The pediatric immune deficiency X-linked proliferative disease-2 (XLP-2) is a unique disease, with patients presenting with either hemophagocytic lymphohistiocytosis (HLH) or intestinal bowel disease (IBD). Interestingly, XLP-2 patients display high levels of IL-18 in the serum even while in stable condition, presumably through spontaneous inflammasome activation. Recent data suggests that LPS stimulation can trigger inflammasome activation through a TNFR2/TNF/TNFR1 mediated loop in xiap−/− macrophages. Yet, the direct role TNFR2-specific activation plays in the absence of XIAP is unknown. We found TNFR2-specific activation leads to cell death in xiap−/− myeloid cells, particularly in the absence of the RING domain. RIPK1 kinase activity downstream of TNFR2 resulted in a TNF/TNFR1 cell death, independent of necroptosis. TNFR2-specific activation leads to a similar inflammatory NF-kB driven transcriptional profile as TNFR1 activation with the exception of upregulation of NLRP3 and caspase-11. Activation and upregulation of the canonical inflammasome upon loss of XIAP was mediated by RIPK1 kinase activity and ROS production. While both the inhibition of RIPK1 kinase activity and ROS production reduced cell death, as well as release of IL-1β, the release of IL-18 was not reduced to basal levels. This study supports targeting TNFR2 specifically to reduce IL-18 release in XLP-2 patients and to reduce priming of the inflammasome components.
Basophil granulocytes and mast cells are recognized for their roles in immunity and are central effectors of diverse immunological disorders. Despite their similarities, there is emerging evidence for non-redundant roles of the circulating yet scarce basophils and tissue-resident mast cells, respectively. Because of their importance in allergic pathogenesis, specific induction of apoptosis in basophils and mast cells may represent an interesting novel treatment strategy. The pro-inflammatory cytokine interleukin-3 serves as a key factor for basophil and mouse mast cell survival. Interleukin-3 increases the expression of anti-apoptotic BCL-2 family members, such as BCL-2, BCL-XL or MCL-1; however, little is known how strongly these individual proteins contribute to basophil survival. Here, we were applying small molecule inhibitors called BH3 mimetics, some of which show remarkable success in cancer treatments, to neutralize the function of anti-apoptotic BCL-2 family members. We observed that expression levels of anti-apoptotic BCL-2 proteins do not necessarily correlate with their respective importance for basophil survival. Whereas naive in vitro-differentiated mouse basophils efficiently died upon BCL-2 or BCL-XL inhibition, interleukin-3 priming rendered the cells highly resistant toward apoptosis, and this could only be overcome upon combined targeting of BCL-2 and BCL-XL. Of note, human basophils differed from mouse basophils as they depended on BCL-2 and MCL-1, but not on BCL-XL, for their survival at steady state. On the other hand, and in contrast to mouse basophils, MCL-1 proved critical in mediating survival of interleukin-3 stimulated mouse mast cells, whereas BCL-XL seemed dispensable. Taken together, our results indicate that by choosing the right combination of BH3 mimetic compounds, basophils and mast cells can be efficiently killed, even after stimulation with potent pro-survival cytokines such as interleukin-3. Because of the tolerable side effects of BH3 mimetics, targeting basophils or mast cells for apoptosis opens interesting possibilities for novel treatment approaches.
Granulocytes are central players of the immune system and, once activated, a tightly controlled balance between effector functions and cell removal by apoptosis guarantees maximal host benefit with least possible collateral damage to healthy tissue.Granulocytes are terminally differentiated cells that cannot be maintained in culture for prolonged times. Isolating primary granulocytes is inefficient and challenging when working with mice, and especially so for the lowly abundant eosinophil and basophil subtypes. Here we describe an in vitro protocol to massively expand mouse derived myeloid progenitors and to differentiate them "on demand" and in large numbers into mature neutrophils or basophils.
BH3-mimetics are small molecule inhibitors that neutralize the function of anti-apoptotic BCL-2 family members. BH3-mimetics have recently gained a lot of popularity in oncology because of their success in cancer treatment. However, BH3-mimetics might have a broader clinical application. Here, we established an ex vivo flow cytometric assay allowing the comparison of the impact of BH3-mimetics (ABT-199, ABT-263, WEHI-539, and S63845) on leukocyte populations of both, healthy human subjects and C57BL/6 J wild type mice. BH3-mimetics were added to freshly drawn blood that was diluted 1/2 in cell medium, and BH3-mimetics-mediated impact on leukocyte count was assessed by flow cytometry. Our results demonstrate that responses towards 1μM of BH3-mimetics can be identical as well as considerably different in leukocytes of humans and mice. For instance, the inhibition of BCL-2 by ABT-199 caused cell death in all types of lymphocytes in mice but was exclusively specific for B cells in humans. Moreover, inhibition of BCL-X L by WEHI-539 affected solely mouse leukocytes while targeting MCL-1 by S63845 resulted in efficient induction of cell death in human neutrophils but not in their mouse counterparts. Our ex vivo assay enables initial identification of analogies and differences between human and mouse leukocytes in response towards BH3-mimetics. Excessive cell death or evasion from apoptosis is associated with the development of autoimmune disorders or cancer 1. Apoptosis is primarily regulated by B cell lymphoma 2 (BCL-2) family members, which differ based on their ability to promote or to suppress apoptosis by affecting the integrity of the mitochondrial outer membrane (MOM). Anti-apoptotic BCL-2 family members (BCL-2, BCL-X L , BCL-W, MCL-1, BFL-1/A1) function by inhibiting their pro-apoptotic counterparts, which, upon their "unleashing", trigger MOM permeabilization followed by activation of apoptotic caspases, culminating in the ultimate, irreversible cascade towards cellular demise 2,3. Previous studies have shown that the interplay of pro-and anti-apoptotic BCL-2 members specifically regulates hematopoiesis and survival of leukocytes in blood and bone marrow 4,5. Importantly, upregulation of certain anti-apoptotic BCL-2 family members, such as BCL-2, B cell lymphoma-extra-large (BCL-X L), and myeloid cell leukemia (MCL-1), are also known to support development and survival of cancerous cells as well as evasion from cancer therapy 6. To overcome such resistance towards apoptosis, small molecule inhibitors called BCL-2 homology domain 3 (BH3)-mimetics have been developed to specifically neutralize anti-apoptotic BCL-2 family members in order to restore normal apoptotic signaling in cancerous cells 7. Most studies involving BH3-mimetics have been carried out in cancer cells. However, with some exceptions, the impact of these compounds on key leukocyte populations from healthy individuals is less clear. As the expression of individual anti-apoptotic BCL-2 family members varies substantially between different leukocyte...
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