Chitinase 3-like-1 Inhibits Innate Antitumor and Tissue Remodeling Immune Responses by Regulating CD47-SIRPα- and CD24-Siglec10-Mediated Phagocytosis.

Innate immune responses such as phagocytosis are critically linked to the generation of adaptive immune responses against the neoantigens in cancer and the efferocytosis that is essential for homeostasis in diseases characterized by lung injury, inflammation, and remodeling as in chronic obstructive pulmonary disease (COPD). Chitinase 3-like-1 (CHI3L1) is induced in many cancers where it inhibits adaptive immune responses by stimulating immune checkpoint molecules (ICPs) and portends a poor prognosis. CHI3L1 is also induced in COPD where it regulates epithelial cell death. In this study, we demonstrate that pulmonary melanoma metastasis inhibits macrophage phagocytosis by stimulating the CD47-SIRPα and CD24-Siglec10 phagocytosis checkpoint pathways while inhibiting macrophage "eat me" signals from calreticulin and HMGB1. We also demonstrate that these effects on macrophage phagocytosis are associated with CHI3L1 stimulation of the SHP-1 and SHP-2 phosphatases and inhibition of the accumulation and phosphorylation of cytoskeleton-regulating nonmuscle myosin IIa. This inhibition of innate immune responses such as phagocytosis provides a mechanistic explanation for the ability of CHI3L1 to stimulate ICPs and inhibit adaptive immune responses in cancer and diseases such as COPD. The ability of CHI3L1 to simultaneously inhibit innate immune responses, stimulate ICPs, inhibit T cell costimulation, and regulate a number of other oncogenic and inflammation pathways suggests that CHI3L1-targeted therapeutics are promising interventions in cancer, COPD, and other disorders.

Identification of Siglec-10 as a new dendritic cell checkpoint for cervical cancer immunotherapy.

BACKGROUND: The occurrence of chronic inflammation resulting from infection with human papillomaviruses is an important factor in the development of cervical cancer (CC); thus, deciphering the crosstalk between the tumor microenvironment and innate immune cells during the establishment of immune tolerance is vital for identifying potential treatment strategies. METHODS: Single-cell RNA sequencing data and primary tumor samples from patients with CC were used to evaluate the functional role of Siglec-10 on dendritic cells (DCs). Patient-derived tumor fragment platforms were used to examine the ability of Siglec-10 blockade to reinvigorate DC-mediate T-cell activation and tumor clearance. RESULTS: Here, we demonstrated that Siglec-10 is a prominent inhibitory checkpoint for DCs infiltrated in CC. CC epithelial cells use their aberrant surface sialylated structures to induce the transformation of conventional DCs into phenotypes characterized by low immunogenicity and high immunotolerance. Additionally, Siglec-10+ DCs suppress the function of adaptive T cells via galectin-9 signaling to strengthen the immunosuppressive CC microenvironment. Disturbance of Siglec-10 signaling restored the DC-mediated tumoricidal response and increased adaptive T cells sensitivity to programmed cell death protein 1 inhibition. CONCLUSION: Our study confirms the checkpoint role of Siglec-10 on DCs and proposes that targeting Siglec-10 may be a promising avenue for immunotherapy against CC.

Siglec7 functions as an inhibitory receptor of non-specific cytotoxic cells and can regulate the innate immune responses in a primitive vertebrate (Oreochromis niloticus).

In mammals, siglec7, an integral component of the siglecs, is principally found on the surface of natural killer (NK) cells, macrophages, and monocytes, where it interacts with various pathogens to perform immunological regulatory activities. Nonetheless, the immune defense and mechanism of siglec7 in early vertebrates remain unknown. In this study, we identified siglec7 from Oreochromis niloticus (OnSiglec7) and revealed its immune functions. Specifically, OnSiglec7 was abundantly expressed in immune-related tissues of healthy tilapia and its transcription level was strongly activated after being challenged with A. hydrophila, S. agalactiae, and Poly: IC. Meanwhile, OnSiglec7 protein was purified and analyzed, which could recognize multiple pathogens through binding and agglutinating activity. Moreover, OnSiglec7-positive cells were mainly distributed in non-specific cytotoxic cells (NCC) of tilapia HKLs and showed cell membrane localization. Furthermore, OnSiglec7 blockage affected multiple innate immune responses (inflammation, apoptosis, and pyroptosis process) by regulating the activation of MAPK, NF-κB, TLR, and JAK-STAT pathways. Finally, OnSiglec7 blockage also greatly enhanced the cytotoxic effect of tilapia NCC. Summarily, this study uncovers immune functions and mechanisms of siglec7 in primitive vertebrates, thereby enhancing our understanding of the systemic evolution and ancient functions of other siglecs within the host's innate immune system (to our knowledge).

