BTK drives neutrophil activation for sterilizing antifungal immunity.

We describe a previously-unappreciated role for Bruton's tyrosine kinase (BTK) in fungal immune surveillance against aspergillosis, an unforeseen complication of BTK inhibitors (BTKi) used for treating B-cell lymphoid malignancies. We studied BTK-dependent fungal responses in neutrophils from diverse populations, including healthy donors, BTKi-treated patients, and X-linked agammaglobulinemia patients. Upon fungal exposure, BTK was activated in human neutrophils in a TLR2-, Dectin-1-, and FcγR-dependent manner, triggering the oxidative burst. BTK inhibition selectively impeded neutrophil-mediated damage to Aspergillus hyphae, primary granule release, and the fungus-induced oxidative burst by abrogating NADPH oxidase subunit p40phox and GTPase RAC2 activation. Moreover, neutrophil-specific Btk deletion in mice enhanced aspergillosis susceptibility by impairing neutrophil function, not recruitment or lifespan. Conversely, GM-CSF partially mitigated these deficits by enhancing p47phox activation. Our findings underline the crucial role of BTK signaling in neutrophils for antifungal immunity and provide a rationale for GM-CSF use to offset these deficits in susceptible patients.

Vaccination against neoantigens induced in cross-priming cDC1 in vivo.

The conventional type 1 dendritic cells (cDC1) play a pivotal role in protective immunity against pathogens and cancer. However, their low frequency in the blood and tissues limits their use in immune therapy. We have recently described a method to vaccinate against neoantigens that are induced in tumor cells by targeted delivery of a TAP siRNA to dendritic cells using a TLR9 binding CpG oligonucleotide. Since TLR9 is also expressed in immune suppressive myeloid populations TLR9 targeting could reduce the effectiveness of this approach. Here, we describe a modular multivalent antibody platform to target the TAP siRNA to resident Clec9a expressing cDC1 and show that it leads to selective and sustained TAP downregulation in cDC1 and inhibits tumor growth in mice more effectively than CpG targeted siRNA. To induce DC maturation an agonistic CD40 antibody was administered to the siRNA treated mice. To obviate the need for a second drug formulation and reduce the risk of toxicity, we exploited the multivalent nature of this targeting platform to co-deliver the TAP siRNA and a DC maturation agent, a CpG containing oligonucleotide, to cDC1 in vivo and show that it was more effective than Clec9a targeting of TAP siRNA in combination with CD40 antibody. This study describes a way to manipulate the function of cDC1 cells in vivo using a broadly applicable antibody-based targeting platform to deliver multiple biological agents to specific cells in vivo to potentiate (immune) therapy and to probe the biology of specific cell types in their natural settings.

Btk Supports Autoreactive B Cell Development and Protects against Apoptosis but Is Expendable for Antigen Presentation.

Bruton's tyrosine kinase (Btk) propagates B cell signaling, and BTK inhibitors are in clinical trials for autoimmune disease. Although autoreactive B cells fail to develop in the absence of Btk, its role in mature cells is unknown. To address this issue, a model of conditional removal (Btk flox/Cre-ERT2 ) was used to excise Btk from mature transgenic B cells that recognize the pathophysiologic autoantigen insulin. Anti-insulin B cells escape central tolerance and promote autoimmune diabetes, mimicking human autoreactive cells. Lifelong Btk deficiency was previously shown to eliminate 95% of anti-insulin B cells, but in this model, mature anti-insulin B cells survived for weeks after targeted Btk deletion, even when competing with a polyclonal repertoire. BCR-stimulated cells could still signal via Syk, PLCy2, and CD22, but failed to upregulate the antiapoptotic protein Bcl-xL, and proliferation was impaired. Surprisingly, Btk-depleted anti-insulin B cells could still present Ag and activate T cells, a critical function in promoting T cell-mediated islet cell destruction. Thus, pharmacologic targeting of Btk may be most effective by blocking expansion of established autoreactive cells, and preventing emergence of new ones.

Impaired B Cell Apoptosis Results in Autoimmunity That Is Alleviated by Ablation of Btk.

