The Role of Innate Cells Is Coupled to a Th1-Polarized Immune Response in Pediatric Nonalcoholic Steatohepatitis

Background

Nonalcoholic steatohepatitis (NASH) is a chronic inflammatory liver disease influenced by risk factors for the metabolic syndrome. In adult patients, NASH is associated with an altered phenotype and functionality of peripheral immune cells, the recruitment of leukocytes and intrahepatic activation, and an exacerbated production of reactive oxygen species (ROS) and cytokines. It remains unclear if the previously described differences between pediatric and adult nonalcoholic fatty liver diseases also reflect differences in their pathogenesis.

Aims

We aimed to investigate the phenotype and functionality of circulating immune cells and the potential contribution of liver infiltrating leukocytes to the immunological imbalance in pediatric NASH.

Results

By a real-time PCR-based analysis of cytokines and immunohistochemical staining of liver biopsies, we demonstrated that the hepatic microenvironment is dominated by interferon-gamma (IFN-γ) but not interleukin-4 and is infiltrated by a higher number of CD8⁺ cells in pediatric NASH. The number of infiltrating neutrophils positively correlated with ROS generation by peripheral polymorphonuclear cells. By a flow cytometric analysis of peripheral blood lymphocytes, a distinctive increase in CD8⁺ CD45RO and CD8⁺ CD45RA subpopulations and an increased production of IFN-γ by CD4⁺ and CD8⁺ cells were shown. The production of ROS following PMA stimulation was augmented in circulating neutrophils but not in monocytes.

Conclusion

In sum, the distinctive phenotype and functionality of infiltrating and circulating cells suggest that the role of innate cells is coupled to a Th1-polarized immune response in pediatric NASH.     

CD4 T Cell Clones Producing both Interferon-γ and Interleukin-10 Predominate in Bronchoalveolar Lavages of Active Pulmonary Tuberculosis Patients

The pattern of cytokine production in T cell clones derived from bronchoalveolar lavages (BAL) of active pulmonary tuberculosis (TB) patients was analyzed in clones obtained by limiting dilution procedures which expand with high efficiency either total T lymphocytes, independently of their antigen-recognition specificity, or Mycobacterium tuberculosis-specific T cells. BAL-derived clones, representative of CD4⁺ cells from five patients with active TB, produced significantly higher amounts of IFN-γ than BAL-derived CD4⁺ clones from three inactive TB donors or four controls (with unrelated, noninfectious pathology). Average IL-4 and IL-10 production did not differ significantly in the three groups. Although these data suggest a predominant Th1 response to M. tuberculosis infection in the lungs, the majority of BAL-derived CD4⁺ clones produced both IFN-γ and IL-10 and the percentage of clones with this pattern of cytokine production was significantly higher in clones derived from BAL of active TB patients than from controls. Only rare clones derived from peripheral blood (PB)-derived CD45RO⁺ CD4⁺ T cells of both patients (nine cases) and controls (four cases) produced both IFN-γ and IL-10; instead, the IL-10-producing clones derived from PB T cells most often also produced IL-4, displaying a typical Th2 phenotype. Higher average amounts of IFN-γ and IL-10 were produced by BAL-derived CD8⁺ clones of four active TB patients than of four controls, although the frequency of CD8⁺ clones producing both IFN-γ and IL-10 was lower than that of CD4⁺ clones. The M. tuberculosis-specific BAL-derived T cell clones from three active TB patients were almost exclusively CD4⁺ and produced consistently high levels of IFN-γ often in association with IL-10, but very rarely with IL-4. Unlike the BAL-derived clones, the M. tuberculosis-specific clones derived from PB CD45RO⁺ CD4⁺ T cells of three different active TB patients and two healthy donors showed large individual variability in cytokine production as well as in the proportion of CD4⁺, CD8⁺, or TCR γ/δ⁺ clones. These results indicate the predominance of CD4⁺ T cells producing both the proinflammatory cytokine IFN-γ and the anti-inflammatory cytokine IL-10 in BAL of patients with active TB.     

