Palmitoylation at Residue C221 of Japanese Encephalitis Virus NS2A Protein Contributes to Viral Replication Efficiency and Virulence.

Palmitoylation of viral proteins is crucial for host-virus interactions. In this study, we examined the palmitoylation of Japanese encephalitis virus (JEV) nonstructural protein 2A (NS2A) and observed that NS2A was palmitoylated at the C221 residue of NS2A. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated the virulence of JEV in mice. NS2A/C221S mutation had no effect on NS2A oligomerization and membrane-associated activities, but reduced protein stability and accelerated its degradation through the ubiquitin-proteasome pathway. These observations suggest that NS2A palmitoylation at C221 played a role in its protein stability, thereby contributing to JEV replication efficiency and virulence. Interestingly, the C221 residue undergoing palmitoylation was located at the C-terminal tail (amino acids 195 to 227) and is removed from the full-length NS2A following an internal cleavage processed by viral and/or host proteases during JEV infection. IMPORTANCE An internal cleavage site is present at the C terminus of JEV NS2A. Following occurrence of the internal cleavage, the C-terminal tail (amino acids 195 to 227) is removed from the full-length NS2A. Therefore, it was interesting to discover whether the C-terminal tail contributed to JEV infection. During analysis of viral palmitoylated protein, we observed that NS2A was palmitoylated at the C221 residue located at the C-terminal tail. Blocking NS2A palmitoylation by introducing a cysteine-to-serine mutation at C221 (NS2A/C221S) impaired JEV replication in vitro and attenuated JEV virulence in mice, suggesting that NS2A palmitoylation at C221 contributed to JEV replication and virulence. Based on these findings, we could infer that the C-terminal tail might play a role in the maintenance of JEV replication efficiency and virulence despite its removal from the full-length NS2A at a certain stage of JEV infection.

Downregulation of miR-296-3p by highly pathogenic porcine reproductive and respiratory syndrome virus activates the IRF1/TNF-α signaling axis in porcine alveolar macrophages.

Porcine reproductive and respiratory syndrome virus (PRRSV, species Betaarterivirus suid 1 or 2) is a major pathogen affecting pigs on farms throughout the world. miR-296-3p is a multifunctional microRNA involved in the regulation of the inflammatory response in mice and humans. However, little is known about the biological functions of miR-296-3p in pigs. In this study, we used a highly pathogenic PRRSV-2 (species Betaarterivirus suid 2) strain to show that PRRSV infection robustly downregulates the expression of miR-296-3p in porcine alveolar macrophages (PAMs). Furthermore, we demonstrated that overexpression of miR-296-3p increases the replication of highly pathogenic (HP)-PRRSV in PAMs. Notably, the overexpression of miR-296-3p inhibited the induction of TNF-α, even with increased viral replication, compared with that in the HP-PRRSV-infected control group. We also demonstrated that miR-296-3p targets IRF1-facilitated viral infection and modulates the expression of TNF-α in PAMs during HP-PRRSV infection and that IRF1 regulates the expression of TNF-α by activating the TNF promoter via IRF1 response elements. In summary, these findings show that HP-PRRSV infection activates the IRF1/TNF-α signaling axis in PAMs by downregulating host miR-296-3p. This extends our understanding of the inflammatory response induced by HP-PRRSV infection.

Identification of Cleavage Sites Proteolytically Processed by NS2B-NS3 Protease in Polyprotein of Japanese Encephalitis Virus.

