IL-17 and IL-23 in lupus nephritis

Renal activity and histopathology

All patients had an active nephritis at baseline with biopsies showing WHO class III
(n?=?15), III/V (n?=?4), IV (n?=?24), V (n?=?8) and one glomerular vasculitis. All
patients had high renal disease activity, 49/52 had renal BILAG A and 3/52 BILAG B.
Forty-six of the patients were female (88%) and 6 were men (12%), mean age was 32 years
(range 18–59).

The patients were treated in accordance with standard therapy for LN; corticosteroids
combined with intravenous cyclophosphamide (n?=?40), mycophenolate mofetil (n?=?9)
or rituximab (n?=?2). One patient was treated with azathioprin. The treatment regimen
for cyclophosphamide was 0.5-1 g/m² monthly as modified from the NIH-protocol 24]. At the time for the first renal biopsy, 36/52 (69%) of the patients were treated
with prednisolone, median dose 20 mg/day (range 2.5-60). At start of immunosuppressive
therapy, all but one (98%) were treated with prednisolone with median dose 40 mg/day,
doses ranging from 2.5 to 80 mg/day as decided by the treating physician, and were
thereafter successively tapered. At repeat biopsies, 98% of the patients were still
treated with prednisolone, median dose 10 mg/day (range 2.5-30).

Follow-up biopsies showed WHO class I (n?=?1), II (n?=?18), III (n?=?9), III/V (n?=?1),
IV (n?=?8) and V (n?=?14) and one had developed a renal vasculitis (seen in a patient
with class III at first biopsy). The patient with a vasculitis pattern at first biopsy
had class II at repeat biopsy. Nineteen patients (36%) were regarded histopathological
responders (class I/II). The renal activity index decreased significantly (0.001)
whereas there was an increase in the chronicity index (p??0.001).

Levels of C3 and C4 increased (p??0.001) and there was a decrease in proteinuria
at follow-up (p??0.001) whereas no overall difference in creatinine levels was found.

At follow-up, 8/52 had renal BILAG A, 21/52 BILAG B, 11/52 BILAG C and 12/52 had BILAG
D. Twenty-two patients (42.3%) were regarded as CR, 20/52 (38.5%) as PR and 10/52
(19.2%) were regarded NR according to BILAG. The clinical characteristics of patients
and nephritis data at baseline and follow-up are presented in Table 1.

Table 1. Patient characteristics at baseline and follow-up

Serum cytokines

Most baseline levels of cytokines were increased in patients vs. controls, this was
highly significant for IL-6, IL-10, IL-17, IL-23 (p??0.001 for all) and also significant
for IFN-? (p?=?0.03). TGF-? was lower in patients vs. controls (p??0.001).

Overall cytokine levels decreased after treatment, this was significant for IL-6 (p??0.001),
IL-10 (p?=?0.02) and IL-17 (p?=?0.01), for IL-23 there was a trend towards lower levels
at follow-up (p?=?0.06). For TNF-? and IFN-? there was an overall decrease in serum
levels although not statistically significant (ns), while for TGF-? an increase was
documented (p?=?0.005). No difference in cytokine levels was found comparing the different
immunosuppressive treatments or doses of prednisolone at first and repeated biopsies
(data not shown). Cytokine levels in patients and controls are presented in Table 2.

Table 2. Baseline levels of cytokines in patients at baseline vs. controls, and baseline vs.
follow-up levels in patients

Cytokines in association to laboratory findings

At baseline there was no correlation between proteinuria and levels of any of the
cytokines whereas at follow-up, a positive correlation was documented for IL-23 and
proteinuria (r?=?0.34, p??0.05). Patients with persisting urine-albumin excretion??0.5
grams/day at follow-up had higher IL-23 as compared to patients with 0.5 grams/day
(median 5.27 vs. 2.29 pg/ml, p?=?0.05). The group of patients with very low-grade
proteinuria at follow-up (urine albumin 0.2 g/day), had more significantly lower
levels of IL-23 (p?=?0.01) compared to patients with???0.2 g/day.

A weak inverse correlation was documented at baseline for C3 and IL-10 (r?=??0.31,
p??0.05) and at follow up for C3 and IL-23 (r?=??0.44, p??0.05), TNF-? (r?=??0.36,
p??0.05) and IFN-? (r?=??0.30, p??0.05) whereas no correlations were found for C4.

There was no correlation between serum creatinine and any of the investigated cytokines.

Cytokines in association to histopathology

First we studied the cytokines in association to histopathological findings at both
first and repeated biopsies. There was no difference in baseline cytokine levels between
patients with class III, IV (proliferative nephritis, PN) vs. class V (membranous
nephritis, MN) at baseline biopsies.

Patients with a poor histopathological response, i.e. with a persisting active nephritis
after immunosuppressive treatment, had significantly higher baseline levels of IL-17
compared to patients with WHO I or II at follow-up (median 111.0 vs. median 53.1 pg/ml)
(p?=?0.03) (Figure 1a). The highest baseline levels of IL-17 were seen in patients with MN at repeat biopsy
(median 146.5 pg/ml) (Figure 1b). No significant differences in baseline levels of any of the other cytokines were
found between histopathological responders and non-responders.