A predominant Th1 polarization is present in synovial fluid of end-stage osteoarthritic knee joints: analysis of peripheral blood,synovial fluid and synovial membrane

Thorough understanding of the complex pathophysiology of osteoarthritis (OA) is necessary in order to open new avenues for treatment. The aim of this study was to characterize the CD4⁺ T cell population and evaluate their activation and polarization status in OA joints. Fifty-five patients with end-stage knee OA (Kellgren–Lawrence grades III–IV) who underwent surgery for total knee arthroplasty (TKA) were enrolled into this study. Matched samples of synovial membrane (SM), synovial fluid (SF) and peripheral blood (PB) were analysed for CD3⁺CD4⁺CD8^– T cell subsets [T helper type 1 (Th1), Th2, Th17, regulatory T cells] and activation status (CD25, CD69, CD45RO, CD45RA, CD62L) by flow cytometry. Subset-specific cytokines were analysed by cytometric bead array (CBA). SM and SF samples showed a distinct infiltration pattern of CD4⁺ T cells. In comparison to PB, a higher amount of joint-derived T cells was polarized into CD3⁺CD4⁺CD8^– T cell subsets, with the most significant increase for proinflammatory Th1 cells in SF. CBA analysis revealed significantly increased immunomodulating cytokines [interferon (IFN)-γ, interleukin (IL)-2 and IL-10] in SF compared to PB. Whereas in PB only a small proportion of CD4⁺ T cells were activated, the majority of joint-derived CD4⁺ T cells can be characterized as activated effector memory cells (CD69⁺CD45RO⁺CD62L^–). End-stage OA knees are characterized by an increased CD4⁺ T cell polarization towards activated Th1 cells and cytokine secretion compared to PB. This local inflammation may contribute to disease aggravation and eventually perpetuate the disease process.     

Elevated circulating CD14<Superscript>low</Superscript>CD16<Superscript>+</Superscript> monocyte subset in primary biliary cirrhosis correlates with liver injury and promotes Th1 polarization

Primary biliary cirrhosis (PBC) is a progressive autoimmune liver disease in which monocytes/macrophages infiltration and skewed T helper type (Th) 1 and Th17 cell responses participate in the development of the disease. Human peripheral blood monocytes are heterogeneous and can be divided into classical CD14^highCD16^?, intermediate CD14^highCD16⁺, and nonclassical CD14^lowCD16⁺ monocyte subsets. Compared to classical monocytes, CD16⁺ monocytes are generally termed pro-inflammatory monocytes and play an important pathogenic role in autoimmune diseases. However, little is known about the immunophenotype and immunopathogenic role of peripheral blood CD16⁺ monocytes in PBC. Thus, we investigated the phenotype and function of these circulating monocyte subsets from PBC patients. The frequencies of circulating CD14^highCD16⁺ and CD14^lowCD16⁺ subpopulation were increased in disease compared with healthy controls. Among them, CD14^lowCD16⁺ monocyte subset positively correlated with disease progress, liver damage indicators and serum C-reactive protein, respectively. Furthermore, the frequencies of Th1 and Th17 cells were upregulated and CD14^lowCD16⁺ monocyte subset was also positively associated with Th1 cell frequency in PBC. Using a vitro coculture model, we further found that CD14^lowCD16⁺ monocytes promoted Th1 cell polarization compared to classical monocytes. Interleukin-12 (IL-12) and direct contact of patient CD4⁺T cell and CD14^lowCD16⁺ monocytes, were responsible for CD14^lowCD16⁺ monocytes promotion of Th1 cells polarization in PBC. Our study demonstrated that the enhanced CD14^lowCD16⁺ monocyte subset participated in fostering liver damage and inflammatory responses, and promoted Th1 cells skewing in PBC.     

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.     

Association of Graves’ Disease and Prevalence of Circulating IFN-γ-producing CD28<Superscript>−</Superscript> T Cells

Background  Peripheral blood CD4⁺ and CD8⁺ T-cell subsets lacking surface CD28 have been suggested to predispose patients to immune-mediated disorders. Materials and Methods  To determine the role of CD28⁻ T-cell subset in Graves’ disease (GD), we characterized peripheral blood CD4⁺CD28⁻ and CD8⁺CD28⁻ T cell from early onset GD patients. Results and Discussion  GD patients had significantly higher percentages of CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells than did healthy donors. Both CD28⁻ T cells expressed mostly CD45RO, suggesting that they are activated and/or are memory T cells. GD patient-derived CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells produced more intracellular IFN-γ than their counterparts from healthy donors. Furthermore, CD4⁺CD28⁻ and CD8⁺CD28⁻ T cells from GD patients with Graves’ ophthalmopathy (GO) secreted higher level of intracellular IFN-γ than those CD28⁻ T cells from GD patients without GO. Retrospective analysis showed that the increased levels of CD4⁺CD28⁻ T cells and their IFN-γ-producing subgroups were positively correlated to the serum anti-thyrotropin receptor (TSHR) autoantibodies (TRAb). Our observations suggest that increased IFN-γ-producing CD28⁻ T cells in GD patients may play an important role in the pathogenesis of GD. Zhiping Sun and Weixue Zhong contributed equally to this paper.     

