Natural IgM Production Research Articles

Sleeve Gastrectomy Regulates Natural IgM Production Independent of Weight and Altered Microbiome

Sleeve Gastrectomy Regulates Natural IgM Production Independent of Weight and Altered Microbiome

Mechanism of differentiation and unique repertoire of IgM secreting B-1 plasma cells

Abstract Natural antibodies are broadly reactive, mostly IgM antibodies that provide a first line of defense against pathogens. Distinct populations of fetal/neonatal-derived IgM-secreting B-1 cells, including non-terminally differentiated B-1 and B-1 plasma cells (PC) are present in bone marrow (BM) and spleen. B-1PC are transcriptionally and phenotypically similar to foreign antigen-induced, long-lived conventional PC, prompting us to hypothesize that both receive similar differentiation signals. To determine the extent to which natural IgM-producing B-1PC depend on T cell help, we studied TCRβδ−/− mice lacking either all T cells, or depleted of CD4 T cells by anti-CD4 mAb treatment. Both showed significantly reduced numbers of BM but not spleen B-1PC and slightly increased populations of B-1 cells. Consistent with those data, reconstitution of RAG-deficient mice with B-1 and CD4+ T cells, but not with B-1 cells alone, restored the BM B-1PC compartment and significantly enhanced serum IgM levels. B-1PC did not bind to PtC-containing liposomes and depletion of B-1 PC had minimal effect on the number of anti-PtC IgM ASC. In contrast, most anti-influenza natural IgM ASC were B-1PC, consistent with reduced serum levels influenza-binding natural IgM in TCRβδ−/− mice. Together, the study reveals that in contrast to previous assumptions, some natural IgM production is dependent on CD4 T cell help, suggesting that multiple mechanisms regulate induction of natural IgM secretion.

A TLR4-TRIF-dependent signaling pathway is required for protective natural tumor-reactive IgM production by B1 cells.

Metastatic cancer involving spread to the peritoneal cavity is referred to as peritoneal carcinomatosis and has a very poor prognosis. Our previous studies demonstrated a toll-like receptor 4 (TLR4) and C-type lectin receptor (CLR; Mincle/MCL) agonist pairing of monophosphoryl lipid A (MPL) and trehalose-6,6'-dicorynomycolate (TDCM) effectively inhibits peritoneal tumor growth and ascites development through a mechanism dependent upon B1a cell-produced natural IgM, complement, and phagocytes. In the current study, we investigated the requirement for TLR4 and Fc receptor common γ chain (FcRγ), required for Mincle/MCL signaling, in the MPL/TDCM-elicited response. MPL/TDCM significantly increased macrophages and Ly6Chi monocytes in the peritoneal cavity of both TLR4-/- and FcRγ-/- mice, suggesting redundancy in the signals required for monocyte/macrophage recruitment. However, B1 cell activation, antibody secreting cell differentiation, and tumor-reactive IgM production were defective in TLR4-/-, but not FcRγ-/- mice. TRIF was required for production of IgM reactive against tumor- and mucin-related antigens, but not phosphorylcholine, whereas TLR4 was required for production of both types of reactivities. Consistent with this, B1 cells lacking TLR4 or TRIF did not proliferate or differentiate into tumor-reactive IgM-producing cells in vitro and did not reconstitute MPL/TDCM-dependent protection against peritoneal carcinomatosis in CD19-/- mice. Our results indicate a TLR4/TRIF-dependent pathway is required by B1 cells for MPL/TDCM-elicited production of protective tumor-reactive natural IgM. The dependency on TRIF signaling for tumor-reactive, but not phosphorylcholine-reactive, IgM production reveals unexpected heterogeneity in TLR4-dependent regulation of natural IgM production, thereby highlighting important differences to consider when designing vaccinesortherapies targeting these specificities.

