The pattern recognition receptors, complement system, inflammasomes, antimicrobial peptides, and cytokines are innate immunity soluble factors. They sense, either directly or indirectly, the potential threats and produce inflammation and cellular death. High interest in their modulation has emerged lately, acknowledging they are involved in many cutaneous inflammatory, infectious, and neoplastic disorders. We extensively reviewed the implication of soluble factors in skin innate immunity. Furthermore, we showed which molecules target these factors, how these molecules work and how they've been used in dermatological practice. Cytokine inhibitors have paved the way to a new era in treating moderate to severe psoriasis and atopic dermatitis.
Cytokines represent a family of small proteins (<40 kDa) released by most of the cells, mainly macrophages and helper T cells, that display a specific effect on the immune response. They can act on distant cells, nearby cells (a paracrine mechanism), or the same cells that secrete them (an autocrine mechanism) . Both pro-inflammatory and anti-inflammatory cytokines are secreted during the immune response, however, their effects depend on the targeted cells .
Keratinocytes also produce many cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), interleukin-20 (IL-20), and tumor necrosis factor-alpha (TNF-α) . It was proved that various environmental molecules such as sodium lauryl sulfate, phenol, and croton oil induce IL-8 and other proinflammatory cytokines expression, production, and release in human keratinocyte cultures, suggesting their importance in skin inflammation . HPV infection, especially high-risk strains, was linked with increased expression of IL-6 in infected keratinocytes promoting local tumorigenesis .
Melanocytes are neuroimmune cells that express type I IFN as an early innate response to viruses. They respond against pathogens using PRRs and can initiate cell death accompanied by IFN-b, TNF-a, IL-6, and IL-8 expression, as a response to some viral analogs . Nevi and thin primary melanomas show little expression of TNF-α, transforming growth factor-β (TGF-β), and IL-8, while a marked-up regulation of cytokines and growth factors can be noticed in thick primary melanomas and metastatic melanomas . The melanoma inhibitor of apoptosis protein (ML-IAP) is an important regulator of apoptosis that inhibits caspase activation and TNF signaling, frequently overexpressed in melanoma cells, but with low expression in normal melanocytes. Expression of ML-IAP has been proved to enhance apoptosis resistance in melanoma by binding caspases 3, 7 and 9, and to provide melanoma cells a higher endurance during tumor progression . Although melanoma cells possess this apoptosis-escape mechanism, the roles of non-apoptotic cell death signaling pathways, such as autophagy-dependent cell death, necroptosis (caspase-independent cell death, similar to necrosis), ferroptosis (iron-dependent lipid-peroxides induced cell death), pyroptosis (via inflammasomes), and parthanatos (mitochondrial accumulation of poly(ADP-ribosyl)ated proteins’ induced cell death) are of major importance as well .
Many cytokines produced by other resident skin cells such as macrophages, NK cells, dendritic cells (Figure 4), or endothelial cells, act at numerous levels to control the cornification process, intercellular adhesion, or the expression of some genes that encode important enzymes. Skin barrier regulation is dictated by the interaction between ligands (cytokines) and cytokine receptor complexes, activating several signaling pathways such as Janus kinase/ Signal Transducer and Activator of Transcription (JAK/ STAT), Phosphoinositide 3-kinase (PI3K/AKT), Mitogen-activated protein kinases (MAPK) and Nuclear Factor kappa-light-chain-enhancer of activated B cells (NFκB) .
Figure 4. Cytokines release during skin immune response and in thick primary melanomas. Keratinocytes, melanocytes, and other innate immunity cells, such as NK cells, dendritic cells and macrophages secrete cytokines that may serve both as pro-inflammatory and anti-inflammatory molecules or may trigger carcinogenesis (IL-6). Thick primary melanomas secrete IL-8, TNF-α, and TGF-β, suggesting a high synthesis rate within neoplastic cells.
In addition to their role in preventing infection, TLRs possess both tumorigenic and anti-neoplasia effects. TLRs expressed on the cancerous cells’ membrane promote invasion and metastasis in some cancers, while they inhibit neoplasia progression in others .