Unravelling CD24-Siglec-10 pathway: Cancer immunotherapy from basic science to clinical studies.

Cancer immunotherapy has revolutionized the treatment landscape by harnessing the power of the immune system to combat malignancies. Two of the most promising players in this field are cluster of differentiation 24 (CD24) and sialic acid-binding Ig-like lectin 10 (Siglec-10), and both of them play pivotal roles in modulating immune responses. CD24, a cell surface glycoprotein, emerges as a convincing fundamental signal transducer for therapeutic intervention, given its significant implication in the processes related to tumour progression and immunogenic evasion. Additionally, the immunomodulatory functions of Siglec-10, a prominent member within the Siglec family of immune receptors, have recently become a crucial point of interest, particularly in the context of the tumour microenvironment. Hence, the intricate interplay of both CD24 and Siglec-10 assumes a critical role in fostering tumour growth, facilitating metastasis and also orchestrating immune evasion. Recent studies have found multiple evidences supporting the therapeutic potential of targeting CD24 in cancer treatment. Siglec-10, on the other hand, exhibits immunosuppressive properties that contribute to immune tolerance within the tumour microenvironment. Therefore, we delve into the complex mechanisms through which Siglec-10 modulates immune responses and facilitates immune escape in cancer. Siglec-10 also acts as a viable target for cancer immunotherapy and presents novel avenues for the development of therapeutic interventions. Furthermore, we examine the synergy between CD24 and Siglec-10 in shaping the immunosuppressive tumour microenvironment and discuss the implications for combination therapies. Therefore, understanding the roles of CD24 and Siglec-10 in cancer immunotherapy opens exciting possibilities for the development of novel therapeutics.

The Role of Ficolins in Lung Injury.

BACKGROUND: Respiratory diseases seriously threaten human health worldwide, and lung injury is an important component of respiratory disease. Complement activation is an important function of the innate immune system. Complement activation helps the body defend against invasion by external microorganisms, whereas excessive complement activation can exacerbate tissue damage or lead to unwanted side effects. Ficolins are a class of immune-related proteins in the lectin pathway that play important roles in the body's immune defense. Although individual ficolins are not well understood, current information suggests that ficolins may play an important regulatory role in lung injury. SUMMARY: Several studies have shown that ficolins are involved in the immune response in the lung, particularly in the response to infectious and inflammatory processes. KEY MESSAGES: This review summarizes the role of ficolins in lung injury. Ficolins may influence the development and repair of lung injury by recognizing and binding pathogenic microorganisms, modulating the inflammatory response, and promoting the clearance of immune cells. In addition, ficolins are associated with the development and progression of lung diseases (such as pneumonia and ARDS) and may have an important impact on the pathophysiological processes of inflammatory diseases.

Insights into the recognition of hypermucoviscous Klebsiella pneumoniae clinical isolates by innate immune lectins of the Siglec and galectin families.

