Innovative solutions are needed for the treatment of bacterial infections, and a range of antibacterial molecules have been explored as alternatives to antibiotics. A different approach is to investigate the immune system of the host for new ways of making the antibacterial defence more efficient. However, the immune system has a dual role as protector and cause of disease: in addition to being protective, increasing evidence shows that innate immune responses can become excessive and cause acute symptoms and tissue pathology during infection. This role of innate immunity in disease suggests that the immune system should be targeted therapeutically, to inhibit over-reactivity. The ultimate goal is to develop therapies that selectively attenuate destructive immune response cascades, while augmenting the protective antimicrobial defence but such treatment options have remained underexplored, owing to the molecular proximity of the protective and destructive effects of the immune response. The concept of innate immunomodulation therapy has been developed successfully in urinary tract infections, based on detailed studies of innate immune activation and disease pathogenesis. Effective, disease-specific, immunomodulatory strategies have been developed by targeting specific immune response regulators including key transcription factors. In acute pyelonephritis, targeting interferon regulatory factor 7 using small interfering RNA or treatment with antimicrobial peptide cathelicidin was protective and, in acute cystitis, targeting overactive effector molecules such as IL-1β, MMP7, COX2, cAMP and the pain-sensing receptor NK1R has been successful in vivo. Furthermore, other UTI treatment strategies, such as inhibiting bacterial adhesion and vaccination, have also shown promise.
Excessive innate immune responses to infection cause symptoms and pathology in acute pyelonephritis and acute cystitis.
Innate immunomodulation therapy is, therefore, a realistic option for treating these conditions.
Targeting excessive innate immune responses at the level of transcription has been successful in animal models.
Innate immunomodulation therapy reduces excessive inflammation and tissue pathology and accelerates bacterial clearance from infected kidneys and bladders in mice.
Innate immunomodulation therapy also accelerates the clearance of antibiotic-resistant bacterial strains.
Innate immunity provides a rapid and selective first line of defence1,2,3,4, preventing pathogens from gaining access to host tissues while sustaining symbiosis with the commensal flora. This impressive level of precision is maintained by specific pathogen recognition mechanisms coupled with the immediate activation of the innate immune system4,5,6,7,8 (Fig. 1). Health is rapidly restored when the innate immune response is efficient and transient, but an imbalanced response can create exaggerated inflammatory states and cause severe acute disease, mortality and chronic sequelae9. By targeting and correcting these weaknesses therapeutically, a functional innate immune response can be restored and the bacteria removed.
The potential of innate immunomodulation therapy is supported by successful studies of its use in urinary tract infection (UTI), in which this concept has been developed10,11,12,13 (Fig. 2). The innate immune response controls the severity of acute pyelonephritis (APN) and acute cystitis (ACY), and genetic screens have identified important transcriptional checkpoints as disease determinants10,11,12,13,14,15,16,17,18,19. For example, transcriptional regulators interferon regulatory factor 3 (IRF3) and IRF7 control disease severity in infected kidneys10,11,20 by regulating the defensive (IRF3) or destructive (IRF7) response cascades (Fig. 3). In the bladder, the inflammasome constituents apoptosis-associated speck-like protein containing a CARD (ASC) and NOD-, LRR- and pyrin domain-containing 3 (NLRP3) serve as transcriptional repressors of the protease matrix metalloproteinase 7 (MMP7) and pain sensor neurokinin 1 receptor (NK1R), controlling the level of inflammation by a non-canonical mechanism of pro-IL-1β processing12,13,21,22,23 (Fig. 4). These findings illustrate the tight genetic control of innate immune activation and disease pathogenesis in UTI.
The adaptive immune response adds antigen specificity and longevity to the host defence (Fig. 1). Antigens on invading pathogens are recognized and memory is created to prevent recurrent infections24. An adaptive immune response is detected in patients with APN, but the protective role of this immune response remains unclear25,26,27. In mice, gene deletions affecting adaptive immunity have not been found to drive disease development but to weaken the defence28,29,30,31. X-linked immunodeficient and RAG1-deficient mice, with attenuated B and T lymphocyte function, were not more susceptible to E. coli-induced UTIs than controls and had low bacterial counts after 24 h (refs15,32). Additionally, αβ T cell-deficient and ɣδ T cell-deficient mice did not show increased susceptibility to kidney infections28. However, some studies have suggested that severe combined immunodeficiency mice have increased bacterial burden compared with wild-type mice during UTI28,33. Additionally, T helper 17 (TH17) cell responses have been proposed to affect bacterial clearance, based on reduced neutrophil and macrophage infiltration in IL-17A-deficient mice17. Sentinel natural killer (NK) cells have been proposed to influence resistance to uropathogenic Escherichia coli (UPEC) infection through mechanisms involving the release of TNF; however, the underlying protective mechanisms of this cell population remain unknown and additional depletion studies will be essential to fully understand the role of NK cells in UTI34. In addition, resident macrophages in the bladder suppress TH1-mediated immunity and macrophage depletion helps the host to clear the infection, but T cell depletion had no effect on bacterial clearance, confirming that myeloid cells rather than lymphocytes are essential for UTI pathogenesis35.
The WHO has declared antimicrobial resistance 1 of the top 10 global public health threats facing humanity; thus, novel antimicrobial therapeutic strategies are urgently needed36. UTIs are among the most prevalent bacterial infectious diseases globally37,38, most often caused by UPEC originating from the gut flora or perineal flora39,40. E. coli infections account for half of the estimated global burden of antibiotic resistance36,41,42, with about 90% of E. coli strains being resistant to at least one antibiotic43. In Europe, UPEC isolates are resistant to third-generation cephalosporins and fluoroquinolones (11.8 % and 22.3% of UPEC strains, respectively)44 and fluoroquinolone-resistant UPEC were 31.3% of the isolates in hospitalized patients with UTI45,46.
In this Review, we discuss innate immune hyperactivation as a disease determinant in UTI and identify targets for innate immunomodulatory therapy. We envisage that this new approach might benefit large patient groups in the future. The use of innate immunomodulation therapy would be similar to antibiotics, aiming to reduce acute inflammation and infection but, in contrast to antibiotics, innate immunomodulation therapy might offer a solution for treating infections caused by antibiotic-resistant strains. Furthermore, efforts to develop UTI vaccines and other prophylactic measures are discussed, as well as investigations into broad anti-inflammatory therapies such as NSAIDs.
