Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8+ T cells in chronic obstructive pulmonary disease

Background

Cigarette smoke is a major risk factor for chronic obstructive pulmonary disease (COPD), an inflammatory lung disorder. COPD is characterized by an increase in CD8⁺ T cells within the central and peripheral airways. We hypothesized that the CD8⁺ T cells in COPD patients have increased Toll-like receptor (TLR) expression compared to control subjects due to the exposure of cigarette smoke in the airways.

Methods

Endobronchial biopsies and peripheral blood were obtained from COPD patients and control subjects. TLR4 and TLR9 expression was assessed by immunostaining of lung tissue and flow cytometry of the peripheral blood. CD8⁺ T cells isolated from peripheral blood were treated with or without cigarette smoke condensate (CSC) as well as TLR4 and TLR9 inhibitors. PCR and western blotting were used to determine TLR4 and TLR9 expression, while cytokine secretion from these cells was detected using electrochemiluminescence technology.

Results

No difference was observed in the overall expression of TLR4 and TLR9 in the lung tissue and peripheral blood of COPD patients compared to control subjects. However, COPD patients had increased TLR4 and TLR9 expression on lung CD8⁺ T cells. Exposure of CD8⁺ T cells to CSC resulted in an increase of TLR4 and TLR9 protein expression. CSC exposure also caused the activation of CD8⁺ T cells, resulting in the production of IL-1β, IL-6, IL-10, IL-12p70, TNFα and IFNγ. Furthermore, inhibition of TLR4 or TLR9 significantly attenuated the production of TNFα and IL-10.

Conclusions

Our results demonstrate increased expression of TLR4 and TLR9 on lung CD8⁺ T cells in COPD. CD8⁺ T cells exposed to CSC increased TLR4 and TLR9 levels and increased cytokine production. These results provide a new perspective on the role of CD8⁺ T cells in COPD.     

Frequency of Regulatory T-Cells in the Peripheral Blood of Patients with Pulmonary Tuberculosis from Shanxi Province,China

Background

Tuberculosis (TB) is a disease caused by the chronic and continuous infection of the pathogen Mycobacterium tuberculosis (M. tuberculosis). M. tuberculosis is an intracellular bacterial pathogen and is eliminated mainly through CD4⁺ effector Th cells. M. tuberculosis induces regulatory T lymphocytes (Tregs) that mediate immune suppression by cell-to-cell contact or by secreting cytokines such as transforming growth factor-β (TGF-β). To understand the role of regulatory T-cells in the pathogenesis of TB, we have measured the in vivo frequency of regulatory T-cells and associated in vivo cytokine production in pulmonary tuberculosis patients.

Methodology/Principal Findings

In this study, we analyzed blood samples from 3 different populations (Group 1: patients with active TB, Group 2: patients recovered from TB and Group 3: healthy controls). We measured natural regulatory T-cell expression in peripheral blood using flow cytometry, and levels of blood serum IFN-γ and TGF-β1 using ELISA. The in vivo function of inductive regulatory T cells was mainly indicated by the expression of IFN-γ, TGF-β1, etc. Frequencyof natural regulatory T cells and inductive regulatory T cells in the peripheral blood samples from Group 1 patients were all significantly higher (P<0.05) than those from Groups 2 and 3.

Conclusion/Significance

Our results indicate that frequency of natural regulatory T cells and inductive regulatory T cells are significantly higher in the peripheral blood of patients with active pulmonary tuberculosis. These findings have potential application in improving TB diagnostic methods.     

Expression of regulatory receptors on γδ T Cells and their cytokine production in Behcet's disease

Introduction

Behcet''s disease (BD) is a multi-systemic disorder with muco-cutaneous, ocular, arthritic, vascular or central nervous system involvement. The role of γδ T cells is implicated in BD. The activation status of γδ T cells and their cytokine secretion against phosphoantigens are evaluated in BD.

