RNA-mediated epigenetic regulation in exercised heart: Mechanisms and opportunities for intervention.

Physical exercise has been widely acknowledged as a beneficial lifestyle alteration and a potent non-pharmacological treatment for heart disease. Extensive investigations have revealed the beneficial effects of exercise on the heart and the underlying mechanisms involved. Exercise is considered one of the key factors that can lead to epigenetic alterations. The increasing number of identified molecules in the exercised heart has led to many studies in recent years that have explored the cellular function of ncRNAs and RNA modifications in the heart. Investigating the regulatory role of RNA-mediated epigenetic regulation in exercised hearts will contribute to the development of therapeutic strategies for the management of heart diseases. This review aims to summarize the positive impact of exercise on cardiac health. We will first provide an overview of the mechanisms through which exercise offers protection to the heart. Subsequently, we will delve into the current understanding of ncRNAs, specifically miRNAs, lncRNAs, and circRNAs, as well as RNA modification, focusing on RNA m6A and RNA A-to-I editing, and how they contribute to exercise-induced benefits for the heart. Lastly, we will explore the emerging therapeutic strategies that utilize exercise-mediated RNA epigenetic regulation in the treatment of heart diseases, while also addressing the challenges faced in this field.

Neutralizing IgG4 antibodies are a biomarker of sustained efficacy after peanut oral immunotherapy.

BACKGROUND: Clinical efficacy of oral immunotherapy (OIT) has been associated with the induction of blocking antibodies, particularly those capable of disrupting IgE-allergen interactions. Previously, we identified mAbs to Ara h 2 and structurally characterized their epitopes. OBJECTIVE: We investigated longitudinal changes during OIT in antibody binding to conformational epitopes and correlated the results with isotype and clinical efficacy. METHODS: We developed an indirect inhibitory ELISA using mAbs to block conformational epitopes on immobilized Ara h 2 from binding to serum immunoglobulins from peanut-allergic patients undergoing OIT. We tested the functional blocking ability of mAbs using passive cutaneous anaphylaxis in mice with humanized FcεRI receptors. RESULTS: Diverse serum IgE recognition of Ara h 2 conformational epitopes are similar before and after OIT. Optimal inhibition of serum IgE occurs with the combination of 2 neutralizing mAbs (nAbs) recognizing epitopes 1.2 and 3, compared to 2 nonneutralizing mAbs (non-nAbs). After OIT, IgG4 nAbs, but not IgG1 or IgG2 nAbs, increased in sustained compared to transient outcomes. Induction of IgG4 nAbs occurs after OIT only in those with sustained efficacy. Murine passive cutaneous anaphylaxis after sensitization with pooled human sera is significantly inhibited by nAbs compared to non-nAbs. CONCLUSIONS: Serum IgE conformational epitope diversity remains unchanged during OIT. However, IgG4 nAbs capable of uniquely disrupting IgE-allergen interactions to prevent effector cell activation are selectively induced in OIT-treated individuals with sustained clinical efficacy. Therefore, the induction of neutralizing IgG4 antibodies to Ara h 2 are clinically relevant biomarkers of durable efficacy in OIT.

Estimating nitric oxide (NO) from MDSCs by Griess method.

The functional importance of nitric oxide (NO) in the fields of immunology concerning its antimicrobial, anti-tumoral, anti-inflammatory, and immunosuppressive effects have made it inevitable to study its secretion from various cells. Nitrogen oxide synthase (NOS) is the enzyme responsible for synthesizing NO and its three isoforms function in a cell-dependent manner. NO is oxidized rapidly to Reactive nitrogen oxide species (RNOS) through which the roles of NO are being carried out. One of the major immune cells secreting NO is myeloid-derived suppressor cells (MDSCs). The function of these MDSCs in the suppression of T-cell proliferation as well as T-cell differentiation is found to be dependent on NO secretion. Apart from T-cell suppressive activity, NO is also known to interfere with natural killer (NK) cell functions. A convenient method to estimate NO secretion is by using Griess reagent named after Johann Peter Griess. In this method, NO reacts with the reagents to form a colored azo dye detectable using a microplate reader at a wavelength of 548nm. In this chapter, we summarized the detailed method of estimating NO from MDSCs by the Griess method.

