Reducing Stress PERKs up Anti-tumor Immunity.

Myeloid-derived suppressor cells (MDSCs) can promote tumor progression. In this issue of Immunity, Mohamed et al. show that the unfolded protein response sensor, PERK, enhances MDSC-mediated immunosuppression through the NRF2 transcription factor, preventing oxidative damage, mitochondrial DNA release, and DNA sensor-STING-dependent type I interferon production.

Heterogeneity of Salmonella enterica lipopolysaccharide counteracts macrophage and antimicrobial peptide defenses.

Salmonella enterica is comprised of over 2,500 serovars, in which non-typhoidal serovars (NTS), Enteritidis (SE), and Typhimurium (STM) are the most clinically associated with human infections. Although NTS have similar genetic elements to cause disease, phenotypic variation including differences in lipopolysaccharide (LPS) composition may control immune evasion. Here, we demonstrate that macrophage host defenses and LL-37 antimicrobial efficacy against SE and STM are substantially altered by LPS heterogeneity. We found that SE evades macrophage killing by inhibiting phagocytosis while STM survives better intracellularly post-phagocytosis. SE-infected macrophages failed to activate the inflammasomes and subsequently produced less interleukin-1ß (IL-1ß), IL-18, and interferon λ. Inactivation of LPS biosynthesis genes altered LPS composition, and the SE LPS-altered mutants could no longer inhibit phagocytosis, inflammasome activation, and type II interferon signaling. In addition, SE and STM showed differential susceptibility to the antimicrobials LL-37 and colistin, and alteration of LPS structure substantially increased susceptibility to these molecules. Collectively, our findings highlight that modification of LPS composition by Salmonella increases resistance to host defenses and antibiotics.

Amino acids suppress macropinocytosis and promote release of CSF1 receptor in macrophages.

The internalization of solutes by macropinocytosis provides an essential route for nutrient uptake in many cells. Macrophages increase macropinocytosis in response to growth factors and other stimuli. To test the hypothesis that nutrient environments modulate solute uptake by macropinocytosis, this study analyzed the effects of extracellular amino acids on the accumulation of fluorescent fluid-phase probes in murine macrophages. Nine amino acids, added individually or together, were capable of suppressing macropinocytosis in murine bone marrow-derived macrophages stimulated with the growth factors colony stimulating factor 1 (CSF1) or interleukin 34, both ligands of the CSF1 receptor (CSF1R). The suppressive amino acids did not inhibit macropinocytosis in response to lipopolysaccharide, the chemokine CXCL12, or the tumor promoter phorbol myristate acetate. Suppressive amino acids promoted release of CSF1R from cells and resulted in the formation of smaller macropinosomes in response to CSF1. This suppression of growth factor-stimulated macropinocytosis indicates that different nutrient environments modulate CSF1R levels and bulk ingestion by macropinocytosis, with likely consequences for macrophage growth and function.

Human neutrophil IL1ß directs intestinal epithelial cell extrusion during Salmonella infection.

Infection of the human gut by Salmonella enterica Typhimurium (STM) results in a localized inflammatory disease that is not mimicked in murine infections. To determine mechanisms by which neutrophils, as early responders to bacterial challenge, direct inflammatory programming of human intestinal epithelium, we established a multi-component human intestinal organoid (HIO) model of STM infection. HIOs were micro-injected with STM and seeded with primary human polymorphonuclear leukocytes (PMN-HIOs). PMNs did not significantly alter luminal colonization of Salmonella, but their presence reduced intraepithelial bacterial burden. Adding PMNs to infected HIOs resulted in substantial accumulation of shed TUNEL+ epithelial cells that was driven by PMN Caspase-1 activity. Inhibition of Caspases-1, -3 or -4 abrogated epithelial cell death and extrusion in the infected PMN-HIOs but only Caspase-1 inhibition significantly increased bacterial burden in the PMN-HIO epithelium. Thus, PMNs promote cell death in human intestinal epithelial cells through multiple caspases as a protective response to infection. IL-1ß was necessary and sufficient to induce cell shedding in the infected HIOs. These data support a critical innate immune function for human neutrophils in amplifying cell death and extrusion of human epithelial cells from the Salmonella-infected intestinal monolayer.

Endoplasmic Reticulum Stress Activates the Inflammasome via NLRP3- and Caspase-2-Driven Mitochondrial Damage.