Influence of a mutation in IFN-γ receptor 2 (IFNGR2) in human cells on the generation of Th17 cells in memory T cells

The T cell subsets involved in inflammatory reactions are mainly the IFN-γ secreting Th1 cells and IL17-producing Th17 cells. Although Th17 cells are primed in the thymus, there is evidence that Th17 cells can be generated from effector memory CD4⁺ T cells. Cytokines as IL-6, TGF-β, IL-21 and IL-23 involved in development of Th17 cells are well described. Here we analyzed the impact of a mutation in the IFN-γ receptor 2 (IFN-γR2) on the induction of Th17 cells. By isolation of T cells and monocytes of a patient with this mutation we could demonstrate an inhibitory role of IFN-γ signaling as IFN-γR2-deficient monocytes induce a higher percentage of IL-17⁺ cells from both healthy and IFN-γR2-deficient CD4⁺ T cells. This data confirm the interference of these two T helper subsets and points to a balance of Th1 and Th17 cells obtained by their own cytokine production and their interplay with APCs.     

Vaccinated C57BL/6 Mice Develop Protective and Memory T Cell Responses to Coccidioides posadasii Infection in the Absence of Interleukin-10

High concentrations of lung tissue-associated interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine, correlate with susceptibility of mice to Coccidioides spp. infection. In this study, we found that macrophages, dendritic cells, neutrophils, and both CD8⁺ and CD4⁺ T cells recruited to Coccidioides posadasii-infected lungs of nonvaccinated and vaccinated mice contributed to the production of IL-10. The major IL-10-producing leukocytes were CD8⁺ T cells, neutrophils, and macrophages in lungs of nonvaccinated mice, while both Foxp3⁺ and Foxp3⁻ subsets of IL-10⁺ CD4⁺ T cells were significantly elevated in vaccinated mice. Profiles of the recruited leukocytes in lungs revealed that only CD4⁺ T cells were significantly increased in IL-10^−/− knockout mice compared to their wild-type counterparts. Furthermore, ex vivo recall assays showed that CD4⁺ T cells isolated from vaccinated IL-10^−/− mice compared to vaccinated wild-type mice produced significantly higher amounts of IL-2, gamma interferon (IFN-γ), IL-4, IL-6, and IL-17A in the presence of a coccidioidal antigen, indicating that IL-10 suppresses Th1, Th2, and Th17 immunity to Coccidioides infection. Analysis of absolute numbers of CD44⁺ CD62L⁻ CD4⁺ T effector memory T cells (T_EM) and IFN-γ- and IL-17A-producing CD4⁺ T cells in the lungs of Coccidioides-infected mice correlated with better fungal clearance in nonvaccinated IL-10^−/− mice than in nonvaccinated wild-type mice. Our results suggest that IL-10 suppresses CD4⁺ T-cell immunity in nonvaccinated mice during Coccidioides infection but does not impede the development of a memory response nor exacerbate immunopathology of vaccinated mice over at least a 4-month period after the last immunization.     

JAK/STAT/PKCδ molecular pathways in synovial fluid T lymphocytes reflect the in vivo T helper-17 expansion in psoriatic arthritis

