Natural killer T cells mediate inflammation in the bile ducts

Cholangiocytes function as antigen-presenting cells with CD1d-dependent activation of natural killer T (NKT) cells in vitro. NKT cells may act both pro- and anti-inflammatory in liver immunopathology. We explored this immune pathway and the antigen-presenting potential of NKT cells in the bile ducts by challenging wild-type and Cd1d^−/− mice with intrabiliary injection of the NKT cell activating agent oxazolone. Pharmacological blocking of CD1d-mediated activation was performed with a monoclonal antibody. Intrabiliary oxazolone injection in wild-type mice caused acute cholangitis with significant weight loss, elevated serum levels of alanine transaminase, aspartate transaminase, alkaline phosphatase and bilirubin, increased histologic grade of cholangitis and number of T cells, macrophages, neutrophils and myofibroblasts per portal tract after 7 days. NKT cells were activated after intrabiliary injection of oxazolone with upregulation of activation markers. Cd1d^−/− and wild-type mice pretreated with antibody blocking of CD1d were protected from disease. These findings implicate that cells in the bile ducts function as antigen-presenting cells in vivo and activate NKT cells in a CD1d-restricted manner. The elucidation of this biliary immune pathway opens up for potentially new therapeutic approaches for cholangiopathies.     

IFN‐γ‐producing NKT cells exacerbate sepsis by enhancing C5a generation via IL‐10‐mediated inhibition of CD55 expression on neutrophils

A role for NKT cells has been implicated in sepsis, but the mechanism by which NKT cells contribute to sepsis remains unclear. Here, we examined WT and NKT‐cell‐deficient mice of C57BL/6 background during cecal ligation and puncture‐induced sepsis. The levels of C5a, IFN‐γ, and IL‐10 were higher in the serum and peritoneal fluid of WT mice than in those of CD1d^?/? mice, while the mortality rate was lower in CD1d^?/? mice than in WT mice. C5a blockade decreased mortality of WT mice during sepsis, whereas it did not alter that of CD1d^?/? mice. As assessed by intracellular staining, NKT cells expressed IFN‐γ, while neutrophils expressed IL‐10. Upon coculture, IL‐10‐deficient NKT cells enhanced IL‐10 production by WT, but not IFN‐γR‐deficient, neutrophils. Meanwhile, CD1d^?/? mice exhibited high CD55 expression on neutrophils during sepsis, whereas those cells from WT mice expressed minimal levels of CD55. Recombinant IL‐10 administration into CD1d^?/? mice reduced CD55 expression on neutrophils. Furthermore, adoptive transfer of sorted WT, but not IFN‐γ‐deficient, NKT cells into CD1d^?/? mice suppressed CD55 expression on neutrophils, but increased IL‐10 and C5a levels. Taken together, IFN‐γ‐producing NKT cells enhance C5a generation via IL‐10‐mediated inhibition of CD55 expression on neutrophils, thereby exacerbating sepsis.     

B-cell-specific-peroxisome proliferator-activated receptor γ deficiency augments contact hypersensitivity with impaired regulatory B cells

Nuclear receptor peroxisome proliferator-activated receptor γ (PPAR-γ) activation can prevent immunoinflammatory disorders and diabetes. B cells play protective roles during inflammation as well. However, the roles of endogenous PPAR-γ in the regulatory properties of B cells to relieve inflammation remain unknown. Here, we developed B-cell-specific PPAR-γ knockout (B-PPAR-γ^−/−) mice and found that the conditional deletion of PPAR-γ in B cells resulted in exaggerated contact hypersensitivity (CHS). Meanwhile, interferon-γ (IFN-γ) of CD4⁺ CD8⁺ T cells was up-regulated in B-PPAR-γ^−/− mice in CHS. This showed that the regulatory function of B cells in B-PPAR-γ^−/− mice declined in vivo. Whereas splenic CD5⁺ CD1d^hi regulatory B-cell numbers and peripheral regulatory T-cell numbers were not changed in naive B-PPAR-γ^−/− mice. Loss of PPAR-γ in B cells also did not affect either CD86 or FasL expression in splenic CD5⁺ CD1d^hi regulatory B cells after activation. Notably, interleukin-10 (IL-10) production in CD5⁺ CD1d^hi regulatory B cells reduced in B-PPAR-γ-deficient mice. In addition, functional IL-10-producing CD5⁺ CD1d^hi regulatory B cells decreased in B-PPAR-γ^−/− mice in the CHS model. These findings were in accordance with augmented CHS. The current work indicated the involvement of endogenous PPAR-γ in the regulatory function of B cells by disturbing the expansion of IL-10-positive regulatory B cells.     

