IFN priming is necessary but not sufficient to turn on a migratory dendritic cell program in lupus monocytes

Abstract

Blood monocytes from children with systemic lupus erythematosus (SLE) behave similar to dendritic cells (DCs), and SLE serum induces healthy monocytes to differentiate into DCs in a type I IFN-dependent manner. In this study, we found that these monocytes display significant transcriptional changes, including a prominent IFN signature, compared with healthy controls. Few of those changes, however, explain DC function. Exposure to allogeneic T cells in vitro reprograms SLE monocytes to acquire DC phenotype and function, and this correlates with both IFN-inducible (IP10) and proinflammatory cytokine (IL-1β and IL6) expression. Furthermore, we found that both IFN and SLE serum induce the upregulation of CCR7 transcription in these cells. CCR7 protein expression, however, requires a second signal provided by TLR agonists such as LPS. Thus, SLE serum "primes" a subset of monocytes to readily (<24 h) respond to TLR agonists and acquire migratory DC properties. Our findings might explain how microbial infections exacerbate lupus.

Figure 1. Phenotypic characterization of blood SLE monocytes

(A) Pediatric SLE patients display similar distribution of CD14⁺CD16⁻ and CD14^dimCD16⁺ cells, while the double positive (CD14⁺/CD16⁺) subpopulation is mildly expanded (p=0.012). (B) HLA-DR expression is mildly dowregulated, especially within the CD14⁺CD16⁻ monocyte fraction. (C) CD81, a co-receptor for HCV and HIV that is known to be downregulated by IFN-α, is significantly downregulated in CD14⁺CD16⁻SLE monocytes.

Figure 2. Gene expression profile of blood SLE monocytes

(A) Supervised hierarchical clustering of transcripts regulated upon culturing healthy blood monocytes with recombinant type I IFN at different type points (1, 6, 24 h, 48, and 72 hrs) and similarly expressed in ex-vivo SLE but not healthy blood monocytes (n=560). (B) Supervised hierarchical clustering of transcripts differentially expressed in ex vivo SLE monocytes that are not regulated by recombinant type I IFN (n=1069). (C) DC maturation markers, such as CD83, and co-stimulatory molecules such as CD40, CD80, and CD86 were not differentially expressed in SLE monocytes. (D) IFN-inducible upregulated transcripts included apoptosis-inducing molecules such as TRAIL and FAS, genes encoding ubiquitination-related molecules, such as culin 1, and chemokines and chemokine receptors, such as CXCL10 (IP10), CCR1, CCR5, CCR7, CCL2 (MCP1) and CCL8 (MCPL2), among others. Among transcripts that we could not formally ascribe to IFN-α, there was a remarkable down-regulation of ribosomal protein-encoding transcripts.

Figure 3. The transcriptional program of blood SLE monocytes overlaps minimally with that of healthy blood mDCs

(A) Healthy blood mDCs differentially express >2,400 transcripts compared to blood monocytes. Of those, only 269 transcripts are differentially expressed between SLE and healthy blood monocytes. (B) Genes related to MHC class II are downregulated in SLE monocytes while they are upregulated in blood mDCs. (C) A few transcripts, including the chemokine receptor CCR5, which is expressed in blood circulating cells, are similarly expressed in SLE monocytes and blood mDCs. (D) Transcripts upregulated in blood SLE monocytes and downregulated in healthy mDCs compared to healthy blood monocytes.

Figure 4. Monocytes from some SLE patients acquire DC function upon co-culture with allogeneic T cells

(A) Experimental design to select SLE monocyte transcripts involved in T cell activation. (B) Differentially regulated genes in SLE monocytes that induced and did not induce CD4 T cell proliferation as measured by CFSE dilution at day 6. Genes induced upon 6 hr co-culture with CD4 T cells (1291 transcripts) that overlap with mDCs (n= 123) are displayed. (C) Some of the transcripts expressed in alloreaction-inducing SLE monocytes include HLA class II and innate immunity-related transcripts. (D) HLA-DR staining of SLE blood monocytes upon 48hr co-culture with allogeneic T cells correlates with T cell proliferation at day 6. E. IL1β and IL6 levels are elevated in 3/4 alloreaction (+) but none of the alloreaction (-) co-cultures.

Figure 5. LPS synergizes with IFN and SLE serum to rapidly (<24 h) induce CCR7 expression on a fraction of healthy blood monocytes

(A)Positively selected monocytes were cultured with autologous serum (AS), recombinant IFN or serum from three pediatric SLE patients for 18–24 h. In the absence of LPS, no CCR7 expression is observed. However, addition of LPS synergizes with IFN and SLE serum to induce CCR7 expression. (B) Negatively selected monocytes replicate the observations made with positively selected monocytes (C) Healthy elutriated monocytes upregulate CCR7 in the presence of some SLE sera without addition of LPS (i.e.SLE-80). CCR7 expression was greatly enhanced by the addition of LPS to these cultures. (D) Combining the three different isolation methods reveals that LPS induces CCR7 expression on monocytes and synergizes with IFN and SLE serum.