CD38 promotes pristane-induced chronic inflammation and increases susceptibility to experimental lupus by an apoptosis-driven and TRPM2-dependent mechanism

In this study, we investigated the role of CD38 in a pristane-induced murine model of lupus. CD38-deficient (Cd38−/−) but not ART2-deficient (Art2−/−) mice developed less severe lupus compared to wild type (WT) mice, and their protective phenotype consisted of (i) decreased IFN-I-stimulated gene expression, (ii) decreased numbers of peritoneal CCR2hiLy6Chi inflammatory monocytes, TNF-α-producing Ly6G+ neutrophils and Ly6Clo monocytes/macrophages, (iii) decreased production of anti-single-stranded DNA and anti-nRNP autoantibodies, and (iv) ameliorated glomerulonephritis. Cd38−/− pristane-elicited peritoneal exudate cells had defective CCL2 and TNF-α secretion following TLR7 stimulation. However, Tnf-α and Cxcl12 gene expression in Cd38−/− bone marrow (BM) cells was intact, suggesting a CD38-independent TLR7/TNF-α/CXCL12 axis in the BM. Chemotactic responses of Cd38−/− Ly6Chi monocytes and Ly6G+ neutrophils were not impaired. However, Cd38−/− Ly6Chi monocytes and Ly6Clo monocytes/macrophages had defective apoptosis-mediated cell death. Importantly, mice lacking the cation channel TRPM2 (Trpm2−/−) exhibited very similar protection, with decreased numbers of PECs, and apoptotic Ly6Chi monocytes and Ly6Clo monocytes/macrophages compared to WT mice. These findings reveal a new role for CD38 in promoting aberrant inflammation and lupus-like autoimmunity via an apoptosis-driven mechanism. Furthermore, given the implications of CD38 in the activation of TRPM2, our data suggest that CD38 modulation of pristane-induced apoptosis is TRPM2-dependent.


Ly6C lo monocytes/macrophages and Ly6G + neutrophils from WT and Cd38 -/mice.
In agreement with previously published studies 2 , pristane-elicited Ly6C hi monocytes expressed higher levels of Tlr7 than any other peritoneal cell subset analysed, including Ly6C lo monocytes (Fig. S8a). However, no difference was observed in Tlr7 gene expression between WT and Cd38 -/mice. Likewise, Ly6C hi monocytes displayed the highest Tlr9 expression out of the 3 PEC populations analysed, with increased expression in Cd38 -/mice (Fig. S8b). In contrast, Gr1 expression was almost exclusively detected in Ly6G + neutrophils as expected (Fig. S8c). Moreover, expression of Tnf-α was higher in neutrophils than in the other cell types and showed an increased expression in WT mice (Fig. S8d). With regards to ISGs, the expression of Isg15 and Irf7 were detected primarily in Ly6G + neutrophils, while Mx1 and Ccl2 were detected in Ly6C hi monocytes with a distinct cell profile (Fig. S8e-h). Thus, expression of Isg15, and to a lesser extent Irf7, was increased in cells from pristane-treated WT mice, while Mx1, and Ccl2 expression was increased in cells from pristane-treated Cd38 -/mice. In contrast, Ly6C lo monocytes showed a relatively low expression of all these genes.
Overall, these results suggest that the observed defective secretion of TNF-α by PECs isolated from 2-weeks pristane-treated Cd38 -/mice in response to TLR7 agonist R848 was not due to a defective transcription of the Tlr7 gene, and defective secretion of CCL2 did not reflect diminished transcription of the Ccl2 gene by Cd38 -/-Ly6C hi monocytes upon pristane treatment.
In vitro chemotaxis of BM Ly6C hi monocytes to CCL2 and CXCL12 is CD38independent. Previous studies have shown that chemotaxis of human monocytes to the FPR ligand fMLP was independent of cADPR, while chemotaxis to the CCR1/CCR5 ligands RANTES and MIP-1, and to the CXCR4 ligand SDF-1, was regulated by cADPR 8 . Unlike the extensive chemotactic studies performed with human monocytes, those with murine monocytes are limited primarily due to the difficulty in obtaining the large numbers of highly purified monocytes needed for such analyses. Altered recruitment of inflammatory Ly6C hi monocytes to sites of Listeria infection in Cd38 -/mice could be explained in part by an impaired migration of these cells 9 , so we assessed the chemotaxis of mouse monocytes to CCL2 and CXCL12.
We purified Ly6C hi monocytes from the bone marrow of WT and Cd38 -/mice by positive selection using anti-CD115 antibody. Purity was assessed by flow cytometry and found to be ≥70% (Fig. S9a). The responses of WT and Cd38 -/monocytes to chemoattractants CCL2 and CXCL12 were measured in vitro, and found to be similar between the two groups ( Fig S9b). Therefore, it is unlikely that the reduced accumulation of Ly6C hi monocytes in the peritoneum of pristane-treated Cd38 -/mice was due to an intrinsically defective chemotactic response to these chemokines.

In vivo migration of Ly6G + neutrophils to the peritoneum in response to zymosan
challenge is CD38-independent. WT and Cd38 -/mice were injected i.p. with 0.5 mg of zymosan A following the model of acute self-limited inflammation 10 , and the in vivo migratory capabilities of Ly6G + neutrophils were assessed 4-hours post treatment. In agreement with previously published data 10 , PECs consisted predominantly of Ly6G + neutrophils during the initiation phase (Fig. S9c). However, no major differences in frequencies of Ly6G + neutrophils were observed between Cd38 -/-(83.8% ± 0.8) and WT (81.9% ± 2.5) mice ( Fig. S9c and S9d). Zymosan-treated mice also had an increased frequency of peritoneal Ly6C hi monocytes (5-10%) compared to untreated control mice (~1%). Moreover, resident macrophages, which in control mice made up >90% of the CD11b + cells (Fig. S9c, left panel), sharply decreased in number and frequency following zymosan treatment. We also measured the frequencies of Ly6G + neutrophils in the spleen and peripheral blood of zymosan-treated mice, and no difference was detected between Cd38 -/and WT mice (data not shown), suggesting a normal mobilization of neutrophils from the BM to the periphery in Cd38 -/mice. Hence, our data suggest that the trafficking of neutrophils in Cd38 -/mice is not impaired in the early phase of this model of acute inflammation. 5 Target gene (  The corresponding band is indicated by an arrow. Each lane represents pooled protein extracts from 3 mice/group.