N6-Methyladenosine and Reader Protein YTHDF2 Enhance the Innate Immune Response by Mediating DUSP1 mRNA Degradation and Activating Mitogen-Activated Protein Kinases during Bacterial and Viral Infections.

Mitogen-activated protein kinases (MAPKs) play critical roles in the induction of numerous cytokines, chemokines, and inflammatory mediators that mobilize the immune system to counter pathogenic infections. Dual-specificity phosphatase 1 (DUSP1) is a member of the dual-specificity phosphatases that inactivates MAPKs through a negative-feedback mechanism. Here, we report that in response to viral and bacterial infections, not only the DUSP1 transcript but also its N6-methyladenosine (m6A) levels rapidly increase together with that of the m6A reader protein YTHDF2, resulting in enhanced YTHDF2-mediated DUSP1 transcript degradation. The knockdown of DUSP1 promotes p38 and Jun N-terminal kinase (JNK) phosphorylation and activation, thus increasing the expression of innate immune response genes, including the interleukin-1ß (IL-1ß), colony-stimulating factor 3 (CSF3), transglutaminase 2 (TGM2), and proto-oncogene tyrosine-protein kinase Src (SRC) genes. Similarly, the knockdown of the m6A eraser ALKBH5 increases the DUSP1 transcript m6A level, resulting in accelerated transcript degradation, the activation of p38 and JNK, and the enhanced expression of IL-1ß, CSF3, TGM2, and SRC. These results demonstrate that m6A and the reader protein YTHDF2 orchestrate optimal innate immune responses during viral and bacterial infections by downregulating the expression of a negative regulator, DUSP1, of the p38 and JNK pathways that are central to innate immune responses against pathogenic infections. IMPORTANCE Innate immunity is central to controlling pathogenic infections and maintaining the homeostasis of the host. In this study, we have revealed a novel mechanism regulating innate immune responses during viral and bacterial infections. We have found that N6-methyladenosine (m6A) and the reader protein YTHDF2 regulate dual-specificity phosphatase 1, a negative regulator of the mitogen-activated protein kinases p38 and JNK, to maximize innate immune responses during viral and bacterial infections. These results provide novel insights into the mechanism regulating innate immunity, which could help in the development of novel approaches for controlling pathogenic infections.

The Role of Bacteria in KSHV Infection and KSHV-Induced Cancers.

The objective of this article is to review the current status of the bacteria-virus interplay in Kaposi's sarcoma-associated herpesvirus (KSHV) infection and KSHV-driven cancers. KSHV is the etiological agent of several cancers, including Kaposi's sarcoma (KS) and primary effusion lymphoma. Due to immunosuppression, patients with KSHV are at an increased risk for bacterial infections. Moreover, among patients coinfected by HIV and KSHV, patients with KS have distinct oral microbiota compared to non-KS patients. Bacterial biomarkers associated with KSHV-driven cancers can provide insights in discerning the mechanisms of KSHV-induced oncogenesis. For example, pathogen-associated molecular patterns and bacterial products of certain bacterial species can regulate the expression of KSHV lytic and latent genes, thereby affecting viral replication and dissemination. In addition, infection with distinct opportunistic bacterial species have been associated with increased cell proliferation and tumorigenesis in KSHV-induced cancers through activation of pro-survival and -mitogenic cell signaling pathways. By elucidating the various mechanisms in which bacteria affect KSHV-associated pathogenesis, we will be able to pinpoint therapeutic targets for KSHV infection and KSHV-related cancers.

Reversible switching of primary cells between normal and malignant state by oncogenic virus KSHV and CRISPR/Cas9-mediated targeting of a major viral latent protein.

