Regulation of Type I Interferon and Autophagy in Immunity against Mycobacterium Tuberculosis: Role of CGAS and STING1.

Mycobacterium tuberculosis (M. tb) is a significant intracellular pathogen responsible for numerous infectious disease-related deaths worldwide. It uses ESX-1 T7SS to damage phagosomes and to enter the cytosol of host cells after phagocytosis. During infection, M. tb and host mitochondria release dsDNA, which activates the CGAS-STING1 pathway. This pathway leads to the production of type I interferons and proinflammatory cytokines and activates autophagy, which targets and degrades bacteria within autophagosomes. However, the role of type I IFNs in immunity against M. tb is controversial. While previous research has suggested a protective role, recent findings from cgas-sting1 knockout mouse studies have contradicted this. Additionally, a study using knockout mice and non-human primate models uncovered a new mechanism by which neutrophils recruited to lung infections form neutrophil extracellular traps. Activating plasmacytoid dendritic cells causes them to produce type I IFNs, which interfere with the function of interstitial macrophages and increase the likelihood of tuberculosis. Notably, M. tb uses its virulence proteins to disrupt the CGAS-STING1 signaling pathway leading to enhanced pathogenesis. Investigating the CGAS-STING1 pathway can help develop new ways to fight tuberculosis.

Activation of the lysosomal damage response and selective autophagy: the coordinated actions of galectins, TRIM proteins, and CGAS-STING1 in providing immunity against Mycobacterium tuberculosis.

Autophagy is a crucial immune defense mechanism that controls the survival and pathogenesis of M. tb by maintaining cell physiology during stress and pathogen attack. The E3-Ub ligases (PRKN, SMURF1, and NEDD4) and autophagy receptors (SQSTM1, TAX1BP1, CALCOCO2, OPTN, and NBR1) play key roles in this process. Galectins (LGALSs), which bind to sugars and are involved in identifying damaged cell membranes caused by intracellular pathogens such as M. tb, are essential. These include LGALS3, LGALS8, and LGALS9, which respond to endomembrane damage and regulate endomembrane damage caused by toxic chemicals, protein aggregates, and intracellular pathogens, including M. tb. They also activate selective autophagy and de novo endolysosome biogenesis. LGALS3, LGALS9, and LGALS8 interact with various components to activate autophagy and repair damage, while CGAS-STING1 plays a critical role in providing immunity against M. tb by activating selective autophagy and producing type I IFNs with antimycobacterial functions. STING1 activates cGAMP-dependent autophagy which provides immunity against various pathogens. Additionally, cytoplasmic surveillance pathways activated by ds-DNA, such as inflammasomes mediated by NLRP3 and AIM2 complexes, control M. tb. Modulation of E3-Ub ligases with small regulatory molecules of LGALSs and TRIM proteins could be a novel host-based therapeutic approach for controlling TB.

Erratum: Piezo1 induces endothelial responses to shear stress via soluble adenylyl Cyclase-IP3R2 circuit.

[This corrects the article DOI: 10.1016/j.isci.2023.106661.].

trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Ubiquitin ligase CHFR mediated degradation of VE-cadherin through ubiquitylation disrupts endothelial adherens junctions.

Vascular endothelial cadherin (VE-cadherin) expressed at endothelial adherens junctions (AJs) is vital for vascular integrity and endothelial homeostasis. Here we identify the requirement of the ubiquitin E3-ligase CHFR as a key mechanism of ubiquitylation-dependent degradation of VE-cadherin. CHFR was essential for disrupting the endothelium through control of the VE-cadherin protein expression at AJs. We observe augmented expression of VE-cadherin in endothelial cell (EC)-restricted Chfr knockout (ChfrΔEC) mice. We also observe abrogation of LPS-induced degradation of VE-cadherin in ChfrΔEC mice, suggesting the pathophysiological relevance of CHFR in regulating the endothelial junctional barrier in inflammation. Lung endothelial barrier breakdown, inflammatory neutrophil extravasation, and mortality induced by LPS were all suppressed in ChfrΔEC mice. We find that the transcription factor FoxO1 is a key upstream regulator of CHFR expression. These findings demonstrate the requisite role of the endothelial cell-expressed E3-ligase CHFR in regulating the expression of VE-cadherin, and thereby endothelial junctional barrier integrity.

An unexpected role of neutrophils in clearing apoptotic hepatocytes in vivo.

