Spinal cord injury-induced neurogenic bowel: A role for host-microbiome interactions in bowel pain and dysfunction.

Background and aims: Spinal cord injury (SCI) affects roughly 300,000 Americans with 17,000 new cases added annually. In addition to paralysis, 60% of people with SCI develop neurogenic bowel (NB), a syndrome characterized by slow colonic transit, constipation, and chronic abdominal pain. The knowledge gap surrounding NB mechanisms after SCI means that interventions are primarily symptom-focused and largely ineffective. The goal of the present studies was to identify mechanism(s) that initiate and maintain NB after SCI as a critical first step in the development of evidence-based, novel therapeutic treatment options. Methods: Following spinal contusion injury at T9, we observed alterations in bowel structure and function reflecting key clinical features of NB. We then leveraged tissue-specific whole transcriptome analyses (RNAseq) and fecal 16S rRNA amplicon sequencing in combination with histological, molecular, and functional (Ca2+ imaging) approaches to identify potential mechanism(s) underlying the generation of the NB phenotype. Results: In agreement with prior reports focused on SCI-induced changes in the skin, we observed a rapid and persistent increase in expression of calcitonin gene-related peptide (CGRP) expression in the colon. This is suggestive of a neurogenic inflammation-like process engaged by antidromic activity of below-level primary afferents following SCI. CGRP has been shown to disrupt colon homeostasis and negatively affect peristalsis and colon function. As predicted, contusion SCI resulted in increased colonic transit time, expansion of lymphatic nodules, colonic structural and genomic damage, and disruption of the inner, sterile intestinal mucus layer corresponding to increased CGRP expression in the colon. Gut microbiome colonization significantly shifted over 28 days leading to the increase in Anaeroplasma, a pathogenic, gram-negative microbe. Moreover, colon specific vagal afferents and enteric neurons were hyperresponsive after SCI to different agonists including fecal supernatants. Conclusions: Our data suggest that SCI results in overexpression of colonic CGRP which could alter colon structure and function. Neurogenic inflammatory-like processes and gut microbiome dysbiosis can also sensitize vagal afferents, providing a mechanism for visceral pain despite the loss of normal sensation post-SCI. These data may shed light on novel therapeutic interventions targeting this process to prevent NB development in patients.

Combined PI3K and MAPK inhibition synergizes to suppress PDAC.

Oncogenic KRAS mutations are nearly ubiquitous in pancreatic ductal adenocarcinoma (PDAC), yet therapeutic attempts to target KRAS as well as its target MAPK pathway effectors have shown limited success due to the difficulty to pharmacologically target KRAS, inherent drug resistance in PDAC cells, and acquired resistance through activation of alternative mitogenic pathways such JAK-STAT and PI3K-AKT. While KRAS canonically drives the MAPK signaling pathway via RAF-MEK-ERK, it is also known to play a role in PI3K-AKT signaling. Our therapeutic study targeted the PI3K-AKT pathway with the drug Omipalisib (p110α/ß/δ/γ and mTORC1/2 inhibitor) in combination with MAPK pathway targeting drug Trametinib (MEK1/2 inhibitor) or SHP099-HCL (SHP099), which is an inhibitor of the KRAS effector SHP2. Western blot analysis demonstrated that application of Trametinib or SHP099 alone selectively blocked ERK phosphorylation (pERK) but failed to suppress phosphorylated AKT (pAKT) and in some instances increased pAKT levels. Conversely, Omipalisib alone successfully inhibited pAKT but failed to suppress pERK. Therefore, we hypothesized that a combination therapeutic comprised of Omipalisib with either Trametinib or SHP099 would inhibit two prominent mitogenic pathways, MEK and PI3K-AKT, to more effectively suppress pancreatic cancer. In vitro studies demonstrated that both Omipalisib/Trametinib and Omipalisib/SHP099 combination therapeutic strategies were generally more effective than treatment with each drug individually at reducing proliferation, colony formation, and cell migration compared to vehicle controls. Additionally, we found that while combination Omipalisib/SHP099 treatment reduced implanted tumor growth in vivo , the Omipalisib/Trametinib treatment was significantly more effective. Therefore, we additionally tested the Omipalisib/Trametinib combination therapeutic in the highly aggressive PKT (Ptf1a cre , LSL-Kras G12D , TGFbR2 fl/fl ) spontaneous mouse model of PDAC. We subsequently found that PKT mice treated with the Omipalisib/Trametinib combination therapeutic survived significantly longer than mice treated with either drug alone, and more than doubled the mean survival time of vehicle control mice. Altogether, our data support the importance of a dual treatment strategy targeting both MAPK and PI3K-AKT pathways.

