Prenatal alcohol exposure promotes NLRP3 inflammasome-dependent immune actions following morphine treatment and paradoxically prolongs nerve injury-induced pathological pain in female mice.

BACKGROUND: Neuroimmune dysregulation from prenatal alcohol exposure (PAE) may contribute to neurological deficits associated with fetal alcohol spectrum disorders (FASD). PAE is a risk factor for developing peripheral immune and spinal glial sensitization and release of the proinflammatory cytokine IL-1ß, which lead to neuropathic pain (allodynia) from minor nerve injury. Although morphine acts on µ-opioid receptors, it also activates immune receptors, TLR4, and the NLRP3 inflammasome that induces IL-1ß. We hypothesized that PAE induces NLRP3 sensitization by morphine following nerve injury in adult mice. METHODS: We used an established moderate PAE paradigm, in which adult PAE and non-PAE control female mice were exposed to a minor sciatic nerve injury, and subsequent allodynia was measured using the von Frey fiber test. In control mice with standard sciatic damage or PAE mice with minor sciatic damage, the effects of the NLRP3 inhibitor, MCC950, were examined during chronic allodynia. Additionally, minor nerve-injured mice were treated with morphine, with or without MCC950. In vitro studies examined the TLR4-NLRP3-dependent proinflammatory response of peripheral macrophages to morphine and/or lipopolysaccharide, with or without MCC950. RESULTS: Mice with standard sciatic damage or PAE mice with minor sciatic damage developed robust allodynia. Blocking NLRP3 activation fully reversed allodynia in both control and PAE mice. Morphine paradoxically prolonged allodynia in PAE mice, while control mice with minor nerve injury remained stably non-allodynic. Allodynia resolved sooner in nerve-injured PAE mice without morphine treatment than in morphine-treated mice. MCC950 treatment significantly shortened allodynia in morphine-treated PAE mice. Morphine potentiated IL-1ß release from TLR4-activated PAE immune cells, while MCC950 treatment greatly reduced it. CONCLUSIONS: In female mice, PAE prolongs allodynia following morphine treatment through NLRP3 activation. TLR4-activated PAE immune cells showed enhanced IL-1ß release with morphine via NLRP3 actions. Similar studies are needed to examine the adverse impact of morphine in males with PAE. These results are predictive of adverse responses to opioid pain therapeutics in individuals with FASD.

RUNX2 regulates leukemic cell metabolism and chemotaxis in high-risk T cell acute lymphoblastic leukemia.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematologic malignancy with inferior outcome compared with that of B cell ALL. Here, we show that Runt-related transcription factor 2 (RUNX2) was upregulated in high-risk T-ALL with KMT2A rearrangements (KMT2A-R) or an immature immunophenotype. In KMT2A-R cells, we identified RUNX2 as a direct target of the KMT2A chimeras, where it reciprocally bound the KMT2A promoter, establishing a regulatory feed-forward mechanism. Notably, RUNX2 was required for survival of immature and KMT2A-R T-ALL cells in vitro and in vivo. We report direct transcriptional regulation of CXCR4 signaling by RUNX2, thereby promoting chemotaxis, adhesion, and homing to medullary and extramedullary sites. RUNX2 enabled these energy-demanding processes by increasing metabolic activity in T-ALL cells through positive regulation of both glycolysis and oxidative phosphorylation. Concurrently, RUNX2 upregulation increased mitochondrial dynamics and biogenesis in T-ALL cells. Finally, as a proof of concept, we demonstrate that immature and KMT2A-R T-ALL cells were vulnerable to pharmacological targeting of the interaction between RUNX2 and its cofactor CBFß. In conclusion, we show that RUNX2 acts as a dependency factor in high-risk subtypes of human T-ALL through concomitant regulation of tumor metabolism and leukemic cell migration.

The LFA-1 antagonist BIRT377 reverses neuropathic pain in prenatal alcohol-exposed female rats via actions on peripheral and central neuroimmune function in discrete pain-relevant tissue regions.

