α‐GalCer ameliorates listeriosis by accelerating infiltration of Gr‐1+ cells into the liver

α‐Galactosylceramide (α‐GalCer) activates invariant (i)NKT cells, which in turn stimulate immunocompetent cells. Although activation of iNKT cells appears critical for regulation of immune responses, it remains elusive whether protection against intracellular bacteria can be induced by α‐GalCer. Here, we show that α‐GalCer treatment ameliorates murine listeriosis, and inhibits inflammation following Listeria monocytogenes infection. Liver infiltration of Gr‐1⁺ cells and γ/δ T cells was accelerated by α‐GalCer treatment. Gr‐1⁺ cell and γ/δ T‐cell depletion exacerbated listeriosis in α‐GalCer‐treated mice, and this effect was more pronounced after depletion of Gr‐1⁺ cells than that of γ/δ T cells. Although GM‐CSF and IL‐17 were secreted by NKT cells after α‐GalCer treatment, liver infiltration of Gr‐1⁺ cells was not prevented by neutralizing mAb. In parallel to the numerical increase of CD11b⁺Gr‐1⁺ cells in the liver following α‐GalCer treatment, CD11b^?Gr‐1⁺ cells were numerically reduced in the bone marrow. In addition, respiratory burst in Gr‐1⁺ cells was enhanced by α‐GalCer treatment. Our results indicate that α‐GalCer‐induced antibacterial immunity is caused, in part, by accelerated infiltration of Gr‐1⁺ cells and to a lesser degree of γ/δ T cells into the liver. We also suggest that the infiltration of Gr‐1⁺ cells is caused by an accelerated supply from the bone marrow.     

Tumor‐induced CD11b+ Gr‐1+ myeloid‐derived suppressor cells exacerbate immune‐mediated hepatitis in mice in a CD40‐dependent manner

Immunosuppressive CD11b⁺Gr‐1⁺ myeloid‐derived suppressor cells (MDSCs) accumulate in the livers of tumor‐bearing (TB) mice. We studied hepatic MDSCs in two murine models of immune‐mediated hepatitis. Unexpectedly, treatment of TB mice with Concanavalin A (Con A) or α‐galactosylceramide resulted in increased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels in comparison to tumor‐free mice. Adoptive transfer of hepatic MDSCs into naïve mice exacerbated Con A induced liver damage. Hepatic CD11b⁺Gr‐1⁺ cells revealed a polarized proinflammatory gene signature after Con A treatment. An IFN‐γ‐dependent upregulation of CD40 on hepatic CD11b⁺Gr‐1⁺ cells along with an upregulation of CD80, CD86, and CD1d after Con A treatment was observed. Con A treatment resulted in a loss of suppressor function by tumor‐induced CD11b⁺Gr‐1⁺ MDSCs as well as enhanced reactive oxygen species (ROS)‐mediated hepatotoxicity. CD40 knockdown in hepatic MDSCs led to increased arginase activity upon Con A treatment and lower ALT/AST serum levels. Finally, blockade of arginase activity in Cd40^?/? tumor‐induced myeloid cells resulted in exacerbation of hepatitis and increased ROS production in vivo. Our findings indicate that in a setting of acute hepatitis, tumor‐induced hepatic MDSCs act as proinflammatory immune effector cells capable of killing hepatocytes in a CD40‐dependent manner.     

IFN‐γ regulates survival and function of tumor‐induced CD11b+Gr‐1high myeloid derived suppressor cells by modulating the anti‐apoptotic molecule Bcl2a1

Myeloid derived suppressor cells (MDSCs) play a critical role in suppression of immune responses in cancer and inflammation. Here, we describe how regulation of Bcl2a1 by cytokines controls the suppressor function of CD11b⁺Gr‐1^high granulocytic MDSCs. Coculture of CD11b⁺Gr‐1^high granulocytic MDSCs with antigen‐stimulated T cells and simultaneous blockade of IFN‐γ by the use of anti‐IFN‐γ blocking antibody, IFN‐γ^?/? effector T cells, IFN‐γR^?/? MDSCs or STAT1^?/? MDSCs led to upregulation of Bcl2a1 in CD11b⁺Gr‐1^high cells, improved survival, and enhanced their suppressor function. Molecular studies revealed that GM‐CSF released by antigen‐stimulated CD8⁺ T cells induced Bcl2a1 upregulation, which was repressed in the presence of IFN‐γ by a direct interaction of phosphorylated STAT‐1 with the Bcl2a1 promotor. Bcl2a1 overexpressing granulocytic MDSCs demonstrated prolonged survival and enhanced suppressor function in vitro. Our data suggest that IFN‐γ/ STAT1‐dependent regulation of Bcl2a1 regulates survival and thereby suppressor function of granulocytic MDSCs.     

