Myeloid‐derived suppressor cell activation by combined LPS and IFN‐γ treatment impairs DC development

Myeloid‐derived suppressor cells (MDSC) and DC are major controllers of immune responses against tumors or infections. However, it remains unclear how DC development and MDSC suppressor activity both generated from myeloid precursor cells are regulated. Here, we show that the combined treatment of BM‐derived MDSC with LPS plus IFN‐γ inhibited the DC development but enhanced MDSC functions, such as NO release and T‐cell suppression. This was not observed by the single treatments in vitro. In the spleens of healthy mice, we identified two Gr‐1^lowCD11b^highLy‐6C^highSSC^lowMo‐MDSC and Gr‐1^highCD11b^lowPMN‐MDSC populations with suppressive potential, whereas Gr‐1^highCD11b^high neutrophils and Gr‐1^lowCD11b^highSSC^low eosinophils were not suppressive. Injections of LPS plus IFN‐γ expanded these populations within the spleen but not LN leading to the block of the proliferation of CD8⁺ T cells. At the same time, their capacity to develop into DC was impaired. Together, our data suggest that spleens of healthy mice contain two subsets of MDSC with suppressive potential. A two‐signal‐program through combined LPS and IFN‐γ treatment expands and fully activates MDSC in vitro and in vivo.     

Gr‐1 antibody induces STAT signaling,macrophage marker expression and abrogation of myeloid‐derived suppressor cell activity in BM cells

The Gr‐1 (RB6‐8C5) Ab binds with high affinity to mouse Ly‐6G molecules and to a lower extent to Ly‐6C and has been widely used for cell depletion in infected or tumor‐bearing mice. Here we found that Gr‐1 treatment of BM cells in vitro and in vivo showed no depleting effects. The epitope recognized by the Gr‐1 Ab overlapped with Ly‐6G (1A8 Ab) but not Ly‐6C (ER‐MP20 Ab). In vitro the Gr‐1 Ab transmitted signals via STAT‐1, STAT‐3 and STAT‐5 into BM cells, similar to GM‐CSF. In healthy mice injected with the Gr‐1 Ab, the Ab remained attached to the surface of myeloid cells for at least four days. Gr‐1 Ab induced myeloid cell expansion, upregulation of macrophage markers, but not the DC marker CD11c. Suppressor activity of two distinct Gr‐1^high and Gr‐1^low expressing BM‐myeloid‐derived suppressor cell subsets was transiently ablated by Gr‐1 Ab injection. Depleting effects of Gr‐1 Ab could only be observed on inflammatory Ly‐6C^intLy‐6G^high neutrophils from the peritoneal cavity, which occurred via apoptosis and was associated with the absence of Mcl‐1 expression. Together, Gr‐1 Ab induces signals leading to myelopoiesis and affects myeloid‐derived suppressor cell activity, suggesting functional roles for Ly‐6C/G molecules in macrophage differentiation and neutrophil apoptosis.     

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.     

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^?Ly6C^high MDSCs and granulocytic CD11b⁺CD115^?Ly6G⁺Ly6C^int 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.     

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.     

Myeloid‐derived suppressor cells in inflammation: Uncovering cell subsets with enhanced immunosuppressive functions

Although originally described in tumor‐bearing hosts, myeloid‐derived suppressor cells (MDSC) have been detected under numerous pathological situations that cause enhanced demand of myeloid cells. Thus, MDSC might be part of a conserved response to different endogenous and exogenous stress signals, including inflammation. Two processes are fundamental for MDSC biology: differentiation from myeloid progenitors and full activation of their immune regulatory program by factors released from activated T cells or present in the microenvironment conditioned by either tumor growth or inflammation. How these two processes are controlled and linked is still an open question. In this issue of the European Journal of Immunology, a paper demonstrates that a combination of the known inflammatory molecules, IFN‐γ and LPS, sustains MDSC expansion and activation while suppressing differentiation of DC from bone marrow precursors. Moreover, this paper contributes to defining the cell subsets that possess immunoregulatory properties within the broad population of CD11b⁺Gr‐1⁺ cells, often altogether referred to as MDSC.     

