Innate Immunity in Mucopolysaccharide Diseases.

Mucopolysaccharidoses are rare paediatric lysosomal storage disorders, characterised by accumulation of glycosaminoglycans within lysosomes. This is caused by deficiencies in lysosomal enzymes involved in degradation of these molecules. Dependent on disease, progressive build-up of sugars may lead to musculoskeletal abnormalities and multi-organ failure, and in others, to cognitive decline, which is still a challenge for current therapies. The worsening of neuropathology, observed in patients following recovery from flu-like infections, suggests that inflammation is highly implicated in disease progression. This review provides an overview of the pathological features associated with the mucopolysaccharidoses and summarises current knowledge regarding the inflammatory responses observed in the central nervous system and periphery. We propose a model whereby progressive accumulation of glycosaminoglycans elicits an innate immune response, initiated by the Toll-like receptor 4 pathway, but also precipitated by secondary storage components. Its activation induces cells of the immune system to release pro-inflammatory cytokines, such as TNF-α and IL-1, which induce progression through chronic neuroinflammation. While TNF-α is mostly associated with bone and joint disease in mucopolysaccharidoses, increasing evidence implicates IL-1 as a main effector of innate immunity in the central nervous system. The (NOD)-like receptor protein 3 inflammasome is therefore implicated in chronic neuroinflammation and should be investigated further to identify novel anti-inflammatory treatments.

O-Acetylation of Capsular Polysialic Acid Enables Escherichia coli K1 Escaping from Siglec-Mediated Innate Immunity and Lysosomal Degradation of E. coli-Containing Vacuoles in Macrophage-Like Cells.

Escherichia coli K1 causes bacteremia and meningitis in human neonates. The K1 capsule, an α2,8-linked polysialic acid (PSA) homopolymer, is its essential virulence factor. PSA is usually partially modified by O-acetyl groups. It is known that O-acetylation alters the antigenicity of PSA, but its impact on the interactions between E. coli K1 and host cells is unclear. In this study, a phase variant was obtained by passage of E. coli K1 parent strain, which expressed a capsule with 44% O-acetylation whereas the capsule of the parent strain has only 3%. The variant strain showed significantly reduced adherence and invasion to macrophage-like cells in comparison to the parent strain. Furthermore, we found that O-acetylation of PSA enhanced the modulation of trafficking of E. coli-containing vacuoles (ECV), enabling them to avoid fusing with lysosomes in these cells. Intriguingly, by using quartz crystal microbalance, we demonstrated that the PSA purified from the parent strain interacted with human sialic acid-binding immunoglobulin-like lectins (Siglecs), including Siglec-5, Siglec-7, Siglec-11, and Siglec-14. However, O-acetylated PSA from the variant interacted much less and also suppressed the production of Siglec-mediated proinflammatory cytokines. The adherence of the parent strain to human macrophage-like cells was significantly blocked by monoclonal antibodies against Siglec-11 and Siglec-14. Furthermore, the variant strain caused increased bacteremia and higher lethality in neonatal mice compared to the parent strain. These data elucidate that O-acetylation of K1 capsule enables E. coli to escape from Siglec-mediated innate immunity and lysosomal degradation; therefore, it is a strategy used by E. coli K1 to regulate its virulence. IMPORTANCE Escherichia coli K1 is a leading cause of neonatal meningitis. The mortality and morbidity of this disease remain significantly high despite antibiotic therapy. One major limitation on advances in prevention and therapy for meningitis is an incomplete understanding of its pathogenesis. E. coli K1 is surrounded by PSA, which is observed to have high-frequency variation of O-acetyl modification. Here, we present an in-depth study of the function of O-acetylation in PSA at each stage of host-pathogen interaction. We found that a high level of O-acetylation significantly interfered with Siglec-mediated bacterial adherence to macrophage-like cells, and blunted the proinflammatory response. Furthermore, the O-acetylation of PSA modulated the trafficking of ECVs to prevent them from fusing with lysosomes, enabling them to escape degradation by lysozymes within these cells. Elucidating how subtle modification of the capsule enhances bacterial defenses against host innate immunity will enable the future development of effective drugs or vaccines against infection by E. coli K1.

Histone H2A Nuclear/Cytoplasmic Trafficking Is Essential for Negative Regulation of Antiviral Immune Response and Lysosomal Degradation of TBK1 and IRF3.

