Th17 cells mediate clade-specific, serotype-independent mucosal immunity.

The interleukin-17 (IL-17) family of cytokines phylogenetically predates the evolution of T cells in jawed vertebrates, suggesting that the ontogeny of the Th17 cell lineage must have arisen to confer an evolutionary advantage to the host over innate sources of IL-17. Utilizing a model of mucosal immunization with the encapsulated bacteria Klebsiella pneumoniae, we found that B cells, which largely recognized polysaccharide capsular antigens, afforded protection to only the vaccine strain. In contrast, memory Th17 cells proliferated in response to conserved outer membrane proteins and conferred protection against several serotypes of K. pneumoniae, including the recently described multidrug resistant New Dehli metallolactamase strain. Notably, this heterologous, clade-specific protection was antibody independent, demonstrating the Th17 cell lineage confers a host advantage by providing heterologous mucosal immunity independent of serotype-specific antibody.

CD4+ T Cell Regulation of Antibodies Cross-Reactive with Fungal Cell Wall-Associated Carbohydrates after Pneumocystis murina Infection.

Pneumocystis pneumonia is a life-threatening opportunistic fungal infection observed in individuals with severe immunodeficiencies, such as AIDS. Molecules with the ability to bind ß-glucan and signal at Fcγ receptors enhance defense against Pneumocystis f. sp. murina, though it is unclear whether antibodies reactive with fungal cell wall carbohydrates are induced during Pneumocystis infection. We observed that systemic and lung mucosal immunoglobulins cross-reactive with ß-glucan and chitosan/chitin are generated after Pneumocystis infection, with increased quantities within the lung mucosal fluid after challenge. While IgG responses against Pneumocystis protein antigens are markedly CD4+ T cell dependent, CD4+ T cell depletion did not impact quantities of IgG cross-reactive with ß-glucan or chitosan/chitin in the serum or mucosa after challenge. Notably, lung mucosal quantities of IgA cross-reactive with ß-glucan or chitosan/chitin are decreased in the setting of CD4+ T cell deficiency, occurring in the setting of concurrent reduced quantities of active transforming growth factor ß, while mucosal IgM is significantly increased in the setting of CD4+ T cell deficiency. Interleukin-21 receptor deficiency does not lead to reduction in mucosal IgA reactive with fungal carbohydrate antigens after Pneumocystis challenge. These studies demonstrate differential CD4+ T cell-dependent regulation of mucosal antibody responses against ß-glucan and chitosan/chitin after Pneumocystis challenge, suggesting that different B cell subsets may be responsible for the generation of these antibody responses, and suggest a potential immune response against fungi that may be operative in the setting of CD4+ T cell-related immunodeficiency.

A Novel CD4+ T Cell-Dependent Murine Model of Pneumocystis-driven Asthma-like Pathology.

RATIONALE: Infection with Pneumocystis, an opportunistic fungal pathogen, can result in fulminant pneumonia in the clinical setting of patients with immunosuppression. In murine models, Pneumocystis has previously been shown to induce a CD4+ T cell-dependent eosinophilic response in the lung capable of providing protection. OBJECTIVES: We sought to explore the role of Pneumocystis in generating asthma-like lung pathology, given the natural eosinophilic response to infection. METHODS: Pneumocystis infection or antigen treatment was used to induce asthma-like pathology in wild-type mice. The roles of CD4+ T cells and eosinophils were examined using antibody depletion and knockout mice, respectively. The presence of anti-Pneumocystis antibodies in human serum samples was detected by ELISA and Western blotting. MEASUREMENTS AND MAIN RESULTS: Pneumocystis infection generates a strong type II response in the lung that requires CD4+ T cells. Pneumocystis infection was capable of priming a Th2 response similar to that of a commonly studied airway allergen, the house dust mite. Pneumocystis antigen treatment was also capable of inducing allergic inflammation in the lung, resulting in anti-Pneumocystis IgE production, goblet cell hyperplasia, and increased airway resistance. In the human population, patients with severe asthma had increased levels of anti-Pneumocystis IgG and IgE compared with healthy control subjects. Patients with severe asthma with elevated anti-Pneumocystis IgG levels had worsened symptom scores and lung parameters such as decreased forced expiratory volume and increased residual volume compared with patients with severe asthma who had low anti-Pneumocystis IgG. CONCLUSIONS: The present study demonstrates for the first time, to our knowledge, that Pneumocystis is an airway allergen capable of inducing asthma-like lung pathology.

