Impaired activation of Stat1 and c‐Jun as a possible defect in macrophages of patients with active tuberculosis

Studies of patients with active tuberculosis (TB) and infected healthy individuals have shown that interferon (IFN)‐γ is present in sites of Mycobacterium tuberculosis infection in comparable levels. This suggests that there is a deficiency in the macrophage response to IFN‐γ in TB patients. We used recombinant human IFN‐γ to stimulate adherent monocyte‐derived macrophages from three groups of people: patients with active tuberculosis (TBP), their healthy household contacts (HHC) and healthy uninfected controls from the community (CC). We then evaluated the ability of the macrophages to inhibit the growth of M. tuberculosis H37Rv as well as their cytokine profile at early in infection (48 h). After IFN‐γ treatment, macrophages of healthy individuals (HHC and CC) controlled M. tuberculosis growth and produced mainly nitric oxide (NO) and interleukin (IL)‐12p70, whereas TBP macrophages did not kill M. tuberculosis. Additionally, TBP macrophages produced low levels of NO and IL‐12p70 and high levels of tumour necrosis factor (TNF)‐α and IL‐10. Transforming growth factor (TGF)‐β levels were similar among all three groups. M. tuberculosis infection had little effect on the cytokine response after IFN‐γ stimulus, but infection alone induced more IL‐10 and TGF‐β in TBP macrophages. There were no differences in Stat1 nuclear translocation and DNA binding between the groups. However, the phosphorylated Stat1 and c‐Jun (AP‐1) in nuclear protein extracts was diminished in TBP macrophages compared to macrophages of healthy individuals. These results indicate an impairment of Stat1‐dependent and Stat1‐independent IFN‐γ signalling in macrophages of people with active tuberculosis, suggesting a different molecular regulation that could impact macrophage functionality and disease outcome.     

An optimized Protocol for Human M2 Macrophages using M‐CSF and IL‐4/IL‐10/TGF‐β Yields a Dominant Immunosuppressive Phenotype

Monocytes are highly abundant circulatory effector cells and play a vital role in driving or resolving inflammatory processes depending on their activation phenotype. We investigated and compared a panel of polarization protocols of blood‐derived monocytes to achieve a stable, optimal and effective regimen for in vitro induction of immunosuppressive human macrophages, evaluating their surface receptor expression, cytokine profile, scavenging function and ability to suppress T‐cell proliferation. Importantly, we assessed the effect of copolarization or secondary pro‐inflammatory stimulation of a primary anti‐inflammatory activation phenotype. A combination of IL‐4/IL‐10/TGF‐β yielded a relatively stable and dominant immunosuppressive phenotype characterized by higher IL‐10 production and down‐regulated TNF‐α, IL‐6, CD86, CD274 and MHC II expression. Functionally, IL‐4/IL‐10/TGF‐β‐stimulated macrophages (M2) had a potent deactivating effect on a subsequent pro‐inflammatory LPS/IFNγ‐activated macrophage (M1) stimulation and significantly suppressed T‐cell proliferation. Monocytes derived from patients with chronic inflammatory diseases could be induced to be anti‐inflammatory using this protocol. Pre‐differentiation with GM‐CSF or M‐CSF was further demonstrated to enhance final M1/M2 activation status. Our findings indicate a robust polarization protocol for generation of specific immunosuppressive human monocyte‐derived macrophages.     

Mycobacterium tuberculosis Sonicate–Induced IFNγ, CXCL10 and IL10 can Differentiate Severity in Tuberculosis

