NADPH oxidase inhibition ameliorates Trypanosoma cruzi-induced myocarditis during Chagas disease

Trypanosoma cruzi, the aetiological agent of Chagas disease, invades nucleated mammalian cells including macrophages. In this study, we investigated the crosstalk between T. cruzi-induced immune activation of reactive oxygen species (ROS) and pro-inflammatory responses, and their role in myocardial pathology. Splenocytes of infected mice (C3H/HeN) responded to Tc-antigenic stimulus by more than a two-fold increase in NADPH oxidase (NOX) activity, ROS generation, cytokine production (IFN-γ > IL-4 > TNFα > IL1-β≈ IL6), and predominant expansion of CD4(+) and CD8(+) T cells. Inhibition of NOX, but not of myeloperoxidase and xanthine oxidase, controlled the ROS (>98%) and cytokine (70-89%) release by Tc-stimulated splenocytes of infected mice. Treatment of infected mice with apocynin (NOX inhibitor) in drinking water resulted in a 50-90% decline in endogenous NOX/ROS and cytokine levels, and splenic phagocytes' proliferation. The splenic percentage of T cells was maintained, though more than a 40% decline in splenic index (spleen weight/body weight) indicated decreased T-cell proliferation in apocynin-treated/infected mice. The blood and tissue parasite burden were significantly increased in apocynin-treated/infected mice, yet acute myocarditis, ie inflammatory infiltrate consisting of macrophages, neutrophils, and CD8(+) T cells, and tissue oxidative adducts (eg 8-isoprostanes, 3-nitrotyrosine, and 4-hydroxynonenal) were diminished in apocynin-treated/infected mice. Consequently, hypertrophy (increased cardiomyocytes' size and β-MHC, BNP, and ANP mRNA levels) and fibrosis (increased collagen, glycosaminoglycans, and lipid contents) of the heart during the chronic phase were controlled in apocynin-treated mice. We conclude that NOX/ROS is a critical regulator of the splenic response (phagocytes, T cells, and cytokines) to T. cruzi infection, and bystander effects of heart-infiltrating phagocytes and CD8(+) T cells resulting in cardiac remodelling in chagasic mice.     

CD8+ T cells and not CD4+ T cells are hyporesponsive to CD28- and CD40L-mediated activation in HIV-infected subjects

T cell dysfunction in HIV-infected subjects could be the consequence of altered sensitivity of CD4⁺ or CD8⁺ T cells to various costimulatory signals. Therefore, we studied proliferation and cytokine production in highly purified CD8⁺ and CD4⁺ T cells from HIV-infected and HIV⁻ subjects, induced by co-activation via cell-bound CD80, CD86 and CD40 or by allo-activation. Regardless of the nature of the first and the costimulatory signal, CD8⁺ T cells from patients proliferated consistently less than controls, while responses from CD4⁺ T cells were similar in patients and controls. This phenomenon was observed after ligation of CD28 combined with anti-CD3 or phorbol myristate acetate (PMA), but also after allogeneic stimulation and after activation by CD40 and anti-CD3. Anti-CD3 combined with CD80 or CD86 induced a mixed Th1/Th2-type cytokine profile in both CD4⁺ and CD8⁺ T cells from controls, whereas anti-CD3 plus CD40 induced only low levels of Th2-type cytokines and no interferon-gamma (IFN-γ) in CD4⁺ T cells. Compared with controls, CD4⁺ T cells from patients produced slightly lower levels of IL-10 but equal amounts of IFN-γ, IL-4 and IL-5, while CD8⁺ T cells from patients produced less of all cytokines tested. In conclusion, responses of purified CD4⁺ T cells from HIV⁺ subjects to various costimulatory pathways are relatively intact, whereas CD8⁺ T cells are hyporesponsive at the level of proliferation and cytokine production. A generalized intrinsic CD8⁺ T cell failure might contribute to viral and neoplastic complications of HIV infection.     

