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Innate lymphoid cells (ILCs) are capable of rapid response to a wide variety of immune challenges, including various respiratory pathogens. Despite this, their role in the immune response against the lethal intracellular bacterium Francisella tularensis is not yet known. In this study, we demonstrate that infection of the airways with F. tularensis results in a significant reduction in lung type 2 ILCs (ILC2s) in mice. Conversely, the expansion of ILC2s via treatment with the cytokine IL-33, or by adoptive transfer of ILC2s, resulted in significantly enhanced bacterial burdens in the lung, liver, and spleen, suggesting that ILC2s may favor severe infection. Indeed, specific reduction of ILC2s in a transgenic mouse model results in a reduction in lung bacterial burden. Using an in vitro culture system, we show that IFN-γ from the live vaccine strain-infected lung reduces ILC2 numbers, suggesting that this cytokine in the lung environment is mechanistically important in reducing ILC2 numbers during infection. Finally, we show Ab-mediated blockade of IL-5, of which ILC2s are a major innate source, reduces bacterial burden postinfection, suggesting that IL-5 production by ILC2s may play a role in limiting protective immunity. Thus, overall, we highlight a negative role for ILC2s in the control of infection with F. tularensis. Our work therefore highlights the role of ILC2s in determining the severity of potentially fatal airway infections and raises the possibility of interventions targeting innate immunity during infection with F. tularensis to benefit the host., (Copyright © 2023 The Authors.)

Radiotherapy (RT) is a highly effective anticancer treatment that is delivered to more than half of all patients with cancer. In addition to the well-documented direct cytotoxic effects, RT can have immunomodulatory effects on the tumour and surrounding tissues. These effects are thought to underlie the so-called abscopal responses, whereby RT generates systemic antitumour immunity outside the irradiated tumour. The full scope of these immune changes remains unclear but is likely to involve multiple components, such as immune cells, the extracellular matrix, endothelial and epithelial cells and a myriad of chemokines and cytokines, including transforming growth factor-β (TGFβ). In normal tissues exposed to RT during cancer therapy, acute immune changes may ultimately lead to chronic inflammation and RT-induced toxicity and organ dysfunction, which limits the quality of life of survivors of cancer. Here we discuss the emerging understanding of RT-induced immune effects with particular focus on the lungs and gut and the potential immune crosstalk that occurs between these tissues., (© 2021. Springer Nature Limited.)

The heterocyclic aldehyde 5-hydroxymethyl-2-furfural (5HMF) interacts allosterically with the abnormal form of haemoglobin (Hb), HbS, in red blood cells (RBCs) from patients with sickle cell disease (SCD), thereby increasing oxygen affinity and decreasing HbS polymerization and RBC sickling during hypoxia. We hypothesized that should 5HMF also inhibit the main cation pathways implicated in the dehydration of RBCs from SCD patients - the deoxygenation-induced cation pathway (Psickle), the Ca(2+)-activated K(+) channel (the Gardos channel) and the K(+)-Cl(-) cotransporter (KCC) - it would have a synergistic effect in protection against sickling, directly through interacting with HbS, and indirectly through maintaining hydration and reducing [HbS]. This study was therefore designed to investigate the effects of 5HMF on RBC volume and K(+) permeability in vitro. 5HMF markedly reduced the deoxygenation-induced dehydration of RBCs whether in response to maintained deoxygenation or to cyclical deoxygenation/re-oxygenation. 5HMF was found to inhibit Psickle, an effect which correlated with its effects on sickling. Deoxygenation-induced activation of the Gardos channel and exposure of phosphatidylserine were also inhibited, probably indirectly via reduced entry of Ca(2+) through the Psickle pathway. Effects of 5HMF on KCC were more modest with a slight inhibition in N-ethylmaleimide (NEM, 1 mm)-treated RBCs and stimulation in RBCs untreated with NEM. These findings support the hypothesis that 5HMF may also be beneficial through effects on RBC ion and water homeostasis., (© 2014 The Authors. The Journal of Physiology © 2014 The Physiological Society.)

