Meng Fu

Novel functions of murine B1 cells: Active phagocytic and microbicidal abilities

B1 cells are evolutionarily conserved innate‐like cells that share many features with macrophages. It has also been established that B1 cells have a close developmental relationship with macrophages. However, whether B1 cells are able to act as professional phagocytic cells is not clear. In this study, we report that mouse peritoneal cavity (PerC) B cells demonstrate in vivo and in vitro phagocytic activities for Staphylococcus aureus, Escherichia coli, and polystyrene fluorescent microspheres. Approximately 5% of PerC B cells, mainly B1b cells, showed phagocytic activity. Ingested microbes were killed efficiently in the phagolysosome. The antigen‐specific B‐cell antigen receptor promoted B‐cell phagocytosis, resulting in antigen presentation to T cells after uptake of bacteria. Our results reveal for the first time that mouse B1 cells have active phagocytic capabilities and thereby act as a bridge linking innate and adaptive immunity.

Identification of poly-reactive natural IgM antibody that recognizes late apoptotic cells and promotes phagocytosis of the cells

AbstractNatural IgM can recognize apoptotic cells, but the molecular structure and the role in macrophage phagocytosis of apoptotic cells remain unclear. Objectives(1) To examine the binding of previously isolated natural IgM (3B4) to apoptotic cells and its effects on phagocytosis of apoptotic cells. (2) To characterize the molecular structure of 3B4. Methods:3B4 binding to apoptotic thymocytes was examined by flow cytometry. Polyreactivity of 3B4 was assayed by ELISA. PKH26-labeled Macrophages were incubated with PKH67-stained apoptotic cells in the presence of 3B4. Macrophages phagocytosis of apoptotic cell was evaluated by flow cytometry. The DNA segments of 3B VH and VK were sequenced and analyzed. Results:3B4 IgM recognized late apoptotic cells. Polyreactive-recognitions of lysophosphatidylcholine (LPC) as well as some autoantigens were observed in 3B4. Phagocytosis of late apoptotic cells was increased in the presence of 3B4. The VH and VK genes of 3B4 showed a germline gene context, while N-sequences and nucleotide loss were observed in CDR3. Conclusion: 3B4 promotes macrophage phagocytosis of late apoptotic cells in a complement-independent process. 3B4 has a germline configuration and is possibly ligand-selected. Out experiments suggest an independent role of natural IgM as opsonin in clearance of late apoptotic cells.

Recovered Patients with Stevens–Johson Syndrome and Toxic Epidermal Necrolysis Maintain Long-Lived IFN-γ and sFasL Memory Response

There is evidence that drug-specific T cells are involved in inducing keratinocyte apoptosis in acute stage of Steven-Johson syndrome (SJS) and Toxic epidermal necrolysis (TEN). However, there are few studies that have attempted to examine T cell memory responses over time. We sought to determine the duration of IFN-γ and sFasL memory response to causal drugs in patients with SJS and TEN after remission. Eight patients with previous SJS and TEN were enrolled. Memory T cells were measured by 10-day cultured IFN-γ enzyme-linked immunosorbent spot-forming cell (ELISpot) assay. Effector T-cell responses were measured by ex vivo IFN-γ ELISpot assay and sFasL ELISA. The sFasL-mediated toxicities of drug-stimulated PBMC supernatants against keratinocyte line were further investigated by MTT proliferation assay and Annexin-V staining. We observed significant cultured and ex vivo IFN-γ ELISpot responses against causal drugs in all 8 patients. In addition, the sFasL levels were specifically increased in the supernatant of PBMCs cultured with causal drugs from 6 of 8 patients. Drug-stimulated PBMC supernatants were cytotoxic against keratinocyte line, which was inhibited by anti-FasL mAb in a dose-dependent manner. Our findings confirmed that drug-specific IFN-γ and sFasL memory response against causal drugs could be sustained over several years and further suggest that patients should avoid causal drug re-exposure after the recovery of TEN and SJS.

Host defence against C. albicans infections in IgH transgenic mice with VH derived from a natural anti-keratin antibody

Fungal infections have been increasing and life‐threatening in recent years, but host immune responses, especially the humoral immunity, to fungi have not been fully understood. In the present study, we report that natural antibodies from unimmunized mice bind to Candida albicans. We established a monoclonal natural antibody, 3B4, which recognized a surface antigen located at germ tubes of C. albicans. The 3B4 antibody protected mice from C. albicans‐induced death in passive immunization, by mechanisms involving suppressing germ tube formation and modulating phagocytosis. Interestingly, 3B4 also bound to a self‐antigen keratin. To further study the generation and anti‐C. albicans activities of natural antibodies in vivo, we constructed a μ chain transgenic mouse (TgVH3B4) using the VH gene from 3B4. TgVH3B4 had elevated serum anti‐keratin/C. albicans IgM, and were resistant to C. albicans infections. Analyses of B cell development showed that in TgVH3B4, B cells secreting the anti‐keratin/C. albicans antibodies were enriched in the B1 B cell compartment. Our findings reveal an important role of keratin‐reactive natural antibodies in anti‐C. albicans immune responses, and suggest that keratin may function in selecting B cells into the B1 B cell compartment, where natural antibodies are made to fight fungal infections.