Endoplasmic reticulum (ER) stress is observed in many human diseases, often associated with inflammation. ER stress can trigger inflammation through nucleotide-binding domain and leucine-rich repeat containing (NLRP3) inflammasome, which might stimulate inflammasome formation by association with damaged mitochondria. How ER stress triggers mitochondrial dysfunction and inflammasome activation is ill defined. Here we have used an infection model to show that the IRE1α ER stress sensor regulates regulated mitochondrial dysfunction through an NLRP3-mediated feed-forward loop, independently of ASC. IRE1α activation increased mitochondrial reactive oxygen species, promoting NLRP3 association with mitochondria. NLRP3 was required for ER stress-induced cleavage of caspase-2 and the pro-apoptotic factor, Bid, leading to subsequent release of mitochondrial contents. Caspase-2 and Bid were necessary for activation of the canonical inflammasome by infection-associated or general ER stress. These data identify an NLRP3-caspase-2-dependent mechanism that relays ER stress to the mitochondria to promote inflammation, integrating cellular stress and innate immunity.

Comparative transcriptional profiling of the early host response to infection by typhoidal and non-typhoidal Salmonella serovars in human intestinal organoids.

Salmonella enterica represents over 2500 serovars associated with a wide-ranging spectrum of disease; from self-limiting gastroenteritis to invasive infections caused by non-typhoidal serovars (NTS) and typhoidal serovars, respectively. Host factors strongly influence infection outcome as malnourished or immunocompromised individuals can develop invasive infections from NTS, however, comparative analyses of serovar-specific host responses have been constrained by reliance on limited model systems. Here we used human intestinal organoids (HIOs), a three-dimensional "gut-like" in vitro system derived from human embryonic stem cells, to elucidate similarities and differences in host responses to NTS and typhoidal serovars. HIOs discriminated between the two most prevalent NTS, Salmonella enterica serovar Typhimurium (STM) and Salmonella enterica serovar Enteritidis (SE), and typhoidal serovar Salmonella enterica serovar Typhi (ST) in epithelial cell invasion, replication and transcriptional responses. Pro-inflammatory signaling and cytokine output was reduced in ST-infected HIOs compared to NTS infections, consistent with early stages of NTS and typhoidal diseases. While we predicted that ST would induce a distinct transcriptional profile from the NTS strains, more nuanced expression profiles emerged. Notably, pathways involved in cell cycle, metabolism and mitochondrial functions were downregulated in STM-infected HIOs and upregulated in SE-infected HIOs. These results correlated with suppression of cellular proliferation and induction of host cell death in STM-infected HIOs and in contrast, elevated levels of reactive oxygen species production in SE-infected HIOs. Collectively, these results suggest that the HIO model is well suited to reveal host transcriptional programming specific to infection by individual Salmonella serovars, and that individual NTS may provoke unique host epithelial responses during intestinal stages of infection.

The IRE1α Stress Signaling Axis Is a Key Regulator of Neutrophil Antimicrobial Effector Function.

Activation of the endoplasmic reticulum stress sensor, IRE1α, is required for effective immune responses against bacterial infection and is associated with human inflammatory diseases in which neutrophils are a key immune component. However, the specific role of IRE1α in regulating neutrophil effector function has not been studied. In this study, we show that infection-induced IRE1α activation licenses neutrophil antimicrobial capacity, including IL-1ß production, formation of neutrophil extracellular traps (NETs), and methicillin-resistant Staphylococcus aureus (MRSA) killing. Inhibition of IRE1α diminished production of mitochondrial reactive oxygen species and decreased CASPASE-2 activation, which both contributed to neutrophil antimicrobial activity. Mice deficient in CASPASE-2 or neutrophil IRE1α were highly susceptible to MRSA infection and failed to effectively form NETs in the s.c. abscess. IRE1α activation enhanced calcium influx and citrullination of histone H3 independently of mitochondrial reactive oxygen species production, suggesting that IRE1α coordinates multiple pathways required for NET formation. Our data demonstrate that the IRE1α-CASPASE-2 axis is a major driver of neutrophil activity against MRSA infection and highlight the importance of IRE1α in neutrophil antibacterial function.

A methodological approach to intra-action reviews - application and adaptation of existing global guidance during the COVID-19 pandemic response in Ireland, 2021.

