ß-Glucan of Candida albicans Cell Wall Extract Inhibits Salmonella Typhimurium Colonization by Potentiating Cellular Immunity (CD8 + and CD4 + T Cells).

INTRODUCTION: Antimicrobial resistance has been reported in the drugs used for the treatment of typhoid fever. The immunomodulatory substance ß-glucan can be used as an alternative therapy as it potentiates host immunity. The aims of this study are to observe the effect of Candida albicans cell wall (CCW) extract towards host immunity (TCD8+ and TCD4+ cells in spleen, intestinal sIgA) and its capacity to kill Salmonella in the intestine and liver of typhoid fever mice models. METHODS: Typhoid fever mice models were created by infecting mice with S. Typhimurium orally. Mice were divided into four groups: the Non-Infected, Infected, CCW (infected mice treated with 300 µg CCW extract/mouse once a day), and Ciprofloxacin groups (infected mice treated with 15 mg/kg BW ciprofloxacin twice a day). RESULTS: Secretory IgA (sIgA) concentrations of mice in the CCW group remained unchanged. However, their TCD4+ and TCD8+ cells increased substantially compared to those in the Non-Infected group. In the Ciprofloxacin group, sIgA concentrations increased markedly compared to those in the Non-Infected and CCW groups; TCD4+ and TCD8+ cells also increased significantly compared to those in the Infected Group, but not significant compared to those in the CCW group. Colonization of S. Typhimurium in the intestine and liver decreased significantly in the CCW and Ciprofloxacin groups compared to that in the Infected group, with the lowest reduction being found in the Ciprofloxacin group. CONCLUSIONS: The inhibition of S. Typhimurium colonization by CCW is associated with the increase in TCD4+ and TCD8+ cells.

Involvement of TNF-Producing CD8+ Effector Memory T Cells with Immunopathogenesis of Erythema Nodosum Leprosum in Leprosy Patients.

Type 2 reaction (T2R) or erythema nodosum leprosum (ENL), a sudden episode of acute inflammation predominantly affecting lepromatous leprosy patients (LL), characterized by a reduced cellular immune response. This possibly indicates a close relationship between the onset of T2R and the altered frequency, and functional activity of T lymphocytes, particularly of memory subsets. This study performed ex vivo and in vitro characterizations of T cell blood subpopulations from LL patients with or without T2R. In addition, the evaluation of activity of these subpopulations was performed by analyzing the frequency of these cells producing IFN-γ, TNF, and IL-10 by flow cytometry. Furthermore, the expression of transcription factors, for the differentiation of T cells, were analyzed by quantitative real-time polymerase chain reaction. Our results showed an increased frequency of CD8+/TNF+ effector memory T cells (TEM) among T2Rs. Moreover, there was evidence of a reduced frequency of CD4 and CD8+ IFN-γ-producing cells in T2R, and a reduced expression of STAT4 and TBX21. Finally, a significant and positive correlation between bacteriological index (BI) of T2R patients and CD4+/TNF+ and CD4+/IFN-γ+ T cells was observed. Thus, negative correlation between BI and the frequency of CD4+/IL-10+ T cells was noted. These results suggest that CD8+/TNF+ TEM are primarily responsible for the transient alteration in the immune response to Mycobacterium leprae in ENL patients. Thus, our study improves our understanding of pathogenic mechanisms and might suggest new therapeutic approaches for leprosy.

PD-1/PD-L1 Pathway Modulates Macrophage Susceptibility to Mycobacterium tuberculosis Specific CD8+ T cell Induced Death.

