Search

Among various TLRs on macrophages, TLR-4, 5, 3, 7,and 9 actively sense spike proteins (N, S or G) or mRNA of NSP-10, S2, and E proteins of SARS-CoV-2 and promote M1 polarization of macrophages ([8]). Out of these, uncontrolled activation of macrophages (also known as double edge component of immunity) leads to Macrophage activation syndrome which is responsible for acute respiratory distress syndrome (ARDS) and subsequent death of COVID-19 patients ([4], [5]). Keywords: COVID-19; macrophage; TLRs (toll-like receptors); inflammasome; miRNA; lncRNAs; MAPK; immune polarization EN COVID-19 macrophage TLRs (toll-like receptors) inflammasome miRNA lncRNAs MAPK immune polarization 1 4 4 10/06/21 20211001 NES 211001 Introduction The current coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has the worst affected the entire population on the earth ([1], [2]). [Extracted from the article]

Protection against Staphylococcus aureus is determined by the polarization of the anti-bacterial immune effector mechanisms. Virulence factors of S. aureus can modulate these and induce differently polarized immune responses in a single individual. We proposed that this may be due to intrinsic properties of the bacterial proteins. To test this idea, we selected two virulence factors, the serine protease-like protein B (SplB) and the glycerophosphoryl diester phosphodiesterase (GlpQ). In humans naturally exposed to S. aureus, SplB induces a type 2-biased adaptive immune response, whereas GlpQ elicits type 1/type 3 immunity. We injected the recombinant bacterial antigens into the peritoneum of S. aureus-naïve C57BL/6N mice and analyzed the immune response. This was skewed by SplB toward a Th2 profile including specific IgE, whereas GlpQ was weakly immunogenic. To elucidate the influence of adjuvants on the proteins’ polarization potential, we studied Montanide ISA 71 VG and Imject™Alum, which promote a Th1 and Th2 response, respectively. Alum strongly increased antibody production to the Th2-polarizing protein SplB, but did not affect the response to GlpQ. Montanide enhanced the antibody production to both S. aureus virulence factors. Montanide also augmented the inflammation in general, whereas Alum had little effect on the cellular immune response. The adjuvants did not override the polarization potential of the S. aureus proteins on the adaptive immune response.

Protection against Staphylococcus aureus is determined by the polarization of the anti-bacterial immune effector mechanisms. Virulence factors of S. aureus can modulate these and induce differently polarized immune responses in a single individual. We proposed that this may be due to intrinsic properties of the bacterial proteins. To test this idea, we selected two virulence factors, the serine protease-like protein B (SplB) and the glycerophosphoryl diester phosphodiesterase (GlpQ). In humans naturally exposed to S. aureus , SplB induces a type 2-biased adaptive immune response, whereas GlpQ elicits type 1/type 3 immunity. We injected the recombinant bacterial antigens into the peritoneum of S. aureus -naïve C57BL/6N mice and analyzed the immune response. This was skewed by SplB toward a Th2 profile including specific IgE, whereas GlpQ was weakly immunogenic. To elucidate the influence of adjuvants on the proteins' polarization potential, we studied Montanide ISA 71 VG and Imject™Alum, which promote a Th1 and Th2 response, respectively. Alum strongly increased antibody production to the Th2-polarizing protein SplB, but did not affect the response to GlpQ. Montanide enhanced the antibody production to both S. aureus virulence factors. Montanide also augmented the inflammation in general, whereas Alum had little effect on the cellular immune response. The adjuvants did not override the polarization potential of the S. aureus proteins on the adaptive immune response. [ABSTRACT FROM AUTHOR]

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization.Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli, and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro, as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model.Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from −5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo, stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21.Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines.

Introduction: Understanding, which factors determine the immunogenicity and immune polarizing properties of proteins, is an important prerequisite for designing better vaccines and immunotherapeutics. While extrinsic immune modulatory factors such as pathogen associated molecular patterns are well-understood, far less is known about the contribution of protein inherent features. Protein fold-stability represents such an intrinsic feature contributing to immunogenicity and immune polarization by influencing the amount of peptide-MHC II complexes (pMHCII). Here, we investigated how modulation of the fold-stability of the grass pollen allergen Phl p 6 affects its ability to stimulate immune responses and T cell polarization. Methods: MAESTRO software was used for in silico prediction of stabilizing or destabilizing point mutations. Mutated proteins were expressed in E. coli , and their thermal stability and resistance to endolysosomal proteases was determined. Resulting peptides were analyzed by mass spectrometry. The structure of the most stable mutant protein was assessed by X-ray crystallography. We evaluated the capacity of the mutants to stimulate T cell proliferation in vitro , as well as antibody responses and T cell polarization in vivo in an adjuvant-free BALB/c mouse model. Results: In comparison to wild-type protein, stabilized or destabilized mutants displayed changes in thermal stability ranging from −5 to +14°. While highly stabilized mutants were degraded very slowly, destabilization led to faster proteolytic processing in vitro. This was confirmed in BMDCs, which processed and presented the immunodominant epitope from a destabilized mutant more efficiently compared to a highly stable mutant. In vivo , stabilization resulted in a shift in immune polarization from TH2 to TH1/TH17 as indicated by higher levels of IgG2a and increased secretion of TNF-α, IFN-γ, IL-17, and IL-21. Conclusion: MAESTRO software was very efficient in detecting single point mutations that increase or reduce fold-stability. Thermal stability correlated well with the speed of proteolytic degradation and presentation of peptides on the surface of dendritic cells in vitro. This change in processing kinetics significantly influenced the polarization of T cell responses in vivo. Modulating the fold-stability of proteins thus has the potential to optimize and polarize immune responses, which opens the door to more efficient design of molecular vaccines. [ABSTRACT FROM AUTHOR]

