Interplay between liver and blood stages of Plasmodium infection dictates malaria severity via γδ T cells and IL-17-promoted stress erythropoiesis.

Plasmodium replicates within the liver prior to reaching the bloodstream and infecting red blood cells. Because clinical manifestations of malaria only arise during the blood stage of infection, a perception exists that liver infection does not impact disease pathology. By developing a murine model where the liver and blood stages of infection are uncoupled, we showed that the integration of signals from both stages dictated mortality outcomes. This dichotomy relied on liver stage-dependent activation of Vγ4+ γδ T cells. Subsequent blood stage parasite loads dictated their cytokine profiles, where low parasite loads preferentially expanded IL-17-producing γδ T cells. IL-17 drove extra-medullary erythropoiesis and concomitant reticulocytosis, which protected mice from lethal experimental cerebral malaria (ECM). Adoptive transfer of erythroid precursors could rescue mice from ECM. Modeling of γδ T cell dynamics suggests that this protective mechanism may be key for the establishment of naturally acquired malaria immunity among frequently exposed individuals.

Multiple Myeloma-Derived Extracellular Vesicles Modulate the Bone Marrow Immune Microenvironment.

Multiple myeloma (MM), the third most frequent hematological cancer worldwide, is characterized by the proliferation of neoplastic plasma cells in the bone marrow (BM). One of the hallmarks of MM is a permissive BM microenvironment. Increasing evidence suggests that cell-to-cell communication between myeloma and immune cells via tumor cell-derived extracellular vesicles (EV) plays a key role in the pathogenesis of MM. Hence, we aimed to explore BM immune alterations induced by MM-derived EV. For this, we inoculated immunocompetent BALB/cByJ mice with a myeloma cell line, MOPC315.BM, inducing a MM phenotype. Upon tumor establishment, characterization of the BM microenvironment revealed the expression of both activation and suppressive markers by lymphocytes, such as granzyme B and PD-1, respectively. In addition, conditioning of the animals with MOPC315.BM-derived EV, before transplantation of the MOPC315.BM tumor cells, did not anticipate the disease phenotype. However, it induced features of suppression in the BM milieu, such as an increase in PD-1 expression by CD4+ T cells. Overall, our findings reveal the involvement of MOPC315.BM-derived EV protein content as promoters of immune niche remodeling, strengthening the importance of assessing the mechanisms by which MM may impact the immune microenvironment.

Immunopathology and Trypanosoma congolense parasite sequestration cause acute cerebral trypanosomiasis.

Trypanosoma congolense causes a syndrome of variable severity in animals in Africa. Cerebral trypanosomiasis is a severe form, but the mechanism underlying this severity remains unknown. We developed a mouse model of acute cerebral trypanosomiasis and characterized the cellular, behavioral, and physiological consequences of this infection. We show large parasite sequestration in the brain vasculature for long periods of time (up to 8 hr) and extensive neuropathology that associate with ICAM1-mediated recruitment and accumulation of T cells in the brain parenchyma. Antibody-mediated ICAM1 blocking and lymphocyte absence reduce parasite sequestration in the brain and prevent the onset of cerebral trypanosomiasis. Here, we establish a mouse model of acute cerebral trypanosomiasis and we propose a mechanism whereby parasite sequestration, host ICAM1, and CD4+ T cells play a pivotal role.

Host lung microbiota promotes malaria-associated acute respiratory distress syndrome.

Severe malaria can manifest itself with a variety of well-recognized clinical phenotypes that are highly predictive of death - severe anaemia, coma (cerebral malaria), multiple organ failure, and respiratory distress. The reasons why an infected individual develops one pathology rather than another remain poorly understood. Here we use distinct rodent models of infection to show that the host microbiota is a contributing factor for the development of respiratory distress syndrome and host mortality in the context of malaria infections (malaria-associated acute respiratory distress syndrome, MA-ARDS). We show that parasite sequestration in the lung results in sustained immune activation. Subsequent production of the anti-inflammatory cytokine IL-10 by T cells compromises microbial control, leading to severe lung disease. Notably, bacterial clearance with linezolid, an antibiotic commonly used in the clinical setting to control lung-associated bacterial infections, prevents MA-ARDS-associated lethality. Thus, we propose that the host's anti-inflammatory response to limit tissue damage can result in loss of microbial control, which promotes MA-ARDS. This must be considered when intervening against life-threatening respiratory complications.

Trypanosoma brucei triggers a broad immune response in the adipose tissue.

