Pathogen-like triggering of monocytes in rheumatoid ...
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INTRODUCTION The initial trigger(s) of the immune system in rheumatoid arthritis (RA) remains unknown. We recently demonstrated that RA is characterised by increased monocyte production in bone marrow, their premature egress into blood (so called left-shift) and rapid infiltration into inflamed joints, where dominant steps of activation and increased turnover of monocytes seem to occur. In contrast to RA, where immunological processes are key steps of joint destruction, in osteoarthritis (OA) joint destruction is related to trauma and cartilage degeneration but limited signs of inflammation. In this study we aimed to dissect inflammation in RA patients with progressive disease by analysing synovial tissues (ST), synovial fluid (SF) and blood from these patients. These were compared to OA to determine the dominant cell type infiltration and the leading molecular immune responses associated wit joint destruction in RA. MATERIAL AND METHOD Synovial tissue transcriptomes from 10 RA and 10 OA patients were compared and differentially expressed genes (DEG) were analysed with GSEA (Gene Set Enrichment Analysis), IPA (Ingenuity pathway analysis), DAVID annotation and by reference transcriptome mapping (RTM). Synovial fluid and blood cells from 6 RA and 6 OA patients were analysed by flow cytometry. Synovial fluid and serum from 18 RA and 15 OA patients were utilised for measuring 28 soluble markers by ELISA and Multiplex technology. RESULTS II) Transcriptome of RA-ST showed a substantial overlap with inflammatory patterns triggered by various microbes and inflammatory mediators in myeloid cells: To analyse activation of innate and adaptive immunity in RA-ST, the coexpression analysis with reference transcriptomes included 1) synovial fibroblast, 2) endothelial cells, 3) platelets, 4) B-cells: naïve-, memory-, germinal centre-B cells and plasma cells; 5) T-cells: naïve-, regulatory-, γδ-Tcells, Th1, Th2, Th17 and 6) myeloid cells activated with: M. tuberculosis, S. aureus, L. rhamnosus, A. fumigatus, F. novicida, Ch. Pneumonia, yellow fiver virus, alarmin S100A8, NOD2L and TLR2/1L, Galectin1, Zymosan, TNF, LPS, IFNα, IFNγ, as well as M1-Mf and M2-Mf. This global analysis dissected the RA-ST and OA-ST transcriptomes in a very comprehensive way and demonstrated that the most prominent patterns in RA-ST were the patterns of pathogen (bacterial and fungal) induced activation of myeloid cells. Pathogen-like triggering of monocytes in rheumatoid arthritis joints contrasts the scavenging-like monocyte response in osteoarthritis 1 Charité - Universitätsmedizin, Berlin, Germany; 2 Deutsches Rheuma–Forschungszentrum Berlin, Germanyin Biljana Smiljanovic 1 , Till Sörensen 1 , Bruno Stuhlmüller 1 , Mark Bonin 1 , Andreas Radbruch 2 , Andreas Grützkau 2 , Gerd R. Burmester 1 , Thomas Häupl 1 III) Flow cytometry and ELISA analyses of immune cells and soluble markers in blood and synovial fluid emphasised activation of innate immunity: Figure 1. In total, 2019 probe-sets were differentially expressed between RA-ST & OA-ST (1010 up- & 1009 down-regulated in RA-ST) as showed by hierarchical clustering (A) and principal component analysis (B). One of 10 dominant networks in RA-ST (C) emphasised activation of TCR, NFkB & STAT1 signalling, as well as cytokine production. A B C RA OA RA-ST OA-ST RESULTS I) Transcriptome of RA-ST portrayed activation of both innate and adaptive immunity: Comparisons between RA-ST and OA-ST transcriptomes identified differential expression of 2019 (1580 genes). Functional analysis of transcriptional changes suggested infiltration and activation of both innate and adaptive immunity in RA-ST. Figure 2. in total, 1010 (A) & 1009 (D) probe-sets were up-regulated in RA & OA, respectively; coexpression matrices (B & E) determined patterns of infiltrated and activated cells in RA & OA determined by 167 reference transcriptomes (C&F). A B C D E F RA-SF OA-SF Freq (% of CD14 Mo) CD14++CD16- CD14++CD16+ CD14+CD16+ CD14++CD16- CD14++CD16+ CD14+CD16+ 0.0 0.2 0.4 0.6 0.8 1.0 Blood SF **0.002 FSC SSC CD14 CD16 