Fc Receptor Function Research Articles

Stellabody: A novel hexamer-promoting mutation for improved IgG potency.

Advances in antibody engineering are being directed at the development of next generation immunotherapeutics with improved potency. Hexamerisation of IgG is a normal physiological aspect of IgG biology and recently described mutations that facilitate this process have a substantial impact upon monoclonal antibody behavior resulting in the elicitation of dramatically enhanced complement-dependent cytotoxicity, Fc receptor function, and enhanced antigen binding effects, such as targeted receptor agonism or microbe neutralization. Whereas the discovery of IgG hexamerisation enhancing mutations has largely focused on residues with exposure at the surface of the Fc-Fc and CH2-CH3 interfaces, our unique approach is the engineering of the mostly buried residue H429 in the CH3 domain. Selective substitution at position 429 forms the basis of Stellabody technology, where the choice of amino acid results in distinct hexamerisation outcomes. H429F results in monomeric IgG that hexamerises after target binding, so called "on-target" hexamerisation, while the H429Y mutant forms pH-sensitive hexamers in-solution prior to antigen binding. Moreover, Stellabody technologies are broadly applicable across the family of antibody-based biologic therapeutics, including conventional mAbs, bispecific mAbs, and Ig-like biologics such as Fc-fusions, with applications in diverse diseases.

Influenza antibody breadth and effector functions are immune correlates from acquisition of pandemic infection of children

Cross-reactive antibodies with Fc receptor (FcR) effector functions may mitigate pandemic virus impact in the absence of neutralizing antibodies. In this exploratory study, we use serum from a randomized placebo-controlled trial of seasonal trivalent influenza vaccination in children (NCT00792051) conducted at the onset of the 2009 H1N1 pandemic (pH1N1) and monitored for infection. We found that seasonal vaccination increases pH1N1 specific antibodies and FcR effector functions. Furthermore, prospective baseline antibody profiles after seasonal vaccination, prior to pH1N1 infection, show that unvaccinated uninfected children have elevated ADCC effector function, FcγR3a and FcγR2a binding antibodies to multiple pH1N1 proteins, past seasonal and avian (H5, H7 and H9) strains. Whereas, children that became pH1N1 infected after seasonal vaccination have antibodies focussed to seasonal strains without FcR functions, and greater aggregated HA-specific profiles for IgM and IgG3. Modeling to predict infection susceptibility, ranked baseline hemagglutination antibody inhibition as the highest contributor to lack of pH1N1 infection, in combination with features that include pH1-IgG1, H1-stem responses and FcR binding to seasonal vaccine and pH1 proteins. Thus, seasonal vaccination can have benefits against pandemic influenza viruses, and some children already have broadly reactive antibodies with Fc potential without vaccination and may be considered ‘elite influenza controllers’.

Enhanced Fc Receptor Expression In Sea Urchin Spermatozoa By Platelet-Activating Factor

Platelet-activating factor (PAF) is a signaling phospholipid that has a variety of reproductive roles, e.g. sperm capacitation and the acrosome reaction (an exocytotic process). PAF’s mechanism action involves G-protein receptor activation induction of intracellular calcium, which enhances sperm motility. In nonreproductive cells, the Fc receptor (FcR) functions in ligand-triggered transmission signals across the membrane which alters secretion, exocytosis, and increased intracellular calcium. This study looked at the impact of PAF on FcR expression in sea urchin spermatozoa. Sea urchin sperm were exposed to exogenous PAF (10-7M; 15 minutes) and evaluated for FcR activity via flow cytometry. A significant (P=0.01) difference (64.30%) in FcR wave activity between the PAF (mean 3.286) and control (2.0) groups was observed. Within waves, there were more (83.31%) high points (crests) in the PAF (mean 1.571) group than the control (mean 0.857) group. Similarly, there were more (49.96%) low (troughs) points in the PAF (mean 1.714) group than the control (mean 1.143) group. This is the first report to demonstrate a relative positive change in spermatozoa FcR expression when exposed to PAF. The FcR functions in ligand-triggered transmission of PAF via a G-protein coupled receptor mediated pathway leading to physiological alterations. Provided the known mechanism of action of PAF on spermatozoa physiology, the data suggests that the interaction of PAF and FcR play a pivotal role in sperm activity and fertilization potential.