Klebsiella pneumoniae is an opportunistic bacterium that frequently colonizes the nasopharynx and gastrointestinal tract and can also cause severe infections when invading other tissues, particularly in immunocompromised individuals. Moreover, K. pneumoniae variants exhibiting a hypermucoviscous (HMV) phenotype are usually associated with hypervirulent strains that can produce invasive infections even in immunocompetent individuals. Major carbohydrate structures displayed on the K. pneumoniae surface are the polysaccharide capsule and the lipopolysaccharide, which presents an O-polysaccharide chain in its outermost part. Various capsular and O-chain structures have been described. Of note, production of a thick capsule is frequently observed in HMV variants. Here we examined the surface sugar epitopes of a collection of HMV and non-HMV K. pneumoniae clinical isolates and their recognition by several Siglecs and galectins, two lectin families of the innate immune system, using bacteria microarrays as main tool. No significant differences among isolates in sialic acid content or recognition by Siglecs were observed. In contrast, analysis of the binding of model lectins with diverse carbohydrate-binding specificities revealed striking differences in the recognition by galactose- and mannose-specific lectins, which correlated with the binding or lack of binding of galectins and pointed to the O-chain as the plausible ligand. Fluorescence microscopy and microarray analyses of galectin-9 binding to entire cells and outer membranes of two representative HMV isolates supported the bacteria microarray results. In addition, Western blot analysis of the binding of galectin-9 to outer membranes unveiled protein bands recognized by this galectin, and fingerprint analysis of these bands identified several proteins containing potential O-glycosylation sites, thus broadening the spectrum of possible galectin ligands on the K. pneumoniae surface. Moreover, Siglecs and galectins apparently target different structures on K. pneumoniae surfaces, thereby behaving as non-redundant complementary tools of the innate immune system.

The macrophage polarization in Entamoeba histolytica infection modulation by the C fragment of the intermediate subunit of Gal/GalNAc-inhibitable lectin.

The protozoan parasite Entamoeba histolytica is the causative agent of amebiasis, with clinical outcomes ranging from asymptomatic infections to severe invasive diseases. The innate immune system, particularly macrophages, is of paramount importance in resisting the invasion of host tissues and organs by the trophozoites of E. histolytica. Parasite-derived pathogenic factors, such as lectins, play a pivotal role in the promotion of macrophage polarization phenotypes that have undergone alteration. Nevertheless, the precise mechanisms by which E. histolytica modulates immune polarization remain largely unknown. The current study focused on the immunomodulatory effects of the Igl-C fragment of E. histolytica Gal/GalNAc lectin on macrophage polarization. These results demonstrated that Igl-C could induce the secretion of IL-1ß, IL-6, and other cytokines, activating a mixed M1/M2 polarization state. M1 polarization of macrophages occurs in the early stages and gradually transitions to M2 polarization in the later stages, which may contribute to the persistence of the infection. Igl-C induces the macrophage M1 phenotype and causes the release of immune effector molecules, including iNOS and cytokines, by activating the NF-κB p65 and JAK-STAT1 transcription factor signaling pathways. Furthermore, Igl-C supports the macrophage M2 phenotype via JAK-STAT3 and IL-4-STAT6 pathways, which activate arginase expression in later stages, contributing to the tissue regeneration and persistence of the parasite. The involvement of distinct signaling pathways in mediating this response highlights the complex interplay between the parasite and the host immune system. These findings enhance our understanding of the Igl-C-mediated pathogenic mechanisms during E. histolytica infection.

A novel lectin with a distinct Gal_Lectin and CUB domain mediates haemocyte phagocytosis in oyster Crassostrea gigas.

Invertebrate lectins exhibit structural diversity and play crucial roles in the innate immune responses by recognizing and eliminating pathogens. In the present study, a novel lectin containing a Gal_Lectin, a CUB and a transmembrane domain was identified from the Pacific oyster Crassostrea gigas (defined as CgGal-CUB). CgGal-CUB mRNA was detectable in all the examined tissues with the highest expression in adductor muscle (11.00-fold of that in haemocytes, p < 0.05). The expression level of CgGal-CUB mRNA in haemocytes was significantly up-regulated at 3, 24, 48 and 72 h (8.37-fold, 12.13-fold, 4.28-fold and 10.14-fold of that in the control group, respectively) after Vibrio splendidus stimulation. The recombinant CgGal-CUB (rCgGal-CUB) displayed binding capability to Mannan (MAN), peptidoglycan (PGN), D-(+)-Galactose and L-Rhamnose monohydrate, as well as Gram-negative bacteria (Escherichia coli, V. splendidus and Vibrio anguillarum), Gram-positive bacteria (Micrococcus luteus, Staphylococcus aureus, and Bacillus sybtilis) and fungus (Pichia pastoris). rCgGal-CUB was also able to agglutinate V. splendidus, and inhibit V. splendidus growth. Furthermore, rCgGal-CUB exhibited the activities of enhancing the haemocyte phagocytosis towards V. splendidus, and the phagocytosis rate of haemocytes was descended in blockage assay with CgGal-CUB antibody. These results suggested that CgGal-CUB served as a pattern recognition receptor to bind various PAMPs and bacteria, and enhanced the haemocyte phagocytosis towards V. splendidus.

