The receptor tyrosine kinase (RTK) MERTK is a paralog of TYRO3 and AXL, and together these receptors are commonly referred to as TAM RTKs. Mertk was named after its expression pattern in monocytes, epithelial tissues and reproductive tissues and for it being a tyrosine kinase (Graham et al., 1994). An understanding of MERTK’s role in molecular and cellular processes, as well as its broader role in mammalian physiology and pathology, in large part, came from the generation of a Mertk knockout (Mertk ^-/-) mouse line established by Camenisch et al., 1999. Use of this Mertk ^-/- mouse line revealed the critical functional role of this RTK in downregulation of inflammatory cytokines such as TNFα, as well as in the phagocytosis and clearance of apoptotic thymocytes (Camenisch et al., 1999; Scott et al., 2001). Subsequently, the Mertk ^-/- mouse line became the fountainhead for the description of Mertk function in a spectrum of phenotypes spanning retinal degeneration, defective adult neurogenesis, neurodegenerative diseases, liver injury, lupus-like autoimmunity, and cancer (Cohen et al., 2002; Cook et al., 2013; Crittenden et al., 2016; Davra et al., 2021; Duncan et al., 2003a; Fourgeaud et al., 2016; Huang et al., 2021; Ji et al., 2013; Lindsay et al., 2021; Stanford et al., 2014; Tormoen et al., 2020; Zagórska et al., 2020).

The Mertk ^-/- mouse line was generated by using the available technology of the time. Specifically, Mertk was targeted in 129P2/OlaHsd (129P2)-derived E14TG2a embryonic stem (ES) cells (Camenisch et al., 1999). ES cells were then microinjected into a C57BL/6 (B6) blastocyst to generate a chimeric mouse with germline transmission of the targeted allele (Figure 1A and B). Subsequently, the chimeric mouse was backcrossed to B6 to obtain Mertk ^-/- mice, henceforth referred to as Mertk ^{-/- V1} (Figure 1A). The Mertk ^-/-V1 mouse line is available through The Jackson Laboratory (strain# 011122). It is typically backcrossed >10 generations into B6 mice by researchers, including us, and has remained the mainstay for MERTK research. Nevertheless, there have been occasional and isolated reports of independently generated Mertk knockout mice that failed to completely recapitulate Mertk ^-/-V1 phenotypes (Maddox et al., 2011). For example, early-onset, severe photoreceptor (PR) degeneration was reported in Mertk ^-/-V1 mice (Duncan et al., 2003a; Duncan et al., 2003b; Prasad et al., 2006). In these mice, the outer nuclear layer (ONL) thickness was significantly reduced by postnatal day (P) 25 (Duncan et al., 2003a). Electroretinogram (ERG) recordings revealed that scotopic a- and b-wave amplitudes were significantly lower in Mertk ^{-/- V1} mice at P20 compared to wildtype (WT) mice at P30 (Duncan et al., 2003a). Photopic amplitudes were also significantly lower in Mertk ^-/-V1 mice versus WT mice at P33 (Duncan et al., 2003a). An independently generated ENU-induced Mertk mutation (Mertk ^{nmf12 or H716R}) in B6 mice caused the substitution of a highly conserved histidine to an arginine and led to a drastic reduction of MERTK in mouse retinas (Maddox et al., 2011). Yet it did not identically phenocopy the Mertk ^-/-V1-associated early-onset, severe retinal degeneration. Since a slow form of retinal degeneration did indeed occur in Mertk ^{nmf12 or H716R} and MERTK expression was not entirely abolished (Maddox et al., 2011), potential problems with Mertk ^-/-V1 mice were not immediately brought to the fore.