Understanding the proteolytic processing of polyprotein mediated by NS2B-NS3 protease contributes to the exploration of the mechanisms underlying infection of Japanese encephalitis virus (JEV), a zoonotic flavivirus. In this study, eukaryotic and prokaryotic cell models were employed to identify the cleavage sites mediated by viral NS2B-NS3 protease in JEV polyprotein. Artificial green fluorescent protein (GFP) substrates that contained the predicted cleavage site sequences of JEV polyprotein were expressed in swine testicle (ST) cells in the presence and absence of JEV infection, or co-expressed in E. coli with the recombinant NS2B-NS3 protease that was generated by fusing the N-terminal protease domain of NS3 to the central hydrophilic domain of NS2B. The cleavage of GFP substrates was examined by western blot. Among twelve artificial GFP substrates containing the cleavage site sequences predictively processed by host cell and/or NS2B-NS3 proteases, all sites were found to be cleaved by host cell proteases with different efficiencies. The sites at internal C, NS2A/NS2B, NS2B/NS3 and NS3/NS4A junctions, but not the sites at internal NS3, internal NS4A and NS4B/NS5 junctions were identified to be cleaved by JEV NS2B-NS3 protease. These data provide insight into the proteolytic processing of polyprotein, which is useful for understanding JEV replication and pathogenesis.

Potential Role of Flavivirus NS2B-NS3 Proteases in Viral Pathogenesis and Anti-flavivirus Drug Discovery Employing Animal Cells and Models: A Review.

Flaviviruses are known to cause a variety of diseases in humans in different parts of the world. There are very limited numbers of antivirals to combat flavivirus infection, and therefore new drug targets must be explored. The flavivirus NS2B-NS3 proteases are responsible for the cleavage of the flavivirus polyprotein, which is necessary for productive viral infection and for causing clinical infections; therefore, they are a promising drug target for devising novel drugs against different flaviviruses. This review highlights the structural details of the NS2B-NS3 proteases of different flaviviruses, and also describes potential antiviral drugs that can interfere with the viral protease activity, as determined by various studies. Moreover, optimized in vitro reaction conditions for studying the NS2B-NS3 proteases of different flaviviruses may vary and have been incorporated in this review. The increasing availability of the in silico and crystallographic/structural details of flavivirus NS2B-NS3 proteases in free and drug-bound states can pave the path for the development of promising antiflavivirus drugs to be used in clinics. However, there is a paucity of information available on using animal cells and models for studying flavivirus NS2B-NS3 proteases, as well as on the testing of the antiviral drug efficacy against NS2B-NS3 proteases. Therefore, on the basis of recent studies, an effort has also been made to propose potential cellular and animal models for the study of flavivirus NS2B-NS3 proteases for the purposes of exploring flavivirus pathogenesis and for testing the efficacy of possible drugs targets, in vitro and in vivo.

Adaptation of a live-attenuated genotype I Japanese encephalitis virus to vero cells is associated with mutations in structural protein genes.

The SD12-F120 is a live-attenuated genotype I strain of Japanese encephalitis virus (JEV) and was obtained by serial passage of wild-type strain SD12 on BHK-21 cells combined with multiple plaque purification and virulence selection in mice. The large scale production and vast clinical trials always demand ideal safety and efficacy profile of live-attenuated vaccines. In the present study, SD12-F120VC has undergone serial passaging of P1-P30 in WHO qualified Vero cells to assess the potential effect of adaptation to growth on Vero cells. The series of experiments showed that vaccine SD12-F120VC (Vero cell adapted) variants have consistently increased in peak virus titer compared to early passages and have good adaptation to growth in Vero cells. The animal experiments showed that Vero cell adapted SD12-F120VC variants have attenuation phenotype in suckling mice and the plaque morphology for all SD12-F120VC variants was small. Vaccination of mice with SD12-F120VC vaccine produced complete protection for homologous SD12 genotype I strain, but failed to give the complete protection of vaccinated mice against the challenge of heterologous N28 genotype III strain. In response to immunization of SD12-F120VC in mice, the neutralizing antibodies titer against homologous SD12-F120VC and SD12 (GI) was higher than heterologous N28 (GIII) strain. The prM protein has 6 amino acid substitutions, of which 5 amino acid changes were confined at the start of the pr domain in the ∼40 amino acids, and some mutations in the pr domain of prM might contribute to Vero cell adaptation. Our findings in this study are important for validation, evaluation and quality control study of live attenuated flaviviruses vaccines and show that Vero cells are a suitable substrate for the production of a safe and stable live-attenuated JEV vaccine.

p53 promotes ZDHHC1-mediated IFITM3 palmitoylation to inhibit Japanese encephalitis virus replication.

