The miR-221/222 cluster regulates hematopoietic stem cell quiescence and multipotency by suppressing both Fos/AP-1/IEG pathway activation and stress-like differentiation to granulocytes.

Throughout life, hematopoietic stem cells (HSCs), residing in bone marrow (BM), continuously regenerate erythroid/megakaryocytic, myeloid, and lymphoid cell lineages. This steady-state hematopoiesis from HSC and multipotent progenitors (MPPs) in BM can be perturbed by stress. The molecular controls of how stress can impact hematopoietic output remain poorly understood. MicroRNAs (miRNAs) as posttranscriptional regulators of gene expression have been found to control various functions in hematopoiesis. We find that the miR-221/222 cluster, which is expressed in HSC and in MPPs differentiating from them, perturbs steady-state hematopoiesis in ways comparable to stress. We compare pool sizes and single-cell transcriptomes of HSC and MPPs in unperturbed or stress-perturbed, miR-221/222-proficient or miR-221/222-deficient states. MiR-221/222 deficiency in hematopoietic cells was induced in C57BL/6J mice by conditional vav-cre-mediated deletion of the floxed miR-221/222 gene cluster. Social stress as well as miR-221/222 deficiency, alone or in combination, reduced HSC pools 3-fold and increased MPPs 1.5-fold. It also enhanced granulopoisis in the spleen. Furthermore, combined stress and miR-221/222 deficiency increased the erythroid/myeloid/granulocytic precursor pools in BM. Differential expression analyses of single-cell RNAseq transcriptomes of unperturbed and stressed, proficient HSC and MPPs detected more than 80 genes, selectively up-regulated in stressed cells, among them immediate early genes (IEGs). The same differential single-cell transcriptome analyses of unperturbed, miR-221/222-proficient with deficient HSC and MPPs identified Fos, Jun, JunB, Klf6, Nr4a1, Ier2, Zfp36-all IEGs-as well as CD74 and Ly6a as potential miRNA targets. Three of them, Klf6, Nr4a1, and Zfp36, have previously been found to influence myelogranulopoiesis. Together with increased levels of Jun, Fos forms increased amounts of the heterodimeric activator protein-1 (AP-1), which is known to control the expression of the selectively up-regulated expression of the IEGs. The comparisons of single-cell mRNA-deep sequencing analyses of socially stressed with miR-221/222-deficient HSC identify 5 of the 7 Fos/AP-1-controlled IEGs, Ier2, Jun, Junb, Klf6, and Zfp36, as common activators of HSC from quiescence. Combined with stress, miR-221/222 deficiency enhanced the Fos/AP-1/IEG pathway, extended it to MPPs, and increased the number of granulocyte precursors in BM, inducing selective up-regulation of genes encoding heat shock proteins Hspa5 and Hspa8, tubulin-cytoskeleton-organizing proteins Tuba1b, Tubb 4b and 5, and chromatin remodeling proteins H3f3b, H2afx, H2afz, and Hmgb2. Up-regulated in HSC, MPP1, and/or MPP2, they appear as potential regulators of stress-induced, miR-221/222-dependent increased granulocyte differentiation. Finally, stress by serial transplantations of miR-221/222-deficient HSC selectively exhausted their lymphoid differentiation capacities, while retaining their ability to home to BM and to differentiate to granulocytes. Thus, miR-221/222 maintains HSC quiescence and multipotency by suppressing Fos/AP-1/IEG-mediated activation and by suppressing enhanced stress-like differentiation to granulocytes. Since miR-221/222 is also expressed in human HSC, controlled induction of miR-221/222 in HSC should improve BM transplantations.

Enhanced Mott cell formation linked with IgM Fc receptor (FcµR) deficiency.

