HIV elite control is associated with reduced TRAILshort expression.

OBJECTIVE: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) dependent apoptosis has been implicated in CD4 T-cell death and immunologic control of HIV-1 infection. We have described a splice variant called TRAILshort, which is a dominant negative ligand that antagonizes TRAIL-induced cell death in the context of HIV-1 infection. HIV-1 elite controllers naturally control viral replication for largely unknown reasons. Since enhanced death of infected cells might be responsible, as might occur in situations of low (or inhibited) TRAILshort, we tested whether there was an association between elite controller status and reduced levels of TRAILshort expression. DESIGN: Cohort study comparing TRAILshort and full length TRAIL expression between HIV-1 elite controllers and viremic progressors from two independent populations. METHODS: TRAILshort and TRAIL gene expression in peripheral blood mononuclear cells (PBMCs) was determined by RNA-seq. TRAILshort and TRAIL protein expression in plasma was determined by antibody bead array and proximity extension assay respectively. RESULTS: HIV-1 elite controllers expressed less TRAILshort transcripts in PBMCs (P = 0.002) and less TRAILshort protein in plasma (P < 0.001) than viremic progressors. CONCLUSION: Reduced TRAILshort expression in PBMCs and plasma is associated with HIV-1 elite controller status.

TRAILshort Protects against CD4 T Cell Death during Acute HIV Infection.

CD4 T cells from HIV-1 infected patients die at excessive rates compared to those from uninfected patients, causing immunodeficiency. We previously identified a dominant negative ligand that antagonizes the TRAIL-dependent pathway of cell death, which we called TRAILshort. Because the TRAIL pathway has been implicated in CD4 T cell death occurring during HIV-1 infection, we used short hairpin RNA knockdown, CRISPR deletion, or Abs specific for TRAILshort to determine the effect of inhibiting TRAILshort on the outcome of experimental acute HIV infection in vitro. Strikingly, all three approaches to TRAILshort deletion/inhibition enhanced HIV-induced death of both infected and uninfected human CD4 T cells. Thus, TRAILshort impacts T cell dynamics during HIV infection, and inhibiting TRAILshort causes more HIV-infected and uninfected bystander cells to die. TRAILshort is, therefore, a host-derived, host-adaptive mechanism to limit the effects of TRAIL-induced cell death. Further studies on the effects of TRAILshort in other disease states are warranted.

HIV Protease-Generated Casp8p41, When Bound and Inactivated by Bcl2, Is Degraded by the Proteasome.

HIV protease is known to cause cell death, which is dependent upon cleavage of procaspase 8. HIV protease cleavage of procaspase 8 generates Casp8p41, which directly binds Bak with nanomolar affinity, causing Bak activation and consequent cell death. Casp8p41 can also bind Bcl2 with nanomolar affinity, in which case cell death is averted. Central memory CD4 T cells express high levels of Bcl2, possibly explaining why those cells do not die when they reactivate HIV. Here, we determine that the Casp8p41-Bcl2 complex is polyubiquitinated and degraded by the proteasome. Ixazomib, a proteasome inhibitor in clinical use, blocks this pathway, increasing the abundance of Casp8p41 and causing more cells to die in a Casp8p41-dependent manner.IMPORTANCE The Casp8p41 pathway of cell death is unique to HIV-infected cells yet is blocked by Bcl2. Once bound by Bcl2, Casp8p41 is polyubiquitinated and degraded by the proteasome. Proteasome inhibition blocks degradation of Casp8p41, increasing Casp8p41 levels and causing more HIV-infected cells to die.

Both HIV-Infected and Uninfected Cells Express TRAILshort, Which Confers TRAIL Resistance upon Bystander Cells within the Microenvironment.