Thymic heterotypic cellular complexes in gene-targeted mice with defined blocks in T cell development and adhesion molecule expression

Thymocytes form unique multicellular complexes with epithelial cells (thymic nurse cells, TNC) and rosettes (ROS) with macrophages, epithelial cells and dendritic cells. To investigate the role of differentiation checkpoints in the formation of the thymic heterotypic complexes in vivo, we used mutant mice which have genetically defined blocks at early and late stages of T cell development. We show that RAG-1^−/−, TCRβ^−/−, and p56^lck−/− mice lack thymocyte ROS formation with epithelial cells, macrophages, or dendritic cells. TNC formation was not affected by TCRβ and p56^lck gene mutations but partially decreased in RAG-1^−/− mice, indicating that TNC are the earliest thymocyte-stromal cell complexes formed in development, whereas ROS only appear after thymocytes have rearranged and expressed a functional TCRβ chain. Genetic blocks in CD8 lineage commitment (CD8^−/− and IFN regulatory factor-1^−/− mice) and positive and negative T cell selection (CD45^−/−, TCRα^−/−, and CD30^−/− mice) did not affect thymocyte-stromal cell complexes. Surprisingly, CD4^−/− mice, but not MHC class II^−/− mice, had significantly reduced numbers of TNC and ROS, in particular, a severe defect in ROS formation with thymic dendritic cells. The CD4^−/− block in ROS and TNC formation was rescued by the introduction of a human CD4 transgene. Moreover, we show that the adhesion receptors CD44 and LFA-1 cooperate in the formation of the thymic microenvironment. These results provide genetic evidence on the role of defined stages in T cell development and adhesion molecules on thymocyte/stromal cell interactions in vitro.     

CD28 controls the development of innate‐like CD8+ T cells by promoting the functional maturation of NKT cells

NK T cells(NKT cells) share functional characteristics and homing properties that are distinct from conventional T cells. In this study, we investigated the contribution of CD28 in the functional development of γδ NKT and αβ NKT cells in mice. We show that CD28 promotes the thymic maturation of promyelocytic leukemia zinc finger⁺ IL‐4⁺ NKT cells and upregulation of LFA‐1 expression on NKT cells. We demonstrate that the developmental defect of γδ NKT cells in CD28‐deficient mice is cell autonomous. Moreover, we show in both wild‐type C57BL/6 mice and in downstream of tyrosine kinase‐1 transgenic mice, a mouse model with increased numbers of γδ NKT cells, that CD28‐mediated regulation of thymic IL‐4⁺ NKT cells promotes the differentiation of eomesodermin⁺ CD44^high innate‐like CD8⁺ T cells. These findings reveal a previously unappreciated mechanism by which CD28 controls NKT‐cell homeostasis and the size of the innate‐like CD8⁺ T‐cell pool.     

CD40‐mediated signalling influences trafficking,T‐cell receptor expression,and T‐cell pathogenesis,in the NOD model of type 1 diabetes

CD40 plays a critical role in the pathogenesis of type 1 diabetes (T1D). The mechanism of action, however, is undetermined, probably because CD40 expression has been grossly underestimated. CD40 is expressed on numerous cell types that now include T cells and pancreatic β cells. CD40⁺ CD4⁺ cells [T helper type 40 (TH40)] prove highly pathogenic in NOD mice and in translational human T1D studies. We generated BDC2.5.CD40^−/− and re‐derived NOD.CD154^−/− mice to better understand the CD40 mechanism of action. Fully functional CD40 expression is required not only for T1D development but also for insulitis. In NOD mice, TH40 cell expansion in pancreatic lymph nodes occurs before insulitis and demonstrates an activated phenotype compared with conventional CD4⁺ cells, apparently regardless of antigen specificity. TH40 T‐cell receptor (TCR) usage demonstrates increases in several Vα and Vβ species, particularly Vα3.2⁺ that arise early and are sustained throughout disease development. TH40 cells isolated from diabetic pancreas demonstrate a relatively broad TCR repertoire rather than restricted clonal expansions. The expansion of the Vα/Vβ species associated with diabetes depends upon CD40 signalling; NOD.CD154^−/− mice do not expand the same TCR species. Finally, CD40‐mediated signals significantly increase pro‐inflammatory Th1‐ and Th17‐associated cytokines whereas CD28 co‐stimulus alternatively promotes regulatory cytokines.     