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In another independent study, Vollrath et al. demonstrated that crossing Mertk ^-/-V1 mice to B6 mice occasionally gave rise to animals with normal retina (Vollrath et al., 2015). The authors further demonstrated that the Mertk ^-/-V1 mice carry an ~40 cM chromosomal segment around the Mertk locus derived from the 129P2 mouse strain. The very low frequency of normal retina phenotype indicates that crossovers are extremely rare within this chromosomal segment around Mertk. Nonetheless, after rare crossover of B6 alleles within this region, Mertk ^-/-V1-dependent retinal degeneration was prevented (Vollrath et al., 2015). Vollrath et al. mapped the suppressor of retinal degeneration to a region that encoded 53 known or predicted open-reading frames (ORFs). Tyro3 was identified a priori as the likely candidate providing the suppressor function since it is a paralog of Mertk. Consistent with this hypothesis, it was observed that TYRO3 expression was at ~33% for Tyro3 ^129/129 (e.g., in Mertk ^{-/- V1}) relative to Tyro3 ^B6/B6 amounts, and associated with retinal degeneration (Vollrath et al., 2015). By contrast, TYRO3 expression was at ~67% for Tyro3 ^B6/129 relative to Tyro3 ^B6/B6 amounts. This ~67% expression of TYRO3 prevented retinal degeneration.

These results indicate that not all phenotypes in Mertk ^-/-V1 mice are solely due to the loss of MERTK function. Such crucial anomalies notwithstanding, the Mertk ^-/-V1 mouse line continues to be used to ascribe pivotal functions to MERTK in wide-ranging diseases such as neurodegeneration and cancer. We investigated whether indeed phenotypes observed in Mertk ^-/-V1 mice can be solely and unambiguously ascribed to the loss of function of Mertk. Here, we show that two independently generated B6 Mertk^-/- mouse lines do not phenocopy the retinal degeneration characteristic of Mertk ^-/-V1 mice. Furthermore, complementary to the genetic evidence that retinal degeneration segregated with Tyro3 ^129/129 but not Tyro3 ^129/B6 reported by Vollrath et al., we demonstrate that the simultaneous ablation of Mertk and Tyro3 in B6 mice is necessary and sufficient for retinal degeneration. MERTK and TYRO3 share the two most well-described TAM functions – phagocytosis and anti-inflammatory signaling (Rothlin et al., 2015). Thus, functional redundancy provided by TYRO3 for the phagocytosis of photoreceptor outer segments (POS) by retinal pigment epithelia (RPE) is still consistent with the well-understood mechanism of retinal homeostasis. Interestingly, the B6 Mertk knockout mouse lines also did not phenocopy the anti-tumor resistance displayed by Mertk ^-/-V1 mice against two syngeneic cancer lines. Even mice with simultaneous ablation of Mertk and Tyro3 did not phenocopy the anti-tumor resistance of Mertk ^-/-V1 mice. Loss of MERTK is proposed to hinder macrophage-dependent phagocytosis of dead or dying cancer cells (efferocytosis). This deficiency in macrophage-mediated disposal of tumor cells, in turn, is postulated to improve availability of tumor antigens for proficient presentation on dendritic cells (DCs) and/or render the tumor microenvironment pro-inflammatory and less immunosuppressive (Davra et al., 2021; Lin et al., 2022; Stanford et al., 2014). Paradoxical to this view, macrophages from Mertk ^-/-V1, Mertk ^-/-V2, or Mertk ^-/-V3 mice all displayed a significant deficit in efferocytosis. RNA sequencing of RPE and bone marrow-derived macrophages (BMDMs) revealed changes in expression of ~12–16 genes located in chromosome 2 in Mertk ^{-/- V1} but not Mertk ^{-/- V2} or Mertk ^{-/- V3} mice. Changes in the expression of additional nonlinked genes beyond chromosome 2 were also observed in Mertk ^-/-V1 but not Mertk ^-/-V2 or Mertk ^-/-V3 mice. This differential gene expression between Mertk ^-/-V1 and Mertk ^-/-V2 or Mertk ^-/-V3 mice was tissue-specific, pointing to the presence of a number of modifier alleles that may function combinatorially in several cell types, and in a variety of biological processes including phagocytosis and beyond, for at least some of the Mertk ^{-/- V1} mouse traits.