Defining the cross-reactivity between peanut allergens Ara h 2 and Ara h 6 using monoclonal antibodies.

In peanut allergy, Arachis hypogaea 2 (Ara h 2) and Arachis hypogaea 6 (Ara h 6) are two clinically relevant peanut allergens with known structural and sequence homology and demonstrated cross-reactivity. We have previously utilized X-ray crystallography and epitope binning to define the epitopes on Ara h 2. We aimed to quantitatively characterize the cross-reactivity between Ara h 2 and Ara h 6 on a molecular level using human monoclonal antibodies (mAbs) and structural characterization of allergenic epitopes. We utilized mAbs cloned from Ara h 2 positive single B cells isolated from peanut-allergic, oral immunotherapy-treated patients to quantitatively analyze cross-reactivity between recombinant Ara h 2 (rAra h 2) and Ara h 6 (rAra h 6) proteins using biolayer interferometry and indirect inhibitory ELISA. Molecular dynamics simulations assessed time-dependent motions and interactions in the antibody-antigen complexes. Three epitopes-conformational epitopes 1.1 and 3, and the sequential epitope KRELRNL/KRELMNL-are conserved between Ara h 2 and Ara h 6, while two more conformational and three sequential epitopes are not. Overall, mAb affinity was significantly lower to rAra h 6 than it was to rAra h 2. This difference in affinity was primarily due to increased dissociation of the antibodies from rAra h 6, a phenomenon explained by the higher conformational flexibility of the Ara h 6-antibody complexes in comparison to Ara h 2-antibody complexes. Our results further elucidate the cross-reactivity of peanut 2S albumins on a molecular level and support the clinical immunodominance of Ara h 2.

Design of an Ara h 2 hypoallergen from conformational epitopes.

INTRODUCTION: Adverse reactions are relatively common during peanut oral immunotherapy. To reduce the risk to the patient, some researchers have proposed modifying the allergen to reduce IgE reactivity, creating a putative hypoallergen. Analysis of recently cloned human IgG from patients treated with peanut immunotherapy suggested that there are three common conformational epitopes for the major peanut allergen Ara h 2. We sought to test if structural information on these epitopes could indicate mutagenesis targets for designing a hypoallergen and evaluated the reduction in IgE binding via immunochemistry and a mouse model of passive cutaneous anaphylaxis (PCA). METHODS: X-ray crystallography characterized the conformational epitopes in detail, followed by mutational analysis of key residues to modify monoclonal antibody (mAb) and serum IgE binding, assessed by ELISA and biolayer interferometry. A designed Ara h 2 hypoallergen was tested for reduced vascularization in mouse PCA experiments using pooled peanut allergic patient serum. RESULTS: A ternary crystal structure of Ara h 2 in complex with patient antibodies 13T1 and 13T5 was determined. Site-specific mutants were designed that reduced 13T1, 13T5, and 22S1 mAbs binding by orders of magnitude. By combining designed mutations from the three major conformational bins, a hexamutant (Ara h 2 E46R, E89R, E97R, E114R, Q146A, R147E) was created that reduced IgE binding in serum from allergic patients. Further, in the PCA model where mice were primed with peanut allergic patient serum, reactivity upon allergen challenge was significantly decreased using the hexamutant. CONCLUSION: These studies demonstrate that prior knowledge of common conformational epitopes can be used to engineer reduced IgE reactivity, an important first step in hypoallergen design.

Chiral Cell Nanomechanics Originated in Clockwise/Counterclockwise Biofunctional Microarrays to Govern the Nuclear Mechanotransduction of Mesenchymal Stem Cells.

