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In dynamic systems like the circulatory system,establishing localized cytokine gradients is challenging. Upon lipopolysaccharide (LPS) stimulation,we observed that monocytes release numerous migrasomes enriched with inflammatory cytokines,such as TNF-α and IL-6. These cytokines are transported into migrasomes via secretory carriers,leading to their immediate exocytosis or eventual release from detached migrasomes. We successfully isolated TNF-α and IL-6-enriched,monocyte-derived migrasomes from the blood of LPS-treated mice. Total secretion analysis revealed a substantial amount of TNF-α and IL-6 released in a migrasome-packaged form. Thus,detached,monocyte-derived migrasomes represent a type of extracellular vesicle highly enriched with cytokines. Physiologically,these cytokine-laden migrasomes rapidly accumulate at local sites of inflammation,effectively creating a concentrated source of cytokines. Our research uncovers novel mechanisms for cytokine release and delivery,providing new insights into immune response modulation. View Publication

The persistence of HIV-1 in long-lived latent reservoirs during suppressive antiretroviral therapy (ART) remains one of the principal barriers to a functional cure. Blocks to transcriptional elongation play a central role in maintaining the latent state,and several latency reversal strategies focus on the release of positive transcription elongation factor b (P-TEFb) from sequestration by negative regulatory complexes,such as the 7SK complex and BRD4. Another major cellular reservoir of P-TEFb is in Super Elongation Complexes (SECs),which play broad regulatory roles in host gene expression. Still,it is unknown if the release of P-TEFb from SECs is a viable latency reversal strategy. Here,we demonstrate that the SEC is not required for HIV-1 replication in primary CD4+ T cells and that a small molecular inhibitor of the P-TEFb/SEC interaction (termed KL-2) increases viral transcription. KL-2 acts synergistically with other latency reversing agents (LRAs) to reactivate viral transcription in several cell line models of latency in a manner that is,at least in part,dependent on the viral Tat protein. Finally,we demonstrate that KL-2 enhances viral reactivation in peripheral blood mononuclear cells (PBMCs) from people living with HIV (PLWH) on suppressive ART,most notably in combination with inhibitor of apoptosis protein antagonists (IAPi). Taken together,these results suggest that the release of P-TEFb from cellular SECs may be a novel route for HIV-1 latency reactivation. Author summarySince the start of the HIV pandemic,it is estimated that nearly 86 million people have been infected with the virus,and about 40 million people have died. Modern antiretroviral therapies potently restrict viral replication and prevent the onset of AIDS,saving millions of lives. However,these therapies are not curative due to the persistence of the virus in a silenced or ‘latent’ state in long-lived cells of the body. One proposed strategy to clear this latent reservoir,termed “shock and kill”,is to activate these silenced viruses such that the infected cells can be cleared from the body by the immune system. While several drugs have been developed that can activate latent viruses,none have proven effective at reducing the size of the latent reservoir in patients in clinical trials. Here,we describe a new method for latency reactivation using a small molecule inhibitor of a human protein complex called the Super Elongation Complex (SEC). Inhibiting the SEC enhances viral transcription during active infection and triggers the reactivation of latent viruses,especially when in combination with other latency reversing agents. These results pave the way for developing more effective strategies to reactivate latent viruses towards a functional cure. View Publication

Glatiramer acetate (GA or Copaxone) is a drug used to treat experimental autoimmune encephalomyelitis in mice and multiple sclerosis in human. Here,we describe a new mechanism of action for this drug. GA enhanced the cytolysis of human NK cells against autologous and allogeneic immature and mature monocyte-derived dendritic cells (DCs). This drug reduced the percentages of mature DCs expressing CD80,CD83,HLA-DR or HLA-I. In contrast,it did not modulate the percentages of NK cells expressing NKG2D,NKp30,or NKp44. Nonetheless,anti-NKp30 or anti-CD86 inhibited GA-enhanced human NK cell lysis of immature DCs. Hence,CD86,and NKp30 are important for NK cell lysis of immature DCs,whereas CD80,CD83,HLA-DR and HLA-I are important for the lysis of mature DCs when GA is used as a stimulus. Further,GA inhibited the release of IFN-gamma 24 h but increased the release of TNF-alpha 48 h after incubation with NK cells. View Publication

The therapeutic monoclonal antibody (mAb) TGN1412 (anti-CD28 superagonist) caused near-fatal cytokine release syndrome (CRS) in all six volunteers during a phase-I clinical trial. Several cytokine release assays (CRAs) with reported predictivity for TGN1412-induced CRS have since been developed for the preclinical safety testing of new therapeutic mAbs. The whole blood (WB) CRA is the most widely used,but its sensitivity for TGN1412-like cytokine release was recently criticized. In a comparative study,using group size required for 90% power with 5% significance as a measure of sensitivity,we found that WB and 10% (v/v) WB CRAs were the least sensitive for TGN1412 as these required the largest group sizes (n = 52 and 79,respectively). In contrast,the peripheral blood mononuclear cell (PBMC) solid phase (SP) CRA was the most sensitive for TGN1412 as it required the smallest group size (n = 4). Similarly,the PBMC SP CRA was more sensitive than the WB CRA for muromonab-CD3 (anti-CD3) which stimulates TGN1412-like cytokine release (n = 4 and 4519,respectively). Conversely,the WB CRA was far more sensitive than the PBMC SP CRA for alemtuzumab (anti-CD52) which stimulates FcγRI-mediated cytokine release (n = 8 and 180,respectively). Investigation of potential factors contributing to the different sensitivities revealed that removal of red blood cells (RBCs) from WB permitted PBMC-like TGN1412 responses in a SP CRA,which in turn could be inhibited by the addition of the RBC membrane protein glycophorin A (GYPA); this observation likely underlies,at least in part,the poor sensitivity of WB CRA for TGN1412. The use of PBMC SP CRA for the detection of TGN1412-like cytokine release is recommended in conjunction with adequately powered group sizes for dependable preclinical safety testing of new therapeutic mAbs. View Publication

