Autosomal dominant hyper-IgE syndrome (AD-HIES), or Job's syndrome, is a rare primary immunodeficiency caused by dominant-negative mutations in STAT3. Patients experience recurrent pulmonary infections and chronic inflammation, leading to severe complications and heightened mortality risk. To investigate whether neutrophil-intrinsic dysfunction contributes to lung pathology in AD-HIES, we used a murine model expressing the STAT3V463Δ mutation, a common disease-associated variant. Following intratracheal infection with Pseudomonas aeruginosa, STAT3V463Δ mice exhibited pronounced alveolar damage, increased vascular congestion, and extensive leukocyte infiltration compared to wild-type (WT) controls. These changes were accompanied by elevated bacterial burden and significantly increased levels of pro-inflammatory cytokines and chemokines in the lung. Neutrophil recruitment to the lungs was markedly elevated, and surface expression of degranulation markers was enhanced in STAT3V463Δ neutrophils in vivo. To determine whether neutrophil hyperactivation was driven by intrinsic defects independent of microbial load, mice were challenged intratracheally with purified lipopolysaccharide (LPS), revealing similarly enhanced neutrophil degranulation and NETosis in STAT3V463Δ mice despite controlled PAMP exposure. Mechanistically, bone marrow-derived neutrophils (BMDNs) from STAT3V463Δ mice displayed heightened degranulation and NETosis in response to PMA or f-MLF stimulation. Together, these findings demonstrate that STAT3V463Δ drives neutrophil hyperresponsiveness, contributing to dysregulated inflammation and pulmonary tissue damage in AD-HIES.

Rationale: Within the lung, efficient gas exchange is determined by the alveolar-capillary membrane. When this membrane is compromised, fluid leaks into the alveolus and disrupts gas exchange. Bacterial pneumonia can promote alveolar-capillary membrane disruption and progress toward ARDS. Infiltration of immune cells into the lung tissue and airspaces is another key feature of ARDS; however, an exacerbated immune response can promote collateral tissue damage and increase lung injury. Soluble adenylyl cyclase isoform 10 (AC10/sAC) is expressed in capillary endothelial cells, and when activated, AC10 disrupts the pulmonary endothelial barrier and induces edema in the isolated lung. Although recently AC10 was implicated in immune cell recruitment in the systemic circulation, its role during lung infection is not fully resolved. Thus, we hypothesized that loss of AC10 would improve survival, attenuate lung edema and alter immune cell recruitment during acute P. aeruginosa-induced lung injury. Methods: Wild type (WT) and AC10 knockout mice were inoculated intratracheally with P. aeruginosa (1E6 to 2.5E6 colony forming units, CFUs) or vehicle control (PBS). Bronchoalveolar lavage fluid (BALF) was collected for protein quantification and cytokine/chemokine analysis. Survival was assessed over 96-hours, and lung injury evaluated histologically. To determine bacterial burden, organs (lungs, kidney, liver, spleen and brain) were harvested 6 and 24 hours after inoculation, homogenized and cultured overnight at 37 ºC on Pseudomonas isolation agar for quantification of CFUs. Finally, to distinguish the contribution of hematopoietic versus non- hematopoietic cells, bone marrow chimera mice were generated by reconstituting lethally irradiated AC10 knockout mice with bone marrow from WT congenic donors, or lethally irradiated WT mice with bone marrow from AC10 knockout donors. Following infection, BALF and lung tissue from WT, AC10 knockout mice, and bone marrow chimera mice, were analyzed by flow cytometry for total CD45+ leukocytes, Ly6G+ neutrophils, and neutrophil CD11b surface expression. Results: Compared to WT controls, AC10 knockout mice had decreased protein content in BALF and a distinct cytokine/chemokine profile, following infection. Genetic ablation of AC10 increased survival at 96-hours and histological analysis suggested attenuated lung injury without altering bacterial clearance at either 6 or 24 hours. The total number of CD45+ white blood cells and Ly6G+ neutrophils were similar between genotypes; however, AC10 knockout mice demonstrated reduced CD11b surface expression on Ly6G+ neutrophils. Importantly, reduced CD11b surface expression on neutrophils was recapitulated in AC10 knockout mice reconstituted with WT bone marrow, indicating that AC10 in non-hematopoietic compartments regulates neutrophil phenotype during infection. Conclusion: AC10 exacerbates lung injury and mortality during acute P. aeruginosa-induced lung injury by promoting alveolar-capillary disruption and enhancing neutrophil CD11b surface expression, rather than altering immune cell recruitment or bacterial clearance. These finding identify AC10 as an “edema factor” that modulates neutrophil phenotype and represents a potential therapeutic target in ARDS associated P. aeruginosa pneumonia. This abstract was presented at the American Physiology Summit 2026 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.