Many countries were under-prepared for the arrival of an emergency such as the COVID-19 pandemic. An intra-action review allows countries, systems and services to reflect on their preparedness and response to date, and revise their policies and approaches as needed. We describe the approach to undertaking an intra-action review of Ireland's Health Protection COVID-19 response during 2021. A project team within National Health Protection developed a project plan, identified key stakeholders, trained facilitators and designed workshop programmes, employing integrated collaborative web tools. Multidisciplinary representatives participated in three half-day, independently facilitated workshops on challenges and solutions within specific response areas: communication, governance and cross-cutting themes such as staff well-being. An all-stakeholder survey sought further in-depth detail. Participants reviewed the ongoing pandemic response in terms of good practice and challenges and recommended implementable solutions. We customised our mixed-methods approach using existing ECDC/WHO guidance, producing consensus recommendations during Ireland's fourth wave of COVID-19, with particular focus on pathways to implementation. Our adaptations may help others in formulating and customising methodological approaches. During an emergency, identifying and reflecting on good practices to retain, and areas for strengthening, with a clear action plan of implementing recommendations, will enhance preparedness now, and for future emergencies.

MRSA in Stealth Mode Evades Antibody Recognition.

In Nature, Gerlach et al. (Nature 2018;563:705-709) report that methicillin-resistant Staphylococcus aureus camouflages its surface by displaying a 'stealth' wall teichoic acid (WTA) isomer. WTA can act as a cloak to limit exposure of surface antigens to the immune system, but this report indicates that even the cloak can become immunologically silent.

Astrovirus replication in human intestinal enteroids reveals multi-cellular tropism and an intricate host innate immune landscape.

Human astroviruses (HAstV) are understudied positive-strand RNA viruses that cause gastroenteritis mostly in children and the elderly. Three clades of astroviruses, classic, MLB-type and VA-type have been reported in humans. One limitation towards a better understanding of these viruses has been the lack of a physiologically relevant cell culture model that supports growth of all clades of HAstV. Herein, we demonstrate infection of HAstV strains belonging to all three clades in epithelium-only human intestinal enteroids (HIE) isolated from biopsy-derived intestinal crypts. A detailed investigation of infection of VA1, a member of the non-canonical HAstV-VA/HMO clade, showed robust replication in HIE derived from different patients and from different intestinal regions independent of the cellular differentiation status. Flow cytometry and immunofluorescence analysis revealed that VA1 infects several cell types, including intestinal progenitor cells and mature enterocytes, in HIE cultures. RNA profiling of VA1-infected HIE uncovered that the host response to infection is dominated by interferon (IFN)-mediated innate immune responses. A comparison of the antiviral host response in non-transformed HIE and transformed human colon carcinoma Caco-2 cells highlighted significant differences between these cells, including an increased magnitude of the response in HIE. Additional studies confirmed the sensitivity of VA1 to exogenous IFNs, and indicated that the endogenous IFN response of HIE to curtail the growth of strains from all three clades. Genotypic variation in the permissiveness of different HIE lines to HAstV could be overcome by pharmacologic inhibition of JAK/STAT signaling. Collectively, our data identify HIE as a universal infection model for HAstV and an improved model of the intestinal epithelium to investigate enteric virus-host interactions.

Barriers to post-placental intrauterine device receipt among expectant minority women.

PURPOSE: We aimed to identify barriers to breastfeeding-compatible post-placental intrauterine devices (IUDs) for expectant predominantly non-Hispanic African-American women. MATERIALS AND METHODS: This cross-sectional survey study, conducted at 3 Cleveland community partner locations, enrolled 119 expectant predominantly unmarried but partnered non-Hispanic African-American women. The survey assessed contraceptive, IUD-specific and breastfeeding attitudes and intentions. Survey responses were described with percentages and frequencies, and compared by feeding intention using 2-sided Chi-Square tests. Factor analysis with Varimax rotation identified 2 potential measures of reluctance to post-placental IUD acceptance. The relationship of factors scores to maternal characteristics was assessed. RESULTS: Feeding intention (breastfeeding versus not) was not related to perceived barriers to post-placental IUD receipt among expectant minority women. A "Personal Risks Reluctance" factor included low risk IUD events (migration and expulsion), misconceptions (delayed fertility return), menstrual changes and partner preference: a higher score was significantly associated with younger age group but no other maternal characteristics. A "Not Me Reasons" factor included provider and insurance barriers, and was not related to any maternal characteristics. CONCLUSIONS: Expectant minority women's perceived barriers to post-placental IUDs are not related to prenatal feeding intentions. We identified two clinically relevant factors that appear to measure barriers to post-placental IUD acceptance.

Preparedness and management of global public health threats at points of entry in Ireland and the EU in the context of a potential Brexit.

