Machine learning identifies clusters of longitudinal autoantibody profiles predictive of systemic lupus erythematosus disease outcomes

OBJECTIVES: A novel longitudinal clustering technique was applied to comprehensive autoantibody data from a large, well-characterised, multinational inception systemic lupus erythematosus (SLE) cohort to determine profiles predictive of clinical outcomes. METHODS: Demographic, clinical and serological data from 805 patients with SLE obtained within 15 months of diagnosis and at 3-year and 5-year follow-up were included. For each visit, sera were assessed for 29 antinuclear antibodies (ANA) immunofluorescence patterns and 20 autoantibodies. K-means clustering on principal component analysis-transformed longitudinal autoantibody profiles identified discrete phenotypic clusters. One-way analysis of variance compared cluster enrolment demographics and clinical outcomes at 10-year follow-up. Cox proportional hazards model estimated the HR for survival adjusting for age of disease onset. RESULTS: Cluster 1 (n=137, high frequency of anti-Smith, anti-U1RNP, AC-5 (large nuclear speckled pattern) and high ANA titres) had the highest cumulative disease activity and immunosuppressants/biologics use at year 10. Cluster 2 (n=376, low anti-double stranded DNA (dsDNA) and ANA titres) had the lowest disease activity, frequency of lupus nephritis and immunosuppressants/biologics use. Cluster 3 (n=80, highest frequency of all five antiphospholipid antibodies) had the highest frequency of seizures and hypocomplementaemia. Cluster 4 (n=212) also had high disease activity and was characterised by multiple autoantibody reactivity including to antihistone, anti-dsDNA, antiribosomal P, anti-Sjögren syndrome antigen A or Ro60, anti-Sjögren syndrome antigen B or La, anti-Ro52/Tripartite Motif Protein 21, antiproliferating cell nuclear antigen and anticentromere B). Clusters 1 (adjusted HR 2.60 (95% CI 1.12 to 6.05), p=0.03) and 3 (adjusted HR 2.87 (95% CI 1.22 to 6.74), p=0.02) had lower survival compared with cluster 2. CONCLUSION: Four discrete SLE patient longitudinal autoantibody clusters were predictive of long-term disease activity, organ involvement, treatment requirements and mortality risk

ChLae1 and ChVel1 Regulate T-toxin Production, Virulence, Oxidative Stress Response, and Development of the Maize Pathogen Cochliobolus heterostrophus

LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel1, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H2O2. Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation

Repression of asexual sporulation in the dark and under cycling conditions is compromised in <i>Chlae1</i> and <i>Chvel1</i> mutants growing vegetatively.

<p><b>A.</b> Quantification of asexual spores from cultures grown under constant light, 12 hour light/dark cycle, and dark conditions. Error bars are standard deviation. Asterisks represent p-value <0.05 in T-test analysis in which each mutant strain was compared with the corresponding WT C4 strain under the same conditions. Asexual sporulation is repressed in WT in the dark or under the light cycling conditions, while this was not observed for the <i>Chlae1</i> mutant. Absence of <i>ChVEL1</i> augments asexual sporulation regardless of the light condition. <b>B.</b> Cultures grown on CMX plates under constant light, 12 hour light/dark cycle and constant dark conditions. Note that in the dark, WT C4 is white and fluffy reflecting aerial hyphal growth and production of very few conidia, while <i>Chlae1</i> and <i>Chvel1</i> mutants are pigmented. Alternating light and dark conidial banding pattern of the WT strain C4 in middle panel indicates that conidiation of the WT strain is responsive to light. This banding pattern is absent or much reduced in the <i>Chvel1</i> mutant, but still evident in the <i>Chlae1</i> mutant. <b>C.</b> Side view of plates of WT strain C4, and the <i>Chlae1</i> and <i>Chvel1</i> mutants grown in constant light or dark on CMX. Note aerial hyphae on plates of WT, especially from the dark. In contrast, the surface of the <i>Chlae1</i> mutant is very flat while the <i>Chvel1</i> mutant shows a small amount of aerial hyphae. Thus Lae1 appears to play a greater role in promoting aerial hyphae growth than Vel1.</p

Overexpression of <i>LAE1</i> and <i>VEL1</i> represses expression of the polyketide synthase gene, <i>PKS18</i>, associated with melanin production.