Background: Understanding macrophage behavior is key to decipher Mycobacterium tuberculosis (Mtb) pathogenesis. We studied the phenotype and ability of human monocyte-derived cells polarized with active vitamin D [1,25(OH)2D3] to control intracellular Mtb infection compared with polarization of conventional subsets, classical M1 or alternative M2.Methods: Human blood-derived monocytes were treated with active vitamin D or different cytokines to obtain 1,25(OH)2D3-polarized as well as M1- and M2-like cells or fully polarized M1 and M2 subsets. We used an in vitro macrophage Mtb infection model to assess both phenotype and functional markers i.e., inhibitory and scavenger receptors, costimulatory molecules, cytokines, chemokines, and effector molecules using flow cytometry and quantitative mRNA analysis. Intracellular uptake of bacilli and Mtb growth was monitored using flow cytometry and colony forming units.Results: Uninfected M1 subsets typically expressed higher levels of CCR7, TLR2, and CD86, while M2 subsets expressed higher CD163, CD200R, and CD206. Most of the investigated markers were up-regulated in all subsets after Mtb infection, generating a mixed M1/M2 phenotype, while the expression of CD206, HLADR, and CD80 was specifically up-regulated (P < 0.05) on 1,25(OH)2D3-polarized macrophages. Consistent with the pro-inflammatory features of M1 cells, Mtb uptake and intracellular Mtb growth was significantly (P < 0.01–0.001 and P < 0.05–0.01) lower in the M1 (19.3%) compared with the M2 (82.7%) subsets 4 h post-infection. However, infectivity rapidly and gradually increased in M1 cells at 24–72 h. 1,25(OH)2D3-polarized monocyte-derived cells was the most potent subset to inhibit Mtb growth at both 4 and 72 h (P < 0.05–0.01) post-Mtb infection. This ability was associated with high mRNA levels of pro-inflammatory cytokines and the antimicrobial peptide LL-37 but also anti-inflammatory IL-10, while expression of the immunosuppressive enzyme IDO (indoleamine 2,3-dioxygenase) remained low in Mtb-infected 1,25(OH)2D3-polarized cells compared with the other subsets.Conclusions: Mtb infection promoted a mixed M1/M2 macrophage activation, and 1,25(OH)2D3-polarized monocyte-derived cells expressing LL-37 but not IDO, were most effective to control intracellular Mtb growth. Macrophage polarization in the presence of vitamin D may provide the capacity to mount an antimicrobial response against Mtb and simultaneously prevent expression of inhibitory molecules that could accelerate local immunosuppression in the microenvironment of infected tissue.

Background: Understanding macrophage behavior is key to decipher Mycobacterium tuberculosis (Mtb) pathogenesis. We studied the phenotype and ability of human monocyte-derived cells polarized with active vitamin D [1,25(OH)2D3] to control intracellular Mtb infection compared with polarization of conventional subsets, classical M1 or alternative M2. Methods: Human blood-derived monocytes were treated with active vitamin D or different cytokines to obtain 1,25(OH)2D3-polarized as well as M1- and M2-like cells or fully polarized M1 and M2 subsets. We used an in vitro macrophage Mtb infection model to assess both phenotype and functional markers i.e., inhibitory and scavenger receptors, costimulatory molecules, cytokines, chemokines, and effector molecules using flow cytometry and quantitative mRNA analysis. Intracellular uptake of bacilli and Mtb growth was monitored using flow cytometry and colony forming units. Results: Uninfected M1 subsets typically expressed higher levels of CCR7, TLR2, and CD86, while M2 subsets expressed higher CD163, CD200R, and CD206. Most of the investigated markers were up-regulated in all subsets after Mtb infection, generating a mixed M1/M2 phenotype, while the expression of CD206, HLADR, and CD80 was specifically up-regulated (P < 0.05) on 1,25(OH)2D3-polarized macrophages. Consistent with the pro-inflammatory features of M1 cells, Mtb uptake and intracellular Mtb growth was significantly (P < 0.01–0.001 and P < 0.05–0.01) lower in the M1 (19.3%) compared with the M2 (82.7%) subsets 4 h post-infection. However, infectivity rapidly and gradually increased in M1 cells at 24–72 h. 1,25(OH)2D3-polarized monocyte-derived cells was the most potent subset to inhibit Mtb growth at both 4 and 72 h (P < 0.05–0.01) post-Mtb infection. This ability was associated with high mRNA levels of pro-inflammatory cytokines and the antimicrobial peptide LL-37 but also anti-inflammatory IL-10, while expression of the immunosuppressive enzyme IDO (indoleamine 2,3-dioxygenase) remained low in Mtb-infected 1,25(OH)2D3-polarized cells compared with the other subsets. Conclusions: Mtb infection promoted a mixed M1/M2 macrophage activation, and 1,25(OH)2D3-polarized monocyte-derived cells expressing LL-37 but not IDO, were most effective to control intracellular Mtb growth. Macrophage polarization in the presence of vitamin D may provide the capacity to mount an antimicrobial response against Mtb and simultaneously prevent expression of inhibitory molecules that could accelerate local immunosuppression in the microenvironment of infected tissue. [ABSTRACT FROM AUTHOR]