Methods

NKG2A, NKG2C, NKG2D, CD16 and CCR7 molecules on γδ T cells were analyzed in 70 BD, 27 tuberculosis (TB) patients and 26 healthy controls (HC). Peripheral γδ T cells were expanded with a phosphoantigen (BrHPP) and IL-2, restimulated with BrHPP and a TLR3 ligand, and cytokine production was measured.

Results

γδ T cells were not increased in both BD and TB patients, but the proportions of TCRVδ2⁺ T cells were lower (58.9 and 50.7 vs. 71.7%, P = 0.04 and P = 0.005) compared to HC. Higher proportion of TCRVδ2⁺ T cells were CD16⁺ (26.2 and 33.9 vs. 16.6%, P = 0.02 and P = 0.001) and CCR7^- (32.2 and 27.9 vs. 17.7%, P < 0.0001 and P = 0.014) in BD and TB patients compared to HC. NKG2C⁺ γδ⁺ T cells were relatively increased (0.5 and 0.6 vs. 0.3%, P = 0.008 and 0.018), whereas NKG2D positivity was decreased in patients with BD and TB (77.7 and 75.8 vs. 87.5%, P = 0.001 and 0.004). Expansion capacity of γδ T cells in BD and TB as well as production of IL-13, IFN-γ, granulocyte monocyte colony stimulating factor (GM-CSF), TNF-α, CCL4 and CCL5 in BD was lower compared to HC, when restimulated by TLR3 ligand and BrHPP.

Conclusion

The changes on γδ T cells of BD as well as TB patients implicate that γδ T cells have already been exposed to regulatory effects, which changed their activity. Lower cytokine response of γδ T cells implicates down modulation of these cells in BD.     

Expansion of Pathogen-Specific Mono- and Multifunctional Th1 and Th17 Cells in Multi-Focal Tuberculous Lymphadenitis

Background

Th1 and Th17 responses are known to play an important role in immunity to pulmonary tuberculosis (PTB), although little is known about their role in extrapulmonary forms of tuberculosis (TB).

Methods

To identify the role of Th1, Th17, and Th22 cells in multi-focal TB lymphadenitis (TBL), we examined mycobacteria–specific immune responses in the whole blood of individuals with PTB (n = 20) and compared them with those with TBL (n = 25).

Results

Elevated frequencies of CD4⁺ T cells expressing IFN- γ, TNF-α, and IL-2 were present in individuals with TBL compared with those with PTB at baseline and in response to ESAT-6 and CFP-10. Similarly, increased frequencies of CD4⁺ T cells expressing IL-17A, IL-17F, and IFN-γ were also present in individuals with TBL at baseline and following ESAT-6 and CFP-10 stimulation although no significant difference in frequency of Th22 cells was observed. Finally, frequencies of Th1 (but not Th17) cells exhibited a significantly negative correlation with natural regulatory T cell frequencies at baseline.

Conclusions

Multi-focal TB lymphadenitis is therefore characterized by elevated frequencies of Th1 and Th17 cells, indicating that Th1 and Th17 responses in TB disease are probably correlates of disease severity rather than of protective immunity.     

Tumor-Derived Microvesicles Induce,Expand and Up-Regulate Biological Activities of Human Regulatory T Cells (Treg)

Background

Tumor-derived microvesicles (TMV) or exosomes are present in body fluids of patients with cancer and might be involved in tumor progression. The frequency and suppressor functions of peripheral blood CD4⁺CD25^highFOXP3⁺ Treg are higher in patients with cancer than normal controls. The hypothesis is tested that TMV contribute to induction/expansion/and activation of human Treg.

Methodology/Principal Findings

TMV isolated from supernatants of tumor cells but not normal cells induced the generation and enhanced expansion of human Treg. TMV also mediated conversion of CD4⁺CD25^neg T cells into CD4⁺CD25^highFOXP3⁺ Treg. Upon co-incubation with TMV, Treg showed an increased FasL, IL-10, TGF-β1, CTLA-4, granzyme B and perforin expression (p<0.05) and mediated stronger suppression of responder cell (RC) proliferation (p<0.01). Purified Treg were resistant to TMV-mediated apoptosis relative to other T cells. TMV also increased phospho-SMAD2/3 and phospho-STAT3 expression in Treg. Neutralizing Abs specific for TGF-β1 and/or IL-10 significantly inhibited TMV ability to expand Treg.