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.     

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.     

Induction of protumoral CD11chigh macrophages by glioma cancer stem cells through GM‐CSF

Cancer stem cells (CSCs) are maintained under special microenvironment called niche, and elucidation and targeting of the CSC niche will be a feasible strategy for cancer eradication. Tumor‐associated macrophages (TAMs) are known to be involved in cancer progression and thus can be a component of CSC niche. Although TAMs are known to play multiple roles in tumor progression, involvement of CSCs in TAM development fully remains to be elucidated. Using rat C6 glioma side population (SP) cells as a model of glioma CSCs, we here show that CSCs induce the TAM development by promoting survival and differentiation of bone marrow‐derived monocytes. CSC‐induced macrophages can be separated into two distinct subsets of cells, CD11c^low and CD11c^high cells. Interestingly, only the CD11c^high subset of cells have protumoral activity, as shown by intracranial transplantation into immune‐deficient mice together with CSCs. These CD11c^high macrophages were observed in the tumor formed by co‐transplantation with CSCs. Furthermore, CSCs produced GM‐CSF and anti‐GM‐CSF antibody inhibited CSC‐induced TAM development. In conclusion, CSCs have the ability to self‐create their own niche involving TAMs through CSC‐derived GM‐CSF, which can thus be a therapeutic target in view of CSC niche disruption.     

Galectin‐9 expands immunosuppressive macrophages to ameliorate T‐cell‐mediated lung inflammation

Galectin‐9 (Gal‐9) plays pivotal roles in the modulation of innate and adaptive immunity to suppress T‐cell‐mediated autoimmune models. However, it remains unclear if Gal‐9 plays a suppressive role for T‐cell function in non‐autoimmune disease models. We assessed the effects of Gal‐9 on experimental hypersensitivity pneumonitis induced by Trichosporon asahii. When Gal‐9 was given subcutaneously to C57BL/6 mice at the time of challenge with T. asahii, it significantly suppressed T. asahii‐induced lung inflammation, as the levels of IL‐1, IL‐6, IFN‐γ, and IL‐17 were significantly reduced in the BALF of Gal‐9‐treated mice. Moreover, co‐culture of anti‐CD3‐stimulated CD4 T cells with BALF cells harvested from Gal‐9‐treated mice on day 1 resulted in diminished CD4 T‐cell proliferation and decreased levels of IFN‐γ and IL‐17. CD11b⁺Ly‐6C^highF4/80⁺ BALF M? expanded by Gal‐9 were responsible for the suppression. We further found in vitro that Gal‐9, only in the presence of T. asahii, expands CD11b⁺Ly‐6C^highF4/80⁺ cells from BM cells, and the cells suppress T‐cell proliferation and IFN‐γ and IL‐17 production. The present results indicate that Gal‐9 expands immunosuppressive CD11b⁺Ly‐6C^high M? to ameliorate Th1/Th17 cell‐mediated hypersensitivity pneumonitis.     

Polysaccharide Agaricus blazei Murill stimulates myeloid derived suppressor cell differentiation from M2 to M1 type,which mediates inhibition of tumour immune‐evasion via the Toll‐like receptor 2 pathway