Cell chirality is extremely important for the evolution of cell morphogenesis to manipulate cell performance due to left-right asymmetry. Although chiral micro- and nanoscale biomaterials have been developed to regulate cell functions, how cell chirality affects cell nanomechanics to command nuclear mechanotransduction was ambiguous. In this study, chiral engineered microcircle arrays were prepared by photosensitive cross-linking synthesis on cell culture plates to control the clockwise/counterclockwise geometric topology of stem cells. Asymmetric focal adhesion and cytoskeleton structures could induce chiral cell nanomechanics measured by atomic force microscopy (AFM) nanoindentation in left-/right-handed stem cells. Cell nanomechanics could be enhanced when the construction of mature focal adhesion and the assembly of actin and myosin cytoskeletons were well organized in chiral engineered stem cells. Curvature angles had a negative effect on cell nanomechanics, while cell chirality did not change cytoskeletal mechanics. The biased cytoskeleton tension would engender different nuclear mechanotransductions by yes-associated protein (YAP) evaluation. The chiral stimuli were delivered into the nuclei to oversee nuclear behaviors. A strong cell modulus could activate high nuclear DNA synthesis activity by mechanotransduction. The results will bring the possibility of understanding the interplay of chiral cell nanomechanics and mechanotransduction in nanomedicines and biomaterials.

Administration of soluble gp130Fc disrupts M-1 macrophage polarization, dendritic cell activation, MDSC expansion and Th-17 induction during experimental cerebral malaria.

Regulatory effect of IL-6 on various immune cells plays a crucial role during experimental cerebral malaria pathogenesis. IL-6 neutralization can restore distorted ratios of myeloid dendritic cells and plasmacytoid dendritic cells as well as the balance between Th-17 and T-regulatory cells. IL-6 can also influence immune cells through classical and trans IL-6 signalling pathways. As trans IL-6 signalling is reportedly involved during malaria pathogenesis, we focused on studying the effects of trans IL-6 signalling blockade on various immune cell populations and how they regulate ECM progression. Results show that administration of sgp130Fc recombinant chimera protein lowers the parasitemia, increases the survivability of Plasmodium berghei ANKA infected mice, and restores the distorted ratios of M1/M2 macrophage, mDC/pDC, and Th-17/Treg. IL-6 trans signalling blockade has been found to affect both expansion of myeloid derived suppressor cells (MDSCs) and expression of inflammatory markers on them during Plasmodium berghei ANKA infection indicating that trans IL-6 signalling might regulate various immune cells and their function during ECM. In this work for the first time, we delineate the effect of sgp130Fc administration on influencing the immunological changes within the host secondary lymphoid organ during ECM induced by Plasmodium berghei ANKA infection.

Immunotherapy-induced neutralizing antibodies disrupt allergen binding and sustain allergen tolerance in peanut allergy.

In IgE-mediated food allergies, exposure to the allergen activates systemic allergic responses. Oral immunotherapy (OIT) treats food allergies through incremental increases in oral allergen exposure. However, OIT only induces sustained clinical tolerance and decreased basophil sensitivity in a subset of individuals despite increases in circulating allergen-specific IgG in all treated individuals. Therefore, we examined the allergen-specific antibodies from 2 OIT cohorts of patients with sustained and transient responses. Here, we compared antibodies from individuals with sustained or transient responses and discovered specific tolerance-associated conformational epitopes of the immunodominant allergen Ara h 2 recognized by neutralizing antibodies. First, we identified what we believe to be previously unknown conformational, intrahelical epitopes using x-ray crystallography with recombinant antibodies. We then identified epitopes only recognized in sustained tolerance. Finally, antibodies recognizing tolerance-associated epitopes effectively neutralized allergen to suppress IgE-mediated effector cell activation. Our results demonstrate the molecular basis of antibody-mediated protection in IgE-mediated food allergy, by defining how these antibodies disrupt IgE-allergen interactions to prevent allergic reactions. Our approach to studying the structural and functional basis for neutralizing antibodies demonstrates the clinical relevance of specific antibody clones in antibody-mediated tolerance. We anticipate that our findings will form the foundation for treatments of peanut allergy using neutralizing antibodies and hypoallergens.

CD4+IL9+ (Th9) cells as the major source of IL-9, potentially modulate Th17/Treg mediated host immune response during experimental cerebral malaria.

