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.     

IL‐11 Expression in Human Term Placental Macrophages

Citation Pavlov OV, Kramareva NL, Selkov SA. IL‐11 Expression in human term placental macrophages. Am J Reprod Immunol 2011; 65: 397–402 Problem IL‐11 is a cytokine with pleiotropic activities, including gestational effects. Whereas IL‐11 production by maternal reproductive tissues is extensively studied, there is poor information about IL‐11 sources in the fetal counterpart of the maternal–fetal unit. Method of study We investigated the expression of IL‐11 in the purified human term placenta macrophages, using flow cytometry and immunoenzyme assay. Results Intracellular IL‐11 was detected in a substantial proportion of cultured CD68⁺ cells (median 38%). IL‐11 secretion by the placental macrophages was observed after 23‐hr cultivation (median 4.3 pg/10⁵ cells). Stimulation with lipopolysaccharide did not significantly change both intracellular expression and secretion of the cytokine. Conclusion Demonstrated IL‐11 expression by placental macrophages suggests that non‐trophoblast cells contribute to IL‐11 production at the maternal–fetal interface and thus account for the reproductive effects of the cytokine.     

Nodal promotes the generation of M2‐like macrophages and downregulates the expression of IL‐12

Nodal, a member of the TGF‐β superfamily, is an embryonic morphogen that is upregulated in different types of tumors. Nodal increases the tumorigenesis by inducing angiogenesis and promoting metastasis. Importantly, Nodal inhibition suppresses the growth and invasion of tumor. Since tumor‐associated macrophages (TAMs) are the major infiltrating leukocytes in most cancers, we investigated whether Nodal is involved in the differentiation of TAMs. Our results revealed that Nodal inhibition in tumor microenvironment upregulated the production of IL‐12 in macrophages and reversed TAMs to classically activated macrophage phenotype. In contrast, treatment with recombinant Nodal (rNodal) decreased the expression of IL‐12 in murine macrophages. Furthermore, rNodal promoted macrophage polarization to an alternatively activated macrophage‐like/TAM phenotype and modulated its function. These results suggest that Nodal may play an important role in macrophage polarization and downregulation of IL‐12. The rescued antitumor function of TAMs via the inhibition of Nodal expression could be a new therapeutic strategy for cancer treatment.     

IL‐1β induces thymic stromal lymphopoietin and an atopic dermatitis‐like phenotype in reconstructed healthy human epidermis

Atopic dermatitis (AD) is a common skin inflammatory disease characterized by the production of thymic stromal lymphopoietin (TSLP) and marked T_H2 polarization. Recent studies suggest that IL‐1β contributes to the development of AD skin inflammation. Here, we have investigated the impact of IL‐1β signalling on the epidermal homeostasis of both healthy subjects and AD patients [with functional filaggrin (FLG) alleles], with particular attention to TSLP production and keratinocyte differentiation. In healthy reconstructed human epidermis (RHE), IL‐1β promoted (i) robust secretion of TSLP in an NF‐κB‐dependent manner and (ii) a significant decrease in the expression of filaggrin and other proteins of the epidermal differentiation complex. These effects were prevented by treatment of RHE with the anti‐IL‐1β mAb canakinumab and by the IL‐1 receptor antagonist anakinra. Interestingly, RHE generated from AD donors behaved like that of healthy individuals and showed comparable responses to IL‐1β signals. Collectively, our results suggest that IL‐1β may be an early key mediator for the acquisition of an AD phenotype through induction of TSLP and alteration of the epidermal homeostasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Association of IL‐1β +3954 C/T and IL‐10‐1082 G/A Cytokine Gene Polymorphisms with Susceptibility to Tuberculosis

