Andrea Hodgson

Mechanisms of sex disparities in influenza pathogenesis

Epidemiological evidence from influenza outbreaks and pandemics reveals that morbidity and mortality are often higher for women than men. Sex differences in the outcome of influenza are age‐dependent, often being most pronounced among adults of reproductive ages (18–49 years of age) and sometimes reflecting the unique state of pregnancy in females, which is a risk factor for severe disease. Small animal models of influenza virus infection illustrate that inflammatory immune responses also differ between the sexes and impact the outcome of infection, with females generating higher proinflammatory cytokine and chemokine responses and experiencing greater morbidity and mortality than males. Males and females also respond differently to influenza vaccines, with women initiating higher humoral immune responses but experiencing more adverse reactions to seasonal influenza vaccines than men. Small animal models further show that elevated immunity following vaccination in females leads to greater cross‐protection against novel influenza viruses in females compared with males. Sex steroid hormones, including estradiol and testosterone, as well as genetic differences between the sexes may play roles in modulating sex differences in immune responses to influenza virus infection and vaccination. Future studies must elucidate the pathways and cellular responses that differ between the sexes and determine how best to use this knowledge to inform public health policy‐makers about prophylaxis and therapeutic treatments of influenza virus infections to ensure adequate protection in both males and females.

Antibody responses and cross protection against lethal influenza A viruses differ between the sexes in C57BL/6 mice.

A mouse model was used to determine if protective immunity to influenza A virus infection differs between the sexes. The median lethal dose of H1N1 or H3N2 was lower for naïve females than males. After a sublethal, primary infection with H1N1 or H3N2, females and males showed a similar transient morbidity, but females generated more neutralizing and total anti-influenza A virus antibodies. Immunized males and females showed similar protection against secondary challenge with a homologous virus, but males experienced greater morbidity and had higher lung viral titers after infection with a lethal dose of heterologous virus. Females develop stronger humoral immune responses and greater cross protection against heterosubtypic virus challenge.

Metalloprotease NleC Suppresses Host NF-κB/Inflammatory Responses by Cleaving p65 and Interfering with the p65/RPS3 Interaction

Attaching/Effacing (A/E) pathogens including enteropathogenic Escherichia coli (EPEC), enterohemorrhagic E. coli (EHEC) and the rodent equivalent Citrobacter rodentium are important causative agents of foodborne diseases. Upon infection, a myriad of virulence proteins (effectors) encoded by A/E pathogens are injected through their conserved type III secretion systems (T3SS) into host cells where they interfere with cell signaling cascades, in particular the nuclear factor kappaB (NF-κB) signaling pathway that orchestrates both innate and adaptive immune responses for host defense. Among the T3SS-secreted non-LEE-encoded (Nle) effectors, NleC, a metalloprotease, has been recently elucidated to modulate host NF-κB signaling by cleaving NF-κB Rel subunits. However, it remains elusive how NleC recognizes NF-κB Rel subunits and how the NleC-mediated cleavage impacts on host immune responses in infected cells and animals. In this study, we show that NleC specifically targets p65/RelA through an interaction with a unique N-terminal sequence in p65. NleC cleaves p65 in intestinal epithelial cells, albeit a small percentage of the molecule, to generate the p651–38 fragment during C. rodentium infection in cultured cells. Moreover, the NleC-mediated p65 cleavage substantially affects the expression of a subset of NF-κB target genes encoding proinflammatory cytokines/chemokines, immune cell infiltration in the colon, and tissue injury in C. rodentium-infected mice. Mechanistically, the NleC cleavage-generated p651–38 fragment interferes with the interaction between p65 and ribosomal protein S3 (RPS3), a ‘specifier’ subunit of NF-κB that confers a subset of proinflammatory gene transcription, which amplifies the effect of cleaving only a small percentage of p65 to modulate NF-κB-mediated gene expression. Thus, our results reveal a novel mechanism for A/E pathogens to specifically block NF-κB signaling and inflammatory responses by cleaving a small percentage of p65 and targeting the p65/RPS3 interaction in host cells, thus providing novel insights into the pathogenic mechanisms of foodborne diseases.

Sam68 modulates the promoter specificity of NF-κB and mediates expression of CD25 in activated T cells

CD25, the alpha chain of the interleukin-2 receptor, is expressed in activated T cells and plays a significant role in autoimmune disease and tumorigenesis; however, the mechanisms regulating transcription of CD25 remain elusive. Here we identify the Src-associated substrate during mitosis of 68kDa (Sam68) as a novel non-Rel component in the nuclear factor-kappaB (NF-κB) complex that confers CD25 transcription. Our results demonstrate that Sam68 plays an essential role in the induction and maintenance of CD25 in T cells. T cell receptor engagement triggers translocation of the inhibitor of NF-κB kinase alpha (IKKα) from the cytoplasm to the nucleus, where it phosphorylates Sam68, causing complex formation with NF-κB in the nucleus. These findings reveal the important roles of KH domain-containing components and their spatial interactions with IKKs in determining the binding targets of NF-κB complexes, thus shedding novel insights into the regulatory specificity of NF-κB.