The tumor suppressor p53 as an innate antiviral regulator contributes to restricting Japanese encephalitis virus (JEV) replication, but the mechanism is still unclear. The interferon-induced transmembrane protein 3 (IFITM3) is an intrinsic barrier to a range of virus infection, whether IFITM3 is responsible for the p53-mediated anti-JEV response remains elusive. Here, we found that IFITM3 significantly inhibited JEV replication in a protein-palmitoylation-dependent manner and incorporated into JEV virions to diminish the infectivity of progeny viruses. Palmitoylation was also indispensible for keeping IFITM3 from lysosomal degradation to maintain its protein stability. p53 up-regulated IFITM3 expression at the protein level via enhancing IFITM3 palmitoylation. Screening of palmitoyltransferases revealed that zinc finger DHHC domain-containing protein 1 (ZDHHC1) was transcriptionally up-regulated by p53, and consequently ZDHHC1 interacted with IFITM3 to promote its palmitoylation and stability. Knockdown of IFITM3 significantly impaired the inhibitory role of ZDHHC1 on JEV replication. Meanwhile, knockdown of either ZDHHC1 or IFITM3 expression also compromised the p53-mediated anti-JEV effect. Interestingly, JEV reduced p53 expression to impair ZDHHC1 mediated IFITM3 palmitoylation for viral evasion. Our data suggest the existence of a previously unrecognized p53-ZDHHC1-IFITM3 regulatory pathway with an essential role in restricting JEV infection and provide a novel insight into JEV-host interaction.

Identification of DNMT3B2 as the Predominant Isoform of DNMT3B in Porcine Alveolar Macrophages and Its Involvement in LPS-Stimulated TNF-α Expression.

DNA methyltransferase 3B (DNMT3B) as one member of the DNMT family functions as a de novo methyltransferase, characterized as more than 30 splice variants in humans and mice. However, the expression patterns of DNMT3B in pig as well as the biological function of porcine DNMT3B remain to be determined. In this study, we first examined the expression patterns of DNMT3B in porcine alveolar macrophages (PAM). We demonstrated that only DNMT3B2 and DNMT3B3 were the detectable isoforms in PAM. Furthermore, we revealed that DNTM3B2 was the predominant isoform in PAM. Next, in the model of LPS (lipopolysaccharide)-activated PAM, we showed that in comparison to the unstimulated PAM, (1) expression of DNTM3B is reduced; (2) the methylation level of TNF-α gene promoter is decreased. We further establish that DNMT3B2-mediated methylation of TNF-α gene promoter restricts induction of TNF-α in the LPS-stimulated PAM. In summary, these findings reveal that DNMT3B2 is the predominant isoform in PAM and its downregulation contributes to expression of TNF-α via hypomethylation of TNF-α gene promoter in the LPS-stimulated PAM.

Mosquito Defensins Enhance Japanese Encephalitis Virus Infection by Facilitating Virus Adsorption and Entry within the Mosquito.

Japanese encephalitis virus (JEV) is a viral zoonosis that can cause viral encephalitis, death, and disability. Although the Culex mosquito is the primary vector of JEV, little is known about JEV transmission by this kind of mosquito. Here, we found that mosquito defensin facilitated the adsorption of JEV on target cells via the defensin/lipoprotein receptor-related protein 2 (LRP2) axis. Mosquito defensin bound the ED III domain of the viral envelope (E) protein and directly mediated efficient virus adsorption on the target cell surface; the receptor LRP2, which is expressed on the cell surface, affected defensin-dependent adsorption. As a result, mosquito defensin enhanced JEV infection in the salivary gland, increasing the possibility of viral transmission by mosquitoes. These findings demonstrate the novel role of mosquito defensin in JEV infection and the mechanisms through which the virus exploits mosquito defensin for infection and transmission.IMPORTANCE In this study, we observed the complex roles of mosquito defensin in JEV infection; mosquito defensin exhibited a weak antiviral effect but strongly enhanced binding. In the latter, defensin directly binds the ED III domain of the viral E protein and promotes the adsorption of JEV to target cells by interacting with lipoprotein receptor-related protein 2 (LRP2), thus accelerating virus entry. Together, our results indicate that mosquito defensin plays an important role in facilitating JEV infection and potential transmission.