Conclusions/Significance

This study suggests that TMV have immunoregulatory properties. They induce Treg, promote Treg expansion, up-regulate Treg suppressor function and enhance Treg resistance to apoptosis. Interactions of TMV with Treg represent a newly-defined mechanism that might be involved in regulating peripheral tolerance by tumors and in supporting immune evasion of human cancers.     

Increased Cytokines Response in Patients with Tuberculosis Complicated with Chronic Obstructive Pulmonary Disease

Objectives

To explore the change and its significance of cytokines in patients with pulmonary tuberculosis complicated with COPD.

Methods

The immune function of 152 cases of pulmonary tuberculosis with COPD was detected to compare with 150 cases of patients with pulmonary tuberculosis, 157 cases of patients with COPD and 50 cases of healthy volunteers who were in the hospital during the same period. T lymphocyte cell population in peripheral blood was detected by flow cytometry. The serum levels of sIL-2R, IL-6, IFN-γ, TNF-α were measured using ELISA.

Results

The percentage of CD4+ T cells in TB patients with or without COPD and COPD patients without TB was significantly lower than that in control group. The percentage of CD4+ T cells in patients with TB and COPD was significantly lower than that in the non-COPD TB patients. The percentage of CD8+ T cells was higher in the TB patients group than that in control group. The CD4+/CD8+ ratio in the TB patients group was significantly lower than that in control group. The concentrations of sIL-2R, IL-6, TNF-α, IFN-γ in TB patients with or without COPD and COPD patients without TB were significantly higher than those in control group. In addition, sIL-2R, IL-6, TNF-α concentrations in the patients with TB and COPD were higher than those in the non-COPD TB patients. The concentrations of sIL-2R, IL-6, TNF-α, IFN-γ in COPD patients with TB were significantly higher than those in COPD patients without TB. There was a significant negative correlation between serum levels of TNF-α, IL-6 and FEV1 (%, predicted) in COPD without TB group.

Conclusions

The patients with pulmonary tuberculosis complicated with COPD were impaired in cellular immunity, and its extent of immune impairment is more serious than those of the patients with pulmonary tuberculosis and the patients with COPD.     

Phosphoantigen/IL2 Expansion and Differentiation of Vγ2Vδ2 T Cells Increase Resistance to Tuberculosis in Nonhuman Primates

Dominant Vγ2Vδ2 T-cell subset exist only in primates, and recognize phosphoantigen from selected pathogens including M. tuberculosis(Mtb). In vivo function of Vγ2Vδ2 T cells in tuberculosis remains unknown. We conducted mechanistic studies to determine whether earlier expansion/differentiation of Vγ2Vδ2 T cells during Mtb infection could increase immune resistance to tuberculosis in macaques. Phosphoantigen/IL-2 administration specifically induced major expansion and pulmonary trafficking/accumulation of phosphoantigen-specific Vγ2Vδ2 T cells, significantly reduced Mtb burdens and attenuated tuberculosis lesions in lung tissues compared to saline/BSA or IL-2 controls. Expanded Vγ2Vδ2 T cells differentiated into multifunctional effector subpopulations capable of producing anti-TB cytokines IFNγ, perforin and granulysin, and co-producing perforin/granulysin in lung tissue. Mechanistically, perforin/granulysin-producing Vγ2Vδ2 T cells limited intracellular Mtb growth, and macaque granulysin had Mtb-bactericidal effect, and inhibited intracellular Mtb in presence of perforin. Furthermore, phosphoantigen/IL2-expanded Vγ2Vδ2 T effector cells produced IL-12, and their expansion/differentiation led to enhanced pulmonary responses of peptide-specific CD4+/CD8+ Th1-like cells. These results provide first in vivo evidence implicating that early expansion/differentiation of Vγ2Vδ2 T effector cells during Mtb infection increases resistance to tuberculosis. Thus, data support a rationale for conducting further studies of the γδ T-cell-targeted treatment of established TB, which might ultimately help explore single or adjunctive phosphoantigen expansion of Vγ2Vδ2 T-cell subset as intervention of MDR-tuberculosis or HIV-related tuberculosis.     

