Epstein–Barr virus‐induced gene 3 suppresses T helper type 1, type 17 and type 2 immune responses after Trypanosoma cruzi infection and inhibits parasite replication by interfering with alternative macrophage activation

The Epstein–Barr virus‐induced gene 3 (EBI3) is a member of the interleukin‐12 (IL)‐12) family structurally related to the subunit p40 of IL‐12 and forms a heterodimer either with the p28 subunit to build IL‐27 or with p35 to form IL‐35. Interleukin‐27 is secreted by antigen‐presenting cells whereas IL‐35 appears to be produced mainly by regulatory T cells and regulatory B cells but both cytokines negatively regulate inflammatory immune responses. We here analysed the function of EBI3 during infection with the intracellular parasite Trypanosoma cruzi. Compared with C57BL/6 wild‐type mice, EBI3‐deficient (EBI3^?/?) mice showed a higher parasitaemia associated with an increased mortality rate. The EBI3^?/? mice displayed an elevated inflammatory immune response with an increased production of T helper type 1 (Th1‐), Th2‐ and Th17‐derived cytokines. The increased Th2 immune response appears to have over‐ridden the otherwise protective Th1 and Th17 immune responses by the induction of arginase‐1‐expressing alternatively activated macrophages in these mice. Hence, neutralization of IL‐4 and arginase‐1 activity partially restored protective immune responses in EBI3^?/? mice. So far, our results demonstrate that EBI3 is an essential general regulator of inflammatory immune responses in experimental Chagas disease and is required for control of T. cruzi infection by inhibiting Th2‐dependent alternative macrophage activation. Further studies are needed to dissect the underlying mechanisms and clarify whether EBI3 association with IL‐27 or/and IL‐35 accounts for its anti‐inflammatory character in parasitic disease.     

Ratio of M2 macrophage expression is closely associated with poor prognosis for Angioimmunoblastic T‐cell lymphoma (AITL)

Angioimmunoblastic T‐cell lymphoma (AITL) is a peripheral T‐cell lymphoma characterized by systemic disease with polymorphous infiltrate including macrophages. Although many studies of tumor‐associated macrophage (TAM) populations in various malignant tumors have been published, only a few have dealt with activation of macrophage phenotypes such as M1 and M2 in tumor tissue. Because M2 macrophages highly express CD163, we suspected that CD163 may be a useful marker for identification of activation of macrophage phenotypes in AITL. We performed a retrospective study of immunohistochemical expression using two markers for macrophages [CD68 (PG‐M1), CD163] and of the correlation of these expressions with overall survival of 42 AITL patients. The number of CD68‐positive cells in AITL tissues did not correlate with overall survival (P= 0.59), whereas the number of CD163‐positive cells and overall survival correlated to some extent (P= 0.08). Meanwhile, a higher ratio of CD163‐positive to CD68‐positive cells in AITL significantly correlated with worse overall survival (P= 0.036). Considering that this ratio reflects the proportion of macrophages polarized to the M2 phenotype, our findings indicate that activation of macrophages towards the M2 phenotype correlates with worse prognosis. Our findings indicate that the ratio of M2 macrophages expressed may be a useful marker for prognosis of AITL.     

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.     

IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

Arginase 1 activity worsens lung‐protective immunity against Streptococcus pneumoniae infection

Type 2 helper cell (Th2) dominated chronic lung diseases such as asthma are characterized by an increased risk for bacterial lung infections. However, the underlying mechanisms are poorly defined. Arginase 1 (Arg1) has been suggested to play an important role in the pathophysiology of asthma, and is rapidly induced in lung macrophages by Th2 cytokines, thereby limiting macrophage‐derived antimicrobial nitric oxide (NO) production. Here we examined the effect of Th2 cytokine induced upregulation or lung myeloid cell specific conditional knockdown of Arg1 on lung resistance against Streptococcus pneumoniae (Spn) in mice. Lung macrophages responded with a profound induction of Arg1 mRNA and protein to treatment with IL‐13 both in vitro and in vivo. IL‐13‐induced Arg1 activity in the lungs of mice led to significantly attenuated lung‐protective immunity against Spn, while conditional Arg1 knockdown had no effect on lung‐protective immunity against Spn. Collectively, the data show that Th2 cytokine induced increased Arg1 activity worsens lung‐protective immunity against Spn, and interventions to block Th2 cytokine induced lung Arg1 activity may thus be a novel immunomodulatory strategy to lower the risk of bacterial infections in asthmatic patients.     