Therapeutic Role of Interleukin 22 in Experimental Intra-abdominal Klebsiella pneumoniae Infection in Mice.

Interleukin 22 (IL-22) is an IL-10-related cytokine produced by T helper 17 (Th17) cells and other immune cells that signals via IL-22 receptor alpha 1 (IL-22Ra1), which is expressed on epithelial tissues, as well as hepatocytes. IL-22 has been shown to have hepatoprotective effects that are mediated by signal transducer and activator of transcription 3 (STAT3) signaling. However, it is unclear whether IL-22 can directly regulate antimicrobial programs in the liver. To test this hypothesis, hepatocyte-specific IL-22Ra1 knockout (Il22Ra1(Hep-/-)) and Stat3 knockout (Stat3(Hep-/-)) mice were generated and subjected to intra-abdominal infection with Klebsiella pneumoniae, which results in liver injury and necrosis. We found that overexpression of IL-22 or therapeutic administration of recombinant IL-22 (rIL-22), given 2 h postinfection, significantly reduced the bacterial burden in both the liver and spleen. The antimicrobial activity of rIL-22 required hepatic Il22Ra1 and Stat3. Serum from rIL-22-treated mice showed potent bacteriostatic activity against K. pneumoniae, which was dependent on lipocalin 2 (LCN2). However, in vivo, rIL-22-induced antimicrobial activity was only partially reduced in LCN2-deficient mice. We found that rIL-22 also induced serum amyloid A2 (SAA2) and that SAA2 had anti-K. pneumoniae bactericidal activity in vitro. These results demonstrate that IL-22, through IL-22Ra1 and STAT3 singling, can induce intrinsic antimicrobial activity in the liver, which is due in part to LCN2 and SAA2. Therefore, IL-22 may be a useful adjunct in treating hepatic and intra-abdominal infections.

Memory CD4+ T cells are required for optimal NK cell effector functions against the opportunistic fungal pathogen Pneumocystis murina.

Little is known about the role of NK cells or their interplay with other immune cells during opportunistic infections. Using our murine model of Pneumocystis pneumonia, we found that loss of NK cells during immunosuppression results in substantial Pneumocystis lung burden. During early infection of C57B/6 CD4(+) T cell-depleted mice, there were significantly fewer NK cells in the lung tissue compared with CD4(+) T cell-intact animals, and the NK cells present demonstrated decreased upregulation of the activation marker NKp46 and production of the effector cytokine, IFN-γ. Furthermore, coincubation studies revealed a significant increase in fungal killing when NK cells were combined with CD4(+) T cells compared with either cell alone, which was coincident with a significant increase in perforin production by NK cells. Finally, however, we found through adoptive transfer that memory CD4(+) T cells are required for significant NK cell upregulation of the activation marker NK group 2D and production of IFN-γ, granzyme B, and perforin during Pneumocystis infection. To the best of our knowledge, this study is the first to demonstrate a role for NK cells in immunity to Pneumocystis pneumonia, as well as to establish a functional relationship between CD4(+) T cells and NK cells in the host response to an opportunistic fungal pathogen.

Novel pneumocystis antigen discovery using fungal surface proteomics.

Pneumonia due to the fungus Pneumocystis jirovecii is a life-threatening infection that occurs in immunocompromised patients. The inability to culture the organism as well as the lack of an annotated genome has hindered antigen discovery that could be useful in developing novel vaccine- or antibody-based therapies as well as diagnostics for this infection. Here we report a novel method of surface proteomics analysis of Pneumocystis murina that reliably detected putative surface proteins that are conserved in Pneumocystis jirovecii. This technique identified novel CD4(+) T-cell epitopes as well as a novel B-cell epitope, Meu10, which encodes a glycosylphosphatidylinositol (GPI)-anchored protein thought to be involved in ascospore assembly. The described technique should facilitate the discovery of novel target proteins for diagnostics and therapeutics for Pneumocystis infection.

