Boram Ham

Sex differences in neuroimmunity and pain.

Differences in the prevalence of chronic pain in women vs. men are well known, and decades of laboratory experimentation have demonstrated that women are more sensitive to pain than are men. Attention has thus shifted to investigating mechanisms underlying such differences. Recent evidence suggests that neuroimmune modulation of pain may represent an important cause of sex differences. The current Review examines the evidence for gonadal hormone modulation of the immune system, immune system modulation of pain, and interactions that might help to explain sex differences in pain.

TNF Receptor-2 Facilitates an Immunosuppressive Microenvironment in the Liver to Promote the Colonization and Growth of Hepatic Metastases

Successful colonization by a cancer cell of a distant metastatic site requires immune escape in the new microenvironment. TNF signaling has been implicated broadly in the suppression of immune surveillance that prevents colonization at the metastatic site and therefore must be blocked. In this study, we explored how TNF signaling influences the efficiency of liver metastasis by colon and lung carcinoma in mice that are genetically deficient for the TNF receptor TNFR2. We found a marked reduction in liver metastases that correlated with a greatly reduced accumulation at metastatic sites of CD11b(+)GR-1(+) myeloid cells with enhanced arginase activity, identified as myeloid-derived suppressor cells (MDSC). Reduced infiltration of MDSC coincided with a reduction in the number of CD4(+)FoxP3(+) T regulatory cells in the tumors. Reconstitution of TNFR2-deficient mice with normal bone marrow, or adoptive transfer of TNFR2-expressing MDSC into these mice, was sufficient to restore liver metastasis to levels in wild-type mice. Conversely, treatment with TNFR2 antisense oligodeoxynucleotides reduced liver metastasis in wild-type mice. Clinically, immunohistochemical analysis of liver metastases from chemotherapy-naïve colon cancer patients confirmed the presence of CD33(+)HLA-DR(-)TNFR2(+) myeloid cells in the periphery of hepatic metastases. Overall, our findings implicate TNFR2 in supporting MDSC-mediated immune suppression and metastasis in the liver, suggesting the use of TNFR2 inhibitors as a strategy to prevent metastatic progression to liver in colon, lung, and various other types of cancer.

T-Cell Mediation of Pregnancy Analgesia Affecting Chronic Pain in Mice

It has been reported consistently that many female chronic pain sufferers have an attenuation of symptoms during pregnancy. Rats display increased pain tolerance during pregnancy due to an increase in opioid receptors in the spinal cord. Past studies did not consider the role of non-neuronal cells, which are now known to play an important role in chronic pain processing. Using an inflammatory (complete Freunds adjuvant) or neuropathic (spared nerve injury) model of persistent pain, we observed that young adult female mice in early pregnancy switch from a microglia-independent to a microglia-dependent pain hypersensitivity mechanism. During late pregnancy, female mice show no evidence of chronic pain whatsoever. This pregnancy-related analgesia is reversible by intrathecal administration of naloxone, suggesting an opioid-mediated mechanism; pharmacological and genetic data suggest the importance of δ-opioid receptors. We also observe that T-cell-deficient (nude and Rag1-null mutant) pregnant mice do not exhibit pregnancy analgesia, which can be rescued with the adoptive transfer of CD4+ or CD8+ T cells from late-pregnant wild-type mice. These results suggest that T cells are a mediator of the opioid analgesia exhibited during pregnancy. SIGNIFICANCE STATEMENT Chronic pain symptoms often subside during pregnancy. This pregnancy-related analgesia has been demonstrated for acute pain in rats. Here, we show that pregnancy analgesia can produce a complete cessation of chronic pain behaviors in mice. We show that the phenomenon is dependent on pregnancy hormones (estrogen and progesterone), δ-opioid receptors, and T cells of the adaptive immune system. These findings add to the recent but growing evidence of sex-specific T-cell involvement in chronic pain processing.