Phenotypic and Genotypic Comparison of a Live-Attenuated Genotype I Japanese Encephalitis Virus SD12-F120 Strain with Its Virulent Parental SD12 Strain.

The phenotypic and genotypic characteristics of a live-attenuated genotype I (GI) strain (SD12-F120) of Japanese encephalitis virus (JEV) were compared with its virulent parental SD12 strain to gain an insight into the genetic changes acquired during the attenuation process. SD12-F120 formed smaller plaque on BHK-21 cells and showed reduced replication in mouse brains compared with SD12. Mice inoculated with SD12-F120 via either intraperitoneal or intracerebral route showed no clinical symptoms, indicating a highly attenuated phenotype in terms of both neuroinvasiveness and neurovirulence. SD12-F120 harbored 29 nucleotide variations compared with SD12, of which 20 were considered silent nucleotide mutations, while nine resulted in eight amino acid substitutions. Comparison of the amino acid variations of SD12-F120 vs SD12 pair with those from other four isogenic pairs of the attenuated and their virulent parental strains revealed that the variations at E138 and E176 positions of E protein were identified in four and three pairs, respectively, while the remaining amino acid variations were almost unique to their respective strain pairs. These observations suggest that the genetic changes acquired during the attenuation process were likely to be strain-specific and that the mechanisms associated with JEV attenuation/virulence are complicated.

Notch signaling contributes to the expression of inflammatory cytokines induced by highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) infection in porcine alveolar macrophages.

Notch signaling, an evolutionarily conserved signal pathway has emerged as a key signal pathway to regulate host immune response but the contribution of Notch signaling to immune response in pigs remains unknown. Infection of porcine alveolar macrophages (PAM) with porcine reproductive and respiratory syndrome virus (PRRSV) triggers expression of Jagged1 mRNA, suggesting that Notch signaling might play a role in the immune response to PRRSV infection. To further explore it, we examined the expression profile of Notch molecules in PAM following a highly pathogenic PRRSV (HP-PRRSV) strain infection. We demonstrated that HP-PRRSV infection resulted in the induction of Notch ligands (Jagged1, Dll3, Dll4), the transcription factor RBP-J, and the target gene Hes1, consistent with activation of Notch signaling. Next, using DAPT treatment and the knockdown of RBP-J illustrated that inhibition of activation of Notch signaling attenuated induction of the inflammatory cytokines (TNF-α and IL-1ß) instead of viral replication in PAM during HP-PRRSV infection. Furthermore, the knockdown of Jagged1, the most induced ligand not only inhibited activation of Notch signaling, but also reduced the expression of inflammatory cytokines without any influence in viral replication. Moreover, our data revealed that several signaling including NF-κB, MAPK and Notch signaling contributed to the induction of Jagged1 in PAM during HP-PRRSV infection. In summary, these findings reveal that Notch as an important signaling pathway could contribute to the regulation of inflammatory response induced by HP-PRRSV infection.

Antiviral activity of phage display-selected peptides against Japanese encephalitis virus infection in vitro and in vivo.