Disease determinants in acute pyelonephritis
APN is a severe, sometimes life-threatening infection37,47,48, initiated by UPEC strains that attack the renal pelvic mucosa and elicit a local innate immune response that is amplified and becomes systemic49,50,51,52. Local symptoms at the site of infection are triggered by excessive kidney inflammation and the systemic spread of inflammatory mediators generates fever and general malaise in the infected patient53,54,55. APN is accompanied by urosepsis in ~30% of adults, and urosepsis remains a major cause of mortality, especially in the elderly56,57,58,59. The mortality rate in APN was estimated to be 10–20% before the introduction of antibiotics, and a frequency of 7.4% was recorded in 2005, after the introduction of antibiotics60. In addition, APN is an important cause of renal growth retardation and permanent kidney damage in childhood, leading to chronic sequelae such as hypertonia, renal insufficiency or renal failure as well as premature delivery61,62,63,64.
Molecular control of the innate immune response in APN
Mechanisms of APN pathogenesis and innate immune activation have been extensively studied and reviewed4,5,6,29. Briefly, pathogen-specific recognition mechanisms activate a rapid innate immune response in infected tissues, leading to inflammation and the mobilization of an antibacterial defence65. Specific bacterial adherence strategies are essential, involving surface fimbriae or non-fimbrial adhesins and host cell receptors16,54,66,67. P fimbriae are expressed by 90–100% of UPEC strains in patients with uncomplicated APN68, with a strong association with disease severity, and have been proposed to facilitate bacterial invasion, leading to urosepsis49,65,69,70. Adherence also facilitates tissue interactions of other virulence factors, including the endotoxin LPS, exotoxins (such as haemolysin and CNF), iron-binding proteins and capsular polysaccharides5,71,72,73,74.
Mechanisms of innate immune activation and essential effector functions in APN have been mapped using cellular infection technology, animal models and clinical studies4,10,11,14,16,18,19,32,75,76,77,78,79,80. Key regulators of UTI severity have been evaluated using gene knockout technology and their relevance to human disease has been verified in clinical studies10,11,29,78. The Toll-like receptor (TLR) family acts as a shared upstream control node of the pathogen-specific innate immune response16,32,81,82,83,84 and TLR4 responds to UPEC virulence factors by engaging different co-receptors, including glycosphingolipid receptors for P fimbriae recognizing Galα1-4-Galβ epitopes65,85,86 or mannosylated glycoproteins recognized by type 1 fimbriae87,88,89,90,91,92. The adaptor proteins regulate the downstream response through phosphorylation cascades10,16,32,93,94 and the activation of transcriptional regulators defines the quality and quantity of the inflammatory cascade10,11, through mediators such as cytokines and chemokines and inflammatory cells recruited to the site of infection95,96 (Fig. 1).
Different arms of the TLR4 signalling cascade can be engaged, depending on the fimbrial adhesins and virulence factors that the pathogens express16,54,66,67,97,98,99. P fimbriated UPEC strains mainly activate the adaptor proteins TIR-domain-containing adapter-inducing interferon-β (TRIF) and TRIF-related adaptor molecule (TRAM)16,85,93,94 and the phosphorylation of mitogen activated protein (MAP) kinases, p38 and cyclic AMP-responsive element-binding protein (CREB) defines signalling cascades downstream of TLR4, which converge on specific transcription factors (including IRF3, IRF7, AP-1 and NF-κB)10,93. The resulting pro-inflammatory cascades include cytokines in the kidneys, such as type I interferons, IL-6 and TNF, and IL-1 in the bladder, as well as neuropeptides (substance P (SP) and galanin) and their receptors. Chemokines (IL-8 (also known as CXCL8), CCL3 (also known as MIP1α), CCL5 (also known as RANTES) and CCL2 (also known as MCP1))12,13,76,95,100,101,102,103,104,105 create the inflammatory cell infiltrate as the recruitment and activation of inflammatory cells are essential for the antibacterial defence14,106,107,108. Other TLRs affecting UTI pathogenesis include TLR1, TLR2 and TLR5, which mainly activate the MyD88 arm of the TLR cascade83,109,110,111,112,113.
The essential role of TLR4 signalling in UTI was first observed in Tlr4–/– mice82. UPEC-infected Tlr4−/− mice do not develop APN or ACY or an inflammatory response32,106. To identify the TLR4-dependent mechanisms of disease, single genes in the TLR4 pathways have been systematically deleted and the resulting phenotypes have been characterized in UPEC-infected mice10. Specific gene deletions have been shown to create an APN or ACY disease phenotype, providing essential insights into the mechanisms of disease pathogenesis. Irf3−/− mice lack the protective IRF3-dependent arm of innate immunity and develop severe APN, characterized by hyperinflammation, urosepsis and massive renal abscess formation10,11. IFNβ is activated downstream of IRF3, and Ifnb1−/− mice showed a similar severe APN disease phenotype to Irf3−/− mice10.
A strong APN phenotype is also observed in Cxcr2−/− mice, deficient in the chemokine receptor CXCR1, which regulates neutrophil activation and neutrophil exit from infected kidneys into the urine14,79,114. The Cxcr2−/− mice develop severe APN with urosepsis and acute mortality, accompanied by renal abscesses14,115. Pathology is caused by massive neutrophil retention in the kidneys, as recruited neutrophils fail to exit into the urine and to scavenge and kill the bacteria14,115,116. The Cxcr2−/− mice also develop renal damage resembling renal scarring in children with APN14,79,116. Neutrophil recruitment is rapid, regulated by the urothelial chemokine response to infection100,117 and release of high levels of TNF from epithelial cells and resident mast cells, which support the neutrophil response54,89,118. Cells in the subepithelial compartment, including dendritic cells, are affected through increased IL-1β and IL-1R expression119 and infiltrating neutrophils have high COX2 expression and release of prostaglandin E2 (PGE2) affects inflammation120. The chemokine receptors are essential determinants of the disease response, by controlling neutrophil activation and migration121.