The Mertk ^-/-V1 mice (Camenisch et al., 1999), heretofore, has remained the workhorse for investigation of the biological consequences of Mertk ablation. Based on studies deploying Mertk ^-/-V1 mice, there is an increasing contemporary interest in the role of this RTK in what are likely to be complex traits, such as anti-tumor response and neurodegeneration (Cook et al., 2013; Crittenden et al., 2016; Davra et al., 2021; Fourgeaud et al., 2016; Huang et al., 2021; Ji et al., 2013; Lindsay et al., 2021; Stanford et al., 2014; Tormoen et al., 2020). There is an emerging concept of MERTK as an innate immune checkpoint and that targeting this RTK might enhance immunotherapy (Akalu et al., 2017; Huelse et al., 2020; Lentz et al., 2021; Liu et al., 2021; Rothlin and Ghosh, 2020). Remarkable anti-tumor resistance was observed in a number of standard mouse tumor models such as MMTV-PyVmT, B16:F10, MC38, E0771, and B78ChOva in Mertk ^{-/- V1} mice (Cook et al., 2013; Davra et al., 2021; Lindsay et al., 2021). Our initial results were entirely consistent with and extended such findings in that we were able to observe an astonishing anti-tumor resistance in hard-to-treat mouse models such as the anti-CTLA-4- and anti-PD-1-refractory YUMM1.7 mouse melanoma. Mertk ^{-/- V1} mice could even be rendered resistant to orthotopic GL261 glioblastoma with post-tumor DC-Vax treatment. Although our use of additional tumor models further validated the paradigm of host anti-tumor resistance in the Mertk ^-/-V1 mice, we surprisingly also discovered that Mertk ablation is not sufficient for resistance against YUMM1.7 and GL261. It is important to acknowledge that the work by Davra et al. and Lindsay et al. not only employed the Mertk ^-/-V1 mouse, but also utilized alternative approaches such as antibodies or small-molecule inhibitors to pharmacologically disable MERTK (Davra et al., 2021; Lindsay et al., 2021). The role of Mertk as an oncogene confounds interpretations of ostensible immune function when using pharmacological agents. It is possible that some of the reported effects are a consequence of inhibiting the oncogenic role of TAM RTKs. Since we did not test identical tumor models as the previous reports, it remains entirely possible that anti-tumor immunity in the models reported before, serendipitously, is dependent exclusively on the loss of MERTK. For instance, macrophage-specific ablation of Mertk in B6 background conferred a statistically significant reduction in tumor growth in the PyMT mouse model (Sekar et al., 2022). Taken together, our findings indicate that the universality of improving anti-tumor immunity through targeting MERTK, irrespective of the tumor model employed, has to be questioned. MERTK inhibition-dependent enhancement of anti-tumor immunity is likely to be contextual.

Similar concerns extend to Mertk ^-/-V1 mouse traits beyond anti-tumor immunity. The conditional ablation of Mertk in microglia led to decreased phagocytosis index in the mouse hippocampus (Fourgeaud et al., 2016). Hippocampal neurogenesis was impaired in Mertk ^-/-V1 mice (Ji et al., 2013) or paradoxically, transiently increased in the absence of Mertk (Fourgeaud et al., 2016), suggesting laboratory-specific strain differences in Mertk ^-/-V1 mice. It remains to be determined if changes in hippocampal neurogenesis in Mertk ^-/-V1 mice are a direct consequence of loss of Mertk or due to strain-specific expression of modifiers. The unequivocal demonstration of independent modifiers of Mertk ^-/-V1 mouse traits calls for re-examination of the molecular and functional basis of phenotypes assigned exclusively to the loss of MERTK. We do not, however, consider all Mertk ^-/-V1 mouse traits to be ambiguous. Using Mertk ^-/-V1 mice, DeBerge et al. demonstrated that post-reperfusion MERTK-dependent phagocytosis is cardioprotective in a mouse model of myocardial ischemia reperfusion injury (DeBerge et al., 2017). Notably, conditional ablation of Mertk in myeloid cells of B6 background confirmed the cardioprotective role for this RTK (DeBerge et al., 2017).

