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.     

L‐Arginine deprivation impairs Leishmania major‐specific T‐cell responses

The amino acid L ‐arginine plays a crucial role in the regulation of immune responses. We have recently shown that uncontrolled replication of Leishmania parasites at the site of pathology correlates with high levels of arginase activity in nonhealing leishmaniasis and that this elevated arginase activity causes local depletion of L ‐arginine. To further our understanding of the impact of L ‐arginine deprivation in experimental leishmaniasis, here we characterize in detail the effects of L ‐arginine deprivation on antigen‐specific T cells and MΦ. The results of our study show that decrease of L ‐arginine levels in the extracellular milieu affects the biological activities of Leishmania major‐specific T cells, both at the level of the magnitude and the quality of their responses. L. major‐specific CD4⁺ T cells rendered hyporesponsive by L ‐arginine deprivation can be partially rescued by addition of exogenous L ‐arginine to produce IL‐4 and IL‐10, but not to produce IFN‐γ. Furthermore, our results show that L ‐arginine deprivation also greatly impacts parasite growth in activated macrophages. In summary, our results suggest that L ‐arginine levels affect both Th cell responses and parasite replication.     

Arginase‐1 is neither constitutively expressed in nor required for myeloid‐derived suppressor cell‐mediated inhibition of T‐cell proliferation

Although previous reports suggest that tumor‐induced myeloid‐derived suppressor cells (MDSC) inhibit T cells by L‐arginine depletion through arginase‐1 activity, we herein show that arginase‐1 is neither inherently expressed in MDSC nor required for MDSC‐mediated inhibition. Employing Percoll density gradients, large expansions of MDSC in the bone marrow of tumor‐bearing mice were isolated and demonstrated potent inhibition in T‐cell proliferation activated by TCR‐ligation, Concanavalin A, PMA plus ionomycin, or IL‐2. Despite demonstrating characteristic immunosuppressive capacity, these MDSC exhibit no arginase‐1 expression and/or exert their inhibitory effects independent of arginase‐1 activity. However, arginase‐1 expression in MDSC can be induced by exposure to TCR‐activated T cells or their culture medium, but not T cells activated by other means or growing tumor cells. Further investigation reveals multiple cytokines secreted by TCR‐activated T cells as orchestrating two signaling‐relay axes, IL‐6‐to‐IL‐4 and GM‐CSF/IL‐4‐to‐IL‐10, leading to arginase‐1 expression in MDSC. Specifically, IL‐6 signaling increases IL‐4R, enabling IL‐4 to induce arginase‐1 expression; similarly, GM‐CSF in concert with IL‐4 induces IL‐10R, allowing IL‐10‐mediated induction. Surprisingly, our study indicates that induction of arginase‐1 expression is not conducive to the critical MDSC‐mediated inhibition toward T cells, which is rather dependent on direct cell contacts undiminished by PD‐L1 blockade or SIRPα deficiency.     

The IL‐13/IL‐4Rα axis is involved in tuberculosis‐associated pathology

Human tuberculosis (TB) is a leading global health threat and still constitutes a major medical challenge. However, mechanisms governing tissue pathology during post‐primary TB remain elusive, partly because genetically or immunologically tractable animal models are lacking. In human TB, the demonstration of a large relative increase in interleukin (IL)‐4 and IL‐13 expression, which correlates with lung damage, indicates that a subversive T helper (TH)2 component in the response to Mycobacterium tuberculosis (Mtb) may undermine protective immunity and contribute to reactivation and tissue pathology. Up to now, there has been no clear evidence regarding whether IL‐4/IL‐13‐IL‐4 receptor‐α (Rα)‐mediated mechanisms may in fact cause reactivation and pathology. Unfortunately, the virtual absence of centrally necrotizing granulomas in experimental murine TB is associated with a poor induction of a TH2 immune response. We therefore hypothesize that, in mice, an increased production of IL‐13 may lead to a pathology similar to human post‐primary TB. In our study, aerosol Mtb infection of IL‐13‐over‐expressing mice in fact resulted in pulmonary centrally necrotizing granulomas with multinucleated giant cells, a hypoxic rim and a perinecrotic collagen capsule, with an adjacent zone of lipid‐rich, acid‐fast bacilli‐containing foamy macrophages, thus strongly resembling the pathology in human post‐primary TB. Granuloma necrosis (GN) in Mtb‐infected IL‐13‐over‐expressing mice was associated with the induction of arginase‐1‐expressing macrophages. Indirect blockade of the endogenous arginase inhibitor l ‐hydroxyarginine in Mtb‐infected wild‐type mice resulted in a strong arginase expression and precipitated a similar pathology of GN. Together, we here introduce an experimental TB model that displays many features of centrally necrotizing granulomas in human post‐primary TB and demonstrate that IL‐13/IL‐4Rα‐dependent mechanisms leading to arginase‐1 expression are involved in TB‐associated tissue pathology. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Tumor‐mediated down‐regulation of MHC class II in DC development is attributable to the epigenetic control of the CIITA type I promoter

