GPR43 regulates marginal zone B-cell responses to foreign and endogenous antigens.

Marginal zone (MZ) B cells are innate-like B cells that produce polyreactive antibodies with an affinity for microbial molecular patterns and carbohydrate ligands. MZ B cells have been shown to be important in mediating immunity to various bacteria including Streptococcus pneumoniae and are also implicated in inflammatory syndromes including lupus erythematosus. The intestinal microbiota is responsible for producing short-chain fatty acids, which can regulate immune cell function by several mechanisms including ligation of the G-protein-coupled receptor (GPR)43. Herein, we show that MZ B cells express Gpr43 messenger RNA and that the absence of this receptor impacts on MZ B-cell surface marker expression and antibody production. In T-cell-independent responses to the hapten 4-hydroxy-3-nitrophenylacetic acid (NP), mice deficient in GPR43 displayed higher serum titers of NP-specific antibodies. Moreover, in response to a pneumococcal polysaccharide vaccine, GPR43-deficient mice developed robust serum antibody responses and had markedly increased numbers of splenic antibody-secreting cells, compared with control mice. Finally, serum immunoglobulin M autoantibodies to double-stranded DNA and phosphatidylcholine were increased in resting 10-15-week-old mice lacking GPR43. Taken together, mice lacking GPR43 have heightened antibody responses to T-cell-independent antigens, which may be a result of impaired regulation of MZ B cells.

BCL-XL and MCL-1 are the key BCL-2 family proteins in melanoma cell survival.

Malignant melanoma is one of the most difficult cancers to treat due to its resistance to chemotherapy. Despite recent successes with BRAF inhibitors and immune checkpoint inhibitors, many patients do not respond or become resistant to these drugs. Hence, alternative treatments are still required. Due to the importance of the BCL-2-regulated apoptosis pathway in cancer development and drug resistance, it is of interest to establish which proteins are most important for melanoma cell survival, though the outcomes of previous studies have been conflicting. To conclusively address this question, we tested a panel of established and early passage patient-derived cell lines against several BH3-mimetic drugs designed to target individual or subsets of pro-survival BCL-2 proteins, alone and in combination, in both 2D and 3D cell cultures. None of the drugs demonstrated significant activity as single agents, though combinations targeting MCL-1 plus BCL-XL, and to a lesser extent BCL-2, showed considerable synergistic killing activity that was elicited via both BAX and BAK. Genetic deletion of BFL-1 in cell lines that express it at relatively high levels only had minor impact on BH3-mimetic drug sensitivity, suggesting it is not a critical pro-survival protein in melanoma. Combinations of MCL-1 inhibitors with BRAF inhibitors also caused only minimal additional melanoma cell killing over each drug alone, whilst combinations with the proteasome inhibitor bortezomib was more effective in multiple cell lines. Our data show for the first time that therapies targeting specific combinations of BCL-2 pro-survival proteins, namely MCL-1 plus BCL-XL and MCL-1 plus BCL-2, could have significant benefit for the treatment of melanoma.

PPAR-γ promotes type 2 immune responses in allergy and nematode infection.

A hallmark of immunity to worm infections and many allergies is a strong type 2 immune response. This is characterized by the production of cytokines interleukin-5 (IL-5) and IL-13 by adaptive T helper 2 (TH2) cells and/or type 2 innate lymphoid cells. Peroxisome proliferator activated receptor-γ (PPAR-γ) is typically regarded as an anti-inflammatory factor. We report that TH2 cells express high levels of PPAR-γ in response to the allergen house dust mite and after infection with the parasite Heligmosomoides polygyrus Mice lacking PPAR-γ in T cells failed to effectively differentiate into IL-5- and IL-13-secreting cells and, hence, did not develop TH2 cell-associated pathologies, including goblet cell metaplasia and eosinophilia, in response to allergen challenge. Furthermore, these mice could not mount protective immune responses to nematode infection. In addition, mice lacking PPAR-γ in T cells had greatly reduced frequencies of TH2 cells in visceral adipose tissue. Mechanistically, PPAR-γ appeared to promote the expression of the IL-33 receptor on the surface of TH2 cells. These results pinpoint PPAR-γ as a factor that drives type 2 responses in allergy, worm infection, and visceral adipose tissue.