Billions of apoptotic cells are removed daily in a human adult by professional phagocytes (e.g. macrophages) and neighboring nonprofessional phagocytes (e.g. stromal cells). Despite being a type of professional phagocyte, neutrophils are thought to be excluded from apoptotic sites to avoid tissue inflammation. Here, we report a fundamental and unexpected role of neutrophils as the predominant phagocyte responsible for the clearance of apoptotic hepatic cells in the steady state. In contrast to the engulfment of dead cells by macrophages, neutrophils burrowed directly into apoptotic hepatocytes, a process we term perforocytosis, and ingested the effete cells from the inside. The depletion of neutrophils caused defective removal of apoptotic bodies, induced tissue injury in the mouse liver, and led to the generation of autoantibodies. Human autoimmune liver disease showed similar defects in the neutrophil-mediated clearance of apoptotic hepatic cells. Hence, neutrophils possess a specialized immunologically silent mechanism for the clearance of apoptotic hepatocytes through perforocytosis, and defects in this key housekeeping function of neutrophils contribute to the genesis of autoimmune liver disease.

Every day, the immune cells clears the remains of billions of old and damaged cells that have undergone a controlled form of death. Removing them quickly helps to prevent inflammation or the development of autoimmune diseases. While immune cells called neutrophils are generally tasked with removing invading bacteria, macrophages are thought to be responsible for clearing dead cells. However, in healthy tissue, the process occurs so efficiently that it can be difficult to confirm which cells are responsible. To take a closer look, Cao et al. focused on the liver by staining human samples to identify both immune and dead cells. Unexpectedly, there were large numbers of neutrophils visible inside dead liver cells. Further experiments in mice revealed that after entering the dead cells, neutrophils engulfed the contents and digested the dead cell from the inside out. This was a surprising finding because not only are neutrophils not usually associated with dead cells, but immune cells usually engulf cells and bacteria from the outside rather than burrowing inside them. The importance of this neutrophil behaviour was shown when Cao et al. studied samples from patients with an autoimmune disease where immune cells attack the liver. In this case, very few dead liver cells contained neutrophils, and the neutrophils themselves did not seem capable of removing the dead cells, leading to inflammation. This suggests that defective neutrophil function could be a key contributor to this autoimmune disease. The findings identify a new role for neutrophils in maintaining healthy functioning of the liver and reveal a new target in the treatment of autoimmune diseases. In the future, Cao et al. plan to explore whether compounds that enhance clearance of dead cells by neutrophils can be used to treat autoimmune liver disease in mouse models of the disease.

Detecting organelle-specific activity of potassium channels with a DNA nanodevice.

Cell surface potassium ion (K+) channels regulate nutrient transport, cell migration and intercellular communication by controlling K+ permeability and are thought to be active only at the plasma membrane. Although these channels transit the trans-Golgi network, early and recycling endosomes, whether they are active in these organelles is unknown. Here we describe a pH-correctable, ratiometric reporter for K+ called pHlicKer, use it to probe the compartment-specific activity of a prototypical voltage-gated K+ channel, Kv11.1, and show that this cell surface channel is active in organelles. Lumenal K+ in organelles increased in cells expressing wild-type Kv11.1 channels but not after treatment with current blockers. Mutant Kv11.1 channels, with impaired transport function, failed to increase K+ levels in recycling endosomes, an effect rescued by pharmacological correction. By providing a way to map the organelle-specific activity of K+ channels, pHlicKer technology could help identify new organellar K+ channels or channel modulators with nuanced functions.

Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis.

Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.

Regulation of autophagy by SARS-CoV-2: The multifunctional contributions of ORF3a.

Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) regulates autophagic flux by blocking the fusion of autophagosomes with lysosomes, causing the accumulation of membranous vesicles for replication. Multiple SARS-CoV-2 proteins regulate autophagy with significant roles attributed to ORF3a. Mechanistically, open reading frame 3a (ORF3a) forms a complex with UV radiation resistance associated, regulating the functions of the PIK3C3-1 and PIK3C3-2 lipid kinase complexes, thereby modulating autophagosome biogenesis. ORF3a sequesters VPS39 onto the late endosome/lysosome, inhibiting assembly of the soluble NSF attachement protein REceptor (SNARE) complex and preventing autolysosome formation. ORF3a promotes the interaction between BECN1 and HMGB1, inducing the assembly of PIK3CA kinases into the ER (endoplasmic reticulum) and activating reticulophagy, proinflammatory responses, and ER stress. ORF3a recruits BORCS6 and ARL8B to lysosomes, initiating the anterograde transport of the virus to the plasma membrane. ORF3a also activates the SNARE complex (STX4-SNAP23-VAMP7), inducing fusion of lysosomes with the plasma membrane for viral egress. These mechanistic details can provide multiple targets for inhibiting SARS-CoV-2 by developing host- or host-pathogen interface-based therapeutics.

