Step-by-Step Immune Activation for Suicide Gene Therapy Reinforcement.

Gene-directed enzyme prodrug gene therapy (GDEPT) theoretically represents a useful method to carry out chemotherapy for cancer with minimal side effects through the formation of a chemotherapeutic agent inside cancer cells. However, despite great efforts, promising preliminary results, and a long period of time (over 25 years) since the first mention of this method, GDEPT has not yet reached the clinic. There is a growing consensus that optimal cancer therapies should generate robust tumor-specific immune responses. The advent of checkpoint immunotherapy has yielded new highly promising avenues of study in cancer therapy. For such therapy, it seems reasonable to use combinations of different immunomodulators alongside traditional methods, such as chemotherapy and radiotherapy, as well as GDEPT. In this review, we focused on non-viral gene immunotherapy systems combining the intratumoral production of toxins diffused by GDEPT and immunomodulatory molecules. Special attention was paid to the applications and mechanisms of action of the granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that is widely used but shows contradictory effects. Another method to enhance the formation of stable immune responses in a tumor, the use of danger signals, is also discussed. The process of dying from GDEPT cancer cells initiates danger signaling by releasing damage-associated molecular patterns (DAMPs) that exert immature dendritic cells by increasing antigen uptake, maturation, and antigen presentation to cytotoxic T-lymphocytes. We hypothesized that the combined action of this danger signal and GM-CSF issued from the same dying cancer cell within a limited space would focus on a limited pool of immature dendritic cells, thus acting synergistically and enhancing their maturation and cytotoxic T-lymphocyte attraction potential. We also discuss the problem of enhancing the cancer specificity of the combined GDEPT-GM-CSF-danger signal system by means of artificial cancer specific promoters or a modified delivery system.

Breast cancer-derived GM-CSF regulates arginase 1 in myeloid cells to promote an immunosuppressive microenvironment.

Tumor-infiltrating myeloid cells contribute to the development of the immunosuppressive tumor microenvironment. Myeloid cell expression of arginase 1 (ARG1) promotes a protumor phenotype by inhibiting T cell function and depleting extracellular l-arginine, but the mechanism underlying this expression, especially in breast cancer, is poorly understood. In breast cancer clinical samples and in our mouse models, we identified tumor-derived GM-CSF as the primary regulator of myeloid cell ARG1 expression and local immune suppression through a gene-KO screen of breast tumor cell-produced factors. The induction of myeloid cell ARG1 required GM-CSF and a low pH environment. GM-CSF signaling through STAT3 and p38 MAPK and acid signaling through cAMP were required to activate myeloid cell ARG1 expression in a STAT6-independent manner. Importantly, breast tumor cell-derived GM-CSF promoted tumor progression by inhibiting host antitumor immunity, driving a significant accumulation of ARG1-expressing myeloid cells compared with lung and melanoma tumors with minimal GM-CSF expression. Blockade of tumoral GM-CSF enhanced the efficacy of tumor-specific adoptive T cell therapy and immune checkpoint blockade. Taken together, we show that breast tumor cell-derived GM-CSF contributes to the development of the immunosuppressive breast cancer microenvironment by regulating myeloid cell ARG1 expression and can be targeted to enhance breast cancer immunotherapy.

Alveolar macrophages rely on GM-CSF from alveolar epithelial type 2 cells before and after birth.

Programs defining tissue-resident macrophage identity depend on local environmental cues. For alveolar macrophages (AMs), these signals are provided by immune and nonimmune cells and include GM-CSF (CSF2). However, evidence to functionally link components of this intercellular cross talk remains scarce. We thus developed new transgenic mice to profile pulmonary GM-CSF expression, which we detected in both immune cells, including group 2 innate lymphoid cells and γδ T cells, as well as AT2s. AMs were unaffected by constitutive deletion of hematopoietic Csf2 and basophil depletion. Instead, AT2 lineage-specific constitutive and inducible Csf2 deletion revealed the nonredundant function of AT2-derived GM-CSF in instructing AM fate, establishing the postnatal AM compartment, and maintaining AMs in adult lungs. This AT2-AM relationship begins during embryogenesis, where nascent AT2s timely induce GM-CSF expression to support the proliferation and differentiation of fetal monocytes contemporaneously seeding the tissue, and persists into adulthood, when epithelial GM-CSF remains restricted to AT2s.