Looking to the sustained psoriatic arthritis (PsA) joint as a model of local human inflammation, this study was designed to assess the T lymphocyte signal transduction pathways potentially involved in this chronic immune-mediated inflammatory process, as characterized by direct ex vivo analysis of T helper (Th)-17 T effector (Teff) cell phenotypes in synovial fluid (SF) and peripheral blood (PB) of clinically active PsA patients. The reverse-phase protein arrays (RPPA) technique was employed to identify STAT3, STAT1, JAK1, JAK2, PKCδ and ERK1/2 phosphoprotein levels on total T cell lysates in SF samples of PsA patients. Frequencies of T CD4⁺IL-17A-F⁺ and T CD4⁺IL-23R⁺ Th17 cells were quantified in SF and matched PB of PsA patients by flow cytometry and compared with PB of healthy controls (HC). Increased levels of JAK1, STAT3, STAT1 and PKCδ phosphoproteins were found in SF T cells of PsA patients, compared with PB of HC. The expansion of T CD4⁺IL-17A-F⁺ cells, as well as of T CD4⁺ cells expressing IL-23Rp19 (T CD4⁺ IL-23R⁺), considered as the pathogenic phenotype of effector Th17 cells, was found to be confined to the joints of PsA patients, as the frequencies of both populations were significantly higher in SF than in matched PB, or in PB of HC. In conclusion, T lymphocyte signal transduction pathway mapping revealed an enhanced activation of JAK1/STAT3/STAT1 and PKCδ phosphoproteins that may drive the local inflammatory process, characterized by the in vivo expansion of T CD4⁺IL-17A-F⁺ and T CD4⁺IL-23R⁺ Th17 Teff cells in SF of clinically active joints of PsA patients.     

Blocking of LFA‐1 enhances expansion of Th17 cells induced by human CD14+CD16++ nonclassical monocytes

Among human peripheral blood (PB) monocyte (Mo) subsets, the classical CD14⁺⁺CD16^? (cMo) and intermediate CD14⁺⁺CD16⁺ (iMo) Mos are known to activate pathogenic Th17 responses, whereas the impact of nonclassical CD14⁺CD16⁺⁺ Mo (nMo) on T‐cell activation has been largely neglected. The aim of this study was to obtain new mechanistic insights on the capacity of Mo subsets from healthy donors (HDs) to activate IL‐17⁺ T‐cell responses in vitro, and assess whether this function was maintained or lost in states of chronic inflammation. When cocultured with autologous CD4⁺ T cells in the absence of TLR‐2/NOD2 agonists, PB nMos from HDs were more efficient stimulators of IL‐17‐producing T cells, as compared to cMo. These results could not be explained by differences in Mo lifespan and cytokine profiles. Notably, however, the blocking of LFA‐1/ICAM‐1 interaction resulted in a significant increase in the percentage of IL‐17⁺ T cells expanded in nMo/T‐cell cocultures. As compared to HD, PB Mo subsets of patients with rheumatoid arthritis were hampered in their T‐cell stimulatory capacity. Our new insights highlight the role of Mo subsets in modulating inflammatory T‐cell responses and suggest that nMo could become a critical therapeutic target against IL‐17‐mediated inflammatory diseases.     

Cord blood dendritic cells prevent the differentiation of naïve T-helper cells towards Th1 irrespective of their subtype

Allogeneic cord blood transplantation is associated with a less severe graft-versus-host disease (GVHD). This observation is thought to be due to immaturity of cord blood cell immune capabilities. Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system capable of initiation and regulation of immune responses. In this investigation, we hypothesized that non-manipulated cord blood dendritic cells (CBDCs) not only differ in their functional maturity from adult peripheral blood DCs (PBDCs) but also differ in their subsets and their preference in promoting Th1 or Th2 immune responses. Non-manipulated fresh DCs were isolated from cord blood (CB) and adult peripheral blood (PB) mononuclear cells as lineage marker negative cells. The differences in expression of costimulatory molecules, the proportion of myeloid and lymphoid DCs subsets, their immunostimulatory characteristics and their influence on promoting the differentiation of naïve T cells towards Th1 or Th2 cells were then investigated in these two populations. Our results showed that freshly isolated CBDCs, similar to cord blood monocyte derived DCs, were poor inducers of IFN-γ secretion while they increased the induction of IL-4 production by T cells in comparison with PBDCs. CBDCs were also poor stimulators of allogenic T cells in mixed leukocyte reaction compared to adult peripheral blood dendritic cells. They also displayed decreased expression of HLA-DR and CD86 molecules. The ratio of lymphoid DCs (CD11c^?, CD123⁺) to myeloid DCs (CD11c⁺, CD123^?) was significantly higher in CB compared to PB. We conclude that CBDCs preferential priming of naive T cells towards Th2 population, seems to be an intrinsic property independent of their subtype. This property along with their functional immaturity should contribute to outcome of cord blood transplantation.     