Impact of cancer stem cell markers Nestin and Sox2 on invasion abilities of epithelial and mesenchymal pancreatic cancer cells

Immunoglobulin G (IgG) antibodies function, in part, through ligation of cell-surface Fc receptors such as FcγRIIIA (also known as CD16A). IgG glycosylation is known to impact antibody function, but the role of FcγRIIIA glycans, if any, is unclear. Patel et al. now reveal that these glycans do impact protein conformation and IgG affinity and display cell-specific glycosylation patterns, leading to a potential model in which the affinity and possibly function of Fc receptors is dictated by the cell type and its surface glycome.

Preclinical characterization and clinical translation of pharmacodynamic markers for MK-5890: a human CD27 activating antibody for cancer immunotherapy

BackgroundImmune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI...

Immunoglobulin Use for the Management of Painful Peripheral Neuropathy: A Systematic Review and Meta-Analysis.

Immunoglobulins (IG) are widely used for the treatment of a variety of immune-mediated diseases. The exact mechanism of action remains unknown, but IG modulate the expression and function of Fc receptors, interfere with complement activation and production of cytokines, neutralize pathogenic autoantibodies, and affect the activation and effector functions of B and T lymphocytes. Immunoglobulins are usually delivered intravenously, and are effective in ameliorating motor symptoms, and/or preventing disease progression in immune-mediated neuropathies, including Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy. The aim of this systematic review and meta-analysis was to study the potential of IG for the treatment of painful peripheral neuropathy (PPN). The outcome of interest was the percentage of patients with PPN who achieved pain relief following IG administration. We performed a systematic literature search on March 17, 2022, in the PubMed database without any publication date restrictions. We also looked for unpublished or ongoing trials in clinicaltrials.org. Pain reduction following IG treatment had to be within the aims (primary or secondary). The aforementioned literature search strategy revealed five studies (two open-label, three randomized placebo-controlled) eligible to be included. The pooled estimate of the percentage of patients with PPN who received immunoglobulins and reported pain relief was found to be 65% (95% CI 58-71%). The likelihood of achieving pain relief with immunoglobulin treatment was 2.9 times higher (95% CI 1.6-5.2) compared to placebo (p = 0.0003). The use of IG for the treatment of pain due to peripheral neuropathy has a potential therapeutic benefit. Further studies across patients with different types of painful peripheral neuropathy are needed to better characterize this effect. Registration number on PROSPERO: CRD42022319614.

Abstract 3425: The CD47-targeting antibody ZL-1201 enhances anti-tumor activity of standard of care therapeutic antibodies by promoting phagocytosis in hematologic and solid tumor models

Abstract Therapeutic monoclonal antibodies (mAbs) comprise the standard of care (SoC) for several hematologic and solid cancer indications. These include rituximab for CD20-expressing lymphomas, trastuzumab for HER2-amplified cancers, and cetuximab for EGFR-driven solid tumors. Although the primary mechanism of action for these therapies is tumor-cell targeting, they are rarely curative, partly because tumor cells employ a variety of mechanisms to evade immune surveillance and blunt anti-tumor immune response. CD47 is upregulated in solid tumors and hematological malignancies and provides a critical anti-phagocytic signal to escape destruction by the innate immune system. Thus, CD47 may represent a promising therapeutic target for activating innate and adaptive immunity and enhancing the therapeutic potential of SoC antibodies in the clinic. Here we present preclinical data supporting the combinatorial effects of the CD47-targeting monoclonal antibody ZL-1201 with SoC mAbs in hematologic and solid tumor models. ZL-1201 potently blocked CD47/SIRPα interaction and enhanced phagocytosis as measured by flow cytometry and high-content imaging analysis. Notably, in combination with trastuzumab, cetuximab, and rituximab, ZL-1201 demonstrated enhanced phagocytosis across hematologic and solid tumor models expressing varying levels of CD47 in in vitro co-culture systems. Neutralizing the Fc receptor function of these SoC mAbs by blocking CD16, CD32, and CD64 abrogated the combinatorial effects, indicating that the CD47 blockade sensitized tumors to pro-phagocytic signals provided by ADCP-inducing mAbs in an Fc receptor-dependent manner. Treatment of xenografts with ZL-1201 also drove tumor-specific immune responses and remodeling of the tumor microenvironment, including increased activation of antigen-presenting cells, decreased frequencies of MDSC, and reduced expression of immune checkpoints such as PD-L1. Further, ZL-1201 in combination with trastuzumab, rituximab, or cetuximab significantly delayed tumor growth and increased survival in in vivo xenograft models, highlighting the pivotal role for CD47 as a resistance mechanism to these therapeutic mAbs and the opportunity to overcome resistance with ZL-1201 combination. In conclusion, ZL-1201 enhances the anti-tumor activity of SoC antibodies and augments the immunologic response by overcoming the CD47/SIRPα “don’t-eat-me” myeloid checkpoint. This study indicates that ZL-1201 may combine with a broad range of SoC mAbs to enhance their clinical benefit across a variety of hematologic and solid tumor indications. ZL-1201 is under Ph1 clinical investigation (NCT04257617). Citation Format: Anthony Cao, Jiaqing Yi, Renyi Wu, Christopher Szeto, Quiping Ye, Bing Wan, Karl Hsu, Omar Kabbarah, Haiying Zhou. The CD47-targeting antibody ZL-1201 enhances anti-tumor activity of standard of care therapeutic antibodies by promoting phagocytosis in hematologic and solid tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3425.