TNF-related apoptosis-inducing ligand (TRAIL) was initially described to induce apoptosis of tumor cells and/or virally infected cells, although sparing normal cells, and has been implicated in the pathogenesis of HIV disease. We previously identified TRAILshort, a TRAIL splice variant, in HIV-infected patients and characterized it as being a dominant negative ligand to subvert TRAIL-mediated killing. Herein, using single-cell genomics we demonstrate that TRAILshort is produced by HIV-infected cells, as well as by uninfected bystander cells, and that the dominant stimulus which induces TRAILshort production are type I IFNs and TLR7, TLR8, and TLR9 agonists. TRAILshort has a short t1/2 by virtue of containing a PEST domain, which targets the protein toward the ubiquitin proteasome pathway for degradation. Further we show that TRAILshort binds preferentially to TRAIL receptors 1 and 2 with significantly reduced interaction with the decoy TRAIL receptors 3 and 4. Recombinant TRAILshort is sufficient to protect cells against TRAIL-induced killing, whereas immunodepletion of TRAILshort with a specific Ab restores TRAIL sensitivity. Importantly we show that TRAILshort is shed in microvesicles into the cellular microenvironment and therefore confers TRAIL resistance not only on the cell which produces it, but also upon neighboring bystander cells. These results establish a novel paradigm for understanding and overcoming TRAIL resistance, in particular how HIV-infected cells escape immune elimination by the TRAIL:TRAILshort receptor axis.

Increasing procaspase 8 expression using repurposed drugs to induce HIV infected cell death in ex vivo patient cells.

HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018) without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches.

Prime, Shock, and Kill: Priming CD4 T Cells from HIV Patients with a BCL-2 Antagonist before HIV Reactivation Reduces HIV Reservoir Size.

UNLABELLED: Understanding how some HIV-infected cells resist the cytotoxicity of HIV replication is crucial to enabling HIV cure efforts. HIV killing of CD4 T cells that replicate HIV can involve HIV protease-mediated cleavage of procaspase 8 to generate a fragment (Casp8p41) that directly binds and activates the mitochondrial proapoptotic protein BAK. Here, we demonstrate that Casp8p41 also binds with nanomolar affinity to the antiapoptotic protein Bcl-2, which sequesters Casp8p41 and prevents apoptosis. Further, we show that central memory CD4 T cells (TCM) from HIV-infected individuals have heightened expression of BCL-2 relative to procaspase 8, possibly explaining the persistence of HIV-infected TCMdespite generation of Casp8p41. Consistent with this hypothesis, the selective BCL-2 antagonist venetoclax induced minimal killing of uninfected CD4 T cells but markedly increased the death of CD4 T cells and diminished cell-associated HIV DNA when CD4 T cells from antiretroviral therapy (ART)-suppressed HIV patients were induced with αCD3/αCD28 to reactivate HIVex vivo Thus, priming CD4 T cells from ART suppressed HIV patients with a BCL-2 antagonist, followed by HIV reactivation, achieves reductions in cell-associated HIV DNA, whereas HIV reactivation alone does not. IMPORTANCE: HIV infection is incurable due to a long-lived reservoir of HIV(+)memory CD4 T cells, and no clinically relevant interventions have been identified that reduce the number of these HIV DNA-containing cells. Since postintegration HIV replication can result in HIV protease generation of Casp8p41, which activates BAK, causing infected CD4 T cell death, we sought to determine whether this occurs in memory CD4 T cells. Here, we demonstrate that memory CD4 T cells can generate Casp8p41 and yet are intrinsically resistant to death induced by diverse stimuli, including Casp8p41. Furthermore, BCL-2 expression is relatively increased in these cells and directly binds and inhibits Casp8p41's proapoptotic effects. Antagonizing BCL-2 with venetoclax derepresses this antagonism, resulting in death, preferentially in HIV DNA containing cells, since only these cells generate Casp8p41. Thus, BCL-2 antagonism is a clinically relevant intervention with the potential to reduce HIV reservoir size in patients.

Casp8p41 generated by HIV protease kills CD4 T cells through direct Bak activation.

