O-Antigen Modulates Infection-Induced Pain States

<div><p>The molecular initiators of infection-associated pain are not understood. We recently found that uropathogenic <em>E. coli</em> (UPEC) elicited acute pelvic pain in murine urinary tract infection (UTI). UTI pain was due to <em>E. coli</em> lipopolysaccharide (LPS) and its receptor, TLR4, but pain was not correlated with inflammation. LPS is known to drive inflammation by interactions between the acylated lipid A component and TLR4, but the function of the O-antigen polysaccharide in host responses is unknown. Here, we examined the role of O-antigen in pain using cutaneous hypersensitivity (allodynia) to quantify pelvic pain behavior and using sacral spinal cord excitability to quantify central nervous system manifestations in murine UTI. A UPEC mutant defective for O-antigen biosynthesis induced chronic allodynia that persisted long after clearance of transient infections, but wild type UPEC evoked only acute pain. <em>E. coli</em> strains lacking O-antigen gene clusters had a chronic pain phenotype, and expressing cloned O-antigen gene clusters altered the pain phenotype in a predictable manner. Chronic allodynia was abrogated in TLR4-deficient mice, but inflammatory responses in wild type mice were similar among <em>E. coli</em> strains spanning a wide range of pain phenotypes, suggesting that O-antigen modulates pain independent of inflammation. Spinal cords of mice with chronic allodynia exhibited increased spontaneous firing and compromised short-term depression, consistent with centralized pain. Taken together, these findings suggest that O-antigen functions as a rheostat to modulate LPS-associated pain. These observations have implications for an infectious etiology of chronic pain and evolutionary modification of pathogens to alter host behaviors.</p> </div

O-antigen modulates pain states.

<p>Mice were infected with NU14Δ<i>wz</i>* bearing a deletion of the O-antigen gene clusters and harboring like or heterologous complementation constructs. (<b>A</b>) NU14 smooth colony morphology (i) is rough in the NU14Δ<i>wz</i>* mutant with a human X chromosome plasmid (ii) or a 83972 <i>wz</i>* plasmid (iv) but is rescued by an NU14<i>wz</i>* plasmid (iii). (<b>B</b>) Tactile allodynia of mice in response to sequential infection with NU14 or NU14Δ<i>wz*</i> containing plasmids with the wz* cluster of 83972, NU14, or a fragment of the human X chromosome (n = 10). (<b>C</b>) Summary of O-antigen modulation of pain responses.</p

DataSheet_1_Immune-related gene-based prognostic index for predicting survival and immunotherapy outcomes in colorectal carcinoma.docx

IntroductionColorectal cancer shows high incidence and mortality rates. Immune checkpoint blockade can be used to treat colorectal carcinoma (CRC); however, it shows limited effectiveness in most patients.MethodsTo identify patients who may benefit from immunotherapy using immune checkpoint inhibitors, we constructed an immune-related gene prognostic index (IRGPI) for predicting the efficacy of immunotherapy in patients with CRC. Transcriptome datasets and clinical information of patients with CRC were used to identify differential immune-related genes between tumor and para-carcinoma tissue. Using weighted correlation network analysis and Cox regression analysis, the IRGPI was constructed, and Kaplan–Meier analysis was used to evaluate its predictive ability. We also analyzed the molecular and immune characteristics between IRGPI high-and low-risk subgroups, performed sensitivity analysis of ICI treatment, and constructed overall survival-related receiver operating characteristic curves to validate the IRGPI. Finally, IRGPI genes and tumor immune cell infiltration in CRC model mice with orthotopic metastases were analyzed to verify the results.ResultsThe IRGPI was constructed based on the following 11 hub genes: ADIPOQ, CD36, CCL24, INHBE, UCN, IL1RL2, TRIM58, RBCK1, MC1R, PPARGC1A, and LGALS2. Patients with CRC in the high-risk subgroup showed longer overall survival than those in the low-risk subgroup, which was confirmed by GEO database. Clinicopathological features associated with cancer progression significantly differed between the high- and low-risk subgroups. Furthermore, Kaplan–Meier analysis of immune infiltration showed that the increased infiltration of naïve B cells, macrophages M1, and regulatory T cells and reduced infiltration of resting dendritic cells and mast cells led to a worse overall survival in patients with CRC. The ORC curves revealed that IRGPI predicted patient survival more sensitive than the published tumor immune dysfunction and rejection and tumor inflammatory signatureDiscussionThus, the low-risk subgroup is more likely to benefit from ICIs than the high-risk subgroup. CRC model mice showed higher proportions of Tregs, M1 macrophages, M2 macrophages and lower proportions of B cells, memory B cell immune cell infiltration, which is consistent with the IRGPI results. The IRGPI can predict the prognosis of patients with CRC, reflect the CRC immune microenvironment, and distinguish patients who are likely to benefit from ICI therapy.</p

<i>E. coli</i> with differential pain phenotypes do not elicit differential inflammation.