C5aR1 has been identified as a determinant of APN disease progression in mice using decreased bacterial counts, lymphocyte infiltration and kidney pathology in the kidneys in C5ar1−/− mice compared with wild-type controls122. Furthermore, bacterial burden and neutrophil and macrophage infiltration was decreased in P2x7−/− mice, suggesting a role of the purinoreceptor P2X7 in APN and associated renal fibrosis123. Finally, increased bacterial burden in the kidneys of Car2−/− mice suggested that carbonic anhydrase 2 in intercalated cells in the kidneys might promote renal bacterial clearance124.
Human relevance of the genetic screens
The human relevance of these findings has been supported by clinical studies10,78,125. IRF3 promoter sequence variants were detected in two APN-prone patient populations10. The homozygous A/A–C/C genotype (nucleotide positions −925 and −776) was prevalent the APN-prone patients (79%), whereas the co-segregating heterozygous single-nucleotide polymorphisms (SNPs) were more common in those with asymptomatic bacteriuria (ABU; 69%)10. The APN haplotype was shown to decrease IRF3 expression in reporter assays, suggesting that IRF3 needs to be fully functional to avoid APN10. CXCR1 mRNA levels are low in children with APN and CXCR1 expression is reduced78. APN-prone patients carry heterozygous CXCR1 polymorphisms affecting receptor expression and the presence of CXCR1 variants has been confirmed in several APN-prone patient groups126,127. CXCL8 SNPs were detected in infected patients with renal involvement confirmed by positive dimercapto succinic acid (DMSA) scans125,127,128,129.
In addition, PTX3 is polymorphic in APN-prone children and in adults who suffer from recurrent cystitis18. IL10 polymorphisms were identified in APN-prone patients80 as well as SNPs in CCL5, encoding the eosinophil and T cell chemoattractant RANTES130, supporting human disease relevance.
The information obtained from these studies is essential, as it provides a framework for distinguishing protective from destructive molecular determinants of the innate immune response and for targeting the cause of disease therapeutically.
IRF7 siRNA innate immunotherapy in APN
The transcription factor IRF7 takes over the innate immune response and drives disease pathology in Irf3−/− mice (Fig. 3), in which IRF7-dependent gene networks are strongly upregulated, including Tlr4, Stat3 and Il6 as well as downstream genes involved in the acute phase response11. Upregulation of IRF7 expression and IRF7-dependent gene networks is also observed in patients with febrile UTI, supporting the human relevance11,99. Direct binding of IRF-7 to promoter DNA fragments (OAS1, CCL5 and INFB1) was demonstrated, supporting the role of IRF7 as a transcriptional regulator, especially in hosts with reduced IRF3 expression99. Further evidence supporting the role of IRF7 was obtained in infected Irf7−/− mice, which were protected from infection and showed no evidence of kidney pathology11. IRF7 was further identified as an important transcriptional mediator during group b streptococcus-induced UTI131.
The protected phenotype in Irf7−/− mice and overactivation of Irf7 in disease-prone Irf3−/− mice identified IRF7 as a potential therapeutic target. Using an short interfering RNA (siRNA)-based strategy of Irf7 inhibition11, siRNA treatment of Irf3−/− mice was shown to inhibit the excessive innate immune response in Irf3−/− mice and to improve bacterial clearance, resulting in resolution of infection by day 7 (Fig. 3). siRNA therapy compared favourably with antibiotic treatment and Irf7 siRNA treatment considerably reduced the disease score11. Importantly, Irf7 silencing immunomodulation therapy accelerated bacterial clearance to the same extent as cefotaxim11. Furthermore, recombinant IFNγ treatment was shown to increase survival, reduce bacterial burden and reduce kidney pathology in a rabbit model of APN, compared with untreated rabbits132.
The IL-6 response to UTI was first described in the 1980s53,133; IL-8, IL-1 and TNF were also detected. The IL-6–STAT3 pathway is activated in the kidneys of infected Irf3−/− mice, shown by transcriptomic analysis and tissue staining11. The IL-6–STAT3 response is IRF7-dependent, and this pathway is inhibited in mice treated with liposomal Irf7 siRNA. The role of IL-6 in an APN mouse model has been investigated19. In Il6-knockout mice with experimental UTI, STAT3 phosphorylation was significantly reduced but total STAT3 expression was unchanged. Furthermore, levels of STAT3 transcriptional targets were reduced. Inhibition of IL-6, using an IL-6 neutralizing antibody in Il6-intact mice, resulted in reduced IL-6 levels and decreased bladder and kidney STAT3 phosphorylation in response to UPEC infection, increased bacterial burden, as well as abscess formation, supporting a role for this cytokine in the host defence19.
The IL-6–STAT3 pathway is also activated in patients infected with P fimbriated bacteria99, and IRF7 has been identified as a key transcription factor driving the response in these patients. IL-6 and other cytokines were subsequently measured in patient urine in a number of studies, in which the levels of these cytokines correlated positively with bacterial virulence and disease severity, fever and CRP75,100,134. A strong effect of P fimbriation, a feature that defines this virulence factor as a host response inducer66,67, was also observed in these studies. The early studies led to further detailed analysis of TLR4 and the TLR4-dependent signalling pathway that activates IL-6–STAT310,53,81.
These results highlight how individual transcription factors regulate beneficial or destructive effects of innate immunity and identify IRF7 as a target for immunoregulation therapy in APN.
Clinical trials support the feasibility of siRNA-based therapies for indications including familial amyloidosis, haemophilia and hepatitis C135,136,137,138. The use of siRNA-based therapies in humans is still developing, and the challenges of off-target effects, pharmacokinetics and immune activation need to be addressed for this approach to be a clinical reality139,140. However, positive effects of siRNA therapy have been observed in human experimental trials141,142, and the successful development of RNA-based vaccines suggests that such technologies should be clinically feasible. The Irf7 siRNA treatment approach is intended to be acute, with similar treatment protocols to those used for antibiotics, which is essential in order to avoid long-term suppression of the immune system and effects on the susceptibility to other infections, including those of viral origin.