Neither are all phenotypes of Mertk ^-/-V1 mice invariably and inviolably linked to loss of phagocytosis in the absence of MERTK. The functional role of MERTK in phagocytosis remains beyond doubt, and we, in fact, validated the dependency of macrophages on MERTK for phagocytosis by using two independently generated mouse knockout lines. Our studies are also consistent with a report by Wanke et al. In mice wherein MERTK signaling was disabled by targeting the sequence coding for the kinase domain of MERTK (Mertk ^K614M/K614M mice), peritoneal macrophages failed to phagocytose apoptotic thymocytes at rates similar to that observed in Mertk ^{-/- V1} mice (Wanke et al., 2021). Importantly, Mertk ^K614M/K614M mice were generated on a B6 background, further indicating that the phagocytic function of macrophages depended on MERTK and is lost in its absence, independent of the 129P2 or B6 ES cell background that Mertk was targeted in. The biological basis for the differences in retinal degeneration phenotype in the Mertk ^-/-V1 versus Mertk ^-/-V2 and Mertk ^-/-V3 mouse lines can also be trivially explained by a redundancy in phagocytosis provided for by TYRO3 in the absence of MERTK. The original Mertk ^-/- mouse line, even after >10 generations of backcrossing to B6 mice in our laboratory, retained an ~15 cM chromosome 2 segment with 129P2 alleles that are genetically linked to the targeted Mertk locus and failed to recombine and segregate from it. A previous report by Vollrath et al. had also reported the presence of an ~40 cM chromosome 2 segment with 129P2 alleles in Mertk ^-/-V1 mice (Vollrath et al., 2015). Vollrath et al. mapped a modifier of the retinal degeneration phenotype to a 2 cM region containing 53 ORFs by genetic linkage mapping (Vollrath et al., 2015). Segregation of the Mertk-linked 129 region with a B6 region that contained Tyro3 restored retinal homeostasis (Vollrath et al., 2015). Since a genetic strategy was used in this previous report, in theory, potential contribution of other genes within the co-segregating region with suppressor function cannot be ruled out. Given that Tyro3 is the only Mertk paralog within this region, such an exception is unlikely. Nonetheless, in a complementary approach, we directly targeted Tyro3 in B6 Mertk ^-/-V2 mice-derived ES cells to unambiguously demonstrate that the simultaneous ablation of Mertk and Tyro3 in B6 mice is necessary and sufficient for retinal degeneration. One of the potential concerns for using MERTK inhibitors in the clinic is the on-target adverse event of rapid retinal degeneration. Vollrath et al., 2015, Maddox et al., 2011, and our results collectively indicate that sparing TYRO3 activity can greatly ameliorate this concern while targeting the oncogenic activity of MERTK.

The phagocytosis paradigm, nevertheless, failed entirely in explaining the remarkable anti-tumor resistance of Mertk ^-/-V1 mice. Loss of/reduced phagocytosis by macrophages and other phagocytes, when Mertk is genetically ablated, is often surmised causal to the manifested phenotype (Davra et al., 2021; Huang et al., 2021; Stanford et al., 2014; Zhou et al., 2020). Tyro3 is not expressed in BMDMs, was not a DEG in BMDM RNAseq experiments, and Mertk ^-/-V1, Mertk ^-/-V2, and Mertk ^-/-V3 BMDMs were equally deficient in efferocytosis in an ex vivo assay. Thus, neither TYRO3 nor any other DEG provided a redundancy to macrophage phagocytosis in the absence of MERTK. This observation seriously challenges the dogma that failure of MERTK-dependent efferocytosis of tumor cells universally improves anti-tumor immunity.