Gr‐1⁺ CD11b⁺ myeloid‐derived suppressor cells (MDSCs) accumulate in tumor‐bearing animals and play a critical negative role during tumor immunotherapy. Strategies for inhibition of MDSCs are expected to improve cancer immunotherapy. Polysaccharide Agaricus blazei Murill (pAbM) has been found to have anti‐cancer activity, but the underlying mechanism of this is poorly understood. Here, pAbM directly activated the purified MDSCs through inducing the expression of interleukin‐6 (IL‐6), IL‐12, tumour necrosis factor and inducible nitric oxide synthase (iNOS), CD86, MHC II, and pSTAT1 of it, and only affected natural killer and T cells in the presence of Gr‐1⁺ CD11b⁺ monocytic MDSCs. On further analysis, we demonstrated that pAbM could selectively block the Toll‐like receptor 2 (TLR2) signal of Gr‐1⁺ CD11b⁺ MDSCs and increased their M1‐type macrophage characteristics, such as producing IL‐12, lowering expression of Arginase 1 and increasing expression of iNOS. Extensive study showed that Gr‐1⁺ CD11b⁺ MDSCs by pAbM treatment had less ability to convert the CD4⁺ CD25^? cells into CD4⁺ CD25⁺ phenotype. Moreover, result from selective depletion of specific cell populations in xenograft mice model suggested that the anti‐tumour effect of pAbM was dependent on Gr‐1⁺ CD11b⁺ monocytes, nether CD8⁺ T cells nor CD4⁺ T cells. In addition to, pAbM did not inhibit tumour growth in TLR2^–/– mice. All together, these results suggested that pAbM, a natural product commonly used for cancer treatment, was a specific TLR2 agonist and had potent anti‐tumour effects through the opposite of the suppressive function of Gr‐1⁺ CD11b⁺ MDSCs.     

IL‐33 promotes DC development in BM culture by triggering GM‐CSF production

Short‐term DC cultures generated with GM‐CSF and other cytokines have markedly improved our ability to study the immunobiology of DC. Here, we tested 65 cytokines individually for their potential to promote the generation of CD11c⁺ cells in a murine BM culture system. In addition to several cytokines known to promote DC survival and/or growth, IL‐33 was found to augment DC development time‐ and dose‐dependently. Although the resulting CD11c⁺ cells generated in the presence of IL‐33 exhibited a typical dendritic morphology, they expressed MHC class II molecules only at modest levels, showed negligible responses to TLR ligands, produced no detectable IL‐12 p70, displayed PD‐L1 and PD‐L2 on the surface, and failed to activate immunologically naïve T cells efficiently. IL‐33‐induced expansion of CD11c⁺ cells was completely blocked by anti‐GM‐CSF mAb, and GM‐CSF mRNA and protein expression in BM culture was markedly elevated by added IL‐33, indicating that IL‐33 promotes in vitro DC generation indirectly by a GM‐CSF‐dependent manner. With regard to the cellular source, IL‐33‐dependent GM‐CSF production was observed exclusively within the CD45⁺/FcεRI⁺ BM population. Not only do our results reinforce the notion that GM‐CSF serves as a primary DC growth factor, but they also reveal a previously unrecognized mechanism supporting DC development.     

Role of myeloid-derived suppressor cells in mouse pre-sensitized cardiac transplant model

Harness of sensitized transplantation remains a clinical challenge particularly in parallel with prolonged cold ischemia time (PCI)-mediated injury. Our present study was to test the role of myeloid-derived suppressor cells (MDSCs) in mouse pre-sensitized transplantation. Our findings revealed that CD11b + Gr1^low MDSC was shown to have strong suppressive activity. MDSCs subsets from the tolerated mice exhibited higher suppressive capacities compared with counterparts from naive (untreated) mice. Depletion of Tregs could not affect splenic CD11b + Gr1^-low MDSC frequency, but increase peripheral and intragraft CD11b + Gr1^-low frequency. Intriguingly, boost of Tregs remarkably caused an increase of CD11b + Gr1^-low frequency in the graft, peripheral blood, and spleen. Furthermore, peripheral CD11b + Gr1^-low cells were massively accumulated at the early stage when allogeneic immune response was enhanced. Taken together, MDSCs could prevent grafts from PCI-mediated injury independent on Tregs in the pre-sensitized transplant recipients. Utilization of MDSC subset particularly CD11b + Gr1^-low might provide a novel insight into improving graft outcome under such clinical scenarios.     

