Pathogen Evasion of Chemokine Response Through Suppression of CXCL10

Abstract

Clearance of intracellular pathogens, such as Leishmania (L.) major, depends on an immune response with well-regulated cytokine signaling. Here we describe a pathogen-mediated mechanism of evading CXCL10, a chemokine with diverse antimicrobial functions, including T cell recruitment. Infection with L. major in a human monocyte cell line induced robust CXCL10 transcription without increasing extracellular CXCL10 protein concentrations. We found that this transcriptionally independent suppression of CXCL10 is mediated by the virulence factor and protease, glycoprotein-63 (gp63). Specifically, GP63 cleaves CXCL10 after amino acid A81 at the base of a C-terminal alpha-helix. Cytokine cleavage by GP63 demonstrated specificity, as GP63 cleaved CXCL10 and its homologs, which all bind the CXCR3 receptor, but not distantly related chemokines, such as CXCL8 and CCL22. Further characterization demonstrated that CXCL10 cleavage activity by GP63 was produced by both extracellular promastigotes and intracellular amastigotes. Crucially, CXCL10 cleavage impaired T cell chemotaxis in vitro, indicating that cleaved CXCL10 cannot signal through CXCR3. Ultimately, we propose CXCL10 suppression is a convergent mechanism of immune evasion, as Salmonella enterica and Chlamydia trachomatis also suppress CXCL10. This commonality suggests that counteracting CXCL10 suppression may provide a generalizable therapeutic strategy against intracellular pathogens.

Importance: Leishmaniasis, an infectious disease that annually affects over one million people, is caused by intracellular parasites that have evolved to evade the host's attempts to eliminate the parasite. Cutaneous leishmaniasis results in disfiguring skin lesions if the host immune system does not appropriately respond to infection. A family of molecules called chemokines coordinate recruitment of the immune cells required to eliminate infection. Here, we demonstrate a novel mechanism that Leishmania (L.) spp. employ to suppress host chemokines: a Leishmania-encoded protease cleaves chemokines known to recruit T cells that fight off infection. We observe that other common human intracellular pathogens, including Chlamydia trachomatis and Salmonella enterica, reduce levels of the same chemokines, suggesting a strong selective pressure to avoid this component of the immune response. Our study provides new insights into how intracellular pathogens interact with the host immune response to enhance pathogen survival.

Keywords: CXCL10; CXCR3; Chlamydia; Leishmania; Salmonella; convergent evolution; gp63; leishmanolysin.

Figure 1

Leishmania major suppresses CXCL10 post-transcriptionally in multiple human cell lines. (A) Cytokine screening of LCLs exposed to L. major demonstrated suppression of CXCL10. Three lymphoblastoid cell lines (LCL), GM07357, GM18524, and GM19203, were infected with L. major. Chemokines secreted into culture supernatants were analyzed by Luminex. Cytokines below the limit of detection were removed from the final analysis. Values are represented as log₂ of the fold change relative to uninfected LCLs. Type-1 associated cytokines are represented in gray. P-value represents Dunnett's post-hoc test compared to 1, after repeated measures one-way ANOVA. (B) CXCL10 suppression by L. major is transcriptionally independent in LCL GM18524. LCL GM18524 was infected with L. major at MOI 10 to confirm the CXCL10 suppression phenotype. Despite significant reduction in CXCL10 protein, there was no change in relative CXCL10 mRNA. CXCL10 mRNA was measured by qRT-PCR TaqMan assay using the ΔΔC_t method with 18s as housekeeping gene, and CXCL10 protein was measured by ELISA. Four experimental replicates were used to calculate mRNA (n = 4) and ELISA (n = 8) fold change relative to uninfected LCL 18524. P-values calculated by Student's t-test. (C) CXCL10 produced by LPS stimulated THP-1 monocytes was suppressed by L. major. THP-1 monocytes were stimulated with LPS prior to L. major infection. Three experimental replicates were used to calculate mRNA (n = 3) and protein (n = 6) fold change relative to unstimulated, uninfected THP-1s. P-values calculated by Student's t-test.

Figure 2

Leishmania major matrix-metalloprotease, glycoprotein-63, is necessary and sufficient to cleave CXCL10. (A) Zinc chelation prevents CXCL10 suppression. Concentration of human recombinant CXCL10 was measured by ELISA after incubation for 12 h with filtered conditioned media from L. major WT promastigote culture and addition of the zinc-chelator 1,10-phenanthroline (n = 8 from 4 experiments). (B) gp63 is required for L. major CXCL10 suppression. Human recombinant CXCL10 concentrations were measured by ELISA after 12 h incubation with conditioned media from L. major WT, Δgp63, or Δgp63+1 (n = 6 from 3 experiments). (C) GP63 expressed and secreted by HEK293Ts is sufficient for CXCL10 suppression. Human recombinant CXCL10 concentrations were measured by ELISA after 12 h incubation with culture supernatant from HEK293Ts transfected with pCDNA3.1-gp63^WT or pCDNA3.1-gp63^E285A (n = 6 from 3 experiments). Concentration is represented as fold change relative to supernatants from YFP transfection control. P-values calculated by one-way ANOVA with Tukey's post-hoc test. Error bars represent standard error of the mean.