SHP2 regulates adipose maintenance and adipocyte-pancreatic cancer cell crosstalk via PDHA1.

Adipocytes are the most abundant cell type in the adipose tissue, and their dysfunction is a significant driver of obesity-related pathologies, such as cancer. The mechanisms that (1) drive the maintenance and secretory activity of adipocytes and (2) mediate the cancer cellular response to the adipocyte-derived factors are not fully understood. To address that gap of knowledge, we investigated how alterations in Src homology region 2-containing protein (SHP2) activity affect adipocyte function and tumor crosstalk. We found that phospho-SHP2 levels are elevated in adipose tissue of obese mice, obese patients, and differentiating adipocytes. Immunofluorescence and immunoprecipitation analyses as well as in-silico protein-protein interaction modeling demonstrated that SHP2 associates with PDHA1, and that a positive association promotes a reactive oxygen species (ROS)-driven adipogenic program. Accordingly, this SHP2-PDHA1-ROS regulatory axis was crucial for adipocyte maintenance and secretion of interleukin-6 (IL-6), a key cancer-promoting cytokine. Mature adipocytes treated with an inhibitor for SHP2, PDHA1, or ROS exhibited an increased level of pro-lipolytic and thermogenic proteins, corresponding to an increased glycerol release, but a suppression of secreted IL-6. A functional analysis of adipocyte-cancer cell crosstalk demonstrated a decreased migration, invasion, and a slight suppression of cell cycling, corresponding to a reduced growth of pancreatic cancer cells exposed to conditioned media (CM) from mature adipocytes previously treated with inhibitors for SHP2/PDHA1/ROS. Importantly, PDAC cell growth stimulation in response to adipocyte CM correlated with PDHA1 induction but was suppressed by a PDHA1 inhibitor. The data point to a novel role for (1) SHP2-PDHA1-ROS in adipocyte maintenance and secretory activity and (2) PDHA1 as a regulator of the pancreatic cancer cells response to adipocyte-derived factors.

Adipose-Tumor Crosstalk contributes to CXCL5 Mediated Immune Evasion in PDAC.