Anti-inflammatory therapies in APN
APN is caused by exaggerated inflammation in infected kidneys. A number of general anti-inflammatory agents have been tested in animal models of APN, with limited benefits120,143,144. Early studies showed that glucocorticoids attenuate inflammation, but they also impaired bacterial clearance145. NSAIDs inhibited the inflammatory response but the infection was not cleared in treated mice145. These results indicate that broad inhibition of the inflammatory response does not equal protection against disease and support the conclusion that drugs with increased specificity are needed to achieve an anti-inflammatory and antibacterial effect.
Antimicrobial peptides (AMPs) are effectors of the innate immune response and resemble antibiotics, in that they mainly target the bacteria rather than the immune system per se146,147. AMPs, including defensins, have been investigated as therapeutics owing to their direct antibacterial effects18,146,147,148. AMPs (including β-defensins, ribonucleases and cathelicidin) are potent effectors of the renal defence against infection147,149,150,151,152,153. RNase7 and cathelicidins act by disrupting the phospholipid membranes of various microorganisms and are constitutively synthesized by the kidney147,151,152. Lipocalin 2 (LCN2; also known as NGAL) is a specific antibacterial protein secreted both in the urine and systemically after Gram-negative infection, kidney injury or urosepsis. LCN2 binds the bacterial siderophore enterochelin, preventing iron transfer and sequestration by the bacteria154. The humoral pattern recognition molecule pentraxin 3 (PTX3) serves as an opsonin and promotes bacterial uptake by neutrophils18.
The cathelicidin AMP (CAMP) LL-37, which is primarily released by macrophages and neutrophils, targets UPEC strains in vitro147. Cramp−/− mice (CRAMP is an LL-37 homologue) showed reduced pathology compared with wild-type controls after infection with the E. coli cystitis strain UTI89 (ref.155), and Cramp−/− mice infected with the pyelonephritis strain E. coli CFT073 were protected147, supporting a role in the antimicrobial defence, similar to antibiotics147,155. Although positive results have been observed in vitro and in knockout mice in vivo, further studies are required to address if purified AMPs can be administered as a therapeutic in disease models.
Immunomodulation in acute cystitis
ACY is mainly caused by bacterial infections of the urinary bladder37,68. The patients experience pain, dysuria and frequency of urination, and the diagnosis is supported by the presence of bacteriuria and pyuria37,156,157. A subset of susceptible patients develop severe ACY with an excessive innate immune response, severe symptoms and pathology158. Recurrent infections are common, and chronic inflammation can lead to sequelae such as interstitial cystitis/bladder pain syndrome (IC/BPS)159,160.
Type 1 fimbriae act as virulence factors in the mouse urinary tract161,162,163, mediating bacterial adherence to the bladder mucosa88,89,90,91. The FimH adhesin binds several mannosylated host cell glycoconjugates and has been proposed to facilitate bacterial invasion of mucosal cells92. TLRs control the innate immune response to E. coli infection in the bladder, and Tlr4−/− mice are protected from disease16,32,106,111. Type 1 fimbriated strains preferentially activate the MyD88 and TIRAP adaptors and NF-κB-dependent effector functions88,162,163,164,165. A pro-inflammatory effect of type 1 fimbriae has been observed in a mouse model, but not in human studies using isogenic strains differing in fimbrial expression99. In contrast to P fimbriae, which triggered an IRF7-driven inflammatory response in the patients, direct effects of type 1 fimbriae on innate immunity were not detected in patients99. Type 1 fimbriae were shown to inhibit basic cellular functions such as RNA translation and effects on neurosensing and solute carriers suggested a potential link to the host reponse99.
IL-1 hyperactivation as a cause of acute cystitis
IL-1β is a potent pro-inflammatory cytokine that amplifies innate immune responses in several chronic infection models, including cystic fibrosis, tuberculosis and inflammatory bowel disease9,166,167,168. ACY has been identified as an IL-1β hyperactivation disorder triggered by E. coli infection of the bladder mucosa in a mouse model12 (Fig. 4). Clinical ACY isolates activated pro-IL-1β expression in human 5637 bladder epithelial cells in vitro, and the processing and release of mature IL-1β were accelerated12. The IL-1 response to E. coli infection is controlled by TLR4 and transcription factors, including ERK, p38 and NF-κB169,170. In addition to IL-1β, a cascade of IL-1-dependent genes is activated, and effectors of the host response include IL-8 and PGE2 as well as SP and NK1R12,13.
Genetic screens further identified a non-canonical mechanism of IL-1β hyperactivation that creates severe ACY12,13 (Fig. 4). The inflammasome controls pro-IL-1β processing in many models; therefore, inactivating gene deletions affecting the inflammasome would be expected to be protective. Instead, a severe ACY disease phenotype was observed in infected mice carrying single-gene deletions of Nlrp3 or Asc12. The excessive, disease-associated IL-1β response in Asc−/− mice was further mapped to the protease MMP7 and the pain sensors NK1R and SP, which were overexpressed12,13. Direct molecular interaction studies attributed the excessive IL-1 response to pro-IL-1β processing by MMP7 (Fig. 4), and included a cascade of IL-1β-dependent, downstream genes12. Importantly, Il1b−/− mice were protected against infection and inflammation, further supporting the importance of IL-1β in this disease12,94. The human relevance of these findings was supported by elevated urine IL-1β levels in patients with ACY, compared with patients with ABU12.
IL-1 receptor antagonist treatment of acute cystitis
IL-1β and its receptor are potential therapeutic targets for immunomodulation therapy in ACY (Fig. 4). The recombinant human IL-1 receptor antagonist (IL-1RA) protein binds to IL-1R1 with similar affinity to IL-1α and IL-1β, inhibiting their binding and the dimerization of IL-1R1 and the IL-1 receptor accessory protein, as well as downstream signalling9. The IL-1RA anakinra was, therefore, investigated as an inhibitor of IL-1β hyperactivation in ACY (Fig. 2). Asc−/− mice, which are susceptible to ACY, were treated with daily injections of anakinra for 7 days and disease severity and bacterial clearance were quantified at sacrifice12. Anakinra treatment reduced tissue pathology by 75–80% compared with untreated mice and the inflammatory response to infection was markedly attenuated, as shown by reduced neutrophil numbers in urine and bladder tissue as well as reduced IL-1-dependent gene expression. Furthermore, bacterial clearance was accelerated, suggesting that correcting the immune imbalance empowers the innate immune response of the host. The efficacy of anakinra treatment was verified in C57Bl/6 wild-type mice with intact inflammasome function13, which exhibit a milder, more transient disease phenotype than Asc−/− mice.
