Distinct Roles of Type I and Type III Interferons during a Native Murine ß Coronavirus Lung Infection.

Coronaviruses are a major health care threat to humankind. Currently, the host factors that contribute to limit disease severity in healthy young patients are not well defined. Interferons are key antiviral molecules, especially type I and type III interferons. The role of these interferons during coronavirus disease is a subject of debate. Here, using mice that are deficient in type I (IFNAR1-/-), type III (IFNLR1-/-), or both (IFNAR1/LR1-/-) interferon signaling pathways and murine-adapted coronavirus (MHV-A59) administered through the intranasal route, we define the role of interferons in coronavirus infection. We show that type I interferons play a major role in host survival in this model, while a minimal role of type III interferons was manifested only in the absence of type I interferons or during a lethal dose of coronavirus. IFNAR1-/- and IFNAR1/LR1-/- mice had an uncontrolled viral burden in the airways and lung and increased viral dissemination to other organs. The absence of only type III interferon signaling had no measurable difference in the viral load. The increased viral load in IFNAR1-/- and IFNAR1/LR1-/- mice was associated with increased tissue injury, especially evident in the lung and liver. Type I but not type III interferon treatment was able to promote survival if treated during early disease. Further, we show that type I interferon signaling in macrophages contributes to the beneficial effects during coronavirus infection in mice. IMPORTANCE The antiviral and pathological potential of type I and type III interferons during coronavirus infection remains poorly defined, and opposite findings have been reported. We report that both type I and type III interferons have anticoronaviral activities, but their potency and organ specificity differ. Type I interferon deficiency rendered the mice susceptible to even a sublethal murine coronavirus infection, while the type III interferon deficiency impaired survival only during a lethal infection or during a sublethal infection in the absence of type I interferon signaling. While treatment with both type I and III interferons promoted viral clearance in the airways and lung, only type I interferons promoted the viral clearance in the liver and improved host survival upon early treatment (12 h postinfection). This study demonstrates distinct roles and potency of type I and type III interferons and their therapeutic potential during coronavirus lung infection.

In silico identification and expression analysis of superoxide dismutases in Tenebrio molitor.

BACKGROUND: Insects encounter various environmental stresses, in response to which they generate reactive oxygen species (ROS). Superoxide dismutase (SOD) is an antioxidant metalloenzyme that scavenges superoxide radicals to prevent oxidative damage. OBJECTIVE: To investigate expressions of SODs under oxidative stress in Tenebrio molitor. METHODS: Here, we investigated the transcriptional expression of SODs by pesticide and heavy metals in Tenebrio moltior. First, we searched an RNA-Seq database for T. molitor SOD (TmSOD) genes and identified two SOD isoforms (TmSOD1-iso1 and iso2). We examined their activities under developmental stage, tissue-specific, and various types (pesticide and heavy metal) of oxidative stress by using qPCR. RESULTS: Our results revealed two novel forms of TmSODs. These TmSODs had a copper/zinc superoxide dismutase domain, active site, Cu2+ binding site, Zn2+ binding site, E-class dimer interface, and P-class dimer interface. TmSODs (TmSOD1-iso1 and iso2) were expressed in diverse developmental phases and tissues. Pesticides and heavy metals caused an upregulation of these TmSODs. CONCLUSION: Our findings suggest that the two TmSODs have different functions in T. molitor, providing insights into the detoxification ability of T. molitor.

A Novel Small-Molecule Inhibitor of Endosomal TLRs Reduces Inflammation and Alleviates Autoimmune Disease Symptoms in Murine Models.

Toll-like receptors (TLRs) play a fundamental role in the inflammatory response against invading pathogens. However, the dysregulation of TLR-signaling pathways is implicated in several autoimmune/inflammatory diseases. Here, we show that a novel small molecule TLR-inhibitor (TAC5) and its derivatives TAC5-a, TAC5-c, TAC5-d, and TAC5-e predominantly antagonized poly(I:C) (TLR3)-, imiquimod (TLR7)-, TL8-506 (TLR8)-, and CpG-oligodeoxynucleotide (TLR9)-induced signaling pathways. TAC5 and TAC5-a significantly hindered the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), reduced the phosphorylation of mitogen-activated protein kinases, and inhibited the secretion of tumor necrosis factor-α (TNF-α) and interleukin-6. Besides, TAC5-a prevented the progression of psoriasis and systemic lupus erythematosus (SLE) in mice. Interestingly, TAC5 and TAC5-a did not affect Pam3CSK4 (TLR1/2)-, FSL-1 (TLR2/6)-, or lipopolysaccharide (TLR4)-induced TNF-α secretion, indicating their specificity towards endosomal TLRs (TLR3/7/8/9). Collectively, our data suggest that the TAC5 series of compounds are potential candidates for treating autoimmune diseases such as psoriasis or SLE.