T Cell Activation and Cytokine Profile of Tuberculosis and HIV-Positive Individuals during Antituberculous Treatment and Efavirenz-Based Regimens

Introduction

The profile of immune activation markers in tuberculosis and HIV-infected patients is already known. The impact of simultaneous infections on the immune parameters is still not fully explored.

Methods

We conducted a prospective study to estimate trajectories of activated T cell subsets and the profile of anti- and pro-inflammatory cytokines in a group of HIV-TB individuals, previously naïve for HAART, recruited from a randomized clinical trial during TB treatment and first antiretroviral therapy with efavirenz. Patients were evaluated according to the immunosuppression levels at baseline as group 1 (CD4<200 cells/mm³) and group 2 (CD4>200 cells/mm³). These parameters were measured at the time of HAART initiation (started about 30 days after the onset of TB treatment) and at the follow-up visits after 30, 60, 90 and 180 days. Trajectories were estimated using least squares estimates of the coefficients of a restricted cubic spline function in time after adjusting for subject effects, bootstrapping it 500 times.

Results

Increase of CD4 T cell counts and suppression of HIV viral load were observed for all patients under HAART and TB treatment. Descendent trajectories were observed for the activated CD8⁺/CD38⁺ and CD3⁺/HLA-DR⁺ T cell subsets, and for plasma concentration of gamma- interferon (IFN-γ). Except for TNF-α and IL-2 discrete variations were observed for the other cytokines. Differences in the trajectories of these parameters were observed for groups 1 and 2. Higher values of IFN-γ, IL-2, IL-6 and IL-10 were observed for group 1 from the baseline to two months after treatment initiation, whereas reduced levels of TNF-α were observed for this group between 60 and 120 days of HAART.

Conclusion

Independent of the immunosuppression profile at baseline, HIV-TB patients under HAART were able to recover the CD4⁺ T cell counts, and control viral replication and immune activation parameters over time.     

A Higher Frequency of Circulating IL-22+CD4+ T Cells in Chinese Patients with Newly Diagnosed Hashimoto’s Thyroiditis

Background

IL-22 and IL-17A are implicated in the pathogenesis of autoimmune diseases. However, the role of IL-22⁺ and IL-17A⁺ CD4⁺ T cells in the pathogenesis of Hashimoto’s thyroiditis (HT) is not fully understood. This study investigates serum IL-22 and IL-17A levels and determines the frequency of circulating IL-22⁺ CD4⁺ T cells in HT patients to understand their roles in the pathogenesis of HT.

Methods

The levels of serum IL-22, IL-17A and IFN-γ and the frequency of circulating IL-22⁺CD4⁺ and IL-17A⁺CD4⁺ T cells in 17 HT patients and 17 healthy controls (HC) were determined by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The levels of serum free triiodothyronine (FT4), free thyroxine (FT3), thyroid stimulating hormone (TSH), anti-thyroid peroxidase (TPO) and anti-thyroglobulin antibodies (TgAb) by chemiluminescent enzyme immunoassay and radioimmunoassay.

Results

The percentages of circulating IL-22⁺CD4⁺ and IL-17⁺CD4⁺ T cells (p<0.0001, p<0.0001) and the levels of serum IL-22, IL-17A and IFN-γ (p<0.0001, p<0.0001, p = 0.0210) in the HT patients were significantly higher than that in the HC. The percentages of IL-22⁺CD4⁺ T cells were positively correlated with Th17 cells (r = 0.8815, p<0.0001) and IL-17A⁺IL-22⁺CD4⁺ T cells (r = 0.8914, p<0.0001), but were negatively correlated with Th1 cells (r = −0.6110, p<0.0092) in the HT patients. The percentages of Th22 cells, Th17 cells and IL-17A⁺IL-22⁺CD4⁺ T cells were negatively correlated with the levels of serum TSH in the HT patients (r = −0.8402, p<0.0001; r = −0.8589, p<0.0001; r = −0.8289 p<0.0001, respectively).