Anti-inflammatory effects of IL-1RA have been demonstrated in several hyper-inflammatory diseases such as rheumatoid arthritis, gout, cryopyrin-associated periodic syndrome171,172,173 and in bacterial and viral infection models including cystic fibrosis175 and COVID-19 (refs174,176), supporting the feasibility of exploring the beneficial effects of IL-1RA as a therapeutic in patients with ACY175,177,178.
The efficiency of IL-1RA therapy was compared with that of antibiotic therapy in Asc−/− mice infected with fully virulent E. coli strains. IL-1RA therapy showed similar efficacy to cefotaxime and both treatments accelerated bacterial clearance179; however, IL-1RA therapy inhibited the hyper-inflammatory response in infected bladders more effectively than cefotaxime, indicating an added benefit. In addition, IL-1RA therapy accelerated the clearance of extended-spectrum β-lactamase (ESBL)-producing E. coli strains against which cefotaxime was inefficient179.
The results of innate immunomodulation therapy in a mouse model of ACY provides a rationale for initiating clinical trials of IL-1RA therapy in ACY.
Therapeutic effects of MMP inhibition in acute cystitis
Mmp7 expression was strongly upregulated in mice that developed severe ACY, as shown by genome-wide transcriptomic analysis of whole-bladder mRNA12. MMP7 was also shown to cleave pro-IL-1β to its mature, active form (Fig. 4). Based on these findings, the broad MMP inhibitor batimastat (also known as BB-94) was tested for therapeutic efficacy using the same protocol as for IL-1RA treatment12 (Fig. 2). The severity of ACY was markedly attenuated in the treated mice, as shown by reduced urine and tissue neutrophil levels and a substantial decrease in pathology scores (Figs 2,4). Furthermore, treatment accelerated bacterial clearance but to a lower extent than IL-1RA treatment12. These results indicate that batimastat or related compounds might be of interest to explore as a potential treatment alternative in ACY.
Pain attenuation in acute cystitis
Pain is a hallmark of ACY and symptom relief is a key result of ACY therapy. Pain is commonly regarded as secondary to inflammation and is defined as one of its hallmarks180. Pro-inflammatory cytokines promote pain by sensitizing nerves and activating transcription and release of pain molecules such as nerve growth factor (NGF), PGE2, MMP9 and the neuropeptide SP, which is an effector molecule of inflammatory pain181,182. SP is released by several cell types and engages neurokinin receptors, particularly NK1R183 (Fig. 5). The interaction between SP and NK1R helps to propagate peripheral pain signals from local afferent nerves to the dorsal roots of the central nervous system184.
SP is spontaneously released within the bladder wall185 and binds to NK1R, triggering the peripheral pain response184. LPS was proposed to cause the pain response during UPEC infection via a non-inflammatory TLR4-dependent mechanism, but the effector mechanisms of this response were not defined186. A direct effect of infection on the nervous system has been detected owing to elevated SP and NK1R levels in UPEC-infected nerve cells in vitro13. Increased levels of SP and NK1R were also detected in the bladder mucosa of UPEC-infected mice and accompanied by increased pain behaviour. The SP–NK1R response was shown to be hyperactivated in Asc−/− and Nlrp3−/− mice through a neuroinflammatory loop controlled by IL-1β and NK1R13 (Fig. 5). Urine SP levels were also found to be elevated in patients with ACY, suggesting the human relevance of this mechanism and a potential for use of SP as a biomarker of symptomatic lower UTIs and ACY13. Interestingly, Nlrp3 and Asc were shown to control the expression of NK1R and SP and the processing of IL-1β, linking inflammation and pain in mice susceptible to ACY. Owing to the strong connection between SP and NK1R expression and pain in experimental ACY, an NK1R antagonist (SR140333) was investigated for therapeutic efficacy in Nlrp3−/− mice13 (Fig. 5). NK1R antagonist treatment reduced NK1R staining in bladder tissue sections as well as Nk1r and Ppt-A mRNA levels in whole-bladder mRNA13. A marked decrease in tissue pathology (oedema, hyperaemia and mucosal integrity) was detected, and treatment inhibited IL-1 superfamily gene expression, inflammation, cytokine production and adaptive immunity13. These results suggest that pain from the urinary tract during ACY involves SP and NK1R signalling and that treatment with an NK1R antagonist can reduce symptoms and inflammation in UPEC-infected mice (Figs 2,5).
Human relevance of innate immunomodulation therapy in the urinary tract
The extent to which these successful treatment strategies in mice can be translated to human ACY needs to be investigated in controlled clinical studies; however, insights into the human relevance of innate immunomodulation therapy have been gained in patients with IC/BPS187. These patients experience severe and debilitating pain during bladder filling, resulting in extreme urgency and frequency188,189. IC/BPS has a prevalence of about 0.1% and affects all aspects of life, as even morphine and morphine analogues fail to provide adequate symptom relief188,189,190. Numerous therapeutic approaches to IC/BPS have been tested in preclinical studies including NK1R antagonists and various chemicals185,191,192, such as protamine sulphate, to induce urothelial cell shedding and facilitate bacterial clearance from the urinary tract193; however, the results of these studies have not been sufficiently convincing for human use194. More specific therapies have been lacking owing to poor understanding of the disease mechanisms.
The IL-1β-dependent and NK1R-dependent pain response in the mouse ACY model suggested that IL-1RA therapy might be an option for this patient group. Patients with severe IC/BPS (n = 17) were, therefore, offered off-label IL-1RA treatment187, and ~70% of the patients showed an initial treatment response, characterized by a reduction in pain and micturition frequency and an increased quality of life, quantified by O’Leary’s symptom score (7.2 versus 17.4)187. Clinical improvement was accompanied by a reduction in urine SP levels and gene expression analysis revealed considerable effects on neuroinflammatory and inflammatory gene sets, including IL-1, IL-6 and IL-8 signalling pathways, which were inhibited187. After the initial treatment cycle, 13 of the patients chose to continue IL-1RA treatment and individual treatment protocols have proven efficient in the long term (>1 year)187.