Defective integration of activating signals derived from the T cell receptor (TCR) and costimulatory molecules in both CD4+ and CD8+ T lymphocytes of common variable immunodeficiency (CVID) patients

CVID is characterized by hypogammaglobulinaemia and impaired antibody production. Previous studies demonstrated defects at the T cell level. In the present study the response of purified CD4⁺ and CD8⁺ T lymphocytes to stimulation with anti-TCR monoclonal antibody (the first signal) in combination with anti-CD4 or anti-CD8, anti-CD2 and anti-CD28 MoAbs (the costimulatory signals) was investigated. Both CD4⁺ and CD8⁺ T cells from the patients showed significantly reduced IL-2 release following stimulation via TCR and costimulation via CD4 or CD8 and CD2, respectively. However, normal IL-2 production following TCR plus phorbol myristate acetate (PMA) costimulation and normal expression of an early activation marker, CD69, after TCR + CD28 stimulation indicated that TCR was able to transduce a signal. Furthermore, both IL-2 and IL-4 release were impaired in CD4⁺ lymphocytes following TCR + CD28 stimulation. In addition, stimulation via TCR + CD28 resulted in significantly decreased expression of CD40 ligand in the patients. These results suggest that the integration of activating signals derived from the TCR and costimulatory molecules is defective in CVID patients; the defect is not confined to costimulation via a single molecule, or restricted to cells producing Thl-type cytokines such as IL-2, and is expressed in both CD4⁺ and CD8⁺T cell subsets.     

Production of tumour necrosis factors by human T cells stimulated by a superantigen,toxic shock syndrome toxin-1

The capacity of human T cell subsets, CD4⁺ or CD8⁺ T cells, to produce tumour necrosis factors (TNF-α and TNF-β) upon stimulation with toxic shock syndrome toxin-l (TSST-I) and the requirement for MHC ctass II molecules on accessory cells (AC) in the response were investigated. The capacity of CD4⁺ T cells was much higher than that of CD8⁺ T cells in TSST-1-induced production of TNF-α and TNF-β. The expression of MHC class II molecules on AC was required in the response.     

Programmed death-1 (PD-1)-dependent functional impairment of CD4+ T cells in recurrent genital papilloma

Genital papilloma is caused by human papilloma virus (HPV) infection and recurs frequently. Although T cells are known to play a critical role in the control of HPV infection and papilloma development, the function and phenotype of these cells in the lesion remain to be elucidated. In the present study, we examined the function and phenotype of CD4⁺ T cells isolated from the lesions of primary (n = 9) and recurrent (n = 11) genital papillomas. In recurrent papillomas, the frequency of proliferating (Ki-67⁺) CD4⁺ T cells was significantly reduced compared with primary papillomas. Cytokine production was evaluated by intracellular cytokine staining in anti-CD3/anti-CD28-stimulated CD4⁺ T cells. CD4⁺ T cells from recurrent lesions showed impaired production of IL-2, IFN-γ, and TNF-α. Of interest, the frequency of cytokine-producing CD4⁺ T cells significantly correlated with the frequency of Ki-67⁺CD4⁺ T cells. We also studied expression of programmed death-1 (PD-1), a T-cell exhaustion marker. The frequency of PD-1⁺CD4⁺ T cells was significantly increased in recurrent lesions and inversely correlated with the frequency of cytokine-producing CD4⁺ T cells. The functional significance of PD-1 expression was determined in blocking assays with anti-PD-L1, which restored cytokine production of CD4⁺ T cells from recurrent lesions. Taken together, in recurrent genital papilloma lesions, proliferation, and cytokine production by CD4⁺ T cells are impaired and the PD-1/PD-L1 interaction is responsible for the functional impairment of CD4⁺ T cells.     

The outer surface lipoprotein OspA of Borrelia burgdorferi provides co-stimulatory signals to normal human peripheral CD4+ and CD8+ T lymphocytes

Studies in man and mice have indicated that T cells induced during Borrelia burgdorferi infection are involved in the pathogenesis of the disease. We analyzed the ability of B. burgdorferi to provide co-stimulatory signals to highly enriched normal human CD2⁺ T lymphocytes in the presence of suboptimal concentrations of immobilized anti-CD3 antibodies. Here we show that the lipid-containing recombinant outer surface lipoprotein A (rlip-OspA) of B. burgdorferi but not its delipidated derivative rNS1-OspA augmented CD3-induced T cell proliferation in a dose-dependent manner and at levels similar to that obtained with anti-CD28 antibodies. Lipopolysaccharide had no effect in this system at any concentration tested, suggesting that the active principle of co-stimulation is associated with the lipid moiety of rlip-OspA and distinct from conventional lipid A. Furthermore, incubation of CD2⁺ T cells or selected CD4⁺ as well as CD8⁺ subpopulations with rlip-OspA, but not with rNS1-OspA led to the production of interferon (IFN)-γ, interleukin (IL)-6 and tumor necrosis factor (TNF)-α, but not IL-4. In contrast, co-stimulation of the respective T cell populations with anti-CD28 antibodies resulted in the generation of IFN-γ, IL-4 and TNF-α, but not IL-6. This indicated that the signal transduction pathway induced by rlip-OspA is distinct from that elicited via the CD28 receptor. Co-stimulation of T cells with rlip-OspA also resulted in the development of cytolytic effector cells. In light of the fact that inflamed tissues of B. burgdorferi-infected hosts contain blood leukocytes together with spirochetes, their degradation products, or both, these results suggest that infiltrating CD4⁺ and CD8⁺ T cells of any specificities, including spirochetes, autoantigens, or both, participate in the pathogenesis of Lyme disease.     