Improved tools are required to study immunopathogenesis of tuberculosis (TB). Mycobacterium tuberculosis antigen‐stimulated T cell‐based assays can detect TB but are less effective when responses are compromised such as in severe disease. We investigated immune responses to M. tuberculosis whole sonicate (MTBs), recombinant antigens ESAT6 and CFP10 in whole blood cells of healthy endemic controls (EC, n = 42) and patients with pulmonary (PTB, n = 36) or extrapulmonary (ETB, n = 41) disease. Biomarkers of T cell activation (IFNγ) or modulation (IL10) and chemokines, CXCL9, CXCL10 and CCL2, secretion were measured. MTBs, ESAT6 and CFP10 all induced IFNγ responses in TB. ESAT6‐induced IFNγ was elevated in TB as compared with EC. MTBs stimulated the highest IFNγ levels but did not differentiate between TB and EC. However, MTBs‐induced CXCL10 (P = 0.004) was reduced, while IL10 (P < 0.001) was raised in TB as compared with EC. Between sites, MTBs‐induced CCL2 (P = 0.001) and IL10 secretion was higher in PTB than ETB (P < 0.001). In comparison of disease severity, MTBs‐induced IFNγ (P = 0.014) and CXCL10 (P = 0.022) levels were raised in moderate as compared with far advanced PTB. In ETB, MTBs‐induced IL10 levels were greater in less‐severe (L‐ETB) than in severe disseminated (D‐ETB) cases, P = 0.035. Within the L‐ETB group, MTBs‐induced IFNγ was greater in patients with tuberculous lymphadenitis than those with pleural TB (P = 0.002). As immune responses to MTBs were differentially activated in TB of different sites and severity, we propose the utility of MTBs‐induced IFNγ, CXCL10 and IL10 as biomarkers in TB.     

The proportion of different interleukin‐17‐producing T‐cell subsets is associated with liver fibrosis in chronic hepatitis C

Studies have suggested the pivotal role of T helper type 1 (Th1) ‐related cytokines on the outcome of hepatitis C virus (HCV) infection. Nevertheless, the role of different interleukin‐17 (IL‐17) ‐secreting T cells on chronic hepatitis C (CHC) is less clear. Here, the in vivo IL‐1β, IL‐6, and IL‐17 levels were positively correlated with both alanine transaminase (ALT) levels and hepatic lesions. When compared with the control group, CHC patients showed a lower proportion of IL‐17‐secreting (CD4⁺ and CD8⁺) T cells capable of simultaneously producing IL‐21. Moreover, the percentage of IL‐10‐secreting Th17 cells was also lower in CHC patients. Notably, advanced liver lesions were observed among those patients with lower percentage levels of IL‐17‐producing T cells positive for IL‐21, interferon‐γ (IFN‐γ) and IL‐10. In contrast, the severity of hepatic damage was associated with peripheral single IL‐17⁺ T cells. The percentage of IL‐17⁺ IL‐21^– IFN‐γ⁺ (CD4⁺ and CD8⁺) T‐cell phenotypes was positively associated with plasma CD14 levels. Finally, elevated levels of circulating CD14 were detected among CHC patients with extensive liver damage. In summary, although preliminary, our results suggest that a balance between different IL‐17‐producing T cells, associated with peripheral levels of CD14, may be a progress marker for liver disease in chronically HCV‐infected patients.     

Dendritic Cells from Rheumatoid Arthritis Patient Peripheral Blood Induce Th17 Cell Differentiation via miR‐363/Integrin αv/TGF‐β Axis

Dendritic cells (DCs) are critical regulators of immune responses. This study was to observe the effect of DCs from peripheral blood on the differentiation of Th17 in patients with rheumatoid arthritis (RA). Peripheral blood samples were collected from 30 patients with RA and 20 healthy controls, respectively. Flow cytometry results showed that in contrast to Treg cells, the proportion of Th17 cells in T cells and the Th17/Treg ratio were both increased in patients with RA. The RT‐PCR results showed that Foxp3、ROR γt and miR‐363 expression in PBMC of patients with RA were reduced, but the ITGAV expression was increased, which was negatively related to miR‐363 expression. IL‐17, TGF‐β and IL‐6 levels detected by ELISA were increased in peripheral blood serum of patients with RA. Moreover, we noted that the CD11C⁺αν⁺/CD11C⁺ DCs ratio was obvious increased in patients with RA and has positive correlation to the Th17/Treg ratio. In cocultured system, Th17 cell differentiation was significantly inhibited in the presence of ITGF‐β suggesting that Th17 cell differentiation was controlled by active TGF‐β (aTGF‐β). After DCs transfecting with miR‐363 mimics and cocultured with T cells, Th17 cell number, IL‐17 level and ROR‐γt expression were significantly reduced in the presence of latent TGF‐β (ITGF‐β). In addition, the integrin αv protein expression was both reduced in the presence of aTGF‐β or ITGF‐β. These data demonstrated that DCs induced Th17 cell differentiation through miR‐363/Integrin αv/TGF‐β pathway in patients with RA.     