While apoptosis plays a role in B-cell self-tolerance, its significance in preventing autoimmunity remains unclear. Here, we report that dysregulated B cell apoptosis leads to delayed onset autoimmune phenotype in mice. Our longitudinal studies revealed that mice with B cell-specific deletion of pro-apoptotic Bim (BBimfl/fl ) have an expanded B cell compartment with a notable increase in transitional, antibody secreting and recently described double negative (DN) B cells. They develop greater hypergammaglobulinemia than mice lacking Bim in all cells and accumulate several autoantibodies characteristic of Systemic Lupus Erythematosus (SLE) and related Sjögren's Syndrome (SS) including anti-nuclear, anti-Ro/SSA and anti-La/SSB at a level comparable to NODH2h4 autoimmune mouse model. Furthermore, lymphocytes infiltrated the tissues including submandibular glands and formed follicle-like structures populated with B cells, plasma cells and T follicular helper cells indicative of ongoing immune reaction. This autoimmunity was ameliorated upon deletion of Bruton's tyrosine kinase (Btk) gene, which encodes a key B cell signaling protein. These studies suggest that Bim-mediated apoptosis suppresses and B cell tyrosine kinase signaling promotes B cell-mediated autoimmunity.

Ibrutinib Potentiates Antihepatocarcinogenic Efficacy of Sorafenib by Targeting EGFR in Tumor Cells and BTK in Immune Cells in the Stroma.

Hepatocellular carcinoma (HCC), the most prevalent primary liver cancer, is a leading cause of cancer-related death worldwide because of rising incidence and limited therapy. Although treatment with sorafenib or lenvatinib is the standard of care in patients with advanced-stage HCC, the survival benefit from sorafenib is limited due to low response rate and drug resistance. Ibrutinib, an irreversible tyrosine kinase inhibitor (TKI) of the TEC (e.g., BTK) and ErbB (e.g., EGFR) families, is an approved treatment for B-cell malignancies. Here, we demonstrate that ibrutinib inhibits proliferation, spheroid formation, and clonogenic survival of HCC cells, including sorafenib-resistant cells. Mechanistically, ibrutinib inactivated EGFR and its downstream Akt and ERK signaling in HCC cells, and downregulated a set of critical genes involved in cell proliferation, migration, survival, and stemness, and upregulated genes promoting differentiation. Moreover, ibrutinib showed synergy with sorafenib or regorafenib, a sorafenib congener, by inducing apoptosis of HCC cells. In vivo, this TKI combination significantly inhibited HCC growth and prolonged survival of immune-deficient mice bearing human HCCLM3 xenograft tumors and immune-competent mice bearing orthotopic mouse Hepa tumors at a dose that did not exhibit systemic toxicity. In immune-competent mice, the ibrutinib-sorafenib combination reduced the numbers of BTK+ immune cells in the tumor microenvironment. Importantly, we found that the BTK+ immune cells were also enriched in the tumor microenvironment in a subset of primary human HCCs. Collectively, our findings implicate BTK signaling in hepatocarcinogenesis and support clinical trials of the sorafenib-ibrutinib combination for this deadly disease.

Combined Systemic and Intratumoral Administration of Human Papillomavirus Vaccine to Treat Multiple Cutaneous Basaloid Squamous Cell Carcinomas.

Importance: Squamous cell carcinoma (SCC) is the second most common form of skin cancer, and its incidence is increasing. When surgical management is not an option, finding a safe and efficacious treatment is a challenge. Mounting evidence suggests that the human papillomavirus (HPV) is involved in the pathogenesis of some SCCs. Objective: To assess whether the 9-valent HPV vaccine could be an effective treatment strategy for cutaneous SCC. Design, Setting, and Participants: A woman in her 90s with multiple, inoperable cutaneous basaloid SCCs was successfully treated at a university-based outpatient dermatology clinic with a combination of systemic and intratumoral delivery of the 9-valent HPV vaccine from March 17, 2016, through February 27, 2017, and then followed up through May 21, 2018. Main Outcomes and Measures: Reduction in tumor size and number after a combination of systemic and intratumoral administration of the HPV vaccine. Results: All tumors resolved 11 months after the first intratumoral injection of the vaccine. The patient remained free of tumors at the end of follow-up. Conclusions and Relevance: This is the first report, to our knowledge, of complete regression of a cutaneous malignant tumor after combined systemic and direct intratumoral injection of the 9-valent HPV vaccine. This report suggests that the HPV vaccine may have therapeutic utility for SCCs in patients who are poor surgical candidates, have multiple lesions, or defer surgery.

Bruton's Tyrosine Kinase Is Not Essential for B Cell Survival beyond Early Developmental Stages.