A cell-based FcRn-dependent recycling assay for predictive pharmacokinetic assessment of therapeutic antibodies.

Aim: Evaluation of suitable pharmacokinetic properties is critical for successful development of IgG-based biotherapeutics. The prolonged half-lives of IgGs depend on the intracellular trafficking function of neonatal Fc receptor, which rescues internalized IgGs from lysosomal degradation and recycles them back to circulation. Results: Here, we developed a novel cell-based assay to quantify recycling of monoclonal antibodiesin a transwell culture system that uses a cell line that stably expresses human neonatal Fc receptor. We tested seven therapeutic antibodies and showed that the recycling output of the assay strongly correlated with the clearance in humans. Conclusion: This recycling assay has potential application as a pharmacokinetic prescreening tool to facilitate development and selection of IgG-based candidate therapeutic monoclonal antibodies.

FCRL4 Is an Fc Receptor for Systemic IgA, but Not Mucosal Secretory IgA.

Fc receptor-like (FCRL) 4 is an immunoregulatory receptor expressed on a subpopulation of human memory B cells of mucosa-associated lymphoid tissue. Fc receptor function of FCRL4 was demonstrated by binding of IgA to FCRL4 following heat aggregation of the Ig. In this study, we demonstrate that FCRL4 recognizes J chain-linked systemic IgA in the absence of heat aggregation. We further demonstrate that mucosal secretory IgA is not recognized by FCRL4 and that systemic IgA binding can be competitively inhibited by recombinant secretory component protein. Finally, we provide evidence that primary FCRL4-bearing human memory B cells are constitutively bound to IgA. Our study provides a mechanism for the negative regulatory activity of FCRL4 on AgR-mediated B cell activation.

Reprogramming the Constant Region of Immunoglobulin G Subclasses for Enhanced Therapeutic Potency against Cancer.

The constant region of immunoglobulin (Ig) G antibodies is responsible for their effector immune mechanism and prolongs serum half-life, while the fragment variable (Fv) region is responsible for cellular or tissue targeting. Therefore, antibody engineering for cancer therapeutics focuses on both functional efficacy of the constant region and tissue- or cell-specificity of the Fv region. In the functional aspect of therapeutic purposes, antibody engineers in both academia and industry have capitalized on the constant region of different IgG subclasses and engineered the constant region to enhance therapeutic efficacy against cancer, leading to a number of successes for cancer patients in clinical settings. In this article, we review IgG subclasses for cancer therapeutics, including (i) IgG1, (ii) IgG2, 3, and 4, (iii) recent findings on Fc receptor functions, and (iv) future directions of reprogramming the constant region of IgG to maximize the efficacy of antibody drug molecules in cancer patients.

Boosting of Markers of Fcγ Receptor Function in Anti-HIV Antibodies During Structured Treatment Interruption.