Initially known as S-26308 or R-837, Imiquimod (1-(2-methyl propyl)-1H-imidazo-[4,5-c]-quinoline-4 amine) is an imidazoquinoline amine analog to guanosine (Figure 5), approved for the treatment of actinic keratosis (AK), superficial basal cell carcinoma (BCC) and anogenital warts (condyloma acuminatum), but is widely used in some other skin disorders as well. Imiquimod binds to the TLR-7 localized on the surface of Langerhans cells, dendritic cells, and monocytes and induces TNF-α and IFN-α secretion, proving its antiviral and antitumor effect . Huang et al. previously showed that Imiquimod could induce immunogenic cell death (ICD) in vivo and in vitro. Imiquimod-induced ICD consists of reactive oxygen species (ROS) production in cancer cells. ROS trigger endoplasmic reticulum stress and subsequently promotes surface exposure of calreticulin, ATP dependent autophagy, and postapoptotic release of high mobility group box-1 protein (HMGB1). HMGB1 eventually promotes maturation of antigen-presenting cells and tumor antigen presentation .
Figure 5. Imiquimod chemical structure (central) and approved and off-label use of Imiquimod in skin diseases. Imiquimod is an amine analog to guanosine, currently approved for topical treatment of BCC, KA, and anogenital warts. Nowadays, in dermatological practice, imiquimod is an off-label drug used to treat many skin diseases, such as inflammatory skin disorders, neoplastic diseases, or skin infections. SCC=squamous cell carcinoma; DLE=discoid lupus erythematosus.
7.1. Imiquimod in basal cell carcinoma (BCC)
A 2019 meta-analysis on 4256 patients compared the safety and efficacy of imiquimod with other treatments in patients with BCC (including vehicle, excisional surgery, cryosurgery, 5-fluorouracil, methylamino levulinate photodynamic therapy). Imiquimod associated significant higher histological clearance rate (RR = 9.28, 95% CI: 5.56, 15.49; p < 0.001) and complete response rate (RR=3.15, 95% CI: 1.55, 6.38; p=0.001). Imiquimod was slightly inferior when comparing the success rates (RR=0.93, 95%CI: 0.90, 0.96; p < 0.001). The authors suggest that imiquimod should be used as the first choice of treatment for BCC . A multicentre, non-inferiority randomized controlled trial (RCT) with 501 participants with nodular and superficial basal cell carcinoma who were divided in two groups (imiquimod group, 254 participants and surgical excision group, with 247 participants) concluded that there is no obvious difference between groups regarding the cosmetic outcome, but treatment successfulness for imiquimod was inferior to surgery (88% in imiquimod group, comparative to 98% in surgery group) . According to the European consensus published in 2019, Imiquimod is particularly useful in low-risk single or multiple superficial BCC, with limited evidence on nodular BCC, but with a better response comparative to methyl-aminolevulinate photodynamic therapy for superficial BCC .
7.2. Imiquimod in actinic keratosis
Ogawa et al. have used an antibody specific to plasmacytoid dendritic cells to investigate whether imiquimod stimulates these cells’ recruitment to AK lesions. Histological staining demonstrated that plasmacytoid dendritic cells were significantly more abundant after the topical treatment and their number inversely correlated with healing duration, indicating that these cells are critically involved in the regression of AK during imiquimod treatment .
In a randomized trial, imiquimod cream 5% effectiveness in AK was compared with other three topical treatments (ingenol mebutate gel 0,015%, fluorouracil cream 5%, methyl aminolevulinate photodynamic therapy). The study concluded that 5% fluorouracil was significantly more effective than imiquimod, MAL-PDT, or ingenol mebutate at 12 months for multiple AKs . In a meta-analysis of five randomized double-blinded trials treating 1293 patients with AK (with almost 90% men) complete clearance occurred in 50% of patients treated with imiquimod and most adverse events were local, most frequently erythema, scabbing, and flaking, but serious adverse events were not significantly different between imiquimod or vehicle control, with a relative risk of 1.2 (0.7–2.0) .
An article from 2015 suggested that imiquimod cream 3,75% is the only effective treatment across a large sun-exposed field (full face or balding scalp), for both clinical and subclinical AK (unmasked by imiquimod during treatment). The median percentage decline in lesions was 92%, and the efficient clearance of both clinical and subclinical lesions led to unremitting lesion clearance for at least 12 months. The authors concluded that imiquimod can reveal patients’ entire burden of disease and SCC risk .
7.3. Imiquimod in condyloma acuminatum
A 2020 meta-analysis included all the RCTs that evaluated topical treatments for external genital warts and revealed podophyllotoxin 0,5% solution is significantly more effective compared to imiquimod 5% cream. However, it was associated with a higher overall adverse event rate. None of the above treatments were significantly different from each other in terms of relapse. .
In genital (penile) warts, imiquimod clearance rates are similar to those of podophyllotoxin, if applied three times per week until total clearance or for a maximum of 16 weeks for imiquimod cream 5%, respectively every night for up to 8 weeks for imiquimod cream 3,75%. The treatment surface should be washed six to ten hours after application to minimize local adverse events . In a RCT comprising 228 patients, an eight-week combination therapy of 400mg oral zinc sulfate with topical imiquimod for vulvar warts significantly decreased relapse after six months .