Linear and Rationally Designed Stapled Peptides Abrogate TLR4 Pathway and Relieve Inflammatory Symptoms in Rheumatoid Arthritis Rat Model.

A mounting evidence exists for the despicable role of the aberrant immune response in the pathogenesis of rheumatoid arthritis (RA), where toll-like receptor 4 (TLR4) can activate synovial fibroblasts that lead to the chronic inflammation and joint destruction, thus making TLR4 a potent drug target in RA. We report that novel TLR4-antagonizing peptide, PIP2, inhibits the induction of inflammatory biomarkers in vitro as well as in vivo. Systemically, PIP2 inhibits the lipopolysaccharide (LPS)-elicited TNF-α, IL-6, and IL-12p40 in a mouse model. The rationally designed cyclic derivative, cPIP2, is capable of inhibiting LPS-induced proinflammatory cytokines at significantly lower concentration as compared to PIP2 (PIP2 IC50 = 20 µM, cPIP2 IC50 = 5 µM). Finally, cPIP2 was able to relieve the inflammatory symptoms and synovial tissue destruction in the RA rat model. Cumulatively, these data suggest that PIP2 and cPIP2 hold strong promise for the development of peptide-based immunotherapeutics that could be of great value in curbing TLR-related immune complications including RA.

A cell-penetrating peptide blocks Toll-like receptor-mediated downstream signaling and ameliorates autoimmune and inflammatory diseases in mice.

Toll-like receptors (TLRs) recognize pathogen/damage-associated molecular patterns and initiate inflammatory signaling cascades. Occasionally, overexpression of TLRs leads to the onset of numerous inflammatory diseases, necessitating the development of selective inhibitors to allow a protective yet balanced immune response. Here, we demonstrate that a novel peptide (TIP1) derived from Toll/interleukin-1 receptor (TIR) domain-containing adapter protein inhibited multiple TLR signaling pathways (MyD88-dependent and MyD88-independent) in murine and human cell lines. TIP1 also inhibited NLRP3-mediated IL-1ß secretion, as we validated at both the protein and mRNA levels. Biophysical experiments confirmed that TIP1 specifically binds to the BB loop of the TLR4-TIR domain. Animal studies revealed that TIP1 inhibited the secretion of lipopolysaccharide (LPS)-induced proinflammatory cytokines in collagen-induced arthritis (CIA) and kaolin/carrageenan-induced arthritis (K/C) rodent models. TIP1 also rescued animals from sepsis and from LPS-induced kidney/liver damage. Importantly, TIP1 ameliorated the symptoms of rheumatoid arthritis in CIA and K/C rodent models, suggesting that TIP1 has therapeutic potential for the treatment of TLR-mediated autoimmune/inflammatory diseases.

Quasi-Stem Cells Derived from Human Somatic Cells by Chemically Modified Carbon Nanotubes.

Surface modification of micro- and nanotopography was employed to alter the surface properties of scaffolds for controlling cell attachment, proliferation, and differentiation. This study reports a method for generating multinucleated colonies as evidenced by spherical colony formation through nanotopography-induced expression of reprogramming factors in human dermal fibroblasts. Colony formation was achieved by subjecting the cells to specific environments such as culturing with single-walled carbon nanotubes and poly-l-lysine (PLL-SWCNTs). We obtained encouraging results showing that PLL-SWCNT treatment transformed fibroblast cells, and the transformed cells expressed the pluripotency-associated factors OCT4, NANOG, and SOX2 in addition to TRA-1-60 and SSEA-4, which are characteristic stem cell markers. Downregulation of lamin A/C, HDAC1, HDAC6, Bcl2, cytochrome c, p-FAK, p-ERK, and p-JNK and upregulation of H3K4me3 and p-p38 were confirmed in the generated colonies, indicating reprogramming of cells. This protocol increases the possibility of successfully reprogramming somatic cells into induced pluripotent stem cells (iPSCs), thereby overcoming the difficulties in iPSC generation such as genetic mutations, carcinogenesis, and undetermined risk factors.

Toll-like receptor 2 antagonists identified through virtual screening and experimental validation.

Toll-like receptor 2 (TLR2) antagonists are key therapeutic targets because they inhibit several inflammatory diseases caused by surplus TLR2 activation. In this study, we identified two novel nonpeptide TLR2 antagonists, C11 and C13, through pharmacophore-based virtual screening. At 10 µm, the level of interleukin (IL)-8 inhibition by C13 and C11 in human embryonic kidney TLR2 overexpressing cells was comparable to the commercially available TLR2 inhibitor CU-CPT22. In addition, C11 and C13 acted in mouse macrophage-like RAW 264.7 cells as TLR2-specific inhibitors and did not suppress the tumor necrosis factor-α induction by TLR3 and TLR4 activators. Moreover, the two identified compounds bound directly to the human recombinant TLR2 ectodomain, during surface plasmon resonance analysis, and did not affect cell viability in a 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium assay. In total, two virtually screened molecules, C11 and C13, were experimentally proven to be effective as TLR2 antagonists, and thus will provide new insights into the structure of TLR2 antagonists, and pave the way for the development of TLR2-targeted drug molecules.