Anti-HIV envelope (Env) antibodies elicit important Fc receptor functions, including FcγRIIIa-mediated natural killer cell killing of opsonized infected targets. How these antibodies evolve during HIV infection and treatment remains poorly understood. We describe changes in anti-HIV Env IgG using longitudinal samples from seroconverter subjects treated soon after infection and later during periods of structured treatment interruption (STI). Our well-validated dimeric rsFcγR binding assays combine effects of opsonizing antibody subclasses, epitopes, and geometries to provide a measure of FcγR (Fcγ receptor)-mediated functionality. IgG1 anti-Env titers diminished rapidly during antiretroviral therapy (ART; t1/2 3.0 ± 0.8 months), while the dimeric rsFcγRIIIa activity persisted longer (t1/2 33 ± 11 months), suggesting that there is maintenance of functional antibody specificities within the diminished pool of anti-HIV Env Abs. The initial antibody response to infection in two subjects was characterized by approximately fivefold higher FcγRIIIa compared with FcγRIIa binding activity. Uncoupling of FcγRIIa and FcγRIIIa activities may be a distinct feature of the early antibody response that preferentially engages FcγRIIIa-mediated effector functions. Two to three STI cycles, even with low viremia, were sufficient to boost dimeric FcγR activity in these seroconverter subjects. We hypothesize that increased humoral immunity induced by STI is a desirable functional outcome potentially achievable by therapeutic immunization during ART. We conclude that controlled viral antigen exposure under the protection of suppressive ART may be effective in eliciting FcγR-dependent function in support of viral reactivation and kill strategies.

FcRn-Dependent Transcytosis of Monoclonal Antibody in Human Nasal Epithelial Cells In Vitro: A Prerequisite for a New Delivery Route for Therapy?

Monoclonal antibodies (mAbs) are promising therapies to treat airway chronic inflammatory disease (asthma or nasal polyps). To date, no study has specifically assessed, in vitro, the potential function of neonatal Fc receptor (FcRn) in IgG transcytosis through the human nasal airway epithelium. The objective of this study was to report the in vitro expression and function of FcRn in nasal human epithelium. FcRn expression was studied in an air–liquid interface (ALI) primary culture model of human nasal epithelial cells (HNEC) from polyps. FcRn expression was characterized by quantitative RT-PCR, western blot, and immunolabeling. The ability of HNECs to support mAb transcytosis via FcRn was assessed by transcytosis assay. This study demonstrates the expression of FcRn mRNA and protein in HNEC. We report a high expression of FcRn in the cytosol of ciliated, mucus, and basal cells by immunohistochemistry with a higher level of FcRn proteins in differentiated HNEC. We also proved in vitro transepithelial delivery of an IgG1 therapeutic mAb with a dose–response curve. This is the first time that FcRn expression and mAb transcytosis has been shown in a model of human nasal respiratory epithelium in vitro. This study is a prerequisite for FcRn-dependent nasal administration of mAbs.

Characterization of human natural killer cells for therapeutic use

As a part of the innate immune system, natural killer (NK) cells are cytotoxic lymphocytes that can exert cytotoxic activity against infected or transformed cells. Furthermore, due to their expression of a functional Fc receptor, they have also been eluded as a major effector fraction in antibody-dependent cellular cytotoxicity. These characteristics have led to multiple efforts to use them for adoptive immunotherapy against various malignancies. There are now at least 70 clinical trials testing the safety and efficacy of NK cell products around the world in early-phase clinical trials. NK cells are also being tested in the context of tumor retargeting via chimeric antigen receptors, other genetic modification strategies, as well as tumor-specific activation strategies such as bispecific engagers with or without cytokine stimulations. One advantage of the use of NK cells for adoptive immunotherapy is their potential to overcome HLA barriers. This has led to a plethora of sources, such as cord blood hematopoietic stem cells and induced pluripotent stem cells, which can generate comparatively high cytotoxic NK cells to peripheral blood counterparts. However, the variety of the sources has led to a heterogeneity in the characterization of the final infusion product. Therefore, in this review, we will discuss a comparative assessment strategy, from characterization of NK cells at collection to final product release by various phenotypic and functional assays, in an effort to predict potency of the cellular product.

Immune Complex Vaccination.