HIV infection and aging: enhanced Interferon- and Tumor Necrosis Factor-alpha production by the CD8+ CD28- T subset

Background  

T cells from HIV⁺ and aged individuals show parallels in terms of suppressed proliferative activity and interleukin-2 (I1-2) production and an increased number of CD8⁺ CD28^- T cells. In order to compare cytokine production from T cells from these two states, CD4⁺ and CD8⁺ T cells from HIV⁺ aged, and normal young donors (controls) were monitored for cytokine production by flow cytometry, quantitative PCR and ELISA upon activation by PMA and anti-CD3. In addition, the CD8⁺ T cell subsets CD28⁺ and CD28^- from the HIV⁺ and the aged groups were evaluated for cytokine production by flow cytometry, and compared with those from young controls.     

Absence of phagocyte NADPH oxidase 2 leads to severe inflammatory response in lungs of mice infected with Coccidioides

Production of reactive oxygen species (ROS) resulting from phagocytic NADPH oxidase (NOX2) activity has been reported to contribute to host defense against numerous microbial pathogens. In this study we explored the role of NOX2 production in experimental coccidioidomycosis, a human respiratory disease caused by a soil-borne fungal pathogen. Activated and non-activated macrophages isolated from either NOX2^−/− knock-out or wild type (WT) mice showed comparable ROS production and killing efficiency in vitro when infected with parasitic cells of Coccidioides. Both mouse strains also revealed similar fungal burden in their lungs and spleen at 7 and 11 days after intranasal challenge with Coccidioides spores, although the NOX2^−/− mice died earlier than the WT strain. Immunization of the NOX2^−/− and WT mice with a live, attenuated vaccine strain of Coccidioides also resulted in comparable reduction of the fungal burden in both lungs and spleen. These combined results initially suggested that NOX2 activity and ROS production are not essential for protection against Coccidioides infection. However, the reduced survival of non-vaccinated NOX2^−/− mice correlated with high, sustained numbers of lung-infiltrated neutrophils on days 7 and 11 postchallenge, an expansion of the regulatory T cell population in infected lungs in the knock-out mice, and elevated concentrations of pro-inflammatory cytokines and chemokines in lung homogenates compared to infected WT mice. Although NOX2-derived ROS appeared to be dispensable for both innate and acquired immunity to pulmonary Coccidioides infection, evidence is presented that NOX2 production plays a role in limiting pathogenic inflammation in this murine model of coccidioidomycosis.     

CD2 and CD8α define porcine γδ T cells with distinct cytokine production profiles

γδ T cells are a remarkably prominent T-cell subset in swine with a high prevalence in blood. Phenotypic analyses in this study showed that CD2⁻ γδ T cells in their vast majority had a CD8α⁻SLA-DR⁻CD27⁺ phenotype. CD2⁺ γδ T cells dominated in spleen and lymph nodes and had a more heterogeneous phenotype. CD8α⁺SLA-DR⁻CD27⁺ γδ T cells prevailed in blood, spleen and lymph nodes whereas in liver a CD8α⁺SLA-DR⁺CD27⁻ phenotype dominated, indicating an enrichment of terminally differentiated γδ T cells. γδ T cells were also investigated for their potential to produce IFN-γ, TNF-α and IL-17A. Within CD2⁺ γδ T cells, IFN-γ and TNF-α single-producers as well as IFN-γ/TNF-α double-producers dominated, which had a CD8α⁺CD27^+/− phenotype. IL-17A-producing γδ T cells were only found within CD2⁻ γδ T cells, mostly co-produced TNF-α and had a rare CD8α⁺CD27⁻ phenotype. However, quantitatively TNF-α single-producers strongly dominated within CD2⁻ γδ T cells. In summary, our data identify CD2 and CD8α as important molecules correlating with functional differentiation.     