Previous studies have shown that human immunodeficiency virus (HIV) protease cleaves procaspase 8 to a fragment, termed Casp8p41, that lacks caspase activity but nonetheless contributes to T cell apoptosis. Herein, we show that Casp8p41 contains a domain that interacts with the BH3-binding groove of pro-apoptotic Bak to cause Bak oligomerization, Bak-mediated membrane permeabilization, and cell death. Levels of active Bak are higher in HIV-infected T cells that express Casp8p41. Conversely, targeted mutations in the Bak-interacting domain diminish Bak binding and Casp8p41-mediated cell death. Similar mutations in procaspase 8 impair the ability of HIV to kill infected T cells. These observations support a novel paradigm in which HIV converts a normal cellular constituent into a direct activator that functions like a BH3-only protein.

Choice of antiretroviral therapy differentially impacts survival of HIV-infected CD4 T cells.

BACKGROUND: HIV eradication strategies are now being evaluated in vitro and in vivo. A cornerstone of such approaches is maximal suppression of viral replication with combination antiretroviral therapy (ART). Since many antiretroviral agents have off target effects, and different classes target different components of the viral life cycle, we questioned whether different classes of ART might differentially affect the survival and persistence of productively HIV-infected CD4 T cells. METHODS: In vitro infections of primary CD4 T cells using clinical isolates of HIV-1 that were either protease inhibitor susceptible (HIV PI-S), or resistant (HIV PI-R) were treated with nothing, lopinavir, efavirenz or raltegravir. Cell viability, apoptosis, and the proportion of surviving cells that were P24 positive was assessed by flow cytometry. RESULTS: In HIV PI-S infected primary cultures, all three antiretroviral agents decreased viral replication, and reduced the total number of cells that were undergoing apoptosis (P < 0.01) similarly. Similarly, in the HIV PI-R infected cultures, both efavirenz and raltegravir reduced viral replication and reduced apoptosis compared to untreated control (P < 0.01), while lopinavir did not, suggesting that HIV replication drives T cell apoptosis, which was confirmed by association by linear regression (P < 0.0001) . However since HIV protease has been suggested to directly induce apoptosis of infected CD4 T cells, and HIV PI are intrinsically antiapoptotic, we evaluated apoptosis in productively infected (HIV P24+) cells. More HIV p24 positive cells were apoptotic in the Efavirenz or raltegravir treated cultures than the lopinavir treated cultures (P = 0.0008 for HIV PI-R and P = 0.06 for the HIV PI-S), indicating that drug class impacts survival of productively infected CD4 T cells. CONCLUSIONS: Inhibiting HIV replication with a PI, NNRTI or INSTI reduces total HIV-induced T cell apoptosis. However, blocking HIV replication with PI but not with NNRTI or INSTI promotes survival of productively HIV-infected cells. Thus, selection of antiretroviral agents may impact the success of HIV eradication strategies.

SDF-1α degrades whereas glycoprotein 120 upregulates Bcl-2 interacting mediator of death extralong isoform: implications for the development of T cell memory.

After a primary immune response, T cell memory occurs when a subset of Ag-specific T cells resists peripheral selection by acquiring resistance to TCR-induced death. Recent data have implicated Bcl-2 interacting mediator of death (Bim) as an essential mediator of the contraction phase of T cell immunity. In this article, we describe that stromal-derived factor-1α (SDF-1α) ligation of CXCR4 on activated T cells promotes two parallel processes that favor survival, phospho-inactivation of Foxo3A, as well as Bim extralong isoform (Bim(EL)) degradation, both in an Akt- and Erk-dependent manner. Activated primary CD4 T cells treated with SDF-1α therefore become resistant to the proapoptotic effects of TCR ligation or IL-2 deprivation and accumulate cells of a memory phenotype. Unlike SDF-1α, gp120 ligation of CXCR4 has the opposite effect because it causes p38-dependent Bim(EL) upregulation. However, when activated CD4 T cells are treated with both gp120 and SDF-1α, the SDF-1α-driven effects of Bim(EL) degradation and acquired resistance to TCR-induced death predominate. These results provide a novel causal link between SDF-1α-induced chemotaxis, degradation of Bim(EL), and the development of CD4 T cell memory.

