Inflammation In SCD Research Articles

Hemolysis Controls Pro-Inflammatory Monocyte Priming Via Induction of and Interaction with Circadian Clock Protein Rev-Erbα in Sickle Cell Disease

Background: Patients with SCD tolerate a systemic pro-inflammatory vascular milieu created by chronic ischemia/reperfusion injury and profound erythrocyte hemolysis. In addition to this chronic low level inflammation, exposure to relatively innocuous, sub-clinical inflammatory stimuli appears to ignite an exaggerated, potentially fatal inflammatory response in patients. The etiology of this inflammatory hyper-reactivity is not well understood. There is ample evidence that, in steady state, a cadre of inflammatory cells, especially monocytes, exhibit a primed phenotype. Such priming, or propensity to activate, likely contributes to baseline inflammation, and is requisite for the inflated inflammatory response. Monocytes are quite unique amongst the leukocytes in that their inflammatory potential, including Il-6 release, is governed by the mammalian circadian clock. A role for the rhythmic oscillation of clock proteins as a controller of inflammation in SCD has never been demonstrated. However, a binding partner for heme, the nuclear receptor rev-erbα, is implicated as a regulator of clock controlled genes.Objective: To test the hypothesis that hemolysis, via heme-induced perturbation of the clock protein Rev-erbα, forms the basis for an enhanced inflammatory response in the monocyte.Methods: Intraperitoneal low dose lipopolysaccharide (LPS) was used to elicit an inflammatory response in the Townes mouse model of SCD. Plasma from the mice was acquired 6 hours after LPS injection. Analysis of 25 cytokines was accomplished using luminex methods. Monocytes were modeled in vitro using THP-1 cells. Simultaneous analysis of 84 induced inflammatory genes was conducted via qRT-PCR using the Qiagen RT Profiler PCR array. Inflammatory cytokine levels in cell supernatants were determined via ELISA.Results: We challenged the mice with low dose LPS (<10ng). Interrogation of the inflammatory cytokines in these mice revealed no change in any cytokine tested in the AA mice, but 20 out of 25 inflammatory cytokines were upregulated in mice with the SS genotype. The monocyte-based cytokines were clearly target of LPS activation in the SS mice. TNF-α and Il-1β were both upregulated 20 fold and 80 fold respectively in the SS mice. KC levels (the murine equivalent of Il-8) levels were increased 80 fold in the SS mice treated with LPS. Il-6 levels, however, were the most pronounced with a 40,000 fold increase over PBS injected SS mice. We then evaluated the role of hemolysis on monocyte inflammatory potential in vitro. Sustained monocyte exposure to physiological levels of heme in SCD alone could induce a low level of inflammatory gene expression and Il-6 release. However, sustained exposure to heme dramatically increased Il-6 release from the monocyte in response to LPS. Expression of the Il-6 gene was also increased, but the peak gene expression was time delayed compared to LPS treatment alone. In fact, we noted this phase shifting of inflammatory gene expression in the heme primed cells. LPS induced the release of significantly more TNF-α and Il-1β into the culture media in the presence of heme - consistent with the notion of heme setting a hyperactive threshold in response to LPS. We also noted that heme induced expression of the clock gene rev-erbα, and that antagonizing the activity of rev-erbα ablated the enhanced inflammatory response induced by LPS in the heme primed cells.Conclusion: These data provide evidence that hemolysis may play an important role in the hyper-inflammatory monocyte response via heme- induced dysregulation of the circadian clock. These novel observations provide entirely new avenues of anti-inflammatory therapy in SCD. DisclosuresNo relevant conflicts of interest to declare.