Previous reports show that moderate prenatal alcohol exposure (PAE) poses a risk factor for developing neuropathic pain following adult-onset peripheral nerve injury in male rats. Recently, evidence suggests that immune-related mechanisms underlying neuropathic pain in females are different compared to males despite the fact that both sexes develop neuropathy of similar magnitude and duration following chronic constriction injury (CCI) of the sciatic nerve. Data suggest that the actions of peripheral T cells play a greater role in mediating neuropathy in females. The goal of the current study is to identify specificity of immune cell and cytokine changes between PAE and non-PAE neuropathic females by utilizing a well-characterized rodent model of sciatic nerve damage, in an effort to unmask unique signatures of immune-related factors underlying the risk of neuropathy from PAE. Cytokines typically associated with myeloid cell actions such as interleukin (IL)-1ß, tumor necrosis factor (TNF), IL-6, IL-4 and IL-10 as well as the neutrophil chemoattractant CXCL1, are examined. In addition, transcription factors and cytokines associated with various differentiated T cell subtypes are examined (anti-inflammatory FOXP3, proinflammatory IL-17A, IL-21, ROR-γt, interferon (IFN)-γ and T-bet). Lymphocyte function associated antigen 1 (LFA-1) is an adhesion molecule expressed on peripheral immune cells including T cells, and regulates T cell activation and extravasation into inflamed tissue regions. A potential therapeutic approach was explored with the goal of controlling proinflammatory responses in neuroanatomical regions critical for CCI-induced allodynia by blocking LFA-1 actions using BIRT377. The data show profound development of hindpaw allodynia in adult non-PAE control females following standard CCI, but not following minor CCI, while minor CCI generated allodynia in PAE females. The data also show substantial increases in T cell-associated proinflammatory cytokine mRNA and proteins, along with evidence of augmented myeloid/glial activation (mRNA) and induction of myeloid/glial-related proinflammatory cytokines, CCL2, IL-1ß and TNF in discrete regions along the pain pathway (damaged sciatic nerve, dorsal root ganglia; DRG, and spinal cord). Interestingly, the characteristic anti-inflammatory IL-10 protein response to nerve damage is blunted in neuropathic PAE females. Moreover, T cell profiles are predominantly proinflammatory in neuropathic Sac and PAE females, augmented levels of Th17-specific proinflammatory cytokines IL-17A and IL-21, as well as the Th1-specific factor, T-bet, are observed. Similarly, the expression of RORγt, a critical transcription factor for Th17 cells, is detected in the spinal cord of neuropathic females. Blocking peripheral LFA-1 actions with intravenous (i.v.) BIRT377 reverses allodynia in Sac and PAE rats, dampens myeloid (IL-1ß, TNF, CXCL1)- and T cell-associated proinflammatory factors (IL-17A and RORγt) and spinal glial activation. Moreover, i.v. BIRT377 treatment reverses the blunted IL-10 response to CCI observed only in neuropathic PAE rats and elevates FOXP3 in pain-reversed Sac rats. Unexpectedly, intrathecal BIRT377 treatment is unable to alter allodynia in either Sac or PAE neuropathic females. Together, these data provide evidence that: 1) fully differentiated proinflammatory Th17 cells recruited at the sciatic nerve, DRGs and lumbar spinal cord may interact with the local environment to shape the immune responses underlying neuropathy in female rats, and, 2) PAE primes peripheral and spinal immune responses in adult females. PAE is a risk factor in females for developing peripheral neuropathy after minor nerve injury.

LFA-1 antagonist (BIRT377) similarly reverses peripheral neuropathic pain in male and female mice with underlying sex divergent peripheral immune proinflammatory phenotypes.