Interferon-gamma negatively regulates Th17-mediated immunopathology during mouse hepatitis virus infection

Fulminant hepatitis can cause acute liver failure and death in both humans and mice. However, the cellular and molecular mechanisms underlying the acute disease are still not well understood. Here, we examine the role of Th17 response in the development of the acute hepatitis following infection with mouse hepatitis virus (MHV). We show that IL-17 levels in serum are rapidly elevated and positively correlated to liver damage and death of the mice. In IFN-γR^−/− mice, Th17 response is enhanced and the elevated IL-17 production contributes to severe liver damage as well as detrimental inflammation because neutralization of IL-17 effectively suppresses inflammation and protects mice from liver injury. We further show that IFN-γ facilitates antigen-induced apoptosis of Th17 cells and adoptive transferred IFN-γR^−/−, but not IFN-γR^+/+; CD4⁺ T cells promotes an enhanced liver damage in wild-type mice. The results demonstrate an essential role of Th17 cells in MHV-induced immunopathology and the importance of IFN-γ in maintaining immune balance between Th1 and Th17 responses during acute viral infection.     

Periodontitis‐associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14+ monocytes leading to enhanced Th17/IL‐17 responses

The Th17/IL‐17 pathway is implicated in the pathogenesis of periodontitis (PD), however the mechanisms are not fully understood. We investigated the mechanism by which the periodontal pathogens Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) promote a Th17/IL‐17 response in vitro, and studied IL‐17⁺ CD4⁺ T‐cell frequencies in gingival tissue and peripheral blood from patients with PD versus periodontally healthy controls. Addition of Pg or Aa to monocyte/CD4⁺ T‐cell co‐cultures promoted a Th17/IL‐17 response in vitro in a dose‐ and time‐dependent manner. Pg or Aa stimulation of monocytes resulted in increased CD40, CD54 and HLA‐DR expression, and enhanced TNF‐α, IL‐1β, IL‐6 and IL‐23 production. Mechanistically, IL‐17 production in Pg‐stimulated co‐cultures was partially dependent on IL‐1β, IL‐23 and TLR2/TLR4 signalling. Increased frequencies of IL‐17⁺ cells were observed in gingival tissue from patients with PD compared to healthy subjects. No differences were observed in IL‐17⁺ CD4⁺ T‐cell frequencies in peripheral blood. In vitro, Pg induced significantly higher IL‐17 production in anti‐CD3 mAb‐stimulated monocyte/CD4⁺ T‐cell co‐cultures from patients with PD compared to healthy controls. Our data suggest that periodontal pathogens can activate monocytes, resulting in increased IL‐17 production by human CD4⁺ T cells, a process that appears enhanced in patients with PD.     

Protective and Pathological Functions of CD8+ T Cells in Leishmania braziliensis Infection

Cutaneous leishmaniasis (CL) caused by Leishmania braziliensis is characterized by a strong Th1 response that leads to skin lesion development. In areas where L. braziliensis transmission is endemic, up to 15% of healthy subjects have tested positive for delayed-type hypersensitivity to soluble leishmania antigen (SLA) and are considered to have subclinical (SC) infection. SC subjects produce less gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) than do CL patients, but they are able to control the infection. The aim of this study was to characterized the role of CD8⁺ T cells in SC infection and in CL. Peripheral blood mononuclear cells (PBMC) were stimulated with SLA to determine the frequencies of CD4⁺ IFN-γ⁺ and CD8⁺ IFN-γ⁺ T cells. Monocytes from PBMC were infected with L. braziliensis and cocultured with CD8⁺ T cells, and the frequencies of infected monocytes and levels of cytotoxicity markers, target cell apoptosis, and granzyme B were determined. The frequency of CD8⁺ IFN-γ⁺ cells after SLA stimulation was higher for SC individuals than for CL patients. The frequency of infected monocytes in SC cells was lower than that in CL cells. CL CD8⁺ T cells induced more apoptosis of infected monocytes than did SC CD8⁺ T cells. Granzyme B production in CD8⁺ T cells was higher in CL than in SC cells. While the use of a granzyme B inhibitor decreased the number of apoptotic cells in the CL group, the use of z-VAD-FMK had no effect on the frequency of these cells. These results suggest that CL CD8⁺ T cells are more cytotoxic and may be involved in pathology.     