TLR4/MD2 specific peptides stalled in vivo LPS-induced immune exacerbation.

Negative regulation of Toll-like receptor-4 (TLR4) is anticipated to control the pathogen-induced exaggerated immune response. However, effective TLR4 antagonists with scarce off-target effects are yet to be developed. To fill this void, we sought to design small peptide-inhibitors of the TLR4/MD2-LPS interaction. Here we report novel TLR4-antagonistic peptides (TAP), identified through phage display, endowed with the LPS-induced proinflammation inhibition, and confirmed in mice. TAPs-attributed TLR4-antagonism were initially evaluated through NF-κB inhibition in HEK-blue hTLR4 and RAW264.7 cells, and further reinforced by the downregulation of MAPKs (mitogen-activated protein kinases), NF-κB, interleukin 6, and suppression of the oxidative-stress products and iNOS in macrophages and human peripheral blood mononuclear cells (hPBMCs). Among these, TAP2 specifically halted the TLR4, but not other TLRs signaling, which was further confirmed by the biophysical kinetic assay. Finally, TAP2 diminished LPS-elicited systemic cytokine response in vivo, suggesting that TAPs, specifically TAP2, have the potential to treat TLR4-mediated immune ailments.

Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells.

Etoposide (ETO) is a commonly used chemotherapeutic drug that inhibits topoisomerase II activity, thereby leading to genotoxicity and cytotoxicity. However, ETO has limited application due to its side effects on normal organs, especially the kidney. Here, we report the mechanism of ETO-induced cytotoxicity progression in human kidney proximal tubule (HK-2) cells. Our results show that ETO perpetuates DNA damage, activates mitogen-activated protein kinase (MAPK), and triggers morphological changes, such as cell and nuclear swelling. When NAC, a well-known reactive oxygen species (ROS) scavenger, is co-treated with ETO, it inhibits an ETO-induced increase in mitochondrial mass, mitochondrial DNA (ND1 and ND4) copy number, intracellular ATP level, and mitochondrial biogenesis activators (TFAM, PGC-1α and PGC-1ß). Moreover, co-treatment with ETO and NAC inhibits ETO-induced necrosis and cell swelling, but not apoptosis. Studies using MAPK inhibitors reveal that inhibition of extracellular signal regulated kinase (ERK) protects ETO-induced cytotoxicity by inhibiting DNA damage and caspase 3/7 activity. Eventually, ERK inhibitor treated cells are protected from ETO-induced nuclear envelope (NE) rupture and DNA leakage through inhibition of caspase activity. Taken together, these data suggest that ETO mediates cytotoxicity in HK-2 cells through ROS and ERK pathways, which highlight the preventive avenues in ETO-induced cytotoxicity in kidney.

Structural and functional analysis of cell adhesion and nuclear envelope nano-topography in cell death.

The cell death mechanisms of necrosis and apoptosis generate biochemical and morphological changes in different manners. However, the changes that occur in cell adhesion and nuclear envelope (NE) topography, during necrosis and apoptosis, are not yet fully understood. Here, we show the different alterations in cell adhesion function, as well as the topographical changes occurring to the NE, during the necrotic and apoptotic cell death process, using the xCELLigence system and atomic force microscopy (AFM). Studies using xCELLigence technology and AFM have shown that necrotic cell death induced the expansion of the cell adhesion area, but did not affect the speed of cell adhesion. Necrotic nuclei showed a round shape and presence of nuclear pore complexes (NPCs). Moreover, we found that the process of necrosis in combination with apoptosis (termed nepoptosis here) resulted in the reduction of the cell adhesion area and cell adhesion speed through the activation of caspases. Our findings showed, for the first time, a successful characterization of NE topography and cell adhesion during necrosis and apoptosis, which may be of importance for the understanding of cell death and might aid the design of future drug delivery methods for anti-cancer therapies.

Activating transcription factor 3 represses inflammatory responses by binding to the p65 subunit of NF-κB.