Tuberculosis (TB) constitutes the major cause of death due to infectious diseases. Cytokines play a major role in defence against Mycobacterium tuberculosis infection. Polymorphisms in the genes encoding various cytokines have been associated with tuberculosis susceptibility. Household contacts (HHC) are at increased risk of developing the disease. In this study, we examined the association of IL‐1β and IL‐10 cytokine gene polymorphisms with risk of developing tuberculosis in TB patients, their HHC and healthy controls (HC) using JavaStat and SPSS. Multifactor dimensionality reduction (MDR) analyses were performed to explore the potential gene–gene interactions. The genotype and allele frequencies of IL‐1β +3954C/T polymorphism did not vary significantly between TB patients and HC. GG (P < 0.005, OR = 0.219 and 95% CI = 0.059–0.735) and GA (P < 0.0001, OR = 2.938 and 95% CI = 1.526–5.696) genotypes of IL‐10‐1082 G/A polymorphism were found to be significantly associated with patients versus HC. HHC with CC (P < 0.03, OR = 1.833 and 95% CI = 1.1–3.35) genotype in IL‐1β and GA (P < 0.0001, OR = 4.612 and 95% CI = 2.225–9.702) genotype in IL‐10 were at increased risk of developing tuberculosis. MDR tests revealed high‐risk genotypes in IL‐1β and IL‐10 based on the association model. Our results demonstrate that the polymorphisms of IL‐1β and IL‐10 genes may be valuable markers to predict the risk for the development of TB in household contacts.     

Significance of IFN‐ɤ/IL‐2 Ratio as a Circulating Diagnostic Biomarker in Extrapulmonary Tuberculosis

Extrapulmonary tuberculosis (EPTB) remains a challenging diagnosis both for clinicians and microbiologists. We hypothesized that the profile of IFN‐?/IL‐2 ratio in clinically diagnosed cases of EPTB would be distinct from that of age‐ and sex‐matched healthy controls. Therefore, in our study, we have assessed the ratio of serum levels of IFN‐? and IL‐2 in clinically diagnosed cases of EPTB to assess their potential role as diagnostic biomarkers. Sixty‐nine (69) clinically confirmed EPTB cases and 69 age‐ and sex‐matched healthy controls were included in the study. All the extrapulmonary specimens were subjected to Ziehl–Neelsen staining for acid‐fast bacilli and culture on Lowenstein–Jensen (LJ) medium. Detection of serum levels of IFN‐? and IL‐2 was carried out using commercially available ELISA kits following manufacturers' instructions. The ratio of serum levels of IFN‐? and IFN‐?/IL‐2 was discriminative for the diagnosis of EPTB cases (p < 0.001), although the same was not observed with IL‐2 (p > 0.05). Distribution of all biomarkers significantly differed between culture‐positive and culture‐negative cases (p < 0.05). Among the smear‐positive and smear‐negative ones, only IFN‐? and IFN‐?/IL‐2 ratio could significantly differentiate (p < 0.05). Ratio of IFN‐? and IL‐2 rather than the individual levels was the best discriminatory biomarker with the highest area under the ROC curve. Although IFN‐? and IFN‐?/ IL‐2 ratio could aid in the diagnosis of EPTB, IL‐2 has a limited utility in the diagnosis of EPTB. Further elaborate studies to validate these results are required.     

Rapid release of cytoplasmic IL‐15 from tumor‐associated macrophages is an initial and critical event in IL‐12‐initiated tumor regression

This study reveals that the IL‐15 rapidly released into serum upon IL‐12 injection into tumor‐bearing mice is critical for the subsequent leukocytic infiltration of the tumor and tumor‐bearing tissue. The increase in serum IL‐15 occurs within 2 h after IL‐12 injection concomitantly with a decrease in cytoplasmic IL‐15 in tumor‐associated M? (TAM). Injection of anti‐IL‐15 one hour prior to IL‐12 abrogates subsequent leukocytic infiltration into the tumor and prevents the IL‐12‐induced reduction of primary tumor mass and the clearance of metastases. Administration of anti‐IL‐15 18 h after IL‐12 did not have a detectable impact on IL‐12‐induced leukocytic infiltration of the tumor. Deletion of NK cells had no impact on the IL‐12‐induced change in the functional phenotype of TAM or on the subsequent initiation of leukocytic infiltration of the tumor. In concert with our previous studies demonstrating that IL‐12 reduces tumor‐supportive activities of TAM, the current study supports the hypothesis that functional re‐programming of TAM not only undermines M? support for tumor growth but also contributes to a critical step in the initiation of anti‐tumor immune responses. In this context, the functional plasticity and pro‐immunogenic potential of TAM may constitute a significant and unappreciated target in existing cytokine therapies.     

