Regulatory effects of IFN-β on the development of experimental autoimmune uveoretinitis in B10RIII mice

Background

Experimental autoimmune uveoretinitis (EAU) serves as a model for human intraocular inflammation. IFN-β has been used in the treatment of certain autoimmune diseases. Earlier studies showed that it ameliorated EAU; however, the mechanisms involved in this inhibition are still largely unknown.

Methodology/Principal Findings

B10RIII mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 161–180 in Complete Freund''s adjuvant. Splenocytes from different time points after immunization were used to evaluate the expression of IFN-β. An increased expression of IFN-β was observed during EAU and its highest expression was observed on day 16, 3 days after the peak of intraocular inflammation. Splenocytes and draining lymph node cells from mice immunized with IRBP_161-180 on day 13 and control mice were activated with anti-CD3/anti-CD28 antibodies or IRBP_161-180 to evaluate the production of IFN-γ and IL-17. The results showed that IFN-γ and IL-17 were significantly higher in immunized mice as compared to the control mice when exposed to anti-CD3/anti-CD28 antibodies. However, the production of IFN-γ and IL-17 was detected only in immunized mice, but not in the control mice when stimulated with IRBP_161-180. Multiple subcutaneous injections of IFN-β significantly inhibited EAU activity in association with a down-regulated expression of IFN-γ, IL-17 and an enhanced IL-10 production. In an in vitro system using cells from mice, IFN-β suppressed IFN-γ production by CD4⁺CD62L⁻ T cells, IL-17 production by CD4⁺CD62L^+/- T cells and proliferation of CD4⁺CD62L^+/- T cells. IFN-β inhibited the secretion of IL-6, but promoted the secretion of IL-10 by monocytes. IFN-β-treated monocytes inhibited IL-17 secretion by CD4⁺CD62L^+/- T cells, but did not influence IFN-γ expression and T cell proliferation.

Conclusions/Significance

IFN-β may exert its inhibitory effect on EAU by inhibiting Th1, Th17 cells and modulating relevant cytokines. IFN-β may provide a potential treatment for diseases mediated by Th1 and Th17 cells.     

IFN-β Inhibits the Increased Expression of IL-9 during Experimental Autoimmune Uveoretinitis

Purpose

It has been shown that IL-9 plays a proinflammatory role in the pathogenesis of certain autoimmune diseases. This study was designed to investigate the possible role of IL-9 in the development of experimental autoimmune uveoretinitis (EAU) and the effect of IFN-β on its expression.

Methods

EAU was induced in B10RIII mice by immunization with interphotoreceptor retinoid-binding protein peptide 161–180 (IRBP_161–180). IFN-β was administered subcutaneously to IRBP_161–180 immunized mice every other day from day one before immunization to the end of the study. Splenocytes and draining lymph node (DLN) cells from EAU mice or control mice or EAU mice treated with IFN-β or PBS were stimulated with anti-CD3/CD28 or IRBP_161–180 for 3 days. Naïve T cells cultured under Th1 or Th17 polarizing conditions were incubated in the presence or absence of IFN-β for 4 days. Effector/memory T cells were activated by anti-CD3/CD28 in the presence or absence of IFN-β for 3 days. IFN-β-treated monocytes were cocultured with naïve T cells or effector/memory T cells for 3 days. Culture supernatants were collected and IL-9 was detected by ELISA.

Results

IL-9 expression in splenocytes and DLN cells was increased in EAU mice during the inflammatory phase and returned back to lower levels during the recovery phase. IFN-β in vivo treatment significantly inhibited EAU activity in association with a down-regulated expression of IL-9. In vitro polarized Th1 and Th17 cells both secreted IL-9 and the addition of IFN-β suppressed production of IL-9 by both Th subsets. Beside its effect on polarized Th cells, IFN-β also suppressed the secretion of IL-9 by effector/memory T cells. However, IFN-β-treated monocytes had no effect on the production of IL-9 when cocultured with naïve or effector/memory T cells.

Conclusion

IL-9 expression is increased during EAU which could be suppressed by IFN-β.     