Background: CXCR1/2 inhibitors are being implemented with immunotherapies in PDAC clinical trials. Cytokines responsible for stimulating these receptors include CXCL ligands, typically secreted by activated immune cells, fibroblasts, and even adipocytes. Obesity has been linked to poor patient outcome and altered anti-tumor immunity. Adipose-derived cytokines and chemokines have been implicated as potential drivers of tumor cell immune evasion, suggesting a possibility of susceptibility to targeting specifically in the context of obesity. Methods: RNA-sequencing of human PDAC cell lines was used to assess differential influences on the cancer cell transcriptome after treatment with conditioned media from peri-pancreatic adipose tissue of lean and obese PDAC patients. The adipose-induced secretome of PDAC cells was then assessed by cytokine arrays and ELISAs. Lentiviral transduction and CRISPR-Cas9 was used to knock out CXCL5 from a murine PDAC cell line for orthotopic tumor studies in diet-induced obese, syngeneic mice. Flow cytometry was used to define the immune profiles of tumors. Anti-PD-1 immune checkpoint blockade therapy was administered to alleviate T cell exhaustion and invoke an immune response, while the mice were monitored at endpoint for differences in tumor size. Results: The chemokine CXCL5 was secreted in response to stimulation of PDAC cells with human adipose conditioned media (hAT-CM). PDAC CXCL5 secretion was induced by either IL-1ß or TNF, but neutralization of both was required to limit secretion. Ablation of CXCL5 from tumors promoted an immune phenotype susceptible to PD-1 inhibitor therapy. While application of anti-PD-1 treatment to control tumors failed to alter tumor growth, knockout CXCL5 tumors were diminished. Conclusions: In summary, our findings show that known adipokines TNF and IL-1ß can stimulate CXCL5 release from PDAC cells in vitro. In vivo , CXCL5 depletion alone is sufficient to promote T cell infiltration into tumors in an obese setting, but requires checkpoint blockade inhibition to alleviate tumor burden. DATA AVAILABILITY STATEMENT: Raw and processed RNAseq data will be further described in the GEO accession database ( awaiting approval from GEO for PRJ number ). Additional raw data is included in the supplemental material and available upon reasonable request. WHAT IS ALREADY KNOWN ON THIS TOPIC: Obesity is linked to a worsened patient outcome and immunogenic tumor profile in PDAC. CXCR1/2 inhibitors have begun to be implemented in combination with immune checkpoint blockade therapies to promote T cell infiltration under the premise of targeting the myeloid rich TME. WHAT THIS STUDY ADDS: Using in vitro/ex vivo cell and tissue culture-based assays with in vivo mouse models we have identified that adipose derived IL-1ß and TNF can promote tumor secretion of CXCL5 which acts as a critical deterrent to CD8 T cell tumor infiltration, but loss of CXCL5 also leads to a more immune suppressive myeloid profile. HOW THIS STUDY MIGHT AFFECT RESEARCH PRACTICE OR POLICY: This study highlights a mechanism and emphasizes the efficacy of single CXCR1/2 ligand targeting that could be beneficial to overcoming tumor immune-evasion even in the obese PDAC patient population.

Synthetic adiponectin-receptor agonist, AdipoRon, induces glycolytic dependence in pancreatic cancer cells.

Obesity creates a localized inflammatory reaction in the adipose, altering secretion of adipocyte-derived factors that contribute to pathologies including cancer. We have previously shown that adiponectin inhibits pancreatic cancer by antagonizing leptin-induced STAT3 activation. Yet, the effects of adiponectin on pancreatic cancer cell metabolism have not been addressed. In these studies, we have uncovered a novel metabolic function for the synthetic adiponectin-receptor agonist, AdipoRon. Treatment of PDAC cells with AdipoRon led to mitochondrial uncoupling and loss of ATP production. Concomitantly, AdipoRon-treated cells increased glucose uptake and utilization. This metabolic switch further correlated with AMPK mediated inhibition of the prolipogenic factor acetyl coenzyme A carboxylase 1 (ACC1), which is known to initiate fatty acid catabolism. Yet, measurements of fatty acid oxidation failed to detect any alteration in response to AdipoRon treatment, suggesting a deficiency for compensation. Additional disruption of glycolytic dependence, using either a glycolysis inhibitor or low-glucose conditions, demonstrated an impairment of growth and survival of all pancreatic cancer cell lines tested. Collectively, these studies provide evidence that pancreatic cancer cells utilize metabolic plasticity to upregulate glycolysis in order to adapt to suppression of oxidative phosphorylation in the presence of AdipoRon.

Effects of skeletal unloading on the antibody repertoire of tetanus toxoid and/or CpG treated C57BL/6J mice.