Leukocyte trafficking to sites of inflammation follows a defined temporal pattern,and evidence suggests that initial neutrophil transendothelial migration modifies endothelial cell phenotype. We tested the hypothesis that preconditioning of human umbilical vein endothelial cells (HUVEC) by neutrophils would also modify the subsequent transendothelial migration of T lymphocytes across cytokine-stimulated HUVEC in an in vitro flow assay. Using fluorescence microscopy,preconditioning of HUVEC by neutrophils was observed to significantly reduce the extent of subsequent stromal cell-derived factor-1alpha (SDF-1alpha [CXCL12])-mediated T lymphocyte transendothelial migration,without reducing accumulation. In contrast,recruitment of a second wave of neutrophils was unaltered. Conditioned medium harvested after transendothelial migration of neutrophils or supernatants from stimulated neutrophils mediated a similar blocking effect,which was negated using a specific neutrophil elastase inhibitor. Furthermore,T lymphocyte transendothelial migration was inhibited by treatment of HUVEC with purified neutrophil elastase,which selectively cleaved the amino terminus of HUVEC-bound SDF-1alpha,which is required for its chemotactic activity. The reduction in T lymphocyte transendothelial migration was not observed using a different chemokine,ELC (CCL19),and was not reversed by replenishment of SDF-1alpha,indicating endothelial retention of the inactivated chemokine. In summary,transmigrating neutrophils secrete localized elastase that is protected from plasma inhibitors,and thereby modulate trafficking of other leukocyte subsets by altering the endothelial-associated chemotactic activities. View Publication

Squalene-based emulsion (SE) adjuvants like MF59 and AS03 are used in protein subunit vaccines against influenza virus (e.g.,Fluad,Pandemrix,Arepanrix) and SARS-CoV-2 (e.g.,Covifenz,SKYCovione). We demonstrate the critical role of uric acid (UA),a damage-associated molecular pattern (DAMP),in triggering immunogenicity by SE adjuvants. In mice,SE adjuvants elevated DAMP levels in draining lymph nodes. Strikingly,inhibition of UA synthesis reduced vaccine-induced innate immunity,subsequently impairing optimal antibody and T cell responses. In vivo treatment with UA crystals elicited partial adjuvant effects. In vitro stimulation with UA crystals augmented the activation of dendritic cells (DCs) and B cells and altered multiple pathways in these cells,including inflammation and antigen presentation in DCs and cell proliferation in B cells. In an influenza vaccine model,UA contributed to protection against influenza viral infection. These results demonstrate the importance of DAMPs,specifically the versatile role of UA in the immunogenicity of SE adjuvants,by regulating DCs and B cells. View Publication

Peroxynitrite toxicity is a major cause of neuronal injury in stroke and neurodegenerative disorders. The mechanisms underlying the neurotoxicity induced by peroxynitrite are still unclear. In this study,we observed that TPEN [N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine],a zinc chelator,protected against neurotoxicity induced by exogenous as well as endogenous (coadministration of NMDA and a nitric oxide donor,diethylenetriamine NONOate) peroxynitrite. Two different approaches to detecting intracellular zinc release demonstrated the liberation of zinc from intracellular stores by peroxynitrite. In addition,we found that peroxynitrite toxicity was blocked by inhibitors of 12-lipoxygenase (12-LOX),p38 mitogen-activated protein kinase (MAPK),and caspase-3 and was associated with mitochondrial membrane depolarization. Inhibition of 12-LOX blocked the activation of p38 MAPK and caspase-3. Zinc itself induced the activation of 12-LOX,generation of reactive oxygen species (ROS),and activation of p38 MAPK and caspase-3. These data suggest a cell death pathway triggered by peroxynitrite in which intracellular zinc release leads to activation of 12-LOX,ROS accumulation,p38 activation,and caspase-3 activation. Therefore,therapies aimed at maintaining intracellular zinc homeostasis or blocking activation of 12-LOX may provide a novel avenue for the treatment of inflammation,stroke,and neurodegenerative diseases in which the formation of peroxynitrite is thought to be one of the important causes of cell death. View Publication

SorLA,encoded by the gene SORL1,is an intracellular sorting receptor of the VPS10P domain receptor gene family. Although SorLA is best recognized for its ability to shuttle target proteins between intracellular compartments in neurons,recent data suggest that also its microglial expression can be of high relevance for the pathogenesis of brain diseases,including glioblastoma (GBM). Here,we interrogated the impact of SorLA on the functional properties of glioma-associated microglia and macrophages (GAMs). In the GBM microenvironment,GAMs are re-programmed and lose the ability to elicit anti-tumor responses. Instead,they acquire a glioma-supporting phenotype,which is a key mechanism promoting glioma progression. Our re-analysis of published scRNA-seq data from GBM patients revealed that functional phenotypes of GAMs are linked to the level of SORL1 expression,which was further confirmed using in vitro models. Moreover,we demonstrate that SorLA restrains secretion of TNFα from microglia to restrict the inflammatory potential of these cells. Finally,we show that loss of SorLA exacerbates the pro-inflammatory response of microglia in the murine model of glioma and suppresses tumor growth. View Publication