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Dendritic cells form the link between the innate and adaptative immune response, particularly on mucosal and epidermal surfaces. The Langerhans, an epidermal dendritic cell subpopulation, play a key role in the skin immune response across several species. Scarse immune cell subpopulations, including Langerhans-like cells, have been identified in endangered green turtles thereby complicating the understanding of the pathogenesis of diseases such as fibropapillomatosis, which induces skin tumours in this species worldwide. In biopsies from green turtle skin, we demonstrated that the polyclonal anti-human Langerin antibodies strongly stained a Langerin+ cell population in epidermal sheets, the suprabasal layer of the epidermis in cryosections and in cells from cytospin preparation of migration assays. The morphology of these cells was round to amoeboid in normal skin; however, in skin with ulcerative dermatitis, Langerin+ cells aggregated around ulcers and adopted a more pleomorphic morphology. To our knowledge, this is the first identification of Langerin+ cells with a molecular marker in a reptile species., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG + PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM + PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67 + ) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Raúl A. Maqueda-Alfaro et al.)

Antigen capturing at the periphery is one of the earliest, crucial functions of antigen-presenting cells (APCs) to initiate immune responses. Langerhans cells (LCs), the epidermal APCs migrate to draining lymph nodes (DLNs) upon acquiring antigens. An arsenal of endocytic molecules is available to this end, including lectins and pathogen recognition receptors (PRRs). However, cutaneous LCs are poorly defined in the early neonatal period. We assessed endocytic molecules expression in situ : Mannose (CD206)-, Scavenger (SRA/CD204)-, Complement (CD2l, CDllb)-, and Fc-Receptors (CD16/32, CD23) as well as CD1d, CD14, CD205, Langerin (CD207), MHCII, and TLR4 in unperturbed epidermal LCs from both adult and early neonatal mice. As most of these markers were negative at birth (day 0), LC presence was revealed with the conspicuous, epidermal LC-restricted ADPase (and confirmed with CD45) staining detecting that they were as numerous as adult ones. Unexpectedly, most LCs at day 0 expressed CD14 and CD204 while very few were MHCII+ and TLR4+. In contrast, adult LCs lacked all these markers except Langerin, CD205, CD11b, MHCII and TLR4. Intriguingly, the CD204+ and CD14+ LCs predominant at day 0, apparently disappeared by day 4. Upon cutaneous FITC application, LCs were reduced in the skin and a CD204+MHCII+FITC+ population with high levels of CD86 subsequently appeared in DLNs, with a concomitant increased percentage of CD3+CD69+ T cells, strongly suggesting that neonatal LCs were able both to ferry the cutaneous antigen into DLNs and to activate neonatal T cells in vivo . Cell cycle analysis indicated that neonatal T cells in DLNs responded with proliferation. Our study reveals that epidermal LCs are present at birth, but their repertoire of endocytic molecules and PRRs differs to that of adult ones. We believe this to be the first description of CDl4, CD204 and TLR4 in neonatal epidermal LCs in situ . Newborns' LCs express molecules to detect antigens during early postnatal periods, are able to take up local antigens and to ferry them into DLNs conveying the information to responsive neonatal T cells., (Copyright © 2020 Becerril-García, Yam-Puc, Maqueda-Alfaro, Beristain-Covarrubias, Heras-Chavarría, Gallegos-Hernández, Calderón-Amador, Munguía-Fuentes, Donis-Maturano, Flores-Langarica and Flores-Romo.)