Spaceflight affects the immune system, but the effects on the antibody repertoire, responsible for humoral immunity, has not been well explored. In particular, the complex gene assembly and expression process; including mutations, might make this process vulnerable. Complementarity determining region 3 (CDR3), composed of parts of the V-(D-)J-gene segments, is very important for antigen binding and can be used as an important measure of variability. Skeletal unloading, and the physiological effects of it, parallel many impacts of space flight. Therefore, we explored the impact of skeletal unloading using the antiorthostatic suspension (AOS) model. Animals were experimentally challenged with tetanus toxoid (TT) and/or the adjuvant CpG. Blood was analyzed for anti-TT antibody and corticosterone concentrations. Whole spleen tissue was prepared for repertoire characterization. AOS animals showed higher levels of corticosterone levels, but AOS alone did not affect anti-TT serum antibody levels. Administration of CpG significantly increased the circulating anti-TT antibody concentrations. AOS did alter constant gene usage resulting in higher levels of IgM and lower levels of IgG. CpG also altered constant gene region usage increasing usage of IgA. Significant changes could be detected in multiple V-, D-, and J-gene segments in both the heavy and light chains in response to AOS, TT, and CpG treatments. Analysis of class-switched only transcripts revealed a different pattern of V-gene segment usage than detected in the whole repertoire and also showed significant alterations in gene segment usage after challenge. Alterations in V/J pairing were also detected in response to challenge. CDR3 amino acid sequence overlaps were similar among treatment groups, though the addition of CpG lowered overlap in the heavy chain. We isolated 3,045 whole repertoire and 98 potentially TT-specific CDR3 sequences for the heavy chain and 569 for the light chain. Our results demonstrate that AOS alters the repertoire response to challenge with TT and/or CpG.

Effects of spaceflight on the immunoglobulin repertoire of unimmunized C57BL/6 mice.

Spaceflight has been shown to suppress the adaptive immune response, altering the distribution and function of lymphocyte populations. B lymphocytes express highly specific and highly diversified receptors, known as immunoglobulins (Ig), that directly bind and neutralize pathogens. Ig diversity is achieved through the enzymatic splicing of gene segments within the genomic DNA of each B cell in a host. The collection of Ig specificities within a host, or Ig repertoire, has been increasingly characterized in both basic research and clinical settings using high-throughput sequencing technology (HTS). We utilized HTS to test the hypothesis that spaceflight affects the B-cell repertoire. To test this hypothesis, we characterized the impact of spaceflight on the unimmunized Ig repertoire of C57BL/6 mice that were flown aboard the International Space Station (ISS) during the Rodent Research One validation flight in comparison to ground controls. Individual gene segment usage was similar between ground control and flight animals, however, gene segment combinations and the junctions in which gene segments combine was varied among animals within and between treatment groups. We also found that spontaneous somatic mutations in the IgH and Igκ gene loci were not increased. These data suggest that space flight did not affect the B cell repertoire of mice flown and housed on the ISS over a short period of time.

Characterization of the naive murine antibody repertoire using unamplified high-throughput sequencing.

Antibody specificity and diversity are generated through the enzymatic splicing of genomic gene segments within each B cell. Antibodies are heterodimers of heavy- and light-chains encoded on separate loci. We studied the antibody repertoire from pooled, splenic tissue of unimmunized, adult female C57BL/6J mice, using high-throughput sequencing (HTS) without amplification of antibody transcripts. We recovered over 90,000 heavy-chain and over 135,000 light-chain immunoglobulin sequences. Individual V-, D-, and J-gene segment usage was uniform among the three mouse pools, particularly in highly abundant gene segments, with low frequency V-gene segments not being detected in all pools. Despite the similar usage of individual gene segments, the repertoire of individual B-cell CDR3 amino acid sequences in each mouse pool was highly varied, affirming the combinatorial diversity in the B-cell pool that has been previously demonstrated. There also was some skewing in the V-gene segments that were detected depending on chromosomal location. This study presents a unique, non-primer biased glimpse of the conventionally housed, unimmunized antibody repertoire of the C57BL6/J mouse.