Yannick Morias

Tumor Hypoxia Does Not Drive Differentiation of Tumor-Associated Macrophages but Rather Fine-Tunes the M2-like Macrophage Population

Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11b(hi)F4/80(hi)Ly6C(lo) TAM subsets, designated as MHC-II(lo) and MHC-II(hi) TAM, both of which were derived from tumor-infiltrating Ly6C(hi) monocytes. MHC-II(lo) TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II(lo) TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II(hi) counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (PHD2)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-II(lo) TAM subset. The same observation in PHD2(+/+) → PHD2(+/-) bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II(lo) TAM.

Tip-DC Development during Parasitic Infection Is Regulated by IL-10 and Requires CCL2/CCR2, IFN-γ and MyD88 Signaling

The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent activation of M1 that produce pathogenic molecules such as TNF and NO contributes to the development of trypanosome infection-associated tissue injury including liver cell necrosis in experimental mouse models. Aiming to identify mechanisms involved in regulation of M1 activity, we have recently documented that during Trypanosoma brucei infection, CD11b+Ly6C+CD11c+ TNF and iNOS producing DCs (Tip-DCs) represent the major pathogenic M1 liver subpopulation. By using gene expression analyses, KO mice and cytokine neutralizing antibodies, we show here that the conversion of CD11b+Ly6C+ monocytic cells to pathogenic Tip-DCs in the liver of T. brucei infected mice consists of a three-step process including (i) a CCR2-dependent but CCR5- and Mif-independent step crucial for emigration of CD11b+Ly6C+ monocytic cells from the bone marrow but dispensable for their blood to liver migration; (ii) a differentiation step of liver CD11b+Ly6C+ monocytic cells to immature inflammatory DCs (CD11c+ but CD80/CD86/MHC-IIlow) which is IFN-γ and MyD88 signaling independent; and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-IIhigh) TNF and NO producing Tip-DCs which is IFN-γ and MyD88 signaling dependent. Moreover, IL-10 could limit CCR2-mediated egression of CD11b+Ly6C+ monocytic cells from the bone marrow by limiting Ccl2 expression by liver monocytic cells, as well as their differentiation and maturation to Tip-DCs in the liver, showing that IL-10 works at multiple levels to dampen Tip-DC mediated pathogenicity during T. brucei infection. A wide spectrum of liver diseases associates with alteration of monocyte recruitment, phenotype or function, which could be modulated by IL-10. Therefore, investigating the contribution of recruited monocytes to African trypanosome induced liver injury could potentially identify new targets to treat hepatic inflammation in general, and during parasite infection in particular.

Adenylate Cyclases of Trypanosoma brucei Inhibit the Innate Immune Response of the Host

Tricky Tryps African trypanosomes, responsible for human sleeping sickness, are known for their powerful strategies of immune evasion, in particular antigenic variation. Adding another facet to this adaptive potential, Salmon et al. (p. 463, published online 14 June; see the cover) now show that early after infection, these parasites subvert the first line of innate host defense by inhibiting tumor necrosis factor-α synthesis in myeloid cells. This occurs through the stress-induced synthesis and release of cyclic adenosine monophosphate by phagocytosed parasites. The findings provide a long-sought function for the abundant and diverse adenylate cyclases in salivarian trypanosomes. Furthermore, this altruistic host colonization strategy, in which a proportion of parasites are sacrificed so that others can thrive, also highlights the selective advantage of population behavior in infection. Parasites release cyclic adenosine monophosphate when swallowed up by myeloid cells, thereby turning off a host defense pathway. The parasite Trypanosoma brucei possesses a large family of transmembrane receptor–like adenylate cyclases. Activation of these enzymes requires the dimerization of the catalytic domain and typically occurs under stress. Using a dominant-negative strategy, we found that reducing adenylate cyclase activity by about 50% allowed trypanosome growth but reduced the parasite’s ability to control the early innate immune defense of the host. Specifically, activation of trypanosome adenylate cyclase resulting from parasite phagocytosis by liver myeloid cells inhibited the synthesis of the trypanosome-controlling cytokine tumor necrosis factor–α through activation of protein kinase A in these cells. Thus, adenylate cyclase activity of lyzed trypanosomes favors early host colonization by live parasites. The role of adenylate cyclases at the host-parasite interface could explain the expansion and polymorphism of this gene family.