Phenotypic differences not accounted for fully by the target gene are not entirely an uncommon occurrence in tumor resistance. For example, the frequency of intestinal adenomas in Apc ^min mice is dramatically modified by the genetic background that the mutation is introduced in. Apc ^min mice with a B6 background were reported to develop 28.5 ± 7.9 tumors and die within 4 months of birth (Dietrich et al., 1993). When these Apc ^min mice were crossed to an AKR mouse, the mixed B6/AKR background had 5.8 ± 4.3 tumors and lived until they were euthanized at 300 days (Dietrich et al., 1993). Crossing the B6/AKR F1 mice with AKR further reduced tumor loads to 1.75 ± 1.7 tumors (Dietrich et al., 1993). The modifier allele was identified as Mom1 mapping to chromosome 4 (Dietrich et al., 1993). We postulate that the anti-tumor resistance of the Mertk ^-/-V1 mouse line also involves modifier alleles. The 129P2 segment in Mertk ^-/-V1 mice not only accounts for a number of gene expression differences within the cis non-B6 flanking region, but also results in differential gene expression in a number of trans loci. It is possible that the anti-tumor resistance ascribed solely to Mertk might actually involve one or more modifier activities encoded within cis or trans loci unique to the Mertk ^-/-V1 mice that are absent in B6 ES cell-derived Mertk ^-/- mouse lines. Furthermore, since gene expression differences between Mertk ^-/-V1 and Mertk ^-/-V2 or Mertk ^{-/- V3} mice are cell type-specific, it is possible that tissue-specific expression quantitative trait loci (eQTLs) may function in combination as modifiers of complex traits, such as anti-tumor immune responses. This makes identification of the crucial modifiers more challenging. Assuming said modifiers are acting in cis, genomic CRISPR/CAS9 screens for loss of YUMM1.7 and GL261 resistance in a series of Mertk ^-/-V1-derived mouse lines ablated for genes within the ~15 cM region of chromosome 2 may reveal the modifiers. Alternatively, if the 129P2 allele is dominant, BAC transgenics using Mertk ^-/-V2 ES cells to express 129P2 variants of genes for the rescue of the anti-tumor response might also be an appropriate strategy.

We cannot also entirely eliminate the possibility that Mertk itself does not have a functional role in some of the traits observed in Mertk ^-/-V1 mice. For example, the DC-intrinsic defect in emigration to inflamed tissue that was initially ascribed to loss of function of Nlrp10 in Nlrp10 ^-/- mice first generated in a B6-BALB/c mixed background was subsequently revealed to be caused by a spontaneous mutation in Dock8, an unexpected by-product of the BALB/c variant of the gene (Eisenbarth et al., 2012; Krishnaswamy et al., 2015). Similarly, Il10 ^-/- mice in a B6 background differed in open-field behavioral tests from Il10 ^-/- mice in a 129-B6 mixed background (Bolivar et al., 2001). The behavioral differences were found to be due to eQTLs Emo4 and Reb1 inherited from flanking region of Il10 in chromosome 1 and not attributable to Il10 itself (de Ledesma et al., 2006).

In conclusion, two newly generated mouse lines with genetic ablation of Mertk reveal that some of the well-established Mertk ^-/-V1 mice traits tested herein were products of epistatic interactions with modifiers in the 129P2 genome. These new Mertk knockout mouse lines should prove useful for validation of phenotypes ascribed exclusively to Mertk based on studies employing Mertk ^-/-V1 mice. Furthermore, these studies also identify a unique anti-tumor resistance in Mertk ^{-/- V1} mice against anti-CTLA-4 and anti-PD-1 refractory YUMM1.7 melanoma as well as against GL261 brain tumors, the molecular basis of which remains unknown. This model may yet prove useful for the discovery of a novel host anti-tumor response that can be therapeutically harnessed for improving outcomes in cancer.

RNA-sequencing data sets and the processed data that support the findings of this study have been deposited to the Gene Expression Omnibus (GEO) under accession ID: GSE205070. All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for all figures included.

