Molecular characteristics and possible functions of innate lymphoid cells in the uterus and gut

With the discovery of innate lymphoid cells (ILCs), which are especially enriched in barrier surfaces, the family of innate lymphocytes has grown. A unique characterization of these cells can provide a phenotypical definition of ILCs and their specific functions in different tissue environments. Although ILCs are part of the innate immune system, they are derived from lymphoid lineages lacking rearranged antigen-specific and pattern-recognition receptors. The International Union of Immunological Societies (IUIS) favors the notion that ILCs can be generally divided into five main groups, namely, NK cells, ILC1s, ILC2s, ILC3s and LTi cells. These cells can be specifically stimulated by environmental and pathogen-derived signals. Upon stimulation, ILCs can rapidly secrete a wide range of soluble cytokines that can modulate the functions of effector cells. Over the last decade, ILCs, especially helper ILCs, which do not include NK cells, have been recognized to be a crucial cell type involved in integrating diverse host immune responses. Recently, emerging research has shown that helper ILCs also play a critical role in promoting tissue restoration and immune responses at barrier surfaces. Notably, helper ILCs act as a double-edged sword, being involved in the inflammatory and reparative responses during homeostasis and disease. Therefore, in this review, we summarize the current findings regarding the molecular characteristics and tissue-specific effector functions of helper ILCs in the uterus during physiological and pathological pregnancy and in the intestine during homeostasis and inflammation.     

CD1d‐mediated activation of group 3 innate lymphoid cells drives IL‐22 production

Innate lymphoid cells (ILCs) are a heterogeneous family of immune cells that play a critical role in a variety of immune processes including host defence against infection, wound healing and tissue repair. Whether these cells are involved in lipid‐dependent immunity remains unexplored. Here we show that murine ILCs from a variety of tissues express the lipid‐presenting molecule CD1d, with group 3 ILCs (ILC3s) showing the highest level of expression. Within the ILC3 family, natural cytotoxicity triggering receptor (NCR)^?CCR6⁺ cells displayed the highest levels of CD1d. Expression of CD1d on ILCs is functionally relevant as ILC3s can acquire lipids in vitro and in vivo and load lipids on CD1d to mediate presentation to the T‐cell receptor of invariant natural killer T (iNKT) cells. Conversely, engagement of CD1d in vitro and administration of lipid antigen in vivo induce ILC3 activation and production of IL‐22. Taken together, our data expose a previously unappreciated role for ILCs in CD1d‐mediated immunity, which can modulate tissue homeostasis and inflammatory responses.     

Rocaglamide enhances NK cell-mediated killing of non-small cell lung cancer cells by inhibiting autophagy

Targeting macroautophagy/autophagy is a novel strategy in cancer immunotherapy. In the present study, we showed that the natural product rocaglamide (RocA) enhanced natural killer (NK) cell-mediated lysis of non-small cell lung cancer (NSCLC) cells in vitro and tumor regression in vivo. Moreover, this effect was not related to the NK cell recognition of target cells or expressions of death receptors. Instead, RocA inhibited autophagy and restored the level of NK cell-derived GZMB (granzyme B) in NSCLC cells, therefore increasing their susceptibility to NK cell-mediated killing. In addition, we further identified that the target of RocA was ULK1 (unc-51 like autophagy activating kinase 1) that is required for autophagy initiation. Using firefly luciferase containing the 5´ untranslated region of ULK1, we found that RocA inhibited the protein translation of ULK1 in a sequence-specific manner. Taken together, RocA could block autophagic immune resistance to NK cell-mediated killing, and our data suggested that RocA was a promising therapeutic candidate in NK cell-based cancer immunotherapy.     