In previous studies, Mott cells, an unusual form of plasma cells containing Ig-inclusion bodies, were frequently observed in peripheral lymphoid tissues in our IgM Fc receptor (FcµR)-deficient (KO) mouse strain. Because of discrepancies in the reported phenotypes of different Fcmr KO mouse strains, we here examined two additional available mutant strains and confirmed that such enhanced Mott-cell formation was a general phenomenon associated with FcµR deficiency. Splenic B cells from Fcmr KO mice clearly generated more Mott cells than those from WT mice when stimulated in vitro with LPS alone or a B-1, but not B-2, activation cocktail. Nucleotide sequence analysis of the Ig variable regions of a single IgMλ+ Mott-hybridoma clone developed from splenic B-1 B cells of Fcmr KO mice revealed the near (VH) or complete (Vλ) identity with the corresponding germline gene segments and the addition of six or five nucleotides at the VH/DH and DH/JH junctions, respectively. Transduction of an FcµR cDNA into the Mott hybridoma significantly reduced cells containing IgM-inclusion bodies with a concomitant increase in IgM secretion, leading to secreted IgM binding to FcµR expressed on Mott transductants. These findings suggest a regulatory role of FcµR in the formation of Mott cells and IgM-inclusion bodies.

Physiological and Pathophysiological Roles of IgM Fc Receptor (FcµR) Isoforms.

IgM is the first antibody to emerge during phylogeny, ontogeny, and immune responses and serves as a first line of defense. Effector proteins interacting with the Fc portion of IgM, such as complement and its receptors, have been extensively studied for their functions. IgM Fc receptor (FcµR), identified in 2009, is the newest member of the FcR family and is intriguingly expressed by lymphocytes only, suggesting the existence of distinct functions as compared to the FcRs for switched Ig isotypes, which are expressed by various immune and non-hematopoietic cells as central mediators of antibody-triggered responses by coupling the adaptive and innate immune responses. Results from FcµR-deficient mice suggest a regulatory function of FcµR in B cell tolerance, as evidenced by their propensity to produce autoantibodies of both IgM and IgG isotypes. In this article, we discuss conflicting views about the cellular distribution and potential functions of FcµR. The signaling function of the Ig-tail tyrosine-like motif in the FcµR cytoplasmic domain is now formally shown by substitutional experiments with the IgG2 B cell receptor. The potential adaptor protein associating with FcµR and the potential cleavage of its C-terminal cytoplasmic tail after IgM binding are still enigmatic. Critical amino acid residues in the Ig-like domain of FcµR for interacting with the IgM Cµ4 domain and the mode of interaction are now defined by crystallographic and cryo-electron microscopic analyses. Some discrepancies on these interactions are discussed. Finally, elevated levels of a soluble FcµR isoform in serum samples are described as the consequence of persistent B cell receptor stimulation, as seen in chronic lymphocytic leukemia and probably in antibody-mediated autoimmune disorders.

Novel Vasoregulatory Aspects of Hereditary Angioedema: the Role of Arginine Vasopressin, Adrenomedullin and Endothelin-1.

The elevation of bradykinin (BK) level during attacks of hereditary angioedema due to C1-Inhibitor deficiency (C1-INH-HAE) is well known. We previously demonstrated that endothelin-1 (ET-1) level also increases during C1-INH-HAE attacks. Although BK and ET-1 are both potent vasoactive peptides, the vasoregulatory aspect of the pathomechanism of C1-INH-HAE has not yet been investigated. Hence we studied the levels of vasoactive peptides in controls and in C1-INH-HAE patients, as well as evaluated their changes during C1-INH-HAE attacks. The levels of arginine vasopressin (AVP), adrenomedullin (ADM) and ET-1 were measured in the plasma of 100 C1-INH-HAE patients in inter-attack periods and of 111 control subjects, using BRAHMS Kryptor technologies. In 18 of the 100 C1-INH-HAE patients, the levels of vasoactive peptides were compared in blood samples obtained during attacks, or in inter-attack periods. AVP, ADM and ET-1 levels were similar in inter-attack samples from C1-INH-HAE patients and in the samples of controls, although cardiovascular risk has an effect on the levels of vasoactive peptides in both groups. The levels of all three vasoactive peptides increased during C1-INH-HAE attacks. Moreover, the levels of ET-1 and ADM as well as their changes during attacks were significantly correlated. This study demonstrated that vascular regulation by vasoactive peptides is affected during C1-INH-HAE attacks. Our results suggest that the cooperation of several vasoactive peptides may be necessary to counterbalance the actions of excess BK, and to terminate the attacks. This may reveal a novel pathophysiological aspect of C1-INH-HAE.

