molecule myeloid differentiation factor 88 (MyD88), and mice with a targeted deletion of MyD88 are highly susceptible to bacterial infections, including primary infection with Listeria monocytogenes (LM). In contrast, herein we demonstrate that MyD88-deficient mice have only a modest impairment in their LM-specific CD4 T cell response, and no impairment in their CD8 T cell response following infection with ActA-deficient LM. Furthermore, CD8 T cells from immunized MyD88-deficient mice protected naive recipient mice following adoptive splenocyte transfer, and immunized MyD88-deficient mice were protected from infection with wild-type LM. These results indicate that adaptive immune responses can be generated and provide protective immunity in the absence of MyD88.     

Toll-like receptor 9 acts at an early stage in host defence against pneumococcal infection

Toll-like receptor 9 (TLR9) induces an inflammatory response by recognition of unmethylated CpG dinucleotides, mainly present in prokaryotic DNA. So far, TLR9-deficient mice have been shown to be more sensitive than wild-type mice to viral, but not to bacterial infections. Here, we show that mice deficient in TLR9 but not in TLR1, TLR2, TLR4 and TLR6 or IL-1R/IL-18R are more susceptible to a respiratory tract bacterial infection caused by Streptococcus pneumoniae. Intranasal challenge studies revealed that TLR9 plays a protective role in the lungs at an early stage of infection prior to the entry of circulating inflammatory cells. Alveolar as well as bone marrow-derived macrophages deficient in either TLR9 or the myeloid adaptor differentiation protein MyD88 were impaired in pneumococcal uptake and in pneumococcal killing. Our data suggest that in the airways, pneumococcal infection triggers a TLR9 and MyD88-dependent activation of phagocytic activity from resident macrophages leading to an early clearance of bacteria from the lower respiratory tract.     

The complex mechanism of antibody-mediated clearance of Bordetella from the lungs requires TLR4

Although the antibacterial effects of Abs are well studied in in vitro systems, the in vivo effects of Abs cannot always be accurately predicted. Complicated cross-talk between different effector functions of Abs and various arms of the immune system can affect their activities in vivo. Using the mouse respiratory pathogen Bordetella bronchiseptica, we examined the mechanisms of Ab-mediated clearance of bacteria from the respiratory tract. Interestingly, although TLR4 was not necessary for protective immunity following infection, it was required for rapid bacterial clearance in mice that were vaccinated or adoptively transferred Abs. TLR4 was important for the rapid recruitment of neutrophils that are necessary for Ab-mediated bacterial clearance via a mechanism that requires both FcgammaR and CR3. These data are consistent with a model in which TLR4-mediated inflammatory responses aid in the recruitment of neutrophils, which phagocytose Ab- and complement-opsonized bacteria via FcgammaRs and CR3. Although pattern recognition receptors are known to be involved in innate immunity and the generation of adaptive immunity, their contributions to specific adaptive immune functions should be considered in ongoing efforts to improve vaccine-induced protective immunity.     

Normal establishment of virus-specific memory CD8 T cell pool following primary infection during pregnancy

Suppression of cell-mediated immunity has been proposed as a mechanism that promotes maternal tolerance of the fetus but also contributes to increased occurrence and severity of certain infections during pregnancy. Despite decades of research examining the effect of pregnancy on Ag-specific T cell responses, many questions remain. In particular, quantitative examination of memory CD8 T cell generation following infection during pregnancy remains largely unknown. To examine this issue, we evaluated the generation of protective immunity following infection during pregnancy with a nonpersistent strain of lymphocytic choriomeningitis virus (LCMV) in mice. The CD8 T cell response to LCMV occurred normally in pregnant mice compared with the nonpregnant cohort with rapid viral clearance in all tissues tested except for the placenta. Despite significant infiltration of CD8 T cells to the maternal-fetal interface, virus persisted in the placenta until delivery. Live pups were not infected and generated normal primary immune responses when challenged as adults. Memory CD8 T cell development in mice that were pregnant during primary infection was normal with regards to the proliferative capacity, number of Ag-specific cells, cytokine production upon re-stimulation, and the ability to protect from re-infection. These data suggest that virus-specific adaptive memory is normally generated in mice during pregnancy.     

Inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection

Secondary bacterial infection often occurs after pulmonary virus infection and is a common cause of severe disease in humans, yet the mechanisms responsible for this viral-bacterial synergy in the lung are only poorly understood. We now report that pulmonary interferon-gamma (IFN-gamma) produced during T cell responses to influenza infection in mice inhibits initial bacterial clearance from the lung by alveolar macrophages. This suppression of phagocytosis correlates with lung IFN-gamma abundance, but not viral burden, and leads to enhanced susceptibility to secondary pneumococcal infection, which can be prevented by IFN-gamma neutralization after influenza infection. Direct inoculation of IFN-gamma can mimic influenza infection and downregulate the expression of the class A scavenger receptor MARCO on alveolar macrophages. Thus, IFN-gamma, although probably facilitating induction of specific anti-influenza adaptive immunity, suppresses innate protection against extracellular bacterial pathogens in the lung.     

