Doris B. Tse

Diesel Exhaust Particle-Exposed Human Bronchial Epithelial Cells Induce Dendritic Cell Maturation

Increased exposure to air pollutants such as diesel exhaust particles (DEP) has been proposed as one mechanism to explain the rise in allergic disorders. However, the immunologic mechanisms by which DEP enhance allergic sensitization and asthma remain unclear. We hypothesized that DEP act as an adjuvant for immature dendritic cell (DC) maturation via its effect on airway epithelial cell-derived microenvironment for DC. Immature monocyte-derived DC (iMDDC) failed to undergo phenotypic (CD80, CD83, CD86) or functional (T cell activation) maturation in response to exposure to DEP (0.001–100 μg/ml). In contrast, primary cultures of human bronchial epithelial cells (HBEC) treated with DEP induced iMDDC phenotypic maturation (2.6 ± 0.1-fold increase in CD83 expression, n = 4, p < 0.05) and functional maturation (2.6 ± 0.2-fold increase in T cell activation, n = 4, p < 0.05). Functional maturation of iMDDC was induced by conditioned medium derived from DEP-treated HBEC, and was inhibited in cultures with DEP-treated HBEC and blocking Abs against GM-CSF, or GM-CSF-targeted small interfering RNA. These data suggest that DEP induce Ag-independent DC maturation via epithelial cell-DC interactions mediated by HBEC-derived GM-CSF. Although additional signals may be required for polarization of DC, these data suggest a novel mechanism by which environmental pollutants alter airway immune responses.

Maximal HIV-1 replication in alveolar macrophages during tuberculosis requires both lymphocyte contact and cytokines.

HIV-1 replication is markedly upregulated in alveolar macrophages (AM) during pulmonary tuberculosis (TB). This is associated with loss of an inhibitory CCAAT enhancer binding protein β (C/EBPβ) transcription factor and activation of nuclear factor (NF)-κB. Since the cellular immune response in pulmonary TB requires lymphocyte–macrophage interaction, a model system was developed in which lymphocytes were added to AM. Contact between lymphocytes and AM reduced inhibitory C/EBPβ, activated NF-κB, and enhanced HIV-1 replication. If contact between lymphocytes and macrophages was prevented, inhibitory C/EBPβ expression was maintained and the HIV-1 long terminal repeat (LTR) was not maximally stimulated although NF-κB was activated. Antibodies that cross-linked macrophage expressed B-7, and vascular cell adhesion molecule and CD40 were used to mimic lymphocyte contact. All three cross-linking antibodies were required to abolish inhibitory C/EBPβ expression. However, the HIV-1 LTR was not maximally stimulated and NF-κB was not activated. Maximal HIV-1–LTR stimulation required both lymphocyte-derived soluble factors, and cross-linking of macrophage expressed costimulatory molecules. High level HIV-1–LTR stimulation was also achieved when IL-1β, IL-6, and TNF-β were added to macrophages with cross-linked costimulatory molecules. Contact between activated lymphocytes and macrophages is necessary to down-regulate inhibitory C/EBPβ, thereby derepressing the HIV-1 LTR. Lymphocyte-derived cytokines activate NF-κB, further enhancing the HIV-1 LTR.

Diesel exhaust particle-exposed human bronchial epithelial cells induce dendritic cell maturation and polarization via thymic stromal lymphopoietin.

Human exposure to air pollutants, including ambient particulate matter, has been proposed as a mechanism for the rise in allergic disorders. Diesel exhaust particles, a major component of ambient particulate matter, induce sensitization to neoallergens, but the mechanisms by which sensitization occur remain unclear. We show that diesel exhaust particles upregulate thymic stromal lymphopoietin in human bronchial epithelial cells in an oxidant-dependent manner. Thymic stromal lymphopoietin induced by diesel exhaust particles was associated with maturation of myeloid dendritic cells, which was blocked by anti-thymic stromal lymphopoietin antibodies or silencing epithelial cell-derived thymic stromal lymphopoietin. Dendritic cells exposed to diesel exhaust particle-treated human bronchial epithelial cells induced Th2 polarization in a thymic stromal lymphopoietin-dependent manner. These findings provide new insight into the mechanisms by which diesel exhaust particles modify human lung mucosal immunity.

Diesel Exhaust Particle-Treated Human Bronchial Epithelial Cells Upregulate Jagged-1 and OX40 Ligand in Myeloid Dendritic Cells via Thymic Stromal Lymphopoietin

Ambient particulate matter, including diesel exhaust particles (DEP), promotes the development of allergic disorders. DEP increase oxidative stress and influence human bronchial epithelial cell (HBEC)–dendritic cell interactions via cytokines, including thymic stromal lymphopoietin (TSLP). Upregulation of TSLP results in Th2 responses. Using primary culture HBEC and human myeloid dendritic cell (mDC) cocultures, we show in this study that DEP upregulation of Th2 responses occurred via HBEC-dependent mechanisms that resulted from oxidative stress. Moreover, DEP-treated HBEC and ambient particulate matter-treated HBEC upregulated OX40 ligand (OX40L) and the Notch ligand Jagged-1 mRNA and expression on mDC. Upregulation of OX40L as well as Jagged-1 on mDC required HBEC and did not occur in the presence of N-acetylcysteine. Furthermore, OX40L and Jagged-1 upregulation was inhibited when HBEC expression of TSLP was silenced. Thus, DEP treatment of HBEC targeted two distinct pathways in mDC that were downstream of TSLP expression. Upregulation of OX40L and Jagged-1 by mDC resulted in mDC-driven Th2 responses. These studies expand our understanding of the mechanism by which ambient pollutants alter mucosal immunity and promote disorders such as asthma.