Histone H2A is a nuclear molecule tightly associated in the form of the nucleosome. Our previous studies have demonstrated the antibacterial property of piscine H2A variants against gram-negative bacteria Edwardsiella piscicida and Gram-positive bacteria Streptococcus agalactiae. In this study, we show the function and mechanism of piscine H2A in the negative regulation of RLR signaling pathway and host innate immune response against spring viremia of carp virus (SVCV) infection. SVCV infection significantly inhibits the expression of histone H2A during an early stage of infection, but induces the expression of histone H2A during the late stage of infection such as at 48 and 72 hpi. Under normal physiological conditions, histone H2A is nuclear-localized. However, SVCV infection promotes the migration of histone H2A from the nucleus to the cytoplasm. The in vivo studies revealed that histone H2A overexpression led to the increased expression of SVCV gene and decreased survival rate. The overexpression of histone H2A also significantly impaired the expression levels of those genes involved in RLR antiviral signaling pathway. Furthermore, histone H2A targeted TBK1 and IRF3 to promote their protein degradation via the lysosomal pathway and impair the formation of TBK1-IRF3 functional complex. Importantly, histone H2A completely abolished TBK1-mediated antiviral activity and enormously impaired the protein expression of IRF3, especially nuclear IRF3. Further analysis demonstrated that the inhibition of histone H2A nuclear/cytoplasmic trafficking could relieve the protein degradation of TBK1 and IRF3, and blocked the negative regulation of histone H2A on the SVCV infection. Collectively, our results suggest that histone H2A nuclear/cytoplasmic trafficking is essential for negative regulation of RLR signaling pathway and antiviral immune response in response to SVCV infection.

Two Distinct Lysosomal Targeting Strategies Afford Trojan Horse Antibodies With Pan-Filovirus Activity.

Multiple agents in the family Filoviridae (filoviruses) are associated with sporadic human outbreaks of highly lethal disease, while others, including several recently identified agents, possess strong zoonotic potential. Although viral glycoprotein (GP)-specific monoclonal antibodies have demonstrated therapeutic utility against filovirus disease, currently FDA-approved molecules lack antiviral breadth. The development of broadly neutralizing antibodies has been challenged by the high sequence divergence among filovirus GPs and the complex GP proteolytic cleavage cascade that accompanies filovirus entry. Despite this variability in the antigenic surface of GP, all filoviruses share a site of vulnerability-the binding site for the universal filovirus entry receptor, Niemann-Pick C1 (NPC1). Unfortunately, this site is shielded in extracellular GP and only uncovered by proteolytic cleavage by host proteases in late endosomes and lysosomes, which are generally inaccessible to antibodies. To overcome this obstacle, we previously developed a 'Trojan horse' therapeutic approach in which engineered bispecific antibodies (bsAbs) coopt viral particles to deliver GP:NPC1 interaction-blocking antibodies to their endo/lysosomal sites of action. This approach afforded broad protection against members of the genus Ebolavirus but could not neutralize more divergent filoviruses. Here, we describe next-generation Trojan horse bsAbs that target the endo/lysosomal GP:NPC1 interface with pan-filovirus breadth by exploiting the conserved and widely expressed host cation-independent mannose-6-phosphate receptor for intracellular delivery. Our work highlights a new avenue for the development of single therapeutics protecting against all known and newly emerging filoviruses.

Keeping the host alive - lessons from obligate intracellular bacterial pathogens.

Mammals have evolved sophisticated host cell death signaling pathways as an important immune mechanism to recognize and eliminate cell intruders before they establish their replicative niche. However, intracellular bacterial pathogens that have co-evolved with their host have developed a multitude of tactics to counteract this defense strategy to facilitate their survival and replication. This requires manipulation of pro-death and pro-survival host signaling pathways during infection. Obligate intracellular bacterial pathogens are organisms that absolutely require an eukaryotic host to survive and replicate, and therefore they have developed virulence factors to prevent diverse forms of host cell death and conserve their replicative niche. This review encapsulates our current understanding of these host-pathogen interactions by exploring the most relevant findings of Anaplasma spp., Chlamydia spp., Rickettsia spp. and Coxiella burnetii modulating host cell death pathways. A detailed comprehension of the molecular mechanisms through which these obligate intracellular pathogens manipulate regulated host cell death will not only increase the current understanding of these difficult-to-study pathogens but also provide insights into new tools to study regulated cell death and the development of new therapeutic approaches to control infection.

