Taehyung Lee

Probiotic L. reuteri Treatment Prevents Bone Loss in a Menopausal Ovariectomized Mouse Model

Estrogen deficiency is a major risk factor for osteoporosis that is associated with bone inflammation and resorption. Half of women over the age of 50 will experience an osteoporosis related fracture in their lifetime, thus novel therapies are needed to combat post‐menopausal bone loss. Recent studies suggest an important role for gut‐bone signaling pathways and the microbiota in regulating bone health. Given that the bacterium Lactobacillus reuteri ATCC PTA 6475 (L. reuteri) secretes beneficial immunomodulatory factors, we examined if this candidate probiotic could reduce bone loss associated with estrogen deficiency in an ovariectomized (Ovx) mouse menopausal model. Strikingly, L. reuteri treatment significantly protected Ovx mice from bone loss. Osteoclast bone resorption markers and activators (Trap5 and RANKL) as well as osteoclastogenesis are significantly decreased in L. reuteri‐treated mice. Consistent with this, L. reuteri suppressed Ovx‐induced increases in bone marrow CD4+ T‐lymphocytes (which promote osteoclastogenesis) and directly suppressed osteoclastogenesis in vitro. We also identified that L. reuteri treatment modifies microbial communities in the Ovx mouse gut. Together, our studies demonstrate that L. reuteri treatment suppresses bone resorption and loss associated with estrogen deficiency. Thus, L. reuteri treatment may be a straightforward and cost‐effective approach to reduce post‐menopausal bone loss. J. Cell. Physiol. 229: 1822–1830, 2014.

G-protein coupled receptor kinase 5 mediates lipopolysaccharide-induced NFκB activation in primary macrophages and modulates inflammation in vivo in mice.

G‐protein coupled receptor kinase‐5 (GRK5) is a serine/threonine kinase discovered for its role in the regulation of G‐protein coupled receptor signaling. Recent studies have shown that GRK5 is also an important regulator of signaling pathways stimulated by non‐GPCRs. This study was undertaken to determine the physiological role of GRK5 in Toll‐like receptor‐4‐induced inflammatory signaling pathways in vivo and in vitro. Using mice genetically deficient in GRK5 (GRK5−/−) we demonstrate here that GRK5 is an important positive regulator of lipopolysaccharide (LPS, a TLR4 agonist)‐induced inflammatory cytokine and chemokine production in vivo. Consistent with this role, LPS‐induced neutrophil infiltration in the lungs (assessed by myeloperoxidase activity) was markedly attenuated in the GRK5−/− mice compared to the GRK5+/+ mice. Similar to the in vivo studies, primary macrophages from GRK5−/− mice showed attenuated cytokine production in response to LPS. Our results also identify TLR4‐induced NFκB pathway in macrophages to be selectively regulated by GRK5. LPS‐induced IκBα phosphorylation, NFκB p65 nuclear translocation, and NFκB binding were markedly attenuated in GRK5−/− macrophages. Together, our findings demonstrate that GRK5 is a positive regulator of TLR4‐induced IκBα–NFκB pathway as well as a key modulator of LPS‐induced inflammatory response. J. Cell. Physiol. 226: 1323–1333, 2011.

Colitis-induced Bone Loss is Gender Dependent and Associated with Increased Inflammation

Background: Patients with inflammatory bowel disease (IBD) are at increase risk for bone loss and fractures. Therefore, in the present study, we examined the effect of experimental IBD on bone health. Methods: We used a murine model of colitis, Helicobacter hepaticus–infected interleukin-10–deficient animals. Molecular and histological properties of bone and intestine were examined to identify the immunopathological consequences of colitis in male and female mice. Results: At 6 weeks postinfection, we observed significant trabecular bone loss in male mice but surprisingly not in female mice. This was true for both distal femur and vertebral locations. In addition, H. hepaticus infection suppressed osteoblast markers only in male mice. Consistent with effects on bone health, male mice with H. hepaticus infection had more severe colitis as determined by histology and elevated levels of inflammatory cytokines in the colon. Although H. hepaticus levels in the stool appeared similar in male and female mice 1 week after infection, by 6 weeks, H. hepaticus levels were greater in male mice, indicating that H. hepaticus survival and virulence within the gastrointestinal tract could be gender dependent. Conclusion: In summary, H. hepaticus–induced colitis severity and associated bone loss is gender regulated, possibly as a result of gender-specific effects on H. hepaticus colonization in the mouse gastrointestinal tract and the consequent immunopathological responses.

