Figure 4.

GP63 produced by L. major promastigotes and amastigotes cleaves CXCL10 and abolishes its chemotactic activity. (A) Leishmania major promastigotes suppress CXCL10 through GP63 activity at 24 and 48 h post-infection. THP-1 monocytes were differentiated using 100 ng/mL of PMA prior to infection, infected at MOI 20 with L. major promastigotes, and extracellular promastigotes were washed away from the differentiated THP-1 monocytes at 24 h post-infection. CXCL10 concentration was assessed in the supernatant by ELISA. Data analyzed by one-way ANOVA with Tukey's post-hoc test (n = 12 from 4 experiments) (B) Leishmania major induces similar levels of CXCL10 mRNA, independent of GP63 genotype. At 48 h post-infection, mRNA was extracted from PMA differentiated THP-1 monocytes and CXCL10 mRNA was measured by qRT-PCR TaqMan assay using the ΔΔC_t method with 18s as housekeeping gene. For (B) data analyzed by one-way ANOVA with Tukey's post-hoc test (n = 9 from 3 experiments). (C) Expression of gp63 mRNA in L. major Δgp63+1 does not fully rescue wildtype gp63 expression in promastigote or amastigotes stages. Promastigote RNA (n = 4 from 4 experiments) was derived from day 5 of parasite culture before preparing for infection, and amastigote RNA (n = 7 from 3 experiments) was derived from intracellular THP-1s as described above. gp63 mRNA was measured by qRT-PCR using Sybr Green and relative expression calculated with the ΔΔC_t method using rRNA45 as housekeeping gene. Data analyzed by two-way ANOVA with Tukey's post-hoc test. (D) GP63 cleavage of CXCL10 only occurs extracellularly. THP-1 macrophages were treated with PMA and infected as described above. At 24 h post-infection, supernatants were collected and cells were removed from the plate by pipetting with cold PBS. The concentration of living cells was determined using 7AAD staining and counting cells with a Guava easyCyte flow cytometer. Cells were then lysed with RIPA buffer supplemented with protease inhibitor tablet and 10 μM 1,10-phenanthroline. CXCL10 in the supernatants and cell lysates (n = 5 from 2 experiments) was measured by ELISA and is expressed per concentration of living cells in each replicate prior to analysis by one-way ANOVA with Tukey's post-hoc test. (E) CXCL10 incubated with GP63 is unable to chemoattract CXCR3+ cells in vitro. Jurkat T cells stably transfected with CXCR3 were seeded on the apical surface of a 5 μm transwell insert, with human recombinant CXCL10 pre-incubated with conditioned media from either L. major WT, Δgp63, or Δgp63+1 in the basal chamber. The number of CXCR3+ Jurkats in the basal chamber after 4 h were counted to assess chemotactic capacity of CXCL10 after exposure to GP63. (F) Proposed model where the host attempts to upregulate CXCL10 in response to infection, but through the activity of GP63 L. major is able to impair signaling through the CXCR3 receptor.