Mycobacterium tuberculosis-Induced CXCR4 and Chemokine Expression Leads to Preferential X4 HIV-1 Replication in Human Macrophages

Opportunistic infections such as pulmonary tuberculosis (TB) increase local HIV-1 replication and mutation. As AIDS progresses, alteration of the HIV-1 gp120 V3 sequence is associated with a shift in viral coreceptor use from CCR5 (CD195) to CXCR4 (CD184). To better understand the effect of HIV/TB coinfection, we screened transcripts from bronchoalveolar lavage cells with high density cDNA arrays and found that CXCR4 mRNA is increased in patients with TB. Surprisingly, CXCR4 was predominately expressed on alveolar macrophages (AM). Mycobacterium tuberculosis infection of macrophages in vitro increased CXCR4 surface expression, whereas amelioration of disease reduced CXCR4 expression in vivo. Bronchoalveolar lavage fluid from TB patients had elevated levels of CCL4 (macrophage inflammatory protein-1β), CCL5 (RANTES), and CX3CL1 (fractalkine), but not CXCL12 (stromal-derived factor-1α). We found that M. tuberculosis infection of macrophages in vitro increased viral entry and RT of CXCR4, using HIV-1, but not of CCR5, using HIV-1. Lastly, HIV-1 derived from the lung contains CD14, suggesting that they were produced in AM. Our results demonstrate that TB produces a permissive environment for replication of CXCR4-using virus by increasing CXCR4 expression in AM and for suppression of CCR5-using HIV-1 by increasing CC chemokine expression. These changes explain in part why TB accelerates the course of AIDS. CXCR4 inhibitors are a rational therapeutic approach in HIV/TB coinfection.

Immunomodulation with Recombinant Interferon-γ1b in Pulmonary Tuberculosis

Background Current treatment regimens for pulmonary tuberculosis require at least 6 months of therapy. Immune adjuvant therapy with recombinant interferon-γ1b (rIFN-γb) may reduce pulmonary inflammation and reduce the period of infectivity by promoting earlier sputum clearance. Methodology/Principal Findings We performed a randomized, controlled clinical trial of directly observed therapy (DOTS) versus DOTS supplemented with nebulized or subcutaneously administered rIFN-γ1b over 4 months to 89 patients with cavitary pulmonary tuberculosis. Bronchoalveolar lavage (BAL) and blood were sampled at 0 and 4 months. There was a significant decline in levels of inflammatory cytokines IL-1β, IL-6, IL-8, and IL-10 in 24-hour BAL supernatants only in the nebulized rIFN-γ1b group from baseline to week 16. Both rIFN-γ1b groups showed significant 3-fold increases in CD4+ lymphocyte response to PPD at 4 weeks. There was a significant (p = 0.03) difference in the rate of clearance of Mtb from the sputum smear at 4 weeks for the nebulized rIFN-γ1b adjuvant group compared to DOTS or DOTS with subcutaneous rIFN-γ1b. In addition, there was significant reduction in the prevalence of fever, wheeze, and night sweats at 4 weeks among patients receiving rFN-γ1b versus DOTS alone. Conclusion Recombinant interferon-γ1b adjuvant therapy plus DOTS in cavitary pulmonary tuberculosis can reduce inflammatory cytokines at the site of disease, improve clearance of Mtb from the sputum, and improve constitutional symptoms. Trial Registration ClinicalTrials.gov NCT00201123

CD40 Contributes to Lethality in Acute Sepsis: In Vivo Role for CD40 in Innate Immunity

ABSTRACT Sepsis induces an early inflammatory cascade initiated by the innate immune response. This often results in the development of multisystem organ failure. We examined the role of CD40, a costimulatory molecule that is integral in adaptive immunity, by using a murine model of polymicrobial sepsis. CD40 knockout (KO) mice had delayed death and improved survival after cecal ligation and puncture (CLP). In addition, they had less remote organ injury as manifested by reduced pulmonary capillary leakage. The improvements in survival and remote organ dysfunction in CD40 KO mice were associated with reduced interleukin-6 (IL-6) and IL-10 levels in serum and bronchoalveolar lavage fluid compared to the levels in wild-type (WT) controls. Furthermore, in contrast to WT mice, CD40 KO mice had no induction of the Th1 cytokines IL-12 and gamma interferon in serum or lungs after CLP. The alterations in cytokine production in CD40 KO mice were associated with similar changes in transcription factor activity. After CLP, CD40 KO mice had attenuated activation of nuclear factor κB and signal transducer and activator of transcription 3 in both the lung and the liver. Finally, WT mice had increased expression of CD40 on their alveolar macrophages. These data highlight the importance of CD40 activation in the innate immune response during polymicrobial sepsis and the subsequent development of remote organ dysfunction.