TLR and CLR agonists induce anti-tumor B cell responses against peritoneal carcinomatosis

Abstract Peritoneal carcinomatosis is a condition where primary tumors metastasize into the peritoneal cavity and induce malignant ascites. The median survival of patients is poor, averaging about twenty weeks post diagnosis. Due to the high mortality rate associated with this disease and the lack of curative treatments available, it is imperative that more effective treatments be developed for peritoneal carcinomatosis. The immune system plays an important role in tumor elimination, and one strategy currently being explored to treat malignancies involves leukocyte activation through pattern recognition receptors (PRRs), including Toll-Like Receptors (TLRs) and C-type Lectin Receptors (CLRs). Our data shows that mice treated with a TLR and CLR agonist are afforded significant protection when challenged with an aggressive ascites-forming mammary tumor cell line (TA3-Ha). However, when mice lack B cells, the protection is lost. Administration of the combined treatment results in a significant increase in proliferating, antibody-producing B cells within the visceral adipose tissue (VAT). Consistent with this, the treatment induces a significant increase in IgM and complement (C′) bound to tumor cells, both of which were determined to be critical players in the anti-tumor effects. In summary, our data demonstrates pathogen associated molecular patterns can induce rapid natural IgM production that elicits anti-tumor protection via C′ activation.

Propionibacterium acnes induces an adjuvant effect in B-1 cells and affects their phagocyte differentiation via a TLR2-mediated mechanism

B-1 lymphocytes are present in large numbers in the mouse peritoneal cavity, as are macrophages, and are responsible for natural IgM production. These lymphocytes migrate to inflammatory foci and are also involved in innate immunity. It was also demonstrated that B-1 cells are able to differentiated into phagocytes (B-1CDP), which is characterized by expression of F4/80 and increased phagocytic activity.B-1 cell responses to antigens and adjuvants are poorly characterized. It has been shown that Propionibacterium acnes suspensions induce immunomodulatory effects in both macrophages and B-2 lymphocytes. We recently demonstrated that this bacterium has the ability to increase B-1 cell populations both in vitro and in vivo. P. acnes induces B-1CDP differentiation, increases the expression of TLR2, TLR4 and TLR9 and augments the expression of CD80, CD86 and CD40 in B-1 and B-1CDP cells.Because P. acnes has been shown to modulate TLR expression, in this study, we investigated the role of TLR2 and TLR4 in B-1 cell population, including B-1CDP differentiation and phagocytic activity in vitro and in vivo. Interestingly, we have demonstrated that TLR2 signaling could be involved in the increase in the B-1 cell population induced by P. acnes. Furthermore, the early differentiation of B-1CDP is also dependent of TLR2. It was also observed that TLR signals also interfere in the phagocytic ability of B-1 cells and their phagocytes. According to these data, it is clear that P. acnes promotes an important adjuvant effect in B-1 cells by inducing them to differentiate into B-1CDP cells and modulates their phagocytic functions both in vivo and in vitro. Moreover, most of these effects are mediated primarily via TLR2. These data reinforce the findings that such bacterial suspensions have powerful adjuvant properties. The responses of B-1 cells to exogenous stimulation indicate that these cells are important to the innate immune response.

An Overview of B-1 Cells as Antigen-Presenting Cells.

The role of B cells as antigen-presenting cells (APCs) has been extensively studied, mainly in relation to the activation of memory T cells. Considering the B cell subtypes, the role of B-1 cells as APCs is beginning to be explored. Initially, it was described that B-1 cells are activated preferentially by T-independent antigens. However, some reports demonstrated that these cells are also involved in a T-dependent response. The aim of this review is to summarize information about the ability of B-1 cells to play a role as APCs and to briefly discuss the role of the BCR and toll-like receptor signals in this process. Furthermore, some characteristics of B-1 cells, such as natural IgM production and phagocytic ability, could interfere in the participation of these cells in the onset of an adaptive response.

RasGRP1 Is an Essential Signaling Molecule for Development of B1a Cells with Autoantigen Receptors.

B1a cells, particularly the PD-L2(+) B1a cell subset, are enriched with autoantigen-specific receptors. However, the underlying molecular mechanism responsible for the skewed selection of autoreactive B1a cells remains unclear. In this study, we find that B1 cells express only Ras guanyl nucleotide-releasing protein (RasGRP) 1, whereas B2 cells express mostly RasGRP3 and little RasGRP1. RasGRP1 is indispensable for transduction of weak signals. RasGRP1 deficiency markedly impairs B1a cell development and reduces serum natural IgM production; in particular, B1a cells that express autoantigen receptors, such as anti-phosphatidylcholine B1a cells, are virtually eliminated. Thus, unlike Btk and other signalosome components, RasGRP1 deficiency selectively affects only the B1a cell population with autoantigen receptors rather than the entire pool of B1a cells.