Endothelial cell activation during edematous attacks of hereditary angioedema types I and II.

BACKGROUND: Hereditary angioedema (HAE) caused by C1-inhibitor (C1-INH) deficiency (HAE-C1-INH) is a potentially life-threatening rare disease caused by the decreased activity of C1-INH. Lack of C1-INH leads to overproduction of bradykinin, a potent vasoactive peptide. Although angioedema is induced by bradykinin, the function and activation of endothelial cells (ECs), the targets of bradykinin, have not yet been studied during HAE attacks. OBJECTIVE: We studied whether EC function is altered during HAE attacks in comparison with attack-free intervals. METHODS: Forty-six consecutive samples obtained during attacks from 18 patients with HAE-C1-INH were compared with inter-attack samples of the same patients. The patients' sera were tested for von Willebrand factor (VWF) antigen, VWF collagen-binding activity, soluble E-selectin, and endothelin-1 levels by using ELISA and BRAHMS Kryptor technologies. RESULTS: Levels of all 4 EC markers (VWF antigen, VWF collagen-binding activity, soluble E-selectin, and endothelin-1) were significantly increased during HAE attacks. Their increases were even more obvious in the subgroup of patients without any pre-existing risk factors for endothelial dysfunction. CONCLUSION: In this study we demonstrated that ECs are activated during HAE attacks. Our results might suggest the need for revising the knowledge on the pathogenesis of HAE-C1-INH and for reconsidering the role of ECs as a possible novel therapeutic target in patients with this disease.

Functions of IgM fc receptor (FcµR) related to autoimmunity.

Unlike Fc receptors for switched immunoglobulin (Ig) isotypes, Fc receptor for IgM (FcµR) is selectively expressed by lymphocytes. The ablation of the FcµR gene in mice impairs B cell tolerance as evidenced by concomitant production of autoantibodies of IgM and IgG isotypes. In this essay, we reiterate the autoimmune phenotypes observed in mutant mice, ie IgM homeostasis, dysregulated humoral immune responses including autoantibodies, and Mott cell formation. We also propose the potential phenotypes in individuals with FCMR deficiency and the model for FcµR-mediated regulation of self-reactive B cells.

Soluble Fc Receptor for IgM in Sera From Subsets of Patients With Chronic Lymphocytic Leukemia as Determined by a New Mouse Monoclonal Antibody.

The FcR for IgM (FcµR) is the newest member of the FcR family, selectively expressed by lymphocytes, and distinct from FcRs for switched Ig isotypes that are expressed by various immune cell types and non-hematopoietic cells. From studies of Fcmr-ablated mice, FcµR was shown to have a regulatory function in B-cell tolerance, as evidenced by high serum titers of autoantibodies of the IgM and IgG isotypes in mutant mice. In our previous studies, both cell-surface and serum FcµR levels were elevated in patients with chronic lymphocytic leukemia (CLL), where antigen-independent self-ligation of BCR is a hallmark of the neoplastic B cells. This was assessed by sandwich ELISA using two different ectodomain-specific mAbs. To determine whether the serum FcµR is derived from cleavage of its cell-surface receptor (shedding) or its alternative splicing to skip the transmembrane exon resulting in a 70-aa unique hydrophilic C-terminus (soluble), we developed a new mouse IgG1κ mAb specific for human soluble FcµR (solFcµR) by taking advantages of the unique nature of transductant stably producing His-tagged solFcµR and of an in vivo differential immunization. His-tagged solFcµR attached to exosomes and plasma membranes, allowing immunization and initial hybridoma screening without purification of solFcµR. Differential immunization with tolerogen (membrane FcµR) and immunogen (solFcµR) also facilitated to generate solFcµR-specific hybridomas. The resultant solFcµR-specific mAb reacted with serum FcµR in subsets of CLL patients. This mAb, along with another ectodomain-specific mAb, will be used for verifying the hypothesis that the production of solFcµR is the consequence of chronic stimulation of BCR.