Pro‐ and anti‐inflammatory cytokine gene single‐nucleotide polymorphisms in inflammatory bowel disease

Anti‐inflammatory cytokines have an important role in disease, tumour and transplant processes. Alterations in the regulation of several cytokines have been implicated in a variety of inflammatory disorders, including IBD (inflammatory bowel disease) [Crohn′s disease (CD) and ulcerative colitis (UC)]. Cytokine polymorphisms are also known to affect the level of gene expression. Thus, the aim of this study was to determine the relationship between cytokine polymorphisms and the IBD pathologies in a Spanish population. Polymorphisms analysis was performed using PCR‐SSOP using a microbeads luminex assay. The following polymorphisms were determined: TNFα [?238G/A (rs361525) and ?308G/A (rs1800629)], IFNγ [+874A/T (rs62559044)], TGFβ [+869C/T (rs1982073) and +915G/C (rs1800471)], IL10 [?1082A/A (rs1800896), ?592A/C (rs1800872), ?819C/T (rs1800871)], IL6 [?174C/G (rs1800795)], IL12p40 [3′UTR ?1188A/C (rs3212227)], IL1α [?889C/T (rs1800587)], IL1β [?511C/T (rs1143634) and +3962C/T (rs1143633)], IL1R [Pst‐1 1970C/T] and IL1RA [Mspa‐1 11100C/T]. No statistical differences in TNFα, IFNγ, TGFβ, IL10, IL6, IL1α, IL1β, IL1R and IL1Ra genotypes and allele distributions between the IBD groups and healthy controls were found. However, we observed significant differences in the 3′UTR ?1188A/C polymorphism of IL12p40. So ?1188A allele was increased in patients with UC and the ?1188C allele (high IL12p40 production) was increased in patients with CD with respect to controls. These data are in concordance with the fact that CD has been shown to be associated with a Th1 T‐cell‐mediated inflammation model and high IL12/IFNγ production at histological affected sites. These data suggest that cytokine polymorphisms in TNFα, IFNγ, TGFβ, IL10, IL6 and IL1α, IL1β, IL1R and IL1Ra cytokine gene do not seem to be relevant in IBD susceptibility and IL12p40 3′UTR ?1188A/C polymorphism seems to be associated with a differential IBD development.     

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T‐cell proliferation

Bone‐marrow‐derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone‐marrow‐derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone‐marrow‐derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4)/IL‐13, respectively. Following in vitro stimulation with lipopolysaccharide, M2‐polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL‐4, IL‐13, transforming growth factor‐β and IL‐10, whereas M1‐polarized peritoneal macrophages expressed negligible amounts of Th1 and pro‐inflammatory cytokines. ELISA showed that M2‐polarized peritoneal macrophages produced significantly more IL‐10 than M1‐polarized peritoneal macrophages. Notably, M2‐polarized peritoneal macrophages contributed more to the suppression of T‐cell proliferation than did M1‐polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL‐4 and IL‐13, increased in T‐cells co‐cultured with M2‐polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T‐cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.     

