IL-23 regulation of myeloid cell biology during inflammation.

Interleukin (IL)-23 is implicated in the pathogenesis of several inflammatory diseases and is usually linked with helper T cell (Th17) biology. However, there is some data linking IL-23 with innate immune biology in such diseases. We therefore examined the effects of IL-23p19 genetic deletion and/or neutralization on in vitro macrophage activation and in an innate immune-driven peritonitis model. We report that endogenous IL-23 was required for maximal macrophage activation by zymosan as determined by pro-inflammatory cytokine production, including a dramatic upregulation of granulocyte-colony stimulating factor (G-CSF). Furthermore, both IL-23p19 genetic deletion and neutralization in zymosan-induced peritonitis (ZIP) led to a specific reduction in the neutrophil numbers, as well as a reduction in the G-CSF levels in exudate fluids. We conclude that endogenous IL-23 can contribute significantly to macrophage activation during an inflammatory response, mostly likely via an autocrine/paracrine mechanism; of note, endogenous IL-23 can directly up-regulate macrophage G-CSF expression, which in turn is likely to contribute to the regulation of IL-23-dependent neutrophil number and function during an inflammatory response, with potential significance for IL-23 targeting particularly in neutrophil-associated inflammatory diseases.

Human chimeric antigen receptor macrophages for cancer immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy has shown promise in hematologic malignancies, but its application to solid tumors has been challenging1-4. Given the unique effector functions of macrophages and their capacity to penetrate tumors5, we genetically engineered human macrophages with CARs to direct their phagocytic activity against tumors. We found that a chimeric adenoviral vector overcame the inherent resistance of primary human macrophages to genetic manipulation and imparted a sustained pro-inflammatory (M1) phenotype. CAR macrophages (CAR-Ms) demonstrated antigen-specific phagocytosis and tumor clearance in vitro. In two solid tumor xenograft mouse models, a single infusion of human CAR-Ms decreased tumor burden and prolonged overall survival. Characterization of CAR-M activity showed that CAR-Ms expressed pro-inflammatory cytokines and chemokines, converted bystander M2 macrophages to M1, upregulated antigen presentation machinery, recruited and presented antigen to T cells and resisted the effects of immunosuppressive cytokines. In humanized mouse models, CAR-Ms were further shown to induce a pro-inflammatory tumor microenvironment and boost anti-tumor T cell activity.

Pain After Whole-Body Vibration Exposure Is Frequency Dependent and Independent of the Resonant Frequency: Lessons From an In Vivo Rat Model.

Occupational whole-body vibration (WBV) increases the risk of developing low back and neck pain; yet, there has also been an increased use of therapeutic WBV in recent years. Although the resonant frequency (fr) of the spine decreases as the exposure acceleration increases, effects of varying the vibration profile, including peak-to-peak displacement (sptp), root-mean-squared acceleration (arms), and frequency (f), on pain onset are not known. An established in vivo rat model of WBV was used to characterize the resonance of the spine using sinusoidal sweeps. The relationship between arms and fr was defined and implemented to assess behavioral sensitivity-a proxy for pain. Five groups were subjected to a single 30-min exposure, each with a different vibration profile, and a sham group underwent only anesthesia exposure. The behavioral sensitivity was assessed at baseline and for 7 days following WBV-exposure. Only WBV at 8 Hz induced behavioral sensitivity, and the higher arms exposure at 8 Hz led to a more robust pain response. These results suggest that the development of pain is frequency-dependent, but further research into the mechanisms leading to pain is warranted to fully understand which WBV profiles may be detrimental or beneficial.

The nuclear transport receptor Importin-11 is a tumor suppressor that maintains PTEN protein.