Th9, a new subgroup of CD4+T cells is characterized by its specific cytokine IL-9, is a critical factor in allergic diseases, cancers and parasitic infections. This study aimed to explore the potential roles of Th9 cells in the immunopathogenesis of ECM. In splenocytes sourced from uninfected, PbA and Py infected mice, Th9 cells were characterised by flow cytometry, cell sorting and qPCR. Enhancement of CD4+IL-9+ (Th9) cells were observed in both the infections, which corroborated with increased expression of the differentiating transcription factors. Moreover, crucial cytokine receptors (IL-4R, TGF-ßR, IL-6R) as well as chemokine receptors (CCR3, CCR6 and CCR7) and activation marker (CD96), demonstrated elevation upon PbA infection in splenic Th9 cells. Furthermore, Neutralization of IL-9 along with IL-6 enhanced host survivability, reduced mean neurological score of ECM. However, anti- IL-9 treatment also down regulated frequency of Th17 cells, and its transcription factors pSTAT3, RORγT along with depleted Il-1ß and Il-6 expression. In sum, understanding how IL-9 producing CD4+ T-cells can alter Th17/Treg ratio and by that modulate host's immune response, could pave the way for developing immunomodulatory interventions against cerebral malaria.

IL-6 dependent expansion of inflammatory MDSCs (CD11b+ Gr-1+) promote Th-17 mediated immune response during experimental cerebral malaria.

Myeloid derived suppressor cells (MDSCs) are a group of heterogeneous cell populations that can suppress T cell responses. Various aspects of MDSCs in regulating immune responses in several cancer and infectious diseases have been reported till date. But the role and regulation of MDSCs have not been systematically studied in the context of malaria. This study depicts the phenotypic and functional characteristics of splenic MDSCs and how they regulate Th-17 mediated immune response during Experimental Cerebral Malaria (ECM). Flow cytometric analysis reveals that MDSCs in the spleen and bone marrow expand at 8 dpi during ECM. Among subtypes of MDSCs, PMN-MDSCs show significant expansion in the spleen but M-MDSCs remain unaltered. Functional analysis of sorted MDSCs from spleens of Plasmodium berghei ANKA (PbA) infected mice shows suppressive nature of these cells and high production of Nitric oxide (NO). Besides, MDSCs were also found to express various inflammatory markers during ECM suggesting the M1 type phenotype of these cells. In-vivo depletion of MDSCs by the use of Anti Gr-1 increases mice survival but doesn't significantly alter the parasitemia. Previously, it has been reported that Treg/Th-17 balance in the spleen is skewed towards Th-17 during ECM. Depletion of MDSCs was found to regulate Th-17 percentages to homeostatic levels and subvert various inflammatory changes in the spleen. Among different factors, IL-6 was found to play an important role in the expansion of MDSCs and expression of inflammatory markers on MDSCs in a STAT3-dependent manner. These findings provide a unique insight into the role of IL-6 in the expansion of the MDSC population which causes inflammatory changes and increased Th-17 responses during ECM.

Pipecolic Acid, a Putative Mediator of the Encephalopathy of Cerebral Malaria and the Experimental Model of Cerebral Malaria.

BACKGROUND: We explored a metabolic etiology of cerebral malaria (CM) coma. METHODS: Plasma metabolites were compared between Malawian children with CM and mild Plasmodium falciparum malaria. A candidate molecule was further studied in animal models of malaria. RESULTS: Clinically abnormal concentrations of pipecolic acid (PA) were present in CM plasma, and nearly normal in mild malaria samples. PA is renally cleared and the elevated PA blood levels were associated with renal insufficiency, which was present only in CM subjects. Prior studies demonstrate that PA has neuromodulatory effects and is generated by malaria parasites. PA brain levels in Plasmodium berghei ANKA-infected animals in the experimental cerebral malaria (ECM) model inversely correlated with normal behavior and correlated with blood-brain barrier (BBB) permeability. Mice infected with malaria species that do not induce neurological abnormalities or manifest BBB permeability had elevated plasma PA levels similar to ECM plasma at 7 days postinfection; however, they had low PA levels in the brain compared to ECM mice brains at 7 days postinfection. CONCLUSIONS: Our model suggests that malaria-generated PA induces coma in CM and in ECM. The role of BBB permeability and the mechanisms of PA neuromodulation in CM will require additional investigation.

Basophil activation test in food allergy: is it ready for real-time?