Mononuclear phagocyte heterogeneity in cancer: Different subsets and activation states reaching out at the tumor site

Mononuclear phagocytes are amongst the most versatile cells of the body, contributing to tissue genesis and homeostasis and safeguarding the balance between pro- and anti-inflammatory reactions. Accordingly, these cells are notoriously heterogeneous, functioning in distinct differentiation forms (monocytes, MDSC, macrophages, DC) and adopting different activation states in response to a changing microenvironment. Accumulating evidence exists that mononuclear phagocytes contribute to all phases of the cancer process. These cells orchestrate the inflammatory events during de novo carcinogenesis, participate in tumor immunosurveillance, and contribute to the progression of established tumors. At the tumor site, cells such as tumor-associated macrophages (TAM) are confronted with different tumor microenvironments, leading to TAM subsets with specialized functions. A better refinement of the molecular and functional heterogeneity of tumor-associated mononuclear phagocytes might pave the way for novel cancer therapies that directly target these tumor-supporting cells.

Pivotal Advance: Arginase-1-independent polyamine production stimulates the expression of IL-4-induced alternatively activated macrophage markers while inhibiting LPS-induced expression of inflammatory genes

In macrophages, basal polyamine (putrescine, spermidine, and spermine) levels are relatively low but are increased upon IL‐4 stimulation. This Th2 cytokine induces Arg1 activity, which converts arginine into ornithine, and ornithine can be decarboxylated by ODC to produce putrescine, which is further converted into spermidine and spermine. Recently, we proposed polyamines as novel agents in IL‐4‐dependent E‐cadherin regulation in AAMs. Here, we demonstrate for the first time that several, but not all, AAM markers depend on polyamines for their IL‐4‐induced gene and protein expression and that polyamine dependency of genes relies on the macrophage type. Remarkably, Arg1‐deficient macrophages display rather enhanced IL‐4‐induced polyamine production, suggesting that an Arg1‐independent polyamine synthesis pathway may operate in macrophages. On the other side of the macrophage activation spectrum, LPS‐induced expression of several proinflammatory genes was increased significantly in polyamine‐depleted CAMs. Overall, we propose Arg1 independently produced polyamines as novel regulators of the inflammatory status of the macrophage. Indeed, whereas polyamines are needed for IL‐4‐induced expression of several AAM mediators, they inhibit the LPS‐mediated expression of proinflammatory genes in CAMs.

M-CSF and GM-CSF Receptor Signaling Differentially Regulate Monocyte Maturation and Macrophage Polarization in the Tumor Microenvironment

Tumors contain a heterogeneous myeloid fraction comprised of discrete MHC-II(hi) and MHC-II(lo) tumor-associated macrophage (TAM) subpopulations that originate from Ly6C(hi) monocytes. However, the mechanisms regulating the abundance and phenotype of distinct TAM subsets remain unknown. Here, we investigated the role of macrophage colony-stimulating factor (M-CSF) in TAM differentiation and polarization in different mouse tumor models. We demonstrate that treatment of tumor-bearing mice with a blocking anti-M-CSFR monoclonal antibody resulted in a reduction of mature TAMs due to impaired recruitment, extravasation, proliferation, and maturation of their Ly6C(hi) monocytic precursors. M-CSFR signaling blockade shifted the MHC-II(lo)/MHC-II(hi) TAM balance in favor of the latter as observed by the preferential differentiation of Ly6C(hi) monocytes into MHC-II(hi) TAMs. In addition, the genetic and functional signatures of MHC-II(lo) TAMs were downregulated upon M-CSFR blockade, indicating that M-CSFR signaling shapes the MHC-II(lo) TAM phenotype. Conversely, granulocyte macrophage (GM)-CSFR had no effect on the mononuclear tumor infiltrate or relative abundance of TAM subsets. However, GM-CSFR signaling played an important role in fine-tuning the MHC-II(hi) phenotype. Overall, our data uncover the multifaceted and opposing roles of M-CSFR and GM-CSFR signaling in governing the phenotype of macrophage subsets in tumors, and provide new insight into the mechanism of action underlying M-CSFR blockade.

Tumor-induced myeloid-derived suppressor cell subsets exert either inhibitory or stimulatory effects on distinct CD8+ T-cell activation events.