Excess sphingomyelin disturbs ATG9A trafficking and autophagosome closure

Sphingomyelin is an essential cellular lipid that traffics between plasma membrane and intracellular organelles until directed to lysosomes for SMPD1 (sphingomyelin phosphodiesterase 1)-mediated degradation. Inactivating mutations in the SMPD1 gene result in Niemann-Pick diseases type A and B characterized by sphingomyelin accumulation and severely disturbed tissue homeostasis. Here, we report that sphingomyelin overload disturbs the maturation and closure of autophagic membranes. Niemann-Pick type A patient fibroblasts and SMPD1-depleted cancer cells accumulate elongated and unclosed autophagic membranes as well as abnormally swollen autophagosomes in the absence of normal autophagosomes and autolysosomes. The immature autophagic membranes are rich in WIPI2, ATG16L1 and MAP1LC3B but display reduced association with ATG9A. Contrary to its normal trafficking between plasma membrane, intracellular organelles and autophagic membranes, ATG9A concentrates in transferrin receptor-positive juxtanuclear recycling endosomes in SMPD1-deficient cells. Supporting a causative role for ATG9A mistrafficking in the autophagy defect observed in SMPD1-deficient cells, ectopic ATG9A effectively reverts this phenotype. Exogenous C12-sphingomyelin induces a similar juxtanuclear accumulation of ATG9A and subsequent defect in the maturation of autophagic membranes in healthy cells while the main sphingomyelin metabolite, ceramide, fails to revert the autophagy defective phenotype in SMPD1-deficient cells. Juxtanuclear accumulation of ATG9A and defective autophagy are also evident in tissues of smpd1-deficient mice with a subsequent inability to cope with kidney ischemia-reperfusion stress. These data reveal sphingomyelin as an important regulator of ATG9A trafficking and maturation of early autophagic membranes.     

Defective early innate immune response to ectromelia virus in the draining lymph nodes of aged mice due to impaired dendritic cell accumulation

It is known that aging decreases natural resistance to viral diseases due to dysfunctional innate and adaptive immune responses, but the nature of these dysfunctions, particularly in regard to innate immunity, is not well understood. We have previously shown that C57BL/6J (B6) mice lose their natural resistance to footpad infection with ectromelia virus (ECTV) due to impaired maturation and recruitment of natural killer (NK) cells to the draining popliteal lymph node (dLN). More recently, we have also shown that in young B6 mice infected with ECTV, the recruitment of NK cells is dependent on a complex cascade whereby migratory dendritic cells (mDCs) traffic from the skin to the dLN, where they produce CCL2 and CCL7 to recruit inflammatory monocytes (iMOs). In the dLN, mDCs also upregulate NKG2D ligands to induce interferon gamma (IFN‐γ) expression by group 1 innate lymphoid cells (G1‐ILCs), mostly NK in cells but also some ILC1. In response to the IFN‐γ, the incoming uninfected iMOs secret CXCL9 to recruit the critical NK cells. Here, we show that in aged B6 mice, the trafficking of mDCs to the dLN in response to ECTV is decreased, resulting in impaired IFN‐γ expression by G1‐ILCs, reduced accumulation of iMOs, and attenuated CXCL9 production by iMOs, which likely contributes to decrease in NK cell recruitment. Together, these data indicate that defects in the mDC response to viral infection during aging result in a reduced innate immune response in the dLN and contribute to increased susceptibility to viral disease in the aged.     

In vivo characterization of transplanted human embryonic stem cell‐derived pancreatic endocrine islet cells

Abstract. Objectives: Islet‐like clusters (ILCs), differentiated from human embryonic stem cells (hESCs), were characterized both before and after transplantation under the kidney capsule of streptozotocin‐induced diabetic immuno‐incompetent mice. Materials and methods: Multiple independent ILC preparations (n = 8) were characterized by immunohistochemistry, flow cytometry and cell insulin content, with six preparations transplanted into diabetic mice (n = 42), compared to controls, which were transplanted with either a human fibroblast cell line or undifferentiated hESCs (n = 28). Results: Prior to transplantation, ILCs were immunoreactive for the islet hormones insulin, C‐peptide and glucagon, and for the ductal epithelial marker cytokeratin‐19. ILCs also had cellular insulin contents similar to or higher than human foetal islets. Expression of islet and pancreas‐specific cell markers was maintained for 70 days post‐transplantation. The mean survival of recipients was increased by transplanted ILCs as compared to transplanted human fibroblast cells (P < 0.0001), or undifferentiated hESCs (P < 0.042). Graft function was confirmed by secretion of human C‐peptide in response to an oral bolus of glucose. Conclusions: hESC‐derived ILC grafts continued to contain cells that were positive for islet endocrine hormones and were shown to be functional by their ability to secrete human C‐peptide. Further enrichment and maturation of ILCs could lead to generation of a sufficient source of insulin‐producing cells for transplantation into patients with type 1 diabetes.     