The type I insulin-like growth factor regulates the liver stromal response to metastatic colon carcinoma cells

Hepatic stellate cells (HSC) play a major role in initiating the liver fibrogenic (wounding) response of the liver and can also orchestrate a pro-metastatic microenvironment in the liver in response to invading cancer cells. Here we explored the role of the hepatic stellate cells in colon carcinoma liver metastasis with emphasis on the contribution of the insulin-like growth factor (IGF) axis to their activation and function. To this end, we used mice with a Tamoxifen inducible liver IGF-I deficiency. We found that in mice with a sustained IGF-I deficiency, recruitment and activation of HSC into tumor-infiltrated areas of the liver were markedly diminished, resulting in decreased collagen deposition and reduced tumor expansion. In addition, IGF-I could rescue HSC from apoptosis induced by pro-inflammatory factors such as TNF-α known to be upregulated in the early stages of liver metastasis. Moreover, in surgical specimens, activated IGF-IR was observed on HSC-like stromal cells surrounding colorectal carcinoma liver metastases. Finally, IGF-targeting in vivo using an IGF-Trap caused a significant reduction in HSC activation in response to metastatic colon cancer cells. Therefore, our data identify IGF as a survival factor for HSC and thereby, a promoter of the pro-metastatic microenvironment in the liver. IGF-targeting could therefore provide a strategy for curtailing the pro-metastatic host response of the liver during the early stages of liver metastasis.Hepatic stellate cells (HSC) play a major role in initiating the liver fibrogenic (wounding) response of the liver and can also orchestrate a pro-metastatic microenvironment in the liver in response to invading cancer cells. Here we explored the role of the hepatic stellate cells in colon carcinoma liver metastasis with emphasis on the contribution of the insulin-like growth factor (IGF) axis to their activation and function. To this end, we used mice with a Tamoxifen inducible liver IGF-I deficiency. We found that in mice with a sustained IGF-I deficiency, recruitment and activation of HSC into tumor-infiltrated areas of the liver were markedly diminished, resulting in decreased collagen deposition and reduced tumor expansion. In addition, IGF-I could rescue HSC from apoptosis induced by pro-inflammatory factors such as TNF-α known to be upregulated in the early stages of liver metastasis. Moreover, in surgical specimens, activated IGF-IR was observed on HSC-like stromal cells surrounding colorectal carcinoma liver metastases. Finally, IGF-targeting in vivo using an IGF-Trap caused a significant reduction in HSC activation in response to metastatic colon cancer cells. Therefore, our data identify IGF as a survival factor for HSC and thereby, a promoter of the pro-metastatic microenvironment in the liver. IGF-targeting could therefore provide a strategy for curtailing the pro-metastatic host response of the liver during the early stages of liver metastasis.

Increased pain sensitivity and decreased opioid analgesia in T cell-deficient mice and implications for sex differences

Abstract The processing of pain in the central nervous system is now known to have an important immune component, including T cells of the adaptive immune system. T cells have been shown to release endogenous opioids, and although it is well known that opioids have effects on T-cell populations, very little attention has been given to the converse: how T cells may affect opioid regulation. We find here that, in addition to displaying significantly increased baseline pain sensitivity across various pain modalities, T-cell-deficient mice (CD-1 nude, Rag1 null mutant, and Cd4 null mutant) exhibit pronounced deficiencies in morphine inhibition of thermal or inflammatory pain. Nude mice are also deficient in endogenous opioid-mediated analgesia, exhibiting no stress-induced analgesia from restraint. The relevant T-cell subpopulation seems to be CD4+ T cells because adoptive transfer of them but not CD8+ cells into nude mice rescues both the pain and morphine analgesia phenotypes. As previously reported, we also observe a sex difference in CD-1 mice, with females requiring 2- to 3-fold more morphine than males to produce equal analgesia. Nude mice display no sex differences in morphine analgesia, and the sex difference is restored in nude mice of either sex receiving CD4+ T cells from CD-1 donor male or female mice. These results suggest that CD4+ T cells play an as yet unappreciated role in opioid analgesia and may be a driver of sex differences therein.

