Urokinase receptor is necessary for adequate host defense against pneumococcal pneumonia

Cell recruitment is a multistep process regulated by cytokines, chemokines, and growth factors. Previous work has indicated that the urokinase plasminogen activator receptor (uPAR) may also play a role in this mechanism, presumably by an interaction with the beta(2) integrin CD11b/CD18. Indeed, an essential role of uPAR in neutrophil recruitment during pulmonary infection has been demonstrated for beta(2) integrin-dependent respiratory pathogens. We investigated the role of uPAR and urokinase plasminogen activator (uPA) during pneumonia caused by a beta(2) integrin-independent respiratory pathogen, Streptococcus pneumoniae. uPAR-deficient (uPAR(-/-)), uPA-deficient (uPA(-/-)), and wild-type (Wt) mice were intranasally inoculated with 10(5) CFU S. pneumoniae. uPAR(-/-) mice showed reduced granulocyte accumulation in alveoli and lungs when compared with Wt mice, which was associated with more S. pneumoniae CFU in lungs, enhanced dissemination of the infection, and a reduced survival. In contrast, uPA(-/-) mice showed enhanced host defense, with more neutrophil influx and less pneumococci in the lungs compared with Wt mice. These data suggest that uPAR is necessary for adequate recruitment of neutrophils into the alveoli and lungs during pneumonia caused by S. pneumoniae, a pathogen eliciting a beta(2) integrin-independent inflammatory response. This function is even more pronounced when uPAR is unoccupied by uPA.     

Accumulation of gamma/delta T cells in the lungs and their roles in neutrophil-mediated host defense against pneumococcal infection

The present study was designed to elucidate the role of Vgamma4(+) gammadelta T cells, a major subset of pulmonary gammadelta T cells, in host defense against infection with Streptococcus pneumoniae. The proportion and number of whole gammadelta T cells, identified as CD3(+) and TCR-delta(+) cells, and Vgamma4(+) gammadelta T cells, identified as CD3(+) and TCR-Vgamma4(+) cells, increased in the lungs at 3, 6 and 12h post-infection. Survival of infected mice and lung bacterial clearance were severely impaired in TCR-Vgamma4(-/-) mice compared with control wild-type (WT) mice. The impaired host protection in TCR-Vgamma4(-/-) mice correlated well with attenuated recruitment of neutrophils in lungs. MIP-2 and TNF-alpha synthesis in the infected tissues was significantly reduced in TCR-Vgamma4(-/-) mice compared with WT mice. Similar results were noted in the synthesis of TNF-alpha, but not clearly of MIP-2, by lung leukocytes stimulated with live bacteria. Our results demonstrate that Vgamma4(+) gammadelta T cells play an important role in the neutrophil-mediated host defense against S. pneumoniae infection by promoting the synthesis of TNF-alpha and possibly of MIP-2 in the lungs.     

TNF-alpha compensates for the impaired host defense of IL-1 type I receptor-deficient mice during pneumococcal pneumonia.

To determine the role of IL-1 in the host defense against pneumonia, IL-1R type I-deficient (IL-1R(-/-)) and wild-type (Wt) mice were intranasally inoculated with Streptococcus pneumoniae. Pneumonia resulted in elevated IL-1alpha and IL-1beta mRNA and protein levels in the lungs. Survival rates did not differ between IL-1R(-/-) and Wt mice after inoculation with 5 x 10(4) or 2 x 10(5) CFU. At early time points (24 and 48 h) IL-1R(-/-) mice had 2-log more S. pneumoniae CFU in lungs than Wt mice; at 72 h bacterial outgrowth in lungs was similar in both groups. Upon histopathologic examination IL-1R(-/-) mice displayed a reduced capacity to form inflammatory infiltrates at 24 h after the induction of pneumonia. IL-1R(-/-) mice also had significantly less granulocyte influx in bronchoalveolar lavage fluid at 24 h after inoculation. Since TNF is known to enhance host defense during pneumonia, we determined the role of endogenous TNF in the early impairment and subsequent recovery of defense mechanisms in IL-1R(-/-) mice. All IL-1R(-/-) mice treated with anti-TNF rapidly died (no survivors (of 14 mice) after 4 days), while 10-day survival in IL-1R(-/-) mice (control Ab), Wt mice (anti-TNF), and Wt mice (control Ab) was 7 of 13, 3 of 14, and 12 of 13, respectively. These data suggest that TNF is more important for host defense against pneumococcal pneumonia than IL-1, and that the impaired early host defense in IL-1R(-/-) mice is compensated for by TNF at a later phase.     