Abstract Herpes stromal keratitis (HSK), caused by herpes simplex virus 1 (HSV-1), is the leading cause of infectious blindness in developed countries. Innate-acting γδ T17 cells are critical for protection against ocular HSV-1 infection. It is well established these cells can facilitate neutrophil influx into the cornea, but whether γδ T17 cells regulate other innate immune cells following ocular HSV-1 infection remains unclear. Natural killer (NK) cells also participate in the innate immune response generated against HSV-1 by directly killing infected cells through secretion of granzymes and promoting the antiviral response by production of IFN-γ. Our data strongly suggest that γδ T17 cell secretion of IL-17A promotes NK cell accumulation in the infected cornea. This is demonstrated in several ways: 1) in mice that lack γδ T cells (TCRδ-/-) there are fewer antiviral NK cells following corneal HSV-1 infection compared to wild-type (WT) mice; 2) administering IL-17A to TCRδ-/- mice restored the NK cell population; and 3) neutralization of IL-17A or 4) inhibiting γδ T17 cell influx to corneas in WT mice diminished NK cell accumulation leading to increased viral titers. In contrast, IL-17A production is enhanced in the absence of IFN-γ. In sum, this study identifies a novel mechanism by which IL-17A production by γδ T17 cells promotes antiviral NK cell responses that, in turn limit further IL-17A production via INF-γ secretion by NK cells in the HSV-1 infected cornea.

Background Sepsis is a life-threatening condition that results from a dysregulated host response to infection, leading to organ dysfunction. Despite the prevalence and associated socioeconomic costs, treatment of sepsis remains limited to antibiotics and supportive care, and a majority of intensive care unit (ICU) survivors develop long-term cognitive complications post-discharge. The present study identifies a novel regulatory relationship between amyloid-β (Aβ) and the inflammasome-caspase-1 axis as key innate immune mediators that define sepsis outcomes. Methods Medical ICU patients and healthy individuals were consented for blood and clinical data collection. Plasma cytokine, caspase-1 and Aβ levels were measured. Data were compared against indices of multiorgan injury and other clinical parameters. Additionally, recombinant proteins were tested in vitro to examine the effect of caspase-1 on a functional hallmark of Aβ, namely aggregation. Results Plasma caspase-1 levels displayed the best predictive value in discriminating ICU patients with sepsis from non-infected ICU patients (area under the receiver operating characteristic curve=0.7080). Plasma caspase-1 and the Aβ isoform Aβx-40 showed a significant positive correlation and Aβx-40 associated with organ injury. Additionally, Aβ plasma levels continued to rise from time of ICU admission to 7 days post-admission. In silico , Aβ harbours a predicted caspase-1 cleavage site, and in vitro studies demonstrated that caspase-1 cleaved Aβ to inhibit its auto-aggregation, suggesting a novel regulatory relationship. Conclusions Aβx-40 and caspase-1 are potentially useful early indicators of sepsis and its attendant organ injury. Additionally, Aβx-40 has emerged as a potential culprit in the ensuing development of post-ICU syndrome.