Piezo1 induces endothelial responses to shear stress via soluble adenylyl Cyclase-IP3R2 circuit.

Endothelial cells (ECs) continuously sense and adapt to changes in shear stress generated by blood flow. Here, we show that the activation of the mechanosensitive channel Piezo1 by defined shear forces induces Ca2+ entry into the endoplasmic reticulum (ER) via the ER Ca2+ ATPase pump. This entry is followed by inositol trisphosphate receptor 2 (IP3R2)-elicited ER Ca2+ release into the cytosol. The mechanism of ER Ca2+ release involves the generation of cAMP by soluble adenylyl cyclase (sAC), leading to IP3R2-evoked Ca2+ gating. Depleting sAC or IP3R2 prevents ER Ca2+ release and blocks EC alignment in the direction of flow. Overexpression of constitutively active Akt1 restores the shear-induced alignment of ECs lacking Piezo1 or IP3R2, as well as the flow-induced vasodilation in endothelial restricted Piezo1 knockout mice. These studies describe an unknown Piezo1-cAMP-IP3R2 circuit as an essential mechanism activating Akt signaling and inducing adaptive changes in ECs to laminar flow.

Endosomal trafficking of two-pore K+ efflux channel TWIK2 to plasmalemma mediates NLRP3 inflammasome activation and inflammatory injury.

Potassium efflux via the two-pore K+ channel TWIK2 is a requisite step for the activation of NLRP3 inflammasome, however, it remains unclear how K+ efflux is activated in response to select cues. Here, we report that during homeostasis, TWIK2 resides in endosomal compartments. TWIK2 is transported by endosomal fusion to the plasmalemma in response to increased extracellular ATP resulting in the extrusion of K+. We showed that ATP-induced endosomal TWIK2 plasmalemma translocation is regulated by Rab11a. Deleting Rab11a or ATP-ligated purinergic receptor P2X7 each prevented endosomal fusion with the plasmalemma and K+ efflux as well as NLRP3 inflammasome activation in macrophages. Adoptive transfer of Rab11a-depleted macrophages into mouse lungs prevented NLRP3 inflammasome activation and inflammatory lung injury. We conclude that Rab11a-mediated endosomal trafficking in macrophages thus regulates TWIK2 localization and activity at the cell surface and the downstream activation of the NLRP3 inflammasome. Results show that endosomal trafficking of TWIK2 to the plasmalemma is a potential therapeutic target in acute or chronic inflammatory states.

Evolution of SARS-CoV-2: BA.4/BA.5 Variants Continues to Pose New Challenges.

The acquisition of a high number of mutations, notably, the gain of two mutations L452R and F486V in RBD, and the ability to evade vaccine/natural infection-induced immunity suggests that Omicron is continuing to use "immune-escape potential" as an evolutionary space to maintain a selection advantage within the population. Despite the low hospitalizations and lower death rate, the surges by these variants may offset public health measures and disrupt health care facilities as seen recently in Portugal and the USA. Interestingly these BA.4/BA.5 variants have been found to be more severe than the earlier-emerged Omicron variants. We believe that aggressive COVID-19 surveillance using affordable testing strategies might actually help understand the evolution and transmission pattern of new variants. The sudden dip in reporting of new cases in some of the low- and middle-income countries is an alarming situation and needs to be addressed as this could lead to undetected transmission of future variants of interest/concern of SARS-CoV-2 in large population settings, including advent of a 'super' virus. It would be interesting to examine the possible role/influence, if any, of the two different kinds of vaccines, the spike protein-based versus the inactivated whole virus, in the evolution of BA.4/BA.5.

An ACE2 decoy can be administered by inhalation and potently targets omicron variants of SARS-CoV-2.