CD8+ cells enhance resistance to pulmonary serotype 3 Streptococcus pneumoniae infection in mice

Despite the success of the pneumococcal conjugate vaccine, pneumococcal pneumonia remains a significant clinical problem, and there is still much to learn about natural resistance and cellular immunity to pneumococcus. We investigated the role of T lymphocytes in resistance to serotype (ST) 3 Streptococcus pneumoniae in an intranasal infection model in C57BL/6 (wild-type [Wt]) and CD8(+) (CD8(-/-))- and CD4(+) (MHC class II(-/-))-deficient mice. CD8(-/-) mice exhibited significantly more bacterial dissemination and lung inflammation and a significantly more lethal phenotype than Wt mice. However, there was no difference in the bacterial dissemination, lung inflammation, or survival of Wt and MHC class II(-/-) mice. Perforin (Pfn)(-/-) and IFN-γ(-/-) mice, which were used to dissect the role of CD8(+) T cells in our model, also exhibited a more lethal survival phenotype than Wt mice. Comparison of lung chemokine/cytokine levels by Luminex and cellular recruitment by FACS in Wt mice and knockout strains revealed that CD8(-/-) and IFN-γ(-/-) mice, which had the most lethal survival phenotype, had more CD4(+)IL-17(+) T (Th17) cells, IL-17, neutrophil chemoattractants, and lung neutrophils, and fewer regulatory T cells than Wt mice. CD4(+) T cell depletion improved the survival of ST-infected CD8(-/-) mice, and survival studies in Th17-deficient mice revealed that the Th17 response was dispensable for ST3 resistance in our model. Taken together, these findings demonstrate that CD8(+) cells are required, but CD4(+) T cells are dispensable for resistance to ST3 pneumonia in mice and suggest a previously unsuspected role for CD8(+) cells in modulating the inflammatory response to ST3.     

TLR2, but not TLR4, is required for effective host defence against Chlamydia respiratory tract infection in early life

Chlamydia pneumoniae commonly causes respiratory tract infections in children, and epidemiological investigations strongly link infection to the pathogenesis of asthma. The immune system in early life is immature and may not respond appropriately to pathogens. Toll-like receptor (TLR)2 and 4 are regarded as the primary pattern recognition receptors that sense bacteria, however their contribution to innate and adaptive immunity in early life remains poorly defined. We investigated the role of TLR2 and 4 in the induction of immune responses to Chlamydia muridarum respiratory infection, in neonatal wild-type (Wt) or TLR2-deficient ((-/-)), 4(-/-) or 2/4(-/-) BALB/c mice. Wt mice had moderate disease and infection. TLR2(-/-) mice had more severe disease and more intense and prolonged infection compared to other groups. TLR4(-/-) mice were asymptomatic. TLR2/4(-/-) mice had severe early disease and persistent infection, which resolved thereafter consistent with the absence of symptoms in TLR4(-/-) mice. Wt mice mounted robust innate and adaptive responses with an influx of natural killer (NK) cells, neutrophils, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, and activated CD4(+) and CD8(+) T-cells into the lungs. Wt mice also had effective production of interferon (IFN)γ in the lymph nodes and lung, and proliferation of lymph node T-cells. TLR2(-/-) mice had more intense and persistent innate (particularly neutrophil) and adaptive cell responses and IL-17 expression in the lung, however IFNγ responses and T-cell proliferation were reduced. TLR2/4(-/-) mice had reduced innate and adaptive responses. Most importantly, neutrophil phagocytosis was impaired in the absence of TLR2. Thus, TLR2 expression, particularly on neutrophils, is required for effective control of Chlamydia respiratory infection in early life. Loss of control of infection leads to enhanced but ineffective TLR4-mediated inflammatory responses that prolong disease symptoms. This indicates that TLR2 agonists may be beneficial in the treatment of early life Chlamydia infections and associated diseases.     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.     