Effects of Pregnancy‐associated Malaria on T Cell Cytokines in Cameroonian Women

Although Th17 cells subsets improve immunity against extra and intracellular pathogens, and in modulating Th1 and other immune responses, its role on pregnancy‐associated malaria (PAM) is unknown. This study aims to investigate the effects of PAM on Th1 (IFN‐γ, TNF‐α), IL‐10 family (IL‐10, IL‐19, IL‐22), Th17 (IL‐17A, IL‐23) cytokines and on CXCL‐10 chemokine profiles in pregnant women. Between 2010 and 2011, venous blood specimens from 107 volunteer pregnant Cameroonian women was used to determine parasitaemia microscopically and haemoglobin levels using HemoCue analyzer. Plasma levels of the biomarkers were determined by ELISA. Parasitaemia was higher in women with low haemoglobin levels, parity and mother's age. IL‐10 and CXCL‐10 plasma levels were higher in the malaria infected and in anaemic women while IFN‐γ and IL‐17A levels were higher in malaria non‐infected and in non‐anaemic women. Parasitaemia correlated positively with IL‐10 and CXCL‐10 levels but inversely with IFN‐γ and IL‐17A. Haemoglobin levels were higher in women with low IL‐10 and CXCL‐10 levels, and in group with high IFN‐γ, IL‐17A and IL‐23 levels. Only IL‐10 levels associated negatively with parity. Positive correlations were observed between Th17 (IL‐17A) and Th1 (IFN‐γ, TNF‐α), IL‐10 family (IL‐19 and IL‐22) and Th17 (IL‐23) cytokines. Multivariate analysis showed association between: mother's age and IFN‐γ levels, parasitaemia and IL‐10 and CXCL‐10 levels and haemoglobin levels, gestational age and IL‐17A levels. In conclusion, during PAM, CXCL‐10 and IL‐10 responses are implicated in the pathogenesis while Th17 and Th1 immune responses, via IL‐17A and IFN‐γ might play protective roles.     

IL‐4 blocks M‐CSF‐dependent macrophage proliferation by inducing p21Waf1 in a STAT6‐dependent way

Macrophages are recruited from the blood stream to the inflammatory loci to carry out their functional activities. In an early phase of the cell cycle, macrophages become activated by Th1‐type cytokines (i.e. IFN‐γ), thereby producing several factors (cytokines, NO, etc.) and developing pro‐inflammatory activities. When bacteria and apoptotic bodies are removed, through the interaction with Th2‐type cytokines (i.e. IL‐4), macrophages become anti‐inflammatory and repair damaged tissues. Incubation of bone‐marrow‐derived macrophages with IFN‐γ or IL‐4 blocked their proliferation. While M‐CSF withdrawal caused cell cycle arrest at the early G₁ phase, treatment of macrophages with IFN‐γ or IL‐4 caused this arrest later, at the G₁/S boundary. Proliferation arrest was not due to an induction of apoptosis. IFN‐γ and IL‐4 induced the expression of the cyclin‐dependent kinase (Cdk) inhibitor p21^Waf1. Using KO mice and iRNA experiments, we found that p21^Waf1is required for IL‐4‐ but not for IFN‐γ‐dependent inhibition of macrophage proliferation. IL‐4 inhibited M‐CSF‐dependent Cdk‐2 and Cdk‐4 activities, which are necessary for entry and passage through the S phase of the cell cycle. The signal transduction used to induce the expression of p21^Waf1after interaction of IL‐4 with the corresponding receptor was mediated by STAT6. Thus, IL‐4 and IFN‐γ blocked M‐CSF‐induced macrophage proliferation through distinct mechanisms.     

Mycobacterial antigen‐induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non‐diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects

Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non‐diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell‐mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non‐diabetic TB patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen‐induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐β], and Th2 (IL‐4, IL‐5, IL‐10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1_pool stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1_pool in healthy subjects. In response to complex mycobacterial antigens, both IFN‐γ and TNF‐β were secreted by PBMC of all groups whereas diabetic TB patients secreted IL‐10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN‐γ and IL‐10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions.     