The central nervous system environment controls effector CD4+ T cell cytokine profile in experimental allergic encephalomyelitis

In experimental allergic encephalomyelitis (EAE), CD4⁺ T cells infiltrate the central nervous system (CNS). We derived CD4⁺ T cell lines from SJL/J mice that were specific for encephalitogenic myelin basic protein (MBP) peptides and produced both Th1 and Th2 cytokines. These lines transferred EAE to naive mice. Peptide-specific cells re-isolated from the CNS only produced Th1 cytokines, whereas T cells in the lymph nodes produced both Th1 and Th2 cytokines. Mononuclear cells isolated from the CNS, the majority of which were microglia, presented antigen to and stimulated MBP-specific T cell lines in vitro. Although CNS antigen-presenting cells (APC) supported increased production of interferon (IFN)-γ mRNA by these T cells, there was no increase in the interleukin (IL)-4 signal, whereas splenic APC induced increases in both IFN-γ and IL-4. mRNA for IL-12 (p40 subunit) was up-regulated in both infiltrating macrophages and resident microglia from mice with EAE. We have thus shown that a Th1 cytokine bias within the CNS can be induced by CNS APC, and that IL-12 is up-regulated in microglial cells within the CNS of mice with EAE. Microglia may therefore control Th1 cytokine responses within the CNS.     

The CD20 homolog Ms4a8a integrates pro‐ and anti‐inflammatory signals in novel M2‐like macrophages and is expressed in parasite infection

Recently, we identified the CD20 homolog Ms4a8a as a novel molecule expressed by tumor‐associated macrophages that directly enhances tumor growth. Here, we analyzed Ms4a8a⁺ macrophages in M2‐associated infectious pathologies. In late‐stage Trypanosoma congolense and Taenia crassiceps infections, Ms4a8a expression was detected in hepatic and peritoneal macrophages respectively. Innate immunity in these infections is modulated by Toll‐like receptor (TLR) signaling and TLR2/4/7 agonists strongly induced Ms4a8a expression in bone marrow derived macrophages (BMDMs) treated with M2 mediators (glucocorticoids/IL‐4). LPS/dexamethasone/IL‐4‐induced Ms4a8a⁺ BMDMs were characterized by strong expression of mRNA of mannose receptor (Mmr), arginase 1, and CD163, and by decreased iNOS expression. Coinduction of Ms4a8a by M2 mediators and TLR agonists involved the classical TLR signaling cascade via activation of MyD88/TRIF and NF‐κB. Forced overexpression of Ms4a8a modulated the TLR4 response of RAW264.7 cells as shown by gene expression profiling. Upregulation of Hdc, Tcfec, and Sla was confirmed both in primary LPS/dexamethasone/IL‐4‐stimulated Ms4a8a⁺ BMDMs and in peritoneal macrophages from late‐stage Taenia crassiceps infection. In conclusion, we show that TLR signaling skews the typical alternative macrophage activation program to induce a special M2‐like macrophage subset in vitro that also occurs in immunomodulatory immune reactions in vivo, a process directly involving the CD20 homolog Ms4a8a.     

Mannan Derivatives Instruct Dendritic Cells to Induce Th1/Th2 Cells Polarization via Differential Mitogen‐Activated Protein Kinase Activation

Mannan derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Immunization with antigen conjugated to oxidized mannan (OM) or reduced mannan (RM) have induced differential immune responses in mice. Yet, the adjuvant effect and differences in molecular profiles of OM and RM on APCs is unresolved. Here, we investigated the response of mouse bone marrow‐derived DCs to OM and RM. OM and RM stimulated DCs to produce differential Th1/Th2‐inducing cytokines in vitro. OM and RM‐activated DCs stimulated allogeneic T‐cell Th1 and Th2 polarization reaction. OM instruct DCs to stimulate Th1 responses via IL‐12p70 production, which depends on the phosphorylation of p38, RM barely induce IL‐12p70, but IL‐10 and IL‐4, and magnitude of ERK phosphorylation, which results in a Th2 bias. These findings indicate that OM and RM were potent adjuvant capable of directly initiating DC activation Th1 and Th2 polarization respectively.     