The role of macrophage inflammatory protein-1 alpha/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection

Macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1alpha is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1alpha(+/+)) mice and MIP-1alpha knockout (MIP-1alpha(-/-)) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1alpha message was detected in the lungs on days 3, 7, and 14 in MIP-1alpha(+/+) mice, but it was undetectable in MIP-1alpha(-/-) mice. On day 16, MIP-1alpha(-/-) mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1alpha(+/+) and MIP-1alpha(-/-) mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1alpha(-/-) mice had a significantly greater number of eosinophils. MIP-1alpha(-/-) mice had extremely high levels of serum IgE. This switch of immune response to a T(2) phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1alpha(-/-) mice compared with MIP-1alpha (+/+) mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T(2) immunity resulted in profound lung damage in MIP-1alpha(-/-) mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1alpha(-/-) mice. These studies, together with our previous studies, demonstrate that MIP-1alpha plays a role in both the afferent (T(1)/T(2) development) and efferent (T(1)-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.     

Blockade of B7-H1 (programmed death ligand 1) enhances humoral immunity by positively regulating the generation of T follicular helper cells

T follicular helper (T(FH)) cells are critical initiators in the development of T cell-dependent humoral immunity and the generation of protective immunity. We demonstrate that T(FH) cell accumulation and Ab production are negatively regulated by B7-H1 (programmed death ligand 1) in response to both helminth infection and active immunization. Following immunization of B7-H1(-/-) mice with keyhole limpet hemocyanin or helminth Ags, there is a profound increase in induction of T(FH) cells as a result of increased cell cycling and decreased apoptosis relative to wild-type mice. The increase in T(FH) cells in the absence of B7-H1 was associated with significant elevations in Ag-specific Ig response. Cotransfer experiments in vivo demonstrated that B7-H1 expression on B cells was required for negatively regulating T(FH) cell expansion and production of Ag-specific Ig. Treatment of immunized wild-type mice with anti-B7-H1 or anti-programmed death 1 mAbs, but not anti-B7-DC, led to a significant expansion of the T(FH) cell population and an enhanced Ag-specific Ig response. Our results demonstrate that the coinhibitory B7-H1/programmed death 1 pathway can limit the expansion of T(FH) cells and constrain Ag-specific Ig responses. This finding has direct implications for investigations examining the feasibility of therapeutically manipulating this pathway and reveals new insights into the regulation of the humoral immune response.     

B7-H3 participates in the development of experimental pneumococcal meningitis by augmentation of the inflammatory response via a TLR2-dependent mechanism

In addition to a well-documented role in regulating T cell-mediated immune responses, B7-H3, a newly discovered member of the B7 superfamily, has been recently identified as a costimulator in the innate immunity-mediated inflammatory response. In this study, we further report that B7-H3 participates in the development of pneumococcal meningitis in a murine model. Exogenous administration of B7-H3 strongly amplified the inflammatory response, exacerbated blood-brain barrier disruption, and aggravated the clinical disease status in Streptococcus pneumoniae-infected C3H/HeN wild-type mice. Consistent with the in vivo findings, B7-H3 substantially augmented proinflammatory cytokine and chemokine production, upregulated NF-κB p65 and MAPK p38 phosphorylation, and enhanced the nuclear transactivation of NF-κB p65 at both TNF-α and IL-6 promoters in S. pneumoniae-stimulated primary murine microglia cells. These B7-H3-associated in vitro and in vivo effects appeared to be dependent on TLR2 signaling, as B7-H3 almost completely lost its amplifying actions in both TLR2-deficient microglial cells and TLR2-deficient mice. Furthermore, administration of the anti-B7-H3 mAb (MIH35) attenuated the inflammatory response and ameliorated blood-brain barrier disruption in S. pneumoniae-infected wild-type mice. Collectively, our results indicate that B7-H3 plays a contributory role in the development of S. pneumoniae infection-induced bacterial meningitis.     