Purpose:To determine whether γδ T cells regulate natural killer (NK) cells in the herpes simplex virus 1 (HSV-1)-infected cornea. Methods:CD57Bl/6 (wild-type [WT]), TCRδ-/-, and IFN-γ-/- mice were infected intracorneally with HSV-1. TCR-/- mice were treated with IL-17A at 24 hours post-infection (PI), and the WT mice received treatments of fingolimod (FTY720) and anti-IL-17A. At 48 hours PI, corneas were excised, and intracellular staining flow cytometry was performed, as well as multiplex analysis. Additionally, single-cell RNA sequencing (scRNAseq) was done to analyze the transcriptome of NK cells from WT and TCRδ-/- mice. Results:In mice lacking γδ T cells, there were significantly fewer NK cells following ocular HSV-1 infection. This reduction of NK cells corresponded with lower levels of cytokines and chemokines associated with the antiviral response. Furthermore, NK cells from WT mice had enriched IL-17A signaling compared to those from TCRδ-/- mice. The NK cell response was partially rescued in TCRδ-/- mice by administration of IL-17A. Correspondingly, the NK cell response could be blunted in WT mice by administration of anti-IL-17A. Finally, IFN-γ-/- mice had significantly less IL-17A production compared to WT mice. Conclusions:γδ T17 cells promote NK cell accumulation in HSV-1-infected corneas. In turn, NK cells secrete IFN-γ, which negatively regulates further IL-17A production by γδ T cells.

Pneumonia is a common cause of end-organ dysfunction, both during and in the aftermath of infection. In particular, pneumonia is a common cause of lung injury, increased risk of myocardial infarction, and neurocognitive dysfunction, although the mechanisms responsible for such increased risk are unknown. Here, we reveal that gamma-secretase activating protein, which contributes to the amyloidogenic pathway, is important for end-organ dysfunction following infection.

To cope with DNA damage, mitochondria have developed a pathway whereby severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules can be discarded and degraded, after which new molecules are synthesized using intact templates. In this unit, we describe a method that harnesses this pathway to eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1) in mitochondria. We also provide alternate protocols for mtDNA elimination using either combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC) or clustered regulatory interspersed short palindromic repeat (CRISPR)-Cas9-mediated knockout of TFAM or other genes essential for mtDNA replication. Support protocols detail approaches for several processes: (1) genotyping ρ0 cells of human, mouse, and rat origin by polymerase chain reaction (PCR); (2) quantification of mtDNA by quantitative PCR (qPCR); (3) preparation of calibrator plasmids for mtDNA quantification; and (4) quantification of mtDNA by direct droplet digital PCR (dddPCR). © 2023 Wiley Periodicals LLC. Basic Protocol: Inducing mtDNA loss with mUNG1 Alternate Protocol 1: Generation of ρ0 cells by mtDNA depletion with EtBr and ddC Alternate Protocol 2: Generation of ρ0 cells by knocking out genes critical for mtDNA replication Support Protocol 1: Genotyping ρ0 cells by DirectPCR Support Protocol 2: Determination of mtDNA copy number by qPCR Support Protocol 3: Preparation of calibrator plasmid for qPCR Support Protocol 4: Determination of mtCN by direct droplet digital PCR (dddPCR).

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A(2) (PLA(2)) that hydrolyses host cell membranes, and P. aeruginosa strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized P. aeruginosa strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA(2)-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA(2)-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with P. aeruginosa strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA(2) activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from P. aeruginosa-infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during P. aeruginosa infection that may inhibit endothelial barrier and inflammatory functions.

The unavailability of tractable reverse genetic analysis approaches represents an obstacle to a better understanding of mitochondrial DNA replication. Here, we used CRISPR-Cas9 mediated gene editing to establish the conditional viability of knockouts in the key proteins involved in mtDNA replication. This observation prompted us to develop a set of tools for reverse genetic analysis in situ, which we called the GeneSwap approach. The technique was validated by identifying 730 amino acid (aa) substitutions in the mature human TFAM that are conditionally permissive for mtDNA replication. We established that HMG domains of TFAM are functionally independent, which opens opportunities for engineering chimeric TFAMs with customized properties for studies on mtDNA replication, mitochondrial transcription, and respiratory chain function. Finally, we present evidence that the HMG2 domain plays the leading role in TFAM species-specificity, thus indicating a potential pathway for TFAM-mtDNA evolutionary co-adaptations.