Induction of systemic tolerance in normal but not in transgenic mice through continuous feeding of ovalbumin

The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA‐T‐cell receptor (OVA‐TCR)‐specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose‐dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naïve cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2‐dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1‐ and Th2‐antibody responses in transgenic mice. Dendritic cells (DC) from tolerant BALB/c mice were less efficient in the induction of the proliferation of cocultured T cells from both BALB/c and DO11.10 mice, as well as Th1 [interleukin (IL)‐2 and interferon (IFN)‐γ] and Th2 (IL‐4 and IL‐10) cytokine production. The DC from DO11.10 transgenic mice were equally efficient in the induction of T‐cell proliferation in both normal and transgenic mice, as well as in the induction of Th1 and Th2 cytokines, whether or not the mice consumed OVA. Transforming growth factor (TGF)‐β secretion was significantly lower in the supernatants of T cells from both normal and transgenic mice cocultured with DC from DO11.10 mice that had consumed OVA, while it was significantly higher in the presence of DC from normal tolerant mice, thus implicating TGF‐β as a regulatory cytokine in oral tolerance in the murine model.     

LIM‐only protein FHL2 regulates experimental pulmonary Schistosoma mansoni egg granuloma formation

LIM‐only protein FHL2 is associated with several immune and inflammatory diseases such as arthritis, influenza A virus infection, and lung inflammation. However, the role of FHL2 in macrophage differentiation and in the development of granuloma formation is unknown. Here, we show that expression of FHL2 is induced in mouse bone marrow derived macrophages (BMMs) following stimulation with M2 cytokines such as IL‐4 and IL‐10. FHL2‐knockout (FHL2‐KO) BMMs exhibit a proinflammatory M1 phenotype after LPS treatment and display a reduced anti‐inflammatory M2 phenotype following IL‐4 treatment. Furthermore, thioglycollate‐induced migration of macrophages and B cells is enhanced in FHL2‐KO mice. To evaluate the importance of FHL2 in the development of pulmonary granuloma formation, FHL2‐KO mice were challenged with Schistosoma mansoni eggs. FHL2‐KO mice show an enhanced number of granulomas and display decreased expression of Th2 markers and an exacerbated Th1 type of inflammation, characterized by enhanced expression of neutrophil markers and Th1 cytokines. Furthermore, the expression of barrier proteins is reduced in FHL2‐KO lung compared to WT. Collectively, these data identify a previously unrecognized role for FHL2 in the pathogenesis of pulmonary granulomatous inflammation, partly through its effect on macrophage polarization, modulation of the Th1/Th2 balance and regulation of permeability in lung.     

Mast cells have no impact on cutaneous leishmaniasis severity and related Th2 differentiation in resistant and susceptible mice

The genus leishmania comprises different protozoan parasites which are causative agents of muco‐cutaneous and systemic, potentially lethal diseases. After infection with the species Leishmania major, resistant mice expand Th1 cells which stimulate macrophages for Leishmania destruction. In contrast, susceptible mice generate Th2 cells which deactivate macrophages, leading to systemic spread of the pathogens. Th‐cell differentiation is determined within the first days, and Th2 cell differentiation requires IL‐4, whereby the initial IL‐4 source is often unknown. Mast cells are potential sources of IL‐4, and hence their role in murine leishmaniasis has previously been studied in mast cell‐deficient Kit mutant mice, although these mice display immunological phenotypes beyond mast cell deficiency. We therefore readdressed this question by infecting Kit‐independent mast cell‐deficient mice that are Th1 (C57BL/6 Cpa^Cre) or Th2 (BALB/c Cpa^Cre) prone with L. major. Using different parasite doses and intra‐ or subcutaneous infection routes, the results demonstrate no role of mast cells on lesion size development, parasite load, immune cell phenotypes expanding in draining lymph nodes, and cytokine production during murine cutaneous leishmaniasis. Thus, other cell types such as ILCs or T cells have to be considered as primary source of Th2‐driving IL‐4.     

Understanding the Regulatory Roles of Natural Killer T Cells in Rheumatoid Arthritis: T Helper Cell Differentiation Dependent or Independent?