Leukotriene B4 receptor 1 is differentially expressed on peripheral T cells of steroid-sensitive and -resistant asthmatics

BackgroundNumbers of CD8⁺ T cells expressing the leukotriene B₄ (LTB4) receptor, BLT1, have been correlated with asthma severity.ObjectiveTo examine the activation and numbers of BLT1-expressing peripheral blood CD4⁺ and CD8⁺ T cells from patients with steroid-sensitive (SS) and steroid-resistant (SR) asthma.MethodsCD4⁺ and CD8⁺ T cells isolated from peripheral blood of healthy human subjects and patients with SS and SR asthma were stimulated in culture with anti-CD3/anti-CD28 followed by analysis of BLT1 surface expression and cytokine production. Activation of CD8⁺ T cells after ligation of BLT1 by LTB4 was monitored by changes in intracellular Ca²⁺ concentrations.ResultsThe number of BLT1-expressing cells was larger in patients with asthma than in controls and larger on activated CD8⁺ than on CD4⁺ T cells. Addition of LTB4 to activated CD8⁺ T cells resulted in increases in intracellular Ca²⁺ concentrations. Expansion of activated CD4⁺ T cells, unlike CD8⁺ T cells, was significantly decreased in the presence of corticosteroid. In patients with SS asthma, numbers of BLT1-expressing CD8⁺ T cells were lower in the presence of corticosteroid, unlike in those with SR asthma in whom cell expansion was maintained. Levels of interleukin-13 were highest in cultured CD8⁺ T cells, whereas interleukin-10 levels were higher in CD4⁺ T cells from controls and patients with SS asthma. Interferon-γ levels were lowest in patients with SR asthma.ConclusionDifferences in BLT1 expression, steroid sensitivity, and cytokine production were demonstrated in T lymphocytes from patients with SS and SR asthma. The LTB4–BLT1 pathway in CD8⁺ cells may play an important role in asthma and serve as an important target in the treatment of patients with SR asthma.     

Costimulation of CD3/TcR complex with either integrin or nonintegrin ligands protects CD4+ allergen-specific T-cell clones from programmed cell death

An optimal stimulation of CD4⁺ cells in an immune response requires not only signals transduced via the TcR/CD3 complex, but also costimulatory signals delivered as a consequence of interactions between T-cell surface-associated costimulatory receptors and their counterparts on antigen-presenting cells ‘APC). The intercellular adhesion molecule-1 ‘ICAM-1, CD54) efficiently costimulates proliferation of resting, but not antigen-specific, T cells. In contrast, CD28 and CD2 support interleukin ‘IL)-2 synthesis and proliferation of antigen-specific T cells more efficiently than those of resting T cells. The molecular basis for this differential costimulation of T cells is poorly understood. Cypress-specific T-cell clones ‘TCC) were generated from four allergic subjects during in vivo seasonal exposure to the allergen. Purified cypress extract was produced directly from fresh collected pollen and incubated with the patients' mononuclear cells. Repeated allergen stimulation was performed in T-cell cultures supplemented with purified extract and autologous APC. The limiting-dilution technique was then adopted to generate allergen-specific TCC, which were also characterized by their cytokine secretion pattern as ThO ‘IL-4 plus interferon-gamma) or Th2 ‘IL-4). Costimulation-induced proliferation or apoptosis was measured by propidium iodide cytofluorometric assay. By cross-linking cypress-specific CD4⁺ and CD8⁺ T-cell clones with either anti-CD3 or anti-CD2, anti-CD28, and anti-CD54 monoclonal antibodies, we demonstrated that CD4+ clones ‘with ThO- or Th2-type cytokine production pattern) undergo programmed cell death only after anti-CD3 stimulation, whereas costimulation with either anti-CD54 or anti-CD28 protects target cells from apoptosis. The costimulation-induced protection from apoptotic death was associated with a significant rise in IL-4 secretion in both Th0 and Th2-type clones. In contrast, cypress-specific Th0 CD8⁺ clones were more susceptible to stimulation-induced apoptosis via either anti-CD3 or anti-CD2, alone or in combination with anti-CD54 or anti-CD28, thus displaying only slight but nonsignificant modifications in the pattern of IL-4 secretion. The death-promoting costimulatory effects were not observed with highly purified normal resting CD4⁺ or CD8⁺ lymphocytes. Taken together, these results suggest that TcR engagement by an allergen in the context of functionally active APC induces activation-dependent cell death of some, perhaps less specific, cells, and this may be an important homeostatic mechanism through which functional expansion of allergen-specific T cells is regulated during an ongoing immune response.     