SOCS3 Attenuates GM-CSF/IFN-γ-Mediated Inflammation During Spontaneous Spinal Cord Regeneration.

SOCS3, a feedback inhibitor of the JAK/STAT signal pathway, negatively regulates axonal regrowth and inflammation in the central nervous system (CNS). Here, we demonstrated a distinct role of SOCS3 in the injured spinal cord of the gecko following tail amputation. Severing the gecko spinal cord did not evoke an inflammatory cascade except for an injury-stimulated elevation of the granulocyte/macrophage colony-stimulating factor (GM-CSF) and interferon gamma (IFN-γ) cytokines. Simultaneously, the expression of SOCS3 was upregulated in microglia, and unexpectedly not in neurons. Enforced expression of SOCS3 was sufficient to suppress the GM-CSF/IFN-γ-driven inflammatory responses through its KIR domain by attenuating the activities of JAK1 and JAK2. SOCS3 was also linked to GM-CSF/IFN-γ-induced cross-tolerance. Transfection of adenovirus overexpressing SOCS3 in the injured cord resulted in a significant decrease of inflammatory cytokines. These results reveal a distinct role of SOCS3 in the regenerating spinal cord, and provide new hints for CNS repair in mammals.

Granulocyte-Macrophage Colony Stimulating Factor As an Indirect Mediator of Nociceptor Activation and Pain.

The interaction between the immune system and the nervous system has been at the center of multiple research studies in recent years. Whereas the role played by cytokines as neuronal mediators is no longer contested, the mechanisms by which cytokines modulate pain processing remain to be elucidated. In this study, we have analyzed the involvement of granulocyte-macrophage colony stimulating factor (GM-CSF) in nociceptor activation in male and female mice. Previous studies have suggested GM-CSF might directly activate neurons. However, here we established the absence of a functional GM-CSF receptor in murine nociceptors, and suggest an indirect mechanism of action, via immune cells. We report that GM-CSF applied directly to magnetically purified nociceptors does not induce any transcriptional changes in nociceptive genes. In contrast, conditioned medium from GM-CSF-treated murine macrophages was able to drive nociceptor transcription. We also found that conditioned medium from nociceptors treated with the well established pain mediator, nerve growth factor, could also modify macrophage gene transcription, providing further evidence for a bidirectional crosstalk.SIGNIFICANCE STATEMENT The interaction of the immune system and the nervous system is known to play an important role in the development and maintenance of chronic pain disorders. Elucidating the mechanisms of these interactions is an important step toward understanding, and therefore treating, chronic pain disorders. This study provides evidence for a two-way crosstalk between macrophages and nociceptors in the peripheral nervous system, which may contribute to the sensitization of nociceptors by cytokines in pain development.

Evaluation of the effect of GM-CSF blocking on the phenotype and function of human monocytes.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multipotent cytokine that prompts the proliferation of bone marrow-derived macrophages and granulocytes. In addition to its effects as a growth factor, GM-CSF plays an important role in chronic inflammatory autoimmune diseases such as multiple sclerosis and rheumatoid arthritis. Reports have identified monocytes as the primary target of GM-CSF; however, its effect on monocyte activation has been under-estimated. Here, using flow cytometry and ELISA we show that GM-CSF induces an inflammatory profile in human monocytes, which includes an upregulated expression of HLA-DR and CD86 molecules and increased production of TNF-α and IL-1ß. Conversely, blockage of endogenous GM-CSF with antibody treatment not only inhibited the inflammatory profile of these cells, but also induced an immunomodulatory one, as shown by increased IL-10 production by monocytes. Further analysis with qPCR, flow cytometry and ELISA experiments revealed that GM-CSF blockage in monocytes stimulated production of the chemokine CXCL-11, which suppressed T cell proliferation. Blockade of CXCL-11 abrogated anti-GM-CSF treatment and induced inflammatory monocytes. Our findings show that anti-GM-CSF treatment induces modulatory monocytes that act in a CXCL-11-dependent manner, a mechanism that can be used in the development of novel approaches to treat chronic inflammatory autoimmune diseases.

Using CRISPR/Cas9 to Knock Out GM-CSF in CAR-T Cells.