Phosphatase and tensin homologue (PTEN) protein levels are critical for tumor suppression. However, the search for a recurrent cancer-associated gene alteration that causes PTEN degradation has remained futile. In this study, we show that Importin-11 (Ipo11) is a transport receptor for PTEN that is required to physically separate PTEN from elements of the PTEN degradation machinery. Mechanistically, we find that the E2 ubiquitin-conjugating enzyme and IPO11 cargo, UBE2E1, is a limiting factor for PTEN degradation. Using in vitro and in vivo gene-targeting methods, we show that Ipo11 loss results in degradation of Pten, lung adenocarcinoma, and neoplasia in mouse prostate with aberrantly high levels of Ube2e1 in the cytoplasm. These findings explain the correlation between loss of IPO11 and PTEN protein in human lung tumors. Furthermore, we find that IPO11 status predicts disease recurrence and progression to metastasis in patients choosing radical prostatectomy. Thus, our data introduce the IPO11 gene as a tumor-suppressor locus, which is of special importance in cancers that still retain at least one intact PTEN allele.

Whole-body vibration induces pain and lumbar spinal inflammation responses in the rat that vary with the vibration profile.

Whole-body vibration (WBV) is linked epidemiologically to neck and back pain in humans, and to forepaw mechanical allodynia and cervical neuroinflammation in a rodent model of WBV, but the response of the low back and lumbar spine to WBV is unknown. A rat model of WBV was used to determine the effect of different WBV exposures on hind paw behavioral sensitivity and neuroinflammation in the lumbar spinal cord. Rats were exposed to 30 min of WBV at either 8 or 15 Hz on days 0 and 7, with the lumbar spinal cord assayed using immunohistochemistry at day 14. Behavioral sensitivity was measured using mechanical stimulation of the hind paws to determine the onset, persistence, and/or recovery of allodynia. Both WBV exposures induce mechanical allodynia 1 day following WBV, but only the 8 Hz WBV induces a sustained decrease in the withdrawal threshold through day 14. Similarly, increased activation of microglia, macrophages, and astrocytes in the superficial dorsal horn of the lumbar spinal cord is only evident after the painful 8 Hz WBV. Moreover, extracellular signal-regulated kinase (ERK)-phosphorylation is most robust in neurons and astrocytes of the dorsal horn, with the most ERK phosphorylation occurring in the 8 Hz group. These findings indicate that a WBV exposure that induces persistent pain also induces a host of neuroimmune cellular activation responses that are also sustained. This work indicates there is an injury-dependent response that is based on the vibration parameters, providing a potentially useful platform for studying mechanisms of painful spinal injuries. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1439-1446, 2016.

Whole-body Vibration at Thoracic Resonance Induces Sustained Pain and Widespread Cervical Neuroinflammation in the Rat.