Rheumatoid arthritis (RA) is the most common chronic systemic autoimmune disease. This disease is thought to be caused by pathogenic T cells. Th1, Th2, Th17 and Treg cells have been implicated in the pathogenesis of RA. These Th cells differentiate from CD4+ T cells primarily due to the effects of cytokines. Natural killer T (NKT) cells are a distinct subset of lymphocytes that can rapidly secrete massive amount of cytokines, including IL‐2, IL‐4, IL‐12 and IFN‐γ. Numerous studies showed that NKT cells can influence the differentiation of CD4+ T cells via cytokines in vitro. These findings suggest that NKT cells play an important role in RA by polarizing Th1, Th2, Th17 and Treg cells. In view of the complexity of RA, we discussed whether NKT cells really influence the development of RA through regulating the differentiation of Th cells.     

Role of T cell TGF‐β signaling in intestinal cytokine responses and helminthic immune modulation

Colonization with helminthic parasites induces mucosal regulatory cytokines, like IL‐10 or TGF‐β, that are important in suppressing colitis. Helminths induce mucosal T cell IL‐10 secretion and regulate lamina propria mononuclear cell (LPMC) Th1 cytokine generation in an IL‐10‐dependent manner in WT mice. Helminths also stimulate mucosal TGF‐β release. As TGF‐β exerts major regulatory effects on T lymphocytes, we investigated the role of T lymphocyte TGF‐β signaling in helminthic modulation of intestinal immunity. T cell TGF‐β signaling is interrupted in TGF‐β receptor II dominant negative (TGF‐βRII DN) mice by T‐cell‐specific over‐expression of a TGF‐βRII DN. We studied LPMC responses in WT and TGF‐βRII DN mice that were uninfected or colonized with the nematode, Heligmosomoides polygyrus. Our results indicate an essential role of T cell TGF‐β signaling in limiting mucosal Th1 and Th2 responses. Furthermore, we demonstrate that helminthic induction of intestinal T cell IL‐10 secretion requires intact T cell TGF‐β‐signaling pathway. Helminths fail to curtail robust, dysregulated intestinal Th1 cytokine production and chronic colitis in TGF‐βRII DN mice. Thus, T cell TGF‐β signaling is essential for helminthic stimulation of mucosal IL‐10 production, helminthic modulation of intestinal IFN‐γ generation and H. polygyrus‐mediated suppression of chronic colitis.     

IL‐37 Expression is Upregulated in Patients with Tuberculosis and Induces Macrophages Towards an M2‐like Phenotype

Interleukin‐37 (IL‐37), a member of the IL‐1 family, primarily functions as an anti‐inflammatory cytokine, reducing inflammation and suppressing the immune response. However, the expression and role of IL‐37 in tuberculosis (TB) remains unknown. We aimed to measure serum levels of IL‐37 and several important cytokines in 25 patients with active TB and to analyse their association with disease activity. We found that IL‐37 levels decreased in patients with TB and recovered after treatment. IL‐37 levels negatively correlated with the serum concentration of IFN‐γ and IL‐12 but positively correlated with IL‐10 and TGF‐β levels. After IL‐37, secretion was blocked in peripheral blood mononuclear cells from active patients with TB, IFN‐γ and IL‐10 production was significantly upregulated; this was not observed in healthy donors or patients after treatment. IL‐37 knockdown significantly enhanced the phagocytic activity of THP1‐derived macrophages towards Mycobacterium tuberculosis (M. tb). M1/M2 polarization‐associated markers were detected simultaneously, and IL‐37 induced a phenotypic shift in THP1‐derived macrophages towards a high CD206⁺ and low CD86⁺ macrophage subtype. Furthermore, this phenotypic shift was accompanied by upregulated mRNA levels of arginase 1, TGF‐β and IL‐10, which are characteristic hallmarks of M2 macrophages. In conclusion, our results suggest that increased levels of IL‐37 in patients with TB are associated with IFN‐γ, IL‐12, IL‐10 and TGF‐β levels and that IL‐37 plays a pathological role in TB infection by inhibiting the production of pro‐inflammatory cytokines and inducing macrophages towards an M2‐like phenotype. Thus, IL‐37 may be a novel research target to understand the pathogenesis of TB infection.     