STAT4 is a critical mediator of early innate immune responses against pulmonary Klebsiella infection

Bacterial pneumonia is a leading cause of morbidity and mortality in the U.S. An effective innate immune response is critical for the clearance of bacteria from the lungs. IL-12, a key T1 cytokine in innate immunity, signals through STAT4. Thus, understanding how STAT4 mediates pulmonary immune responses against bacterial pathogens will have important implications for the development of rational immunotherapy targeted at augmenting innate immunity. We intratracheally administered Klebsiella pneumoniae to wild-type BALB/c and STAT4 knockout (STAT4-/-) mice. Compared with wild-type controls, STAT4-/- mice had decreased survival following intratracheal Klebsiella administration, which was associated with a higher lung and blood bacterial burden. STAT4-/- animals also displayed impaired pulmonary IFN-gamma production and decreased levels of proinflammatory cytokines, including the ELR- CXC chemokines IFN-gamma-inducible protein-10 and monokine induced by IFN-gamma. Although total lung leukocyte populations were similar between STAT4-/- and wild-type animals following infection, alveolar macrophages isolated from infected STAT4-/- mice had decreased production of proinflammatory cytokines, including IFN-gamma, compared with infected wild-type mice. The intrapulmonary overexpression of IFN-gamma concomitant with the systemic administration of IFN-gamma partially reversed the immune deficits observed in STAT4-/- mice, resulting in improved bacterial clearance from the blood. Collectively, these studies demonstrate that STAT4 is required for the generation of an effective innate host defense against bacterial pathogens of the lung.     

Helical Carbon Nanotubes Enhance the Early Immune Response and Inhibit Macrophage-Mediated Phagocytosis of Pseudomonas aeruginosa

Aerosolized or aspirated manufactured carbon nanotubes have been shown to be cytotoxic, cause pulmonary lesions, and demonstrate immunomodulatory properties. CD-1 mice were used to assess pulmonary toxicity of helical carbon nanotubes (HCNTs) and alterations of the immune response to subsequent infection by Pseudomonas aeruginosa in mice. HCNTs provoked a mild inflammatory response following either a single exposure or 2X/week for three weeks (multiple exposures) but were not significantly toxic. Administering HCNTs 2X/week for three weeks resulted in pulmonary lesions including granulomas and goblet cell hyperplasia. Mice exposed to HCNTs and subsequently infected by P. aeruginosa demonstrated an enhanced inflammatory response to P. aeruginosa and phagocytosis by alveolar macrophages was inhibited. However, clearance of P. aeruginosa was not affected. HCNT exposed mice depleted of neutrophils were more effective in clearing P. aeruginosa compared to neutrophil-depleted control mice, accompanied by an influx of macrophages. Depletion of systemic macrophages resulted in slightly inhibited bacterial clearance by HCNT treated mice. Our data indicate that pulmonary exposure to HCNTs results in lesions similar to those caused by other nanotubes and pre-exposure to HCNTs inhibit alveolar macrophage phagocytosis of P. aeruginosa. However, clearance was not affected as exposure to HCNTs primed the immune system for an enhanced inflammatory response to pulmonary infection consisting of an influx of neutrophils and macrophages.     

Resident Vdelta1+ gammadelta T cells control early infiltration of neutrophils after Escherichia coli infection via IL-17 production

Neutrophils infiltrate the site of infection and play critical roles in host defense, especially against extracellular bacteria. In the present study, we found a rapid and transient production of IL-17 after i.p. infection with Escherichia coli, preceding the influx of neutrophils. Neutralization of IL-17 resulted in a reduced infiltration of neutrophils and an impaired bacterial clearance. Ex vivo intracellular cytokine flow cytometric analysis revealed that gammadelta T cell population was the major source of IL-17. Mice depleted of gammadelta T cells by mAb treatment or mice genetically lacking Vdelta1 showed diminished IL-17 production and reduced neutrophil infiltration after E. coli infection, indicating an importance of Vdelta1(+) gammadelta T cells as the source of IL-17. It was further revealed that gammadelta T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23, which was induced rapidly after E. coli infection in a TLR4 signaling-dependent manner. Thus, although gammadelta T cells are generally regarded as a part of early induced immune responses, which bridge innate and adaptive immune responses, our study demonstrated a novel role of gammadelta T cells as a first line of host defense controlling neutrophil-mediated innate immune responses.     

Plasmacytoid Dendritic Cells Mediate the Regulation of Inflammatory Type T Cell Response for Optimal Immunity against Respiratory Chlamydia Pneumoniae Infection

Chlamydia pneumoniae (Cpn) infection is a leading cause for a variety of respiratory diseases and has been implicated in the pathogenesis of chronic inflammatory diseases. The regulatory mechanisms in host defense against Cpn infection are less understood. In this study, we investigated the role of plasmacytoid dendritic cells (pDCs) in immune regulation in Cpn respiratory tract infection. We found that in vivo depletion of pDCs increased the severity of infection and lung pathology. Mice depleted of pDC had greater body weight loss, higher lung bacterial burden and excessive tissue inflammation compared to the control mice. Analysis of specific T cell cytokine production pattern in the lung following Cpn infection revealed that pDC depleted mice produced significantly higher amounts of inflammatory cytokines, especially TNF-α, but lower IL-10 compared to the controls. In particular, pDC depleted mice showed pathogenic T cell responses characterized by inflammatory type-1 (CD8 and CD4) and inflammatory Th2 cell responses. Moreover, pDC depletion dramatically reduced CD4 regulatory T cells (Tregs) in the lungs and draining lymph nodes. Furthermore, pDC-T cell co-culture experiments showed that pDCs isolated from Cpn infected mice were potent in inducing IL-10 producing CD4 Tregs. Together, these findings provide in vivo evidence for a critical role of pDCs in homeostatic regulation of immunity during Cpn infection. Our findings highlight the importance of a ‘balanced’ immune response for host protective immunity and preventing detrimental immunopathology during microbial infections.