TRAIL dependent fratricidal killing of gp120 primed hepatocytes by HCV core expressing hepatocytes.

The mechanism by which HIV and HCV cooperatively accelerate hepatocyte damage is not clearly understood; however, each virus affects the TRAIL: TRAIL-receptor system. We, therefore, questioned whether the independent effects of HCV and HIV combine to synergistically result in TRAIL dependent hepatocyte killing. We describe that Huh7 hepatocytes treated with HIV gp120 results in both increase TRAIL-R2 expression and an acquired sensitivity to TRAIL mediated killing. Moreover HCV infection and HCV core expression alone in Huh7 cells upregulates TRAIL. Co-incubation of HIV gp120 primed hepatocytes with HCV core expressing hepatocytes results in the selective death of the HIV gp120 primed hepatocytes that is selectively blocked by TRAIL-R2-Fc fusion protein. Liver biopsies from HIV mono-infected patients have increased TRAIL-R2; biopsies from HCV infected patients have increased TRAIL, while co-infected liver biopsies have increased PARP cleavage within hepatocytes indicating enhanced apoptosis. These findings suggest a pathogenic model to understand why HIV/HCV co-infection accelerates liver injury.

Human immunodeficiency virus envelope protein Gp120 induces proliferation but not apoptosis in osteoblasts at physiologic concentrations.

Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05), which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism.

Isolation of a TRAIL antagonist from the serum of HIV-infected patients.

Virus-host interactions are characterized by the selection of adaptive mechanisms by which to evade pathogenic and defense mechanisms, respectively. In primary T cells infected with HIV, HIV infection up-regulates TNF-related apoptosis inducing ligand (TRAIL) and death-inducing TRAIL receptors, but blockade of TRAIL:TRAIL receptor interaction does not alter HIV-induced cell death. Instead, HIV infection results in a novel splice variant that we call TRAIL-short (TRAIL-s), which antagonizes TRAIL-R2. In HIV patients, plasma TRAIL-s concentration increases with increasing viral load and renders cells resistant to TRAIL-induced death. Knockdown of TRAIL-s abrogates this resistance. We propose that TRAIL-s is a novel adaptive mechanism of apoptosis resistance acquired by HIV-infected cells to avoid their elimination by TRAIL-dependent effector mechanism.

The HIV-1-specific protein Casp8p41 induces death of infected cells through Bax/Bak.

Casp8p41, a novel protein generated when HIV-1 protease cleaves caspase 8, independently causes NF-κB activation, proinflammatory cytokine production, and cell death. Here we investigate the mechanism by which Casp8p41 induces cell death. Immunogold staining and electron microscopy demonstrate that Casp8p41 localizes to mitochondria of activated primary CD4 T cells, suggesting mitochondrial involvement. Therefore, we assessed the dependency of Casp8p41-induced death on Bax/Bak and caspase 9. In wild-type (WT) mouse embryonic fibroblast (MEF) cells, Casp8p41 causes rapid mitochondrial depolarization (P < 0.001), yet Casp8p41 expression in Bax/Bak double-knockout (DKO) MEF cells does not. Similarly, caspase 9-deficient T cells (JMR cells), which express Casp8p41, undergo minimal cell death, whereas reconstituting these cells with caspase 9 (F9 cells) restores Casp8p41 cytotoxicity (P < 0.01). The infection of caspase 9-deficient cells with a green fluorescent protein (GFP) HIV-1 reporter virus results in cell death in 32% of infected GFP-positive cells, while the restoration of caspase 9 expression in these cells restores infected-cell killing to 68% (P < 0.05), with similar levels of viral replication between infections. Our data demonstrate that Casp8p41 requires Bax/Bak to induce mitochondrial depolarization, which leads to caspase 9 activation following either Casp8p41 expression or HIV-1 infection. This understanding allows the design of strategies to interrupt this form of death of HIV-1-infected cells.