Dectin immunoadhesins and pneumocystis pneumonia.

The opportunistic pathogen Pneumocystis jirovecii is a significant cause of disease in HIV-infected patients and others with immunosuppressive conditions. Pneumocystis can also cause complications in treatment following antiretroviral therapy or reversal of immunosuppressive therapy, as the newly reconstituted immune system can develop a pathological inflammatory response to remaining antigens or a previously undetected infection. To target ß-(1,3)-glucan, a structural component of the Pneumocystis cell wall with immune-stimulating properties, we have developed immunoadhesins consisting of the carbohydrate binding domain of Dectin-1 fused to the Fc regions of the 4 subtypes of murine IgG (mIgG). These immunoadhesins bind ß-glucan with high affinity, and precoating the surface of zymosan with Dectin-1:Fc can reduce cytokine production by macrophages in an in vitro stimulation assay. All Dectin-1:Fc variants showed specificity of binding to the asci of Pneumocystis murina, but effector activity of the fusion molecules varied depending on Fc subtype. Dectin-1:mIgG2a Fc was able to reduce the viability of P. murina in culture through a complement-dependent mechanism, whereas previous studies have shown the mIgG1 Fc fusion to increase macrophage-dependent killing. In an in vivo challenge model, systemic expression of Dectin-1:mIgG1 Fc significantly reduced ascus burden in the lung. When administered postinfection in a model of immune reconstitution inflammatory syndrome (IRIS), both Dectin-1:mIgG1 and Dectin-1:mIgG2a Fc reduced hypoxemia despite minimal effects on fungal burden in the lung. Taken together, these data indicate that molecules targeting ß-glucan may provide a mechanism for treatment of fungal infection and for modulation of the inflammatory response to Pneumocystis and other pathogens.

Trp53 negatively regulates autoimmunity via the STAT3-Th17 axis.

Emerging evidence suggests that the tumor suppressor p53 is also a crucial regulator for many physiological processes. Previous observations indicate that p53 suppresses inflammation by inhibiting inflammatory antigen-presenting cells. To investigate the potential role of p53 in autoimmune effector T cells, we generated p53(null)CD45.1 mice by crossing p53(null)CD45.2 and CD45.1 mice. We demonstrate that p53(null)CD45.1 mice spontaneously developed autoimmunity, with a significant increase in IL-17-producing Th17 effectors in their lymph nodes (4.7 ± 1.0%) compared to the age-matched counterparts (1.9 ± 0.8% for p53(null)CD45.2, 1.1 ± 0.2% for CD45.1, and 0.5 ± 0.1% for CD45.2 mice). Likewise, p53(null)CD45.1 mice possess highly elevated serum levels of inflammatory cytokines IL-17 and IL-6. This enhanced Th17 response results largely from an increased sensitivity of p53(null)CD45.1 T cells to IL-6-induced STAT3 phosphorylation. Administration of STAT3 inhibitor S31-201 (IC50 of 38.0 ± 7.2 µM for IL-6-induced STAT3 phosphorylation), but not PBS control, to p53(null)CD45.1 mice suppressed Th17 effectors and alleviated autoimmune pathology. This is the first report revealing that p53 activity in T cells suppresses autoimmunity by controlling Th17 effectors. This study suggests that p53 serves as a guardian of immunological functions and that the p53-STAT3-Th17 axis might be a therapeutic target for autoimmunity.

Role of interleukin-23-dependent antifungal immune responses in dendritic cell-vaccinated mice.