IL‐1α induces CD11blow alveolar macrophage proliferation and maturation during granuloma formation

Macrophages play a central role in immune and tissue responses of granulomatous lung diseases induced by pathogens and foreign bodies. Circulating monocytes are generally viewed as central precursors of these tissue effector macrophages. Here, we provide evidence that granulomas derive from alveolar macrophages serving as a local reservoir for the expansion of activated phagocytic macrophages. By exploring lung granulomatous responses to silica particles in IL‐1‐deficient mice, we found that the absence of IL‐1α, but not IL‐1β, was associated with reduced CD11b^high phagocytic macrophage accumulation and fewer granulomas. This defect was associated with impaired alveolar clearance and resulted in the development of pulmonary alveolar proteinosis (PAP). Reconstitution of IL‐1α^?/? mice with recombinant IL‐1α restored lung clearance functions and the pulmonary accumulation of CD11b^high phagocytic macrophages. Mechanistically, IL‐1α induced the proliferation of CD11b^low alveolar macrophages and differentiated these cells into CD11b^high macrophages which perform critical phagocytic functions and organize granuloma. We newly discovered here that IL‐1α triggers lung responses requiring macrophage proliferation and maturation from tissue‐resident macrophages. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

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.     

IL‐15 modulates the balance between Bcl‐2 and Bim via a Jak3/1‐PI3K‐Akt‐ERK pathway to promote CD8αα+ intestinal intraepithelial lymphocyte survival

IL‐15 is an essential survival factor for CD8αα⁺ intestinal intraepithelial lymphocytes (iIELs) in vitro and in vivo. However, the IL‐15‐induced survival signals in primary CD8αα⁺ iIELs remains elusive. Although Bcl‐2 level in CD8αα⁺ iIELs positively correlates with IL‐15Rα expression in the intestinal epithelial cells, overexpression of Bcl‐2 only moderately restores CD8αα⁺ γδ iIELs in Il15^?/? mice. Here, we found that IL‐15 promptly activated a Jak3‐Jak1‐PI3K‐Akt pathway that led to the upregulation of Bcl‐2 and Mcl‐1. This pathway also induced a delayed but sustained ERK1/2 activation, which not only was necessary for the maintenance of Bcl‐2 but also resulted in the phosphorylation of extra‐long Bim at Ser⁶⁵. The latter event facilitated the dissociation of Bim from Bcl‐2 without affecting Bim abundance in IL‐15‐treated CD8αα⁺ iIELs. Using an adoptive cell transfer approach, we found that either overexpression of Bcl‐2 or removal of Bim from CD8αα⁺ iIELs promoted their survival in Il15ra^?/? mice. Taken together, IL‐15 promotes CD8αα⁺ iIEL survival by both increasing Bcl‐2 levels and dissociating Bim from Bcl‐2 through activation of a Jak3‐Jak1‐PI3K‐Akt‐ERK1/2 pathway, which differs from a previously reported IL‐15‐induced survival signal.     

Cytokine‐dependent regulation of dendritic cell differentiation in the splenic microenvironment

The DC‐derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady‐state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α^?CD11b⁺ LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α‐galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α^? and CD8α⁺ splenic DC subsets and enhances cross‐presentation of exogenous antigens. Based on genome‐wide expression profiling, we now show that splenic CD11b⁺ DCs are susceptible to IFN‐γ‐mediated suppression of CCL17, whereas LN CD11b⁺CCL17⁺ DCs downregulate the IFN‐γR and are much less responsive to IFN‐γ. Under inflammatory conditions, particularly in the absence of IFN‐γ signaling in IFN‐γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM‐CSF in concert with IL‐4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression.     