Adipose tissue is one of the major reservoirs of Trypanosoma brucei parasites, the causative agent of sleeping sickness, a fatal disease in humans. In mice, the gonadal adipose tissue (AT) typically harbors 2-5 million parasites, while most solid organs show 10 to 100-fold fewer parasites. In this study, we tested whether the AT environment responds immunologically to the presence of the parasite. Transcriptome analysis of T. brucei infected adipose tissue revealed that most upregulated host genes are involved in inflammation and immune cell functions. Histochemistry and flow cytometry confirmed an increasingly higher number of infiltrated macrophages, neutrophils and CD4+ and CD8+ T lymphocytes upon infection. A large proportion of these lymphocytes effectively produce the type 1 effector cytokines, IFN-γ and TNF-α. Additionally, the adipose tissue showed accumulation of antigen-specific IgM and IgG antibodies as infection progressed. Mice lacking T and/or B cells (Rag2-/-, Jht-/-), or the signature cytokine (Ifng-/-) displayed a higher parasite load both in circulation and in the AT, demonstrating the key role of the adaptive immune system in both compartments. Interestingly, infections of C3-/- mice showed that while complement system is dispensable to control parasite load in the blood, it is necessary in the AT and other solid tissues. We conclude that T. brucei infection triggers a broad and robust immune response in the AT, which requires the complement system to locally reduce parasite burden.

Bringing Macrophages to the Frontline against Cancer: Current Immunotherapies Targeting Macrophages.

Macrophages are found in all tissues and display outstanding functional diversity. From embryo to birth and throughout adult life, they play critical roles in development, homeostasis, tissue repair, immunity, and, importantly, in the control of cancer growth. In this review, we will briefly detail the multi-functional, protumoral, and antitumoral roles of macrophages in the tumor microenvironment. Our objective is to focus on the ever-growing therapeutic opportunities, with promising preclinical and clinical results developed in recent years, to modulate the contribution of macrophages in oncologic diseases. While the majority of cancer immunotherapies target T cells, we believe that macrophages have a promising therapeutic potential as tumoricidal effectors and in mobilizing their surroundings towards antitumor immunity to efficiently limit cancer progression.

Elimination of Hepatic Rodent Plasmodium Parasites by Amino Acid Supplementation.

Plasmodium parasites, causative agents of malaria, scavenge host nutrients to sustain their intracellular replication. Modulation of the host's nutritional status can potentially help control infection by limiting the parasite's access to nutrients, or by boosting the immune system. Here, we show that dietary supplementation of mice employing a combination of arginine (R) with two additional amino acids, lysine (K) and valine (V), termed RKV, significantly decreases Plasmodium liver infection. RKV supplementation results in the elimination of parasites at a late stage of their development in the liver. Our data employing genetic knockout mouse models and in vivo depletion of specific cell populations suggest that RKV supplementation boosts the host's overall innate immune response, and that parasite elimination is dependent on MyD88 signaling in immune cells. The immunostimulatory effect of RKV supplementation opens a potential role for dietary supplementation as an adjuvant for prophylaxis or immunization strategies against Plasmodium infection.

Chitosan/γ-PGA nanoparticles-based immunotherapy as adjuvant to radiotherapy in breast cancer.

Radiotherapy (RT) is an essential treatment modality for several types of cancer. Despite its therapeutic potential, RT is frequently insufficient to overcome the immunosuppressive nature of the tumor microenvironment, failing to control tumor metastases. Innovative immunomodulatory strategies, like immunostimulatory biomaterials could be used to boost the immunogenic effects of RT. Herein, we addressed the synergistic potential of immunostimulatory chitosan/poly(γ-glutamic acid) nanoparticles (Ch/γ-PGA NPs) combined with RT to induce antitumor immunity in the 4T1 orthotopic breast tumor mouse model. Non-treated animals had progressive primary tumor growth and developed splenomegaly and lung metastases. While RT decreased primary tumor burden, Ch/γ-PGA NPs-treatment decreased systemic immunosuppression and lung metastases. The combination therapy (RT + Ch/γ-PGA NPs) synergistically impaired 4T1 tumor progression, which was associated with a significant primary tumor growth and splenomegaly reduction, a decrease in the percentage of splenic immunosuppressive myeloid cells and an increase in antitumoral CD4+IFN-γ+ population. Notably, animals from the combination therapy presented less and smaller lung metastatic foci and lower levels of the systemic pro-tumor cytokines IL-3, IL-4, IL-10, and of the CCL4 chemokine, in comparison to non-treated animals. Overall, these results evidenced that Ch/γ-PGA NPs potentiate and synergize with RT, headlining their promising role as adjuvant anticancer strategies.