The Suppressive Function of Human CD8+ iTregs is Inhibited by IL‐1β and TNFα

CD8⁺ Tregs display an immunoregulatory activity and may play an essential role in the immunopathology of several diseases. Therefore, their therapeutic potential is exquisite and further studies on their differentiation and function are essential. The aim of this study was to evaluate the role of the innate immune system in CD8⁺ iTreg differentiation and function. Naive human CD8⁺CD25^?CD45RA⁺ T cells were cultured in Treg‐inducing conditions with or without IL‐1β, TNFα or monocyte‐derived dendritic cells (DCs). The differentiation of CD8⁺CD127^?CD25^hiFoxP3^hi‐induced Tregs (CD8⁺ iTregs) is dependent on TGF‐β1 and IL‐2, which had synergistic effect upon their differentiation. CD8⁺ iTregs were also induced in a coculture with allogeneic mature DCs (mDCs). The CD8⁺ iTregs suppressive function was confirmed, which was diminished in the presence of IL‐1β and TNFα. The IL‐1β‐prevented suppressive function was associated with reduced secretion of IL‐10 and IFNγ, whereas the presence of TNFα did not affect their secretion. Furthermore, the presence of TNFα reduced IL‐10 and TGF‐β1 secretion by CD8⁺ iTregs, whereas only IL‐10 secretion was decreased by IL‐1β. Together, these results suggest that IL‐1β and TNFα prevent IL‐2‐ and TGF‐β1‐driven CD8⁺ iTregs suppressive function in human T cells. Such pro‐inflammatory innate immune response possibly mediates its negative tolerogenic effect through reduced IFNγ‐, IL‐10‐ and TGF‐β1‐driven mechanism.     

Effect of enterohaemorrhagic Escherichia coli O157:H7‐specific enterohaemolysin on interleukin‐1β production differs between human and mouse macrophages due to the different sensitivity of NLRP3 activation

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection in humans can cause acute haemorrhagic colitis and severe haemolytic uraemic syndrome. The role of enterohaemolysin (Ehx) in the pathogenesis of O157:H7‐mediated disease in humans remains undefined. Recent studies have revealed the importance of the inflammatory response in O157:H7 pathogenesis in humans. We previously reported that Ehx markedly induced interleukin‐1β (IL‐1β) production in human macrophages. Here, we investigated the disparity in Ehx‐induced IL‐1β production between human and mouse macrophages and explored the underlying mechanism regarding the activation of NOD‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasomes. In contrast to the effects on human differentiated THP‐1 cells and peripheral blood mononuclear cells, Ehx exerted no effect on IL‐1β production in mouse macrophages and splenocytes because of a disparity in pro‐IL‐1β cleavage into mature IL‐1β upon caspase‐1 activation. Additionally, Ehx significantly contributed to O157:H7‐induced ATP release from THP‐1 cells, which was not detected in mouse macrophages. Confocal microscopy demonstrated that Ehx was a key inducer of cathepsin B release in THP‐1 cells but not in mouse IC‐21 cells upon O157:H7 challenge. Inhibitor experiments indicated that O157:H7‐induced IL‐1β production was largely dependent upon caspase‐1 activation and partially dependent upon ATP signalling and cathepsin B release, which were both involved in NLRP3 activation. Moreover, inhibition of K⁺ efflux drastically diminished O157:H7‐induced IL‐1β production and cytotoxicity. The findings in this study may shed light on whether and how the Ehx contributes to the development of haemolytic uraemic syndrome in human O157:H7 infection.     

CD4+ CD25+ GARP+ regulatory T cells display a compromised suppressive function in patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a lethal inflammatory heart disease and closely connected with dysfunction of the immune system. Glycoprotein A repetitions predominant (GARP) expressed on activated CD4⁺ T cells with suppressive activity has been established. This study aimed to investigate the frequency and function of circulating CD4⁺   CD25⁺ GARP⁺ regulatory T (Treg) cells in DCM. Forty‐five DCM patients and 46 controls were enrolled in this study. There was a significant increase in peripheral T helper type 1 (Th1) and Th17 number and their related cytokines [interferon‐γ (IFN‐γ), interleukin (IL‐17)], and an obvious decrease in Treg number, transforming growth factor‐β₁ (TGF‐β₁) levels and the expression of forkhead box P3 (FOXP3) and GARP in patients with DCM compared with controls. In addition, the suppressive function of CD4⁺ CD25⁺ GARP⁺ Treg cells was impaired in DCM patients upon T‐cell receptor stimulation detected using CFSE dye. Lower level of TGF‐β₁ and higher levels of IFN‐γ and IL‐17 detected using ELISA were found in supernatants of the cultured CD4⁺ CD25⁺ GARP⁺ Treg cells in DCM patients compared with controls. Together, our results indicate that CD4⁺ CD25⁺ GARP⁺ Treg cells are defective in DCM patients and GARP seems to be a better molecular definition of the regulatory phenotype. Therefore, it might be an attractive stategy to pay more attention to GARP in DCM patients.     