IL‐18Rα‐deficient CD4+ T cells induce intestinal inflammation in the CD45RBhi transfer model of colitis despite impaired innate responsiveness

IL‐18 has been implicated in inflammatory bowel disease (IBD), however its role in the regulation of intestinal CD4⁺ T‐cell function remains unclear. Here we show that murine intestinal CD4⁺ T cells express high levels of IL‐18Rα and provide evidence that IL‐18Rα expression is induced on these cells subsequent to their entry into the intestinal mucosa. Using the CD45RB^hi T‐cell transfer colitis model, we show that IL‐18Rα is expressed on IFN‐γ⁺, IL‐17⁺, and IL‐17⁺IFN‐γ⁺ effector CD4⁺ T cells in the inflamed colonic lamina propria (cLP) and mesenteric lymph node (MLN) and is required for the optimal generation and/or maintenance of IFN‐γ‐producing cells in the cLP. In the steady state and during colitis, TCR‐independent cytokine‐induced IFN‐γ and IL‐17 production by intestinal CD4⁺ T cells was largely IL‐18Rα?dependent. Despite these findings however, IL‐18Rα?deficient CD4⁺ T cells induced comparable intestinal pathology to WT CD4⁺ T cells. These findings suggest that IL‐18‐dependent cytokine induced activation of CD4⁺ T cells is not critical for the development of T‐cell‐mediated colitis.     

T‐cell receptor activation of human CD4+ T cells shifts the innate TLR response from CXCL8hiIFN‐γnull to CXCL8loIFN‐γhi

Toll‐like receptors (TLRs) play a major part in providing innate immunity against pathogenic microorganisms. Recent studies show that these receptors are also expressed on T cells, which are the sentinels of adaptive immunity. Here, we have investigated the regulatory role of the T‐cell receptor in the functioning of these innate receptors in T cells. We show that freshly isolated human CD4⁺ T cells readily secrete the neutrophil chemoattractant CXCL8 upon activation with the TLR ligands Pam3CSK and flagellin. In contrast, TCR‐activated cells secrete considerably less CXCL8 but start producing IFN‐γ upon stimulation with TLR agonists in the absence of concomitant TCR engagement. These T cells show increased activation of p38 and JNK MAP‐kinases in response to TLR stimulation, and inhibition of p38 abrogates TLR‐induced IFN‐γ secretion. The shifting of the T‐cell innate immune response from CXCL8^hiIFN‐γ^null in freshly isolated to CXCL8^loIFN‐γ^hi in activated T cells is also observed in response to endogenous innate stimulus, IL‐1. These results suggest that the innate immune response of human CD4⁺ T cells switches from a proinflammatory to an effector type following activation of these cells through the antigen receptor.     

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.     

Type I interferon potentiates T‐cell receptor mediated induction of IL‐10‐producing CD4+ T cells

Type I interferons (IFNs) have the dual ability to promote the development of the immune response and exert an anti‐inflammatory activity. We analyzed the integrated effect of IFN‐α, TCR signal strength, and CD28 costimulation on human CD4⁺ T‐cell differentiation into cell subsets producing the anti‐ and proinflammatory cytokines IL‐10 and IFN‐γ. We show that IFN‐α boosted TCR‐induced IL‐10 expression in activated peripheral CD45RA⁺CD4⁺ T cells and in whole blood cultures. The functional cooperation between TCR and IFN‐α efficiently occurred at low engagement of receptors. Moreover, IFN‐α rapidly cooperated with anti‐CD3 stimulation alone. IFN‐α, but not IL‐10, drove the early development of type I regulatory T cells that were mostly IL‐10⁺ Foxp3^? IFN‐γ^? and favored IL‐10 expression in a fraction of Foxp3⁺ T cells. Our data support a model in which IFN‐α costimulates TCR toward the production of IL‐10 whose level can be amplified via an autocrine feedback loop.     