Hepatocyte growth factor renders BRAF mutant human melanoma cell lines resistant to PLX4032 by downregulating the pro-apoptotic BH3-only proteins PUMA and BIM.

A large proportion of melanomas harbour the activating BRAFV600E mutation that renders these cells dependent on MAPK signalling for their survival. Although the highly specific and clinically approved BRAFV600E kinase inhibitor, PLX4032, induces apoptosis of melanoma cells bearing this mutation, the underlying molecular mechanisms are not fully understood. Here, we reveal that PLX4032-induced apoptosis depends on the induction of the pro-apoptotic BH3-only protein PUMA with a minor contribution of its relative BIM. Apoptosis could be significantly augmented when PLX4032 was combined with an inhibitor of the pro-survival protein BCL-XL, whereas neutralization of the pro-survival family member BCL-2 caused no additional cell death. Although the initial response to PLX4032 in melanoma patients is very potent, resistance to the drug eventually develops and relapse occurs. Several factors can cause melanoma cells to develop resistance to PLX4032; one of them is the activation of the receptor tyrosine kinase cMET on melanoma cells by its ligand, hepatocyte growth factor (HGF), provided by the tumour microenvironment or the cancer cells themselves. We found that HGF mediates resistance of cMET-expressing BRAF mutant melanoma cells to PLX4032-induced apoptosis through downregulation of PUMA and BIM rather than by increasing the expression of pro-survival BCL-2-like proteins. These results suggest that resistance to PLX4032 may be overcome by specifically increasing the levels of PUMA and BIM in melanoma cells through alternative signalling cascades or by blocking pro-survival BCL-2 family members with suitable BH3 mimetic compounds.

Spontaneous retrotransposon insertion into TNF 3'UTR causes heart valve disease and chronic polyarthritis.

Rheumatoid arthritis (RA) and ankylosing spondylitis (AS) are chronic inflammatory diseases that together affect 2-3% of the population. RA and AS predominantly involve joints, but heart disease is also a common feature in RA and AS patients. Here we have studied a new spontaneous mutation that causes severe polyarthritis in bone phenotype spontaneous mutation 1 (BPSM1) mice. In addition to joint destruction, mutant mice also develop aortic root aneurism and aorto-mitral valve disease that can be fatal depending on the genetic background. The cause of the disease is the spontaneous insertion of a retrotransposon into the 3' untranslated region (3'UTR) of the tumor necrosis factor (TNF), which triggers its strong overexpression in myeloid cells. We found that several members of a family of RNA-binding, CCCH-containing zinc-finger proteins control TNF expression through its 3'UTR, and we identified a previously unidentified regulatory element in the UTR. The disease in BPSM1 mice is independent of the adaptive immune system and does not appear to involve inflammatory cytokines other than TNF. To our knowledge, this is the first animal model showing both polyarthritis and heart disease as a direct result of TNF deregulation. These results emphasize the therapeutic potential of anti-TNF drugs for the treatment of heart valve disease and identify potential therapeutic targets to control TNF expression and inflammation.

Foxo-mediated Bim transcription is dispensable for the apoptosis of hematopoietic cells that is mediated by this BH3-only protein.

The BH3-only protein Bim is a critical initiator of apoptosis in hematopoietic cells. Bim is upregulated in response to growth factor withdrawal and in vitro studies have implicated the transcription factor Foxo3a as a critical inducer. To test the importance of this regulation in vivo, we generated mice with mutated Foxo-binding sites within the Bim promoters (Bim(ΔFoxo/ΔFoxo)). Contrary to Bim-deficient mice, Bim(ΔFoxo/ΔFoxo) mice had a normal hematopoietic system. Moreover, cytokine-dependent haematopoietic cells from Bim(ΔFoxo/ΔFoxo) and wt mice died at similar rates. These results indicate that regulation of Bim by Foxo transcription factors is not critical for the killing of hematopoietic cells.