A Promising Intracellular Protein-Degradation Strategy: TRIMbody-Away Technique Based on Nanobody Fragment.

Most recently, a technology termed TRIM-Away has allowed acute and rapid destruction of endogenous target proteins in cultured cells using specific antibodies and endogenous/exogenous tripartite motif 21 (TRIM21). However, the relatively large size of the full-size mAbs (150 kDa) results in correspondingly low tissue penetration and inaccessibility of some sterically hindered epitopes, which limits the target protein degradation. In addition, exogenous introduction of TRIM21 may cause side effects for treated cells. To tackle these limitations, we sought to replace full-size mAbs with the smaller format of antibodies, a nanobody (VHH, 15 kDa), and construct a new type of fusion protein named TRIMbody by fusing the nanobody and RBCC motif of TRIM21. Next, we introduced enhanced green fluorescent protein (EGFP) as a model substrate and generated αEGFP TRIMbody using a bispecific anti-EGFP (αEGFP) nanobody. Remarkably, inducible expression of αEGFP TRIMbody could specifically degrade intracellular EGFP in HEK293T cells in a time-dependent manner. By treating cells with inhibitors, we found that intracellular EGFP degradation by αEGFP TRIMbody relies on both ubiquitin-proteasome and autophagy-lysosome pathways. Taken together, these results suggested that TRIMbody-Away technology could be utilized to specifically degrade intracellular protein and could expand the potential applications of degrader technologies.

The lysosome as an imperative regulator of autophagy and cell death.

Lysosomes are single membrane-bound organelles containing acid hydrolases responsible for the degradation of cellular cargo and maintenance of cellular homeostasis. Lysosomes could originate from pre-existing endolysosomes or autolysosomes, acting as a critical juncture between autophagy and endocytosis. Stress that triggers lysosomal membrane permeabilization can be altered by ESCRT complexes; however, irreparable damage to the membrane results in the induction of a selective lysosomal degradation pathway, specifically lysophagy. Lysosomes play an indispensable role in different types of autophagy, including microautophagy, macroautophagy, and chaperone-mediated autophagy, and various cell death pathways such as lysosomal cell death, apoptotic cell death, and autophagic cell death. In this review, we discuss lysosomal reformation, maintenance, and degradation pathways following the involvement of the lysosome in autophagy and cell death, which are related to several pathophysiological conditions observed in humans.

Role of autophagy on cancer immune escape.

Autophagy is catabolic process by degradation of intracellular components in lysosome including proteins, lipids, and mitochondria in response to nutrient deficiency or stress such as hypoxia or chemotherapy. Increasing evidence suggests that autophagy could induce immune checkpoint proteins (PD-L1, MHC-I/II) degradation of cancer cells, which play an important role in regulating cancer cell immune escape. In addition to autophagic degradation of immune checkpoint proteins, autophagy induction in immune cells (macrophages, dendritic cells) manipulates antigen presentation and T cell activity. These reports suggest that autophagy could negatively or positively regulate cancer cell immune escape by immune checkpoint protein and antigens degradation, cytokines release, antigens generation. These controversial phenomenon of autophagy on cancer cell immune evasion may be derived from different experimental context or models. In addition, autophagy maybe exhibit a role in regulating host excessive immune response. So rational combination with autophagy could enhance the efficacy of cancer immunotherapy. In this review, the current progress of autophagy on cancer immune escape is discussed. Video Abstract.

Lysosomal amino acid transporters as key players in inflammatory diseases.

Controlling inflammation can alleviate immune-mediated, lifestyle-related and neurodegenerative diseases. The endolysosome system plays critical roles in inflammatory responses. Endolysosomes function as signal transduction hubs to convert various environmental danger signals into gene expression, enabling metabolic adaptation of immune cells and efficient orchestration of inflammation. Solute carrier family 15 member A3 (SLC15A3) and member A4 (SLC15A4) are endolysosome-resident amino acid transporters that are preferentially expressed in immune cells. These transporters play essential roles in signal transduction through endolysosomes, and the loss of either transporter can alleviate multiple inflammatory diseases because of perturbed endolysosome-dependent signaling events, including inflammatory and metabolic signaling. Here, we summarize the findings leading to a proof-of-concept for anti-inflammatory strategies based on targeting SLC15 transporters.

Gatekeepers of the Gut: The Roles of Proteasomes at the Gastrointestinal Barrier.