Interference with nuclear factor kappaB signaling pathway by pathogen-encoded proteases: Global and selective inhibition

Pathogens have evolved a myriad of ways to abrogate and manipulate the host response to infections. Of the various mechanisms involved, pathogen‐encoded and sometimes host‐encoded proteases are an important category of virulence factors that cause robust changes on the host response by targeting key proteins along signaling cascades. The nuclear factor kappaB (NF‐κB) signaling pathway is a crucial regulatory mechanism for the cell, controlling the expression of survival, immune and proliferation genes. Proteases from pathogens of almost all types have been demonstrated to target and cleave members of the NF‐κB signaling pathway at nearly every level. This review provides discussion of proteases targeting the most abundant NF‐κB subunit, p65, and the impact of protease‐mediated p65 cleavage on the immune responses and survival of the infected host cell. After examining various examples of protease interference, it becomes evident that the cleavage fragments produced by pathogen‐driven proteolytic processing should be further characterized to determine whether they have novel and unique functions within the cell. The selective targeting of p65 and its effect on gene transcription reveals unique mechanisms by which pathogens acutely alter their microenvironment, and further research may open new opportunities for novel therapeutics to combat pathogens.

Interference with NF‐κB signaling pathway by pathogen‐encoded proteases: global and selective inhibition

Pathogens have evolved a myriad of ways to abrogate and manipulate the host response to infections. Of the various mechanisms involved, pathogen‐encoded and sometimes host‐encoded proteases are an important category of virulence factors that cause robust changes on the host response by targeting key proteins along signaling cascades. The nuclear factor kappaB (NF‐κB) signaling pathway is a crucial regulatory mechanism for the cell, controlling the expression of survival, immune and proliferation genes. Proteases from pathogens of almost all types have been demonstrated to target and cleave members of the NF‐κB signaling pathway at nearly every level. This review provides discussion of proteases targeting the most abundant NF‐κB subunit, p65, and the impact of protease‐mediated p65 cleavage on the immune responses and survival of the infected host cell. After examining various examples of protease interference, it becomes evident that the cleavage fragments produced by pathogen‐driven proteolytic processing should be further characterized to determine whether they have novel and unique functions within the cell. The selective targeting of p65 and its effect on gene transcription reveals unique mechanisms by which pathogens acutely alter their microenvironment, and further research may open new opportunities for novel therapeutics to combat pathogens.

Caspase-3 cleaved p65 fragment dampens NF-κB-mediated anti-apoptotic transcription by interfering with the p65/RPS3 interaction

Caspase‐3‐mediated p65 cleavage is believed to suppress nuclear factor‐kappa B (NF‐κB)‐mediated anti‐apoptotic transactivation in cells undergoing apoptosis. However, only a small percentage of p65 is cleaved during apoptosis, not in proportion to the dramatic reduction in NF‐κB transactivation. Here we show that the p651‐97 fragment generated by Caspase‐3 cleavage interferes with ribosomal protein S3 (RPS3), an NF‐κB “specifier” subunit, and selectively retards the nuclear translocation of RPS3, thus dampening the RPS3/NF‐κB‐dependent anti‐apoptotic gene expression. Our findings reveal a novel cell fate determination mechanism to ensure cells undergo programed cell death through interfering with RPS3/NF‐κB‐conferred anti‐apoptotic transcription by the fragment from partial p65 cleavage by activated Caspase‐3.

Ultrasound imaging of splenomegaly as a proxy to monitor colon tumor development in Apcmin716/+ mice

Animal models of colon cancer are widely used to understand the molecular mechanisms and pathogenesis of the disease. These animal models require a substantial investment of time and traditionally necessitate the killing of the animal to measure the tumor progression. Several in vivo imaging techniques are being used in both human clinics and preclinical studies, albeit at high cost and requiring particular expertise. Here, we report that the progression of splenomegaly coincides with and positively correlates to colon tumor development in Apcmin716/+ mice expressing a mutant gene encoding an adenomatous polyposis coli protein truncated at amino acid 716. Ultrasound image‐based spleen size measurement precisely mirrors splenomegaly development in vivo in the tumor‐laden Apcmin716/+ mice. Moreover, the spleen dimensions extracted from the ultrasound sonograms are positively correlated with normalized spleen weight and the number and area of colon tumors. Hence, we propose measuring the spleen size in vivo by ultrasound imaging as a novel approach to estimate splenomegaly development and to indirectly monitor colon tumor development in Apcmin716/+ mice. The widespread use of ultrasound machines in the laboratory setting, coupled with the fact that it is a noninvasive method, make it a straightforward and useful tool for monitoring the experimental progress of colon cancer in mice and determining end points without killing animals strictly for diagnostics purposes.

Sam68/KHDRBS1-dependent NF-κB activation confers radioprotection to the colon epithelium in γ-irradiated mice

Previously we reported that Src-associated-substrate-during-mitosis-of-68kDa (Sam68/KHDRBS1) is pivotal for DNA damage-stimulated NF-κB transactivation of anti-apoptotic genes (Fu et al., 2016). Here we show that Sam68 is critical for genotoxic stress-induced NF-κB activation in the γ-irradiated colon and animal and that Sam68-dependent NF-κB activation provides radioprotection to colon epithelium in vivo. Sam68 deletion diminishes γ-irradiation-triggered PAR synthesis and NF-κB activation in colon epithelial cells (CECs), thus hampering the expression of anti-apoptotic molecules in situ and facilitating CECs to undergo apoptosis in mice post whole-body γ-irradiation (WBIR). Sam68 knockout mice suffer more severe damage in the colon and succumb more rapidly from acute radiotoxicity than the control mice following WBIR. Our results underscore the critical role of Sam68 in orchestrating genotoxic stress-initiated NF-κB activation signaling in the colon tissue and whole animal and reveal the pathophysiological relevance of Sam68-dependent NF-κB activation in colonic cell survival and recovery from extrinsic DNA damage. DOI: http://dx.doi.org/10.7554/eLife.21957.001

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production.