Notch signaling suppresses CD14+ monocytes cells activity in patients with chronic hepatitis C

Hepatitis C virus (HCV) infection always leads to chronic hepatitis via dysregulation of host immunity. Notch signaling also modulates the response of monocytes/macrophages. Thus, we aimed to investigate the regulatory role of Notch signaling to CD14⁺ monocytes. Forty patients with chronic hepatitis C and twenty normal controls (NC) were enrolled. CD14⁺ monocytes and CD4⁺ T cells were purified from peripheral bloods. Notch receptors' mRNA expression in CD14⁺ monocytes was semi‐quantified by real‐time PCR. Cytokine production by CD14⁺ monocytes in response to γ‐secretase inhibitor (GSI) was investigated by ELISA. GSI‐induced CD14⁺ monocytes activity to HCV clearance in Huh7.5 cells and to CD4⁺ T cell differentiation was also assessed in direct and indirect contact co‐culture system. Notch1 mRNA relative level was approximately 10‐fold elevated in CD14⁺ monocytes from chronic hepatitis C patients when compared with NC. GSI stimulation resulted in enhanced cytokines production by CD14⁺ monocytes from chronic hepatitis C patients. GSI‐stimulated CD14⁺ monocytes from chronic hepatitis C patients induced suppression of HCV RNA replication in both direct and indirect contact co‐culture system of CD14⁺ monocytes and HCVcc‐infected Huh7.5 cells, and this process was accompanied by elevation of interferon‐γ production but not increased target cell death. Moreover, GSI stimulation also enhanced CD14⁺ monocytes‐induced Th1 and Th17 cells activation, and this process required direct cell‐to‐cell contact. Effective antiviral therapy down‐regulated Notch1 mRNA expression and promoted cytokine production by CD14⁺ monocytes from chronic hepatitis C. Current data revealed an important immunoregulatory property of Notch signaling to CD14⁺ monocytes in chronic HCV infection.     

Oral glucocorticoid treatment decreases interleukin‐10 receptor expression on peripheral blood leucocyte subsets

Glucocorticoids (GCS) are capable of stimulating the secretion of interleukin (IL)‐10 by leucocytes; however, the potential of GCS to modulate leucocyte susceptibility to IL‐10‐mediated actions has not yet been studied. In the current paper, we performed a detailed cross‐sectional analysis of IL‐10 receptor (IL‐10R) expression by CD4⁺ and CD8⁺ T cells, natural killer (NK) cells, monocytes and neutrophils. Next, we analysed the effects of short‐term oral GCS treatment on surface IL‐10R expression by various leucocyte subpopulations in asthmatic patients. All leucocyte subsets studied presented with substantial levels of surface IL‐10R. The highest levels of IL‐10R were found on monocytes, predominantly with CD14²⁺CD16⁺ and CD14⁺CD16⁺ phenotypes, and on CD4⁺CD25^high T cells. In contrast, levels of IL‐10R on CD8⁺ T cells, NK cells and neutrophils were significantly lower and similar to each other in intensity. GCS treatment resulted in a significant decrease of IL‐10R expression on all analysed peripheral blood leucocyte subsets. Our data suggest that down‐regulation of IL‐10R could counterbalance the otherwise suppressive action of GCS.     

Quantification and phenotype of regulatory T cells in rheumatoid arthritis according to Disease Activity Score-28

Here we studied and characterized different peripheral blood (PB) regulatory T cell (Treg) subsets in rheumatoid arthritis (RA) patients and tested the hypothesis that changes in these cells can be linked to the degree of inflammation and relapsing/remission periods. PB cells were examined from RA subjects (n = 60) with different disease activity score-28 (DAS28) and from healthy controls (n = 40). Frequencies of Treg subsets expressing characteristic membrane antigens, FoxP3 or intracellular cytokines were quantified by flow cytometry. We observed a decrease in the percentages of CD4⁺CD25^high, CD4⁺CD25^int, CD4⁺CD25^int/highFoxP3⁺, CD4⁺CD38⁺, CD4⁺CD62L⁺, CD8⁺CD25^highCD45RA⁺ and CD8⁺CD25^intCD45RA⁺ T cells in PB of RA patients compared to healthy controls. In addition, we found increased percentages of cells expressing membrane/intracellular regulatory antigens such as OX40 (CD134), CD45RB^low or CTLA-4 (CD152), and a higher proportion of other T cell subsets including CD4⁺CTLA-4⁺, CD4⁺IL10⁺, CD4⁺CD25^intIL10⁺, CD4⁺CD25^int TGFβ⁺, CD4⁺CD25^low TGFβ⁺ and CD8⁺CD28^? . We show that most of these changes parallel the intensity of inflammation, with lowest or highest values in patients with moderately/very active disease compared to healthy controls and at times to patients with inactive RA. The balance between these cell subsets and their antigen expression would determine the inflammation levels and could thus be linked to the relapsing/remission periods of the disease.     