Japanese Encephalitis virus (JEV) is a zoonotic flavivirus that is the most significant etiological agent of childhood viral neurological infections. However, no specific antiviral drug is currently available to treat JEV infections. The JEV envelope (E) protein is a class II viral fusion protein that mediates host cell entry, making interference with the interaction between the E protein of JEV and its cognate receptors an attractive strategy for anti-JEV drug development. In this study, we identified a peptide derived from a phage display peptide library against the E protein of JEV, designated P1, that potentially inhibits in vitro and in vivo JEV infections. P1 inhibits JEV infection in BHK-21 cells with 50% inhibitory capacity at a concentration of 35.9 µM. The time-of-addition assay indicates that JEV replication is significantly inhibited during pre-infection and co-infection of P1 with JEV while post-infection treatments with P1 have very little impact on JEV proliferation, showing that P1 inhibits JEV infection at early stages and indicating the potential prophylactic effect of P1. We adapted an in vitro BiFC assay system and demonstrated that P1 interacts with JEV E proteins and blocks their entry into cells. We also evaluated the therapeutic efficacy of P1 in a lethal JEV mouse model exhibiting systemic and brain infections. Interestingly, P1 treatment protected C57BL/6 mice against mortality, markedly reduced the viral loads in blood and brain, and diminished the histopathological lesions in the brain cells. In addition to controlling systemic infection, P1 has a very low level of cytotoxicity and acts in a sequence-specific manner, as scrambled peptide sP1 does not show any antiviral activity. In conclusion, our in vitro and in vivo experimental findings show that P1 possesses antiviral activity against JEV infections, is safe to use, and has potential for further development as an antiviral treatment against JEV infections.

Porcine reproductive and respiratory syndrome virus counteracts type I interferon-induced early antiviral state by interfering IRF7 activity.

Porcine reproductive and respiratory syndrome (PRRS) is an economically important disease with a significant impact on the pig industry. It is caused by PRRS virus (PRRSV), which predominantly infects and replicates in porcine pulmonary alveolar macrophages (PAMs). We pretreated PAMs with porcine interferon (IFN)-α to induce an antiviral state within the cells and subsequently infected them with highly pathogenic PRRSV. Changes in global gene expression in IFN-α-pretreated PAMs in response to PRRSV infection were determined by RNA-sequence analysis and confirmed by real-time PCR. We found that IRF7 and other antiviral interferon stimulating genes (ISG)s were suppressed by PRRSV infection. Further studies demonstrated that PRRSV could down-regulate the expression of IRF7 by the non-structure protein 7 (nsp7). In conclusion, PRRSV infection had a strong immunosuppressive effect of IFN. PRRSV nsp7 inhibits the expression of IRF7, thereby down-regulating the expression of IFN and downstream ISGs and facilitated the virus to replicate.

Acidity/Alkalinity of Japanese Encephalitis Virus E Protein Residue 138 Alters Neurovirulence in Mice.

The Japanese encephalitis virus (JEV) envelope (E) protein, as one of mediators of virus entry into host cells, plays a critical role in determining virulence. The Glu-to-Lys mutation of residue 138 in E protein (E138) plays an important role in attenuating JEV vaccine strain SA14-14-2. However, it is not clear how E138 attenuates JEV. Here, we demonstrate that the Glu-to-Arg mutation of E138 also determines the attenuation of JEV strain 10S3. Likewise, for its parent strain (HEN0701), a virulence strain, the mutations of E138 are responsible for virulence alteration. Furthermore, we demonstrated that mutations of alkaline residues in E138 contributed to the attenuation of neurovirulence; in contrast, mutations of acidic residues enhanced the neurovirulence of the strains. Moreover, acidity in residue E47 had a similar effect on neurovirulence. Furthermore, the alkaline E138 residue enhanced susceptibility to heparin inhibition in vitro and limited JEV diffusion in mouse brain. These results suggest that the acidity/alkalinity of the E138 residue plays an important role in neurovirulence determination.IMPORTANCE The E protein is the only glycoprotein in mature JEV, and it plays an important role in viral neurovirulence. E protein mutations attenuate JEV neurovirulence through unclear mechanisms. Here, we discovered that E138 is a predominant determinant of JEV neurovirulence. We demonstrated that the alkalinity/acidity of E138 determines JEV neurovirulence. These data contribute to the characterization of the E protein and the rational development of novel JEV vaccines.

A conjugate protein containing HIV TAT, ISG20, and a PRRSV polymerase binding inhibits PRRSV replication and may be a novel therapeutic platform.