AIM: The majority of preclinical studies investigating aberrant glial-neuroimmune actions underlying neuropathic pain have focused on male rodent models. Recently, studies have shown peripheral immune cells play a more prominent role than glial cells in mediating pathological pain in females. Here, we compared the onset and duration of allodynia in males and females, and the anti-allodynic action of a potentially novel therapeutic drug (BIRT377) that not only antagonizes the action of lymphocyte function-associated antigen-1 (LFA-1) to reduce cell migration in the periphery, but may also directly alter the cellular inflammatory bias. METHODS: Male and female mice were subjected to peripheral nerve injury chronic constriction injury (CCI) applying two methods, using either 4-0 or 5-0 chromic gut suture material, to examine potential sex differences in the onset, magnitude and duration of allodynia. Hindpaw sensitivity before and after CCI and application of intravenous BIRT377 was assessed. Peripheral and spinal tissues were analyzed for protein (multiplex electrochemiluminescence technology) and mRNA expression (quantitative real-time PCR). The phenotype of peripheral T cells was determined using flow cytometry. RESULTS: Sex differences in proinflammatory CCL2 and IL-1ß and the anti-inflammatory IL-10 were observed from a set of cytokines analyzed. A profound proinflammatory T cell (Th17) response in the periphery and spinal cord was also observed in neuropathic females. BIRT377 reversed pain, reduced IL-1ß and TNF, and increased IL-10 and transforming growth factor (TGF)-ß1, also an anti-inflammatory cytokine, in both sexes. However, female-derived T cell cytokines are transcriptionally regulated by BIRT377, as demonstrated by reducing proinflammatory IL-17A production with concurrent increases in IL-10, TGF-ß1 and the anti-inflammatory regulatory T cell-related factor, FOXP3. CONCLUSION: This study supports that divergent peripheral immune and neuroimmune responses during neuropathy exists between males and females. Moreover, the modulatory actions of BIRT377 on T cells during neuropathy are predominantly specific to females. These data highlight the necessity of including both sexes for studying drug efficacy and mechanisms of action in preclinical studies and clinical trials.

111In-DANBIRT In Vivo Molecular Imaging of Inflammatory Cells in Atherosclerosis.

Atherosclerosis-related morbidity and mortality remain a global concern. Atherosclerotic disease follows a slow and silent progression, and the transition from early-stage lesions to vulnerable plaques remains difficult to diagnose. Inflammation is a key component of the development of atherosclerotic plaque and consequent life-threatening complications. This study assessed 111In-DANBIRT as an in vivo, noninvasive SPECT/CT imaging probe targeting an inflammatory marker, Lymphocyte Function Associated Antigen-1 (LFA-1), in atherosclerotic plaques. Methods. Selective binding of 111In-DANBIRT was assessed using Sprague-Dawley rats exposed to filtered air and ozone (1 ppm) by inhalation for 4 hours to induce a circulating leukocytosis and neutrophilia in peripheral blood. After 24 hours, whole blood was collected and incubated with radiolabeled DANBIRT (68Ga-DANBIRT and 111In-DANBIRT). Isolated cell component smeared slides using cytospin technique were stained with Wright-Giemsa stain. Apolipoprotein E-deficient (apoE-/-) mice were fed either a normal diet or a high-fat diet (HFD) for 8 weeks. Longitudinal SPECT/CT imaging was performed 3 hours after administration at baseline, 4, and 8 weeks of HFD diet, followed by tissue harvesting for biodistribution, serum lipid analysis, and histology. 3D autoradiography was performed in both groups 24 hours after administration of 111In-DANBIRT. Results. Increased specific uptake of radiolabeled DANBIRT by neutrophils in the ozone-exposed group was evidenced by the acute immune response due to 4-hour ozone exposure. Molecular imaging performed at 3 hours using SPECT/CT imaging evidenced an exponential longitudinal increase in 111In-DANBIRT uptake in atherosclerosis lesions in HFD-fed mice compared to normal-diet-fed mice. Such results were consistent with increased immune response to vascular injury in cardiovascular and also immune tissues, correlated by 24 hours after administration of 3D autoradiography. Histologic analysis confirmed atherosclerotic disease progression with an increased vascular lesion area in HFD-fed mice compared to normal-diet-fed mice. Conclusion. 111In-DANBIRT is a promising molecular imaging probe to assess inflammation in evolving atheroma and atherosclerotic plaque.

Improved safety and efficacy of 213Bi-DOTATATE-targeted alpha therapy of somatostatin receptor-expressing neuroendocrine tumors in mice pre-treated with L-lysine.