Neisserial adhesin A (NadA) binds human Siglec-5 and Siglec-14 with high affinity and promotes bacterial adhesion/invasion.

Neisserial adhesin A (NadA) is a meningococcal surface protein included as recombinant antigen in 4CMenB, a protein-based vaccine able to induce protective immune responses against Neisseria meningitidis serogroup B (MenB). Although NadA is involved in the adhesion/invasion of epithelial cells and human myeloid cells, its function in meningococcal physiology is still poorly understood. To clarify the role played by NadA in the host-pathogen interaction, we sought to identify its cellular receptors. We screened a protein microarray encompassing 2,846 human and 297 mouse surface/secreted recombinant proteins using recombinant NadA as probe. Efficient NadA binding was revealed on the paired sialic acid-binding immunoglobulin-type lectins receptors 5 and 14 (Siglec-5 and Siglec-14), but not on Siglec-9 therein used as control. The interaction was confirmed by biochemical tools with the determination of the KD value in the order of nanomolar and the identification of the NadA binding site by hydrogen-deuterium exchange coupled to mass spectrometry. The N-terminal domain of the Siglec-5 that recognizes the sialic acid was identified as the NadA binding domain. Intriguingly, exogenously added recombinant soluble Siglecs, including Siglec-9, were found to decorate N. meningitidis surface in a NadA-dependent manner. However, Siglec-5 and Siglec-14 transiently expressed in CHO-K1 cells endorsed NadA binding and increased N. meningitidis adhesion/invasion while Siglec-9 did not. Taken together, Siglec-5 and Siglec-14 satisfy all features of NadA receptors suggesting a possible role of NadA in the acute meningococcal infection.IMPORTANCEBacteria have developed several strategies for cell colonization and immune evasion. Knowledge of the host and pathogen factors involved in these mechanisms is crucial to build efficacious countermoves. Neisserial adhesin A (NadA) is a meningococcal surface protein included in the anti-meningococcus B vaccine 4CMenB, which mediates adhesion to and invasion of epithelial cells. Although NadA has been shown to bind to other cell types, like myeloid and endothelial cells, it still remains orphan of a defined host receptor. We have identified two strong NadA interactors, Siglec-5 and Siglec-14, which are mainly expressed on myeloid cells. This showcases that NadA is an additional and key player among the Neisseria meningitidis factors targeting immune cells. We thus provide novel insights on the strategies exploited by N. meningitidis during the infection process, which can progress to a severe illness and death.

Changes in serum immunoglobulin G glycosylation patterns for antiphospholipid syndrome patients with lectin microarray.

Antiphospholipid syndrome is a rare autoimmune disease characterized by persistent antiphospholipid antibodies. Immunoglobulin G plays a vital role in disease progression, with its structure and function affected by glycosylation. We aimed to investigate the changes in the serum immunoglobulin G glycosylation pattern in antiphospholipid syndrome patients. We applied lectin microarray on samples from 178 antiphospholipid syndrome patients, 135 disease controls (including Takayasu arteritis, rheumatoid arthritis and cardiovascular disease) and 100 healthy controls. Lectin blots were performed for validation of significant differences. Here, we show an increased immunoglobulin G-binding level of soybean agglutinin (p = 0.047, preferring N-acetylgalactosamine) in antiphospholipid syndrome patients compared with healthy and disease controls. Additionally, the immunoglobulin G from antiphospholipid syndrome patients diagnosed with pregnancy events had lower levels of fucosylation (p = 0.001, recognized by Lotus tetragonolobus) and sialylation (p = 0.030, recognized by Sambucus nigra I) than those with simple thrombotic events. These results suggest the unique serum immunoglobulin G glycosylation profile of antiphospholipid syndrome patients, which may inform future studies to design biomarkers for more accurate diagnosis of antiphospholipid syndrome and even for the prediction of clinical symptoms in patients.