Porcine Reproductive and Respiratory Syndrome (PRRS), which is caused by Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection, has caused substantial economic losses for the global swine industry. To date, there are limited commercially available measures to control the spread of PRRSV. The available vaccines are unstable and there is no anti-PRRSV therapeutic available. Therefore, this study designed a novel recombinant antiviral protein that included a novel polypeptide that binds to the PRRSV polymerase (p9), the transduction ability of the HIV TAT, and the nucleotide degradation activity of interferon stimulated gene 20 (ISG20). The recombinant proteins TAT-p9-ISG20 and p9-ISG20 were expressed in MARC-145 cells by transient transfection and then tested for antiviral activity and entry efficiency. The p9-ISG20 construct had greater antiviral activity than either p9 alone (1.37-fold) or ISG20 alone (1.9-fold). Addition of the HIV TAT protein increased the entry efficiency of p9-ISG20 by 1.57-fold, which was associated with increased anti-viral activity (1.52-fold) compared to P9-ISG20. In summary, TAT-p9-ISG20 achieved a synergistic effect by combining three different antiviral proteins and may be a novel PRRSV therapeutic platform.

mosGCTL-7, a C-Type Lectin Protein, Mediates Japanese Encephalitis Virus Infection in Mosquitoes.

Japanese encephalitis virus (JEV) is an arthropod-borne flavivirus prevalent in Asia and the Western Pacific and is the leading cause of viral encephalitis. JEV is maintained in a transmission cycle between mosquitoes and vertebrate hosts, but the molecular mechanisms by which the mosquito vector participates in transmission are unclear. We investigated the expression of all C-type lectins during JEV infection in Aedes aegypti The C-type lectin mosquito galactose-specific C-type lectin 7 (mosGCTL-7) (VectorBase accession no. AAEL002524) was significantly upregulated by JEV infection and facilitated infection in vivo and in vitro mosGCTL-7 bound to the N-glycan at N154 on the JEV envelope protein. This recognition of viral N-glycan by mosGCTL-7 is required for JEV infection, and we found that this interaction was Ca2+ dependent. After mosGCTL-7 bound to the glycan, mosPTP-1 bound to mosGCTL-7, promoting JEV entry. The viral burden in vivo and in vitro was significantly decreased by mosPTP-1 double-stranded RNA (dsRNA) treatment, and infection was abolished by anti-mosGCTL-7 antibodies. Our results indicate that the mosGCTL-7/mosPTP-1 pathway plays a key role in JEV infection in mosquitoes. An improved understanding of the mechanisms underlying flavivirus infection in mosquitoes will provide further opportunities for developing new strategies to control viral dissemination in nature.IMPORTANCE Japanese encephalitis virus is a mosquito-borne flavivirus and is the primary cause of viral encephalitis in the Asia-Pacific region. Twenty-four countries in the WHO Southeast Asia and Western Pacific regions have endemic JEV transmission, which exposes >3 billion people to the risks of infection, although JEV primarily affects children. C-type lectins are host factors that play a role in flavivirus infection in humans, swine, and other mammals. In this study, we investigated C-type lectin functions in JEV-infected Aedes aegypti and Culex pipiens pallens mosquitoes and cultured cells. JEV infection changed the expression of almost all C-type lectins in vivo and in vitro, and mosGCTL-7 bound to the JEV envelope protein via an N-glycan at N154. Cell surface mosPTP-1 interacted with the mosGCTL-7-JEV complex to facilitate virus infection in vivo and in vitro Our findings provide further opportunities for developing new strategies to control arbovirus dissemination in nature.

Cryptococcal heat shock protein 70 homolog Ssa1 contributes to pulmonary expansion of Cryptococcus neoformans during the afferent phase of the immune response by promoting macrophage M2 polarization.