CD8+T cells contribute to tuberculosis (TB) infection control by inducing death of infected macrophages. Mycobacterium tuberculosis (Mtb) infection is associated with increased PD-1/PD-L1 expression and alternative activation of macrophages. We aimed to study the role of PD-1 pathway and macrophage polarization on Mtb-specific CD8+T cell-induced macrophage death. We observed that both PD-L1 on CD14+ cells and PD-1 on CD8+T cells were highly expressed at the site of infection in pleurisy TB patients' effusion samples (PEMC). Moreover, a significant increase in CD8+T cells' Mtb-specific degranulation from TB-PEMC vs. TB-PBMC was observed, which correlated with PD-1 and PDL-1 expression. In an in vitro model, M1 macrophages were more susceptible to Mtb-specific CD8+T cells' cytotoxicity compared to M2a macrophages and involved the transfer of cytolytic effector molecules from CD8+T lymphocytes to target cells. Additionally, PD-L1 blocking significantly increased the in vitro Ag-specific CD8+T cell cytotoxicity against IFN-γ-activated macrophages but had no effect over cytotoxicity on IL-4 or IL-10-activated macrophages. Interestingly, PD-L1 blocking enhanced Mtb-specific CD8+ T cell killing of CD14+ cells from human tuberculous pleural effusion samples. Our data indicate that PD-1/PD-L1 pathway modulates antigen-specific cytotoxicity against M1 targets in-vitro and encourage the exploration of checkpoint blockade as new adjuvant for TB therapies.

Β-Glucan of candida albicans cell wall extract inhibits salmonella typhimurium colonization by potentiating cellular immunity (cd8 + and cd4 + t cells)

Abstract INTRODUCTION: Antimicrobial resistance has been reported in the drugs used for the treatment of typhoid fever. The immunomodulatory substance β-glucan can be used as an alternative therapy as it potentiates host immunity. The aims of this study are to observe the effect of Candida albicans cell wall (CCW) extract towards host immunity (TCD8+ and TCD4+ cells in spleen, intestinal sIgA) and its capacity to kill Salmonella in the intestine and liver of typhoid fever mice models. METHODS: Typhoid fever mice models were created by infecting mice with S. Typhimurium orally. Mice were divided into four groups: the Non-Infected, Infected, CCW (infected mice treated with 300 µg CCW extract/mouse once a day), and Ciprofloxacin groups (infected mice treated with 15 mg/kg BW ciprofloxacin twice a day). RESULTS: Secretory IgA (sIgA) concentrations of mice in the CCW group remained unchanged. However, their TCD4+ and TCD8+ cells increased substantially compared to those in the Non-Infected group. In the Ciprofloxacin group, sIgA concentrations increased markedly compared to those in the Non-Infected and CCW groups; TCD4+ and TCD8+ cells also increased significantly compared to those in the Infected Group, but not significant compared to those in the CCW group. Colonization of S. Typhimurium in the intestine and liver decreased significantly in the CCW and Ciprofloxacin groups compared to that in the Infected group, with the lowest reduction being found in the Ciprofloxacin group. CONCLUSIONS The inhibition of S. Typhimurium colonization by CCW is associated with the increase in TCD4+ and TCD8+ cells.

BCG constitutively expressing the adenylyl cyclase encoded by Rv2212 increases its immunogenicity and reduces replication of M. tuberculosis in lungs of BALB/c mice.

Mycobacterium tuberculosis remains as a threat to public health around the world with 1.7 million cases of TB-associated deaths during 2016. Despite the use of Bacillus Calmette-Guerin (BCG) vaccine, control of the infection has not been successful. Because of this, several efforts have been made in order to develop new vaccines capable of boosting previous immunization or attempted for replacing current BCG. We previously showed that over expression of the M. tuberculosis adenylyl cyclase encoding gene Rv2212 in BCG bacilli (BCG-Rv2212), induced an attenuated phenotype when administered in BALB/c mice. Moreover, two-dimensional proteomic analysis showed that heat shock proteins such as GroEL2 and DnaK were overexpressed in this BCG-Rv2212. In this report, we show that immunization of mice with BCG-Rv2212 significantly increments IFN-γ+ CD4+ and CD8+ T-lymphocytes after PPD stimulation in comparison with BCG vaccinated mice. Mice vaccinated with BCG-Rv2212 significantly reduced the bacterial load in lungs after four-month post infection with M. tuberculosis H37Rv but was similar to BCG after 6 month-post-challenge. Survival experiment showed that both vaccines administered separately in mice induce similar levels of protection after 20-week post-challenge with M. tuberculosis H37Rv. Virulence experiments developed in nude mice, showed that BCG-Rv2212 and BCG bacilli were equally safe. Our results suggest that BCG-Rv2212 is capable of stimulating cellular immune response effectively and reduce bacterial burden in lungs of mice after challenge. Particularly, it seems to be more effective in controlling bacterial burden during the first steps of infection.