These results provide clinical evidence that IL-1RA therapy might be useful and effective in patients with IC/BPS, but controlled clinical trials should be performed to validate these effects. In addition, studies of IL-1RA therapy might be of interest to treat IL-1β-induced symptoms and pathology in ACY, but clinical data are not yet available.
Additional approaches to inhibiting innate immunity in acute cystitis
A variety of anti-inflammatory regimens have been tested as therapeutics in ACY47. Numerous studies have shown that broad anti-inflammatory agents such as corticosteroids and NSAIDs reduce inflammation in UTI models145,195,196, but have adverse effects on bacterial clearance.
Controlled clinical studies have compared diclofenac or ibuprofen with antibiotics, but no significant benefits were detected for bacterial clearance or symptom relief144,197,198. In a study of 181 women with ACY, comparing a 3-day ibuprofen course with pivmecillinam, approximately half of the patients who received ibuprofen had persisting or worsening symptoms during the 4-week follow-up period compared with 10% of patients treated with the antibiotic198. In studies comparing ibuprofen with fosfomycin treatment, symptom burden was increased in 34% in the ibuprofen group143,144. In one study, NSAID treatment with diclofenac was associated with an increased risk of APN197,198.
Another mechanism by which ACY, specifically recurrent cystitis, is mediated in mice is through cyclooxygenase 2 (COX2), which catalyses the rate-limiting step in the conversion of omega-6 arachidonic acid to prostanoids and is involved in the development of acute inflammation. In mice sensitized to develop recurrent cystitis, Ptgs2, which encodes COX2, showed a 50-fold increased expression in bladder tissues from UPEC-infected mice, and immunofluorescent antibody staining of bladder sections showed robust expression of COX2 by urothelial cells in bladders exhibiting severe inflammation120.
Specific inhibition of COX2, using a selective COX2 inhibitor (SC-236), was shown to reduce bladder inflammation and bacterial load in a mouse model of ACY, consistent with the role of inflammation in the disease process120; by contrast, a COX1 inhibitor did not significantly affect inflammation or bacterial counts120. Furthermore, SC-236-treated mice had lower bladder bacterial titres 24 h after infection than those treated with the COX1 inhibitor, suggesting that bacterial clearance was facilitated by the blockade of COX2.
Forskolin regulates exocytosis of E. coli in bladder epithelial cells
In a mouse model in which mice were catheterized and intravesically instilled with the type 1 fimbriated UPEC strain CI5, bacterial invasion into bladder epithelial cells was found to be mediated through fusiform vesicles199. Further in vitro investigations showed that E. coli infection of 5637 bladder epithelial cells initiated bacterial incorporation into secretory lysosomes and release of the secretory lysosomes. Secretory lysosomes are stimulated through intracellular Ca2+ levels and cyclic AMP (cAMP) flux, and discharge their contents in response to fluctuations in these factors. E. coli infection was observed to induce Ca2+-sensitive and cAMP-sensitive exocytosis of secretory lysosomes from bladder epithelial cells. Further investigation showed E. coli inside the exocytosed lysosomes199.
Forskolin is a labdane diterpene that stimulates adenylate cyclase and increases intracellular levels of cAMP. Using forskolin in combination with gentamicin in BALB/c mice, a reduction in intracellular bacterial numbers was detected in bladder tissue, and urine IL-6 levels were lowered199.
These results suggest that broad anti-inflammatory agents lack specificity as they affect both the protective and the destructive arms of innate immunity.
Immunomodulation by molecules of bacterial origin
Bacteria are an interesting source of molecules that regulate innate immunity in the host. In contrast to virulence factors, which can have detrimental effects, bacterial molecules have been shown to target the transcriptional machinery or TLR signalling and inhibit the innate immune response, resulting in a protective effect. These molecules have an interesting potential as candidates for innate immunomodulation therapy.
Bacterial NlpD inhibits Pol II-dependent gene expression
In early experiments, ABU strains were shown to inhibit host gene expression by targeting the RNA Pol II phosphorylation machinery200,201 (Fig. 6). The RNA Pol II cycle controls RNA synthesis through numerous precisely regulated steps202. Productive RNA elongation requires phosphorylation of the RPB1 subunit, and this step was inhibited by most ABU strains200. The bacterial protein NlpD has been identified as an active inhibitor released by ABU and faecal E. coli isolates under normal growth conditions201,203,204. NlpD is internalized by host cells and inhibits gene expression broadly, including effects on several pro-inflammatory mediators.
NlpD was further shown to act as an innate immune inhibitor in UPEC-infected mice treated with the recombinant NlpD protein. Inflammation was suppressed and bacterial clearance accelerated following intraperitoneal NlpD administration201,204 (Fig. 6). The results identify NlpD as an efficient immunomodulator with therapeutic efficacy in a mouse UTI model. Further studies are required to determine the suitability of NlpD for human trials.
Bacterial TIR domain homologues inhibit innate immunity
The evolution of mechanisms that specifically interfere with TLR-mediated immune responses in bacteria is not surprising given the central role of the TLRs in host defence16,82,83,84. TIR domain homologues (TIR-containing proteins (TCPs)) have been detected in bacteria and viruses205,206,207, and ~20% of clinical urinary tract isolates belonging to the B2 clade express the TIR-containing protein TcpC94,206,208. TcpC attenuates signalling cascades defined by the TIR domain of molecules such as MYD88, TIRAP, TRIF, TRAM and IL-1R, inhibiting TLR signalling, IL-1 expression and the STAT–IL-6 signalling pathway94,207,209. TcpC-deletion mutants showed reduced virulence in a mouse UTI model206, suggesting that bacteria are capable of attenuating the TLR-dependent host defence, but the therapeutic potential of TIR domain homologues has not been further explored.
The bacterial Lon protease is a MYC inhibitor
Transcriptional control of the innate immune response is essential, and targeting upstream transcriptional regulators such as IRF7 has a therapeutic potential. The pleiotropic transcription factor MYC controls the expression of about 60–70% of all genes, affecting metabolism, cell growth and survival and inflammatory networks210,211,212 (Fig. 7). MYC is essential for renal development, guiding the fusion of ectoderm and endoderm and regulating renal growth213,214. Infants and children suffering from APN in childhood often show renal growth retardation, consistent with MYC inhibition by infection215 (Fig. 7).