Antibody/antigen binding results in immune complexes (IC) that have a variety of regulatory functions. One important feature is the enhanced host immune activation against antigen contained in the complex. ICs play important roles at several critical steps that lead to B and T cell activation, including antigen targeting/retention, facilitated antigen uptake, antigen presenting cell activation and proper balancing of positive and negative stimulatory signals. In both poultry industry and clinical health care, ICs have been used as preventive and therapeutic vaccines. With our deepening understanding of antibody biology, particularly in light of new revelations of regulatory functions of Fc receptors, mechanistically more precise engineering has spearheaded tailored use of this tool for infection control and cancer therapy. IC-based treatment and prophylaxis have been tested to different extents in HBV, HIV and influenza viral infection control and are actively examined as an alternative treatment for several forms of tumor. As a part of this book series, this chapter aims to discuss the mechanistic aspects of IC signaling and their impact on immune cells. We give samples how this old technology has been used by practitioners over the last several decades and suggest potential paths for future development of IC-based immune therapy.

Comparative functional and pharmacological characterization of Sandoz proposed biosimilar adalimumab (GP2017): rationale for extrapolation across indications

ABSTRACTBackground: Biosimilars are approved biologics that match reference medicine in quality, safety, and efficacy. The development of Sandoz proposed biosimilar adalimumab (SPBA; GP2017) involved a target-directed, iterative state-of-the-art quality-by-design development program. Here, we describe the functional and pharmacological characterization of SPBA and its proposed mechanism of action in immune-mediated inflammatory diseases.Methods: Sensitive in vitro binding and functional characterization studies, and nonclinical evaluations (pharmacokinetics, pharmacodynamics, and safety/toxicology) were performed as part of a stepwise approach to confirm the biosimilarity of SPBA with reference adalimumab.Results: Matching values were reported for SPBA and reference adalimumab in binding assays involving tumor necrosis factor (TNF)-α, complement 1q and human immune effector cell Fcγ receptor subtypes in cell-based bioassays for Fc receptor function (complement- and antibody-dependent cytotoxicity), and in apoptosis inhibition. Furthermore, SPBA and reference adalimumab were equivalent in terms of membrane TNF binding and induction of reverse signaling. Pharmacokinetics of SPBA and reference adalimumab were comparable in rabbits, and the two biologics were equally effective in a human TNF transgenic mouse model of polyarthritis.Conclusion: SPBA matches reference adalimumab with regards to target binding, functional, pharmacokinetic, and pharmacodynamic properties at the nonclinical level supporting its approval in all indications of the reference adalimumab.

NK cells inhibit Plasmodium falciparum growth in red blood cells via antibody-dependent cellular cytotoxicity.

Antibodies acquired naturally through repeated exposure to Plasmodium falciparum are essential in the control of blood-stage malaria. Antibody-dependent functions may include neutralization of parasite-host interactions, complement activation, and activation of Fc receptor functions. A role of antibody-dependent cellular cytotoxicity (ADCC) by natural killer (NK) cells in protection from malaria has not been established. Here we show that IgG isolated from adults living in a malaria-endemic region activated ADCC by primary human NK cells, which lysed infected red blood cells (RBCs) and inhibited parasite growth in an in vitro assay for ADCC-dependent growth inhibition. RBC lysis by NK cells was highly selective for infected RBCs in a mixed culture with uninfected RBCs. Human antibodies to P. falciparum antigens PfEMP1 and RIFIN were sufficient to promote NK-dependent growth inhibition. As these results implicate acquired immunity through NK-mediated ADCC, antibody-based vaccines that target bloodstream parasites should consider this new mechanism of action.

Antibody receptors steal the sweet spotlight

Immunoglobulin G (IgG) antibodies function, in part, through ligation of cell-surface Fc receptors such as FcγRIIIA (also known as CD16A). IgG glycosylation is known to impact antibody function, but the role of FcγRIIIA glycans, if any, is unclear. Patel et al. now reveal that these glycans do impact protein conformation and IgG affinity and display cell-specific glycosylation patterns, leading to a potential model in which the affinity and possibly function of Fc receptors is dictated by the cell type and its surface glycome.

Molecular engineering and plant expression of an immunoglobulin heavy chain scaffold for delivery of a dengue vaccine candidate.