The expression of mouse CLEC‐2 on leucocyte subsets varies according to their anatomical location and inflammatory state

Expression of mouse C‐type lectin‐like receptor 2 (CLEC‐2) has been reported on circulating CD11b^high Gr‐1^high myeloid cells and dendritic cells (DCs) under basal conditions, as well as on a variety of leucocyte subsets following inflammatory stimuli or in vitro cell culture. However, previous studies assessing CLEC‐2 expression failed to use CLEC‐2‐deficient mice as negative controls and instead relied heavily on single antibody clones. Here, we generated CLEC‐2‐deficient adult mice using two independent approaches and employed two anti‐mouse CLEC‐2 antibody clones to investigate surface expression on hematopoietic cells from peripheral blood and secondary lymphoid organs. We rule out constitutive CLEC‐2 expression on resting DCs and show that CLEC‐2 is upregulated in response to LPS‐induced systemic inflammation in a small subset of activated DCs isolated from the mesenteric lymph nodes but not the spleen. Moreover, we demonstrate for the first time that peripheral blood B lymphocytes present exogenously derived CLEC‐2 and suggest that both circulating B lymphocytes and CD11b^high Gr‐1^high myeloid cells lose CLEC‐2 following entry into secondary lymphoid organs. These results have significant implications for our understanding of CLEC‐2 physiological functions     

Myeloid cells expressing high level of CD45 are associated with a distinct activated phenotype in glioma

Glioblastoma multiforme is characterized by high accumulation of microglia/macrophages. The function of these tumor-infiltrating myeloid cells is not sufficiently elucidated. Therefore, a better understanding of the precise immune cell composition and function in brain tumors is required. In rodent glioma models, two different myeloid cell populations exist, determined by the expression level of CD45, namely CD11b⁺CD45^low and CD11b⁺CD45^high. Previous analyses of cytokine and marker expression profiles were almost exclusively performed on the entire myeloid cell fraction. Consequently, described pro- and anti-tumoral characteristics were not assigned to the evident subpopulations. In the present study, we used a syngeneic glioblastoma mouse model and subsequent flow cytometric analyses to demonstrate the distinct properties of CD11b⁺CD45^high and the CD11b⁺CD45^low cells. First, the majority of CD11b⁺CD45^high cells expressed high level of GR1 and around 6% of IL10 representing in part features of myeloid-derived suppressor cells, while the CD11b⁺CD45^low fraction displayed no upregulation of these molecules. Second, we detected that specifically the CD11b⁺CD45^high population showed antigen-presenting, co-stimulatory, and inflammatory features. Here, we identified up to 80% of MHCII and approximately 50% of CD86 and TNFα-expressing cells. Investigation of MHCI and CD80 revealed a moderate upregulation. By contrast, in the CD11b⁺CD45^low cell fraction, merely MHCII and TNFα were marginally overexpressed. In summary, these data emphasize the specific phenotype of CD11b⁺CD45^high cells in glioma with suppressive as well as pro-inflammatory characteristics whereas the CD11b⁺CD45^low cells were almost unaffected. Hence, primarily, the subpopulation consisting of CD45^high-expressing cells is activated by the tumor and should be considered as therapeutic target.     

Immunotherapy using lipopolysaccharide‐stimulated bone marrow‐derived dendritic cells to treat experimental autoimmune encephalomyelitis

Lipopolysaccharide (LPS) produced by Gram‐negative bacteria induces tolerance and suppresses inflammatory responses in vivo; however, the mechanisms are poorly understood. In this study we show that LPS induces apoptosis of bone marrow‐derived dendritic cells (DCs) and modulates phenotypes of DCs. LPS treatment up‐regulates expression of tolerance‐associated molecules such as CD205 and galectin‐1, but down‐regulates expression of Gr‐1 and B220 on CD11c⁺ DCs. Moreover, LPS treatment regulates the numbers of CD11c⁺CD8⁺, CD11c⁺CD11b^low and CD11c⁺CD11b^hi DCs, which perform different immune functions in vivo. Our data also demonstrated that intravenous transfer of LPS‐treated DCs blocks experimental autoimmune encephalomyelitis (EAE) development and down‐regulates expression of retinoic acid‐related orphan receptor gamma t (ROR‐γt), interleukin (IL)‐17A, IL‐17F, IL‐21, IL‐22 and interferon (IFN)‐γ in myelin oligodendrocyte glycoprotein (MOG)‐primed CD4⁺ T cells in the peripheral environment. These results suggest that LPS‐induced apoptotic DCs may lead to generation of tolerogenic DCs and suppress the activity of MOG‐stimulated effector CD4⁺ T cells, thus inhibiting the development of EAE in vivo. Our results imply a potential mechanism of LPS‐induced tolerance mediated by DCs and the possible use of LPS‐induced apoptotic DCs to treat autoimmune diseases such as multiple sclerosis.     