Figure 1. Baseline levels of IL-17 were higher in patients with an unfavourable histopathological
outcome. (a) Baseline levels were significantly higher in patients who had a persisting active
nephritis (WHO class III, IV or V) at follow-up, i.e. WHO non-responders, vs.WHO responders
(WHO I or II). (b) Baseline levels of IL-17 in patients in relation to histopathology at follow-up.
The patient with vasculitis at repeat biopsy was here included in the class III-IV
group. The highest levels were seen in patients with a memranous nephritis, WHO class
V, at follow-up. Boxes limits show 25^th to 75^th percentile and median values are marked inside the boxes.

Patients displayed higher IL-17 both at baseline (p??0.001) and follow-up (p?=?0.01)
compared to controls (Figure 2a). Patients with the highest levels of IL-17 at baseline (arbitrarily defined as
165 pg/ml and encompassing the upper quartile), also had higher levels of IL-23 (p?=?0.007),
TNF-? (p?=?0.005) and IFN-? (ns) vs. those with IL-17 levels 165 pg/ml (Figure 2b, c and d). Of these patients, 11/13 (85%) were histopathological non-responders.
At follow-up, 8/52 patients had persistently high IL-17 levels as defined above (Figure 2a), this subgroup also had higher levels of TNF-? (p?=?0.0001) and IFN-? (p?=?0.0006)
(Figure 3a and b) and were all histopathological non-responders (WHO III n?=?4, WHO V n?=?3
and one with glomerular vasculitis).

Figure 2. Serum levels of IL-17 in patients and controls. Patients with the highest baseline
levels had higher levels of IL-23, TNF-? and IFN-?. (a) Serum levels of IL-17 in patients at baseline and follow-up in patients and levels
in controls (medians 97.4, 48.0 and 3.3 pg/ml respectively). The dotted line denotes
the upper 25% of IL-17 levels (above 165 pg/ml). The patients with the highest baseline
levels of IL-17 (165) pg/ml had higher levels of IL-23 as shown in (b), TNF-? (c) and IFN-? (d) vs. those with IL-17 levels??165. Boxes limits show 25^th to 75^th percentile and median values are marked inside the boxes.

Figure 3. Patients with persisting high levels of IL-17 at follow-up had higher levels of TNF-?
and IFN-?. Patients with persisting high levels of IL-17 (165 pg/ml) had significantly higer
levels of TNF-? (a) and IFN-? (b) vs those with IL-17??165 pg/ml. Boxes limits show 25^th to 75^th percentile and median values are marked inside the boxes.

Cytokines in association to clinical response

We also analysed cytokine levels in association to clinical response according to
the definition used.

Patients displayed higher IL-23 levels compared to controls both at baseline (p??0.001)
and follow-up (p??0.001) (Figure 4a). At follow-up, BILAG-non-responders (NR) had significantly higher levels of IL-23
(median 10.6 pg/ml) vs. complete responders (CR) (median 2–6 pg/ml, p?=?0.02) (Figure 4b). This was even more pronounced in the subgroup of patients with MN at follow-up
(n?=?14), in which the non-responding patients had higher IL-23 (median 10.9 pg/ml)
vs. both PR (median 5.0 pg/ml, p?=?0.05) and CR (median 1.2 pg/ml, p?=?0.01) (Figure 4c).

Figure 4. Follow-up levels of IL-23 were higher in patients with an unfavourable BILAG response.
(a) Serum levels of IL-23 in patients at baseline and follow-up in patients and levels
in controls (medians 5.5, 3.3 and 0.7 pg/ml respectively). (b) Levels of of IL-23 in all patients were significantly higher in BILAG non-responder
(NR) patients vs. partial- (PR) and complete responders (CR), this was most pronounced
in the group of patients with WHO class V at follow-up as shown in (c). Boxes limits show 25^th to 75^th percentile and median values are marked inside the boxes.

No significant difference between CR-, PR- and NR-patients was found for any of the
other cytokines.

Correlations between cytokines

At baseline, IL-17 correlated with TNF-? (r?=?0.63, p??0.05), IFN-? (r?=?0.49, p??0.05)
and IL-23 (r?=?0.30, p??0.05). There was also a correlation between TNF-? and IFN-?
(r?=?0.71, p??0.05).

At follow up, IL-17 correlated with IFN-? (r?=?0.80, p??0.05) and TNF-? (r?=?0.79,
p??0.05). There was also a correlation between TNF-? and IFN-? (r?=?0.80, p??0.05).

Immunohistochemistry

Immunostaining demonstrated expression of IL-17 in all the 6 examined biopsies from
LN patients (4 with class V and 2 with class IV). The staining was most pronounced
in areas of inflammatory infiltrates of CD3+ T-cells (Figure 5). There was no clear difference observed in the amount of IL-17 comparing PN and
MN. In renal tissue from control kidney biopsies, IL-17 staining was negative (data
not shown).

Figure 5. Immunostaining of IL-17 in renal tissue. The figure demonstrates a kidney biopsy from a patient with lupus nephritis WHO class
V. Representative micrographs displaying (A) an inflammatory infiltrate with T cells as demonstrated by a positive CD3-staining
and in (B) the same infiltrate from a consecutive section stained with irrelevant isotype control
antibody. In (C), IL-17 staining is demonstrated, predominantly found in the inflammatory infiltrate
shown in A, and (D) demonstrates staining with the corresponding isotype control antibody. Original magnifications:
12.5×.