SummaryIn order to enhance vaccine uptake by the immune cells in vivo, molecular engineering approach was employed to construct a polymeric immunoglobulin G scaffold (PIGS) that incorporates multiple copies of an antigen and targets the Fc gamma receptors on antigen‐presenting cells. These self‐adjuvanting immunogens were tested in the context of dengue infection, for which there is currently no globally licensed vaccine yet. Thus, the consensus domain III sequence (cEDIII) of dengue glycoprotein E was incorporated into PIGS and expressed in both tobacco plants and Chinese Ovary Hamster cells. Purified mouse and human cEDIII‐PIGS were fractionated by HPLC into low and high molecular weight forms, corresponding to monomers, dimers and polymers. cEDIII‐PIGS were shown to retain important Fc receptor functions associated with immunoglobulins, including binding to C1q component of the complement and the low affinity Fcγ receptor II, as well as to macrophage cells in vitro. These molecules were shown to be immunogenic in mice, with or without an adjuvant, inducing a high level IgG antibody response which showed a neutralizing potential against the dengue virus serotype 2. The cEDIII‐PIGS also induced a significant cellular immune response, IFN‐γ production and polyfunctional T cells in both the CD4+ and CD8+ compartments. This proof‐of‐principle study shows that the potent antibody Fc‐mediated cellular functions can be harnessed to improve vaccine design, underscoring the potential of this technology to induce and modulate a broad‐ranging immune response.

The expression and function of the neonatal Fc receptor in thyrocytes of Hashimoto's thyroiditis

ObjectiveThyroglobulin (Tg) and thyroid peroxidase (TPO) antibodies (TgAb and TPOAb), which are primarily of the immunoglobulin G (IgG) class, can mediate antibody-dependent cell-mediated cytotoxicity in vitro. However, it is unclear whether any thyrocyte molecules can facilitate the transport and elimination of TgAb and TPOAb. The IgG transport receptor neonatal Fc receptor (FcRn) is a candidate mediator of these processes. In this study, we aimed to evaluate FcRn expression and function in normal and Hashimoto's thyroiditis (HT) thyrocytes. MethodsFcRn expression in primary thyrocyte cultures (four normal and four HT groups) was examined by polymerase chain reaction (PCR) and Western blotting. Localization of FcRn was demonstrated by immunoelectron microscopy. A double immunofluorescence staining method was adopted to detect FcRn and internalized human TgAb IgG. Stimulation experiments were performed to assess the regulation of FcRn expression by T helper cell 1 (Th1) (IFN-γ and TNF-α) and Th2 cytokines (IL-10 and IL-4). ResultsFcRn expression was lower in HT thyrocytes than in normal thyrocytes. FcRn was located in the cytoplasm, membranes, mitochondria and transport vesicles of thyrocytes. Both human IgG and TgAb IgG were internalized by thyrocytes in a pH-dependent manner and co-localized with FcRn in thyrocytes. FcRn expression was downregulated by Th1 and Th2 cytokines in both normal and HT thyrocytes in a dose-dependent manner. ConclusionsOur results suggest that FcRn may be associated with the transport and metabolism of IgG in thyrocytes and that transport is independent of IgG type. FcRn may be involved in HT pathogenesis.

Nrf2 Modulates Host Defense during Streptococcus pneumoniae Pneumonia in Mice

Nrf2 regulates the transcriptional response to oxidative stress. These studies tested the role of Nrf2 during Streptococcus pneumoniae pneumonia and identified Nrf2-dependent genes and pathways in lung tissue and in recruited neutrophils. Nrf2 null and wild type (WT) mice were studied at 6 and 24 h after instillation of S. pneumoniae or PBS. At 6 h, fewer neutrophils were recruited and the number of bacteria remaining in the lungs tended to be less (p = 0.06) in the Nrf2 null compared with WT mice. In uninfected lungs, 53 genes were already differentially expressed in Nrf2 null compared with WT mouse lungs, and gene sets involved in phagocytosis, Fc receptor function, complement, and Ig regulation are enhanced in PBS-treated Nrf2 null gene profiles compared with those of WT mice. These results suggest that initial host defense is enhanced in Nrf2 null mice, resulting in less recruitment of neutrophils. At 24 h, neutrophil recruitment was greater. The percentages of early apoptotic and late apoptotic/necrotic neutrophils were similar. At increasing inoculum numbers, mortality rates strikingly increased from 15 to 31 and 100% in Nrf2 null mice, whereas all WT mice survived, and Nrf2 null mice had a defect in clearance, particularly at the intermediate dose. The mortality was due to enhanced lung injury and greater systemic response. Gene profiling identified differentially regulated genes and pathways in neutrophils and lung tissue, including those involved in redox stress response, metabolism, inflammation, immunoregulatory pathways, and tissue repair, providing insight into the mechanisms for the greater tissue damage and increased neutrophil accumulation.