Patients with discordant responses to antiretroviral therapy have impaired killing of HIV-infected T cells.

In medicine, understanding the pathophysiologic basis of exceptional circumstances has led to an enhanced understanding of biology. We have studied the circumstance of HIV-infected patients in whom antiretroviral therapy results in immunologic benefit, despite virologic failure. In such patients, two protease mutations, I54V and V82A, occur more frequently. Expressing HIV protease containing these mutations resulted in less cell death, caspase activation, and nuclear fragmentation than wild type (WT) HIV protease or HIV protease containing other mutations. The impaired induction of cell death was also associated with impaired cleavage of procaspase 8, a requisite event for HIV protease mediated cell death. Primary CD4 T cells expressing I54V or V82A protease underwent less cell death than with WT or other mutant proteases. Human T cells infected with HIV containing these mutations underwent less cell death and less Casp8p41 production than WT or HIV containing other protease mutations, despite similar degrees of viral replication. The reductions in cell death occurred both within infected cells, as well as in uninfected bystander cells. These data indicate that single point mutations within HIV protease which are selected in vivo can significantly impact the ability of HIV to kill CD4 T cells, while not impacting viral replication. Therefore, HIV protease regulates both HIV replication as well as HIV induced T cell depletion, the hallmark of HIV pathogenesis.

CXCR4 Tropic HIV-1 gp120 Inhibition of SDF-1α-Induced Chemotaxis Requires Lck and is Associated with Cofilin Phosphorylation.

OBJECTIVE: HIV gp120 is a pleiotropic protein present in the plasma and tissues of HIV-infected patients, which affects a variety of homeostatic functions. In this report, we examine the mechanism of how gp120 blocks CD4 T cells from migrating towards SDF-1α. METHODS: In vitro treatment of primary CD4 T cells with CXCR4 tropic gp120, SDF, and measurement of chemotaxis and cell signaling. RESULTS: gp120 signaling through CD4 receptor and Lck are required for its ability to inhibit chemotaxis induced by SDF, as demonstrated by CD4 receptor decoys, Lck inhibitors, as well as cells deficient in Lck, in which Lck expression is restored. Blocking Lck abrogates the ability of CXCR4 tropic gp120 to antagonize SDF-1α-induced chemotaxis. This inhibition is associated with cofilin phosphorylation, thereby providing a potential mechanism. CONCLUSION: We conclude that the ability of gp120 to inhibit SDF-1α-induced chemotaxis is mediated by the CD4 receptor and Lck signaling, potentially by promoting cofilin phosphorylation.

Intracellular Casp8p41 content is inversely associated with CD4 T cell count.

Casp8p41 is a protein fragment generated by cleavage of procaspase 8 by human immunodeficiency virus (HIV) protease. We measured Casp8p41 content in memory CD4 T cells and analyzed the association of Casp8p41 content with CD4 T cell count, cross-sectionally and longitudinally. Casp8p41 content was inversely correlated with CD4 T cell count, and change in Casp8p41 content was associated with absolute CD4 T cell count with change over time. Casp8p41 change was a better predictor of CD4 T cell count change than activated CD8 T cell percentage or viral load and was comparable to bacterial 16s DNA levels. This suggests that Casp8p41 is a relevant mediator of CD4 T cell death during HIV infection.

Casp8p41 expression in primary T cells induces a proinflammatory response.