7.4. Imiquimod off-label use
Off-label use of imiquimod is not rare in our daily practice. Imiquimod possesses an immunomodulatory effect in neoplastic, infectious, and inflammatory skin disorders .
Several clinical studies and case reports investigated imiquimod efficiency in Bowen disease. A retrospective study of 49 patients (96% males, 96% Caucasians, various body locations) concluded that 86% had a complete clinical response with topical imiquimod . In an open-label study sixteen patients with a single biopsy-proven lesion that was 1 cm or larger in diameter (up to 5,4 cm) assigned to once-daily self-application of imiquimod 5% cream for 16 weeks. Histological clearance was obtained in their 6-week posttreatment biopsy specimens in 93% of the lesions . However, more extensive studies are necessary to compare imiquimod with other nonsurgical therapies in Bowen disease.
Resembling condyloma acuminatum, but with the histopathological appearance of Bowen’s disease, Bowenoid papulosis of the genital area can display a good response to topical imiquimod 5% , but the results are scarce, in few case reports. An almost complete regression to topical imiquimod in monotherapy was obtained in HIV patients . Favorable results are obtained in Bowenoid papulosis patients who are HIV positive, when combining oral acitretin with topical 5% imiquimod .
With the cost of moderate burning sensation, imiquimod 5% can be an alternative for surgical and invasive procedures, which may provide poor cosmetic and functional outcomes since this type of in situ carcinoma is usually located on the glans of the penis . In negative polymerase chain reaction (PCR) testings for HPV, imiquimod efficiency is most likely explained by cytokines’ activation, with antitumor effect, as shown in BCC .
Complete surgical excision is the treatment of choice, but poor cosmetic outcomes may arise. Thus, conservative treatments using podophyllin, 5-fluorouracil, imiquimod, or even intralesional injection of methotrexate or corticosteroids may be beneficial. Topical imiquimod 5% was successfully used in some solitary facial KA , and low-dose acitretin association was documented to promote complete resolution in multiple KAs patients . Contrariwise, a retrospective study showed that conservative treatments led to significantly higher recurrence rates when compared to surgical treatment .
The available studies do not currently endorse topical treatments in SCC; therefore, surgical excision is the preferred treatment . When applied over large surfaces, inflammation may occur, decreasing topical imiquimod usefulness in field cancerization. 5-fluorouracil is preferred instead .
Topical imiquimod 5% can be used as a second-line treatment in lentigo maligna, particularly in large tumors, which are located on the face, ears, or scalp, especially in poor surgical candidates and old patients. Adjuvant imiquimod after complete surgical excision showed improved outcomes in terms of local recurrence . Other skin malignancies that may respond to topical imiquimod include extramammary Paget disease (alternative treatment to surgery) , the classical form of Kaposi disease (safe and suitable for home application) , folliculotropic mycosis fungoides , or melanoma skin metastases (palliative therapy, favors metastases resolution) .
On the one hand, a recent case-control study on 48 pediatric patients recommends topical imiquimod as a first-line treatment in children with disseminated lesions . On the other hand, some scientists found the use of imiquimod in children with molluscum contagiosum not only inefficient but also hazardous, considering high systemic absorption, hematological abnormalities , and even pemphigus-like eruption as side-effects .
Acyclovir is a drug extensively used to treat HSV infections. Some recent studies have even investigated vaginal acyclovir delivery methods . In few case reports, immunocompromised patients with acyclovir-resistant anogenital herpes simplex were reported to respond to topical administration of imiquimod, thus avoiding the intravenous administration of toxic antiviral drugs, such as cidofovir or foscarnet . Imiquimod interacts with the adenosine receptor A1, activating the protein kinase A pathway and upregulating cystatin A synthesis in infected nonimmune cells, proving an IFN-independent antiviral mechanism . Herpes simplex virus (HSV) can manipulate TLR and evade the innate immune response. Recent studies showed that HSV‐mediated TLR signaling enables virus replication and suppresses interferon production via NF‐κB signaling . Another double-stranded DNA virus, varicella-zoster virus, can induce proinflammatory cytokines upon activation of TLR-2, while TLR-2 abnormalities were associated with an increased number of local recurrences in patients with genital HSV infection .