<p><b>A.</b> qPCR analyses of <i>PKS18</i>, <i>ChVEL1</i> and <i>ChLAE1</i>. Expression of the genes was determined for the cultures grown for 48 hrs in CM with PGA as the carbon source. WT strain C4, and three independent <i>ChVEL1</i> (left to right: OEVEL1-3, OEVEL1-4 and OEVEL1-7) and <i>ChLAE1</i> (left to right: OELAE1-1, OELAE1-3 and OELAE1-4) overexpression strains each were examined, and expression level relative to the WT sample at 48 hrs is shown. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Single asterisks indicate p-value <0.05, double asterisks indicate p-value <0.001 in T-test analysis in which each strain was compared with WT C4. <i>ChLAE1</i> and <i>ChVEL1</i> are overexpressed in each OE strains and <i>PKS18</i> is repressed in these strains, except for strain OEVEL1-7. The data confirm that ChLae1 and ChVel1 negatively regulate melanin biosynthesis at the transcriptional level. Note that expression of <i>ChLAE1</i> is up in <i>ChVEL1</i> overexpression strains. <b>B.</b> C4, and three independent <i>ChVEL1</i> and <i>ChLAE1</i> (left to right: as above) overexpression strains each, grown as in <b>A</b>. Note that <i>ChVEL1</i> and <i>ChLAE1</i> overexpression strains displayed less pigmentation than the WT C4 strains with <i>ChLAE1</i> OE having the least melanization (compare to <i>Chlae1</i> mutant strains <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002542#ppat-1002542-g007" target="_blank">Figure 7</a>). This indicates that these proteins are negative regulators of melanization of mycelia and that ChLae1 plays a larger role.</p

ChLae1 and ChVel1 positively regulate T-toxin biosynthesis genes.

<p><b>A.</b> RT-PCR analysis of the genes known to be involved in T-toxin production in WT and mutant strains. Expression of <i>ACT1</i> indicates relative RNA quantity in each sample. All <i>Tox1</i> genes are up-regulated in the dark, relative to in the light in WT. Evidence of light regulation is erased in <i>Chvel1</i> mutants. Most genes are weakly up-regulated in <i>Chlae1</i> mutants, except for <i>RED1</i>, <i>RED2</i> and <i>RED3</i>. <b>B.</b> Quantification of band intensity. Band intensity in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002542#ppat-1002542-g002" target="_blank">Figure 2A</a> was quantified by Image J. The band intensity ratio of each <i>Tox1</i> gene and that of the corresponding control <i>ACT1</i> gene was calculated and normalized to that of WT strain C4 in light. Note that band intensity of all genes is elevated in the dark in WT, while band intensity of all genes is minimal in the <i>Chvel1</i> mutant grown in both the light and dark, and minimally elevated in the <i>Chlae1</i> mutant grown in the dark. <b>C</b>. qPCR of <i>PKS1</i> and <i>PKS2</i>. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.001 in T-test analysis in which all the strains grown in the dark were compared to their corresponding strain grown in constant light. <b>D.</b> RT-PCR analysis of <i>Tox1</i> genes in overexpression strains. Overexpression of <i>ChLAE1</i> results in drastic up-regulation of all genes in the light and a moderate increase in the dark compared to WT. In contrast, <i>ChVEL1</i> overexpression caused up-regulation of <i>Tox1</i> gene expression in the dark but not the light. <b>E.</b> qPCR of <i>ChLAE1</i> and <i>ChVEL1</i>. cDNA samples are the same as A. Error bars represent range of fold change calculated according to standard deviation of ΔΔCt. Asterisks indicate p-value <0.01 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition. <b>F</b>. qPCR of <i>PKS1</i> and <i>PKS2</i>. Same as B. Asterisks indicate p-value <0.05 in T-test analysis in which both overexpression strains were compared to WT grown in the same condition.</p

<i>Cochliobolus heterostrophus</i> strains used in this study.

<p><i>Cochliobolus heterostrophus</i> strains used in this study.</p

<i>Chlae1</i> and <i>Chvel1</i> mutants show reduced virulence to maize with T- and N-cytoplasms.