Figure 3

CXCL10 cleavage by GP63 occurs between positions A81 and I82. (A) CXCL9/10/11 share significant homology at the amino-acid level. Multisequence alignment demonstrates that physical characteristics of amino acids are conserved across the CXCL10 family of chemokines. There are three putative GP63 cleavage sites (underlined) based on the consensus sequence of polar (P1), hydrophobic (P1′), basic (P2′) (Bouvier et al., 1990). (B) GP63 selectively cleaves chemokine ligands of the CXCR3 receptor. Conditioned media from L. major WT, Δgp63, and Δgp63+1 was incubated with human recombinant chemokines for 12 h and product detected by western blot. Cleavage is only detected for the CXCL9/10/11 family. Representative blots are shown from at least two independent experiments. (C) Cleavage by GP63 generates a smaller molecular weight protein. A time course of cleavage of human CXCL10 by heterologously expressed GP63 demonstrated an intermediate cleavage product, resolved by PAGE and Coomassie staining. (D) Cleavage by GP63 results in a change in CXCL10 molecular weight of 2.2 kD. Capillary electrophoresis-Mass Spectrometry (CE-MS) determined the molecular weight of the uncleaved (CXCL10^Hi) and cleaved (CXCL10^Lo) bands as 8.8 and 6.6 kD, respectively. (E) Comparative analysis by trypsin digest of cleaved and uncleaved CXCL10 reveals cleavage occurring between A81-I82. Liquid chromatography-mass spectrometry (LC-MS) following trypsin digest of CXCL10^Hi and CXCL10^Lo identified peptide ending at A81, exclusively in the CXCL10^Lo band, and a corresponding lack of peptide coverage from AA84-91. (F) Mutation of A81F significantly impairs GP63 cleavage of CXCL10. In the presence of GP63, CXCL10^A81F (n = 4 from 4 experiments) remains stable for up to 45 min whereas CXCL10^WT (n = 3 from 3 experiments) degradation is nearly complete by15 min. Percentage of GP63 remaining at 15min is plotted. P-value calculated by Student's t-test. (G) The GP63 cleavage site is found on the C-terminal alpha-helix loop of CXCL10. Based on the NMR crystal structure of CXCL10 (Booth et al., 2002), the A81, I82, K83 (P1, P1′, P2′) GP63 cleavage motif maps to an exposed alpha-helical region.

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.

Figure 5

Multiple intracellular pathogens have evolved a mechanism for CXCL10 suppression. (A) Leishmania spp. with diverse geographic origin, genetic background, and clinical manifestations suppress CXCL10. 1 × 10⁶ live promastigotes from day 5 cultures of L. major Seidman WT (p = 0.0001), L. major Seidman Δgp63 (p = 0.81), L. major Seidman Δgp63+1 (p = 0.0001), L. major Friedlin (p = 0.0001), L. tropica (p = 0.0001), L. donovani (p = 0.0001), L. venezuelensis (p = 0.0001), and L. braziliensis (p = 0.0001) were incubated in 50 μl of M199 supplemented with 1 ng/μl of human recombinant CXCL10 at 37°C for 24 h. (B) LCL 18524 was used to screen L. major (p = 0.0001), P. berghei (p = 0.99), T. gondii I (RH) (p = 0.44), T. gondii II (Prugniaud A7) (p = 0.011), S. enterica serovar Typhimurium (p = 0.0001), S. aureus (p = 0.12), C. trachomatis (p = 0.0001), M. marinum (p = 0.37), M. smegmatis (p > 0.99), and C. neoformans (p = 0.010) for CXCL10 suppressing activity (n = 2–4 for each pathogen). For (A,B) CXCL10 concentration was measured by ELISA and is represented as the log₂ of fold change relative to uninfected controls. P-values calculated by one-way ANOVA with Dunnett's post-hoc test comparing non-log transformed values to 1, which would represent no change relative to uninfected *p < 0.01. (C,D) S. Typhimurium and C. trachomatis suppress CXCL10 in a second LCL. Infections were performed in the LCL HG02647 for S. Typhimurium (n = 6; two experiments) and C. trachomatis (n = 5; three experiments). Mean ± standard error of the mean is plotted and P-values calculated by Student's t-test. (E) S. Typhimurium suppresses production of CXCL10 in THP-1 monocytes. THP-1 monocytes were stimulated with 1 μg/mL of purified LPS from S. Typhimurium at the time of infection. CXCL10 concentration in culture supernatant at 24 hpi was assayed by ELISA. Mean ± standard error the mean is plotted, and P-values calculated by two-way ANOVA with Tukey's post-hoc test. (F) Chlamydia trachomatis suppresses CXCL10 in the human endocervical epithelial cell line A2EN. CXCL10 concentration in culture supernatant at 72 hpi was assayed by ELISA. Mean ± standard error of the mean is plotted and P-values calculated by Student's t-test.