Health security in the European Union (EU) aims to protect citizens from serious threats to health such as biological agents and infectious disease outbreaks- whether natural, intentional or accidental. Threats may include established infections, emerging diseases or chemical and radiological agents. Co-ordinated international efforts attempt to minimize risks and mitigate the spread of infectious disease across borders.We review the current situation (March 2019) with respect to detection and management of serious human health threats across Irish borders- and what may change for Ireland if/when the United Kingdom (UK) withdraws from the EU (Brexit).Specifically, this paper reviews international legislation covering health threats, and its national transposition; and EU legislation and processes, especially the relevant European Decision No. 1082/2013/EU of the European Parliament and of the Council on serious cross border threats to health with repeal of Decision No 2119/98/EC. We enumerate European surveillance systems and agencies which relate to port health security; we consider consortia and academic arrangements within the EU framework and established collaboration with the World Health Organization. We describe current Health Services Executive port health structures in Ireland which address preparedness and management of human health threats at points of entry. We appraise risks which Brexit could bring, reviewing literature on shared concerns about these risks, and we evaluate post-Brexit challenges for the EU, and potential opportunities to remain within current structures in shared health threat preparedness and response.It is imperative that the UK, Ireland and the EU work together to mitigate these risks using some agreed joint coordination mechanisms for a robust, harmonised approach to global public health threats at points of entry.

Impact of Rifabutin or Rifampin on Bedaquiline Safety, Tolerability, and Pharmacokinetics Assessed in a Randomized Clinical Trial with Healthy Adult Volunteers.

Bedaquiline is a diarylquinoline that specifically inhibits mycobacterial ATP synthase. Bedaquiline has been used to effectively treat tuberculosis (TB) caused by drug-susceptible and drug-resistant Mycobacterium tuberculosis Rifamycins are a cornerstone of combination drug regimens for the treatment of TB. This phase 1, open-label, randomized, controlled trial evaluated the effect of steady-state dosing of rifabutin or rifampin on the safety, tolerability, and pharmacokinetics of bedaquiline given as a single dose. Thirty-three healthy subjects were enrolled to receive a 400-mg single oral dose of bedaquiline at two time points, on study days 1 and 29. Subjects were randomly assigned to once daily oral doses of rifabutin (300 mg/day, n = 17) or rifampin (600 mg/day, n = 16) during period 2 from days 20 to 41. Serial blood sampling for bedaquiline measurement occurred on days 1 and 29 through 336 h after bedaquiline administration. The day 29 bedaquiline pharmacokinetic parameter estimates were compared to the corresponding day 1 estimates for each rifamycin group. Steady-state rifampin reduced bedaquiline AUC0-336 approximately 45%, from 47.69 h·µg/ml in period 1 to 26.33 h·µg/ml in period 2. Bedaquiline apparent clearance accelerated 24% in rifampin-treated subjects from 6.59 liters/h in period 1 to 8.19 liters/h in period 2. Steady-state rifabutin resulted in little quantitative impact on bedaquiline exposure but was associated with grade 3 and 4 adverse events before and after the day 29 bedaquiline dose. Dosage adjustments may therefore be necessary to ensure that bedaquiline plasma concentrations reach therapeutic levels safely when combining bedaquiline and rifamycins in TB treatment regimens. (This single-site, randomized, open-label, prospective study in healthy adult volunteers was registered at Clinicaltrials.gov under registration no. NCT01341184.).

A near death experience: Shigella manipulates host death machinery to silence innate immunity.

Release of mitochondrial contents often triggers inflammation and cell death, and modulating this process can be advantageous to invading pathogens. In this issue of The EMBO Journal, Andree and colleagues reveal new findings that an intracellular bacterial pathogen exploits apoptotic machinery to suppress host immune signaling, yet avoids cell death. This study emphasizes the need to expand our understanding of the roles played by pro­apoptotic proteins in non­death scenarios.

Safety, Tolerability, Systemic Exposure, and Metabolism of CRS3123, a Methionyl-tRNA Synthetase Inhibitor Developed for Treatment of Clostridium difficile, in a Phase 1 Study.

Clostridium difficile causes antibiotic-associated diarrhea and is a major public health concern. Current therapies disrupt the protective intestinal flora, do not reliably prevent recurrent infections, and will be decreasingly effective should less susceptible strains emerge. CRS3123 is an oral agent that inhibits bacterial methionyl-tRNA synthetase and has potent activity against C. difficile and aerobic Gram-positive bacteria but little activity against Gram-negative bacteria, including anaerobes. This first-in-human, double-blind, placebo-controlled, dose escalation study evaluated the safety and systemic exposure of CRS3123 after a single oral dose in healthy adults. Five cohorts of eight subjects each received CRS3123 or placebo in a 3:1 ratio. Doses for the respective active arms were 100 mg, 200 mg, 400 mg, 800 mg, and 1,200 mg. Blood and urine were collected for pharmacokinetic analysis. CRS3123 concentrations were measured with validated LC-MS/MS techniques. There were no serious adverse events or immediate allergic reactions during administration of CRS3123. In the CRS3123-treated groups, the most frequent adverse events were decreased hemoglobin, headache, and abnormal urine analysis; all adverse events in the active-treatment groups were mild to moderate, and their frequency did not increase with dose. Although CRS3123 systemic exposure increased at higher doses, the increase was less than dose proportional. The absorbed drug was glucuronidated at reactive amino groups on the molecule, which precluded accurate pharmacokinetic analysis of the parent drug. Overall, CRS3123 was well tolerated over this wide range of doses. This safety profile supports further investigation of CRS3123 as a treatment for C. difficile infections. (This study has been registered at ClinicalTrials.gov under identifier NCT01551004.).