Creation of the first monoclonal antibody recognizing an extracellular epitope of hABCC6.

Mutations in the ABCC6 gene result in calcification diseases such as pseudoxanthoma elasticum or Generalized Arterial Calcification of Infancy. Generation of antibodies recognizing an extracellular (EC) epitope of ABCC6 has been hampered by the short EC segments of the protein. To overcome this limitation, we immunized bovine FcRn transgenic mice exhibiting an augmented humoral immune response with Human Embryonic Kidney 293 cells cells expressing human ABCC6 (hABCC6). We obtained a monoclonal antibody recognizing an EC epitope of hABCC6 that we named mEChC6. Limited proteolysis revealed that the epitope is within a loop in the N-terminal half of ABCC6 and probably spans amino acids 338-347. mEChC6 recognizes hABCC6 in the liver of hABCC6 transgenic mice, verifying both specificity and EC binding to intact hepatocytes.

SARS-CoV-2 in severe COVID-19 induces a TGF-ß-dominated chronic immune response that does not target itself.

The pathogenesis of severe COVID-19 reflects an inefficient immune reaction to SARS-CoV-2. Here we analyze, at the single cell level, plasmablasts egressed into the blood to study the dynamics of adaptive immune response in COVID-19 patients requiring intensive care. Before seroconversion in response to SARS-CoV-2 spike protein, peripheral plasmablasts display a type 1 interferon-induced gene expression signature; however, following seroconversion, plasmablasts lose this signature, express instead gene signatures induced by IL-21 and TGF-ß, and produce mostly IgG1 and IgA1. In the sustained immune reaction from COVID-19 patients, plasmablasts shift to the expression of IgA2, thereby reflecting an instruction by TGF-ß. Despite their continued presence in the blood, plasmablasts are not found in the lungs of deceased COVID-19 patients, nor does patient IgA2 binds to the dominant antigens of SARS-CoV-2. Our results thus suggest that, in severe COVID-19, SARS-CoV-2 triggers a chronic immune reaction that is instructed by TGF-ß, and is distracted from itself.

A rheostat sets B-cell receptor repertoire selection to distinguish self from non-self.

In bone marrow VDJ-recombination continuously generates original repertoires of immature B cells expressing IgM-B cell receptor (BcR), in which each cell recognizes the wide variety of self and non-self antigens with an individually different spectrum of avidities. High avidity self-reactive B cells try to edit their BcRs by secondary or multiple VL-rearrangements to JL-rearrangements. If they do not manage to change their self reactivity, they are deleted by apoptosis. Low avidity self-reactive B cells are anergized, while B cells with no avidity to self are ignored. A rheostat crosslinking antigen-binding BcRs, self antigen complexed with pentameric IgM and Fcµ-receptor monitors high, low or no binding. PI3K and PTEN are the effectors of this self antigen-sensing device. In mature B cells this rheostat continues to function in the activation of resting B cells by foreign antigens which crosslink BcR, antigen and pentameric IgM with Fcµ-receptors.

FcRn Overexpression Expands Diversity of the Humoral Immune Response in bFcRn Transgenic Mice.

The neonatal Fc receptor (FcRn) plays key roles in IgG and albumin homeostasis, maternal IgG transport, and antigen presentation of IgG-opsonized antigens. Previously, we reported that transgenic (Tg) mice that overexpress bovine FcRn (bFcRn) have augmented T-dependent humoral immune response with increased IgG protection, higher level of antigen-specific antibodies, greater number of antigen-specific B cells, and effective immune response even against weakly immunogenic epitopes. In this study we analyzed the diversity of the humoral immune response of bFcRn Tg mice, using a length distribution analysis (spectratyping) and next generation sequencing (NGS) of the immunoglobulin heavy chain variable regions. Our analysis showed that in response to immunization with ovalbumin or transfected cells that expressed a unique membrane protein, our Tg animals developed a more diverse plasma cell repertoire than controls, which manifested in greater numbers of different clones, and clusters with fewer highly expanded large clones, as identified by the variable region (CDR3) of the immunoglobulin heavy chain. The increased antibody diversity in Tg mice after immunization was observed at both IgM and IgG levels, indicating that the increased humoral immune diversity in Tg mice is due to a higher number of both activated, antigen-specific naïve and isotype switched B cells. We thus demonstrated that the BCR repertoire of the immunized bFcRn Tg animals is more diverse compared to wild type mice, which likely makes these Tg mice a better choice for monoclonal antibody production against challenging antigens, including the extracellular regions of cell membrane proteins.