Membrane blebs are released from Gram-negative bacteria, however, little is known about Brucella blebs. This work pursued two objectives, the first was to determine and identify the proteins in the membrane blebs by proteomics and in silico analysis. The second aim was to evaluate the use of membrane blebs of Brucella abortus 2308 and B. abortus RB51 as an acellular vaccine in vivo and in vitro . To achieve these aims, membrane blebs from B. abortus 2308 and RB51 were obtained and then analyzed by liquid chromatography coupled to mass spectrometry. Brucella membrane blebs were used as a "vaccine" to induce an immune response in BALB/c mice, using the strain B. abortus RB51 as a positive vaccine control. After subsequent challenge with B. abortus 2308, CFUs in spleens were determined; and immunoglobulins IgG1 and IgG2a were measured in murine serum by ELISA. Also, activation and costimulatory molecules induced by membrane blebs were analyzed in splenocytes by flow cytometry. Two hundred and twenty eight proteins were identified in 2308 membrane blebs and 171 in RB51 blebs, some of them are well-known Brucella immunogens such as SodC, Omp2b, Omp2a, Omp10, Omp16, and Omp19. Mice immunized with membrane blebs from rough or smooth B. abortus induced similar protective immune responses as well as the vaccine B. abortus RB51 after the challenge with virulent strain B. abortus 2308 ( P < 0.05). The levels of IgG2a in mice vaccinated with 2308 membrane blebs were higher than those vaccinated with RB51 membrane blebs or B. abortus RB51 post-boosting. Moreover, mice immunized with 2308 blebs increased the percentage of activated B cells (CD19 + CD69 + ) in vitro. Therefore, membrane blebs are potential candidates for the development of an acellular vaccine against brucellosis, especially those derived from the rough strains so that serological diagnostic is not affected., (Copyright © 2019 Araiza-Villanueva, Avila-Calderón, Flores-Romo, Calderón-Amador, Sriranganathan, Qublan, Witonsky, Aguilera-Arreola, Ruiz-Palma, Ruiz, Suárez-Güemes, Gómez-Lunar and Contreras-Rodríguez.)

Ethnopharmacological Relevance: Buddleja cordata Humb. Bonpl. & Kunth, known by the population as Tepozán blanco, is a shrub plant used in traditional herbal medicine in Mexico for the treatment of tumors, cancer, sores, skin burns, rheumatic pains and diseases related to inflammatory processes such as arthritis; authors adjudicate this etno-medicinal effect to the presence of secondary metabolites in the plant such as verbascoside, however due to its low concentration in recent years biotechnological tools are applied as cell culture to biosynthesize these pharmacological active metabolites in greater quantities., Aim of the Study: Evaluate the possible toxic effect after a daily administration of MeOH extracts from wild plant leaves (Bc-Wp), and cell culture (Bc-Cc) of B. cordata for 28 days, and after their anti-edematous and antioxidant activities in vivo, as well their effect on the cytokines profile during experimental arthritis induced by complete Freund's adjuvant., Materials and Methods: Both extracts were evaluated in CD1 male mice first in a toxicity test of repeated dose administrations (1 g/kg) for 28 days, after which pharmacological activity of both extracts was measure during experimental induced arthritis where three doses were tested, at the end of the study edema formation, body weight gain and antioxidant activity were measure in edema and ganglionic tissues. Finally, dose that exerted the best protective effect (250 mg/kg) was evaluated to quantify its effect over ganglionic tissue concentration of lymphocytes T CD4 + , and cytokines (IL-1β, TNF-α and IL-10), as well histological analysis in arthritic mice., Results: Both extracts (Bc-Wp and Bc-Cc) did not exert lethality, however body weight gain and food in-take were lower than in healthy mice administered with vehicles, also extract-treated animals showed a decrease in serum lipid concentration and only Bc-Wp extract treated animals decrease serum alkaline phosphatase after 28 daily administration compared to healthy un-treated mice. During experimental arthritis best inhibition effect over edema development was observed in those animals administered with both extracts at dose of 250 mg/kg (Bc-Wp and Bc-Cc) on day 28, compared to CFA un-treated mice. Also both extracts reduce oxidative damage over lipids and proteins at the same dose, in both ganglionic and edema tissue, as well antioxidant enzymatic response was reduced in both tissues compared to arthritic un-treated group. In ganglionic tissue of arthritic mice, CD4 + lymphocytes concentration was reduced by Bc-Wp and Bc-Cc treatment (250 mg/kg) respectively, as well IL-1β, and TNF-α levels. Only arthritic animals treated with Bc-Cc extract at 250 mg/kg generated a significant increase of IL-10 doubling the levels compared to CFA un-treated group. Histological analysis of popliteal ganglion showed that both extracts decrease the incidence of lytic lesions, lipid inclusions and leukocyte infiltration., Conclusion: Methanolic extracts of wild Buddleja cordata and its cell cultures did not generated lethality after a daily administration for 28 days at 1 g/kg, but it was observed that both showed a lipid-lowering effect. Also at dose of 250 mg/kg both extracts exerted anti-edematous, protection against the oxidation of lipid and proteins, regulation on antioxidant enzymatic response, down-regulation on lymphocytes CD4 + producers of IL-1β and TNF-α, an increase in IL-10 levels, which caused a decrease in leukocyte infiltration in ganglionic tissue during experimental arthritis., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Gram-negative bacteria release outer membrane vesicles (OMVs) into the extracellular environment. OMVs have been studied extensively in bacterial pathogens, however, information related with the composition of Aeromonas hydrophila OMVs is missing. In this study we analyzed the composition of purified OMVs from A. hydrophila ATCC ® 7966 TM by proteomics. Also we studied the effect of OMVs on human peripheral blood mononuclear cells (PBMCs). Vesicles were grown in agar plates and then purified through ultracentrifugation steps. Purified vesicles showed an average diameter of 90-170 nm. Moreover, 211 unique proteins were found in OMVs from A. hydrophila ; some of them are well-known as virulence factors such as: haemolysin Ahh1, RtxA toxin, extracellular lipase, HcpA protein, among others. OMVs from A. hydrophila ATCC ® 7966 TM induced lymphocyte activation and apoptosis in monocytes, as well as over-expression of pro-inflammatory cytokines. This work contributed to the knowledge of the composition of the vesicles of A. hydrophila ATCC ® 7966 TM and their interaction with the host cell.