Activating transcription factor 3 (ATF3) is induced by inflammatory responses, cell death, cytokines, and oxidative stress conditions. ATF3 is a negative regulator in the Toll-like receptor 4 signalling pathway. The principal molecule in this pathway is nuclear factor κB (NF-κB) that translocates into the nucleus to initiate the transcription of inflammatory mediators. However, scarce data are available regarding the interaction of ATF3 and p65, a part of the NF-κB dimer. Therefore, we studied the mechanism of regulation of p65 by ATF3 in RAW 264.7 cells. First, LPS-mediated NF-κB activation was confirmed, and then the direct interaction of ATF3 and p65 was observed through immunoprecipitation (IP). The presence of histone deacetylase 1 (HDAC1) was also detected in the complex. In ATF3 deficient cells, NF-κB activity was up-regulated and HDAC1 was not detected by IP. These observations suggest that p65 is attenuated by ATF3 such that ATF3 recruits HDAC1 to the ATF3/p65 complex and facilitates the deacetylation of p65. Likewise, inflammatory response genes were induced by translocated NF-κB in ATF3-deficient cells. Cumulatively, we uncovered a novel mechanism for the negative regulation of NF-κB by ATF3 via direct interaction with p65.

Doxorubicin-induced necrosis is mediated by poly-(ADP-ribose) polymerase 1 (PARP1) but is independent of p53.

Necrosis, unregulated cell death, is characterized by plasma membrane rupture as well as nuclear and cellular swelling. However, it has recently been reported that necrosis is a regulated form of cell death mediated by poly-(ADP-ribose) polymerase 1 (PARP1). PARP1 is thought to mediate necrosis by inducing DNA damage, although this remains unconfirmed. In this study, we examined the mechanisms of PARP1-mediated necrosis following doxorubicin (DOX)-induced DNA damage in human kidney proximal tubular (HK-2) cells. DOX initiated DNA damage response (DDR) and upregulated PARP1 and p53 expression, resulting in morphological changes similar to those observed during necrosis. Additionally, DOX induced mitochondrial hyper-activation, as evidenced by increased mitochondrial respiration and cytosolic ATP (cATP) production. However, DOX affected mitochondrial mass. DOX-induced DNA damage, cytosolic reactive oxygen species (cROS) generation, and mitochondrial hyper-activation decreased in cells with inhibited PARP1 expression, while generation of nitric oxide (NO) and mitochondrial ROS (mROS) remained unaffected. Moreover, DOX-induced DNA damage, cell cycle changes, and oxidative stress were not affected by p53 inhibition. These findings suggest that DNA damage induced necrosis through a PARP1-dependent and p53-independent pathway.

Etoposide Induces Necrosis Through p53-Mediated Antiapoptosis in Human Kidney Proximal Tubule Cells.

The p53 protein is an important transcription factor that modulates signaling pathways for both cell death and survival. Its antiapoptotic mechanisms that correlate with necrotic and apoptotic cell death are not well understood. Here, we report that etoposide promotes progression of the DNA damage response as well as necrotic morphological changes including plasma membrane rupture using carbon nanotube-tipped/atomic force microscopy (CNT/AFM) probes in human kidney proximal tubule (HK-2) cells. Inhibition of p53 abrogated cell cycle arrest and led to a decrease in the expression levels of repair proteins that were induced by DNA damage. Mitochondrial biogenesis and cytosolic production of reactive oxygen species were also reduced after p53 inhibition; the latter change induced mitochondrial superoxide accumulation and mitochondrial damage, which triggered the activation of caspase 3. Inhibition of p53 also led to a loss of cell adhesion and converted necrotic cell death to apoptotic cell death, with appreciable cell shrinkage and appearance of apoptotic bodies that were observed using CNT/AFM probes. Thus, our study demonstrated that p53 protects against apoptosis, and leads to etoposide-induced necrosis. These results are expected to aid in the understanding of mechanism of antiapoptosis and its relationship to cell death.

Doxorubicin induces cytotoxicity through upregulation of pERK-dependent ATF3.

Although doxorubicin is commonly used in the treatment of many cancer types, its use in chemotherapy has been limited, largely because of its severe side effects, including cardiotoxicity and nephrotoxicity. In this study, we aimed to identify the mechanism of doxorubicin-induced cytotoxicity by using the human kidney proximal tubule cell line HK-2. Furthermore, we investigated the role of activating transcription factor 3 (ATF3) as a mediator of doxorubicin-induced cytotoxicity by using wild-type mouse embryonic fibroblasts (MEF) cells and ATF3 knockout (KO) cells. In HK-2 cells, doxorubicin decreased cell viability in a dose-dependent manner and induced an increase in cells in the sub G1 and G2/M phases at all doses. Doxorubicin treatment showed the following dose-dependent effects: increase in the secretion of tumor necrosis factor alpha; decrease in the expression of phosphorylated protein kinase A and Bcl-2; and increase in the expression of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal-regulated kinase (ERK), and ATF3. Based on these results, we suggest that doxorubicin induces cytotoxicity through an ERK-dependent pathway, and ATF3 plays a pivotal role as a transcriptional regulator in this process.