Increased Inflammatory Response in Cytomegalovirus Seropositive Patients with Alzheimer’s Disease

Alzheimer’s disease (AD) has been associated with increased local inflammation in the affected brain regions, and in some studies also with elevated levels of proinflammatory cytokines in peripheral blood. Cytomegalovirus (CMV) is known to promote a more effector-oriented phenotype in the T-cell compartment, increasing with age. The aim of this study was to investigate the inflammatory response of peripheral blood mononuclear cells (PBMCs) from AD patients and non-demented (ND) controls. Using a multiplex Luminex xMAP assay targeting GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IP-10 and TNF-α, cytokine profiles from PBMCs were analysed after stimulation with anti-CD3/CD28 beads, CMV pp65 peptide mix or amyloid β (Aβ) protofibrils, respectively. CMV seropositive AD subjects presented with higher IFN-γ levels after anti-CD3/CD28 and CMV pp65 but not after Aβ stimulation, compared to CMV seropositive ND controls. When analysing IFN-γ response to anti-CD3/CD28 stimulation on a subgroup level, CMV seropositive AD subjects presented with higher levels compared to both CMV seronegative AD and CMV seropositive ND subjects. Taken together, our data from patients with clinically manifest AD suggest a possible role of CMV as an inflammatory promoter in AD immunology. Further studies of AD patients at earlier stages of disease, could provide better insight into the pathophysiology.     

The Pronounced Th17 Profile in Systemic Sclerosis (SSc) Together with Intracellular Expression of TGFβ and IFNγ Distinguishes SSc Phenotypes

Background

Systemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc.

Methodology and Principal Findings

Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 12) or diffuse cutaneous SSc (dcSSc, n = 24). A further arbitrary subdivision was made between early dcSSc (n = 11) and late dcSSc (n = 13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGFβ and IFNγ using flow cytometry. Levels of IL-17, IL-6, IL-1α and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFNγ and TGFβ were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1α were increased in all or subsets of SSc patients.

Conclusion and Significance

The combination of IL-17, IFNγ and TGFβ levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc.     

Th1 and Th17 Responses to Helicobacter pylori in Bangladeshi Infants,Children and Adults

Both Th1 and Th17 cells are important components of the immune response to Helicobacter pylori (Hp) in adults, but less is known about T cell responses to Hp during early childhood, when the infection is often acquired. We investigated Th1 and Th17 type responses to Hp in adults, children and infants in Bangladesh, where Hp is highly endemic. IL-17 and IFN-γ mRNA levels in gastric biopsies from Hp-infected Bangladeshi adults were analyzed and compared to levels in infected and uninfected Swedish controls. Since biopsies could not be collected from infants and children, cytokine responses in Bangladeshi infants (6–12 months), children (3–5 years) and adults (>19 years) were instead compared by stimulating peripheral blood mononuclear cells (PBMCs) with a Hp membrane preparation (MP) and analyzing culture supernatants by ELISA and cytometric bead array. We found significantly higher expression of IL-17 and IFN-γ mRNA in gastric mucosa of Hp-infected Bangladeshi and Swedish adults compared to uninfected Swedish controls. PBMCs from all age groups produced IL-17 and IFN-γ after MP stimulation, but little Th2 cytokines. IL-17 and IFN-γ were primarily produced by CD4⁺ T cells, since CD4⁺ T cell depleted PBMCs produced reduced amounts of these cytokines. Infant cells produced significantly more IL-17, but similar levels of IFN-γ, compared to adult cells after MP stimulation. In contrast, polyclonal stimulation induced lower levels IL-17 and IFN-γ in infant compared to adult PBMCs and CD4⁺ T cells. The strong IL-17 production in infants after MP stimulation was paralleled by significantly higher production of the IL-17 promoting cytokine IL-1β from infant compared to adult PBMCs and monocytes. In conclusion, these results show that T cells can produce high levels of IL-17 and IFN-γ in response to Hp from an early age and indicate a potential role for IL-1β in promoting Th17 responses to Hp during infancy.     