HLA -DR FSC SSC CD14 CD16 HLA -DR RA-SF_MMP3 OA-SF_MMP3 RA-S_MMP3 OA-S_MMP3 HD-S_MMP3 RA-SF_S100A8/9 OA-SF_S100A8/9 RA-S_S100A8/9 OA-S_S100A8/9 HD-S_S100A8/9 RA-SF_S100P OA-SF_S100P RA-S_S100P OA-S_S100P HD-S_S100P RA-SF_CCL18 OA-SF_CCL18 RA-S_CCL18 OA-S_CCL18 HD-S_CCL18 RA-SF_CXCL13 OA-SF_CXCL13 RA-S_CXCL13 OA-S_CXCL13 HD-S_CXCL13 10 -3 10 -2 10 -1 10 0 10 1 10 2 10 3 10 4 10 5 10 6 Conc (ng/mL) RA-SF_MIF OA-SF_MIF RA-S_MIF OA-S_MIF HD-S_MIF RA-SF_CXCL10 OA-SF_CXCL10 RA-S_CXCL10 OA-S_CXCL10 HD-S_CXCL10 RA-SF_CXCL9 OA-SF_CXCL9 RA-S_CXCL9 OA-S_CXCL9 HD-S_CXCL9 RA-SF_CCL2 OA-SF_CCL2 RA-S_CCL2 OA-S_CCL2 HD-S_CCL2 10 -2 10 -1 10 0 10 1 10 2 10 3 Conc (ng/mL) Figure 3. Three monocyte subsets were identified in blood (A) classical (CD14 ++ CD16 - ), intermediate (CD14 ++ CD16 + ) and non- classical (CD14 + CD16 ++ ), while in SF (B), intermediate subset & myeloid like cells were identified. Soluble molecules elevated in (D) RA-SF & RA-serum & (E) elevated only in RA-SF, when compared to OA related samples. RA-SF OA-SF RA-PB OA-PB HD-PB A B C D E CONCLUSION Monocytes play an essential role in RA pathogenesis by responding to pathogen trigger(s) that initiate innate immune response, drive chronicity as well as activate adaptive immunity. In OA these cells play an important role in scavenging, tissue regeneration and wound healing. LITERATUR Smiljanovic et al. (2018), Ann Rheum Dis. 77(2):300-308 CD14 CD16 MDC GMC CMP CLP HSC DC Granulocyte Monocyte Blood Bone marrow CD14 ++ CD16 - (classical ) CD14 ++ CD16 + (intermediate) CD14 + CD16 ++ (non-classical) healthy RA Left-shift monocytopoiesis & monocyte differentiation Immune cells: infiltration, activation, „consumption“
Transcript of Pathogen-like triggering of monocytes in rheumatoid ...
INTRODUCTION The initial trigger(s) of the immune system in rheumatoid arthritis (RA) remains unknown. We recently demonstrated that RA is characterised by increased monocyte production in bone marrow, their premature egress into blood (so called left-shift) and rapid infiltration into inflamed joints, where dominant steps of activation and increased turnover of monocytes seem to occur. In contrast to RA, where immunological processes are key steps of joint destruction, in osteoarthritis (OA) joint destruction is related to trauma and cartilage degeneration but limited signs of inflammation. In this study we aimed to dissect inflammation in RA patients with progressive disease by analysing synovial tissues (ST), synovial fluid (SF) and blood from these patients. These were compared to OA to determine the dominant cell type infiltration and the leading molecular immune responses associated wit joint destruction in RA.
MATERIAL AND METHOD Synovial tissue transcriptomes from 10 RA and 10 OA patients were compared and differentially expressed genes (DEG) were analysed with GSEA (Gene Set Enrichment Analysis), IPA (Ingenuity pathway analysis), DAVID annotation and by reference transcriptome mapping (RTM). Synovial fluid and blood cells from 6 RA and 6 OA patients were analysed by flow cytometry. Synovial fluid and serum from 18 RA and 15 OA patients were utilised for measuring 28 soluble markers by ELISA and Multiplex technology.
RESULTS II) Transcriptome of RA-ST showed a substantial overlap with inflammatory patterns triggered by various microbes and inflammatory mediators in myeloid cells: To analyse activation of innate and adaptive immunity in RA-ST, the coexpression analysis with reference transcriptomes included 1) synovial fibroblast, 2) endothelial cells, 3) platelets, 4) B-cells: naïve-, memory-, germinal centre-B cells and plasma cells; 5) T-cells: naïve-, regulatory-, γδ-Tcells, Th1, Th2, Th17 and 6) myeloid cells activated with: M. tuberculosis, S. aureus, L. rhamnosus, A. fumigatus, F. novicida, Ch. Pneumonia, yellow fiver virus, alarmin S100A8, NOD2L and TLR2/1L, Galectin1, Zymosan, TNF, LPS, IFNα, IFNγ, as well as M1-Mf and M2-Mf. This global analysis dissected the RA-ST and OA-ST transcriptomes in a very comprehensive way and demonstrated that the most prominent patterns in RA-ST were the patterns of pathogen (bacterial and fungal) induced activation of myeloid cells.