Chimeric antigen receptor T (CAR-T) cell therapy is a cutting edge and potentially revolutionary new treatment option for cancer. However, there are significant limitations to its widespread use in the treatment of cancer. These limitations include the development of unique toxicities such as cytokine release syndrome (CRS) and neurotoxicity (NT) and limited expansion, effector functions, and anti-tumor activity in solid tumors. One strategy to enhance CAR-T efficacy and/or control toxicities of CAR-T cells is to edit the genome of the CAR-T cells themselves during CAR-T cell manufacturing. Here, we describe the use of CRISPR/Cas9 gene editing in CAR-T cells via transduction with a lentiviral construct containing a guide RNA to granulocyte macrophage colony-stimulating factor (GM-CSF) and Cas9. As an example, we describe CRISPR/Cas9 mediated knockout of GM-CSF. We have shown that these GM-CSFk/o CAR-T cells effectively produce less GM-CSF while maintaining critical T cell function and result in enhanced anti-tumor activity in vivo compared to wild type CAR-T cells.

The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model.

Pattern recognition receptors (PRRs) are key elements in the innate immune response. Formyl peptide receptor (FPR) 2 is a PRR that, in addition to proinflammatory, pathogen-derived compounds, also recognizes the anti-inflammatory endogenous ligand annexin A1 (AnxA1). Because the contribution of this signaling axis in viral infections is undefined, we investigated AnxA1-mediated FPR2 activation on influenza A virus (IAV) infection in the murine model. AnxA1-treated mice displayed significantly attenuated pathology upon a subsequent IAV infection with significantly improved survival, impaired viral replication in the respiratory tract, and less severe lung damage. The AnxA1-mediated protection against IAV infection was not caused by priming of the type I IFN response but was associated with an increase in the number of alveolar macrophages (AMs) and enhanced pulmonary expression of the AM-regulating cytokine granulocyte-M-CSF (GM-CSF). Both AnxA1-mediated increase in AM levels and GM-CSF production were abrogated when mouse (m)FPR2 signaling was antagonized but remained up-regulated in mice genetically deleted for mFPR1, an mFPR2 isoform also serving as AnxA1 receptor. Our results indicate a novel protective function of the AnxA1-FPR2 signaling axis in IAV pathology via GM-CSF-associated maintenance of AMs, expanding knowledge on the potential use of proresolving mediators in host defense against pathogens.-Schloer, S., Hübel, N., Masemann, D., Pajonczyk, D., Brunotte, L., Ehrhardt, C., Brandenburg, L.-O., Ludwig, S., Gerke, V., Rescher, U. The annexin A1/FPR2 signaling axis expands alveolar macrophages, limits viral replication, and attenuates pathogenesis in the murine influenza A virus infection model.

Tubular GM-CSF Promotes Late MCP-1/CCR2-Mediated Fibrosis and Inflammation after Ischemia/Reperfusion Injury.

BACKGROUND: After bilateral kidney ischemia/reperfusion injury (IRI), monocytes infiltrate the kidney and differentiate into proinflammatory macrophages in response to the initial kidney damage, and then transition to a form that promotes kidney repair. In the setting of unilateral IRI (U-IRI), however, we have previously shown that macrophages persist beyond the time of repair and may promote fibrosis. METHODS: Macrophage homing/survival signals were determined at 14 days after injury in mice subjected to U-IRI and in vitro using coculture of macrophages and tubular cells. Mice genetically engineered to lack Ccr2 and wild-type mice were treated ±CCR2 antagonist RS102895 and subjected to U-IRI to quantify macrophage accumulation, kidney fibrosis, and inflammation 14 and 30 days after the injury. RESULTS: Failure to resolve tubular injury after U-IRI results in sustained expression of granulocyte-macrophage colony-stimulating factor by renal tubular cells, which directly stimulates expression of monocyte chemoattractant protein-1 (Mcp-1) by macrophages. Analysis of CD45+ immune cells isolated from wild-type kidneys 14 days after U-IRI reveals high-level expression of the MCP-1 receptor Ccr2. In mice lacking Ccr2 and wild-type mice treated with RS102895, the numbers of macrophages, dendritic cells, and T cell decreased following U-IRI, as did the expression of profibrotic growth factors and proimflammatory cytokines. This results in a reduction in extracellular matrix and kidney injury markers. CONCLUSIONS: GM-CSF-induced MCP-1/CCR2 signaling plays an important role in the cross-talk between injured tubular cells and infiltrating immune cells and myofibroblasts, and promotes sustained inflammation and tubular injury with progressive interstitial fibrosis in the late stages of U-IRI.