PURPOSE OF REVIEW: Utilization of basophil activation in the diagnosis and monitoring of food allergy has gained increasing recognition. An ex-vivo functional assay, basophil activation reflects clinical reactivity, thereby providing clinically relevant insights. Moreover, as a biomarker of reactivity and tolerance, basophil activation testing (BAT) may provide a useful tool for management of food allergies. Despite its utility, significant limitations of BAT have prevented widespread use. Addressing these limitations will increase the future application and adoption of BAT in food allergy. RECENT FINDINGS: A number of clinical trials in the past few years have demonstrated the use of BAT in the diagnosis and treatment of food allergy. Specifically, BAT has been found to be a biomarker of tolerance. SUMMARY: Basophil activation testing is an effective biomarker for diagnosis and monitoring of food allergy.

Plasmodium yoelii Uses a TLR3-Dependent Pathway to Achieve Mammalian Host Parasitism.

Malaria is associated with complicated immunopathogenesis. In this study, we provide evidence for an unexpected role of TLR3 in promoting the establishment of Plasmodium yoelii infection through delayed clearance of parasitemia in wild type C57BL/6jRj (B6) compared with TLR3 knockout mice. In this study, we confirmed an increased expression of Tlr3, Trif, Tbk1, and Irf7/Irf3 in the liver 42 h postinfection and the initiation of an early burst of proinflammatory response such as Ifng, NF-kB, and Tnfa in B6 mice that may promote parasite fitness. Interestingly, in the absence of TLR3, we showed the involvement of high IFN-γ and lower type I IFN response in the early clearance of parasitemia. In parallel, we observed an increase in splenic NK and NKT cells expressing TLR3 in infected B6 mice, suggesting a role for TLR sensing in the innate immune response. Finally, we find evidence that the increase in the frequency of CD19+TLR3+ B cells along with reduced levels of total IgG in B6 mice possibly suggests the initiation of TLR3-dependent pathway early during P. yoelii infection. Our results thus reveal a new mechanism in which a parasite-activated TLR3 pathway promotes blood stage infection along with quantitative and qualitative differences in Ab responses.

Partial impairment of late-stage autophagic flux in murine splenocytes leads to sqstm1/p62 mediated nrf2-keap1 antioxidant pathway activation and induced proteasome-mediated degradation in malaria.

Splenomegaly, a major symptom in Plasmodium infection, is extensively studied for its immunopathological role in mice malaria model infected with Plasmodium berghei ANKA. The status of autophagic regulation in hosts in malaria pathogenesis remains unreported till date. This study demonstrated the autophagy, proteasomal degradation and NRF2-KEAP1 antioxidant pathway status in the host during Plasmodium infection taking murine spleen as our organ of interest. Initial staining and autophagic gene expression indicate a possibility of autophagic pathway activation. Although the conversion of LC3A to LC3B and lysosome-autophagosome fusion increases, the final degradation step remains incomplete. Resultant upregulation of p62 and its altered phosphorylated status enhances its binding to keap1 causing NRF2 translocation to the nucleus. NRF2 act as transcription factor upregulating p62 level itself leading to an autoinduction loop of p62 expression. Interestingly, enhancement of P62 interaction with proteasome subunit RPT1 indicates a possible role in transporting ubiquitinated cargo to proteasome complex. Ubiquitination level increased with subsequent upregulation of all three modes of proteasomal degradation i.e trypsin-like, caspase-like and especially chymotrypsin-like. Sqstm1/p62 plays a critical central role in regulating autophagy, proteasomal degradation, and NRF2-KEAP1 pathway. The incomplete autophagic flux in the final step may be a key therapeutic target, as autophagic degradation and subsequent pathogenic peptide presentation is of utmost necessity for downstream immune response.

Expression of CD300lf by microglia contributes to resistance to cerebral malaria by impeding the neuroinflammation.