Pathogen-like triggering of monocytes in rheumatoid arthritis joints contrasts the scavenging-like monocyte response in osteoarthritis
1Charité - Universitätsmedizin, Berlin, Germany; 2Deutsches Rheuma–Forschungszentrum Berlin, Germanyin
Biljana Smiljanovic1, Till Sörensen1, Bruno Stuhlmüller1, Mark Bonin1, Andreas Radbruch2, Andreas Grützkau2, Gerd R. Burmester1, Thomas Häupl1
III) Flow cytometry and ELISA analyses of immune cells and soluble markers in blood and synovial fluid emphasised activation of innate immunity:
Figure 1. In total, 2019 probe-sets were differentially expressed between RA-ST & OA-ST (1010 up- & 1009 down-regulated in RA-ST) as showed by hierarchical clustering (A) and principal component analysis (B). One of 10 dominant networks in RA-ST (C) emphasised activation of TCR, NFkB & STAT1 signalling, as well as cytokine production.
A B C
RA
OA
RA-ST OA-ST
RESULTS I) Transcriptome of RA-ST portrayed activation of both innate and adaptive immunity: Comparisons between RA-ST and OA-ST transcriptomes identified differential expression of 2019 (1580 genes). Functional analysis of transcriptional changes suggested infiltration and activation of both innate and adaptive immunity in RA-ST.
Figure 2. in total, 1010 (A) & 1009 (D) probe-sets were up-regulated in RA & OA, respectively; coexpression matrices (B & E) determined patterns of infiltrated and activated cells in RA & OA determined by 167 reference transcriptomes (C&F).
A B C
D E F
RA-SF
OA-SF
Freq
(% o
f CD
14 M
o)
CD14++
CD16-
CD14++
CD16+
CD14+C
D16+
CD14++
CD16-
CD14++
CD16+
CD14+C
D16+
0.0
0.2
0.4
0.6
0.8
1.0
Blood SF
**0.002
FSC
SSC
CD14
CD16
HLA-DR
FSC
SSC
CD14
CD16
HLA-DR
RA
-SF_
MM
P3O
A-S
F_M
MP3
RA
-S_M
MP3
OA
-S_M
MP3
HD
-S_M
MP3
RA
-SF_
S100
A8/
9O
A-SF
_S10
0A8/
9R
A-S
_S10
0A8/
9O
A-S_
S100
A8/9
HD
-S_S
100A
8/9
RA
-SF_
S100
P O
A-SF
_S10
0PRA
-S_S
100P
OA-
S_S1
00P
HD-S
_S10
0PR
A-S
F_C
CL1
8O
A-S
F_C
CL1
8R
A-S
_CC
L18
OA
-S_C
CL1
8H
D-S
_CC
L18
RA
-SF_
CXC
L13
OA
-SF_
CXC
L13
RA
-S_C
XCL1
3O
A-S
_CXC
L13
HD
-S_C
XCL1
310-310-210-1100101102103104105106
Con
c (n
g/m
L)
RA
-SF_
MIF
OA
-SF_
MIF
RA
-S_M
IFO
A-S
_MIF
HD
-S_M
IFR
A-S
F_C
XCL1
0O
A-S
F_C
XCL1
0R
A-S
_CXC
L10
OA
-S_C
XCL1
0H
D-S
_CXC
L10
RA
-SF_
CXC
L9O
A-S
F_C
XCL9
RA
-S_C
XCL9
OA
-S_C
XCL9
HD
-S_C
XCL9
RA
-SF_
CC
L2O
A-S
F_C
CL2
RA
-S_C
CL2
OA
-S_C
CL2
HD
-S_C
CL2
10-2
10-1
100
101
102
103
Con
c (n
g/m
L)
Figure 3. Three monocyte subsets were identified in blood (A) classical (CD14++CD16-), intermediate (CD14++CD16+) and non-classical (CD14+CD16++), while in SF (B), intermediate subset & myeloid like cells were identified. Soluble molecules elevated in (D) RA-SF & RA-serum & (E) elevated only in RA-SF, when compared to OA related samples.
RA-SF OA-SF RA-PB OA-PB HD-PB
A B C
D E
CONCLUSION Monocytes play an essential role in RA pathogenesis by responding to pathogen trigger(s) that initiate innate immune response, drive chronicity as well as activate adaptive immunity. In OA these cells play an important role in scavenging, tissue regeneration and wound healing.
LITERATUR Smiljanovic et al. (2018), Ann Rheum Dis. 77(2):300-308
CD
14
CD16
MDC$
GMC$
CMP$
CLP$HSC$
DC$
Granulocyte$
Monocyte$Synovial fluid
Blood Bone marrow
TNFα%
Macrophage%
DC%
B%cell%
IL15$IL12$
IL17$IFNγ%
IL6$
T%cell%
Y%
IL23$
Y%
MMP$
Synovial tissue
CD14++CD16- (classical )
CD14++CD16+ (intermediate)
CD14+CD16++ (non-classical)
healthy
RA Left-shift
monocytopoiesis & monocyte differentiation Immune cells: infiltration, activation, „consumption“