Endoplasmic reticulum stress induced LOX‐1+ CD15+ polymorphonuclear myeloid‐derived suppressor cells in hepatocellular carcinoma

A recent study indicated that Lectin‐type oxidized LDL receptor‐1 (LOX‐1) was a distinct surface marker for human polymorphisms myeloid‐derived suppressor cells (PMN‐MDSC). The present study was aimed to investigate the existence LOX‐1 PMN‐MDSC in hepatocellular carcinoma (HCC) patients. One hundred and twenty‐seven HCC patients, 10 patients with mild active chronic hepatitis B, 10 liver cirrhosis due to hepatitis B, 10 liver dysplastic node with hepatitis B and 50 health control were included. LOX‐1⁺ CD15⁺ PMN‐MDSC were significantly elevated in HCC patients compared with healthy control and patients with benign diseases. LOX‐1⁺ CD15⁺ PMN‐MDSC in circulation were positively associated with those in HCC tissues. LOX‐1⁺ CD15⁺ PMN‐MDSCs significantly reduced proliferation and IFN‐γ production of T cells with a dosage dependent manner with LOX‐1^? CD15⁺ PMNs reached negative results. The suppression on T cell proliferation and IFN‐γ production was reversed by ROS inhibitor and Arginase inhibitor. ROS level and activity of arginase of LOX‐1 ⁺CD15⁺ PMN were higher in LOX‐1⁺ CD15⁺ PMN‐MDSCs than LOX‐1^? CD15⁺ PMNs, as well as the expression of the NADPH oxidase NOX2 and arginase I. RNA sequence revealed that LOX‐1⁺ CD15⁺ PMN‐MDSCs displayed significantly higher expression of spliced X‐box ‐binding protein 1 (sXBP1), an endoplasmic reticulum (ER) stress marker. ER stress inducer induced LOX‐1 expression and suppressive function for CD15⁺ PMN from health donor. For HCC patients, LOX‐1⁺ CD15⁺ PMN‐MDSCs were positively related to overall survival. Above all, LOX‐1⁺ CD15⁺ PMN‐MDSC were elevated in HCC patients and suppressed T cell proliferation through ROS/Arg I pathway induced by ER stress. They presented positive association with the prognosis of HCC patients.     

CD40 ligand-positive CD8+ T cell clones allow B cell growth and differentiation

A fraction of activated CD8⁺ T cells expresses CD40 ligand (CD40L), a molecule that plays a key role in T cell-dependent B cell stimulation. CD8⁺ T cell clones were examined for CD40L expression and for their capacity to allow the growth and differentiation of B cells, upon activation with immobilized anti-CD3. According to CD40L expression, CD8⁺ clones could be grouped into three subsets. CD8⁺ T cell clones expressing high levels of CD40L (≥80% CD40L⁺ cells) were equivalent to CD4⁺ T cell clones with regard to induction of tonsil B cell proliferation and immunoglobulin (Ig) production, provided the combination of interleukin (IL)-2 and IL-10 was added to cultures. CD8⁺ T cell clones, with intermediate levels of CD40L expression (10 to 30% CD40L⁺ cells), also stimulated B cell proliferation and Ig secretion with IL-2 and IL-10. B cell responses induced by these CD8⁺ T cell clones were neutralized by blocking monoclonal antibodies specific for either CD40L or CD40. By contrast, CD40L^?? T cell clones (?5 % CD40L⁺ cells), only induced marginal B cell responses even with IL-2 and IL-10. All three clone types were able to activate B cells as shown by up-regulation of CD25, CD80 and CD86 expression. A neutralizing anti-CD40L antibody indicated that T cell-dependent B cell activation was only partly dependent on CD40-CD40L interaction. These CD40L^?? clones had no inhibitory effects on B cell proliferation induced by CD40L-expressing CD8⁺ T cell clones. Taken together, these results indicate that CD8⁺ T cells can induce B cell growth and differentiation in a CD40L-CD40-dependent fashion.     

