Sara A. Gibson

Protective Effect of Suppressing STAT3 Activity in LPS-induced Acute Lung Injury

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are diseases with high mortality. Macrophages and neutrophils are responsible for inflammatory responses in ALI and ARDS, which are characterized by excessive production of proinflammatory mediators in bronchoalveolar lavage fluid (BALF) and plasma. Aberrant activation of the JAK/STAT pathway is critical for persistent inflammation in many conditions such as infection and autoimmunity. Given the importance of the STAT3 transcription factor in activating macrophages and neutrophils and augmenting inflammation, we investigated the therapeutic potential of inhibiting STAT3 activity using the small-molecule STAT3 inhibitor, LLL12. Our results demonstrate that LPS induces STAT3 activation in macrophages in vitro and in CD45+CD11b+ cells from BALF in the LPS-induced ALI model in vivo. LLL12 treatment inhibits LPS-induced lung inflammation in the ALI model, which is accompanied by suppression of LPS-induced STAT3 activation and an inhibition of macrophage and inflammatory cell infiltration in lung and BALF. LLL12 treatment also suppresses expression of proinflammatory genes including IL-1β, IL-6, TNF-α, iNOS, CCL2, and MHC class II in macrophages and inflammatory cells from BALF and serum as determined by ELISA. Furthermore, hyperactivation of STAT3 in LysMCre-SOCS3fl/fl mice accelerates the severity of inflammation in the ALI model. Both pre- and post-LPS treatment with LLL12 decrease LPS-induced inflammatory responses in mice with ALI. Importantly, LLL12 treatment attenuates STAT3 phosphorylation in human peripheral blood mononuclear cells induced by plasma from patients with ARDS, which suggests the feasibility of targeting the STAT3 pathway therapeutically for patients with ALI and ARDS.

Attenuation of PKR-like ER Kinase (PERK) Signaling Selectively Controls Endoplasmic Reticulum Stress-induced Inflammation Without Compromising Immunological Responses

Inflammation and endoplasmic reticulum (ER) stress are associated with many neurological diseases. ER stress is brought on by the accumulation of misfolded proteins in the ER, which leads to activation of the unfolded protein response (UPR), a conserved pathway that transmits signals to restore homeostasis or eliminate the irreparably damaged cell. We provide evidence that inhibition or genetic haploinsufficiency of protein kinase R-like endoplasmic reticulum kinase (PERK) can selectively control inflammation brought on by ER stress without impinging on UPR-dependent survival and adaptive responses or normal immune responses. Using astrocytes lacking one or both alleles of PERK or the PERK inhibitor GSK2606414, we demonstrate that PERK haploinsufficiency or partial inhibition led to reduced ER stress-induced inflammation (IL-6, CCL2, and CCL20 expression) without compromising prosurvival responses. In contrast, complete loss of PERK blocked canonical PERK-dependent UPR genes and promoted apoptosis. Reversal of eIF2α-mediated translational repression using ISRIB potently suppressed PERK-dependent inflammatory gene expression, indicating that the selective modulation of inflammatory gene expression by PERK inhibition may be linked to attenuation of eIF2α phosphorylation and reveals a previously unknown link between translational repression and transcription of inflammatory genes. Additionally, ER-stressed astrocytes can drive an inflammatory M1-like phenotype in microglia, and this can be attenuated with inhibition of PERK. Importantly, targeting PERK neither disrupted normal cytokine signaling in astrocytes or microglia nor impaired macrophage phagocytosis or T cell polarization. Collectively, this work suggests that targeting PERK may provide a means for selective immunoregulation in the context of ER stress without disrupting normal immune function.

Role of the JAK/STAT signaling pathway in regulation of innate immunity in neuroinflammatory diseases.

The Janus Kinase/Signal Transducers and Activators of Transcription (JAK/STAT) signaling pathway is utilized by numerous cytokines and interferons, and is essential for the development and function of both innate and adaptive immunity. Aberrant activation of the JAK/STAT pathway is evident in neuroinflammatory diseases such as Multiple Sclerosis and Parkinsons Disease. Innate immunity is the front line defender of the immune system and is composed of various cell types, including microglia, macrophages and neutrophils. Innate immune responses have both pathogenic and protective roles in neuroinflammation, depending on disease context and the microenvironment in the central nervous system. In this review, we discuss the role of innate immunity in the pathogenesis of neuroinflammatory diseases, how the JAK/STAT signaling pathway regulates the innate immune response, and finally, the potential for ameliorating neuroinflammation by utilization of JAK/STAT inhibitors.

Protein Kinase CK2 Controls the Fate between Th17 Cell and Regulatory T Cell Differentiation

CK2 is a highly conserved and pleiotropic serine/threonine kinase that promotes many prosurvival and proinflammatory signaling pathways, including PI3K/Akt/mTOR and JAK/STAT. These pathways are essential for CD4+ T cell activation and polarization, but little is known about how CK2 functions in T cells. In this article, we demonstrate that CK2 expression and kinase activity are induced upon CD4+ T cell activation. Targeting the catalytic activity of CK2 using the next-generation small molecule inhibitor CX-4945 in vitro significantly and specifically inhibited mouse and human Th17 cell differentiation while promoting the generation of Foxp3+ regulatory T cells (Tregs). These findings were associated with suppression of PI3K/Akt/mTOR activation and STAT3 phosphorylation upon CX-4945 treatment. Furthermore, we demonstrate that CX-4945 treatment inhibits the maturation of Th17 cells into inflammatory IFN-γ–coproducing effector cells. The Th17/Treg axis and maturation of Th17 cells are major contributing factors to the pathogenesis of many autoimmune disorders, including multiple sclerosis. Using a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis, we demonstrate that in vivo administration of CX-4945 targets Akt/mTOR signaling in CD4+ T cells and the Th17/Treg axis throughout disease. Importantly, CX-4945 treatment after disease initiation significantly reduced disease severity, which was associated with a significant decrease in the frequency of pathogenic IFN-γ+ and GM-CSF+ Th17 cells in the CNS. Our data implicate CK2 as a regulator of the Th17/Treg axis and Th17 cell maturation and suggest that CK2 could be targeted for the treatment of Th17 cell–driven autoimmune disorders.