Immunoproteasome Subunits Are Required for CD8+ T Cell Function and Host Resistance to Brucella abortus Infection in Mice.

The immunoproteasome is a specific proteasome isoform composed of three subunits, termed ß1i, ß2i, and ß5i. Its proteolytic activity enhances the quantity and quality of peptides to be presented by major histocompatibility complex class I (MHC-I) molecules to CD8+ T cells. However, the role of the combined deficiency of the three immunoproteasome subunits in protective immunity against bacterial pathogens has not been investigated. In this study, we addressed the role of the immunoproteasome during infection by Brucella abortus, an intracellular bacterium that requires CD8+ T cell responses for the control of infection. Here, we demonstrate that immunoproteasome triple-knockout (TKO) mice were more susceptible to Brucella infection. This observed susceptibility was accompanied by reduced interferon gamma (IFN-γ) production by mouse CD4+ and CD8+ T lymphocytes. Moreover, the absence of the immunoproteasome had an impact on MHC-I surface expression and antigen presentation by dendritic cells. CD8+ T cell function, which plays a pivotal role in B. abortus immunity, also presented a partial impairment of granzyme B expression and, consequently, reduced cytotoxic activity. In conclusion, these results strongly suggest that immunoproteasome subunits are important components in host resistance to B. abortus infection by impacting both the magnitude and quality of CD8+ T cell responses.

Inhibition of MHC-I by Brucella abortus is an early event during infection and involves EGFR pathway.

Brucella abortus is able to persist inside the host despite the development of potent CD8+ T-cell responses. We have recently reported the ability of B. abortus to inhibit the interferon-γ-induced major histocompatibility complex (MHC)-I cell surface expression on human monocytes. This phenomenon was due to the B. abortus-mediated retention of MHC-I molecules within the Golgi apparatus and was dependent on bacterial viability. However, the implications of bacterial virulence or replicative capacity and the signaling pathways remained unknown. Here we demonstrated that the B. abortus mutant strains RB51 and virB10- are able to inhibit MHC-I expression in the same manner as wild-type B. abortus, even though they are unable to persist inside human monocytes for a long period of time. Consistent with this, the phenomenon was triggered early in time and could be observed at 8 h postinfection. At 24 and 48 h, it was even stronger. Regarding the signaling pathway, targeting epidermal growth factor (EGF) receptor (EGFR), ErbB2 (HER2) or inhibition of tumor necrosis factor-α-converting enzyme, one of the enzymes which generates soluble EGF-like ligands, resulted in partial recovery of MHC-I surface expression. Moreover, recombinant EGF and transforming growth factor-α as well as the combination of both were also able to reproduce the B. abortus-induced MHC-I downmodulation. Finally, when infection was performed in the presence of an extracellular signal-regulated kinase 1/2 (Erk1/2) inhibitor, MHC-I surface expression was significantly recovered. Overall, these results describe how B. abortus evades CD8+ T-cell responses early during infection and exploits the EGFR-ERK signaling pathway to escape from the immune system and favor chronicity.

CD11c(+) CD103(+) cells of Mycobacterium tuberculosis-infected C57BL/6 but not of BALB/c mice induce a high frequency of interferon-γ- or interleukin-17-producing CD4(+) cells.