Bruton's tyrosine kinase (Btk) is a crucial regulator of B cell signaling and is a therapeutic target for lymphoma and autoimmune disease. BTK-deficient patients suffer from humoral immunodeficiency, as their B cells fail to progress beyond the bone marrow. However, the role of Btk in fully developed, mature peripheral B cells is not well understood. Analysis using BTK inhibitors is complicated by suboptimal inhibition, off-target effects, or failure to eliminate BTK's adaptor function. Therefore a Btkflox/Cre-ERT2 mouse model was developed and used to excise Btk after B cell populations were established. Mice lacking Btk from birth are known to have reduced follicular (FO) compartments, with expanded transitional populations, suggesting a block in development. In adult Btkflox/Cre-ERT2 mice, Btk excision did not reduce FO B cells, which persisted for weeks. Autoimmune-prone B1 cells also survived conditional Btk excision, contrasting their near absence in global Btk-deficient mice. Therefore, Btk supports BCR signaling during selection into the FO and B1 compartments, but is not needed to maintain these cell populations. B1-related natural IgM levels remained normal, contrasting global Btk deficiency, but B cell proliferation and T-independent type II immunization responses were blunted. Thus, B cells have nuanced signaling responses that are differentially regulated by Btk for development, survival, and function. These findings raise the possibility that Btk may also be expendable for survival of mature human B cells, therefore requiring prolonged dosing to be effective, and that success of BTK inhibitors may depend in part on off-target effects.

The eIF2α Kinase Heme-Regulated Inhibitor Protects the Host from Infection by Regulating Intracellular Pathogen Trafficking.

The host employs both cell-autonomous and system-level responses to limit pathogen replication in the initial stages of infection. Previously, we reported that the eukaryotic initiation factor 2α (eIF2α) kinases heme-regulated inhibitor (HRI) and protein kinase R (PKR) control distinct cellular and immune-related activities in response to diverse bacterial pathogens. Specifically for Listeria monocytogenes, there was reduced translocation of the pathogen to the cytosolic compartment in HRI-deficient cells and consequently reduced loading of pathogen-derived antigens on major histocompatibility complex class I (MHC-I) complexes. Here we show that Hri-/- mice, as well as wild-type mice treated with an HRI inhibitor, are more susceptible to listeriosis. In the first few hours of L. monocytogenes infection, there was much greater pathogen proliferation in the liver of Hri-/- mice than in the liver of Hri+/+ mice. Further, there was a rapid increase of serum interleukin-6 (IL-6) levels in Hri+/+ mice in the first few hours of infection whereas the increase in IL-6 levels in Hri-/- mice was notably delayed. Consistent with these in vivo findings, the rate of listeriolysin O (LLO)-dependent pathogen efflux from infected Hri-/- macrophages and fibroblasts was significantly higher than the rate seen with infected Hri+/+ cells. Treatment of cells with an eIF2α kinase activator enhanced both the HRI-dependent and PKR-dependent infection phenotypes, further indicating the pharmacologically malleability of this signaling pathway. Collectively, these results suggest that HRI mediates the cellular confinement and killing of virulent L. monocytogenes in addition to promoting a system-level cytokine response and that both are required to limit pathogen replication during the first few hours of infection.

Specialized Proresolving Mediators Rescue Infant Mice from Lethal Citrobacter rodentium Infection and Promote Immunity against Reinfection.

Infants are generally highly susceptible to oral pathogens. Intestinal infection and the associated diarrhea are significant global causes of morbidity and mortality in infants. Among the enteric pathogens, enteropathogenic Escherichia coli (EPEC) stands out as showing the highest risk for infection-induced death in infants ≤12 months old. We have developed an experimental model of infant infection with EPEC, using the mouse-specific pathogen Citrobacter rodentium Our murine infant model is similar to EPEC infection in human infants since infant mice are much more susceptible to C. rodentium infection than adult mice; infants infected with 50-fold fewer bacteria than the standard adult dose uniformly succumbed to the infection. Infant infection is characterized by high early and sustained bacterial titers and profound intestinal inflammation associated with extensive necrosis and systemic dissemination of the bacteria. Therefore, it seems likely that infant deaths result from sepsis secondary to intestinal damage. Recently, specialized proresolving mediators (SPM) have been found to exert profound beneficial effects in adult models of infection. Thus, we investigated the actions of two proresolving lipid mediators, resolvin D1 (RvD1) and resolvin D5 (RvD5), on the course of infection in infants. Strikingly, postinfection treatment with RvD1 and RvD5 reduced bacterial loads, mitigated inflammation, and rescued the infants from death. Furthermore, postinfection treatment with RvD1 and RvD5 led to protection from reinfection associated with C. rodentium-specific IgG responses comparable to those in adults. These results indicate that SPM may provide novel therapeutic tools for the treatment of pathological intestinal infections in infants.