Frontiers in B-cell immunology

Frontiers in B-cell immunology

Adjuvant effect of killed Propionibacterium acnes on mouse peritoneal B-1 lymphocytes and their early phagocyte differentiation.

B-1 lymphocytes are the predominant cells in mouse peritoneal cavity. They express macrophage and lymphocyte markers and are divided into B-1a, B-1b and B-1c subtypes. The role of B-1 cells is not completely clear, but they are responsible for natural IgM production and seem to play a regulatory role. An enriched B-1b cell population can be obtained from non-adherent peritoneal cell cultures, and we have previously demonstrated that these cells undergo differentiation to acquire a mononuclear phagocyte phenotype upon attachment to the substrate in vitro. Nevertheless, the B-1 cell response to antigens or adjuvants has been poorly investigated. Because killed Propionibacterium acnes exhibits immunomodulatory effects on both macrophages and B-2 lymphocytes, we analyzed whether a killed bacterial suspension or its soluble polysaccharide (PS) could modulate the absolute number of peritoneal B-1 cells in BALB/c mice, the activation status of these cells and their ability to differentiate into phagocytes in vitro. In vivo, P. acnes treatment elevated the absolute number of all B-1 subsets, whereas PS only increased B-1c. Moreover, the bacterium increased the number of B-1b cells that were positive for MHC II, TLR2, TLR4, TLR9, IL-4, IL-5 and IL-12, in addition to up-regulating TLR9, CD80 and CD86 expression. PS increased B-1b cell expression of TLR4, TLR9, CD40 and CD86, as well as IL-10 and IL-12 synthesis. Both of the treatments decreased the absolute number of B-1b cells in vitro, suggesting their early differentiation into B-1 cell-derived phagocytes (B-1CDP). We also observed a higher phagocytic activity from the phagocytes that were derived from B-1b cells after P. acnes and PS treatment. The adjuvant effect that P. acnes has on B-1 cells, mainly the B-1b subtype, reinforces the importance of B-1 cells in the innate and adaptive immune responses.

Natural IgM in Immune Equilibrium and Harnessing Their Therapeutic Potential

Natural IgM Abs are the constitutively secreted products of B1 cells (CD5(+) in mice and CD20(+)CD27(+)CD43(+)CD70(-) in humans) that have important and diverse roles in health and disease. Whereas the role of natural IgM as the first line of defense for protection against invading microbes has been extensively investigated, more recent reports have highlighted their potential roles in the maintenance of tissue homeostasis via clearance of apoptotic and altered cells through complement-dependent mechanisms, inhibition of inflammation, removal of misfolded proteins, and regulation of pathogenic autoreactive IgG Abs and autoantibody-producing B cells. These observations have provided the theoretical underpinnings for efforts that currently seek to harness the untapped therapeutic potential of natural IgM either by boosting in vivo natural IgM production or via therapeutic infusions of monoclonal and polyclonal IgM preparations.

