MiRNA Exporter HuR Clears  Macrophage Internalized Pathogens by Promoting Pro-inflammatory Response (2020)
Figure 8

Figure 8 Requirement of simultaneous activation of HuR and deactivation of PP2A driven processes in mammalian macrophage to prevent infection by antimonial drug resistant Leishmania parasite.

A-D Effect of antimonial drug resistant and sensitive forms of the Ld (LdR and LdS) on cellular cytokine level as well as on phospho-ERK and MDR1 level. RAW 264.7 cells were infected with Antimony sensitive (LdS-Ag83) and antimony resistant (LdR-BHU569) form of Ld for 24 hours along with uninfected cells kept as control. IL-10 (A) and TNF-α (B) were measured at mRNA level by qRT-PCR after infection (mean+/- s.e.m. and, n=4). p-ERK, ERK and MDR1 level were also measured by western blot (D). Densitometry analysis was done for p-ERK level by ImageJ and relative values were plotted (C). β-Actin was used as loading control Values are mean+/- s.e.m. and, n=3.

E Effect of Antimony sensitive and antimony resistant form of the Ld on HuR and PP2A. RAW 264.7 cells were infected with LdS and LdR for 12 hrs and HuR level was measured by western blot (left panel). PP2A level was also measured by western blot after 4 and 12 hrs of LdS and LdR infection (right panel). β-Actin was used as loading control.

F Effect of ectopic expression of HuR and PP2A inhibitor OA treatment on LdR infection of macrophage cell. RAW 264.7 cells were transfected with HA-HuR or pCIneo followed by 2 hrs of pretreatment with OA. These cells were then infected with LdR for 24 hrs. Pro-inflammatory cytokines TNF-α (right panel; mean+/- s.e.m., n=3) and IL-1β (left panel; mean+/- s.e.m. and, n=4) were measured at mRNA level and relative values are plotted. Values without OA treatment was set as control.

G-I Effect of HuR over expression on LdR infection in mice liver. A scheme for animal experiment done with LdR infection followed by HA-HuR expression and OA treatment is shown (G). Pro-inflammatory cytokines TNF-α (left panel) and IL-1β (right panel) level were measured at mRNA level in liver (H). Parasite load was measured in infected liver tissue by using specific primer against kinetoplastid minicircle DNA of Ld (I). Values are mean+/- s.e.m. and, n=6).

J The model depicts inter-regulatory balance between PP2A and HuR in controlling pro-inflammatory and anti-inflammatory response via altering Ago2 phosphorylation-dephosphorylation cycle and exosomal export of miRNAs in mammalian macrophage cells. Left half of the model shows Lipopolysaccharide (LPS) induced PP2A upregulation via TLR4 pathway during late hours of treatment which results in Ago2 dephosphorylation. Right half of the scheme represents Leishmania induced PP2A increase mediated by membrane glycolipid Lipophosphoglycan (LPG). Interestingly, the RNA binding protein HuR plays a central role in both the contexts. Inhibitory effect of HuR on PP2A ensures phosphorylated form of Ago2 to dominate which in turn facilitates miRNA unbinding to Ago2 and miRNA export via exosome thereby promoting pro-inflammatory cytokine response. On the contrary, Ld induced downregulation of HuR via a zinc-metallopotease, GP63 which results in PP2A upregulation that facilitates anti-inflammatory response necessary for the parasite survival and proliferation.

Data information: In all experimental data, ns; non-significant and *, ** and *** represent P value of <0.05, <0.01 and <0.001 respectively quantified by using student's t test. For statistical analysis, all experiments are done three times. Exact P-values against each experimental set are presented in the Appendix Table S2. Positions of molecular weight markers are marked and shown in the western blots used in different panels.

Figure legends for Expanded View Figures