Conclusions

A higher frequency of circulating IL-22⁺CD4⁺ and IL-17A⁺CD4⁺ T cells may be associated with the development of HT in Chinese patients.     

Combined Antigen-Specific Interferon-γ and Interleukin-2 Release Assay (FluoroSpot) for the Diagnosis of Mycobacterium tuberculosis Infection

Background

To evaluate interleukin (IL)-2 and interferon (IFN)-γ secreting T-cells in parallel for the differentiation of latent infection with Mycobacterium tuberculosis infection (LTBI) from active tuberculosis.

Methods

Following ex-vivo stimulation of peripheral blood mononuclear cells (PBMC) with M. tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein (CFP)-10, immune responses were assessed by enzyme-linked immunospot IFN-γ release assay (EliSpot-IGRA) and a novel dual cytokine detecting fluorescence-linked immunospot (FluoroSpot) in 18 patients with pulmonary tuberculosis, 10 persons with previously cured tuberculosis, 25 individuals with LTBI and 16 healthy controls.

Results

Correlation of IFN- γ⁺ spot-forming cells in EliSpot-IGRA and FluoroSpot were R² = 0.67 for ESAT-6 and R² = 0.73 for CFP-10. The number of IL-2^- IFN- γ⁺ producing cells was higher in patients with tuberculosis compared with past tuberculosis (CFP-10-induced p = 0.0068) or individuals with LTBI (ESAT-6-induced p = 0.0136). A cutoff value of >16 CFP-10-induced IFN-γ⁺ secreting cells/200.000 PBMC in the EliSpot-IGRA discriminated with highest sensitivity and specificity (89% and 76%, respectively). However, overlap in cytokine responses precludes distinction between the cohorts on an individual basis.

Conclusions

Combined analysis of IFN-γ and IL-2 secretion by antigen specific T-cells does not allow a reliable differentiation between different states of M. tuberculosis infection in clinical practice.     

Altered expression of T cell Immunoglobulin-Mucin (TIM) molecules in bronchoalveolar lavage CD4+ T cells in sarcoidosis

Background

Activated T helper (Th)-1 pulmonary CD4⁺ cells and their mediators are essential for the inflammation and granulomatous process in sarcoidosis. Recently, T-cell immunoglobulin and mucin domain (TIM) molecules were suggested to be important regulators of immune function. In this study, we wanted to investigate whether TIM molecules could play a role in sarcoidosis.

Methods

We used real-time polymerase chain reaction to investigate the differential gene expression of TIM-1 and TIM-3 as well as a few Th1 and Th2 cytokines (IL-2, IFN-γ, IL-4, IL-5 and IL-13) in CD4⁺ T cells isolated from bronchoalveolar lavage fluid (BALF) of patients (n = 28) and healthy controls (n = 8). Using flow cytometry, we were also able to analyse TIM-3 protein expression in 10 patients and 6 healthy controls.

Results

A decreased TIM-3 mRNA (p < 0.05) and protein (p < 0.05) expression was observed in patients, and the level of TIM-3 mRNA correlated negatively with the CD4/CD8 T cell ratio in BALF cells of patients. Compared to a distinct subgroup of patients i.e. those with Löfgren''s syndrome, BALF CD4⁺ T cells from non- Löfgren''s patients expressed decreased mRNA levels of TIM-1 (p < 0.05). mRNA expression of IL-2 was increased in patients (p < 0.01) and non-Löfgren''s patients expressed significantly higher levels of IFN-γ mRNA (p < 0.05) versus patients with Löfgren''s syndrome.

Conclusion

These findings are the first data on the expression of TIM-1 and TIM-3 molecules in sarcoidosis. The reduced TIM-3 expression in the lungs of patients may result in a defective T cell ability to control the Th1 immune response and could thus contribute to the pathogenesis of sarcoidosis. The down-regulated TIM-1 expression in non-Löfgren''spatients is in agreement with an exaggerated Th1 response in these patients.     

