A microsatellite DNA-derived oligodeoxynucleotide attenuates lipopolysaccharide-induced acute lung injury in mice by inhibiting the HMGB1-TLR4-NF-κB signaling pathway.

Acute lung injury (ALI) with uncontrolled inflammatory response has high morbidity and mortality rates in critically ill patients. Pathogen-associated molecular patterns (PAMPs) are involved in the development of uncontrolled inflammatory response injury and associated lethality. In this study, we investigated the inhibit effect of MS19, a microsatellite DNA-derived oligodeoxynucleotide (ODN) with AAAG repeats, on the inflammatory response induced by various PAMPs in vitro and in vivo. In parallel, a microsatellite DNA with AAAC repeats, named as MS19-C, was used as controls. We found that MS19 extensively inhibited the expression of inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α induced by various PAMPs stimulation, including DNA viruses, RNA viruses, bacterial components lipopolysaccharide (LPS), and curdlan, as well as the dsDNA and dsRNA mimics, in primed bone marrow-derived macrophage (BMDM). Other than various PAMPs, MS19 also demonstrated obvious effects on blocking the high mobility group box1 (HMGB1), a representative damage-associated-molecular pattern (DAMP), nuclear translocation and secretion. With the base substitution from G to C, MS19-C has been proved that it has lost the inhibitory effect. The inhibition is associated with nuclear factor kappa B (NF-κB) signaling but not the mitogen-activated protein kinase (MAPK) transduction. Moreover, MS19 capable of inhibiting the IL-6 and TNF-α production and blocking the HMGB1 nuclear translocation and secretion in LPS-stimulated cells was used to treat mice ALI induced by LPS in vivo. In the ALI mice model, MS19 significantly inhibited the weight loss and displayed the dramatic effect on lessening the ALI by reducing consolidation, hemorrhage, intra-alveolar edema in lungs of the mice. Meanwhile, MS19 could increase the survival rate of ALI by downregulating the inflammation cytokines HMGB1, TNF-a, and IL-6 production in the bronchoalveolar lavage fluid (BALF). The data suggest that MS19 might display its therapeutic role on ALI by inhibiting the HMGB1-TLR4-NF-κB signaling pathway.

TRIM18 is a critical regulator of viral myocarditis and organ inflammation.

BACKGROUND: Infections by viruses including severe acute respiratory syndrome coronavirus 2 could cause organ inflammations such as myocarditis, pneumonia and encephalitis. Innate immunity to viral nucleic acids mediates antiviral immunity as well as inflammatory organ injury. However, the innate immune mechanisms that control viral induced organ inflammations are unclear. METHODS: To understand the role of the E3 ligase TRIM18 in controlling viral myocarditis and organ inflammation, wild-type and Trim18 knockout mice were infected with coxsackievirus B3 for inducing viral myocarditis, influenza A virus PR8 strain and human adenovirus for inducing viral pneumonia, and herpes simplex virus type I for inducing herpes simplex encephalitis. Mice survivals were monitored, and heart, lung and brain were harvested for histology and immunohistochemistry analysis. Real-time PCR, co-immunoprecipitation, immunoblot, enzyme-linked immunosorbent assay, luciferase assay, flow cytometry, over-expression and knockdown techniques were used to understand the molecular mechanisms of TRIM18 in regulating type I interferon (IFN) production after virus infection in this study. RESULTS: We find that knockdown or deletion of TRIM18 in human or mouse macrophages enhances production of type I IFN in response to double strand (ds) RNA and dsDNA or RNA and DNA virus infection. Importantly, deletion of TRIM18 protects mice from viral myocarditis, viral pneumonia, and herpes simplex encephalitis due to enhanced type I IFN production in vivo. Mechanistically, we show that TRIM18 recruits protein phosphatase 1A (PPM1A) to dephosphorylate TANK binding kinase 1 (TBK1), which inactivates TBK1 to block TBK1 from interacting with its upstream adaptors, mitochondrial antiviral signaling (MAVS) and stimulator of interferon genes (STING), thereby dampening antiviral signaling during viral infections. Moreover, TRIM18 stabilizes PPM1A by inducing K63-linked ubiquitination of PPM1A. CONCLUSIONS: Our results indicate that TRIM18 serves as a negative regulator of viral myocarditis, lung inflammation and brain damage by downregulating innate immune activation induced by both RNA and DNA viruses. Our data reveal that TRIM18 is a critical regulator of innate immunity in viral induced diseases, thereby identifying a potential therapeutic target for treatment.