Identification of immune subsets with distinct lectin binding signatures using multi-parameter flow cytometry: correlations with disease activity in systemic lupus erythematosus.

Objectives: Cell surface glycosylation can influence protein-protein interactions with particular relevance to changes in core fucosylation and terminal sialylation. Glycans are ligands for immune regulatory lectin families like galectins (Gals) or sialic acid immunoglobulin-like lectins (Siglecs). This study delves into the glycan alterations within immune subsets of systemic lupus erythematosus (SLE). Methods: Evaluation of binding affinities of Galectin-1, Galectin-3, Siglec-1, Aleuria aurantia lectin (AAL, recognizing core fucosylation), and Sambucus nigra agglutinin (SNA, specific for α-2,6-sialylation) was conducted on various immune subsets in peripheral blood mononuclear cells (PBMCs) from control and SLE subjects. Lectin binding was measured by multi-parameter flow cytometry in 18 manually gated subsets of T-cells, NK-cells, NKT-cells, B-cells, and monocytes in unstimulated resting state and also after 3-day activation. Stimulated pre-gated populations were subsequently clustered by FlowSOM algorithm based on lectin binding and activation markers, CD25 or HLA-DR. Results: Elevated AAL, SNA and CD25+/CD25- SNA binding ratio in certain stimulated SLE T-cell subsets correlated with SLE Disease Activity Index 2000 (SLEDAI-2K) scores. The significantly increased frequencies of activated AALlow Siglec-1low NK metaclusters in SLE also correlated with SLEDAI-2K indices. In SLE, activated double negative NKTs displayed significantly lower core fucosylation and CD25+/CD25- Siglec-1 binding ratio, negatively correlating with disease activity. The significantly enhanced AAL binding in resting SLE plasmablasts positively correlated with SLEDAI-2K scores. Conclusion: Alterations in the glycosylation of immune cells in SLE correlate with disease severity, which might represent potential implications in the pathogenesis of SLE.

Potential Involvement of the South American Lungfish Intelectin-2 in Innate-Associated Immune Modulation.

Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (Lepidosiren paradoxa), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 ß-sheets: an anti-parallel ß-sheet, a ß-hairpin, and a disordered ß-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in L. paradoxa samples. Overall, our results show the potential relevance of LpITLN2-B in L. paradoxa immune defense against pathogens.

Glycans in melanoma: Drivers of tumour progression but sweet targets to exploit for immunotherapy.

Aberrant glycosylation recently emerged as an unmissable hallmark of cancer progression in many cancers. In melanoma, there is growing evidence that the tumour 'glycocode' plays a major role in promoting cell proliferation, invasion, migration, but also dictates the nature of the immune infiltrate, which strongly affects immune cell function, and clinical outcome. Aberrant glycosylation patterns dismantle anti-tumour defence through interactions with lectins on immune cells, which are crucial to shape anti-tumour immunity but also to trigger immune evasion. The glycan/lectin axis represents a new immune subversion pathway that is exploited by melanoma to hijack immune cells and escape from immune control. In this review, we describe the glycosylation features of melanoma tumour cells, and further gather findings related to the role of glycosylation in melanoma tumour progression, deciphering in detail its impact on immunity. We also depict glycan-based strategies aiming at restoring a functional anti-tumour response in melanoma patients. Glycans/lectins emerge as key immune checkpoints with promising translational properties. Exploitation of these pathways could reshape potent anti-tumour immunity while impeding immunosuppressive circuits triggered by aberrant tumour glycosylation patterns, holding great promise for cancer therapy.

Angiogenic factors and the lectin pathway of complement in women with secondary recurrent pregnancy loss.

