Transcriptomic profiling implicates PAF1 in both active and repressive immune regulatory networks.

BACKGROUND: Sitting at the interface of gene expression and host-pathogen interaction, polymerase associated factor 1 complex (PAF1C) is a rising player in the innate immune response. The complex localizes to the nucleus and associates with chromatin to modulate RNA polymerase II (RNAPII) elongation of gene transcripts. Performing this function at both proximal and distal regulatory elements, PAF1C interacts with many host factors across such sites, along with several microbial proteins during infection. Therefore, translating the ubiquity of PAF1C into specific impacts on immune gene expression remains especially relevant. RESULTS: Advancing past work, we treat PAF1 knockout cells with a slate of immune stimuli to identify key trends in PAF1-dependent gene expression with broad analytical depth. From our transcriptomic data, we confirm PAF1 is an activator of traditional immune response pathways as well as other cellular pathways correlated with pathogen defense. With this model, we employ computational approaches to refine how PAF1 may contribute to both gene activation and suppression. Specifically focusing on transcriptional motifs and regulons, we predict gene regulatory elements strongly associated with PAF1, including those implicated in an immune response. Overall, our results suggest PAF1 is involved in innate immunity at several distinct axes of regulation. CONCLUSIONS: By identifying PAF1-dependent gene expression across several pathogenic contexts, we confirm PAF1C to be a key mediator of innate immunity. Combining these transcriptomic profiles with potential regulatory networks corroborates the previously identified functions of PAF1C. With this, we foster new avenues for its study as a regulator of innate immunity, and our results will serve as a basis for targeted study of PAF1C in future validation studies.

Systems Biology of Virus-Host Protein Interactions: From Hypothesis Generation to Mechanisms of Replication and Pathogenesis.

As obligate intracellular parasites, all viruses must co-opt cellular machinery to facilitate their own replication. Viruses often co-opt these cellular pathways and processes through physical interactions between viral and host proteins. In addition to facilitating fundamental aspects of virus replication cycles, these virus-host protein interactions can also disrupt physiological functions of host proteins, causing disease that can be advantageous to the virus or simply a coincidence. Consequently, unraveling virus-host protein interactions can serve as a window into molecular mechanisms of virus replication and pathogenesis. Identifying virus-host protein interactions using unbiased systems biology approaches provides an avenue for hypothesis generation. This review highlights common systems biology approaches for identification of virus-host protein interactions and the mechanistic insights revealed by these methods. We also review conceptual innovations using comparative and integrative systems biology that can leverage global virus-host protein interaction data sets to more rapidly move from hypothesis generation to mechanism.

Let's Get Physical: Flavivirus-Host Protein-Protein Interactions in Replication and Pathogenesis.

Flaviviruses comprise a genus of viruses that pose a significant burden on human health worldwide. Transmission by both mosquito and tick vectors, and broad host tropism contribute to the presence of flaviviruses globally. Like all viruses, they require utilization of host molecular machinery to facilitate their replication through physical interactions. Their RNA genomes are translated using host ribosomes, synthesizing viral proteins that cooperate with each other and host proteins to reshape the host cell into a factory for virus replication. Thus, dissecting the physical interactions between viral proteins and their host protein targets is essential in our comprehension of how flaviviruses replicate and how they alter host cell behavior. Beyond replication, even single interactions can contribute to immune evasion and pathogenesis, providing potential avenues for therapeutic intervention. Here, we review protein interactions between flavivirus and host proteins that contribute to virus replication, immune evasion, and disease.

Nuclear dengue virus NS5 antagonizes expression of PAF1-dependent immune response genes.

Dengue virus (DENV) disruption of the innate immune response is critical to establish infection. DENV non-structural protein 5 (NS5) plays a central role in this disruption, such as antagonism of STAT2. We recently found that DENV serotype 2 (DENV2) NS5 interacts with Polymerase associated factor 1 complex (PAF1C). The primary members of PAF1C are PAF1, LEO1, CTR9, and CDC73. This nuclear complex is an emerging player in the immune response. It promotes the expression of many genes, including genes related to the antiviral, antimicrobial and inflammatory responses, through close association with the chromatin of these genes. Our previous work demonstrated that NS5 antagonizes PAF1C recruitment to immune response genes. However, it remains unknown if NS5 antagonism of PAF1C is complementary to its antagonism of STAT2. Here, we show that knockout of PAF1 enhances DENV2 infectious virion production. By comparing gene expression profiles in PAF1 and STAT2 knockout cells, we find that PAF1 is necessary to express immune response genes that are STAT2-independent. Finally, we mapped the viral determinants for the NS5-PAF1C protein interaction. We found that NS5 nuclear localization and the C-terminal region of the methyltransferase domain are required for its interaction with PAF1C. Mutation of these regions rescued the expression of PAF1-dependent immune response genes that are antagonized by NS5. In sum, our results support a role for PAF1C in restricting DENV2 replication that NS5 antagonizes through its protein interaction with PAF1C.