TGF‐β interactions with IL‐1 family members trigger IL‐4‐independent IL‐9 production by mouse CD4+ T cells

TGF‐β and IL‐4 were recently shown to selectively upregulate IL‐9 production by naïve CD4⁺ T cells. We report here that TGF‐β interactions with IL‐1α, IL‐1β, IL‐18, and IL‐33 have equivalent IL‐9‐stimulating activities that function even in IL‐4‐deficient animals. This was observed after in vitro antigenic stimulation of immunized or unprimed mice and after polyclonal T‐cell activation. Based on intracellular IL‐9 staining, all IL‐9‐producing cells were CD4⁺ and 80–90% had proliferated, as indicated by reduced CFSE staining. In contrast to IL‐9, IL‐13 and IL‐17 were strongly stimulated by IL‐1 and either inhibited (IL‐13) or were unaffected (IL‐17) by addition of TGF‐β. IL‐9 and IL‐17 production also differed in their dependence on IL‐2 and regulation by IL‐1/IL‐23. As IL‐9 levels were much lower in Th2 and Th17 cultures, our results identify TGF‐β/IL‐1 and TGF‐β/IL‐4 as the main control points of IL‐9 synthesis.     

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.     

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.     

Drug‐specific in vitro release of IL‐2, IL‐5, IL‐13 and IFN‐γ in patients with delayed‐type drug hypersensitivity

Background: The most prevalent drug hypersensitivity reactions are T‐cell mediated. The only established in vitro test for detecting T‐cell sensitization to drugs is the lymphocyte transformation test, which is of limited practicability. To find an alternative in vitro method to detect drug‐sensitized T cells, we screened the in vitro secretion of 17 cytokines/chemokines by peripheral blood mononuclear cells (PBMC) of patients with well‐documented drug allergies, in order to identify the most promising cytokines/chemokines for detection of T‐cell sensitization to drugs. Methods: Peripheral blood mononuclear cell of 10 patients, five allergic to β‐lactams and five to sulfanilamides, and of five healthy controls were incubated for 3 days with the drug antigen. Cytokine concentrations were measured in the supernatants using commercially available 17‐plex bead‐based immunoassay kits. Results: Among the 17 cytokines/chemokines analysed, interleukin‐2 (IL‐2), IL‐5, IL‐13 and interferon‐γ (IFN‐γ) secretion in response to the drugs were significantly increased in patients when compared with healthy controls. No difference in cytokine secretion patterns between sulfonamide‐ and β‐lactam‐reactive PBMC could be observed. The secretion of other cytokines/chemokines showed a high variability among patients. Conclusion: The measurement of IL‐2, IL‐5, IL‐13 or IFN‐γ or a combination thereof might be a useful in vitro tool for detection of T‐cell sensitization to drugs. Secretion of these cytokines seems independent of the type of drug antigen and the phenotype of the drug reaction. A study including a higher number of patients and controls will be needed to determine the exact sensitivity and specificity of this test.     

Interleukin‐21 (IL‐21) synergizes with IL‐2 to enhance T‐cell receptor‐induced human T‐cell proliferation and counteracts IL‐2/transforming growth factor‐β‐induced regulatory T‐cell development

Interleukin‐2 (IL‐2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL‐2 facilitates regulatory T (Treg) cell development. IL‐21 is a type I cytokine acting as a potent T‐cell co‐mitogen but less efficient than IL‐2 in sustaining T‐cell proliferation. Using various in vitro models for T‐cell receptor (TCR)‐dependent human T‐cell proliferation, we found that IL‐21 synergized with IL‐2 to make CD4⁺ and CD8⁺ T cells attain a level of expansion that was impossible to obtain with IL‐2 alone. Synergy was mostly evident in naive CD4⁺ cells. IL‐2 and tumour‐released transforming growth factor‐β (TGF‐β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin‐21 hampered Treg cell expansion induced by IL‐2/TGF‐β combination in naive CD4⁺ cells by facilitating non‐Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL‐21 did not modulate the conversion of naive activated CD4⁺ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down‐modulation of IL‐2/TGF‐β‐induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL‐21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4⁺ and CD8⁺ T cells. Present data provide proof‐of‐concept for evaluating a combinatorial approach that would reduce the IL‐2 needed to sustain T‐cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T‐cell response.