Comparative Analysis of the Morphological, Cytochemical, Immunophenotypical, and Functional Characteristics of Normal Human Peripheral Blood Lineage/CD16/HLA-DR/CD14 Cells, CD14 Monocytes, and CD16 Dendritic Cells

Human peripheral blood (PB) CD14^lo/HLA-DR⁺ cells were initially described as a subset of mature monocytes. Recently, it has been suggested that these represent a part of a new subset of dendritic cells (DC), characterized by the coexpression of MDC-8/HLA-DR/CD16. The aim of the present paper was to analyze the morphological, cytochemical, phenotypical, and functional characteristics of PB CD16⁺/HLA-DR⁺ cells compared to both PB CD14⁺ monocytes and CD16⁻ DC. In contrast to CD14⁺ monocytes, purified CD16⁺/HLA-DR⁺ cells displayed cytoplasmic veils and lacked cytoplasmic myeloperoxidase and α-naphthyl acetate esterase. Normal human PB CD16⁺/HLA-DR⁺ cells also displayed phenotypic characteristics different from those of CD14⁺ monocytes: they lacked the CD64 Fcγ receptor, showed lower levels of CD32, and expressed higher amounts of CD16 compared to CD14⁺ monocytes. They also displayed a different pattern of expression of other antigens, including CD14, HLA-DR, CD45RA, CD45RO, complement receptors and complement regulatory surface proteins, adhesion and costimulatory molecules, and cytokine receptors, among others. When compared to CD16⁻ DC, CD16⁺/HLA-DR⁺ cells showed reactivity for CD16, dim positivity for CD14, higher expression of both Ig- and complement-receptors and lower reactivity for HLA-DR, adhesion, and costimulatory molecules (with the exception of CD86). The CD16⁺/HLA-DR⁺ cell subset displayed a higher Ig/complement-mediated phagocytic/oxidative activity than CD16⁻ DC, although this activity was significantly lower than that of mature monocytes. Regarding cytokine production at the single cell level, LPS plus IFN-γ-stimulated PB CD16⁺/HLA-DR⁺ cells produced significant amounts of IL1β, IL6, IL12, TNFα, and IL8; however, the percentage of cytokine-producing cells and the amount of cytokine/cell were lower in CD16⁺/HLA-DR⁺ cells than in CD14⁺ monocytes. In addition, upon comparing CD16⁺/HLA-DR⁺ cells with CD33⁺⁺⁺/CD16⁻ DC, we found that the percentage of cytokine-producing cells and the amount of cytokine/cell were significantly different in both cell subsets. In summary, our results show that CD16⁺/HLA-DR⁺ cells clearly display different morphologic, cytochemical, immunophenotypical, and functional characteristics compared to both mature monocytes and CD16⁻ DC. Interestingly, these cells are more frequent than other DC in normal human adult PB and cord blood samples, while they are less represented in normal bone marrow.     

All roads lead to Rome: pathways of NKT cells promoting asthma

NKT cells are the prominent manipulator in asthma development. Asthmatic NKT cells migrate from thymus, spleen, liver and bone marrow into blood vessels, and then concentrate in airway bronchi mucosa. This recruitment is dependent on high expression of CCR9 and engagement of CCL25/CCR9. NKT cells promote asthma in two different pathways. One is an indirect pathway. NKT cells contact with CD3⁺ T cells and induce them secreting large quantity of Th2 cytokines (IL-4, IL-13), which requires the participation of dentritic cells and the synergic signaling of CCL25/CCR9 and CD226. The other is a direct pathway. Circulating asthmatic NKT cells selectively highly express Th1 cytokines (IFN-γ). Once reached airway epithelium, most NKT cells shift to Th2-bias, highly expressing IL-4, IL-13, but not IFN-γ. Both pathways lead to airway hyperresponsiveness and inflammation, asthma development. Comparing to the well documented suppressive regulatory T cells, CD4⁺CD25⁺ T cells, NKT cells perform as a novel active regulator in asthma. These recent understanding of NKT cells performance in the development of asthma might unveil new therapy targets and management strategies for asthma.