Loss of neutrophil polarization in colon carcinoma liver metastases of mice with an inducible, liver-specific IGF-I deficiency

The growth of cancer metastases in the liver depends on a permissive interaction with the hepatic microenvironment and neutrophils can contribute to this interaction, either positively or negatively, depending on their phenotype. Here we investigated the role of IGF-I in the control of the tumor microenvironment in the liver, using mice with a conditional, liver-specific, IGF-I deficiency (iLID) induced by a single tamoxifen injection. In mice that had a sustained (3 weeks) IGF-I deficiency prior to the intrasplenic/portal inoculation of colon carcinoma MC-38 cells, we observed an increase in neutrophil accumulation in the liver relative to controls. However, unlike controls, these neutrophils did not acquire the (anti-inflammatory) tumor-promoting phenotype, as evidenced by retention of high ICAM-1 expression and nitric oxide production and low CXCR4, CCL5, and VEGF expression and arginase production, all characteristic of the (pro-inflammatory) phenotype. This coincided with an increase in apoptotic tumor cells and reduced metastasis. Neutrophils isolated from these mice also had reduced IGF-IR expression levels. These changes were not observed in iLID mice with a short-term (2 days) IGF-I depletion, despite a 70% reduction in their circulating IGF-I levels, indicating that a sustained IGF-I deficiency was necessary to alter the neutrophil phenotype. Similar results were obtained with the highly metastatic Lewis lung carcinoma subline H-59 cells and in mice injected with an IGF-Trap that blocks IGF-IR signaling by reducing ligand bioavailability. Our results implicate the IGF axis in neutrophil polarization and the induction of a pro-metastatic microenvironment in the liver.

Modulation of social behavior and dominance status by chronic pain in mice

The potential influence of pain on social behavior in laboratory animals has rarely been evaluated. Using a new assay of social behavior, the tube co‐occupancy test (TCOT), we assess propinquity—the tendency to maintain close physical proximity—in mice exposed to pain using subcutaneous zymosan or spared nerve injury as noxious stimuli. Our previous experience with the TCOT showed that outbred mouse sibling dyads show higher levels of tube co‐occupancy than stranger dyads. We find here that long‐lasting pain from spared nerve injury given to both mice in the dyad abolishes this effect of familiarity, such that strangers also display high levels of propinquity. We performed a separate experiment to assess the effect on dominance behavior of nerve injury to one or both mice of a dyad in which relative dominance status had been previously established via the confrontation tube test. We find that neuropathic pain given only to the dominant mouse reverses the relationship in male but not female mice, such that the previously subordinate mouse becomes dominant. These observations bolster the scant but growing evidence that pain can robustly affect social behavior in animals.

Abstract # 1775 Attenuation of opioid analgesia in T cell deficient mice

It is now known that neurons are not the only cell type involved in pain processing, which involves Schwann cells, satellite cells, and cells of the immune system, such as microglia, macrophages, and T cells. Many pain researchers have adopted the use of T-cell deficient mice in their experimental methods to elucidate the role of T-cells in neuropathic pain (Fitzgerald et al., 2009; Zuo et al., 2013), and T cells have been shown to release endogenous opioids (Dietrich et al., 2011). While it is well known that opioids have varying effects on the immune system, very little attention has been given to how the immune system may affect opioid regulation. We now have evidence that T-cell deficient mice (CD-1 nude and Rag1 null mutant) exhibit pronounced deficiencies in morphine analgesia, measured using the tail withdrawal or formalin test. Furthermore, T-cell deficient mice do not exhibit stress-induced analgesia after restraint. Adoptive transfer of splenocytes from CD-1 mice into T-cell deficient mice rescues opioid analgesia. These results suggest that T-cells play a role in opioid-mediated analgesia, not previously observed. Current experiments are investigating the mechanism behind this phenomenon.

