Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization through LC3-Dependent Autophagy

Brucellosis is caused by infection with Brucella species and exhibits diverse clinical manifestations in infected humans. Monocytes and macrophages are not only the first line of defense against Brucella infection but also a main reservoir for Brucella. In the present study, we examined the effects of Brucella infection on human peripheral monocytes and monocyte-derived polarized macrophages. We showed that Brucella infection led to an increase in the proportion of CD14++CD16− monocytes and the expression of the autophagy-related protein LC3B, and the effects of Brucella-induced monocytes are inhibited after 6 weeks of antibiotic treatment. Additionally, the production of IL-1β, IL-6, IL-10, and TNF-α from monocytes in patients with brucellosis was suppressed through the LC3-dependent autophagy pathway during Brucella infection. Moreover, Brucella infection inhibited macrophage polarization. Consistently, the addition of 3-MA, an inhibitor of LC3-related autophagy, partially restored macrophage polarization. Intriguingly, we also found that the upregulation of LC3B expression by rapamycin and heat-killed Brucella in vitro inhibits M2 macrophage polarization, which can be reversed partially by 3-MA. Taken together, these findings reveal that Brucella dysregulates monocyte and macrophage polarization through LC3-dependent autophagy. Thus, targeting this pathway may lead to the development of new therapeutics against Brucellosis

Characterization of Full-Length Enterovirus 71 Strains from Severe and Mild Disease Patients in Northeastern China

Human enterovirus 71 (EV71)-associated hand, foot, and mouth disease (HFMD) has been a leading cause of childhood infection in China since 2008. Epidemic and molecular characteristics of HFMD have been examined in many areas of China, including the central and southern regions. However, clinical and genetic characterization of EV71 in the northeastern region of China is scarce. In this study, a series of analyses were performed on seven full-length EV71 sequences from HFMD patients who had either severe or mild disease. We have determined that these seven circulating EV71 viruses from Changchun, China are actually complex recombinant viruses involving multiple type A human enterovirus (HEV). Classified as EV71 subtype C4 (EV71 C4), these Changchun EV71 viruses contain genetic recombination events between the CA4, CA5, EV71B4 and EV71C1 strains. Most of the structural protein region (P1) of these viruses resembled that of the prototype EV71 C1 strains. The non-structural protein domains (P2 and P3) showed a high degree of similarity with CA4, CA5 and EV71 B4 in different regions. The 5′UTR had unclassified recombination,while partial 3D region of these viruses showed a high degree of similarity to CA16. Phylogenetic analysis of full-length or partial sequences of isolates from severe or mild disease patients in Changchun always formed a single cluster in various phylogenetic analyses of different genomic regions, suggesting that all seven strains originated from one single common ancestor. There was no correlation between viral genomic sequence and virulence. Thus, we found that circulating recombinant forms of EV71 are prevalent among HFMD patients in Northeastern China. The existence of a unique cluster of EV71 related viruses in Northeast China has important implications for vaccine development that would address the increasing prevalence of HFMD

Phylogenetic analysis of the complete genome and VP1 protein-coding region of the Changchun strains.

<p>Phylogenetic trees were generated by the neighbor-joining method with 1000 bootstraps for 7 representative Changchun strains and other EV71 strains of known subgenotypes. The poliovirus 1 strain was used as the outlier. The ▪ icon indicates the fatal cases; ▴ indicates the severe cases; • indicates the mild cases. A: Phylogenetic tree based on the whole genome sequences. B: Phylogenetic tree based on the VP1 region (891 bp).</p

Identification of recombination in the Changchun strains.

<p>A: Similarity plot and bootscan analysis for Changchun011. B: Similarity plot and bootscan analysis for Changchun077. C: Similarity and bootscan analysis for Changchun103 (others not shown). A window size of 500 nucleotides in increments of 20 nucleotides at a time was used. Positions containing gaps were not excluded from the comparison.</p

Phylogenetic analysis for partial genomes from the Changchun strains.

<p>A: Based on the entire P1 gene region of the Changchun strains, B: Based on the P2 region of the Changchun strains, C: Based on the 2A region of the Changchun strains, D: Based on the 3C region of the Changchun strains The ▪ icon indicates the fatal cases; ▴ indicates the severe cases; • indicates the mild cases.</p

Phylogenetic analysis of seven Changchun strains and GenBank database sequences of the EV71 strains from other provinces in China.

<p>A: Based on the complete genome sequence, B: Based on the VP1 region sequence, C: Based on the 5′-UTR region sequence, D: Based on the P2 region sequence,.. The ▪ icon indicates the fatal cases; ▴ indicates the severe cases; • indicates the mild cases.</p

Bootscan and phylogenetic analyses of partial genomes from the Changchun strains.

<p>A: Identification of the recombinant sequences in the 5′-UTR region of the Changchun011 genome. The window size of 100 nucleotides slides in increments of 20 nucleotides at a time. B: The neighbor-joining tree was established from alignments of the entire 5′-UTR region for the seven Changchun strains. C: Bootscan analysis of a 3D region in the Changchun011 genome. The window size included 200 nucleotides slides in increments of 20 nucleotides at a time. D: The neighbor-joining tree was established from sequence alignments of the entire 3D region for the seven Changchun strains. The ▪ icon indicates the fatal cases; ▴ indicates the severe cases; • indicates the mild cases.</p

Brucella Dysregulates Monocytes and Inhibits Macrophage Polarization through LC3-Dependent Autophagy

Brucellosis is caused by infection with Brucella species and exhibits diverse clinical manifestations in infected humans. Monocytes and macrophages are not only the first line of defense against Brucella infection but also a main reservoir for Brucella. In the present study, we examined the effects of Brucella infection on human peripheral monocytes and monocyte-derived polarized macrophages. We showed that Brucella infection led to an increase in the proportion of CD14++CD16− monocytes and the expression of the autophagy-related protein LC3B, and the effects of Brucella-induced monocytes are inhibited after 6 weeks of antibiotic treatment. Additionally, the production of IL-1β, IL-6, IL-10, and TNF-α from monocytes in patients with brucellosis was suppressed through the LC3-dependent autophagy pathway during Brucella infection. Moreover, Brucella infection inhibited macrophage polarization. Consistently, the addition of 3-MA, an inhibitor of LC3-related autophagy, partially restored macrophage polarization. Intriguingly, we also found that the upregulation of LC3B expression by rapamycin and heat-killed Brucella in vitro inhibits M2 macrophage polarization, which can be reversed partially by 3-MA. Taken together, these findings reveal that Brucella dysregulates monocyte and macrophage polarization through LC3-dependent autophagy. Thus, targeting this pathway may lead to the development of new therapeutics against Brucellosis