Differential effects of stromal derived factor‐1α (SDF‐1α) on early and late stages of human megakaryocytic development

Stromal derived factor‐1α (SDF‐1α), the high‐affinity ligand of CXC‐chemokine receptor 4 (CXCR4), was added to human CD34⁺ hematopoietic progenitor cells that can be induced to differentiate along the monocytic or megakaryocytic lineages. In control liquid cell cultures supplemented with two different cytokine cocktails: stem cell factor (SCF), interleukin‐3 (IL‐3), macrophage‐colony stimulating factor (M‐CSF), and 10% fetal calf serum (FCS), or, SCF and thrombopoietin (TPO), the expression of surface CXCR4 progressively increased in both the CD14⁺ monocytic and CD41⁺ megakaryocytic lineages. While SDF‐1α caused only modest effects on cells of the monocytic lineage, it induced profound down‐regulation of CXCR4 in megakaryocytic cells at all stages of differentiation. Moreover, while SDF‐1α initially up‐regulated the early megakaryocytic antigen CD41, at later time points (days 12–16) it induced down‐regulation of the late megakaryocytic antigen CD42b. Consistently, at day 16, the number of mature megakaryocytes was significantly decreased in cultures supplemented with SDF‐1α. These findings indicate that, besides its primary role in regulating the retention of precursor cells in hematopoietic tissues, the SDF‐1α/CXCR4 system participates in the regulation of megakaryocytic development by stimulating the formation of immature megakaryoblasts and inhibiting the formation of mature megakaryocytes. Anat Rec 260:141–147, 2000. © 2000 Wiley‐Liss, Inc.     

TGF‐β induces the differentiation of human CXCL13‐producing CD4+ T cells

In the ectopic lymphoid‐like structures present in chronic inflammatory conditions such as rheumatoid arthritis, a subset of human effector memory CD4⁺ T cells that lacks features of follicular helper T (Tfh) cells produces CXCL13. Here, we report that TGF‐β induces the differentiation of human CXCL13‐producing CD4⁺ T cells from naïve CD4⁺ T cells. The TGF‐β‐induced CXCL13‐producing CD4⁺ T cells do not express CXCR5, B‐cell lymphoma 6 (BCL6), and other Tfh‐cell markers. Furthermore, expression levels of CD25 (IL‐2Rα) in CXCL13‐producing CD4⁺ T cells are significantly lower than those in FoxP3⁺ in vitro induced Treg cells. Consistent with this, neutralization of IL‐2 and knockdown of STAT5 clearly upregulate CXCL13 production by CD4⁺ T cells, while downregulating the expression of FoxP3. Furthermore, overexpression of FoxP3 in naïve CD4⁺ T cells downregulates CXCL13 production, and knockdown of FoxP3 fails to inhibit the differentiation of CXCL13‐producing CD4⁺ T cells. As reported in rheumatoid arthritis, proinflammatory cytokines enhance secondary CXCL13 production from reactivated CXCL13‐producing CD4⁺ T cells. Our findings demonstrate that CXCL13‐producing CD4⁺ T cells lacking Tfh‐cell features differentiate via TGF‐β signaling but not via FoxP3, and exert their function in IL‐2‐limited but TGF‐β‐rich and proinflammatory cytokine‐rich inflammatory conditions.     

IL‐17‐producing γδ T cells

IL‐17 is produced not only by CD4⁺ αβ T cells, but also CD8⁺ αβ T cells, NKT cells, and γδ T cells, plus some non‐T cells, including macrophages and neutrophils. The ability of IL‐17 to deploy neutrophils to sites of inflammation imparts this cytokine with a key role in diseases of several types. Surprisingly, γδ T cells are responsible for much of the IL‐17 produced in several disease models, particularly early on.     

Adipose tissue derived stromal stem cell therapy in murine ConA‐derived hepatitis is dependent on myeloid‐lineage and CD4+ T‐cell suppression

Mesenchymal stromal stem cells (MSCs) are an attractive therapeutic model for regenerative medicine due to their pluripotency. MSCs are used as a treatment for several inflammatory diseases, including hepatitis. However, the detailed immunopathological impact of MSC treatment on liver disease, particularly for adipose tissue derived stromal stem cells (ADSCs), has not been described. Here, we investigated the immuno‐modulatory effect of ADSCs on hepatitis using an acute ConA C57BL/6 murine hepatitis model. i.v. administration of ADSCs simultaneously or 3 h post injection prevented and treated ConA‐induced hepatitis. Immunohistochemical analysis revealed higher numbers of CD11b⁺, Gr‐1⁺, and F4/80⁺ cells in the liver of ConA‐induced hepatitis mice was ameliorated after the administration of ADSCs. Hepatic expression of genes affected by ADSC administration indicated tissue regeneration‐related biological processes, affecting myeloid‐lineage immune‐mediating Gr‐1⁺ and CD11b⁺ cells. Pathway analysis of the genes expressed in ADSC‐treated hepatic inflammatory cells revealed the possible involvement of T cells and macrophages. TNF‐α and IFN‐γ expression was downregulated in hepatic CD4⁺ T cells isolated from hepatitis livers co‐cultured with ADSCs. Thus, the immunosuppressive effect of ADSCs in a C57BL/6 murine ConA hepatitis model was dependent primarily on the suppression of myeloid‐lineage cells and, in part, of CD4⁺ T cells.     