B cells are required for generation of protective effector and memory CD4 cells in response to Pneumocystis lung infection

B cell-deficient mice are susceptible to infection by Pneumocystis carinii f. sp. muris (PC). To determine whether this susceptibility is due to a requirement for B cells to prime T cells, we compared CD4 T cell responses to PC in bone marrow chimeric mice that express MHC class II (MHCII) on all APCs (wild-type (WT) chimeras) and in bone marrow chimeric mice that express MHCII on all APCs except B cells (MHCII(-/-) chimeras). Although PC was rapidly cleared by WT chimeric mice, PC levels remained high in chimeric mice that lacked MHCII on B cells. In addition, although T cells were primed in the draining lymph nodes of MHCII(-/-) chimeric mice, the number of activated CD4 T cells infiltrating the lungs of these mice was reduced relative to the number in the lungs of WT chimeras. We also adoptively transferred purified CD4 T cells from the draining lymph nodes of PC-infected normal or B cell-deficient mice into SCID mice. Mice that received CD4 cells from normal mice were able to mount a response to infection in the lungs and clear PC. However, mice that received CD4 cells from B cell-deficient mice had a delayed T cell response in the lungs and failed to control the infection. These data indicate that B cells play a vital role in generation of CD4(+) memory T cells in response to PC infection in the lungs.     

Selectin ligand-independent priming and maintenance of T cell immunity during airborne tuberculosis

Immunity to Mycobacterium tuberculosis infection is critically dependent on the timely priming of T effector lymphocytes and their efficient recruitment to the site of mycobacterial implantation in the lung. E-, P-, and L-selectin counterreceptors control lymphocyte homing to lymph nodes and leukocyte trafficking to peripheral sites of acute inflammation, their adhesive function depending on fucosylation by fucosyltransferases (FucT) IV and VII. To address the relative importance of differentially glycosylated selectin counterreceptors for priming of T cell effector functions in a model of mycobacteria-induced granulomatous pulmonary inflammation, we used aerosol-borne M. tuberculosis to infect FucT-IV-/-, FucT-VII-/-, FucT-IV-/-/FucT-VII-/-, or wild-type control mice. In lymph nodes, infected FucT-IV-/-/FucT-VII-/- and, to a lesser extent, FucT-VII-/- mice had severely reduced numbers of T cells and reduced Ag-specific effector responses. By contrast, recruitment of activated T cells into the lungs was similar in all four groups of mice during infection and expression of T cell, and macrophage effector functions were only delayed in lungs of FucT-IV-/-/FucT-VII-/- mice. Importantly, lungs from all groups expressed CXCL13, CCL21, and CCL19 and displayed organized follicular neolymphoid structures after infection with M. tuberculosis, which suggests that the lung served as a selectin ligand-independent priming site for immune responses to mycobacterial infection. All FucT-deficient strains were fully capable of restricting M. tuberculosis growth in infected organs until at least 150 days postinfection. Our observations indicate that leukocyte recruitment functions dictated by FucT-IV and FucT-VII-dependent selectin ligand activities are not critical for inducing or maintaining T cell effector responses at levels necessary to control pulmonary tuberculosis.     

Infection of B cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lung pathology

Long-term survival of mice infected with Mycobacterium tuberculosis is dependent upon IFN-gamma and T cells, but events in early phases of the immune response are not well understood. In this study, we describe a role for B cells during early immune responses to infection with a clinical isolate of M. tuberculosis (CDC 1551). Following a low-dose infection with M. tuberculosis CDC 1551, similar numbers of bacteria were detected in the lungs of both B cell knockout (IgH 6-, BKO) and C57BL/6J (wild-type) mice. However, despite comparable bacterial loads in the lungs, less severe pulmonary granuloma formation and delayed dissemination of bacteria from lungs to peripheral organs were observed in BKO mice. BKO mice reconstituted with naive B cells, but not those given M. tuberculosis-specific Abs, before infection developed pulmonary granulomas and dissemination patterns similar to wild-type animals. Further analysis of lung cell populations revealed greater numbers of lymphocytes, especially CD8+ T cells, macrophages, and neutrophils in wild-type and reconstituted mice than in BKO mice. Thus, less severe lesion formation and delayed dissemination of bacteria found in BKO mice were dependent on B cells, not Abs, and were associated with altered cellular infiltrate to the lungs. These observations demonstrate an important, previously unappreciated, role for B cells during early immune responses to M. tuberculosis infections.     