Dietary Polyunsaturated Fatty Acids Promote Neutrophil Accumulation in the Spleen by Altering Chemotaxis and Delaying Cell Death.

Neutrophils are the most abundant circulating leukocytes in humans and are essential for the defense against invading pathogens. Like many other cells of an organism, neutrophils can be highly influenced by the diet. We have previously described that mice fed a high-fat diet rich in polyunsaturated fatty acids (HFD-P) present a higher frequency of neutrophils in bone marrow than mice fed a high-fat diet rich in saturated fatty acids (HFD-S). Interestingly, such an increase correlated with improved survival against bacterium-induced sepsis. In this study, we aimed to investigate the effects of dietary polyunsaturated and saturated fatty acids on neutrophil homeostasis. We found that HFD-P specifically induced the accumulation of neutrophils in the marginal pools of the spleen and liver. The accumulation of neutrophils in the spleen was a result of a dual effect of polyunsaturated fatty acids on neutrophil homeostasis. First, polyunsaturated fatty acids enhanced the recruitment of neutrophils from the circulation into the spleen via chemokine secretion. Second, they delayed neutrophil cell death in the spleen. Interestingly, these effects were not observed in mice fed a diet rich in saturated fatty acids, suggesting that the type of fat rather than the amount of fat mediates the alterations in neutrophil homeostasis. In conclusion, our results show that dietary polyunsaturated fatty acids have a strong modulatory effect on neutrophil homeostasis that may have future clinical applications.

Role of GM-CSF in a mouse model of experimental autoimmune prostatitis.

The etiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is still unknown. Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to play an important role in the development of autoimmune and inflammatory diseases. Here, we investigated the expression and function of GM-CSF in patients with CP/CPPS and in a mouse model of experimental autoimmune prostatitis (EAP). GM-CSF mRNA levels were detected in expressed prostatic secretions samples from patients with CP/CPPS and in prostate tissue from a mouse model of EAP. The expression of GM-CSF receptor in mouse prostate and dorsal root ganglia were determined using PCR and immunohistochemistry. Behavioral testing and inflammation scoring were performed to evaluate the role of GM-CSF in disease development and symptom severity of EAP using GM-CSF knockout mice. mRNA levels of putative nociceptive and inflammatory markers were measured in the prostate after the induction of EAP. Elevated GM-CSF mRNA levels were observed in expressed prostatic secretions samples from patients with CP/CPPS compared with healthy volunteers. GM-CSF mRNA was also significantly increased in prostate tissue of the EAP mice model. The expression of GM-CSF receptors was confirmed in mouse prostate and dorsal root ganglia. GM-CSF knockout mice showed fewer Infiltrating leukocytes and pain symptoms after the induction of EAP. Deletion of GM-CSF significantly diminished EAP-induced increases of chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 3, and nerve growth factor mRNA expression. The results indicated that GM-CSF plays a functional role in the pathogenesis of EAP. GM-CSF may function as a signaling mediator for both inflammation and pain transduction in CP/CPPS.

MCP-1, KC-like and IL-8 as critical mediators of pathogenesis caused by Babesia canis.

Canine babesiosis caused by the intraerythrocytic protozoan parasite Babesia canis is a tick-borne disease characterized by a host response that involves both cellular and humoral immunity. This study focuses on the secretion of cytokines Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Keratinocyte Chemotactic-like (KC-like), Interleukins (IL)-2, IL-7, IL-8, IL-10, IL-15, IL-18 and Monocyte Chemotactic Protein-1 (MCP-1) in babesiosis caused by Babesia canis upon treatment with Imizol®. We assessed time dependent changes in cytokine levels and tested whether these changes correlate with pathogenesis of the disease. Sixteen healthy dogs and 31 dogs infected with Babesia canis, of which 18 showed complications, were treated with Imizol®. One dog died during the study (3.2%). Longitudinal study was perfomed by monitoring dogs at the first day of presentation (day 1) and 6 days later (day 7). Our results show that higher MCP-1 levels on day 1 are positively associated with the occurrence of complications, (complicated vs. uncomplicated; p = 0.00016). A similar pattern was observed for KC-like on day 1 (p = 0.0326) and day 7 (p = 0.044). Moreover, babesiosis caused by B. canis produced a steady increase in IL-8 levels with a moderate to strong negative correlation with erythrocyte counts and hematocrit in uncomplicated diseased dogs only (Spearman's rank correlation coefficient rs = -0.582 and rs = -0.598 respectively). Like for MCP-1, KC-like levels also differed in complicated and uncomplicated diseased dogs on day 1 (p = 0.03236) and day 7 (p = 0.044). Furthermore, KC-like levels were strongly correlated with IL-8 levels (rs = 0.663-0.7) and non-segmented neutrophil counts (rs = 0.572-0.732) in both diseased groups. Analysis of ROC suggests the use of serum levels of MCP-1 and IL-7 as predictors of the occurrence of complications with an AUC of 0.906 and 0.896 respectively and linear combinations of MCP-1, KC-Like, IL-7 and GM-CSF with values up to AUC = 0.983. Cytokine cluster analysis presented in this study can contribute to a better understanding of the pathogenesis of babesiosis and serve as a prognostic tool for the early detection of cases with highest likelihood of developing complications. Overall, our studies show that infection by B. canis elicits a cytokine pattern that is distinct from that observed with B. rossi, and that some of the inflammatory mediators can be useful to predict complications. Our results also suggest targets for the development of novel therapeutic strategies in babesiosis caused by B. canis.