Characteristics of Schistosoma japonicum infection induced IFN‐γ and IL‐4 co‐expressing plasticity Th cells

Schistosoma japonicum infection can induce granulomatous inflammation and cause tissue damage in the mouse liver. The cytokine secretion profile of T helper (Th) cells depends on both the nature of the activating stimulus and the local microenvironment (e.g. cytokines and other soluble factors). In the present study, we found an accumulation of large numbers of IFN‐γ⁺ IL‐4⁺ CD4⁺ T cells in mouse livers. This IFN‐γ⁺ IL‐4⁺ cell population increased from 0·68 ± 0·57% in uninfected mice to 7·05 ± 3·0% by week 4 following infection and to 9·6 ± 5·28% by week 6, before decreasing to 6·3 ± 5·9% by week 8 in CD4 T cells. Moreover, IFN‐γ⁺ IL‐4⁺ Th cells were also found in mouse spleen and mesenteric lymph nodes 6 weeks after infection. The majority of the IFN‐γ⁺ IL‐4⁺ Th cells were thought to be related to a state of immune activation, and some were memory T cells. Moreover, we found that these S. japonicum infection‐induced IFN‐γ⁺ IL‐4⁺ cells could express interleukin‐2 (IL‐2), IL‐9, IL‐17 and high IL‐10 levels at 6 weeks after S. japonicum infection. Taken together, our data suggest the existence of a population of IFN‐γ⁺ IL‐4⁺ plasticity effector/memory Th cells following S. japonicum infection in C57BL/6 mice.     

TGF‐β indirectly favors the development of human Th17 cells by inhibiting Th1 cells

Human Th17 clones and circulating Th17 cells showed lower susceptibility to the anti‐proliferative effect of TGF‐β than Th1 and Th2 clones or circulating Th1‐oriented T cells, respectively. Accordingly, human Th17 cells exhibited lower expression of clusterin, and higher Bcl‐2 expression and reduced apoptosis in the presence of TGF‐β, in comparison with Th1 cells. Umbilical cord blood naïve CD161⁺CD4⁺ T cells, which contain the precursors of human Th17 cells, differentiated into IL‐17A‐producing cells only in response to IL‐1β plus IL‐23, even in serum‐free cultures. TGF‐β had no effect on constitutive RORγt expression by umbilical cord blood CD161⁺ T cells but it increased the relative proportions of CD161⁺ T cells differentiating into Th17 cells in response to IL‐1β plus IL‐23, whereas under the same conditions it inhibited both T‐bet expression and Th1 development. These data suggest that TGF‐β is not critical for the differentiation of human Th17 cells, but indirectly favors their expansion because Th17 cells are poorly susceptible to its suppressive effects.     

Multi‐functional flow cytometry analysis of CD4+ T cells as an immune biomarker for latent tuberculosis status in patients treated with tumour necrosis factor (TNF) antagonists