Circulating immunity protects the female reproductive tract from Chlamydia infection.

Anatomical positioning of memory lymphocytes within barrier tissues accelerates secondary immune responses and is thought to be essential for protection at mucosal surfaces. However, it remains unclear whether resident memory in the female reproductive tract (FRT) is required for Chlamydial immunity. Here, we describe efficient generation of tissue-resident memory CD4 T cells and memory lymphocyte clusters within the FRT after vaginal infection with Chlamydia Despite robust establishment of localized memory lymphocytes within the FRT, naïve mice surgically joined to immune mice, or mice with only circulating immunity following intranasal immunization, were fully capable of resisting Chlamydia infection via the vaginal route. Blocking the rapid mobilization of circulating memory CD4 T cells to the FRT inhibited this protective response. These data demonstrate that secondary protection in the FRT can occur in the complete absence of tissue-resident immune cells. The ability to confer robust protection to barrier tissues via circulating immune memory provides an unexpected opportunity for vaccine development against infections of the FRT.

NOD1/NOD2 and RIP2 Regulate Endoplasmic Reticulum Stress-Induced Inflammation during Chlamydia Infection.

The inflammatory response to Chlamydia infection is likely to be multifactorial and involve a variety of ligand-dependent and -independent recognition pathways. We previously reported the presence of NOD1/NOD2-dependent endoplasmic reticulum (ER) stress-induced inflammation during Chlamydia muridarum infection in vitro, but the relevance of this finding to an in vivo context is unclear. Here, we examined the ER stress response to in vivo Chlamydia infection. The induction of interleukin 6 (IL-6) production after systemic Chlamydia infection correlated with expression of ER stress response genes. Furthermore, when tauroursodeoxycholate (TUDCA) was used to inhibit the ER stress response, an increased bacterial burden was detected, suggesting that ER stress-driven inflammation can contribute to systemic bacterial clearance. Mice lacking both NOD1 and NOD2 or RIP2 exhibited slightly higher systemic bacterial burdens after infection with Chlamydia Overall, these data suggest a model where RIP2 and NOD1/NOD2 proteins link ER stress responses with the induction of Chlamydia-specific inflammatory responses.IMPORTANCE Understanding the initiation of the inflammatory response during Chlamydia infection is of public health importance given the impact of this disease on young women in the United States. Many young women are chronically infected with Chlamydia but are asymptomatic and therefore do not seek treatment, leaving them at risk of long-term reproductive harm due to inflammation in response to infection. Our manuscript explores the role of the endoplasmic reticulum stress response pathway initiated by an innate receptor in the development of this inflammation.

Unexpected Role of CD8 T Cells in Accelerated Clearance of Salmonella enterica Serovar Typhimurium from H-2 Congenic mice.

Salmonella infection can cause gastroenteritis in healthy individuals or a serious, systemic infection in immunocompromised patients and has a global impact. CD4 Th1 cells represent the main lymphocyte population that participates in bacterial clearance during both primary and secondary infections in mice of the H-2b haplotype. Previous studies have used congenic mice to examine the function of major histocompatibility complex (MHC) molecules in elimination of this pathogen from the host. In this study, we further characterized the ability of H-2b, H-2k, and H-2u molecules to influence adaptive immunity to Salmonella in MHC congenic mice. By depleting different cell populations during infection, we unexpectedly found that CD8 T cells, in addition to CD4 T cells, play a major role in accelerated clearance of bacteria from H-2k congenic hosts. Our data suggest that CD8 T cells accelerate clearance in some MHC congenic mouse strains and could therefore represent an unexpected contributor to the protective efficacy of Salmonella vaccines outside the typical studies in C57BL/6 mice.

Optimal protection against Salmonella infection requires noncirculating memory.

While CD4 Th1 cells are required for resistance to intramacrophage infections, adoptive transfer of Th1 cells is insufficient to protect against Salmonella infection. Using an epitope-tagged vaccine strain of Salmonella, we found that effective protection correlated with expanded Salmonella-specific memory CD4 T cells in circulation and nonlymphoid tissues. However, naive mice that previously shared a blood supply with vaccinated partners lacked T cell memory with characteristics of tissue residence and did not acquire robust protective immunity. Using a YFP-IFN-γ reporter system, we identified Th1 cells in the liver of immunized mice that displayed markers of tissue residence, including P2X7, ARTC2, LFA-1, and CD101. Adoptive transfer of liver memory cells after ARTC2 blockade increased protection against highly virulent bacteria. Taken together, these data demonstrate that noncirculating memory Th1 cells are a vital component of immunity to Salmonella infection and should be the focus of vaccine strategies.

T cell expression of IL-18R and DR3 is essential for non-cognate stimulation of Th1 cells and optimal clearance of intracellular bacteria.