Secreted IL‐1α promotes T‐cell activation and expansion of CD11b+Gr1+ cells in carbon tetrachloride‐induced liver injury in mice

Interleukin‐1α is mainly expressed on the cell membrane, but can also be secreted during inflammation. The roles of secreted and membrane IL‐1α in acute liver inflammation are still not known. Here, we examined the functions of secreted and membrane IL‐1α in a mouse model of carbon tetrachloride‐induced acute liver injury. We show that secreted IL‐1α aggravates liver damage and membrane IL‐1α slightly protects mice from liver injury. Further studies showed that secreted IL‐1α promotes T‐cell activation. It also increased the expansion of CD11b⁺Gr1⁺ myeloid cells, which may serve as a negative regulator of acute liver inflammation. Moreover, secreted IL‐1α induced IL‐6 production from hepatocytes. IL‐6 neutralization reduced the proliferation of CD11b⁺Gr1⁺ myeloid cells in vivo. CCL2 and CXCL5 expression was increased by secreted IL‐1α in vitro and in vivo. Antagonists of the chemokine receptors for CCL2 and CXCL5 significantly reduced the migration of CD11b⁺Gr1⁺ myeloid cells. These results demonstrate that secreted and membrane IL‐1α play different roles in acute liver injury. Secreted IL‐1α could promote T‐cell activation and the recruitment and expansion of CD11b⁺Gr1⁺ myeloid cells through induction of CCL2, CXCL5, and IL‐6. The controlled release of IL‐1α could be a critical regulator during acute liver inflammation.     

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.     

Flt3 ligand‐eGFP‐reporter expression characterizes functionally distinct subpopulations of CD150+ long‐term repopulating murine hematopoietic stem cells

The pool of hematopoietic stem cells (HSCs) in the bone marrow is a mixture of resting, proliferating, and differentiating cells. Long‐term repopulating HSCs (LT‐HSC) are routinely enriched as Lin^?Sca1⁺c‐Kit⁺CD34^?Flt3^?CD150⁺CD48^? cells. The Flt3 ligand (Flt3L) and its receptor Flt3 are important regulators of HSC maintenance, expansion and differentiation. Using Flt3L‐eGFP reporter mice, we show that endogenous Flt3L‐eGFP‐reporter RNA expression correlates with eGFP‐protein expression. This Flt3L‐eGFP‐reporter expression distinguishes two LT‐HSC populations with differences in gene expressions and reconstituting potential. Thus, Flt3L‐eGFP‐reporter^low cells are identified as predominantly resting HSCs with long‐term repopulating capacities. In contrast, Flt3L‐eGFP‐reporter^high cells are in majority proliferating HSCs with only short‐term repopulating capacities. Flt3L‐eGFP‐reporter^low cells express hypoxia, autophagy‐inducing, and the LT‐HSC‐associated genes HoxB5 and Fgd5, while Flt3L‐eGFP‐reporter^high HSCs upregulate genes involved in HSC differentiation. Flt3L‐eGFP‐reporter^low cells develop to Flt3L‐eGFP‐reporter^high cells in vitro, although Flt3L‐eGFP‐reporter^high cells remain Flt3L‐eGFP‐reporter^high. CD150⁺Flt3L‐eGFP‐reporter^low cells express either endothelial protein C receptor (EPCR) or CD41, while Flt3L‐eGFP‐reporter^high cells do express EPCR but not CD41. Thus, FACS‐enrichment of Flt3/ Flt3L‐eGFP‐reporter negative, Lin^?CD150⁺CD48^? EPCR⁺CD41⁺ HSCs allows a further 5‐fold enrichment of functional LT‐HSCs.