In patients with cancer, DC express significantly lower amounts of MHC class II compared with those of normal individuals. However, the underlying mechanisms for this have not yet been fully defined. In the present study, we found that IL‐10 plays a major role in the tumor‐conditioned medium (TCM)‐mediated decrease of MHC class II expression on BM‐derived DC. IL‐10 inhibited the expression of type I CIITA during DC differentiation. GM‐CSF‐mediated histone (H3 and H4) acetylation at the type I promoter (pI) locus of the CIITA gene was markedly increased during DC differentiation and this increase was blocked by IL‐10. We also found that STAT5 bound to the CIITA pI locus during DC differentiation, and the binding was markedly attenuated by TCM or IL‐10. Altogether, these findings suggest that (i) the down‐regulation of MHC class II in tumor microenvironments is most likely attributable to IL‐10 in the TCM and (ii) IL‐10‐mediated MHC class II down‐regulation results from the inhibition of type I CIITA expression. This inhibition is most likely due to blocking of the STAT5‐associated epigenetic modifications of the CIITA pI locus during the entire period of DC differentiation from BM cells, as opposed to a simple inhibition of MHC class II expression at the DC stage.     

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.     

DLK1 is a novel inflammatory inhibitor which interferes with NOTCH1 signaling in TLR‐activated murine macrophages

Delta‐like protein 1 (DLK1) is a noncanonical ligand that inhibits NOTCH1 receptor activity and regulates multiple differentiation processes. In macrophages, NOTCH signaling increases TLR‐induced expression of key pro‐inflammatory mediators. We have investigated the role of DLK1 in macrophage activation and inflammation using Dlk1‐deficient mice and Raw 264.7 cells overexpressing Dlk1. In the absence of Dlk1, NOTCH1 expression is increased and the activation of macrophages with TLR3 or TLR4 agonists leads to higher production of IFN‐β and other pro‐inflammatory cytokines, including TNF‐α, IL‐12, and IL‐23. The expression of key proteins involved in IFN‐β signaling, such as IRF3, IRF7, IRF1, or STAT1, as well as cRel, or RelB, which are responsible for the generation of IL‐12 and IL‐23, is enhanced in Dlk1 KO macrophages. Consistently, Dlk1 KO mice are more sensitive to LPS‐induced endotoxic shock. These effects seem to be mediated through the modulation of NOTCH1 signaling. TLR4 activation reduces DLK1 expression, whereas increases NOTCH1 levels. In addition, DLK1 expression diminishes during differentiation of human U937 cells to macrophages. Overall, these results reveal a novel role for DLK1 as a regulator of NOTCH‐mediated, pro‐inflammatory macrophage activation, which could help to ensure a baseline level preventing constant tissue inflammation.     

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 blood vessel formation in tumors by IL‐18‐polarized M1 macrophages

We previously showed that interleukin (IL)‐18 produced by NFSA cells induced the M1 type of macrophages in NFSA tumors, caused the destruction of endothelial cells in vitro and may have resulted in the necrosis of NFSA tumors by enhancing macrophage phagocytosis and cytotoxicity. However, the effect of IL‐18 on blood vessel formation in vivo has not been elucidated. MS‐K cells do not express il‐18, and they form tumors with well‐developed blood vessels. Here, we established IL‐18‐over‐expressing MS‐K cell clones (MS‐K‐IL‐18) to address the roles of IL‐18 in angiogenesis. The over‐expression of IL‐18 inhibited the proliferation rate of the MS‐K‐IL‐18 cells in vitro and blood vessel formation in the MS‐K‐IL‐18 tumors. Interestingly, CD14‐positive cells from the MS‐K‐IL‐18 tumor had up‐regulated expression of the M1‐type macrophage marker il‐6 and down‐regulated expression of interferon (ifn)‐γ. Furthermore, FACS analysis showed more accumulation of CD11b+/CD80+ M1 macrophages in the MS‐K‐IL‐18 tumors than in the parental MS‐K tumor. Moreover, an in vitro coculture assay showed that MS‐K‐IL‐18‐conditioned medium (CM) stimulated macrophages to induce the apoptosis of endothelial cells. Cumulatively, our data showed that IL‐18 inhibited tumor blood vessel formation in vivo.     