A newly identified novel variant in the CSF2RA gene in a child with pulmonary alveolar proteinosis: a case report.

BACKGROUND: The congenital form of pulmonary alveolar proteinosis due to colony stimulating factor 2 receptor alpha gene mutations is a rare disease with only a few cases reported worldwide. In this study we report a new case of pulmonary alveolar proteinosis with a novel variant in colony stimulating factor 2 receptor alpha gene. CASE PRESENTATION: A 5-year-old Saudi boy presented with a history of progressive dyspnea over 6 months; he was diagnosed as having pulmonary alveolar proteinosis. A molecular study revealed a novel variation in colony stimulating factor 2 receptor alpha gene. His clinical condition showed significant improvement after whole lung lavage. CONCLUSIONS: This case has the typical presentation of congenital pulmonary alveolar proteinosis due to colony stimulating factor 2 receptor alpha defect with a novel variant in this gene likely to be pathogenic.

GR-independent down-modulation on GM-CSF bone marrow-derived dendritic cells by the selective glucocorticoid receptor modulator Compound A.

Dendritic cells (DC) initiate the adaptive immune response. Glucocorticoids (GCs) down-modulate the function of DC. Compound A (CpdA, (2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride) is a plant-derived GR-ligand with marked dissociative properties. We investigated the effects of CpdA on in vitro generated GM-CSF-conditioned bone marrow-derived DC (BMDC). CpdA-exposed BMDC exhibited low expression of cell-surface molecules and diminution of the release of proinflammatory cytokines upon LPS stimulation; processes associated with BMDC maturation and activation. CpdA-treated BMDC were inefficient at Ag capture via mannose receptor-mediated endocytosis and displayed reduced T-cell priming. CpdA prevented the LPS-induced rise in pErk1/2 and pP38, kinases involved in TLR4 signaling. CpdA fully inhibited LPS-induced pAktSer473, a marker associated with the generation of tolerogenic DC. We used pharmacological blockade and selective genetic loss-of-function tools and demonstrated GR-independent inhibitory effects of CpdA in BMDC. Mechanistically, CpdA-mediated inactivation of the NF-κB intracellular signaling pathway was associated with a short-circuiting of pErk1/2 and pP38 upstream signaling. Assessment of the in vivo function of CpdA-treated BMDC pulsed with the hapten trinitrobenzenesulfonic acid showed impaired cell-mediated contact hypersensitivity. Collectively, we provide evidence that CpdA is an effective BMDC modulator that might have a benefit for immune disorders, even when GR is not directly targeted.

GM-CSF Induces Inflammatory Macrophages by Regulating Glycolysis and Lipid Metabolism.