The poor remodeling of placental spiral arteries seen in preeclampsia is also discussed to contribute to recurrent pregnancy loss (RPL) preceded by abnormal angiogenesis and excessive complement activation. Low levels of Mannose-binding-lectin (MBL), a pattern recognition molecule (PRM) of the lectin pathway, have been found in women with RPL. We propose that pregnancy loss is connected to defective angiogenesis with reperfusion damage in the placenta and decreased levels of PRM in the lectin pathway in women with RPL. In this cohort study, we investigate the angiogenic factors and the lectin complement pathway in early pregnancy and their time-dependent relationship with pregnancy outcomes in 76 women with secondary RPL (sRPL) who have at least four prior pregnancy losses and a live birth. We evaluated levels of Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), Vascular Endothelial Growth Factor (VEGF), soluble fms-like tyrosine kinase-1 (sFlt-1), and the PRMs, MBL, ficolin-1, -2, -3 and an additional soluble PRM, Pentraxin-3, during the 5th, 6th, and 7th gestational weeks. Our results showed that, compared to live births, pregnancies that ended in loss were associated with elevated VEGF levels and decreased levels of the Ang-2/Ang-1 ratio. Also, increasing levels of ficolin-2 were significantly associated with pregnancy loss, with MBL showing no association. Our research suggests that women with sRPL may have inadequate placentation with impaired angiogenesis in pregnancies ending in a loss.

A pair of G-type lectin receptor-like kinases modulates nlp20-mediated immune responses by coupling to the RLP23 receptor complex.

Plant cells recognize microbial patterns with the plasma-membrane-localized pattern-recognition receptors consisting mainly of receptor kinases (RKs) and receptor-like proteins (RLPs). RKs, such as bacterial flagellin receptor FLS2, and their downstream signaling components have been studied extensively. However, newly discovered regulatory components of RLP-mediated immune signaling, such as the nlp20 receptor RLP23, await identification. Unlike RKs, RLPs lack a cytoplasmic kinase domain, instead recruiting the receptor-like kinases (RLKs) BAK1 and SOBIR1. SOBIR1 specifically works as an adapter for RLP-mediated immunity. To identify new regulators of RLP-mediated signaling, we looked for SOBIR1-binding proteins (SBPs) in Arabidopsis thaliana using protein immunoprecipitation and mass spectrometry, identifying two G-type lectin RLKs, SBP1 and SBP2, that physically interacted with SOBIR1. SBP1 and SBP2 showed high sequence similarity, were tandemly repeated on chromosome 4, and also interacted with both RLP23 and BAK1. sbp1 sbp2 double mutants obtained via CRISPR-Cas9 gene editing showed severely impaired nlp20-induced reactive oxygen species burst, mitogen-activated protein kinase (MAPK) activation, and defense gene expression, but normal flg22-induced immune responses. We showed that SBP1 regulated nlp20-induced immunity in a kinase activity-independent manner. Furthermore, the nlp20-induced the RLP23-BAK1 interaction, although not the flg22-induced FLS2-BAK1 interaction, was significantly reduced in sbp1 sbp2. This study identified SBPs as new regulatory components in RLP23 receptor complex that may specifically modulate RLP23-mediated immunity by positively regulating the interaction between the RLP23 receptor and the BAK1 co-receptor.

An update on Ym1 and its immunoregulatory role in diseases.

Ym1 is a rodent-specific chitinase-like protein (CLP) lacking catalytic activity, whose cellular origins are mainly macrophages, neutrophils and other cells. Although the detailed function of Ym1 remains poorly understood, Ym1 has been generally recognized as a fundamental feature of alternative activation of macrophages in mice and hence one of the prevalent detecting targets in macrophage phenotype distinguishment. Studies have pointed out that Ym1 may have regulatory effects, which are multifaceted and even contradictory, far more than just a mere marker. Allergic lung inflammation, parasite infection, autoimmune diseases, and central nervous system diseases have been found associations with Ym1 to varying degrees. Thus, insights into Ym1's role in diseases would help us understand the pathogenesis of different diseases and clarify the genuine roles of CLPs in mammals. This review summarizes the information on Ym1 from the gene to its expression and regulation and focuses on the association between Ym1 and diseases.

Complement Activation via the Lectin and Alternative Pathway in Patients With Severe COVID-19.