Inhibition of Interferon Regulatory Factor 4 Suppresses Th1 and Th17 Cell Differentiation and Ameliorates Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease that is characterized by recurrent episodes of T‐cell‐mediated immune attack on central nervous system (CNS) myelin, leading to axon damage and progressive disability. Interferon regulatory factor 4 (IRF4) is expressed predominantly in the immune system and plays an important role in its development and function. Recent study demonstrated that IRF4 was critical for the generation of IL‐17‐producing Th17 cells. However, the effect of IRF4 on experimental autoimmune encephalomyelitis (EAE), an animal model of MS, needs to be further investigated. In our current study, inhibition of IRF4 with IRF4 siRNA (SiIRF4) decreases EAE scores and infiltration of Th1 and Th17 cells, but increases Treg infiltration. SiIRF4 inhibits Th1 and Th17 cell differentiation in vivo and in vitro. In our DC‐T‐cell coculture system, SiIRF4‐treated DCs resulted in significantly less IFN‐γ and IL‐17 production from T cells. Next, we adoptively transfer CD11c⁺ DCs from SiIRF4‐treated mice into recipient mice and found that these CD11c⁺ DCs ameliorated EAE. Furthermore, CD11c⁺ DCs from SiIRF4‐treated naive mice exhibited significantly reduced expression of pro‐inflammatory cytokines TNF‐α, IL‐1β, IL‐6 and IL‐12/IL‐23 (p40), and a corresponding increase in anti‐inflammatory IL‐10 expression. In conclusion, inhibition of IRF4 suppresses Th1 and Th17 cell differentiation and ameliorates EAE, via a direct regulation of DCs.     

Plasmacytoid dendritic cells induce a distinct cytokine pattern in virus-specific CD4+ memory T cells that is modulated by CpG oligodeoxynucleotides

Inherent properties of dendritic cell (DC) subsets are important in the regulation of naïve T‐cell differentiation (e.g. Th1 versus Th2 cells), whereas effector memory T cells are believed to produce a fixed cytokine repertoire independent of the type of antigen presenting cell. Here we show that two distinct human DC subsets, plasmacytoid DC (PDC) and myeloid CD11c+ DC, induced autologous mumps virus‐specific memory CD4⁺ T cells to produce markedly different cytokine patterns upon antigen stimulation. PDC stimulated the T cells to produce γ‐interferon (IFN‐γ) and interleukin‐(IL)‐10, whereas CD11c+ DC induced lower levels of IFN‐γ, virtually no IL‐10, but significant levels of IL‐5. Analysis of intracellular cytokine production showed simultaneous production of IL‐10 and IFN‐γ in mumps‐specific T cells activated by PDC, a cytokine pattern similar to that described for Th1‐like regulatory cells. Introduction of CpG oligodeoxynucleotides in PDC/T‐cell co‐cultures had synergistic effect on virus‐dependent IFN‐γ production, whereas the other cytokines remained unchanged. Together, our results show that the type of DC involved in reactivation of previously primed T cells may have significant impact on the resulting cytokine response and suggest that targeting of viral antigens and adjuvant to specific DC subsets should be considered in the design of therapeutic antiviral vaccines.     

Alloreactive regulatory T cells generated with retinoic acid prevent skin allograft rejection

CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells mediate immunological self‐tolerance and suppress immune responses. Retinoic acid (RA), a natural metabolite of vitamin A, has been reported to enhance the differentiation of Treg cells in the presence of TGF‐β. In this study, we show that the co‐culture of naive T cells from C57BL/6 mice with allogeneic antigen‐presenting cells (APCs) from BALB/c mice in the presence of TGF‐β, RA, and IL‐2 resulted in a striking enrichment of Foxp3⁺ T cells. These RA in vitro‐induced regulatory T (RA‐iTreg) cells did not secrete Th1‐, Th2‐, or Th17‐related cytokines, showed a nonbiased homing potential, and expressed several cell surface molecules related to Treg‐cell suppressive potential. Accordingly, these RA‐iTreg cells suppressed T‐cell proliferation and inhibited cytokine production by T cells in in vitro assays. Moreover, following adoptive transfer, RA‐iTreg cells maintained Foxp3 expression and their suppressive capacity. Finally, RA‐iTreg cells showed alloantigen‐specific immunosuppressive capacity in a skin allograft model in immunodeficient mice. Altogether, these data indicate that functional and stable allogeneic‐specific Treg cells may be generated using TGF‐β, RA, and IL‐2. Thus, RA‐iTreg cells may have a potential use in the development of more effective cellular therapies in clinical transplantation.