The gut epithelial barrier provides the first line of defense protecting the internal milieu from the environment. To circumvent the exposure to constant challenges such as pathogenic infections and commensal bacteria, epithelial and immune cells at the gut barrier require rapid and efficient means to dynamically sense and respond to stimuli. Numerous studies have highlighted the importance of proteolysis in maintaining homeostasis and adapting to the dynamic changes of the conditions in the gut environment. Primarily, proteolytic activities that are involved in immune regulation and inflammation have been examined in the context of the lysosome and inflammasome activation. Yet, the key to cellular and tissue proteostasis is the ubiquitin-proteasome system, which tightly regulates fundamental aspects of inflammatory signaling and protein quality control to provide rapid responses and protect from the accumulation of proteotoxic damage. In this review, we discuss proteasome-dependent regulation of the gut and highlight the pathophysiological consequences of the disarray of proteasomal control in the gut, in the context of aberrant inflammatory disorders and tumorigenesis.

Antifungal activity of dendritic cell lysosomal proteins against Cryptococcus neoformans.

Cryptococcal meningitis is a life-threatening disease among immune compromised individuals that is caused by the opportunistic fungal pathogen Cryptococcus neoformans. Previous studies have shown that the fungus is phagocytosed by dendritic cells (DCs) and trafficked to the lysosome where it is killed by both oxidative and non-oxidative mechanisms. While certain molecules from the lysosome are known to kill or inhibit the growth of C. neoformans, the lysosome is an organelle containing many different proteins and enzymes that are designed to degrade phagocytosed material. We hypothesized that multiple lysosomal components, including cysteine proteases and antimicrobial peptides, could inhibit the growth of C. neoformans. Our study identified the contents of the DC lysosome and examined the anti-cryptococcal properties of different proteins found within the lysosome. Results showed several DC lysosomal proteins affected the growth of C. neoformans in vitro. The proteins that killed or inhibited the fungus did so in a dose-dependent manner. Furthermore, the concentration of protein needed for cryptococcal inhibition was found to be non-cytotoxic to mammalian cells. These data show that many DC lysosomal proteins have antifungal activity and have potential as immune-based therapeutics.

Dendritic Cells Require TMEM176A/B Ion Channels for Optimal MHC Class II Antigen Presentation to Naive CD4+ T Cells.

Intracellular ion fluxes emerge as critical actors of immunoregulation but still remain poorly explored. In this study, we investigated the role of the redundant cation channels TMEM176A and TMEM176B (TMEM176A/B) in retinoic acid-related orphan receptor γt+ cells and conventional dendritic cells (DCs) using germline and conditional double knockout mice. Although Tmem176a/b appeared surprisingly dispensable for the protective function of Th17 and group 3 innate lymphoid cells in the intestinal mucosa, we found that they were required in conventional DCs for optimal Ag processing and presentation to CD4+ T cells. Using a real-time imaging method, we show that TMEM176A/B accumulate in dynamic post-Golgi vesicles preferentially linked to the late endolysosomal system and strongly colocalize with HLA-DM. Taken together, our results suggest that TMEM176A/B ion channels play a direct role in the MHC class II compartment of DCs for the fine regulation of Ag presentation and naive CD4+ T cell priming.

ß2-microglobulin triggers NLRP3 inflammasome activation in tumor-associated macrophages to promote multiple myeloma progression.

As substantial constituents of the multiple myeloma (MM) microenvironment, pro-inflammatory macrophages have emerged as key promoters of disease progression, bone destruction, and immune impairment. We identify beta-2-microglobulin (ß2m) as a driver in initiating inflammation in myeloma-associated macrophages (MAMs). Lysosomal accumulation of phagocytosed ß2m promotes ß2m amyloid aggregation in MAMs, resulting in lysosomal rupture and ultimately production of active interleukin-1ß (IL-1ß) and IL-18. This process depends on activation of the NLRP3 inflammasome after ß2m accumulation, as macrophages from NLRP3-deficient mice lack efficient ß2m-induced IL-1ß production. Moreover, depletion or silencing of ß2m in MM cells abrogates inflammasome activation in a murine MM model. Finally, we demonstrate that disruption of NLRP3 or IL-18 diminishes tumor growth and osteolytic bone destruction normally promoted by ß2m-induced inflammasome signaling. Our results provide mechanistic evidence for ß2m's role as an NLRP3 inflammasome activator during MM pathogenesis. Moreover, inhibition of NLRP3 represents a potential therapeutic approach in MM.