Although monitoring tuberculosis (TB) infection during long‐term treatment with tumour necrosis factor (TNF) antagonists is of great importance, no monitoring strategy has yet proved successful. Indeed, even the newly proposed interferon‐gamma release assays (IGRAs) are known to produce dynamic changes in IFN‐γ plasma levels, making them unreliable indicators of patients' pathological/clinical status. We used intracellular cytokine flow cytometry (ICCFC) to investigate the performance of multi‐functional CD4⁺ T cells producing IFN‐γ, interleukin (IL)‐2 and/or TNF in response to Mycobacterium tuberculosis‐specific antigens in subjects treated with TNF antagonists. Patients were classified into three groups based on their TB status before commencement of treatment and on IFN‐γ level fluctuations evaluated by IGRA during a 36‐month follow‐up period. The cytokine profile of M. tuberculosis‐specific CD4⁺ T cells showed that latent tuberculosis infection (LTBI) subjects had a higher frequency of double‐positive IFN‐γ⁺ IL‐2⁺ CD4⁺ T cells and triple‐positive IFN‐γ⁺ IL‐2⁺ TNF⁺ CD4⁺ T cells compared to those without LTBI, who showed IFN‐γ‐level fluctuations over time. In contrast, this latter group of patients showed similar proportions of cells producing IFN‐γ alone, IL‐2 alone and IL‐2 in combination with TNF in response to M. tuberculosis‐specific antigens. It therefore appears that patients with and without LTBI infection are characterized by different intracellular cytokine profiles. This is the first study evaluating ICCFC in patients treated with TNF antagonists, and suggests that multi‐functional analysis of CD4⁺ T cells could be useful for ruling out TB infection in patients classified at screening as LTBI‐negative but who show IGRA fluctuations under long‐term TNF antagonist treatment.     

Decreased Plasma IL‐22 Levels and Correlations with IL‐22‐Producing T Helper Cells in Patients with New‐Onset Systemic Lupus Erythematosus

Interleukin‐22 (IL‐22) and IL‐22‐producing T helper (Th) cells are involved in the pathogenesis of autoimmune diseases. However, the roles of IL‐22 and IL‐22‐producing T helper cells in systemic lupus erythematosus (SLE) remain unclear. Plasma levels of IL‐22 were measured in 41 patients with SLE (19 new‐onset and 22 relapsing patients) and 20 healthy controls by enzyme‐linked immunosorbent assay (ELISA). Meanwhile, the percentages of CD4⁺IFN‐γ⁺ (Th1), CD4⁺IL‐17⁺ (Th17) and CD4⁺IFN‐γ^?IL‐17^? IL‐22⁺ (Th22) cells in peripheral lymphocytes were determined by flow cytometry, and plasma IL‐22 autoantibodies were detected by ELISA in 19 new‐onset SLE patients and 20 healthy controls. Plasma IL‐22 levels in new‐onset SLE patients were significantly decreased compared with relapsing SLE patients and healthy controls. After treatment with prednisone and hydroxychloroquine, the levels of plasma IL‐22 in new‐onset SLE patients were obviously increased but still lower than healthy controls. There was a positive correlation between plasma IL‐22 levels and the percentages of Th22 cells, but not Th1 and Th17 cells. Moreover, plasma IL‐22 levels as well as peripheral Th17 and Th22 cells correlated with SLE disease activity index (SLEDAI) scores and erythrocyte sedimentation rate (ESR). High frequencies of plasma IL‐22 autoantibodies were detected in new‐onset SLE patients. However, IL‐22 levels did not correlate with IL‐22 autoantibody. Decreased plasma IL‐22 levels and correlation with Th22 cells may be distinct features in new‐onset SLE. Moreover, IL‐22 and Th22 cell correlated with SLE disease activity.     

Engulfment of Hb‐activated platelets differentiates monocytes into pro‐inflammatory macrophages in PNH patients

The distinct response shown by different phenotypes of macrophages and monocytes under various clinical conditions has put the heterogeneity of these cells into focus of investigation for several diseases. Recently, we have described that after engulfing hemoglobin (Hb)‐activated platelets, classical monocytes differentiated into pro‐inflammatory phenotypes, which were abundant in the circulation of paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease patients. Our current study shows that upon engulfment of Hb‐activated platelets, monocytes differentiate into M1‐macrophages under M1‐polarization stimulus (GM‐CSF, IFN‐γ + LPS). When grown under M2‐polarization stimulus (M‐CSF, IL‐4 + IL13), the cells exhibited an M1‐like phenotype, secreted elevated levels of pro‐inflammatory cytokines including TNF‐α and IL‐1β, and displayed loss of the secretion of cytokine such as IL‐10 and also phagocytic ability unlike the conventional M2 macrophages. Interestingly, when differentiated under the above polarization stimulus, monocytes from PNH patients expressed high levels of CD80 and phospho‐STAT1, like M1 macrophages. Hemolytic mice also exhibited a gradual increase in monocyte–platelet aggregates in circulation and accumulation of CD80^high macrophages in thioglycollate‐induced inflamed peritoneum. The spleen of the mice was also populated by CD80^high macrophages with compromised phagocytic capacity. Our findings suggest that the hemolytic environment and specifically the Hb‐activated platelets, which are abundant in circulation during intravascular hemolysis, closely regulate monocyte differentiation.     