Corrigendum: Distinct Transcriptomic Features Are Associated with Transitional and Mature B-Cell Populations in the Mouse Spleen.

[This corrects the article on p. 30 in vol. 6, PMID: 25717326.].

Perforin-2 Protects Host Cells and Mice by Restricting the Vacuole to Cytosol Transitioning of a Bacterial Pathogen.

The host-encoded Perforin-2 (encoded by the macrophage-expressed gene 1, Mpeg1), which possesses a pore-forming MACPF domain, reduces the viability of bacterial pathogens that reside within membrane-bound compartments. Here, it is shown that Perforin-2 also restricts the proliferation of the intracytosolic pathogen Listeria monocytogenes Within a few hours of systemic infection, the massive proliferation of L. monocytogenes in Perforin-2(-/-)mice leads to a rapid appearance of acute disease symptoms. We go on to show in cultured Perforin-2(-/-)cells that the vacuole-to-cytosol transitioning of L. monocytogenesis greatly accelerated. Unexpectedly, we found that in Perforin-2(-/-)macrophages,Listeria-containing vacuoles quickly (≤ 15 min) acidify, and that this was coincident with greater virulence gene expression, likely accounting for the more rapid translocation of L. monocytogenes to its replicative niche in the cytosol. This hypothesis was supported by our finding that aL. monocytogenes strain expressing virulence factors at a constitutively high level replicated equally well in Perforin-2(+/+)and Perforin-2(-/-)macrophages. Our findings suggest that the protective role of Perforin-2 against listeriosis is based on it limiting the intracellular replication of the pathogen. This cellular activity of Perforin-2 may derive from it regulating the acidification of Listeria-containing vacuoles, thereby depriving the pathogen of favorable intracellular conditions that promote its virulence gene activity.

In aged mice, low surrogate light chain promotes pro-B-cell apoptotic resistance, compromises the PreBCR checkpoint, and favors generation of autoreactive, phosphorylcholine-specific B cells.

In aged mice, new B-cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B-cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5(low) B-cell precursors generate new B cells which show increased reactivity to the self-antigen/bacterial antigen phosphorylcholine (PC). Pro-B cells in old bone marrow as well as pro-B cells from young adult λ5-deficient mice are resistant to cytokine-induced apoptosis (TNFα; TGFß), indicating that low λ5 expression in pro-B cells is sufficient to cause increased survival. Transfer of TNFα-producing 'age-associated B cells' (ABC; CD21/35(-) CD23(-)) or follicular (FO) B cells from aged mice into RAG-2 KO recipients led to preferential loss of λ5(high) pro-B cells, but retention of λ5(low), apoptosis-resistant pro-B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα-producing B cells, results in preferential loss of SLC(high) pro-B cells within the bone marrow. Further B-cell development then occurs via an 'SLC(low)' pathway that not only impairs B-cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery.

Distinct Transcriptomic Features are Associated with Transitional and Mature B-Cell Populations in the Mouse Spleen.

Splenic transitional B-cells (T1 and T2) are selected to avoid self-reactivity and to safeguard against autoimmunity, then differentiate into mature follicular (FO-I and FO-II) and marginal zone (MZ) subsets. Transcriptomic analysis by RNA-seq of the five B-cell subsets revealed T1 cell signature genes included RAG suggesting a potential for receptor revision. T1 to T2 B-cell differentiation was marked by a switch from Myb to Myc, increased expression of the PI3K adapter DAP10 and MHC class II. FO-II may be an intermediate in FO-I differentiation and may also become MZ B-cells as suggested by principle component analysis. MZ B-cells possessed the most distinct transcriptome including down-regulation of CD45 phosphatase-associated protein (CD45-AP/PTPRC-AP), as well as upregulation of IL-9R and innate molecules TLR3, TLR7, and bactericidal Perforin-2 (MPEG1). Among the endosomal TLRs, stimulation via TLR3 further enhanced Perforin-2 expression exclusively in MZ B-cells. Using gene-deleted and overexpressing transgenic mice we show that IL-9/IL-9R interaction resulted in rapid activation of STAT1, 3, and 5, primarily in MZ B-cells. Importantly, CD45-AP mutant mice had reduced transitional and increased mature MZ and FO B-cells, suggesting that it prevents premature entry of transitional B-cells to the mature B-cell pool or their survival and proliferation. Together, these findings suggest, developmental plasticity among splenic B-cell subsets, potential for receptor revision in peripheral tolerance whereas enhanced metabolism coincides with T2 to mature B-cell differentiation. Further, unique core transcriptional signatures in MZ B-cells may control their innate features.

DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers.

UNLABELLED: Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. IMPORTANCE: Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans.

Bruton's tyrosine kinase promotes persistence of mature anti-insulin B cells.

Autoreactive B lymphocytes are essential for the development of T cell-mediated type 1 diabetes (T1D). Cytoplasmic Bruton's tyrosine kinase (BTK) is a key component of B cell signaling, and its deletion in T1D-prone NOD mice significantly reduces diabetes. However, the role of BTK in the survival and function of autoreactive B cells is not clear. To evaluate the contributions of BTK, we used mice in which B cells express an anti-insulin BCR (125Tg) and promote T1D, despite being anergic. Crossing Btk deficiency onto 125Tg mice reveals that, in contrast to immature B cells, mature anti-insulin B cells are exquisitely dependent upon BTK, because their numbers are reduced by 95%. BTK kinase domain inhibition reproduces this effect in mature anti-insulin B cells, with less impact at transitional stages. The increased dependence of anti-insulin B cells on BTK became particularly evident in an Igκ locus site-directed model, in which 50% of B cells edit their BCRs to noninsulin specificities; Btk deficiency preferentially depletes insulin binders from the follicular and marginal zone B cell subsets. The persistent few Btk-deficient anti-insulin B cells remain competent to internalize Ag and invade pancreatic islets. As such, loss of BTK does not significantly reduce diabetes incidence in 125Tg/NOD mice as it does in NOD mice with a normal B cell repertoire. Thus, BTK targeting may not impair autoreactive anti-insulin B cell function, yet it may provide protection in an endogenous repertoire by decreasing the relative availability of mature autoreactive B cells.

B-lymphocyte tolerance and effector function in immunity and autoimmunity.

B-lymphocytes are integral to host defense against microbial pathogens and are associated with many autoimmune diseases. The B-cell receptor implements B-cell self-tolerance based on the antigen specificity, and B-cell-activating factor receptor (BAFF-R) imposes homeostatic control. While shaping the repertoire, the immune tolerance process also culls mature B cells into distinct populations. The activation response of B cells is tailored to the type of pathogen attack and is facilitated by T-cell help via CD40/CD40L interaction and/or innate cell help via toll-like receptors in conjunction with BAFF receptors and ligands. Activated effector B cells not only produce antibodies, but also produce a variety of cytokines to enhance and suppress the immune response. Not surprisingly, B cells play multiple roles in both humoral and cellular immune responses during infection and autoimmune pathogenesis. Here, we discuss how gene expression and signaling networks regulate peripheral B-cell tolerance, B-cell effector functions and emerging therapies targeting B-cell signaling in autoimmune diseases.

A20-mediated negative regulation of canonical NF-κB signaling pathway.

The nuclear factor kappa B (NF-κB) plays vital role in the immune system by regulating innate and adaptive immunity, development and survival of lymphocytes, and lymphoid organogenesis. All known NF-κB activators converge on the IkappaB kinase (IKK) complex to activate the canonical and non-canonical NF-κB pathways. The IKK complex contains two catalytic subunits (IKKα and IKKß) and a regulatory subunit NEMO/IKKγ that regulates the canonical NF-κB pathway, whereas IKKα regulates the non-canonical pathway. The process of IKKα activation and its role in the regulation of canonical NF-κB activation remain elusive. The canonical pathway is rapidly activated and produces a potent inflammatory response to bacterial and viral infections as well as different types of stress; however, uncontrolled NF-κB activation can lead to autoimmune diseases and cancers. Therefore, to keep the inflammatory response in check, elaborate negative regulatory mechanisms operate to terminate NF-κB activation at multiple levels by de novo synthesis of NF-κB inhibitory proteins, and orchestration of protein ubiquitination and deubiquitination. The NF-κB target genes, IκBα and A20, play critical roles in termination of the active canonical NF-κB pathway. In this review, we discuss our recent findings describing a novel function for IKKα in nucleating the ubiquitin-editing enzyme A20 complex, a major negative regulator of canonical NF-κB signaling. Consistently with an inhibitory function of IKKα, it is targeted by the human T-cell leukemia virus 1 (HTLV-1) oncoprotein, Tax, to prevent assembly of the A20 complex to maintain persistent NF-κB activation that promotes transformation and survival of virus-transformed cells.

The host-encoded Heme Regulated Inhibitor (HRI) facilitates virulence-associated activities of bacterial pathogens.

Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria. For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.