Genetic mapping and genome-wide studies provide evidence for the association of several genetic polymorphisms with malaria, a complex pathological disease with multiple severity degrees. We have previously described Berr1and Berr2 as candidate genes identified in the WLA/Pas inbreed mouse strain predisposing to resistance to cerebral malaria (CM) induced by P. berghei ANKA. We report in this study the phenotypic and functional characteristics of a congenic strain we have derived for Berr2WLA allele on the C57BL/6JR (B6) background. B6.WLA-Berr2 was found highly resistant to CM compared to C57BL/6JR susceptible mice. The mechanisms associated with CM resistance were analyzed by combining genotype, transcriptomic and immune response studies. We found that B6.WLA-Berr2 mice showed a reduced parasite sequestration and blood-brain barrier disruption with low CXCR3+ T cell infiltration in the brain along with altered glial cell response upon P. berghei ANKA infection compared to B6. In addition, we have identified the CD300f, belonging to a family of Ig-like encoding genes, as a potential candidate associated with CM resistance. Microglia cells isolated from the brain of infected B6.WLA-Berr2 mice significantly expressed higher level of CD300f compared to CMS mice and were associated with inhibition of inflammatory response.

Autophagic induction modulates splenic plasmacytoid dendritic cell mediated immune response in cerebral malarial infection model.

Splenic plasmacytoid dendritic cells (pDC) possess the capability to harbor live replicative Plasmodium parasite. Isolated splenic pDC from infected mice causes malaria when transferred to naïve mice. Incomplete autophagic degradation might cause poor antigen processing and poor immune response. Induction of autophagic flux by rapamycin treatment led to better prognosis by boosting pDC centered immune response against the pathogen. Splenic pDC from rapamycin-treated infected mice, caused less parasitemia in naïve mice. The downregulation of adhesion with unaltered phagocytic potential of the cells post autophagic induction restricted excessive parasite burden within them. Rapamycin-treated pDC played a better role in antigen presentation. They showed higher expression of co-stimulatory molecules CD80, CD86, DEC205, MHCI. Rapamycin-treated pDC induced CD28 expression on CD8+ T cells and suppressed FasL level. This cells also influenced differentiation of effector, memory T cell population. The increase in IL10: TNFα ratio, Treg: Th17 ratio and lowering of myeloid DC: plasmacytoid DC ratio was observed. It shifted the overaggressive inflammation mediated Th1 pathway that is reported to incur host damage, to a better well-balanced cytokine profile exhibiting Th2 pathway. Autophagic flux induction within pDC proved to be beneficial in combating malarial pathogenicity.

Impact of autophagic regulation on splenic red pulp macrophages during cerebral malarial infection.

Splenic red pulp macrophages play a critical role infiltration of infected RBC and elimination of pathogens during malarial infection. However, the efficiency of pathogenic processing and the intricate pathway followed by them to boost the downstream immune response has not been studied in details. We checked the status of autophagic regulation within the cells both before and after the infection and also modulated the autophagic flux with either its inducer or inhibitor. We found that the upregulation of autophagic gene and the corresponding pathway is correlated with better parasite clearance and survivability, with an enhanced downstream immune response. It also increases their phagocytic potential with better Lysosomal associated protein I and II synthesis. The autophagolysosome formation increases as well, and more vacuole bound LC3B protein are detected. Chemokine synthesized from Red Pulp macrophage helps in mediating the induction for recruiting neutrophil and CD4 + T cells to the splenic red pulp region. The skewing of M1 macrophage polarity is observed post autophagic induction with a better costimulatory molecule like CD80, CD86 expression and antigen presenting molecule MHC I, MHC II is observed. This study shows the possibility of an alternative or adjuvant therapy regimen for the malarial patient by inducing the autophagic pathway that targets the red pulp macrophages. This might be helpful for better pathogen degradation and processing. The subsequent clearance of parasite will result in a better outcome for the patients.

Differential modulation of glial cell mediated neuroinflammation in Plasmodium berghei ANKA infection by TGF ß and IL 6.