Th1 cells can be activated by TCR-independent stimuli, but the importance of this pathway in vivo and the precise mechanisms involved require further investigation. Here, we used a simple model of non-cognate Th1 cell stimulation in Salmonella-infected mice to examine these issues. CD4 Th1 cell expression of both IL-18R and DR3 was required for optimal IFN-γ induction in response to non-cognate stimulation, while IL-15R expression was dispensable. Interestingly, effector Th1 cells generated by immunization rather than live infection had lower non-cognate activity despite comparable IL-18R and DR3 expression. Mice lacking T cell intrinsic expression of MyD88, an important adapter molecule in non-cognate T cell stimulation, exhibited higher bacterial burdens upon infection with Salmonella, Chlamydia or Brucella, suggesting that non-cognate Th1 stimulation is a critical element of efficient bacterial clearance. Thus, IL-18R and DR3 are critical players in non-cognate stimulation of Th1 cells and this response plays an important role in protection against intracellular bacteria.

NOD1 and NOD2 signalling links ER stress with inflammation.

Endoplasmic reticulum (ER) stress is a major contributor to inflammatory diseases, such as Crohn disease and type 2 diabetes. ER stress induces the unfolded protein response, which involves activation of three transmembrane receptors, ATF6, PERK and IRE1α. Once activated, IRE1α recruits TRAF2 to the ER membrane to initiate inflammatory responses via the NF-κB pathway. Inflammation is commonly triggered when pattern recognition receptors (PRRs), such as Toll-like receptors or nucleotide-binding oligomerization domain (NOD)-like receptors, detect tissue damage or microbial infection. However, it is not clear which PRRs have a major role in inducing inflammation during ER stress. Here we show that NOD1 and NOD2, two members of the NOD-like receptor family of PRRs, are important mediators of ER-stress-induced inflammation in mouse and human cells. The ER stress inducers thapsigargin and dithiothreitol trigger production of the pro-inflammatory cytokine IL-6 in a NOD1/2-dependent fashion. Inflammation and IL-6 production triggered by infection with Brucella abortus, which induces ER stress by injecting the type IV secretion system effector protein VceC into host cells, is TRAF2, NOD1/2 and RIP2-dependent and can be reduced by treatment with the ER stress inhibitor tauroursodeoxycholate or an IRE1α kinase inhibitor. The association of NOD1 and NOD2 with pro-inflammatory responses induced by the IRE1α/TRAF2 signalling pathway provides a novel link between innate immunity and ER-stress-induced inflammation.

Contaminated water delivery as a simple and effective method of experimental Salmonella infection.

AIM: In most infectious disease models, it is assumed that gavage needle infection is the most reliable means of pathogen delivery to the GI tract. However, this methodology can cause esophageal tearing and induces stress in experimental animals, both of which have the potential to impact early infection and the subsequent immune response. MATERIALS & METHODS: C57BL/6 mice were orally infected with virulent Salmonella Typhimurium SL1344 either by intragastric gavage preceded by sodium bicarbonate, or by contamination of drinking water. RESULTS: We demonstrate that water contamination delivery of Salmonella is equivalent to gavage inoculation in providing a consistent model of infection. Furthermore, exposure of mice to contaminated drinking water for as little as 4 h allowed maximal mucosal and systemic infection, suggesting an abbreviated window exists for natural intestinal entry. CONCLUSION: Together, these data question the need for gavage delivery for infection with oral pathogens.

Protective host immune responses to Salmonella infection.

Salmonella enterica serovars Typhi and Paratyphi are the causative agents of human typhoid fever. Current typhoid vaccines are ineffective and are not widely used in endemic areas. Greater understanding of host-pathogen interactions during Salmonella infection should facilitate the development of improved vaccines to combat typhoid and nontyphoidal Salmonellosis. This review will focus on our current understanding of Salmonella pathogenesis and the major host immune components that participate in immunity to Salmonella infection. In addition, recent findings regarding host immune mechanisms in response to Salmonella infection will be also discussed, providing a new perspective on the utility of improved tools to study the immune response to Salmonella infections.

Toll-like receptor and inflammasome signals converge to amplify the innate bactericidal capacity of T helper 1 cells.

T cell effector functions can be elicited by noncognate stimuli, but the mechanism and contribution of this pathway to the resolution of intracellular macrophage infections have not been defined. Here, we show that CD4(+) T helper 1 (Th1) cells could be rapidly stimulated by microbe-associated molecular patterns during active infection with Salmonella or Chlamydia. Further, maximal stimulation of Th1 cells by lipopolysaccharide (LPS) did not require T-cell-intrinsic expression of toll-like receptor 4 (TLR4), interleukin-1 receptor (IL-1R), or interferon-γ receptor (IFN-γR) but instead required IL-18R, IL-33R, and adaptor protein MyD88. Innate stimulation of Th1 cells also required host expression of TLR4 and inflammasome components that together increased serum concentrations of IL-18. Finally, the elimination of noncognate Th1 cell stimulation hindered the resolution of primary Salmonella infection. Thus, the in vivo bactericidal capacity of Th1 cells is regulated by the response to noncognate stimuli elicited by multiple innate immune receptors.