Paper II we show that LPS is another UPEC virulence factor that can be sensed by the sensory nervous system. The neural sensing of LPS results in both neural and immune responses, indicating that UPEC LPS both can trigger sensory signals as well as complement renal epithelial cells in mounting immune responses to combat the infection. Our analyses with various cell types show a differentiated role of each cell type in responding to components present at the infection site, as well as their role in initiating further local and distal signals.
Our work thus contributes to the understanding of initial phase of host-pathogen interactions, a critical phase wherein a well-balanced immune response is crucial for clearing the infection without causing massive collateral damage. Identifying new host-pathogen interactions and mechanisms resulting in infection-associated activation of neural reflexes, as well as deciphering the currently known neuro-immune reflexes can help us develop better diagnostics and adjunctive or new treatments of infections.
6.1.2 Role of neuro-immune reflexes arising from neural sensing of UPEC We demonstrate the involvement of two different neuro-immune reflexes during UPEC kidney infection. In Paper I we show that infection with bacteria expressing HlyA results in nerve-driven inter-organ communication that culminates in the activation of the spleen and splenic IFNγ release. Our results from Paper II indicate that this inter-organ communication seems to be dynamic, and more prominent during the initial phases of UPEC kidney infection.
At a later timepoint of infection we instead find indications that an axon-axon reflex is still at play, with infection resulting in CGRP release in the infected renal tissue. Together our in vivo and in vitro findings suggest that this signal is not dependent on HlyA, but instead LPS appears to be a catalyst for stimulating this CGRP release from sensory nerves.
With regards to the role of the observed neuro-immune reflexes we show that the splenic IFNγ has an immune-modulatory effect in the infected kidney, where it seems to modulate inflammatory (IL-6 and IL-8) signaling generated by infected renal epithelial cells. CGRP is also known to have both pro-inflammatory and anti-inflammatory effects130,283. Thus, the observed CGRP release in infected kidneys may alter the host response to infection. The role of neuro-immune reflexes in inflammation and infection is becoming more apparent. The long term immune-modulatory effects and impact on infection outcome of both the observed inter-organ (splenic activation) and intra-organ (CGRP release) reflexes remains to be investigated. Future research will also delineate if this immune-modulation is beneficial to the host or the bacteria as the infection progresses. However, we hypothesize that the nerve-mediated immune modulation acts to fine-tune immune responses, so that pro-inflammatory signals are not perpetuated when no longer needed, and thus excessive collateral tissue damage is prevented.
Mapping of neural reflex circuits during infection can potentially help in both diagnostics and treatment of infections. Reduction of heart rate variability, an indirect indicator of a dysfunctional inflammatory reflex, has been found as an early indicator of multiple organ dysfunction syndrome in patients with sepsis284. Being able to measure neuro-immune reflexes might be particularly helpful for patient populations that are especially fragile or not
able to interpret or communicate their visceral pain (e.g. infants, elderly with dementia, sedated patients in the intensive care unit) or for patient populations with higher basal inflammatory signaling (e.g. cancer patients or patients with chronic systemic inflammation).
Recordings of neuro-immune reflexes would add to current diagnostic tools, as the nerve reflexes are instantaneous, compared to e.g. the rise of acute phase reactants such as C-reactive protein (where levels do not rise until after approximately 24 h). Further, such recordings could aid in differentiating between inflammation due to sterile injury or infection.
If recordings of neuro-immune reflexes are to be used in a clinical setting, further mapping of these signals are required. To reach a good specificity and sensitivity of such recordings we need to clarify where and how these signals should be recorded and understand more about the difference between normal and disturbed neural signaling.