<p><b>A.</b> Virulence on T-toxin sensitive T-cytoplasm corn. Leaves spray-inoculated with WT race T strain C4, the <i>nps6</i> mutant lacking extracellular siderophores (strain Chnps6-3), <i>Chvel1</i> mutant (strain ChW7), <i>Chlae1</i> mutant (strain ChW5), and T-toxin⁻, race O control strain C5. WT C4 shows chlorotic halos around beige lesion cores and chlorotic streaking (red arrow) due to T-toxin production. Control race O strain C5 makes defined beige-light brown lesions and no chlorotic streaking. The <i>nps6</i> mutant makes tiny beige lesions, but lots of chlorosis (red arrow) due to T-toxin production. <i>Chvel1</i> and <i>Chlae1</i> mutants show reduced lesion size compared to WT and the amount of chlorosis is less than that of WT or the <i>nps6</i> strain (red arrows). <b>B.</b> Virulence on T-toxin insensitive N-cytoplasm corn leaves. Leaves spray-inoculated with the same set of strains as in <b>A</b>, except for strain C5. On N-cytoplasm, <i>Chvel1</i> and especially <i>Chlae1</i>, mutants show reduced lesion size compared to WT but in both cases lesions are less reduced in size than those of the <i>nps6</i> strain. <b>C.</b> Quantification of disease on T-cytoplasm leaves shown in <b>A</b>. The length of the chlorosis and necrosis symptom associated with individual lesions was measured and the average chlorosis/necrosis ratio plotted. Error bars are standard deviation. Asterisks represent p-value <0.05 in T-test analysis when each mutant was compared with WT C4. <b>D.</b> Quantification of lesion sizes of N-cytoplasm leaves shown in <b>B</b>. The length of the necrosis lesion symptom associated with individual lesions was measured and the average plotted. Error bars are standard deviation. Asterisks represent p-value <0.05 in T-test analysis when each mutant was compared with WT C4. <b>E.</b> Quantification of disease on T-cms corn leaves caused by <i>ChVEL1</i> and <i>ChLAE1</i> overexpression strains, using methods described in <b>C</b> above. <b>F.</b> Quantification of lesion sizes on N-cytoplasm corn leaves caused by <i>ChVEL1</i> and <i>ChLAE1</i> overexpression strains, using methods described in <b>D</b> above.</p

<i>Chlae1</i> and <i>Chvel1</i> mutants make less, or less active, T-toxin and <i>ChVEL1</i> and <i>ChLAE1</i> overexpression strains make more T-toxin in constant dark than wild-type strains.

<p><b>A.</b> Microbial assay plates for T-toxin production. Plates were spread with <i>E. coli</i> cells carrying the <i>URF13</i> gene that confers sensitivity to T-toxin. Plugs of each fungal strain grown for eight days at 19°C on CMX medium under constant dark and constant light conditions, were placed on the plates, mycelium side up and incubated overnight. Clear area (halo) indicates T-toxin production and killing of <i>E. coli</i> cells. On each plate, the bottom single plug is a race O, T-toxin⁻ control (strain C5, no halo). Second row from bottom is race T, T-toxin⁺ control (strain C4, halos). Top three rows are three replicates of the mutant strain indicated. From left to right in each row are plugs of mycelium taken from the center to the edge of the colony. Note that the halo sizes around WT strain C4 are comparable in light, however, in the dark they are larger when plugs were taken from the outer (younger) edges of the colony. Both mutants form smaller halos compared to WT grown in the dark, howeve,r halos around <i>Chvel1</i> mutants are almost as big as those of WT grown in the light. <b>B. </b><i>ChVEL1</i> and <i>ChLAE1</i> overexpression strains grown on CMX medium supplemented with polygalacturonic acid (PGA) as described in A. Plates were set up as in A, except that overexpression strains were assayed instead of mutants. The <i>LAE1</i> overexpressing strain produced more T-toxin than WT under both light and dark conditions while the <i>VEL1</i> overexpressing strain produced more T-toxin than WT in the dark, but not in the light.</p

<i>Chlae1</i> and <i>Chvel1</i> mutants are hypersensitive to oxidative stress.

<p><b>A</b>. Hypersensitivity of <i>Chlae1</i> and <i>Chvel1</i> mutants to H₂O₂. Serial dilutions of conidial suspensions (left to right: 4, 2 and 1 µl) prepared from WT strain C4, <i>nps6</i>, <i>Chvel1</i>, <i>Chlae1</i> mutants, and <i>Chvel1</i> (<i>vel1</i>[<i>VEL1</i>]) and <i>Chlae1</i> (<i>lae1</i>[<i>LAE1</i>]) complemented strains were placed on complete medium (CM) with and without indicated concentrations of H₂O₂. The <i>Chvel1</i> and <i>Chlae1</i> mutants are more sensitive to oxidative stress mediated by H₂O₂ than WT and the complemented strains, but not as sensitive as the extracellular siderophore <i>nps6</i> mutant <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002542#ppat.1002542-Oide2" target="_blank">[32]</a>. <b>B</b>. qPCR analysis of <i>CAT3</i>, one of the three catalase-encoding genes in <i>C. heterostrophus</i>. <i>CAT3</i> expression was examined in the same set of strains as in <b>A</b>. Expression level relative to WT C4 at time 0 is shown. Error bars show range of fold change calculated according to standard deviation of ΔΔCt. Asterisks represent p-value <0.001 in T-test analysis in which each strain was compared with the corresponding WT C4 strain at the same time point. Deletion of <i>ChLAE1</i> does not affect expression of <i>CAT3</i>, while reduced expression was observed for <i>Chvel1</i> mutant (20 fold at time 0 and 4 fold at 30 min after H₂O₂ addition).</p