The present work investigates the contribution of various second messenger systems to Ca(2+)-induced phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients. The Ca(2+) dependence of PS exposure was confirmed using the Ca(2+) ionophore bromo-A23187 to clamp intracellular Ca(2+) over 4 orders of magnitude in high or low potassium-containing (HK or LK) saline. The percentage of RBCs showing PS exposure was significantly increased in LK over HK saline. This effect was reduced by the Gardos channel inhibitors, clotrimazole and charybdotoxin. Nevertheless, although Ca(2+) loading in the presence of an outwardly directed electrochemical gradient for K(+) stimulated PS exposure, substantial exposure still occurred in HK saline. Under the conditions used inhibitors of other second messenger systems (ABT491, quinacrine, acetylsalicylic acid, 3,4-dichloroisocoumarin, GW4869 and zVAD-fmk) did not inhibit the relationship between [Ca(2+)] and PS exposure. Inhibitors of phospholipase A2, cyclooxygenase, platelet-activating factor, sphingomyelinase and caspases, therefore, were without effect on Ca(2+)-induced PS exposure in RBCs, incubated in either HK or LK saline.

Aromatic aldehydes like o-vanillin were designed to reduce the complications of sickle cell disease (SCD) by interaction with HbS, to reduce polymerisation and RBC sickling. Present results show that o-vanillin also directly affects RBC membrane permeability. Both the K(+)-Cl(-) cotransporter (KCC) and the Ca(2+)-activated K(+) channel (or Gardos channel) were inhibited with IC50 of about 0.3 and 1 mM, respectively, with activities almost completely abolished by 5 mM. Similar effects were observed in RBCs treated with the thiol reacting reagent N-ethylmaleimide or with the Ca(2+) ionophore A23187, to circumvent any action via HbS polymerisation. The deoxygenation-induced cation conductance (sometimes termed P(sickle)) was partially inhibited, whilst deoxygenation-induced exposure of phosphatidylserine was completely abrogated. Na(+)/K(+) pump activity was also reduced. Notwithstanding, o-vanillin stimulated K(+) efflux through an unidentified pathway and resulted in reduction in cell volume (as measured by wet weight-dry weight). These actions are relevant to understanding how aromatic aldehydes may affect RBC membrane permeability per se as well as HbS polymerisation and thereby inform design of compounds most efficacious in ameliorating the complications of SCD., (Copyright © 2014. Published by Elsevier Inc.)

Phosphatidylserine (PS) exposure in red blood cells (RBCs) from sickle cell disease (SCD) patients is increased compared to levels in normal individuals and may participate in the anaemic and ischaemic complications of SCD. Exposure is increased by deoxygenation and occurs with elevation of intracellular Ca²⁺ to low micromolar levels. The Ca²⁺ entry step has not been defined but a role for the deoxygenation-induced pathway, Psickle, is postulated. Partial Psickle inhibitors 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), 4,4'-dithiocyano-2,2'-stilbene-disulphonic acid (DIDS) and dipyridamole inhibited deoxygenation-induced PS exposure (DIDS IC50, 118 nM). Inhibitors and activators of other pathways (including these stimulated by depolarisation, benzodiazepines, glutamate and stretch) were without effect. Zn²⁺ and Gd³⁺ stimulated PS exposure to high levels. In the case of Zn²⁺, this effect was independent of oxygen (and hence HbS polymerisation and RBC sickling) but required extracellular Ca²⁺. The effect was completely abolished when Zn²⁺ (100 μM) was added to RBCs suspended in autologous plasma, implying a requirement of high levels of free Zn²⁺.

Phosphatidylserine (PS) is usually confined to the inner leaflet of the red blood cell (RBC) membrane. It may become externalised in various conditions, however, notably in RBCs from patients with sickle cell disease (SCD) where exposed PS may contribute to anaemic and ischaemic complications. PS externalisation requires both inhibition of the aminophospholipid translocase (or flippase) and activation of the scramblase. Both may follow from elevation of intracellular Ca(2+). Flippase inhibition occurs at low [Ca(2+)](i), about 1μM, but [Ca(2+)](i) required for scrambling is reported to be much higher (around 100μM). In this work, FITC-labelled lactadherin and FACS were used to measure externalised PS, with [Ca(2+)](i) altered using bromo-A23187 and EGTA/Ca(2+) mixtures. Two components of Ca(2+)-induced scrambling were apparent, of high (EC(50) 1.8±0.3μM) and low (306±123μM) affinity, in RBCs from normal individuals and the commonest SCD genotypes, HbSS and HbSC. The high affinity component was lost in the presence of unphysiologically high [Mg(2+)] but was unaffected by high K(+) (90mM) or vanadate (1mM). The high affinity component accounted for PS scrambling in ≥2/3rd RBCs. It is likely to be most significant in vivo and may be involved in the pathophysiology of SCD or other conditions involving eryptosis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)