Kallistatin levels in HIV-infected patients and effects of statin therapy.

BACKGROUND: Kallistatin, a serine proteinase inhibitor, has vasodilatory and anti-inflammatory properties and is increased in other inflammatory conditions. We measured kallistatin in HIV for the first time, examined its relationship with inflammation, and determined if statin therapy affected levels. METHODS: Kallistatin levels were measured in subjects from a randomized, double-blinded, placebo-controlled trial. RESULTS: One hundred and thirty-five HIV-infected subjects were included. Kallistatin levels were 28.4 µg/mL at baseline and not affected by rosuvastatin. Levels were correlated with high-sensitivity C-reactive protein (hsCRP), interleukin-6, fibrinogen and insulin resistance. CONCLUSIONS: Kallistatin levels were correlated with some markers of systemic inflammation and should be further explored in the HIV population.

Anti-infective Activity of 2-Cyano-3-Acrylamide Inhibitors with Improved Drug-Like Properties against Two Intracellular Pathogens.

Due to the rise of antibiotic resistance and the small number of effective antiviral drugs, new approaches for treating infectious diseases are urgently needed. Identifying targets for host-based therapies represents an emerging strategy for drug discovery. The ubiquitin-proteasome system is a central mode of signaling in the eukaryotic cell and may be a promising target for therapies that bolster the host's ability to control infection. Deubiquitinase (DUB) enzymes are key regulators of the host inflammatory response, and we previously demonstrated that a selective DUB inhibitor and its derivative promote anti-infective activities in host cells. To find compounds with anti-infective efficacy but improved toxicity profiles, we tested a library of predominantly 2-cyano-3-acrylamide small-molecule DUB inhibitors for anti-infective activity in macrophages against two intracellular pathogens: murine norovirus (MNV) and Listeria monocytogenes We identified compound C6, which inhibited DUB activity in human and murine cells and reduced intracellular replication of both pathogens with minimal toxicity in cell culture. Treatment with C6 did not significantly affect the ability of macrophages to internalize virus, suggesting that the anti-infective activity interferes with postentry stages of the MNV life cycle. Metabolic stability and pharmacokinetic assays showed that C6 has a half-life in mouse liver microsomes of ∼20 min and has a half-life of approximately 4 h in mice when administered intravenously. Our results provide a framework for targeting the host ubiquitin system in the development of host-based therapies for infectious disease. Compound C6 represents a promising tool with which to elucidate the role of DUBs in the macrophage response to infection.

Membrane damage during Listeria monocytogenes infection triggers a caspase-7 dependent cytoprotective response.

The cysteine protease caspase-7 has an established role in the execution of apoptotic cell death, but recent findings also suggest involvement of caspase-7 during the host response to microbial infection. Caspase-7 can be cleaved by the inflammatory caspase, caspase-1, and has been implicated in processing and activation of microbial virulence factors. Thus, caspase-7 function during microbial infection may be complex, and its role in infection and immunity has yet to be fully elucidated. Here we demonstrate that caspase-7 is cleaved during cytosolic infection with the intracellular bacterial pathogen, Listeria monocytogenes. Cleavage of caspase-7 during L. monocytogenes infection did not require caspase-1 or key adaptors of the primary pathways of innate immune signaling in this infection, ASC, RIP2 and MyD88. Caspase-7 protected infected macrophages against plasma membrane damage attributable to the bacterial pore-forming toxin Listeriolysin O (LLO). LLO-mediated membrane damage could itself trigger caspase-7 cleavage, independently of infection or overt cell death. We also detected caspase-7 cleavage upon treatment with other bacterial pore-forming toxins, but not in response to detergents. Taken together, our results support a model where cleavage of caspase-7 is a consequence of toxin-mediated membrane damage, a common occurrence during infection. We propose that host activation of caspase-7 in response to pore formation represents an adaptive mechanism by which host cells can protect membrane integrity during infection.