Macrophage migration inhibitory factor (MIF) is essential for Type 2 effector cell immunit y t o an int est inal helmint h parasite

Immunity to intestinal helminths is known to require both innate and adaptive components of the immune system activated along the Type 2 IL-4R/ STAT6-dependent pathway. We have found that macrophage migration inhibitory factor (MIF) is essential for the development of effective immunity to the intestinal helminth Heligmosomoides polygyrus, even following vaccination which induces sterile immunity in wild-type mice. A chemical inhibitor of MIF, 4-IPP, was similarly found to compromise anti-parasite immunity. Cellular analyses found that the adaptive arm of the immune response, including IgG1 antibody responses and Th2-derived cytokines, was intact and that Foxp3+ T regulatory cell responses were unaltered in the absence of MIF. However, MIF was found to be an essential cytokine for innate cells, with ablated eosinophilia and ILC2 responses, and delayed recruitment and activation of ΡΕΗΧΤΥΜΕΛΙΨŋΪΤŋΪΜΙŋŹ⁷ŋΥΜΙΣΤΪﬃΥΙŋŝΙήΥΧΙΨΨΝΣΛŋẃΧΛΝΣΕΨΙŋ⁶ũŋẅΜΝΠ⁸ũŋΕΣΘŋR(cid:324)űŹŪΕΠΥΜΕŦŋΫΥΤΣŋΝΣΚΙΗΪΝΤΣŋΤΚŋŹ″țŪΘΙΚΝΗΝΙΣΪŋΡΝΗΙ₆ŋΕ macrophage deficit was also seen in wild-type BALB/ c mice exposed to 4-IPP. Gene expression analysis of intestinal and lymph node tissues from MIF-deficient and -sufficient infected mice indicated significantly reduced levels of Arl2bp, encoding a factor involved in nuclear localization of STAT3. We further found that STAT3-deficient macrophages expressed less Arginase-1, and that mice lacking STAT3 in the myeloid compartment (LysMCrexSTAT3fl/ fl) were unable to reject a secondary infection with H. polygyrus. Abstract Immunity to intestinal helminths is known to require both innate and adaptive components of the 20 immune system activated along the Type 2 IL-4R/STAT6-dependent pathway. We have found 21 that macrophage migration inhibitory factor (MIF) is essential for the development of effective 22 immunity to the intestinal helminth Heligmosomoides polygyrus , even following vaccination 23 which induces sterile immunity in wild-type mice. A chemical inhibitor of MIF, 4-IPP, was 24 similarly found to compromise anti-parasite immunity. Cellular analyses found that the adaptive 25 arm of the immune response, including IgG1 antibody responses and Th2-derived cytokines, was 26 intact and that Foxp3 + T regulatory cell responses were unaltered in the absence of MIF. 27 However, MIF was found to be an essential cytokine for innate cells, with ablated eosinophilia 28 and ILC2 responses, and delayed recruitment and activation of macrophages to the M2 29 phenotype (expressing Arginase 1, Chil3, and R Ε LM- α ) upon infection of MIF-deficient mice; a 30 macrophage deficit was also seen in wild-type BALB/c mice exposed to 4-IPP. Gene expression 31 analysis of intestinal and lymph node tissues from MIF-deficient and -sufficient infected mice 32 indicated significantly reduced levels of Arl2bp , encoding a factor involved in nuclear 33 localization of STAT3. We further found that STAT3-deficient macrophages expressed less 34 Arginase-1, and that mice lacking STAT3 in the myeloid compartment (LysM Cre xSTAT3 fl/fl ) 35 were unable to reject a secondary infection with H. polygyrus . We thus conclude that in the 36 context of a Type 2 infection, MIF plays a critical role in polarizing macrophages into the 37 protective alternatively-activated phenotype, and that STAT3 signaling may make a previously 38 unrecognized contribution to immunity to helminths. (PMSF) (Sigma) a TissueLyser (Qiagen). Samples were centrifuged at 12,000 rpm for 10 mins to remove debris and supernatants added to ELISAs, at a 1:10 dilution, to measure RELM- α (Peprotech) and Chil3 (R&D). Levels were normalised to total protein content measured using a Bradford assay. The same ELISA sets were used to analyse peritoneal lavage levels of RELM- α and Chi3. - / mice do not have an intrinsic defect in ILC2 induct ion . IL - 5 + ILC2s as a proportion of live cells in digested lung tissue of BALB/c or MIF - / - m ice treated intranasally with PBS or rIL - 33, measured by flow cytometry. Data are representative of two independent experiments, and were analyzed by nonparametric statistics. normal ability to release the key alarmin IL - 33 upon stimulation. Levels of IL mice r after intranasal administration of 10 µ g Alternaria antigen, measured by ELISA. Results are combined from two experiments with similar results.