Virulent UPEC strains have been shown to trigger MYC protein degradation and to inhibit the expression of MYC and MYC-related genes in a wide range of human cells216. MYC degradation was shown to be executed by the bacterial Lon protease, which enters human cells and animal tissues216. Treatment with recombinant Lon protease delayed cancer progression in models of bladder and colon cancer and increased long-term survival (Fig. 7), seemingly without any toxic response216. Furthermore, MYC regulated the renal response to infection by affecting the IRF3 and IRF7 transcription factors, suggesting that the protective potential of the MYC inhibitor in APN should be investigated216.
Competitive adherence inhibitors
Fimbriae-specific adherence to host cell receptors is competitively inhibited by soluble receptor analogues. Oligosaccharides that competitively inhibit adherence have shown protective effects in rodent UTI models217,218. Soluble glycolipid receptor antagonists have been shown to inhibit UPEC adhesion by occupying the P fimbrial adhesin PapG and have been shown to reduce bacterial numbers in the mouse UTI model219,220. Using a high-affinity inhibitory mannoside in C3H mice, a reduction in intestinal colonization of FimH and F17 fimbriated UPEC strains was shown while simultaneously protecting against UTIs221. No adverse effects on the intestinal microbiota were observed, suggesting a novel therapeutic approach to treating UTIs while leaving the microbiota unchanged. In early studies, α-methyl-d-mannose was shown to inhibit type 1 fimbrial binding to the bladder mucosa and affect cell shedding161,217,222. Inhibition of the FimH type 1 pilus lectin, was proposed to affect bacterial adherence, immune cell activation and the formation of intracellular bacterial communities in the mouse bladder epithelium223. An earlier study in a rat UTI model showed that low molecular-weight mannosides inhibit the bacterial adherence and persistence218. Oral treatment with active FimH inhibitors was non-inferior to trimethoprim-sulphamen toxazol in mediating bacterial clearance from bladders in an infected UTI mouse model using C3H/HeN mice224. Thus, blocking bacterial adhesion might provide an interesting therapeutic strategy. Nearly all clinical cystitis isolates are FimH positive, but translation to clinical treatment has not been reported.
A number of antibacterial vaccination strategies have been successfully implemented, demonstrating that adaptive immunity can prevent infection by highly virulent organisms225. Vaccines prevent tetanus and diphtheria, as well as infections caused by Haemophilus influenzae and Streptococcus pneumoniae225. The awareness of vaccines and their crucial role for public health has increased dramatically, inspiring further attempts to prevent or treat infections by boosting adaptive immunity. Vaccination strategies are mostly designed to prevent infection, in contrast to innate immunomodulation therapy, which has been examined for therapeutic use.
Vaccination studies in UTI have a long history with varying results226,227,228,229. Early UTI vaccine studies targeted bacterial O-antigens expressed on LPS by the most virulent UPEC strains226,230,231. Clinical studies detected antibodies to a limited number of O-antigens in patients with APN but despite extensive studies in animal models, strong protective effects of vaccination were not observed230. Capsular polysaccharides were also tested as vaccine antigens, in analogy to the Haemophilus influenzae vaccine approach, but substantial protective effects were not achieved231. Antigenic heterogeneity and poor immunogenicity of UPEC capsular polysaccharides complicated vaccine design. Additional vaccine antigens that have been tested in animal models of UTI include the FimH adhesin228, the PapDG protein232, α-haemolysin227 and iron acquisition molecules229,233,234,235 as antigens, but no licensed UTI vaccines are available for use in the USA.
Overall, four vaccines have been tested in human clinical trials, Uro-Vaxom, Urovac, ExPEC4V and Uromune. Uro-Vaxom (OM-89), which contains lyophilized UPEC lysates and is administered as a daily oral tablet, was first approved in Switzerland in 1988 for the prevention of recurrent cystitis236. Urovac (StroVac) is an intramuscular injection containing heat-killed uropathogenic bacteria, including E. coli, Proteus vulgaris, Klebsiella pneumoniae, Morganella morganii and Enterococcus faecalis, and is approved for human use in Europe237. ExPEC4V consists of four conjugated O-antigens from E. coli serotypes O1A, O2, O6A and O25B, common to UPEC strains238. Uromune (MV140) consists of a sublingual preparation of inactivated strains of E. coli, P. vulgaris, K. pneumoniae and E. faecalis239. Retrospective observational studies showed a reduced number of recurrences240, but no randomized controlled trial results have been reported yet. In a study comparing the efficacies of Uro-Vaxom, Urovac and ExPEC4V vaccines in adults with recurrent UTI, Uro-Vaxom but not ExPEC4V reduced the UTI recurrence rate241. However, the daily regimen and toxic effects have limited the widespread use of Uro-Vaxom242.
Interesting vaccine studies have identified siderophores and their receptors as vaccine antigens that trigger a mucosal and systemic immune response and show promising results in mouse UTI models229,234,243. Siderophores are essential iron-acquisition molecules in UPEC strains and have an important role in UTI pathogenesis, enhancing bacterial virulence71,154,244. The potential of siderophores as vaccine antigens was investigated by adding the siderophores Ybt or Aer to the carrier protein cBSA229. A robust adaptive immune response was observed, with protection against bladder and kidney infection in the mouse229.
Potential vaccine candidates have further been selected from a pool of bacterial cell surface proteins expressed during growth in human urine, mouse infection models and confirmed in human infections245,246. Using an immunoproteomics approach, 23 outer-membrane proteins were shown to be immunoreactive247, including four that were prevalent among UPEC isolates248,249. Intranasal immunization with Hma, IutA, FyuA, or IreA, conjugated to cholera toxin, considerably reduced the bacterial burden in the bladder or kidneys after transurethral challenge with UPEC234. As cholera toxin is unsuitable for human use250, other mucosal adjuvants were tested for efficacy, including a double mutant heat-labile E. coli enterotoxin, dmLT251. Intranasal immunization with dmLT-Hma and dmLT-IutA induced antigen-specific antibody production and provided robust protection in immunized mice following transurethral challenge with UPEC243.