G-Protein-Coupled Receptor Kinase-5 Mediates Inflammation but Does Not Regulate Cellular Infiltration or Bacterial Load in a Polymicrobial Sepsis Model in Mice

NFκB-dependent signaling is an important modulator of inflammation in several diseases including sepsis. G-protein-coupled receptor kinase-5 (GRK5) is an evolutionarily conserved regulator of the NFκB pathway. We hypothesized that GRK5 via NFκB regulation plays an important role in the pathogenesis of sepsis. To test this we utilized a clinically relevant polymicrobial sepsis model in mice that were deficient in GRK5. We subjected wild-type (WT) and GRK5 knockout (KO) mice to cecal ligation and puncture (CLP)-induced polymicrobial sepsis and assessed the various events in sepsis pathogenesis. CLP induced a significant inflammatory response in the WT and this was markedly attenuated in the KO mice. To determine the signaling mechanisms and the role of NFκB activation in sepsis-induced inflammation, we assessed the levels of IκBa phosphorylation and expression of NFκB-dependent genes in the liver in the two genotypes. Both IκBa phosphorylation and gene expression were significantly inhibited in the GRK5 KO compared to the WT mice. Interestingly, however, GRK5 did not modulate either immune cell infiltration (to the primary site of infection) or local/systemic bacterial load subsequent to sepsis induction. In contrast GRK5 deficiency significantly inhibited sepsis-induced plasma corticosterone levels and the consequent thymocyte apoptosis in vivo. Associated with these outcomes, CLP-induced mortality was significantly prevented in the GRK5 KO mice in the presence of antibiotics. Together, our studies demonstrate that GRK5 is an important regulator of inflammation and thymic apoptosis in polymicrobial sepsis and implicate GRK5 as a potential molecular target in sepsis.

Lithium Chloride Induces TNFα in Mouse Macrophages Via MEK–ERK‐Dependent Pathway

Lithium (Li) is one of the currently prescribed drugs for bipolar disorders (BPDs) and has many neuro‐regulatory and immune‐modulating properties. Because many neuro‐pathological diseases including BPDs have been associated with some level of inflammation, Lis effect on inflammation may have some crucial consequences. Even though Li has been shown to have pro‐ and anti‐inflammatory activities in different cell models, mechanisms involved in these effects are not well understood. Moreover, Lis effect on inflammation in the presence of activators of Toll‐like receptors (TLRs), especially TLR‐2 (that activates MyD88‐dependent pathway) and TLR‐3 (that activates TRIF‐dependent pathway) is not known. Here we tested the role of Li in the presence and absence of TLR2, and TLR3 on MAPK and NFκB pathways and the consequent production of tumor necrosis factor‐α (TNFα) in Raw264.7 macrophages. Our results indicate that Li enhances TNFα production both in the absence and presence of TLR stimulation. Interestingly, Li differentially modulates MAPK and NFκB pathways in the absence and presence of TLR2/3 ligands. Our results further indicate that the effect of Li on TNFα occurs at the post‐transcriptional level. Together, these studies demonstrate that Li induces TNFα production in macrophages and that it modulates signaling at different levels depending on the presence or absence of TLR2/3 stimulation. J. Cell. Biochem. 115: 71–80, 2014.