Cytokine Diversity in the Th1-Dominated Human Anti-Influenza Response Caused by Variable Cytokine Expression by Th1 Cells,and a Minor Population of Uncommitted IL-2+IFNγ- Thpp Cells

Within overall Th1-like human memory T cell responses, individual T cells may express only some of the characteristic Th1 cytokines when reactivated. In the Th1-oriented memory response to influenza, we have tested the contributions of two potential mechanisms for this diversity: variable expression of cytokines by a uniform population during activation, or different stable subsets that consistently expressed subsets of the Th1 cytokine pattern. To test for short-term variability, in vitro-stimulated influenza-specific human memory CD4+ T cells were sorted according to IL-2 and IFNγ expression, cultured briefly in vitro, and cytokine patterns measured after restimulation. Cells that were initially IFNγ+ and either IL-2+ or IL-2- converged rapidly, containing similar proportions of IL-2-IFNγ+ and IL-2+IFNγ+ cells after culture and restimulation. Both phenotypes expressed Tbet, and similar patterns of mRNA. Thus variability of IL-2 expression in IFNγ+ cells appeared to be regulated more by short-term variability than by stable differentiated subsets. In contrast, heterogeneous expression of IFNγ in IL-2+ influenza-specific T cells appeared to be due partly to stable T cell subsets. After sorting, culture and restimulation, influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ cells maintained significantly biased ratios of IFNγ+ and IFNγ- cells. IL-2+IFNγ- cells included both Tbet^lo and Tbet^hi cells, and showed more mRNA expression differences with either of the IFNγ+ populations. To test whether IL-2+IFNγ-Tbet^lo cells were Thpp cells (primed but uncommitted memory cells, predominant in responses to protein vaccines), influenza-specific IL-2+IFNγ- and IL-2+IFNγ+ T cells were sorted and cultured in Th1- or Th2-generating conditions. Both cell types yielded IFNγ-secreting cells in Th1 conditions, but only IL-2+IFNγ- cells were able to differentiate into IL-4-producing cells. Thus expression of IL-2 in the anti-influenza response may be regulated mainly by short term variability, whereas different T cell subsets, Th1 and Thpp, may contribute to variability in IFNγ expression.     

Interferon-β Suppresses Murine Th1 Cell Function in the Absence of Antigen-Presenting Cells

Interferon (IFN)-β is a front-line therapy for the treatment of the relapsing-remitting form of multiple sclerosis. However, its immunosuppressive mechanism of function remains incompletely understood. While it has been proposed that IFN-β suppresses the function of inflammatory myelin antigen-reactive T cells by promoting the release of immunomodulatory cytokines such as IL-27 from antigen-presenting cells (APCs), its direct effects on inflammatory CD4⁺ Th1 cells are less clear. Here, we establish that IFN-β inhibits mouse IFN-γ⁺ Th1 cell function in the absence of APCs. CD4⁺ T cells express the type I interferon receptor, and IFN-β can suppress Th1 cell proliferation under APC-free stimulation conditions. IFN-β-treated myelin antigen-specific Th1 cells are impaired in their ability to induce severe experimental autoimmune encephalomyelitis (EAE) upon transfer to lymphocyte-deficient Rag1^-/- mice. Polarized Th1 cells downregulate IFN-γ and IL-2, and upregulate the negative regulatory receptor Tim-3, when treated with IFN-β in the absence of APCs. Further, IFN-β treatment of Th1 cells upregulates phosphorylation of Stat1, and downregulates phosphorylation of Stat4. Our data indicate that IFN-γ-producing Th1 cells are directly responsive to IFN-β and point to a novel mechanism of IFN-β-mediated T cell suppression that is independent of APC-derived signals.     

Immune Alterations in Patients with Anti-Interferon-γ Autoantibodies

Autoantibodies against interferon-gamma (IFN-γ) can cause immunodeficiency and are associated with various opportunistic infections. In the present study, we investigated other cellular immune parameters for a better understanding of the immunodeficiency condition in the patients. The numbers of WBC, monocytes and NK cells were increased in patients with anti-IFN-γ autoantibodies (AAbs). Upon TCR activation, T cell proliferation and IL-2 receptor of the patients remained intact. Nonetheless, the Th1 cytokine (IFN-γ and TNF-α) production was up-regulated. The production of Th2 (IL-4) and Th17 (IL-17) cytokines was unchanged. We suggest that, in addition to the presence of anti-IFN-γ autoantibodies, alterations in the cellular immune functions may also contribute to this immunodeficiency.     