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Author details

1. Yemsratch T Akalu

   Department of Immunobiology, Yale School of Medicine, New Haven, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Maria E Mercau

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-0829-7972

2. Maria E Mercau

   Department of Immunobiology, Yale School of Medicine, New Haven, United States

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Visualization, Writing – original draft, Writing – review and editing

   Contributed equally with

   Yemsratch T Akalu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6971-8676

3. Marleen Ansems

   Department of Immunobiology, Yale School of Medicine, New Haven, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

4. Lindsey D Hughes

   Department of Immunobiology, Yale School of Medicine, New Haven, United States

   Contribution

   Formal analysis, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1764-4553

5. James Nevin

   Department of Immunobiology, Yale School of Medicine, New Haven, United States

   Contribution

   Writing – review and editing

   Competing interests

   No competing interests declared

6. Emily J Alberto

   Department of Immunobiology, Yale School of Medicine, New Haven, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Xinran N Liu

   Department of Cell Biology, Center for Cellular and Molecular Imaging, Yale School of Medicine, New Haven, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

8. Li-Zhen He

   Celldex Therapeutics, New Haven, United States

   Contribution

   Resources, Methodology

   Competing interests

   is affiliated with Celldex Therapeutics. The author has no financial interests to declare

9. Diego Alvarado

   Celldex Therapeutics, New Haven, United States

   Contribution

   Resources, Methodology

   Competing interests

   is affiliated with Celldex Therapeutics. The author has no financial interests to declare

10. Tibor Keler

    Celldex Therapeutics, New Haven, United States

    Contribution

    Resources, Methodology

    Competing interests

    is affiliated with Celldex Therapeutics. The author has no financial interests to declare

11. Yong Kong

    Department of Molecular Biophysics and Biochemistry, W. M. Keck Foundation Biotechnology Resource Laboratory, School of Medicine, Yale University, New Haven, United States

    Contribution

    Formal analysis

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2881-5274

12. William M Philbrick

    Center on Endocrinology and Metabolism, Yale Genome Editing Center, School of Medicine, Yale University, New Haven, United States

    Contribution

    Methodology

    Competing interests

    No competing interests declared

13. Marcus Bosenberg

    Departments of Dermatology, Pathology and Immunobiology, Yale School of Medicine, New Haven, United States

    Contribution

    Resources

    Competing interests

    No competing interests declared

14. Silvia C Finnemann

    Center for Cancer, Genetic Diseases and Gene Regulation, Department of Biological Sciences, Fordham University, Bronx, United States

    Contribution

    Conceptualization, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0001-9298-0736

15. Antonio Iavarone

    Departments of Neurology and Pathology and Cell Biology, Institute for Cancer Genetics, Columbia Medical Center, New York, United States

    Contribution

    Conceptualization, Writing – review and editing

    Competing interests

    No competing interests declared

16. Anna Lasorella

    Departments of Pediatrics and Pathology and Cell Biology, Institute for Cancer Genetics, Columbia University, New York, United States

    Contribution

    Conceptualization, Writing – review and editing

    Competing interests

    No competing interests declared

17. Carla V Rothlin

    Departments of Immunobiology and Pharmacology, Yale School of Medicine, New Haven, United States

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Visualization, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    carla.rothlin@yale.edu

    Competing interests

    Senior editor, eLife

    "This ORCID iD identifies the author of this article:" 0000-0002-5693-5572

18. Sourav Ghosh

    Departments of Neurology and Pharmacology, Yale School of Medicine, New Haven, United States

    Contribution

    Conceptualization, Resources, Supervision, Funding acquisition, Visualization, Writing – original draft, Project administration, Writing – review and editing

    For correspondence

    sourav.ghosh@yale.edu

    Competing interests

    has received grant support from Mirati Therapeutics

    "This ORCID iD identifies the author of this article:" 0000-0001-5990-8708

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