BACKGROUND: Whole-body vibration (WBV) is associated with back and neck pain in military personnel and civilians. However, the role of vibration frequency and the physiological mechanisms involved in pain symptoms are unknown. QUESTIONS/PURPOSES: This study asked the following questions: (1) What is the resonance frequency of the rat spine for WBV along the spinal axis, and how does frequency of WBV alter the extent of spinal compression/extension? (2) Does a single WBV exposure at resonance induce pain that is sustained? (3) Does WBV at resonance alter the protein kinase C epsilon (PKCε) response in the dorsal root ganglia (DRG)? (4) Does WBV at resonance alter expression of calcitonin gene-related peptide (CGRP) in the spinal dorsal horn? (5) Does WBV at resonance alter the spinal neuroimmune responses that regulate pain? METHODS: Resonance of the rat (410 ± 34 g, n = 9) was measured by imposing WBV at frequencies from 3 to 15 Hz. Separate groups (317 ± 20 g, n = 10/treatment) underwent WBV at resonance (8 Hz) or at a nonresonant frequency (15 Hz). Behavioral sensitivity was assessed throughout to measure pain, and PKCε in the DRG was quantified as well as spinal CGRP, glial activation, and cytokine levels at Day 14. RESULTS: Accelerometer-based thoracic transmissibility peaks at 8 Hz (1.86 ± 0.19) and 9 Hz (1.95 ± 0.19, mean difference [MD] 0.290 ± 0.266, p < 0.03), whereas the video-based thoracic transmissibility peaks at 8 Hz (1.90 ± 0.27), 9 Hz (2.07 ± 0.20), and 10 Hz (1.80 ± 0.25, MD 0.359 ± 0.284, p < 0.01). WBV at 8 Hz produces more cervical extension (0.745 ± 0.582 mm, MD 0.242 ± 0.214, p < 0.03) and compression (0.870 ± 0.676 mm, MD 0.326 ± 0.261, p < 0.02) than 15 Hz (extension, 0.503 ± 0.279 mm; compression, 0.544 ± 0.400 mm). Pain is longer lasting (through Day 14) and more robust (p < 0.01) after WBV at the resonant frequency (8 Hz) compared with 15 Hz WBV. PKCε in the nociceptors of the DRG increases according to the severity of WBV with greatest increases after 8 Hz WBV (p < 0.03). However, spinal CGRP, cytokines, and glial activation are only evident after painful WBV at resonance. CONCLUSIONS: WBV at resonance produces long-lasting pain and widespread activation of a host of nociceptive and neuroimmune responses as compared with WBV at a nonresonance condition. Based on this work, future investigations into the temporal and regional neuroimmune response to resonant WBV in both genders would be useful. CLINICAL RELEVANCE: Although WBV is a major issue affecting the military population, there is little insight about its mechanisms of injury and pain. The neuroimmune responses produced by WBV are similar to other pain states, suggesting that pain from WBV may be mediated by similar mechanisms as other neuropathic pain conditions. This mechanistic insight suggests WBV-induced injury and pain may be tempered by antiinflammatory intervention.

Burst and Tonic Spinal Cord Stimulation Differentially Activate GABAergic Mechanisms to Attenuate Pain in a Rat Model of Cervical Radiculopathy.

OBJECTIVE: Spinal cord stimulation (SCS) is widely used to treat neuropathic pain. Burst SCS, an alternative mode of stimulation, reduces neuropathic pain without paresthesia. However, the effects and mechanisms of burst SCS have not been compared to conventional tonic SCS in controlled investigations. This study compares the attenuation of spinal neuronal activity and tactile allodynia, and the role of γ-aminobutyric acid (GABA) signaling during burst or tonic SCS in a rat model of cervical radiculopathy. METHODS: The effects of burst and tonic SCS were compared by recording neuronal firing before and after each mode of stimulation at day 7 following a painful cervical nerve root compression. Neuronal firing was also recorded before and after burst and tonic SCS in the presence of the GABAB receptor antagonist, CGP35348. RESULTS: Burst and tonic SCS both reduce neuronal firing. The effect of tonic SCS, but not burst SCS, is blocked by CGP35348. In a separate study, spinal cord stimulators were implanted to deliver burst or tonic SCS beginning on day 4 after painful nerve root compression; allodynia and serum GABA concentration were measured through day 14. Burst and tonic SCS both reduce allodynia. Tonic SCS attenuates injury-induced decreases in serum GABA, but GABA remains decreased from baseline during burst SCS. CONCLUSION AND SIGNIFICANCE: Together, these studies suggest that burst SCS does not act via spinal GABAergic mechanisms, despite its attenuation of spinal hyperexcitability and allodynia similar to that of tonic SCS; understanding other potential spinal inhibitory mechanisms may lead to enhanced analgesia during burst stimulation.

Stimulation parameters define the effectiveness of burst spinal cord stimulation in a rat model of neuropathic pain.