The influence of BCR density on the differentiation of natural poly-reactive B cells begins at an early stage of B cell development

B cell antigen receptor (BCR) density plays a role in the differentiation of immature B cells to their mature compartments; however, the exact strategy of its influence on the development of natural autoreactive B cells is still unclear. In the present study, we explored the role of BCR surface density in autoreactive B cell development by studying two lines of mice containing distinct copy numbers of an IgH transgene with V(H) derived from a poly-reactive natural antibody 3B4. Surface BCR levels were found to be related to the transgene copy number in these mice. In mice with higher copy numbers of the transgene, the BCRs were found to promote the remaining of autoreactive B cells into marginal zone (MZ) and B-1a subsets; meanwhile, elevated surface BCR levels were correlated with a significant decrease of follicular (Fo) B cell numbers and a reduction in the number of multiple stages of immature B cells both in spleen and bone marrow (BM). Interestingly, no difference in the ratio of cell apoptosis and proliferation was found in all stages of B cell development between two lines, except that more severely aberrant proliferation of pro/pre-B cells in BM was found in mice with higher transgene copies. This data supports the idea that natural poly-reactive B cells can be positively selected into MZ and B-1 cells, and high BCR surface density favors this selection. More importantly, our data suggests that the influence by receptor expression on the differentiation of natural poly-reactive B cells begins at an early stage of B cell development.

Clinicopathological and Ultrastructural Study of Multiple Lobular Capillary Hemangioma after Scalding

We herein report 2 cases of multiple lobular capillary hemangiomas after scalding. The patients exhibited papules and nodules on the scalded areas after healing. Histopathological examination of the lesions showed capillary proliferation in the upper dermis with edematous stroma containing inflammatory infiltrates predominantly composed of neutrophils. Biopsy tissue and secretion specimens from lesions of case 1 were cultured for bacteria, and both grew Enterobacter cloacae. Ultrastructural examination revealed features typical of a lobular capillary hemangioma and viral inclusion bodies in the epidermis of case 1. Multiple lobular capillary hemangiomas after scalding are rarely reported. Trauma may play an important role in the development of this rare condition. Accumulation of similar cases and its precise observation is needed to confirm the associations and to establish an etiological link between the disease and the pathogens.

Activation of Langerhans cells promotes the inflammation in imiquimod-induced psoriasis-like dermatitis

BACKGROUND Langerhans cells (LCs) are epidermis-resident dendritic cells that sense and mediate stimuli from skin and outside world, and participate in various skin diseases, playing either pro-inflammatory or regulatory roles. However, the exact function of LCs in the pathogenesis of psoriasis remains unclear, and the conclusions of previous studies are controversial. OBJECTIVES To explore the role of LCs in mouse model of imiquimod (IMQ)-induced psoriasis-like dermatitis using langerin-diphtheria toxin A (DTA) mice that are constitutively deficient in LCs. METHODS IMQ (Aldara) was painted on the skin of mice to produce psoriasis-like dermatitis, and inflammation was evaluated by gross ear thickness, histopathology, flow cytometry and cytokine production. Bone marrow transplantation and fluorescein isothiocyanate tracing were applied to access the migration of LCs. RESULTS The severity of IMQ-induced dermatitis in langerin-DTA mice was significantly lower than that of wild-type mice, as evidenced by decreased level of ear thickness, inflammatory cell infiltration (γδ T cells and neutrophils) and inflammatory cytokine expression (IL-17, IL-22, IL-23 and tumor necrosis factor-α). After application with IMQ, LCs expanded in epidermis and showed increased expression of CD80 and CD86, and migrated to draining lymph node within 48h. LCs in the lymph node 48h after application with IMQ expressed increased level of CD80, CD86, CD40 and CC chemokine receptor 7. CONCLUSION LCs were activated upon application with IMQ, and promoted the inflammatory responses in psoriasis-like dermatitis.

Plexin-B1 and semaphorin 4D cooperate to promote cutaneous squamous cell carcinoma cell proliferation, migration and invasion.