A disintegrin and metalloproteinase domain-15 deficiency leads to exaggerated cigarette smoke-induced chronic obstructive pulmonary disease (COPD)-like disease in mice

A disintegrin and metalloproteinase domain-15 (ADAM15) is expressed by cells implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD), but its contributions to COPD are unknown. To address this gap, ADAM15 levels were measured in samples from cigarette smoke (CS)-versus air-exposed wild-type (WT) mice. CS-induced COPD-like disease was compared in CS-exposed WT, Adam15^−/−, and Adam15 bone marrow chimeric mice. CS exposure increased Adam15 expression in lung macrophages and CD8⁺ T cells and to a lesser extent in airway epithelial cells in WT mice. CS-exposed Adam15^−/− mice had greater emphysema, small airway fibrosis, and lung inflammation (macrophages and CD8⁺ T cells) than WT mice. Adam15 bone marrow chimera studies revealed that Adam15 deficiency in leukocytes led to exaggerated pulmonary inflammation and COPD-like disease in mice. Adam15 deficiency in CD8⁺ T cells was required for the exaggerated pulmonary inflammation and COPD-like disease in CS-exposed Adam15^−/− mice (as assessed by genetically deleting CD8⁺ T cells in Adam15^−/− mice). Adam15 deficiency increased pulmonary inflammation by rendering CD8⁺ T cells and macrophages resistant to CS-induced activation of the mitochondrial apoptosis pathway by preserving mTOR signaling and intracellular Mcl-1 levels in these cells. These results strongly link ADAM15 deficiency to the pathogenesis of COPD.     

Anti-tumour synergy of cytotoxic chemotherapy and anti-CD40 plus CpG-ODN immunotherapy through repolarization of tumour-associated macrophages

We studied the effectiveness of monoclonal anti‐CD40 + cytosine–phosphate–guanosine‐containing oligodeoxynucleotide 1826 (CpG‐ODN) immunotherapy (IT) in mice treated with multidrug chemotherapy (CT) consisting of vincristine, cyclophosphamide and doxorubicin. Combining CT with IT led to synergistic anti‐tumour effects in C57BL/6 mice with established B16 melanoma or 9464D neuroblastoma. CT suppressed the functions of T cells and natural killer (NK) cells, but primed naïve peritoneal macrophages (Mφ) to in vitro stimulation with lipopolysaccharide (LPS), resulting in augmented nitric oxide (NO) production. IT, given after CT, did not restore the responsiveness of T cells and NK cells, but further activated Mφ to secrete NO, interferon‐γ (IFN‐γ) and interleukin (IL)‐12p40 and to suppress the proliferation of tumour cells in vitro. These functional changes were accompanied by immunophenotype alterations on Mφ, including the up‐regulation of Gr‐1. CD11b⁺ F4/80⁺ Mφ comprised the major population of B16 tumour‐infiltrating leucocytes. CT + IT treatment up‐regulated molecules associated with the M1 effector Mφ phenotype [CD40, CD80, CD86, major histocompatibility complex (MHC) class II, IFN‐γ, tumour necrosis factor‐α (TNF‐α) and IL‐12] and down‐regulated molecules associated with the M2 inhibitory Mφ phenotype (IL‐4Rα, B7‐H1, IL‐4 and IL‐10) on the tumour‐associated Mφ compared with untreated controls. Together, the results show that CT and anti‐CD40 + CpG‐ODN IT synergize in the induction of anti‐tumour effects which are associated with the phenotypic repolarization of tumour‐associated Mφ.     

Inhibition of R5‐tropic HIV type‐1 replication in CD4+ natural killer T cells by γδ T lymphocytes

After the development of highly active anti‐retroviral therapy, it became clear that the majority of emergent HIV‐1 is macrophage‐tropic and infects CD4⁺, CCR5‐expressing cells (R5‐tropic). There are three distinct cell populations, R5‐tropic, HIV‐1‐susceptible CD4⁺ cells: (i) natural killer T (NKT) cells, (ii) dendritic cells and macrophages, and (iii) tissue‐associated T cells residing primarily at mucosal surfaces. We have confirmed that CD4⁺ NKT cells derived from peripheral blood mononuclear cells (PBMCs) predominantly express CCR5 rather than CXCR4, whereas the reverse is true for CD4⁺ T cells derived from circulating PBMCs, and that R5‐tropic HIV‐1 expands efficiently in the CD4⁺ NKT cells. Moreover, when PBMCs depleted of CD8α⁺ cells were stimulated in the presence of α‐galactosylceramide (α‐GalCer) and R5‐tropic HIV‐1 [NL(AD8)], the production of HIV‐1 virions was not suppressed, whereas, similar to the untreated PBMCs, depletion of CD8β⁺ cells from PBMCs significantly inhibited virion production. These findings suggest that CD8αα⁺ but not CD8αβ⁺ cells may have the ability to inhibit R5‐tropic HIV‐1 replication in CD4⁺ NKT cells. Here, we show that co‐culturing R5‐tropic HIV‐1‐infected CD4⁺ NKT cells with CD8αα⁺ γδ T cells, in particular Vγ1Vδ1 cells, but not with CD8αα⁺ NKT cells or CD8αα⁺ dendritic cells, inhibits HIV‐1 replication mainly by secreting chemokines, such as macrophage inflammatory proteins 1α and 1β and RANTES. Collectively, these results indicate the importance of CD8αα⁺ γδ T cells in the control of R5‐tropic HIV‐1 replication and persistence in CD4⁺ NKT cells.     