Impaired TRIM16-Mediated Lysophagy in Chronic Obstructive Pulmonary Disease Pathogenesis.

Insufficient autophagic degradation has been implicated in accelerated cellular senescence during chronic obstructive pulmonary disease (COPD) pathogenesis. Aging-linked and cigarette smoke (CS)-induced functional deterioration of lysosomes may be associated with impaired autophagy. Lysosomal membrane permeabilization (LMP) is indicative of damaged lysosomes. Galectin-3 and tripartite motif protein (TRIM) 16 play a cooperative role in recognizing LMP and inducing lysophagy, a lysosome-selective autophagy, to maintain lysosome function. In this study, we sought to examine the role of TRIM16-mediated lysophagy in regulating CS-induced LMP and cellular senescence during COPD pathogenesis by using human bronchial epithelial cells and lung tissues. CS extract (CSE) induced lysosomal damage via LMP, as detected by galectin-3 accumulation. Autophagy was responsible for modulating LMP and lysosome function during CSE exposure. TRIM16 was involved in CSE-induced lysophagy, with impaired lysophagy associated with lysosomal dysfunction and accelerated cellular senescence. Airway epithelial cells in COPD lungs showed an increase in lipofuscin, aggresome and galectin-3 puncta, reflecting accumulation of lysosomal damage with concomitantly reduced TRIM16 expression levels. Human bronchial epithelial cells isolated from COPD patients showed reduced TRIM16 but increased galectin-3, and a negative correlation between TRIM16 and galectin-3 protein levels was demonstrated. Damaged lysosomes with LMP are accumulated in epithelial cells in COPD lungs, which can be at least partly attributed to impaired TRIM16-mediated lysophagy. Increased LMP in lung epithelial cells may be responsible for COPD pathogenesis through the enhancement of cellular senescence.

Teleost-Specific MxG, a Traitor in the Mx Family, Negatively Regulates Antiviral Responses by Targeting IPS-1 for Proteasomal Degradation and STING for Lysosomal Degradation.

IFN-ß promoter stimulator-1 (IPS-1)- and stimulator of IFN genes (STING)-mediated type I IFNs play a critical role in antiviral responses. Myxovirus resistance (Mx) proteins are pivotal components of the antiviral effectors induced by IFNs in many species. An unprecedented expansion of Mx genes has occurred in fish. However, the functions and mechanisms of Mx family members remain largely unknown in fish. In this study, we found that grass carp (Ctenopharyngodon idella) MxG, a teleost-specific Mx protein, is induced by IFNs and viruses, and it negatively regulates both IPS-1- and STING-mediated antiviral responses to facilitate grass carp reovirus, spring viremia of carp virus, and cyprinid herpesvirus-2 replication. MxG binds and degrades IPS-1 via the proteasomal pathway and STING through the lysosomal pathway, thereby negatively regulating IFN1 antiviral responses and NF-κB proinflammatory cytokines. MxG also suppresses the phosphorylation of STING IFN regulatory factor 3/7, and it subsequently downregulates IFN1 and NF-κB1 at the promoter, transcription, and protein levels. GTPase and GTPase effector domains of MxG contribute to the negative regulatory function. On the contrary, MxG knockdown weakens virus replication and cytopathic effect. Therefore, MxG can be an ISG molecule induced by IFNs and viruses, and degrade IPS-1 and STING proteins in a negative feedback manner to maintain homeostasis and avoid excessive immune responses after virus infection. To our knowledge, this is the first identification of a negative regulator in the Mx family, and our findings clarify a novel mechanism by which the IFN response is regulated.

Distinct antigen uptake receptors route to the same storage compartments for cross-presentation in dendritic cells.

An exclusive feature of dendritic cells (DCs) is their capacity to present exogenous antigens by MHC class I molecules, called cross-presentation. Here, we show that protein antigen can be conserved in mature murine DCs for several days in a lysosome-like storage compartment, distinct from MHC class II and early endosomal compartments, as an internal source for the supply of MHC class I ligands. Using two different uptake routes via Fcγ receptors and C-type lectin receptors, we could show that antigens were routed towards the same endolysosomal compartments after 48 h. The antigen-containing compartments lacked co-expression of molecules involved in MHC class I processing and presentation including TAP and proteasome subunits as shown by single-cell imaging flow cytometry. Moreover, we observed the absence of cathepsin S but selective co-localization of active cathepsin X with protein antigen in the storage compartments. This indicates cathepsin S-independent antigen degradation and a novel but yet undefined role for cathepsin X in antigen processing and cross-presentation by DCs. In summary, our data suggest that these antigen-containing compartments in DCs can conserve protein antigens from different uptake routes and contribute to long-lasting antigen cross-presentation.