CD40 ligand (CD40L) transduction of antigen-pulsed dendritic cells (DCs) can result in antigen-specific humoral immune responses even in CD4(+) T-cell-depleted settings. Here, we show that CD40L transduction of DCs results in the induction of interleukin-12p40 (IL-12p40), IL-12p70, and IL-23. Using DCs that were deficient in IL-12p40, IL-12p35, or IL-23p19, we show that these molecules are dispensable for primary IgG1 responses to Pneumocystis, but IgG2c was dependent on IL-12p40 and IL-23p19 but not IL-12p35. Antigen-specific recall responses in CD4-deficient mice were critically dependent on IL-12p40 and IL-23p19 expression in DCs and were not affected by the lack of IL-12p35. To confirm that this defect in recall was due to IL-23, transduction of IL-12p40(-/-) DCs with a recombinant adenovirus expressing functional IL-23 restored recall responses in DC-vaccinated CD4-deficient mice. These data show that DC-produced IL-23 is critical for vaccine-induced antigen-specific IgG2c and recall antibody responses in the setting of CD4(+) T-cell depletion.

Divergent roles of IL-23 and IL-12 in host defense against Klebsiella pneumoniae.

Interleukin (IL)-23 is a heterodimeric cytokine that shares the identical p40 subunit as IL-12 but exhibits a unique p19 subunit similar to IL-12 p35. IL-12/23 p40, interferon gamma (IFN-gamma), and IL-17 are critical for host defense against Klebsiella pneumoniae. In vitro, K. pneumoniae-pulsed dendritic cell culture supernatants elicit T cell IL-17 production in a IL-23-dependent manner. However, the importance of IL-23 during in vivo pulmonary challenge is unknown. We show that IL-12/23 p40-deficient mice are exquisitely sensitive to intrapulmonary K. pneumoniae inoculation and that IL-23 p19-/-, IL-17R-/-, and IL-12 p35-/- mice also show increased susceptibility to infection. p40-/- mice fail to generate pulmonary IFN-gamma, IL-17, or IL-17F responses to infection, whereas p35-/- mice show normal IL-17 and IL-17F induction but reduced IFN-gamma. Lung IL-17 and IL-17F production in p19-/- mice was dramatically reduced, and this strain showed substantial mortality from a sublethal dose of bacteria (10(3) CFU), despite normal IFN-gamma induction. Administration of IL-17 restored bacterial control in p19-/- mice and to a lesser degree in p40-/- mice, suggesting an additional host defense requirement for IFN-gamma in this strain. Together, these data demonstrate independent requirements for IL-12 and IL-23 in pulmonary host defense against K. pneumoniae, the former of which is required for IFN-gamma expression and the latter of which is required for IL-17 production.

Interleukin-22 treatment ameliorates alcoholic liver injury in a murine model of chronic-binge ethanol feeding: role of signal transducer and activator of transcription 3.

UNLABELLED: Interleukin-22 (IL-22), a recently identified member of the IL-10 family of cytokines that is produced by Th17 and natural killer cells, plays an important role in controlling bacterial infection, homeostasis, and tissue repair. Here, we tested the effect of IL-22 on alcohol-induced liver injury in a murine model of chronic-binge ethanol feeding. Feeding male C57BL/6 mice with a Lieber-DeCarli diet containing 5% ethanol for 10 days, followed by a single dose of ethanol (5 g/kg body weight) by gavage, induces significant fatty liver and liver injury with peak serum levels of approximately 250 IU/L alanine aminotransferase and 420 IU/L aspartate aminotransferase 9 hours after gavage. Moreover, chronic-binge ethanol administration increases expression of hepatic and serum inflammatory cytokines and hepatic oxidative stress. Using this model, we demonstrate that treatment with IL-22 recombinant protein activates hepatic signal transducer and activator of transcription 3 (STAT3) and ameliorates alcoholic fatty liver, liver injury, and hepatic oxidative stress. Administration with IL-22 adenovirus also prevents alcohol-induced steatosis and liver injury. Deletion of STAT3 in hepatocytes abolishes the hepatoprotection provided by IL-22 in alcoholic liver injury. In addition, IL-22 treatment down-regulates the hepatic expression of fatty acid transport protein, but up-regulates several antioxidant, antiapoptotic, and antimicrobial genes. Finally, expression of IL-22 receptor 1 is up-regulated whereas IL-22 is undetectable in the livers from mice with chronic-binge ethanol feeding or patients with alcoholic hepatitis. CONCLUSION: Chronic-binge ethanol feeding may be a useful model to study the early stages of alcoholic liver injury. IL-22 treatment could be a potential therapeutic option to ameliorate alcoholic liver disease, due to its antioxidant, antiapoptotic, antisteatotic, proliferative, and antimicrobial effects with the added benefit of potentially few side effects.