Identification of poly(ADP-ribose) polymerase 9 (PARP9) as a noncanonical sensor for RNA virus in dendritic cells.

Innate immune cells are critical in protective immunity against viral infections, involved in sensing foreign viral nucleic acids. Here we report that the poly(ADP-ribose) polymerase 9 (PARP9), a member of PARP family, serves as a non-canonical sensor for RNA virus to initiate and amplify type I interferon (IFN) production. We find knockdown or deletion of PARP9 in human or mouse dendritic cells and macrophages inhibits type I IFN production in response to double strand RNA stimulation or RNA virus infection. Furthermore, mice deficient for PARP9 show enhanced susceptibility to infections with RNA viruses because of the impaired type I IFN production. Mechanistically, we show that PARP9 recognizes and binds viral RNA, with resultant recruitment and activation of the phosphoinositide 3-kinase (PI3K) and AKT3 pathway, independent of mitochondrial antiviral-signaling (MAVS). PI3K/AKT3 then activates the IRF3 and IRF7 by phosphorylating IRF3 at Ser385 and IRF7 at Ser437/438 mediating type I IFN production. Together, we reveal a critical role for PARP9 as a non-canonical RNA sensor that depends on the PI3K/AKT3 pathway to produce type I IFN. These findings may have important clinical implications in controlling viral infections and viral-induced diseases by targeting PARP9.

EphA2 phosphorylates NLRP3 and inhibits inflammasomes in airway epithelial cells.

Inflammasomes are intracellular complexes that form in the cytosol of inflammatory cells. NLRP3 is one of the sensor proteins in the complex that can recognize a wide variety of stimuli ranging from microbial components to environmental particulates. Here, we report that in mouse airway epithelial cells (AECs), inflammasome activation is inhibited by EphA2, a member of the transmembrane tyrosine kinase receptor family, via tyrosine phosphorylation of NLRP3 in a model of reovirus infection. We find that EphA2 depletion markedly enhances interleukin-1ß (IL-1ß) and interleukin-18 (IL-18) production in response to the virus. EphA2-/- mice show stronger inflammatory infiltration and enhanced inflammasome activation upon viral infection, and aggravated asthma symptoms upon ovalbumin (ova) induction. Mechanistically, EphA2 binds to NLRP3 and induces its phosphorylation at Tyr132, thereby interfering with ASC speck formation and blocking the activation of the NLRP3-inflammasome. These data demonstrate that reovirus employs EphA2 to suppress inflammasome activation in AECs and that EphA2 deficiency causes a pathological exacerbation of asthma in an ova-induced asthma model.

The protective effect of interfering TLR9-IRF5 signaling pathway on the development of CVB3-induced myocarditis.

Since toll-like receptor 9 (TLR9) or interferon regulatory factor 5 (IRF5) was reported to be associated with the development of myocarditis, we wondered if the TLR9-IRF5 pathway could contribute to the development of coxsackievirus B3 (CVB3)-induced myocarditis. We detected signaling molecules of TLR9-IRF5 pathway in CVB3-infected patients and mice. The results showed that TLR9, IRF5 and its downstream molecules such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were significantly increased, and the increase was correlated with the severity of heart injury during CVB3 infection. In addition, we demonstrated that an AAAG ODN with IRF5 interfering activities significantly decreased the levels of the TLR9-IRF5 pathway molecules in hearts, spleens as well as white blood cells, and alleviated the myocarditis in CVB3-infected mice. The data suggest that interfering TLR9-IRF5 pathway could be an approach to treat CVB3-induced myocarditis.

Attribution of NKG2DL to the inhibition of early stage allogeneic tumors in mice.

Allogeneic tumors are eventually rejected by adaptive immune responses, however, little is known about how allogeneic tumors are eradicated at the early stage of tumor development. In present study, we found that NKG2DL low expressing cancer cells were developed into palpable allogeneic tumors in mice within a week after the inoculation, while NKG2DL high expressing cancer cells failed to. The NKG2DL high expressing cancer cells could increase NKG2D+ NK cells in the allogeneic mice after being inoculated for 3 days. Artificially up-regulating NKG2DL on cancer cells with low level expressed NKG2DL by a CpG ODN resulted in the retardation and rejection of the allogeneic tumors at the early stage. The contribution of up-regulated NKG2DL to the early rejection was further confirmed by the results that the development of allogeneic tumors from cancer cells transfected with NKG2DL genes was significantly inhibited in mice at the early stage. Overall, hopefully, the data may provide insights for combining the allogeneic NK cell adoptive transfer with the approaches of up-regulating NKG2DL to treat cancer patients.