Monoclonal antibodies targeting the SARS-CoV-2 spike (S) neutralize infection and are efficacious for the treatment of COVID-19. However, SARS-CoV-2 variants, notably sublineages of B.1.1.529/omicron, have emerged that escape antibodies in clinical use. As an alternative, soluble decoy receptors based on the host entry receptor ACE2 broadly bind and block S from SARS-CoV-2 variants and related betacoronaviruses. The high-affinity and catalytically active decoy sACE22 .v2.4-IgG1 was previously shown to be effective against SARS-CoV-2 variants when administered intravenously. Here, inhalation of aerosolized sACE22 .v2.4-IgG1 increased survival and ameliorated lung injury in K18-hACE2 mice inoculated with P.1/gamma virus. Loss of catalytic activity reduced the decoy's therapeutic efficacy, which was further confirmed by intravenous administration, supporting dual mechanisms of action: direct blocking of S and turnover of ACE2 substrates associated with lung injury and inflammation. Furthermore, sACE22 .v2.4-IgG1 tightly binds and neutralizes BA.1, BA.2, and BA.4/BA.5 omicron and protects K18-hACE2 mice inoculated with a high dose of BA.1 omicron virus. Overall, the therapeutic potential of sACE22 .v2.4-IgG1 is demonstrated by the inhalation route and broad neutralization potency persists against highly divergent SARS-CoV-2 variants.

Can Mycobacterium tuberculosis infection lead to cancer? Call for a paradigm shift in understanding TB and cancer.

Infections are known to cause tumours though more attributed to viruses. Strong epidemiological links suggest association between bacterial infections and cancers as exemplified by Helicobacter pylori and Salmonella spp. Infection with Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), has been reported to predispose patients to lung cancers and possibly in other organs as well. While this etiopathogenesis warrant inclusion of M. tb in IARC's (International Agency for Research on Cancer) classified carcinogenic agents, the lack of well-defined literature and direct experimental studies have barred the research community from accepting the role of M. tb as a carcinogen. The background research, case studies, and experimental data extensively reviewed in Roy et al., 2021; provoke the debate for elucidating carcinogenic properties of M. tb. Moreover, proper, timely and correct diagnosis of both diseases (which often mimic each other) will save millions of lives that are misdiagnosed. In addition, use of Anti Tubercular therapy (ATT) in misdiagnosed non-TB patients contributes to drug resistance in population thereby severely impacting TB disease control measures. Research in this arena can further aid in saving billions of dollars by preventing the superfluous use of cancer drugs. In order to achieve these goals, it is imperative to identify the underlying mechanism of M. tb infection acting as major risk factor for cancer.

Systematic Development of Solid Lipid Nanoparticles of Abiraterone Acetate with Improved Oral Bioavailability and Anticancer Activity for Prostate Carcinoma Treatment.

In the present work, an attempt was undertaken to improve the oral bioavailability and anticancer activity of abiraterone acetate. Solid lipid nanoparticles (SLNs) were developed using the quality by design (QbD) principles and evaluated through in vitro, ex vivo, and in vivo studies. Solid lipid suitability was evaluated by equilibrium solubility study, while surfactant and cosurfactant were screened based on the ability to form microemulsion with the selected lipid. SLNs were prepared by emulsion/solvent evaporation method using glyceryl monostearate, Tween 80, and Poloxamer 407 as the solid lipid, surfactant, and cosurfactant, respectively. Box-Behnken design was applied for optimization of material attributes and evaluating their impact on particle size, polydispersity index, zeta potential, and entrapment efficiency of the SLNs. In vitro drug release study was evaluated in simulated gastric and intestinal fluids. Cell culture studies on PC-3 cells were performed to evaluate the cytotoxicity of the drug-loaded SLNs in comparison to the free drug suspension. Qualitative uptake was evaluated for Rhodamine B-loaded SLNs and compared with free dye solution. Ex vivo permeability was evaluated on Wistar rat intestine and in vivo pharmacokinetic evaluation on Wistar rats for SLNs and free drug suspension. Concisely, the SLNs showed potential for significant improvement in the biopharmaceutical performance of the selected drug candidate over the existing formulations of abiraterone acetate.

Emerging trends in immunotoxin targeting cancer stem cells.

Cancer stem cells (CSCs) are self-renewing multipotent cells that play a vital role in the development of cancer drug resistance conditions. Various therapies like conventional, targeted, and radiotherapies have been broadly used in targeting and killing these CSCs. Among these, targeted therapy selectively targets CSCs and leads to overcoming disease recurrence conditions in cancer patients. Immunotoxins (ITs) are protein-based therapeutics with selective targeting capabilities. These chimeric molecules are composed of two functional moieties, i.e., a targeting moiety for cell surface binding and a toxin moiety that induces the programmed cell death upon internalization. Several ITs have been constructed recently, and their preclinical and clinical efficacies have been evaluated. In this review, we comprehensively discussed the recent preclinical and clinical advances as well as significant challenges in ITs targeting CSCs, which might reduce the burden of drug resistance conditions in cancer patients from bench to bedside.