Superantigen activation of CD4+ and CD8+ T cells from HIV-infected subjects: role of costimulatory molecules and antigen-presenting cells (APC)

T cell receptor (TCR) triggering via superantigens induces decreased proliferative responses and increased apoptosis in T cells from HIV-infected patients compared with controls. Our aim was to delineate the role of intrinsic T cell defects, of APC dysfunction and of cytokines and costimulatory signal dysregulation in the deficient responses of CD4⁺and CD8⁺ T cells from HIV⁺ subjects to the superantigen Staphylococcus enterotoxin A (SEA). Proliferation and IL-2Rα up-regulation on SEA-stimulated CD4⁺and CD8⁺T cells in whole blood were reduced in HIV⁺ subjects with CD4 counts < 500, compared with controls. Neither addition of IL-2, IL-12 or phorbol myristate acetate (PMA) nor neutralization of endogenous IL-10, tumour necrosis factor-alpha (TNF-α), TNF-β or transforming growth factor-beta (TGF-β) could restore the decreased activation by SEA. Possible intrinsic T cell defects were studied by presenting SEA on HLA-DR-transfected Chinese hamster ovary (CHO) cells, co-expressing LFA3 and/or CD80, to purified T cells. In this system CD8⁺T cells from most HIV⁺ patients were hyporesponsive with regard to IL-2 production, IL-2Rα up-regulation and proliferation, whereas clearly reduced responses were only shown in CD4⁺T cells from AIDS patients. Similarly, apoptosis was increased in CD8⁺T cells from all patients, but only in CD4⁺T cells from AIDS patients. During HIV infection, the responses to TCR triggering through SEA are deficient in both T cell subsets. The intrinsic defect appears earlier during disease progression in purified CD8⁺T than in CD4⁺T cells, it occurs in conjunction with both CD2 and CD28 costimulation, and it is correlated with increased levels of apoptosis.     

Prostaglandin E2 primes naive T cells for the production of anti-inflammatory cytokines

In addition to their capacity to induce pain, vasodilatation and fever, prostaglandins E (PGE) exert anti-inflammatory activities by inhibiting the release of pro-inflammatory cytokines by macrophages and T cells, and by increasing interleukin (IL)-10 production by macrophages. We here report that PGE₂, the major arachidonic acid metabolite released by antigen-presenting cells (APC), primes naive human T cells for enhanced production of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. Unfractionated as well as CD45RO^?CD31⁺ sort-purified neonatal CD4 T cells acquire the capacity to produce a large spectrum of cytokines after priming with anti-CD3 and anti-CD28 monoclonal antibodies (mAb), in the absence of both APC and exogenous cytokines. PGE₂ primes naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10 and IL-13, and very low levels of IL-2, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and TNF-β. PGE₂ does not significantly increase IL-4 production in priming cultures, whereas it suppresses IL-2 and IFN-γ. Addition of a neutralizing mAb to IL-4 receptor in primary cultures, supplemented or not with PGE₂, prevents the development of IL-4-producing cells but does not abolish the effects of PGE₂ on IL-10 and IL-13 as well as T helper (Th)1-associated cytokines. Addition of exogenous IL-2 in primary cultures does not alter the effects of PGE₂ on naive T cell maturation. Thus PGE₂ does not act by increasing IL-4 production in priming cultures, and its effects are partly IL-4 independent and largely IL-2 independent. Together with the recent demonstration that PGE₂ suppresses IL-12 production, our results strongly suggest that this endogenously produced molecule may play a significant role in Th subset development and that its stable analogs may be considered for the treatment of Th1-mediated inflammatory diseases.     