Delivery System of CpG Oligodeoxynucleotides through Eliciting an Effective T cell Immune Response against Melanoma in Mice.

PURPOSE: In order to improve the immunogenicity of whole tumor cell lysate for tumor vaccine, we have designed a series of CpG ODNs to study their transport and to evaluate their anti-tumor activity in B16 melanoma mouse models. METHODS: In this study, we investigated whether C-class CpG ODN (CpG ODN-685) could facilitate tumor cell lysate to induce vigorous anti-tumor activity against tumors in mice both prophylactically and therapeutically. RESULTS: It was found that the combination of tumor cell lysate and CpG ODN-685 could inhibit the growth of B16 melanoma and prolong the survival of tumor-bearing mice. Moreover CpG ODN-685 with the addition of tumor cell lysate can also cause the generation of tumor specific immune memory by inducing specific cytotoxic T lymphocytes and helper T lymphocytes in mice. CONCLUSION: The results suggest that CpG ODN-685 could be developed as an efficient adjuvant for tumor vaccines against melanoma.

The therapeutic potency of HSP65-GTL in GL261 glioma-bearing mice.

Gliomas are the most common type of brain tumor with poor prognosis. Even after combination treatments including surgery, radiation, and chemotherapy, the median survival is around 15 months, calling for novel approaches such as immunotherapy. To develop novel therapeutic approaches, we tried to prepare a candidate vaccine by mixing the recombinant mycobacterial heat-shock protein 65 (HSP65) with GL261 glioma tissue lysate (GTL). Our data showed that HSP65-GTL induced potent cytotoxic T lymphocyte and prolonged the survival of mice bearing GL261 gliomas. Furthermore, HSP65 or HSP65-GTL upregulated mRNA expressions of RORγt and interleukin-17A in spleen cells or draining lymph node cells, respectively, and enhanced the ratios of brain-infiltrating Th17 cells and inflammatory cells, indicating that the antitumor effect of HSP65-GTL was associated with Th17-type immunity.

miRNA profiling reveals a potential role of milk stasis in breast carcinogenesis.

The tumor microenvironment plays an important role in breast carcinogenesis. Milk acts as an important microenvironment of breast cancer, but its role in breast carcinogenesis is largely unknown. Milk stasis may exist in the breast for a number of years after breastfeeding. In the present study, we reported the first microRNA (miRNA) profiling of milk from patients with milk stasis. We identified 266 known miRNAs and 271 novel miRNAs in 10 milk stasis only samples, 271 known miRNAs and 140 novel miRNAs in 10 milk stasis plus breast neoplasm samples by deep sequencing. miRNA profiles were different between the two groups. Furthermore, nine tumor suppressor miRNAs such as miR-29a, miR-146 and miR-223 were significantly downregulated, while seven oncogenic miRNAs such as miR-451, miR-486, miR-107, miR-92 and miR-10 were significantly upregulated in the milk of milk stasis plus neoplasm patients. Three of the identified miRNAs (miR-140, miR-21 and let-7a) were selected using real-time PCR, confirming that these miRNAs were highly expressed. The results also showed that the three miRNAs detected were more abundant in the milk than in the blood. In summary, the data suggested that miRNAs in milk from milk stasis patients may contribute to breast carcinogenesis and that they are more sensitive biomarkers for breast cancer than miRNAs in the blood.

MF59 formulated with CpG ODN as a potent adjuvant of recombinant HSP65-MUC1 for inducing anti-MUC1+ tumor immunity in mice.