The Responses of Multiple Cytokines Following Incubation of Whole Blood from TB Patients,Latently Infected Individuals and Controls with the TB Antigens ESAT‐6, CFP‐10 and TB7.7

The development of clinically relevant biomarkers is important for diagnosing latent tuberculosis infection (LTBI) and active tuberculosis (TB) and predicting their prognoses. This study examined whether the responses of multiple cytokines can be used as a biomarker to distinguish the TB infection status and mycobacterial load. We analysed the responses of multiple cytokines (IFN‐γ, IL‐2, IL‐10, IL‐13, IL‐17 and TNF‐α) in the supernatant from the QuantiFERON‐TB Gold In‐Tube assay following stimulation of whole blood from the TB group (n = 32), LTBI group (n = 19) and healthy controls (n = 30) with TB antigens (ESAT‐6, CFP‐10 and TB7.7). The median responses of IFN‐γ, IL‐2, IL‐10 and IL‐13 were higher in the LTBI and active TB groups than in the non‐TB control group (IFN‐γ, P < 0.001; IL‐2, P < 0.001; IL‐10, P = 0.012; IL‐13, P < 0.001). The median IL‐2/IFN‐γ ratio of the LTBI group was higher than that of the active TB group (P = 0.014) and differed significantly between patients with LTBI, patients with smear‐negative TB and patients with smear‐positive TB (P = 0.027). This difference was especially evident between the patients with LTBI and patients with smear‐positive TB (P = 0.047). In conclusion, IFN‐γ, IL‐2, IL‐10 and IL‐13 can serve as biomarkers for distinguishing TB infection. In addition, the IL‐2/IFN‐γ ratio appears to be a biomarker for diagnosing LTBI and may be useful as a prognostic factor and for evaluating treatment responses.     

TLR2‐activated human langerhans cells promote Th17 polarization via IL‐1β, TGF‐β and IL‐23

The cytokines IL‐6, IL‐1β, TGF‐β, and IL‐23 are considered to promote Th17 commitment. Langerhans cells (LC) represent DC in the outer skin layers of the epidermis, an environment extensively exposed to pathogenic attack. The question whether organ‐resident DC like LC can evoke Th17 immune response is still open. Our results show that upon stimulation by bacterial agonists, epidermal LC and LC‐like cells TLR2‐dependently acquire the capacity to polarize Th17 cells. In Th17 cells, expression of retinoid orphan receptor γβ was detected. To clarify if IL‐17⁺cells could arise per se by stimulated LC we did not repress Th1/Th2 driving pathways by antibodies inhibiting differentiation. In CD1c⁺/langerin⁺ monocyte‐derived LC‐like cells (MoLC), macrophage‐activating lipopeptide 2, and peptidoglycan (PGN) induced the release of the cytokines IL‐6, IL‐1β, and IL‐23. TGF‐β, a cytokine required for LC differentiation and survival, was found to be secreted constitutively. Anti‐TLR2 inhibited secretion of IL‐6, IL‐1β, and IL‐23 by MoLC, while TGF‐β was unaffected. The amount of IL‐17 and the ratio of IL‐17 to IFN‐γ expression was higher in MoLC‐ than in monocyte‐derived DC‐cocultured Th cells. Anti‐IL‐1β, ‐TGF‐β and ‐IL‐23 decreased the induction of Th17 cells. Interestingly, blockage of TLR2 on PGN‐stimulated MoLC prevented polarization of Th cells into Th17 cells. Thus, our findings indicate a role of TLR2 in eliciting Th17 immune responses in inflamed skin.