Lymph nodes (LNs) have evolved to maximize antigen (Ag) collection and presentation as well as lymphocyte proliferation and differentiation-processes that are spatially regulated by stromal cell subsets, including fibroblastic reticular cells (FRCs) and follicular dendritic cells (FDCs). Here, we showed that naïve neonatal mice have poorly organized LNs with few B and T cells and undetectable FDCs, whereas adult LNs have numerous B cells and large FDC networks. Interestingly, immunization on the day of birth accelerated B cell accumulation and T cell recruitment into follicles as well as FDC maturation and FRC organization in neonatal LNs. However, compared to adults, the formation of germinal centers was both delayed and reduced following immunization of neonatal mice. Although immunized neonates poorly expressed activation-induced cytidine deaminase (AID), they were able to produce Ag-specific IgGs, but with lower titers than adults. Interestingly, the Ag-specific IgM response in neonates was similar to that in adults. These results suggest that despite an accelerated structural maturation of LNs in neonates following vaccination, the B cell response is still delayed and reduced in its ability to isotype switch most likely due to poor AID expression. Of note, naïve pups born to Ag-immunized mothers had high titers of Ag-specific IgGs from day 0 (at birth). These transferred antibodies confirm a mother-derived coverage to neonates for Ags to which mothers (and most likely neonates) are exposed, thus protecting the neonates while they produce their own antibodies. Finally, the type of Ag used in this study and the results obtained also indicate that T cell help would be operating at this stage of life. Thus, neonatal immune system might not be intrinsically immature but rather evolutionary adapted to cope with Ags at birth.

Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1 - , CD19 - , CD138 + ) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD - , CD19 + , PNA + ) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220 + , Blimp1 + ) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers.

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen's naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.