Tumor growth coincides with an accumulation of myeloid‐derived suppressor cells (MDSCs), which exert immune suppression and which consist of two main subpopulations, known as monocytic (MO) CD11b+CD115+Ly6G−Ly6Chigh MDSCs and granulocytic CD11b+CD115−Ly6G+Ly6Cint polymorphonuclear (PMN)‐MDSCs. However, whether these distinct MDSC subsets hamper all aspects of early CD8+ T‐cell activation — including cytokine production, surface marker expression, survival, and cytotoxicity — is currently unclear. Here, employing an in vitro coculture system, we demonstrate that splenic MDSC subsets suppress antigen‐driven CD8+ T‐cell proliferation, but differ in their dependency on IFN‐γ, STAT‐1, IRF‐1, and NO to do so. Moreover, MO‐MDSC and PMN‐MDSCs diminish IL‐2 levels, but only MO‐MDSCs affect IL‐2Rα (CD25) expression and STAT‐5 signaling. Unexpectedly, however, both MDSC populations stimulate IFN‐γ production by CD8+ T cells on a per cell basis, illustrating that some T‐cell activation characteristics are actually stimulated by MDSCs. Conversely, MO‐MDSCs counteract the activation‐induced change in CD44, CD62L, CD162, and granzyme B expression, while promoting CD69 and Fas upregulation. Together, these effects result in an altered CD8+ T‐cell adhesiveness to the extracellular matrix and selectins, sensitivity to FasL‐mediated apoptosis, and cytotoxicity. Hence, MDSCs intricately influence different CD8+ T‐cell activation events in vitro, whereby some parameters are suppressed while others are stimulated.

The tumour microenvironment harbours ontogenically distinct dendritic cell populations with opposing effects on tumour immunity

Various steady state and inflamed tissues have been shown to contain a heterogeneous DC population consisting of developmentally distinct subsets, including cDC1s, cDC2s and monocyte-derived DCs, displaying differential functional specializations. The identification of functionally distinct tumour-associated DC (TADC) subpopulations could prove essential for the understanding of basic TADC biology and for envisaging targeted immunotherapies. We demonstrate that multiple mouse tumours as well as human tumours harbour ontogenically discrete TADC subsets. Monocyte-derived TADCs are prominent in tumour antigen uptake, but lack strong T-cell stimulatory capacity due to NO-mediated immunosuppression. Pre-cDC-derived TADCs have lymph node migratory potential, whereby cDC1s efficiently activate CD8+ T cells and cDC2s induce Th17 cells. Mice vaccinated with cDC2s displayed a reduced tumour growth accompanied by a reprogramming of pro-tumoural TAMs and a reduction of MDSCs, while cDC1 vaccination strongly induces anti-tumour CTLs. Our data might prove important for therapeutic interventions targeted at specific TADC subsets or their precursors.

A Trypanosoma brucei Kinesin Heavy Chain Promotes Parasite Growth by Triggering Host Arginase Activity

Background In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. Methodology/Principal findings By screening a cDNA library of T. brucei with an antibody neutralizing the arginase-inducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptor-dependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Rα-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1-mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. Conclusion A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

IL-10 limits production of pathogenic TNF by M1 myeloid cells through induction of nuclear NF-κB p50 member in Trypanosoma congolense infection-resistant C57BL/6 mice

A balance between parasite elimination and control of infection‐associated pathogenicity is crucial for resistance to African trypanosomiasis. By producing TNF and NO, CD11b+ myeloid cells with a classical activation status (M1) contribute to parasitemia control in experimental Trypanosoma congolense infection in resistant C57BL/6 mice. However, in these mice, IL‐10 is required to regulate M1‐associated inflammation, avoiding tissue/liver damage and ensuring prolonged survival. In an effort to dissect the mechanisms behind the anti‐inflammatory activity of IL‐10 in T. congolense‐infected C57BL/6 mice, we show, using an antibody blocking the IL‐10 receptor, that IL‐10 impairs the accumulation and M1 activation of TNF/iNOS‐producing CD11b+Ly6C+ cells in the liver. Using infected IL‐10flox/floxLysM‐Cre+/+ mice, we show that myeloid cell‐derived IL‐10 limits M1 activation of CD11b+Ly6C+ cells specifically at the level of TNF production. Moreover, higher production of TNF in infected IL‐10flox/floxLysM‐Cre+/+ mice is associated with reduced nuclear accumulation of the NF‐κB p50 subunit in CD11b+ M1 cells. Furthermore, in infected p50−/− mice, TNF production by CD11b+Ly6C+ cells and liver injury increases. These data suggest that preferential nuclear accumulation of p50 represents an IL‐10‐dependent anti‐inflammatory mechanism in M1‐type CD11b+ myeloid cells that regulates the production of pathogenic TNF during T. congolense infection in resistant C57BL/6 mice.