Interactions among myeloid regulatory cells in cancer.

Mounting evidence has accumulated on the critical role of the different myeloid cells in the regulation of the cancerous process, and in particular in the modulation of the immune reaction to cancer. Myeloid cells are a major component of host cells infiltrating tumors, interacting with each other, with tumor cells and other stromal cells, and demonstrating a prominent plasticity. We describe here various myeloid regulatory cells (MRCs) in mice and human as well as their relevant therapeutic targets. We first address the role of the monocytes and macrophages that can contribute to angiogenesis, immunosuppression and metastatic dissemination. Next, we discuss the differential role of neutrophil subsets in tumor development, enhancing the dual and sometimes contradicting role of these cells. A heterogeneous population of immature myeloid cells, MDSCs, was shown to be generated and accumulated during tumor progression as well as to be an important player in cancer-related immune suppression. Lastly, we discuss the role of myeloid DCs, which can either contribute to effective anti-tumor responses or play a more regulatory role. We believe that MRCs play a critical role in cancer-related immune regulation and suggest that future anti-cancer therapies will focus on these abundant cells.

Interferon-Gamma at the Crossroads of Tumor Immune Surveillance or Evasion.

Interferon-gamma (IFN-γ) is a pleiotropic molecule with associated antiproliferative, pro-apoptotic and antitumor mechanisms. This effector cytokine, often considered as a major effector of immunity, has been used in the treatment of several diseases, despite its adverse effects. Although broad evidence implicating IFN-γ in tumor immune surveillance, IFN-γ-based therapies undergoing clinical trials have been of limited success. In fact, recent reports suggested that it may also play a protumorigenic role, namely, through IFN-γ signaling insensitivity, downregulation of major histocompatibility complexes, and upregulation of indoleamine 2,3-dioxygenase and of checkpoint inhibitors, as programmed cell-death ligand 1. However, the IFN-γ-mediated responses are still positively associated with patient's survival in several cancers. Consequently, major research efforts are required to understand the immune contexture in which IFN-γ induces its intricate and highly regulated effects in the tumor microenvironment. This review discusses the current knowledge on the pro- and antitumorigenic effects of IFN-γ as part of the complex immune response to cancer, highlighting the relevance to identify IFN-γ responsive patients for the improvement of therapies that exploit associated signaling pathways.

Tumor-associated neutrophils suppress pro-tumoral IL-17+ γδ T cells through induction of oxidative stress.

Interleukin 17 (IL-17)-producing γδ T cells (γδ17 T cells) have been recently found to promote tumor growth and metastasis formation. How such γδ17 T-cell responses may be regulated in the tumor microenvironment remains, however, largely unknown. Here, we report that tumor-associated neutrophils can display an overt antitumor role by strongly suppressing γδ17 T cells. Tumor-associated neutrophils inhibited the proliferation of murine CD27- Vγ6+ γδ17 T cells via induction of oxidative stress, thereby preventing them from constituting the major source of pro-tumoral IL-17 in the tumor microenvironment. Mechanistically, we found that low expression of the antioxidant glutathione in CD27- γδ17 T cells renders them particularly susceptible to neutrophil-derived reactive oxygen species (ROS). Consistently, superoxide deficiency, or the administration of a glutathione precursor, rescued CD27- Vγ6+ γδ17 T-cell proliferation in vivo. Moreover, human Vδ1+ γδ T cells, which contain most γδ17 T cells found in cancer patients, also displayed low glutathione levels and were potently inhibited by ROS. This work thus identifies an unanticipated, immunosuppressive yet antitumoral, neutrophil/ROS/γδ17 T-cell axis in the tumor microenvironment.

MicroRNA-146a controls functional plasticity in γδ T cells by targeting NOD1.

γδ T cells are major providers of proinflammatory cytokines. They are preprogrammed in the mouse thymus into distinct subsets producing either interleukin-17 (IL-17) or interferon-γ (IFN-γ), which segregate with CD27 expression. In the periphery, CD27- γδ (γδ27-) T cells can be induced under inflammatory conditions to coexpress IL-17 and IFN-γ; the molecular basis of this functional plasticity remains to be determined. On the basis of differential microRNA (miRNA) expression analysis and modulation in γδ T cell subsets, we identified miR-146a as a thymically imprinted post-transcriptional brake to limit IFN-γ expression in γδ27- T cells in vitro and in vivo. On the basis of biochemical purification of Argonaute 2-bound miR-146a targets, we identified Nod1 to be a relevant mRNA target that regulates γδ T cell plasticity. In line with this, Nod1-deficient mice lacked multifunctional IL-17+ IFN-γ+ γδ27- cells and were more susceptible to Listeria monocytogenes infection. Our studies establish the miR-146a/NOD1 axis as a key determinant of γδ T cell effector functions and plasticity.