Viral infection has been implicated in the pathogenesis of a plethora of human diseases. Although antiviral therapies effectively confront the viral spread and infection, how to completely eradicate the viral genome from infected cells remains a challenge. In this study, we demonstrated the reversible switching of primary cells between normal and malignant states by an oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) and CRISPR/Cas9-mediated targeting of a major viral latent protein. Primary cells can be transformed into malignant status by infection of KSHV, while elimination of the KSHV genome from latent KSHV-infected cells reverses KSHV-transformed primary cells back to a "normal state" by CRISPR/Cas-mediated knockout of viral major latent gene LANA. As a proof of concept, we demonstrated efficient elimination of KSHV episome in KSHV-associated primary effusion lymphoma cells resulting in the induction of apoptosis by liposome-encapsulated CRISPR/Cas9 ribonucleoprotein complexes (Lipo/Cas9-LANAsgRNA). Our work illustrates CRISPR/Cas as a promising technology for eliminating oncogenic viruses from persistently infected cells by taking advantage of the genetic differences between viral and cellular genomes. Compared to traditional antiviral therapy, our study offer an approach for antagonizing human oncogenic virus-related cancers by directly targeting as well as clearing viral genomes.

Pseudomonas aeruginosa Stimulates Inflammation and Enhances Kaposi's Sarcoma Herpesvirus-Induced Cell Proliferation and Cellular Transformation through both Lipopolysaccharide and Flagellin.

Inflammation triggered by innate immunity promotes carcinogenesis in cancer. Kaposi's sarcoma (KS), a hyperproliferative and inflammatory tumor caused by Kaposi's sarcoma-associated herpesvirus (KSHV) infection, is the most common cancer in AIDS patients. KSHV infection sensitizes cells to pathogen-associated molecular patterns (PAMPs). We examined the role of Pseudomonas aeruginosa, an opportunistic bacterium that can affect AIDS patients, in inflammation and cell proliferation of KSHV-transformed cells. P. aeruginosa stimulation increased cell proliferation and efficiency of colony formation in soft agar of KSHV-transformed rat primary mesenchymal precursor (KMM) cells but had no significant effect on the untransformed (MM) cells. P. aeruginosa stimulation also increased cell proliferation of KSHV-infected human B cells, BJAB, but not the uninfected cells. Mechanistically, P. aeruginosa stimulation resulted in increased inflammatory cytokines and activation of p38, ERK1/2, and JNK mitogen-activated protein kinase (MAPK) pathways in KMM cells while having no obvious effect on MM cells. P. aeruginosa induction of inflammation and MAPKs was observed with and without inhibition of the Toll-like receptor 4 (TLR4) pathway, while a flagellin-deleted mutant of P. aeruginosa required a functional TLR4 pathway to induce inflammation and MAPKs. Furthermore, treatment with either lipopolysaccharide (LPS) or flagellin alone was sufficient to induce inflammatory cytokines, activate MAPKs, and increase cell proliferation and efficiency of colony formation in soft agar of KMM cells. These results demonstrate that both LPS and flagellin are PAMPs that contribute to P. aeruginosa induction of inflammation in KSHV-transformed cells. Because AIDS-KS patients are susceptible to P. aeruginosa infection, our work highlights the preventive and therapeutic potential of targeting P. aeruginosa infection in these patients.IMPORTANCE Kaposi's sarcoma (KS), caused by infection with Kaposi's sarcoma-associated herpesvirus (KSHV), is one of the most common cancers in AIDS patients. KS is a highly inflammatory tumor, but how KSHV infection induces inflammation remains unclear. We have previously shown that KSHV infection upregulates Toll-like receptor 4 (TLR4), sensitizing cells to lipopolysaccharide (LPS) and Escherichia coli In the current study, we examined the role of Pseudomonas aeruginosa, an opportunistic bacterium that can affect AIDS patients, in inflammation and cell proliferation of KSHV-transformed cells. P. aeruginosa stimulation increased cell proliferation, inflammatory cytokines, and activation of growth and survival pathways in KSHV-transformed cells through two pathogen-associated molecular patterns, LPS and flagellin. Because AIDS-KS patients are susceptible to P. aeruginosa infection, our work highlights the preventive and therapeutic potential of targeting P. aeruginosa infection in these patients.