Autophagic flux,supported by toll-like receptor 2 activity,defends against the carcinogenesis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the most common primary malignant liver tumor, is the third leading cause of cancer deaths. The pathogenesis of HCC is closely associated with chronic liver inflammation fired by a variety of stimulates such as virus infection and metabolic stress. Recent work indicates that autophagy, a homeostatic self-degradation process, which decides cell survival or death upon stress, acts as an effector machinery of immune systems in defending microbial invasion and carcinogenesis. SQSTM1 is a selective target and receptor of autophagy, and the protein content of SQSTM1 reflects the level of autophagic flux in cells. Through degrading SQSTM1, decreasing SQSTM1 aggregates, and therefore interrupting the positive feedback between SQSTM1 aggregates and ROS production, autophagy plays a protective role against hepatocellular carcinoma. Indeed, our studies indicate that toll-like receptor 2 (TLR2)-mediated immune activities in the genotoxic carcinogen diethylnitrosamine (DEN)-injured liver tissue provide essential nutrient stimulates to induce intracellular senescence, which can ensure the activation and maturation of autophagy in liver cells. Loss of TLR2-mediated immune activity and senescence leads to the attenuation of autophagic flux, which cannot eliminate SQSTM1 aggregates, ROS accumulation, and DNA damage, and facilitates the development and progression of HCC.     

Autophagy impairment with lysosomal and mitochondrial dysfunction is an important characteristic of oxidative stress-induced senescence

Macroautophagy/autophagy has profound implications for aging. However, the true features of autophagy in the progression of aging remain to be clarified. In the present study, we explored the status of autophagic flux during the development of cell senescence induced by oxidative stress. In this system, although autophagic structures increased, the degradation of SQSTM1/p62 protein, the yellow puncta of mRFP-GFP-LC3 fluorescence and the activity of lysosomal proteolytic enzymes all decreased in senescent cells, indicating impaired autophagic flux with lysosomal dysfunction. The influence of autophagy activity on senescence development was confirmed by both positive and negative autophagy modulators; and MTOR-dependent autophagy activators, rapamycin and PP242, efficiently suppressed cellular senescence through a mechanism relevant to restoring autophagic flux. By time-phased treatment of cells with the antioxidant N-acetylcysteine (NAC), the mitochondria uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and ambroxol, a reagent with the effect of enhancing lysosomal enzyme maturation, we found that mitochondrial dysfunction plays an initiating role, while lysosomal dysfunction is more directly responsible for autophagy impairment and senescence. Interestingly, the effect of rapamycin on autophagy flux is linked to its role in functional revitalization of both mitochondrial and lysosomal functions. Together, this study demonstrates that autophagy impairment is crucial for oxidative stress-induced cell senescence, thus restoring autophagy activity could be a promising way to retard senescence.     

Autophagy modulates cell migration and β1 integrin membrane recycling

Cell migration is dependent on a series of integrated cellular events including the membrane recycling of the extracellular matrix receptor integrins. In this paper, we investigate the role of autophagy in regulating cell migration. In a wound-healing assay, we observed that autophagy was reduced in cells at the leading edge than in cells located rearward. These differences in autophagy were correlated with the robustness of MTOR activity. The spatial difference in the accumulation of autophagic structures was not detected in rapamycin-treated cells, which had less migration capacity than untreated cells. In contrast, the knockdown of the autophagic protein ATG7 stimulated cell migration of HeLa cells. Accordingly, atg3^?/? and atg5^?/? MEFs have greater cell migration properties than their wild-type counterparts. Stimulation of autophagy increased the co-localization of β1 integrin-containing vesicles with LC3-stained autophagic vacuoles. Moreover, inhibition of autophagy slowed down the lysosomal degradation of internalized β1 integrins and promoted its membrane recycling. From these findings, we conclude that autophagy regulates cell migration, a central mechanism in cell development, angiogenesis, and tumor progression, by mitigating the cell surface expression of β1 integrins.     

A novel method for autophagy detection in primary cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8⁺ T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.     

A novel method for autophagy detection in primary cells: impaired levels of macroautophagy in immunosenescent T cells

Autophagy is a conserved constitutive cellular process, responsible for the degradation of dysfunctional proteins and organelles. Autophagy plays a role in many diseases such as neurodegeneration and cancer; however, to date, conventional autophagy detection techniques are not suitable for clinical samples. We have developed a high throughput, statistically robust technique that quantitates autophagy in primary human leukocytes using the Image stream, an imaging flow cytometer. We validate this method on cell lines and primary cells knocked down for essential autophagy genes. Also, using this method we show that T cells have higher autophagic activity than B cells. Furthermore our results indicate that healthy primary senescent CD8(+) T cells have decreased autophagic levels correlating with increased DNA damage, which may explain features of the senescent immune system and its declining function with age. This technique will allow us, for the first time, to measure autophagy levels in diseases with a known link to autophagy, while also determining the contribution of autophagy to the efficacy of drugs.     