INTRODUCTION: Although burst spinal cord stimulation (SCS) has been reported to reduce neuropathic pain, no study has explicitly investigated how the different parameters that define burst SCS may modulate its efficacy. The effectiveness of burst SCS to reduce neuronal responses to noxious stimuli by altering stimulation parameters was evaluated in a rat model of cervical radiculopathy. METHODS: Neuronal firing was recorded in the spinal dorsal horn before and after burst SCS on day 7 following painful cervical nerve root compression (N = 8 rats). The parameters defining the stimulation (number of pulses per burst, pulse frequency, pulse width, burst frequency, amplitude) were individually varied in separate stimulation trials while holding the remaining parameters constant. The percent reduction of firing of wide-dynamic-range (WDR) and high-threshold neurons after SCS and the percentage of neurons responding to SCS were quantified for each parameter and correlated to the charge per burst delivered during stimulation. RESULTS: Pulse number, pulse width, and amplitude each were significantly correlated (p <0.009) to suppression of neuronal firing after SCS. Pulse frequency and amplitude significantly affected (p <0.05) the percentage of responsive neurons. Charge per burst was correlated to a reduction of WDR neuronal firing (p <0.03) and had a nonlinear effect on the percentage of neurons responding to burst SCS. CONCLUSIONS: Burst SCS can be optimized by adjusting relevant stimulation parameters to modulate the charge delivered to the spinal cord during stimulation. The efficacy of burst SCS is dependent on the charge per burst.

Upregulation of BDNF and NGF in cervical intervertebral discs exposed to painful whole-body vibration.

STUDY DESIGN: In vivo study defining expression of the neurotrophins, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), in cervical intervertebral discs after painful whole-body vibration (WBV). OBJECTIVE: The goal of this study is to determine if BDNF and NGF are expressed in cervical discs after painful WBV in a rat model. SUMMARY OF BACKGROUND DATA: WBV is a possible source of neck pain and has been implicated as increasing the risk for disc disorders. Typically, aneural regions of painful human lumbar discs exhibit hyperinnervation, suggesting nerve ingrowth as potentially contributing to disc degeneration and pain. BDNF and NGF are upregulated in painfully degenerate lumbar discs and hypothesized to contribute to this pathology. METHODS: Male Holtzman rats underwent 7 days of repeated WBV (15 Hz, 30 min/d) or sham exposures, followed by 7 days of rest. Cervical discs were collected for analysis of BDNF and NGF expression through RT-qPCR and Western blot analysis. Immunohistochemistry also evaluated their regional expression in the disc. RESULTS: Vibration significantly increases BDNF messenger ribonucleic acid (mRNA) levels (P=0.036), as well as total-NGF mRNA (P=0.035). Protein expression of both BDNF (P=0.006) and the 75-kDa NGF (P=0.045) increase by nearly 4- and 10-fold, respectively. Both BDNF mRNA (R=0.396; P=0.012) and protein (R=0.280; P=0.035) levels are significantly correlated with the degree of behavioral sensitivity (i.e., pain) at day 14. Total-NGF mRNA is also significantly correlated with the extent of behavioral sensitivity (R=0.276; P=0.044). Both neurotrophins are most increased in the inner annulus fibrosus and nucleus pulposus. CONCLUSION: The increases in BDNF and NGF in the cervical discs after painful vibration are observed in typically aneural regions of the disc, consistent with reports of its hyperinnervation. Yet, the induction of nerve ingrowth into the disc was not explicitly investigated. Neurotrophin expression also correlates with behavioral sensitivity, suggesting a role for both neurotrophins in the development of disc pain. LEVEL OF EVIDENCE: N/A.

Identification of PHLPP1 as a tumor suppressor reveals the role of feedback activation in PTEN-mutant prostate cancer progression.

Hyperactivation of the PI 3-kinase/AKT pathway is a driving force of many cancers. Here we identify the AKT-inactivating phosphatase PHLPP1 as a prostate tumor suppressor. We show that Phlpp1-loss causes neoplasia and, on partial Pten-loss, carcinoma in mouse prostate. This genetic setting initially triggers a growth suppressive response via p53 and the Phlpp2 ortholog, and reveals spontaneous Trp53 inactivation as a condition for full-blown disease. Surprisingly, the codeletion of PTEN and PHLPP1 in patient samples is highly restricted to metastatic disease and tightly correlated to deletion of TP53 and PHLPP2. These data establish a conceptual framework for progression of PTEN mutant prostate cancer to life-threatening disease.