Accumulation of gammadelta T cells in the lungs and their regulatory roles in Th1 response and host defense against pulmonary infection with Cryptococcus neoformans

The present study was designed to elucidate the role of gammadelta T cells in the host defense against pulmonary infection with Cryptococcus neoformans. The gammadelta T cells in lungs commenced to increase on day 1, reached a peak level on day 3 or 6, and then decreased on day 10 after intratracheal infection. The increase of these cells was similar in monocyte chemoattractant protein (MCP)-1-deficient mice, although that of NK and NKT cells was significantly reduced. The number of live microorganisms in lungs on days 14 and 21 was significantly reduced in mice depleted of gammadelta T cells by a specific mAb compared with mice treated with control IgG. Similarly, elimination of this fungal pathogen was promoted in gammadelta T cell-deficient (TCR-delta(-/-)) mice compared with control littermate mice. Finally, lung and serum levels of IFN-gamma on days 7 and 14 and on day 7 postinfection, respectively, were significantly higher in TCR-delta(-/-) mice than in littermate mice, whereas levels of TGF-beta showed the opposite results. IL-4 and IL-10 were not different between these mice. IFN-gamma production by draining lymph node cells upon restimulation with cryptococcal Ags was significantly higher in the infected TCR-delta(-/-) mice than in control mice. Our results demonstrated that gammadelta T cells accumulated in the lungs in a manner different from NK and NKT cells after cryptococcal infection and played a down-modulatory role in the development of Th1 response and host resistance against this fungal pathogen.     

Macrophages, CD4+ or CD8+ cells are each sufficient for protection against Chlamydia pneumoniae infection through their ability to secrete IFN-gamma

By using a T, B, or NK cell-deficient mouse strain (recombinase-activating gene (RAG)-1(-/-)/common cytokine receptor gamma-chain (gamma(C)R)), and T and B cell and IFN-gamma-deficient (RAG-1(-/-)/IFN-gamma(-/-)) mice, we have studied the generation of immunity against infection by Chlamydia pneumoniae. We found that IFN-gamma secreted by innate-cell populations protect against C. pneumoniae infection. However, NK cells were not needed for such IFN-gamma-dependent innate immune protection. Inoculation of wild type, but not IFN-gamma(-/-) bone marrow-derived macrophages protected RAG-1(-/-)/IFN-gamma(-/-) mice against C. pneumoniae infection. In line, pulmonary macrophages from RAG-1(-/-) C. pneumoniae-infected mice expressed IFN-gamma mRNA. Reconstitution of RAG-1(-/-)/gamma(c)R(-/-) or RAG-1(-/-)/IFN-gamma(-/-) mice with CD4(+) or CD8(+) cells by i.v. transfer of FACS sorted wild type spleen cells (SC) increased resistance to C. pneumoniae infection. On the contrary, no protection was observed upon transfer of IFN-gamma(-/-) CD4(+) or IFN-gamma(-/-) CD8(+) SC. T cell-dependent protection against C. pneumoniae was weaker when IFN-gammaR(-/-) CD4(+) or IFN-gammaR(-/-) CD8(+) SC were inoculated into RAG-1(-/-)/IFN-gamma(-/-) mice. Thus both nonlymphoid and T cell-derived IFN-gamma can play a central and complementary role in protection against C. pneumoniae. IFN-gamma secreted by nonlymphoid cells was not required for T cell-mediated protection against C. pneumoniae; however, IFN-gamma regulated T cell protective functions.     

The integrins Mac-1 and alpha4beta1 perform crucial roles in neutrophil and T cell recruitment to lungs during Streptococcus pneumoniae infection

Neutrophils and T cells play an important role in host protection against pulmonary infection caused by Streptococcus pneumoniae. However, the role of the integrins in recruitment of these cells to infected lungs is not well understood. In this study we used the twin approaches of mAb blockade and gene-deficient mice to investigate the relative impact of specific integrins on cellular recruitment and bacterial loads following pneumococcal infection. We find that both Mac-1 (CD11b/CD18) and α(4)β(1) (CD49d/CD29) integrins, but surprisingly not LFA-1 (CD11a/CD18), contribute to two aspects of the response. In terms of recruitment from the circulation into lungs, neutrophils depend on Mac-1 and α(4)β(1), whereas the T cells are entirely dependent on α(4)β(1). Second, immunohistochemistry results indicate that adhesion also plays a role within infected lung tissue itself. There is widespread expression of ICAM-1 within lung tissue. Use of ICAM-1(-/-) mice revealed that neutrophils make use of this Mac-1 ligand, not for lung entry or for migration within lung tissue, but for combating the pneumococcal infection. In contrast to ICAM-1, there is restricted and constitutive expression of the α(4)β(1) ligand, VCAM-1, on the bronchioles, allowing direct access of the leukocytes to the airways via this integrin at an early stage of pneumococcal infection. Therefore, integrins Mac-1 and α(4)β(1) have a pivotal role in prevention of pneumococcal outgrowth during disease both in regulating neutrophil and T cell recruitment into infected lungs and by influencing their behavior within the lung tissue itself.     