<p>(<b>A</b>) Hematoxylin-eosin stained sections of bladders from mice instilled with saline, 83972, NU14, Δ<i>waaL</i>, and SΦ874 appeared similar at 6 hours and 14 days. Calibration mark is 100 µm. (<b>B</b>) Inflammation that was scored by a blinded reviewer and expressed as arbitrary units (AU) was significantly elevated for 83972-, NU14-, Δ<i>waaL-</i>, and SΦ874-infected bladders harvested at 6 hours, relative to saline (P<0.001) but was not significantly different among <i>E. coli</i>. (<b>C</b>) Inflammation scores were not significantly different for 83972-, NU14-, Δ<i>waaL-</i>, and SΦ874-infected bladders harvested at 14 days (P = 0.11). (<b>D</b>) Myeloperoxidase (MPO) was quantified in mouse urine by ELISA. Urines were collected at 6 h and 14 d following instillation of saline, 83972, or SΦ874. (<b>E</b>) MPO was quantified in mouse urines obtained at baseline or at 6 h, 24 h, and 14 d following serial instillation of saline (—, n = 4), NU14 (N, n = 4), or Δ<i>waaL</i> (Δ, n = 7). *P<0.05. No significant differences were observed in urinary MPO of mice with treated with NU14 or Δ<i>waaL.</i></p

Purified LPS mimics effects of intact <i>E. coli</i>, and chronic pain is TLR4-dependent.

<p>(<b>A</b>) Mice (n = 8) were instilled 25 µl of 2 µg/ml of LPS purified from NU14, 83972, Δ<i>waaL</i>, or SΦ874 and then evaluated for pelvic allodynia. (<b>B</b>) SΦ874-induced pain in +/+ mice is reduced in TLR4^−/− mice (n = 5; P<0.05 Days 4–14). (<b>C</b>) +/+ mice (C3H/HeJOuJ, “OuJ") or TLR4-deficient mice (C3H/HeJ, “HeJ") were used as bone marrow donors for γ-irradiated recipients; legend arrow indicates donor bone marrow into recipient (n = 9, 9, 14 and 15 respectively). C3H/HeJOuJ recipients exhibited SΦ874-induced pain that was reduced in C3H/HeJ recipients (P<0.01 Days 3–14).</p

TLR4 mediates chronic pain.

<p>UTI was induced in female mice by instilling 10⁸<i>E. coli</i> into the bladder, and tactile allodynia and bladder colonization were quantified. (<b>A</b>) Mice were instilled repeatedly with saline, NU14, or Δ<i>waaL</i> (n = 9). NU14 induced resolving acute pain (P<0.001 Days 2–5, P<0.01 Days 1 and 6, P<0.05 Days 7–10), but Δ<i>waaL</i> induced chronic pain following the second infection. (<b>B</b>) Bladders from mice in (A) had detectable NU14 colonization but not Δ<i>waaL</i> colonization (P<0.044). (<b>C</b>) SΦ874 pain (n = 10) was abrogated in mice infected with SΦ874 pWQ288 (n = 10). (<b>D</b>) SΦ874 is cleared rapidly from the bladder; open circle indicates inoculum (n = 5 Days 1–14, n = 10 Day 35).</p

Pelvic pain behavior is associated with sacral spinal cord excitability.

<p>Spontaneous action potentials and evoked potentials were quantified in sacral spinal cords ex vivo at ventral roots S1–S3. (<b>A</b>) A sacral spinal cord is mounted in a recording chamber (upper panel), and spontaneous activity is recorded from the ventral root (lower panel). (<b>B</b>) Representative action potentials from spontaneous firing of individual neurons identified by pCLAMP. (<b>C</b>) Firing activity in sacral spinal cords is higher in Δ<i>waaL</i> at 14 d (n = 5, P = 0.0099), SΦ874 at 20 days (n = 9, P = 0.0001), and NU14 mice at 2 d (n = 6, P = 0.0298) than in saline controls (n = 7) or resolved NU14 (n = 5). (<b>D</b>) Evoked ventral root responses to dorsal root current at 2× current intensity for spinal cord of saline mouse at 2 d (upper trace) and Δ<i>waaL</i>-infected mouse after serial infection. (<b>E</b>) Normalized responses at P2–P5 relative to P1 in ventral roots of mice instilled with saline (n = 23), NU14 (n = 26), SΦ874 (n = 21), or Δ<i>waaL</i> (n = 12). *P<0.05 and **P<0.01 by Student <i>t</i> test relative to saline. (<b>F</b> and <b>G</b>) Responses across stimulus intensities at P3 and P4, respectively.</p