Detection of vasostatin‐1‐specific CD8+ T cells in non‐obese diabetic mice that contribute to diabetes pathogenesis

Chromogranin A (ChgA) is an antigenic target of pathogenic CD4⁺ T cells in a non‐obese diabetic (NOD) mouse model of type 1 diabetes (T1D). Vasostatin‐1 is a naturally processed fragment of ChgA. We have now identified a novel H2‐K^d‐restricted epitope of vasostatin‐1, ChgA 36‐44, which elicits CD8⁺ T cell responses in NOD mice. By using ChgA 36‐44/K^d tetramers we have determined the frequency of vasostatin‐1‐specific CD8⁺ T cells in pancreatic islets and draining lymph nodes of NOD mice. We also demonstrate that vasostatin‐1‐specific CD4⁺ and CD8⁺ T cells constitute a significant fraction of islet‐infiltrating T cells in diabetic NOD mice. Adoptive transfer of T cells from ChgA 36‐44 peptide‐primed NOD mice into NOD/severe combined immunodeficiency (SCID) mice led to T1D development. These findings indicate that vasostatin‐1‐specific CD8⁺ T cells contribute to the pathogenesis of type 1 diabetes in NOD mice.     

Disruption of the ATP/adenosine balance in CD39−/− mice is associated with handling‐induced seizures

Seizures are due to excessive, synchronous neuronal firing in the brain and are characteristic of epilepsy, the fourth most prevalent neurological disease. We report handling‐induced and spontaneous seizures in mice deficient for CD39, a cell‐surface ATPase highly expressed on microglial cells. CD39^−/− mice with handling‐induced seizures had normal input–output curves and paired‐pulse ratio measured from hippocampal slices and lacked microgliosis, astrogliosis or overt cell loss in the hippocampus and cortex. As expected, however, the cerebrospinal fluid of CD39^−/− mice contained increased levels of ATP and decreased levels of adenosine. To determine if immune activation was involved in seizure progression, we challenged mice with lipopolysaccharide (LPS) and measured the effect on microglia activation and seizure severity. Systemic LPS challenge resulted in increased cortical staining of Iba1/CD68 and gene array data from purified microglia predicted increased expression of interleukin‐8, triggering receptor expressed on myeloid cells 1, p38, pattern recognition receptors, death receptor, nuclear factor‐κB , complement, acute phase, and interleukin‐6 signalling pathways in CD39^−/− versus CD39^+/+ mice. However, LPS treatment did not affect handling‐induced seizures. In addition, microglia‐specific CD39 deletion in adult mice was not sufficient to cause seizures, suggesting instead that altered expression of CD39 during development or on non‐microglial cells such as vascular endothelial cells may promote the seizure phenotype. In summary, we show a correlation between altered extracellular ATP/adenosine ratio and a previously unreported seizure phenotype in CD39^−/− mice. This work provides groundwork for further elucidation of the underlying mechanisms of epilepsy.     

The Role of CD1d-Restricted NKT Cells in the Clearance of Pseudomonas aeruginosa from the Lung Is Dependent on the Host Genetic Background

Pseudomonas aeruginosa is an important human opportunistic pathogen, accounting for a significant fraction of hospital-acquired lung infections. CD1d-restricted NKT cells comprise an unusual innate-like T cell subset that plays important roles in both bacterial and viral infections. Previous reports have differed in their conclusions regarding the role of NKT cells in clearance of P. aeruginosa from the lung. Since there is significant strain-dependent variation in NKT cell number and function among different inbred strains of mice, we investigated whether the role of NKT cells was dependent on the host genetic background. We found that NKT cells did indeed play a critical role in the clearance of P. aeruginosa from the lungs of BALB/c mice but that they played no discernible role in clearance from the lungs of C57BL/6 mice. We found that the strain-dependent role of NKT cells was associated with significant strain-dependent differences in cytokine production by lung NKT cells and that impaired clearance of P. aeruginosa in BALB/c CD1d^−/− mice was associated with an increase in neutrophil influx to the lung and increased levels of proinflammatory cytokines and chemokines after infection. Finally, we found that the role of alveolar macrophages was also dependent on the genetic background. These data provide further support for a model in which the unusually high level of variability in NKT cell number and function among different genetic backgrounds may be an important contributor to infectious-disease susceptibility and pathology.     