Relationship between the anti-inflammatory properties of salmeterol/fluticasone and the expression of CD4+CD25+Foxp3+ regulatory T cells in COPD

Background

Salmeterol and fluticasone combination (SFC) has anti-inflammatory effects and improves clinical symptoms in patients with chronic obstructive pulmonary disease (COPD). However, the anti-inflammatory mechanism of SFC remains unclear. In this study, we investigated the inflammatory responses of COPD, as well as the relationship of the inflammatory factors with the levels of CD4⁺CD25⁺Foxp3⁺ regulatory T cells (Foxp3⁺Tregs) after SFC therapy.

Methods

Twenty-one patients with moderate or severe COPD received treatment with 50/500 μg of SFC twice a day for 12 weeks. Before and after treatment, the patients were evaluated using the Modified Medical Research Council (MMRC) dyspnea scale and by conducting a 6-min walk test. The number of neutrophils, monocytes and lymphocytes in induced sputum were counted. Levels of cytokines, including pre-inflammatory IL-8, TNF-α, IL-17A and cytokine IL-10, in the sputum supernatant and peripheral blood were measured by ELISA. The proportion of Foxp3⁺Tregs in the total CD4⁺ T cell of the peripheral blood was determined by flow cytometry. The relationship between IL-17A levels and the percentage of Foxp3⁺Tregs was analyzed by statistical analysis.

Results

After treatment with SFC, the forced expiratory volume in 1 s as a percentage of predicted values (FEV1%) and the 6-min walk distance in the COPD patients significantly increased, while dyspnea scores decreased. The total number of cells, neutrophils, and the percentage of neutrophils in induced sputum reduced notably, while the proportion of monocytes was significantly increased. Levels of the inflammatory cytokines IL-8, TNF-α, and IL-17A in the sputum supernatant and in the blood were markedly lowered, while IL-10 levels were unchanged. The proportion of Foxp3⁺Tregs in the total CD4⁺T cell population in the peripheral blood was drastically higher than that before treatment. The level of IL-17A was negatively correlated with the proportion of Foxp3⁺Tregs in CD4⁺T cells.

Conclusion

SFC can reduce the levels of inflammatory factors and improve symptoms of COPD. The levels of inflammatory factors are associated with the variation of Foxp3⁺Tregs in COPD.

Trial registration

This study was registered with http://www.chictr.org (Chinese Clinical Trial Register) as follows: ChiCTR-TNC-10001270     

A Novel Mechanism of Soluble HLA-G Mediated Immune Modulation: Downregulation of T Cell Chemokine Receptor Expression and Impairment of Chemotaxis

Background

In recent years, many immunoregulatory functions have been ascribed to soluble HLA-G (sHLA-G). Since chemotaxis is crucial for an efficient immune response, we have investigated for the first time the effects of sHLA-G on chemokine receptor expression and function in different human T cell populations.

Methodology/Principal Findings

T cell populations isolated from peripheral blood were stimulated in the presence or absence of sHLA-G. Chemokine receptors expression was evaluated by flow cytometry. sHLA-G downregulated expression of i) CCR2, CXCR3 and CXCR5 in CD4⁺ T cells, ii) CXCR3 in CD8⁺ T cells, iii) CXCR3 in Th1 clones iv) CXCR3 in TCR Vδ2γ9 T cells, and upregulated CXCR4 expression in TCR Vδ2γ9 T cells. sHLA-G inhibited in vitro chemotaxis of i) CD4⁺ T cells towards CCL2, CCL8, CXCL10 and CXCL11, ii) CD8⁺ T cells towards CXCL10 and CXCL11, iii) Th1 clones towards CXCL10, and iv) TCR Vδ2γ9 T cells towards CXCL10 and CXCL11. Downregulation of CXCR3 expression on CD4+ T cells by sHLA-G was partially reverted by adding a blocking antibody against ILT2/CD85j, a receptor for sHLA-G, suggesting that sHLA-G downregulated chemokine receptor expression mainly through the interaction with ILT2/CD85j. Follicular helper T cells (T_FH) were isolated from human tonsils and stimulated as described above. sHLA-G impaired CXCR5 expression in T_FH and chemotaxis of the latter cells towards CXCL13. Moreover, sHLA-G expression was detected in tonsils by immunohistochemistry, suggesting a role of sHLA-G in local control of T_FH cell chemotaxis. Intracellular pathways were investigated by Western Blot analysis on total extracts from CD4+ T cells. Phosphorylation of Stat5, p70 s6k, β-arrestin and SHP2 was modulated by sHLA-G treatment.