BACKGROUND OBJECTIVES Semaphorin 4D (Sema4D) and its receptor, Plexin-B1, are involved in the pathogenesis of squamous cell carcinoma (SCC) by mediating angiogenesis or perineural invasion through the interaction between Sema4D expression on SCC cells and Plexin-B1 expression on endothelial cells or nerves. Plexin-B1 was also recently found to be expressed on SCC cells. Plexin-B1 expression on several types of tumor cells could mediate various, and occasionally opposing, effects, including tumor cell survival, proliferation, angiogenesis, invasion, and metastasis. However, whether Sema4D exerts paracrine or autocrine effects on SCC via Plexin-B1 remains unclear. OBJECTIVES The aim of this study is to explore the effects of Sema4D/Plexin-B1 interaction on SCC via Plexin-B1 expressed on the tumor cells. METHODS In the present study, we detected the expression of Plexin-B1 and Sema4D in cutaneous SCC (cSCC) tissues and in the cSCC cell line A431 and analyzed the effects of the Sema4D/Plexin-B1 interaction on cSCC cell proliferation, migration, and invasion, as well as on the signaling pathway downstream of Plexin-B1. RESULTS We observed significantly increased Plexin-B1 and Sema4D expression in keratinocytes in cSCC lesions and in A431 cells compared with that in normal skin tissue and in non-malignant keratinocytes. Plexin-B1 silencing reduced the growth, proliferation, migration, and invasion of A431 cells and inhibited the phosphorylation of Akt and extracellular signal-regulated protein kinase (Erk). Soluble recombinant Sema4D promoted the growth, proliferation, migration, and invasion of A431 cells; Akt and Erk phosphorylation is also involved in these processes with a Plexin-B1 dependent manner. CONCLUSION Plexin-B1 induces cSCC cell proliferation, migration, and invasion by interacting with Sema4D. Plexin-B1 might serve as a useful biomarker and/or as a novel therapeutic target for cSCC.

CD100–Plexin-B2 Promotes the Inflammation in Psoriasis by Activating NF-κB and the Inflammasome in Keratinocytes

PlxnB2 and its ligand, CD100, were originally identified as axon-guidance molecules that function during neuronal development; however, studies also showed that CD100-plexins participate in various immune responses. In this study, we found that the expression of PlxnB2 on keratinocytes was specifically increased in lesional skin of psoriasis patients but not atopic dermatitis. Levels of soluble CD100 and membrane-bound CD100 were elevated in sera of psoriasis patients and on keratinocytes of psoriatic skin, respectively. By binding to PlxnB2, soluble CD100 promoted the production of CXCL-1, CCL-20, IL-1β, and IL-18 by keratinocytes and activated the NLRP3 inflammasome. Moreover, CD100-PlxnB2 stimulated the NF-κB signaling pathway in keratinocytes through activation of small GTPase RhoA and Rac1. Our data showed that cooperation of CD100 and PlxnB2 promoted the inflammatory responses in keratinocytes by activating NF-κB and the NLRP3 inflammasome and participated in the pathogenesis of psoriasis. CD100/PlxnB2 might be a potential therapeutic target for psoriasis.

Sema4D is required in both the adaptive and innate immune responses of contact hypersensitivity.

Originally recognized as a regulator of axon guidance in the nervous system, Semaphorin 4D (Sema4D, CD100) also participates in various immune responses and many immune-related diseases. However, whether Sema4D is involved in the pathogenesis of contact hypersensitivity (CHS) remains unclear. In this study, we explored the role of Sema4D in oxazolone-induced CHS using Sema4D knockout (KO) mice. We found that Sema4D KO mice developed attenuated CHS responses, as indicated by milder ear-swelling, lower expression of IL-1β, IL-6, CXCL2 and CXCL5, and decreased recruitment of neutrophils, CD8+ T cells and CD4+ T cells. CHS was impaired in the wide type (WT) mice reconstituted with bone marrow from Sema4D KO mice, indicating that deletion of Sema4D gene in hematopoietic cells played a key role in the alleviated CHS in Sema4D KO mice. CHS was also attenuated in the WT mice transferred with draining lymph nodes (dLNs) cells from oxazolone-sensitized Sema4D KO mice, and the activation and differentiation of hapten-specific CD8+ T cells were impaired in Sema4D KO mice. Furthermore, Sema4D KO mice expressed less IL-1β and CXCL2 than WT mice after oxazolone sensitization, and after transferred with dLNs cells from oxazolone-sensitized WT mice, naïve Sema4D KO mice showed attenuated CHS responses upon oxazolone challenge, indicating that the innate immune response of CHS in Sema4D KO mice was also abrogated. Taken together, our findings revealed for the first time that Sema4D positively regulated both the adaptive and innate immune responses in CHS.