Implication of a lysosomal antigen in the pathogenesis of lupus erythematosus.

Naturally-occurring autoantibodies to certain components of autophagy processes have been described in a few autoimmune diseases, but their fine specificity, their relationships with clinical phenotypes, and their potential pathogenic functions remain elusive. Here, we explored IgG autoantibodies reacting with a panel of cytoplasmic endosomal/lysosomal antigens and individual heat-shock proteins, all of which share links to autophagy. Sera from autoimmune patients and from MRL/lpr and NZB/W lupus-prone mice reacted with the C-terminal residues of lysosome-associated membrane glycoprotein (LAMP)2A. No cross-reaction was observed with LAMP2B or LAMP2C variants, with dsDNA or mononucleosomes, or with heat-shock protein A8. Moreover, administering chromatography-purified LAMP2A autoantibodies to MRL/lpr mice accelerated mortality. Furthermore, flow cytometry revealed elevated cell-surface expression of LAMP2A on MRL/lpr B cells. These findings reveal the involvement of a new class of autoantibodies targeting the C-terminus of LAMP2A, a receptor for cytosolic proteins targeted for degradation via chaperone-mediated autophagy. These autoantibodies could affect the autophagy process, which is abnormally upregulated in lupus. The data presented support a novel connection between autophagy dysregulation, autoimmune processes and pathophysiology in lupus.

Modulation of lactate-lysosome axis in dendritic cells by clotrimazole potentiates antitumor immunity.

BACKGROUND: Dendritic cells (DCs) play a critical role in antitumor immunity, but the therapeutic efficacy of DC-mediated cancer vaccine remains low, partly due to unsustainable DC function in tumor antigen presentation. Thus, identifying drugs that could enhance DC-based antitumor immunity and uncovering the underlying mechanism may provide new therapeutic options for cancer immunotherapy. METHODS: In vitro antigen presentation assay was used for DC-modulating drug screening. The function of DC and T cells was measured by flow cytometry, ELISA, or qPCR. B16, MC38, CT26 tumor models and C57BL/6, Balb/c, nude, and Batf3-/- mice were used to analyze the in vivo therapy efficacy and impact on tumor immune microenvironment by clotrimazole treatment. RESULTS: By screening a group of small molecule inhibitors and the US Food and Drug Administration (FDA)-approved drugs, we identified that clotrimazole, an antifungal drug, could promote DC-mediated antigen presentation and enhance T cell response. Mechanistically, clotrimazole acted on hexokinase 2 to regulate lactate metabolic production and enhanced the lysosome pathway and Chop expression in DCs subsequently induced DC maturation and T cell activation. Importantly, in vivo clotrimazole administration induced intratumor immune infiltration and inhibited tumor growth depending on both DCs and CD8+ T cells and potentiated the antitumor efficacy of anti-PD1 antibody. CONCLUSIONS: Our findings showed that clotrimazole could trigger DC activation via the lactate-lysosome axis to promote antigen cross-presentation and could be used as a potential combination therapy approach to improving the therapeutic efficacy of anti-PD1 immunotherapy.

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι.

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2-infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.

Disruption of Cxcr3 chemotactic signaling alters lysosomal function and renders macrophages more microbicidal.

Chemotaxis and lysosomal function are closely intertwined processes essential for the inflammatory response and clearance of intracellular bacteria. We used the zebrafish model to examine the link between chemotactic signaling and lysosome physiology in macrophages during mycobacterial infection and wound-induced inflammation in vivo. Macrophages from zebrafish larvae carrying a mutation in a chemokine receptor of the Cxcr3 family display upregulated expression of vesicle trafficking and lysosomal genes and possess enlarged lysosomes that enhance intracellular bacterial clearance. This increased microbicidal capacity is phenocopied by inhibiting the lysosomal transcription factor EC, while its overexpression counteracts the protective effect of chemokine receptor mutation. Tracking macrophage migration in zebrafish revealed that lysosomes of chemokine receptor mutants accumulate in the front half of cells, preventing macrophage polarization during chemotaxis and reaching sites of inflammation. Our work shows that chemotactic signaling affects the bactericidal properties and localization during chemotaxis, key aspects of the inflammatory response.