Dengue virus (DENV) has four serotypes, which can cause from asymptomatic disease to severe dengue. Heterologous secondary infections have been associated to a greater risk of potentially fatal dengue due to non-neutralizing memory antibodies (Abs), which facilitate the infection, such as anti-precursor membrane (prM) Abs, among other mechanisms. Usually, class-switched memory Abs are generated mainly through germinal centers (GCs). However, the cellular events underlying these Ab responses to DENV, especially during repeated/secondary infections, have been poorly studied. We wanted to know whether there is involvement of GC reactions during cutaneous DENV infection and whether there is any sort of preferential Ab responses to defined viral proteins. Intradermal DENV inoculation at a relatively low dose efficiently infects immune-competent BALB/c mice, inducing higher quantities of DENV-specific GC B cells and larger GCs than the control conditions. Interestingly, GCs exhibited as much prM as envelope (E) and non-structural 3 viral proteins in situ. Intriguingly, despite the much larger abundance of E protein than of prM protein in the virions, infected animals showed similar amounts of circulating Abs and Ag-specific GC B cells both for prM and for E proteins, even significantly higher for prM. To the best of our knowledge, there are no reports of the GC responses during DENV infection. This relatively stronger anti-prM response could be triggered by DENV to preferentially promote Abs against certain viral proteins, which might favor infections by facilitating DENV invasion of host cells. It is thus conceivably that DENV might have evolved to induce this kind of Ab responses.

Neutrophils are one the earliest, crucial innate defenses against innumerable pathogens. Their main microbicidal activities include phagocytosis and degranulation, with many pharmacologically active molecules contributing to inflammation. Recently, a novel antimicrobial mechanism was discovered; the Neutrophil Extracelullar Traps (NETs) formed by extrusion of DNA and associated molecules (histones, elastase, antimicrobial peptides, among others) which trap and kill microorganisms. Since NETs were recently described, research has focused on their induction and microbicidal properties, and recently on disease involvement. However, the functional consequences of NETs interacting with other immune cells, either resident or recruited during early inflammation, have not been assessed. We therefore investigated the consequences of exposing two major APCs, macrophages (Mfs) and conventional Dendritic Cells (cDCs) to NETs. Our data revealed that at early times (30 min), both Antigen Presenting Cells (APCs) showed induction of important costimulatory molecules (CD80, CD86). Unexpectedly, however, at later times (6 and 24 hours) NETs apparently triggered a cell death process in these APCs by a caspase- and Apoptosis induced factor (AIF)-dependent pathway, suggesting mitochondrial damage. By rhodamine-123 labelling we found that in both APCs, relatively prolonged exposure to NETs or their components importantly decreased the mitochondrial membrane potential. Ultrastructural analysis confirmed mitochondrial alterations in both APCs. Our results would suggest that early in inflammation, NETs can activate the two main APCs (Mfs and cDCs), but as the process continues, NETs can then initiate apoptosis of these cells through mitochondrial harm. Conceivable, this "late" induction of cell death in these two APCs might start limiting an ongoing inflammatory process to control it.

The intestinal mucosa is exposed to a vast antigenic contact. Several antigen presenting cell (APCs) have been described within the gut associated lymphoid tissue (GALT) (Peyer's patches, lamina propria, mesenteric lymph nodes, muscular layer); however, this has been done almost exclusively in adult organisms. As there is no characterization of intestinal muscular layer's APCs during early neonate development we adapted the conventional technique used in adults, to the neonate intestine. We obtained the intestinal muscular layer from early neonates (days 0-3 upon birth) and from young mice (2 and 3 weeks after birth). A planar network of CD45(+), MHC-II(+), DEC-205(+) cells with irregular, some with prominent dendritic morphology was found at birth under basal physiological conditions, whereas Langerin(+) DCs appeared after two weeks. The variations seen in CD45(+), MHC-II(+) and DEC-205(+) cells along the early neonatal development, could be related to the new challenges by intestinal antigen exposure from the newborn diet (breast milk, solid food), and to important environmental changes (start walking, exploring the surroundings, etc). Our study reveals the presence of APCs in intestinal muscular layer at birth, and their subsequent changes in physiological, non-induced conditions, contributing basic information about these cells in the neonate intestinal immune system.