Abstract # 1773 Cytotoxic T cells may induce pain hypersensitivity in female but not male mice after nerve injury in an interferon-g-receptor-dependent manner

In our previous study, we observed that female mice, unlike males, do not require microglia to produce pain hypersensitivity after neuropathic or inflammatory injury. Using T-cell deficient mice, we found that female mutant mice “switch” to the “male” microglial system. Important questions include how microglial or T cell systems are recruited, how and which T cells are infiltrating into the spinal cord, and what T cells are releasing that can sensitize pain-relevant neurons. Immune cells were extracted from lumbar spinal cord of WT mice and IFNgRKO mice post SNI and CFA injection, and analyzed using flow cytometry. Additionally, mechanical allodynia was assessed. Analyzing the spinal cord microenvironment after SNI revealed that female mice displayed: (1) a 2-fold increase in CD8+ cytotoxic T cells, (2) upregulation of the anti-inflammatory microglia population, and (3) downregulation of anti-inflammatory T regulatory cells and pro-inflammatory microglia. In contrast, males showed: (1) a 2-fold decrease in CD8+ cytotoxic T cells, and (2) an increase in pro-inflammatory microglia. IFNgRKO female mice showed the same pain behavioural response as WT males wherein minocycline reversed allodynia. Consistent with this, IFNgRKO female, like WT male, mice did not show upregulation of CD8+ cytotoxic T cells. Our current data further suggest that female mice, unlike males, are using cytotoxic CD8+ T cells to induce neuropathic pain, and that this mechanism appears to be dependent on IFNgRs.

Abstract 163: Identification of a bone marrow-derived MDSC subset that promotes the growth of colon carcinoma liver metastases

The hepatic microenvironment can play an important role in promoting liver metastasis. We and others have shown that when metastatic carcinoma cells enter the liver, a rapid inflammatory response mediated by TNF-α is triggered, enabling tumor transendothelial migration and metastasis. TNF-α binds to the cell surface through two receptors; the p55/p60 TNFR1 and the p75/p80 TNFR2. To elucidate the roles of these receptors in the inflammatory cascade and in liver colonization by tumor cells, we used mice with tnfr1, tnfr2 and combined tnfr1/tnfr2 gene deficiencies and investigated parameters of the host response and the outcome of metastasis in these mice using the highly metastatic mouse colon carcinoma MC-38 and lung carcinoma H-59 cells. We found that experimental liver metastasis following intrasplenic/portal inoculation of the tumor cells was markedly reduced in TNFR2-/- and even further in TNFR1-/-/TNFR2-/- female mice as compared to wild-type controls but no reduction was observed in TNFR1-/- mice. To identify the underlying mechanism(s), we investigated changes in the liver microenvironment following tumor inoculation using a combination of immunohistochemistry, confocal microscopy, qRT-PCR, and cytokine/chemokines profiling. We found that while in wild-type and TNFR1-/- mice, VCAM-1 expression on the sinusoidal endothelium was significantly upregulated within 16 hr post tumor injection, it was unchanged in TNFR2-/- and double negative female mice. In addition, qRT-PCR analysis revealed that the production of the neutrophil chemokine CXCL1, while upregulated in control mice, was suppressed in TNFR2-/- mice. These changes were associated with a parallel decrease in Ly6G+ neutrophil recruitment into extravascular, pro-metastatic niches 16-48 hr post tumor inoculation. Interestingly, these reductions were not observed in male TNFR2-/- mice and correspondingly, their metastatic burden was not reduced relative to control mice. Taken together, the results suggest that TNFR2 plays a critical and gender-specific role in Ly6G+ cell recruitment into the tumor-microenvironment by regulating sinusoidal endothelial VCAM-1 expression and chemokine production. In turn, the recruitment of these cells into extravascular pro-metastatic niches appears to promote liver metastasis. Citation Format: Boram Ham. Identification of a bone marrow-derived MDSC subset that promotes the growth of colon carcinoma liver metastases. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 163. doi:10.1158/1538-7445.AM2014-163