GM-CSF induces proinflammatory macrophages, but the underlying mechanisms have not been studied thus far. In this study, we investigated the mechanisms of how GM-CSF induces inflammatory macrophages. First, we observed that GM-CSF increased the extent of LPS-induced acute glycolysis in murine bone marrow-derived macrophages. This directly correlates with an inflammatory phenotype because glycolysis inhibition by 2-deoxyglucose abolished GM-CSF-mediated increase of TNF-α, IL-1ß, IL-6, and IL-12p70 synthesis upon LPS stimulation. Increased glycolytic capacity is due to de novo synthesis of glucose transporter (GLUT)-1, -3, and -4, as well as c-myc. Meanwhile, GM-CSF increased 3-hydroxy-3-methyl-glutaryl-CoA reductase, which is the rate-limiting enzyme of the mevalonate pathway. Inhibition of acute glycolysis or 3-hydroxy-3-methyl-glutaryl-CoA reductase abrogated the inflammatory effects of GM-CSF priming in macrophages. Finally, mice with inflamed colons exposed to dextran sodium sulfate containing GLUT-1high macrophages led to massive uptake of [18F]-fluorodeoxyglucose, but GM-CSF neutralization reduced the positron-emission tomography signal in the intestine and also decreased GLUT-1 expression in colonic macrophages. Collectively, our results reveal glycolysis and lipid metabolism created by GM-CSF as the underlying metabolic constructs for the function of inflammatory macrophages.

GM-CSF Down-Regulates TLR Expression via the Transcription Factor PU.1 in Human Monocytes.

Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding-and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions.

Role of Serum Amyloid A, Granulocyte-Macrophage Colony-Stimulating Factor, and Bone Marrow Granulocyte-Monocyte Precursor Expansion in Segmented Filamentous Bacterium-Mediated Protection from Entamoeba histolytica.

Intestinal segmented filamentous bacteria (SFB) protect from ameba infection, and protection is transferable with bone marrow dendritic cells (BMDCs). SFB cause an increase in serum amyloid A (SAA), suggesting that SAA might mediate SFB's effects on BMDCs. Here we further explored the role of bone marrow in SFB-mediated protection. Transient gut colonization with SFB or SAA administration alone transiently increased the H3K27 histone demethylase Jmjd3, persistently increased bone marrow Csf2ra expression and granulocyte monocyte precursors (GMPs), and protected from ameba infection. Pharmacologic inhibition of Jmjd3 H3K27 demethylase activity during SAA treatment or blockade of granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling in SFB-colonized mice prevented GMP expansion, decreased gut neutrophils, and blocked protection from ameba infection. These results indicate that alteration of the microbiota and systemic exposure to SAA can influence myelopoiesis and susceptibility to amebiasis via epigenetic mechanisms. Gut microbiota-marrow communication is a previously unrecognized mechanism of innate protection from infection.

Depletion of CD4+ CD25+ regulatory T cells confers susceptibility to experimental autoimmune encephalomyelitis (EAE) in GM-CSF-deficient Csf2-/- mice.

Previous studies established that GM-CSF-deficient (Csf2-deficient) mice exhibit profound resistance to experimental autoimmune encephalomyelitis. This study addressed whether the resistance of Csf2-deficient mice was a result of a requirement for GM-CSF in controlling the functional balance between effector and regulatory T cell subsets during experimental autoimmune encephalomyelitis. The main observation was that treatment with the anti-CD25 mAb PC61 rendered Csf2-deficient mice fully susceptible to severe, chronic experimental autoimmune encephalomyelitis, with disease incidences and severities equivalent to that of C57BL/6 mice. When both donors and recipients were treated with PC61 in a passive model of experimental autoimmune encephalomyelitis, adoptive transfer of myelin-specific Csf2-deficient T cells into Csf2-deficient recipients resulted in a nonresolving chronic course of severe paralytic experimental autoimmune encephalomyelitis. The peripheral Csf2-deficient T cell repertoire was marked by elevated CD3+ T cell frequencies that reflected substantial accumulations of naïve CD44null-low CD4+ and CD8+ T cells but essentially normal frequencies of CD4+ CD25+ forkhead box P3+ T cells among the CD3+ T cell pool. These findings suggested that Csf2-deficient mice had secondary deficiencies in peripheral T cell sensitization to environmental antigens. In accordance, myelin oligodendrocyte glycoprotein 35-55/CFA-sensitized Csf2-deficient mice exhibited deficient peripheral sensitization to myelin oligodendrocyte glycoprotein, whereas pretreatment of Csf2-deficient mice with PC61 enabled the robust induction of myelin oligodendrocyte glycoprotein-specific T cell responses in the draining lymphatics. In conclusion, the experimental autoimmune encephalomyelitis resistance of Csf2-deficient mice, at least in part, reflects a deficient induction of effector T cell function that cannot surmount normal regulatory T cell barriers. Experimental autoimmune encephalomyelitis effector responses, however, are unleashed upon depletion of regulatory CD25+ T cells.