Lipocalin 2 is required for pulmonary host defense against Klebsiella infection.

Antimicrobial proteins comprise a significant component of the acute innate immune response to infection. They are induced by pattern recognition receptors as well as by cytokines of the innate and adaptive immune pathways and play important roles in infection control and immunomodulatory homeostasis. Lipocalin 2 (siderocalin, NGAL, 24p3), a siderophore-binding antimicrobial protein, is critical for control of systemic infection with Escherichia coli; however, its role in mucosal immunity in the respiratory tract is unknown. In this study, we found that lipocalin 2 is rapidly and robustly induced by Klebsiella pneumoniae infection and is TLR4 dependent. IL-1beta and IL-17 also individually induce lipocalin 2. Mucosal administration of IL-1beta alone could reconstitute the lipocalin 2 deficiency in TLR4 knockout animals and rescue them from infection. Lipocalin 2-deficient animals have impaired lung bacterial clearance in this model and mucosal reconstitution of lipocalin 2 protein in these animals resulted in rescue of this phenotype. We conclude that lipocalin 2 is a crucial component of mucosal immune defense against pulmonary infection with K. pneumoniae.

Alveolar macrophage-mediated killing of Pneumocystis carinii f. sp. muris involves molecular recognition by the Dectin-1 beta-glucan receptor.

Innate immune mechanisms against Pneumocystis carinii, a frequent cause of pneumonia in immunocompromised individuals, are not well understood. Using both real time polymerase chain reaction as a measure of organism viability and fluorescent deconvolution microscopy, we show that nonopsonic phagocytosis of P. carinii by alveolar macrophages is mediated by the Dectin-1 beta-glucan receptor and that the subsequent generation of hydrogen peroxide is involved in alveolar macrophage-mediated killing of P. carinii. The macrophage Dectin-1 beta-glucan receptor colocalized with the P. carinii cyst wall. However, blockage of Dectin-1 with high concentrations of anti-Dectin-1 antibody inhibited binding and concomitant killing of P. carinii by alveolar macrophages. Furthermore, RAW 264.7 macrophages overexpressing Dectin-1 bound P. carinii at a higher level than control RAW cells. In the presence of Dectin-1 blockage, killing of opsonized P. carinii could be restored through FcgammaRII/III receptors. Opsonized P. carinii could also be efficiently killed in the presence of FcgammaRII/III receptor blockage through Dectin-1-mediated phagocytosis. We further show that Dectin-1 is required for P. carinii-induced macrophage inflammatory protein 2 production by alveolar macrophages. Taken together, these results show that nonopsonic phagocytosis and subsequent killing of P. carinii by alveolar macrophages is dependent upon recognition by the Dectin-1 beta-glucan receptor.

Transcriptomic and Proteomic Approaches to Finding Novel Diagnostic and Immunogenic Candidates in Pneumocystis.