Modulation of pro-inflammatory and anti-inflammatory axis and orientation of glial cell function towards neuroinflammation, were hallmark signs of cerebral malaria (CM). CM pathogenesis was concerned with the circulating levels of Interleukin 6 (IL 6) and Transforming growth factor ß (TGF ß). Definite roles of these two cytokines in brain related pathology remained largely unexplored. To study the effect of these two cytokines, we have examined changes in morphology and in activation profile of the glial cells after TGF ß and IL 6 neutralization during CM in cortex and cerebellum of the Plasmodium berghei ANKA (PbA) infected male swiss albino mice. PbA infection caused severe inflammation by inducing changes in morphological features as well as in activation profile of the astrocytes and microglia. Similar inflammatory signs were evident in Anti TGF ß treated set. Interestingly in the Anti IL 6 treated set, reduced level of activation of these glial cells corresponds to the reduced level of inflammatory profile. Microglial activation was found to be synchronous with TLR4 engagement. Neuronal death was triggered by neuroinflammatory milieu seen in PbA and PbA+Anti TGF ß treated set. In conclusion, it can be said that IL 6 and TGF ß perform essential role in CM pathogenesis by modulating the level of glial cell induced neuroinflammation.

Role of TGF-ß and IL-6 in dendritic cells, Treg and Th17 mediated immune response during experimental cerebral malaria.

The role of cytokines in Plasmodium infection have been extensively investigated, but pro and anti inflammatory cytokines mediated imbalance during malaria immune-pathogenesis is still unrevealed. Malaria is associated with the circulating levels of Interleukin-6 (IL-6) and transforming growth factor ß (TGF-ß), but association between these two cytokines in immune response remains largely obscured. Using mouse model, we proposed that IL-6 and TGF-ß are involved in immune regulation of dendritic cells (DC), regulatory T cells (Treg), T-helper cells (Th17) during P. berghei ANKA (PbA) infection. Association between the cytokines and the severity of malaria was established with anti-TGF-ß treatment resulting in increased parasitemia and increased immunopathology, whereas; anti-IL-6 treatment delays immunopathology during PbA infection. Further, splenocytes revealed differential alteration of myeloid DC (mDC), plasmocytoid DC (pDC), Treg, Th17 cells following TGF-ß and IL-6 neutralization. Interestingly anti-TGF-ß reduces CD11c+CD8+ DC expression, whereas anti-IL-6 administration causes a profound increase during PbA infection in Swiss mice. We observed down regulation of TGF-ß, IL-10, NFAT, Foxp3, STAT-5 SMAD-3 and upregulation of IL-6, IL-23, IL-17 and STAT-3 in splenocytes during PbA infection. The STAT activity probably plays differential role in induction of Th17 and Treg cells. Interestingly we found increase in STAT-3 and decrease in STAT-5 expression during PbA infection. This pattern of STAT indicates that possibly TGF-ß and IL-6 play a crucial role in differentiation of DCs subsets and Treg/Th17 imbalance during experimental cerebral malaria (ECM).

Suppression of CD4+ Effector Responses by Naturally Occurring CD4+ CD25+ Foxp3+ Regulatory T Cells Contributes to Experimental Cerebral Malaria.

The role of naturally occurring CD4(+) CD25(+) Foxp3(+) regulatory T cells (nTreg) in the pathogenesis of cerebral malaria (CM), which involves both pathogenic T cell responses and parasite sequestration in the brain, is still unclear. To assess the contribution and dynamics of nTreg during the neuropathogenesis, we unbalanced the ratio between nTreg and naive CD4(+) T cells in an attenuated model of Plasmodium berghei ANKA-induced experimental CM (ECM) by using a selective cell enrichment strategy. We found that nTreg adoptive transfer accelerated the onset and increased the severity of CM in syngeneic C57BL/6 (B6) P. berghei ANKA-infected mice without affecting the level of parasitemia. In contrast, naive CD4(+) T cell enrichment prevented CM and promoted parasite clearance. Furthermore, early during the infection nTreg expanded in the spleen but did not efficiently migrate to the site of neuroinflammation, suggesting that nTreg exert their pathogenic action early in the spleen by suppressing the protective naive CD4(+) T cell response to P. berghei ANKA infection in vivo in both CM-susceptible (B6) and CM-resistant (B6-CD4(-/-)) mice. However, their sole transfer was not sufficient to restore CM susceptibility in two CM-resistant congenic strains tested. Altogether, these results demonstrate that nTreg are activated and functional during P. berghei ANKA infection and that they contribute to the pathogenesis of CM. They further suggest that nTreg may represent an early target for the modulation of the immune response to malaria.