In Situ Activation of Helper T Cells in the Lung

ABSTRACT To better understand the lung and systemic responses of helper T cells mediating memory immunity to Mycobacterium tuberculosis, we used three- and four-color flow cytometry to study the surface phenotype of CD4+ lymphocytes. Bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) samples were obtained from a total of 25 subjects, including 10 tuberculosis (TB)-infected subjects, 8 purified-protein-derivative-negative subjects, and 7 purified-protein-derivative-positive subjects. In marked contrast to CD4+ lymphocytes from PB (9% ± 5% expressing CD45RA and CD29), the majority (55% ± 16%) of CD4+ lymphocytes in BAL (ALs) simultaneously expressed CD45RA, a naı̈ve T-cell marker, and CD29, members of the very late activation family. Further evaluation revealed that CD4+ ALs expressed both CD45RA and CD45RO, a memory T-cell marker. In addition, the proportion of CD4+ lymphocytes expressing CD69, an early activation marker, was drastically increased in BAL fluid (83% ± 9%) compared to PB (1% ± 1%), whereas no significant difference was seen in the expression of CD25, the low-affinity interleukin 2 receptor (34% ± 15% versus 40% ± 16%). More importantly, we identified a minor population of CD69bright CD25bright CD4+lymphocytes in BAL (10% ± 6%) that were consistently absent from PB (1% ± 1%). Thus, CD4+ lymphocytes in the lung paradoxically coexpress surface molecules characteristic of naı̈ve and memory helper T cells as well as surface molecules commonly associated with early and late stages of activation. No difference was observed for ALs obtained from TB-infected and uninfected lung segments in this regard. It remains to be determined if these surface molecules are induced by the alveolar environment or if CD4+ lymphocytes coexpressing this unusual combination of surface molecules are selectively recruited from the circulation. Our data suggest that ex vivo experiments on helper T-cell subsets that display distinctive phenotypes may be pivotal to studies on the human immune response to potential TB vaccines.

Host-dependent Lewis (Le) antigen expression in Helicobacter pylori cells recovered from Leb-transgenic mice

Variation of surface antigen expression is a mechanism used by microbes to adapt to and persist within their host habitats. Helicobacter pylori, a persistent bacterial colonizer of the human stomach, can alter its surface Lewis (Le) antigen expression. We examined H. pylori colonization in mice to test the hypothesis that host phenotype selects for H. pylori (Le) phenotypes. When wild-type and Leb-expressing transgenic FVB/N mice were challenged with H. pylori strain HP1, expressing Lex and Ley, we found that bacterial populations recovered after 8 mo from Leb-transgenic, but not wild-type, mice expressed Leb. Changes in Le phenotype were linked to variation of a putative galactosyltransferase gene (β-(1,3)galT); mutagenesis and complementation revealed its essential role in type I antigen expression. These studies indicate that H. pylori evolves to resemble the hosts gastric Le phenotype, and reveal a bacterial genetic locus that is subject to host-driven selection pressure.

Improving immunogenicity of HIV-1 envelope gp120 by glycan removal and immune complex formation

HIV-1 envelope (Env) gp120 is an important target for neutralizing antibody (Ab) responses against the virus; however, developing gp120 vaccines that elicit potent and broad neutralizing Abs has proven to be a formidable challenge. Previously, removal of an N-linked glycan at residue 448 by an N to Q mutation (N448Q) has been found to enhance the in vitro antigenicity of neutralizing epitopes in the V3 loop. In this study the mutated gp120 was first compared with wild type gp120 for immunogenicity in mice using a DNA prime and protein boost immunization regimen. The N448Q mutant did not elicit higher titers of anti-gp120 serum Abs and failed to generate anti-V3 Abs. The sera also had no virus-neutralizing activity, even though the mutant induced higher levels of lymphoproliferation and cytokine production. Subsequently, the N448Q mutant was used to construct an immune complex vaccine with the anti-CD4 binding site monoclonal antibody (mAb) 654. The N448Q/654 complex stimulated comparably high levels of serum Abs to gp120 and V3 as the wild type complex. However, Abs against the C1 and C2 regions in the gp120 core were more elevated. Importantly, the mutant complex also elicited higher titers of neutralizing Abs activity than the wild type counterpart. Similar results were achieved with a complex made with gp120 bearing an N448E mutation, confirming the importance of the N448-linked glycan in modulating gp120 immunogenicity. Neutralizing activity was directed to V3 and other undefined neutralizing epitopes. Improved immunogenicity of the immune complexes correlated with alterations in exposure of V3 and other Ab epitopes and their stability against proteases. These data demonstrate the advantage of combining site-specific N-glycan removal and immune complex formation as a novel vaccine strategy to improve immunogenicity of targeted Ab epitopes on critical regions of HIV-1 gp120.