Enhancement of Neutrophil Function by Interleukin-18 Therapy Protects Burn-Injured Mice from Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) infection is a grave concern in burn-injured patients. We investigated the efficacy of interleukin-18 (IL-18) treatment in postburn MRSA infection. Alternate-day injections of IL-18 into burn-injured C57BL/6 mice significantly increased their survival after MRSA infection and after methicillin-sensitive S. aureus infection. Although IL-18 treatment of burn-injured mice augmented natural IgM production before MRSA infection and gamma interferon (IFN-γ) production after MRSA infection, neither IgM nor IFN-γ significantly contributed to the improvement in mouse survival. IL-18 treatment increased/restored the serum tumor necrosis factor (TNF), IL-17, IL-23, granulocyte colony-stimulating factor (G-CSF), and macrophage inflammatory protein (MIP-2) levels, as well as the neutrophil count, after MRSA infection of burn-injured mice; it also improved impaired neutrophil functions, phagocytic activity, production of reactive oxygen species, and MRSA-killing activity. However, IL-18 treatment was ineffective against MRSA infection in both burn- and sham-injured neutropenic mice. Enhancement of neutrophil functions by IL-18 was also observed in vitro. Furthermore, when neutrophils from IL-18-treated burn-injured mice were adoptively transferred into nontreated burn-injured mice 2 days after MRSA challenge, survival of the recipient mice increased. NOD-SCID mice that have functionally intact neutrophils and macrophages (but not T, B, or NK cells) were substantially resistant to MRSA infection. IL-18 treatment increased the survival of NOD-SCID mice after burn injury and MRSA infection. An adoptive transfer of neutrophils using NOD-SCID mice also showed a beneficial effect of IL-18-activated neutrophils, similar to that seen in C57BL/6 mice. Thus, although neutrophil functions were impaired in burn-injured mice, IL-18 therapy markedly activated neutrophil functions, thereby increasing survival from postburn MRSA infection.

Osteopontin-Igm as a Marker for the Diagnosis of Hepatocellular Carcinoma

Introduction Biomarkers for early hepatocellular carcinoma (HCC) detection are still a clinical need. Recent data indicate that cancer-associated antigens lead to the formation of circulating IgM-linked immune complexes as the result of natural IgM production by the innate immune response. The SCCA-IgM immune complex has already been described for primary liver cancer detection. Osteopontin (OPN) is a member of the SIBLING family of proteins recently shown to be related to tumorigenesis, progression and metastasis in various cancer types. Aim To evaluate the serum levels of the OPN-IgM complex in comparison to the SCCA-IgM complex in patients with HCC. Patients A total of 256 patients were analyzed including 151 patients with HCC (M/F 115/36; mean age ± SD: 67 ± 12 years) and 106 patients with cirrhosis (M/F 68/38; mean age ± SD: 62 ± 12 years). HCC, tested before any therapeutic approach, was mainly of viral etiology (58% HCV, 11% HBV), while alcohol abuse (22%) was the main risk factor for the remaining cases. A similar distribution was found in patients with cirrhosis (46% HCV, 15% HBV, 38% alcohol). Methods Serum levels of OPN-IgM were measured using a homemade ELISA assay with a polyclonal anti-human OPN antibody (Biodesign Int, USA). SCCA-IgM was detected in serum using an ELISA assay kit (Hepa-IC, Xeptagen). Results OPN-IgM was positive in 64/151 (42%) patients with HCC and in 45/106 (42%) patients with cirrhosis (specificity 58%), while the reference biomarker SCCA-IgM showed 35% sensitivity (49/139) and 71 % specificity. When patients were stratified on the basis of etiology, the sensitivity values for OPN-IgM and SCCA-IgM were 51% vs 44% in HCV patients, and 36% vs 23% in the other patients, while the specificity was lower for OPN-IgM (55% vs 69% in HCV patients; 62% vs 72% in the other patients). Combination of the two biomarkers resulted in an increase in sensitivity to 63% for viral etiology and to 40% for nonviral etiology. Conclusion Different IgM-linked biomarkers are detectable in primary liver cancer. OPN-IgM and SCCA-IgM showed a similar behavior, while the combination of these biomarkers increased the diagnostic performance for primary liver cancer.

Osteopontin-IgM in the diagnosis of HCC

Introduction. Biomarkers for early hepatocellular carcinoma (HCC) detection are still an unmet clinical need. Osteopontin (OPN) is a molecule strongly involved in cancer progression. Cancer-associated antigens lead to the formation of circulating IgM-linked immune complexes, as the result of natural IgM production by the innate immune response. SCCA-IgM immune complex has been already described for primary liver cancer detection.