Pneumocystis pneumonia is the most common serious opportunistic infection in patients with HIV/AIDS. Furthermore, Pneumocystis pneumonia is a feared complication of the immunosuppressive drug regimens used to treat autoimmunity, malignancy, and posttransplantation rejection. With an increasing at-risk population, there is a strong need for novel approaches to discover diagnostic and vaccine targets. There are multiple challenges to finding these targets, however. First, Pneumocystis has a largely unannotated genome. To address this, we evaluated each protein encoded within the Pneumocystis genome by comparisons to proteins encoded within the genomes of other fungi using NCBI BLAST. Second, Pneumocystis relies on a multiphasic life cycle, as both the transmissible form (the ascus) and the replicative form (the trophozoite [troph]) reside within the alveolar space of the host. To that end, we purified asci and trophs from Pneumocystis murina and utilized transcriptomics to identify differentially regulated genes. Two such genes, Arp9 and Sp, are differentially regulated in the ascus and the troph, respectively, and can be utilized to characterize the state of the Pneumocystis life cycle in vivoGsc1, encoding a ß-1,3-glucan synthase with a large extracellular domain previously identified using surface proteomics, was more highly expressed on the ascus form of Pneumocystis GSC-1 ectodomain immunization generated a strong antibody response that demonstrated the ability to recognize the surface of the Pneumocystis asci. GSC-1 ectodomain immunization was also capable of reducing ascus burden following primary challenge with Pneumocystis murina Finally, mice immunized with the GSC-1 ectodomain had limited fungal burden following natural transmission of Pneumocystis using a cohousing model.IMPORTANCE The current report enhances our understanding of Pneumocystis biology in a number of ways. First, the current study provided a preliminary annotation of the Pneumocystis murina genome, addressing a long-standing issue in the field. Second, this study validated two novel transcripts enriched in the two predominant life forms of Pneumocystis These findings allow better characterization of the Pneumocystis life cycle in vivo and could be valuable diagnostic tools. Furthermore, this study outlined a novel pipeline of -omics techniques capable of revealing novel antigens (e.g., GSC-1) for the development of vaccines against Pneumocystis.

Interleukin-12 and host defense against murine Pneumocystis pneumonia.

Little is known about the role of the cytokine interleukin-12 (IL-12) in Pneumocystis pneumonia or its potential use as immunotherapy. We asked whether release of IL-12 is part of the normal host response to this infection and whether local treatment with IL-12 or gene transfer of IL-12 could accelerate clearance of infection. IL-12 was assayed by enzyme-linked immunosorbent assay in normal mice and in mice deficient in IL-12 after inoculation of Pneumocystis carinii. P. carinii-infected mice were treated with local instillation of IL-12 and gene transfer of the IL-12 gene. Inoculation of P. carinii into normal mice evoked a brisk release of IL-12 into lung tissue, and IL-12 P35-deficient mice showed delayed clearance of infection measured by PCR for P. carinii rRNA. In control mice, intranasal recombinant IL-12 accelerated clearance of infection, and this was associated with increased recruitment of inflammatory cells into lavage fluid and increased release of tumor necrosis factor alpha, IL-12, and gamma interferon. Similar results were observed in infected mice depleted of CD4+ lymphocytes by using in vivo transfer of the IL-12 gene in a replication-deficient adenoviral vector. IL-12 is part of the normal host response to infection with P. carinii. IL-12 therapy can enhance host resistance to infection in both normal mice and mice depleted of CD4+ T lymphocytes. A treatment effect of IL-12 is mediated through enhanced inflammatory cell recruitment into lung tissue and increased tissue concentrations of proinflammatory cytokines.

CD4+ T cell-independent DNA vaccination against opportunistic infections.

Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modified DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.

Murine models of Pneumocystis infection recapitulate human primary immune disorders.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

Critical role of IL-17 receptor signaling in acute TNBS-induced colitis.

BACKGROUND: Inflammatory bowel diseases (IBDs) such as Crohn's disease and ulcerative colitis are characterized by recurrent inflammation in the gastrointestinal tract. Infiltration of CD4 lymphocytes and neutrophils is one of the predominant features of IBD. MATERIALS AND METHODS: Recently, interleukin (IL)-23 and the downstream T cell-derived cytokine IL-17 have been found to be elevated in intestinal tissue and serum of IBD patients. However, the role of IL-17 and IL-17R signaling in gut inflammation is unknown. To examine this role, we investigated gut inflammation in wild-type or IL-17R knockout mice. RESULTS: Using a model of acute trinitrobenzenesulfonic acid (TNBS)-induced colitis, we found that IL-17 was produced in colon tissue at 24 and 48 hours and that IL-17R knockout mice were significantly protected against TNBS-induced weight loss, IL-6 production, colonic inflammation, and local macrophage inflammatory protein-2 induction. This protection occurred in the presence of equivalent induction of local IL-23 and higher levels of IL-12p70 and interferon-gamma in IL-17R knockout mice compared with wild-type mice. Moreover, IL-17R knockout mice showed reduced tissue myeloperoxidase activity. Furthermore, overexpression of an IL-17R IgG1 fusion protein significantly attenuated colonic inflammation after acute TNBS. CONCLUSIONS: These results demonstrate that IL-17R signaling plays a critical role in the development of TNBS-induced colitis and may represent a target for therapeutic intervention for IBD.

