Search

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

Colorectal cancer is the second most commonly diagnosed cancer worldwide, and may develop sporadically or due to chronic colitis. Macrophages are a major component of the colorectal tumour microenvironment and may be broadly classified as classically-activated (CAM) ‘inflammatory’ or alternatively-activated (AAM) ‘pro-angiogenic’ macrophages. Hematopoietic Cell Kinase (Hck) is a myeloid-specific Src family kinase involved in alternative macrophage polarisation, which correlates with a poor prognosis in human colorectal cancer. To investigate the role of Hck in colorectal tumourigenesis, Hck mutant mice that express a constitutively active form of Hck (HckCA) were subjected to models of sporadic and colitis-associated colorectal cancer. In both models, HckCA mice demonstrated increased tumour size and multiplicity compared to wild-type animals. FACS analysis of tumour immune-cell infiltrates did not reveal a difference in the number of tumour-associated macrophages (TAMs) between genotypes; however, CD206+ alternatively-activated TAMs were significantly greater in HckCA mice compared to wild-type. Furthermore, qPCR analysis highlighted a significant increase in genes associated with alternative-macrophage activation in HckCA TAMs. To confirm the role of macrophages in HckCA-driven tumourigenesis, wild-type mice were reconstituted with HckCA bone-marrow, and subjected to sporadic or colitis-associated colon cancer induction, after which we observed an increased tumour burden and an AAM phenotype compared to wild-type bone-marrow recipients. Finally, we have shown that the inhibition of Hck using a small molecule inhibitor reduces alternative macrophage polarisation. Collectively, our results suggest that excessive Hck activation enhances colorectal tumourigenesis by facilitating alternative macrophage polarisation, which may be alleviated through the therapeutic targeting of this kinase.

Gastric cancer has a high mortality rate associated with its late stage diagnosis. Although Gp130 knock-in mutant mice (gp130 F / F ) develop benign gastric tumours dependent upon IL-11-induced Stat3/mTORC activation with hallmarks of early stage intestinal-type gastric cancer in humans [1] , [2] , there is an urgent need to develop mouse models that mimic the progression from adenoma to carcinoma and metastasis. Macrophages are a major component of the tumour micro-environment, and their increased numbers and alternative activation is commonly associated with tumour progression and metastasis. Haematopoietic Cell Kinase (Hck) is a myeloid-specific Src family kinase that is involved in polarization of alternatively activated macrophages, which correlate with a poor prognosis for human gastric cancer. To establish whether aberrant Hck activation would induce tumourigenesis, Hck mutant mice that exhibit constitutive kinase activation (Hck Up / Up ) were crossed with gp130 F /+ mice that are predisposed to gastric tumour development. Surprisingly, the resulting gp130 F /+ ;Hck Up / Up compound mutants developed tumours similar to gp130[F/F] animals, while gp130 F /+ mice remained tumour-free. Furthermore, tumours in 9 month old gp130 F /+ ;Hck Up / Up mutants showed sub-mucosal invasion, which was not observed in gp130 F / F mice. Western blotting and immunohistochemical analysis revealed a significant increase in tumour cell proliferation in gp130 F /+ ;Hck Up / Up mutants compared to gp130 F / F animals, consistent with an increased abundance of the activated forms of Stat3, Erk, Akt and S6 cytoplasmic signalling proteins. While the total number of macrophages remained unaffected, qPCR analysis of purified tumour-associated macrophages from gp130 F /+ ; Hck Up / Up mice showed a significant increase in markers of alternative macrophage activation, including Il4 , Il13 , Arg1 and Ym1 when compared to cells from gp130 F / F animals. Furthermore, reciprocal adoptive transfer of Hck Up / Up bone-marrow into gp130 mutant recipients was sufficient to increase tumour burden and promote alternative macrophage polarisation. Collectively, our results demonstrate that aberrant Hck activation promotes polarisation towards alternative macrophage activation and confers an invasive gastric cancer phenotype.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the world [1] and our current understanding of the biology underpinning this disease is poor. Polymorphisms and mutations in haematopoietic cell kinase (Hck) have been observed in patients with COPD and lung cancer while aberrant activation of Hck promotes inflammatory disease and airspace enlargement in the lungs of mice similar to COPD in humans [2] . To investigate the role of Hck in pulmonary inflammation and cancer in mice, we used genetic complementation to investigate the contribution of the immune systems to inflammation in Hck mutant mice that harbour a constitutive active kinase ( Hck Up / Up ). Adult Hck Up / Up mice developpulmonary inflammation as indicated by an increased macrophage, neutrophil and eosinophil numbers in bronchoalveolar lavage fluid (BALF) compared to Wt mice, which progressed to emphysema from 12 weeks of age. A contribution of the adaptive immune system to disease was discounted since lymphocyte ( Hck Up / Up ; Rag1 −/− ) and Th 2 -deficient ( Hck Up / Up ; Stat6 −/− ) mice retained abundant macrophages in BALF. Mutant macrophages were implicated in promoting disease after adoptive transfer of Wt bone marrow alleviated disease in Hck Up / Up host mice and haploinsufficiency for Csf1r ( Hck Up / Up ; Csfr1 +/− ) culminated in reduced macrophage numbers in the BALF. Surprisingly, adoptive transfer of Wt alveolar macrophages rescued COPD-like disease in Hck Up / Up mice. Genetic depletion of TNF-α or MyD88 also partially rescued the inflammation phenotype of Hck Up / Up mice, while IL-6 was causally linked to disease after no inflammation was observed in Hck Up / Up ; Il6 −/− mice. We detected a striking bias towards expression of genes associated with alternatively activated macrophages in BALF cells and bone marrow-derived macrophages of Hck Up / Up mice. Furthermore, lung tumour burden was significantly larger in Hck Up / Up ; Kras LSL-G12D /+ mice than in Kras LSL-G12D /+ , consistent with alternative macrophage polarisation promoting cancer progression and COPD as an independent risk factor for lung cancer development. Hck activity within alveolar macrophages promotes alternative macrophage polarisation to drive a COPD-like inflammatory pulmonary disease and lung tumour progression in mice.