Several case reports proved that porokeratosis of Mibelli has a favorable response to topical imiquimod 5%, both in children and elders, including some severe cases, nonresponsive to prior cryotherapy or 5-fluorouracil . It is also beneficial in some patients with disseminated or linear porokeratosis. Some authors suggest that imiquimod may modulate p53 tumor suppression protein and have reported overexpressed or mutant p53 protein in porokeratosis lesions; therefore, a p53-dependent mechanism may explain imiquimod efficiency in porokeratosis .
Imiquimod's effectiveness in morphea is attributed to its capacity to induce local interferon synthesis, which inhibits collagen production by human fibroblasts, as shown in other sclerodermoid disorders. A prospective, open-label, double-baseline study from 2011 showed that topical imiquimod 5% is effective and safe in children with plaque morphea. A decrease in the thickness of the plaques was quantified using ultrasound . Contrarily, a 2015 study that enrolled 25 adult patients with plaque morphea showed no ultrasonographic differences in thicknesses between the treatment and vehicle groups, but induration demonstrated a favorable response .
Following imiquimod 5% treatment, patients with generalized DLE showed remission, after 20, respectively 24 applications, in two case reports . Nevertheless, imiquimod-induced cutaneous lupus erythematosus has been reported in the literature .
Complement pathways are associated with several skin disorders, such as bullous pemphigoid, psoriasis, urticaria, urticarial vasculitis, hidradenitis suppurativa, vasculitis, and lupus erythematosus. Consequently, the interest in producing complement-specific molecules has been increasing . Intravenous immunoglobulin (IVIG) binds C1q, C3, and C4, preventing C1 attack and C3 and C4 in situ deposition, respectively. IVIG also downregulates C3 convertase activity, therefore it is successfully used in some small-medium-vessel vasculitides . Eculizumab and its long-lasting form, ravulizumab, are monoclonal antibodies that bind to C5 and inhibit its cleavage by C5a, both approved for paroxysmal nocturnal hemoglobinuria . IFX-1, an anti-C5a inhibitor, was evaluated in a trial involving 12 participants with hidradenitis suppurativa (NCT03001622); however, no results are currently available .
Canakinumab is an interleukin-1 monoclonal human antibody that can successfully control and prevent flares in patients with colchicine-resistant familial Mediterranean fever, mevalonate kinase deficiency, and tumor necrosis factor receptor-associated periodic syndrome, as proved in a 2018 study . Canakinumab is additionally effective in Schnitzler syndrome, with the cost of infection as a frequently encountered adverse event , and can also represent a therapeutic option for steroid-resistant pyoderma gangrenosum . Some cases of pyoderma gangrenosum remain refractory to this treatment . Anakinra is an IL-1 inhibitor that demonstrated a decreased disease activity of moderate to severe hidradenitis suppurativa in a five-patient open-label study .
Other interleukin inhibitors were approved for atopic dermatitis and psoriasis (Figure 6). Dupilumab is an IL-4/ IL-13 inhibitor approved in moderate-to-severe atopic dermatitis . Risankizumab, guselkumab, tildrakizumab (p19 subunit of IL-23 inhibitors), ustekinumab (IL-12/ 23 inhibitor), ixekizumab, brodalumab, and secukinumab (IL-17 inhibitors) are the modern biologics approved in moderate-to-severe plaque psoriasis. TNF-α inhibitors, such as infliximab, adalimumab, certolizumab, and etanercept, may similarly represent a good alternative in patients with moderate-to-severe psoriasis . Most biologics used in psoriasis show no difference regarding short-term efficacy and tolerability .
Figure 6. Drugs that target innate immunity soluble factors (others than PRRs). Complement modulators and biologics target innate immunity soluble factors. Dupilumab inhibits IL-4/IL-13 and is approved for use in moderate-to-severe atopic dermatitis. IL-23, IL-17, IL-12/ IL-23 inhibitors, and TNF-α inhibitors are approved for use in moderate-to-severe plaque psoriasis. Canakinumab and anakinra inhibit IL-1. Eculizumab, ravulizumab, IFX-1, and IVIG inhibit complement pathways.
Imiquimod, a TLR-7 ligand, is currently approved for BCC, AK, and condyloma acuminatum, but there is an emerging interest in using imiquimod for other skin cancers, infections, and inflammatory skin conditions. Nevertheless future prospective studies are warranted to provide clear guidelines regarding the off-label uses of Imiquimod. Newly discovered IL-1 inhibitors, canakinumab and anakinra, exhibit a consistent inhibition of the abnormal inflammasome assembly, providing optimistic results in managing patients with CAPS and Schnitzler syndrome. Cytokine inhibitors have opened the door to a new era in treating moderate to severe psoriasis and atopic dermatitis. Given their role in a variety of dermatological diseases, further research is warranted.