Association of Mu Opioid Receptor with Neurogenic Inflammation in Sickle Mic

Abstract 895Sickle cell disease (SCD) is accompanied by chronic inflammation and severe pain for which available therapies are unsatisfactory. We found that pain was accompanied by increased peripheral expression of pro-inflammatory and vasoactive neuropeptides, substance P (SP) and calcitonin gene related peptide (CGRP), and decreased expression of mu opioid receptor (MOR) in the skin of BERK mice expressing sickle hemoglobin (HbSS) compared to control HbA-BERK mice expressing normal human hemoglobin (Kohli et al., Blood 2010). Neuropeptide modulations are key features of neurogenic inflammation in chronic pain states. Tryptase released from mast cells is known to activate the release of neuropeptides from peripheral nerve endings, which in turn act on target cells including mast cells, inflammatory cells, endothelial cells and smooth muscle cells, resulting in increased vascular flow, inflammation and plasma extravasation collectively termed neurogenic inflammation. MOR influences neuropeptide release and perhaps modulates neurogenic inflammation. We hypothesize that neurogenic inflammation contributes to chronic pain and inflammation in SCD. We first examined the quantitative expression of MOR-, SP-, CGRP- and tryptase-immunoreactivity (ir) using laser scanning confocal microscopy (LSCM) in thick skin sections of BERK sickle and HbA-BERK control mice. Significantly decreased MOR-ir (p<0.001), but increased SP-, CGRP- (p<0.01 for both) and tryptase-ir (p<0.001) was observed in the skin of BERK as compared to HbA-BERK. Decreased MOR-ir co-localized with epidermal and dermal layers in the skin of BERK, but blood vessels showed robust MOR-ir in BERK similar to HbA-BERK. Tryptase-ir was more pronounced in the epidermal layer, indicative of its proximity to peripheral nerve endings. Increased tryptase co-localized with CD117/c-kit, confirming the mast cell origin. Next, we analyzed if MOR downregulation contributes to these alterations, by using mice with deletion of mu-, delta- and kappa-opioid receptors (MOR, DOR and KOR, respectively) and wild type (WT), 129S6 mice. SP-, CGRP- and tryptase-ir were increased about 2-fold in the skin of MOR, DOR and KOR knockout as compared to WT mice, suggesting that downregulation of MOR in sickle mice may contribute to increased neuropeptide and tryptase expression. Consistent with an increase in cutaneous neuropeptides, BERK and MOR knockout mice showed ∼2.5- and ∼1.5-fold increase in dorsal skin blood flow using Laser Doppler blood perfusion monitor, as compared to HBA-BERK and WT control mice, respectively. Thermography measurements revealed a correlative increase in temperature in dorsal skin of BERK as compared to HbA-BERK (p<0.05), indicative of increased inflammation. These neurochemical and physiological alterations in the skin were accompanied by activation of dorsal root ganglion (DRG) neuron and glial cells. LSCM of whole DRG revealed increased-ir for activating transcription factor-3 (ATF3), a marker of neuropathic and inflammatory insult; and increased glial fibrillary acidic protein (GFAP)-ir, a marker of inflammation, in BERK relative to HbA-BERK mice. It is likely that chronic pain and ischemia-reperfusion injury in SCD activates ATF3 in DRG leading to the release of mast cell tryptase followed by release of neuropeptides in the DRG and their ‘antidromic’ release from the nociceptive C-fibers in the skin. In addition, recurrent/chronic pain and inflammation in SCD may cause peripheral nociceptor sensitization mediated by mast cell-tryptase, and neuropeptide release from nerve endings. Nociceptor sensitization together with decrease in MOR, in turn may activate mast cell tryptase, promoting a vicious cycle between components of central nervous system and inflammation that may exacerbate neurogenic inflammation in SCD. It is likely that the pro-inflammatory neuropeptide-enhanced microenvironment has a compounding effect on inflammation and pain in SCD. We speculate that therapies targeting neuropeptides and their release mechanism(s) may potentiate the effect of analgesics and anti-inflammatory therapies for treating pain in SCD. Disclosures:No relevant conflicts of interest to declare.

Increased Levels and Activities of Matrix Metalloproteinases in Sickle Cell Disease.

Sickle cell disease is characterized by a chronic inflammatory state, however, the mechanism underlying this inflammation is unclear. Elevated basal leukocyte counts, endothelial activation, altered availability of vaso-active factors, increased adhesion molecule expression and cytokines are found in this disease. Matrix metalloproteinases (MMPs) play an important role in physiological and pathological processes, participating in the extracellular matrix turnover as well as tissue degradation, repair mechanisms and cell migration. Since MMPs are important modulators of inflammation and the activity of cytokines and chemokines, our aim was to compare MMP and tissue inhibitor of matrix metalloproteinases (TIMP; inhibitors of MMP) levels and the activities of MMPs in the plasma of healthy subjects and in SCD patients on or off HU therapy (20–30 mg/kg/day, 3 month minimum duration). Gelatin zymography was used to measure MMP-2 and MMP-9 activities and ELISA was used for MMPs and TIMPs determination. After densitometric analysis of zymograms, a significant increase (p=0.03) in the activity of pro-MMP-9 was observed in the plasma of SCD patients (27.09 ± 1.99 average pixel, n=30) compared with the control group (20.96 ± 1.35 average pixel, n=29). Pro-MMP-9 activity in the plasma of SCD patients on HU (SCDHU) was greater (27.2 ± 1.35 average pixel, n=17); however this difference was not quite significant (p=0.07). With regard to MMP-2 activity, no significant difference (P>0.05, data not shown) between groups of patients and control individuals was observed. MMP-9 levels were significantly increased in the plasma of SCD patients (20.99 ± 1.52, n=32) compared to healthy controls (13.96 ± 1.64, n=16, p=0.02), although no effect of HU therapy on these augmented levels was observed (SCDHU; 23.66 ± 2.93, n=22, P>0.05). MMP-9 levels correlated significantly with total WBC counts (r=0.4221, p=0.01) in SCD patients (on and off HU), as well as with neutrophil counts (r=0.4436, p<0.001), but not with mononuclear cell counts. TIMP-1 and TIMP-2 levels were significantly (p<0.0001) higher in SCD patients not on HU therapy (83.69 ± 6.73, n=15; 95.49 ± 4.99, n=14, respectively) compared to control individuals (41.26 ± 3.75, n=11; 62.84 ± 3.43, n=10, respectively). HU therapy had no significant effect on increased TIMP-1 levels (84.14 ± 4.69, n=12, p>0.05) when compared to SCD patients not on HU. TIMP-2 levels were higher in SCD patients treated with HU (87.62 ± 9.39, n=12) than those observed in control subjects, however this difference was not quite significant (p=0.059). In contrast, MMP-2 and MMP-8 plasma concentrations were not significantly different in SCD individuals compared to controls (P>0.05). Data demonstrate, for the first time, that the level and activity of plasma MMP-9 are significantly increased in SCD, and that these levels are not affected by HU therapy. Importantly, MMP-9 levels correlate with leukocyte counts in SCD, indicating that MMP-9 may be a useful marker for inflammation in SCD.