Regulatory role of B-1 B cells in chronic colitis

According to the 'hygiene hypothesis', enhanced microbial exposure due to early childhood infections leads to a reduction of T(h)2-mediated allergic diseases and inflammatory bowel disease. To begin to elucidate the mechanisms underlying this hypothesis, we studied development of T(h)2-mediated colitis of the TCRalpha knockout (KO) mouse in both a specific pathogen-free (SPF) facility and a conventional (CV) facility. After more than five generations in each facility, TCRalpha KO mice kept in the CV facility developed dramatically less colitis than mice that were kept in the SPF facility. Surprisingly, the suppression of colitis in the CV facility correlated with a significant increase in natural IgM production by B-1 B cells. In contrast, B cell-deficient TCRalpha double-knockout (alphamu DKO) mice maintained in the CV facility continued to develop severe colitis, strongly suggesting that B-1 B cells contributed to the suppression of colitis. Indeed, the adoptive transfer of B-1 B cells isolated from the peritoneal cavity of TCRalpha KO mice (SPF) into alphamu DKO mice (CV) suppressed the development of colitis in the recipient mice. We conclude that B-1 cells play a regulatory role in T(h)2-mediated colitis under non-hygienic conditions, possibly by generating natural antibodies in response to microbial flora.

Restoration of Natural IgM Production from Liver B Cells by Exogenous IL-18 Improves the Survival of Burn-Injured Mice Infected with Pseudomonas aeruginosa

Pseudomonas aeruginosa is the most common bacterium of postburn infection. In the present study we investigated the immune mechanism of susceptibility to this type of postburn infection and also examined the efficacy of IL-18 treatment. C57BL/6 mice were challenged with P. aeruginosa on day 7 after burn injury. Although the burn-injured mice showed a poor survival rate after bacterial challenge, they retained their IFN-gamma production. The burned mice showed lower serum IgM levels and a poor IgM response following P. aeruginosa challenge in comparison with the sham mice, whereas IL-18 treatment after burn injury (alternate day injections for 1 wk) greatly improved the serum IgM levels, which are P. aeruginosa-independent natural IgM before bacterial challenge, thereby increasing the survival rate after the challenge. IL-18 treatment also induced specific IgM to P. aeruginosa in the sera 5 days after bacterial challenge in the burned mice. Interestingly, CD43(+)CD5(-)CD23(-)B220(dim) cells, namely B-1b cells, increased in the liver after the IL-18 treatment and were found to actively produce IgM in vitro without any additional stimulation. Furthermore, the IL-18 treatment up-regulated the neutrophil count and the C3a levels in the blood as a result of the increased IgM level, which may thus play a critical role in the opsonization and elimination of any invading bacteria. IL-18 treatment for the burned mice and their resultant natural IgM production were thus found to strengthen the host defense against P. aeruginosa infection.

Origin of CD5+ B cells and natural IgM-secreting cells: reconstitution potential of adult bone marrow, spleen and peritoneal cells.

The bulk of natural IgM secretion is currently attributed to peritoneal CD5+ B cells and their progeny, believed to be independent of adult bone marrow precursors. We have compared the capacity of peritoneal or splenic cells from normal adult mice to generate serum IgM after transfer into allotype-congenic, irradiated and bone marrow-protected mice. Recipients of either cell population produced donor-allotype IgM-secreting cells in the spleen, and had donor-derived serum IgM. In both cases as well, recipient IgM secretion recovered to control levels. Since the spleen cell-derived natural IgM production could result from expansion of CD5+ B cells present in the inoculum, we next investigated the ability of Ig- bone marrow (BM) cells (Ig- BM) to reconstitute natural IgM secretion in irradiated mice. This cell population was most efficient in reconstituting donor-derived IgM secretion. The origin and phenotype (IgM, CD5) of B cells present in spleen and peritoneum of recipient mice were also analyzed. In agreement with the high level of donor IgM-secreting cells, transfers of splenic and Ig- BM cells fully reconstitute donor B cells in spleen and peritoneum and inhibit reconstitution from host origin. In contrast, donor peritoneal cells reconstitute B cells very poorly in spleen and allow for reconstitution by host cells. Furthermore, Ig- BM cells as well as splenic or peritoneal donor cells, all reconstitute CD5+ B cells in the peritoneum of recipient mice. Interestingly, the fraction of IgM+ cells of each allotype that differentiate to IgM secretion varies widely, but normal levels of IgM are established even when the number of donor B cells present in the animal is very limited.