Induction of Regulatory T Cells by High-Dose gp96 Suppresses Murine Liver Immune Hyperactivation

Immunization with high-dose heat shock protein gp96, an endoplasmic reticulum counterpart of the Hsp90 family, significantly enhances regulatory T cell (Treg) frequency and suppressive function. Here, we examined the potential role and mechanism of gp96 in regulating immune-mediated hepatic injury in mice. High-dose gp96 immunization elicited rapid and long-lasting protection of mice against concanavalin A (Con A)-and anti-CD137-induced liver injury, as evidenced by decreased alanine aminotransaminase (ALT) levels, hepatic necrosis, serum pro-inflammatory cytokines (IFN-γ, TNF-α, and IL-6), and number of IFN-γ ⁺ CD4⁺ and IFN-γ ⁺ CD8⁺ T cells in the spleen and liver. In contrast, CD4⁺CD25⁺Foxp3⁺ Treg frequency and suppressive function were both increased, and the protective effect of gp96 could be generated by adoptive transfer of Treg cells from gp96-immunized mice. In vitro co-culture experiments demonstrated that gp96 stimulation enhanced Treg proliferation and suppressive function, and up-regulation of Foxp3, IL-10, and TGF-β1 induced by gp96 was dependent on TLR2- and TLR4-mediated NF-κB activation. Our work shows that activation of Tregs by high-dose gp96 immunization protects against Con A- and anti-CD137-induced T cell-hepatitis and provides therapeutic potential for the development of a gp96-based anti-immune hyperactivation vaccine against immune-mediated liver destruction.     

IL-36α Exerts Pro-Inflammatory Effects in the Lungs of Mice

Interleukin (IL-) 36 cytokines (previously designated as novel IL-1 family member cytokines; IL-1F5– IL-1F10) constitute a novel cluster of cytokines structurally and functionally similar to members of the IL-1 cytokine cluster. The effects of IL-36 cytokines in inflammatory lung disorders remains poorly understood. The current study sought to investigate the effects of IL-36α (IL-1F6) and test the hypothesis that IL-36α acts as a pro-inflammatory cytokine in the lung in vivo. Intratracheal instillation of recombinant mouse IL-36α induced neutrophil influx in the lungs of wild-type C57BL/6 mice and IL-1αβ^−/− mice in vivo. IL-36α induced neutrophil influx was also associated with increased mRNA expression of neutrophil-specific chemokines CXCL1 and CXCL2 in the lungs of C57BL/6 and IL-1αβ^−/− mice in vivo. In addition, intratracheal instillation of IL-36α enhanced mRNA expression of its receptor IL-36R in the lungs of C57BL/6 as well as IL-1αβ^−/− mice in vivo. Furthermore, in vitro incubation of CD11c⁺ cells with IL-36α resulted in the generation of neutrophil-specific chemokines CXCL1, CXCL2 as well as TNFα. IL-36α increased the expression of the co-stimulatory molecule CD40 and enhanced the ability of CD11c⁺ cells to induce CD4⁺ T cell proliferation in vitro. Furthermore, stimulation with IL-36α activated NF-κB in a mouse macrophage cell line. These results demonstrate that IL-36α acts as a pro-inflammatory cytokine in the lung without the contribution of IL-1α and IL-1β. The current study describes the pro-inflammatory effects of IL-36α in the lung, demonstrates the functional redundancy of IL-36α with other agonist cytokines in the IL-1 and IL-36 cytokine cluster, and suggests that therapeutic targeting of IL-36 cytokines could be beneficial in inflammatory lung diseases.     

Mycobacterium tuberculosis-Specific IL-21+IFN-γ+CD4+ T Cells Are Regulated by IL-12

In the current study of Mycobacterium tuberculosis (MTB)-specific T and B cells, we found that MTB-specific peptides from early secreted antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) induced the expression of IL-21 predominantly in CD4⁺ T cells. A fraction of IL-21-expressing CD4⁺ T cells simultaneously expressed Th1 cytokines but did not secrete Th2 or Th17 cytokines, suggesting that MTB-specific IL-21-expressing CD4⁺ T cells were different from Th1, Th2 and Th17 subpopulations. The majority of MTB-specific IL-21-expressing CD4⁺ T cells co-expressed IFN-γ and IL-21⁺IFN-γ⁺CD4⁺ T cells exhibited obviously polyfunctionality. In addition, MTB-specific IL-21-expressing CD4⁺ T cells displayed a CD45RO⁺CD62L^lowCCR7^lowCD40L^highICOS^high phenotype. Bcl-6-expression was significantly higher in IL-21-expressing CD4⁺ T cells than IL-21^-CD4⁺ T cells. Moreover, IL-12 could up-regulate MTB-specific IL-21 expression, especially the frequency of IL-21⁺IFN-γ⁺CD4⁺ T cells. Taken together, our results demonstrated that MTB-specific IL-21⁺IFN-γ⁺CD4⁺ T cells from local sites of tuberculosis (TB) infection could be enhanced by IL-12, which have the features of both Tfh and Th1 cells and may have an important role in local immune responses against TB infection.     