MF59 is an oil-in-water emulsion adjuvant approved for influenza vaccines for human use in Europe. Due to its Th2 inducing properties, MF59 is seldom tested for cancer vaccines. In this study, MF59 formulated with a C-type CpG oligodeoxynucleotide (YW002) was tested for its Th1 adjuvant activity to induce immune responses to HSP65-MUC1, a recombinant fusion protein incorporating a mycobacterial heat shock protein (HSP65) and mucin 1, cell surface associated (MUC1) derived peptide. Combination of YW002 with MF59 (MF59-YW002) could confer a potent Th1 biasing property to the adjuvant, which enhanced the immunogenicity of HSP65-MUC1 to induce significantly higher levels of specific IgG2c, increased IFN-γ mRNA expression in splenocytes and the generation of antigen-specific cytotoxic T lymphocytes in mice. When prophylactically applied, MF59-YW002 adjuvant containing HSP65-MUC1 inhibited the growth of MUC1+ B16 melanoma and prolonged the survival of tumor-bearing mice. In contrast, adjuvant containing MF59 with HSP65-MUC1 in the absence of YW002, promoted the growth of MUC1+ B16 melanoma in mice. These results suggest that MF59 plus CpG oligodeoxynucleotide might be developed as an efficient adjuvant for tumor vaccines against melanoma, and possibly other tumors.

An oligodeoxynucleotide capable of lessening acute lung inflammatory injury in mice infected by influenza virus.

Infection of influenza virus could induce acute lung inflammatory injury (ALII) that was at least partially caused by excessive innate immune responses. To study whether down-regulating Toll-like receptor (TLR)-mediated innate immune response could lessen influenza virus-induced ALII, a microsatellite DNA mimicking oligodeoxynucleotide (MS ODN), named as SAT05f capable of inhibiting TLR7/9-activation in vitro, was used to treat mice infected with FM1 virus. In parallel, two MS ODNs confirmed with less or no in vitro activities, named as MS19 and MS33, were used as controls. Unexpectedly, SAT05f failed to lessen ALII in the mice, whereas MS19 significantly inhibited the weight loss and displayed dramatic effect on lessening the ALII by reducing consolidation, hemorrhage, intra-alveolar edema and neutrophils infiltration in lungs of the mice. Meanwhile, MS19 could decrease the mortality of influenza virus infected mice and down-regulate TNF-α production in their lungs. The data suggest that MS19 might display its therapeutic role on ALII induced by influenza virus by reducing over-production of TNF-α.

Purification of clinical-grade recombinant HSP65-MUCI fusion protein.

HSP65-MUCI is a fusion protein between BCG (Bacille Calmette-Guerin)-derived HSP65 (heat-shock protein 65) and human MUCI (mucin I) VNTR (variable number of tandem repeats)-domain peptides that has shown antitumour efficacy. China's Food and Drug Administration has recently approved a Phase I clinical trial using HSP65-MUCI for the treatment of MUCI-positive breast cancer. In order to produce sufficient quantities of clinical-grade HSP65-MUCI, we established a pilot-scale purification scheme comprising two steps of column chromatography: HIC (hydrophobic-interaction chromatography) and IEX (ion-exchange chromatography). The pH values of the buffers used in homogenization and HIC were adjusted to pH 9.0 to maintain protein stability and prevent protein degradation. Using this manufacturing process, we obtained clinical-grade HSP65-MUCI with a yield of 400 mg per 70 g of wet cell pellet and >96% purity.

Purification of a non-tagged recombinant BCG heat shock protein 65-Her2 peptide fusion protein from Escherichia coli.

Bacille Calmette-Guerin (BCG)-derived heat shock protein 65 (HSP65) has been demonstrated capable of assisting a fused peptide to generate the peptide-specific cellular immunity. Various HSP65 fusion proteins have been developed as therapeutic cancer vaccines. Purifying a recombinant HSP65 fusion protein with no purification tags for human use is routinely a challenge. Here, we report a scheme for purifying a non-tagged recombinant HSP65-Her2 peptide fusion protein (HSP65-Her2) from Escherichia coli. The HSP65-Her2 is being developed as an immunotherapeutic for the treatment of Her2-positive tumors. After fermentation in a 10-L fermentor, the HSP65-Her2 expressing E. coli were harvested and lysed by sonication. The recombinant HSP65-Her2 was then purified with four successive steps including Butyl-Sepharose FF, DEAE-Sepharose FF, 1% Triton X-114 phase separation and Sephadex G-25. Results showed that HSP65-Her2 was purified up to 97% purity and was able to generate Her2-specific cytotoxic T lymphocytes (CTLs), suggesting that the scheme is efficient for purifying the non-tagged HSP65-Her2 fusion protein with biological activity.