A CCL24-dependent pathway augments eosinophilic airway inflammation in house dust mite-challenged Cd163−/− mice

CD163 is a macrophage scavenger receptor with anti-inflammatory and pro-inflammatory functions. Here, we report that alveolar macrophages (AMΦs) from asthmatic subjects had reduced cell-surface expression of CD163, which suggested that CD163 might modulate the pathogenesis of asthma. Consistent with this, house dust mite (HDM)-challenged Cd163^−/− mice displayed increases in airway eosinophils and mucous cell metaplasia (MCM). The increased airway eosinophils and MCM in HDM-challenged Cd163^−/− mice were mediated by augmented CCL24 production and could be reversed by administration of a neutralizing anti-CCL24 antibody. A proteomic analysis identified the calcium-dependent binding of CD163 to Dermatophagoides pteronyssinus peptidase 1 (Der p1). Der p1-challenged Cd163^−/− mice had the same phenotype as HDM-challenged Cd163^−/− mice with increases in airway eosinophils, MCM and CCL24 production, while Der p1 induced CCL24 secretion by bone marrow-derived macrophages (BMMΦs) from Cd163^−/− mice, but not BMMΦs from wild-type (WT) mice. Finally, airway eosinophils and bronchoalveolar lavage fluid CCL24 levels were increased in Der p1-challenged WT mice that received adoptively transferred AMΦ's from Cd163^−/− mice. Thus, we have identified CD163 as a macrophage receptor that binds Der p1. Furthermore, we have shown that HDM-challenged Cd163^−/− mice have increased eosinophilic airway inflammation and MCM that are mediated by a CCL24-dependent mechanism.     

Regulation of HLA-DR and co-stimulatory molecule expression on natural killer T cells by granulocyte-macrophage colony-stimulating factor

A subset of mononuclear cells present in most tissues coexpresses receptors of both natural killer (NK) and T cells. Although linked to antiviral immunity, the function of these putative NKT cells is uncertain. We present evidence that human CD56⁺ DR^? NKT cells exhibit hybrid adaptive and innate immune functions. These cells spontaneously lysed tumour cell targets and upon engagement of T‐cell antigen receptors secreted the cytokines interferon‐γ and granulocyte–macrophage colony‐stimulating factor (GM‐CSF). Conversely, GM‐CSF treatment transformed the NKT cells into dendritic cells, inducing rapid expression of HLA‐DR and the co‐stimulatory molecules CD80 and CD86. The ability to stimulate tetanus toxoid‐specific responses from naïve T cells was acquired within 3 days of activating CD56⁺ NKT cells with GM‐CSF. These results suggest a potential role for NKT cells in the initiation and control of primary immunity during the acute phase of infection.     

CD46 accelerates macrophage‐mediated host susceptibility to meningococcal sepsis in a murine model

CD46, a membrane cofactor expressed on all nucleated human cells, plays an essential role in suppressing autoimmune reactions and protecting host cells from complement‐mediated attack. Human transgenic CD46 homozygous mice (CD46^+/+) are prone to lethal sepsis upon infection with Neisseria meningitidis (N. meningitidis). However, the underlying mechanisms are poorly understood. Here, we determined thatCD46^+/+ mice produce large numbers of M1 type macrophages with enhanced surface expression of MHC II and production of pro‐inflammatory mediators such as IL‐6, TNF, IL‐12, and IL‐1β In the presence of M‐CSF or GM‐CSF, CD46 signaling enhances monocyte‐macrophage differentiation. Additionally, CD46^+/+ macrophages rapidly undergo apoptosis upon LPS challenge or meningococcal infection, which could contribute to uncontrolled bacterial dissemination in vivo. Adoptive transfer of CD46^+/+ peritoneal macrophages aggravated septic responses in wild‐type mice, but the depletion of macrophages partially alleviated septic reactions in CD46^+/+ mice after N. meningitidis infection. Our findings reveal a novel role of CD46 in accelerating inflammatory responses upon meningococcal infection or LPS stimulation by regulating the functional polarization and survival of macrophages.