Imbalanced Frequencies of Th17 and Treg Cells in Acute Coronary Syndromes Are Mediated by IL-6-STAT3 Signaling

Aims

Extensive evidence suggests inflammatory components participate in the pathogenic processes of acute coronary syndromes (ACS). In this study, we aimed to elucidate the role and mechanism underlying the imbalance of Th17 and Treg cell peripheral populations in the pathogenesis of ACS.

Methods and Results

Using a flow cytometric analysis, we observed a significantly increased frequency of Th17 cells and a concurrently decreased CD4⁺CD25⁺Foxp3⁺ Treg cells in patients with ACS. To elucidate the mechanism of Th17/Treg imbalance in ACS, 22 inflammatory cytokines were measured using multiplexed immunobead-based assays. Of six elevated cytokines in ACS patients, only IL-6 was positively correlated with a higher Th17 cell level (r = 0.39, P<0.01). Relying on IL-6 stimulating and neutralizing studies, we demonstrated a direct role for IL-6 in sera from ACS patients with an increased frequency of Th17 cells. IL-6 induces the differentiation of Th17 cells from naïve CD4⁺ T cells through STAT3 activation and RORγt induction. However, we observed that high levels of TGF-β1 inhibited IL-6-dependent Th17 cell differentiation, indicating a complex interplay between the two cytokines in the control of Th17 and Treg cell populations.

Conclusions

Our results demonstrate the role of IL-6-STAT3 signaling in ACS through increased Th17 cell differentiation. These findings indicate that IL-6 neutralizing strategies could present novel therapeutic avenues in the treatment of ACS.     

IL-17/Th17 Pathway Is Activated in Acne Lesions

The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1β, IL-6, TGF-β, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-β) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.     

Treg/IL-17 Ratio and Treg Differentiation in Patients with COPD

Background

Chronic obstructive pulmonary disease (COPD) is characterized by chronic pulmonary and systematic inflammation. An abnormal adaptive immune response leads to an imbalance between pro- and anti-inflammatory processes. T-helper (Th), T-cytotoxic (Tc) and T-regulatory (Treg) cells may play important roles in immune and inflammatory responses. This study was conducted to clarify the changes and imbalance of cytokines and T lymphocyte subsets in patients with COPD, especially during acute exacerbations (AECOPD).

Methods

Twenty-three patients with stable COPD (SCOPD) and 21 patients with AECOPD were enrolled in the present study. In addition, 20 age-, sex- and weight-matched non-smoking healthy volunteers were included as controls. The serum levels of selected cytokines (TGF-β, IL-10, TNF-α, IL-17 and IL-9) were measured by enzyme-linked immunosorbent assay (ELISA) kits. Furthermore, the T lymphocyte subsets collected from peripheral blood samples were evaluated by flow cytometry after staining with anti-CD3-APC, anti-CD4-PerCP, anti-CD8- PerCP, anti-CD25-FITC and anti-FoxP3-PE monoclonal antibodies. Importantly, to remove the confounding effects of inflammatory factors, the authors introduced a concept of “inflammation adjustment” and corrected each measured value using representative inflammatory markers, such as TNF-α and IL-17.

Results

Unlike the other cytokines, serum TGF-β levels were considerably higher in patients with AECOPD relative to the control group regardless of adjustment. There were no significant differences in the percentages of either CD4⁺ or CD8⁺ T cells among the three groups. Although Tregs were relatively upregulated during acute exacerbations, their capacities of generation and differentiation were far from sufficient. Finally, the authors noted that the ratios of Treg/IL-17 were similar among groups.

Conclusions

These observations suggest that in patients with COPD, especially during acute exacerbations, both pro-inflammatory and anti-inflammatory reactions are strengthened, with the pro-inflammatory reactions dominating. Although the Treg/IL-17 ratios were normal, the regulatory T cells were still insufficient to suppress the accompanying increases in inflammation. All of these changes suggest a complicated mechanism of pro- and anti-inflammatory imbalance which needs to be further investigated.