Complement plays an important role in the direct defense to pathogens, but can also activate immune cells and the release of pro-inflammatory cytokines. However, in critically ill patients with COVID-19 the immune system is inadequately activated leading to severe acute respiratory syndrome (SARS) and acute kidney injury, which is associated with higher mortality. Therefore, we characterized local complement deposition as a sign of activation in both lungs and kidneys from patients with severe COVID-19. Using immunohistochemistry we investigated deposition of complement factors C1q, MASP-2, factor D (CFD), C3c, C3d and C5b-9 as well as myeloperoxidase (MPO) positive neutrophils and SARS-CoV-2 virus particles in lungs and kidneys from 38 patients who died from COVID-19. In addition, tissue damage was analyzed using semi-quantitative scores followed by correlation with complement deposition. Autopsy material from non-COVID patients who died from cardiovascular causes, cerebral hemorrhage and pulmonary embolism served as control (n=8). Lung injury in samples from COVID-19 patients was significantly more pronounced compared to controls with formation of hyaline membranes, thrombi and edema. In addition, in the kidney tubular injury was higher in these patients and correlated with lung injury (r=0.361*). In autopsy samples SARS-CoV-2 spike protein was detected in 22% of the lungs of COVID-19 patients but was lacking in kidneys. Complement activation was significantly stronger in lung samples from patients with COVID-19 via the lectin and alternative pathway as indicated by deposition of MASP-2, CFD, C3d and C5b9. Deposits in the lung were predominantly detected along the alveolar septa, the hyaline membranes and in the alveolar lumina. In the kidney, complement was significantly more deposited in patients with COVID-19 in peritubular capillaries and tubular basement membranes. Renal COVID-19-induced complement activation occurred via the lectin pathway, while activation of the alternative pathway was similar in both groups. Furthermore, MPO-positive neutrophils were found in significantly higher numbers in lungs and kidneys of COVID-19 patients and correlated with local MASP-2 deposition. In conclusion, in patients who died from SARS-CoV-2 infection complement was activated in both lungs and kidneys indicating that complement might be involved in systemic worsening of the inflammatory response. Complement inhibition might thus be a promising treatment option to prevent deregulated activation and subsequent collateral tissue injury in COVID-19.

Immunoadjuvant and Humoral Immune Responses of Garlic (Allium sativum L.) Lectins upon Systemic and Mucosal Administration in BALB/c Mice.

Dietary food components have the ability to affect immune function; following absorption, specifically orally ingested dietary food containing lectins can systemically modulate the immune cells and affect the response to self- and co-administered food antigens. The mannose-binding lectins from garlic (Allium sativum agglutinins; ASAs) were identified as immunodulatory proteins in vitro. The objective of the present study was to assess the immunogenicity and adjuvanticity of garlic agglutinins and to evaluate whether they have adjuvant properties in vivo for a weak antigen ovalbumin (OVA). Garlic lectins (ASA I and ASA II) were administered by intranasal (50 days duration) and intradermal (14 days duration) routes, and the anti-lectin and anti-OVA immune (IgG) responses in the control and test groups of the BALB/c mice were assessed for humoral immunogenicity. Lectins, co-administered with OVA, were examined for lectin-induced anti-OVA IgG response to assess their adjuvant properties. The splenic and thymic indices were evaluated as a measure of immunomodulatory functions. Intradermal administration of ASA I and ASA II had showed a four-fold and two-fold increase in anti-lectin IgG response, respectively, vs. the control on day 14. In the intranasal route, the increases were 3-fold and 2.4-fold for ASA I and ASA II, respectively, on day 50. No decrease in the body weights of animals was noticed; the increases in the spleen and thymus weights, as well as their indices, were significant in the lectin groups. In the adjuvanticity study by intranasal administration, ASA I co-administered with ovalbumin (OVA) induced a remarkable increase in anti-OVA IgG response (~six-fold; p < 0.001) compared to the control, and ASA II induced a four-fold increase vs. the control on day 50. The results indicated that ASA was a potent immunogen which induced mucosal immunogenicity to the antigens that were administered intranasally in BALB/c mice. The observations made of the in vivo study indicate that ASA I has the potential use as an oral and mucosal adjuvant to deliver candidate weak antigens. Further clinical studies in humans are required to confirm its applicability.

In silico construction of a multiepitope Zika virus vaccine using immunoinformatics tools.