6.1.3 Infection-associated mechanisms that promote infection-mediated coagulation during experimental kidney infection
In paper III we set out to probe for potential infection-associated products that might contribute to the activation of coagulation during UPEC kidney infection. By combining in vitro and in vivo models we found that the acylation state of the lipid A moiety of LPS alters the kinetics of infection-mediated coagulation, and identify CD147 released from host epithelial cells as a potential activator of infection-mediated coagulation. The epithelial CD147 release was found to be dependent on UPEC expression of HlyA. Collectively our results indicate that different bacterial virulence factors contribute to the dynamics of infection-mediated coagulation through both direct and indirect stimulation of endothelial cells.
Further probing regarding the role of both other virulence factors, as well as CD147 during kidney infection in vivo could elucidate important details regarding the role of infection-mediated coagulation. We have been able to translate the Tissue Microbiology in vivo model of kidney infection from rats to mice90. Future studies could utilize transgenic mice in this model to study the specific roles of different host factors (e.g. CD147) in infection-mediated coagulation in vivo. In a similar way the role of different receptors and signaling molecules in neuro-immune reflexes could be studied (e.g. in mice with knocked-out expression of IFNγ, CGRP or their respective receptor).
Ultimately it would be very interesting to investigate if neuro-immune signals are involved in triggering infection-mediated coagulation. In addition to immune cells, receptors involved in the inflammatory reflex (α7nAChR) are expressed by endothelial cells and platelets285, and incubation of human platelets with an a7nAChR agonist has been shown to decrease platelet aggregation285. This may represent an important linkage between Neuroimmunity and Immunothrombosis285.
6.1.4 Association between antithrombotic treatment and infection severity in humans with acute pyelonephritis
In Paper IV we investigated if reduced clotting leads to higher risk of bacteremia or lower risk of acute kidney injury in a clinical setting. Thankfully, patients with acute pyelonephritis and antithrombotic treatment do not appear to have a higher risk of developing bacteremia.
In contrast to our initial hypothesis, we found a slightly lower risk of bacteremia among patients with LMWH treatment at prophylactic doses. For acute kidney injury, concordant with our hypothesis, we found that treatment with LMWH at prophylactic doses also seem to have protective effects. While our results might indicate that coagulation has a role during acute pyelonephritis, future studies validating the potential protective effects, and investigating possible causative relationships are needed. Collectively, our findings suggest that it is safe to continue antithrombotic treatments in patients with acute pyelonephritis if there are no other contraindications.
Identification of novel risk factors of bacteremia or acute kidney injury during acute pyelonephritis is important as it can help clinicians recognize high-risk patients. A potential protective effect of antithrombotic therapy is also of high interest. It could indicate a role for potential adjunctive treatments to antibiotic treatment of acute pyelonephritis, to prevent complications such as bacteremia and acute kidney injury. One yet unexplored theory is if inhibition of clot formation in vessels surrounding the infection might enable better penetration of antibiotics, and thus enhanced bacterial killing and reduced complications of acute pyelonephritis. However, the role of clotting might go beyond thrombus formation, and involve the immune part of Immunothrombosis. Heparin and heparin-like anticoagulants (such as LMWH) are especially interesting to study, as they are known to have multiple biological effects95. Apart from anticoagulant effects, heparin also has described immunomodulatory effects, including inhibition of platelet activation, disruption of NETs286, interaction with complement proteins and regulation of multiple steps in the complement cascade287,288. Further, by binding to heparin binding protein, it dampens inflammatory signaling289,290, and reduces procoagulant feedback291. Heparin binding protein contributes to vascular leakage during acute pyelonephritis, which might facilitate dissemination of bacteria from urine to blood290. Thus, the inhibitory effect of heparin might also attenuate this vascular leakage and reduce the risk of bacteria passing over to the vascular site. The multifaceted effects of heparin and LMWH opens for a plethora of theories regarding what role it might play during a localized kidney infection. Future exciting studies could investigate the effects of heparin and LMWH, but also of other anticoagulants, and of Sevuparin, a heparinoid with low anticoagulant activity292. Such studies could map the role of Immunothrombosis during localized kidney infection.