OBJECTIVE: HIV infection of CD4 T cells can lead to HIV protease-mediated cleavage of procaspase 8 generating a novel, HIV-specific peptide called Casp8p41. Casp8p41 has at least two biologic functions: induction of cell death via mitochondrial depolarization and release of cytochrome C, as well as activation of nuclear factor kappa B (NFkappaB). We have previously shown that Casp8p41-induced NFkappaB activation enhances HIV LTR transcription and consequently increases HIV replication. Herein, we questioned whether Casp8p41-induced NFkappaB activation impacts the cytokine profile of cells expressing Casp8p41. DESIGN: Analysis of cells expressing Casp8p41 and HIV-infected T cells. METHODS: We assessed whether host genes are transcriptionally activated following Casp8p41 production, using microarray analysis, cytokine quantification, followed by western blot and flow cytometry. RESULTS: Microarray analysis identified 259 genes significantly upregulated following expression of Casp8p41. Furthermore, Casp8p41 expression in primary CD4 T cells results in increased production of interleukin (IL)-2, IL-15 and tumor necrosis factor (TNF), as well as IL-1RA; whereas levels of granulocyte macrophage colony-stimulating factor and interferon (IFN)-gamma were reduced in the Casp8p41 expressing cells. Intracellular flow cytometry confirmed the co-association of Casp8p41 with elevated TNF in HIV-infected cells. CONCLUSION: These data indicate that the expression of Casp8p41 in HIV-infected CD4 T cells in addition to promoting apoptosis and enhancing HIV replication also promotes a proinflammatory cytokine milieu, which is characteristic of untreated HIV infection.

CD4 T Cells Treated with gp120 Acquire a CD45R0+/CD45RA+ Phenotype.

HIV-infected patients exhibit quantitative and qualitative defects in CD4 T cells, including having increased numbers of CD4+CD45R0+/CD45RA+ T cells, although it remains unclear how these cells arise. Here we demonstrate that gp120 treatment of activated but not resting primary human CD4 T cells decreases number of cells with single positive CD45R0+/CD45RA- effector memory phenotype while proportionally increasing the subset of cells with double positive CD45R0+/CD45RA+ mixed phenotype. We found that double positive CD45R0+/CD45RA+CD4 T cells preferentially undergo apoptosis while single positive CD45R0+/CD45RA- and CD45R0-/CD45RA+ do not. Blocking gp120-CD4 interaction with sCD4 or inhibition Lck activity reverses gp120 induced increase in double positive CD45R0+/CD45RA+CD4 T cells and subsequently diminishes the apoptosis of double positive CD45R0+/CD45RA+ cells. Altogether these data indicate that gp120 ligation of the CD4 receptor increases the number of double positive CD45R0+/CD45RA+ CD4 T cells which subsequently undergo apoptosis in a CD4 dependent manner.

Nelfinavir/ritonavir reduces acinar injury but not inflammation during mouse caerulein pancreatitis.

There is no clinical treatment that reduces acinar injury during pancreatitis. Human immunodeficiency virus (HIV) protease inhibitors (PI), including nelfinavir (NFV) and ritonavir (RTV), may reduce the rate of pancreatitis in HIV-infected patients. Since permeability transition pore (PTPC)-mediated mitochondrial dysfunction occurs during pancreatitis, and we have shown that PI prevents PTPC opening, we studied its effects in a model of pancreatitis. The effect of NFV plus RTV (NFV/RTV) or vehicle on caerulein-induced pancreatitis in mice was compared by measuring changes in mitochondrial membrane potential in vitro and cytochrome c leakage in vivo. Histological and inflammatory makers were also compared. NFV/RTV improved DiOC6 retention in acini exposed to caerulein in vitro. In vivo NFV prevented cytosolic leakage of cytochrome c and reduced pancreatic acinar injury, active caspase-3 staining, TUNEL-positive acinar cells, and serum amylase (P < 0.05). Conversely, trypsin activity, serum cytokine levels, and pancreatic and lung inflammation were unaffected. NFV/RTV reduces pancreatic injury and acinar cell death in experimental mouse caerulein-induced pancreatitis but does not impact inflammation.