Conclusions/Significance

Our data demonstrated that sHLA-G impairs expression and functionality of different chemokine receptors in T cells. These findings delineate a novel mechanism whereby sHLA-G modulates T cell recruitment in physiological and pathological conditions.     

IL-2 Regulates SEB Induced Toxic Shock Syndrome in BALB/c Mice

Background

Toxic Shock Syndrome (TSS) is characterized by fever, rash, hypotension, constitutional symptoms, and multi-organ involvement and is caused by Staphylococcus aureus enterotoxins such as Staphylococcal Enterotoxin B (SEB). SEB binds to the MHC-IIα chain and is recognized by the TCRβ chain of the Vβ8 TCR⁺ T cells. The binding of SEB to Vβ chain results in rapid activation of T cells and production of inflammatory cytokines, such as Interleukin-2 (IL-2), Interferon-γ and Tumor Necrosis Factor-α which mediate TSS. Although IL2 was originally identified as the T cell growth factor and was proposed to contribute to T cell differentiation, its role in TSS remains unexplored.

Methodology/Principal Findings

Mice were injected with D-Gal (25 mg/mouse). One hour after D-Galactosamine (D-Gal) injection each mouse was injected with SEB (20 µg/mouse. Mice were then observed for 72 hrs and death was recorded at different times. We tested Interleukin-12, IFNγ, and IL-2 deficient mice (IL-2^−/−), but only the IL-2 deficient mice were resistant to SEB induced toxic shock syndrome. More importantly reconstitution of IL-2 in IL-2 deficient mice restored the shock. Interestingly, SEB induced IL-2 production from T cells was dependent on p38MAPK activation in macrophages as inhibition of it in macrophages significantly inhibited IL-2 production from T cells.

Conclusion

This study shows the importance of IL -2 in TSS which has not been previously explored and it also shows that regulating macrophages function can regulate T cells and TSS.     

The Role of Interleukin-1 Receptor-Associated Kinases in Vogt-Koyanagi-Harada Disease

Purpose

To explore whether IRAK1 and IRAK4 are involved in the pathogenesis of Vogt-Koyanagi-Harada (VKH) disease.

Methods

Thirty-nine VKH patients and thirty-two healthy controls were included in this study. The mRNA levels of IRAK1 and IRAK4 from active VKH patients, inactive VKH patients, and normal controls in peripheral blood mononuclear cells (PBMCs) were detected using real-time quantitative PCR. CD4⁺T cells were purified from PBMCs obtained from active VKH patients and normal controls. The effect of IRAK1/4 inhibition on CD4⁺T cell proliferation following stimulation with IL-18 or IL-1β was measured using a modified MTT assay. CD4⁺T cell expression of IFN-γ and IL-17 were detected by flow cytometry (FCM) and enzyme-linked immunosorbent assay (ELISA). The effect of IRAK1/4 inhibition on NF-κB, STAT1, and STAT3 activation was detected by FCM.

Results

The mRNA levels of IRAK1 and IRAK4 were both significantly increased in active VKH patients compared to inactive VKH patients and healthy controls. No difference in the IRAK1 or IRAK4 mRNA level could be detected between inactive patients and healthy controls. After incubation with IRAK1/4 inhibitor, the proliferation of CD4⁺T cells was inhibited both in the active VKH patients and in the healthy controls. IRAK1/4 inhibition was also associated with a decreased expression of IFN-γ and IL-17. Phosphorylation of NF-κB, STAT1, and STAT3 in CD4⁺T from healthy controls was significantly decreased after inhibition of IRAK1/4.