Primary Tumors Limit Metastasis Formation through Induction of IL15-Mediated Cross-Talk between Patrolling Monocytes and NK Cells.

Metastases are responsible for the vast majority of cancer-related deaths. Although tumor cells can become invasive early during cancer progression, metastases formation typically occurs as a late event. How the immune response to primary tumors may dictate this outcome remains poorly understood, which hampers our capacity to manipulate it therapeutically. Here, we used a two-step experimental model, based on the highly aggressive B16F10 melanoma, that temporally segregates the establishment of primary tumors (subcutaneously) and the formation of lung metastases (from intravenous injection). This allowed us to identify a protective innate immune response induced by primary tumors that inhibits experimental metastasis. We found that in the presence of primary tumors, increased numbers of natural killer (NK) cells with enhanced IFNγ, granzyme B, and perforin production were recruited to the lung upon metastasis induction. These changes were mirrored by a local accumulation of patrolling monocytes and macrophages with high expression of MHC class II and NOS2. Critically, the protective effect on metastasis was lost upon patrolling monocyte or NK cell depletion, IL15 neutralization, or IFNγ ablation. The combined analysis of these approaches allowed us to establish a hierarchy in which patrolling monocytes, making IL15 in response to primary tumors, activate NK cells and IFNγ production that then inhibit lung metastasis formation. This work identifies an innate cell network and the molecular determinants responsible for "metastasis immunosurveillance," providing support for using the key molecular mediator, IL15, to improve immunotherapeutic outcomes. Cancer Immunol Res; 5(9); 812-20. ©2017 AACR.

Low-dose ionizing radiation induces therapeutic neovascularization in a pre-clinical model of hindlimb ischemia.

AIMS: We have previously shown that low-dose ionizing radiation (LDIR) induces angiogenesis but there is no evidence that it induces neovascularization in the setting of peripheral arterial disease. Here, we investigated the use of LDIR as an innovative and non-invasive strategy to stimulate therapeutic neovascularization using a model of experimentally induced hindlimb ischemia (HLI). METHODS AND RESULTS: After surgical induction of unilateral HLI, both hindlimbs of female C57BL/6 mice were sham-irradiated or irradiated with four daily fractions of 0.3 Gy, in consecutive days and allowed to recover. We demonstrate that LDIR, significantly improved blood perfusion in the murine ischemic limb by stimulating neovascularization, as assessed by laser Doppler flow, capillary density, and collateral vessel formation. LDIR significantly increased the circulating levels of VEGF, PlGF, and G-CSF, as well as the number of circulating endothelial progenitor cells (EPCs) mediating their incorporation to ischemic muscles. These effects were dependent upon LDIR exposition on the ischemic niche (thigh and shank regions). In irradiated ischemic muscles, these effects were independent of the recruitment of monocytes and macrophages. Importantly, LDIR induced a durable and simultaneous up-regulation of a repertoire of pro-angiogenic factors and their receptors in endothelial cells (ECs), as evident in ECs isolated from the irradiated gastrocnemius muscles by laser capture microdissection. This specific mechanism was mediated via vascular endothelial growth factor (VEGF) receptor signaling, since VEGF receptor inhibition abrogated the LDIR-mediated gene up-regulation and impeded the increase in capillary density. Finally, the vasculature in an irradiated non-ischemic bed was not affected and after 52 week of LDIR exposure no differences in the incidence of morbidity and mortality were seen. CONCLUSIONS: These findings disclose an innovative, non-invasive strategy to induce therapeutic neovascularization in a mouse model of HLI, emerging as a novel approach in the treatment of critical limb ischemia patients.

Developmental and Functional Assays to Study Murine and Human γδ T Cells.

The key roles played by gamma-delta (γδ) T cells in immunity to infection and tumors critically depend on their differentiation into effectors capable of secreting cytokines (such as interferon-γ or interleukin-17), and killing infected or transformed cells. Here we detail the main methods used to investigate the differentiation of γδ T cells from murine or human origin. We describe developmental assays, such as thymic organ cultures (TOCs) and coculture of progenitors cells with OP9-DL1 stomal cells, as well as functional assays typically employed to evaluate γδ T cell cytotoxicity and cytokine production.