Investigation of Pneumocystis jirovecii colonization in patients with chronic pulmonary diseases in the People's Republic of China.

BACKGROUND: The detection of Pneumocystis jirovecii DNA in respiratory specimen from individuals who do not have signs or symptoms of pneumonia has been defined as colonization. The role of P. jirovecii colonization in the development or progression of various lung diseases has been reported, but little information about P. jirovecii colonization in patients is available in the People's Republic of China. OBJECTIVE: To determine the prevalence of P. jirovecii colonization in patients with various pulmonary diseases, including the acute and stable stage of COPD, interstitial lung diseases, cystic fibrosis, and chronic bronchiectasis. MATERIALS AND METHODS: A loop-mediated isothermal amplification (LAMP) and a conventional polymerase chain reaction (PCR) method for detecting P. jirovecii were developed. Ninety-eight HIV-negative patients who were followed-up and who had undergone bronchoscopy for diagnosis of various underlying respiratory diseases were included in the study. Sputa of these patients were analyzed with LAMP amplification of P. jirovecii gene. In addition, conventional PCR, Giemsa and Gomori's methenamine silver nitrate staining assays were applied to all specimens. RESULTS: The sensitivity and specificity test showed that there was no cross-reaction with other fungi or bacteria in detecting the specific gene of P. jirovecii by LAMP, and the minimum detection limits by LAMP was 50 copies/mL. P. jirovecii DNA was detected in 62 of 98 (63.3%) sputa specimens by LAMP assay and 22.45% (22/98) by conventional PCR. However, no P. jirovecii cysts were found by Giemsa and Gomori's methenamine silver nitrate in all of gene-positive specimens. CONCLUSION: The results of our study showed that prevalence of P. jirovecii colonization is particularly high in patients with chronic pulmonary diseases in the People's Republic of China, and the LAMP method is better for evaluation of the colonization of P. jirovecii in sputum specimen than conventional PCR.

Ethanol and LPS modulate NF-kappaB activation, inducible NO synthase and COX-2 gene expression in rat liver cells in vivo.

Ethanol and LPS are immunomodulators, whose actions are associated with the activation of the transcription factor, NF-kappaB, that mediates the expression of a number of rapid response genes involved in the whole body inflammatory response to injury, including transcriptional regulation of iNOS and COX-2. We investigated modulation by acute ethanol (EtOH) intoxication, LPS and LPS tolerance of NF-kappaB activation in hepatocytes, Kupffer cells and sinusoidal endothelial cells (SEC), concurrent regulation of iNOS and COX-2 gene expression and the influence of gender on these mechanisms. In vivo EtOH alone or with LPS significantly activates NF-kappaB in Kupffer cells and SEC. iNOS gene expression in these cells is modulated by LPS+EtOH in a gender- dependent manner. Acute EtOH administration enhanced iNOS mRNA in hepatocytes and Kupffer cells.LPS tolerance decreased LPS-induced NF-kappaB activation in Kupffer cells, but markedly raised iNOS mRNA in all three cell types with gender differences (females being higher). In LPS tolerant rats EtOH decreased elevated iNOS mRNA in all cells studied. LPS tolerance significantly reduced LPS-induced COX-2 mRNA in SEC, but only moderately in Kupffer cells of females, and not at all in males. Since NO is a known scavenger of superoxide and therefore protective against oxidative injury associated with LPS and acute EtOH intoxication, the gender differential effect of LPS+EtOH on iNOS gene expression (reduced only in females) may contribute to the greater susceptibility of females to alcoholic liver disease. Suppression of COX-2 gene expression in SEC may cause detrimental effects in the hepatic microcirculation, associated with cirrhosis.