Phosphorylated STAT3 and PD‐1 regulate IL‐17 production and IL‐23 receptor expression in Mycobacterium tuberculosis infection

We studied the factors that regulate IL‐23 receptor expression and IL‐17 production in human tuberculosis infection. Mycobacterium tuberculosis (M. tb)‐stimulated CD4⁺ T cells from tuberculosis patients secreted less IL‐17 than did CD4⁺ T cells from healthy tuberculin reactors (PPD⁺). M. tb‐cultured monocytes from tuberculosis patients and PPD⁺ donors expressed equal amounts of IL‐23p19 mRNA and protein, suggesting that reduced IL‐23 production is not responsible for decreased IL‐17 production by tuberculosis patients. Freshly isolated and M. tb‐stimulated CD4⁺ T cells from tuberculosis patients had reduced IL‐23 receptor and phosphorylated STAT3 (pSTAT3) expression, compared with cells from PPD⁺ donors. STAT3 siRNA reduced IL‐23 receptor expression and IL‐17 production by CD4⁺ T cells from PPD⁺ donors. Tuberculosis patients had increased numbers of PD‐1⁺ T cells compared with healthy PPD⁺ individuals. Anti‐PD‐1 antibody enhanced pSTAT3 and IL‐23R expression and IL‐17 production by M. tb‐cultured CD4⁺ T cells of tuberculosis patients. Anti‐tuberculosis therapy decreased PD‐1 expression, increased IL‐17 and IFN‐γ production and pSTAT3 and IL‐23R expression. These findings demonstrate that increased PD‐1 expression and decreased pSTAT3 expression reduce IL‐23 receptor expression and IL‐17 production by CD4⁺ T cells of tuberculosis patients.     

Reciprocal modulation of C/EBP‐α and C/EBP‐β by IL‐13 in activated microglia prevents neuronal death

In response to aggravation by activated microglia, IL‐13 can significantly enhance ER stress induction, apoptosis, and death via reciprocal signaling through CCAAT/enhancer‐binding protein alpha (C/EBP‐α) and C/EBP‐beta (C/EBP‐β). This reciprocal signaling promotes neuronal survival. Since the induction of cyclooxygenase‐2 (COX‐2) and peroxisome proliferator‐activated receptor gamma/heme oxygenase 1 (PPAR‐γ/HO‐1) by IL‐13 plays a crucial role in the promotion of and protection from activated microglia, respectively; here, we investigated the role of IL‐13 in regulating C/EBPs in activated microglia and determined its correlation with neuronal function. The results revealed that IL‐13 significantly enhanced C/EBP‐α/COX‐2 expression and PGE₂ production in LPS‐treated microglial cells. Paradoxically, IL‐13 abolished C/EBP‐β/PPAR‐γ/HO‐1 expression. IL‐13 also enhanced ER stress‐evoked calpain activation by promoting the association of C/EBP‐β and PPAR‐γ. SiRNA‐C/EBP‐α effectively reversed the combined LPS‐activated caspase‐12 activation and IL‐13‐induced apoptosis. In contrast, siRNA‐C/EBP‐β partially increased microglial cell apoptosis. By NeuN immunochemistry and CD11b staining, there was improvement in the loss of CA3 neuronal cells after intrahippocampal injection of IL‐13. This suggests that IL‐13‐enhanced PLA2 activity regulates COX‐2/PGE₂ expression through C/EBP‐α activation. In parallel, ER stress‐related calpain downregulates the PPAR‐γ/HO‐1 pathway via C/EBP‐β and leads to aggravated death of activated microglia via IL‐13, thereby preventing cerebral inflammation and neuronal injury.