Altered phenotype and functionality of circulating immune cells characterize adult patients with nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a chronic inflammatory liver disease associated with insulin resistance and its metabolic consequences. Leukocyte mobilization, intrahepatic activation, and an exacerbated production of reactive oxygen species (ROS) and cytokines contribute to the development of NASH. Though alterations in peripheral blood (PB) T cell proportions and functionality remain unidentified, they might play a main role in NASH progression. We have compared the phenotype and Th1/Th2 commitment of peripheral immune cell reservoirs in adult patients and controls as well as the ability of neutrophils and monocytes to handle an ex vivo challenge. Also, we correlated those parameters with the main histological characteristics in NASH. Compared with controls, patients showed increased numbers of CD4⁺ cells and both CD4⁺ and CD8⁺ CD45RO subsets together with a higher frequency of IFN-γ-producing CD4⁺ and CD8⁺ T cells. We also found a decreased number of CD4⁺ and CD8⁺ CD45RA subsets. The distinctive production of IFN-γ highlights the significance of the observed skewed frequencies of PB T cells. Whereas ROS production by monocytes from NASH patients did not differ from controls, circulating neutrophils displayed a particularly higher phorbol myristate acetate-induced production of ROS. A negative correlation between oxidative burst and fibrosis grade was observed. This study reveals the presence of a characteristic profile of peripheral immune cells in NASH. We also discuss the probable influence of obesity on some of our present findings.     

Regulation of 4-1BB expression by cell-cell interactions and the cytokines,interleukin-2 and interleukin-4

4-1BB expression increased gradually following T cell activation, and by day 3 post-stimulation with immobilized anti-CD3 (anti-CD3i) or concanavalin A (Con A), splenic T cells were routinely 35–45% 4-1BB⁺ by flow cytometric analysis. 4-1BB was expressed on activated CD8⁺, CD4⁺, CD28⁺ and CD45RB⁺ T cells. Optimal 4-1BB expression was seen by day 6 post-stimulation and was cell density dependent. When T cells were cultured for 6 days at 1 × 10⁶/well in a 24-well plate with anti-CD3i, 82% of the cells were 4-1BB⁺. In contrast, at lower cell densities (4 × 10⁵, 2 × 10⁵ and 1 × 10⁵), optimal 4-1BB expression was observed only if the cultures were supplemented with recombinant interleukin-2 (IL-2) or recombinant IL-4 (IL-4). In agreement, with these results, modes of inducing endogenous IL-2 production such as cross-linking the costimulatory molecule, CD28, or the addition of syngeneic accessory cells to T cells activated with anti-CD3i, resulted in high levels of 4-1BB expression. The addition of interleukin-1α(IL-1α) or interferon-γ (IFN-γ) did not increase 4-1BB expression on anti-CD3i-activated T cells. In addition, if T cells were incubated with IL-2, IL-4, IL-1α, IFN-γ or anti-CD28 alone, no 4-1BB expression was induced. T cells activated with soluble anti-CD3 (anti-CD3s) in the presence of IL-2, IL-4, or accessory cells, did not express higher levels of 4-1BB on their cell surface. These data suggest that initial events crucial for efficient T cell activation, such as signals delivered through the T cell receptor/CD3 complex and the CD28 molecule, are instrumental in regulating subsequent 4-1BB expression.     

Presence,function, and regulation of IL-17F-expressing human CD4+ T cells

The pro-inflammatory cytokine IL-17A has been implicated in the immunopathology of inflammatory arthritis. IL-17F bears 50% homology to IL-17A and has recently been suggested to play a role in inflammation. We investigated the induction and cytokine profile of IL-17F⁺ CD4⁺ T cells, and how IL-17F may contribute to inflammation. Upon culture of healthy donor CD4⁺ T cells with IL-1β, IL-23, anti-CD3, and anti-CD28 mAb, both IL-17A and IL-17F-expressing cells were detected. In comparison to IL-17A⁺IL-17F⁻ CD4⁺ T cells, IL-17F⁺IL-17A⁻ and IL-17A⁺IL-17F⁺ CD4⁺ T cells contained lower proportions of IL-10-expressing and GM-CSF-expressing cells and higher proportions of IFN-γ-expressing cells. Titration of anti-CD28 mAb revealed that strong co-stimulation increased IL-17F⁺IL-17A⁻ and IL-17A⁺IL-17F⁺ CD4⁺ T cell frequencies, whereas IL-17A⁺IL-17F⁻ CD4⁺ T cell frequencies decreased. This was partly mediated via an IL-2-dependent mechanism. Addition of IL-17A, IL-17F, and TNF-α to synovial fibroblasts from patients with inflammatory arthritis resulted in significant production of IL-6 and IL-8, which was reduced to a larger extent by combined blockade of IL-17A and IL-17F than blockade of IL-17A alone. Our data indicate that IL-17A and IL-17F are differentially regulated upon T cell co-stimulation, and that dual blockade of IL-17A and IL-17F reduces inflammation more effectively than IL-17A blockade alone.