Numerous virulence factors expressed by Cryptococcus neoformans modulate host defenses by promoting nonprotective Th2-biased adaptive immune responses. Prior studies demonstrate that the heat shock protein 70 homolog, Ssa1, significantly contributes to serotype D C. neoformans virulence through the induction of laccase, a Th2-skewing and CNS tropic factor. In the present study, we sought to determine whether Ssa1 modulates host defenses in mice infected with a highly virulent serotype A strain of C. neoformans (H99). To investigate this, we assessed pulmonary fungal growth, CNS dissemination, and survival in mice infected with either H99, an SSA1-deleted H99 strain (Δssa1), and a complement strain with restored SSA1 expression (Δssa1::SSA1). Mice infected with the Δssa1 strain displayed substantial reductions in lung fungal burden during the innate phase (days 3 and 7) of the host response, whereas less pronounced reductions were observed during the adaptive phase (day 14) and mouse survival increased only by 5 d. Surprisingly, laccase activity assays revealed that Δssa1 was not laccase deficient, demonstrating that H99 does not require Ssa1 for laccase expression, which explains the CNS tropism we still observed in the Ssa1-deficient strain. Lastly, our immunophenotyping studies showed that Ssa1 directly promotes early M2 skewing of lung mononuclear phagocytes during the innate phase, but not the adaptive phase, of the immune response. We conclude that Ssa1's virulence mechanism in H99 is distinct and laccase-independent. Ssa1 directly interferes with early macrophage polarization, limiting innate control of C. neoformans, but ultimately has no effect on cryptococcal control by adaptive immunity.

Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans-containing lung cells compared with C. neoformans-free cells. Among C. neoformans-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-γ abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.

Type I interferon-mediated immune response against influenza A virus is attenuated in the absence of p53.

Influenza A virus (IAV) infection induces secretion of type I interferon (IFN) and activation of p53, which play essential roles in the host defense against tumor development and viral infection. In this study, we knocked down p53 expression by RNA interference. The expression levels of IFN-stimulated genes (ISGs) including IFN regulatory factor (IRF) 5, IRF9, ISG15, ISG20, guanylate-binding protein 1, retinoic acid-inducible gene-I and 2'-5'-oligoadenylate synthetase 1 were significantly attenuated in response to IAV infection and IFN-α stimulation in p53-knockdown cells. This attenuated expression of ISGs was associated with enhanced replication of IAV. Pretreatment of p53-knockdown cells with IFN-α failed to inhibit IAV replication, indicating impaired antiviral activity. These findings indicate that p53 plays an essential role in the enhancement of the type I IFN-mediated immune response against IAV infection.

Macrophage M1/M2 polarization dynamically adapts to changes in cytokine microenvironments in Cryptococcus neoformans infection.

The outcome of cryptococcal pneumonia correlates with local macrophage polarization status, as M1 and M2 polarization marks protective and nonprotective responses, respectively. Overall, pulmonary macrophage polarization status changes over time during a cryptococcal infection. This could have been caused by repolarization of individual macrophages or by a replacement of M2-polarized cells by new M1-polarized cells. To explore the ability of macrophages to change between polarization states, we conducted a series of experiments using in vitro macrophages. Coculture of macrophages with Cryptococcus neoformans resulted in development of a weak M1-like phenotype, with modestly increased inducible nitric oxide synthase (iNOS) but lacking interleukin 6 (IL-6) induction. The C. neoformans-induced M1-like polarization state was plastic, as macrophages stimulated first with C. neoformans and then with gamma interferon (IFN-γ) or IL-4 expressed mRNA polarization patterns similar to those stimulated with cytokines alone. To further evaluate macrophage polarization plasticity, cytokine stimulatory conditions were established which fully polarized macrophages. IFN-γ and IL-4 stimulation differentially induced complete M1 and M2 polarization, defined by differential expression of marker mRNA panels, surface marker expression, and tumor necrosis factor alpha (TNF-α) protein production. Switching IFN-γ- to IL-4-stimulating conditions, and vice versa, resulted in uniform changes in profiles of polarization marker genes consistent with the most recent cytokine environment. Furthermore, the ability of sequentially stimulated macrophages to inhibit C. neoformans reflected the most recent polarizing condition, independent of previous polarization. Collectively, these data indicate that M1/M2 macrophage polarization phenotypes are highly plastic to external signals, and interventions which therapeutically repolarize macrophages could be beneficial for treatment of cryptococcosis.