Non-Hematopoietic β-Arrestin1 Confers Protection Against Experimental Colitis

β‐Arrestins are multifunctional scaffolding proteins that modulate G protein‐coupled receptor (GPCR)‐dependent and ‐independent cell signaling pathways in various types of cells. We recently demonstrated that β‐arrestin1 (β‐arr1) deficiency strikingly attenuates dextran sodium sulfate (DSS)‐induced colitis in mice. Since DSS‐induced colitis is in part dependent on gut epithelial injury, we examined the role of β‐arr1 in intestinal epithelial cells (IECs) using a colon epithelial cell line, SW480 cells. Surprisingly, we found that knockdown of β‐arr1 in SW480 cells enhanced epithelial cell death via a caspase‐3‐dependent process. To understand the in vivo relevance and potential cell type‐specific role of β‐arr1 in colitis development, we generated bone marrow chimeras with β‐arr1 deficiency in either the hematopoietic or non‐hematopoietic compartment. Reconstituted chimeric mice were then subjected to DSS‐induced colitis. Similar to our previous findings, β‐arr1 deficiency in the hematopoietic compartment protected mice from DSS‐induced colitis. However, consistent with the role of β‐arr1 in epithelial apoptosis in vitro, non‐hematopoietic β‐arr1 deficiency led to an exacerbated colitis phenotype. To further understand signaling mechanisms, we examined the effect of β‐arr1 on TNF‐α‐mediated NFκB and MAPK pathways. Our results demonstrate that β‐arr1 has a critical role in modulating ERK, JNK and p38 MAPK pathways mediated by TNF‐α in IECs. Together, our results show that β‐arr1‐dependent signaling in hematopoietic and non‐hematopoietic cells differentially regulates colitis pathogenesis and further demonstrates that β‐arr1 in epithelial cells inhibits TNF‐α‐induced cell death pathways. J. Cell. Physiol. 231: 992–1000, 2016.

Role of G protein-coupled receptor kinase-6 in Escherichia coli lung infection model in mice

G protein-coupled receptor kinase-6 (GRK6) is a serine/threonine kinase that is important in inflammatory processes. In this study, we examined the role of GRK6 in Escherichia coli-induced lung infection and inflammation using GRK6 knockout (KO) and wild-type (WT) mice. Intratracheal instillation of E. coli significantly enhanced bacterial load in the bronchoalveolar lavage (BAL) of KO compared with WT mice. Reduced bacterial clearance in the KO mice was not due to an intrinsic defect in neutrophil phagocytosis or killing but as a result of reduced neutrophil numbers in the KO BAL. Interestingly, neutrophil numbers in the lung were increased in the KO compared with WT mice, suggesting a potential dysfunction in transepithelial migration of neutrophils from the lungs to the bronchoalveolar space. This effect was selective for lung tissue because peritoneal neutrophil numbers were similar between the two genotypes following peritoneal infection. Although neutrophil expression of CXCR2/CXCR3 was similar between WT and KO, IL-17A expression was higher in the KO compared with WT mice. These results suggest that enhanced neutrophil count in the KO lungs but reduced numbers in BAL are likely due to transepithelial migration defect and/or altered chemokines/cytokines. Together, our studies suggest a previously unrecognized and novel role for GRK6 in neutrophil migration specific to pulmonary tissue during bacterial infection.

G-Protein Coupled Receptor Kinase-2 Deficient Mice are Protected from Dextran Sodium Sulfate-Induced Acute Colitis

G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase and plays a key role in different disease processes. Previously, we showed that GRK2 knockdown enhances wound healing in colonic epithelial cells. Therefore, we hypothesized that ablation of GRK2 would protect mice from dextran sodium sulfate (DSS)-induced acute colitis. To test this, we administered DSS to wild-type (GRK2+/+) and GRK2 heterozygous (GRK+/-) mice in their drinking water for 7 days. As predicted, GRK2+/- mice were protected from colitis as demonstrated by decreased weight loss (20% loss in GRK2+/+ vs. 11% loss in GRK2+/-). lower disease activity index (GRK2+/+ 9.1 vs GRK2+/- 4.1), and increased colon lengths (GRK2+/+ 4.7 cm vs GRK2+/- 5.3 cm). To examine the mechanisms by which GRK2+/- mice are protected from colitis, we investigated expression of inflammatory genes in the colon as well as immune cell profiles in colonic lamina propria, mesenteric lymph node, and in bone marrow. Our results did not reveal differences in immune cell profiles between the two genotypes. However, expression of inflammatory genes was significantly decreased in DSS-treated GRK2+/- mice compared with GRK2+/+. To understand the mechanisms, we generated myeloid-specific GRK2 knockout mice and subjected them to DSS-induced colitis. Similar to whole body GRK2 heterozygous knockout mice, myeloid-specific knockout of GRK2 was sufficient for the protection from DSS-induced colitis. Together our results indicate that deficiency of GRK2 protects mice from DSS-induced colitis and further suggests that the mechanism of this effect is likely via GRK2 regulation of inflammatory genes in the myeloid cells.