Conclusions

High mRNA levels of IRAK1 and IRAK4 correlated with VKH disease activity. IRAK1 and IRAK4 play a role in the activation and proliferation of CD4⁺T cells and the higher expression observed in VKH may contribute to the pathogenesis of this blinding condition.     

Cigarette smoke-induced accumulation of lung dendritic cells is interleukin-1α-dependent in mice

Background

Evidence suggests that dendritic cells accumulate in the lungs of COPD patients and correlate with disease severity. We investigated the importance of IL-1R1 and its ligands IL-1α and β to dendritic cell accumulation and maturation in response to cigarette smoke exposure.

Methods

Mice were exposed to cigarette smoke using a whole body smoke exposure system. IL-1R1-, TLR4-, and IL-1α-deficient mice, as well as anti-IL-1α and anti-IL-1β blocking antibodies were used to study the importance of IL-1R1 and TLR4 to dendritic cell accumulation and activation.

Results

Acute and chronic cigarette smoke exposure led to increased frequency of lung dendritic cells. Accumulation and activation of dendritic cells was IL-1R1/IL-1α dependent, but TLR4- and IL-1β-independent. Corroborating the cellular data, expression of CCL20, a potent dendritic cells chemoattractant, was IL-1R1/IL-1α-dependent. Studies using IL-1R1 bone marrow-chimeric mice revealed the importance of IL-1R1 signaling on lung structural cells for CCL20 expression. Consistent with the importance of dendritic cells in T cell activation, we observed decreased CD4⁺ and CD8⁺ T cell activation in cigarette smoke-exposed IL-1R1-deficient mice.

Conclusion

Our findings convey the importance of IL-1R1/IL-1α to the recruitment and activation of dendritic cells in response to cigarette smoke exposure.     

The Contribution of Non-Conventional T Cells and NK Cells in the Mycobacterial-Specific IFNγ Response in Bacille Calmette-Guérin (BCG)-Immunized Infants

Background

The Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine is given to >120 million infants each year worldwide. Most studies investigating the immune response to BCG have focused on adaptive immunity. However the importance of TCR-gamma/delta (γδ) T cells and NK cells in the mycobacterial-specific immune response is of increasing interest.

Methods

Participants in four age-groups were BCG-immunized. Ten weeks later, in vitro BCG-stimulated blood was analyzed for NK and T cell markers, and intracellular IFNgamma (IFNγ) by flow cytometry. Total functional IFNγ response was calculated using integrated median fluorescence intensity (iMFI).

Results

In infants and children, CD4 and CD4-CD8- (double-negative (DN)) T cells were the main IFNγ-expressing cells representing 43-56% and 27-37% of total CD3+ IFNγ+ T cells respectively. The iMFI was higher in DN T cells compared to CD4 T cells in all age groups, with the greatest differences seen in infants immunized at birth (p=0.002) or 2 months of age (p<0.0001). When NK cells were included in the analysis, they accounted for the majority of total IFNγ-expressing cells and, together with DN Vδ2 γδ T cells, had the highest iMFI in infants immunized at birth or 2 months of age.

Conclusion

In addition to CD4 T cells, NK cells and DN T cells, including Vδ2 γδ T cells, are the key populations producing IFNγ in response to BCG immunization in infants and children. This suggests that innate immunity and unconventional T cells play a greater role in the mycobacterial immune response than previously recognized and should be considered in the design and assessment of novel tuberculosis vaccines.     

Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients

Background

Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25⁺FOXP3⁺ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples.

Methodology/Principle Findings

Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3⁺, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4⁺CD25⁺FOXP3⁺ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25⁺ cells of the CD4⁺ but not that of CD8⁺ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients.

Conclusions/Significance

Our results indicate that FOXP3⁺ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.