Effector γδ T Cell Differentiation Relies on Master but Not Auxiliary Th Cell Transcription Factors.

γδ T lymphocytes are programmed into distinct IFN-γ-producing CD27(+) (γδ27(+)) and IL-17-producing CD27(-) (γδ27(-)) subsets that play key roles in protective or pathogenic immune responses. Although the signature cytokines are shared with their αß Th1 (for γδ27(+)) and Th17 (for γδ27(-)) cell counterparts, we dissect in this study similarities and differences in the transcriptional requirements of murine effector γδ27(+), γδ27(-)CCR6(-), and γδ27(-)CCR6(+) γδ T cell subsets and αß T cells. We found they share dependence on the master transcription factors T-bet and RORγt for IFN-γ and IL-17 production, respectively. However, Eomes is fully dispensable for IFN-γ production by γδ T cells. Furthermore, the Th17 cell auxiliary transcription factors RORα and BATF are not required for IL-17 production by γδ27(-) cell subsets. We also show that γδ27(-) (but not γδ27(+)) cells become polyfunctional upon IL-1ß plus IL-23 stimulation, cosecreting IL-17A, IL-17F, IL-22, GM-CSF, and IFN-γ. Collectively, our in vitro and in vivo data firmly establish the molecular segregation between γδ27(+) and γδ27(-) T cell subsets and provide novel insight on the nonoverlapping transcriptional networks that control the differentiation of effector γδ versus αß T cell subsets.

γδ T cells in cancer.

With the promise of T cell-based therapy for cancer finally becoming reality, this Review focuses on the less-studied γδ T cell lineage and its diverse responses to tumours. γδ T cells have well-established protective roles in cancer, largely on the basis of their potent cytotoxicity and interferon-γ production. Besides this, recent studies have revealed a series of tumour-promoting functions that are linked to interleukin-17-producing γδ T cells. Here, we integrate the current knowledge from both human and mouse studies to highlight the potential of γδ T cell modulation to improve cancer immunotherapy.

Soluble flagellin coimmunization attenuates Th1 priming to Salmonella and clearance by modulating dendritic cell activation and cytokine production.

Soluble flagellin (sFliC) from Salmonella Typhimurium (STm) can induce a Th2 response to itself and coadministered antigens through ligation of TLR5. These properties suggest that sFliC could potentially modulate responses to Th1 antigens like live STm if both antigens are given concurrently. After coimmunization of mice with sFliC and STm there was a reduction in Th1 T cells (T-bet(+) IFN-γ(+) CD4 T cells) compared to STm alone and there was impaired clearance of STm. In contrast, there was no significant defect in the early extrafollicular B-cell response to STm. These effects are dependent upon TLR5 and flagellin expression by STm. The mechanism for these effects is not related to IL-4 induced to sFliC but rather to the effects of sFliC coimmunization on DCs. After coimmunization with STm and sFliC, splenic DCs had a lower expression of costimulatory molecules and profoundly altered kinetics of IL-12 and TNFα expression. Ex vivo experiments using in vivo conditioned DCs confirmed the effects of sFliC were due to altered DC function during a critical window in the coordinated interplay between DCs and naïve T cells. This has marked implications for understanding how limits in Th1 priming can be achieved during infection-induced, Th1-mediated inflammation.

Molecular Mechanisms of Differentiation of Murine Pro-Inflammatory γδ T Cell Subsets.

γδ T cells are unconventional innate-like lymphocytes that actively participate in protective immunity against tumors and infectious organisms including bacteria, viruses, and parasites. However, γδ T cells are also involved in the development of inflammatory and autoimmune diseases. γδ T cells are functionally characterized by very rapid production of pro-inflammatory cytokines, while also impacting on (slower but long-lasting) adaptive immune responses. This makes it crucial to understand the molecular mechanisms that regulate γδ T cell effector functions. Although they share many similarities with αß T cells, our knowledge of the molecular pathways that control effector functions in γδ T cells still lags significantly behind. In this review, we focus on the segregation of interferon-γ versus interleukin-17 production in murine thymic-derived γδ T cell subsets defined by CD27 and CCR6 expression levels. We summarize the most recent studies that disclose the specific epigenetic and transcriptional mechanisms that govern the stability or plasticity of discrete pro-inflammatory γδ T cell subsets, whose manipulation may be valuable for regulating (auto)immune responses.