A critical role for ABCG1 in macrophage inflammation and lung homeostasis

ATP-binding cassette transporter G1 (ABCG1) effluxes cholesterol from macrophages and plays an important role in pulmonary lipid homeostasis. We hypothesize that macrophages from Abcg1(-/-) mice have increased inflammatory activity, thereby promoting acceleration of pulmonary disease. We herein demonstrate increased numbers of inflammatory cytokines and infiltrating neutrophils, eosinophils, dendritic cells, T cells, and B cells into lungs of Abcg1(-/-) mice before the onset of severe lipidosis. We further investigated the role of macrophages in causing pulmonary disease by performing bone marrow transplantations using B6 and Abcg1(-/-) bone marrow. We found that it was the macrophage, and not pneumocyte type II cells or other nonhematopoietic cells in the lung, that appeared to be the primary cell type involved in the onset of both pulmonary lipidosis and inflammation in the Abcg1(-/-) mice. Additionally, our results demonstrate that Abcg1(-/-) macrophages had elevated proinflammatory cytokine production, increased apoptotic cell clearance, and were themselves more prone to apoptosis and necrosis. However, they were quickly repopulated by monocytes that were recruited to Abcg1(-/-) lungs. In conclusion, we have shown that ABCG1 deletion in macrophages causes a striking inflammatory phenotype and initiates onset of pulmonary lipidosis in mice. Thus, our studies reveal a critical role for macrophage ABCG1 in lung inflammation and homeostasis.     

Blockade of PD-1/B7-H1 interaction restores effector CD8+ T cell responses in a hepatitis C virus core murine model

The impaired function of CD8(+) T cells is characteristic of hepatitis C virus (HCV) persistent infection. HCV core protein has been reported to inhibit CD8(+) T cell responses. To determine the mechanism of the HCV core in suppressing Ag-specific CD8(+) T cell responses, we generated a transgenic mouse, core(+) mice, where the expression of core protein is directed to the liver using the albumin promoter. Using a recombinant adenovirus to deliver Ag, we demonstrated that core(+) mice failed to clear adenovirus-LacZ (Ad-LacZ) infection in the liver. The effector function of LacZ-specific CD8(+) T cells was particularly impaired in the livers of core(+) mice, with suppression of IFN-gamma, TNF-alpha, and granzyme B production by CD8(+) T cells. In addition, the impaired CD8(+) T cell responses in core(+) mice were accompanied by the enhanced expression of the inhibitory receptor programmed death-1 (PD-1) by LacZ-specific CD8(+) T cells and its ligand B7-H1 on liver dendritic cells following Ad-LacZ infection. Importantly, blockade of the PD-1/B7-H1 inhibitory pathway (using a B7-H1 blocking antibody) in core(+) mice enhanced effector function of CD8(+) T cells and cleared Ad-LacZ-infection as compared with that in mice treated with control Ab. This suggests that the regulation of the PD-1/B7-H1 inhibitory pathway is crucial for HCV core-mediated impaired T cell responses and viral persistence in the liver. This also suggests that manipulation of the PD-1/B7-H1 pathway may be a potential immunotherapy to enhance effector T cell responses during persistent HCV infection.     

Murine model of BCG lung infection: Dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes

C57B1/6 female mice were infected with an intrapulmonary dose of 2.5 × 10⁴ BCG(Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1,2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4–6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3⁺ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1⁺) per lung increased by about 50%, 5–6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNγ producing non-T cells outnumbered T cells producing IFNγ. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNγ producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNγ response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNγ, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine     

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.     

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

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