Ablation of PDE4A produces detrimental effects in this model of acute PA-lung infection. Current studies aim to determine whether an impaired bacterial killing, and the resulting increase in bacterial load, are responsible for the hyperinflammation, or whether these are independent effects of PDE4A ablation. PAN-PDE4 inhibition mimics some of the beneficial effects of selective PDE4B ablation, but also some of the detrimental effects of PDE4A ablation. These findings suggest that selective inactivation of PDE4B may be a more effective therapeutic approach, compared to PAN-PDE4 inhibition, in settings of acute PA-lung infection.

Pseudomonas aeruginosa is a frequent cause of hospital‐acquired lung infections characterized by hyperinflammation, antibiotic resistance, and high morbidity/mortality. Here, we show that the genetic ablation of one cAMP‐phosphodiesterase 4 subtype, PDE4B, is sufficient to protect mice from acute lung injury induced by P aeruginosa infection as it reduces pulmonary and systemic levels of pro‐inflammatory cytokines, as well as pulmonary vascular leakage and mortality. Surprisingly, despite dampening immune responses, bacterial clearance in the lungs of PDE4B‐KO mice is significantly improved compared to WT controls. In wildtypes, P aeruginosa‐infection produces high systemic levels of several cytokines, including TNF‐α, IL‐1β, and IL‐6, that act as cryogens and render the animals hypothermic. This, in turn, diminishes their ability to clear the bacteria. Ablation of PDE4B curbs both the initial production of acute response cytokines, including TNF‐α and IL‐1β, as well as their downstream signaling, specifically the induction of the secondary‐response cytokine IL‐6. This synergistic action protects PDE4B‐KO mice from the deleterious effects of the P aeruginosa‐induced cytostorm, while concurrently improving bacterial clearance, rather than being immunosuppressive. These benefits of PDE4B ablation are in contrast to the effects resulting from treatment with PAN‐PDE4 inhibitors, which have been shown to increase bacterial burden and dissemination. Thus, PDE4B represents a promising therapeutic target in settings of P aeruginosa lung infections.

Rationale Non‐selective inhibitors of Type 4 cAMP‐phosphodiesterases (PDE4s) exert well established anti‐inflammatory effects in non‐infectious models of lung inflammation, such as upon exposure to lipopolysaccharide or ovalbumin, and have demonstrated clinical efficacy in the treatment of COPD. However, they also produce side effects, including emesis, nausea, diarrhea, and weight loss, that limit their clinical utility. The PDE4 family comprises 4 subtypes (PDE4A‐D). As each plays unique and non‐overlapping physiological roles, targeting individual subtypes may serve to separate the therapeutically beneficial from side effects resulting from non‐selective PDE4 inhibition. PDE4B has been proposed as a primary target by which PAN‐PDE4 inhibitors exert anti‐inflammatory effects. Here, we assessed whether genetic ablation of PDE4B is per se sufficient to protect mice infected with live P.aeruginosa from acute lung injury and whether PDE4B ablation can dampen immune responses without worsening the bacterial infection. Methods PDE4B knockout (PDE4BKO) mice and their wildtype (WT) littermates were infected intranasally with P.aeruginosa lab strain PA01 and bronchoalveolar lavage fluid (BALF) and lungs were harvested 16 h later to assess lung infection/inflammation. Results At 16 h post‐infection, the levels of pro‐inflammatory cytokines in BALF and lung tissue (TNFα, IL1β, IL‐6, and KC) as well as lung histopathological scores were reduced in PDE4BKO mice compared to their WT littermates indicating anti‐inflammatory benefits of PDE4B ablation, whereas the total number of leukocytes or neutrophils in BALF was unchanged. Unexpectedly, despite dampening the immune response, bacterial load in BALF and lungs was significantly decreased in PDE4BKO compared to WT controls. The ability of PDE4BKO mice to clear the bacteria more efficiently correlates with their ability to alleviate the significant PA ‐induced hypothermia observed in WT controls. Indeed, when differences in hypothermia between WT and PDE4BKO mice are equalized by externally warming the animals, the bacterial load in WT and KO mice are comparable whereas anti‐inflammatory benefits, such as reduced cytokines levels in PDE4BKO mice, are retained. Conclusions Our results suggest that ablation of PDE4B per se is sufficient to alleviate lung inflammation in a model of acute P.aeruginosa infection without worsening the bacterial infection. Thus, PDE4B may represent a therapeutic target for inflammatory lung diseases associated with P.aeruginosa infections, such as ventilator associated pneumonia or cystic fibrosis. Support or Funding Information Supported by grants from the Cystic Fibrosis Foundation (SALEH18H0, RICHTE16GO) and the NIH (HL76125, HL141473, HL066299).