Functional Roles of the IgM Fc Receptor in the Immune System.

It is now evident from studies of mice unable to secrete IgM that both non-immune "natural" and antigen-induced "immune" IgM are important for protection against pathogens and for regulation of immune responses to self-antigens. Since identification of its Fc receptor (FcµR) by a functional cloning strategy in 2009, the roles of FcµR in these IgM effector functions have begun to be explored. Unlike Fc receptors for switched Ig isotypes (e.g., FcγRs, FcεRs, FcαR, Fcα/µR, pIgR, FcRn), FcµR is selectively expressed by lymphocytes: B, T, and NK cells in humans and only B cells in mice. FcµR may have dual signaling ability: one through a potential as yet unidentified adaptor protein non-covalently associating with the FcµR ligand-binding chain via a His in transmembrane segment and the other through its own Tyr and Ser residues in the cytoplasmic tail. FcµR binds pentameric and hexameric IgM with a high avidity of ~10 nM in solution, but more efficiently binds IgM when it is attached to a membrane component via its Fab region on the same cell surface (cis engagement). Four different laboratories have generated Fcmr-ablated mice and eight different groups of investigators have examined the resultant phenotypes. There have been some clear discrepancies reported that appear to be due to factors including differences in the exons of Fcmr that were targeted to generate the knockouts. One common feature among these different mutant mice, however, is their propensity to produce autoantibodies of both IgM and IgG isotypes. In this review, we briefly describe recent findings concerning the functions of FcµR in both mice and humans and propose a model for how FcµR plays a regulatory role in B cell tolerance.

Transcriptome analysis of inflammation-related gene expression in endothelial cells activated by complement MASP-1.

Mannan-binding lectin-associated serine protease 1 (MASP-1), the most abundant enzyme of the complement lectin pathway, is able to stimulate human umbilical vein endothelial cells (HUVECs) to alter the expression of several cytokines and adhesion molecules. This study has assessed to what extent MASP-1 is able to modify the transcriptional pattern of inflammation-related (IR) genes in HUVECs. We utilized Agilent microarray to analyse the effects of recombinant MASP-1 (rMASP-1) in HUVECs, on a set of 884 IR genes. Gene Set Enrichment Analysis showed an overall activation of inflammation-related genes in response to rMASP-1. rMASP-1 treatment up- and down-regulated 19 and 11 IR genes, respectively. Most of them were previously unidentified, such as genes of chemokines (CXCL1, CXCL2, CXCL3), inflammatory receptors (TLR2, BDKRB2) and other inflammatory factors (F3, LBP). Expression of IR genes changed early, during the first 2 hours of activation. Both p38-MAPK inhibitor and NFκB inhibitor efficiently suppressed the effect of rMASP-1. We delineated 12 transcriptional factors as possible regulators of rMASP-1-induced IR genes. Our microarray-based data are in line with the hypothesis that complement lectin pathway activation, generating active MASP-1, directly regulates inflammatory processes by shifting the phenotype of endothelial cells towards a more pro-inflammatory type.

Complement MASP-1 enhances adhesion between endothelial cells and neutrophils by up-regulating E-selectin expression.