The recognition of bacterial adherence as a virulence factor, and secretory IgA antibodies as potent anti-adhesives in patient urine, suggested that mucosal vaccination could be feasible, using fimbriae as antigens27,87,252. Not limited by antigen recognition, secretory IgA possesses broad antibacterial function through carbohydrate receptors for the mannose-specific lectin of type 1-fimbriated E. coli and resulting in agglutination of the bacteria and inhibition of attachment to epithelial cells87. Urinary secretory IgA levels are elevated during symptomatic UTIs and are low in children susceptible to recurrent UTIs in the absence of infection, making low urinary sIgA values a possible marker for recurrent symptomatic infections27,252.
The vaccine concept has been advanced by using the FimH adhesin as the antigen253. In an open-label, dose-escalation phase I trial, 67 women with or without history of recurrent UTIs received intramuscular injections of FimH adhesin on four occasions. The drug was well tolerated with no serious adverse events reported and women with a history of UTIs had a 150-fold increase in FimH antibodies254. These preliminary positive data have led to FDA permission for compassionate use of the vaccine in patients with UTIs caused by multi drug-resistant E. coli6; however, no data on therapeutic efficacy have been disclosed.
Classical questions and obstacles in triggering an adaptive immune response are the antigenic variation of the infecting bacterial strains, problems relating to defining pathogen-specific vaccine antigens, avoiding detrimental effects on the normal flora and identifying target populations suitable for different vaccine candidates. Most vaccination studies focus on creating a successful antibody response, and T cell responses in the bladder have previously received less attention7. However, macrophage depletion has been shown to promote TH1-mediated responses and subsequent bacterial clearance while not affecting TH2 responses35. These observations are supported by the results of a study that showed that bladder infection triggers a robust TH2 cell response, causing re-epithelialization with a limited capacity to clear infection255. Furthermore, immunization with UPEC antigens combined with the TH1-skewing adjuvant CpG was observed to protect mice from developing both single-episode and recurrent UTIs compared with untreated mice256. Intravesical vaccination provided a substantially better response than subcutaneous vaccination and was accompanied by an increase in local TH1 cells. These results suggest that boosting TH1 responses in the urinary tract might enhance protection. Controlled clinical trials are needed to define the protective potential of the different vaccination strategies.
This Review highlights the importance of innate immune control in UTI and the potential of innate immunomodulation therapy to target ‘bad inflammation’ as a cause of disease. This approach differs from the use of broad anti-inflammatory compounds, in that it seeks to correct specific innate immune defects in susceptible hosts. Potent therapeutic effects have been achieved in animal models, and patients with IC/BPS have responded favourably to off-label treatment with an IL-1RA187, but further studies are needed to understand the clinical potential of this approach. Controlled clinical trials are being initiated to improve understanding of the potential of IL-1RA treatment for ACY and important future target populations include patients with infections caused by antibiotic-resistant strains, in whom the need for new therapeutic options is immediately obvious.
Most previous studies of UTI pathogenesis and therapy have been conducted in animals without a clear disease phenotype or established human relevance. Genetic screens of mice with single gene deletions have now established that APN and ACY can be recreated, using single-gene deletions affecting innate immunity. Different innate immune defects distinguish APN from ACY, illustrating the molecular specificity for each organ system. Specific innate immunomodulatory therapeutic approaches have been designed to correct these defects and restore the defence. The disease phenotypes in mice were shown to share important features with human disease, suggesting that the translation of some of these findings into the clinic is of interest.
The finding of accelerated bacterial clearance in the kidneys of mice receiving Irf7 siRNA interference therapy is intriguing and partly unexpected. A similar effect was seen after IL-1RA treatment of ACY, suggesting that reducing inflammation restores immune balance and the antibacterial defence. The mechanisms are not entirely clear, but the findings suggest that the defence is impaired or overwhelmed by the excessive inflammatory response that accompanies disease. Despite a massive neutrophil infiltrate and hyperactive IRF7 or IL-1-dependent genes, the functionality of the defence seems to be lost. This effect was first observed in mCxcr2−/− mice, in which neutrophil activation is impaired and defective exit across the mucosa into the urine creates massive neutrophil retention in the kidneys, leading to massive tissue damage14,79,116. The results also suggest that in addition to restored homeostasis, as yet undefined mechanisms of bacterial clearance might contribute to the resistance to infection in Irf7−/− or Il1b−/− mice and immediate bacterial clearance from their tissues, a fascinating topic for further studies.
The strong effects of innate immunotherapy are promising, as they suggest new potential therapeutic solutions for the treatment of UTI and other bacterial infections. The early work on bacterial adherence and the identification of specific host cell receptors resulted in the realization that mucosal cells respond to infection and a number of host response parameters were identified, establishing the importance of innate immunity in UTI. Further characterization of signalling pathways and transcriptional regulators of the innate immune response led to the identification of specific defence dysfunctions and pinpointed exaggerated inflammation as the cause of acute, severe disease and tissue pathology. The control of these processes at the level of transcription and the involvement of individual transcription factors as arbitrators of protection or disease adds a new perspective. Adding the perspective that these mechanisms can be targeted to treat infection has provided convincing evidence that controlling the innate immune responses can be a potent alternative to antibiotics.
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We gratefully acknowledge the support of the Swedish Research Council, Swedish Cancer Society, HJ Forssman Foundations, Medical Faculty (Lund University), Österlund, Royal Physiographic Society, Network of Excellence: Infect-ERA and ALF grants from the Medical Faculty and Regional Laboratories (Labmedicin Skåne), the European Union’s Horizon 2020 research and innovation programme (grant No 954360) for support to the laboratory infrastructure.
Patents have been filed with the scientists as inventors for the therapeutic use of the NlpD protein, IRF7 siRNA, IL-1R antagonists, MMP and NK1R inhibitors for treating urinary tract infections. The rights to develop these patents are held by SelectImmune Pharma, where the scientists hold shares. Patents for the MYC inhibitor have also been filed for treating cancer and infections.
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Butler, D., Ambite, I., Wan, M.L.Y. et al. Immunomodulation therapy offers new molecular strategies to treat UTI. Nat Rev Urol 19, 419–437 (2022). https://doi.org/10.1038/s41585-022-00602-4