The magnitude of the cellular adaptive immune response is critical for the control of Mycobacterium tuberculosis infection in the chronic phase. In addition, the genetic background is equally important for resistance or susceptibility to tuberculosis. In this study, we addressed whether lung populations of dendritic cells, obtained from genetically different hosts, would play a role in the magnitude and function of CD4(+) populations generated after M. tuberculosis infection. Thirty days post-infection, C57BL/6 mice, which generate a stronger interferon-γ (IFN-γ)-mediated immune response than BALB/c mice, exhibited a higher number and frequency of lung CD11c(+)  CD11b(-)  CD103(+) cells compared with BALB/c mice, which exhibited a high frequency of lung CD11c(+)  CD11b(+)  CD103(-) cells. CD11c(+)  CD11b(-)  CD103(+) cells, purified from lungs of infected C57BL/6 mice, but not from infected BALB/c mice, induced a higher frequency of IFN-γ-producing or interleukin-17 (IL-17)-producing CD4(+) cells. Moreover, CD4(+) cells also arrive at the lung of C57BL/6 mice faster than in BALB/c mice. This pattern of immune response seems to be associated with higher gene expression for CCL4, CCL19, CCL20 and CCR5 in the lungs of infected C57BL/6 mice compared with infected BALB/c mice. The results described here show that the magnitude of IFN-γ-producing or IL-17-producing CD4(+) cells is dependent on CD11c(+)  CD11b(-)  CD103(+) cells, and this pattern of immune response is directly associated with the host genetic background. Therefore, differences in the genetic background contribute to the identification of immunological biomarkers that can be used to design human assays to predict progression of M. tuberculosis infection.

Expression of regulatory T cells in jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum.

Using flow cytometry, we evaluated the frequencies of CD4(+) and CD8(+) T cells and Foxp3(+) regulatory T cells (Tregs) in mononuclear cells in the jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum and in uninfected controls. All infected dogs showed chronic lymphadenitis and enteritis. Despite persistent parasite loads, no erosion or ulcers were evident in the epithelial mucosa. The colon harbored more parasites than the jejunum. Frequencies of total CD4(+), total Foxp3, and CD4(+) Foxp3(+) cells were higher in the jejunum than in the colon. Despite negative enzyme-linked immunosorbent assay (ELISA) serum results for cytokines, levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), transforming growth factor beta (TGF-ß), and tumor necrosis factor alpha (TNF-α) were higher in the jejunum than in the colon for infected dogs. However, IL-4 levels were higher in the colon than in the jejunum for infected dogs. There was no observed correlation between clinical signs and histopathological changes or immunological and parasitological findings in the gastrointestinal tract (GIT) of canines with visceral leishmaniasis. However, distinct segments of the GIT presented different immunological and parasitological responses. The jejunum showed a lower parasite load, with increased frequencies and expression of CD4, Foxp3, and CD8 receptors and IL-10, TGF-ß, IFN-γ, and TNF-α cytokines. The colon showed a higher parasite load, with increasing expression of IL-4. Leishmania infantum infection increased expression of CD4, Foxp3, IL-10, TGF-ß, IFN-γ, and TNF-α and reduced CD8 and IL-4 expression in both the jejunum and the colon.

Induction of cell-mediated immunity to Staphylococcus aureus in the mouse mammary gland by local immunization with a live attenuated mutant.

The efficacy of intramammary (Ima) immunization with a live attenuated (la) Staphylococcus aureus mutant to protect the mouse mammary gland from infection has previously been established. The present study was aimed at evaluating whether Ima immunization with la-S. aureus can induce cell-mediated immune responses to the pathogen within the mammary gland. Mice were immunized by Ima route with la-S. aureus, and regional lymph node mononuclear cells were obtained thereafter. A higher expression of the interleukin-2 receptor was found on B and T cells from immunized mice when they were compared with control mice. Immunization with la-S. aureus induced strong proliferative responses to S. aureus. Moreover, significantly increased levels of gamma interferon (IFN-gamma) were produced by CD4+ T cells when lymphocytes from immunized mice, but not from control mice, were cultured in the presence of staphylococcal antigens. Moreover, a significant increase in the percentage of IFN-gamma-producing CD4+ and CD8+ T cells was observed after S. aureus Ima challenge in immunized mice compared to challenged control mice. Our results demonstrated that Ima immunization with la-S. aureus induced primed lymphocyte populations capable of responding against staphylococcal antigens during in vitro stimulation, as well as during in vivo infection by S. aureus. CD4+ and CD8+ T cells appear to be the main lymphocyte subpopulations involved in this response. It is suggested that IFN-gamma production induced by Ima immunization may play a pivotal role in the eradication of intracellular staphylococci.