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

<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