The complement system and neutrophil granulocytes are indispensable in the immune response against extracellular pathogens such as bacteria and fungi. Endothelial cells also participate in antimicrobial immunity largely by regulating the homing of leukocytes through their cytokine production and their pattern of cell surface adhesion molecules. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), a complement lectin pathway enzyme, is able to activate endothelial cells by cleaving protease activated receptors, which leads to cytokine production and enables neutrophil chemotaxis. Therefore, we aimed to investigate how recombinant MASP-1 (rMASP-1) can modify the pattern of P-selectin, E-selectin, ICAM-1, ICAM-2, and VCAM-1 adhesion molecules in human umbilical vein endothelial cells (HUVEC), and whether these changes can enhance the adherence between endothelial cells and neutrophil granulocyte model cells (differentiated PLB-985). We found that HUVECs activated by rMASP-1 decreased the expression of ICAM-2 and increased that of E-selectin, whereas ICAM-1, VCAM-1 and P-selectin expression remained unchanged. Furthermore, these changes resulted in increased adherence between differentiated PLB-985 cells and endothelial cells. Our finding suggests that complement MASP-1 can increase adhesion between neutrophils and endothelial cells in a direct fashion. This is in agreement with our previous finding that MASP-1 increases the production of pro-inflammatory cytokines (such as IL-6 and IL-8) and chemotaxis, and may thereby boost neutrophil functions. This newly described cooperation between complement lectin pathway and neutrophils via endothelial cells may be an effective tool to enhance the antimicrobial immune response.

Serum MASP-1 in complex with MBL activates endothelial cells.

The complement system plays an important role in the induction of inflammation. In this study we demonstrate that the initiation complexes of the lectin pathway, consisting of mannose-binding lectin (MBL) and associated serine proteases (MASPs) elicit Ca(2+) signaling in cultured endothelial cells (HUVECs). This is in agreement with our previous results showing that the recombinant catalytic fragment of MASP-1 activates endothelial cells by cleaving protease activated receptor 4. Two other proteases, MASP-2 and MASP-3 are also associated with MBL. Earlier we showed that recombinant catalytic fragment of MASP-2 cannot activate HUVECs, and in this study we demonstrate that the same fragment of MASP-3 has also no effect. We find the same to be the case if we use recombinant forms of the N-terminal parts of MASP-1 and MASP-2 which only contain non-enzymatic domains. Moreover, stable zymogen mutant form of MASP-1 was also ineffective to stimulate endothelial cells, which suggests that in vivo MASP-1 have the ability to activate endothelial cells directly as well as to activate the lectin pathway simultaneously. We show that among the components of the MBL-MASPs complexes only MASP-1 is able to trigger response in HUVECs and the proteolytic activity of MASP-1 is essential. Our results strengthen the view that MASP-1 plays a central role in the early innate immune response.

MASP-1 induces a unique cytokine pattern in endothelial cells: a novel link between complement system and neutrophil granulocytes.

Microbial infection urges prompt intervention by the immune system. The complement cascade and neutrophil granulocytes are the predominant contributors to this immediate anti-microbial action. We have previously shown that mannan-binding lectin-associated serine protease-1 (MASP-1), the most abundant enzyme of the complement lectin pathway, can induce p38-MAPK activation, NFkappaB signaling, and Ca(2+)-mobilization in endothelial cells. Since neutrophil chemotaxis and transmigration depends on endothelial cell activation, we aimed to explore whether recombinant MASP-1 (rMASP-1) is able to induce cytokine production and subsequent neutrophil chemotaxis in human umbilical vein endothelial cells (HUVEC). We found that HUVECs activated by rMASP-1 secreted IL-6 and IL-8, but not IL-1alpha, IL-1ra, TNFalpha and MCP-1. rMASP-1 induced dose-dependent IL-6 and IL-8 production with different kinetics. rMASP-1 triggered IL-6 and IL-8 production was regulated predominantly by the p38-MAPK pathway. Moreover, the supernatant of rMASP-1-stimulated HUVECs activated the chemotaxis of neutrophil granulocytes as an integrated effect of cytokine production. Our results implicate that besides initializing the complement lectin pathway, MASP-1 may activate neutrophils indirectly, via the endothelial cells, which link these effective antimicrobial host defense mechanisms.