Zika virus (ZIKV) is an arbovirus from the Flaviviridae family and Flavivirus genus. Neurological events have been associated with ZIKV-infected individuals, such as Guillain-Barré syndrome, an autoimmune acute neuropathy that causes nerve demyelination and can induce paralysis. With the increase of ZIKV infection incidence in 2015, malformation and microcephaly cases in newborns have grown considerably, which suggested congenital transmission. Therefore, the development of an effective vaccine against ZIKV became an urgent need. Live attenuated vaccines present some theoretical risks for administration in pregnant women. Thus, we developed an in silico multiepitope vaccine against ZIKV. All structural and non-structural proteins were investigated using immunoinformatics tools designed for the prediction of CD4 + and CD8 + T cell epitopes. We selected 13 CD8 + and 12 CD4 + T cell epitopes considering parameters such as binding affinity to HLA class I and II molecules, promiscuity based on the number of different HLA alleles that bind to the epitopes, and immunogenicity. ZIKV Envelope protein domain III (EDIII) was added to the vaccine construct, creating a hybrid protein domain-multiepitope vaccine. Three high scoring continuous and two discontinuous B cell epitopes were found in EDIII. Aiming to increase the candidate vaccine antigenicity even further, we tested secondary and tertiary structures and physicochemical parameters of the vaccine conjugated to four different protein adjuvants: flagellin, 50S ribosomal protein L7/L12, heparin-binding hemagglutinin, or RS09 synthetic peptide. The addition of the flagellin adjuvant increased the vaccine's predicted antigenicity. In silico predictions revealed that the protein is a probable antigen, non-allergenic and predicted to be stable. The vaccine's average population coverage is estimated to be 87.86%, which indicates it can be administered worldwide. Peripheral Blood Mononuclear Cells (PBMC) of individuals with previous ZIKV infection were tested for cytokine production in response to the pool of CD4 and CD8 ZIKV peptide selected. CD4 + and CD8 + T cells showed significant production of IFN-γ upon stimulation and IL-2 production was also detected by CD8 + T cells, which indicated the potential of our peptides to be recognized by specific T cells and induce immune response. In conclusion, we developed an in silico universal vaccine predicted to induce broad and high-coverage cellular and humoral immune responses against ZIKV, which can be a good candidate for posterior in vivo validation.

Elevated CD19+Siglec-10+ B cell levels are correlated with systemic lupus erythematosus disease activity.

BACKGROUND: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by B cell dysregulation and the breakdown of self-tolerance, leading to pathogenic autoantibody production. Human Siglec-10 is a member of the sialic acid-binding immunoglobulin-type lectin (Siglec) family and a B cell surface coreceptor that inhibits B cell receptor-induced signalling. However, to date, no report has investigated CD19+Siglec-10+ B cells in SLE patients. Thus, this study aimed to measure the population of CD19+Siglec-10+ B cells in patients with SLE and its correlation with disease activity. METHODS: Flow cytometry was employed to measure the population of CD19+Siglec-10+ B cells in peripheral blood mononuclear cells (PBMCs) of both SLE patients and healthy controls (HCs). The correlation of the proportion of CD19+Siglec-10+ B cells with the values of SLE disease activity was analysed. PBMCs from HCs were challenged with serum from active SLE, inactive SLE, or HCs, and the proportion of CD19+Siglec-10+ B cells was then assessed. The effect of dexamethasone (DEX) or hydroxychloroquine (HCQ) treatment on the proportion of CD19+Siglec-10+ B cells in PBMCs from SLE patients was also determined. RESULTS: The proportion of CD19+Siglec-10+ B cells in SLE patients was significantly elevated (P < 0.05), correlated positively with the SLEDAI score (r = 0.304; P = 0.018) and negatively with complement component 3 (C3) (r = -0.283; P = 0.04). In vitro assays indicated that sera from active SLE patients could significantly enhance the proportion of CD19+Siglec-10+ B cells (P < 0.05), while HCQ treatment significantly attenuated their proportions (P < 0.01). CONCLUSIONS: The elevation of CD19+Siglec-10+ B cells and their correlation with disease activity may suggest a role for Siglec-10 in the pathogenesis and progression of SLE and provide a serum biomarker for SLE activity.