BACKGROUND: Targeted alpha therapy (TAT) offers advantages over current ß-emitting conjugates for peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors. PRRT with 177Lu-DOTATATE or 90Y-DOTATOC has shown dose-limiting nephrotoxicity due to radiopeptide retention in the proximal tubules. Pharmacological protection can reduce renal uptake of radiopeptides, e.g., positively charged amino acids, to saturate in the proximal tubules, thereby enabling higher radioactivity to be safely administered. The aim of this preclinical study was to evaluate the therapeutic effect of 213Bi-DOTATATE with and without renal protection using L-lysine in mice. Tumor uptake and kinetics as a function of injected mass of peptide (range 0.03-3 nmol) were investigated using 111In-DOTATATE. These results allowed estimation of the mean radiation absorbed tumor dose for 213Bi-DOTATATE. Pharmacokinetics and dosimetry of 213Bi-DOTATATE was determined in mice, in combination with renal protection. A dose escalation study with 213Bi-DOTATATE was performed to determine the maximum tolerated dose (MTD) with and without pre-administration of L-lysine as for renal protection. Neutrophil gelatinase-associated lipocalin (NGAL) served as renal biomarker to determine kidney injury. RESULTS: The maximum mean radiation absorbed tumor dose occurred at 0.03 nmol and the minimum at 3 nmol. Similar mean radiation absorbed tumor doses were determined for 0.1 and 0.3 nmol with a mean radiation absorbed dose of approximately 0.5 Gy/MBq 213Bi-DOTATATE. The optimal mass of injected peptide was found to be 0.3 nmol. Tumor uptake was similar for 111In-DOTATATE and 213Bi-DOTATATE at 0.3 nmol peptide. Lysine reduced the renal uptake of 213Bi-DOTATATE by 50% with no effect on the tumor uptake. The MTD was <13.0 ± 1.6 MBq in absence of L-lysine and 21.7 ± 1.9 MBq with L-lysine renal protection, both imparting an LD50 mean renal radiation absorbed dose of 20 Gy. A correlation was found between the amount of injected radioactivity and NGAL levels. CONCLUSIONS: The therapeutic potential of 213Bi-DOTATATE was illustrated by significantly decreased tumor burden and improved overall survival. Renal protection with L-lysine immediately prior to TAT with 213Bi-DOTATATE prolonged survival providing substantial evidence for pharmacological nephron blockade to mitigate nephrotoxicity.

Muscle RING finger-1 promotes a maladaptive phenotype in chronic hypoxia-induced right ventricular remodeling.

Exposure to chronic hypoxia (CH) induces elevated pulmonary artery pressure/resistance, leading to an eventual maladaptive right ventricular hypertrophy (RVH). Muscle RING finger-1 (MuRF1) is a muscle-specific ubiquitin ligase that mediates myocyte atrophy and has been shown to play a role in left ventricular hypertrophy and altered cardiac bioenergetics in pressure overloaded hearts. However, little is known about the contribution of MuRF1 impacting RVH in the setting of CH. Therefore, we hypothesized that MuRF1 deletion would enhance RVH compared to their wild-type littermates, while cardiac-specific overexpression would reduce hypertrophy following CH-induced pulmonary hypertension. We assessed right ventricular systolic pressure (RVSP), right ventricle to left ventricle plus septal weight ratio (RV/LV+S) and hematocrit (Hct) following a 3-wk isobaric CH exposure. Additionally, we conducted dual-isotope SPECT/CT imaging with cardiac function agent 201Tl-chloride and cell death agent 99mTc-annexin V. Predictably, CH induced pulmonary hypertension, measured by increased RVSP, RV/LV+S and Hct in WT mice compared to normoxic WT mice. Normoxic WT and MuRF1-null mice exhibited no significant differences in RVSP, RV/LV+S or Hct. CH-induced increases in RVSP were also similar between WT and MuRF1-null mice; however, RV/LV+S and Hct were significantly elevated in CH-exposed MuRF1-null mice compared to WT. In cardiac-specific MuRF1 overexpressing mice, RV/LV+S increased significantly due to CH exposure, even greater than in WT mice. This remodeling appeared eccentric, maladaptive and led to reduced systemic perfusion. In conclusion, these results are consistent with an atrophic role for MuRF1 regulating the magnitude of right ventricular hypertrophy following CH-induction of pulmonary hypertension.