CD11c+ macrophages and levels of TNF‐α and MMP‐3 are increased in synovial and adipose tissues of osteoarthritic mice with hyperlipidaemia

To understand more clearly the link between osteoarthritis and hyperlipidaemia, we investigated the inflammatory macrophage subsets and macrophage‐regulated matrix metalloprotease‐3 (MMP‐3) and A disintegrin and metalloprotease with thrombospondin motifs‐4 (ADAMTS4) in synovial (ST) and adipose tissues (AT) of osteoarthritic mice with hyperlipidaemia (STR/Ort). CD11c⁺F4/80⁺CD11b⁺ macrophage populations in the ST and AT of 9‐month‐old STR/Ort and C57BL/6J mice were characterized and compared by flow cytometry and real‐time polymerase chain reaction (PCR) analyses. Expression of tumour necrosis factor (TNF)‐α, MMP‐3 and ADAMTS4, and the response of these factors to anionic liposomal clodronate induced‐macrophage depletion were also evaluated by real‐time PCR. Expression of TNF‐α in CD11c⁺ cells, which were isolated by magnetic beads, was compared to CD11c^– cells. In addition, the effect of TNF‐α on cultured synovial fibroblasts and adipocytes was investigated. CD11c⁺F4/80⁺CD11b⁺ macrophages were increased in ST and AT of STR/Ort mice. The CD11c⁺ cell fraction highly expressed TNF‐α. Expression of TNF‐α and MMP3 was increased in ST and AT, and was decreased upon macrophage depletion. TNF‐α treatment of cultured synovial fibroblasts and adipocytes markedly up‐regulated MMP‐3. CD11c⁺F4/80⁺CD11b⁺ macrophages were identified as a common inflammatory subset in the AT and ST of STR/Ort mice with hyperlipidaemia. The induction of inflammation in AT and ST may be part of a common mechanism that regulates MMP3 expression through TNF‐α. Our findings suggest that increased numbers of CD11c⁺ macrophages and elevated levels of TNF‐α and MMP‐3 in AT and ST may explain the relationship between hyperlipidaemia and OA.     

Recruitment of hepatic macrophages from monocytes is independent of IL‐4Rα but is associated with ablation of resident macrophages in schistosomiasis

Alternatively activated Mφs (AAMφ) accumulate in hepatic granulomas during schistosomiasis and have been suggested to originate in the bone marrow. What is less understood is how these Mφ responses are regulated after S. mansoni infection. Here, we investigated the role of IL‐4 receptor α‐chain (IL‐4Rα)‐signalling in the dynamics of liver Mφ responses. We observed that IL‐4Rα signalling was dispensable for the recruitment of Ly6C^hi monocytes and for their conversion into F4/80^hiCD64^hiCD11b^hiMφ. Moreover, while IL‐4Rα provided an AAMφ phenotype to liver F4/80^hiCD64^hiCD11b^hiMφ that was associated with regulation of granuloma formation, it was dispensable for host survival. Resident F4/80^hiCD64^hiCD11b^loMφ did not upregulate the AAMφ signature gene Ym1. Rather, resident Mφ nearly disappeared by week 8 after infection and artificial ablation of resident Mφ in CD169^DTR mice did not affect the response to S. mansoni infection. Interestingly, ablation of CD169⁺ cells in naive mice resulted in the accumulation of F4/80^hiCD64^hiCD11b^hiMφ, which was amplified when ablation occurred during schistosomiasis. Altogether, our results suggest the ablation of resident KCs after S. mansoni infection to be associated with the recruitment and accumulation of F4/80^hiCD64^hiCD11b^hiMφ with lyz2‐dependent IL‐4Rα contributing to the regulation of granuloma inflammation but being dispensable for host survival.