Autophagy, nutrition and immunology

Turnover of cellular components in lysosomes or autophagy is an essential mechanism for cellular quality control. Added to this cleaning role, autophagy has recently been shown to participate in the dynamic interaction of cells with the surrounding environment by acting as a point of integration of extracellular cues. In this review, we focus on the relationship between autophagy and two types of environmental factors: nutrients and pathogens. We describe their direct effect on autophagy and discuss how the autophagic reaction to these stimuli allows cells to accommodate the requirements of the cellular response to stress, including those specific to the immune responses.     

Presence of innate lymphoid cells in allogeneic hematopoietic grafts correlates with reduced graft-versus-host disease

BackgroundAllogeneic hematopoietic cell transplantation (HCT) can be devastating when graft-versus-host disease (GvHD) develops. GvHD is characterized by mucosal inflammation due to breaching of epithelial barriers. Innate lymphoid cells (ILCs) are immune modulatory cells that are important in the maintenance of epithelial barriers, via their production of interleukin (IL)-22 and their T cell suppressive properties. After chemo- and radiotherapy, ILCs are depleted, and recovery after remission-induction therapy and after allogeneic HCT is slow and incomplete in a significant number of patients, which is associated with an increased risk to develop acute GvHD.ObjectiveTo investigate whether the presence of mature ILCs within G-CSF–mobilized HCT grafts is correlated with the development of acute GvHD after allogeneic HCT.Study DesignWe analyzed ILCs in a cohort of 36 patients who received allogeneic HCT for a hematologic malignancy, by flow-cytometric immune-phenotyping of prospectively collected, cryopreserved peripheral blood mononuclear cells (PBMCs) and donor-derived HCT grafts collected for the same patients. Biased analysis, with ILCs defined as CD3^?lineage^?CD45⁺CD127⁺CD161⁺ lymphocytes, was performed using FlowJo version 10 software. Unbiased analysis was done using FlowSOM, which uses a self-organizing map (SOM) with a minimal spanning tree (MST) to define and visualize different clusters present in the samples.ResultsRemission-induction therapy significantly depleted ILCs from the blood, and patients who had a relatively low percentage of ILCs before allogeneic HCT were significantly more prone to develop acute GvHD, confirming previous findings in a separate cohort. Allogeneic HCT grafts, which were all obtained from the blood of G-CSF–mobilized healthy donors, contained ILCs at a frequency very similar to the peripheral blood of healthy individuals. The ILC subset composition was also comparable to that of the blood of healthy individuals, with the exception of NKp44⁺ ILC3s, which were significantly more abundant in HCT grafts. The relative ILC content of the graft tended to correlate with ILC reconstitution after allogeneic HCT, suggesting that peripheral expansion of transplanted mature ILCs may contribute to early ILC reconstitution after allogeneic HCT. Patients who received a relatively ILC-poor HCT graft had a significantly increased risk to develop acute GvHD, compared with patients who received relatively ILC-rich allogeneic HCT grafts. Unbiased phenotypic analysis with the FlowSOM algorithm confirmed that allogeneic HCT grafts of patients who developed acute GvHD contained a lower frequency of ILCs that clustered in NKp44⁺ ILC3 signature groups.ConclusionThe presence of ILCs in allogeneic HCT grafts is associated with a reduced risk to develop acute GvHD. These data suggest that enhancement of ILC reconstitution of ILC3s in particular, for example via adoptive transfer of ILCs, may prevent acute GvHD and has the potential to improve outcome of allogeneic HCT recipients.     

Autophagic cell death: Loch Ness monster or endangered species?

The concept of autophagic cell death was first established based on observations of increased autophagic markers in dying cells. The major limitation of such a morphology-based definition of autophagic cell death is that it fails to establish the functional role of autophagy in the cell death process, and thus contributes to the confusion in the literature regarding the role of autophagy in cell death and cell survival. Here we propose to define autophagic cell death as a modality of non-apoptotic or necrotic programmed cell death in which autophagy serves as a cell death mechanism, upon meeting the following set of criteria: (i) cell death occurs without the involvement of apoptosis; (ii) there is an increase of autophagic flux, and not just an increase of the autophagic markers, in the dying cells; and (iii) suppression of autophagy via both pharmacological inhibitors and genetic approaches is able to rescue or prevent cell death. In light of this new definition, we will discuss some of the common problems and difficulties in the study of autophagic cell death and also revisit some well-reported cases of autophagic cell death, aiming to achieve a better understanding of whether autophagy is a real killer, an accomplice or just an innocent bystander in the course of cell death. At present, the physiological relevance of autophagic cell death is mainly observed in lower eukaryotes and invertebrates

such as Dictyostelium discoideum and Drosophila melanogaster. We believe that such a clear definition of autophagic cell death will help us study and understand the physiological or pathological relevance of autophagic cell death in mammals.