Protein kinase CK2 is important for the function of glioblastoma brain tumor initiating cells

Protein kinase CK2 is a ubiquitously expressed serine/threonine kinase composed of two catalytic subunits (α) and/or (α′) and two regulatory (β) subunits. The expression and kinase activity of CK2 is elevated in many different cancers, including glioblastoma (GBM). Brain tumor initiating cells (BTICs) are a subset of cells that are highly tumorigenic and promote the resistance of GBM to current therapies. We previously reported that CK2 activity promotes prosurvival signaling in GBM. In this study, the role of CK2 signaling in BTIC function was examined. We found that expression of CK2α was increased in CD133+ BTICs compared to CD133− cells within the same GBM xenolines. Treatment with CX-4945, an ATP-competitive inhibitor of CK2, led to reduced expression of Sox2 and Nestin, transcription factors important for the maintenance of stem cells. Similarly, inhibition of CK2 also reduced the frequency of CD133+ BTICs over the course of 7 days, indicating a role for CK2 in BTIC persistence and survival. Importantly, using an in vitro limiting dilution assay, we found that inhibition of CK2 kinase activity with CX-4945 or siRNA knockdown of the CK2 catalytic subunits reduced neurosphere formation in GBM xenolines of different molecular subtypes. Lastly, we found that inhibition of CK2 led to decreased EGFR levels in some xenolines, and combination treatment with CX-4945 and Gefitinib to inhibit CK2 and EGFR, respectively, provided optimal inhibition of viability of cells. Therefore, due to the integration of CK2 in multiple signaling pathways important for BTIC survival, CK2 is a promising target in GBM.

Protein Kinase CK2: An Emerging Regulator of Immunity

Although it has historically been studied in the context of cancer, recent literature has highlighted the importance of the highly conserved serine/threonine kinase casein kinase II (CK2) in inflammatory disorders. Most strikingly, CK2 is a major regulator of the Th17-Treg axis relevant to many T cell-driven autoimmune disorders including multiple sclerosis (MS).

Opportunities for Translation from the Bench: Therapeutic Intervention of the JAK/STAT Pathway in Neuroinflammatory Diseases.

Pathogenic CD4+ T cells and myeloid cells play critical roles in the pathogenesis of multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. These immune cells secrete aberrantly high levels of pro-inflammatory cytokines that pathogenically bridge the innate and adaptive immune systems and damage neurons and oligodendrocytes. These cytokines include interleukin-2 (IL-2), IL-6, IL-12, IL-21, IL-23, granulocyte macrophage-colony stimulating factor (GM-CSF), and interferon-γ (IFN-γ). It is, therefore, not surprising that both the dysregulated expression of these cytokines and the subsequent activation of their downstream signaling cascades is a common feature in MS/EAE. The Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway is utilized by numerous cytokines for signal transduction and is essential for the development and regulation of immune responses. Unbridled activation of the JAK/STAT pathway by pro-inflammatory cytokines has been demonstrated to be critically involved in the pathogenesis of MS/EAE. In this review, we discuss recent advancements in our understanding of the involvement of the JAK/STAT signaling pathway in the pathogenesis of MS/EAE, with a particular focus on therapeutic approaches to target the JAK/STAT pathway.

CK2 Controls Th17 and Regulatory T Cell Differentiation Through Inhibition of FoxO1

Growing evidence demonstrates that the highly conserved serine/threonine kinase CK2 promotes Th17 cell differentiation while suppressing the generation of Foxp3+ regulatory T cells (Tregs); however, the exact mechanism by which CK2 regulates the Th17/Treg axis remains unclear. CK2 can be composed of three distinct subunits: two catalytic subunits, CK2α and CK2α′, and the regulatory subunit CK2β. We generated mice that lack the major catalytic subunit of CK2, CK2α, specifically in mature T cells using the distal Lck-Cre (CK2α−/−). Importantly, CK2α deficiency resulted in a significant decrease in the overall kinase activity of CK2. Further, CK2α deficiency resulted in a significant defect in Th17 cell polarization and a reciprocal increase in Tregs both in vitro and in vivo in the context of autoimmune neuroinflammation. The transcription factor forkhead box protein O1 (FoxO1) directly inhibits Th17 cell differentiation and is essential for the generation of Tregs. CK2α−/− CD4+ T cells exhibit less phosphorylated FoxO1 and a corresponding increase in the transcription of FoxO1-regulated genes. Treatment of CK2α−/− CD4+ T cells with the FoxO1 inhibitor AS1842856 or short hairpin RNA knockdown of FoxO1 is sufficient to rescue Th17 cell polarization. Through use of a genetic approach to target CK2 kinase activity, the current study provides evidence of a major mechanism by which CK2 regulates the Th17/Treg axis through the inhibition of FoxO1.