Caspase-3 and -7 are executioner caspases whose enzymatic activity is necessary to complete apoptotic cell death. Here, we questioned whether endothelial cell infection leads to caspase-3/7-mediated cell death. Pulmonary microvascular endothelial cells (PMVECs) were infected with Pseudomonas aeruginosa (PA103). PA103 caused cell swelling with a granular appearance, paralleled by intracellular caspase-3/7 activation and cell death. In contrast, PMVEC infection with ExoY+ (PA103 ΔexoUexoT::Tc pUCPexoY) caused cell rounding, but it did not activate intracellular caspase-3/7 and it did not cause cell death. However, ExoY+ led to a time-dependent accumulation of active caspase-7, but not caspase-3, in the supernatant, independent of apoptosis. To study the function of extracellular caspase-7, caspase-7- and caspase-3-deficient PMVECs were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. Caspase-7 activity was significantly reduced in supernatants from infected caspase-7-deficient cells but was unchanged in supernatants from infected caspase-3 deficient cells, indicating an uncoupling in the mechanism of activation of these two enzymes. Because ExoY+ leads to the release of heat stable amyloid cytotoxins that are responsible for transmissible cytotoxicity, we next questioned whether caspase-7 contributes to the severity of this process. Supernatants obtained from infected caspase-7-deficient cells displayed significantly reduced transmissible cytotoxicity when compared with supernatants from infected wild-type controls, illustrating an essential role for caspase-7 in promoting the potency of transmissible cytotoxicity. Thus, we report a mechanism whereby ExoY+ infection induces active caspase-7 accumulation in the extracellular space, independent of both caspase-3 and cell death, where it modulates ExoY+-induced transmissible cytotoxicity.

To cope with DNA damage, mitochondria developed a pathway by which severely damaged or unrepairable mitochondrial DNA (mtDNA) molecules are abandoned and degraded, and new molecules are resynthesized using intact templates, if available. In this unit, we describe a method that harnesses this pathway to completely eliminate mtDNA from mammalian cells by transiently overexpressing the Y147A mutant of human uracil-N-glycosylase (mUNG1). We also provide an alternate protocol for mtDNA depletion using combined treatment with ethidium bromide (EtBr) and dideoxycytidine (ddC). Support protocols detail approaches for (1) genotyping ρ° cells of human, mouse, and rat origin by PCR; (2) quantitation of mtDNA by quantitative PCR (qPCR); and (3) preparation of calibrator plasmids for mtDNA quantitation. © 2018 by John Wiley & Sons, Inc.

Due to the essential role played by mitochondrial DNA (mtDNA) in cellular physiology and bioenergetics, methods for establishing cell lines with altered mtDNA content are of considerable interest. Here, we report evidence for the existence in mammalian cells of a novel, low- efficiency, presequence-independent pathway for mitochondrial protein import, which facilitates mitochondrial uptake of such proteins as Chlorella virus ligase (ChVlig) and Escherichia coli LigA. Mouse cells engineered to depend on this pathway for mitochondrial import of the LigA protein for mtDNA maintenance had severely (up to >90%) reduced mtDNA content. These observations were used to establish a method for the generation of mouse cell lines with reduced mtDNA copy number by, first, transducing them with a retrovirus encoding LigA, and then inactivating in these transductants endogenous Lig3 with CRISPR-Cas9. Interestingly, mtDNA depletion to an average level of one copy per cell proceeds faster in cells engineered to maintain mtDNA at low copy number. This makes a low-mtDNA copy number phenotype resulting from dependence on mitochondrial import of DNA ligase through presequence-independent pathway potentially useful for rapidly shifting mtDNA heteroplasmy through partial mtDNA depletion.